Blackburn Martial Arts and Kickboxing club in Lower Darwen have raised £310 to mark Professor Tony Smith's 50 years in the Nippon Dai Budo Kai.
The Hospice is close to the clubs heart of both Professor Tony Smith and Simon Jones, who knew people who were cared for at the hospice.
Professor Tony Smith said: "I've known people in the hospice and it is an important part of that time in their lives.
Thank you Tony and the rest of the club.
Pictured is Leanne Green, Fundraiser, with Professor Tony Smith and head coach Simon Jones and Blackburn Martial Arts and Kickboxing Club.


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The University of British Columbia's Appropriation of and Attack on Critical Discourse; & Why Postmodernism Fails to Protect Animals
by Aylon • June 15, 2012 • 2 Comments
Fun Fact: In addition to bisexual and homosexual behavior, male dolphins carry pieces of plants and twigs to impress females, rather than simply playful behaviour. The discovery could provide proof of the existence of dolphin culture – defined as a non-hereditary, complex skill taught to some members of a population by others and passed down through generations.
On today's show we start with a review of the cookbook "The Happy Herbivore: Over 175 Delicious Fat-Free & Low-Fat Vegan Recipes" by Lindsay S. Nixon. Then we chat with former University of British Columbia students about the university's attempt to appropriate a radical speaker series on animals that challenges the university's governance on animals. We also talk to Dr. Gary Steiner, John Howard Harris Professor of Philosophy at Bucknell University, about his forthcoming book Animals and the Limits of Postmodernism.
Defusing the Challenge of Critique: The University of British Columbia's Attempts at Appropriating a Critical Speaker Series
Darren Chang and Viara Gioreva are two former UBC students who were part of the organizing collective that created the speaker series "Bringing the Collective Together: Nonhuman Animals, Humans and Practice at the University."
The series initiated meaningful interdisciplinary, scholarly deliberation about the use of nonhuman animals in university teaching and research. It brought together scholars from the humanities, social sciences and sciences who otherwise have scant occasion to interact.
UBC was impressed, but also scared that the series that the potential to fundamentally question and alter their practices of animal testing. UBC has a bad reputation of being extremely opaque when it comes to animal testing and saw the series as attempt to prove their "openness." Viara and Darren are not happy, initially calling the university out in the university student newspaper. However, the UBC administration is at it again, using the series as evidence, it seems, of the openness and public discussion on campus around the question of animals. Of course the inventors of the series are made invisible; rendered invisible is what has motivated them to invent the series: that there was sorely inadequate public discussion and scholarly exchange on campus around the question of animals.
The Vegan Imperative and Postmodernism's disservice to Animals
We chat with Dr. Gary Steiner, John Howard Harris Professor of Philosophy, about his work in the philosophy of animal ethics and rights.
Steiner believes that that philosophers such as Peter Singer, Jeremy Bentham, and Tom Regan ultimately fail to truly support animals in their plight as they remain caught within a humanist framework.
In response to the failures of humanism, a number of writers who could be called postmodernists have taken up the cause of animals. But Dr. Steiner believes that postmodernists are not very helpful either when it comes to animals.
In this episode, we discuss what is wrong with the current model of humanist philosophy, why postmodernism is not the way to fight for animals, and what a truly non-anthropocentric humanism would look like.
Click here to listen to this show.
Tags: Animals and the Limits of Postmodernism Bringing the Collective Together darren chang gary steiner happy herbivore viara gioreva
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Anthony Marr, Campaign to Ban Shark Fin; & Shannon Keith, Beagle Freedom Project →
2 comments for "The University of British Columbia's Appropriation of and Attack on Critical Discourse; & Why Postmodernism Fails to Protect Animals"

redpajama_set_name: RedPajamaCommonCrawl


text:
Deiphobe är ett släkte av bönsyrsor. Deiphobe ingår i familjen Mantidae.

Kladogram enligt Catalogue of Life:

Källor

Externa länkar

Bönsyrsor
Deiphobe

redpajama_set_name: RedPajamaWikipedia


Centrally situated, the Kroon Boma is 8km south of Kroonstad, Free State on the N1 towards Bloemfontein.
Major transport routes pass on our doorstep, with very easy access.
This world class auction house boasts 7000m2 under roof facility, which includes 100 closed game bomas and 100 livestock pens, fitted with water and food troughs.
The air conditioned auction room seats 600-800 people and has modern audiovisual equipment with Wi-Fi and live internet streaming during auctions.
A well designed auction ring and crush speeds up livestock management during sales with good loading ramps and a livestock scale.
There is also open bomas for game and holding paddocks for cattle.
There is a large bar area adjacent to the auction hall as well as a catering area to seat 400 people, a full kitchen and ample toilets.
We are able to host large, medium and small game and livestock auctions and work in conjunction with all game and livestock auctioneering/marketing companies.
This facility is close to Kroonstad Airport and other private landing strips and is surrounded by many places for accommodation of all types.


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redpajama_set_name: RedPajamaGithub


Israeli bulldozers demolished, Thursday, the al-Araqib 'unrecognized' Bedouin village, in the Negev, for the 132th consecutive time, and abducted three Palestinians.
Media sources said dozens of officers surrounded the village, before demolishing and removing residential structures and tents, in addition to barns.
They added that the police also abducted Sheikh Siyam at-Touri, and his son Aziz, in addition to Salim Abu Ashraf, for obstructing the work of the officers.
Israeli demolitions of al-Araqib are carried out in the attempt to force the Bedouin population to relocate to government-zoned townships.
Like the 34 other Bedouin villages "unrecognized" by Israel, al-Araqib does not receive any services from the Israeli government, and is constantly subjected to the threats of expulsion and home demolition.
Many Bedouins were forcibly transferred to the village sites during the 17-year period when Palestinians inside Israel were governed under Israeli military law, which ended shortly before Israel's military takeover of Gaza and the West Bank, including East Jerusalem, in 1967.
Now, more than 60 years later, the Bedouin villages have yet to be legally recognized by Israel and live under constant threat of demolition and forcible removal.


This summer, there are at least six major cookery books on Italy – all beautifully illustrated with scenes of Italian street life, covering different regions of the country from the far south of Sicily to the far north of the Veneto.
It's no surprise to see publishers concentrate on Italy: the country's passion for food and the simplicity of its cooking is perfectly in tune with today – and not a quinoa recipe in sight.
All these books have things to recommend them, but which one – or ones – should you buy? Leslie Geddes-Brown takes a look.
First choice must be Rachel Roddy's Two Kitchens, her kitchens being one in her home town of Rome, the second in Sicily.
Her first book on Rome won two awards and this one has attracted another two.
These are well deserved, as Miss Roddy chooses simple recipes that are unusual and tempting. Definitely one for the shelves.
In this book Melissa Muller concentrates on an island that well deserves the attention, having a cuisine derived from its Greek, Norman and Moorish invaders.
Its recipes are heavy on spices, lemons and wild herbs such as oregano and fennel; pasta includes saffron and, of course, fish is a major ingredient. Sadly, her writing is a bit clonky.
Moving a bit further north, the Silver Spoon writers tackle Naples and the Amalfi Coast (Phaidon, £24.95), where the food is pretty much the same as Sicily (the Greeks were here, too), but with such famous dishes as pizza and espresso coffee being typical.
The book is beautifully produced, full of moody photos (I loved the glowering black pig), but has only 30 recipes, so it is, perhaps, more a travel book than a cookery book.
The only celeb book in this list is this effort by the main who mentored Jamie Oliver and teamed up with Antonio Carluccio for the BB2 series Two Greey Italians, Gennaro Contaldo was brought up on the Amalfi coast and loves the food and the fun; he recalls meals at which 25 diners were not uncommon and being sent out by his father to hunt and fish and by his mother to forage for herbs. She, apparently, was considered 'a white witch' who could forecast the future.
The book has an awful front cover and interior design (why choose a typeface that's so hard to read?), but has more than 100 satisfying recipes.
Further north still, up to the Maremma on the coast of southern Tuscany, and we have the latest book by Emiko Davies, whose last book profiled Florentine cooking.
This region has also had different influences: a heavy Jewish input and, from way back in history, the legacy of the Etruscans. It's now known for its flocks of sheep and fierce, but dutiful Maremma sheep dogs.
The Maremma regional style is, once again, based on the land and its hunters, farmers and fishermen. A speciality is one-pot cooking (a result of the people's poverty). Acquacotta (cooked water) is actually a soup made with vegetables seethed in water and is a great deal more appetising than it sounds. The recipe here uses wild chicory, wild fennel and calamint, but substitutes are given.
The book is full of seductive photographs: if the Maremma was a well-kept secret, it won't be now.
From the north of Italy, Veneto emphasises that this is not about Venice, but the countryside around the city.
The recipes are delicious and delightful and the excellent pictures are the author's own.


This entry was posted on perjantai, huhtikuu 5th, 2013 at 14:08	and is filed under Musta hevonen. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.


text:
Another Sony Smear: Nintendo DSi A 'Kiddie' Console
Eric Krangel Apr. 7, 2009, 1:58 AM
Another day, another bizarre attack from Sony (SNE) on the gaming competition. (See also: Sony attacks the iPhone or Sony's claim the PS2 will steal market share from the Wii.)
The latest: Sony exec John Koller blasts Nintendo's (NTDOY) updated DSi portable console as not appealing to anyone outside the "kids market."
But it's just not true. The DS (in Japan at least) actually appeals to a wide array of demographics, with gamers in their early 30s the DS' second biggest demographic outside of the 10-12 year old set.
And then there's the success of Take-Two's (TTWO) DS title Grand Theft Auto: Chinatown Wars. Critics who thought Nintendo's DS line was only for kids predicted the title the M-rated title — one segment features the protagonist involved a menage à trois while on a ketamine binge — wouldn't sell. Wrong: The game is ranked the #10 selling title globally by the respected vgzhartz site.
But regardless of who's playing the consoles, the numbers speak for themselves: The DS line has sold about 101 million units to the PSP's 47 million.
Perhaps instead of Nintendo needing to aim older, Sony needs to aim younger.
John's statement in full.
If Nintendo is really committed to reaching a broader, more diverse audience of gamers beyond the "kids" market that they've always engaged, there isn't much new with the DSi to support that. Significant gamer demographic groups are being ignored, and there continues to be limited opportunities for games from external publishers to do well on the DSi. Compare that with the PSP platform, where we have many blockbuster franchises from our publishing partners launching this year, representing a wide variety of genres and targeting diverse demographics. Games such as Rock Band Unplugged from MTV Games, Assassin's Creed from Ubisoft, Dissidia Final Fantasy from Square Enix, and Hannah Montana from Disney demonstrate the commitment that publishers have to the PSP. From our own first-party studios, we're launching unique versions of LittleBigPlanet and MotorStorm, and we're also planning a steady stream of downloadable games — both new titles and PSone classics — to add to the content that PSP owners can already purchase wirelessly through PlayStation Store.
Eric Krangel


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In this instalment of our Creative Capital series, the co-founder and music director of the Telok Ayer Arts Club tells CNA Lifestyle what he's been up to since the pandemic started and what music lovers can look forward to from him.
Like many other artistes, Mr Has yearns for the return of live entertainment. (Photo: Hasnor Sidik)
28 Nov 2021 07:47AM (Updated: 17 Dec 2021 11:10AM)
For music fans and club kids of all ages, Hasnor Sidik needs no introduction, except that most would know him by his professional moniker, Mr Has. Hasnor is one of Singapore's most influential DJs and a key influencer in the music and lifestyle scene. He's played gigs from Bali to Berlin and spent time working as the in-house music director at the W Hotel and also at the Lo & Behold Group.
He is a co-founder and music director of the Telok Ayers Art Club, a Mr Porter Style Council member (one of only three from Singapore), and founder of his own music label Rawjak.
Mr Has is yearning for a time in which live entertainment can come back and has an awesome idea for what he calls the Covid Closing Party.
HI HAS, WERE YOU ALWAYS INTO MUSIC, EVEN WHEN REALLY YOUNG?
I have always enjoyed music since a very young age. Back in the late '80s when breakdancing came onto our shores, I remember dancing to Kraftwerks' Tour De France in the HDB common corridor with some other kids with our track suits on.
WHEN DID YOU FIRST TRY YOUR HAND AT DJING?
The real reason why I wanted to DJ came about in the late 90s; it was because of drum and bass music that came from the UK at that time. I thought it would be really cool to play that sound in a club, so I bought myself a pair of turntables and a mixer with the money that I saved from working after my O Levels and just started practising at home.
Has says he wishes he had spent more time record digging and less time hunting down gigs as a young DJ. (Photo: Hasnor Sidik)
IF YOU WERE TO LOOK BACK, HOW WOULD THE MR HAS OF TODAY CRITIQUE THE YOUNG DJ YOU WERE WHEN YOU FIRST STARTED OUT?
I would encourage more record digging and collecting, and not to hurry to get a gig. It is a beautiful part of the process which takes a lot of time and practice, so I would tell my younger self to slow down.
WHEN DID YOU DECIDE MUSIC COULD BE A REAL CAREER FOR YOU?
When the gigs started coming in! That obviously was a big factor. At that time, I told myself if I wanted to have a DJ career, I must deejay at Zouk at some point. So things started falling into place and pretty soon, I had a part-time residency there and more gigs kept coming.
WAS IT HARD TO GET RECOGNISED AND TO GET GIGS?
Yes, I had to do a lot of networking apart from just practising at home. Getting to know the scene more and sharing my mixtapes with other fellow DJs and friends helped in getting my music out there.
WHAT ADVICE DO YOU GIVE YOUR DJS WHO ASK YOU HOW THEY CAN STAND OUT AND BUILD THEIR OWN CAREERS?
I suggest going analog to start with, work on the turntables and record digging first before going with the current software and hardware solutions. You need to understand the fundamentals of DJing. Also, gain some experience producing your own music – it's a key part in building a music career.
Some of Has' most memorable gigs were those organised by the W Hotel. (Photo: Hasnor Sidik)
WHAT IS THE MOST MEMORABLE PERFORMANCE TO DATE?
I have done many amazing gigs but the one gig where I truly enjoyed myself was the courtyard party that I did with W Hotels and Singapore Art Museum. A gathering of Singapore's best DJs playing to a thousand-strong crowd under the stars. Quite magical!
AND WHAT WAS THE MOST CHALLENGING?
They are all challenging. The most challenging part, as a DJ, is that connection and control on the dance floor. It's a powerful energy that you have to keep up for a good four to six hours, sometimes more at a party. Not an easy task.
WHAT IS BIGGEST MISCONCEPTION YOU GET ABOUT WHAT YOU DO?
That it's all fun and travel.
ANY OPINION ON CELEBRITIES WHO ALSO DJ?
I'm pretty sure there are some out there that can really deejay.
WHAT WOULD YOUR DREAM GIG BE?
My dream gig right now would be called The CCP – Covid Closing Party – located in a beautiful green, open jungle space in Singapore with plenty of fresh air and not many mosquitoes. It has 24 hours of music and art programming where everyone can come and have fun from day to night to celebrate the end of this pandemic. DJs, musicians, artists, chefs and F&B operators would get together to celebrate the start of "the new endemic". Mask, optional. I need to talk to the Singapore Tourism Board about this actually. Maybe they will like the idea enough to help me make this a reality.
Mr Has playing a gig at the Singapore Art Museum before COVID-19 hit. (Photo: Hasnor Sidik)
YOU HAVE WORKED IN-HOUSE AT COMPANIES LIKE LO & BEHOLD AND W HOTEL. HOW DIFFERENT IS WORKING FOR COMPANIES LIKE THESE FROM PLAYING GIGS?
It was a totally different scope. Those were both management roles for brands that involved budgets, strategy, planning, producing, curating & executing on a corporate level. Very different from gigs.
TELL ME ABOUT TELOK AYER ARTS CLUB? HOW DID THAT COME ABOUT?
My partner Sue-Shan told me about the idea when she had a space on McCallum Street and was figuring out what to do with it. After much discussion and realising there weren't many art 'offerings' in the CBD, Telok Ayer Arts Club was born. We were also trying to pay homage to the Telok Ayer Performing Arts Centre, which used to be nearby but was sadly demolished.
WHAT PROJECTS AND PROGRAMMES DO YOU DRIVE AT THERE?
Our club night is called Kelab Malam. It is a monthly party where we have DJs and friends get together for a fun night of food, music and drinks. Makan Matters is our guest chef nights where we introduce new individuals showcasing their personal recipes. TAAC:LIVE is our local live electronic music showcase. Our arts programming is handled by the arts manager, Anmari, and we have collaborated and worked with artistes like Ashidiq Ghazali, Dawn Ng, Aiwei Foo and more. It's really great to have this kind of space that drives collaborations with local creatives.
Has on what gets him going these days: "Family, friends, ping pong. And of course, music." (Photo: Hasnor Sidik)
WHAT EXCITES YOU RIGHT NOW ABOUT THE LOCAL MUSIC SCENE?
The talent that is coming out right now excites me. We are more expressive these days so we will see more musicians and artistes doing their thing.
Family, friends, ping pong. And of course, music.
HOW HAS THE PANDEMIC AFFECTED TELOK AYER ARTS CLUB AND YOUR OTHER GIGS?
We had to end our lease and regroup during this difficult time. We just launched #TAACAWAY, an online takeaway platform featuring new Singaporean cuisine. Check it out! Every order comes with a playlist and an NFT.
As for the other gigs, they will only come back when the entertainment industry is back on.
ANY EXCITING PLANS FOR 2022?
Look out for more updates on Telok Ayer Arts Club and the rebranding of my label Rawjak.
Creative Capital: This Singaporean multi-disciplinary artist take visual arts to the next level
Source: CNA/hs
Entertainment Music Creative Capital


Comments: Broadleaf deciduous tree native to eastern Canada and eastern US (from Maine to Texas. Variable leaves--oval, ovate and sometimes lobed--on the same tree. Low growing and delicate branching in tiers. An attractive ornamental with aromatic leaves with good Fall color. Member of the laurel family.


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STATE OF ARIZONA, Appellee, v. RAYMOND DANIEL ROBBINS, Appellant.
¶1 Robbins appeals the superior court's preclusion of evidence regarding his possession of a registry identification card ("AMMA Card") pursuant to the Arizona Medical Marijuana Act ("AMMA"). See Ariz. Rev. Stat. ("A.R.S") §§ 36-2801 to -2819 (2014). He argues the court committed reversible error by excluding evidence of his AMMA Card because doing so precluded an affirmative defense under the AMMA. The State argues the court did not err because evidence of Robbins' AMMA Card was not relevant to Robbins' conviction under § A.R.S. 28-1381(A)(1) (2014) ("Subsection (A)(1)"), and that any error regarding his conviction under A.R.S. § 28-1381(A)(3) ("Subsection (A)(3)") was harmless. For the reasons stated below, we affirm.
We cite to the current statutes unless changes material to this decision have occurred.
¶2 In June 2014 at approximately 1:45 a.m., police officers M and R observed Robbins' vehicle veer into the bicycle lane while making a left turn. After following Robbins and noticing that he continued to weave in and out of his lane, the officers conducted a traffic stop and noted a slight odor of marijuana. When the officers asked Robbins for identification, he presented his AMMA Card because he could not find his driver's license. Robbins stated he had smoked marijuana approximately two hours before the encounter, but he denied having ingested alcohol and showed officers he had an interlock device on his vehicle. An officer trained in DUI detection arrived and administered field sobriety tests, noting that Robbins' eyes were bloodshot and watery and that Robbins was unable to successfully complete several of the tests. Based on Robbins' driving, physical signs, and poor performance on the tests, the officers arrested him and obtained a blood sample. Blood testing revealed methamphetamine, amphetamine, marijuana, and marijuana's metabolites.
¶3 The State indicted Robbins on two counts of aggravated driving or actual physical control while under the influence of intoxicating liquor or drugs, both class 4 non-dangerous, repetitive felonies. The State brought Count 1 pursuant to A.R.S. § 28-1381(A)(1), which criminalizes driving while under the influence of any drug "if the person is impaired to the slightest degree." The State brought Count 2 pursuant to A.R.S. § 28-1381(A)(3), which criminalizes driving "[w]hile there is any drug defined in § 13-3401 or its metabolite in the person's body." The State also alleged Robbins had nine prior felonies, was on probation at the time of the offense, and that eight aggravating factors applied.
Section 13-3401 includes marijuana, amphetamine, and methamphetamine. A.R.S. § 13-3401(6), (19) (Supp. 2015).
¶4 Before trial, the State sought to preclude testimony that Robbins held an AMMA Card as a defense to Count 2, arguing the evidence was not relevant to the Subsection (A)(3) charge. Although Robbins conceded that the evidence would not affect the jury's determination as to Count 2, he nevertheless argued knowledge of his AMMA Card "would take the sting out of the fact that he might be impaired." The court granted the State's motion, with the caveat that the State could not suggest that Robbins possessed marijuana illegally and had to treat marijuana in the same manner it would treat any type of prescription drug.
¶5 The jury found Robbins guilty on both counts. At sentencing, the State proved two prior felony convictions and all eight aggravating factors. The superior court sentenced Robbins to an aggravated sentence of twelve years' imprisonment and community supervision for both counts, the sentences to be served concurrently.
¶6 Robbins timely appealed. We have jurisdiction pursuant to A.R.S. §§ 12-120.21(A)(1) (2016), 13-4031 (2010), and 13-4033 (2010).
¶8 We review rulings on the admissibility of evidence for an abuse of discretion, and we consider only the facts presented at the suppression hearing. State v. Newell, 212 Ariz. 389, 396, ¶ 22 (2006). We view the disputed evidence in the "light most favorable to its proponent, maximizing its probative value and minimizing its prejudicial effect." State v. Harrison, 195 Ariz. 28, 33, ¶ 21 (App. 1998) (citation omitted). "When a judge commits an error of law . . . in the process of reaching [a] discretionary conclusion, he may be regarded as having abused his discretion." Twin City Fire Ins. Co. v. Burke, 204 Ariz. 251, 254, ¶ 10 (2003) (quotations and citations omitted). Interpreting rules, statutes, and constitutional provisions raises questions of law that we review de novo. E.g. Pima County v. Pima Cty. Law Enforcement Merit Sys. Council, 211 Ariz. 224, 227, ¶ 13 (2005) (rule and statute); Duncan v. Scottsdale Med. Imaging, Ltd., 205 Ariz. 306, 308, ¶ 2 (2003) (statute and constitution).
¶9 Arizona law prohibits driving or being in actual physical control of a vehicle while under the influence of certain substances. See A.R.S. § 28-1381. The controlling statute, A.R.S. § 28-1381, addresses various categories of DUI; Subsection (A)(1) focuses on impairment, forbidding driving under the influence of any drug "if the person is impaired to the slightest degree," whereas Subsection (A)(3) focuses on the presence of certain drugs or their metabolite in the person's body. Id.; see also Dobson v. McClennan, 238 Ariz. 389, 391, ¶ 10 (2015).
¶11 Although Robbins concedes the affirmative defense does not apply to Subsection (A)(1), he maintains the superior court erred by precluding evidence regarding his possession of an AMMA Card. He argues any evidence that went to his tolerance for drugs was relevant to determining "impairment" pursuant to Subsection (A)(1). We disagree.
¶12 Under Subsection (A)(1), the State must only show that the defendant took "any drug" that caused impairment. State v. George, 233 Ariz. 400, 403, ¶ 12 (App. 2013) (citation omitted). "Whether a particular drug may cause a driver to be impaired is the driver's responsibility," and the driver bears personal responsibility for taking a drug and driving. Id. (citations omitted). Evidence of erratic driving, poor performance on field sobriety tests, and physical signs of impairment constitute evidence of impairment. See State ex rel. McDougall v. Albrecht, 168 Ariz. 128, 132 (App. 1991).
¶14 Robbins argues the superior court erred by precluding the evidence showing his possession of an AMMA card because he was entitled to assert an affirmative defense against the Subsection (A)(3) charge. We agree.
¶15 An AMMA patient may establish the affirmative defense to Subsection (A)(3) by showing that: (1) the patient's use was authorized by the AMMA, and (2) the marijuana or its metabolite was in a concentration insufficient to cause impairment. Dobson, 238 Ariz. at 393, ¶ 20. Possession of an AMMA Card creates a presumption that a qualifying patient is engaged in the use of marijuana pursuant to the AMMA, so long as the patient does not possess more than the permitted quantity of marijuana. Id. at ¶ 19; A.R.S. § 36-2811(A)(1). Evidence of possession of an AMMA Card is generally admissible in a Subsection (A)(3) prosecution to invoke this presumption. Dobson, 238 Ariz. at 393, ¶ 22.
¶16 When this issue was before the superior court, neither the parties nor the court had the benefit of the supreme court's decision in Dobson. However, under Dobson, Robbins was entitled to present an affirmative defense to Subsection (A)(3) and to present evidence in support of that defense. See A.R.S. § 13-205(A) (2010) ("[A] defendant shall prove any affirmative defense raised by a preponderance of the evidence."). The court therefore erred by excluding evidence of Robbins' AMMA Card. See Twin City Fire, 204 Ariz. at 254, ¶ 10 (stating a court abuses its discretion when it commits an error of law in making a discretionary conclusion).
¶17 Because the superior court erred in precluding evidence relating to Robbins' possession of an AMMA Card and Robbins objected to such preclusion, we employ harmless error review. Harmless error review places the burden on the State to prove beyond a reasonable doubt that the error did not contribute to or affect the verdict. State v. Valverde, 220 Ariz. 582, 585, ¶ 11 (2009). "The inquiry . . . is not whether, in a trial that occurred without the error, a guilty verdict would surely have been rendered, but whether the guilty verdict actually rendered in this trial was surely unattributable to the error." State v. Bible, 175 Ariz. 549, 588 (1993) (citations omitted). We consider the error in light of all the evidence. Id. (citation omitted).
¶18 The State argues any error regarding the Subsection (A)(3) conviction was harmless because substantial other evidence supported Robbins' conviction. It asserts the jury would still have concluded Robbins had drugs in his system because sufficient evidence showed his blood tested positive for methamphetamine and amphetamine. Robbins counters that knowledge of his AMMA Card and status may have contributed to or affected the jury's deliberation and the factual allegation of whether traces of amphetamine and methamphetamine were true. We agree with the State.
¶19 To convict Robbins under Subsection (A)(3), the State had to prove beyond a reasonable doubt that Robbins had methamphetamine, amphetamine, marijuana, or marijuana's metabolites in his body while driving. See A.R.S. §§ 28-1381, 13-3401. Subsection (A)(3) prohibits driving with any amount of an impairing substance in the body, regardless of impairment. State ex rel. Montgomery v. Harris, 234 Ariz. 343, 347, ¶ 24 (2014).
¶20 We conclude sufficient evidence supported Robbins' conviction under Subsection (A)(3) and, accordingly, that the superior court's error regarding Robbins' AMMA Card was harmless. In addition to a blood test showing Robbins' blood contained methamphetamine and amphetamine, the State presented two expert witnesses who testified regarding the methods used to test and confirm the blood results. Regardless of the presence of marijuana and its metabolites in Robbins' blood, sufficient evidence of the presence of methamphetamine and amphetamine in Robbins' blood supported the conviction under Subsection (A)(3). We therefore conclude the court's error was harmless.
¶21 For the foregoing reasons, we affirm Robbins' convictions and sentences.


text:
Q: Summing "masked" datetime ranges using overlapping datetimes in SQL Server I'm having trouble summing up date differences (in minutes) across overlapping date times, using the second set of datetimes as more of a "mask".
WHAT I HAVE
My table might look something like this:
ID         StartDateTime           EndDateTime            Type
1          9-16-2013 12:00:00      9-16-2013 16:00:00     A
2          9-16-2013 12:00:00      9-16-2013 13:00:00     A
3          9-16-2013 14:00:00      9-16-2013 15:00:00     A
4          9-16-2013 12:00:00      9-16-2013 13:00:00     C
5          9-16-2013 14:00:00      9-16-2013 17:00:00     C

A visual timeline would look like this:
      12      13      14      15      16      17
A     |-------------------------------|                    (ID 1)
A     |-------|       |-------|                            (IDs 2 and 3)
C     |-------|       |-----------------------|

WHAT I NEED
What I need to do is sum type A only if type B.  So the set I'm looking to generate and sum is (ID included for reference):
ID         StartDateTime           EndDateTime
1          9-16-2013 12:00:00      9-16-2013 13:00:00
1          9-16-2013 14:00:00      9-16-2013 16:00:00
2          9-16-2013 12:00:00      9-16-2013 13:00:00
3          9-16-2013 14:00:00      9-16-2013 15:00:00

The new "masked" timeline would look like this:
      12      13      14      15      16      17
A     |-------|       |---------------|                    (ID 1)
A     |-------|       |-------|                            (ID 2 and 3)

The problem that I am having is splitting the first ID into 2 different records because of the break in type C.  I've looked at a lot of the overlap posts on this site, but none seem to address this particular need.  Any help would be greatly appreciated.  Please let me know if you need more information or if I can be clearer in any way.  I'm using SQL Server 2008 R2.

A: One possible solution:
--set up test data
DECLARE @t TABLE
(ID INT,StartDateTime DATETIME,EndDateTime DATETIME,[Type] CHAR(1))

INSERT @t
(ID,StartDateTime,EndDateTime,[Type])
VALUES
(1,'9-16-2013 12:00:00','9-16-2013 16:00:00','A'),
(2,'9-16-2013 12:00:00','9-16-2013 13:00:00','A'),
(3,'9-16-2013 14:00:00','9-16-2013 15:00:00','A'),
(4,'9-16-2013 12:00:00','9-16-2013 13:00:00','C'),
(5,'9-16-2013 14:00:00','9-16-2013 17:00:00','C')

-- solution
SELECT  a.Id,
        CASE WHEN a.StartDateTime < c.StartDateTime THEN c.StartDateTime ELSE a.StartDateTime END AS StartDateTime,
        CASE WHEN a.EndDateTime < c.EndDateTime THEN a.EndDateTime ELSE c.EndDateTime END AS EndDateTime,
        a.[Type]
FROM    (SELECT * FROM @t WHERE [Type] = 'A') AS a
JOIN    (SELECT * FROM @t WHERE [Type] = 'C') AS c
ON      c.StartDateTime < a.EndDateTime
AND     c.EndDateTime > a.StartDateTime
ORDER BY a.Id, a.StartDateTime

redpajama_set_name: RedPajamaStackExchange


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text:
Notice of Available Officer and Director Positions for 2021
by Dallas Tschida | Jul 14, 2020 | Featured, Get Involved, News & Events, What's New | 0 comments
According to our Policy and Procedures, the board is to notify our members of the upcoming officer and director positions that need to be filled during the 2021 Calendar year. Our elections will be held in accordance with our Policies and Procedures via electronic ballot.
The following positions will be on the ballot in August.
Open Officer Positions
Open Director Positions
Malheur County Director
MLS Director
Nominations Now Open
Are you interested in joining our Board of Directors or do you know somebody that would be a great addition to our Leadership Team?
Please send all nominations to social@fraor.org by Friday, July 24, 2020 so we can prepare canidate information and the ballot for publication.
Scroll down to see the chief duties of each available position.
Must be a local REALTOR®
Term: 3 Years: (One-Year as President-Elect, One-Year as President and One-Year as Immediate Past President)
Chief Duties of the President-Elect are as follows:
Assume and perform the duties of the President in his/her abense.
Represent the President at conferences and meetings when directed to do so by the President or Board of Directors
Assist the President in the performance of his/her duties
Perform such other duties as may be delegated to him/her by President or the Board of Directors
The President-Elect shall automatically succeed to the office of President upon completion of his/her term.
The President-Elect shall take office of the President upon resignation, death, incapacitation or impeachment of the President and shall complete the remaining term unless otherwise determinded by the Board of Directors.
Term: 2 Years
Chief Duties of the Secretary are as follows:
Assist the President and officiate in keeping correct minutes of all meetings of the Association.
Distribute meeting minutes as appropriate to the Board and General Membership bodies.
Meeting minutes are to be distributed to the appropriate body within one week of each meeting.
Mail out paper billings via USPS as necessary.
Perform any other duties as may be delegated to him/her by the President or the Board of Directors.
When retiring from office immediately deliver all books, papers and other property in his/her charge belonging to the Association to his/her successor in office, or whomever the Board of Directors may appoint.
Chief Duties of the Treasurer are as follows:
Oversee the management of Association funds by the designated account manager.
Facilitate and assist with the monthly reconciliation of the bank statements monthly.
Review the monthly Balance Sheet to ensure funds are appropriated as dictated by the Board of Directors.
Review the Profit and Loss Statements to ensure expenses have been assessed to the appropriate accounts.
Report the financial status of the Association monthly to the Board of Directors.
Monitor the status of each account closely to ensure none incurs a deficit.
Should an account become deficient in funds the Treasurer will immediately report it to the Board of Directors for action as deemed appropriate and/or necessary.
Co-sign checks as necessary.
Handle and account for funds received during events, as per Article V, Section 5.09 Cash Control and Handling of these Policies and Procedures.
Deposit funds received on the day of receipt, or as soon as practical within the following three (3) business days.
Any funds that cannot be deposited the day they are received are to be held in the safe in the Treasurer's possession until they can be deposited.
All deposits and the source of funds are to be immediately reported to the account manager.
Serves a three (3) year term
Direct committee(s) assigned to him/her.
Serve as a member of assigned committee(s).
Report to the President and Association on all Association activities handled by him/her.
Be responsible for the general conduct of all affairs of the Association and assist in enacting such rules and regulations for its government as may be deemed practical, providing that same shall not be inconsistent with the Bylaws and Policies of the Association.
Keep the membership reasonably and properly informed on all matters of particular interest and concern with respect to the Real Estate profession and business.
Perform such other duties as are prescribed by the President or Association.
Attend not less than two meetings annually for each assigned committee.
MLS County Director
Serves a two (2) year term
Chief Duties of the MLS Director are as follows:
Attend all meetings of the IMLS as required by IMLS for the MLS Directors.
Be responsible for the general conduct and affairs of the Association.
Assist in enacting such rules and regulations for its government as may be deemed practical, providing the same shall not be inconsistent with the Bylaws or Policies of the Association.
Keep the membership reasonably and properly informed on all matters of particular interest and concern with respect to the Real Estate profession and business, more specifically information concerning the IMLS.
Perform other duties as are prescribed by the President or Association.


Barrie Downsizing Online Auction - Creswick Court in Barrie, Ontario by MaxSold Inc.
This online auction features Military Surplus Winter Parkas, Flight Suits, Winter Overalls, Winter Gloves, mitts, Classic Rock Albums Beatles Led Zeppelin, comics, vintage collectibles and much more!
Please view catalog and pictures online at MaxSold. Everything will SELL BY Tuesday February 12, 2019 8:00 PM regardless of price - everything starts at just $1. For more information, please visit our website.


text:
7 Mindfuck Movies on Netflix Canada That Will Mess You Right Up
These movies will make you question *everything*
by JP Larocque
(Photo: Netflix, Illustration: Joel Louzado)
Reality sucks, amirite? The monotony of the day-to-day, with bills, deadlines and the endless hamster wheel of food prep. (I bet 10-year-old you didn't think your life would require this much food prep, but that's neither here nor there as you attempt to make chicken breast exciting for the third day in a row.) So, when you get home after a long day, and retreat to the comfort of your couch and your trusty Netflix account, you're ready for something a little… different.
What you need is a good ol' fashioned mindfuck. Something that can turn your world upside down. Something that can shake things up and make you forget all about that boring chicken breast. And you're in luck, because Netflix Canada has a number of films on hand that will plunge you into alternative realities and absolutely blow your mind. And whether we're talking about a blindfolded Sandra Bullock wandering around the wilderness (Birdbox), Toni Collette coming to terms with some unfortunate truths about her family (Hereditary) or Jake Gyllenhaal getting stalked by artwork (Velvet Buzzsaw), there is a little something for everyone.
Synopsis: When strange paintings by an unknown artist are discovered in a dead man's apartment, a supernatural entity begins to murder anyone who dares to profit off the artwork.
Cast: Jake Gyllenhaal, Rene Russo, Toni Collette, John Malcovich
What makes it a mindfuck: Things don't get more reality-bending than artwork that murders a bunch of greedy art dealers. And with vivid visuals, a stellar cast and twists and turns that keep you on your toes, this film will leave you worried that the art print in your bathroom isn't just boring… it could be trying to kill you.
Synopsis: While attending a dinner party as his ex-wife's home, a man begins to suspect that his former lover may have sinister plans for him and the other guests.
Cast: Logan Marshall-Green, Tammy Blanchard, Michiel Huisman, Emayatzy Corinealdi
What makes it a mindfuck: Like the best films on this list, The Invitation starts as one thing and quickly evolves into something… very different. Much like the lead character, Will (Logan Marshall-Green), your paranoia will be off the charts after this one—and it may give you the strength to not text that ex you've been thinking about lately. Don't do it. Trust me.
Eternal Sunshine of a Spotless Mind
Synopsis: When a man discovers his ex-girlfriend has undergone a procedure to erase memories of him from her mind, he decides to do the same thing…only for the two former lovers to continue to cross each other's paths in surprising ways.
Cast: Jim Carrey, Kate Winslet, Kirsten Dunst
What makes it a mindfuck: It's a classic mindfuck—and for a reason. Aside from putting Michel Gondry on the map as a film director (he won an Academy Award for the screenplay) and solidifying Jim Carrey as a dramatic lead, the astounding visuals place you directly in the minds and memories of two lovers falling out of love. It's a depressing feast for the senses.
Synopsis: In a world where malevolent supernatural entities make you harm yourself if you see them, a woman and two children must journey to a safe zone—alone, blindfolded and relying on their other senses to make their way.
Cast: Sandra Bullock, Trevante Rhodes, John Malkovich, Sarah Paulson
What makes it a mindfuck: A sensory horror film in the vein of A Quiet Place, this Netflix hit inspired the "Bird Box Challenge" meme that had people getting blindfolded and putting themselves in danger for a laugh. Like, maybe don't do that, but you can be thrilled by a stellar cast navigating terrible circumstances. And there are also a few surprising twists along the way…
OtherLife
Synopsis: When a researcher invents a biological form of virtual reality that can play with human memory and perception of time, she attracts the attention of shady government officials who see more nefarious uses for her invention.
Cast: Jessica De Gouw, T. J. Power, Thomas Cocquerel
What makes it a mindfuck: Building off of a grim-but-believable tech premise, this Australian sci-fi thriller plays with your sense of reality and will make you question the reliability of the lead character Ren Amari (Jessica De Gouw)'s perspective at just about every turn. Definitely worth a watch.
Synopsis: In the future, the world's atmosphere has been rendered toxic, and most of humanity has left the planet to live on one of Jupiter's moons, Io. Still, a young scientist works in a far-flung outpost on Earth, determined to find a way to make the planet liveable, when she crosses paths with a stranger…
Cast: Margaret Qualley, Anthony Mackie, Danny Huston
Streaming: Yes.
What makes it a mindfuck: Well, it's not twisty so much as post-apocalyptic-y, but as a love story between two lone wolves on a deserted planet, it makes for quite the diversion and a cautionary tale on what could happen if we don't get our act together on the global warming front. Also, Anthony Mackie is sexy on a pants-warming front.
Synopsis: When her mother dies, a woman uncovers some unsettling information about her sinister bloodline and the dark fate that awaits her children.
Cast: Toni Collette, Alex Wolff, Ann Dowd, Gabriel Byrne
What makes it a mindfuck: Writer and director Ari Aster is currently breaking brains with his latest film, Midsommar, but Hereditary remains his first and true masterpiece—a tense psychological horror film that unravels slowly and makes you feel as uncertain of what's going on as the characters themselves. Critics loved it, and I suspect you will, too—although it will make you question everything you know about your Nana.
Synopsis: A thief with the ability to enter dreams and steal important information is tasked with a seemingly impossible job—plant information in a man's subconscious. And the prize? Having his criminal record cleared once and for all.
Cast: Leonardo DiCaprio, Ken Watanabe, Joseph Gordon-Levitt, Marion Cotillard
What makes it a mindfuck: Writer and director Christopher Nolan is known for his fair share of complex narrative puzzles (see also: Memento), but this film put him in the big leagues. An A-list cast running through dreams and into dreams from dreams by dreams… you'll probably need to draw a bit of a flow chart to make sense of it all, but it's worth the watch and quite the visual spectacle.
Full disclosure: Writer JP Larocque writes for Netflix shows Slasher and Another Life.
What's New on CBC Gem in August 2019
12 Totally Kick-Ass Action Movies to Watch on Netflix
What's New on Netflix Canada in August 2019
Streaming Round-Up


Gift registries are a convenient way of letting your guests know what you'd like to have as your wedding present. But before you set it up and send the shopping invites, make sure you think about the selection of shops and items you'd really enjoy. There's no point in creating a registry if you just fill up the list with many items and later on regretting having put them on the list. You surely don't want to start your newly wed-lifestyle with a bunch of things that you don't need in your home. So here are some ideas for gifts that could help you create your wish list for the wedding gift registry.
Think about your hobbies. What do you like to do in your free time? What do you enjoy doing together with your partner? These will most likely turn out to be the best gifts!
Many times an experience is better than (more) things. If you and your partner have been thinking about going skydiving or taking a diving class, wouldn't that be the perfect gift? Not only can you do it together with your partner, you can cherrish the beautiful memories together for ever.
Probably the classic among wedding gifts. It comes from the tradition of moving into a new home with your partner after the wedding. Moving brings costs along the way and you don't have things of your own. Your friends and family are here to help you out with that.
Well, nowadays you might have been living together before the wedding. Or not. Either way some home appliances that you can use in your home are always welcome, aren't they?
Make a list of all the things you've been wishing for but never managed to look for. Whether it's a simple toaster or an ice cream maker, think about it, talk with your partner and write it down.
There are always a few things you would like to change in your home. And there's always that coffee table that you don't quite like anymore but don't have the heart to change it because there's nothing really wrong with it. But if you happen to stumble upon a new one among your wedding gifts, it's the perfect time to make a few changes around the house. New married life, new furniture. If it's something larger that you wish for, make a registry for a contribution, so several of your guests can pitch in on one item. They help you out and you get your perfect gift.
The post Wedding registry ideas and suggestions appeared first on Her Wedding Planner.


Abstract : We examine initial-boundary value problems for diffusion equations with distributed order time-fractional derivatives. We prove existence and uniqueness results for the weak solution to these systems, together with its continuous dependency on initial value and source term. Moreover, under suitable assumption on the source term, we establish that the solution is analytic in time.


text:
Fotoizomeryzacja – reakcja polegająca na przemianie danego związku chemicznego w jego inny izomer pod wpływem światła.

Związek ulegający fotoizomeryzacji absorbuje światło. Zjawisko fotoizomeryzacja można zaobserwować dla barwników organicznych np. stilben, czy azobenzen. Wykorzystywane jest ono między innymi do tworzenia optycznych nośników pamięci.

Zobacz też
 izomeryzacja
 izomerazy

Reakcje chemiczne
Stereochemia
Fotochemia
Izomeria


We went to another Pacers game last week! This time it was Cody Zeller night, as the Pacers were playing the Charlotte Bobcats. The IU promotion was the Lucas Oil Family Night promotion, and our nose-bleed tickets (~$15) also included a free gift (a t-shirt) and a food voucher good for a hot dog, bag of chips, and Pepsi product. I'd say it was a pretty good deal!
Boomer came down from the ceiling before introductions, and he went back up to the rafters during the game with this Get Loud banner. Awesome!
Halftime this game was pretty cool (although the trick dogs might win forever). First they had a tug-of-war shooting contest. The two shooters below are connected with a rope around their waists, and each is trying to make as many shots as he can. It was funny to watch a shooter leave the ground to shoot and be tugged backwards while the other guy was running forward!
The other part of halftime were these two trampoline jumpers. They were awesome. I took an Instagram video of them doing some tricks.
We had another great time at the game! Was anyone else at the game last Friday? Did you have any troubles with the snow? My dad tried to convince us to not go since it was supposed to snow so much and we don't have a 4-wheel-drive vehicle, but we convinced him to go anyway and it turned into a great time (and the snow wasn't too bad).


Chemical cross-linking is used for protein-protein contacts mapping and for structural analysis. One of the difficulties in cross-linking studies is the analysis of mass-spectrometry data and the assignment of the site of cross-link incorporation. The difficulties are due to higher charges of fragment ions, and to the overall low-abundance of cross-link species in the background of linear peptides. Cross-linkers non-specific at one end, such as photo-inducible diazirines, may complicate the analysis further. In this report, we design and validate a novel cross-linked peptide mapping algorithm (XLPM) and compare it to StavroX, which is currently one of the best algorithms in this class.
We have designed a novel cross-link search algorithm -XLPM - and implemented it both as an online tool and as a downloadable archive of scripts. We designed a filter based on an observation that observation of a b-ion implies observation of a complimentary y-ion with high probability (b-y filter). We validated the b-y filter on the set of linear peptides from NIST library, and demonstrate that it is an effective way to find high-quality mass spectra. Next, we generated cross-linked data from an ssDNA binding protein, Rim1with a specific cross-linker disuccinimidyl suberate, and a semi-specific cross-linker NHS-Diazirine, followed by analysis of the cross-linked products by nanoLC-LTQ-Orbitrap mass spectrometry. The cross-linked data were searched by XLPM and StavroX and the performance of the two algorithms was compared. The cross-links were mapped to the X-ray structure of Rim1 tetramer. Analysis of the mixture of NHS-Diazirine cross-linked 15N and 14N-labeled Rim1 tetramers yielded 15N-labeled to 14N-labeled cross-linked peptide pairs, corresponding to C-terminus-to-N-terminus cross-linking, demonstrating interaction between different two Rim1 tetramers. Both XLPM and StavroX were successful in identification of this interaction, with XLPM leading to a better annotation of higher-charged fragments. We also put forward a new method of estimating specificity and sensitivity of identification of a cross-linked residue in the case of a non-specific cross-linker.
The novel cross-link mapping algorithm, XLPM, considerably improves the speed and accuracy of the analysis compared to other methods. The quality selection filter based on b-to-y ions ratio proved to be an effective way to select high quality cross-linked spectra.
Mapping of protein-protein interactions (PPIs) is an important, yet challenging analytical problem. This is also a potential clinical need, as having a detailed PPI information may aid in finding novel targets for therapeutic intervention . Thus far, mainstream methods of PPI mapping include affinity-purification-mass-spectrometry (AP-MS), yeast-two-hybrid (Y2H), or mammalian-two hybrid methods. PPI information obtained from these experiments is deposited into online databases, such as BioGRID . However, it takes a lot of effort to map the full interactome using these approaches. Also, as researches are starting to ask questions about interactome dynamics , we need faster and more effecting ways to map PPI networks. We suggest that PPI sampling using chemical cross-linking is an attractive alternative to AP-MS and Y2H methods.
In our recent review article on the subject, we presented a case for the use of short-length cross-linkers of broad specificity for the purpose of sampling PPIs and discussed existing challenges and limitations . We argued that a short, broadly specific cross-linker could yield a more comprehensive interactome analysis and, in some cases, the mapping of individual PPI interfaces. The main difficulties include identification of cross-linked peptides in the background of non-cross-linked species and also, discriminating between inter- and intra-protein cross-links and assessing the false positive rate of cross-linked site identification. The other technical problem is the computational expense of allowing for non-specific cross-linking (i.e. cross-linked to any amino acid). We proposed the following steps to overcome these difficulties. Step 1 - starting at the scale of few proteins, develop an efficient filtering and scoring system to analyze cross-linking data, allowing for non-specific cross-linking at one end. Step 2 - expand to the large scale removing all specificity requirements. In this publication we complete the Step 1 and build a foundation to move onto Step 2.
For a scale of few proteins, it is easy to design a cross-link search algorithm, when one is relying on a database consisting of pair-wise combinations of peptides from interacting proteins. Next, from this database the list of possible cross-linked precursors is prepared. There are many software tools that use this approach: CLPM , xComb , GPMAW , X!Link , StavroX , MassMatrix . However, all of them perform rather poorly when non-specificity is allowed. Also, not all of those algorithms take a full advantage of MS/MS spectra for defining the position of the cross-linked amino acid residues.
In this paper, we present a new algorithm, XLPM, which uses a novel method to allow effective use of the tandem MS information for the cross-link analysis without dramatically affecting the speed when non-specificity is allowed. We used StavroX as a benchmark to evaluate the XLPM performance, because, in our hands, StavroX produced the most robust and reliable data amongst all the programs we evaluated. We used the photo labile diazirine cross-linker SDA  on a Rim1 tetramer to evaluate the XLPM. Rim1 is a single strand DNA binding protein localizing to yeast mitochondria. Our data suggest a previously unreported tetramer-to-tetramer interaction. The functional analysis of this interaction and its biological implications will be presented elsewhere. Here, we focused mainly on the computational analysis. Additionally we propose a novel method to validate the cross-linked site assignment: by relaxing specificity of a known, selective cross-linker. For this purpose we used the Rim1 cross-linked dataset obtained using DSS.
The algorithm and the program workflow of XLPM are shown in Additional file 1. XLPM receives information of protein sequences, a spectra file in mgf format, digestion enzyme, and cross-linker, static and variable modifications of amino acids, missed cleavage level, precursor ion tolerance in ppm and fragment ion tolerance in Daltons. XLPM generate temporary database of cross-linked pairs from given protein sequences. First, amino acids that may be cross-linked are marked in the protein sequence. A list of digested fragment sequences is generated. A missed cleavage is forced. In other words, digested fragments having only one amino acid at the C terminal, which can be cross-linked, are removed from the list. The digested fragments devoid of any amino acids that can be cross-linked are also filtered out. A database of cross-linked pairs is generated from the list of digested fragments including the change of the mass due to any amino acid modification.
A file in a mascot generic format (mgf) is uploaded and read; data are extracted from it and parsed into precursor ions and fragment ions. The precursor ion masses are compared with the masses of cross-linked pairs in the database generated previously with the user defined error tolerance. Each matched cross-linked product is further analyzed using its MS/MS fragmentation pattern and scored.
XLPM considers b and y ions while analyzing MS/MS fragmentation pattern. Cross-linked amino acids carry an additional mass of cross-linker and the second fragment. Thus, it can be treated as a modification on that amino acid while calculating the theoretical masses of the b and y ions. The algorithm calculates the mass of each b or y ion with and without the cross-linked second fragment. The algorithm then compares all calculated b ions (with and without the cross-linked fragment) with the MS/MS spectra. Complementary y ions of matched b ions are then compared with MS/MS spectra (Figure 1).
XLPM implements a unique filter based on high probability to observe a complimentary y-ion, given a b-ion. For each candidate pair of cross-linked peptides and for each possible cross-link position theoretical masses of b and y ions are computed. Next, the candidate experimental spectrum is checked for the presence of b-ions. In every case when charge of b-ion is less than the charge of the precursor, the experimental spectrum is searched for the complementary y-ion.
XLPM is a library of Perl scripts. All of the scripts are interdependent on one another. Each script serves a specific function in the course of the algorithm. Thus, a small modification in any of the scripts can tweak the algorithm to do a variety of analyses. The program uses a MySQL database to store the user input and the processed information at each stage of the analysis, which again aids in making the scripts efficient and flexible. XLPM can be accessed through a simple and intuitive web interface (http://binf-app.host.ualr.edu/~mihir/cgi-bin/xlpm.cgi) which allows the user to submit jobs to the system. A web form allows variables to be set and input files to be uploaded. Jobs are placed in a queue and upon completion results are emailed to the user. The workflow is depicted in Figure 2.
The workflow of XLPM web server. The job manager manages the submitted jobs. Once a completed job is detected by the job manager, it submits the next job in the queue to XLPM for the analysis. The results are mailed to the users and errors are mailed to the administrator, if any.
The database contains pre-compiled data of digestive enzymes, cross-linkers, modifications, and amino acids. A new enzyme, cross-linker or custom modification can easily be added to the database and used in subsequent analyses. The algorithm is compatible with digestive enzym cleaving at either end of one or more amino acids. The algorithm can also handle all kinds of amino acid modifications for which precise changes in the masses are known. XLPM is better than many existing tools, as it can accommodate specific, semi-specific and non-specific cross-linkers. The algorithm can also perform isotopic analysesusing15N-labled proteins.
XLPM score = N mb N tb + N my N ty 1 R yb .
Nmb and Nmy are numbers of theoretical b ions and y ions matched to the CID spectrum, respectively. Ntb and Nty are numbers of total possible b ions and y ions, respectively. Ryb is the median ratio of complementary y ions to b ions for the charge of the precursor ion as calculated from NIST the data, described in the previous segment.
The following materials were purchased from Thermo Fisher Scientific or its subsidiaries: HPLC-grade acetonitrile, formic acid, HEPES, Tris, NaCl, EDTA, MgCl2, SDS, KOH, β-mercaptoethanol, acrylamide, bisacrylamide, formamide, xylene cyanol, bromphenol blue, urea, glycerol, SDA cross-linker, formaldehyde, DSS, Gel-Code blue stain, and Zeba-Spin Desalting columns for buffer exchange.
Recombinant helicase domain of Pif1, full-length Pif1, and Rim1-C-terminal 6xHis proteins used in this work were purified as described in  K29A Rim1-C-terminal 6xHis mutant was purified from E. Coli, using the same isolation protocols as for wild-type Rim1, established in . Throughout the article, we call Rim1-C-terminal 6xHis as wild type Rim1, and K29A Rim1-C-terminal -6xHis as K29A Rim1. The proteins without 6xHis tag as labeled as "no-tag", when appropriate.
To metabolically label Rim1, E. Coli expressing Rim1 was grown on media prepared with 15N-ammonium chloride as the sole source of nitrogen . Next, the 15N-labeled Rim1 was purified according to the previously established protocol .
To the 2X stock of Rim1 protein (6 µM of Rim1 tetramer), was added equal volume of 5 mM DSS in a cross-linking reaction buffer. Final concentrations of the reagents in the reaction mix were: 25 mM HEPES, 100 mMNaCl, 10% Glycerol, 3 µM of Rim1 tetramer, 2.5 mM DSS, pH = 7.5. The reaction was incubated at room temperature for 40 min, with mild agitation. Next, the quenching buffer (Tris, pH 8) was added to the final concentration of 50 mM. Using Zeba Spin Desalting columns (Thermo Fisher), the buffer was exchanged into 25 mM HEPES, 100 mMNaCl, pH 7.5. 90% protein recovery was verified by Bradford assay. The mixture was boiled in SDS loading buffer, and the products were resolved on 5-15% gradient SDS-PAGE.
Prior to the cross-linking experiments, the proteins were thawed on ice, and using Zeba Spin Desalting columns, the storage buffer was exchanged to the cross-linking reaction buffer (25 mM HEPES, 100 mM NaCl, 10% Glycerol, pH 7.5). In all of the cross-linking reactions, the final concentrations of Rim1 (tetramer) were 3 µM. To the 2X stock of Rim1 protein in the cross-linking buffer (6 µM of Rim1), SDA stock solution in DMSO was added (50 µM SDA and 0.5% DMSO final concentrations). The NHS reaction was carried out at 25 °C for 30 min followed by the addition of 1M Tris, pH 8.0 (50 mM final Tris) and the additional incubation at 25 °C for 5 min. Next, to remove the unreacted cross-linker, the buffer was exchanged with fresh cross-linking buffer using the Zeba Spin Desalting columns. A 90-95% percent protein recovery was verified by Bradford assay. For the second step of the SDA-cross-linking reaction, an equal volume of the 2X Rim1 protein or 2X Pif1 protein, to be reacted, was added to the NHS-reacted mix. UV-cross-linking was performed in a Strata linker 1800 using coated bulbs (365 nm emission maximum) at a 2 cm distance from the bulbs in black, flat-bottomed 96-well plates. 4X SDS buffer was added to the reactions and the reaction products were resolved on a5-15% gradient SDS-PAGE. The cross-linked band was excised, in-gel digested and analyzed by the NanoLC-LTQ-Orbitrap-Velos mass spectrometry, as described below.
Gel slices were destained in 50% methanol, 100 mM ammonium bicarbonate, followed by reduction in 10 mM Tris[2-carboxyethyl] phosphine and alkylation in 50 mM iodoacetamide. Gel slices were then dehydrated in acetonitrile, followed by addition of 100 ng porcine trypsin in 100 mM ammonium bicarbonate and incubation at 37°C for 12-16 hours. Peptide products were then acidified in 0.1% formic acid. Tryptic peptides were separated by reverse phase Jupiter Proteo resin (Phenomenex) on a 100 × 0.075 mm column using a nano Acquity UPLC system (Waters). Peptides were eluted using a 30 min gradient from 98:2 to 40:60 buffers A:B ratio. [Buffer A = 0.1% formic acid, 0.05% acetonitrile; buffer B = 0.1% formic acid, 75% acetonitrile.] Eluted peptides were ionized by electrospray (1.9 kV) followed by MS/MS analysis using collision induced dissociation on an LTQ Orbitrap Velos mass spectrometer (Thermo).
To detect the cross-linked peptides, an LTQ-Orbitrap-Velos was used with the following parameters: 1) the three most intense precursors were subjected to MS/MS analysis - each of the precursors was analyzed consecutively by HCD (7,500 resolution), CID with the analysis of the fragments in OrbiTrap (FT-CID 7,500 resolution), and regular CID; 2) each sample was run twice, the first time allowing all of the charges, and the second time allowing only precursors of charge 4 and higher to be fragmented; 3) the precursor selection intensity threshold was set to 5000; 4) for the 15N-labeled samples, only the HCD fragmentation of the 10 most intense precursor ions at 7,500 resolution was used.
Peaks lists in a mascot generic format were generated from the raw files using Proteo Wizard tools. The same sets of parameters were used for both XLPM and StavroX. An input file with tags and values was made for XLPM. To evaluate a possible impact of charge deconvolution of fragment spectra we used spectral refinement function available in the PEAKS v7 software suite (Bioinformatics Solutions Inc.). The spectral deconvolution did increase the scores obtained through StavroX by annotating more highly-charge fragments, while no improvement was observed in the case of XLPM, because XLPM already had most of the highly charged fragments (data not shown).
To construct, manipulate, and visualize the X-ray structure we used PyMOL (The PyMOL Molecular Graphics System, Version 1.3.0.0 Schrödinger, and LLC).
XLPM is implemented both as a stand-alone program, with the source code freely available, and an online tool. The online interface is user friendly and very straightforward (Figure 3).
The algorithm, comparing MS/MS spectra of precursor ion with all b ions and complementary y ions, uses b-y ion filter and is based on the observation, "Detection of a particular b ion with the charge less than that of the precursor implies a high probability of detection of the complementary y-ion with the remaining charge." As the amino acid sequences of proteins and the details of cross-linker are known, the algorithm can analyse the results without matching the entire MS/MS spectra. Matching all b ions and complementary y ions reduces the work of the algorithm considerably, making it fast and efficient.
We analysed the NIST (National Institute of Standard and Technology) (http://peptide.nist.gov/) library of annotated ion trap spectra to validate the b-y filter. For an MS/MS spectrum of each precursor, b-ions having charges less than that of the corresponding precursor ion were identified, and the spectrum was then scanned for the complementary y ions with the remaining charge. The ratio of the number of such y ions to the number of b ions was calculated for the spectrum of each precursor ion. All the ratios were plotted separately for each of the charges; two, three, four, five and higher.
The graphs in Figure 4 show the histograms of the above mentioned ratios for precursors with charges two, three, four, five and higher. As the distributions of the ratios are non-zero, the probability of the complementary y ion with the remaining charge being present when a b ion is present is non-zero, which supports the observation. In addition, the histograms show a negative correlation with the charge. As the charge increases, the distribution shifts to the left (the ratio moves towards zero).
Histograms of complementary y ions with remaining charge to b ions ratio derived from NIST data set of annotated peptides. The number in the chart is the median value of the distribution.
Rim1 tetramer was cross-linked using DSS, a 11.4A-long cross-linker, cross-linking primary amino groups. Figure 5, left panel shows the migration of protein bands corresponding to Rim1 tetramer (~60 kDa), Rim1 dimer (~30 kDa), and monomer (~15 kDa) on the gel. The mass spectra were analysed using XLPM and StavroX and the feasibility of the cross-links was assessed by mapping of the cross-links onto the available Rim1 X-ray structure. The monomer band did not yield any cross-links. The dimer and tetramer bands showed a similar pattern of interactions. Mainly, four lysines - 29, 64, 86, 105 - were found to be involved in various cross-links. Furthermore, lysine 105 cross-linked with lysine 64 was the highest scoring cross-linked pair in the dimer. A cross-link between this pair was also present in the tetramer band. Cross-linking of lysine 29 with lysine 86 was also a prominent interaction found by both XLPM and StavroX. False discovery rate was controlled by search against database based of reverse Rim1 sequence. With the XLPM found 33 and 18 cross-linked pairs from tetramer and dimer of Rim1 with better than 5% FDR . Similar cross-linking sites were found by StavroX. Tables 1, 2, 3, 4 provide summary of StavroX and XLPM-identified cross-links. The major difference between StavroX and XLPM results were cross-linking between C terminal beta sheets in StavroX. However, the rigid beta sheet structure at C terminal in the Rim1 makes it improbable site for DSS cross-linking, DSS being a cross-linker with long spacer arm (11.4 Ă) (Additional file 2A), making cross-linking between any pair of lysines 95,101 and 104 unlikely. In addition, four or higher charge is expected of cross-linked species. Most of the cross-link pairs involving lysines 95, 101, 104 identified by StavroX have charge less than four, decreasing the feasibility of these cross-links (as we expect such species to ionize at charge 4+ and higher). While, all the cross-linked pairs identified by XLPM have been of charge four and higher. Figure 6 shows one of the fragment spectra, from DSS-cross-linked dataset, annotated by XLPM. The x-ray crystal structure for Rim1 is solved for residues 2-104. Additional file 3 depicts the highest scoring cross-link determined by XLPM between lysines 64 and lysines 105. The red circle shows possible position of lysine 105 (as this lysine is absent from in the crystal structure). Additional file 4 shows another predicted cross-link pair of lysine 29 and lysine 86. Furthermore, a cross-link between lysine 105 and N-terminal is also plausible as predicted by both XLPM and StavroX.
Products of cross-linked Rim1 tetramer resolved on SDS-PAGE. Left panel - results of the DSS cross-linking. Right panel - results of SDA cross-linking. 60kDA band corresponds to fully assembled Rim1 tetramer. 45 kDa band - to trimer, 30 kDa - to dimer, and the 15kDa band - to monomer.
A representative MS/MS spectra derived from DSS-cross-linked peptide annotated by XLPM. The header in the graph depicts the scan number in the spectra. Fragment sequence 1: KGALVYVEADAANYVFERDDGSKGTTLSLVQK. Fragment sequence 2: YLKYSIASQPRR. Mass to charge: 1013.544006. Charge: 5+.
While the false discovery rate of finding a cross-linked peptide pairs can be estimated using decoy databases, it is not apparent how to estimate the false positive rate of the cross-link site assignment within already identified peptide pair. In this work, for the first time, we proposed the relaxed specificity approach: in analysing the false positive rate of cross-linked site assignment a specific cross-linker (e.g. Lys-to-Lys cross-linker DSS) is treated as non-specific (e.g. Lys-to-any) by XLPM. Any sites of cross-linked attachment outside the range of known cross-linker's specificity found in such way are considered false-positives. Figure shows such ROC curves. Figure A shows ROC curve when no filter is applied on the XLPM score, considering all positive scored results as positives. Figure B shows ROC curve for the results at 5% FDR (Figure 7).
ROC curves of the DSS analysis. The specificity of DSS at the second end was removed. A, No score cutoff, AUC: 0.6727.B, Score cutoff at 5% FDR, AUC: 0.9066.
SDA cross-linker connects lysine at one end to any amino acid at the other. We developed XLPM to include non-specific analysis with no limitations. Rim1 tetramer was cross-linked using the SDA (Figure 5, right panel ) and the cross-linked products were analysed using XLPM. XLPM successfully identified nine cross-linked candidates in each of the two bands. This result illustrates the ability of XLPM not only to identify the peptides cross-linked using a non-specific cross-linker, but also XLPM's ability to distinguish between the sites of cross-linking. Two confident sites were identified by XLPM from the SDA-cross-linked spectra: N-terminus MDFSK to YLKSIAPQR peptide (Additional file 5), and N-terminus MDFSK to C-terminal peptide LEADAEGQENAASSEHHHHHH.
To investigate a possibility of Rim1 tetramers interacting with each other, we prepared separately 14N-labeled Rim1 tetramer and 15N-labeled Rim1 tetramer, mixed them at 1:1 ratio and subjected to the cross-linking analysis using SDA and XLPM. Multiple spectra were identified corresponding to the N-terminal MDFSK peptide cross-linked to C-terminal LEDAEGQENAASSEHHHHHH peptide. Cross-linked species, where one peptide chain is 15N-labeled and the other is 14N-labeled were clearly visible. Figure 8 shows the most visible 15N-14N interaction, verified and annotated by PEAKS v7 software suit. The spectra were analyzed in PEAKS with MDFSK peptide specified as variable modification to validate the results of XLPM. In contrast, the other site of SDA-cross-ling MDFSK to YLKSAIPQR did not show mixed 14N-15N cross-linked - only 14N-14N and 15N-15N species were observed. These results clearly indicate that the N-terminus to C-terminus SDA-derived cross-link arises due to interaction between different subunits of Rim1, either from direct interactions between Rim1 tetramers, or from dissociation and reassembly of Rim1 tetramers.
N-terminus to C-terminus interaction occurs between different subunits of Rim1. The spectra consisting of 14N-labeled C-terminus and 15N-labeled N-terminus is shown. After identification with XLPM, the result was verified independently with PEAKS v7, encoding 15N-labeled peptide, MFDSK as variable modification. The spectrum as preprocessed and annotate by PEAKS is shown.
The b-y filter implemented in the current version of XLPM, makes the cross-link identification considerably faster compared to existing algorithms. This is because only a sub-set of fragment ions is calculated in theoretical spectra and considered for identification. Even for linear peptides, using the analysis of the set of NIST MS/MS spectra, we showed that there is a high correlation between the presence of a b-ion and the presence of the complementary y-ion in the same spectrum. This is in line with the observations by Frank et. al. that b and y ions are highly correlated in doubly charged peptides . Furthermore, Tabb et. al. noted that y-ions are usually higher in intensity than the complementary b ions, in the case of tryptic peptides. Therefore, if a b-ion is observed in an MS/MS spectrum, it is highly likely that the complimentary y-ion will also be observed. While it is certainly true that having a complimentary set of b and y ions is not a requirement for peptide identification, what is likely - is that when such condition occurs it indicates that the MS/MS spectrum is of very high quality, leading to a high-scoring identification. The b-y filter, therefore, could be thought as a quality filter. Our analysis of NIST data suggests that even a regular data-base search of a proteomics dataset may benefit from such a filter. We also expect that for the non-linear, cross-linked peptides, the b-y filter should perform just as well as for linear peptides. Indeed, a fragmentation spectrum of a regular linear peptide precursor in a typical experiment using collisional excitation, conforms to a "mobile proton model": when peptide is collisionally excited, a proton from its protonated N-terminus is free to travel along the peptide backbone and to form intermediates with protonated amide nitrogens, with protons on the side chains of arginines and lysins (e,g, in the case of tryptic ends) remaining immobile. For the most type of cross-linkers it is safe to assume that mobile protons will not jump through the cross-link and will remain localized to their original peptide chains. We should therefore, expect that the fragmentation spectrum of a typical charge 4+ cross-linked pair of tryptic peptides will resemble a sum of fragment spectra of individual charge 2+ peptides, as opposed to a fragment spectrum of a charge 4+ linear peptide. The b-y filter could be easily modified, if needed, to accommodate different types of experiments - e.g. when intensity of b-ions is enhanced or suppressed through modification of peptide N-termini. It can also be easily implemented as a c-z filter for spectra obtained using electron transfer dissociation; however, we have not yet tested its effectiveness in this setting due to the lack of standard annotated datasets of ETD spectra.
XLPM also does better job at annotating higher-charged fragments compared to other cross-linked identification algorithms. Partially, this is due to b-y filter, as the XLPM searches for a complimentary y-ion having a charge such, that the sum of charges is equal to the charge of the precursor. In fact we have not found much improvement in XLPM performance upon deconvolution of highly-charged fragments into 1+ fragments (data not shown).
XLPM performs well, without sacrificing much of the analysis speed, when one of the cross-linker's functionality is non-specific and cross-linking at any amino-acid is possible. Finding cross-linked peptides in mass spectra is similar to finding a needle in a haystack. The mass spectra contain majority of non cross-linked peptides and a small number of cross-linked peptides. Target-decoy method of finding FDR has been widely acceptable in protein mass spectrometry data analysis. However, this method should be used with extra caution in cross-linking mass spectra analysis. As the expected correct matches of cross-linked peptides with the mass spectra are small, few hits from decoy may shoot up the FDR significantly. By putting stringent FDR requirements may eliminate some of the matches as false negatives.
In case of XLPM, we have used target-decoy method to calculate FDR and successfully validated the results. However, target and decoy sequences were analyzed separately, which may have boosted the score of some decoy sequences. In creating the database from protein sequences, XLPM removes database entries that do not contain any site of cross-linking, contains only one possible site of cross-linking at the N terminal of the digested fragment (forcing the miss cleavage), and digested fragments less than three amino acids. These filters decrease the difference in the characteristics of the target and decoy database. Thus, use of FDR requires caution in case of cross-linking analysis.
To validate XLPM's effectiveness in cross-linking assignment, we plotted the ROC curves with relaxed specificity of cross-linker at the second end. We plotted the ROC curves without any cut-off and 5% FDR. With the improvement in FDR, the area under curve for the ROC curve was also increased. Thus, relaxed specificity analysis can be an effective method for validation of cross-linking assignment and its usefulness at least as an auxiliary tool to the target-decoy method can not be ruled out.
XLPM is a fast and reliable algorithm for the identification of cross-linked peptide pairs. In XLPM we implemented, validated, and demonstrated utility of the unique spectral filter, which is based on the expected ratio of y-to-b ions in the fragment spectra. Furthermore, XLPM performs well in identification of the specific sites of the cross-linking, as demonstrated by the relaxed specificity ROC analysis.
The work presented in this paper is supported by NIH Grant R01 GM098922 (to Kevin Raney), and by a UAMS Cancer Pilot Grant (to Boris Zybailov), the Arkansas IDeA Network for Biomedical Research Excellence P20GM103429 (to the UAMS Proteomics core), the UA Center for Protein Structure and Function (P30GM103450), and the UAMS Center for Microbial Pathogenesis and Host Inflammatory Responses (P20GM103625), Arkansas Tobacco Settlement grant and the INBRE grants NCRR (5P20RR016460-11) and NIGMS (8 P20 GM103429-11) from NIH. We acknowledge NIH R01 grant GM098922 for the funding of the publication of this article.
XLPM is available to use on our web server at http://binf-app.host.ualr.edu/~mihir/cgi-bin/xlpm.cgi. Web server has limited options. However, if a user wants to use some other digestion enzyme, cross-linker or amino acids modifications, the user can email us with the details and we will update the web server to include the additional enzyme, cross-linker or amino acid modification. We also provide Perl code and MySQL dumps of XLPM upon request. One can customize the scripts and/or MySQL database to perform specific analysis. The code works with wide variety of enzymes, cross-linkers and amino acid modifications.
We thank Dr. K.I. Varughese for providing the coordinates for the x-ray crystal structure of Rim1.
Mihir Jaiswal and Nathaniel Mark Crabtree wrote the bulk of the code. Mihir Jaiswal wrote the manuscript, supervised XLPM development, performed the computational analyses and validations. Mihir Jaiswal also proposed and validated the y-to-b ratio filter for the purpose of the cross-link analysis. Michael A. Bauer participated in the web-site development and the implementation of the online access. Roger Hall consulted the XLPM team and provided additional software development support. Boris Zybailov performed the cross-linking experiments and proteomics analysis of cross-linked products. Boris Zybailov and Kevin Raney designed the study and participated in the data analysis and interpretation.