A senior Israeli official on Thursday reportedly criticized US President Donald Trump for appearing to give Iran free rein to further entrench militarily in Syria.

The US president, on Wednesday, said of Iranian forces in Syria: “They can do what they want there, frankly,” while suggesting Tehran was removing its troops from the country.

“It is sad that he is not attentive to intelligence materials,” the unnamed Israeli source said, according to a Thursday report by the Ynet news website.

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“I am quite simply in shock,” the source continued. “Trump simply does not know what is happening in Syria and the Iranian entrenchment there.”

Trump’s comments came two weeks after he rattled Jerusalem by announcing that he would pull all American troops out of Syria. US soldiers had been leading the coalition against the Islamic State terror group, while also helping to thwart the establishment of permanent Iranian military infrastructure in Syria.

Israel has repeatedly warned in recent years that Iran is seeking to establish a military presence in Syria, where it is fighting alongside its Lebanese proxy Hezbollah and Russia to restore the regime of Syrian President Bashar Assad.

Israeli officials have also warned that America’s absence would open the door for Tehran to create a so-called “land bridge” from Iran, through Iraq and Syria, into Lebanon and to the Mediterranean Sea.

Over the last several years, Israel has carried out hundreds of airstrikes in Syria against targets linked to Iran.

Yet Trump, on Wednesday, said at a cabinet meeting that Tehran, like the US, was withdrawing its forces from Syria.

The American president went on to say that in pulling out of the nuclear deal with Iran last year, Washington had changed Tehran’s calculus and stymied its efforts to destabilize the region.

“Iran is no longer the same country,” he said. “Iran is pulling people out of Syria. They can do what they want there, frankly, but they’re pulling people out. They’re pulling people out of Yemen. Iran wants to survive now.”

Trump’s decision to pull America’s 2,000 troops out from Syria caused a major shakeup within his own administration; his secretary of defense, James Mattis, resigned over the withdrawal.

Trump offered a stark take on the situation in Syria Wednesday, summing it up in two words — “sand and death” — while remaining vague about the timing of the US troop withdrawal.

“So Syria was lost long ago. It was lost long ago. And besides that, I don’t want — we’re talking about sand and death. That’s what we’re talking about,” Trump said during a cabinet meeting. “We’re not talking about vast wealth. We’re talking about sand and death.”

On when US forces would leave Syria, Trump said: “I don’t want to be in Syria forever.”

He added: “I never said we are getting out overnight… We’re withdrawing… over a period of time.”

The US president’s announcement of the Syrian withdrawal was the first significant point of contention between Washington and Jerusalem since he took office — Prime Minister Benjamin Netanyahu reportedly pleaded with him to rethink the decision — and has fortified the perception that he views the US relationship with Israel as transactional.

On Tuesday, US Secretary of State Mike Pompeo told Netanyahu that the planned withdrawal of US ground forces from Syria will not alter America’s commitment to countering Iranian aggression and maintaining Israel’s security.

“The decision by the president on Syria in no way changes anything that this administration is working on alongside Israel,” Pompeo said at a joint press conference with Netanyahu before they held talks in Brazil.

Trump said last week that he did not think America’s removing its troops from Syria would endanger Israel.

Agencies contributed to this report.


Souhila Abdelkader

Souhila Abdelkader (born 21 April 1978) is an Algerian team handball goalkeeper. She plays for the club GS Pétroliers, and on the Algerian national team. She competed at the 2013 World Women's Handball Championship in Serbia, where Algeria placed 22nd.

References

Category:1978 births
Category:Living people
Category:Algerian female handball players


Imaging connected porosity of crystalline rock by contrast agent-aided X-ray microtomography and scanning electron microscopy.
We set out to study connected porosity of crystalline rock using X-ray microtomography and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) with caesium chloride as a contrast agent. Caesium is an important radionuclide regarding the final deposition of nuclear waste and also forms dense phases that can be readily distinguished by X-ray microtomography and SEM-EDS. Six samples from two sites, Olkiluoto (Finland) and Grimsel (Switzerland), where transport properties of crystalline rock are being studied in situ, were investigated using X-ray microtomography and SEM-EDS. The samples were imaged with X-ray microtomography, immersed in a saturated caesium chloride (CsCl) solution for 141, 249 and 365 days and imaged again with X-ray microtomography. CsCl inside the samples was successfully detected with X-ray microtomography and it had completely penetrated all six samples. SEM-EDS elemental mapping was used to study the location of caesium in the samples in detail with quantitative mineral information. Precipitated CsCl was found in the connected pore space in Olkiluoto veined gneiss and in lesser amounts in Grimsel granodiorite. Only a very small amount of precipitated CsCl was observed in the Grimsel granodiorite samples. In Olkiluoto veined gneiss caesium was found in pinitised areas of cordierite grains. In the pinitised areas caesium was found in notable excess compared to chloride, possibly due to the combination of small pore size and negatively charged surfaces. In addition, elevated concentrations of caesium were found in kaolinite and sphalerite phases. The findings concerning the location of CsCl were congruent with X-ray microtomography.


Evaluation of glycoprotein gII ISCOMs subunit vaccine for pseudorabies in pig.
One of the pseudorabies virus glycoproteins, gII, was integrated into Quil A matrix and formed an antigenic presentation structure known as immunostimulating complexes (ISCOMs). The gII ISCOMs protected mice from a lethal infection of pseudorabies virus (PrV). Subsequently, the efficacy of gII ISCOMs as a vaccine was evaluated in pigs, a natural host species of PrV. Immunization of pigs with gII ISCOMs protected them from PrV-induced acute symptoms and death. The serum obtained from immunized pigs was clearly discriminated from that of infected pigs by immunoprecipitation. The data indicated that the ISCOM technique was useful in PrV subunit vaccine preparation and demonstrated the importance of glycoprotein gII as a component of PrV vaccine.


//
//  LYNavWeChatPopAnimator.h
//  LYCustomTransitionDemo
//
//  Created by liyang on 2017/2/22.
//  Copyright © 2017年 liyang. All rights reserved.
//

#import <UIKit/UIKit.h>

@interface LYNavWeChatPopAnimator : NSObject<UIViewControllerAnimatedTransitioning>

@property (nonatomic, strong) UIImageView *transitionImgView;

@property (nonatomic, assign) CGRect transitionBeforeImgFrame;  //转场前图片的frame

@property (nonatomic, assign) CGRect transitionAfterImgFrame;   //转场后图片的frame

@end


People complain about negative advertising, but it never goes away. That's because it works.

PayPal has taken this conventional wisdom to heart, launching an attack ad against its latest and most formidable competitor, Apple Pay, before the new service even lands in the hands of consumers. Appearing in multiple newspapers, the full-page spread is meant to sow distrust in Apple by reminding the world of this month's massive iCloud celebrity selfie hack.

PayPal

The ad may or may not be fair. It might not even have the desired effect. But it indisputably shows what happens when Apple muscles into a new market: the incumbents immediately become the underdogs. And how they react becomes a measure of how powerful Apple really is. In the case of the payments business, PayPal has clear reason to be worried.

When Microsoft made Internet Explorer the default Windows web browser, it quickly shot to the top of the list of most popular browsers, where it stayed for years, despite the existence of other, arguably better browsers that came before it. Apple has the power to do the same for payments with Apple Pay, but with notable differences that put it in an even better position than Microsoft. First, Apple controls the hardware as well as the software. Second, Apple's demonstrated design chops suggest a company that will never let its products sink into the kind of user-experience decrepitude that sank IE.

The analogy also breaks down a bit around the sheer duration of PayPal's incumbency. PayPal has been synonymous with online payments for more than a decade, while the web was still in its infancy when IE came out. Integrating Apple Pay across the online and offline retail worlds will take time. PayPal users wouldn't be able to make any kind of full-time switch even if they wanted to go all-Apple. PayPal has reason to be hopeful. But in the long run, the threat is real.

Apple Isn't the Other

If any company has the power to hasten its own ubiquity, it's Apple. Apple Pay will spread quickly once the iPhone 6 and iOS 8 are released this week. Even PayPal's own Braintree division has released a way for developers to integrate Apple Pay into their online apps. And Apple is restricting the use of the iPhone 6's NFC chip to Apple Pay, which means PayPal won't be able to take advantage of the new phones' power to act as in-store credit card substitutes.

Those factors leave PayPal with little choice but to seek to undermine Apple's advantages through marketing. The new PayPal ad tries to sound a populist note: "We the people want our money safer than our selfies." Aiming at Apple's apparent negligence in locking down iCloud is the obvious target. If Apple can't be trusted with photos, can it really be trusted with credit card numbers?

A crucial difference, however, appears to be that Apple Pay's tokenization system doesn't appear to store a user's credit card number at all, locally or online, once that card is scanned. If nothing else, that makes Apple Pay more secure than physical credit cards themselves.

Even granting that Apple has yet to make up the trust deficit the iCloud hack incurred, however, portraying Apple as the overlord aligned against the "people" seems unlikely to succeed. In polls, Apple consistently ranks near the top among the world's most trusted and admired brands. And Apple users relate to their devices. However much control they really have, people who carry their iPhones everywhere they go feel like the devices belong to them. Apple isn't the Other. Apple is what's in your pocket, a digital extension of yourself.

The Omnivore Dilemma

That closeness users feel to Apple hints at a broader problem for PayPal: it's a niche platform, while Apple is an omnivore. Apple has the power to try to absorb nearly any function or feature into itself. To escape Apple's predations, PayPal could make itself another omnivore's prey.

Last week, trading in PayPal parent company eBay's shares spiked on rumors Google planned to buy a major stake in the company, a rumor eBay quickly shot down. But a Google acquisition might not be such a bad thing for PayPal, especially if Google decided to make it the Apple Pay of Android. In mobile, Google's reach is much broader than Apple's, if less focused. And for Google, PayPal is already a much more recognized brand than any of Google's own payment efforts.

Going with Google, another corporate behemoth, wouldn't exactly make PayPal look more populist. But when you're up against Apple, it's one of the moves most likely to help you stay popular.


46,XY individuals born with conditions of intersexuality can be reared as either male or female, depending upon an individual's medical, familial and cultural situation. Currently, very little information exists on the long-term outcome of such patients. The goal of the present study is to determine the endocrinological, surgical and psychological outcome of adult 46,XY intersex patients. Specifically, the question of whether it is optimal to raise such individuals as males or females will be investigated. Subjects include all 46,XY intersex patients treated in the Johns Hopkins Pediatric Endocrine Clinic over the past fifty years. Only subjects 21 years and older are eligible to participate. Subjects are first asked to complete a Life Inventory Survey to assess their adult adjustment to their condition. Then, subjects are asked to participate in a urological/gynecological and endocrine exam.


Amphiphilic p-sulfonatocalix[4]arene-coated CdSe/ZnS quantum dots for the optical detection of the neurotransmitter acetylcholine.
Water-soluble CdSe/ZnS (core-shell) semiconductor quantum dots surface-modified with tetrahexyl ether derivatives of p-sulfonatocalix[4]arene were synthesized for the optical detection of the neurotransmitter acetylcholine.


Changing an ether to a ketone within the framework of a bis-urea macrocycle has little effect on the supramolecular assembly of this building block into porous crystals but introduces a triplet sensitizer into the framework that dramatically alters the photochemical reactions of included guests.


The Marine Strategy Framework Directive and the ecosystem-based approach – pitfalls and solutions.
The European Marine Strategy Framework Directive aims at good environmental status (GES) in marine waters, following an ecosystem-based approach, focused on 11 descriptors related to ecosystem features, human drivers and pressures. Furthermore, 29 subordinate criteria and 56 attributes are detailed in an EU Commission Decision. The analysis of the Decision and the associated operational indicators revealed ambiguity in the use of terms, such as indicator, impact and habitat and considerable overlap of indicators assigned to various descriptors and criteria. We suggest re-arrangement and elimination of redundant criteria and attributes avoiding double counting in the subsequent indicator synthesis, a clear distinction between pressure and state descriptors and addition of criteria on ecosystem services and functioning. Moreover, we suggest the precautionary principle should be followed for the management of pressures and an evidence-based approach for monitoring state as well as reaching and maintaining GES.


In a surprising move, the Kansas state Senate Republican leadership announced on Friday that it was killing – at least, in its current form – a controversial religious liberty measure, harpooned for opening the door to broad anti-gay discrimination.

House Bill 2453, as drafted and passed by the Republican-controlled state House of Representatives this week, would give any individual, business, group, or government official the right to deny same-sex couples a host of basic goods, services, benefits, or employment – on the grounds of a conflicting “sincerely held religious belief.” Lawmakers in the state House approved the measure on Wednesday by a vote of 72-49.

The bill was widely expected to pass the state Senate, too, as Republicans outnumber Democrats 32-8 in that chamber. But Republican state Sen. Susan Wagle, the Senate’s president, poured cold water on its prospects Thursday night when she released a statement saying that a majority of her caucus opposed the bill’s potential green light to discriminate. On Friday, she reaffirmed that position.

“I believe that when you hire police officers or a fireman that they have no choice in who they serve,” Wagle said at a news conference Friday, according to the Wichita Eagle. “They serve anyone who’s vulnerable, any age, any race, any sexual orientation.”

“Public service needs to remain public service for the entire public,” she added.

Read more: Religious liberty bill opens door for LGBT discrimination

One of the strongest criticisms of the bill was that it gave state employees license to ignore legally valid same-sex marriages. Opponents feared officials could cite the law’s protections in refusing to intervene in a domestic violence dispute between a gay couple, for example, or in denying a gay couple a marriage license, should a judge overturn the state’s 2005 voter-approved ban on gay marriage.

Supporters insisted the purpose of the measure was to ensure the free exercise of religion, and pointed to the bill’s stipulation that an employer find another employee to perform a service if one refused on religious grounds.

Wagle said that provision would increase the cost of doing business and place a burden on employers.

“I believe the intent of the House was to protect religious liberties. We respect that,” she said, according to the Associated Press. “But the business implications are going to harm the practice of employment in Kansas.”

As the bill began to pick up steam, it fueled a chorus of growing opposition. The Kansas Chamber of Commerce and the newly formed Kansas Employers for Liberty Coalition released statements saying that the bill posed legal problems for the business community, and that it would strain employer-employee relationships. And a Facebook page titled “Stop Kansas House Bill 2453” has netted more than 50,000 “likes” as of Friday evening.

“Political pages here, if they get 1,000 ‘likes,’ they’re pretty successful,” Thomas Witt, executive director of the Kansas Equality Coalition, told msnbc. “This has exploded in this state. And it blew up in their faces.”

At an afternoon press conference Friday, state House Speaker Ray Merrick said that if the vote were held again, it probably wouldn’t pass. He promised to work with the Senate until they reached a consensus on what to do with the bill.


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Is There Emancipatory Potential in Israeli Municipal Politics?: An Interview with Matan Kaminer

By Max Ajl, Jadaliyya
December 03, 2013

Max Ajl: Start by telling us a little about the Ir Lekulanu campaign.

Matan Kaminer: IrLeKulanu, “city for all of us” in Hebrew, is a municipal political party in Tel Aviv-Jaffa. It was founded six years ago by a number of activists who had been involved in different local struggles. Among the groups represented at the founding conference were the Palestinian community in Jaffa and the working-class Mizrahi Jewish community in Kfar Shalem – formerly Salameh – both of which were facing eviction from homes they had been settled in after 1948; residents in the centre of town who were fighting for a public lot to be turned into a park rather than residential towers, a group struggling against privatization of the seashore, and more.

During the previous election, in 2008, our candidate for mayor was Dov Khenin, a member of parliament from the non-Zionist left Democratic Front for Peace and Equality (Hadash/al-Jabhah)*. Khenin won 35% of the vote and we got 5 seats in the city council, which was a huge and surprising success. This time, our candidate was Aharon Maduel, who has led the struggle in Kfar Shalem for two decades. There was also another candidate for mayor, Nitzan Horovitz of the left-Zionist party Meretz. Results were less encouraging than last time: Maduel got 9% of the vote for mayor (with 38% for Horovitz and 53% for Huldai), and we garnered 6% of the vote and three seats (out of 31) in the city council.

MA: Aharon Maduel is a Mizrahi candidate linked to Likud. This is an interesting political position– can you say more about it?

Ir Lekulanu is not formally linked to any national political party and it includes the range of political views at the national level. There is a strong contingent of non-Zionist leftists like me, and there is a group from the South of Tel Aviv, many of whom are Likud members or sympathizers at the national level – but have a deep animosity towards the local Likud leadership in Tel Aviv. In addition, this time we were joined by the Jaffa section of Hadash, which is almost exclusively Palestinian, and its representative Amir Badran was number four on our list.

What makes this coalition possible is that Ir Lekulanu doesn’t deal with issues at what is called the “state-political”(medini) level– that is, it doesn’t have a position on the occupation and other issues that are decided at the national level. Our platform and programme don’t deal with anything beyond the city limits.

I want to emphasize that Ir Lekulanu ran against the Likud in Tel Aviv-Jaffa, and helped to contribute to its pathetic showing – only one city council seat, for the party governing the country! The Likud ran a racist campaign promising to “silence the Muezzin” in Jaffa, which we strongly condemned; and then they smeared Aharon, printing copies of our leaflets in Arabic and handing them out in the South, with a gloss warning voters that Ir Lekulanu was running with “the Islamic movement”.

What was the role of Hadash in this campaign?

The role played by Hadash as such was more minor than last time. Hadash is a front, in which the Communist Party of Israel and Tarabut** are two components. One can speak of two major camps within Ir Lekulanu – the CPI camp and the camp formed by Tarabut and the Southerners around Maduel. After Maduel was elected chairman and the city council list was chosen, it was clear that the Southern camp had gained the upper hand. At this point the CPI chose to focus all their efforts and energies on ensuring that the representative of Hadash-Jaffa would be included in the list – a worthy target, of course, but in so doing they unfortunately marginalized themselves. Tarabut remains very influential –number two on the list is Yael Ben-Yefet, a leading member of Tarabut as well as chairwoman of the “Mizrahi Democratic Rainbow”.

It is worth mentioning that this election has been disastrous for Hadash across the country. The municipality of Nazareth, which Hadash has held since the 1970s, fell to an apolitical local boss, ‘Ali Salam. I have been a member of Hadash for about fifteen years, and it seems to me that the front is at a watershed. The current leadership is sclerotic and conservative and has fought off change for many years.There has to be a major internal shakeup in Hadash. If this doesn’t happen, I don’t think it has much of a future left.

You have written that “municipal political party IrLeKulanu is not considered “left” in Israeli terms, it embodies one of the greatest successes of the non-Zionist left in Israeli history.” Can you expand on what that means? By “Left” do you mean its socio-economic stance or its position on the colonial question?

Yes, that was before the election and perhaps I would not be so triumphal about it today. In Israel “left” and “right” are used exclusively to refer to positions on the conflict, and that’s what I was referring to when I mentioned “Israeli terms.” Of course I reject such a limited definition, and for me a consistent left position is necessarily both anti-capitalist and anti-colonialist. Ir Lekulanu was, and is, such an important project because it unites Palestinians with Jews of both middle-class, Ashkenazi backgrounds and working-class, Mizrahi backgrounds, and in this it is totally unique. I don’t claim that Ir Lekulanu is itself anti-colonial; that would be absurd, given that its current head is a Zionist. I do claim that the strong position held by radicals, and now also by Palestinians, within the movement is a major achievement that should be consolidated.

Yemeni Jews – Mizrahi – are at the bottom of the Jewish Israeli class structure but often reluctant to work with Arabs because ‘their only privilege is their Jewishness’.

You have also written, “the radical leftists who form an important component of IrLeKulanu should not pass up the opportunity to throw their weight behind a Mizrahi urban leadership in coalition with Palestinians, Ashkenazis, refugees and migrants.” Does this risk subordinating the colonial question to internal class issues?

The risk exists; it always does. There are a lot of internal tensions within Ir Lekulanu that can be traced to this problem. An example is the issue of the African asylum-seekers whom the state has channeled into two or three neighbourhoods in South Tel Aviv over the last two years. These neighbourhoods, which were already underprivileged, have had to absorb about 30,000 new residents. Our position as a party is that the state has to take responsibility for the asylum-seekers; we don’t take a position on whether this means accepting or rejecting their claims.

Ibrahim, a refugee from Sudan, holds a document given to him by the UN in Egypt, as he takes part in a protest held by refugees and activists outside the UN offices in Tel Aviv, June 10, 2012. Photo by Oren Ziv/Activestills.org

But at a more fundamental level, I don’t see how the “colonial question” and “internal issues” are separable, such that we can speak of one being “subordinated” to the other. This separation reinforces a naïve theory that puts Israelis on a continuum from those “nearer” to a good position on the conflict (the Zionist left) to those“farther”(the right). This theory supposes that those on the left, which the Communist Party used to call “the democratic forces”, can be won over to the anti-colonial cause. The strategy based on it has proven completely inefficacious, over and over. Election results in Tel Aviv are a good example: 44,000 Tel Avivis voted for Khenin for mayor – most of them belonging to the stratum traditionally represented by the Zionist left. In the next national elections, where voting for Khenin would have meant voting for Hadash, an explicitly non-Zionist movement led by Arabs, only about 10% of those voters (4,000) did so. In fact, even among those Jews who have the least to lose – those who have class and ethnic privileges in addition to their national Jewish privilege – it seems that only a tiny minority are willing to step over the line and reject Zionism.

When that is the case, it would be hypocritical to expect those whose only privilege is their Jewishness – those mostly Mizrahi Jews with no property, little cultural capital, no European passport – to be the first to give up Zionism. But even the meagre privilege that being Jewish affords this group is being rapidly eroded. The fact that Shas, which represents this constituency, has been locked out of government by a middle-upper-class alliance uniting the right (Naftali Bennett) and “centre-left” (Yair Lapid), is decisive. What will happen when the Jewish privilege of disenfranchised Israeli Jews disappears entirely? Will they turn to the anti-colonial cause, or to fascist reaction? The answer to this question is not foreordained. Anti-colonial forces must have a foothold in this community. Many Palestinian-Israeli activists are in agreement with me on this point.

What kind of reception did the campaign receive amongst anti-Zionists? And can you say more about Maduel’s position on Palestinian issues?

Anti- or non-Zionists in Tel Aviv-Jaffa were split during this election. The Islamic Movement did not participate in it at all; the National Democratic Union (Balad/Tajama’u) ran under the name “Yaffa”; both the Jaffa and Tel Aviv sections of Hadash supported Ir Lekulanu. Most of the anti-Zionist criticism came from the Balad quarter. They accused us of splitting the Palestinian community in Jaffa and preventing its representation in the city council. But we didn’t split Yaffa; it split a few months before we were involved, and Hadash-Jaffa approached us and asked to enter Ir Lekulanu.

Maduel has been to Jaffa very often in the past five years and has always upheld the right of the Palestinian community there to individual and collective sumoud***, though not in those terms of course. People in Jaffa are aware of this; even some activists for Balad, which did not officially endorse any candidate for mayor and supposedly reject Zionist candidates, told me they were voting for him.

Maduel did not do very well. Can you give some reasons for this?

Ir Lekulanu began as an alliance between middle-class, young Ashkenazi Jews from central Tel Aviv and working-class Mizrahi families from the south of the city. In the previous campaign, the first group was the senior partner. This time, after Maduel took over as leader, the centre of gravity shifted. It was quite fascinating to see the subtle operations of hegemony between the two campaigns: Khenin, an Ashkenazi academic and parliamentarian, found it easy to take up the mantle of the universal, representing the common interest of all groups in the alliance; and I say this to his credit. Maduel, a Mizrahi autodidact who makes a living as a home repair contractor, found the cards stacked against him. Racism and classism definitely hurt his chances.

Another huge challenge was the fact that this was a three-way race. Nitzan Horovitz is an Ashkenazi journalist and Knesset member. Whereas in 2008 Khenin was heading a unique alliance, Horovitz targeted his home constituency in a very well-funded campaign. Because we represent all sorts of constituencies, we were spread very thin all over the city, and everywhere we were vulnerable to attack: in the South, Aharon was attacked for running with leftists and Arabs and being soft on African “infiltrators”; in the centre, he was attacked as a Likudnik racist, often as a thin veil for racist attacks on him as a Mizrahi.

So what is the next move given this defeat?

I think “defeat” is too harsh a word. It’s a setback, certainly, but Ir Lekulanu has proven that it’s here to stay and that as an organization it is resilient enough to survive the change of leadership. We are the only consistently anti-neo-liberal, anti-racist movement in the city, and hopefully we will continue to attract new activists as we did during this campaign.

There will definitely have to be organizational changes, however. Compared to other municipal parties, which are funded by national parties or by rich donors, we are dirt poor. So if we want to win the next election, we have to develop deeper roots in the community so that we can bring out the vote.

Notes and links

* Hadash is an acronym for HaHazit HaDemokratit LeShalom VeLeShivion,the Democratic Front for Peace and Equality. It was founded in 1977 and is a socialist party of Arabs and Jews.


INTRODUCTION
============

Deep RNA sequencing has enabled profiling the transcriptional landscape of the cell at unprecedented resolution \[e.g. ([@gkt211-B1],[@gkt211-B2])\]. Technological advances have dramatically increased the read coverage and the dynamic range of RNA-Seq, facilitating a wide range of analyses to answer pertinent questions. One of the most fundamental analyses is comparative transcriptome analysis of samples that have been exposed to different environmental conditions or have variable genetic background. The development of computational tools to carry out such pairwise comparisons is a field of active research and the subject of this work.

For single isoform genes, the true mRNA isoform abundance is tightly coupled to the number of reads that map to exonic regions of the corresponding gene ([@gkt211-B2]). A widely used model to explain the number of mapping reads as a function of the unknown abundance is the binomial model and its Poisson limit. Several early methods have directly used such idealized statistics to test for differential expression between samples from the raw read count information \[e.g. ([@gkt211-B3],[@gkt211-B4])\]. More recent extensions ([@gkt211-B5; @gkt211-B6; @gkt211-B7; @gkt211-B8]) generalize the basic Poisson model to a more flexible class of distributions, such as negative binomial (NB) models. In contrast to Poisson-based tests, these models account for so-called overdispersion, i.e. the empirical variability of counts because of biological or technical factors.

The large majority of genes of higher eukaryotes have multiple annotated isoforms that are the result of alternative usage of transcription starts, splice sites, RNA editing sites or polyadenylation sites. Defining gene expression in the case of multiple isoforms becomes conceptually difficult and testing for differential gene expression can easily be confounded by differential RNA processing events, such as alternative splicing. In particular, the number of observed RNA-Seq reads may significantly change, even if the total number of RNA molecules remains constant. This may occur, for instance, if a significant part of the RNA molecule is excised during splicing. An alternative is to test for differential expression of an isoform in multiple samples. However, if one of the samples is subject to a significant increase of transcriptional activity of a gene, under this test, all alternative isoforms would be detected as differentially expressed.

In this work, we are interested in an alternative formulation. We seek to identify significant differences in relative isoform expression. Importantly, these relative abundances are insensitive to overall gene expression changes, but they reflect changes because of differential RNA processing. See [Figure 1](#gkt211-F1){ref-type="fig"} for an illustration. Figure 1.On the top, two transcripts are shown together with the read density one would observe if they were present isolated from each other. On the bottom, the read densities for two mixtures of the transcripts are shown. The mixture for the conditions A (light gray) and B (dark gray) is different, which is reflected by the difference of the read densities.

Recently, several algorithms for inferring the abundance of a given set of isoforms based on the observed read coverages have been proposed ([@gkt211-B9; @gkt211-B10; @gkt211-B11; @gkt211-B12; @gkt211-B13]). These approaches solve the problem of deconvolving the observed read coverage and implicitly or explicitly assigning reads to individual isoforms. The difficulty of assigning reads to isoforms comes from the fact that these are often near-identical and a read from an overlapping region cannot be assigned to a specific isoform without additional information. Perhaps the most advanced approaches are those carrying out full Bayesian inference, such as MISO ([@gkt211-B13]) or BitSeq ([@gkt211-B14]), propagating and accounting for uncertainty and covariation of expression estimates from multiple overlapping isoforms. In general, for all of these methods, the estimated abundances typically correlate well but far from perfectly with other experimental data, such as qPCR and NanoString measurements of RNA isoform abundances, in particular when many isoforms are present (A. Mortazavi, personal communication).

A natural and appealing strategy is to combine methods to estimate isoform abundance for different RNA-Seq experiments with a statistical test for differential expression of isoforms. However, the solution to the quantification problem may not be unique \[see, for instance, discussions in Lacroix *et al.* and Hiller *et al.* ([@gkt211-B15],[@gkt211-B16])\]. This problem can be partially alleviated by estimating confidence intervals for the abundance estimates, either by evaluating the Fisher information matrix ([@gkt211-B9]) or by conducting full Bayesian inference ([@gkt211-B13],[@gkt211-B14]), but the estimation of this correlation structure is technically challenging and depends on a number of assumptions that may not always be satisfied in practical settings. Most sophisticated approaches, such as characterizing the non-unique solutions using Markov Chain Monte Carlo methods ([@gkt211-B13],[@gkt211-B14]), circumvent some of these weaknesses, at the price of considerable computational cost. Further, if one is only interested in which genes or isoforms are differentially expressed, first quantifying and then testing for differential expression might be an unnecessary detour, solving a harder task than actually required.

In this work, we seek alternative strategies for detecting differential abundances of RNA isoforms in pairs of biological samples. We focus on the case where the sum of the abundances of all isoforms either remains constant or as such is irrelevant, and the abundances of the isoforms between two conditions vary ([Figure 1](#gkt211-F1){ref-type="fig"}). This setting is particularly interesting for analyzing RNA-Seq experiments with the aim to gain a deeper understanding of RNA modifying processes (such as alternative splicing or polyadenylation).

The devised approaches are simple and implemented as a single step, avoiding the need to quantify isoform abundances first. Importantly, they operate on the level of isoforms and are not restricted to differences on the level of the overall expression of a given gene. First, we propose a test called rDiff.parametric, extending established Poisson and NB-based tests for detecting differential expression of genes to testing for differential isoform abundance. The idea is to identify genomic regions based on the given isoform annotation that are not shared among all isoforms and detect differences in the read coverage in these informative regions (compare regions marked in light green in [Figure 2](#gkt211-F2){ref-type="fig"}a). We show how this principle can be used to build efficient statistical tests to identify regions with alternative isoform expression. Second, we propose rDiff.nonparametric, an approach that can detect differential isoform abundance without depending on any knowledge of the underlying isoform structure. To avoid the need to quantify within known regions, the approach directly assesses differences of the read mapping distribution at a predefined genomic locus. This test is especially useful for the large number of newly sequenced genomes where the gene structure is often only determined by homology to already annotated species. The parametric variant, rDiff.parametric, shares many ideas and concepts with recent work, such as DEXSeq ([@gkt211-B17]). However, DEXSeq is aimed at modeling the exon-specific abundance rather than transcripts and does not extend to settings without transcript annotation. Conceptually, the non-parametric testing approach has previously been described in Stegle et al. ([@gkt211-B18]), and related ideas have later been proposed in ([@gkt211-B19]). There the authors followed a similar idea but concentrated on counts on splice junctions in a constructed splicing graph. Importantly, their approach does not consider a variance model as used in rDiff and DEXSeq ([@gkt211-B17]) \[as well as in DESeq ([@gkt211-B7]) and edgeR ([@gkt211-B20])\]. Figure 2.(**a**) The alternative regions used by rDiff.parametric. Alternative regions are defined as regions in the genome that are not contained in all transcripts of a gene but at least one, according to the gene structure. In a second step, all regions are merged, which are in the same subgroups of transcripts, to obtain the so-called alternative regions. (**b**) Test statistic used by rDiff.nonparametric. Shown are the two read densities in the two conditions A and B and their difference in gray and the underlying gene structure in light green.

We perform a detailed simulation study to comprehensively compare rDiff.nonparametric and rDiff.parametric with existing methodology and to elucidate the strengths and limitations of the algorithms. Moreover, we illustrate the algorithms' practical use in a realistic setting of three RNA-Seq libraries from *Arabidopsis thaliana* and four libraries from *Drosophila melanogaster*. We find that the detection of alternative events is reliable and in concordance with results from RT--qPCR (reverse transcription--quantitative polymerase chain reaction), even when the gene structure is not used.

MATERIALS AND METHODS
=====================

We start by introducing the statistical read model and present a practical scheme to estimate biological variability on splicing data. Building on this description, we introduce a first statistical test that exploits complete information on the gene annotation. Finally, we provide a non-parametric variant that can be used when the isoform annotation is incomplete or missing.

Read statistics
---------------

When doing inference from read counts it is important to account for the fact that reads are generated by a random sequencing procedure. Thus, read counts should not be treated as fixed values but instead as draws from a suitable distribution to capture random fluctuation.

Previous work on differential testing of whole-gene expression established the duality of types of noise variation that are dominant in specific regimes ([@gkt211-B6; @gkt211-B7; @gkt211-B8],[@gkt211-B20]). First, read data are subject to shot noise because of the nature of sequencing data from random sampling. This noise is dominant for low read counts. Second, overdispersion because of biological variation increases the expected noise level as empirically observed between biological replicates. The first type of variance is well described by a linear relationship between mean and variances, whereas the second type is characterized by a quadratic component. Contrary to the shot noise, the effect of overdispersion is strongest for high counts. The variance caused by different barcodes or the use of different mappers for the samples also has a quadratic component and can for simplicity be considered as part of the biological variance. Here, we follow largely the approaches proposed previously ([@gkt211-B7],[@gkt211-B20]) and build on NB distributions to model the read counts. A major difference to these approaches is that we do not model the counts for gene expression but for smaller regions that are indicative of a change in relative isoform abundance. Throughout, we assume that the variance of the distribution for a given read count is a function of the expression abundance. This empirical variance function estimates the variance to be expected for different expression levels. For a detailed discussion of our statistical model we refer to [Supplemental Section S1](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1).

### Variance estimation

The estimation of biological variance is an integral building block to differentiate true differences from fluctuations caused by biological or technical variation. Let in the following *G* be the set of genes and *R* be a biological sample that consists of a set of replicates . For all genes and replicates , we assume to have an estimate of the gene expression and read counts for each region , where *J~g~* is the set of regions in gene *g*. We estimated the biological variance by using replicate data, to get the means and variances of tuples of normalized read counts in the replicates. To detect changes in the relative transcript abundances and not changes in absolute abundance, we computed a normalizing constant The normalization makes counts comparable across the replicates when having variability in gene expression (which may have different total numbers of reads).

We then computed normalized counts . For each region in gene *g*, we then estimated the mean of the normalized counts as well as their empirical variance: Finally, we performed a local regression on the set of points \[similar to the procedure proposed previously ([@gkt211-B7])\] to obtain a functional mapping *f~R~* between the empirical mean to the expected variance. This was done using the Locfit ([@gkt211-B21]) package.

Working without replicates.
---------------------------

If replicate data are not available, conservative estimates of the variance function can be obtained from between-sample fits. Following ([@gkt211-B7]), one can consider the two samples *A* and *B* as replicates to fit the variance function. If there are no differential sites, this approximation is fully legitimate, whereas in the presence of true differences, one can expect an overestimation of the variance fits, leading to a conservative approximation. Alternatively, one can use an estimated variance function from a similar sample as the ones under investigation.

Statistical testing with known gene structure
---------------------------------------------

### Defining alternative regions

Given a known and complete gene annotation, differential isoform abundance can be detected by differential comparison of a set of restricted exonic regions, denoted alternative regions in the following ([Figure 2](#gkt211-F2){ref-type="fig"}a). These regions are defined as isoform-specific loci, i.e. those positions where reads map that can only stem from a non-empty strict subset of all isoforms. Relative changes of the abundance between isoforms can in principle be only observed at those positions; hence, the remainder of exonic loci can be left aside.

To avoid explicitly solving the deconvolution problem of multiple overlapping isoforms, we grouped the alternative regions into areas that are absent or present in the same isoforms. The resulting grouped regions are the regions on which we tested for differences in relative abundance between isoform with respect to the total gene expression.

### Testing for changes

Statistical testing is carried out in each alternative region of a gene *g*. As the testing is performed for one gene at a time, we omit the index *g* for simplicity of notation. Our null hypothesis *H*~0~ is that there is no differential expression in a particular region. Formally, this corresponds to the read in intensity relative to the gene expression being the same in a region *j* for samples and , where *u* is the number of replicates in sample *A* and *v* the number of replicates in sample *B*. Under this hypothesis, the number of counts we expect to observe in sample *A* in region *j* can be calculated by the normalized mean expression *q~j~* for both samples, i.e. by averaging the normalized reads in all samples: where *r* runs over the replicates from either sample *A* and *B*, *N^r^* is the gene expression in replicate *r* and is the number of reads mapping to region *j* in replicate *r*. Using the normalized expression, we then calculated the average number of counts we expect to see under the null hypothesis as . The calculations for were analogous. The distribution under the null hypothesis was computed as follows. Let and be the rounded up average number of observed reads in a region *j*. We assumed that the observed counts are drawn from an NB distribution and , where *f~A~* is the variance function estimated for sample *A* and analogous for *f~B~*. For brevity denote by , the joint probability of observing *k* reads in sample *A* and *l* reads in sample *B*. Denote furthermore the total read counts in region *j* as . Then the *P*-value *p~j~* of the observed counts and under the null hypothesis *H*~0~ is given by: where *I~T~* is an indicator function that is 1 if *T* is true and 0 otherwise. Finally, we combined the *P*-values across regions into a genewise *P*-value of relative transcript abundance variability using a conservative Bonferroni correction ([@gkt211-B22]): We refer to this method as rDiff.parametric. Alternatively, the information as to which specific testing region is differentially expressed can be used directly, which is similar as the approach taken previously ([@gkt211-B17]).

Testing with unknown gene structure
-----------------------------------

In many cases, the gene annotation is not available; hence, alternative regions cannot be defined *a priori*. We propose an alternative strategy to test changes in the read density at the whole-genomic locus. Our approach builds on the non-parametric Maximum Mean Discrepancy (MMD) test ([@gkt211-B23],[@gkt211-B24]).

This flexible two-sample test for high-dimensional vectors is well suited for our setting, as it poses few assumptions on the distribution of the reads. The basic idea of this test applied to our setting is to represent the reads *A~g~* and *B~g~* that map to gene *g* in samples *A* and *B* in the space , where *l~g~* is the length of g. This is done by representing each read *i* in sample *r* as a vector of length *l~g~*. The entry of at the *j*th dimension is 1 if the read covers the *j*th position of the gene and 0 otherwise. In this space, the mean for the sample *r* is given by: where *K^r^* is the number of reads in the sample *r*. The difference between the two read densities *A~g~* and *B~g~* is then computed and used as the test statistic (see [Figure 3](#gkt211-F3){ref-type="fig"} for an illustration). To determine the significance of the distance *D*, this value is compared with an empirical null distribution, estimated from *T* differences between two random samples from the joint read distribution . Figure 3.Illustration of variance of the read density difference (see gray area in [Figure 2](#gkt211-F2){ref-type="fig"}b) between random samples from the null distribution. The distribution difference between two biological samples is shown as a dashed black curve, the one between two random samples when not correcting for biological variance in dark gray dashed and when correcting for biological variance in light gray dashed. The resulting *P*-value for rDiff.nonparametric corresponds to the gray area of surface, which is the fraction of random samples that have a bigger difference than the difference observed between the two conditions. For highly expressed genes, when not correcting for biological variance, the density difference between random samples converges to zero, thus leading to an unrealistically small *P*-value.

The basic MMD strategy described earlier in the text was extended in two different ways: (i) we accounted for biological variance by sampling such that the variance of the random samples was in concordance with the empirically fitted biological variance model.

This extension of the original bootstrapping procedure leads to an appropriate variance of the null distribution as illustrated in [Figure 3](#gkt211-F3){ref-type="fig"}, thereby avoiding an oversensitivity on highly expressed genes. The *P*-value was estimated by the number of times the observed difference *D* is larger than *D~t~*: . For a detailed description, see [Supplementary Section S2.1](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1). (ii) We increased the power by preferentially focusing on regions in the gene that could potentially reflect a differentially processing. We observed empirically that one can increase the power of the MMD test, when only considering genomic positions that have a lower than maximal read coverage in one of the samples. This observation can be explained by the fact that the regions with large relative coverage are unlikely to be differentially covered between samples (as this would require them to be not fully covered in at least one sample and thus cannot have a maximal coverage). Exploiting this characteristic, we perform several tests on the subsets of the positions where the coverage is below different thresholds. More specifically, we applied the MMD test on the 10% of the positions that have the lowest positive coverage to obtain a *P*-value *p*~10~. Subsequently, we repeated the procedure for 20% and so forth until we had 10 *P*-values . These *P*-values were combined, Bonferroni corrected and reported as the result of rDiff.nonparametric.

Data sets used for evaluation
-----------------------------

We considered a data set from *A. thaliana* to apply and compare the proposed methods.

For this study, *A. thaliana* Wt seedlings were grown in darkness and exposed to light for 0, 1 or 6 h. Furthermore, we used *cry1cry2* seedlings ([@gkt211-B25],[@gkt211-B26]) grown under the same conditions as the 0 h Wt seedlings.

The mRNA libraries were prepared using the Illumina mRNA-Seq 8-sample Prep kit. We sequenced 80 bp reads on the Illumina GAIIx platform using a single-end flow cell, resulting in ∼3.9 × 10^7^ reads per lane on average. In each library, a variable fraction between 86.3 and 87.4% of reads could be uniquely aligned to the genome using Palmapper ([@gkt211-B27]), resulting in an average coverage of the transcriptome of ∼51-fold per lane. For ∼24% of the reads, the best alignment obtained was a spliced alignment, i.e. it spanned an exon--exon border. Full details on the experimental design and implementation can be found in [Supplementary Section S5](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1).

### Simulation approach

In addition to empirical data, we also created two artificial data sets to have data sets with exact ground truth expression levels. We focused on the 5875 mRNA coding genes in the *A. thaliana* TAIR10 reference annotation that have at least two splice variants. Both artificial data sets consisted of samples for two different conditions with two simulated biological replicates each. To simulate a realistic extent of biological variance, we estimated the gene expression variance on experimental data described before. In a first simulated setting, we used the two samples grown with 0 h light exposition to estimate the biological variance and an additional sample from the seedlings 1 h to get realistic gene expressions for the simulated conditions. The true simulated isoform abundances were drawn from a uniform distribution, and the absolute gene expression abundance was drawn from the expressions measurements. For half of the genes, we simulated a differential relative isoform expression. Furthermore, we simulated a biological variance in both samples by drawing isoform abundances such that the resulting variance of the reads matched the estimated biological variance.

To assess the effect and relevance of biological variance, we repeated the same simulation procedure with increased biological variability. In this second simulated data set, we considered the variation between the samples at 0 h and 1 h to simulate the biological variance. Full details of the read simulation can be found in [Supplementary Section S3](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1).

### False discovery rate estimation

As a measure of the genome wide significance of the findings, we used the false discovery rate (FDR). The FDR was calculated as described previously ([@gkt211-B28]).

RESULTS
=======

Evaluation on synthetic data
----------------------------

### Benchmark data and alternative methods

For objective comparison of alternative methods, we considered two realistic simulated data sets (see 'Materials and Methods'). We used the proposed models either explicitly using the gene annotation (rDiff.parametric) or not using the annotation (rDiff.nonparametric). For comparative purposes, we also considered two state-of-the-art methods that explicitly quantify transcript isoforms to test for differences, MISO ([@gkt211-B13]) and cuffDiff ([@gkt211-B29]). To assess the impact of modeling biological variance, we also applied the simplified variant of the parametric test, called rDiff. poisson, which is based on the Poisson distribution instead of the NB distribution. A detailed description of how the competing methods were applied is found in [Supplementary Section S4](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1).

### Ranking of differentially expressed genes

First, we evaluated the ranking of differentially expressed genes produced by alternative methods. To quantify their respective performances, we used the receiver operating characteristics (ROC), depicting the true-positive rate (TPR) of predictions for different false-positive rates (FPR). For biological applications, the most confident predictions with a moderate FPR are most relevant; thus, we restrict the interval of considered FPR to at most 0.2. The ROC curves for each method evaluated on the synthetic data set are shown in [Figure 4](#gkt211-F4){ref-type="fig"}, and a tabular summary of the area under the ROC curve is given in [Table 1](#gkt211-T1){ref-type="table"}. rDiff.parametric consistently outperformed cuffDiff and MISO with the differences being most striking for most confident calls, where rDiff.parametric achieved a substantially higher TPR. The Poisson-based parametric model (rDiff.poisson) was slightly, but consistently, outperformed by its NB counterpart. rDiff.nonparametric performed as well as MISO and cuffDiff, which is surprising, given the fact that our approach does not use the gene annotation and is conceptually much simpler. This finding highlights the applicability and practical use of the simple one-step methods, both in settings where the genome annotation is available but also if it is incomplete or missing. Figure 4.Comparison of rDiff with MISO and CuffDiff. (**a**) ROC curve for rDiff, MISO and CuffDiff. (**b**) Comparison of the empirical false discovery rate (empFDR) and the FDR based on *P*-values provided by the methods, for rDiff and CuffDiff. This was not possible for MISO, as it did not provide *P*-values. (**c**) Number of detected genes as a function of the FDR cut-off. Table 1.Area under the ROC curve in the interval (0, 0.2) (auROC20) for rDiff, cuffDiff and MISOMethodauROC20 for small biol. varianceauROC20 for large biol. variancerDiff.nonparametric0.0770.073rDiff.parametric0.1010.093rDiff.poisson0.0990.082cuffDiff0.0850.055MISO0.0890.061[^1]

To investigate the robustness of the different methods with respect to biological variability, we considered a second synthetic data set with larger biological variation ([Supplementary Figure S2a](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1)). Although the previously observed trends still hold, the differences between the respective methods were more pronounced. The performance of MISO, rDiff.poisson (which does not model biological variance) and cuffDiff decreased dramatically, in particular for low FPR. rDiff.parametric and rDiff.nonparametric both consider biological variability for computing significance levels and perform best, in particular for the most confident cases. This emphasizes the relevance of modeling biological variability.

### Calibration of test statistics

It is important that the tests deliver meaningful significance levels and false discovery estimates. Therefore, we tested the statistical calibration of the calling confidences provided by the different methods by comparing the estimated FDRs with the empirical FDRs (empFDR). The latter is known because we simulated the data. The empirical FDR was calculated as the fraction of false positives in the number of genes having *P*-values below a certain threshold. [Figure 4](#gkt211-F4){ref-type="fig"}b shows the calibration curves for all methods on the first synthetic data set. rDiff.parametric was the most conservative approach, and the empirical FDR was about three times smaller than the estimated FDR (at 0.2). rDiff.nonparametric was less conservative (empirical FDR ∼1.3 times smaller than estimated FDR) and overall achieved an acceptable level of calibration. cuffDiff and rDiff.Poisson, however, seemed to be overoptimistic by calling a large number of false positives for small FDRs: the most confident predictions were false. This behavior is likely caused by the lack of control for biological variance. MISO could not be considered in this evaluation, as the method does not yield *P*-values. Another interesting observation is that the number of genes that are reported is different as shown in [Figure 4](#gkt211-F4){ref-type="fig"}c. One can see that for a small FDR cut-off, cuffDiff and rDiff.poisson report many more genes than rDiff.nonparametric and rDiff.parametric.

Differential RNA processing in *A. thaliana*
--------------------------------------------

### Data and set-up

To illustrate how rDiff can be applied in a typical experimental setting, we investigated a data set from *A. thaliana*. We obtained RNA-Seq data from seedlings, grown in darkness before light exposure (0 h; two samples, Wt and *cry1cry2*), as well as 1 and 6 h after light exposure. We estimated the variance function between two 0 h samples and used the same parameters for the methods as before.

### Detected events

Both methods, rDiff.parametric and rDiff.nonparametric, identified the largest number of differential genes when comparing the sample 0 h with the sample 6 h ([Table 2](#gkt211-T2){ref-type="table"}). The non-parametric model found a substantially larger number of events, retrieving between 2.7 and 5.4 times as many significant events (at FDR 0.1). The overlaps between the findings retrieved were surprisingly low. This suggests that the non-parametric model provides an orthogonal view of events that cannot be explained when restricting to the annotation. Visual inspection suggested that the great majority of the exclusive hits retrieved by rDiff.nonparametric were plausible (see [Figure 6](#gkt211-F6){ref-type="fig"} for representative examples). Figure 5.Plot of the −log(*P*-value) against the log(fold-change) measured by RT-qPCR. The *P*-values for rDiff.nonparametric are shown in light gray, for rDiff.parametric in dark gray and for CuffDiff in black. Spearman's correlation coefficient ρ for the two methods is given in the legend. Table 2.Overlap between methods for 1 h versus 1 h/ 0 h versus 6 h/ 1 h versus 6 h for genes with an FDRMethodrDiff.parametricrDiff.nonparametricrDiff.parametric**39/80/54**Diff.nonparametric18/29/16**213/219/138**[^2]

These results suggest that the predictions by rDiff.nonparametric can indeed be used to obtain an unbiased view with respect to alternative splicing, without annotation bias. Overall, we found that ∼60% of the detected genes had only one transcript annotated. Furthermore, we performed a classification of the events found by rDiff.nonparametric by the type of region where the biggest change was observed. The exact technical details of this annotation step are described in [Supplementary Section S5.6](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1). The result of the classification of the changes between 0 h and 1 h can be found in [Figure 7](#gkt211-F7){ref-type="fig"} and [Supplementary Table S1](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1). In particular, changes in the 3′-UTR, 5′-UTR and introns were overrepresented (see [Figure 6](#gkt211-F6){ref-type="fig"} for examples). As the libraries were prepared in parallel using the same reagents, we are confident that the observed coverage differences reflect the changes of the transcripts structure. Figure 6.Examples of two genes detected by rDiff.nonparametric with a minimal *P*-value of 0.01. Shown is the read density on top. The gray area indicates the region in which the change was detected, and the black bar in the upper part of the plot shows the 100-bp region which showed the biggest difference. Below the read densities is the splice graph in dark gray and the transcripts in black. The light gray indicates the UTRs.

### RT--qPCR validation

To have an objective comparison on real data, we measured relative isoform levels using RT--qPCR for five genes in the three samples. This validation allowed us to verify whether the isoforms predicted to be differentially expressed have indeed a varying abundance. The protocol is described in [Supplementary Section S5.4](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1).

As a measure of correspondence between the *P*-value and the fold-change we used Spearman's correlation between the negative log-*P*-value and the log-fold-change. We chose this correlation measure, as it is invariant under monotone transformation. We removed one outlier that led to an overly optimistic correlation for all methods. We have found a good correlation of 0.84 for rDiff.parametric, 0.68 for cuffDiff and 0.66 for rDiff.nonparametric ([Figure 5](#gkt211-F5){ref-type="fig"}). These correlations are well in line with the results on the artificial data set and support that the proposed methods retrieve accurate results. Figure 7.Categorization of the most differential 100 bp between the time points 0 h and 1 h according to the gene structure, in genes detected by rDiff.nonparametric with an FDR smaller than 10%. The width of the boxes is the average length of those regions, and the area equals the total number of detected differential cases.

Differential RNA processing in *D. melanogaster*
------------------------------------------------

We also analyzed a *D. melanogaster* data set presented and thoroughly analyzed previously ([@gkt211-B30]). It consists of two samples, one from wild-type and the other from Pasilla knockdown mutant flies, each containing two paired-end libraries and one single-end library. The authors derived multiple read counts for different types of alternative splicing events (exon skipping, intron retention and so forth) and used Fisher's exact test (corrected *P* ≤ 0.05) to find significant differences in the contingency tables for the two libraries \[methodology conceptually similar to a previous study ([@gkt211-B31])\]. Differential splicing in 323 genes was found to be significantly different, of which 16 were experimentally validated.

We applied rDiff.parametric and rDiff.nonparametric (FDR 0.1) after aligning the reads from the paired-end libraries using TopHat ([@gkt211-B32]) (more details in [Supplementary Section S6](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1)). To have a small variance in the samples, we chose to exclude the single-end libraries from our analysis. Overall, rDiff.parametric and rDiff.nonparametric found 71 and 278 genes with differential relative isoform expression, respectively. Although it is reassuring that the numbers are somewhat similar, the degree calibration of the methods, including the one from Brooks *et al.* ([@gkt211-B30]), will significantly influence the number of detected events. We, therefore, concentrated on the top 323 genes \[the number of significant events found in Brooks *et al.* ([@gkt211-B30])\]. We find that of the 16 cases that were experimentally validated previously ([@gkt211-B30]), rDiff.parametric found 12 and rDiff.nonparametric found 11 genes. For three of the remaining genes, the read coverage was too low to detect significant changes for both of our methods because of the strict alignment settings used and using only the paired-end libraries. Nonetheless, the fact that rDiff.nonparametric found a large fraction of the cases is noteworthy, as it does not make use of the gene annotation.

DISCUSSION AND CONCLUSIONS
==========================

Our results on the artificial data and the study on *A. thaliana* and on *D. melanogaster* show that the proposed one-step methods outperform alternative approaches and are generally applicable. We believe that one reason that the methods perform better in practice is due to fewer assumptions made compared with other methods. In particular, quantification of alternative isoforms is a challenging task, and the predictions are often unstable and suffer from multiple possible solutions. As a consequence, the achieved FDRs are typically higher, in particular for highly confident cases. On the contrary, the proposed methods are simple, robust and can operate in complex settings while yielding statistically better calibrated estimates than other methods.

Notably, this high level of accuracy extends to the cases where genome annotations are missing. In these settings, existing quantification-based methods cannot be applied at all; hence, for the first time, we provide a workable and sufficiently accurate approach to deal with these instances. In particular, the non-parametric version of rDiff will facilitate early quantitative characterizations of transcriptomes of newly sequenced species. This finding also highlights the value of non-parametric methods that extend beyond classical uni-variate tests, such as Kolmogorov--Smirnov or the Mann--Whitney U test. rDiff.nonparametric is implemented in a flexible manner and can be used to incorporate additional features to assess the differential behavior, such as splice site or paired-end information.

We would like to note that the proposed rDiff.nonparametric method was designed to test for differential relative isoform expression. However, the method solves a more general problem ubiquitous in deep sequencing data analysis. It detects differential read coverages or other read-dependent properties that are the result of biological circumstances that one has set out to understand. For instance, the method may also be applicable for analysis of data from RNA structure probing ([@gkt211-B33],[@gkt211-B34]), ChIP-seq for differential chromatin binding in different samples (G. Schweikert, personal communication) and whole-genome sequencing for testing of highly polymorphic regions (D. Weigel, personal communication). However, accounting for confounding factors in those analyses is topic of ongoing research.

In summary, we have proposed two complementary statistical tests to detect differential isoform abundances from RNA-Seq. We have shown that the methods perform better than other quantification-based methods and yield reliable predictions of differential relative isoform expression. These tools can be used in a wide range of settings, using existing gene model annotations or solely the observed read data. The NB-based rDiff.parametric test performs considerably better than the rDiff.Poisson test, as it takes the biological variance into account. The rDiff.nonparametric test is based on permutations, and taking biological variance into account is technically less straightforward. We developed the method of limited re-sampling to match the sampling variance to the biological variance. The resulting algorithm rDiff.nonparametric is significantly more robust against biological variability. Our experiments underline that biological replicates are an essential prerequisite to accurately estimate significance levels. Therefore, we advocate measurement of at least two biological replicates to estimate the variance function. Additionally, we recommend using the same sequencing method, as well as the same mapping method, to reduce systematic biases, which could lead to many false positives.

SUPPLEMENTARY DATA
==================

[Supplementary Data](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gkt211/-/DC1) are available at NAR Online: Supplementary Table 1, Supplementary Figure 1 and Supplementary Methods.

FUNDING
=======

Volkswagen foundation and Marie Curie FP7 fellowship (OS); German Research Foundation \[WA2167/4-1 to A.W. and L.H.; RA1894/1-1 and RA1894/2-1 to G.R. and P.D.\]; Emmy Noether fellowship \[WA2167/2-1 to A.W.\]. MSKCC Center for Translational Cancer Genomic Analysis \[U24 CA143840 to P.D. and A.K.\]; Sloan-Kettering Institute core funding (to G.R., P.D., A.K.). Funding for open access charge: German Research Foundation \[RA1894/2-1\].

*Conflict of interest statement*. None declared.

Supplementary Material
======================

###### Supplementary Data

The authors acknowledge fruitful discussions with Wolfgang Huber, Detlef Weigel, Arthur Gretton and Gabriele Schweikert. G.R., O.S., K.B. and P.D. conceived the study, P.D., O.S. and G.R. designed computational study, G.R., L.H. and A.W. designed RNA-Seq and validation experiments, P.D. and O.S. developed statistical tests, P.D. (re-)implemented algorithms and performed computational experiments, A.K. and G.R. performed RNA-Seq alignments, L.H. performed RNA-Seq and validation experiments, P.D., O.S., G.R. and L.H. wrote and A.W. and K.B. revised the article.

[^1]: The comparison is shown on the two artificial data sets with a small and large biological variance (see 'Materials and Methods' section).

[^2]: The events written in bold are the number of events predicted by the corresponding method.


Diagnostic Criteria of Major Sexual Dysfunctions

For this Discussion, you analyze the diagnostic criteria of major sexual dysfunctions and disorders in the DSM. Review the case study below and reflect on which DSM sexual dysfunction/disorder might be the most reflective of the client's symptoms. Then consider a counterargument as to why this dysfunction/disorder might not be appropriate for this client.

Susan, age 34, is a married mother of two preschool-aged children (ages 4 and 2). Her husband, Steve, age 35, works full-time, and Susan works part-time on the weekends but primarily is a stay-at-home mother to her children.

The couple sought couples counseling, and their primary presenting concern was that Susan has not had any interest in sex with Steve for the past year. The couple reports that they have had intercourse about 3 times in the past year ("always at Steve's initiation"); they rarely display physical affection toward one another; and they fought frequently about their lack of sex for about the first six months of the past year, but lately they have not fought often about the issue.

The partners indicate that, up until a year ago, Susan was very interested in sex, and they had sex approximately 3 times per week throughout their marriage (aside from immediately following the birth of their children). Susan states that she can't pinpoint any particular reason for her lack of interest in sex and says, "Really, I just don't want it anymore. I'm not sure if I ever will again." Steve says that he is very frustrated but doesn't want to fight about it. He says, "I just want my wife back."
With these thoughts in mind:

Post by Day 4 an explanation of which DSM sexual dysfunction or disorder might be the most reflective of the client's symptoms. Provide a counter-argument for why the dysfunction or disorder you selected might not be appropriate for this client. Justify your response with references to the Learning Resources and the DSM.

Solution Preview

In this situation it appears that Susan suffers from hypoactive sexual desire disorder. This is due to the fact that she exhibits the very strong symptom of not having any desire for sexual intercourse, as well as not having any form of fantasies concerning sexual intercourse as well. In addition, what is supportive of ...


Giant cell tumour of the temporal bone: case report and review of the literature.
This paper presents an expansile lesion of the temporal bone, in a 14-year-old boy, that was initially diagnosed as intra-osseous meningioma from CT appearance. At histopathology a final diagnosis of giant cell tumour was made. A brief review of the literature is presented for this rare case.


LLVM Weekly - #198, Oct 16th 2017

Welcome to the one hundred and ninety-eighth issue of LLVM Weekly, a weekly newsletter (published every Monday) covering developments in LLVM, Clang, and related projects. LLVM Weekly is brought to you by Alex Bradbury. Subscribe to future issues at http://llvmweekly.org and pass it on to anyone else you think may be interested. Please send any tips or feedback to asb@asbradbury.org, or @llvmweekly or @asbradbury on Twitter.

Like many of you, I'll be attend the LLVM Dev Meeting this week - be sure to say hi!

News and articles from around the web

The call for speakers has been issued for the LLVM Performance Workshop at CGO 2018. The deadline for submissions is December 1st, 2017.

The call for papers / participation has been posted for the LLVM toolchain devroom at FOSDEM 2018. This will take place on Feb 4th 2018 in Brussels. The proposal deadline is Nov 26th, 2017.

The CppCheck GUI has been extended with support for clang-tidy. The author hopes to add support for the Clang static analyzer in the future too. See here for some screenshots.

On the mailing lists

LLVM commits

LLVM now has much more detailed documentation about the various fuzzers available for fuzzing LLVM itself. r315544.

.Lcfi labels are no longer emitted when producing textual assembly. r315259.

The ARM backend now gives better diagnostics for floating point and general purpose register operands. r315303, r315304.

The NumFixedArgs field of CallLoweringInfo is now correctly updated. As noted in the commit message, there is a small chance this could affect your backend if you were directly accessing this field or the IsFixed property. r315457.

MergeFunctions has been improved. r315852, r315853.

createElfObjectWriter now takes a std::unique_ptr<MCElfObjectTargetWriter> and returns a std::unique_ptr<MCObjectWriter> . If you have an out-of-tree backend, it will need updating. The MCObjectStreamer prototype has also changed. r315254, r315327, r315531.

The loop invariant code motion pass will no longer make the illegal optimisation of sinking an unordered atomic load into a loop. r315438.

Clang commits

Clang gained support for destroying operator delete, as proposed for C+++2a. r315662.

The -Wtautological-constant-compare warning has been reintroduced. r315875.

Other project commits

LLD has seen a large number of code cleanups. e.g. r315525, r315617.

LLDB learnt to search for split DWARF files in a specified search path. r315387.

Review Corner

The LLVM Weekly review corner serves to highlight patches that are stuck waiting awaiting review, or work from first-time contributors. See here for more information and how to submit you work for inclusion. Of course the hope is that highlighting these patches will enable LLVM Weekly readers will step up and help to get them merged. I'll be reporting back each week on any activity generated on these patches, as well as sharing a new batch. If you want your patch included you must submit it via the linked form.

Last week featured three patches, with two out of three seeing fresh review activity after inclusion. D36471 sadly has seen no additional feedback. Thanks to Jessica Paquette and Anna Zaks for contributing review comments to last week's patches.

There is just one patch submission this week:


// Copyright (c) Microsoft Corporation. All rights reserved.
// Licensed under the MIT license.

#include "CameraRenderer.h"

namespace AzureSpatialAnchors {

const GLfloat QuadVertices[] = {
        -1.0f, -1.0f, 0.0f,
        +1.0f, -1.0f, 0.0f,
        -1.0f, +1.0f, 0.0f,
        +1.0f, +1.0f, 0.0f
};
const GLfloat QuadUvs[] {
        0.0f, 1.0f,
        1.0f, 1.0f,
        0.0f, 0.0f,
        1.0f, 0.0f
};

void CameraRenderer::Initialize(AAssetManager* assetManager) {
    m_program = Util::CreateProgram("shaders/camera.vert", "shaders/camera.frag", assetManager);
    if (!m_program) {
        LOGE("Failed to create program.");
    }

    m_texture = static_cast<GLuint>(glGetUniformLocation(m_program, "uTexture"));
    m_vertices = static_cast<GLuint>(glGetAttribLocation(m_program, "aPosition"));
    m_uvs = static_cast<GLuint>(glGetAttribLocation(m_program, "aTextureCoord"));

    glGenTextures(1, &m_textureId);
    glBindTexture(GL_TEXTURE_EXTERNAL_OES, m_textureId);
    glTexParameteri(GL_TEXTURE_EXTERNAL_OES, GL_TEXTURE_MIN_FILTER, GL_LINEAR);
    glTexParameteri(GL_TEXTURE_EXTERNAL_OES, GL_TEXTURE_MAG_FILTER, GL_LINEAR);
}

void CameraRenderer::Draw(const ArSession* session, const ArFrame* frame) {
    int32_t displayGeometryChanged = 0;
    ArFrame_getDisplayGeometryChanged(session, frame, &displayGeometryChanged);
    if (displayGeometryChanged != 0) {
        // Transform the given texture coordinates to correctly show the background image.
        ArFrame_transformDisplayUvCoords(session, frame, 8, QuadUvs, m_transformedUvs);
    }

    int64_t frameTimestamp;
    ArFrame_getTimestamp(session, frame, &frameTimestamp);
    if (frameTimestamp == 0) {
        // Skip rendering if no frame is produced.
        return;
    }

    RenderQuad();
}

void CameraRenderer::RenderQuad() {
    if (!m_program) {
        LOGE("program is null.");
        return;
    }

    glUseProgram(m_program);
    glDepthMask(GL_FALSE);

    glUniform1i(m_texture, 1);
    glActiveTexture(GL_TEXTURE1);

    glEnableVertexAttribArray(m_vertices);
    glVertexAttribPointer(m_vertices, 3, GL_FLOAT, GL_FALSE, 0, QuadVertices);

    glEnableVertexAttribArray(m_uvs);
    glVertexAttribPointer(m_uvs, 2, GL_FLOAT, GL_FALSE, 0, m_transformedUvs);

    glDrawArrays(GL_TRIANGLE_STRIP, 0, 4);

    glUseProgram(0);
    glDepthMask(GL_TRUE);
    Util::CheckGlError("CameraRenderer::RenderQuad() error");
}

GLuint CameraRenderer::GetTextureId() const {
    return m_textureId;
}

}


Thursday, 3 December 2009

Well Said And Couldn't Have Said It Better.

Jeniffer Lopez, Britany Spears, Elizabeth Taylor, Donald Trump Angelina Jolie take note. On November 20th 2008 I became the legal partner to on Brendan Bernard Aloysios McCann over on King's Road Brydon Room Chelsea Old Town Hall, where many a rock star had their ceremony. After 7 years together I am still madly deeply truly in love with him, although I might annoy the shit out of him from time to time. I know hand over heart he does love me for me. My heart and love is his forever. Although he jokes about me being the wife, Pooch is my second wife. And it's a shame that governments in America still find the word marriage a sacred trust. It wasn't till I saw this speech spoken by NY Senator Diane Savino that she made a clear concise point, that hit the nail on the head. Home girl should get a bigger office. I believe it's not just people whom pledge their love so often that they forget the words for better or worse. Most mean till something better comes along, or till I Quit This Bitch! Good relationships are hard and difficult, bad relationships are easy to get. Simply look at the divorce rate. That being said I have my parents to thank, because they are still together and I know witnessing first hand, their relationship in close quarters. Good and bad through the years there is actual love. So when honest with my partner as scary as it seems sometimes I know I will be honest with him always. Yet, I vow to make him laugh and try my dammedest to make him happy. For the most part any way. So to celebrate our one year together as a betrothed couple we went to Paris to celebrate. In the seven years me and my partner have been together there have been alot of laughter, tears and hope for the future. I love the man with all my heart. Above is my favorite photo taken on the day we got hitched taken In Holland Park 3 hours before. So if your reading this Bren, I love you still, always and forever. Perhaps one day America will catch up with the rest of the free thinking civilised world.


Pieve delle Sante Flora e Lucilla

The Pieve delle Sante Flora e Lucilla is a Roman Catholic parish church in the comune of Santa Fiora, Province of Grosseto, region of Tuscany, Italy.

The first documentation of a church here dates to 1144. Above the stone portal is a Romanesque window. The interior has three naves and a wooden ceiling. The inside of the church has terra cotta decorations by Andrea della Robbia; these depict a Madonna della cintola, The Baptism of Jesus, a Last Supper and a triptych with a Coronation of the Virgin flanked by Saints Francis and Jerome.

References

Category:Churches in the province of Grosseto
Category:Romanesque architecture in Tuscany
Category:12th-century Roman Catholic church buildings


Tristram Hunt's resignation is sudden but hardly unexpected. Bright, young and ambitious, the historian entered politics with the hope of becoming a cabinet minister. But Labour has never been further from power. His party is led by Jeremy Corbyn, to whom he is implacably opposed, and his Stoke seat is due to be abolished in the boundary changes (posing the prospect of a tricky selection contest). In these circumstances, only a masochist would have declined the chance to become director of the V&A.

Hunt's announcement follows that of Jamie Reed, who is resigning his Copeland seat to become Head of Development and Community Relations for Sellafield power station. Labour MPs believe that many more will follow. The party's parlous poll ratings, Jeremy Corbyn's re-election (in defiance of 80 per cent of MPs), the boundary review (the first since 2010) and opportunities outside parliament all mean that Westminster is an increasingly unattractive berth.

Under the boundary changes, some Labour MPs will see their seats become notionally Conservative. As UK Polling Report's Anthony Wells has calculated, Andy Slaughter will face a Tory majority of 14 per cent in the new Hammersmith and Fulham seat, Gareth Thomas will face one of 11 per cent in the new Harrow and Stanmore seat, Ruth Cadbury will face one of 10 per cent Brentford and Chiswick and Tulip Siddiq will face one of 9 per cent in Hampstead and Golders Green. Alison McGovern and Margaret Greenwood are both eligible to seek selection for the new Bebington and Heswall seat but it is also notionally Tory.

Labour MPs whose seats would be vulnerable based on current poll ratings include Rupa Huq (274 majority), Wes Streeting (589), John Woodcock (795), Karen Buck (1,977), Tom Blenkinsop (2,268), Mary Creagh (2,613), Gisela Stuart (2,706), Kerry McCarthy (3,980) and Ian Austin (4,181). "There's not a safe seat north of Islington," an MP told me recently. Others, such as Stella Creasy and Peter Kyle, are at risk of deselection by local activists (through the trigger ballot mechanism).

Then there are those already seeking lifeboats. Andy Burnham and Steve Rotheram are bidding to become the mayors of Greater Manchester and Liverpool respectively. Brexit will create new opportunities for those with political and European expertise as businesses seek to equip themselves for the upheaval.

Labour has already suffered a severe brain drain at adviser level. It is now facing one of its MPs.


Tuesday, October 25, 2011

Bunkered Barack Obama Awaits World War

As The Denver Post reported
Sunday, today marks the second time in less than a month that Barack
Obama has come to the mile-high city, headquarters of the nation's
state-of-emergency command apparatus, where he will remain through
tomorrow.

The last time he came was four weeks ago, when
he arrived on Sept 27 to command massive terror exercises occurring
during the window of opportunity for an Israeli attack against Iran, as
reported by Haaretz in Netanyahu must be stopped from attacking Iran. The timing of the "continuity of government" training seemed so suspicious that I posted America Awakens to Denver False Flag Danger.
Just five days before, Big Oil shenanigans had raised false flag fears
in Ron Paul's toxic home turf of Texas City, which I described in my
article the following morning, Texas City Terror Scare.

This
time around BO's stated mission is the less menacing promotion of his
$447 billion Jobs Bill, but who knows what evil lurks in the heart of
the Kenya Kid? Observers find him to be quite as malicious as his
predecessor, King George, so much so that my friend Lori Price, editor
of Citizens for Legitimate Government regularly lampoons him as Obusha.

Despite
the reassuring White House press release, duly regurgitated by the mass
media for public consumption, there are unsettling current events that
make this presidential appearance in Denver seem like another command
post exercise ready to go live for a false flag, world war or both.

I offer a few points of particular concern for the penetrating reader:

Yesterday
Obama was in Las Vegas, where the false flag establishment has a score
to settle, and expects Harry Reid to make up for his past failure by
helping it do so:

'Terrorist attack' on Las Vegas canceled
At
Sen. Harry Reid's request, the Obama administration canceled a mock
'dirty bomb' terrorist attack exercise in Las Vegas. Critics say the
administration is playing politics with national preparedness, but
others say a shift to secret surprise tests is a better way to prepare
for the worst. -- Christian Science Monitor, 3/30/10

Then there is the release last week of a Hamas-captured Israeli soldier:

477 for 1 Gilad Shalit
Israel
has published the names of 477 Palestinian prisoners who are to be
released in exchange for captured Israeli soldier Gilad Shalit. A
further 550 prisoners are to be released after Sgt Shalit returns home.
-- NowPublic.com, 10/16/11

The number 477 is
a numeric harmonic, a variation, of the $447 billion in the Obama Jobs
Bill. It's likely that the Israelis have encrypted a go code for a false
flag and world war even as they have removed their boy from the reach
of the expected Hamas backlash.

The
only other time Barack Obama came to Denver with an economic agenda was
on Feb 17, 2009, for the signing of the Stimulus Bill, of which the
Jobs Bill is a close cousin. Few recall that within a day of his
mile-high triumph he was looking at a cartoon brutally depicting him as a
chimp, shot dead for his Stimulus Bill failure. How could this be?

I answered the riddle in a 2010 article:
the Stimulus Bill then, like the Jobs Bill now, was a code, the
underlying reality of which was a diabolic deal with world finance to
nurture the ruinous national debt in exchange for a false flag attack on
America. Thanks to a vigorous Internet Resistance, we prevented a
Chicago attack then. Let's pray that we can do the same for whatever
U.S. city is the target of treason now.