text:
The yuletide has been a traditional time for ghostly tales... for it is associated with the winter solstice, the time the ancients feared the sun would be completely extinguished.
As the dying yellow light grows weaker... we are pleased to offer for your consideration a selection of the macabre lots for auction from the Carcosa Collection.
Auction Start December 20th 2010 Midnight PST
Auction End December 31st Midnight PST
LOT#: YiY10-00
TITLE: Conversation with Castaigne
CATEGORY: Coaching
ORIGIN: Carcosa
CIRCA: Nowhen
RESERVE BID: Unknown
DESCRIPTION: 3 hours of coaching from the dark minds of Carcosa
LOT#: YiY10-01, YiY10-02, YiY10-03
TITLE: The King in Yellow 1st EditionCATEGORY: BookORIGIN: Alternative Time LineCIRCA: 19th Century?CONDITION: Ancient Tome
OPENING BID: $200RESERVE BID: A Mystery
DETAILS: The King in Yellow Play. First Edition. Author Anonymous. Bound in a serpentine hide of unknown origin, upon which is emblazoned the Yellow Sign. The King in Yellow has two acts that describe the coming of the King to Carcosa. Act I as quite ordinary, but reading Act II drives the reader mad with the "irresistible" revealed truths. "The very banality and innocence of the first act only allowed the blow to fall afterward with more awful effect." Even seeing of the first page of the second act is enough to draw the reader in: "If I had not caught a glimpse of the opening words in the second act I should never have finished it …"
Filled with poetry, prose and incantations, with images from dark artists, the play is designed to be a utility mystery book, for use to do readings, predictions, pegasii pages, and more. You can tear and burn out pages… and then replace them before the next time you are ready to unleash the horrors bound betwixt the covers. Each is one of a kind in its patterns & colours –ranging from billious yellows to serpentine green to boa brown to black adder… its scaly horrors each unique. and only 13 official plays will be auctioned.
The current 3 lots consist of 4/13, 5/13, and 6/13.
LOT #: YiY10-5, YiY10-6, YiY10-7
TITLE: Arcana Carcosa and the Oracular Eye CaseCATEGORY: Tarot DeckORIGIN: UnknownCIRCA: 21st CenturyCONDITION: MintOPENING BID: $100RESERVE BID: A Mystery
DETAILS: Arcana Carcosa: The Major Arcana of Carcosa, rendered by mad artists to create gateways to the realms of nightmare. The images are from the accursed play The King in Yellow, and images within that tome resonate with the ones upon the cards. With hypnotic patterns and embedded mysterious sigils, the pack can be used for many mysterious presentations. It is said when used with the actual book of the play, the enigmas of the Hyades can be unlocked. The pack comes with an Oracular case which has its own secrets to be discovered by its future owner…various mystery practioners have contributed routines that will be shared with obtainers of these diabolical doorways to the unknown. A maximum of 13 packs with the Oracular Case and its sinister extras will be auctioned. 66 packs over time will be offered in various accompanied vessels of darkness.
The following are #3/13, 4/13, 5/13
LOT #: YiY10-04
TITLE: The Scrapbook of Canon AlbericCATEGORY: BookORIGIN: Multiple illuminated manuscripts robbed from across the medival world.CIRCA: 17th CenturyCONDITION: Ancient Tome
OPENING BID: $250RESERVE BID: A Mystery
DETAILS: Ancient Medieval tome, bound in ancient leather, with the seals of Albéric de Mauléon. Almost every page has a stolen illuminated manuscript upon it, looted by the Canon for his own collection. One such acquistion is rumored to be cursed, upon which a terrying scene is portrayed as descbried by this account: The picture in question was a sepia drawing at the end of the seventeenth century, representing, one would say at first sight, a Biblical scene; for the architecture (the picture represented an interior) and the figures had that semi-classical flavour about them which the artists of two hundred years ago thought appropriate to illustrations of the Bible. On the right was a King on his throne, the throne elevated on twelve steps, a canopy overhead, lions on either side -- evidently King Solomon. He was bending forward with outstretched sceptre, in attitude of command; his face expressed horror and disgust, yet there was in it also the mark of imperious will and confident power. The left half of the picture was the strangest, however. The interest plainly centred there. On the pavement before the throne were grouped four soldiers, surrounding a crouching figure which must be described in a moment. A fifth soldier lay dead on the pavement, his neck distorted, and his eyeballs starting from his head. The four surrounding guards were looking at the King. In their faces the sentiment of horror was intensified; they seemed, in fact, only restrained from flight by their implicit trust in their master. All this terror was plainly excited by the being that crouched in their midst. I entirely despair of conveying by any words the impression which this figure makes upon anyone who looks at it. I recollect once showing the photograph of the drawing to a lecturer on morphology -- a person of, I was going to say, abnormally sane and unimaginative habits of mind. He absolutely refused to be alone for the rest of that evening, and he told me afterwards that for many nights he had not dared to put out his light before going to sleep. However, the main traits of the figure I can at least indicate. At first you saw only a mass of coarse, matted black hair; presently it was seen that this covered a body of fearful thinness, almost a skeleton, but with the muscles standing out like wires. The hands were of a dusky pallor, covered, like the body, with long, coarse hairs, and hideously taloned. The eyes, touched in with a burning yellow, had intensely black pupils, and were fixed upon the throned King with a look of beast-like hate. Imagine one of the awful bird-catching spiders of South America translated into human form, and endowed with intelligence just less than human, and you will have some faint conception of the terror inspired by this appalling effigy. One remark is universally made by those to whom I have shown the picture: 'It was drawn from the life.'
Each book is OOAK and a maximum of 13 will be made depending on demand. This is #2/3. It too is a utility book using images for mystery effects.
LOT #: YiY10-11, YiY10-12, YiY10-13
TITLE: Ye Essential SaltesCATEGORY: Mystical RemainsORIGIN: From the lab of Joseph CurwenCIRCA: 15th Century… saltes unknownCONDITION: N/AOPENING BID: $120RESERVE BID: A Mystery
DETAILS: "The essential Saltes of Animals may be so prepared and preserved, that an ingenious Man may have the whole Ark of Noah in his own Studie, and raise the fine Shape of an Animal out of its Ashes at his Pleasure; and by the lyke Method from the essential Saltes of humane Dust, a Philosopher may, without any criminal Necromancy, call up the Shape of any dead Ancestour from the Dust whereinto his Bodie has been incinerated". – BorellusBeware of raising up that which you cannot put back down into the dust… and only the liveliest awefullness may await you… for here we have samples of the essential saltes rendered from the remains from the plundered tombs chosen by Curwen and his brothers in dark sorcery. These saltes have various uses, but its major effect is to make it appear that a small amount of ash transforms into a sputtering bloody mass of bloody gore to which you can fish out an eye or finger to use for necromatic purposes. The saltes are sealed in an alchemical containter and some other extra items needed for a gory climax to your conjurings. Warning: things can get red and messy if you are not careful. 13 packets of essential saltes will be auctioned over time – but almost every set will be unique in that the type of vessels and extras that come with it will vary. The following are for these auctions 3/13, 4/13, 5/13.
LOT #: YiY10-14, YiY10-15, YiY10-16
TITLE: Wind Walker ensembleCATEGORY: Ritual tools / OraclesORIGIN: Great Wilds of CanadaCIRCA: 19th CenturyCONDITION: MintOPENING BID: $225
RESERVE BID: A Mystery
DETAILS: The Wendigo is a mythical creature appearing in the mythology of the Algonquian people. It is a malevolent cannibalistic spirit into which humans could transform, or which could possess humans. Those who indulged in cannibalism were at particular risk,[ and the legend appears to have reinforced this practice as taboo. They were strongly associated with the Winter, the North, and coldness, as well as with famine and starvation. It is claimed by some occultists that Ithaqua the Wind Walker, one of the Great Old Ones, is the master of the Wendgio. The entity appears as a horrifying giant with a roughly human shape and glowing red eyes. He has been reported from as far north as the Arctic to the Sub-Arctic, where Native Americans first encountered him. He is believed to prowl the Arctic waste, hunting down unwary travelers and slaying them gruesomely.
Wendigos are said to be beings with hearts of ice encased in a monstrous form: The Wendigo was gaunt to the point of emaciation, its desiccated skin pulled tautly over its bones. With its bones pushing out against its skin, its complexion the ash gray of death, and its eyes pushed back deep into their sockets, the Weendigo looked like a gaunt skeleton recently disinterred from the grave. What lips it had were tattered and bloody [....] Unclean and suffering from suppurations of the flesh, the Weendigo gave off a strange and eerie odor of decay and decomposition, of death and corruption.All cultures in which the Wendigo myth appeared shared the belief that human beings could turn into Wendigos if they ever resorted to cannibalism[ or, alternately, become possessed by the demonic spirit of a Wendigo, often in a dream. Once transformed into a being with a heart of ice and mishapen mosntrous features, a person would become violent and obsessed with eating human flesh. They could ride the winds and search for prey, howling as they sought their victims. The most frequent cause of transformation into a Wendigo was if a person had resorted to cannibalism, consuming the body of another human in order to keep from starving to death during a time of extreme hardship or famine.
During a time of great famine, a shaman help convince his people to transgress the taboo to save their own tribe. Having been decimated by enemies, forced off their land, and their culture shattered, he led and uprising against the enemies of the tribe – and consumed many captured victims. The tribe had decided to seek vengence on humanity as a whole for leaving stealing not only their physical possesions, but their culture and spirituality. Thus they transformed into wendigos. When the local authorities arrived, their coventional weapons and experiences did not prepare them for the slaughter of their troops. Eventually, other shamans helped defeat these monsters, for the sake of the innocent.
The Carcosa Collection has obtained a few relics and artifacts from that event, including some of the dark bad medicine bags of the corrupted shamans, the weirdling bone oracles that were kept within them, and pendulums made of wendigo molars that can be used to seek warm human flesh. Or so the legends go. Each bag has the ability to be used for mystery effects to obtain knowledge, produce secret writing, and the like. The molar is suspended by a chain for pendulum use. The bone runes are designed for multiple effects ranging from forces of multiple types, Karatsuke, to dark oracular readings.
#2/13. #3/13, #4/13
LOT #: YiY10-18, YiY10-19, YiY10-20, YiY10-21, YiY10-22
TITLE: Second Sight CaseCATEGORY: WalletORIGIN: Mythic Venice
CIRCA: 20th CenturyCONDITION: Mint
OPENING BID: $75RESERVE BID: A Mystery
Description: A sinister scaled wallet, rumored to be made by a galleria artisan in the courts of the King in the palace of Ythill. Some of these have appeared in the shadowy shops of Venice... and owners of the leathery cases are often stalked by a small figure in a red rain coat...
Refurbished SUC Gen I (and one Gen II) (no one wants to get rid of their Gen II cases, it appears). The artisans of the collection have redone the surface of the cases to give them a more colorful and serpentine look… in keeping with the primal evil of the King in Yellow (yes, they ended up two toned - black leather on the inside of the original wallet). In replacing the worn surface, a new concept was conceived to allow some extra abilities new to such a case. Hence, this is a Second Sight case – and it also conjures up the concept of Second Sight which one can simulate using this wallet.. Note – the keepers have only obtained 6 so far, and thus only 5 will be auctioned (as one will be kept for the permanent collection). The keepers will not make any new cases from Scratch as the rights for the cases belong to Mark Strivings, who has given no one permission to make any. Thus, only ones that actually existed that we could buy used and then refurbished and modified were used by our artisans… as many other used magic items have been. Due to the pain of trying to get these items and then taking them apart to redo, these will likely be the only ones sold - it took 3 years to get these alone... The SSC will come along with a special book from Piers, who has offered it as part of the lot and thus the entire amount from these sales go 100% to charity.
YiY10-18 – Black Subtle scales Gen IYiY10-19 – Dark Brown subtle scales Gen IYiY10-20 – Red Lava Scales Gen IYiY10-21 – Big Brown Crocodile Gen IYiY10-22 – Lizard Emerald Green Gen II
LOT #: YiY10-8, YiY10-9, YiY10-10
TITLE: Lovecraftian ShipmatesCATEGORY: Spirit TrapsORIGIN: South PacificCIRCA: UnknownCONDITION: MintOPENING BID: $50RESERVE BID: A Mystery
DETAILS: " … These folk say that on a table in a bare room on the ground floor are many peculiar bottles, in each a small piece of lead suspended pendulum-wise from a string. And they say that the Terrible Old Man talks to these bottles, addressing them by such names as Jack, Scar-Face, Long Tom, Spanish Joe, Peters, and Mate Ellis, and that whenever he speaks to a bottle the little lead pendulum within makes certain definite vibrations as if in answer."Now you can own a set of 6 pendulums which are spirit traps to contain the souls of those you wish to communicate with! Based on classic techniques for multi-pendulum use, as well as some gimmicked options, conduct a Lovecraftian séance when you wish. Each of the 6 pendulums is named after one of the Terrible Old Man's shipmates. Bottles are not supplied to keep costs of shipping down – but the pendulums can be used in a bottle or not. The key is to allow the vibrations to flow…
Each set is OOAK, and only 13 will be auctioned. The following are numbers 4/13, 5/13, 6/13.
The Spirit Traps... carved with ancient glyphs from a prehuman language...
LOT #: YiY10-17
TITLE: A Music Box of Erich ZannCATEGORY: Music BoxORIGIN: ParisCIRCA: 19th CenturyCONDITION: Fine-MintOPENING BID: $150RESERVE BID: A Mystery
DETAILS: The Musing box redone, with the blessings of Lee Earle. A university student is forced, by his lack of funds, to take the only lodging he can afford. In a strange part of the city he had never seen before, on a street named "Rue d'Auseil", he finds an apartment in an almost empty building. One of the few other tenants is an old German man named Erich Zann. The old man is mute and plays the viol with a local orchestra. He lives on the top floor and when alone at night, plays strange melodies never heard before. Over time, the student gains Zann's trust, and eventually learns of his secret, that the old man has discovered melodies and rhythms of sound of an almost otherworldly nature. Zann plays these sounds to keep back unknown and unseen creatures from Zann's window, which is said to look out into a black abyss, most likely another dimension. Well, the Collection has obtained a box that contains the trapped essence of one of these alien entities. Open at your own risk… only 13 of these creatures have been captured by Zann in his day. This box is #3/13.
LOT: YiY10-23, YiY10-24, YiY10-25, YiY10-26
TITLE: The Caskets of Cries (Lost Souls Box Compositions)
CATEGORY: Reliquary of the damned?ORIGIN: EuropeCIRCA: 20th CenturyCONDITION: Mint
DETAILS: Please see the LeMarchand and Munch gallery for further details. These particular boxes were made early on in their collaborations. They are rumored to be able to perform various transdimensional gateway effects... including Bilocation, transmutation, and other occult effects. In other words, they are refurbished and stylized m******c l*****g c**d b***s. Now do Mephisto's Journey or the Original Voodoo effects in horrific style... or darksome tarot deck predictions... a utility device with the faces of the screaming lost souls...
LOT #: YiY10-37
TITLE: The Stone TapeCATEGORY: Spirit RecordORIGIN: TaskerlandsCIRCA: 20th CenturyCONDITION: Mint
OPENING BID: $666RESERVE BID: A Mystery
DETAILS: One of the first to promulgate the hypothesis of residual haunting, that ghosts may be recordings of past events made by the physical environment, was Thomas Charles Lethbridge in books such as Ghost and Ghoul, written in 1961. Since the broadcast of the play The Stone Tape by Nigel Kneale, this hypothesis has come to be known as the "Stone Tape Theory" by parapsychological researchers. The recording medium of the future – organic based stone, can capture events… explaining ghostly phenomena… but is it just a tape? Or is it sentient?
Castaigne would love to send you a brick. Be glad we are not calling it an iPhone5.
LOT #: YiY10-44
TITLE: Belasco's WorldCATEGORY: Haunted photographs in hollow bookORIGIN: North AmericaCIRCA: 20th CenturyCONDITION: Mint
DETAILS: A dark form of OOTW based on some of the artifacts found in Hell House. Similar to a living and dead test in the vien of Whitestar and Brown's Undertaker… this one involves even darker psychological factors and, honestly, likely would never be used by the obtainer of this lot… unless they have become possessed by the spirit of Evil Emeric. You have been warned.
Lot: YiY10-40
TITLE: Hellshard
CATEGORY: Scrying Crystal
ORIGIN: Yuggoth
CIRCA: Prehuman
CONDITION: Sharp
DESCRIPTION: Sinister byproducts of the creation process of the Shining Trapezohedron. These darksome gems are said to allow one to scry and use them for oracular activities. A Dichromatic sinsiter pendulum #3/13
LOT: YiY10-666
TITLE: Toynbee ConvectorCATEGORY: Dimensional TransporterORIGIN: ?CIRCA: ?CONDITION: Mint
OPENING BID: SPECIAL - $1000 but $ will not be enough
DETAILS: The Toynbee Convector – change the future or alter the past. OOAK.
Yuletide in Yellow 2010
Tome Tribute
"They say foul beings of Old Times still lurk in dark forgotten corners of the world, And Gates stll gape to loose, on certain nights, Shapes pent in Hell."
From Unaussprechlichen Kulten
Credits and Copyrights
Carcosa Collection and Website Bruce Ballon © 2010-2015 , Web Design Luke Shield and Bruce Ballon, Art / Graphics / Photos by Steve Lines and Bruce Ballon, music clip by Ennio Morricone
Thought of the Eon
There is a concept that is the corrupter and destroyer of all others. I speak not of Evil, whose limited empire is that of ethics; I speak of the infinite.
-Jorge Luis Borges


text:
The first prototype of the purported Galaxy S6 has been spotted, snapped by an iPhone 5C by the looks of the reflection in the shot above. The first thing you'll notice, is the super in-your-face fluorescent yellow colour, which we hope isn't standard.
Judging from the leaked shots and specs, we should expect a 5.5-inch display, Android 4.4 KitKat on board and Qualcomm Snapdragon 801 processor. The first thing that should jump out at you is the mention of Android 4.4 and not Android 5.0, but this could be an early production model that doesn't have Lollipop just yet, or completely fake.
The 5.5-inch display has a resolution of 1920×1080, while the rear-facing camera is a huge 20MP. We should expect costs of the new Galaxy S6 to be around $700-$800 as usual.
Source: Chiphell.

subset: commoncrawl


text:
Transcription Elongation and Tissue-Specific Somatic CAG Instability
The expansion of CAG/CTG repeats is responsible for many diseases, including Huntington's disease (HD) and myotonic dystrophy 1. CAG/CTG expansions are unstable in selective somatic tissues, which accelerates disease progression. The mechanisms underlying repeat instability are complex, and it remains unclear whether chromatin structure and/or transcription contribute to somatic CAG/CTG instability in vivo. To address these issues, we investigated the relationship between CAG instability, chromatin structure, and transcription at the HD locus using the R6/1 and R6/2 HD transgenic mouse lines. These mice express a similar transgene, albeit integrated at a different site, and recapitulate HD tissue-specific instability. We show that instability rates are increased in R6/2 tissues as compared to R6/1 matched-samples. High transgene expression levels and chromatin accessibility correlated with the increased CAG instability of R6/2 mice. Transgene mRNA and H3K4 trimethylation at the HD locus were increased, whereas H3K9 dimethylation was reduced in R6/2 tissues relative to R6/1 matched-tissues. However, the levels of transgene expression and these specific histone marks were similar in the striatum and cerebellum, two tissues showing very different CAG instability levels, irrespective of mouse line. Interestingly, the levels of elongating RNA Pol II at the HD locus, but not the initiating form of RNA Pol II, were tissue-specific and correlated with CAG instability levels. Similarly, H3K36 trimethylation, a mark associated with transcription elongation, was specifically increased at the HD locus in the striatum and not in the cerebellum. Together, our data support the view that transcription modulates somatic CAG instability in vivo. More specifically, our results suggest for the first time that transcription elongation is regulated in a tissue-dependent manner, contributing to tissue-selective CAG instability.
Published in the journal:
. PLoS Genet 8(11): e32767. doi:10.1371/journal.pgen.1003051
Category: Research Article
doi: 10.1371/journal.pgen.1003051
The expansion of CAG/CTG repeats is responsible for many diseases, including Huntington's disease (HD) and myotonic dystrophy 1. CAG/CTG expansions are unstable in selective somatic tissues, which accelerates disease progression. The mechanisms underlying repeat instability are complex, and it remains unclear whether chromatin structure and/or transcription contribute to somatic CAG/CTG instability in vivo. To address these issues, we investigated the relationship between CAG instability, chromatin structure, and transcription at the HD locus using the R6/1 and R6/2 HD transgenic mouse lines. These mice express a similar transgene, albeit integrated at a different site, and recapitulate HD tissue-specific instability. We show that instability rates are increased in R6/2 tissues as compared to R6/1 matched-samples. High transgene expression levels and chromatin accessibility correlated with the increased CAG instability of R6/2 mice. Transgene mRNA and H3K4 trimethylation at the HD locus were increased, whereas H3K9 dimethylation was reduced in R6/2 tissues relative to R6/1 matched-tissues. However, the levels of transgene expression and these specific histone marks were similar in the striatum and cerebellum, two tissues showing very different CAG instability levels, irrespective of mouse line. Interestingly, the levels of elongating RNA Pol II at the HD locus, but not the initiating form of RNA Pol II, were tissue-specific and correlated with CAG instability levels. Similarly, H3K36 trimethylation, a mark associated with transcription elongation, was specifically increased at the HD locus in the striatum and not in the cerebellum. Together, our data support the view that transcription modulates somatic CAG instability in vivo. More specifically, our results suggest for the first time that transcription elongation is regulated in a tissue-dependent manner, contributing to tissue-selective CAG instability.
The expansion of trinucleotide repeats (TNRs) is the causative mutation of more than fifteen neurodegenerative, neurological and neuromuscular genetic diseases, including Huntington's disease (HD), several dominant spinocerebellar ataxias (SCAs), myotonic dystrophy 1 (DM1), Friedreich ataxia (FRDA) and fragile X syndrome (FXS) [1]. TNR expansions become unstable and toxic above a threshold of 30 to 50 repeat units. TNR instability leads to repeat size variation, often biased towards further expansion, in the germline and in somatic tissues over time [2]–[4]. Remarkably, the toxicity and the instability of TNRs are tissue-selective in a disease-specific manner. In some diseases, including HD, DM1 and FRDA, the affected and 'unstable' tissues partially coincide, thereby accelerating disease progression [1], [5]–[7]. In HD, somatic instability of CAG repeats is most important in the striatum, a tissue that selectively degenerates [4], [6]. In DM1, somatic CTG instability is also most extensive in affected tissues, including the muscle and some brain regions [8]–[10]. Similarly, somatic GAA instability in FRDA is important in dorsal root ganglia and cerebellum, two tissues primarily affected in the disease [11], [12]. Thus, it is essential to decipher the mechanisms underlying tissue-selective instability of TNRs to better define the relationship between toxicity and instability.
CAG/CTG repeats as well as other TNRs form stable DNA secondary structures in vitro, and several studies support the view that the error-prone repair of these structures by overwhelmed DNA repair machineries contributes to TNR instability [1], [13]–[19]. Studies based on cell models also suggest that transcription is involved in CAG/CTG instability [20], [21] and formation of stable DNA:RNA hybrids (R-loops) at repeat sites might contribute to the process [22]–[24]. Furthermore, all TNR loci associated with diseases are located in transcribed regions of the genome, supporting the idea that transcription and TNR instability are linked. However, the role of transcription in TNR instability in vivo remains unclear [1]. First, if transcription contributes to TNR instability, one would expect that transcriptional activity at TNR genes correlates with tissue-selective repeat instability, which seems to be contradicted by previous studies [25]–[27]. For instance, Huntingtin (HTT) mRNA in the striatum of HD patients or rodent models was not increased when compared to tissues exhibiting minimal CAG instability, such as the cerebellum [25], [26]. Similarly, somatic CTG instability in selective tissues did not correlate with increased transcription levels in DM1 mice [27]. Second, it is unclear whether the transcriptional process would contribute per se to TNR instability or whether transcription would only facilitate access of transacting-factors such as DNA repair proteins, due to increased accessibility of chromatin at transcribed regions [28]. In the latter hypothesis, chromatin structure rather than transcriptional activity is expected to influence TNR instability.
It has been shown that TNRs, together with specific cis-elements, have the capacity to modify the chromatin environment by inducing a heterochromatinization process, eventually leading to transcriptional silencing such as in FRDA and FXS, which are caused by GAA and CGG expansions, respectively [29]–[31]. Thus, heterochromatin rather than accessible chromatin has been associated with unstable TNRs, pointing to an apparent paradox.
Here, we have investigated the relationship between chromatin structure, transcription and tissue-selective CAG instability by using R6/1 and R6/2 HD transgenic mouse lines. These two lines have been generated using the same HD transgenic construct, which consists of ≈1000 bp of the human HTT promoter, the entire HTT exon-1, including a CAG repeat expansion, and 262 bp of HTT intron-1 [32]. The transgene is integrated in different genomic loci, recently mapped to chromosome 3 and 4 in R6/1 and R6/2 lines, respectively [33], [34]. R6/1 and R6/2 mice develop a comparable HD-like phenotype. However, progression of the disease is much faster in R6/2 than in R6/1 mice, dying at ≈12 and ≈40 weeks, respectively. Importantly, somatic CAG instability in R6 mice exhibits a similar tissue-selectivity profile as in HD patients, indicating that the mechanisms underlying tissue selectivity are conserved between the two species [35]. Specifically, CAG instability in R6 lines and in humans is most marked in the striatum and minimal in the cerebellum. We previously showed that the level of somatic instability was higher in R6/2 than in R6/1 mice of similar ages, indicating that the instability rate might be increased in R6/2 mice [36]. CAG instability was also higher in R6/2 mice, irrespective of the repeat size (100 or 160 CAG repeats), than in R6/1 mice (130 CAG repeats) [36], suggesting that it is the chromatin context at the site of transgene integration and possibly the expression level of HD transgene, rather than the initial expansion size that underlies the different somatic instability levels.
To test the hypothesis that the chromatin structure and/or transcriptional activity at the HD locus might contribute to somatic CAG instability, we have performed chromatin immunoprecipitation experiments and expression analyses from the striatum and the cerebellum of R6/1 and R6/2 mice. Our results indicate that the HD transgene is integrated in a more euchromatinized and transcriptionally active region in R6/2 mice than in R6/1 mice, supporting the view that an active chromatin structure results in increased CAG instability. However, chromatin accessibility and transcription initiation at the HD locus were similar in the striatum and cerebellum of R6 mice, indicating that tissue-selective CAG instability is unlikely to be simply explained by an accessible or transcriptionally active chromatin state. Strikingly, H3K36 trimethylation and elongating RNA Pol II were increased at the HD locus in the striatum of both R6/1 and R6/2 mice. Together, our in vivo data suggest that transcription elongation mediates tissue-specific somatic CAG instability in HD.
CAG instability rates in striatum and cerebellum of R6/1 and R6/2 mice
To correlate the chromatin state and the transcriptional activity at the HD locus with the level of CAG instability in R6/1 and R6/2 striatum and cerebellum, we determined CAG instability rates. The level of instability of CAG repeats in mouse striatum and cerebellum was assessed in this study and in previous work [36]. R6/1 mice with 130 repeats were analyzed at 3, 6, 12 and 38 weeks and R6/2 mice with 160 repeats at 3, 6 and 12 weeks (R6/1 and R6/2 mice die around 40 and 12 weeks, respectively). To determine instability rates, we represented the level of CAG instability in R6/1 and R6/2 striatum and cerebellum, as measured by the variation of CAG repeat size, according to age (Figure 1). Interestingly, instability rates over time could be modeled using linear regression analyses (Figure 1). CAG instability rates, as measured by the regression coefficient (slope), ranked as follows: R6/2 striatum>R6/1 striatum>R6/2 cerebellum>R6/1 cerebellum (Figure 1).
Chromatin at the HD locus is less accessible in R6/1 tissues compared to R6/2 tissues
We investigated the hypothesis that the chromatin structure at the HD locus might be different in R6/1 and R6/2 tissues, which might underlie the different CAG instability rates. To explore this idea, we performed chromatin immunoprecipitation-PCR (ChIP-PCR) experiments and quantitatively assessed the level of histone marks associated with heterochromatin, euchromatin or transcriptionally active chromatin around the CAG expansion, as previously done for other TNRs associated to diseases [31], [37]–[39]. More specifically, we analyzed the striatum and the cerebellum of R6/1 and R6/2 mice. Tissue samples were sonicated to <500 bp to increase resolution of ChIP experiments (Figure S1). Primer pairs around the CAG tract, located ≈300 bp upstream of the repeats, within HTT proximal promoter (HD promoter), and ≈300 bp downstream of the repeats (HD intron-1), were selected to analyze the HD locus (Figure 2A). As controls, we included primers targeting regions expected to be euchromatic/transcriptionally active (the endogenous murine huntingtin gene Hdh, Hdh promoter and Hdh intron-1, Figure 2A) and heterochromatic/transcriptionally inactive (the ICRH19 locus). We used R6/2 mice carrying 160 CAGs at late-pathological stage (12 week-old) and R6/1 mice carrying 130 CAGs at both early- and late-pathological stages (13 and 36 week-old, respectively).
We first assessed the level of H3K9 dimethylation (Me2H3K9), a hallmark of heterochromatin. Me2H3K9 was 3- to 4-fold more elevated at HD intron-1 in R6/1 striatum and cerebellum when compared to R6/2 tissue-matched samples, regardless of age, and reached levels comparable to those measured at the ICRH19 locus (Figure 2B). Me2H3K9 at the HD promoter was also increased in R6/1 cerebella with respect to R6/2 cerebella, though it was similar in striata (Figure 2B). Globally, Me2H3K9 level at the HD locus, as assessed by calculating the mean level at the HD promoter and HD intron-1, was significantly increased in R6/1 tissues as compared to R6/2 tissues (Figure 2C). The increased level was not dependent upon age. Moreover, the higher level of Me2H3K9 did not result from increased nucleosome occupancy, as measured by the level of total H3 at the HD locus, which was similar in R6/1 and R6/2 tissues (Figure 3A). Noticeably, unmodified H3 was lower at the HD promoter when compared to HD intron-1, consistent with reduced nucleosome occupancy at the region upstream of the transcription start site (TSS), a feature shared by many promoters [40]. We also assessed Me2H3K9 at the HD locus and at a control heterochromatic region (the N-myc locus) in HD and control human lymphoblasts. The level of Me2H3K9 at the HD locus was low as compared to the level at the N-myc locus, and similar in HD and control cells (Figure 2E). In addition, it was comparable to the level measured using R6/2 tissues. Together, these results suggest that the 3′ end of the HD transgene is heterochromatinized in R6/1 mice, due to position effect imposed on the transgene.
We then examined the correlation between somatic CAG instability and Me2H3K9 levels at the HD locus (Figure 2D). The R2 value was only 0.31. This is because Me2H3K9 level was similar in striatum and cerebellum, despite the fact that it was increased in R6/1 tissues relative to R6/2 tissues. Thus, Me2H3K9 level at the HD locus inversely correlates with mouse-line dependent instability, but not with tissue-selective instability.
Chromatin is in a more transcriptionally active state at the HD locus in R6/2 tissues compared to R6/1 tissues
To further explore the chromatin structure at the HD locus of R6/1 and R6/2 mice, we assessed the level of the euchromatic mark acetyl H3K9 (AcH3K9) (Figure 3B). AcH3K9 was lowest at the ICRH19 locus, which is consistent with heterochromatin. AcH3K9 at HD intron-1 was lower in R6/1 cerebellum with respect to R6/2 cerebellum but not significantly different in R6/1 and R6/2 striatum. AcH3K9 was consistently low at the HD promoter, likely resulting from reduced H3 levels (Figure 3A). No significant difference between tissues and mouse lines was seen for AcH3K9 at the HD promoter (Figure 3B). Globally, AcH3K9 at the HD locus was similar in the different tissues and mice, though it was slightly decreased in R6/1 cerebellum as compared to R6/2 cerebellum, and did not correlate with CAG instability rates (R2 = 0.11, Figure 3C).
The euchromatic state is also characterized by histone post-translational modifications associated with transcription, including Me3H3K4, which is enriched at the TSS of active genes [41]. We assessed by ChIP the level of Me3H3K4 at the HD locus in the striatum and cerebellum of R6/1 and R6/2 mice (Figure 4). R6/1 mice were analyzed at 13 and 36 weeks of age and R6/2 mice at 12 weeks. To closely target the HTT TSS region, we included an amplicon corresponding to a fragment located between the TSS and CAG repeats (HD exon-1, Figure 4A). Consistent with the idea that nucleosome occupancy is low upstream of the TSS, Me3H3K4 was low at the HD promoter (Figure 4B). In contrast, Me3H3K4 at HD exon-1 and HD intron-1 was elevated, particularly in R6/2 tissues when compared to R6/1 tissues. Globally, Me3H3K4 at the HD locus was significantly increased in R6/2 tissues relative to R6/1 matched-tissues (Figure 4C), and this was not dependent upon mouse age. These results support the view that the chromatin at the HD locus is in a more transcriptionally active state in R6/2 than in R6/1 mice, and suggest that the transcriptional activity of the HD transgene is increased in R6/2 mice when compared to R6/1 mice. This is consistent with the results of Figure 2 suggesting that the chromatin at the HD locus is more heterochromatinized in R6/1 mice than in R6/2 mice. Thus, high Me3H3K4 and low Me2H3K9 levels at the HD locus correlate with high CAG instability levels of R6/2 mice. However, the correlation the R2 value between Me3H3K4 levels at the HD locus and CAG instability rates in striatum and cerebellum of R6/1 and R6/2 mice was only 0.40, due to comparable Me3H3K4 levels in mouse striatum and cerebellum (Figure 4C). Our results suggest that Me3H3K4 level does not correlate with tissue-selective instability, though it correlates with mouse line-dependent instability.
Transcriptional activity of the HD transgene is increased in R6/2 mice compared to R6/1 mice
The above results suggest that HD transgene expression might be increased in R6/2 mice when compared to R6/1 mice. We measured the levels of HD transgene mRNA in R6 tissues by using quantitative RT-PCR (qRT-PCR) (Figure 5). The striatum and cerebellum of R6/1 mice and R6/2 mice were analyzed at both early- and end-pathological stages (i.e. 6, 13 and 36 weeks and 6 and 12 weeks, respectively). We selected primers targeting a region upstream of CAG repeats in HD exon-1 (Figure S2). Expression values were normalized relative to expression of Gapdh or 18S expression (Figure 5A and Figure S3), which led to comparable results. At 6 weeks, transgene expression was 3- to 5-fold higher in R6/2 tissues in comparison to R6/1 tissue-matched samples, in accordance with the results above showing that chromatin structure at the HD locus is transcriptionally more active in R6/2 mice than in R6/1 mice. The differential transgene expression between R6/1 and R6/2 tissues was reduced over time, since HD transcript levels showed a tendency for decrease in R6/2 tissues. Transgene expression was not significantly different between the striatum and the cerebellum of either R6/1 or R6/2 mice. As a result, the correlation value R2 between HD transgene mRNA levels and CAG instability rates was only 0.40 (Figure 5B).
We further assessed transgene expression in striatum and cerebellum of 6 week-old R6/1 and R6/2 mice using an absolute quantification method with the corresponding genomic DNA as standards. This allowed for comparison between PCR product levels amplified with the above primers (located upstream of the repeats) and with primers encompassing the CAG repeats ("CAG" primers) (Figure 5C and S4). The level of transgene transcripts were comparable using both sets of primers, and increased in R6/2 tissues when compared to R6/1 tissue-matched samples using both sets of primers.
RNA polymerase II level at the HD locus correlates with somatic CAG instability
To investigate the relationship between transcription and somatic CAG instability, we performed ChIP from the striatum and cerebellum of R6/1 and R6/2 mice at late-pathological stages with the RNA Pol II 7C2 antibody, which recognizes phosphorylated and non phosphorylated forms of the polymerase [42]. RNA Pol II was distributed along the HD locus, peaking downstream of the TSS, at HD exon-1 (Figure 6A). Interestingly, RNA Pol II at the HD locus was higher in the striatum than in the cerebellum of both R6/1 and R6/2 mice, suggesting that the level of RNA Pol II at the HD region is regulated in a tissue-specific manner. Moreover, as previously found with Me2H3K9 and Me3H3K4 marks, RNA Pol II at the HD locus was increased (2- to 3-fold) in R6/2 tissues when compared to R6/1 tissue-matched samples (Figures 6A and 6B). As a result, RNA Pol II at the HD locus was highly correlated with CAG instability in R6 mice (R2 = 0.91, Figure 6B). RNA Pol II at control regions, including at Hdh regions, was also more abundant in the striatum than in the cerebellum of R6/1 and R6/2 mice (Figure 6A), further indicating that increased RNA Pol II at genes in striatum relative to cerebellum is tissue-specific. Together, these results suggest that a high level of RNA Pol II at the HD locus might contribute to CAG instability in vivo.
Elongating RNA polymerase at the HD locus correlates with somatic CAG instability
The activity of RNA Pol II during transcription is regulated by phosphorylation [43]–[45]. Ser5 phosphorylation of RNA Pol II is involved in transcription initiation, while Ser2 phosphorylation promotes transcription elongation. To discriminate between initiating and elongating RNA Pol II at the HD locus, we performed ChIP with antibodies to phosphorylated Ser5 RNA Pol II and phosphorylated Ser2 RNA Pol II (Figure 7A). As for Me3H3K4, which is associated with transcription initiation, the level of initiating RNA Pol II at the HD locus was increased downstream of the TSS, was comparable in the striatum and cerebellum of HD mice and was globally increased in R6/2 mice when compared to R6/1 mice (Figure 7A and 7B). As a result, initiating RNA Pol II was poorly correlated with somatic CAG instability in R6 mice (R2 = 0.30; Figure 7B). Elongating RNA Pol II at the HD locus peaked downstream of the TSS, particularly at the HD intron-1, but was more elevated in striata than in cerebella of both R6/1 and R6/2 mice (Figure 7C). In addition, the increase was more pronounced in R6/2 striatum relative to R6/1 striatum (Figures 7C and 7D). As a result, CAG instability rates were highly correlated with the level of elongating RNA Pol II at the HD locus (R2 = 0.86; Figure 7D). These results suggest that the dynamics of transition from initiation to elongation is tissue-specific and might contribute to tissue-selective CAG instability. Elongating RNA Pol II at the HD locus appeared abnormally high because we did not detect such levels at similar regions of control active genes, including Hdh (Figure 7C and S5). This might suggest that CAG repeats impair the dynamics of transcription. Together, these results suggest that high elongating RNA Pol II levels, resulting from tissue-specific regulation of elongation dynamics, and high transcriptional activity at the HD locus, as seen in R6/2 striatum, increase the propensity for repeat instability.
We further examined the correlation between transcription elongation and somatic CAG instability by performing ChIP with an antibody to Me3H3K36, a histone mark associated with transcription elongation and termination [41], [46]. Me3H3K36 is particularly elevated at the 3′ ends of transcribed genes, and, accordingly, was elevated at the last Hdh exon (Figure 8A). Me3H3K36 at HD intron-1 was lower than at Hdh last exon, but interestingly was significantly increased in striatal tissues in both R6/1 and R6/2 mice when compared to corresponding cerebellar tissues. Globally, Me3H3K36 level at the HD locus was increased in striatum when compared to cerebellum (Figure 8B), and strongly correlated with somatic CAG instability in HD mice (R2 = 0.94; Figure 8B). Since the tissue selectivity of somatic CAG instability is conserved between R6 mice and HD patients, these results, taken together, suggest that transcription elongation regulation might contribute to HD somatic CAG instability in vivo.
In vitro or cell-based studies support a role for transcription in CAG/CTG instability [20]–[24]. However, the expression level of TNR genes does not correlate with tissue-selective somatic instability in vivo [26], [27], [47], [48]. Thus, the role of transcription in somatic CAG instability has remained unclear (reviewed in [1]). Our results show that despite HD transgene expression being similar between striatum and cerebellum from both R6/1 and R6/2 mouse lines, these tissues present very different levels of CAG instability (Figure 5). In addition, the level of histone modifications associated with heterochromatin (Me2H3K9), euchromatin (AcH3K9) and transcription initiation (Me3H3K4) at the HD locus was comparable in the striatum and cerebellum of HD mice (Figure 2, Figure 3 and Figure 4). Remarkably however, the level of a histone mark and RNA Pol II form associated with transcription elongation (e.g. Me3H3K36 and phosphorylated Ser2 RNA Pol II, respectively) correlated strongly with CAG instability in tissues of R6/1 and R6/2 mice (Figure 7 and Figure 8). Thus, our data suggest a specific role for transcription elongation regulation in modulating tissue-selective CAG/CTG instability in HD. Moreover, comparison between mouse lines shows that increased CAG instability in R6/2 tissues relative to R6/1 matched-tissues correlates with increased chromatin accessibility and transcription of the HD transgene (Figure 2, Figure 4 and 5). This suggests that integration site of the transgene influences repeat instability, affecting chromatin structure and transcriptional activity.
Previous studies have shown that TNRs can induce a heterochromatinization process, consisting in formation and spreading of heterochromatin at TNR loci. This process has been described in the case of DM1, FRDA and FXS [29], [31], [37], [38], [49]–[51]. Me2H3K9 was not significantly increased at the HD locus in lymphoblasts derived from HD patients, suggesting absence or limited heterochromatinization of the locus (Figure 2). CAG expansion implicated in HD rarely exceeds 80 repeats, whereas TNRs associated with DM1, FRDA and FXS are typically larger (often reaching several hundred to thousand units), which could contribute to their high propensity to induce heterochromatinization, and to transcriptional silencing in the case of FRDA and FXS [29], [51]–[53]. Consistent with transcriptional repression, the levels of histone marks and RNA Pol II associated with transcription initiation and elongation are decreased at the FRDA locus in FRDA cells [37], [38], [54]. However, the effect of heterochromatinization on TNR instability has remained unclear. Our results suggest that heterochromatinization of the HD locus, a situation mimicked in R6/1 tissues, correlates with decreased somatic CAG instability. Whether heterochromatinization would limit TNR instability at the expense of transcription in FRDA or FXS is an intriguing possibility. If true, reversal of heterochromatinization through histone deacetylase (HDAC) inhibition, a process currently considered to alleviate GAA-repeat induced gene silencing in FRDA [55], might in turn increase repeat instability. Surprisingly, depletion of HDACs reduced TNR expansion in yeast and a plasmid-based cell model [56]. However, the effect was likely mediated through alteration of non-histone enzyme activities, including the Sae2 nuclease.
Factors involved in various DNA repair mechanisms, including MMR, BER and NER, contribute to CAG/CTG instability in vivo, most likely through aberrant processing of stable secondary structures formed at repeats, such as hairpins or slip-outs [1], [14], [16], [17], [19], [57]–[60]. Accessibility of DNA to repair factors increases when chromatin is in an open conformation. We therefore tested the hypothesis that CAG/CTG instability and chromatin accessibility might be correlated. Although Me2H3K9 at the HD locus was increased in R6/2 tissues when compared to R6/1 tissues, it was similar in the striatum and in the cerebellum of R6 mice, indicating that chromatin accessibility is unlikely to underlie tissue-selective HD somatic CAG instability (Figure 2). Instead, our data suggest a role for transcription, and more specifically for transcription elongation, in somatic CAG instability in HD. Events associated with transcription initiation were only partially correlated with CAG instability rates in R6 mouse tissues, since Me3H3K4 and phosphorylated Ser5 RNA Pol II levels at the HD locus were increased in R6/2 when compared to R6/1 tissues, but were comparable in striatum and cerebellum (Figure 4 and Figure 7). In contrast, events associated with transcription elongation were strongly correlated with somatic CAG instability. Specifically, Me3H3K36 and phosphorylated Ser2 RNA Pol II levels at the HD locus were increased in striatum relative to cerebellum as well as in R6/2 tissues as compared to R6/1 tissues (Figure 7 and Figure 8). Interestingly, a previous study based on a cell model allowing for detection of contraction events at CAG/CTG repeats showed that the elongation factor TFIIS contributes to repeat instability [15]. In addition, inhibition of the proteasome and downregulation of BRCA1/BARD1 proteins, which modulate RNA Pol II activity and transcription elongation, decreased CAG/CTG instability in the treated cells [15]. It remains to be determined whether these proteins as well as other factors controlling the dynamics of transcription elongation, including positive transcription-elongation factor-b (P-TEFb), negative elongating factor (NELF) or DRB sensitivity-inducing factor (DSIF), a factor composed of SPT4 and SPT5 [61]–[63], contribute to tissue-selective CAG instability in HD. Interestingly, a recent study showed that depletion of SPT4 in yeast and mammalian cells selectively prevents transcription of CAG-expanded genes, thereby reducing toxicity of the gene products [64]. Thus, targeting transcription elongation might reduce both repeat instability and toxicity.
Total RNA Pol II at the HD locus and at the promoter-proximal region of the murine Hdh gene were increased in the striatum when compared to the cerebellum (Figures 5). This suggests that transcriptional activity at the Huntingtin gene is differently regulated in the striatum and in the cerebellum in both mice and humans. However, and in accordance with previous studies showing that normal and mutant Huntingtin expression is widespread in many tissues and comparable in striatum and cerebellum, HD transgene expression was not different in the striatum and cerebellum, irrespective of mouse age or line (Figure 5) [26], [27], [47], [48], [65]. HTT mRNA processing might be regulated in a tissue-specific manner, as previously suggested [66], [67]. As a result, despite increased transcriptional activity in the striatum as compared to the cerebellum, HTT mRNA levels could be similar in the two tissues, due to increased HTT mRNA stability in the cerebellum. Alternatively, the dynamics of HTT transcription might be different in striatum and cerebellum, which cannot be examined by measuring bulk mRNA levels. Increasing evidence shows that genes can be expressed through alternative modes, ranging from bursts of transcription to more continuous smooth mode [68], [69]. Whether transcriptional mode can be regulated in a tissue-specific manner is currently unknown and cannot be easily assessed. The differential levels of RNA Pol II at HTT promoter-proximal region might suggest that the regulation of the transition from initiation to productive elongation is different in striatum and cerebellum, supporting the view that the dynamics of HTT transcription is different in the two tissues [70]. Whether HTT is expressed through a bursting mode in the striatum -promoting CAG instability, and a more continuous mode in the cerebellum –limiting repeat instability, is an attractive hypothesis.
The dynamics of transcription across CAG expansion might be impaired, particularly in the striatum where elongating RNA Pol II at the HD locus is elevated (Figure 7). Using an in vitro approach, it was recently shown that G/C-rich sequences induce transcription stalling, due to formation of stable R-loops during elongation [71]. Cell-based studies showed that increased transcriptional activity at genes with TNRs or bidirectional transcription across CAG/CTG repeats promotes formation of R-loops, leading to increased TNR instability [22]–[24], [72]. How then would R-loops promote TNR instability is currently unknown, but it has been hypothesized that arrest of elongating RNA Pol II at non canonical DNA structures might result in gratuitous transcription-coupled nucleotide excision repair (TCR) [73]. Supporting this view, it was shown that proteins of the NER/TCR pathway, including CSB, XPG and XPA, reduce transcription-mediated instability of CAG/CTG repeats in a cell model allowing for detection of contraction events [15], [20]. Accordingly, somatic CAG instability was reduced in post-mitotic tissues, including brain tissues, in spinocerebellar ataxia type 1 mice deficient for Xpa [19]. The idea that the propensity for formation of stable R-loops is increased in somatic tissues presenting high CAG instability rates, such as the striatum, is an intriguing hypothesis.
In conclusion, our results indicate that increased chromatin accessibility and transcription provide a context favoring somatic CAG instability but do not underlie the tissue selectivity of the process. Our data suggest that transcription elongation specifically contributes to tissue-specific CAG instability. It is tempting to speculate that the dynamics of transcription elongation of the HTT gene is regulated in a tissue-specific manner, influencing the risk for CAG instability.
Materials and Methods
Mouse lines and breeding
Hemizygous R6/1 (130 CAG) and R6/2 (160 CAG) mice from the Jackson Laboratory were both maintained on a mixed CBAxC57BL/6 genetic background. The experiments were approved by the ethical committee C.R.E.M.E.A.S (Comité Régional d'Ethique en Matière d'Expérimentation Animale de Strasbourg).
Cell lines
Lymphoblasts from control individuals (GM14907, GM06903) and HD patients (GM04868, GM04282, GM04724, GM04846) were obtained from the Coriell Cell Repository. The size of CAG repeats in HD cell lines is 49/13, 74/17, 68/18 and 50/48, respectively. Cell lines were grown in RPMI 1640 medium without Hepes supplemented with 15% fetal calf serum and gentamycin under standard conditions.
CAG repeat sizing and analysis
CAG repeat size was determined as previously described [35], [36]. Briefly, PCR products containing the CAG repeats were analyzed using the ABI Prism 3100 DNA analyzer instrument and GeneScan and Genotyper softwares. The variation of repeat size corresponds to the amplitude of Genescan profile and was determined by calculating the number of peaks above 10% of the maximum fluorescent peak intensity. The striatum and cerebellum of the same mice were analyzed at each time point and 4 to 8 different mice at each time point were analyzed.
ChIP experiments were performed essentially as described [36]. Briefly, for each experiment, striata and cerebella from 3 to 5 transgenic mice were pooled, cut into small fragments, fixed in 1% formaldehyde and incubated for 10 min at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M. Tissue fragments were washed with cold PBS supplemented with protease inhibitors [36]. Phosphatase inhibitors were included when using antibody to phospho Ser2 or Ser5 RNA Pol II. The tissues were then homogenized in sonication buffer, and lysates were sonicated to obtain DNA fragments <500 bp and centrifuged, as described [36]. The soluble chromatin fraction was pretreated with protein A Agarose/Salmon Sperm DNA (Millipore) for 1 h at 4°C. Subsequently, samples were incubated overnight at 4°C with antibodies to AcH3K9 (ab10812, Abcam), Me2H3K9 (ab1220, Abcam), H3 (ab1791, Abcam), Me3H3K4 (ab1012, Abcam), Me3H3K36 (ab9050, Abcam), phospho S5 RNA Pol II (ab5131, Abcam), phospho S2 RNA Pol II (ab5095, Abcam) and 7C2 RNA Pol II antibody [42]. Protein A Agarose/Salmon Sperm DNA was then added and the mixture was incubated for 3 h at 4°C. Agarose beads were washed, protein-DNA complexes were eluted from the beads and decrosslinked, and DNA recovered by phenol chloroform extraction and ethanol precipitation after treatment with ribonuclease A (Abcam) and proteinase K, as described [36]. Typically, 2 to 3 antibodies were used per experiment, and immunoprecipitation with each antibody was duplicated. 2 to 4 independent ChIP experiments were performed with each antibody. The results are expressed as percentage of input, and correspond to mean values calculated from the different experiments. ChIP experiments from cell lines were performed using 5×106 cells per ChIP and the procedure described above. A typical ChIP experiment was performed using 4 HD cell lines and 2 control cell lines. Immunoprecipitations with each cell line were performed with antibodies to Me2H3K9 (ab1220, Abcam) and RNA Pol II (7C2) in duplicates, and 2 independent ChIP experiments were performed. The results, expressed as percentage of input, correspond to a representative experiment and are calculated by averaging values from the 2 control cells and from the 4 HD cells. The PCR primers used are listed in Figure S2.
RNA extraction and real-time RT–PCR analysis
Striata and cerebella were dissected, snap frozen and stored at −80°C. Total RNA was prepared from frozen tissues using the RNeasy Mini Kit (Qiagen). cDNA were prepared using 1 mg of total RNA, random hexamers and SuperScriptII reverse transcriptase (Invitrogen) according to the manufacturer instructions. Quantitative RT-PCRs were performed on a Light-Cycler instrument (Roche) to assess expression levels of HD transgene. The SYBR PCR master mix (Qiagen) was used to assess the level of HD transgene mRNA using the primers upstream of CAG repeats. Specific conditions were used to assess the level of HD transgene mRNA using primers amplifying the repeats, as previously described [36]. Briefly, the Herculase Hotstart DNA Polymerase (Stratagene) was used according to recommendation of manufacturer, and 8% DMSO and Sybr green (Molecular probe) were included in the reaction. The PCR reactions were performed on a Light Cycler instrument (Roche). DNA was inactivated for 3 min at 98°C, and 45 cycles consisting in 40 seconds at 98°C, 30 seconds at 60°C and 2 min at 72°C were performed. As a control, PCR reactions were also performed in the absence of reverse transcriptase. Data were normalized to expression of Gapdh or 18S housekeeping genes. For absolute quantifications, standard curves were generated using genomic DNA and used to calculate amounts of PCR products. cDNA samples prepared from the striatum and cerebellum of R6/1 and R6/2 mice were analyzed using corresponding genomic DNA. All experiments were performed at least three times in biological triplicates. The PCR primers used are provided in Figure S2.
Statistical analyses
ANOVA followed by Newman Keuls tests were used for statistical analyses.
Supporting Information
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33. ChiangC, JacobsenJC, ErnstC, HanscomC, HeilbutA, et al. (2012) Complex reorganization and predominant non-homologous repair following chromosomal breakage in karyotypically balanced germline rearrangements and transgenic integration. Nat Genet 44: 390–397, S391.
34. CowinRM, BuiN, GrahamD, GreenJR, GrueningerS, et al. (2011) Onset and progression of behavioral and molecular phenotypes in a novel congenic R6/2 line exhibiting intergenerational CAG repeat stability. PLoS ONE 6: e28409 doi:10.1371/journal.pone.0028409
35. MangiariniL, SathasivamK, MahalA, MottR, SellerM, et al. (1997) Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation [see comments]. Nat Genet 15: 197–200.
36. GoulaAV, BerquistBR, WilsonDM3rd, WheelerVC, TrottierY, et al. (2009) Stoichiometry of base excision repair proteins correlates with increased somatic CAG instability in striatum over cerebellum In Huntington's disease transgenic mice. PLoS Genet 5: e1000749 doi:10.1371/journal.pgen.1000749
37. KimE, NapieralaM, DentSY (2011) Hyperexpansion of GAA repeats affects post-initiation steps of FXN transcription in Friedreich's ataxia. Nucleic Acids Res 39: 8366–8377.
38. KumariD, BiacsiRE, UsdinK (2011) Repeat expansion affects both transcription initiation and elongation in friedreich ataxia cells. J Biol Chem 286: 4209–4215.
39. KumariD, UsdinK (2010) The distribution of repressive histone modifications on silenced FMR1 alleles provides clues to the mechanism of gene silencing in fragile X syndrome. Hum Mol Genet 19: 4634–4642.
40. KaplanN, MooreIK, Fondufe-MittendorfY, GossettAJ, TilloD, et al. (2009) The DNA-encoded nucleosome organization of a eukaryotic genome. Nature 458: 362–366.
41. BarskiA, CuddapahS, CuiK, RohTY, SchonesDE, et al. (2007) High-resolution profiling of histone methylations in the human genome. Cell 129: 823–837.
42. BesseS, VigneronM, PichardE, Puvion-DutilleulF (1995) Synthesis and maturation of viral transcripts in herpes simplex virus type 1 infected HeLa cells: the role of interchromatin granules. Gene Expr 4: 143–161.
43. NechaevS, AdelmanK (2011) Pol II waiting in the starting gates: Regulating the transition from transcription initiation into productive elongation. Biochim Biophys Acta 1809: 34–45.
44. MuseGW, GilchristDA, NechaevS, ShahR, ParkerJS, et al. (2007) RNA polymerase is poised for activation across the genome. Nat Genet 39: 1507–1511.
45. GuentherMG, LevineSS, BoyerLA, JaenischR, YoungRA (2007) A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130: 77–88.
46. BannisterAJ, SchneiderR, MyersFA, ThorneAW, Crane-RobinsonC, et al. (2005) Spatial distribution of di- and tri-methyl lysine 36 of histone H3 at active genes. J Biol Chem 280: 17732–17736.
47. StrongTV, TagleDA, ValdesJM, ElmerLW, BoehmK, et al. (1993) Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nature Genet 5: 259–265.
48. LiS-H, SchillingG, YoungWSIII, LiX-J, MargolisRL, et al. (1993) Huntington's disease gene (IT15) is widely expressed in human and rat tissues. Neuron 11: 985–993.
49. KumariD, UsdinK (2009) Chromatin remodeling in the noncoding repeat expansion diseases. J Biol Chem 284: 7413–7417.
50. Al-MahdawiS, PintoRM, IsmailO, VarshneyD, LymperiS, et al. (2008) The Friedreich ataxia GAA repeat expansion mutation induces comparable epigenetic changes in human and transgenic mouse brain and heart tissues. Hum Mol Genet 17: 735–746.
51. HermanD, JenssenK, BurnettR, SoragniE, PerlmanSL, et al. (2006) Histone deacetylase inhibitors reverse gene silencing in Friedreich's ataxia. Nat Chem Biol 2: 551–558.
52. De BiaseI, ChutakeYK, RindlerPM, BidichandaniSI (2009) Epigenetic silencing in Friedreich ataxia is associated with depletion of CTCF (CCCTC-binding factor) and antisense transcription. PLoS ONE 4: e7914 doi:10.1371/journal.pone.0007914
53. BiacsiR, KumariD, UsdinK (2008) SIRT1 inhibition alleviates gene silencing in Fragile X mental retardation syndrome. PLoS Genet 4: e1000017 doi:10.1371/journal.pgen.1000017
54. PungaT, BuhlerM (2010) Long intronic GAA repeats causing Friedreich ataxia impede transcription elongation. EMBO Mol Med 2: 120–129.
55. GottesfeldJM (2007) Small molecules affecting transcription in Friedreich ataxia. Pharmacol Ther 116: 236–248.
56. DebackerK, FrizzellA, GleesonO, Kirkham-McCarthyL, MertzT, et al. (2012) Histone deacetylase complexes promote trinucleotide repeat expansions. PLoS Biol 10: e1001257 doi:10.1371/journal.pbio.1001257
57. PearsonCE, SindenRR (1996) Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci. Biochemistry 35: 5041–5053.
58. WheelerVC, LebelLA, VrbanacV, TeedA, te RieleH, et al. (2003) Mismatch repair gene Msh2 modifies the timing of early disease in Hdh(Q111) striatum. Hum Mol Genet 12: 273–281.
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60. van den BroekWJ, NelenMR, WansinkDG, CoerwinkelMM, te RieleH, et al. (2002) Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch-repair proteins. Hum Mol Genet 11: 191–198.
61. SaundersA, CoreLJ, LisJT (2006) Breaking barriers to transcription elongation. Nat Rev Mol Cell Biol 7: 557–567.
62. AdelmanK, MarrMT, WernerJ, SaundersA, NiZ, et al. (2005) Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS. Mol Cell 17: 103–112.
63. YamadaT, YamaguchiY, InukaiN, OkamotoS, MuraT, et al. (2006) P-TEFb-mediated phosphorylation of hSpt5 C-terminal repeats is critical for processive transcription elongation. Mol Cell 21: 227–237.
64. LiuCR, ChangCR, ChernY, WangTH, HsiehWC, et al. (2012) Spt4 is selectively required for transcription of extended trinucleotide repeats. Cell 148: 690–701.
65. StineOC, LiSH, PleasantN, WagsterMV, HedreenJC, et al. (1995) Expression of the mutant allele of IT-15 (the HD gene) in striatum and cortex of Huntington's disease patients. HumMolGenet 4: 15–18.
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68. ZenklusenD, LarsonDR, SingerRH (2008) Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15: 1263–1271.
69. KaernM, ElstonTC, BlakeWJ, CollinsJJ (2005) Stochasticity in gene expression: from theories to phenotypes. Nat Rev Genet 6: 451–464.
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71. BelotserkovskiiBP, LiuR, TornalettiS, KrasilnikovaMM, MirkinSM, et al. (2010) Mechanisms and implications of transcription blockage by guanine-rich DNA sequences. Proc Natl Acad Sci U S A 107: 12816–12821.
72. LinY, LengM, WanM, WilsonJH (2010) Convergent transcription through a long CAG tract destabilizes repeats and induces apoptosis. Mol Cell Biol 30: 4435–4451.
73. HanawaltPC, SpivakG (2008) Transcription-coupled DNA repair: two decades of progress and surprises. Nat Rev Mol Cell Biol 9: 958–970.
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PLOS Genetics
2012 Číslo 11
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Combine a rock song from the 1990's, a rap song from the 2000's & a tune from the 2010's you get 3 decades into one...
David Bowie vs. Smashing Pumpkins (Negative Mix)
Justice – Genesis vs. Smashing Pumpkins – Zero
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MashupCit features mashup from DJ's and bedroom producers from around ze world. I listen to DOZENS of mashups every day and promote those that float to the top.
If you are a first time listener, search for your favorite artist to see if I have a mashup of them.
If you are a DJ and want your mashup reviewed, click on the submit to send in your mashup.
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Welcome to the 361st edition of Phish.Net's Mystery Jam Monday, the first* and easiest of February! The winner will receive an MP3 download code courtesy of our friends at LivePhish.com / Nugs.Net. To win, be the first person to identify the song and date of the mystery clip. Each person gets one guess to start – if no one answers correctly in the first 24 hours, I'll post a hint. After the hint, everyone gets one more guess before Wednesday at 10 AM PT / 1 PM ET. Good luck!
*Reminder: For the first MJM of each month, only folks who have never won an MJM are allowed to answer before the hint. If you have never won an MJM, please answer as a blog comment below. If you have previously won an MJM, but you'd like to submit a guess before the hint, you may do so by PMing me; once the hint has been posted, everyone should answer on the blog. If that's confusing to you, check out the handy decision tree that @ucpete threw together to help guide you. If you're not sure if you've won before, check in the MJM Results spreadsheet linked below.
Answer: In back-to-back weeks, we have fresh MJMers with their first wins! @ckess22 chimed in not too long before the hint went live, correctly identifying the 8/10/97 Split Open and Melt - depsite the show being well known for another jam... no matter, he has earned himself a download code and enters the fray of newcomers seeking seven wins and the fabled emeritus status. Tune back in next week on the same bat-time, same bat-channel, for another offering in which the difficulty and number of jams doubles!
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Skip to 0 minutes and 0 seconds And could we say that in China these textbooks also lead to more coherence in the way that mathematics is taught? Because I hear a uniform curriculum, I hear textbooks that provide the content. So there is freedom to put your own stamp on it, but there also is a very solid, let's say standardised or that sounds awful, but an element that is the same across the country which means that every student gets at least a good solid foundation. (Dr.
Skip to 0 minutes and 42 seconds Zhao:) Yes. The unified textbooks ensure the coherence of mathematical content. However, another coherence I should tell you is the coherence of teaching, coherence of instruction. In China, many mathematics teachers think the coherence is very important for their, for the implementing a smooth lesson and also helpful for student and what the teacher is teaching. They will try their best to organise the content into a package, different knowledge into a package, knowledge package.
Skip to 1 minute and 39 seconds Besides, they also will outline the lesson structure in many aspects, such as how to inspire students' interest, how to review the content to make a good prepare for the new content, how to divide the new content into different parts, one to a short brief summary and how to give overall summary… All of these could help the lesson structure appears very clear, logical and smooth, because of the coherence of instruction. (Christian:) So, just a sort of round-off with two final questions. So if you would pick one of these things, because we lots of countries are looking towards China, and they say well there are good things there are bad things.
Skip to 3 minutes and 0 seconds If there is one thing that you think would be the most realistic point for other countries to adopt, what point do you think would be would be the most important one? A difficult question I realise. (Dr.
Skip to 3 minutes and 15 seconds Zhao:) It's very difficult for me to answer. As we all know, the teaching is a cultural practice, cultural activity. So the features I mentioned about all rooted in the Chinese background, Chinese cultural background. So I'm not sure which one and how these features could be adopted in other countries. For me, in my opinion, what the Chinese experience could provide to the educators in other countries, maybe not a pattern or a model, but a mirror to help them to understand and reflect the classroom practice of their own. (Christian:) By looking at other experiences and other ways of doing education, rather than say oh let's copy this. (Dr.
Skip to 4 minutes and 25 seconds Zhao: Yes.) You use it as a way to reflect on why you're doing, what you're doing, and then every country decide for themselves what best fits. (Dr.
Skip to 4 minutes and 33 seconds Zhao: Yes.) Are there things in China you think that could be, what do you think is, that's the last question, what do you think it will be the most important development or thing that China will change or look at in the next ten years? What feature do you think is important, that also what feature do you think China wants to pay more attention to? (Dr.
Skip to 4 minutes and 59 seconds Zhao:) In my personal opinion, there are many things that need to be reformed or to be improved for Chinese mathematics education, although we have many good experiences, we have got many outcomes. The two challenge for us I think may be balance the, between the solid foundation and the students' creativity, balance between teachers' dominance, teachers' guidance and students' initiative. Why? I think the education, including the mathematics education, should educate for future generation, not just to master knowledge. They should learn how to think critically. They should learn by themselves independently, not just push by their teacher or push by the examination. This is my opinion. (Christian:) Thank you very much for this enjoyable interview. (Dr.
Skip to 6 minutes and 31 seconds Zhao:) It's my pleasure.
Learning maths in China - interview with Dr Dongchen Zhao - part 3
This is part 3 of the interview with Dr Dongchen Zhao.
Dongchen Zhao is research and teaching staff in the School of Education, Harbin Normal University, China. His research interests focus on the mathematics classroom practice and teachers' knowledge development. Between August 2017 and August 2018, he has been at the University of Southampton as a visiting academic. He received his primary school education in a small village in northeastern China in the 1980s. He received his master's degree and doctoral degree in Northeast Normal University, China. Both his master thesis and PhD dissertation were about China's primary school mathematics classrooms.
© 2018 University of Southampton


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Bob Kolar illustrated this busy worksite for our Construction Jumbo Puzzle from Mudpuppy. In addition to a backhoe, dump trucks, bulldozer, asphalt spreader, and line painter, you can spot bluebirds!
Jumbo Puzzles are a fun way to work on hand and eye coordination and to have fun together.
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Systems and Control for Robotic Applications in Ocean and Space
posted in STEM on November 3rd, 2015
All are invited to attend the next NASA STEM seminar on Tuesday, November 10, 2015 from 12:15 – 1:15pm in the mid-café. Dr. May-Win Thein, Associate Professor of Mechanical and Ocean Engineering at UNH, will present a seminar on "Systems and Control for Robotic Applications in Ocean and Space". The STEM Seminar Series is funded by NASA and the Maine Space Grant Consortium.


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Red-Tigers-Eye Properties
- 1 Red-Tigers-Eye Properties
- 2 Why Would You Use Red-Tigers-Eye?
- 3 How Will Red-Tigers-Eye Help You?
- 4 How to Use Red-Tigers-Eye for the Best Results
- 5 Red-Tigers-Eye and Spiritual Energy
- 6 My Final Thoughts on the Power of Red-Tigers-Eye
Red-Tigers-Eye is also known as Dragon's Eye.
It's a stone that's brownish red in color and has both light and dark lines running through it.
It belongs to the oxide mineral class and the quartz group.
It has a trigonal crystal system, and the shine and shimmer that can be seen on the stone are created during its formation process.
The red color in Red-Tigers-Eye is because of the level of oxidation that occurs.
Red-Tigers-Eye's most notable locations include South Africa, Namibia, India, Canada, Brazil, and the USA.
Why Would You Use Red-Tigers-Eye?
Red-Tigers-Eye is a very stimulating stone that will give you inspiration and motivation in all aspects of your life.
It's a highly calming stone that will keep you relaxed and centered even if you lead a very busy or chaotic life.
When you work with the energies of this stone, you will have peace and harmony wherever you are, whoever you're with, and whatever you're doing.
The energies of Red-Tigers-Eye will also motivate you to achieve all your goals and complete all your tasks with energy and enthusiasm.
This stone will infuse you with confidence so that you can fulfill any role, perform any task, and speak to anyone with wisdom and grace.
Red-Tigers-Eye has a strong grounding energy that will make sure your physical body is connected to the earth.
This will ensure that you have all the skills that you need to survive and preserve yourself.
This will ensure that you have all the skills that you need to survive and preserve yourself.
This is why Red-Tigers-Eye is also called the survival stone!
Red-Tigers-Eye will protect your integrity. It will make sure that you will not be trapped in situations where you need to sacrifice your integrity and all the other values that you uphold.
This stone will also promote self-care and self-love. You will do what's best for you, and you will be the champion of your own self.
You will engage in pursuits that will make you a better, wiser, and stronger person, especially if you combine it with the July Birthstone.
Red-Tigers-Eye will also encourage you to have a healthy passion for any project, pursuit, or endeavor.
It will keep your mind occupied and your hands busy, and it will stimulate your creativity.
It will boost your energies and get your creative juices flowing. It will also help you relax when you need to slow down and take a breather.
The energies of this stone will have a slowing effect on your system, which will get rid of any excess energy!
Red-Tigers-Eye will strike a balance between the light and dark aspects of yourself, your Yin and Yang, your mind and heart, and your earthly and spiritual aspects.
This stone will enhance your gift of psychic visions, and it will give you spiritual clarity. It will also promote balance in your etheric fields!
How Will Red-Tigers-Eye Help You?
Red-Tigers-Eye, Healing and Health
The healing properties of Red-Tigers-Eye are believed to be very effective against blood disorders, such as anemia.
It can also help in the treatment of eye infections and enhance night vision.
This stone can give physical strength and vitality. It can work to regulate the natural rhythms of the body. It can also accelerate metabolism.
Red-Tigers-Eye is helpful in relieving congestion in the digestive system.
It can give relief to pains and soothe the nervous system and overstimulated adrenal glands.
It can also help with sexual performance and other sexual dysfunctions.
The healing energies of Red-Tigers-Eye can benefit bones and muscle tissues. It can also improve the health of the teeth and nails.
Red-Tigers-Eye can regulate blood pressure. It can also have a calming effect on the heart for those who suffer from heart conditions.
Red-Tigers-Eye is an excellent post-surgery stone that will speed up your recovery period and restore the body back to good health.
Red-Tigers-Eye and Wealth
The energies of Red-Tigers-Eye will add passion and vitality to any project that you're working on or any pursuit that you are involved with.
It will also encourage you to make practical decisions and choices.
It says a lot about your creativity and resourcefulness when you are able to achieve something extraordinary using limited or budgeted resources.
Red-Tigers-Eye will strengthen your willpower to stay committed to your projects, to overcome your challenges, and to have backup plans in case the original one doesn't work.
It will keep your motivation high because you will be filled with energy and enthusiasm. You will also not run out of self-confidence and positivity.
Red-Tigers-Eye will improve your luck and give you a boost in abundance and prosperity.
You will enjoy good fortune and good luck, and you will not run out of new ideas to keep you busy.
Red-Tigers-Eye is a wonderful money charm, so always keep it in your wallet or purse to keep the money flow moving. It's also a great success and power charm.
Red-Tigers-Eye, Love and Relationships
Red-Tigers-Eye and Picasso Marble will promote a more active sex life. It will help you manifest your sexual ideas, desires, and fantasies.
It will also give you answers how you can resolve your sexual issues, and it will give you a clear idea what's causing the problem.
It will enhance your self-confidence and self-esteem. It will make you feel very attractive and proud of your assets. You will fully embrace even your flaws.
Red-Tigers-Eye will make you feel very connected to your partner physically, emotionally, intellectually, and spiritually.
Your relationship will be strengthened, and it will only get better with the guidance of this stone.
If you pair it with Covellite, this stone will enhance your personal energy fields and inspire you to open up to unconditional love.
It will inspire you to be more welcoming and accepting of the love that will come to you and the love that you will give to your partner.
Red-Tigers-Eye will create a balance between your interactions with your significant other.
You will enjoy fun and wonderful moments, and you will also share serious and life-changing moments.
This stone will also give you wisdom and courage to face any trial and overcome any challenge in your love life. You will be strong and faithful in love!
It will release you from your emotional grief. It will also remove all the hurt, sadness, and anger that you have in your heart.
You will experience emotional healing, and you will become a stronger and better person for it.
This stone will also show you how you can be discerning when it's the right time to do something good for yourself, your partner, and your relationship.
It will give you the ability to see ahead and plan your next steps.
Red-Tigers-Eye will promote mental clarity and help focus your mind. It will help you concentrate on the things that matter in your relationship.
It will also solve your problems in an objective manner without relying too much on your emotions. It will also remove your fears and anxieties about love.
Red-Tigers-Eye will promote a passion for your partner and your relationship. It will make you desire to create unforgettable moments and unique experiences with the person you love.
How to Use Red-Tigers-Eye for the Best Results
Red-Tigers-Eye accompanied by Pearl can be your companion stone during difficult moments. Think of it as your counsel stone that you can consult for wisdom and guidance.
Keep a piece of Red-Tigers-Eye stone in your bag, inside your car, or in your medicine kit. Make sure that you're never without it!
Whether you're going to an awkward family gathering, a stressful client meeting, a nerve-wracking public performance, or a first date with someone you're in love with, keep this stone close to you so that you can harness its loving and stabilizing energies!
Red-Tigers-Eye can also be used to increase your libido and boost your sexual appetite.
Having this stone with you can help you ground your sexual fantasies to reality. It will also help you resolve your sexual issues that are affecting the quality of your relationship.
You can also attach it to your cat's collar because it will emit protective energies for your cat.
Red-Tigers-Eye and Spiritual Energy
The spiritual energy of Red-Tigers-Eye helps raise your life's vibrations, enhancing your experiences and the way you live your life.
It also connects the stone's spiritual energy to your chakras, which prevents you from feeling like you are disconnected when you're working on your higher goals.
Enjoy the benefits of Red-Tigers-Eye's spiritual energies when you carry a piece of this stone with you on a daily basis.
It will add passion and vitality to anything that you do. It will also enhance your energy field and encourage you to open yourself up to unconditional love.
Because Red-Tigers-Eye is connected energetically to your root chakra, your sacral chakra, and your solar plexus chakra, this stone will also help you stay grounded to the earth.
It's important to have a connection between the earth and your physical body because it will keep your survival and self-preservation skills strong.
This is why Red-Tigers-Eye is also known as the survival stone.
The spiritual energy of this stone will help you enhance your sense of practicality, strengthen your willpower and integrity, boost your motivation, and work on your self-care.
It will also encourage you to adopt a healthy passion for anything that you're involved with or any opportunity that may present itself.
While Red-Tigers-Eye will boost your energies, it will also help you pause for a while and slow down.
When it's needed, this stone can slow down its effect on your third chakra and get rid of any excess energies.
Because this stone is a type of Tigers Eye, it will create balance within your interactions with other people.
It will also strike a balance in many areas, like your dark and light aspects, your yin and yang, your feelings, and your awareness's spiritual and earthly aspects.
Aside from increasing your motivation, self-confidence, strength, and enthusiasm, Red-Tigers-Eye will also calm your mind and ease your anxieties over things to come.
It will also give you spiritual protection and improve your luck. This stone's spiritual energy will give you a boost towards prosperity.
Red-Tigers-Eye will provide support to your base chakra and strengthen your connection to the earth.
This stone will help you maintain your focus on what you need, which are always provided for you with the right intention.
If you wish to be successful at everything you do, channel the spiritual energy of Red-Tigers-Eye. When it comes to negative emotions, Red-Tigers-Eye will help you keep everything in perspective.
With a beauty as bold and powerful as its spiritual energy, Red-Tigers-Eye will help balance the harmful emotions by focusing your mind on your genuine desires.
Make sure that you keep or carry this stone with you always so that you will always have mental clarity, focus, and good luck.
The spiritual energy of Red-Tigers-Eye will help you find ways to resolve personal challenges, balance and stabilize mood swings, willpower and a sense of purpose, and energize while releasing tensions, fears, and anxieties.
It's truly one of the most loving and supportive stones out there!
My Final Thoughts on the Power of Red-Tigers-Eye
Red-Tigers-Eye will make sure that you stay motivated in all your pursuits, and that you are filled with energies of passion and motivation.
It's a particularly stimulating stone that will help you when you're feeling slow or lethargic.
It will give you the strength that you need to overcome your fatigue.
It will also help ground and protect you.
Red-Tigers-Eye will deflect any unwanted negative energy.
Instead, it will attract good luck and good fortune in your life!
This stone will promote a passion for life and love.
It will show you how you can be sympathetic and compassionate to the needs of the people you love.
Red-Tigers-Eye will also help you in letting go of thoughts that you are not worthy of love.
It will foster a healthy and positive self-image of yourself which will reflect to everyone around you. It's an excellent stone that will help you with your self-realization goals.
Red-Tigers-Eye will give you good judgment and insight, and it will keep away all the bad energies and negative elements!


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Two days consultation meeting for Human Rights Situation in Puntland organized by PUNSAA and Office of Puntland Human Rights Defender started on 22 March, 2018 and ended on 23 March, 2018. The purpose of this consultation meeting was to discuss deeply the Human Rights Situation in Puntland.
The participants of workshop were 35 members from the different human right stakeholders including representatives from the Office of Puntland Human Rights Defender, together with members from civil society organisations. During this consultation meeting the participants were shared the latest report (2016/2017) on the Puntland human rights situation by the Office of Puntland human rights defender and given a chance to raise concerns and questions that focus on Human right issues, the chairman of the Puntland human right defender answered all raised questions related to the current human right situation in puntland.
The consultation meeting ended with success and the participants were suggested valuable recommendations that can improve the Human rights situation in Puntland.


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Grade Point Average Problems
- Author:
- David Gurney
- Topic:
- Arithmetic Mean, Statistics
Last updated 6/3/2019 Move the slider to see a new problem. Check the box to see the answer.
There will be a problem similar to this on test 2 and the final exam. Updated 5/29/2012, 9/6/2013, 2/4/2019, and 6/3/2019.


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