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[ "Timing, it would appear, sometimes gets in the way of progress for Apple. ", "The company experienced failure with its personal digital assistant Newton in the early 1990s, only to find overwhelming success with the iPad in 2010.", "\n\nAnd now, recently unearthed concept art by Hartmut Esslinger — whose design firm frog helped conceptualize many successful Apple products — proves this observation true again. ", "It appears that Apple was dreaming of an iPhone way back in 1983 — meaning it took 24 years for the company to figure out the right timing for its flagship mobile device.", "\n\nOSXDaily, an Apple-focused blog, shared the photos from the concept art from Esslinger’s 1983 iPhone, which were published in an article on fudder.de, a German news site.", "\n\nThe concept art for the ’83 iPhone actually looks like an iPad crossed with a car phone. ", "It includes a stylus and shows off Apple’s vision for electronic banking, with a hand signing an electronic check.", "\n\nSo given that Apple first thought of the iPhone in 1980s and the iPad in the 1990s, its next breakthrough device has likely long been in the pipeline as well. ", "The question is, how long will we have to wait to actually hold it?" ]

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[ "Q:\n\nTYPO3 sys_language_mode = content_fallback not working as expected\n\nHere is a little follow-up question to this question of mine: Language switching in Typo3 v6.", "\nThis is my current setup:\nconfig {\n tx_realurl_enable = 1\n simulateStaticDocuments = 0\n sys_language_uid = 1\n language = de\n locale_all = de_DE\n htmlTag_langKey = de\n linkVars := addToList(L)\n uniqueLinkVars = 1\n sys_language_mode = content_fallback\n sys_language_overlay = 1\n}\n\n[globalVar = GP:L = 1]\nconfig.sys_language_uid = 1\nconfig.language = de\nconfig.locale_all = de_DE\nconfig.htmlTag_langKey = de\n[global]\n\n[globalVar = GP:L = 2]\nconfig.sys_language_uid = 2\nconfig.language = en\nconfig.locale_all = en_EN\nconfig.htmlTag_langKey = en\n[global]\n\nIn the backend, German has the ID 1 and English the ID 2. ", "Up to now, everything worked fine. ", "But now there is a site where some German content has no direct translated element in the English content. ", "In other words: I need an element only to appear on the German version.", "\nUp to now, the German content sat in the \"default\" column and the English content was added in the \"english\" column. ", "So I thought all I have to do is add a german translation to the site and add the element there. ", "But it is not working as expected:\n\nThis is the content I added. ", "The left column (marked green) is the default content. ", "The middle column (pink) is the German content. ", "The right column (blue) is the English content.", "\nBut in the frontend, on the English site the blue (English) content is show and on the German site, the green (default) content is shown, not the pink (German) content, as I would have expected and desired.", "\nWhere did I go wrong?", "\nI already confirmed that L=1 by messing around with the htmlTag_langKey. ", "It definitely is set by the first conditional block. ", "So why is sys_language_uid = not displaying the German content. ", "Is any additional configuration needed?", "\n\nA:\n\nYou are adding a german translation to a german text. ", "This can't go well.", "\nIf your main language is german, it should stay the default language. ", "You can rename the label via TSCONFIG in the Backend for editor's convenience. ", "\nmod.", "SHARED{\n defaultLanguageFlag = de.gif\n defaultLanguageLabel = Deutsch\n}\n\nThe default language has the ID 0, so you can call index.php without a language parameter. ", "So you just have to add the foreign languages on page 0, default is already there\nI think you will run into problems if you try to force another localisation to be the default language, so you might want to go back to 2 languages quickly. ", "\nBy the way, I find the most logical setup for TYPO3 language fallback to be:\nIn TS:\nconfig.sys_language_overlay =1\n// don't set sys_language_mode\n\nIn TSCONFIG:\nmod.web_layout.defLangBinding = 1\n\nwhich will bind each article with it's translations\n\n" ]

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[ "AutoGuide.com\n\nAudi is previewing more than its design future at the 2015 Geneva Motor Show.", "\n\nThe German automaker has spilled the details on its new Prologue Avant concept, which will make its official debut in Switzerland early next week. ", "Featuring a high-performance hybrid powertrain with 455 hp and 553 lb-ft of torque, Audi claims that the technology is “close to series production” and hints at the potential of what is already powering current Audi models. ", "The plug-in hybrid drive actually comes from the existing Q7 e-tron quattro, albeit with more power, using a 3.0-liter diesel engine with an electric motor to blast the concept to 62 MPH (100 km/h) in 5.1 seconds. ", "The concept also has an all-electric range of 33.6 miles.", "\n\nThe Prologue Avant concept measures in at 16.8 feet long and 6.5 feet wide while riding on a 10-foot wheelbase. ", "All in all, it’s a little larger than the two-door Prologue concept that debuted at the 2014 LA Auto Show but sports four individual seats, four doors and a large tailgate.", "\n\n2015 Geneva Motor Show Preview\n\nAudi is also previewing future technologies inside the concept with a full width front dashboard that is designed as a display surface that integrates three touch displays. ", "Aluminum frames the two driver-faced displays flanking the steering wheel with the left display controlling the assist functions and the right display for multimedia management. ", "Even in the rear, Audi is incorporating flexible OLED displays that can bend to adapt its angle to the rear seat backrests’ angle of inclination.", "\n\n“With our show car for Geneva, we now translate the design language of the Audi prologue into a new, dynamic and stretched form. ", "We are merging it with a concept of high variability,” Audi product development boss Ulrich Hackenberg said. “", "The Audi prologue Avant is progressive, emotion-filled and versatile, it reflects Audi’s technological competence and quality claim perfectly.”", "\n\nDiscuss this story at our Audi forum" ]

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[ "Running a successful Education Technology Project\n\nThat well-known expression applies as much to running an ed tech project successfully as to anything else. ", "In other words, for an ed tech project to succeed, you need to think about more than just the technology, or even the pedagogy. ", "You have to think about management as well.", "\n\nNext week at the Reform Symposium Conference I’ll be giving a presentation on this very topic. ", "How’s that for a coincidence?! ", "I’ll be looking at 14 factors that ought to be considered if a major project is to succeed.", "\n\nGet ready to be inspired! ", "Now? ", "Now! ", "by James Vauhn http://www.flickr.com/photos/x-ray_delta_one/" ]

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[ "The response to stress in Brazilian children and adolescents with attention deficit hyperactivity disorder.", "\nThis study assessed the function of the hypothalamic-pituitary-adrenal (HPA) axis, during response to stress, through the measurement of salivary cortisol in 38 children with attention deficit hyperactivity disorder (ADHD) and its subtypes, who were matched to 38 healthy control subjects. ", "These measures were made at four time intervals: 15 min before exposing the subjects to a stressor - the Continuous Performance Test (CPT) - and 20, 40, and 60 min after such exposure. ", "The baseline cortisol levels were statistically similar in both groups. ", "The mean values of cortisol at the four time intervals were not statistically different between the three subtypes of ADHD (inattentive, hyperactive-impulsive and combined); thus, the ADHD group was treated as a single group. ", "Following the stressor test, the ADHD group had significantly higher levels of salivary cortisol than the control group at time intervals of 20 and 40 min, whereas in this latter group exposure to the CPT did not induce an increase of cortisol. ", "These results suggest that the increased cortisol levels in the ADHD group could be due to the lack of comorbidities. ", "In addition, these patients, when facing a computerized test, might have responded with a motivational pathway with an increase of cortisol." ]

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[ "Cardiovascular magnetic resonance: myocardial perfusion.", "\nThere is growing evidence that the noninvasive assessment of myocardial perfusion with cardiovascular magnetic resonance is a valid and accurate tool for the assessment of ischemic heart disease and its introduction into routine clinical evaluation of patients is rapidly expected. ", "Magnetic resonance measurements allow the evaluation of reversible and irreversible myocardial ischemia, the assessment of acute myocardial infarction, as well as the recognition and detection of viable myocardium. ", "Magnetic resonance perfusion measurements are mainly performed with T1-shortening contrast agents such as gadolinium-DTPA either by visual analysis or based on the analyses of signal intensity time curves. ", "For the detection of myocardial ischemia the first pass kinetics of a gadolinium-DTPA bolus and for the detection of myocardial necrosis and the definition of viable myocardium steady state distribution kinetics are assessed. ", "Quantitative analysis of myocardial perfusion can be performed but requires complex modeling due to the characteristics of gadolinium-DTPA. ", "Thus, semi-quantitative parameters are preferred. ", "There is accumulating evidence in the literature that magnetic resonance imaging can be used for the detection of coronary artery stenosis with high diagnostic accuracy both with semi-quantitative or visual analysis. ", "Myocardial infarction can be reliably detected and the infarcted area determined. ", "Non-reperfused infarcted myocardium can be differentiated from reperfused myocardium by different enhancement patterns that correlates with viability. ", "Cardiac magnetic resonance is a promising technique that can combine different functional studies during one examination, such as the assessment of wall motion and perfusion at rest and stress. ", "With further improvements in analysis software magnetic resonance perfusion measurement may rapidly become a routine tool for the assessment of patients with coronary artery disease." ]

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[ "Q:\n\nWhy Kalki Avatar will come at the end of the yuga not in between like all other Avatars of Vishnu?", "\n\nAs there are 4 Yugas. ", "\nHow many Avatars in each yuga? ", "Why different in number of Avatars?", "\nWhy only one avatar in Kali Yug? ", "\nLike lord Rama and lord Krishna have come in between of those Yugas. ", "But why Kalki Avatar will come at the end?", "\nIt is said that whenever Papa increases then an Avatar comes to restore the Dharma. ", "And now also in Kali Yug many Papa are done by many Ravana and Kansas from each family. ", "In Ramayan there was only one Sita was abducted by one Ravan, in Kali Yug also many Sita are there who are abducted by many Ravans.", "\nso Is it not the time for Vishnu to take an Avatar to restore the Dharma? ", "\nIf Kalki Avatar comes at the end of Kali Yug and start of Satya Yug and no people of Kali Yug will have experience with this Avatar, then can we say Kalki is not an Avatar of Kali Yug but of Satya Yug And no Avatars in Kaliyuga?", "\n\nA:\n\nHow many Avatars in each yuga? ", "Why different in number of Avatars?", "\nAs Lord's incarnations are infinite, so hard to say but if you believe only in 24 or 10 incarnations then this can be answered. ", "Number of Avataras depends on incidents, in previous Yugas, Dharma was more but mighter Asuras were also more.", "\nThis is partially answered in What is chronological timeline (in Manvantara and Mahayuga) of Lord Vishnu incarnations?", "\nWhy only one avatar in Kali Yug?", "\nBuddha avatar already happened in Kali Yuga and Kalki is yet to be happened.", "\nLike lord Rama and lord Krishna have come in between of those Yugas. ", "But why Kalki Avatar will come at the end?", "\nWrong, Lord Krishna incarnated at the end of 28th Dwapara Yuga or in the conjunction time of Dwapara and Kali Yuga to be more correct.", "\nKaliyuga started from king Parikshit who was next king after Yudhisthira. - ", "as per Bhagvata Purana\nSo Is it not the time for Vishnu to take an Avatar to restore the Dharma?", "\nOpinion based question. ", "May be the pot of sins yet to be filled. ", "\nThis is partially answered in What is the apex of Adharma and When will the Kalki avatar be born?.", "\nIf Kalki Avatar comes at the end of Kali Yug and start of Satya Yug and no people of Kali Yug will have experience with this Avatar, then can we say Kalki is not an Avatar of Kali Yug but of Satya Yug And no Avatars in Kaliyuga?", "\nOne avatar (Budhdha) has already incarnated. ", "And if you believe in this theory then you should consider Lord Krishna as an avatara of Kali Yuga.", "\n\n" ]

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[ "Back to the Satoshi Nakamoto Bitcoin affair Rory Cellan-Jones\n\nTechnology correspondent\n\n@BBCRoryCJon Twitter Published duration 20 June 2016\n\nimage caption Craig Wright failed to deliver on his promise of \"extraordinary proof\" that we was Bitcoin's inventor\n\nAt the beginning of May, it seemed that a great mystery had been solved.", "\n\nAn Australian academic and entrepreneur, Craig Wright, identified himself as Satoshi Nakamoto, the creator of the crypto-currency Bitcoin.", "\n\nBut, over the following days, the proof he offered was torn apart, not just by angry people on the Bitcoin forums but by a number of authorities on cryptography.", "\n\nAfter first promising more evidence, Dr Wright then wrote a blog saying he did not have the strength to continue and retired from public view.", "\n\nNakamoto's true identity appeared as opaque as ever - the Australian had not proved his case, but nor had it been disproved.", "\n\nNow, though, we have a whole new collection of evidence, in the form of a 35,000-word article about the Nakamoto affair in the London Review of Books\n\nThe author is the journalist and novelist Andrew O'Hagan, whose work includes a fascinating and hilarious account of his time trying to be the ghost writer of Julian Assange's autobiography.", "\n\nIt seems Mr O'Hagan has an appetite for driven and difficult high-performing geeky types, because he spent six months with Craig Wright.", "\n\nimage copyright Getty Images image caption The value of Bitcoin has strengthened against \"real\" currencies since Dr Wright disappeared from view\n\nThat meant he was there right through the period when Dr Wright was first outed as a potential Nakamoto last December, then dismissed as a fraud, and later through his own \"self-outing\" and the aftermath.", "\n\nIt is a gripping and beautifully written tale, more a short novel than a magazine article.", "\n\nWe learn all sorts of new details about Dr Wright's career, his claims about his involvement in the creation of Bitcoin, and his reasons for choosing to identify himself as Nakamoto seven years after the currency was created.", "\n\nTo me, the key revelation is about this motivation.", "\n\nHe had told the BBC that he had not wanted to come out into the spotlight but needed to dispel damaging rumours affecting his family, friends and colleagues.", "\n\nBut O'Hagan shows us something rather different - a man under intense pressure from business associates who stood to profit from him if he could be shown to be Nakamoto.", "\n\nThese people had signed a deal with Dr Wright in June last year, which saw them pay off his debts, including legal fees incurred in a battle with the Australian tax authorities.", "\n\nThen, they had a plan for him.", "\n\n\"They would bring Wright to London and set up a research and development centre for him, with around 30 staff working under him,\" O'Hagan writes.", "\n\n\"They would complete the work on his inventions and patent applications - he appeared to have hundreds of them - and the whole lot would be sold as the work of Satoshi Nakamoto, who would be unmasked as part of the project.\"", "\n\nThe intellectual property, which he had already created and would now augment, would be worth as much as $1bn (£700m) with the Nakamoto name attached, so they would all end up very rich indeed.", "\n\nThat may sound fanciful, but Dr Wright's research was into the block chain, the technology underlying Bitcoin, and the world's big banks are rushing to invest in exploring its potential.", "\n\nNow, they may have had dollar signs in their eyes, but Dr Wright's backers certainly seemed to believe he was Nakamoto, and O'Hagan outlines some new evidence that supports that view.", "\n\nHe spends days talking to Dr Wright about his relationship with Dave Kleiman, an American who died in 2013 and is thought by many to have played a big part in the creation of Bitcoin.", "\n\nWith some reluctance, Dr Wright eventually supplies some emails that seem to suggest the two worked together on the 2008 Nakamoto white paper explaining the idea of the \"peer-to-peer electronic cash system\".", "\n\nimage caption Dr Wright gave interviews to only the BBC, the Economist and GQ magazine, in addition to speaking to Mr O'Hagan\n\nO'Hagan goes to meet Dr Wright's ex-wife, who stands up a story about the two men meeting at a conference in Orlando in 2009 - because she came along too.", "\n\nShe gives a detailed account of their meeting and the way Dr Wright hero-worshipped Mr Kleiman, which rings true coming from someone who has no reason to lie.", "\n\nThere is a document about a trust fund set up by Dr Wright to hold some bitcoins for Mr Kleiman, along with a promise not to reveal the identity behind Nakamoto's email address.", "\n\nAnd there are minutes of a meeting between the Australian tax authorities and Dr Wright's business, where his advisor appears to suggest that he possessed 1.1m bitcoins - worth nearly £600m at the current exchange rate.", "\n\nThere is also an intriguing tale about a meeting between Dr Wright and Ross Ulbricht, the man now serving a life sentence in the United States for setting up the Silk Road, the bitcoin-fuelled drug and gun trading site.", "\n\nAnd, then, we get a behind-the-scenes account of what happened when this brilliant, difficult, angry man - described by one colleague as \"like Steve Jobs but only worse\" - went public, in separate meetings with the BBC, the Economist and GQ, and the days afterwards, when everything fell apart.", "\n\nAs someone who played a part in that saga - and still asks himself whether he should have asked better questions of Dr Wright - I was just a little disappointed with this climax to the story.", "\n\nThe author seems more removed from the action at this point than in the months before the denouement.", "\n\nHere is an example: on the Wednesday after the big reveal on Monday 2 May, Dr Wright agreed to undertake a much simpler proof of his identity than he had offered before.", "\n\nHe asked me, and the two Bitcoin experts who had verified his story - Jon Matonis and Gavin Andresen - to send a small amount of bitcoins to an address known to be linked to Nakamoto.", "\n\nHe would then send it back, proving his claims to the identity.", "\n\nimage copyright Blockchain.info image caption Others have also transferred small amounts of bitcoins to Nakamoto's address without them being returned\n\nThe money was sent but never returned.", "\n\nWhat was going on in the hours between us being invited to take part in this proof and the moment when we were told it was \"on hold\"?", "\n\nThe only reason we get for Dr Wright's bizarre behaviour , which sabotaged everything he had worked for, comes in the form of an email to O'Hagan.", "\n\nHe sends the writer a news story that suggests the father of Bitcoin might be arrested for helping to facilitate terrorism by allowing people to buy weapons anonymously.", "\n\nDr Wright, it seems, decided he would prefer to be called a fraud than risk spending years in jail.", "\n\nThis seems unconvincing.", "\n\nAfter all, he had been planning to out himself for months - albeit under pressure from his backers - so why this sudden fear of the consequences now?", "\n\nThe other question left hanging is why the backers who had invested so much in their Nakamoto had not demanded more proof from him at an earlier stage.", "\n\nIn the end, we are left uncertain about Dr Wright's true role in the creation of Bitcoin.", "\n\nIt seems very likely he was involved, perhaps as part of a team that included Dave Kleiman and Hal Finney, the recipient of the first transaction with the currency.", "\n\nHe may have exaggerated his contribution, he may have constructed a very elaborate fantasy - or this fragile personality may have lost his nerve as he realised that his life would never be the same again once he was Craig \"Nakamoto\" Wright.", "\n\nIf you are expecting O'Hagan to reveal the truth behind Bitcoin's creation myth, you will be left disappointed.", "\n\nBut if you enjoy a ripping yarn - with some piercing insights into geek culture - then you will find the Satoshi Affair an engrossing way to spend a couple of hours." ]

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[ "Q:\n\nAlternate definition of singular homology.", "\n\nInstead of defining the spaces $\\Delta^n=\\{(x_0,...x_{n}) \\in \\mathbb R^{n+1}:\\Sigma_i x_i=1 \\ \\text{&} \\ x_m \\geq0\\ \\forall m\\}$ define $s\\Delta^n =\\{(x_0,...x_{n}) \\in \\mathbb R^{n+1}:\\Sigma_i x_i=1\\}$ with the face maps given by $d_i:(x_0,..x_n) \\mapsto (x_0,...x_{i-1},0,x_i...,x_n)$. Does this define an isomorphic version of homology? ", "As in if X is a space, do the two definitions coincide. ", "Not necessarily naturally.", "\nI've been struggling with this problem for over two weeks now. ", "I could elaborate on methods I have tried but none have been succesful so I don't see the point. ", "I really only need the result for open subsets of $\\mathbb R^n$ so make any assumptions you want about the space X. Thank you in advance!", "\n\nA:\n\nYes, this is naturally isomorphic to singular homology. ", " To prove it, first construct a sequence of retractions $r_n:s\\Delta^n\n\\to\\Delta^n$ which are compatible with the face maps. ", " For instance, $r_1$ will take the line $s\\Delta^1$ and contract the ends of it down to just the bounded interval $\\Delta^1$. Then $r_2$ will contract the plane $s\\Delta^2$ down to the triangle $\\Delta^2$, contracting each line bounding the triangle down to the edge of the triangle according to $r_1$. In general, having constructed $r_i$ for $i<n$, the existence of such an $r_n$ follows from the Tietze extension theorem (or more geometrically, from obstruction theory, if you triangulate $s\\Delta^n$ such that each of the faces are subcomplexes).", "\nThese maps $r_n$ will induce a map of chain complexes $r^*:C_*(X)\\to sC_*(X)$ where $C_*(X)$ is the ordinary singular chain complex and $sC_*(X)$ is the singular chain complex using $s\\Delta^n$ instead of $\\Delta^n$. There is also a map $i^*:sC_*(X)\\to C_*(X)$ given simply by restricting from $s\\Delta^n$ to $\\Delta^n$, and $i^*r^*=1$. To prove that $i^*$ induces isomorphisms on homology, it thus suffices to show that $r^*i^*:sC_*(X)\\to sC_*(X)$ is chain-homotopic to the identity.", "\nTo construct the chain homotopy, we just extend the $r_n$'s to a sort of deformation retraction. ", " Specifically, let $P^n$ be the space obtained by gluing together $n$ copies of $s\\Delta^n$ the same way that $n$ copies of $\\Delta^n$ are glued together to form the standard triangulation of $\\Delta^n\\times[0,1]$. In particular, $P^n$ has two \"start and end\" faces which correspond to the faces $\\Delta^n\\times\\{0\\}$ and $\\Delta^n \\times\\{1\\}$ of $\\Delta^n\\times[0,1]$, and \"side\" copies of $P^{n-1}$ which correspond to $F\\times[0,1]$ for each face $F$ of $\\Delta^n$. We can now construct a sequence of maps $h_n:P^{n+1}\\to s\\Delta^n$ such that $h_n$ is given by $h_{n-1}$ on each side face (mapping to the corresponding face of $s\\Delta^n$), is given by the identity on the bottom face, and is given by $r_n$ on the top face. ", " Indeed, given $h_i$ for each $i<n$, the existence of $h_n$ with the required properties follows from the Tietze extension theorem.", "\nThese maps $h_n$ then induce maps $h_n^*:sC_n(X)\\to sC_{n+1}(X)$, sending each simplex to the alternating sum of the $n+1$ simplices of $P^{n+1}$ which are mapped into $X$ by composing with $h_n$. Since $h_n$ is given by $h_{n-1}$ on the side faces, these maps $h_n^*$ will define a chain homotopy between what the $h_n$'s do on the bottom and top faces, i.e. between the identity and $r^*i^*$.\n\nMore generally, similar arguments show that if you replace the simplices $\\Delta^n$ with any sufficiently nice levelwise contractible cosimplicial space, the homology you get will be naturally isomorphic to singular homology (or more strongly, the \"singular set\" you get by mapping out of your cosimplicial space will be naturally weak equivalent to the usual singular set). ", " Here \"sufficiently nice\" turns out to mean \"Reedy cofibrant\"; more generally, if I'm not mistaken, then the following is true:\n\nLet $C$ be a model category and $A$ be a fibrant object in $C$. ", " Define $h_A:C^{\\Delta}\\to(\\mathtt{Set}^{\\Delta^{op}})^{op}$ by $h_A(X)_n=\\operatorname{Hom}(X_n,A)$. Then $h_A$ is a left Quillen functor for the Reedy model structure on $C^{\\Delta}$, and in particular it preserves weak equivalences between Reedy cofibrant objects.", "\n\nIn other words, working very generally in a model category, you could define a \"singular homology\" theory using maps out of a cosimplicial object, and then the singular homology theories given by weak equivalent Reedy cofibrant cosimplicial objects agree on fibrant objects.", "\n\n" ]

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[ "Q:\n\nProgrammatically fireEvent in TextField - JavaFX\n\nLet's say a have a TextField\nTextField tf = new TextField();\n\nand I want to simulate the user pressing a key. ", "\nKeyEvent ke = new KeyEvent(KeyEvent.", "KEY_RELEASED, \n KeyCode.", "A.toString(), KeyCode.", "A.toString(), \n KeyCode.", "A, false, false, false, false);\ntf.fireEvent(ke);\n\nif I examine the text contents of tf, I will see this state hold true\ntf.getText() == \"\"\n\nWhat happened to my KeyEvent and why didn't the text equal \"a\"? ", "I have tried this with KEY_RELEASED, KEY_PRESSED, and KEY_TYPED\nContained Example:\nimport org.junit.", "Assert;\nimport org.junit.", "Rule;\nimport org.junit.", "Test;\n\nimport javafx.scene.control.", "TextField;\nimport javafx.scene.input.", "KeyCode;\nimport javafx.scene.input.", "KeyEvent;\n\nimport java.util.concurrent.", "CountDownLatch;\n\nimport javax.swing.", "SwingUtilities;\n\nimport javafx.application.", "Platform;\nimport javafx.embed.swing.", "JFXPanel;\n\nimport org.junit.rules.", "TestRule;\nimport org.junit.runner.", "Description;\nimport org.junit.runners.model.", "Statement;\n\npublic class TextFieldExample {\n\n @Rule public JavaFXThreadingRule javafxRule = new JavaFXThreadingRule();\n @Test\n public void onlyAcceptValidInputs() {\n TextField tf = new TextField();\n KeyEvent ke = new KeyEvent(KeyEvent.", "KEY_RELEASED, \n KeyCode.", "A.toString(), KeyCode.", "A.toString(), \n KeyCode.", "A, false, false, false, false);\n tf.fireEvent(ke);\n Assert.assertEquals(\"A\", tf.getText());\n }\n}\n\nclass JavaFXThreadingRule implements TestRule { \n /**\n * Flag for setting up the JavaFX, we only need to do this once for all tests.", "\n */\n private static boolean jfxIsSetup;\n\n @Override\n public Statement apply(Statement statement, Description description) {\n\n return new OnJFXThreadStatement(statement);\n }\n\n private static class OnJFXThreadStatement extends Statement {\n\n private final Statement statement;\n\n private OnJFXThreadStatement(Statement aStatement) {\n statement = aStatement;\n }\n\n private Throwable rethrownException = null;\n\n @Override\n public void evaluate() throws Throwable {\n\n if(!jfxIsSetup) {\n setupJavaFX();\n\n jfxIsSetup = true;\n }\n\n final CountDownLatch countDownLatch = new CountDownLatch(1);\n\n Platform.runLater(new Runnable() {\n @Override\n public void run() {\n try {\n statement.evaluate();\n } catch (Throwable e) {\n rethrownException = e;\n }\n countDownLatch.countDown();\n }});\n\n countDownLatch.await();\n\n // if an exception was thrown by the statement during evaluation,\n // then re-throw it to fail the test\n if(rethrownException !", "= null) {\n throw rethrownException;\n }\n }\n\n protected void setupJavaFX() throws InterruptedException {\n final CountDownLatch latch = new CountDownLatch(1); \n SwingUtilities.invokeLater(new Runnable() {\n public void run() {\n // initializes JavaFX environment\n new JFXPanel(); \n latch.countDown();\n }\n });\n\n latch.await();\n }\n\n }\n}\n\nA:\n\nIt seems the text field is listening for KEY_TYPED events. ", "Note that for KEY_TYPED events, the text should be the empty string, and the code should be KeyCode.", "UNDEFINED.", "\nNote also that in your test case, the text field is never added to a live scene. ", "Because of this, it doesn't install its skin, which contains the logic for actually processing events. ", "So you need to force the installation of the skin by calling setSkin(...), passing in the result of the protected method createDefaultSkin():\nimport javafx.application.", "Application;\nimport javafx.geometry.", "Pos;\nimport javafx.scene.", "Scene;\nimport javafx.scene.control.", "Button;\nimport javafx.scene.control.", "TextField;\nimport javafx.scene.input.", "KeyCode;\nimport javafx.scene.input.", "KeyEvent;\nimport javafx.scene.layout.", "VBox;\nimport javafx.stage.", "Stage;\n\npublic class TextFieldFireEvent extends Application {\n\n @Override\n public void start(Stage primaryStage) {\n TextField tf = new TextField() {\n {\n setSkin(createDefaultSkin());\n }\n };\n\n Button button = new Button(\"A\");\n button.setOnAction(e -> {\n KeyEvent ke = new KeyEvent(KeyEvent.", "KEY_TYPED, \n \"a\", \"\", \n KeyCode.", "UNDEFINED, false, false, false, false);\n tf.fireEvent(ke);\n System.out.println(tf.getText());\n });\n VBox root =new VBox(10, /*tf,*/ button);\n\n root.setAlignment(Pos.", "CENTER);\n primaryStage.setScene(new Scene(root, 400, 400));\n primaryStage.show();\n }\n\n public static void main(String[] args) {\n launch(args);\n }\n}\n\n" ]

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[ "INTRODUCTION {#SEC1}\n============\n\nA wide spectrum of functionally different mRNAs and protein isoforms can be obtained from a single primary transcript by way of alternative pre-mRNA splicing, an active process in the majority of human genes ([@B1]). ", "Intron excision is performed by the spliceosome ([@B2]), which precisely recognizes the exon--intron borders guided by a multitude of *cis*-acting sequence motifs and *trans*-acting factors like regulatory proteins---altogether termed the 'splicing code' ([@B3],[@B4]). ", "In particular, splice site selection is equally controlled both by the proper 5′ and 3′ splice sites (5′ss and 3′ss) and by nearby activating or repressing splicing regulatory elements (SREs) ([@B5],[@B6]).", "\n\nDisease-associated aberrant splicing can e.g. be caused by mutations as well in splice sites as in SREs, which often are 6--8-nucleotide long binding sites for SR-proteins (exonic splicing enhancers) or hnRNP-proteins (intronic splicing enhancers). ", "These SREs have signature positional distributions ([@B7]) and their enhancing or silencing effects on splice site usage depend on their localization with respect to the splice site ([@B8]). ", "In addition, weaker splice sites can be compensated by stronger neighboring splicing enhancers, thus avoiding aberrant splicing ([@B8]).", "\n\nComputational identification of SREs is highly important e.g. in human genetics, and several current bioinformatics algorithms for SRE identification provide sets of hexamers ([@B9]) as binding sites for splicing regulatory proteins. ", "Aggregating SREs across available algorithms currently yields 979 exonic splicing enhancers (ESE) motifs and 496 exonic splicing silencer (ESS) motifs ([@B16]), as well as recently described position-dependent enrichment of multivalent tetramer motifs ([@B17]). ", "In a systematic experimental evaluation, Ke *et al.* ", "inserted all 4096 hexamers at five positions in two different internal exons of a 3-exon minigene, obtaining 1182 ESE and 1090 ESS motifs ([@B15]). ", "Thus, in genomic sequences SRE searches frequently detect entire arrays of motif 'hits', and most mutations in the vicinity of a splice site alter at least one putative SRE, which renders mutation assessment difficult ([@B18]). ", "Recently, a machine learning approach using a random forest algorithm yielded a mutation classifier using a variety of SNP-, exon- and gene-based features, which takes into account mutational changes both in splice site and SRE sequences ([@B16]). ", "While this approach attempts to unify the effects of intrinsic splice site properties and of neighboring regulatory elements and thus presents a step toward a 'functional splice site score', it also includes highly non-local information like evolutionary conservation and properties of the entire gene. ", "In contrast, it is our goal to derive a scoring for SREs that only uses properties of the splice site neighborhood.", "\n\nWe selected the HIV-1 genome as a model system for several reasons: (i) it is ∼10 000 nt small and its splicing patterns during early and late phases of the replication cycle are well characterized, (ii) it contains highly regulated splice sites dependent on many known SREs ([@B19]) and (iii) HIV-1 splicing is easily experimentally accessible both in subgenomic splicing reporter constructs and in replication competent virus.", "\n\nWithin HIV-1-infected cells, more than 40 different viral mRNAs are spliced from a single primary RNA transcript. ", "Depending on introns retained, these RNAs can be separated into three distinct classes: intronless (2 kb), intron-containing (4 kb) and unspliced (9 kb) viral mRNAs ([@B40],[@B41]). ", "The sophisticated splicing pattern is derived from alternatively used subsets of at least four viral 5′ss and eight 3′ss. ", "Splice site selection is controlled by SREs, which can either activate or repress functional recognition of a nearby splice site ([@B40]). ", "Disruption of only one of these viral SREs can severely interfere with the viral splicing balance, which has to be maintained for proper replication ([@B19],[@B20]). ", "For instance, exon 3 splicing is repressed by the *vpr* exonic splicing silencer ESSV ([@B19],[@B21]), and inactivation of vpr exonic splicing silencer (ESSV) results in dramatically increased levels of exon 3 inclusion, abolishing unspliced viral mRNAs and thus suppressing virus particle production ([@B19],[@B21]).", "\n\nIn this study, we defined and validated a 'HEXplorer score' for every nucleotide in a genomic sequence, based on all overlapping hexamers rather than only on dedicated SRE motifs. ", "We hypothesize this HEXplorer score to capture the splice enhancing and silencing properties of genomic regions in the vicinity of splice sites. ", "Using the HIV-1 pre-mRNA as a model system highly dependent on SREs, we found an excellent correlation in 29 mutations between splicing activity and HEXplorer score. ", "We successfully predicted and confirmed five novel ESEs and optimized mutations inactivating the known silencer ESSV. ", "The HEXplorer score ([www.uni-duesseldorf.de/rna](http://www.uni-duesseldorf.de/rna)) allows landscaping of splicing regulatory regions, provides a quantitative measure of mutation effects on splice enhancing and silencing properties, and permits calculation of the mutationally most effective nucleotide.", "\n\nMATERIALS AND METHODS {#SEC2}\n=====================\n\nOligonucleotides {#SEC2-1}\n----------------\n\nOligonucleotides were obtained from Metabion GmbH (Martinsried, Germany).", "\n\nPrimers used for site-directed mutagenesis (see Supplementary Tables S1 and S2).", "\n\nPrimers used for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analyses (see Supplementary Table S3).", "\n\nHIV-1-based subgenomic splicing reporter {#SEC2-2}\n----------------------------------------\n\nThe LTR ex2 ex3 minigenes were generated as described previously ([@B20],[@B23]). ", "Exon 3 mutants were constructed by PCR mutagenesis. ", "For construction, the *Alw*NI/*Spe*I fragment of LTR ex2 ex3 was replaced by the respective PCR products using the appropriate forward PCR primer (see Supplementary Table S1) and \\#2588 as a reverse PCR primer containing *Alw*NI and *Spe*I restriction sites. ", "After cloning, all PCR amplicons were validated by sequencing.", "\n\nSV-*env* ([@B24]) derived splicing reporters (see Supplementary Table S2) were constructed by replacing the *Eco*RI/*Sac*I fragment with respective PCR products: exon 3 and exon 3 ESSV^−^ (\\#1906/\\#1907) or linkers: part I (\\#1958/\\#1959), part II (\\#1960/\\#1961), part II ESSV^−^ (\\#1962/\\#1963) and part III (\\#1964/\\#1965).", "\n\nSV-*env/eGFP* reporters ([@B42]) were generated by substitution of the *Eco*RI/*Nde*I fragment with respective PCR products using the appropriate forward PCR primer (see Supplementary Table S2) and \\#640 as a reverse primer. ", "SV-SD4-*env*/*eGFP* reporters (corresponding to SV-*env*/*eGFP* with HIV-1 D4 sequence instead of D1) were cloned by replacing the *EcoR*I/*Sac*I fragment with the indicated linker sequences.", "\n\nProviral HIV-1 plasmids {#SEC2-3}\n-----------------------\n\npNL4-3 mutants were constructed by replacing the region between *Pfl*MI and *Eco*RI with mutated LTR ex2 ex3 fragments as described previously ([@B20]).", "\n\nCell culture and RT-PCR analysis {#SEC2-4}\n--------------------------------\n\nHeLa and HEK 293T cells were maintained in Dulbecco\\'s high glucose-modified Eagle\\'s medium (Invitrogen) supplemented with 10% fetal calf serum and 50 μg/ml of each penicillin and streptomycin (Invitrogen). ", "Transfections were done in 6-well plates with 2.5 × 10^5^ cells per plate using TransIT®-LT1 reagent (Mirus) according to the manufacturer\\'s instructions. ", "Total RNA samples were collected 48 h after transfection from either HeLa or HEK 293T cells transfected with subgenomic or proviral constructs and pXGH1 to control transfection efficiency. ", "For reverse transcription, 4 μg of RNA were subjected to DNA digestion with 10 U of DNase I (Roche). ", "DNase I was heat-inactivated at 70°C for 5 min and cDNA synthesis occurred for 1 h at 50°C and 15 min at 72°C by using 200 U Superscript III RNAse H^−^ Reverse Transcriptase (Invitrogen), 7.5-pmol oligo(dT)~12--18~ (Invitrogen) as primer, 20 U of RNAsin (Promega) and 10 mM of each deoxynucleoside triphosphate (Qiagen). ", "For semi-quantitative analysis of LTR ex2 ex3 minigene mRNAs, cDNA was used as a template for a PCR reaction with forward primer \\#1544 and reverse primer \\#2588 (see Supplementary Table S3). ", "For semi-quantitative analysis of SV-*env/eGFP*-derived reporter mRNAs, cDNA was used as a template for a PCR reaction with forward primer \\#3210 and reverse primer \\#3211. ", "For transfection control, PCR was performed with primers \\#1224 and \\#1225 to specifically detect GH1 mRNA. ", "For analysis of exon 3 inclusion in viral *tat* mRNAs and *vpr* mRNA splicing, a PCR reaction was carried out using primers \\#1544 (E1) and \\#3632 (E4). ", "For the analysis of intronless 1.8-kb HIV-1 mRNAs, PCR reaction was carried out with forward primer \\#1544 (E1) and reverse primer \\#3392 (E7). ", "Finally, intron containing 4.0-kb HIV-1 mRNAs were detected with primers \\#1544 (E1) and \\#640 (I4). ", "PCR products were separated on 8% non-denaturing polyacrylamide gels and stained with ethidium bromide for visualization.", "\n\nAntibodies {#SEC2-5}\n----------\n\nThe following primary antibodies were used for immunoblot analysis: mouse antibody against α-actin (A2228) was obtained from Sigma-Aldrich. ", "Sheep antibody against HIV-1 p24 was purchased from Biochrom AG. ", "For detection, we used a horseradish peroxidase (HRP)-conjugated anti-mouse antibody (NA931) from GE Healthcare and an HRP-conjugated anti-sheep antibody from Jackson Immunoresearch Laboratories Inc.\n\nProtein analysis {#SEC2-6}\n----------------\n\nTransfected cells were lysed in radio immunoprecipitation assay (RIPA) buffer (25-mM Tris·HCl pH 7.6, 150-mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), protease inhibitor cocktail (Roche)). ", "Proteins were separated by SDS polyacrylamide gel electrophoresis, transferred on a nitrocellulose membrane and subjected to immunoblotting procedure. ", "Membranes were probed with the respective primary and secondary antibodies and developed with ECL chemiluminescence reagents (GE Healthcare).", "\n\nHexamer score calculation {#SEC2-7}\n-------------------------\n\nFollowing the RESCUE concept ([@B9]), hexamer frequencies were determined in the four data sets of up to 100-nt long exonic and intronic sequences up- and downstream of 10 359 weak and 10 407 strong constitutive canonical 5′ splice sites, respectively. ", "All 11 nucleotides (three exonic, eight intronic) of the 5′ splice site consensus sequence were excluded from the analysis to avoid hexamer count bias from splice site motif conservation. ", "Weak and strong 5′ss were selected as lower (HBS ≤ 13.5) and upper (HBS \\> 17.0) quartiles of the HBond score (HBS) distribution ([@B43]).", "\n\nTo compare the relative occurrences in both data sets of a given hexamer that occurred *f~E~* times in the exonic and *f~I~* times in the intronic sequence data set, the normal distributed z-score *Z*~EI~ was calculated as $\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$Z_{{\\rm EI}} = (f_{\\rm E} - f_{\\rm I} )/\\sqrt {(1/N_{\\rm E} + {\\rm 1}/N_{\\rm I}{\\rm {\\rm }} ) \\cdot g \\cdot (1 - g)}$\\end{document}$.\n\nHere, *N*~E~ and *N*~I~ denote the total numbers of exonic and intronic positions in the respective data sets, and $\\documentclass[12pt]{minimal}\n\\usepackage{amsmath}\n\\usepackage{wasysym} \n\\usepackage{amsfonts} \n\\usepackage{amssymb} \n\\usepackage{amsbsy}\n\\usepackage{upgreek}\n\\usepackage{mathrsfs}\n\\setlength{\\oddsidemargin}{-69pt}\n\\begin{document}\n}{}$g = (N_{\\rm E} \\cdot f_{\\rm E} + N_{\\rm I} \\cdot f_{\\rm I} )/(N_{\\rm E} + N_{\\rm I} )$\\end{document}$ is the weighted average frequency. ", "The 'weak--strong' Z-score *Z*~WS~ was calculated accordingly, using hexamer frequencies *f*~W~ and *f*~S~ in exons upstream weak and strong 5′ splice sites, respectively.", "\n\nDetection of SREs by other available algorithms {#SEC2-8}\n-----------------------------------------------\n\nThe comparison of the novel HEXplorer score with other available algorithms for the identification of SREs was performed on 50 nucleotides upstream of each 5′ss in the HIV-1 genome. ", "These sequences were screened for enhancer and silencer motifs, using web resources for the following available algorithms ([@B9]): ESEfinder 3.0 (<http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home>), RESCUE-ESE (<http://genes.mit.edu/burgelab/rescue-ese/>), FAS-ESS-hex3 (<http://genes.mit.edu/fas-ess/>), PESX (<http://cubweb.biology.columbia.edu/pesx/>) and ESRsearch (<http://esrsearch.tau.ac.il/>). ", "Where appropriate, the respective default settings for SRE detection were applied.", "\n\nFrom all these algorithms, motifs were identified in reference and mutated sequences, and a weight of +1 or −1 was assigned to a sequence for each predicted enhancer or silencer motif that overlapped with a mutated nucleotide. ", "From the change in the number of ESE- and ESS-motifs induced by these mutations, we calculated an overall 'exonic splicing motif difference' (ESMD) similar to Ke *et al.* ([", "@B44]), capturing the summary effect of ESR gain and loss predicted by the various algorithms.", "\n\nSystematic comparison of HEXplorer score and ESRseq score was performed by extracting hexamer weights for 1182 ESS and 1090 ESS hexamer motifs provided by Ke *et al.* ([", "@B15]). ", "Using these hexamer weights instead of the weights obtained from our data set, we calculated the average ESRseq score of all hexamers overlapping with any index nucleotide in a genomic sequence. ", "In this way, we obtained a metric based on the ESRseq score that permitted a direct comparison to the HEXplorer score.", "\n\nRESULTS {#SEC3}\n=======\n\nDerivation of HEXplorer score---an *in silico* approach for SREs {#SEC3-1}\n----------------------------------------------------------------\n\nThe HEXplorer score presented in this work was based on hexamer weights calculated by a RESCUE-type approach ([@B9]). ", "Splice site recognition depends more strongly on the presence of SREs for weak than for strong 5′ss ([@B8]). ", "Therefore, hexamers associated with ESEs are generally assumed to be over-represented in exonic sequences upstream of weak 5′ss compared to sequences upstream of strong 5′ss, as well as in exonic versus intronic sequences. ", "Here, RESCUE-type hexamer weights were derived from hexamer frequencies in four sets of exonic and intronic sequences taken from the human 5′ splice site data set described in ([@B45]). ", "This data set contained 43 464 constitutively spliced internal human exons with canonical 5′ss collected from the ENSEMBL database (chromosomes 6, 7, 9, 10, 13, 14, 20, 22, X). ", "Intrinsic strength of 5′ splice sites was assessed by the HBS describing 5′ss complementarity with all 11 nucleotides of the free 5′ end of U1 snRNA ([@B43]). ", "In the whole set of 43 464 constitutive canonical 5′ splice sites, the HBS was approximately Gaussian distributed with mean 15.0 and standard deviation (SD) 2.59 (range 1.80--23.8; Figure [1A](#F1){ref-type=\"fig\"}). ", "Weak and strong 5′ss were selected as upper and lower quartiles of the HBS distribution, resulting in 10 359 weak 5′ss with HBS ≤ 13.5 and 10 407 strong 5′ss with HBS \\> 17.0. ", "Subsequently, hexamer frequencies were counted in 100-nt long exonic and intronic sequences up- and downstream of these weak and strong 5′ss, respectively. ", "Each 5′ss could maximally contribute 179 different hexamer positions, and a total of 1 728 912 exonic and 1 835 932 intronic hexamer occurrences were counted in these four sequence data sets.", "\n\n{#F1}\n\nFollowing the RESCUE concept, two normalized Z-scores were subsequently determined for all 4096 hexamers ([@B9]). ", "Each score reflects the difference in hexamer occurrence between two sets of 5′ss neighborhoods: for each hexamer, its exon--intron Z-score *Z*~EI~ is the scaled difference of hexamer frequency between 100-nt long sequences up- and downstream of 5′ss, while its weak--strong Z-score *Z*~WS~ measures the difference in hexamer occurrence between exons upstream of weak and strong 5′ss, respectively. ", "Figure [1](#F1){ref-type=\"fig\"} B shows the scatterplot of *Z*~WS~ versus *Z*~EI~ for all individual 4096 hexamers. ", "Hexamers plotted e.g. in the upper right hand corner were found more frequently in exons compared to introns, and more often in exons of weak than of strong splice sites. ", "Within the RESCUE framework, such hexamers are considered as candidate sequences for exonic splicing enhancers. ", "Both hexamer scores *Z*~EI~ and *Z*~WS~ were significantly correlated, having Pearson\\'s *r* = 0.70 (*P* \\< 0.0001), as suggested by the elongated shape of the scatterplot cloud. ", "However, hexamer frequencies differed more widely between exons and introns than between exons of weak and strong 5′ss. ", "This was quantitatively reflected in the different hexamer score ranges: across all 4096 hexamers, *Z*~EI~ had a range of −73.3--34.4 (mean of 0.746, SD 8.76, median 1.92), while *Z*~WS~ had a six times smaller range of −7.34--10.5 (mean of −0.167, SD 2.50, median −0.25).", "\n\nDistinct sets of splicing regulatory proteins have been found to support U1 snRNP binding in a strictly position-dependent manner. ", "In particular, exonic or intronic enhancer sequences frequently act as silencers in their respective position opposite the 5′ss---they have a signature positional distribution around the splice site ([@B7],[@B8]). ", "Therefore, sequences acting as exonic splicing silencers that are statistically depleted upstream of functional 5′ss are at the same time enriched downstream as intronic enhancers. ", "Thus, we assumed the exon--intron hexamer score *Z*~EI~ to also capture enhancer/silencer sequence properties beyond general exon--intron differences, and selected *Z*~EI~ for our further studies. ", "Furthermore, we expected *Z*~EI~ to have higher discriminatory power due to its larger range of values across hexamers.", "\n\nAiming at developing a RESCUE-concept-based score for each nucleotide position in a given sequence, we next calculated the HEXplorer score HZ~EI~ for every index nucleotide as the average hexamer score of all six hexamers overlapping with the index nucleotide. ", "Beyond the index nucleotide, this HEXplorer score HZ~EI~ depends on 5 nt to both sides (up- and downstream). ", "In the exemplary sequence GTGAA**[T]{.ul}**ATCAA, e.g. the index nucleotide **[T]{.ul}** (HZ~EI~ = −1.93) is located at the last position in hexamer GTGAA**[T]{.ul}** (*Z*~EI~ = −1.93), at the fourth position in hexamer GAA**[T]{.ul}**AT (*Z*~EI~ = −0.69) and at the first position in **[T]{.ul}**ATCAA (*Z*~EI~ = 2.62). ", "A schematic representation of HZ~EI~ calculation from all six hexamers containing the index nucleotide **[T]{.ul}** in this exemplary sequence is depicted in Figure [1C](#F1){ref-type=\"fig\"}. ", "By its construction as a moving average, the HEXplorer score varies more slowly along the sequence than the individual contributing hexamer scores *Z*~EI~, similar to the effect of a low-pass filter.", "\n\nHEXplorer score values were graphically represented by a bar graph along a sequence (Figure [1D](#F1){ref-type=\"fig\"}). ", "In this picture, HZ~EI~-positive areas above the horizontal axis indicated regions with exonic enhancer properties, while areas below the axis corresponded to sequences with exonic silencer properties. ", "Upstream of 5′ss, HZ~EI~-positive sequence stretches that contained more nucleotides and/or had higher HEXplorer scores were expected to possess stronger exonic enhancing property and more efficiently support splicing.", "\n\nIt is important to note that within this framework we associated 'exonic splice enhancing' with being a property of an entire sequence 'region' rather than that of a single hexamer. ", "Correspondingly, the HEXplorer score was constructed to take into account 'all' hexamer frequencies in any given sequence rather than just a small subset of 'Z-extreme' hexamers indicating specific protein binding sites. ", "Quantitatively, the splice enhancing property of a region in the context of a given splice site was measured by the area under the HZ~EI~ graph in this region.", "\n\nHEXplorer score plots provide distinct exonic enhancer/silencer profiles for all HIV exons {#SEC3-2}\n------------------------------------------------------------------------------------------\n\nIn order to validate the HEXplorer score, we systematically examined HZ~EI~ in all exons of the entire HIV-1 genome, which is known to contain a large number of SREs ([@B40]). ", "Figure [2](#F2){ref-type=\"fig\"} presents an overview of the HIV-1 genome with all known exonic splicing regulatory motifs marked by green (enhancer) or red (silencer) boxes.", "\n\n![", "Schematic drawing of known and HEXplorer-predicted splicing regulatory elements (SREs) in the HIV-1 genome. ", "Top: open reading frames are indicated by open boxes. ", "The long terminal repeats (LTRs) are located at both ends of the provirus. ", "Center: all HIV-1 proteins are encoded by a single primary RNA. ", "Alternative splicing leads to more than 40 different mRNA transcripts enabling efficient translation of all open reading frames within the host cell. ", "Intrinsic strength of the 5′ss (D1--D4) and 3′ss (A1--A7) is indicated in the parentheses (5′ss: HBond Score, <http://www.uni-duesseldorf.de/rna>; 3′ss: MaxEntScore, <http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.html>). ", "Bottom: positions of the SREs within the HIV-1 pre-mRNA: known splicing enhancers (green) and silencers (red) are indicated together with HEXplorer-predicted enhancers (dashed green). ", "\\[ESE-Ld2 ([@B25]); ESE-Vif ([@B27]); ESEM ([@B26]); guanosine (G)-rich silencer G4 ([@B27]); G~I2~--1 ([@B23]); ESSV ([@B19],[@B21]); ESE*~vpr~* ([@B20]); ESS2p ([@B28]); ESE2 ([@B29],[@B30]); ESS2 ([@B31]); guanosine-adenosine-rich (GAR) ESE ([@B24],[@B34],[@B46]); E42 fragment ([@B34]); ISS ([@B35]); ESE3 ([@B36]); ESS3 ([@B36]) (adapted to ([@B34],[@B39])\\].](gku736fig2){#F2}\n\nWe then calculated and plotted HZ~EI~ HEXplorer profiles for splicing relevant sequences in HIV-1 exons 1, 2, 2b, 3, 4, 5 and 7 (Figure [3](#F3){ref-type=\"fig\"}). ", "The examined exonic sequences were between 50-nucleotides (for exon 2) and 97 nucleotides (for exon 7) long. ", "In all seven HIV-1 exons, HZ~EI~ graphs slowly varied between peak values of −11 and 33 and contained both HZ~EI~-positive and -negative sequence stretches. ", "In six out of seven exons, HZ~EI~ graphs were mostly dominated by large positive areas, and only exon 3 contained a long HZ~EI~-negative sequence stretch corresponding to the known silencer ESSV. ", "In all seven exons, known exonic enhancers and silencers correlated well with HZ~EI~-positive and -negative sequence stretches, respectively.", "\n\n{#F3}\n\nIn particular, the exonic splicing silencers G~I2~--1 (exon 2b), ESSV (exon 3), ESS2p and ESS2 (exon 4) as well as ESS3 (exon 7) mostly lie in HZ~EI~-negative regions, whereas the exonic splicing enhancers ESE-Ld2 (exon 1), ESE-Vif, ESEM1 and ESEM2 (exon 2), ESE2 (exon 4), GAR (exon 5), as well as ESE3 (exon 7) lie in HEXplorer score positive regions (Figure [3](#F3){ref-type=\"fig\"}).", "\n\nFrom a helicopter view, these findings confirmed the general qualitative association of HEXplorer score positive and negative sequence stretches with all known HIV-1 exonic enhancers and silencers, respectively. ", "To examine this association in more detail, we studied HIV-1 exon 3 containing both the well-known splicing silencer ESSV ([@B19]) and the recently found enhancer ESE*~vpr~* ([@B20]).", "\n\nSplice enhancing and silencing properties of exon 3 fragments correlate with HEXplorer score {#SEC3-3}\n--------------------------------------------------------------------------------------------\n\nThe HZ~EI~-plot along the pNLA1-derived HIV-1 exon 3 sequence ([@B47]) clearly depicted HZ~EI~-positive and -negative regions (Figure [4A](#F4){ref-type=\"fig\"}). ", "For a closer examination, exon 3 was partitioned into three consecutive parts (I--III) of equal sequence length. ", "With respect to the HEXplorer score, these three parts corresponded to (I) a HZ~EI~ heterogeneous region, (II) a predominantly HZ~EI~-negative region and (III) a mainly HZ~EI~-positive region. ", "The entire exon 3 sequence and the three individual parts I--III were tested in the context of the HIV-1-based splicing reporter (SV-*env*; Figure [4B](#F4){ref-type=\"fig\"}). ", "SV-*env* is a single intron splicing reporter that contains an enhanced green fluorescent protein (eGFP) coding region downstream of the splice acceptor, permitting fluorescence monitoring and western blot analysis of HIV-1 glycoprotein expression depending on U1 snRNA binding to 5′ss D4. ", "In addition, a proximal enhancer is required for binding of the U1 snRNP to the splicing reporter\\'s 5′ss D4. ", "Moreover, only in the presence of the HIV-1 regulatory protein Rev, U1 snRNP binding at the reporter\\'s 5′ss D4 correlates with the expression of viral glycoprotein gp160 and gp120 ([@B24],[@B43],[@B46]).", "\n\n![", "Splice enhancing and silencing properties of exon 3 fragments correlate with HEXplorer score. (**", "A**) HZ~EI~-score plot of the HIV-1 exon 3 sequence partitioned into three consecutive fragments (parts I--III) of equal sequence length. ", "HZ~EI~-positive and -negative regions are indicated in green and red, respectively. (**", "B**) Schematic of the SV-*env* expression plasmid carrying the *env* open reading frame (ORF). ", "HIV-1 exon 3-derived sequences that were inserted upstream of the splicing enhancer dependent 5′ss D4 are enlarged below. ", "ESSV^−^ is known to disrupt the silencer ESSV. ", "Either single part I, part II, part II ESSV^−^ or part III was inserted, but no combinations. (**", "C**) Western blot analysis of cell lysates from HeLa cells transiently transfected with 1 μg of each of the constructs together with 1-μg SVcrev and 1 μg of pGL3 (Promega) to control for equal transfection efficiencies. ", "Forty-eight hours post transfection proteins were extracted and separated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ", "Samples were normalized for equal protein and luciferase amounts as described previously ([@B46]). ", "After transfer to a nitrocellulose membrane, samples were probed with a mouse monoclonal antibody specifically detecting splicing-dependent gp120 expression within the transfected cells (87--133/026; kindly provided by Dade Behring). (", "SV40: simian virus 40 early promoter; RRE: Rev responsive element.)](gku736fig4){#F4}\n\nWhile the wild-type exon 3 sequence failed to enhance glycoprotein expression, inactivation of the repressing ESSV activity as described previously ([@B21]) led to considerable expression of gp160 (and the proteolytic cleavage product gp120) within the cells (Figure [4C](#F4){ref-type=\"fig\"}, upper panel, cf. ", "lanes 1 and 2). ", "The HZ~EI~-heterogeneous part I alone slightly enhanced glycoprotein expression (Figure [4C](#F4){ref-type=\"fig\"}, upper panel, lane 3) consistent with its weak enhancing property predicted by HZ~EI~. Similarly, both in the presence and absence of an intact ESSV, the HZ~EI~-negative part II did not support glycoprotein expression (Figure [4C](#F4){ref-type=\"fig\"}, upper panel, lanes 4 and 5). ", "In contrast, the HZ~EI~-positive part III expected to support U1 snRNP binding strongly increased the levels of gp160 and gp120, confirming ESE*~vpr~* activity ([@B20]). ", "RT-PCR analysis showed the same pattern for the spliced message (Figure [4C](#F4){ref-type=\"fig\"}, lower panel). ", "However, a high amplification had to be chosen in order to detect even low levels of the spliced message (cf., ", "e.g. Figure [4C](#F4){ref-type=\"fig\"}, lower panel, lane 5), leading to an overamplification of the unspliced message in the nonlinear range.", "\n\nFor each part, the HEXplorer score-based prediction of exonic enhancer/silencer property correlated well with glycoprotein expression as surrogate marker of U1 snRNP binding. ", "Therefore, the HEXplorer score may be a valuable screening tool for narrowing down regions with enhancer or silencer property as candidates for more detailed analysis by mutagenesis.", "\n\nHIV-1 exon 3 inclusion correlates well with HEXplorer score change in ESE*~vpr~* mutation analysis {#SEC3-4}\n--------------------------------------------------------------------------------------------------\n\nIn order to examine HEXplorer predictions of mutation effects on splice site usage, we systematically introduced single- and double-point mutations in the recently described Tra2α-/Tra2β-dependent ESE*~vpr~* ([@B20]). ", "We used a subgenomic pNLA1-derived four exon splicing reporter (Figure [5A](#F5){ref-type=\"fig\"}) with an inactivated ESSV resulting in an intermediate exon 3 splicing phenotype (Figure [5E](#F5){ref-type=\"fig\"}, cf. ", "lanes 1 and 2). ", "This reporter permitted determining positive and negative effects on the degree of exon 3 inclusion. ", "All 16 mutants tested in the ESE*~vpr~* region are given in Figure [5B](#F5){ref-type=\"fig\"} and their positions are denoted by arrows in the HEXplorer graph shown in Figure [5C](#F5){ref-type=\"fig\"}. ", "Semi-quantitative RT-PCR of RNA isolated from transiently transfected HeLa cells was carried out to measure the level of exon 3 splicing for each mutated minigene.", "\n\n![", "Detailed HEXplorer-guided mutational analysis of ESE*~vpr~*. (**", "A**) Schematic of the HIV-1-based LTR ex2 ex3 splicing reporter. ", "Positions of the RT-PCR primers (\\#1544/\\#2588) are indicated by arrows. (**", "B**) ESE*~vpr~* reference (pNLA1) and mutant sequences. ", "Nucleotide residues are denoted by their position relative to the GT-dinucleotide. ", "Exon--intron border of exon 3 is denoted by '/' and 5′ss D3 sequence is underlined. (**", "C**) HEXplorer score profile plotted along the 3' end of exon 3 containing ESE*~vpr~* (green bar). ", "Positions of mutated nucleotides are indicated by arrows. (**", "D**) HEXplorer score profiles for reference ESE*~vpr~* (gray) and exemplary point mutation −17A\\>T (red, indicated by arrow) plotted along the same sequence as in (C). (**", "E**) RT-PCR of RNA from HeLa cells transfected with mutants described in (B). ", "HEXplorer score difference ΔHZ~EI~ between mutant and reference (ESE*~vpr~* (ESSV^−^) lane 2) as well as exon 3 inclusion (upper band Tat3 \\[1/3/4\\]) level is given below each lane. ", "Mean inclusion level and standard deviation (±SD) were calculated from triplicates. ", "GH1 (growth hormone) serves as control for transfection efficiency. ", "2.5 × 10^5^ HeLa cells were transiently transfected with 1 μg of each of the constructs together with 0.2 μg of SVctat and 1 μg of pXGH5. ", "Thirty hours after transfection RNA was isolated from the cells and subjected to RT-PCR analysis using primer pairs \\#1544/\\#2588 and \\#1224/\\#1225 (GH1). ", "PCR products were separated by 8% non-denaturating polyacrylamide gel electrophoresis and stained with ethidium bromide.](gku736fig5){#F5}\n\nIn order to assess the point mutations' effects on the enhancer property of the ESE*~vpr~* region, we calculated the difference between the two HEXplorer score areas of mutant and reference (intact ESE*~vpr~* with ESSV^−^) sequences in a sufficiently long window upstream of 5′ss D3. ", "We provide the HEXplorer score algorithm in an Excel file as Supplementary material (cf. ", "Supplementary XLSM-file) and for download at [www.uni-duesseldorf.de/rna](http://www.uni-duesseldorf.de/rna). ", "Any such point mutation affected only the HEXplorer scores of 11 nucleotides centered on the point mutation itself (5 nt up- and downstream; Figure [5D](#F5){ref-type=\"fig\"}). ", "A HEXplorer score difference of zero thus corresponded to comparable enhancer property levels in mutant and reference, which in this case exhibited partial exon inclusion. ", "Figure [5E](#F5){ref-type=\"fig\"} shows the exon 3 inclusion ratio and HEXplorer score difference ΔHZ~EI~ for wild type, reference (intact ESE*~vpr~* with ESSV^−^) and all 16 mutants.", "\n\nObserved changes both for higher and lower levels of exon 3 inclusion were mostly consistent with the HEXplorer score differences ΔHZ~EI~ mutant--reference. ", "A large positive or slightly negative HEXplorer score difference was found, if the mutant sequence had a stronger enhancer property than the reference (intact ESE*~vpr~* with ESSV^−^), and such mutants were associated with a higher degree of ESE*~vpr~*-dependent exon inclusion (e.g. Figure [5E](#F5){ref-type=\"fig\"}, lanes 12--18). ", "Correspondingly, negative HEXplorer score differences were mostly found in mutant sequences with considerably weaker splice enhancing property than the reference (e.g. Figure [5E](#F5){ref-type=\"fig\"}, lanes 3--5 and 9, 10). ", "Thus, mutants with large negative ΔHZ~EI~ were prone to disrupt the enhancer ESE*~vpr~*.", "\n\nThese experiments showed a good semi-quantitative correlation between inclusion ratio and HEXplorer score difference in a series of mutations in the splicing enhancer ESE*~vpr~*. ", "In the next step, we applied the HEXplorer score to ESSV mutation optimization.", "\n\nHEXplorer pre-screening identifies minimal point mutations disrupting ESSV {#SEC3-5}\n--------------------------------------------------------------------------\n\nUsing the HEXplorer score-based mutation assessment, automatic screening of entire sequence regions for point mutations with maximal effects became possible. ", "To this end, every single nucleotide in the region was systematically replaced with each of the three other possible nucleotides by the HEXplorer algorithm. ", "From the 3·N alternative sequences obtained from an N nucleotide long sequence, point mutations with similar HEXplorer score differences were suspected to alter the sequence\\'s enhancing/silencing property to a comparable degree.", "\n\nWe tested this procedure on the splicing silencer ESSV. ", "Starting from the known mutation ESSV^−^ (pNEU), which inactivates ESSV by substituting 7 nucleotides ([@B19]) (Figure [6A](#F6){ref-type=\"fig\"}), we searched for a functionally equivalent but smaller set of point mutations. ", "The known mutation ESSV^−^ (pNEU) had a higher HEXplorer score compared to the reference sequence pNL4-3 (ΔHZ~EI~ = 144.0). ", "From a thorough scrutiny of the HEXplorer score table we identified the double mutation −29G\\>C and −36A\\>C that together increased the pNL4-3 HEXplorer score in a similar order of magnitude (ΔHZ~EI~ = 157.9). ", "From Figure [6B](#F6){ref-type=\"fig\"} it is evident that this double mutation ESSV^−^ (dm) changed the shape of the HEXplorer graph in a similar way as ESSV^−^ (pNEU). ", "In comparison, each single-point mutation had a smaller effect on the HEXplorer score: ΔHZ~EI~ = 92.8 for −29G\\>C and 65.0 for −36A\\>C, respectively.", "\n\n![", "HEXplorer screening identifies minimal point mutations disrupting ESSV. (**", "A**) pNL4-3-derived wild-type and mutated HIV-1 exon 3 sequences. ", "Nucleotide coordinates refer to the 3' end of exon 3. (", "dm) refers to the double mutation −29G\\>C and -36A\\>C. (**B**) HZ~EI~ score profiles of HIV exon 3 reference pNL4--3 (gray) versus mutant sequences ESSV^−^ (pNEU), ESSV^−^ −29G\\>C, ESSV^−^ −36A\\>C and ESSV^−^ (dm) (red). ", "HEXplorer score differences are given below the profile graphs, and point mutations are indicated by black arrows and corresponding positions. (**", "C**) RT-PCR analysis of splicing patterns for different classes of viral RNAs. ", "2.5 × 10^5^ HEK 293T cells were transiently transfected with 1 μg of each of the proviral DNAs. ", "Thirty hours after transfection, total RNA was isolated from the cells and subjected to RT-PCR analyses with different sets of primer pairs covering intronless and intron-containing mRNA classes described elsewhere ([@B20]). ", "RT-PCR products were separated by 8% non-denaturing polyacrylamide gel electrophoresis and visualized using ethidium bromide staining. ", "HIV-1 mRNA isoforms are indicated on the right and correspond to the nomenclature published previously ([@B41]). ", "HZ~EI~-score differences (ΔHZ~EI~) with respect to the reference pNL4-3 are given below the gel. (**", "D**) Western blot analysis of Gag expressed by reference and mutant proviruses. ", "2.5 × 10^5^ HEK 293T cells were transiently transfected with 1 μg of each of the proviral DNAs. ", "Forty-eight hours post transfection viral supernatants were collected, layered onto 20% sucrose solution and centrifuged at 28 000 rpm for 1.30 h at 4°C to pellet the released viral particles. ", "In addition, cells were harvested and resuspended in lysis buffer. ", "Supernatants and cellular lysates were resolved in 12% SDS-PAGE and electroblotted on nitrocellulose membranes. ", "To determine virus particle production and the expression of viral proteins, samples were probed with a primary antibody against HIV-1 p24 (Biochrom AG). ", "Equal amounts of cell lysates were controlled by the detection of α-actin (Sigma-Aldrich, A2228).](gku736fig6){#F6}\n\nThese mutations were now experimentally analyzed for their ability to disrupt the ESSV activity within the infectious molecular clone NL4-3 (GenBank Accession No. ", "M19921). ", "Following transfection of HEK 293T cells with pNL4-3 or the ESSV mutants, the HIV-1 splicing pattern was determined by semi-quantitative RT-PCR using primer pairs spanning intronless or intron-containing HIV-1 mRNA classes (Figure [6C](#F6){ref-type=\"fig\"}). ", "In NL4-3, only low levels of exon 3-including viral mRNA species could be detected (Figure [6C](#F6){ref-type=\"fig\"}, lane 2, e.g. Tat3 or Nef4) due to the silencing activity of ESSV. ", "As expected from preceding studies ([@B19],[@B20]) and in consistence with the higher HEXplorer score (ΔHZ~EI~ = 144.0) relative to pNL4-3, the ESSV^−^ (pNEU) mutation led to an almost complete exon 3 inclusion (Figure [6C](#F6){ref-type=\"fig\"}, cf. ", "lanes 2 and 3, e.g. Tat3 or Nef4) obviously at the expense of the respective exon 3-lacking isoforms (Figure [6C](#F6){ref-type=\"fig\"}, cf. ", "lanes 2 and 3, e.g. Tat1 or Nef2). ", "In agreement with their increased HEXplorer scores (ΔHZ~EI~ = 92.8 for −29G\\>C and 65.0 for −36A\\>C), each of the HEXplorer-identified single-point mutations was already capable to substantially increase the levels of exon 3 inclusion (Figure [6C](#F6){ref-type=\"fig\"}, lanes 4 and 5, e.g. Tat3 or Nef4). ", "However, both point mutations still retained some mRNAs lacking exon 3 (Figure [6C](#F6){ref-type=\"fig\"}, cf. ", "lanes 4 and 5, e.g. Tat1 or Nef2), and were thus less efficient in including exon 3 than ESSV^−^ (pNEU). ", "The same observations were made within the 4-kb class mRNAs (Figure [6C](#F6){ref-type=\"fig\"}; 4-kb mRNAs). ", "Both in 2-kb and 4-kb mRNA classes, the double-mutation ESSV^−^ (dm) was as efficient in promoting exon 3 inclusion as ESSV^−^ (pNEU) (Figure [6C](#F6){ref-type=\"fig\"}, cf. ", "lanes 3 and 6), which was consistent with a HEXplorer score difference ΔHZ~EI~ = 157.9 similar to ESSV^−^ (pNEU), but clearly exceeding the single-point mutations' ΔHZ~EI~.\n\nTransfection experiments were complemented by western blot analyses of intracellular Gag protein levels (Figure [6D](#F6){ref-type=\"fig\"}). ", "To evaluate the importance of ESSV for virus particle production, cell-free supernatants were harvested from HEK 293T cells transfected with proviral DNAs, and viral release for each sample was detected via p24 levels. ", "Western blot results revealed that ESSV^−^ (pNEU) led to low amounts of intracellular Gag protein (Figure [6D](#F6){ref-type=\"fig\"}, cf. ", "lanes 2 and 3), as expected from excessive exon 3 splicing causing a replication defect ([@B19]). ", "While the single-point mutations ESSV^−^ (−29G\\>C) and ESSV^−^ (−36A\\>C) partially inactivated ESSV leading to slight replication defects as seen in Gag protein and virus particle production (Figure [6D](#F6){ref-type=\"fig\"}, cf. ", "lanes 2, 4 and 5), the double mutation ESSV^−^ (dm) showed Gag protein levels not exceeding those detected in ESSV^−^ (pNEU) both within cells and supernatants (Figure [6D](#F6){ref-type=\"fig\"}, cf. ", "lanes 3 and 6).", "\n\nIn these experiments, we have exemplified the HEXplorer-score-guided mutagenesis inactivating the exonic splicing silencer ESSV with just two point mutations, obtaining an efficiency comparable to the known 7-nucleotide mutation ESSV^−^ (pNEU).", "\n\nA HEXplorer-based screening of the HIV-1 genome uncovers novel SREs {#SEC3-6}\n-------------------------------------------------------------------\n\nIn the next step, we aimed at discovering novel ESEs guided by the HEXplorer profiles of all HIV-1 exons shown in Figure [3](#F3){ref-type=\"fig\"}. ", "To this end, we first identified exonic sequences with uninterrupted positive HEXplorer scores flanked by valley regions with lower positive or negative HEXplorer scores. ", "In these typically 20-40-nucleotide long regions with supposed exonic enhancer property we searched for point mutations that disrupted the putative enhancer sequences. ", "The mutation positions were selected inside the HEXplorer positive regions, and the substituted nucleotide was chosen to maximize the HEXplorer score difference ΔHZ~EI~.\n\nBoth the putative ESE containing reference and mutated sequences were tested in the context of the ESE-dependent SV-*env* splicing reporter (([@B8],[@B46]); Figure [4B](#F4){ref-type=\"fig\"}). ", "The exonic splicing enhancer SRSF7 binding site and the splicing neutral sequence were used for calibration of ESE strength ([@B8],[@B48]). ", "Since this analysis covers different exons, we denote mutated positions with a single coordinate counting from the start of the NL4-3 genome.", "\n\nIn exon 1, we narrowed down the recently published enhancer region upstream of the major 5′ss D1 ([@B25]) by identifying the double mutation 708G\\>T, 718C\\>G that effectively suppressed splice site usage in a degree comparable to the neutral sequence (Figure [7A](#F7){ref-type=\"fig\"}, cf. ", "lanes 2--4).", "\n\n{ref-type=\"fig\"}. ", "For each HIV-1 exon, experimentally tested reference and mutated sequences are given below a schematic representation of their location (green bars). ", "All positions in HEXplorer-predicted enhancer and mutant sequences are counted from the start of the HIV-1 NL4-3 genome. ", "HEXplorer profiles span the entire experimentally tested sequences. ", "HEXplorer score differences are given below the profile graphs, and mutated nucleotides are indicated by arrows and corresponding positions (dm: double mutation; tm: triple mutation). ", "2.5 × 10^5^ HeLa cells were transiently transfected with 1 μg of each of the splicing reporters and 1 μg of pXHG5 for normalization. ", "Thirty hours post transfection, RNA was extracted and reverse transcribed using the resultant cDNA in a PCR reaction with primer pair \\#3210/\\#3211. ", "To control for equal transfection efficiency, we also performed a separate PCR reaction with primer pair \\#1224/\\#1225 detecting constitutively spliced GH1 mRNA. ", "RT-PCR products were resolved on 8% non-denaturing polyacrylamide gels and stained with ethidium bromide.](gku736fig7){#F7}\n\nIn exon 2, we discovered a novel enhancer between ESEM1 and ESEM2 ([@B26]) that was inactivated by the double mutation 4942C\\>T, 4947A\\>T. This novel enhancer was weaker than the SRSF7 binding site, but was still functional as part of an array of consecutive enhancer motifs possibly compensating for its lack of strength (ESE-Vif, ESEM1, ESEM2; Figure [7B](#F7){ref-type=\"fig\"}, cf. ", "lanes 1--4).", "\n\nBy mutation of the G-run splicing silencer G~I2~--1, usage of alternative 5′ss D2b can be significantly increased ([@B23]). ", "In this exon 2b, we discovered the novel enhancer ESE^5005--5032^ upstream of G~I2~--1, which was almost completely inactivated by the point mutation 5015A\\>T, and fully disabled by the double mutation 5015A\\>T, 5025A\\>T (Figure [7C](#F7){ref-type=\"fig\"}, cf. ", "lanes 1--6). ", "The different degree of ESE^5005--5032^ inactivation was reflected by a larger HEXplorer score difference ΔHZ~EI~ = −267.4 for the double mutation compared to ΔHZ~EI~ = −178.4 for 5015A\\>T or ΔHZ~EI~ = −89.0 for 5025A\\>T.\n\nA particularly interesting example was discovered in exon 4: the HEXplorer score profile indicated an exonic region with strong enhancing property wedged between the known silencer ESS2p and the known enhancer ESE2 (Figure [3](#F3){ref-type=\"fig\"}). ", "The enhancing property of this region was confirmed by the two single-point mutations 5816G\\>T (ΔHZ~EI~ = −79.1) and 5827G\\>T (ΔHZ~EI~ = −119.4), and the corresponding double mutation (ΔHZ~EI~ = −198.6) that reduced D4 splice site usage to a similar degree as the neutral sequence (Figure [7D](#F7){ref-type=\"fig\"}, cf. ", "lanes 2 and 6). ", "The single-point mutations achieved only an intermediate reduction of D4 usage in line with the respective HEXplorer score differences (Figure [7D](#F7){ref-type=\"fig\"}, cf. ", "lanes 3--5). ", "Furthermore, the additional third mutation 5821G\\>T (ΔHZ~EI~ = −263.1) slightly reduced both D4 usage and HEXplorer score below the levels of the double mutation. ", "By analyzing the same RNA samples with primer pairs also detecting the unspliced messages, we additionally confirmed that the reduced amount of spliced messages was indeed due to mutation of an SRE (Supplementary Figure S1).", "\n\nTaken together, HEXplorer score profiling permitted us to firstly identify novel putative splice enhancing regions in HIV-1 exons 1, 2, 2b and 4 and to secondly find specific enhancer inactivating mutations inside these regions, which can serve to identify enhancer binding proteins. ", "In all cases, splicing reporter experiments qualitatively confirmed the HEXplorer predicted effects of enhancer mutations.", "\n\nHEXplorer score difference quantitatively correlates with HIV-1 splicing activity {#SEC3-7}\n---------------------------------------------------------------------------------\n\nIn order to quantitatively compare HEXplorer score differences with changes in exon recognition or splice site usage even across different reporter systems, we plotted splicing activity versus HEXplorer score difference ΔHZ~EI~ between pairs of mutated and reference sequences. ", "In experiments using the 4-exon splicing reporter we measured splicing activity by exon inclusion rate defined as inclusion/(inclusion + exclusion), while in the single-intron splicing reporter experiments we used the ratio of 5′ splice site usage/GH1 transfection control instead. ", "To account for different reference splicing activity levels across different exons and reporter systems, we separately normalized reference splicing activity to 100% in each reporter. ", "In Figure [8](#F8){ref-type=\"fig\"}, all 16 point mutations examined in the 4-exon splicing reporter (Figure [5](#F5){ref-type=\"fig\"}) are represented by red squares, while data from four point mutations within ESSV^−^ (pNEU) (Figure [6](#F6){ref-type=\"fig\"}, and one additional mutation not shown) and nine point mutations obtained from exons 1, 2, 2b and 4 (Figure [7](#F7){ref-type=\"fig\"}) are denoted by individual symbols. ", "Across all these experiments, we obtained an excellent correlation of *r* = 0.85 (*P* \\< 0.001) between splicing activity and HEXplorer score difference. ", "These promising results suggest that the HEXplorer score can be effectively used to (i) determine the splice enhancing or silencing property of an entire region and (ii) predict the effects of point mutations on splice site usage.", "\n\n{ref-type=\"fig\"}), exon 3 inclusion ratio was determined in triplicate for 16 single or double mutations in the ESE*~vpr~* region upstream 5'ss D3. ", "The exon 3 inclusion ratio showed a linear correlation of *r* = 0.75 with changes in hexamer score HZ~EI~ (red squares). ", "Adding 13 mutations from a series of control experiments involving HIV exons 1--4 (colored circles and triangles denote exons; cf. ", "Figure [7](#F7){ref-type=\"fig\"}) even improved the correlation for the entire data set (*r* = 0.85, 16 + 13 mutations; error bars denote standard deviations from triplicates; *N* denotes the number of mutations per exon).](gku736fig8){#F8}\n\nHEXplorer score difference correlates well with various available SRE algorithms {#SEC3-8}\n--------------------------------------------------------------------------------\n\nWe additionally applied two different approaches to compare the HEXplorer score with other available algorithms for the identification of SREs, using the data set of 29 experimentally tested mutations.", "\n\nFirst, for each of the 29 mutations shown in Figure [8](#F8){ref-type=\"fig\"}, we determined all ESR motifs predicted to be overlapping with the mutated positions by the ESEfinder ([@B10],[@B12]), RESCUE-ESE ([@B9],[@B49]), FAS-ESS-hex3 ([@B13]), PESX ([@B11]) or ESRsearch ([@B14]) algorithms. ", "We aggregated the results of the different ESR-identifying algorithms by assigning +1 for each ESE and −1 for each ESS motif, similar to the approach chosen in ([@B44]). ", "Totaling these ±1-weights for every pair of reference and mutant sequences, we obtained the 'mutant--reference' ESMD, an integer number measuring the overall net gain and loss of ESE and ESS. ", "For all 29 mutations, we obtained an excellent correlation of *r* = 0.93 (*P* \\< 0.001) between ESMD and HEXplorer score difference (see Supplementary Figure S2).", "\n\nSecond, we compared the individual exon--intron and weak--strong hexamer scores (*Z*~EI~ and *Z*~WS~) to the ESRseq score available for all 1182 ESE and 1090 ESS hexamers identified as exonic SREs in ([@B15]). ", "From the scatterplots in Supplementary Figure S3 we obtained a good correlation of *r* = 0.72 with both *Z*~EI~ and *Z*~WS~. Finally, for all 29 mutations shown in Figure [8](#F8){ref-type=\"fig\"}, we calculated the ESRseq difference between mutant and reference sequences, using the same averaging approach as in the HEXplorer score definition, but with the ESRscore hexamer weights instead of *Z*~EI~. We again found an excellent correlation of *r* = 0.93 between the ESRseq difference and HEXplorer score difference (mutant--reference) as shown in Supplementary Figure S4.", "\n\nAlthough predictions of individual currently available ESR-identifying algorithms are not necessarily consistent with each other, our results indicate that there is a considerable agreement between the HEXplorer score prediction of SREs---in particular mutation effects---and the average of all other algorithms for ESR motif identification examined in this context.", "\n\nDISCUSSION {#SEC4}\n==========\n\nHexamer over-representation between different data sets of exonic and intronic sequences has been successfully used as a basis for the identification of exonic SREs ([@B9],[@B11]). ", "In the present study, we extended this approach by incorporating all hexamer frequencies of an entire sequence region into a novel HEXplorer score HZ~EI~ that was calculated as the average exon--intron hexamer *Z*~EI~-score of all six hexamers overlapping with any given index nucleotide. ", "Capturing the overall hexamer content of an entire sequence region, HEXplorer score profiles plotted along exonic sequences exhibited a continuous spectrum of exonic enhancing and silencing properties in the context of a given splice site, rather than the presence or absence of individual predicted SRE hexamer binding sites.", "\n\nGenome wide analyses ([@B7],[@B17],[@B50]) as well as systematic splicing reporter experiments ([@B8]) have confirmed that SREs act in a position-dependent manner: classical splice enhancing SR-proteins bind upstream of a 5′ss, but inhibit splicing from an intronic position. ", "In a similar way, the typical exonic splice silencing hnRNP H/F proteins act as enhancers from intronic positions. ", "Based upon the respective different exonic and intronic hexamer distributions, the HEXplorer score HZ~EI~ presumably captures these position-dependent splicing regulatory sequence properties in the context of any given splice site.", "\n\nFor accurate splicing, nearby splice regulatory elements often modulate proper splice site motif recognition, and they may be evolutionarily adapted to particular splice site properties (e.g. intrinsic strength). ", "Therefore, splice enhancing or silencing sequence properties must always be rated in the context of the actual splice site, and this rating can be different in the vicinity of weak splice sites than near strong ones. ", "Thus, while HEXplorer score 'differences' upstream of a given 5′ss have proven quantitatively valid in mutation analyses, the comparative validity of 'absolute' HEXplorer area size was limited to its 5′ss context, and care must be taken in comparing different splice sites. ", "It is therefore natural that the absolute HEXplorer score area size should be most meaningful together with the intrinsic 5′ss strength, measured e.g. by its maxent or HBS. ", "A systematic examination of the interplay between intrinsic splice site strength and neighboring HEXplorer score profile and their merging into a 'functional splice site score' could further the understanding of normal and pathological splice site usage.", "\n\nUsing the HIV-1 genome heavily relying on SREs ([@B40]) as a model system, we experimentally examined whether the HEXplorer score faithfully represented the respective enhancing or silencing properties of genomic regions. ", "In a helicopter view, we first generated HEXplorer score profiles of each HIV-1 exon exhibiting specific HEXplorer score positive and negative regions that generally coincided with known exonic enhancers or silencers.", "\n\nIn a set of systematic point mutations of the exemplarily chosen exonic enhancer ESE*~vpr~*, the HEXplorer score correlated well with exon inclusion in a four-exon splicing reporter. ", "Outside this splicing reporter, ESE*~vpr~* was previously examined in a proviral context. ", "In the absence of the repressing ESSV, strong exon 3 splice site activation led to reduced levels of unspliced viral mRNA and a deficiency in virus particle production ([@B19]), while the release of virus particles into the supernatant could be rescued by an ESE*~vpr~* double mutation that was originally suggested by the HEXplorer algorithm ([@B20]). ", "These experiments confirmed that the four-exon splicing reporter reliably captured HIV-1 splice site usage.", "\n\nBased on HEXplorer profile prediction, we discovered five novel splicing enhancers in HIV-1 exons and experimentally confirmed all of them by mutagenesis. ", "Furthermore, we were able to design a novel, reduced set of point mutations disrupting the splicing silencer ESSV to a similar degree as the established ESSV^−^ (pNEU). ", "Based on the relative occurrences of all hexamers, the HEXplorer score permitted optimizing mutagenesis by *a priori* determining the point mutation(s) with the largest effects on the exonic enhancing or silencing property of a given sequence. ", "Moreover, using HEXplorer score predictions of point mutation effects on splicing now opens the perspective to design silent mutations interfering with splice site usage while not altering the amino acid sequence of the encoded protein.", "\n\nThe HEXplorer score definition was derived from a specific set of exonic and intronic sequences extracted from the ENSEMBL database, and it is therefore based on pooled sequences expressed in different human tissues. ", "Hexamer weights derived in this way cannot take into account tissue-specific protein expression and possibly fail to detect SRE motifs bound by splicing regulatory proteins that are not ubiquitously expressed ([@B54]). ", "On the contrary, HEXplorer score predictions of regulatory properties may be invalid for a specific tissue not expressing a required splicing regulatory protein. ", "This principal deficiency, however, holds for all algorithms identifying SREs from pooled sequence data. ", "Future applications may overcome these limitations by deriving hexamer weights from cell-type-specific transcriptome data sets obtained from RNA deep sequencing.", "\n\nIn the HEXplorer score definition, the choice of exonic/intronic neighborhoods entering into the RESCUE-like hexamer weight calculation was somewhat arbitrary. ", "Since the median size of a middle exon in human genes is 123 nucleotides ([@B58]), we chose 100-nt wide regions (or the entire exon if it was shorter). ", "These sets of exonic and intronic sequences were sufficiently large that all hexamers were multiply represented, but shorter than the sequences used in the original work ([@B9]). ", "Experimentally, most splice enhancers and silencers can be expected to lie within a 100-nucleotide splice site neighborhood. ", "We comparatively evaluated an even smaller neighborhood size of only 30 nucleotides (data not shown) and found an excellent correlation of *r* = 0.96 between the respective exon--intron scores of all 4096 hexamers. ", "Thus, within the range of values covered, the size of the splice site neighborhoods used for the HEXplorer score definition has only limited impact on the HEXplorer score values obtained.", "\n\nWe chose to derive the HEXplorer score from exon--intron hexamer scores *Z*~EI~ based on heuristic arguments that splicing enhancing or silencing properties differ more widely between exonic and intronic splice site neighborhoods than between exons of strong and weak splice sites. ", "To examine the validity of this choice, we also calculated analogous *Z*~WS~-based HEXplorer score differences for all 29 mutations shown in Figure [8](#F8){ref-type=\"fig\"}. ", "From these *Z*~WS~-based HEXplorer scores, we obtained a weaker correlation of *r* = 0.75 with experimentally determined splicing activity than from ΔHZ~EI~, which was consistent with our assumption.", "\n\nThe novel computational HEXplorer score profiles provide a global landscaping of splicing regulatory regions, as well as a quantitative measure of mutation effects on their splice enhancing and silencing properties in the vicinity of a given splice site. ", "In particular, HEXplorer score calculation may significantly alleviate mutational analyses by reliably predicting possibly silent point mutations most effectively disrupting or even creating SREs. ", "This is especially helpful for the identification of proteins binding to regulatory elements. ", "HEXplorer score analyses may also further the computational prediction of pathogenic mutation effects in human genetics.", "\n\nSUPPLEMENTARY DATA {#SEC5}\n==================\n\n[Supplementary Data](http://nar.oxfordjournals.org/lookup/suppl/doi:10.1093/nar/gku736/-/DC1) are available at NAR Online.", "\n\n###### SUPPLEMENTARY DATA\n\nWe thank Björn Wefers, Sarah Otten and Sebastian Wittich for excellent technical assistance.", "\n\nFUNDING {#SEC6}\n=======\n\nDeutsche Forschungsgemeinschaft (DFG) \\[SCHA 909/3-1\\]; Stiftung für AIDS-Forschung, Düsseldorf \\[to H.S.\\]; Jürgen Manchot Stiftung \\[to M.W., J.O.P. and H.S.\\].", "\n\n*Conflict of interest statement*. ", "None declared.", "\n\n[^1]: The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.", "\n" ]

{ "pile_set_name": "PubMed Central" }

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[ "Nettles anger at Bergerac repeats ban: Actor criticises BBC for refusing to show re-runs because show featured children's home linked to Jimmy Savile\n\nSeries used Haut de la Garenne on Jersey as a police HQ\n\nThe building has since been dubbed the Jersey House of Horrors\n\nIt f ollows alleg ations that child abuse to ok place there\n\nSavile visited the home and it is feared he abused youngsters there\n\nActor John Nettles has criticised the BBC for cancelling re-runs of Bergerac\n\nJohn Nettles criticised the BBC yesterday for cancelling re-runs of Bergerac – because they feature a children's home linked to Jimmy Savile.", "\n\nThe 1980s detective series used Haut de la Garenne – dubbed the Jersey House of Horrors – as police HQ.", "\n\nA child abuse investigation found that former residents had been molested. ", "Savile visited the home and it is feared he abused youngsters there.", "\n\nBBC2 said it had postponed re-runs of the show because of 'the sensitive nature of the public hearing into incidents at Haut de la Garenne'.", "\n\nNettles fumed: 'It seems to be a little bizarre that exterior Haut de la Garenne shots can't be used. ", "It means you can't photograph or film anywhere where there is a suspicion that a crime has been committed.", "\n\n'It just shows how far the BBC is bending over backwards to apologise for the Jimmy Savile scandal.", "\n\n'How it can be connected by viewers with Jimmy Savile and the Yewtree inquiry is beyond belief. ", "It's just unfortunate that viewers have to suffer for it.'", "\n\nThe Haut de la Garenne home, which was unoccupied at the time, featured as a police station in six of the show's nine series.", "\n\nBBC2 bosses had planned to re-run the entire back catalogue of Bergerac, which ran from 1981 to 1991 and made Nestles, who played Jim Bergerac, a household name.", "\n\nBut after running the first three series, corporation chiefs have 'postponed' the remaining six, which all feature Haut de la Garenne as the fictional HQ.", "\n\nA fresh inquiry looking at allegations of abuse in children's homes and fostering services in Jersey from 1960 to the present day is due to start this year.", "\n\nThe former Haut de la Garenne children's home in Jersey has been the subject of a major child abuse investigation. ", "It was used as a police HQ in Bergerac\n\nA spokesman for the BBC said: 'Due to the sensitive nature of the public hearing into incidents at Haut de la Garenne and out of respect for the victims, the BBC has decided, for the moment, to postpone episodes of Bergerac.'", "\n\nHaut de la Garenne began in 1867 as an industrial school for 'young people of the lower classes of society and neglected children'.", "\n\nIt closed in 1986 and was the centre of a huge police inquiry in 2008 which revealed numerous instances of child abuse against past residents." ]

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[ "916 F.2d 1239\nDorothy A. WILSON and Louis P. Wilson, Plaintiffs-Appellees,v.HUMPHREYS (CAYMAN) LIMITED, a corporation, and Holiday Inns,Inc., a corporation, Defendants-Appellants.", "\nNos. ", "88-2495, 88-2496.", "\nUnited States Court of Appeals,Seventh Circuit.", "\nArgued Oct. 31, 1989.Decided Oct. 24, 1990.Rehearing Denied Nov. 21, 1990.", "\n\nRobert L. Gibson, Alfred H. Kreckman, Jr., Massey, Anderson & Gibson, Paris, Ill., Phillip A. Terry, McHale, Cook & Welch, Indianapolis, Ind., for plaintiffs-appellees.", "\nJames A. McDermott, Jan M. Carroll, Barnes & Thornburg, Indianapolis, Ind., Theodore J. Nowacki, Bose, McKinney & Evans, Indianapolis, Ind., for defendants-appellants.", "\nBefore BAUER, Chief Judge, and RIPPLE, Circuit Judge, and WILL, Senior District Judge.*", "\nRIPPLE, Circuit Judge.", "\n\n\n1\nDorothy and Louis Wilson initiated this diversity action in 1986. ", " The Wilsons asserted that the two defendants involved in this appeal, together with a third defendant, were liable to them for damages in connection with a serious injury suffered by Mrs. Wilson while staying as a guest in appellant Humphreys' hotel.1 Humphreys and Holiday Inns moved to dismiss the complaint due to lack of subject matter and personal jurisdiction, and asserted that the forum selected by the plaintiffs violated the doctrine of forum non conveniens. ", " The district court denied these motions. ", " Upon application by the appellants, the district court certified the questions of jurisdiction and forum non conveniens to this court on May 25, 1988 (for Humphreys), and July 7, 1988 (for Holiday Inns). ", " This court accepted the appeal on July 13, 1988. ", " We now affirm the judgment of the district court in part and remand for further consideration.", "\n\n\n2\n* BACKGROUND\n\n\n3\nHumphreys (Cayman) Limited is incorporated under the laws of the Cayman Islands. ", " Humphreys is a licensee of appellant Holiday Inns, Inc. and operates a hotel in the Cayman Islands under authority of that license. ", " Humphreys maintains a corporate office in Memphis, Tennessee and has operated a reservations office in Miami, Florida. ", " Holiday Inns, Inc. is a Tennessee corporation and is registered to do business in Indiana. ", " The record does not reveal the nature or extent of its activities within the state.", "\n\n\n4\nSince 1976, Humphreys has participated in a continuing commercial relationship with American Trans Air (American) to provide accommodations for tours run by American to the Cayman Islands. ", " As part of this relationship, Humphreys sent officers to Indianapolis on at least one occasion to meet with representatives of American. ", " During a trip to Indiana in the summer of 1981, Humphreys' representatives negotiated a long-term contract with American by which a certain number of rooms at Humphreys' hotel would be guaranteed at a reduced rate for American-run tours, with American tour leaders receiving free accommodations. ", " American frequently has listed trips to the Cayman Islands in its regular advertising brochures and has included specific references to Humphreys' hotel. ", " In addition, Humphreys advertises extensively in national magazines directed both at travel agents and potential travellers. ", " Humphreys printed rate cards and brochures that it sent for further distribution to American in Indiana and to various travel agents around the country.", "\n\n\n5\nDorothy and Louis Wilson participated in a tour organized by American that left Indianapolis International Airport on October 28, 1984. ", " The group stayed at Humphreys' hotel in the Cayman Islands. ", " According to Mrs. Wilson's affidavit, arrangements for the trip were made as a result of advertisements for the tour that appeared in American's travel bulletin. ", " Arrangements were made with American's tour subsidiary, Ambassadair, and payment was made to Ambassadair in Indianapolis, Indiana.", "\n\n\n6\nOn October 30, 1984, Mrs. Wilson was assaulted by an intruder in her hotel room. ", " The intruder attempted to rob and rape Mrs. Wilson, and she suffered serious injuries. ", " She spent a week in a hospital on the Cayman Islands and then returned to Indianapolis to be treated at Methodist Hospital.", "\n\n\n7\nThe complaint contains thirteen counts against the two appellants in this case. ", " The counts charge the appellants with negligence, breach of express and implied warranties, breach of contract, and negligent infliction of emotional distress. ", " The appellants moved to dismiss the complaint on the grounds of lack of subject matter or personal jurisdiction, and forum non conveniens. ", " The district court denied these motions, but subsequently certified them for interlocutory appeal pursuant to 28 U.S.C. Sec. ", "1292(b). ", " We accepted the appeal and now consider each issue in turn.", "\n\nII\nANALYSIS\nA. Subject Matter Jurisdiction\n\n8\nDiversity jurisdiction in this suit is based on 28 U.S.C. Sec. ", "1332(a)(3), which provides for jurisdiction when the suit is between \"citizens of different States and in which citizens or subjects of a foreign state are additional parties.\" ", " Humphreys argues that the district court cannot exercise subject matter jurisdiction over it because it is a company incorporated in the Cayman Islands, and the Cayman Islands is not a \"foreign state.\" ", " Humphreys reaches this conclusion by noting that the Cayman Islands is a dependency of Great Britain and that the United States does not regard the Cayman Islands as an independent sovereign. ", " See The World Factbook 1989 at 56.", "\n\n\n9\nThe power to exercise jurisdiction over a foreign citizen under 28 U.S.C. Sec. ", "1332 has been referred to as \"alienage jurisdiction.\" ", " Sadat v. Mertes, 615 F.2d 1176, 1182 (7th Cir.1980). ", " This type of jurisdiction \"was intended to provide the federal courts with a form of protective jurisdiction over matters implicating international relations where the national interest was paramount.\" ", " Id.\n\n\n10\n\"The dominant considerations which prompted the provision for such jurisdiction appear to have been:\n\n\n11\n(1) Failure on the part of individual states to give protection to foreigners under treaties; ... [and]\n\n\n12\n(2) Apprehension of entanglements with other sovereigns that might ensue from failure to treat the legal controversies of aliens on a national level.\"", "\n\n\n13\nId. (quoting Blair Holdings Corp. v. Rubinstein, 133 F.Supp. ", "496, 500 (S.D.N.Y.1955)).", "\n\n\n14\nOur inquiry therefore must be whether the policies supporting alienage jurisdiction permit a United States District Court to assume jurisdiction over a citizen of the Cayman Islands.2 The Cayman Islands is a British Dependent Territory.3 A citizen of a British Dependent Territory is a \"citizen of the United Kingdom and Colonies.\" ", " British Nationality Act 1981 Sec. ", "51(3)(a)(ii). ", " According to an affidavit of a Cayman Islands attorney submitted by Humphreys, the court system in the Cayman Islands is patterned after that of Great Britain. ", " In addition, \"[t]he Common Law of England ... is recognised and applied by the Courts in all cases where there has not been a specific local enactment.\" ", " Humphreys' App. ", "at 66. ", " The Cayman Islands is administered by a governor who is appointed by the British monarch. ", " The United Kingdom represents Caymanian diplomatic interests and is responsible for its military defense. ", " The World Factbook 1989 at 56.", "\n\n\n15\nSeveral federal courts have determined, although generally without discussion, that subject matter jurisdiction existed in suits between citizens of the United States and Cayman Island corporations. ", " See Bally Export Corp. v. Balicar, Ltd., 804 F.2d 398, 399-400 (7th Cir.1986); Philan Ins. ", "Ltd. v. Frank B. Hall & Co., Inc., 712 F.Supp. ", "339, 345 (S.D.N.Y.1989); Rolls Royce (Canada) Ltd. v. Cayman Airways, Ltd., 617 F.Supp. ", "17, 18 (S.D.Fla.1985). ", " In addition, the Second Circuit concluded that alienage jurisdiction existed over a company incorporated in Bermuda, also a British Dependent Territory. ", " Netherlands Shipmortgage Corp. v. Madias, 717 F.2d 731, 735 (2d Cir.1983); see also British Nationality Act 1981, 31 Halsbury's Statutes 172 (4th ed. ", "1987) (Bermuda identified as a British Dependent Territory).", "\n\n\n16\nHumphreys relies on one unpublished district court decision for support of its assertion that alienage jurisdiction does not apply to this case. ", " St. Germain v. West Bay Leasing, Ltd., CV-81-3945, order (E.D.N.Y.1982). ", " That court in fact did conclude that subject matter jurisdiction was lacking when one of the parties was a Cayman Islands corporation. ", " The court relied in part on an earlier decision denying diversity jurisdiction over a business incorporated in Hong Kong, a British colony. ", " Windert Watch Co. v. Remex Elecs. ", " Ltd., 468 F.Supp. ", "1242 (S.D.N.Y.1979). ", " However, the force of the Windert decision has been eroded by a more recent case from the same court. ", " In Tetra Finance (HK) Ltd. v. Shaheen, 584 F.Supp. ", "847 (S.D.N.Y.1984), the court noted that corporations should not be denied access to American courts simply because of the status of the country in which they are incorporated. ", " \"It would seem hypertechnical to preclude Hong Kong corporations from asserting claims in our courts simply because Hong Kong has not been formally recognized by the United States as a foreign sovereign in its own right.\" ", " Id. at 848.4\n\n\n17\nWe agree with the district court that subject matter jurisdiction under section 1332 is present. ", " We see no reason to depart from the weight of authority. ", " Certainly, the exercise of American judicial authority over the citizens of a British Dependent Territory implicates this country's relationship with the United Kingdom--precisely the raison d'etre for applying alienage jurisdiction. ", " Not applying alienage jurisdiction in this case would allow \"form rather than substance\" to govern. ", " Murarka v. Bachrack Bros., 215 F.2d 547, 552 (2d Cir.1954) (Harlan, J.).5\n\nB. Personal Jurisdiction\n\n18\nIn a diversity case, a federal district court has personal jurisdiction over a nonresident defendant \"only if a court of the state in which it sits would have such jurisdiction.\" ", " Turnock v. Cope, 816 F.2d 332, 334 (7th Cir.1987). ", " Therefore, review of decisions regarding jurisdiction of a federal court in a diversity suit requires a two-part inquiry: (1) whether the state statute allows jurisdiction, and (2) whether the assertion of jurisdiction complies with constitutional due process standards. ", " See Heritage House Restaurants, Inc. v. Continental Funding Group, Inc., 906 F.2d 276, 279 (7th Cir.1990); John Walker & Sons, Ltd. v. DeMert & Dougherty, Inc., 821 F.2d 399, 401-02 (7th Cir.1987) (Illinois long-arm statute); 4 C. Wright & A. Miller, Federal Practice and Procedure Sec. ", "1069 at 347 (1987). ", " In this case, these twin inquiries collapse into one, because, as this court has noted, Indiana's long-arm statute extends personal jurisdiction to the limit allowed under the due process clause of the fourteenth amendment. ", " Wallace v. Herron, 778 F.2d 391, 393 (7th Cir.1985) (and cases cited therein), cert. ", "denied, 475 U.S. 1122, 106 S.Ct. ", "1642, 90 L.Ed.2d 187 (1986). ", " Accordingly, we shall focus on whether assertion of jurisdiction in this case violates due process.", "\n\n\n19\nDue process requires that the defendant have \"minimum contacts with [the forum] such that the maintenance of the suit does not offend 'traditional notions of fair play and substantial justice.' \" ", " International Shoe Co. v. Washington, 326 U.S. 310, 316, 66 S.Ct. ", "154, 158, 90 L.Ed. ", "95 (1945) (quoting Milliken v. Meyer, 311 U.S. 457, 463, 61 S.Ct. ", "339, 342, 85 L.Ed. ", "278 (1940)); FMC Corp. v. Varonos, 892 F.2d 1308, 1313 (7th Cir.1990). ", " The minimum contacts must be established by the purposeful acts of the defendant. ", " Burger King Corp. v. Rudzewicz, 471 U.S. 462, 475, 105 S.Ct. ", "2174, 2183, 85 L.Ed.2d 528 (1985). ", " Examples of acts that, depending on the circumstances, might support a determination of jurisdiction include \"designing the product for the market in the forum State, advertising in the forum State, establishing channels for providing regular advice to customers in the forum State, or marketing the product through a distributor who has agreed to serve as the sales agent in the forum State.\" ", " Asahi Metal Indus. ", "Co. v. Superior Court, 480 U.S. 102, 112, 107 S.Ct. ", "1026, 1033, 94 L.Ed.2d 92 (1987).", "\n\n\n20\nThere are two types of jurisdiction that can be exercised over an out-of-state defendant--specific and general. ", " When the defendant's activities in the state with respect to the transaction underlying the litigation serve as the basis for jurisdiction, the assertion of jurisdiction is said to be \"specific.\" ", " See Helicopteros Nacionales de Columbia v. Hall, 466 U.S. 408, 414 n. 8, 104 S.Ct. ", "1868, 1872 n. 8, 80 L.Ed.2d 404 (1984); Giotis v. Apollo of the Ozarks, Inc., 800 F.2d 660, 666 n. 3 (7th Cir.1986) (discussing difference between general and specific jurisdiction), cert. ", "denied, 479 U.S. 1092, 107 S.Ct. ", "1303, 94 L.Ed.2d 158 (1987); Wallace, 778 F.2d at 393 (same). ", " By contrast, when the defendant's overall activity in the state, not simply its actions with respect to the underlying transaction, are the basis, the jurisdiction is said to be \"general.\" ", " See Helicopteros Nacionales de Colombia, 466 U.S. at 414 n. 9, 104 S.Ct. ", "at 1872 n. 9.", "\n\n1. ", "specific jurisdiction over Humphreys\n\n21\nConsidering Humphreys first, we shall examine whether the contacts it had with the state satisfy the assertion of specific jurisdiction.6 Jurisdiction based on such contacts comports with due process only if Humphreys reasonably should have anticipated being haled into court in Indiana. ", " See Burger King Corp. v. Rudzewicz, 471 U.S. 462, 474, 105 S.Ct. ", "2174, 2183, 85 L.Ed.2d 528 (1985); World-Wide Volkswagen Corp. v. Woodson, 444 U.S. 286, 297, 100 S.Ct. ", "559, 567, 62 L.Ed.2d 490 (1980); FMC Corp. v. Varonos, 892 F.2d 1308, 1313 (7th Cir.1990); Wallace, 778 F.2d at 393-94. ", " Humphreys must purposefully have availed itself of the \" 'privilege of conducting activities' \" in Indiana. ", " Burger King, 471 U.S. at 475, 105 S.Ct. ", "at 2183 (quoting Hanson v. Denckla, 357 U.S. 235, 253, 78 S.Ct. ", "1228, 1239, 2 L.Ed.2d 1283 (1958)); see also Giotis, 800 F.2d at 666; Wallace, 778 F.2d at 395.", "\n\n\n22\n\"Specific jurisdiction turns on a particularized assessment of the 'relationship among the defendant, the forum, and the litigation.' \" ", " Saylor v. Dyniewski, 836 F.2d 341, 344 (7th Cir.1988) (quoting Shaffer v. Heitner, 433 U.S. 186, 204, 97 S.Ct. ", "2569, 2579, 53 L.Ed.2d 683 (1977)). ", " The conduct engaged in by Humphreys in Indiana is certainly sufficient to satisfy specific jurisdiction. ", " Humphreys advertised in the state of Indiana, entered into a long-term arrangement with an Indiana corporation to provide a guaranteed number of rooms to be used in connection with tour packages that departed from Indiana, and sent representatives to Indiana to negotiate the contract. ", " In short, Humphreys established a continuing commercial relationship with an Indiana tour company with the expectation that its service (providing accommodation) would be purchased by residents of Indiana (and other states). ", " These contacts convince us that the district court correctly determined that Humphreys is subject to personal jurisdiction in Indiana. ", " Humphreys, by its own business conduct, had purposefully availed itself \"of the privilege of conducting activities within the forum State, thus invoking the benefits and protections of its laws.\" ", " Hanson v. Denckla, 357 U.S. 235, 253, 78 S.Ct. ", "1228, 1240, 2 L.Ed.2d 1283 (1958). ", " It should have \"reasonably anticipate[d] being haled into court there.\" ", " World-Wide Volkswagen v. Woodson, 444 U.S. at 297, 100 S.Ct. ", "at 567; see Shute v. Carnival Cruise Lines, 897 F.2d 377, 380, 386 (9th Cir.1990) (in plaintiff's personal injury suit, specific jurisdiction over corporation was found where corporation advertised in forum, sent brochures to forum, solicited business in forum, and sold tickets on a commission basis through travel agents in forum) (as amended and refiled); Rainbow Travel Serv., ", "Inc. v. Hilton Hotels Corp., 896 F.2d 1233, 1238 (10th Cir.1990) (contacts sufficient to meet due process requirements when hotel solicited business in forum state, carried out negotiations there, and sent contracts there for execution); Neiman v. Rudolf Wolff & Co., 619 F.2d 1189, 1193 (7th Cir.) ", " (personal jurisdiction established where employee of defendant [an English company] came to forum for a lunch meeting, and meeting constituted bulk of negotiations that led to formation of contract; \"A defendant's participation in the state in substantial preliminary negotiations leading to the contract in issue has been held a sufficient basis for long-arm jurisdiction.\"), ", "cert. ", "denied, 449 U.S. 920, 101 S.Ct. ", "319, 66 L.Ed.2d 148 (1980).7\n\n2. ", "general jurisdiction over Holiday Inns\n\n23\nOn this record, personal jurisdiction over Holiday Inns must be based, if at all, on general jurisdiction, because the only connection Holiday Inns had with the injury was its franchise arrangement with Humphreys. ", " Jurisdiction based on a party's generally affiliating nexus with the state, as previously noted, does not offend due process when there are \"continuous and systematic general business contacts\" of the defendant in the state. ", " Helicopteros Nacionales de Columbia, 466 U.S. at 416, 104 S.Ct. ", "at 1873. ", " \"This is a fairly high standard in practice.\" ", " Fields v. Sedgwick Associated Risks Ltd., 796 F.2d 299, 301 (9th Cir.1986); see Shute, 897 F.2d at 380-81 (collecting cases). ", " The Wilsons argue that the Indiana registration act, Indiana Code 23-1-11-6,8 is sufficient to support general jurisdiction. ", " Registering to do business is a necessary precursor to engaging in business activities in the forum state. ", " However, it cannot satisfy ... standing alone ... the demands of due process. ", " Such an interpretation of the Indiana registration statute would render it constitutionally suspect and, accordingly, we decline to give it such a reading. ", " Cf. ", "NLRB v. Catholic Bishops of Chicago, 440 U.S. 490, 500, 99 S.Ct. ", "1313, 1318, 59 L.Ed.2d 533 (1979).", "\n\n\n24\nOn this record, we cannot determine whether Holiday Inns had a sufficient level of conduct and activity in Indiana to justify the characterization \"continuous and systematic.\" ", " Helicopteros Nacionales de Colombia, 466 U.S. at 416, 104 S.Ct. ", "at 1873. ", " Accordingly, we remand this aspect of the case to the district court for a more plenary inquiry into the contacts and conduct of Holiday Inns in Indiana.", "\n\nC. Forum Non Conveniens\n\n25\nWe review under an abuse of discretion standard the district court's decision to deny a motion to dismiss for forum non conveniens. ", " Piper Aircraft Co. v. Reyno, 454 U.S. 235, 257, 102 S.Ct. ", "252, 266, 70 L.Ed.2d 419 (1981); FMC Corp. v. Varonos, 892 F.2d 1308, 1314 (7th Cir.1990). ", " As the Supreme Court has instructed,\n\n\n26\n[W]here the court has considered all relevant public and private interest factors, and where its balancing of these factors is reasonable, its decision deserves substantial deference.", "\n\n\n27\nPiper Aircraft Co., 454 U.S. at 257, 102 S.Ct. ", "at 266. ", " Furthermore, the district court is granted \"substantial flexibility\" in deciding a forum non conveniens argument. ", " Van Cauwenberghe v. Biard, 486 U.S. 517, 529, 108 S.Ct. ", "1945, 1953, 100 L.Ed.2d 517 (1988).", "\n\n\n28\n\"There is ordinarily a strong presumption in favor of the plaintiff's choice of forum, which may be overcome only when the private and public interest factors clearly point towards trial in the alternative forum.\" ", " Macedo v. Boeing Co., 693 F.2d 683, 688 (7th Cir.1982); see Piper Aircraft Co., 454 U.S. at 255, 102 S.Ct. ", "at 265; Koster v. (American) Lumbermens Mutual Casualty, 330 U.S. 518, 524, 67 S.Ct. ", "828, 831, 91 L.Ed. ", "1067 (1947); Gulf Oil v. Gilbert, 330 U.S. 501, 508, 67 S.Ct. ", "839, 843, 91 L.Ed. ", "1055 (1947). ", " \"Courts routinely give less weight to a foreign plaintiff's choice of forum. ", " Citizens or residents deserve somewhat more deference than foreign plaintiffs.\" ", " Macedo, 693 F.2d at 688; see Piper Aircraft, 454 U.S. at 256, 102 S.Ct. ", "at 266. ", " Nevertheless, the district court must be able to police misuse, such as actions brought in a particular forum to harass a party. ", " There is no rigid test upon which a district court must focus when analyzing a forum non conveniens argument; instead, as articulated by the Supreme Court in Gulf Oil Corp. v. Gilbert, there are several public and private interest factors that may be considered.9 Private interests include\n\n\n29\nthe relative ease of access to sources of proof; availability of compulsory process for attendance of unwilling, and the cost of obtaining attendance of willing, witnesses; possibility of view of premises, if view would be appropriate to the action; and all other practical problems that make trial of a case easy, expeditious and inexpensive. ", " There may also be questions as to the enforceability of a judgment if one is obtained. ", " The court will weigh relative advantages and obstacles to fair trial.", "\n\n\n30\n330 U.S. at 508, 67 S.Ct. ", "at 843. ", " Public interests include judicial administration, trying cases in a forum that has a relation to the litigation, and having matters of state law decided by a court sitting in that state. ", " Id. at 508-09, 67 S.Ct. ", "at 843.", "\n\n\n31\nIn this case, the district court relied exclusively on the reasoning found in Lehman v. Humphrey Cayman, Ltd., 713 F.2d 339 (8th Cir.1983), cert. ", "denied, 464 U.S. 1042, 104 S.Ct. ", "708, 79 L.Ed.2d 172 (1984). ", " We recognize, as did the district court, the substantial similarity between the forum-related factors present in this suit and those present in the Lehman suit. ", " As is apparent from the case name, Lehman involves the same Cayman Islands hotel as is involved here. ", " Similarly, the injury occurred on the Cayman Islands to a United States resident who responded to a Humphreys' advertisement in the United States. ", " The Eighth Circuit evaluated many of the Gilbert factors. ", " The court first determined that the location of witnesses in the Cayman Islands did not militate strongly for applying forum non conveniens, because many of the witnesses were Holiday Inns employees (thus enabling Humphreys to obtain their cooperation in travelling to testify), and other modes of obtaining the evidence--such as admissions and depositions--could be used. ", " Lehman, 713 F.2d at 343. ", " The court also concluded that Iowa and the United States had an interest in the suit because the injured party was an Iowa resident and had made the travel arrangements in Iowa. ", " Id. at 344. ", " The Eighth Circuit placed some weight on the fact that the procedures for litigating in the Cayman Islands would disadvantage the plaintiff. ", " Attorneys there do not accept work for contingent fee arrangements; it is likely that the plaintiff could not receive a jury trial for her injury action; and she would have been required to post a $1,000 bond with the court because she was a foreigner. ", " Id. at 345-46. ", " Accordingly, the Eighth Circuit determined that the district court abused its discretion in dismissing the action on the ground of forum non conveniens. ", " Id. at 347.", "\n\n\n32\nWe cannot say that the district court abused its discretion in declining to dismiss the suit. ", " Great weight must be given the Wilsons' choice of forum. ", " The inconvenience for Humphreys of litigating the case in Indiana must be balanced against the disadvantageous procedures available in the Cayman Islands for the Wilsons,10 the interest of Indiana in providing a remedy for an injury stemming from a tour booked in Indiana for a group that left from Indiana, and the hardships that the Wilsons would encounter in litigating a case at such a great distance from their home. ", " For these reasons, we conclude that the district court did not abuse its discretion in denying the forum non conveniens motion.", "\n\nConclusion\n\n33\nFor the foregoing reasons, we affirm the orders of the district court regarding Humphreys, but remand to the district court for further consideration on whether Holiday Inns has subjected itself to general jurisdiction in Indiana.", "\n\n\n34\nAFFIRMED IN PART AND REMANDED.", "\n\n\n35\nWILL, Senior District Judge, concurring in part and dissenting in part.", "\n\n\n36\nI concur in the remand as to Holiday Inns. ", " I dissent, however, from the assertion of \"specific\" jurisdiction over Humphreys and from the majority's affirmance that Indiana is a proper forum.", "\n\n\n37\nThe Wilsons are residents of Illinois. ", " Their trip to the Caymans was a gift from their son. ", " He planned it and paid for it. ", " Neither the son nor the Wilsons had any contacts with Humphreys, in Indiana or anywhere else in the United States. ", " He booked the trip through American, and there is no evidence that he was influenced in any way by anything Humphreys said or did, that he selected Humphreys or even knew that it was to be the hotel. ", " In fact, the record is clear, as the majority recognizes, that \"arrangements for the trip were made as a result of advertisements for the tour that appeared in American's travel bulletin. ", " Arrangements were made with American's tour subsidiary, Ambassadair, and payment was made to Ambassadair....\" Majority op. ", "at 1241.", "\n\nI. Personal Jurisdiction\n\n38\nJurisdiction over an unwilling, non-resident defendant conforms to the requirements of due process only if the \"defendant's conduct and connection with the forum State are such that he should reasonably anticipate being haled into court there.\" ", " World-Wide Volkswagen Corp. v. Woodson, 444 U.S. 286, 297, 100 S.Ct. ", "559, 567, 62 L.Ed.2d 490 (1980). ", " A defendant who has \"purposefully established 'minimum contacts' in the forum State\" has impliedly submitted to jurisdiction in the forum. ", " Burger King Corp. v. Rudzewicz, 471 U.S. 462, 474, 105 S.Ct. ", "2174, 2183, 85 L.Ed.2d 528 (1985). ", " But merely \" 'random,' 'fortuitous,' or 'attenuated' \" contacts with a forum are not enough. ", " Id. at 475, 105 S.Ct. ", "at 2183. ", " Further, any \"assertion of personal jurisdiction [must] comport with 'fair play and substantial justice.' \" ", " Id. at 476, 105 S.Ct. ", "at 2184, quoting International Shoe Co. v. Washington, 326 U.S. 310, 66 S.Ct. ", "154, 90 L.Ed. ", "95 (1945). ", " And, finally, in the special case of \"specific\" jurisdiction, a defendant's contacts with the forum serve as a basis for personal jurisdiction only for suits \"arising out of or related to\" those contacts. ", " Helicopteros Nacionales de Colombia v. Hall, 466 U.S. 408, 414 n. 8, 104 S.Ct. ", "1868, 1872 n. 8, 80 L.Ed.2d 404 (1984).", "\n\n\n39\nHumphreys here had no purposeful contacts with the plaintiffs or with Indiana related to this suit, which arises from acts and events that took place entirely in the Cayman Islands. ", " Humphreys could not reasonably have anticipated being haled into court in Indiana, and forcing Humphreys to defend this case in an Indiana courtroom is far from fair or substantially just.", "\n\n\n40\nAny and all contacts with the Wilsons' son in Indiana related to this lawsuit were American's, not Humphreys', and American admittedly did not act as Humphreys' agent. ", " American had no authority to confirm reservations and bind Humphreys and it did not purport to do so. ", " American had to submit all reservations to Humphreys for confirmation.1\n\n\n41\nI can understand how Humphreys could reasonably have expected to be haled into an Indiana court, state or federal, to resolve a dispute arising from its contract with American Trans Air, which contract was negotiated in Indiana, or if it had sold products in Indiana. ", " But this case arises from a dispute between Humphreys and the Wilsons, not Humphreys and American. ", " Humphreys did not sell products in Indiana, and Humphreys had no contacts whatsoever with the Wilsons in Indiana or anywhere else in the United States.", "\n\n\n42\nThe majority's suggestion that Indiana courts have jurisdiction here because \"Humphreys established a continuing commercial relationship with an Indiana tour company with the expectations that its services would be purchased by residents of Indiana (and other states),\" majority op. ", "at 1244, and \"Humphreys reasonably should have anticipated being haled into court in Indiana,\" id. at 1244, strikes me as wholly inconsistent with the facts as well as the requirements of due process.", "\n\n\n43\nHumphreys undoubtedly serves hundreds or even thousands of U.S. tourist guests each year as the result of reservations made by American and other U.S. tour operators and travel agencies. ", " Every U.S. tour organizer or travel agency has innumerable foreign hotels, bus lines, guide services, etc., ", "with which it maintains continuing commercial arrangements. ", " It would be surprising, however, if Humphreys or any other foreign service provider anticipated that, merely as a result of its contacts with U.S. travel agents and without more, it might wind up defending lawsuits brought by its U.S. guests in U.S. courts.", "\n\n\n44\nTo hold, as the majority does today, that Indiana courts may properly exercise in personam jurisdiction over Humphreys is to hold that any foreign hotel, restaurant, museum or other facility or service provider, which confirms reservations for U.S. travel agents and tour operators and compensates those travel agents and tour operators for arranging reservations (a frequent and typical practice), is subject to the jurisdiction of U.S. courts and should expect to be haled into them to defend tort suits or any other claims brought by U.S. guests. ", " This, notwithstanding that the hotel or other service provider has had no dealings with the guest in any U.S. forum, that there was no principal/agent relationship between it and the travel agent or tour operator with whom the guest solely dealt, and even though all the acts and events alleged in the guest's complaint occurred in Timbuktu, Bali, Japan, Australia, Majorca, Tahiti or where-have-you, where, in addition, all the witnesses and relevant records are located.", "\n\n\n45\nThe kind of global long-arm jurisdiction which the majority endorses, in my opinion, both fails the minimum contacts test and is at odds with the fundamental due process requirement that jurisdiction comport with \"fair play and substantial justice.\" ", " I believe that compelling Humphreys to litigate in Indiana is unanticipated, unfair and beyond the bounds of due process. ", " Cf. ", "Asahi Metal Indus. ", "Co. v. Superior Court, 480 U.S. 102, 114, 107 S.Ct. ", "1026, 1034, 94 L.Ed.2d 92 (1987) (\"The unique burdens placed upon one who must defend oneself in a foreign legal system should have significant weight in assessing the reasonableness of stretching the long arm of personal jurisdiction over national borders.", "\").II. ", "Forum Non Conveniens\n\n\n46\nIn addition to holding that Indiana's long-arm statute reaches Humphreys, the majority also finds, as did the Eighth Circuit, in Lehman v. Humphrey Cayman, Ltd., 713 F.2d 339 (8th Cir.1983), that the fact that the hotel and all the witnesses are located in the Cayman Islands, where all the events giving rise to this case took place, is outweighed by other considerations such as the allegedly limited availability of contingent fee arrangements and jury trials in the Cayman Islands, the necessity of a bond, the plaintiffs' prerogative to choose their forum, and the hardship for the plaintiffs of litigating in the Cayman Islands (where their alleged cause of action arose). ", " I respectfully disagree.", "\n\n\n47\nThere are a number of serious problems with trying this case in Indiana. ", " First, the site of the alleged tort, the defendant, all the witnesses other than the plaintiffs and all the records are in the Cayman Islands. ", " The inconvenience to Humphreys far outweighs any inconvenience to the Wilsons to try the case where it arose.", "\n\n\n48\nSecond, Indiana citizens undoubtedly have an interest in a convenient home forum. ", " And, if due process permits, Indiana may have an interest in providing such a forum for its citizens who choose to litigate there. ", " But it is difficult to conceive of what real interest either Indiana or its citizens have in this case. ", " The Wilsons are citizens of Illinois. ", " In Lehman, by contrast, the plaintiffs were, at least, citizens of the forum state.", "\n\n\n49\nThird, while it would undoubtedly be to the Wilsons' advantage to try this case under Indiana law (and before an Indiana jury)--which is, presumably, why they chose to file it there--the differences the parties have identified between Indiana law and the law of the Cayman Islands are not in themselves a justification either for Indiana courts to assert due process long-arm jurisdiction or for finding that the balance of inconvenience in this case favors an Indiana trial. ", " Both the majority and the district court have given too much weight to these differences. ", " \"The possibility of a change in substantive law should ordinarily not be given conclusive or even substantial weight in the forum non conveniens inquiry.\" ", " Piper Aircraft Co. v. Reyno, 454 U.S. 235, 247, 102 S.Ct. ", "252, 261, 70 L.Ed.2d 419 (1981) (availability of strict liability in Pennsylvania but not in Scotland did not, by itself, bar dismissal for forum non conveniens ). ", " See also id. at 252 n. 19, 102 S.Ct. ", "at 264 n. 19.", "\n\n\n50\nI would note, in addition, that although the Wilsons have apparently been assuming that in Indiana, local law would apply, that may or may not be true. ", " Although the majority does not discuss the issue, it is an important conflict of laws question, and possibly a difficult one, whether Cayman Islands or Indiana law should govern at trial. ", " See Piper Aircraft, 454 U.S. at 251, 102 S.Ct. ", "at 263 (\"The doctrine of forum non conveniens ... is designed in part to help courts avoid conducting complex exercises in comparative law.\"). ", " The answer to that question, which depends on Indiana's choice-of-law rules, may determine not only the applicable law of liability and the right to a jury but also Mr. Wilson's right to sue. ", " If the majority is holding, as it seems to suggest by referring to trial by jury, not only that Indiana courts have jurisdiction but also that Indiana substantive law applies, then I believe the opinion ought not to assume it, but should say so and explain why.", "\n\n\n51\nA fourth problem with allowing this case to proceed in Indiana is that it is likewise unclear and troubling to me how an Indiana judgment against Humphreys, should that be the outcome, would be enforced--an issue which, again, the majority's opinion overlooks. ", " I would certainly hope that Humphreys, desiring to continue to attract U.S. tourists, would recognize and respond to any judgment that might be entered against it. ", " But asserting jurisdiction over and entering judgments against foreign entities, many of whom have no assets in the United States, is at best a speculative practice.", "\n\nIII. ", "Conclusion\n\n52\nIt is worth noting the probable consequences of the majority's decision. ", " Many Americans are frequent foreign travelers. ", " If U.S. courts have jurisdiction every time an American tourist has a dispute with a foreign service provider which has an ongoing contact with an American tour organizer or travel agent, we are going to see a lot more lawsuits such as this one, and foreign service providers will likely protect themselves from their increased exposure by charging higher rates and/or insisting on exculpatory provisions in their contracts with Americans.", "\n\n\n53\nI recognize that finding no U.S. jurisdiction in cases like this might require American travelers like Mrs. Wilson to return to places where they have had traumatic experiences in order to pursue remedies for alleged wrongs. ", " Those are not considerations, however, that are relevant to jurisdiction, which can only be stretched so far as due process allows and has, I believe, been stretched far too far in this case.", "\n\n\n54\nIt is undisputed that U.S. tourists traveling voluntarily in foreign countries are subject to the laws and jurisdiction of the courts of those countries. ", " Consistent with that principle, the long-arm jurisdiction of the fifty states cannot be applied globally so as to give U.S. courts specific jurisdiction over foreign defendants in tort cases arising from events that took place entirely on foreign soil and are unrelated to the defendants' contacts with any U.S. forum.", "\n\n\n55\nBelieving that the bounds of due process have been far exceeded in this case, I would reverse the finding of jurisdiction as to Humphreys. ", " I believe, in addition, that denying Humphreys' motion for a dismissal for forum non conveniens constituted an abuse of discretion and would also reverse on that basis, even if Humphreys' contacts with Indiana satisfied due process. ", " I concur, however, with the majority's conclusions as to Holiday Inns.", "\n\n\n\n*\n The Honorable Hubert L. Will of the United States District Court for the Northern District of Illinois, Eastern Division, is sitting by designation\n\n\n1\n One of the original defendants, American Trans Air, Inc., moved for summary judgment, which was granted by the district court on April 6, 1988. ", " The district court entered final judgment pursuant to Rule 54(b) of the Federal Rules of Civil Procedure and that portion of the case was appealed immediately. ", " This court affirmed the district court's judgment regarding the air carrier in Wilson v. American Trans Air, Inc., 874 F.2d 386 (7th Cir.1989)\n\n\n2\n Corporations are considered citizens of the country in which they are incorporated. ", " National Steamship Co. v. Tugman, 106 U.S. (16 Otto) 118, 120-21, 27 L.Ed. ", "87 (1882); Panalpina Welttransport GMBH v. Geosource, Inc., 764 F.2d 352, 354 (5th Cir.1985)\n\n\n3\n British Nationality Act 1981, 31 Halsbury's Statutes 172 (4th ed.1987)\n\n\n4\n Windert also was rejected by Judge Kocoras in Creative Distributors, Ltd. v. Sari Niketan, Inc., Mem. ", "op., ", "1989 WL 105210, 1989 U.S. Dist. ", "LEXIS 10436 at (N.D. Ill.1989) (finding the reasoning of Tetra Finance persuasive), and Judge Luongo in Timco Eng'g, Inc. v. Rex & Co., 603 F.Supp. ", "925, 930 n. 8 (E.D.Pa.1985) (same)\n\n\n5\n In Murarka v. Backrack Bros., Inc., 215 F.2d 547 (2d Cir.1954), the plaintiff was a citizen of India who filed his suit while India was still a British colony. ", " The defendant claimed that, because India was not a sovereign nation when the complaint was filed, the court did not have alienage jurisdiction. ", " Then-Judge Harlan determined that the district court did have jurisdiction, because the United States had granted de facto recognition of India by accepting an ambassador from that country. ", " Id. at 552\n\n\n6\n It is not argued that Humphreys' business in Indiana constitutes \"the kind of continuous and systematic general business contacts,\" Helicopteros Nacionales de Colombia v. Hall, 466 U.S. 408, 416, 104 S.Ct. ", "1868, 1873, 80 L.Ed.2d 404 (1984), necessary for general jurisdiction\n\n\n7\n The Neiman court also noted that convenience for the parties is a factor that is often considered as part of a due process minimum contacts analysis. ", " In that case, the fact that the defendant was an English company and that litigation in Illinois might have been inconvenient was not dispositive, for \"it would also be inconvenient for plaintiff to sue overseas.\" ", " 619 F.2d at 1195 n. 8. ", " Because of the \"purposeful acts\" in the forum, the inconvenience did not amount to denial of due process. ", " Id\n\n\n8\n This section was repealed in 1986 and superceded by the foreign corporations chapter of the Indiana Code, sections 23-1-49-1 to -10\n\n\n9\n The Supreme Court never has ruled whether the criteria governing the forum non conveniens decision are governed by federal or state law under the rule of Erie R.R. Co. v. Tompkins, 304 U.S. 64, 58 S.Ct. ", "817, 82 L.Ed. ", "1188 (1938). ", " See Piper Aircraft Co. v. Reyno, 454 U.S. 235, 248 n. 13, 102 S.Ct. ", "252, 262, 70 L.Ed.2d 419 (1981). ", " Because the parties appear to agree that federal and Indiana criteria are the same, we need not decide the matter. ", " Humphreys' Br. ", "at 24\n\n\n10\n We recognize that, to the extent these disadvantages are grounded in differences in the substantive law of the Cayman Islands, they may be given some, but not substantial or conclusive, weight. ", " See Piper Aircraft Co. v. Reyno, 454 U.S. 235, 247, 102 S.Ct. ", "252, 261, 70 L.Ed.2d 419 (1981); Macedo v. Boeing Co., 693 F.2d 683, 688 (7th Cir.1982)\n\n\n1\n See Fordyce v. Round Hill Developments, Ltd., 585 F.2d 30 (2d Cir.1978) (Jamaican hotel did not subject itself to jurisdiction of New York courts by contracting with a travel agent, where the travel agent was an independent contractor which leased rooms from the hotel at its own risk); Kopolowitz v. Deepdene Hotel & Tennis Club, 464 F.Supp. ", "677 (S.D.N.Y.1979) (Bermuda hotel did not submit to jurisdiction of New York courts by engaging a sales representative to promote the hotel and handle inquiries and requests for reservations, in New York and elsewhere in the United States, where the sales representative did not have the power to confirm reservations). ", " Compare Gelfand v. Tanner Motors Tours, Ltd., 385 F.2d 116 (2d Cir.1967) (involving a nonforum defendant whose agent in New York had power to confirm reservations)\n\n\n" ]

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[ "Structure and function of the oxidoreductase DsbA1 from Neisseria meningitidis.", "\nNeisseria meningitidis encodes three DsbA oxidoreductases (NmDsbA1-NmDsbA3) that are vital for the oxidative folding of many membrane and secreted proteins, and these three enzymes are considered to exhibit different substrate specificities. ", "This has led to the suggestion that each N. meningitidis DsbA (NmDsbA) may play a specialized role in different stages of pathogenesis; however, the molecular and structural bases of the different roles of NmDsbAs are unclear. ", "With the aim of determining the molecular basis for substrate specificity and how this correlates to pathogenesis, we undertook a biochemical and structural characterization of the three NmDsbAs. ", "We report the 2.0-A-resolution crystal structure of the oxidized form of NmDsbA1, which adopted a canonical DsbA fold similar to that observed in the structures of NmDsbA3 and Escherichia coli DsbA (EcDsbA). ", "Structural comparisons revealed variations around the active site and candidate peptide-binding region. ", "Additionally, we demonstrate that all three NmDsbAs are strong oxidases with similar redox potentials; however, they differ from EcDsbA in their ability to be reoxidized by E. coli DsbB. Collectively, our studies suggest that the small structural differences between the NmDsbA enzymes and EcDsbA are functionally significant and are the likely determinants of substrate specificity." ]

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[ "Anniversary Guide\n\nOur Anniversary Guide can help you find that perfect piece of jewelry for your loved one.", "\n\nNeed Ideas for an Anniversary Gift?", "\n\nOur list contains both traditional and modern gifts for each year of your anniversary. ", "But it doesn't always have to be a special occasion to give the gift of jewelry. ", "Not sure what to get? ", "Ask our staff at J. Gowen Jewelry Artistes for some great gift ideas! ", "Contact us today." ]

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[ "const fs = require('fs');\nconst tokensDirectory = './src/tokens';\nconst request = require('request');\n\nfunction checkTokenLogos() {\n let invalidImgArray = [];\n fs.readdirSync(tokensDirectory).forEach(folder => {\n fs.readdirSync(`${tokensDirectory}/${folder}`).forEach(file => {\n const fullPath = `${tokensDirectory}/${folder}/${file}`;\n const obj = JSON.parse(fs.readFileSync(fullPath, 'utf8'));\n\n if (obj.logo.src === '') {\n return;\n }\n\n request({method: 'HEAD', uri: obj.logo.src}, function(err, response) {\n if (err) {\n invalidImgArray.push(obj.logo.src);\n }\n\n if (!", "err && response.statusCode === 400) {\n invalidImgArray.push(obj.logo.src);\n }\n \n fs.writeFileSync('./invalidLogoSrc.js', JSON.stringify(invalidImgArray))\n })\n }) \n })\n}\n\ncheckTokenLogos();\n" ]

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[ "<?", "php // only copy if needed\n\n/**\n * Forces tax recalculation for Subscriptions renewal orders when created.", "\n *\n * NOTE THAT this snippet can ONLY be used when all Subscriptions gateways support payment total changes!", "\n * Without this, a change in the tax amount can cause your renewals to fail.", "\n */\n\n\n/**\n * Forces taxes to be recalculated on renewal orders from Subscriptions.", "\n *\n * @param \\WC_Order $renewal_order the newly-created renewal order\n * @param int|\\WC_Subscription $subscription Post ID of a 'shop_subscription' post, or instance of a WC_Subscription object\n * @return \\WC_Order updated renewal order\n */\nfunction sv_wc_avatax_recalculate_renewal_taxes( $renewal_order, $subscription ) {\n\n\tif ( function_exists( 'wc_avatax' ) ) {\n\n\t\twc_avatax()->get_order_handler()->calculate_order_tax( $renewal_order );\n\t}\n\n\treturn $renewal_order;\n}\nadd_filter( 'wcs_renewal_order_created', 'sv_wc_avatax_recalculate_renewal_taxes', 20, 2 );" ]

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[ "Q:\n\nShould we generalize this old build order question?", "\n\nToday I came across this question:\n\nIn Age of Empires 3, what is a good build order for the British?", "\n\nWhile this topic has been discussed before with a seeming end result of \"don't touch them\", I'm not entirely convinced we shouldn't touch this question.", "\nThe question displays very little effort, and could easily apply to any civilization in the game, and the accepted answer seems a bit shallow, only stating one way to play the British, and even that could be called simply a list of British strengths.", "\nIf I were to ask a series of nearly identical questions pertaining to every other civ in AoE3, would those questions be acceptable? ", " I rather doubt it, but I don't see what makes the British so special.", "\nShould we do anything here?", "\n\nA:\n\nQuite frankly, I'm not sure what the problem would be with a question about every other civ in AoE3? ", "Spamming bad questions to prove your point wouldn't be productive; after all, they'd be bad questions that don't reflect research effort or experience or an understanding of what concerns the question ought to address most likely, but that doesn't mean that we can't have lots of them. ", "Don't focus on how many variants of a question might exist (different divs, or build types or whatever). ", "Consider the fact that the answers would be so wildly different that they don't start from the same place.", "\nWhat problem exactly, are you trying to solve here? ", "What I see is a reasonably specific question with a number of answers with quite a bit of specific detail in their responses. ", "Are those responses appreciably similar to the answers you'd get for a question about playing AoE3 as the French or the Spanish or the Cree or the Iroquois? ", "I don't know. ", "I don't know enough about AoE 3 to make that judgment.", "\nI can certainly say though, that if you'd linked say... a question about a build order for playing as Protoss in SC2, and asked if we should broaden it to include Zerg and Terran, I'd laugh in your face. ", "If you'd linked a question about playing Civ 5 as the French, I'd probably argue to broaden it.", "\nWhat I'm getting at here is that my answer to the previous question holds. ", "Categorical statements are a bad idea:\n\nNow, Build questions in general are a bit thornier, but again, a blanket ban is not the appropriate solution. ", "Yes, a lot of character build questions are really bad questions; cf. ", "skyrim. ", "This does not make questions about character builds inherently bad. ", "The main thing is that the question needs to reflect some actual experience - a concrete problem faced by the player. \"", "I want a build that is totally cool and sneaky and uses big swords and magic lasers and stuff\" is not a good question. ", "It's unfocused, vague, and doesn't really provide any useful information for answerers to provide a good answer. ", "The thing that gets tricky here, is that the criteria that separates a good build question from a bad one is very often specific to the game in question. ", "A good build question for diablo-3 is generally going to specify a class, any skills that are 'mandatory' to the asker and must be built around, and a desired play style (tanky, hardcore, low-gear dependency magic-find, whatever). ", "A good build question for mass-effect-3 by contrast, can generally be a bit more vague, and will generally expect it's answers to be as much or more focused on play style as it is on skill selection. ", "Good build questions for skyrim tend to be either extremely specific (and focus on whether certain specific skills or spells are useful or synergize as they appear to), or very general and focused more on the meta-strategy of how to pick skills, rather than on particular skill choices. ", "Good build questions about an MMO like world-of-warcraft will tend to be fairly mathy and depend on optimal DPS/HPS/Threat/Effective Health numbers - however, even with these, there may be multiple answers as different builds will make different tradeoffs and a multiplicity of answers arguing for these many variations is a good thing.", "\n\nIs this question a bad question? ", "I don't know enough about AoE3 to say. ", "Do you? ", "If so, make the case that it's a bad question, and that it ought to be fixed by being made broader. ", "At the moment, you haven't done so at all - and the number and diversity of answers speaks to the fact that it probably shouldn't be, unless being made broader doesn't appreciably change those answers. ", "Keep the usual heuristic for whether a question is too broad. ", "Yes, some people like to, and are able to, write novels in response to questions. ", "But any answer to a question ought to be able to stand on it's own; questions which compel either an extensive, extremely long comprehensive answer, or a 'one-per-answer' situation often end up being problematic.", "\n\n" ]

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[ "Q:\n\nInsert element into stream\n\nis there a way to insert an element T into a Stream<T>?", "\n ArrayList<Foo> foos = new ArrayList<>();\n Foo foo = new Foo();\n\n Stream<Foo> stream = Stream.concat(foos.stream(), Stream.of(foo));\n\nIs there another way? ", "basically a kind of foo.stream().add(foo)... - of course add() doesn't exist. -", "\n\nA:\n\nNo, there's no other way to add elements to the given stream in standard Java Stream API. ", "Some third-party libraries including my StreamEx library provide additional convenient methods to do this:\nStream<Foo> stream = StreamEx.of(foos).append(foo);\n\nInternally it uses the same concat method.", "\nSimilar method is available in jOOL library:\nStream<Foo> stream = Seq.seq(foos).concat(foo);\n\n" ]

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[ "{\n \"$schema\": \"https://dev.office.com/json-schemas/core-build/tslint.schema.json\",\n // Display errors as warnings\n \"displayAsWarning\": true,\n // The TSLint task may have been configured with several custom lint rules\n // before this config file is read (for example lint rules from the tslint-microsoft-contrib\n // project). ", "If true, this flag will deactivate any of these rules.", "\n \"removeExistingRules\": true,\n // When true, the TSLint task is configured with some default TSLint \"rules.\":", "\n \"useDefaultConfigAsBase\": false,\n // Since removeExistingRules=true and useDefaultConfigAsBase=false, there will be no lint rules\n // which are active, other than the list of rules below.", "\n \"lintConfig\": {\n // Opt-in to Lint rules which help to eliminate bugs in JavaScript\n \"rules\": {\n \"class-name\": false,\n \"export-name\": false,\n \"forin\": false,\n \"label-position\": false,\n \"member-access\": true,\n \"no-arg\": false,\n \"no-console\": false,\n \"no-construct\": false,\n \"no-duplicate-case\": true,\n \"no-duplicate-variable\": true,\n \"no-eval\": false,\n \"no-function-expression\": true,\n \"no-internal-module\": true,\n \"no-shadowed-variable\": true,\n \"no-switch-case-fall-through\": true,\n \"no-unnecessary-semicolons\": true,\n \"no-unused-expression\": true,\n \"no-use-before-declare\": true,\n \"no-with-statement\": true,\n \"semicolon\": true,\n \"trailing-comma\": false,\n \"typedef\": false,\n \"typedef-whitespace\": false,\n \"use-named-parameter\": true,\n \"valid-typeof\": true,\n \"variable-name\": false,\n \"whitespace\": false\n }\n }\n}" ]

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[ "WASHINGTON — By making federal appeals court Judge Brett Kavanaugh his Supreme Court pick, President Donald Trump selected someone who already once secured votes from GOP swing Sens. ", "Susan Collins, R-Maine, and Lisa Murkowski, R-Alaska (back in 2006). ", "What’s more, key Democratic red-state senators like Joe Donnelly, D-Ind., Heidi Heitkamp, D-N.D., and Joe Manchin, D-W.V., released statements Monday saying they’d keep an open mind about Kavanaugh.", "\n\nAdd it all up and it’s very possible that, in the current 51-49 Senate, Kavanaugh could match the 54-45 confirmation vote that Neil Gorsuch got in 2017.", "\n\nBut there’s one significant wild card to Trump picking Kavanaugh: the Mueller probe.", "\n\nAnd if there’s a big development in the investigation — we haven’t really had one since April, when the FBI raided Trump lawyer Michael Cohen’s offices — then Kavanaugh’s 2009 law review article could be an impediment to confirmation.", "\n\nIn the article, Kavanaugh argues that a sitting president shouldn’t be distracted by civil lawsuits or criminal investigations while in office. “", "Having seen first-hand how complex and difficult that job [of president] is, I believe it vital that the president be able to focus on his never-ending tasks with as few distractions as possible,” he wrote. “", "Looking back to the late 1990s, for example, the nation certainly would have been better off if President Clinton could have focused on Osama bin Laden without being distracted by the Paula Jones sexual harassment case and its criminal-investigation offshoots.” ", "And he called for Congress to consider passing a law to defer any civil lawsuit or criminal investigation against a president until after he leaves office.", "\n\nNotably, Kavanaugh doesn’t pass constitutional judgment on this matter. “", "The result the Supreme Court reached in Clinton v. Jones — that presidents are not constitutionally entitled to deferral of civil suits — may well have been entirely correct; that is beyond the scope of this inquiry.” ", "But he does make his personal opinion clear: “The indictment and trial of a sitting president, moreover, would cripple the federal government, rendering it unable to function with credibility in either the international or domestic arenas. ", "Such an outcome would ill serve the public interest, especially in times of financial or national security crisis.”", "\n\nWhat’s especially striking about Kavanaugh’s opinion in that 2009 law review article is that it contradicts his work for Ken Starr, who investigated President Bill Clinton in the Whitewater/Lewinsky probes.", "\n\nAs we wrote after Justice Anthony Kennedy announced his retirement late last month, what makes this Supreme Court fight different from the ones during the Bush or Obama years is the Mueller probe. ", "The president's 2016 campaign — and the president himself — is under investigation for its possible ties to Russian interests.", "\n\nAnd if there's a significant development in the next month (another indictment, guilty plea, or battle over a subpoena), Democrats could argue that the president shouldn't be able to appoint a justice to the court who will probably have to rule on some aspect of the Mueller probe. ", "And that becomes even more potentially explosive given Kavanaugh’s article in 2009.", "\n\nHere’s what the swing GOP and Democratic senators said about Kavanaugh’s nomination\n\nAs mentioned above, here are the statements that key GOP and Democratic senators released last night on Kavanaugh’s nomination, per NBC’s Frank Thorp, Garrett Haake and Rebecca Shabad:\n\nSusan Collins: “Judge Kavanaugh has impressive credentials and extensive experience, having served more than a decade on the D.C. Circuit Court of Appeals. ", "I will conduct a careful, thorough vetting of the President’s nominee to the Supreme Court, as I have done with the five previous Supreme Court Justices whom I have considered.”", "\n\nLisa Murkowski: “I intend to review Judge Kavanaugh’s decisions on the bench and writings off the bench, and pay careful attention to his responses to questions posed by my colleagues on the Senate Judiciary Committee.”", "\n\nJoe Donnelly: He said before the Kavanaugh pick that he declined an invitation to appear at last night’s White House announcement \"so that I can meet first with the nominee in a setting where we can discuss his or her experience and perspectives.\"", "\n\nHeidi Heitkamp (per a spokeswoman): “She has made clear — as she said to the president in person two weeks ago — that she considers fully vetting Supreme Court nominees one of the most important jobs of any U.S. senator, and she plans to fulfill that critical duty.”", "\n\nJoe Manchin: “I will evaluate Judge Kavanaugh’s record, legal qualifications, judicial philosophy and particularly, his views on healthcare. ", "The Supreme Court will ultimately decide if nearly 800,000 West Virginians with pre-existing conditions will lose their health care. ", "This decision will directly impact almost 40% of my state, so I’m very interested in his position on protecting West Virginians with pre-existing conditions.”", "\n\nInterest groups plan to spend millions on the confirmation fight\n\nNBC’s Leigh Ann Caldwell: “The Charles Koch-backed organization Americans for Prosperity announced Monday that it will spend \"seven figures\" on grassroots organizing, paid media and door-to-to door canvassing in the ten states that Democrats are trying to defend in November. ", "And other political interest groups are planning a barrage of campaign ads as well as on-the-ground grassroots activities. ", "Among those Democrats bearing the brunt will be Sens. ", "Joe Donnelly of Indiana, Heidi Heitkamp of North Dakota and Joe Manchin of West Virginia.”", "\n\nMore: “Before Monday's announcement, those [Democrats] were already the targets of at least $1.4 million worth of advertising from the conservative Judicial Crisis Network, an umbrella organization pushing for the confirmation of Trump’s nominee. ", "The ad says, ‘extremists will lie and attack the nominee. ", "But don’t be fooled.’”", "\n\nTrump continues to criticize NATO — in a harsher way than he’s ever criticized Vladimir Putin\n\nBefore embarking to Brussels, Belgium for the NATO conference, Trump fired off these tweets:\n\n“Getting ready to leave for Europe. ", "First meeting - NATO. ", "The U.S. is spending many times more than any other country in order to protect them. ", "Not fair to the U.S. taxpayer. ", "On top of that we lose $151 Billion on Trade with the European Union. ", "Charge us big Tariffs (& Barriers)!”", "\n\n“NATO countries must pay MORE, the United States must pay LESS. ", "Very Unfair!”", "\n\nThis NATO meeting has the potential to be a replay of that G-7 meeting in Canada — after which Trump criticized Canadian Prime Minister Justin Trudeau and refused to sign the joint communique.", "\n\n“In the past, Europe did not doubt that U.S. interests and values were fundamentally aligned with theirs,” Daniel M. Price, an international economic adviser to President George W. Bush, tells the New York Times. “", "Now they wonder whether they can count on us in times of crisis without our first checking to see if they are current on their rent or royalty payments. ", "The decline in confidence is palpable.”", "\n\nAnd European Council President Donald Tusk said this, per the AP: “Dear America, appreciate your allies, after all you don’t have all that many.”", "\n\nAnother secret recording jolts the Cagle-vs.-Kemp gubernatorial runoff in Georgia\n\n“Lt. ", "Gov. Casey Cagle's campaign was already rocked last month by the release of a secretly recorded conversation in which Cagle said he backed what he called ‘bad public policy’ for political gain. ", "Cagle's runoff opponent, Secretary of State Brian Kemp, released another snippet of that conversation Monday,” the AP writes. “", "In this 50-second piece, Cagle can be heard candidly discussing the GOP primary's sharp turn to the right, saying the five-man race came down to ‘who had the biggest gun, who had the biggest truck and who could be the craziest.’ ", "Kemp said in a statement to the AP on Monday that the newly released recording ‘exposes Cagle's real opinion of Republican voters in Georgia.’”", "\n\nThe runoff is on July 24, and the winner faces Democrat Stacey Abrams.", "\n\nLooking at the history of baseball’s All-Star Game in Washington D.C.\n\nFinally, next week brings us Major League Baseball’s All-Star Game, which takes place in D.C. And writing for NBC Washington, Frederic Frommer looks at the history of the All-Star Game in the nation’s capital. “", "On a hot sticky July day in Washington 81 years ago, President Franklin D. Roosevelt made his way to the tiny ballpark two miles northeast of the White House, escaping a political storm of his own making. ", "On his heels over his unpopular plan to ‘pack’ the U.S. Supreme Court with extra justices to overcome a court that had stymied some of his New Deal legislation, FDR found campaign-style adoration at the 1937 All-Star Game.”", "\n\n“That afternoon marked the first time Washington hosted the All-Star Game, and it would do so again in 1956, 1962, and 1969, with presidents playing a central role in three of the four games. ", "In July, the Midsummer Classic returns to DC for the first time in nearly a half-century. ", "The White House has not yet said whether President Trump will throw out the first pitch on July 17. ", "If so, he'd be continuing a summer tradition started by FDR, who died a year before Trump was born in 1946.”" ]

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[ "Most Trusted Professional Moving Company\n\nOur Services\n\nLong Distance\n\nLocal Moving\n\nCommercial\n\nStorage\n\nSelf storage 10020\n\nThis is the ultimate guide to find the most convenient Self storage 10020 based on instant quotes, an affordable price, excellent customer service, a prominent record of genuine five stars reviews, and located close to my area, street, neighborhood, ZIP, borough, city, or county. ", "In the next paragraphs, I will enumerate the most important attributes to select the best Self storage 10020.", "\n\nThe five characteristics of Self storage 10020 must have are: instant quotes, maximum of 5 minutes response time, and 100% response rate; affordable price based on an average of $130 per hour for local moves, and $4.90 per cubic foot in long-distance or interstate operations; excellent customer service with full support for disassembling, packing, storage, moving, unpacking, and reassembling furniture; positive feedback, reviews are a good source that provide details about the moving company; and, location, the area of operations is easily identifiable in the company’s website.", "\n\nThere are so many moving companies, some are better than others, and all of them are trying to appear as the right choice for you. ", "The first thing we have to check is which companies serve the area that our furniture is located for pick-up. ", "This will impact directly the amount of the estimate we will request. ", "For example, Self storage 10020 operate in almost every state in the US.", "\n\nMoving Estimates\n\nThe first thing we need from the Self storage 10020 is the Moving Estimate. ", "There is a misconception around this. ", "An estimate is not the same thing than a quote, let’s see the difference between one to another:\n\nAn Moving Estimate is an educated guess that is based on what the job may cost. ", "It includes our first thoughts on costs and can change drastically when you get further information, while a Quote or Quotation is an exact price for the offered job and cannot be changed. ", "Generally, a quote is issued after an on-site visit where you are confident that you have established what you need, in contrast to an estimate that is issued generally online. ", "In summary, an estimate is a rough idea of our move while a quote is a legally binding document.", "\n\nAffordable Price\n\nThe Moving Industry involves two kinds of operations: Local Moves, and Long Distance Moves. ", "Everyone has their characteristics. ", "Let’s check one by one.", "\n\nLocal Moves\n\nOn a Local Move, you can be expected to be asked to pay around $130 per hour. ", "To count a move as Local it has to be inside the tree-state area —New York, New Jersey, Connecticut. ", "A local move requires to hire three men with a truck. ", "The rate will be higher at the beginning and the end of the month and in summer because of the increased demand for this service. ", "Travel time is included as well, one hour is the average time required to go to your home. ", "Remember that the hourly rate is applied only to Local Moves, that means any relocation inside the three-state area.", "\n\nLong Distance Moves\n\nA Long Distance Relocation is any operation between the Three-State area and the rest of the US. ", "There are multiple expenses on a Long Distance move such as the size of your move, packing services, date of the move, storage, travel fees, among others. ", "Hiring a top-rated Moving Company brings multiple benefits such as consolidated shipping —a practice that consists in combine multiple shipments in one (multi-stop truckload) shipment. ", "Another factor to consider is the date of the move, this will impact directly in your final invoice. ", "Moving at the beginning, or the end of the month could be expensive; the same occurs in summer." ]

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[ "Kazagići (Kiseljak)\n\nKazagići is a village in the municipality of Kiseljak, in central Bosnia and Herzegovina.", "\n\nReferences\n\nCategory:Populated places in Kiseljak" ]

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[ "Check out our new site Makeup Addiction\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\nadd your own caption\n\na link to a scientific article upvote to feel smart" ]

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[ "NEW ORLEANS – The NOPD is seeking the public’s assistance in identifying and locating a vehicle of interest in an armed robbery and shooting which occurred June 3 at the intersection of St. Charles Avenue and Lee Circle.", "\n\nThe male victim in this incident was in the area when he was approached by an unknown male suspect armed with a handgun. ", "The suspect demanded the victim's property. ", "The male victim refused and was shot.", "\n\nThe perpetrator in this incident is observed via video surveillance being dropped off at the location where the incident occurred.", "\n\nThe vehicle is described as gold in color pickup truck with a red stripe down the center. ", "A photo of the vehicle captured from surveillance video can be found below.", "\n\nAnyone with information regarding this vehicle or this incident is asked to contact the Eighth District Detective Unit at 504-658-6080.", "\n\nCitizens with information that can help solve a crime are asked to call Crimestoppers at 822-1111 or toll-free 1-877-903-STOP. ", "You could receive a cash reward of up to $2,500 for the information leading only to the arrest, except on homicides, where Crimestoppers will pay half of the reward upon the arrest and half on the indictment of the responsible person(s). ", "You do not have to give your name nor testify to receive the reward but you must contact Crimestoppers directly with your information. ", "Citizens can also submit an anonymous tip online to Crimestoppers at www.crimestoppersgno.org." ]

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[ "Mitch McConnell recently attributed a major drop in unemployment and growth of the U.S. economy to \"the expectation of a Republican congress.\" ", "Oh, hell no, said Jon Stewart on Thursday evening's edition of \"The Daily Show.\"", "\n\n\"Oh, come on! ", "You've been in control of the Senate for two days!\" ", "Stewart said. \"", "Unless my calendar is broken, Senator, which is possible, since I bought it at a discount calendar factory, the general economic improvement trend started before the November elections.\"", "\n\nAdvertisement:\n\n\"Thankfully, the Republican Senate majority under Mitch McConnell came to our rescue and saved the economy retroactively,\" Stewart continued. \"", "It's easy to assume that Mitch McConnell is saying that people felt so inspired by the moment, he switched the nameplate on his office door, then he invented time travel and went back to 2009 and started spending and hiring again, but the truth is actually much simpler. ", "The truth is this, and it'll blow your minds: If you rub Mitch McConnell's shell, you will receive five years of business growth.\"" ]

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[ "If you never noticed that Alpha DSLR/T are more \"creative skills\" oriented than other competitors (I mean the opposite to techno-geek oriented), so it's you that you missed something. (", "Same things for the e-mount)\n\nThe questions here is: « Who is the idiot ?»", "Those who bought a DSLR and using it permanently in [Auto] mode, or what? ", "And maybe « Who's next?»" ]

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[ "Q:\n\nFinding partial strings using str.find() then replace values from dictionary\n\nI need to replace the values from a column. ", "The values from the column need not to be exact match, so I use str.find(). ", "Once it scanned the string, it should replace the values from the dictionary.", "\nI achieved the desired result for one instance, but I need to do it multiple times.", "\nI tried creating a function but it didn't work. ", "It only worked for the last dictionary value. ", "\ndictionary = {\"AA\" : \"111\", \"BB\" : \"222\", \"CC\": \"333,444\"}\n\n#result = []\nfor k, v in dictionary.items():\n df[\"renamed\"] = np.nan\n df.loc[(df[\"combined_topic\"].str.find(k) !", "= -1), \"renamed\"] = v\n #result.extend(df[\"renamed\"].to_dict(orient=\"records\"))\n\nHow should I fix my code? ", "or can you suggest more efficient way to replace multiple values.", "\nExpected output:\ncombined_topic renamed\nAA, harvard 111\nDiliman, Technology, BB 222\nCat, Dog, CC, Bull 333, 444\n``\n\nA:\n\nUse Series.str.extract for get first matched value of dictionary and then Series.map by dict:\npat = '|'.join(dictionary)\ndf['renamed'] = df['combined_topic'].str.extract('('+ pat + ')', expand=False).map(dictionary)\nprint (df)\n combined_topic renamed\n0 AA, harvard 111\n1 Diliman, Technology, BB 222\n2 Cat, Dog, CC, Bull 333,444\n\nYour solution houl be used with Series.str.contains, but mainly remove df[\"renamed\"] = np.nan, because data are always overwritten in each loop:\nfor k, v in dictionary.items():\n df.loc[df[\"combined_topic\"].str.contains(k), \"renamed\"] = v\n\nOr:\nfor k, v in dictionary.items():\n df.loc[(df[\"combined_topic\"].str.find(k) !", "= -1), \"renamed\"] = v\n\n" ]

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[ "Q:\n\nHow to find absolute index() with jQuery?", "\n\n<nav>\n <section>\n <aside>\n <div class=\"findthis\">111111</div>\n <div class=\"findthis\">222222</div>\n <div class=\"findthis\">333333</div> \n <div class=\"findthis\">444444</div>\n </aside>\n </section>\n</nav>\n\n$('.findthis').each( function(){\n var index = $(this).index()+1;\n $(this).append(\" ( this element = \"+index+\" )\")\n});\n\nthis is working! (", "http://jsfiddle.net/3c5TZ/)\nbut.... when i do/insert more HTML-tags like this...\n<nav>\n <section>\n <aside>\n <div class=\"findthis\">111111</div>\n <h2><h2>\n <div class=\"findthis\">222222</div>\n <span></span>\n <div class=\"findthis\">333333</div>\n <b></b> \n <div class=\"findthis\">444444</div>\n </aside>\n </section>\n</nav>\n\nit fails! (", "http://jsfiddle.net/3c5TZ/1/)\nAny ideas?", "\n\nA:\n\nThe index is passed to the .each() method you don't have to reinvent the wheel:\n$('.findthis').each( function(index){\n $(this).append(\" ( this element = \" + (index + 1) + \" )\")\n});\n\nUpdated fiddle.", "\n\nA:\n\nPlease consult the documentation of index(). ", "In your example, jQuery cannot really know what kind of index you are looking for, relative to what?", "\nOne way of doing this is providing the selector for .index():\n$('.findthis').each( function(){\n var index = $(this).index('.findthis')+1;\n $(this).append(\" ( this element = \"+index+\" )\")\n});\n\njsFiddle Demo\nAnother (in this case maybe more effective) way is to run index() on the whole collection, and pass the element as a parameter:\nvar $collection = $('.findthis');\n$collection.each( function(){\n var index = $collection.index(this)+1;\n $(this).append(\" ( this element = \"+index+\" )\")\n});\n\njsFiddle Demo\n\nA:\n\nYou have accidentally nested elements inside each other.", "\nChange this:\n<h2><h2>\n\nto this:\n<h2></h2>\n\nThen it works just fine. ", "Demo: http://jsfiddle.net/Guffa/3c5TZ/14/\n\n" ]

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[ "Quantification of regional ventilation-perfusion ratios with PET.", "\nThe topographic matching of alveolar ventilation (V(A)) and perfusion (Q) is the main determinant of gas exchange efficiency of the lung. ", "However, no pulmonary functional imaging technique has been shown to predict whole-lung gas exchange in health and disease. ", "This study aims to present a PET-based method to estimate regional alveolar ventilation-to-perfusion ratios (V(A)/Q) predictive of arterial blood gases. ", "The method is based on the regional tracer kinetics of (13)N-nitrogen ((13)NN) after an intravenous bolus injection during a breath-hold period and subsequent washout from the lungs with resumption of breathing. ", "The method takes into account the presence of inter- and intraregional nonuniformities at length scales smaller than the imaging spatial resolution. ", "An algorithm used regional tracer washout to classify regional V(A)Q/ uniformity. ", "Intraregional V(A)/Q mismatch in nonuniform regions was described with a 2-compartment model. ", "Regional V(A)/Q estimates were combined into a whole-lung distribution of V(A)/Q ratios and were used to compute global arterial blood gases. ", "The method was applied to 3-dimensional PET data from anesthetized and mechanically ventilated sheep before and after methacholine bronchoconstriction (n = 3) and pulmonary embolism (n = 3) and after saline lung lavage (n = 3). ", "PET images revealed regional changes in ventilation and perfusion consistent with the different disease models. ", "Quantification of the images using PET-derived V(A)Q/ distributions showed unimodal and narrow distributions in control conditions that became wider and unimodal after pulmonary embolism and saline lung lavage and bimodal after bronchoconstriction. ", "Images of regional gas exchange allowed for visualization of regional gas exchange. ", "Arterial blood gases estimated from the PET-based V(A)/Q distributions closely agreed with measured values (partial pressure of oxygen, arterial [PaO(2)]: r(2) = 0.97, P < 0.001; partial pressure of carbon dioxide, arterial [PaCO(2)]: r(2) = 0.96, P < 0.001). ", "Tracer kinetics analysis of PET images after an intravenous injection of (13)NN provides a quantitative assessment of regional V(A)/Q heterogeneity including that corresponding to length scales smaller than the spatial resolution of the imaging method. ", "Quantification of V(A)/Q mismatch obtained with the presented technique is directly related to severity of gas exchange impairment as determined by arterial blood gases." ]

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[ "On the podium with U.S. national team head coach Jill Ellis at the World Cup final press conference on Saturday, Megan Rapinoe didn’t even want to wait for the question about the NWSL to be finished.", "\n\n\n\n“Shout-out to the Seattle Reign!” ", "she crowed, referencing Friday night’s Reign FC 1-0 victory over Cascadia rivals Portland Thorns FC. “", "Me and Allie (Long) were talking so much shit this morning, it was so good. ", "Very quiet from the Portland players, by the way.”", "\n\n\n\nIt was another delightful moment in the Rapinoe media availability show that’s been the theme through the last month in France, but it was also a reminder that the USWNT players are still deeply invested in the domestic results happening back home in the NWSL. ", "They’ve also been paying attention to all the news coming out of the league.", "\n\n\n\nOn Thursday, the league announced a 14-game television deal with ESPN, which will include the entire postseason being..." ]

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