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With the launch of the Apprenticeship Levy drawing ever nearer, much has been made of its impact on businesses, but its impact on educational institutions has been somewhat overlooked.
In this guide, we'll take an in-depth look at the effects of the Apprenticeship Levy on schools and trusts in England.
The Apprenticeship Levy, multi-academy trusts and large schools
As covered in our detailed Apprenticeship Levy Guide, the legislation will affect any organisation that has a payroll of more than £3 million. Those who fall into this bracket will be required to pay 0.5 per cent of their annual pay bill into the Levy fund (minus a £15,000 allowance).
For organisations like multi-academy trusts, which may encompass several schools, this also means that they'll only get one £15,000 allowance to share between their constituent parts. While there is a degree of flexibility in terms of how this can be used, it puts connected organisations like these at something of a disadvantage when compared to stand-alone conventional educational institutions.
The government has enacted this initiative as part of its target of generating three million apprenticeship starts by the end of the decade. It's a response to the findings of the Richard and Sainsbury reviews, which highlighted a lack of productivity in the UK, as well as an developing skills gap that extant apprenticeships were failing to address.
Although the Levy aims to get employers to contribute to apprenticeship training, those that pay in will be able to access their funds, plus a ten per cent top-up from the government, via the Digital Apprenticeship Service and spend this sum on apprenticeship-based training.
A raft of new apprenticeship standards are also being unveiled, which aim to better-meet the needs of employers and are being designed with direct input from industry bodies. Similarly, previous barriers to eligibility (like age and prior qualifications) are being broken down, which means there'll be a wealth of ways to utilise apprenticeship training across a variety of fields.
Public sector apprenticeship targets
In addition to the Levy, schools and multi-academy trusts with 250 or more staff will also be required to take on new apprentices, or utilise new standards to upskill existing employees via apprenticeship-based training each year.
These regulations, which are part of the Enterprise Act and were confirmed following a consultation last year, will come into force in April 2017 - coinciding with the launch of the Levy - and will require organisations within their scope to employ the equivalent of 2.3 per cent of their workforce as apprentices each year.
This target will apply to all bodies that are classed as being within the public sector, however, there are some notable exceptions, including the BBC, Channel 4 and the Post Office. Financial institutions are also exempt from the rules although it's not clear what exact definition the government is using on this front.
The consultation also omitted colleges from its list of public sector organisations, since these were reclassified in 2012 from government-controlled bodies to non-profit institutions.
What educational institutions will be affected by the Levy?
Any organisation with more than £3 million in annual payroll will have to contribute to the Levy. The government estimates that this will only affect around two per cent of employers in the UK, however, it's likely that the majority of maintained schools, whose employer is classed as the local authority, multi-academy trusts and stand-alone academies at the larger end of the scale will be affected.
As detailed in our Apprenticeship Levy Infographic, affected organisations will have to work out what they need to pay and let HMRC know on a monthly basis before contributing via PAYE. The first payment will be due in May 2017, although depending on exact payroll figures, it's possible that some organisations won't have to pay the Levy consistently month on month.
What options do schools and trusts have?
While this double whammy of legislation comes at a time when many educational institutes are already tightening their fiscal belts, the Levy could present a cost-saving opportunity for at least some schools.
However, the NAHT union warned that many schools were not aware that they fell within the scope of the Levy and emphasised the need for them to take action:
"With this new obligation on schools and academies, it is critical that they mitigate the impact by ensuring that the money they're paying out comes back into their institution. Schools and academies need to start thinking now about how they can best utilise the apprenticeship levy," it said.
By fully engaging with the Levy and using funds paid in to offset current training spend, it's possible for educational institutes to get out more than they put in. Levy payers will have 24 months to use their funds, which will sit in their Digital Apprenticeship Service account, and can direct these to the training provider of their choice via the platform.
Levy funds can be used to cover any apprenticeship-related training costs (subject to an upper limit on each standard set by the government), however, they will not cover salaries. There's also a range of additional incentives and funding additions available for taking on younger apprentices, those from deprived areas or troubled backgrounds.
As mentioned, barriers to eligibility are also being broken down, which means that taking on an apprentice doesn't necessarily mean hiring new entry-level staff. There's already a variety of apprenticeships that schools can utilise to train up non-teaching staff, like business managers, teaching assistants and custodial employees - and more of these Trailblazer standards are currently in development, with all existing frameworks set to be replaced by 2020.
And you?
Hopefully, we've managed to shed some light on how the Apprenticeship Levy will work for schools and academies, but if you've got any specific questions - be sure to let us know via Twitter or LinkedIn.
And if you're a Levy payer in the public sector looking for further advice, be sure to get in touch with our team of Levy experts for an obligation-free chat today:


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Genetics of hypertriglyceridemia
Nature Genetics
July 26, 2010
Common and rare genetic variants associated with hypertriglyceridemia ― where the concentration of triglyceride blood lipid is above the ninety-fifth percentile ― are reported online this week in Nature Genetics, The study suggests that both common and rare genetic variants found in the population contribute to susceptibility to hypertriglyceridemia.
Robert Hegele and colleagues report a genome-wide association study in which they identify association of hypertriglyceridemia with common variants at four genes. They sequenced these four genes in individuals with hypertriglyceridemia and in controls and identified a significant excess of rare variants in individuals with hypertriglyceridemia compared to controls.

subset: commoncrawl


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If I continue to blog at this rate I will average an impressive two photos a year. Considering the fact that I have thousands of them on my hard drive I've made a new years resolution to increase output a little. I'll be posting photos of the last six months of 2008 mainly. I took one in Rinville park.
One Comment
Nice shot Ken. Good to see new pics going up on the site!! (I don't think two a year is a great target to have )


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The West is raising the threat of possible use of Strategic Weapons of Mass Destruction (WMD) by bin Laden brigade since bin Laden and his deputy have issued edicts to justify the use on the basis of certain interpretations of Quran and Hadith. Journal of International Security Affairs was probably the first to give an in-depth analysis of Islamic reaching and the designs of al Qaeda duo to acquire and use WMD. As far as their edict is concerned, they do not qualify to issue edicts as per Islamic teachings. It is, however, clear from their edict that they could not find any clear justification from Islamic traditions and basic injunctions; they only twisted some historic events in support of their designs. The last sermon delivered on the conclusion of Hajj this week clearly issued a formal edict that terrorist activities are not sanctioned by Islam and that those who indulge themselves in the acts of terror cease to be Muslims. This should take away the justification, whatsoever that al Qaeda duo have in support of their designs.
The world should understand the fact that the only way to render al Qaeda ineffective is to strip it of the justification it carries for its acts of slaughtering innocent citizens; unjust support by USA of Israel at the cost of Palestinians and of autocratic rulers in the Muslim world. It was discussed separately while listing down 7Deadly Scenarios that USA will always remain exposed to the risk of troubles due to its failure to recognize that its international meddling has unintended consequences. Basil & Spice in its review of the book on 7 Deadly Scenarios urges USA to recognize that Israel has already brought it the Arab Oil Embargo of the 1970s and 9/11, and repeatedly embarrassed the U.S. by its abuse of the Palestinians. Similarly, USA's nuclear support for India to 'balance' China and Pakistan creates new trip-wire risks of serious military conflict.
An article appeared in the latest issue of Foreign Policy Magazine saying that evidence for al Qaeda's intentions aren't hidden in encoded communications or classified intelligence. Quite the opposite: They're hidden in plain sight. Just as Osama bin Laden issued a fatwa to declare war on the United States in 1998, his deputy, Ayman al-Zawahiri, issued a fatwa a decade later to herald a prospective next stage in the conflict. If we take him at his word, some day jihadists will use weapons of mass destruction to change history once and for all. Of course, al Qaeda leaders have spoken of acquiring weapons of mass destruction for well over a decade. They have had little observable success in achieving their goals of producing a nuclear bomb or biological weapon capable of producing mass casualties. Fortunately, it is extremely difficult, but not impossible, for a terrorist group to acquire a strategic weapon of mass destruction (WMD). Nonetheless, the al Qaeda core has kept at it over the years, in the hopes that time and opportunity will enable it to overcome the daunting challenges in this regard.
According to the article Zawahiri chose to release a book in 2008 titled Exoneration. In it, he resurrects a fatwa issued by senior Saudi cleric Nasir al-Fahd in May 2003 -- notoriously, the only such treatise that ever endorsed the use of WMD. Zawahiri adopts Fahd's ideas wholesale. He uses the same ideas, thoughts, examples, and scholarly citations to reach the same conclusion: The use of nuclear weapons would be justified as an act of equal retaliation, "repaying like for like."
Zawahiri raises key Quranic themes to sweep away all potential objections to the use of WMD. He offers answers to questions about the legality of killing women, children, and the elderly; the justice of environmental destruction; the morality of harming noncombatants; the tactical prudence of attacking at night; and analyses of deterrence. Zawahiri adopts Fahd's examples verbatim: The Prophet Mohammed's attack on the village of al-Taif using a catapult, for instance, permits the use of weapons of "general destruction" incapable of distinguishing between innocent civilians and combatants.
The take-away from Zawahiri's book is that the use of weapons of mass destruction should be judged on intent rather than on results; if the intent to use WMD is judged to be consistent with the Quran, then the results are justifiable, even if they clearly violate specific prohibitions under Islam. The same reasoning is applied in a detailed explanation of such matters as loyalty to the state, contracts, obligations, and treaties; the permissibility of espionage; and deception and trickery. For example, on the topic of Muslims killed in combat unintentionally in the fight against infidels: "When Muslims fight nonbelievers, any Muslim who is killed is a martyr."
The article goes on to say that Zawahiri is at pains to prove his judiciousness. He cites a variety of viewpoints from the Quran and hadiths (sayings of the Prophet Mohammed), some of which support his judgments, others which do not. At times, he dramatically prefaces his conclusion with the words "I say ..." to draw attention to the fact that his judgments digress from the views held by some Islamic scholars; it is also a way for Zawahiri -- a medical doctor, not a religious scholar by training -- to assume authority for himself as an arbiter of Islamic law. Second, al Qaeda has reckoned with the horrific scale of a nuclear attack; indeed, Zawahiri sees mass casualties as a point in WMDs' favor. Zawahiri's book explicitly justifies a potential attack that could kill 10 million Americans. Again, that enormous figure is not merely tossed off casually by Zawahiri. He believes that such a plan requires justification, and he is satisfied, at the conclusion of his book, that he has done so.
It is notable that Zawahiri repeatedly uses the phrase "artillery bombardment" in the context of discussing the wide-scale destruction of a WMD attack. For al Qaeda, it seems, modern weapons of mass destruction are simply a form of weapon that cannot distinguish between civilians and combatants. Nuclear weapons, Zawahiri wants to argue, are no more morally significant than the catapult often cited in the Quran and hadiths. Here Zawahiri quotes Fahd once again: "If a bomb were dropped on them, destroying 10 million of them and burning as much of their land as they have burned of Muslim land, that would be permissible without any need to mention any other proof."
Needless to say, Zawahiri's approach goes against all Western theories of just war and Islamic ethics of war. He will have to find his justification for use of WMD outside the teachings of Islam.


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Celebrating Six Years!
This month, Rebecca Rose Events celebrates its sixth birthday, and what's an amazing six years it has been. We're looking forward to some exciting growth and new opportunities in this next year and we are so grateful for the love and support of our incredible clients, industry partners, family and friends who help make it all possible. Thank you, from the bottom of our hearts!


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3. Eligibility and Limitations. Participants and winner(s) must be United States residents, 18 years or older or have the permission of a parent or guardian to enter if a minor. Each photo counts as one (1) entry per person and multiple photos may be submitted. Employees of Seattle Rep, their advertising agencies, and members of the immediate family of any such persons are not eligible to win.4. Contest Rules and Decisions. By participating in the Contest, each participant agrees to be bound by the official contest rules and the decisions of Seattle Rep. Persons who violate any rule, gain unfair advantage in participating in the Contest, or obtain winner status using fraudulent means may be disqualified. Unsportsmanlike, disruptive, annoying, harassing or threatening behavior is prohibited. Seattle Rep will interpret these rules and resolve any disputes, conflicting claims or ambiguities concerning the rules or the Contest and Seattle Rep's decisions concerning such disputes shall be final. If the conduct or outcome of the Contest is affected by human error, any mechanical malfunctions or failures of any kind, intentional interference or any event beyond the control of Seattle Rep, the Sponsors reserve the right to terminate this Contest, or make such other decisions regarding the outcome as the Sponsors deem appropriate. Seattle Rep may waive any of these rules in its sole discretion.
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I do read the Daily Telegraph quite often on line, for its international affairs coverage. However, its Middle East correspondent, Robert Tait, who ought to know what he is talking about, wrote this:
the fate of almost five million Arab refugees expelled to neighbouring countries during Israel's 1948 war of independence,
which is a complete untruth. About 711,000 Arab people became refugees following 1948, which is less than the number of Jewish refugees from Arab countries between 1948 and 1970 (about 850,000). Not all of those who became refugees were "expelled" from anywhere.
Lies and the lying liars, hein? But let's be kind and say that Mr Tait just didn't know, and got it wrong, and now that it has been pointed out he, and the Telegraph, will - oh, never mind.


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[Note: I've had the pleasure of seeing The Force Awakens twice already, so I'm here to fill you in on all you need to know to have peace of mind when you book your tickets, without a single plot spoiler. This is a film that is best served fresh first time, so even if you get tempted, don't read or believe any spoilers that may crop up – I guarantee it'll be worth it.]
Chances are if you haven't seen Star Wars: The Force Awakens, you're currently avoiding any and all Star Wars talk like the plague in fear of the dreaded plot spoilers. Chances are you're also concerned, like many others, as to whether this new entry in the series would be the shining star in a galaxy far, far away we need it to be. It's safe to say this should not be a concern – the production team has crafted a Star Wars entry that, whilst it has its flaws like any other film, is certainly among the best in the series.
Whilst Episode VII takes place several years after Return of the Jedi, it hasn't gotten lost in the vast reaches of the Star Wars universe. Right from the opening crawl and then continued through early dialogue, the writing does everything possible to catch you up on the story since RotJ. This does, however, come at the price of some moderately weak early lines of dialogue that are very clearly out of flow with the rest of the film, worded to fill viewers in on specific details. With such a jump through the Star Wars timeline, it's an inevitable flaw to be expected and certainly not one to ruin the experience.
On a more positive note, The Force Awakens does an exceptional job of making its plot accessible to new audiences, not just through dialogue but overall plot setup. Not a lot of knowledge about the prior trilogies is needed, if any, to understand the plot of TFA thanks to superb introduction and sustained use of all-new characters. Effectively, the film can be contained in its own little plot "bubble", with virtually all ties to earlier films explained in-house and the rest of the content brand new – characters, concepts, entities, all of it perfectly understandable to a new viewer.
Speaking of characters, their implementation within the story is possibly one of the greatest positives of the film. I had two main reservations when going in to seeing The Force Awakens – that the returning characters would be overused, and that the new main characters wouldn't fit neatly into place amongst legends like Han and Leia. I'm glad to say they were both completely unnecessary concerns, with our returning favourites having both a worthwhile and well-measured presence whilst the new characters slot in so smoothly they almost make the original trilogy look weak. In fact, not only do they slot in well, they actually create some of the most memorable (and hilarious) moments in the entire saga. An important note about the comic moments present throughout the film – these are not of the Jar Jar Binks form, but rather a more genuine set of laughs placed well to catch the viewer off-guard.
The plot flows from strength to strength, always keeping the viewer gripped (and in my case, grinning throughout) and engaged – something not so easily done in a new story arc. I have to admit, certain events within the film felt like they were over-and-done with all too quickly, but the beauty of its momentum was that I was never given much more than a moment to recognise the issue before something more exciting came along.
When JJ Abrams committed to using as many practical effects as possible, he wasn't lying. It's clear as the film progresses that the community's many voicings over the use of CGI vs. practical effects has been heard and acted upon, with many of the creatures, objects and effects are both of the practical variety and extremely well detailed. This doesn't mean, however, that CGI has no place, as there is a good helping of digital effects, but the majority of these have also been carefully crafted to look as genuine as possible and suit the needs of the scene. When I returned for my second viewing I consciously made an effort to spot and potential weak CGI and I'm pleased to say I had a seriously hard time trying to spot anything of concern.
I'd love to write a paragraph here on the phenomenal work of John Williams in creating the musical score for The Force Awakens, but I'd be doing it a disservice. If you're a Star Wars veteran, you know it already, if you're new to the universe, there isn't any amount of words that can fully realise the experience created by merging the on-screen action with Williams' nostalgic yet fresh musical masterpiece.
Something has to be said for a film that can take an audience through such a rollercoaster of emotions in the space of 140 minutes. Through well-measured doses of wonder, nostalgia, fear, laughter, anger, sadness, solemnity, serenity and more (in no particular order), Star Wars: The Force Awakens begins a new chapter in the history of filmmaking with one of the strongest entries in the saga to date. JJ Abrams and the phenomenally talented team behind this production have not only created a fantastic film, but an homage to all that makes Star Wars great. A seasoned Star Wars fan will be able to spot elements of both the original and prequel trilogies, with a spark of something that is new and fresh which makes the experience that much more enthralling. It is a new step in the journey of characters we have grown to love, through the eyes of new characters we will soon come to love just as much, if not more.
tl;dr – Star Wars: The Force Awakens is the film you are looking for. It's not perfect, but it's not far off it. Well done to all involved in crafting this work of art, you have made, and are going to make a lot of fans very happy indeed.


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Hip pain
Encompassing a variety of conditions ranging from soft tissue such as trochanteric bursitis, hip tendonitis and labral tears to bone conditions such as femoro-acetabular impingement and hip osteoarthritis. Treatment is aimed at the underlying cause of pain and assessment would be made by a consultant. Further investigations such as scans may be necessary to make a diagnosis. Treatment can include physiotherapy, injections and surgery.


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How to Find from which Server a GPO Created
November 18, 2013 Leave a comment
If you didn't change the domain controller it will automatically point to PDC in all GPMC by default.
But in case if you are using Different servers for GPMC and created by different Admins and you want to find from where and which server the GPO created use the below command.
repadmin /showmeta . "CN={insertgpoguid},CN=policies,CN=system,dc=domain,dc=com"
Check when created and originating DSA for that.


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Oh yum. As if Marshmallow Treats aren't good enough - add peanut butter to them, along with peanut butter cups, and you have a fabulously decadent treat. This is my second favorite bar cookie: Special Kay Bars are in first place.
These no bake bar cookies are a classic recipe that everyone knows about. But the peanut butter, peanut butter cups, and the optional frosting kick this recipe up to the next level.
As with all bar cookie recipes, measuring accurately is important. I add extra marshmallows to my Rice Krispie Bars because I like the bars to be chewy and not dry, although the peanut butter fixes that!
Enjoy every bite of these wonderful bar cookies.
What You'll Need
- 3 tablespoons butter
- 1/3 cup peanut butter
- 1 bag large marshmallows plus 12 more marshmallows
- 6 cups crisp rice cereal
- 24 1" peanut butter cups (unwrapped)
How to Make It
Grease a 13 x 9" pan with butter. I don't like using cooking spray when making these bars because I don't like the flavor.
Combine the butter, peanut butter, and marshmallows in a large microwave safe bowl. Microwave uncovered on high for 1-1/2 to 3 minutes, stirring after 1 minute, until the mixture is melted and smooth. Stir in the cereal.
Press the mixture into prepared pan, using buttered hands (be careful, the mixture is pretty warm).
Immediately make 24 indentations in rows in the cereal mixture using handle of wooden spoon. Press the peanut butter cups into the indentations. Let cool. Cut into squares with a candy in the center of each square.
Or you can omit the candy, but top the bars with a mixture of 2 cups semisweet chocolate chips and 1/2 cup peanut butter, melted together, as I did in the picture. Or use the candies AND the frosting for the ultimate indulgent treat. It's still a five ingredient recipe!


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- Method
- Open Access
Identification of cis-regulatory mutations generating de novo edges in personalized cancer gene regulatory networks
- Zeynep Kalender Atak†1, 2,
- Hana Imrichova†1, 2,
- Dmitry Svetlichnyy1, 2,
- Gert Hulselmans1, 2,
- Valerie Christiaens1, 2,
- Joke Reumers3,
- Hugo Ceulemans3 and
- Stein Aerts1, 2Email author
© The Author(s). 2017
- Received: 24 April 2017
- Accepted: 2 August 2017
- Published: 30 August 2017
The identification of functional non-coding mutations is a key challenge in the field of genomics. Here we introduce μ-cisTarget to filter, annotate and prioritize cis-regulatory mutations based on their putative effect on the underlying "personal" gene regulatory network. We validated μ-cisTarget by re-analyzing the TAL1 and LMO1 enhancer mutations in T-ALL, and the TERT promoter mutation in melanoma. Next, we re-sequenced the full genomes of ten cancer cell lines and used matched transcriptome data and motif discovery to identify master regulators with de novo binding sites that result in the up-regulation of nearby oncogenic drivers. μ-cisTarget is available from http://mucistarget.aertslab.org.
- cis-regulatory mutations
- Whole-genome sequencing
- Gene regulatory networks
- Cancer genomics
Oncogenic programs are characterized by aberrant gene expression profiles. A gene regulatory network underlying a cancer transcriptome can be considered as a perturbed stable network configuration, or as a cancer attractor state [1]. Gene expression changes leading from a normal cell to a malignant state are generally due to a series of acquired somatic mutations, which often affect proteins playing a key role in transcriptional regulation [2]. These can include mutations, amplifications, or translocations leading to an altered function or expression of transcription factors (e.g., MYC, TAL1, MITF, TP53), co-factors (EZH2, RB1, IDH1, MLL), or signaling molecules that lead to downstream alterations in transcription factor activity (e.g., RAS/RAC/RAF, KIT, PTEN, CDKN2A). More subtle changes can also occur in gene regulatory networks, which may cause fine-tuning of the emerging transcriptome rather than necessarily yielding a different attractor state. Such local network changes can involve the addition or removal of an edge in the network, affecting a single interaction between a transcription factor and a target gene. Edge perturbations can be caused by a mutation of a transcription factor binding site in a promoter or enhancer, leading to a de novo gain or loss of the binding site and a consequential expression change of a nearby target gene. Several examples of such perturbations are known to be associated with oncogenic programs, such as the gain of an ETS family binding site in the TERT promoter, the gain of a MYB binding site in a 7.5-kb upstream TAL1 enhancer [3–5], and the recently identified gain of a MYB binding site 4 kb upstream of the LMO1 oncogene [6]. Note that whereas these three examples occur recurrently across melanoma or liver cancer (for TERT) or across T-cell acute lymphoblastic leukemia (for TAL1 and LMO1), they represent exceptional cases, since whole-genome sequencing, even across large cohorts such as 560 breast cancer genomes [7], failed to identify additional binding site changes that are significantly recurrent [8] (recently reviewed in [9–11]). This suggests either that cis-acting mutations are usually passenger mutations rather than driver mutations, or that they can occur as drivers at diverse positions, spread across hundreds of kilobases affecting the regulation of a target gene. The latter would render current cohort sizes underpowered and would require different approaches to identify causal cis-regulatory mutations and their downstream consequences.
Computational predictions of a gain or loss of a transcription factor binding site can be performed by scoring the reference and mutated sequence with a position weight matrix (PWM) of the candidate factor [12, 13]. This results in a "delta" PWM score, to which an arbitrary threshold can be applied to decide whether the gain or loss is strong enough. Such an approach is implemented in various bioinformatics tools, such as FunSeq2 [14] and OncoCis [15]. However, as position weight matrices are notorious for producing false positive predictions, the delta score also results in an excess of false positive gains or losses of binding sites. A possible solution to this problem is to take the context of the binding site into account, i.e., the encompassing regulatory region (promoter or enhancer). For example, the gain of a MYB binding site in a random genomic position may not lead to de novo enhancer activity, whereas such a gain in the context of RUNX binding sites (MYB and RUNX bind together to leukemia enhancers) may cause ectopic enhancer activity [16]. Computationally, this solution depends on training more complex enhancer models, for example, based on k-mer support vector machines [17], random forests [16], or deep learning (deepSEA [18]). The main limitation of this approach is the dependence on high-quality training data to construct accurate enhancer models [16].
Here we investigate how "personalised" gene regulatory network reconstruction can be used to identify specific candidate cis-regulatory driver mutations in cancer genomes. Gene regulatory network inference is a common technique that has provided insight into master regulators in many cancer types [19–21] and the targets they regulate. Here, we exploit gene regulatory networks for the prioritization of non-coding mutations. Particularly, by first identifying the master regulators operating in a cancer sample, we can identify those non-coding mutations that generate de novo targets of these master regulators. We have developed an online tool to streamline this process, called μ-cisTarget, and we demonstrate the use of μ-cisTarget on known cases of TERT promoter and TAL1 enhancer mutations. Finally, we predict new cis-regulatory mutations in ten cancer cell lines for which we sequenced the genome, transcriptome, and epigenome.
Analysis of melanoma whole genomes for TERT promoter mutations
Expression data (Z-scores across all sequenced cancers in The Cancer Genome Atlas (TCGA)) for seven melanoma samples (Additional file 1: Table S1) with TERT promoter mutations based on [22] were downloaded from Cosmic (v74). The signatures per sample (i.e., genes that have an expression Z-score above 1) were analyzed by iRegulon to build the personalized gene regulatory networks (using the following parameters: motif collection 19 K, putative regulatory region centered around TSS [20 kb, 10 kb, 500 bp], motif rankings database across 10 and 7 species, NES threshold = 3, ROC = 0.03, rank threshold = 5000).
Raw sequence data (bam files) were available for six out of seven melanoma samples via the GDC Data Portal (Legacy Archive). We performed somatic mutation calling with VarScan [23] (command somatic with minimum variant allele frequency of 0.1, and a minimum coverage of five and two reads in tumor and normal samples, respectively). Non-coding mutations associated with melanoma-related or cancer driver genes were scored with MotifLocator for master transcription factors obtained in the previous step. The list of candidate cis-GoF mutations was filtered further using topologically associating domains (TADs) from 21 human cell lines and tissues [24].
Whole-genome-sequencing on ten cell lines
A549, COLO-205, and PC-3 cell lines were sequenced with Complete Genomics (CG), DU-145, OVCAR-3, SKOV-3 were sequenced with Illumina, and HCT-116, HT-29, MDA-MB-231, and SK-MEL-5 were sequenced with both technologies. CG sequencing was performed by the service provider using a proprietary sequencing-by-ligation technology. CG also performed primary data analysis, including image analysis, base calling, alignment, and variant calling. CG variants were further filtered with a depth of coverage threshold of 10, mutation coverage threshold of 5, and variant allele frequency threshold of 0.20. Illumina sequencing was done in accordance with the manufacturer's protocol. Primary data analysis was performed with the manufacturer's software Casava (v1.8.2). Illumina variants were further filtered with a depth of coverage threshold of 10 and mutation coverage threshold of 5. Variant calls were intersected for samples that were sequenced with both technologies. Coding variants were subtracted using protein coding exon locations from GENCODE (v19). Variants were annotated as non-SNPs or SNPs using dbSNP build 144 [25].
Cell line-specific regulatory data
H3K27ac ChIP-seq data for six of ten cell lines were obtained from the Gene Expression Omnibus (GEO) and literature: A549 and HCT-116 (GSE31755) [26], HT-29 (GSE53602), PC-3 [27], MDA-MB-231 [28], SK-MEL-5 (GSE60666) [29]. For the remaining four cell lines (OVCAR-3, SKOV-3, DU-145, Colo-205), ChIP-seq was performed in this study. These cell lines were a kind gift from the following laboratories: OVCAR-3 and SKOV-3 from the laboratory of Gynaecological Oncology, KU Leuven (head, Frédéric Amant); COLO-205 from the laboratory of Molecular Digestive Oncology, KU Leuven (head, Sabine Tejpar); and DU-145 from the laboratory of Lipid Metabolism and Cancer, KU Leuven (head, Johan Swinnen). The cell lines were grown to ~ 85% confluence per 15-cm dish. A total of 20 million cells per sample were collected, yielding ~ 20 fractions of chromatin. ChIP samples were prepared following the Magna ChIP-Seq preparation kit using at least two chromatin fractions and 2–2.5 μg of antibody per fraction. Anti-histone H3 acetyl K27 antibody (ab4729, Abcam) was used for ChIP. Per sample, 5–30 ng of precipitated DNA or input was used to perform library preparation according to the Illumina TruSeq DNA Sample preparation guide. In brief, the immunoprecipitated DNA was end-repaired, A-tailed, and ligated to diluted sequencing adapters (1/100). After PCR amplification (15–18 cycles) and bead purification (Agencourt AmpureXp, Analis), the libraries with fragment size of 300–500 bp were sequenced using the HiSeq 2500 (Illumina). Sequence reads were mapped to the reference genome (hg19-Gencode v18) using Bowtie2 2.1.0 and narrow peaks were called using MACS2 algorithm (q-value < 0.001) [30]. Then the peaks less than 350 bp from each other were merged. Additionally, we used peak calls from the ChIP-Atlas database for 433 regulatory datasets (transcription factor and chromatin ChIP-seq) across seven cell lines (Additional file 1: Table S2; http://chip-atlas.org).
Selection of cancer type-specific transcription factors and target genes
Lists of cancer type-specific genes were extracted from NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene; Additional file 1: Table S3). Next, a collection of 1050 known cancer driver genes compiled from different resources [31–36] was used to further annotate the cancer type-related genes (Additional file 1: Table S3).
Cell line-specific gene signatures and identification of sample-specific master regulators
Sample-specific master regulators predicted from sample-specific gene signatures
Cell line
Cancer type
Number of master TFs/motifs
Master TFsa
Number of cancer-type relevant or driver genes associated with master TFs
Detection of candidate mutations
The non-coding mutations were assigned to genes using the GREAT tool (up to 1 Mb) [37]. Only the non-coding mutations associated with genes that are a) over-expressed in the cell line (expression Z-score above 1) and b) relevant to the cancer type or on the list of known cancer drivers were scored by MotifLocator [13].
All the motifs corresponding to master transcription factors from the personalized gene regulatory networks were tested to reveal if any mutation from the matched cell line caused a gain of any of these motifs.
Annotation of mutations using TADs
The assignment of the mutations to the potential target genes within 1 Mb is further annotated using a large dataset of TAD boundaries generated for 21 samples (14 human tissues and seven human cell lines) [24]. In the output file, information on whether the mutation and the gene fall between the boundaries of a certain sample is provided, i.e., if the mutation is in the same TAD region as the promoter of the gene. This annotation can be used to further filter mutation–gene associations.
Genome-wide screening of DNA sequences by MotifLocator
A collection of 8053 unique PWMs directly annotated to 1628 TFs was used for scoring with MotifLocator [13]. Scoring was performed at the mutation sites with a window size between 20 and 60 bp depending on the motif size (20 bp if the motif size is 15 bp or less, 30 bp if the motif size is between 16 and 25 bp, and 60 bp if the motif size is larger than 25 bp). The variants with mutant MotifLocator score ≥ 0.90 and delta ≥ 0.1 were selected (where delta represents the difference between MotifLocator scores of mutant and wild-type sequences).
Validation of candidate mutations using matched transcriptome and epigenome data
RNA-seq data (bam files) were downloaded for eight (A549, COLO-205, DU-145, HCT-116, HT-29, MDA-MB-231, PC-3, SK-MEL-5) cell lines from the GDC Data Portal (Legacy Archive). Heterozygous SNPs per cell line were obtained from whole genome mutation calls by requiring at least five reads for the reference and variant allele, as well as a variant allele frequency of at least 0.10. Heterozygous SNPs were intersected for samples that were sequenced with both Illumina and CG. Allele-specific expression per cell line was calculated using MBASED one-sample analysis [38]. Any gene with an adjusted p value ≤ 0.05 and estimated mean allele frequency ≥ 0.6 was annotated as exhibiting allele-specific expression.
A small number of non-coding mutations generate de novo oncogenic edges in driver gene regulatory networks
Application of μ-cisTarget to ten re-sequenced cancer cell lines
After validating μ-cisTarget on the TAL1, LMO1, and TERT mutations, we analyzed ten widely used cancer cell lines as a discovery set (Additional file 1: Table S6). We essentially implemented the same strategy as in the validation cases. Namely, we first identified cell line-specific master regulators that are relevant genes per cell line using motif enrichment analysis. Next, we identified non-coding mutations in the genome of that cell line that create de novo binding sites for any of these master regulators, and that are near oncogenic drivers (relevant or driver genes; Additional file 1: Table S3). For the first step we obtained gene expression profiles of these cell lines from the COSMIC Cell Lines Project [40] and predicted master regulators using gene signatures of these cell lines (Table 1). For each sample, all genes expressed with Z-score above 1 (compared to all other cell lines in Cosmic Cell Line Project) are used for motif enrichment-based master regulator discovery. The initial set of predicted transcription factors was filtered for their own overexpression (Z-score above 1) and their cancer type specificity (based on Additional file 1: Table S3). Interestingly, our motif-based predictions of master regulators is supported by ChIP-seq data (when available) for 6/33 master regulators across the ten cell lines (Additional file 1: Table S6).
Transcription factors identified at this step can be linked to several signaling pathways [41] (Additional file 2: Figure S2) but two functional classes of transcription factors emerge at this step: lineage-associated transcription factors and EMT-associated transcription factors. Lineage associated transcription factors include MITF for the melanoma cell line SK-MEL-5 [42], TP63 for the lung cancer line A549 [43], KLF5 for the colon cancer line HT-29 [44], and ETS family transcription factors for prostate, colon, ovarian, and breast cancer cell lines [45–49]. This class of transcription factors is expressed at an earlier developmental stage and is reactivated during tumorigenesis. Another group of transcription factors are the EMT associated factors: FOSL1 for MDA-MB-231 and DU-145, ZEB1 for SKOV-3, FOS for OVCAR-3, and SNAI2 for SK-MEL-5. The majority of cell lines with these transcription factors as master regulators are derived from metastatic sites (SK-MEL-5, DU-145, PC-3). Of the remaining two cell lines, OVCAR-3 is derived from a chemoresistant patient [50] and MDA-MB-231 demonstrates mesenchymal cell morphology [51] and is regarded as invasive in vitro [52]. Master regulators obtained at this step also corroborate well with what is known about these cell lines. For instance, the predicted master regulators for the lung cancer cell line A549 include NFE2L2 (NRF2), which is an essential gene for cell proliferation and chemoresistance in lung cancers, and specifically in A549 since knock-down of NFE2L2 in A549 inhibits proliferation [53]. Another example involves the MDA-MB-231 cell line for which ETS factors ETS1 and ETV1, as well as FOSL1 and STAT5A/B (the motif is directly annotated for both STAT5A and STAT5B), are found as master regulators. Gene knock-down studies involving these four transcription factors in this cell line demonstrated that each of these transcription factors is essential for growth, migration, and metastatic potential of this cell line [54–57]. And lastly, it has been shown for the ovarian cancer cell line SKOV-3 that inhibition of ZEB1, which is predicted as a master regulator, hampers migration in vivo and tumor growth in vitro when xenografted in mice [58]. In conclusion, master regulator predictions seem to capture and represent oncogenic processes ongoing in these cell lines.
Evaluation of predicted cis-regulatory mutations using matched epigenomes and allele-specific expression
Next, we investigated whether the predicted cis-regulatory mutations were present in an allele-specific manner in the expression data; in other words we checked if the mutation is associated with a gene with allele-specific expression. Using coding heterozygous SNPs from whole genome sequencing calls and RNA-seq data (which was not available for SKOV-3 and OVCAR-3), we identified genes with allele-specific expression, and this revealed that 154 of 468 candidate cis-GoF mutations show allelic bias in expression data (Additional file 1: Table S8). Note that our effort to identify mutations showing allelic bias in regulatory data failed since the coverage of H3K27Ac data was too low to determine variant allele frequency (80/98 candidate cis-GoF mutations in peaks have a depth of coverage below 5). When we expanded our search to also include SNPs, we identified 1304 SNPs in H3K27Ac peaks with a motif gain for a master transcription factor, and of these, 41 show allelic bias in the regulatory data (Additional file 1: Table S12). This illustrates that gain of important motifs can yield allele-specific regulatory activity, but very few non-dbSNP, i.e., candidate somatic mutations, were identified with this property across the ten cell lines. On the other hand, by combining regulatory activity information and RNA-seq-based allele-specific expression we found evidence of selection for 217 of 468 candidate cis-GoF mutations (Fig. 4). In conclusion, μ-cisTarget can be applied to matched genome–transcriptome data, or to matched genome–epigenome data, to obtain non-coding gain-of-function mutations resulting in binding site gains for subtype-specific master regulators near overexpressed oncogenes.
Whole-genome re-sequencing of cancer genomes is taking a prominent place in research and the clinic. The identification and prioritization of candidate driver mutations in the non-coding genome is therefore a key challenge. Indeed, several recent studies indicate an important role for cis-regulatory mutations in disease, not only in cancer (e.g., TERT, TAL1 [3–5, 61]), but also in complex diseases (e.g., type II diabetes FTO locus [62], Parkinson's disease [63]) and in familial disorders (e.g., preaxial polydactyly [64]). However, other studies have highlighted that cancer cis-regulatory mutations are usually not recurrent across patients, except for a few exceptions such as the TERT promoter mutation. To reconcile these two opposing directions, we decided to step away from recurrence calculations in a cohort, but to work under the assumption that cis-regulatory mutations may be rarer than expected. A consequence of this assumption, if each sample harbors none or only few cis-regulatory mutations, is that statistical enrichment analyses may fail to provide meaningful results. Rather, the identification of functional cis-regulatory mutations may require a more ad hoc biological approach, which we explored in this study.
We are not the first to score and prioritize candidate mutations based on their putative gain (or loss) of transcription factor binding sites. In fact, most previously existing methods, such as FunSeq2, OncoCis, or RegulomeDB, use an ad hoc approach to annotate and filter candidate mutations based on motif loss/gain (FunSeq2, OncoCis) or on regulatory data from publicly available databases such as ENCODE (RegulomeDB). However, several important pieces of information are not utilized by previously existing tools, and are explored in our study. Firstly, the gain (or loss) of a motif is expected to be functionally relevant if the transcription factor itself is expressed in the cancer cells under study. If the transcription factor is (or was) not expressed, the gain of a binding site is not expected to be under positive selection.
Secondly, the motif gain (for an expressed transcription factor) should preferably represent a new binding site for a master regulator, meaning that this (over-expressed) transcription factor regulates other genes in the cancer cell through this motif. The approach we present here is, to our knowledge, the first one to take this criterion into account. Practically, to address this challenge we use a patient-specific, or subtype-specific, gene signature, in which over-represented motifs represent candidate motifs of master regulators.
Thirdly, the motif gain should either yield de novo regulatory activity of the encompassing enhancer or strengthen/amplify that enhancer. Such a gain of function may be visible as allele-specific bias of regulatory activity, whereby, for example, the ChIP-seq reads are homozygous for the variant.
Fourthly, the motif and enhancer gain should result in the up-regulation of a nearby oncogene so that it provides a growth advantage to the cancer cell and can be positively selected. Moreover, under this fourth piece of information, we expect that the predicted target gene is actually a known oncogene. Indeed, it is rather unlikely that previously unknown oncogenes could be discovered that are only up-regulated by a cis-regulatory mutation, and not by any other means (e.g., duplication, translocation, or mutation).
Taken together, our approach allows the selection of candidate mutations from whole genome sequencing data in a relatively short time since the filtering removes a large portion of the mutations (for instance, the total number of JURKAT mutations drops from 4.3 million to 200 K when we focus only on those mutations that are close to expressed and relevant genes from the predicted gene regulatory network specific for this cell line). On top of that, only motifs annotated for the transcription factors predicted as master regulators of the specific sample are used for scoring these mutations. Specifically, the time required to score one mutation and motifs directly annotated for one TF is approximately 2.5 s (e.g., 29 motifs for MYB factor); scoring 10, 100, and 1000 mutations for these motifs takes 3.6, 16, and 135 seconds, respectively.
Our approach currently focuses on gain-of-function mutations that generate new binding sites for over-expressed activators, yielding up-regulation of a nearby oncogene. Clearly, several other scenarios are not covered by our proof-of-concept analyses. These include, for example, the loss of an activator binding site near a tumor suppressor (e.g., loss of p53 binding site), the loss of a repressor binding site near an oncogene, or the gain of a repressor binding site near a tumor suppressor. We have focused in this study on the gain of an activator site near an oncogene because the currently known cis-regulatory driver mutations are all of this class, and this is the most conceivable and most pragmatic way to work within the context of motif discovery and gene regulatory networks. Future work is needed to address the other categories, and may reveal new types of cis-regulatory mutations. Currently we focus on transcription factors that are expressed in the sample and this may lead to problems since (i) the cut-off for expression status of a transcription factor is user-dependent (and arbitrary) and (ii) mRNA expression levels might be misleading for some transcription factors that are regulated at the protein level. This is exemplified in the TERT promoter case study where we identify ETS family transcription factors collectively but not the GABPA transcription factor specifically because it is only expressed (according to our cut-off) in one out of seven samples. A possible increase in the sensitivity would come from allowing motif gains for relevant transcription factors that are related to the cancer type even though the motif was not enriched in the input gene signature or TF was not expressed in the sample.
Our method provides a handle on understanding non-coding mutations in the context of regulatory genomics; thus, we envisioned μ-cisTarget not as a method for a final analysis but rather a starting point for in-depth analysis. The typical use of a strategy like we depict here can be either to annotate a cancer genome with functional information regarding cis-regulatory mutations or in a research context to generate a list of candidate mutations that can be further tested in a targeted screen, for example, using massively parallel enhancer-reporter assays or CRISPR-Cas9 based modulation/mutation of the candidate mutations.
We present a computational framework inspired by a cancer biological viewpoint on oncogenic driver mutations. Our method can be used to identify candidate cis-regulatory mutations using sequence information alone, but works best on samples with combined genome and transcriptome data; while optimal results can be obtained if matching epigenome data are also available. Overall our results suggest the presence of only few cis-regulatory driver mutations per genome in cancer genomes that may alter the expression levels of specific oncogenes.
Not applicable.
This work is funded by The Research Foundation - Flanders (FWO; http://www.fwo.be/; G.0791.14), Special Research Fund (BOF) KU Leuven (http://www.kuleuven.be/research/funding/bof/; grant PF/10/016), Foundation Against Cancer (http://www.cancer.be; 2012-F2 to SA), European Research Council (ERC; 724226_cis-CONTROL to SA). ZKA is funded by a research grant from Kom of tegen kanker. HI has a PhD Fellowships from the agency for Innovation by Science and Technology (IWT; https://www.iwt.be/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
The data generated for this study (H3K27ac ChIP-seq for OVCAR-3, SKOV-3, DU-145, COLO-205) have been submitted to the NCBI's GEO (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE101408. Genome data are available at the European Genome-phenome Archive (EGA; http://www.ebi.ac.uk/ega/) under accession number EGAS00001002571. The code for μ-cisTarget is freely available at https://github.com/aertslab/mucistarget and a web-implementation is available at http://mucistarget.aertslab.org.
Authors' contributions
ZKA, HI, and SA designed the study. VC performed the experiments. ZKA and HI performed all the bioinformatics analyses with the help of GH and DS. JR and HC contributed materials. ZKA, HI, and SA wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
JR and HC are employees of a private company, Janssen Pharmaceutica NV. The remaining authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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The Good Samaritan Supper Club has cranked up again this year and is starting out with a bang. Located at the Eighth Street Church of God, 906 8th St., Bessemer City, organizers are featuring some outstanding talent and a great meal every first Saturday of the month. The meals, served at the tables, begin at 5 p.m. and singings begin promptly at 6 p.m. The cost is just $10 per person for both concert and meal. Concert only is $5.
This Saturday you can hear Jaidyn�s Call from Dawsonville, Ga. Although I have not heard this trio, a number who have say they are really good. One of the members, Debra Perry, was the original soprano for The Perrys and another member, Chris Reed, is originally from Gaston County.
Pastor Ronnie and Faye Pendleton encourage you to come out for an evening of Christian fellowship in a clean, safe atmosphere and, ��get your physical and spiritual souls fed.�
Call 704-860-3492 or 704-884-6530 for additional information. Go and enjoy!
David Harvell, senior adult pastor at Restoring Hope Foursquare Church in Dallas, is available for singing at churches, senior citizens� groups, etc., days or evenings. Harvell , a fine vocalist, sang professionally and semi-professionally with several groups over the years. His health problems are mostly behind him now and he is, ��ready to get out and sing again.� Call 704-674-6955.
The Watking Family recently was inducted into the Atlanta Country Music Hall of Fame.
Consider this: Simply going to church doesn�t make you a Christian any more than standing in a garage makes you a car!


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For the 2006 LA Auto Show Italian design house Italdesign, led by Fabrizio Giugiaro (Giorgetto Giugairo's son – see Ferrari GG50 concept), decided to reinterpret the contemporary Ford Mustang.
In 2005 Guigiaro had approached J Mays, Ford Motor Company's group vice president, Design and chief creative officer, with the idea to create an American icon with Italian flair powered by Ford Racing products.
It took just 4 months to create an exterior model of the Guigiaro Mustang in the Turin, Italy, based studio.
The Ford Mustang by Giugiaro appears to be more compact than the standard production car, thanks to a reduction of the rear overhang and the signature Giugiaro style of tapering the angles on the car to the limit of its mechanical outlines.
The Giugiaro styled Mustang is 30 millimeters longer in the nose, and wider by a full 80 millimeters in the rear.
Ford Racing improved the Guigiaro Mustang's all-aluminum 4.6-liter 3-valve V-8 engine by adding an intercooled twin-screw supercharger, fuel injectors from the Ford GT and a unique engine calibration. Engine air intake was increased with a larger 95 mm mass air meter and a conical air filter. Exhaust performance of the Guigiaro Mustang was enhanced with new Ford Racing mufflers and an X-pipe. The upgrades allow for an estimated 500 horsepower at a boost level of 11 psi from the Ford Racing supercharger.
To keep the Guigiaro Ford Mustang handling as good as it looks, the FR500C-inspired chassis tuning is accomplished with a tailored Ford Racing Handling Pack. This package, available through Ford Racing for the Mustang GT, adds new Dynamic-tuned dampers, lowering springs and anti-sway bars. The changes give the car a lower stance while sharpening handling response. The car rides approximately 1.5 inches lower than the production Mustang GT.
Features of the Ford Mustang Concept by Guigiaro:
- The interior – featuring a dramatic instrument panel that sweeps the width of the car; circular gauges that project from behind the sports steering wheel; dark brown horsehide-covered headrests with horse logo accents; and seat cushions and backrests elaborately upholstered in dark brown mottled horse hides.
- The single curved glass panel that bridges the windshield and rear window, serving as the concept's roof. Produced by Solutia of Detroit, the panel is made from a special type of crystal that filters out 100 percent of UVA rays while providing unfettered vista views.
- Doors hinged at the base of the upright A-pillar and open vertically at the touch of a button.
- Bespoke taillights, which are three separate elements as found on the 1964 Mustang – but reinterpreted into a more dramatic arrow shape that links to the louver panels that replace the rear side windows.
- The visible curl that sweeps into the crest of the concept's carbon fiber fenders, hinting at tail fins that defined American cars of the '50s.
- The sporting-but-elegant unique 20-inch rims, fitted with 275/40 tires on the front and larger 315/35 tires on the rear.
By: diseno-art


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"What's a 'sensitive?'" Agent Paul Cohen asked nervously.
"Some humans can pick up on our energy, thoughts, and emotions," Todd explained, "although not necessarily all three."
"Yeah, well," Master Chief said, sounding dismissive, "We have more important things to talk about." He motioned towards the agents. "Cohen, Gibson, what do you have for us right now."
Agent Randall Gibson cleared his throat. "We have a small problem," he said. "It appears that the apartment complex we set you up in is now under scrutiny."
"Oh?" Todd leaned forward in his seat. "How is that?"
"Well, complaints are starting to come in for discrimination," Cohen responded.
"Oh," Amanda said quietly, "right."
Todd looked at her, waiting for an explanation.
"Well," Amanda said, "Where we are from, there's not enough sunlight to cause our skin to develop enough melanin to darken us."
"But we know that there are some here that are not full Inersiens," Gibson said, "You couldn't find someone that has an ethnic family background?"
Brian scoffed.
Amanda and Todd looked at him.
"Well," Todd said, "We only found a few. It was a short search." He looked back at the agents. "We need more time."
"I'm afraid there is no more time," Gibson said. "The city wants to do an investigation on Carter for unfair housing practices."
"This is ridiculous!" Brian exclaimed, "Humans are humans. There's no difference! Why would you force them to be-"
"Brian!" Amanda said sternly.
Brian stopped and looked at her in shock. Todd and Stanley were also surprised at her commanding tone. Roger sat back with his arms crossed, looking smugly at Brian.
Amanda's head started to hurt. She put her hand on her head.
"I know that there are empty apartments," Amanda said, "maybe we can get a few in there now to curb the suspicion."
"Amanda," Todd said, "We run the risk of being discovered. We don't know if they're sensitives until after we exchange thoughts."
Amanda nodded, "We can move." She was starting to feel sleepy again. She can feel Todd put his arm on her back. She can feel a little energy come her way.
She took a deep breath. "Maybe to a town that's dying in the Midwest."
Gibson nodded. "I could arrange for one of our cover companies to start buying houses. Then we can start moving your people out there."
"I already have a town in mind," Cohen said, "It's in Texas."
"Well, that is a bit hot," Brian said.
"Why don't we look at our options first," Amanda said, "I know we don't have a lot of time, but I don't want to move only to have to move again."
Master Chief Taylor cleared his throat.
"What happens when there are a lot of you together," Taylor said. "You don't plan on …"
"Again," Brian interrupted, annoyed. "We are not like that. We are here because of that assumption-"
Brian stopped and looked at Amanda who was looking wearily at him.
"With all due respect," Amanda said turning back to Taylor, "you watch too many super heroes, villain movies."
"Well then," Master Chief said, "You will be home soon. Hopefully, we'll get this sorted out soon. Y'all will wait in your quarters until we come get you."
All of the people in the room stood up. Amanda could sense the Master Chief's hostility. She grabbed Todd's arm.
"Get these people an escort to their quarters," Taylor said to the Petty Officer.
"I wanted to be able to talk to you alone," She thought to Todd.
"Humor him," Todd thought back, "We'll be home soon."
They both looked at Paul who was staring at them. Then he shook his head and headed out of the room.
Five sailors walked the room and escorted each of the Inersiens to their quarters on different parts of the ship.
When Amanda got to her room, she wasn't sleepy anymore. Todd had given her enough energy to wake her up.
She sat on her bed. She had no phone, no internet, and no paper and pencil.
Then she realized that she was trying to "run" from what's important again. She laid down on her bed, closed her eyes, and tried to call up as many memories as she could.


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Heart disease affects millions of people and is America's leading cause of death. The good news however, is that following a heart healthy diet can reduce your risk for both developing and dying from heart disease by helping you maintain a healthy weight, improve your cholesterol levels and control high blood pressure. Here are some dietary strategies to help you follow a heart healthy diet:
1) Follow a DASH style eating plan: The Dietary Approaches to Stop Hypertension (DASH) Eating Plan helps to prevent and control high blood pressure, which can increase your risk for heart disease and stroke. Following a DASH style plan means eating a diet that is and rich in fruits, vegetables and fat-free or low-fat dairy foods. The DASH plan also emphasizes whole grain products, fish, lean meats and nuts. It is rich in potassium, magnesium, and calcium, as well as protein and fiber and low in total and saturated fat and cholesterol. To learn more about how to follow the DASH Diet, visit: www.nhlbi.nih.gov.
2) Reduce your sodium intake: The 2010 Dietary Guidelines for Americans recommends a daily sodium intake of less than 2,300 milligrams. Those who are 51 and older, African American or have hypertension, diabetes, or chronic kidney disease should aim for a daily sodium intake of no more than 1500 milligrams. To lower your sodium intake limit the amount of sodium that you add to food on your plate and when cooking and instead add flavor to foods by using herbs and spices. Also when buying processed and prepared foods, make sure to read the food label and look for "reduced sodium" or "no salt added" products.
3) Choose your fats wisely: Limit saturated fat intake to less than 10% of your total calorie intake and eat as few trans fats as possible. Instead choose healthy fats including mono and polyunsaturated sources. To do this, try substituting butter, lard, shortening and stick margarine for olive or canola oil. Also choose lean meats (beef loin and round cuts, boneless skinless chicken breast, turkey cutlets, pork loin, etc.) and eat more seafood, especially the higher fat varieties like salmon and tuna which are rich in heart healthy omega 3 fatty acids. Choose low-fat or fat free dairy products which have the same nutrients as the full fat varieties minus the fat. To reduce trans fats, check food labels for trans fats and limit your intake of foods with hydrogenated or partially hydrogenated vegetable oils as one of the main ingredients.
4) Limit intake of dietary cholesterol- Keep your dietary cholesterol intake to less than 300 milligrams per day. Cholesterol is found in foods from animal sources such as meat and dairy foods so choose lean meats and low fat or fat free dairy foods. Also pay attention to portion sizes and trim visible fat from meat whenever possible. When cooking, instead of frying, choose leaner cooking methods such as grilling, baking or broiling. When it comes to eggs, despite containing some fat and cholesterol, they are packed with nutrients and can fit into a heart healthy diet. In fact, new research shows they are lower in cholesterol than we once thought. According to the USDA, one large egg contains about 185 mg of cholesterol so it is possible to eat an egg each day and still stay below the recommended 300 mg of cholesterol per day. If you want to include an egg in your daily meal plan, make sure to choose low cholesterol foods throughout the day and if you want more than one egg, consider combining one whole egg with egg whites.
5) Fiber up- Fiber, especially soluble fiber, may help to lower cholesterol by binding to fatty substances in the body and helping to eliminate them. To increase your intake of fiber rich foods, eat more fruits, vegetables, whole grains, nuts, seeds, beans and lentils.
Please note: The information provided here is for educational purposes only. It is not intended to diagnose or treat any particular illness or condition, nor is it intended to support any particular product or service. You should always consult your healthcare provider prior to making any changes to your healthcare routine.
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Last week, a Tennessee woman named Kimberly Jones posted a video of her son Keaton online. It went mega-viral.
The video, which has been viewed on Facebook more than 20 million times since posting, shows Keaton in tears over being bullied at school. There's a sense of despair and helplessness in his voice that no child should have to feel, but too many have.
"Just out of curiosity, why do they bully?" a distraught Keaton asks his mom. "What's the point of it? Why do they find joy in taking innocent people and finding a way to be mean to them?"
This is Keaton Jones, he lives in Knoxville and he has a little something to say about bullying.pic.twitter.com/coyQxFp33V
— Everything TN (@Everything_TN) December 9, 2017
The video clearly resonated with people — some who have been bullied, some who have been the bully — and within hours, words of support began to roll in from around the world, including some notes from some high profile people.
Hollywood has Keaton's back.
Avengers Chris Evans and Mark Ruffalo came up big for the little guy.
Stay strong, Keaton. Don't let them make you turn cold. I promise it gets better. While those punks at your school are deciding what kind of people they want to be in this world, how would you and your mom like to come to the Avengers premiere in LA next year? https://t.co/s1QwCQ3toi
— Chris Evans (@ChrisEvans) December 10, 2017
Little buddy, I was bullied when I was a kid. You are right #ItGetsBetter! You are my own personal super hero. Protect Yo Heart. You got a pal in the Hulk. https://t.co/fRTAENcmV4
— Mark Ruffalo (@MarkRuffalo) December 10, 2017
As did Eleven from "Stranger Things," offering her friendship.
Keaton, this is so accurate. Why do people do this? I think your sooo cool Keaton! I wanna be your friend ( but srsly) ur freakin awesome ❤ https://t.co/LD7Q762bL9
— Millie Bobby Brown (@milliebbrown) December 9, 2017
The delightful Tom Cavanagh of "The Flash" voiced his support for Jones and against bullies everywhere.
Hey Keaton
We here on "The Flash" are on your side – and against bullies everywhere.⚡ #TheFlash @CW_TheFlash #StandWithKeaton https://t.co/svn8FTZmaT
— Tom Cavanagh (@CavanaghTom) December 11, 2017
Same with Beth Behrs of "Two Broke Girls."
Keaton Jones you are amazing. So brave and strong and a beautiful human! Sending love from California! https://t.co/AALuA27r0Z
— Beth Behrs (@BethBehrs) December 10, 2017
He got some love from members of "The Walking Dead" cast.
Hey #KeatonJones Just saw your video about the tough time you're going through. Not sure if you're a Walking Dead fan but if it happens that you are then please be in touch as I would love to see you and your family @WalkrStalkrCon show in Nashville next year. On me. X
— Tom Payne (@justanactor) December 10, 2017
My dude. Lotta folks got your back. Me included. I'll come down and have a chat with anybody getting on your nerves. You're awesome my man. Be strong. Always. You will win because you are the better man. Believe that. Xojd
— Jeffrey Dean Morgan (@JDMorgan) December 10, 2017
Even Gaston and LeFou (a couple of fiction's most famous bullies) weren't having it.
I was bullied my whole childhood. Bullying makes me sick. Keaton, it will get better. I promise. These bullies will be a story you will tell one day when you are big and strong and mightier than all of them. https://t.co/8yEqalWfHd
— Luke Evans (@TheRealLukevans) December 10, 2017
This is my message for Keaton. Love you buddy pic.twitter.com/pdYK5E7JRk
— Josh Gad (@joshgad) December 10, 2017
Broadway star Ben Platt offered a few words of support.
Keaton- your vulnerability, honesty and emotional intelligence already show how much more powerful, valuable, unique and human you are than any of the bullies you talked about. I have no doubt it will get better and we will all do our part to make sure it does. We love you. https://t.co/JN9hCU49pY
— Ben Platt (@BenSPLATT) December 10, 2017
And so did voice actors Susan Eisenberg and Kevin Conroy, who provided the voices for Wonder Woman and Batman, respectively, on the animated "Justice League" TV show.
Stay strong, Keaton… You are not alone!! #KeatonJones #StopBullying pic.twitter.com/ASTUxPlgD4
— Susan Eisenberg (@susaneisenberg1) December 10, 2017
Keaton you are not alone. Please believe that it gets better in time. Bullies are really very weak and not worthy of your tears. I was bullied and grew up to play Batman, how cool is that?! https://t.co/wVWj7PkqLe
— Kevin Conroy (@RealKevinConroy) December 10, 2017
"Coco" director Lee Unkrich and "Ghostbusters" mastermind Paul Feig stepped up.
.@Lakyn_jones Hi! I directed Coco and Toy Story 3. If Keaton ever wants to visit Pixar, he has an open invitation! pic.twitter.com/Xv43RONyDr
— Lee Unkrich (@leeunkrich) December 11, 2017
Keaton, you are an amazing kid, a beautiful unique person. You are one thousand times better than any bully. You are right that it will get better one day. You will thrive. Keep learning, stay positive, laugh at how small-minded they are & keep being yourself. @STOMPOutBullyng https://t.co/1rjbarEYDO
— Paul Feig (@paulfeig) December 11, 2017
Some of the biggest stars in professional sports showed up, as well.
LeBron James called bullies "straight up wack, corny, cowards, chumps."
Damn right! Bullies are straight up wack, corny, cowards, chumps, etc, etc! Keaton keep your head up buddy and push forward! You're the best https://t.co/pqDgay77l9
— LeBron James (@KingJames) December 11, 2017
Cubs slugger Anthony Rizzo and Pirates outfielder Andrew McCutchen both offered words of kindness and comfort.
Keaton,U are not ugly, U are amazing. U r my hero for sharing ur story & continuing the conversation about the harm of bullying.Stay Strong! https://t.co/HaO2NqP4SP
— Anthony Rizzo (@ARizzo44) December 11, 2017
This breaks my heart. I stand with you Keaton. You are 1 million times stronger than those bullies. https://t.co/XmGwVYLUjF
— andrew mccutchen (@TheCUTCH22) December 10, 2017
Former Green Bay Packers cornerback Bernard Blake urged Jones to "never be ashamed of who you are." Former NFL star Antonio Cromartie stepped in to say that bullies are often just insecure about themselves, asking him to be strong.
Keaton I, along with my @CSUFootball and @packers brothers wanted you to know that we stand against bullying. @Lakyn_Jones #StandWithKeaton pic.twitter.com/qReeMlDp71
— Bernard Blake (@BamBam_Blake23) December 10, 2017
Keaton you are a handsome young man who is made in the image God. The kids that bully you are very insecure about themselves. And it's a shame that the parents of those kids won't be accountable for their kids actions. Keaton Jones I'm here for you buddy. Be Strong young man
— ANTONIO CROMARTIE (@CRO31) December 10, 2017
Former NFL wide receiver Donté Stallworth urged caution for people suggesting that the bullies be confronted with hostility, asking people who really want to make a difference to try to do it through lessons of love.
We all know kids can be little shits at times—especially when it's a group of them. We have to teach them to be better people as well through love and leading by example. They are kids, they can change their behavior through love.
— Donté Stallworth (@DonteStallworth) December 9, 2017
"Bullying is bullshit," summed up World Cup champion Ali Krieger. "We need to start coming together, supporting each other and most importantly, standing up for beautiful kids in this world like Keaton."
This brings tears to my eyes..bullying is bullshit! We need to start coming together, supporting each other and most importantly, standing up for beautiful kids in this world like Keaton. https://t.co/PxGHusq0Dp
— Ali Krieger (@alikrieger) December 11, 2017
Similarly, the music world had words of encouragement and support for Jones.
Demi Lovato predicted that Jones would come out of this experience much stronger than he entered it. Enrique Iglesias called the video "heartbreaking."
I #StandwithKeaton. Keaton baby, just know you're not alone. There are so many people who come out of bullying so much stronger and you will be one of them!! God bless you sweetheart. https://t.co/LdCAy2lDHu
— Demi Lovato (@ddlovato) December 11, 2017
Heartbreaking to see this. Keaton you're strong to say these things and care so much for others. #TeamKeaton https://t.co/hgp0ehnn8Y
— Enrique Iglesias (@enriqueiglesias) December 10, 2017
"This extremely raw and real moment has brought hope and truth to so many people," wrote Kevin Jonas. Nickelback called Jones "a brave young man," asking if there was anything the band could do for them.
Keaton jones you are a hero. This extremely raw and real moment has brought hope and truth to so many people. Be you and be forever fearless. Never give up. You are loved!
— kevin jonas (@kevinjonas) December 10, 2017
"Courage is fire, and bullying is smoke." Stay strong Keaton, you're a very brave young man and a lot of great folks have your back. @Lakyn_Jones if there is anything we can do for Keaton, please let us know. #StandWithKeaton #StopBullying
— Nickelback (@Nickelback) December 11, 2017
Justin Bieber and Snoop Dogg posted words of support on Instagram. "The fact that he still has the sympathy and compassion for other people when he's going through it himself is a testament to who he is," said Bieber.
Anti-bullying activists, models, and YouTube sensations all got in on the act as well.
Monica Lewinsky offered a few kind words, saying that she's sorry Jones is being treated this way, saying that other kids "would be lucky to be friends with [Jones]."
keaton, i'm so sorry you're being treated this way. you don't deserve one ounce of this pain. they would be 💫lucky💫 to be friends with you. it'll get better. #KeatonJones #KeatonJonesIsAwesome https://t.co/IZoJQ60eY2
— Monica Lewinsky (@MonicaLewinsky) December 10, 2017
Model Mia Kang said Jones is her "absolute hero," offering to fly out and visit him at school for lunch.
Keaton Jones you are an absolute hero. Anyone know how I can get in touch with this kid? I'll fly out n have lunch with him at school any day. https://t.co/FmTqdvh8hK
— Mia Kang (@MissMiaKang) December 10, 2017
Logan Paul offered to chat with Jones on FaceTime and send some gear his way.
Hey @Lakyn_Jones I messaged your mother yesterday about Keaton… send me a DM, let's get me on a FaceTime with Keaton and get him some Maverick gear 🤗
— Logan Paul (@LoganPaul) December 11, 2017
Politicians across the political spectrum offered words of kindness and courage.
Senator Bob Corker (R-Tennessee) thanked the young man for his courage, and Representative Joe Kennedy III (D-Massachusetts) asked others to look to Jones as a positive example.
Keaton, I know Tennesseans and people across our country join me in saying thank you for your courage. Bullying isn't acceptable, especially in our schools. We're all with you! #StandWithKeaton
— Senator Bob Corker (@SenBobCorker) December 11, 2017
We can all learn from Keaton Jones. #StandWithKeaton #StopBullying https://t.co/VkiN5cMWCO
— Rep. Joe Kennedy III (@RepJoeKennedy) December 11, 2017
Responding to an offer from UFC head Dana White to visit the organization's headquarters, Donald Trump Jr. offered the Jones family a place to stay. Jane O'Meara Sanders of the Sanders Institute urged action over platitudes, calling on the country to "stand up to bullies — in our schools and communities, on social media and in politics and the White House."
This boy is incredibly brave and the video really got to me. @danawhite, If he takes you up on your offer to see UFC Headquarters, I would be honored to host him and his family at our place if they need somewhere to stay. https://t.co/EWx05o0yI0
— Donald Trump Jr. (@DonaldJTrumpJr) December 11, 2017
💔 It will only "get better someday" if we ALL stand up to bullies – in our schools & communities, on social media & in politics & the White House. Keaton & every other child, teen & adult deserves to be defended against aggression. Please speak up. 💔 https://t.co/JCMPsaBjMd
— Jane O'Meara Sanders (@janeosanders) December 10, 2017
Media personalities joined the chorus with offers of support and workplace tours.
Jemele Hill and Sean Hannity offered Jones and his family tours of ESPN and Fox News, respectively. NBC's Stephanie Ruhle pointed to Jones as a motivation for a more honest, brave, and kind world.
Hey Keaton, unfortunately you aren't alone. A lot of kids are bullied, but they will be so uplifted by your courage. You're a hero, young man. You are treasured and valued. Let me know if you'd ever like to visit ESPN. You can be my special guest. https://t.co/N8mkUTYnEy
— Jemele Hill (@jemelehill) December 11, 2017
If someone can get to me the name of the school, the principal, school board members i will gladly make the calls myself and fix this. Also if anyone knows Keaton and his parents, i will gladly fly them to NYC to be my guest & visit Fox News. https://t.co/uFyp2r2Yn7
— Sean Hannity (@seanhannity) December 10, 2017
At a time when hate wants to reign supreme, let #KeatonJones be our #mondaymotivation – be honest, be brave & be kind. Have a great day at school Keaton! #yougotthis
— Stephanie Ruhle (@SRuhle) December 11, 2017
HLN's S.E. Cupp shared a story about being bullied as a child, saying, "It's got nothing to do with you and everything to do with them." Sunny Hostin, from "The View," ended with a reminder that "being different makes you special."
Keaton buddy, I was bullied just like you. It's got nothing to do with you and everything to do with them. We love you, little man. Your squad's BETTER than theirs! 👍🏻💪🏻😘 https://t.co/Z4U0soTCcT
— S.E. Cupp (@secupp) December 10, 2017
The bullies are the problem Keaton. Not you. Always remember being different makes you special. @Everything_TN https://t.co/K9VBbrYSQx
— Sunny Hostin (@sunny) December 10, 2017
It's wonderful to see so many people, from so many backgrounds, come together in support of this one boy.
It's worth remembering, however, that he's not the only child in the world being bullied.
According to StopBullying.gov, 28% of U.S. students in grades 6 through12 have experienced bullying. 30% of students have admitted to being a bully to others. School bullying creates a hostile environment not conducive to learning and puts students' physical, emotional, and mental health at risk.
If Keaton Jones' story inspired you to take action, check out the StopBullying prevention toolkits for students, parents, teachers, and community members.
Read more: http://www.upworthy.com/39-of-the-best-celebrity-responses-to-keaton-jones-powerful-video-about-bullying