Here is a simple LDAP schema for storing Secure Remote Password (SRP-6a) authentication credentials. It defines an object class srp6Account which can be attached to any directory entry to enable SRP-6a authentication for it. The SRP salt and verifier are stored in a text attribute called srp6Verifier.
The salt and the verifier are hex encoded (to save space and avoid ambiguity) , separated by a semicolon.
Check out Nimbus SRP if you're looking for a solid and well documented Secure Remote Password library.


text:
Q: Understanding how images are served and cached So I'm wondering how browsers treat requests for images. I'm hoping to use a cdn for serving product images on my website. I'd also like to use the cdn for serving button images and images used in my css.
The problem with this is that I don't have control over the expires headers (Rackspace files is what I'm looking into).
See, say I have a large image file as a background on my home page. So the page is accessed often, but the image stays the same. Is the browser going to request this image every time?
Or should I just use a cdn for my product images?

A: caching is quite a broad subject. I suggest you start by reading about the different kinds of caching here http://www.mnot.net/cache_docs/#BROWSER and how caching works here http://www.web-caching.com/mnot_tutorial/how.html
Now, to answer your question: assuming the user has caching enabled and the cdn response headers are properly configured a user visiting your page multiple times will only request that background image once until the cache expires or those files are cleaned.

A: No, AFAIK you need necessarily to add the 'cache' header to your images to enable browser caching. This is a great tutorial about it.
Additionally you can read this article from Yahoo to get a very brief view of the topics.
Review specially these topics of the article:

*

*Minimize HTTP Requests

*Add an Expires or a Cache-Control Header

*Use a Content Delivery Network

Hope it helps you

redpajama_set_name: RedPajamaStackExchange


Patrick M. Marrs is president and owner of a marine electronics company and an American Bureau of Shipping technical specialist. He serves as a director of Cook Inlet Tribal Council and an advisory board member of the Seward branch of Wells Fargo Bank, and previously served as chair of the CIRI Alaska Tourism Corporation Board. Marrs served on the CIRI Board from 1977 to 2000 and has been a Board member from 2001 to present.


text:
Q: INSERT statement for one column to populate each row I have a table called books that I just altered to have a column called yearPUB. I'm trying to populate each row with a year but it's not working. This is the INSERT statement I'm using.
INSERT INTO books (yearPub)
    VALUES (2002),
           (2006),
           (1999),
           (2005),
           (2003),
           (2001),
           (1998),
           (1968),
           (2009),
           (1988),

Can someone tell me why it doesn't work?

A: You need to insert them one at a time, e.g.:
INSERT INTO books (yearPub) VALUES (2002);
INSERT INTO books (yearPub) VALUES (2006);

Alternatively, you could insert using a subquery with a select statement.  The syntax may differ depending on what database you are using.  For example, you could follow an example here and write something like:
INSERT INTO books (yearPub)
SELECT yearNumber FROM othertablename;


nesaralaw.us ⭐ 9 out of 10 based on 216 ratings. 2757 user reviews.
Download Ez Go Battery Diagram Pdf, ePub, Mobi.
As an manager of a car, I obtain personally turning into knowledgeable on the subject matter of motor vehicle cabling. Since I am almost the final person that needs to be tinkering with whatever that has electrical latest working by means of it, I had been reluctant to seek to sort out the matter of motor vehicle cabling.
However my vehicle resolved otherwise in the event it begun to randomly decide on electrical methods that it planned to me tamper with. When the electric power seating straps begun to glide lower back and forth with no reason as I drove downward the freeway, I decided I much better discover ways to tamper with the electric power methods, contemplating the exuberant price of motor vehicle repair service.
I came across my own self with a decision to either, break-down and devote $100 an hour for a so-named skilled to consider it and try and restoration it, or I really could find out how the electro-mechanical process of my car been working. Thinking of my degree of ignorance I perhaps was going to try and conquer the out of the question. Of course the only point I realized about electro-mechanical devices is that Air conditioning/DC was a rock band and that beginners and wiring typically failed to get on.
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text:
The initial of return-based event studies as put forward by Fama, Fisher, Jensen, and Roll in 1969 captures short-term effects of events on stock prices. For longer term effects, a related methodology has been developed that captures if an event has had a persistant impact on stock prices over periods of time as long as several months or years after the event. This method hence measures more effect persistence than the size of the short term effect. Common applications of long-term event studies include investigations of stock performances following M&A transactions or public offerings of equity. There are two related approaches for such analyses: (1) The buy-and-hold abnormal return approach, and (2) the calendar-time portfolio method, also refered to as Jensen's alpha approach. Both approaches involve the construction of stock portfolios.
(1) Buy-and-hold abnormal return approach (BHAR)
Buy and hold is an investment strategy where an investor buys stocks and holds them for a long time. The BHAR is based on this principle and calculates abnormal returns by deducting the normal buy-and-hold return from the realized buy-and-hold return.
(2) Calendar-time portfolio approach (CTIME)/Jensen's alpha approach
The CTIME approach calculates the abnormal return of a portfolio consisting of all firms that experienced the event of interest. The abnormal return is defined as the excess return that cannot be explained by expected return models, such as the CAPM or other factor models. The CTIME approach is also named "alpha approach" since "alpha", both in a statistical and an investment theory sense, closely relates to the excess return concept of the CTIME approach. Statistically, alpha refers to the intercept in a time-series regression on actual returns that statistically tests whether these models do explain the actual returns. If they do not explain the actual returns sufficiently, an intercept significantly different from zero results that represents the abnormal returns occured at the event date. Within the context of investments, this alpha reflects the risk-adjusted return measure that expresses the so-called active return on an investment, the return that would be attributable to the investment manager. In event studies, the event substitutes the "investment manager" in being responsible for the abnormal returns. The CTIME approach thus reflects an investor's actual investment experience in the respective stock portfolio. While the method has typically been applied in investment and behavioral and corporate finance research, it has become adopted also in fields like marketing (e.g., Moorman et al., 2012).

redpajama_set_name: RedPajamaCommonCrawl


Orange County Congressman Rohrabacher met with accused Russian agent in St. Petersburg… Story.
Kimberly Guilfoyle laments Gavin Newsom's 'far left' turn, praises Donald Trump Jr. in interview… Story.
Pro-PG&E wildfire bill written by lawmaker whose son works at PG&E…Story.
Voter Registration for Non-Citizens Begins in S.F. School Board Election…Story.
Jerry Brown to Supreme Court: Hurry up and hear my pension law case…Story.
Assault weapon registrations in California are up 43% under new law…Story.
California's top court to decide what officers must do to escape liability in pursuit accidents…Story.
Massive tree die-off brings unprecedented danger as wildfire burns near Yosemite…Story.
What, no refund? A vacationer's guide to California fire season…Story.
Is Lafayette's Utilities System for sale? -Lafayette Utilities System's electric division could be managed by or sold to an outside company for the right price, the mayor said.
Hackers account for 90% of login attempts at online retailers - Selling stolen personal data is a big business for hackers: Somewhere on the dark web, your e-mail address and a few passwords are probably for sale (hopefully, old ones).
There were a couple of new stories published overnight about Mayor Don Tatzin's decision not to run for reelection in November. See, Top Stories, above.


text:
Q: Replace a micro switch with the correct type of transistor Below is a micro switch used on an existing circuit, which turns on/off a load in a DC circuit.  I'd like to replace the micro switch with a transistor, since I'm controlling the load using an Arduino which uses a high/low, and not a tristate.  But I don't know how to look for a compatible transistor.  The specifications on this micro switch seems to work for both AC and DC, despite having "VDC" listed in the specifications.  The circuit I'm working with has a load which only needs 3V.
What type of transistor should I use instead of this micro switch?
Micro Switch:
L62,Micro Switch 15A,125 or 250 VAC 3/4 HP 125 VAC 1 1/2 HP 250 VAC NC
https://www.ebay.com/itm/L62-Micro-Switch-15A-125-or-250-VAC-3-4-HP-125-VAC-1-1-2-HP-250-VAC-NC-1PL29750-/182962174497


text:
Зграда је грађевина која се састоји од спољних зидова и кровне конструкције, с једном или више затворених просторија којим је омогућен приступ. Може служити као заштита људи, животиња или имовине.
Зграде могу бити различитих врста и намена - од сложених комплекса као што су болнице, дворане, куће или стамбене зграде. У згради се може регулисати температура, светло, доток ваздуха или путеви кретања људи. Обликовање зграда је старо као само човечанство.

Дефиниције

У енглеском језику реч building је именица и глагол: сама структура и чин њеног стварања. Као именица, зграда је 'грађевина која има кров и зидове и стоји мање-више трајно на једном месту'; "на углу је била троспратна зграда"; "било је то импозантно здање". У најширем тумачењу ограда или зид је зграда. Међутим, реч структура се користи шире од изградње укључујући природне и вештачке формације и не мора да има зидове. Структура ће се вероватније користити за ограду. Стургисов речник је укључио да се "[зградња] разликује од архитектуре по томе што искључује сваку идеју уметничког третмана; а разликује се од грађења по идеји искључивања научног или веома вештог третмана." Као глагол, градња је чин изградње.

Висина конструкције у техничкој употреби је висина до највишег архитектонског детаља на згради од нивоа улице. У зависности од тога како су класификовани, торњеви и јарболи могу или не морају бити укључени у ову висину. Торњеви и јарболи који се користе као антене углавном нису укључени. Дефиниција ниске зграде у односу на вишеспратницу је предмет дебате, али се генерално три или мање спрата сматрају ниским зградама.

Историја

Постоје јасни докази о градњи домова око 18.000 година пре нове ере. Грађевине су постале уобичајене током неолита (погледајте неолитска архитектура).

Врсте зграда

По намени могу бити стамбене, индустријске, пословне, јавне, здравствене, културне, сакралне, спортске зграде ... У зависности од материјала, могу бити на пример зидане, камене, дрвене или металне зграде.

Комплекс

Понекад се група међусобно повезаних зграда назива комплексом – на пример стамбени комплекс, образовни комплекс, болнички комплекс, итд.

Оштећење у згради

Објекти могу бити оштећени током изградње објекта или током одржавања. Постоји неколико других разлога иза оштећења зграда као што су несреће као што су олује, експлозије, слегање узроковано минирањем, повлачење воде или лоши темељи и клизишта. Зграде такође могу претрпети оштећења од пожара и поплаве у посебним околностима. Оне такође могу постати оронули због недостатка одговарајућег одржавања или неправилног извођења радова.

Види још
 Архитектура
 Конструкција
 Грађевинарство
 Облакодер
 Тврђава
 Замак
 Торањ
 Кућа
 Викендица
 Палата
 Дворац

Референце

Литература

Спољашње везе

European Architectural History Network web site
Western Architecture Timeline
Extensive collection of source documents in the history, theory and criticism of 20th-century architecture

Архитектура
Архитектонски речник

redpajama_set_name: RedPajamaWikipedia


But unpalatable for all that.
This entry was posted in Abuse of Color, Floral Patterns, Paisley and Ornamental Patterns, Summer Floral, Ugly-Pants Alert! and tagged abuse of color, blue, brown, carmine, floral, green, iPhone, lavender, leaves, multicolored, mustard, orange, paisley, peach, pink, purple, red, sea greens, seafoam green, smartphone, summer floral, sunglasses, ugly pants, ugly pattern, ugly skirt, ugly sweater, white, yellow on May 22, 2016 by firstshade.
She looks like a giant pipe cleaner.
This entry was posted in Animal Print and Fur, Pictures and Illustrations and tagged acid wash, black, blue, drawings, faux fur, fleece, gray, grey, hoodie, hoody, illustrations, lips, mass transit, pink, red, red violet, skinny jeans, ugly bag, ugly jacket, ugly sweater, ugly vest on May 18, 2016 by firstshade.
Now just stand there and think about what you've done.
This entry was posted in Camo, Clashing Patterns, Splotches, Ugly-Pants Alert! and tagged acid wash, black and white, camo, gray, grey, pattern mismatch, pool, reflecting pool, splotches, tucked pants, ugly backpack, ugly jeans, ugly pants, ugly pattern, ugly sweater, yellow on April 24, 2016 by firstshade.
This entry was posted in Abuse of Color, Felonious Footwear, Floral Patterns, I'm Going to Hell, Summer Floral, Ugly-Pants Alert! and tagged abuse of color, baggy, black, floral, mass transit, studs, summer floral, ugly pants, ugly pattern, ugly sneakers, ugly sweater, wide-leg pants, yellow on March 4, 2016 by firstshade.
A Thai restaurant exploded on her legs.
This entry was posted in Clashing Patterns, Splotches, Ugly-Pants Alert! and tagged aqua, black and white, cyan, mass transit, multicolored, orange, pattern mismatch, red, sparkles, splotches, teal, turquoise, ugly bag, ugly pants, ugly pattern, ugly sweater, yellow on February 25, 2016 by firstshade.
This calls for serious disciplinary measures.
This entry was posted in Abuse of Color, Clashing Patterns, Felonious Footwear, Floral Patterns, Plaid and Check Patterns, Socks and Leg Warmers and tagged abuse of color, black and white, blue, brown, burgundy, floral, green, mass transit, olive, olive green, pattern mismatch, plaid, red, rust, sneakers, tapestry, ugly bag, ugly pattern, ugly skirt, ugly socks, ugly sweater on February 21, 2016 by firstshade.


text:
Archive for Fiat Money
Where Does Your Tax Money Go?
Posted in Attack on Freedom, Economic Hitmen, economic tyranny, Economy, End The Fed, Fiat Currency, Grace Commission, News, Ron Paul, Taxes, The Banking Cartel, The Federal Reserve with tags Debt, Debt based Money, Fiat Money, Illegal, Income Tax, News, Ron Paul, Ronald Reagan, Sound Money, The Federal Reserve, The Grace Commission on November 28, 2010 by truthwillrise
TheTruthwillrise | November 17, 2010 | 0 likes, 0 dislikes
Everything you have been told about the income tax is wrong. I share the information contained in the Grace Commission Report.
Search Engine " Grace Commission Report"
"IRS Publication 6200"
follow on twitter at http://www.twitter.com/truthwillrise
Check the blog out at https://thetruthwillrise.wordpress.com
What You Need To Know About Money
Posted in "Terrorism", Aaron Russo, Attack on Freedom, Attack on the Republic, economic tyranny, Economy, End The Fed, Fiat Currency, International Bankers, Life Improvement, New World Order, News, The Banking Cartel, The Federal Reserve with tags Aaron Russo, America: Freedom to Fascism, Audit the Fed, End the Fed, Fiat Money, Gold Standard, Hr 2750, Money, New World Order, News, Ron Paul, Sound Money, The Federal Reserve, TheTruthWillRise, War on November 16, 2010 by truthwillrise
TheTruthwillrise | October 13, 2010
Most people do not know how money really works or where it comes from. In this video, I shed some light on that.
G.Edward Griffin-Inflation
Posted in Attack on Freedom, Attack on the Republic, economic tyranny, Economy, End The Fed, International Bankers, Multi-National Corporations, New World Order, News, Stupid Government Tricks, The Banking Cartel, The Constitution, The Federal Reserve, Truth/Freedom, Tyranny, Unconstitutional with tags Col. E. Mandel House, Fiat Money, Franklin Delano Roosevelt, G. Edward Griffin, Gold Standard, Inflation, International Bankers, New World Order, The Banking Cartel, The Federal Reserve, Weimar Republic on August 16, 2010 by truthwillrise
If you have ever wondered what exactly inflation is, and how it's created, then this informative video, by Edward Griffin, done back in 1969 will explain it to you. Learn how institutions which can legally print money are artificially creating inflation every second. Those central banks are the root cause for decreasing the value our money and also the value of our work.
Inflation: The Tax You Don't Notice
Posted in Attack on Freedom, economic tyranny, Economy, Fiat Currency, International Bankers with tags Audit the Fed, End the Fed, Executive Order 11110, Fiat Currency, Fiat Money, Fraud, International Bankers, My Thoughts, New World Order, News, Ron Paul, Sound Money, The Federal Reserve, truth, Truth/Freedom on June 16, 2010 by truthwillrise
It is often said " money doesn't go as far as it used to" . Most people chalk it up to rising prices or greedy companies and take it no further. However that is not truly the case. If people realized they were in actuality paying a hidden tax, they may ask some serious questions.
The American people pay scores of taxes on everything from gasoline to gurdles and everything in between. One of the least known and most insidious is known as inflation.
To understand inflation,one must have a basic understanding of how our banking and monetary system works. In short, our money does not in fact come from the treasury, but is loaned to the government by a private corporation known as the Federal Reserve. As with any loan, there is interest attached to it, which is why we have the income tax. This private corporation has a monopoly over the money supply and because of that the only place the "money" can come from to pay for the debt is from the Fed, they have to constantly increase the money supply to pay for it. Since our money is no longer backed by anything (i.e gold, silver), putting more money into the circulation only decreases the value of the currency.
A simple illustration of this is you buy a pound of meat for $1 and then more currency is put into circulation, that additional currency has decreased its buying power, thus it takes more of it to buy the same amount of goods and to reflect that diminished value the "price" of the meat increases to $1.50.
What this means for you is you must work more to maintain even your current standard of living. The cost of living adjustments that people receive are mere pittance, designed to appease and pacify a population that, for the most part, have no idea what is befalling them.
This is why it is imperative that sound money be restored. It would halt the erosion of our currency's buying power and severely stifle inflation. Executive Order 11110, which allows the treasury to issue silver backed United States notes rather than fiat Federal Reserve notes should be reactivated. Bills such as HR 1207 which audits the Federal Reserve, HR 2750, which abolishes the Fed, need to be supported and passed in their original form.
The alternative is an ever decreasing currency, having to work even more and eventually a collapse of the currency and economy. The choice is yours.
Barack Obama, Barry Soetoro
Posted in Aaron Russo, Alex Jones, Attack on Freedom, economic tyranny, Fiat Currency, International Bankers, New World Order, News, Poison Foods and Products, Police State/Martial Law, Secret Societies, Shadow Government, Stupid Government Tricks, The Constitution, The Federal Reserve, Truth/Freedom, Tyranny, Unconstitutional with tags Alex Jones, Attack on Free Speech, Attack on the Republic, Barack Hussein Obama, Command and Control, economic tyranny, Economy, Fiat Money, Government Corruption, International Bankers, New World Order, Police State, Stupid Government Tricks, The Banking Cartel, Truth/Freedom, Tyranny on June 4, 2010 by truthwillrise
Don't be fooled by the slick charismatic puppet that can charm the back legs off a donkey, Obama is not who he claims to be, watch this video and the picture will become more clear. For those of you who voted for the pimp, do not blame yourself, you can make real change from here on out.
SMASHING THE AXIS OF FINANCIAL FRAUD
Posted in economic tyranny, Economy, Fiat Currency, International Bankers, New World Order, News, The Federal Reserve with tags Dr. Edwin Viera, Fiat Money, International Bankers, The Federal Reserve on December 19, 2009 by truthwillrise
By Dr. Edwin Vieira, Jr., Ph.D., J.D.
[The following is the full text of a somewhat shortened address presented to the Committee for Monetary Research and Education on 15 October 2009…]
The more things change, the more they remain the same. In 1814, in an address to the House of Representatives, Daniel Webster observed that
public credit, the last reliance of government, * * * does not exist. This is a state of things calling for the soberest counsels, and yet it seems to meet only the wildest speculations. Nothing is talked of but banks, and a circulating paper medium, and exchequer notes, and the thousand other contrivances which ingenuity, vexed and goaded by the direst necessity, can devise, with the vain hope of giving value to mere paper. All these things are not revenue, nor do they produce it. * * * [N]or is there a device more shallow or more mischievous, than to pour forth new floods of paper without credit as a remedy for the evils which paper without credit has already created.[1]
Even earlier, Thomas Jefferson had predicted the reason for such a sorry state of affairs:
From the conclusion of the [W]ar [of Independence] we shall be going down hill. It will not then be necessary to resort every moment to the people for support. They will be forgotten, therefore, and their rights disregarded. They will forget themselves, but in the sole faculty of making money, and will never think of uniting to effect a due respect for their rights.[2]
Jefferson was all too prescient. Ever since his day, the political class has looked elsewhere than to the American people for support—and always found it from the financial class.
The financial class has arrayed itself on the side of the political class, and the political class has arrayed itself on the side of the financial class—not just in an incestuous coupling, but in the veritable fusion of a political-cum-financial hermaphrodite: the full integration and consolidation of bank and state.
This unholy alliance has always centered around a mechanism by means of which the financial class can create ersatz "money" out of nothing tangible—through a monopolistic national bank (the First and Second Banks of the United States), then a national banking conglomerate (the National Banks of the Civil War), and finally a fully corporative-state banking apparatus (the modern Federal Reserve System), all operating on the basis of "reserves" so increasingly fractional that they have now become essentially fictional. Through the General Government, the political class has guaranteed the continuance of this scheme, in one form or another, for more than two hundred years.
By so doing, the political class has always been able to count on the support of the financial class—but only at the cost of enabling the financial class to exercise exorbitant influence over the General Government, and through the General Government over the American people themselves.
The true name of this system is financial fascism.
As with all fascistic arrangements, it involves an axis of coöperation between big private special-interest groups and rogue public officials—in this case, the Axis of Financial Fraud that runs from Lower Manhattan in New York City to Washington, in the Disgrace of Columbia.
Whenever and wherever a scheme of this type has been put into operation, it has rested upon a threefold fraud:
First, the falsehood that the purpose of "money" is to serve some disembodied entity—"the economy"; or some institution—"the government"; or some self-selected élite—"the financial community", rather than the people as a whole.
Second, the falsehood that "money" should be created and managed by self-styled "experts" who are politically independent of the people.
Third, and most important, the falsehood that "money" should be based upon debt, whether public or private, not upon any tangible commodity, such as silver or gold, the supply and the value of which the free market determines.
Currency generated out of debt is not an attempt to create a medium of exchange "out of nothing", such as the irredeemable, but debt-free, legal-tender Lincoln Greenbacks of the Civil War. Rather, it is an attempt to create a medium of exchange out of something worse than nothing. For, all other things being equal, debt is always less desirable than the absence of debt. At base, currency generated out of debt is a contradiction in terms, because it purports to transform liabilities into assets. Indeed, it is more of a fantasy even than the fabled Philosopher's Stone. For that was supposedly able to transform one asset—lead—into a more valuable asset—gold, not to turn something that was not an asset at all into something else that was.
Currency generated out of debt rests upon the delusion that increases in the stock of society's medium of exchange will cause corresponding increases in society's real wealth—that is, that debt can be the source of and foundation for wealth because real economic development can always be "stimulated" with new doses of debt. Of course, this naive notion that "having more money equals having more wealth" ignores the questions of whether increases in the supply of money decrease the purchasing power of all money, and of whether such increases actually undermine or even sabotage the operation of the free market, thereby decreasing real wealth. It also ignores the reality that every emission of new currency results in a redistribution of real wealth to the currency's issuer from the society that initially accepts the new currency at its full face value and then continues to use it as it depreciates in purchasing power—that is, that the issuers of currency generated from debt are in fact embezzling wealth from everyone else.
The generation of currency out of debt is a confidence game, because it depends for its continuation upon the average citizen's misplaced confidence in the efficacy and especially the stability of the system emitting that currency. As with every confidence game, however, reality eventually supplants illusion. As time goes by, society suffers an ever-increasing dependence upon ever-expanding increments of debt in order to generate ever-contracting increments of purported "economic growth". The economy supersaturates with debt. Then, like the drug addict who chokes to death on his own vomit as a result of an overdose, the economy finds itself strangled by the ever-tightening cords of unpayable debt—and slips into the fatal coma of stagnation, then depression, and perhaps hyperinflation, too.
The sole constitutional power of Congress with respect to money is "[t]o coin Money, regulate the Value thereof, and of foreign Coin, and fix the Standard of Weights and Measures".[3] Observe that this clause links as cognate powers "regulat[ing] the Value [of Money]" and "fix[ing] the Standard of Weights and Measures", because the Constitution intends for "Money" itself to be a scientific "Standard"—rational, objective, verifiable, or falsifiable—akin to every other known or knowable "Standard of Weights and Measures".
Although emitted under a purported delegation of power from Congress, the contemporary Federal Reserve Note, in contrast, is a standard of nothing tangible or even theoretical—as John Exter used to say, it is truly "an IOU nothing currency". The Federal Reserve System provides this country with neither a rational, nor an objective, nor a verifiable or falsifiable standard—not even a standard predictable in its changeability—let alone anything that could be called a constitutional legal standard. Operating without a scientific monetary standard, the Federal Reserve System interferes with the free market's formation of prices—indeed, the System's manipulations under the rubric of "monetary policy" intentionally falsify prices, causing widespread and ineradicable economic confusion and the monumental waste of human efforts and natural resources.
Of course, America's financial class is not composed entirely of fools devoid of foresight. Anticipating all of these problems, the financial class arranged for the political class to provide three forms of protection for different levels of systemic risk in the Federal Reserve System:
at the lowest level, the concept of "deposit insurance", designed to gull the victims of the fractional-reserve scam into believing that the banking system can and will secure its clients against a relatively few especially imprudent operators;
at the intermediate level, the concept of "the lender of last resort", the spigot of "liquidity" within the banking system itself which enables the system to keep afloat some (albeit not all) large financial institutions when their mismanagement threatens to sink them in bankruptcy; and
at the highest level, the concept of "institutions too big to fail", the ultimate safety-valve to be turned on whenever the financial class's irresponsible speculations endanger one or more segments of the economy so sizeable that only the political class can marshal an adequate "bail out" through the General Government.
The term "institutions too big to fail" is a rather sorry misnomer—because under contemporary financial fascism the big financial institutions—and, it seems, the big industrial concerns as well—are proving too rotten with corruption not to fail. They cannot help but fail, they will fail, and for rogue public officials to provide them with "bail outs" merely transfers the real burden of failure from the pocketbooks of the financial class onto the backs of the American people. Having put across the swindle of "institutions too big to fail", though, the financial class now holds the people of the United States hostage to its manipulations. Without "bail out" after "bail out", the financial class warns, the economy will collapse, social chaos will break out, and the invocation of "martial law" will be necessary to restore order. Thus, common Americans have been made the unwilling, but perpetual guarantors of a gigantic Ponzi scheme in which well-organized racketeers in the financial class rake in the illicit profits—their cronies in the political class secure the votes, the pensions, and the golden parachutes—and average citizens must swallow the ever-increasing losses in jobs, in productivity, in income, in standards of living, and in every other measure of economic well-being.
Americans must also suffer the loss of their fundamental freedoms, because, politically, financial fascism is neither a benign nor a static system. Its vicious principles are such that their application will drive this country to a centralized financial police state—and then to a full-blown police state in every horrific sense of that term. Already in 1791 this potential for political disaster was recognized in the debate in the House of Representatives on the First Bank of the United States, during which Representative Giles warned the country that
"all the arguments adduced in favor of [a national bank], from whatever source they arise, if pursued, will be found to rush into the great one of expediency, to bear down all Constitutional provisions, and to end themselves in the unlimited ocean of despotism."[4]
The question then becomes: Must the Axis of Financial Fraud be suffered to "crucify America on a cross of debt"?
The answer is NO! There is a way out. The purveyors of the culture of debt at both ends of the Axis of Financial Fraud have forgotten that their pyramids of phony paper promises are still subject to constitutional law. To smash the Axis once and for all, Americans must:
(i) restore commodity money of silver and gold as the only official media of exchange for the General Government and the States;
(ii) gradually displace and replace currency generated from debt with commodity money through competition in the free market;
(iii) enforce the absolute separation of bank and state, so that common people are no longer compelled to underwrite the financial class's Ponzi schemes; and
(iv) declare uncollectible all unconstitutionally incurred debt.
The last entry in this list bears repetition and explanation: To declare uncollectible all unconstitutionally incurred debt is entirely different from "repudiating debt". Repudiation of debt presumes that the debt was originally lawful, but that now, for some reason, the law must be set aside, or disregarded, or changed ex post facto. In contrast, declaring uncollectible all unconstitutionally incurred debt presumes that the debt was never valid at all. But how can this be accomplished? In at least two ways:
First, under the doctrine of the Supreme Court's decision in Craig v. Missouri,[5] all contracts, agreements, or other arrangements in which any part of the consideration consists or consisted of the emission of unconstitutional "bills of credit" through or under the auspices of any Federal Reserve Bank, "member bank", or "depositary institution" within the Federal Reserve System are declared to be void ab initio and unenforceable in any court of the United States or of any State.
Second, all public debt obligations of the United States, howsoever made and in whatever form, that have been incurred for the purpose of raising revenue to be expended from the general fund of the Treasury in payment of costs arising under a particular budget of the United States are declared to be void ab initio and unenforceable in any court of the United States or of any State in the same percentage that the unconstitutional programs, activities, or expenditures in that budget bear to the total programs, activities, or expenditures therein. Every lender must be presumed to know the constitutional limits on the expenditures of money the General Government borrows, to the same extent that every official of that government knows those limits. So, if a lender extends a loan to public officials, knowing that his loan will be used for unconstitutional purposes, or with willful blindness to or reckless disregard of the unconstitutionality of those purposes, he is thereby a participant in a fraud against the American people. And for the repayment of such a loan, the American people cannot be held liable.
Plainly enough, though, the present Congress, President, and Supreme Court will never reform the contemporary monetary and banking systems, and reduce average Americans' burden of debt, along these lines. That leaves it to the victims of the swindle: WE THE PEOPLE themselves. Which is quite appropriate.
After all, the real "federal government" is not the General Government alone—it is not the General Government and the States alone—rather, it is the General Government, and the States, and most importantly WE THE PEOPLE. Most importantly, because the real "federal government" is not a pyramid in which power flows up from the bottom to concentrate at the top, with an all-seeing eye at its apex. For the strength of any pyramid is found not at its apex, but in its base. WE THE PEOPLE are the essential support of the entire structure, the source of its strength, the font of political power, the ultimate sovereigns.
All too many Americans for all too long have been conditioned to think of "the federal government" in terms of "higher" and "lower" levels as on some crude bureaucratic organizational chart: with Washington, D.C. at the top, the States and THE PEOPLE at the bottom. The correct description, however, takes into account degrees of authority: The truly "highest" level is the one closest to the well-spring of sovereignty, the "lower" levels the ones increasingly removed from that source. WE THE PEOPLE occupy the "highest" level in the real "federal government", because WE THE PEOPLE not only originally "ordain[ed] and establish[ed] th[e] Constitution", but also sustain—and must enforce—its authority every day. As the Supreme Court has held, "[t]he power to enact carries with it final authority to declare the meaning of the legislation".[6] Moreover, as Sir William Blackstone taught the Founding Fathers, "whenever a question arises between the society at large and any magistrate vested with powers originally delegated by that society, it must be decided by the voice of the society itself: there is not upon earth any other tribunal to resort to".[7]
To be sure, the partisans of the Federal Reserve System will contend that the System has been declared constitutional on numerous occasions, or at least is generally treated as constitutional, and that therefore its validity cannot now be contested. In fact, however, the Supreme Court has never heard a challenge to the central-banking scheme, or its irredeemable paper currency, on the numerous grounds of their greatest constitutional vulnerability. In any event, the very same legal gurus who tout the alleged legitimacy of the Federal Reserve System—as well as the propriety of the ever-expanding expenditures of the General Government that the Federal Reserve System finances through the banks' "monetization" of public debt—also claim that America has a so-called "living constitution", the meaning of which can change from time to time to meet differing circumstances. If that is true, then no matter how many times in the past the Federal Reserve System and the General Government's expenditures have supposedly been declared constitutional, WE THE PEOPLE can decide tomorrow that circumstances demand reinterpretation of this "living constitution" in order to strike down the Federal Reserve System and those expenditures, and thereby to save this country from economic disaster. Or, WE THE PEOPLE can decide on the basis of the Constitution's "original intent" that the Federal Reserve System and expenditures of those kinds were never even arguably lawful in the first place. In either case, the Federal Reserve System and all such expenditures—and the huge mountain of ostensible public and private debt associated with them—can be eliminated.
Why WE THE PEOPLE will take this course is obvious. As the old saw has it, "Nothing focuses a man's mind more than his impending hanging!" THE PEOPLE have been asleep for a long time. Now, however, as the economic screws tighten on every side, they have finally awakened to the danger confronting them. Everyone with an IQ even a single point higher than his age recognizes the utter and irremediable corruption of the Federal Reserve System and the entire financial and political régime centered around and dependent upon it: Consider the gargantuan "bail outs" that amount to the most mammoth, brazen, and insolent financial looting any society has ever suffered in the history of the world. Consider the régime's refusal to accept responsibility or to provide for accountability or even minimal transparency. Consider the régime's grab for ever-more-abusive powers, not only over the financial sector of the economy, but also over what remains of the industrial sector. In short, ordinary Americans are witnessing—and know they are witnessing—a war being waged against them by the racketeers of the financial class and rogue public officials in the outlaw city-states of New York and Washington, D.C. So far, it is a war in which only the aggressors are doing any fighting. But that disparity will not persist much longer.
As Jefferson predicted, now that THE PEOPLE are no longer making money, but instead are losing it hand over fist—and with it their jobs, their prosperity, their economic security, and their hopes for decent retirement—they have but one other alternative: They must "think of uniting to effect a due respect for their rights". When they do, THE PEOPLE will recognize that the only way to restore their national independence, societal prosperity, and individual liberty is to break the links once and for all between bank and state and between currency and debt, using the industrial-strength tools the Constitution supplies.
How will they do it? WE THE PEOPLE must first declare and then secure their economic and legal independence from the Federal Reserve System's régime of fiat currency and central banking within their own States and Localities. They can succeed in this endeavor because:
(i) THE PEOPLE do not need the advice, or the help, or the interference, or the direction, or least of all the dictation of anyone in Washington, D.C. or New York City to live as free and prosperous Americans in their own States and Localities.
(ii) THE PEOPLE vastly outnumber the totality of all public officials, politicians, financiers, bankers, and these parasites' clients, partisans, hangers-on, and touts.
(iii) THE PEOPLE physically control most of the property in this country—and actual "possession is nine-tenths of the law". If THE PEOPLE en masse and through properly organized and authorized resistance simply refused to relinquish their physical possession in defiance of the financial class's merely paper claims, precisely what could the financial class do about it?
(iv) THE PEOPLE can still exercise an effective franchise in many States. And, most consequentially in the final analysis,
(v) THE PEOPLE constitute the Militia, which the Constitution declares to be "necessary to the security of a free State", and to which institution alone the Constitution explicitly assigns the responsibility and the authority "to execute the Laws of the Union".[8] Combining THE PEOPLE'S actual possession of most of the property throughout America with their authority through the Militia "to execute the Laws" would mean that the financial class could not possibly maintain its stranglehold over the economy for a single minute after THE PEOPLE decided to declare uncollectible all unconstitutionally incurred debt.
The initial step on the long march to reform is to introduce, State by State, an alternative currency of silver and gold that can compete with, and shortly replace, the Federal Reserve Note in each State's public finances and private economy. This process must take place through the States, because:
(i) the States are large enough politically and economically to make it work;
(ii) the States have the undoubted legal authority to do it;
(iii) with respect to the choice of a currency for the performance of their own governmental functions, the States enjoy absolute legal immunity from interference by rogue public officials in the General Government;[9] and
(iv) the Militia, through which the alternative currency will quickly move into each State's private economy, are "the Militia of the several States".[10]
This plan is not some pie-in-the-sky vision. For several States—New Hampshire, Montana, Indiana, and Georgia, for instance—have already set out in the right direction (albeit only with halting steps so far). True enough, they and other States still have a long road to travel—but, as the Chinese say, even a journey of a thousand li begins with but a single step.
An alternative currency—introduced through the States' governments and spread throughout the economy by THE PEOPLE themselves in their Militia—is not simply an idea whose time has come. More than that, true constitutional monetary reform will be the new "shot heard 'round the world", the announcement of a new American declaration of independence from the corrupt alliance of politicians and financiers that has driven this country to the brink of irretrievable disaster.
1, 9 December 1814
2, Quoted in Merrill D. Peterson, Thomas Jefferson and the New Nation (New York, New York: Oxford University Press, 1970), at 99
3, Article I, Section 8, Clause 5
4, The Debates and Proceedings in the Congress of the United States (J. Gales compilation, 1834), Volume 2, at 1942-1943.
5, 29 U.S. (4 Peters) 410 (1830).
6, Propper v. Clark, 337 U.S. 472, 484 (1949)
7, William Blackstone, Commentaries on the Laws of England, Volume 1, at 212.
8, U.S. Const. art. I, § 8, cl. 15 and amend. II.
9, Lane County v. Oregon, 74 U.S. (7 Wallace) 71 (1869).
10, U.S. Const. art. I, § 8, cls. 15 and 16, and art. II, § 2, cl. 1.
© 2009 Edwin Vieira, Jr. – All Rights Reserved
Putting Obama on Hold, in a Hint of Who's Boss
Posted in 1 with tags Banker Bailout/Takeover, Barack Hussein Obama, Citi, Fiat Money, Goldman Sachs, Henry Paulson, International Bankers, New World Order, News, Shadow Government, TARP, The Banking Cartel on December 5, 2009 by truthwillrise
By ANDREW ROSS SORKIN
President Obama didn't exactly look thrilled as he stared at the Polycom speakerphone in front of him. "Well, I appreciate you guys calling in," he began the meeting at the White House with Wall Street's top brass on Monday.
He was, of course, referring to the three conspicuously absent attendees who were being piped in by telephone: Lloyd C. Blankfein, the chief executive of Goldman Sachs; John J. Mack, chairman of Morgan Stanley; and Richard D. Parsons, chairman of Citigroup.
Their excuse? "Inclement weather," according to the White House. More precisely, fog delayed flights into Reagan National Airport. (In the "no good deed goes unpunished" category, the absent bankers were at least self-aware enough to try to fly commercial.)
That awkward moment on speakerphone in the White House, for better or worse, spoke volumes about how the balance of power between Wall Street and Washington has shifted again, back in Wall Street's favor.
Now that Citigroup has given back its bailout money — and Wells Fargo announced late on Monday that it would, too — whatever leverage Washington had over the financial services industry seems to be quickly eroding.
Executive compensation, leverage limits and lending standards were all issues that Washington said it planned to change — and when the taxpayers were the shareholders of these firms, it probably could have done so. But now the White House has been left in the position of extending invitations, rather than exercising its clout. And in the figurative and literal sense, it is getting stood up.
Those who attended the meeting — Jamie Dimon of JPMorgan flew down on a private jet and didn't take any heat for it — seemed to talk a good game, but even President Obama acknowledged they might have been just toying with him.
"The problem is there's a big gap between what I'm hearing here in the White House and the activities of lobbyists on behalf of these institutions or associations of which they're a member up on Capitol Hill," he said after the discussion.
Are we making too much of this meeting and its grounded attendees?
The meeting was always just going to be political theater. Wall Street bankers were supposed to play their part on the public stage in Washington, and submit to a scolding from the president about bonuses and the need to start lending more to help get the economy moving.
But inevitably public perception will issue its harsh ruling, and it goes something like this: If the meeting were really that important to Mr. Blankfein, Mr. Mack and Mr. Parsons, they would have found a way to get there.
They would have left the night before, or they would have flown out at the crack of dawn, or better yet, taken Amtrak (I called customer service, and the Acela was running only a couple of minutes late).
In fairness, there is little question that they wanted to be there and seemed genuinely disappointed they couldn't make it. (You could hear it in Mr. Mack and Mr. Blankfein's voice when they got on the call. "Mr. President, we're upset we're not able to be there, but we're on line with you now," Mr. Mack said. "It's certainly not for a lack of effort," Mr. Blankfein quickly followed up.)
But this missed meeting clearly didn't help their case.
After all, they sure hoofed it down there last year, when Henry M. Paulson Jr. ordered them to meet him in Washington with less than 24 hours of notice. Most of them got there early, and went home with $10 billion to $25 billion of taxpayer money.
Upon hearing the news Monday morning of the airplane delays, Mark Haines, an anchor at CNBC, went on the air and, in a Howard Beale moment, said what many Americans were probably thinking: "These guys are such little girls! Give me a break. What a bunch of wimps! Thanks for all that taxpayer money … and, ah, gee, there are delays at the airport!"
But extra effort may have been a lot to ask given the blasting headwinds they were flying into down in Washington.
President Obama's "60 Minutes" interview Sunday night eviscerating Wall Street laid down the not-so-welcome mat. "I did not run for office to be helping out a bunch of fat-cat bankers," he said.
Inside the Obama administration, there were bruised feelings about the need for a conference call to have a meeting.
"It was pretty nervy," one staff member told me.
That's not to say that Mr. Blankfein, Mr. Mack and Mr. Parsons have not been trying to be constructive.
Mr. Mack has been particularly outspoken about the need for serious financial reform on Wall Street. Mr. Parsons, too, has been trying to act as a liaison with Washington and has not pushed back on legislation.
And Mr. Blankfein, who is under perhaps the hottest spotlight, has been saying many of the right things, though he probably can't say enough of them at the moment.
But as President Obama has said, it is not what those leaders say to him that really matters.
"The way I see it, having recovered with the help of the American government and the American taxpayers, our banks now have a greater obligation to the goal of a wider recovery, a more stable system, and more broadly shared prosperity," Mr. Obama said.
There's an expression that many bankers already know, and might want to keep in mind if they are summoned to Washington again. The saying is often trotted out on Wall Street when people need to be reminded of the importance of getting on a plane and seeing a client: "You can't fax a handshake."


text:
Q: Remove columns of equal variance after dplyr::group_by and before prcomp I am performing pcas for multiple groups in a large data set using dplyr. When the data is split into groups using the group_by function, some variables have equal variance, so the pca can't run. How can I remove any columns of equal variance and then perform prcomp on what remains? Dummy data below. Thank you.
Add equal variance for setosa - Sepal.Length.
library(dplyr)

iris[1:50,1]<-0

Attempt to run pcas with equal variance
> iris%>%
+   group_by(Species)%>%
+   group_map(~prcomp(.[,1:4], scale.=T))
Error in prcomp.default(.[, 1:4], scale. = T) :
  cannot rescale a constant/zero column to unit variance

Check for equal variance
> iris%>%
+   group_by(Species)%>%
+   group_map(~names(.[,1:4][, sapply(.[,1:4], function(v) var(v, na.rm=TRUE)==0)]))
[[1]]
[1] "Sepal.Length"

[[2]]
character(0)

[[3]]
character(0)

Attempt to exclude equal variance column and run pcas
> iris%>%
+   group_by(Species)%>%
+   group_map(~sapply(.[,1:4], function(v) var(v, na.rm=TRUE)>0))%>%
+   group_map(~prcomp(.[,1:4], scale.=T))
Error in UseMethod("group_split") :
  no applicable method for 'group_split' applied to an object of class "list"

A: We can use map_if which checks for a condition and then applies the function.
library(tidyverse)

iris %>%
   group_split(Species, keep = FALSE) %>%
   map_if(~all(map_dbl(.x, var) != 0), ~prcomp(.x, scale. = TRUE),
          .else = function(x) return(NULL))

#[[1]]
#NULL

#[[2]]
#Standard deviations (1, .., p=4):
#[1] 1.7106550 0.7391040 0.6284883 0.3638504

#Rotation (n x k) = (4 x 4):
#                    PC1        PC2        PC3        PC4
#Sepal.Length -0.4823284 -0.6107980  0.4906296  0.3918772
#Sepal.Width  -0.4648460  0.6727830  0.5399025 -0.1994658
#Petal.Length -0.5345136 -0.3068495 -0.3402185 -0.7102042
#Petal.Width  -0.5153375  0.2830765 -0.5933290  0.5497778

#[[3]]
#Standard deviations (1, .., p=4):
#[1] 1.5667601 0.9821979 0.6725116 0.3581596

#Rotation (n x k) = (4 x 4):
#                   PC1        PC2         PC3         PC4
#Sepal.Length 0.5544765 -0.4324382  0.01239569  0.71091442
#Sepal.Width  0.4755317  0.4401787  0.75272551 -0.11626101
#Petal.Length 0.5501112 -0.4296642 -0.20236407 -0.68688796
#Petal.Width  0.4047258  0.6592637 -0.62633812  0.09627561

If we want to remove only the columns which have 0 variance and not the entire group, we can use select_if to select columns
iris %>%
  group_split(Species, keep = FALSE) %>%
  map(~.x %>% select_if(~var(.) != 0) %>% prcomp(scale. = TRUE))


This self-catering resort is absolutely perfect for families or young adults looking for an active getaway close to the best beaches, walks and cycle routes Jersey has to offer, whilst St Helier is only a 15 minute drive away. The resort has a variety of one, two and three bedroom lodges and apartments as well as more bespoke accommodation at Les Ormes Farm and Les Ormes Farmstead, both of which are pet friendly. Clustered together in small groups, the one bedroom lodges are stylishly furnished and boast their own hot tub on the decking area. Les Ormes two bedroom Terrasses are two-storey granite or cedar clad units with a decked area while Les Ormes three bedroom Lizard Apartments have open living areas and a veranda or decking and overlook the golf course. There is an enormous choice of leisure facilities, including a children's indoor Fun Zone, tennis, a health and leisure club along with a 9 hole, par 32 golf course. In addition, a variety of high adrenalin activities suchas coasteering, abseiling, blokarting and RIB boat tours are available to book locally. For those that prefer not to spend their holiday cooking, Les Ormes also offers great value meal plans at its restaurant, alternatively take away meals are available along with packs for the BBQ area.
All feature a fully equipped kitchen with cooker or stove top, microwave, fridge/freezer, dishwasher, washer/dryer and lounge with satellite television.
Contemporary open plan lodges which are either detached or semi-detached consist of one double/twin bedroom with sofa bed in the lounge (max 4 adults or 2 adults and 2 children) and an outdoor hot tub on the deck. Some lodges can be linked to sleep 8 (max 8 adults or 4 adults and 4 children).
Two bedroom lodges are ideal for families or couples.(max 4 adults or 2 adults and 2 children) They are scattered within a picturesque landscaped environment, have an open plan lounge, two ensuite bedrooms and a hot tub outside.
Two-storey units offer one double/twin bedroom with shower on the ground floor and one double/twin bedroom with one extra bed upstairs, bathroom with bath/shower (max 4 adults and 1 child) and a private decked area.
Two-storey building with each apartment occupying one floor. Each apartment has one double/twin master bedroom, one double/twin bedroom and one bedroom with bunk beds (max 6 adults or 4 adults and 2 children), open living areas and a veranda overlooking the golf course or swimming pool. Further villas and apartments are available at Les Ormes Resort.


text:
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Sequence-specific PCR is the most common approach for virus identification in diagnostic laboratories. However, as specific PCR only detects pre-defined targets, novel virus strains or viruses not included in routine test panels will be missed. Recently, advances in high-throughput sequencing allow for virus-sequence-independent identification of entire virus populations in clinical samples, yet standardized protocols are needed to allow broad application in clinical diagnostics. Here, we describe a comprehensive sample preparation protocol for high-throughput metagenomic virus sequencing using random amplification of total nucleic acids from clinical samples.
In order to optimize metagenomic sequencing for application in virus diagnostics, we tested different enrichment and amplification procedures on plasma samples spiked with RNA and DNA viruses. A protocol including filtration, nuclease digestion, and random amplification of RNA and DNA in separate reactions provided the best results, allowing reliable recovery of viral genomes and a good correlation of the relative number of sequencing reads with the virus input. We further validated our method by sequencing a multiplexed viral pathogen reagent containing a range of human viruses from different virus families. Our method proved successful in detecting the majority of the included viruses with high read numbers and compared well to other protocols in the field validated against the same reference reagent. Our sequencing protocol does work not only with plasma but also with other clinical samples such as urine and throat swabs.
The workflow for virus metagenomic sequencing that we established proved successful in detecting a variety of viruses in different clinical samples. Our protocol supplements existing virus-specific detection strategies providing opportunities to identify atypical and novel viruses commonly not accounted for in routine diagnostic panels.
To date, sequence-specific PCR is the most common approach for virus identification and quantification in diagnostic laboratories as it is highly sensitive, rapid, and cost effective. However, specific PCR requires prior knowledge of the virus sequence, and a separate assay needs to be designed for each individual virus or virus type. Recently, high-throughput or next generation sequencing (NGS) technologies enabled metagenomic-based identification of viruses by sequencing random fragments of all genomes present in a clinical or environmental sample [1–3]. As viral metagenomics is virus-sequence independent, potentially any virus, cultivable or uncultivable, known or novel, can be readily detected and the method can be applied to all types of virus genomes, including single-stranded DNA and RNA. Several research studies have used this technology in recent years to explore the breadth of the virome in diverse biological and environmental samples including human and animal feces [4–12], blood [13, 14], animal and human tissues [15–17], and human respiratory tract secretions [18–22] and highlighted the validity of the approach to detect rare and novel viruses.
Despite the numerous promising attempts to apply metagenomics to virology, direct sequencing of nucleic acids obtained from biological samples results in a high background of genetic material mainly derived from the host and bacteria hampering the detection of viruses [22, 23]. Sample type greatly influences the composition of sequencing reads, and due to the complexity of clinical materials, sample preparation and virus enrichment methods need to be specifically adapted. Common steps in sample preparation for unbiased metagenomic sequencing are virus enrichment, extraction of nucleic acids, reverse transcription, and unbiased amplification.
Various virus enrichment methods for clinical samples have been suggested. Commonly employed methods include filtration, ultracentrifugation, and nuclease treatment [1, 24–31]. These methods rely on the small size of viruses and the stability of their capsid. Several approaches for virus genome amplification do exist: Multiple displacement amplification (MDA) is often used for whole genome amplification [32–34], e.g., in the VIDISCA method for virus discovery . Linker-amplified shotgun library (LASL) was applied for virus sequencing from marine water samples . The anchored random PCR approach, as used in our study, has been frequently used and described in detail in previous studies [16, 36–42].
Here, we tested various conditions and methods for virus enrichment, nucleic acid extraction, and unbiased amplification with the goal to create a sensitive and robust workflow for metagenomic sequencing of clinical samples in virus diagnostics. We spiked several virus types into plasma from healthy donors to cover different classes of viruses: small non-enveloped DNA (Human adenovirus), large enveloped DNA (Human herpesvirus 4/EBV or Human herpesvirus 5/CMV), small non-enveloped RNA virus (Poliovirus), and enveloped RNA virus (influenzavirus A, Additional file 1: Table S1). We validated our approach using a highly multiplexed viral pathogen reagent containing 25 different viruses. Finally, in experiments with different ratios of spiked viruses, we show a good correlation of the relative number of sequencing reads with the virus input.
Human adenovirus 7, Human poliovirus 1 (strain LSa), and Human herpesvirus 5 (HHV-5, ATCC VR AD 169) were propagated on MRC-5 cells (human fetal lung fibroblasts) obtained from the European Collection of Cell Cultures (Salisbury, UK). Viruses were cultivated for 14 days, cells were sonicated for 5 s, centrifuged (5 min at 1200 rpm), and the supernatant harvested and filtered (0.45 μm). Human herpesvirus 4 (HHV-4) was propagated on B-95 cells (marmoset B-lymphoblastoid cell line) centrifuged (5 min at 1200 rpm), and the supernatant harvested and filtered (0.45 μm). Influenzavirus A/WSN/33 was propagated on A549 cells (human alveolar basal epithelial cells) and Influenzavirus A/H1N1/PR8 on MDCK cells (canine kidney epithelial cells) for up to 4 days, and the supernatants were centrifuged (10 min at 1200 rpm). Aliquots were stored at −80 °C.
Viral loads in spiked samples and amplicon concentrations after random amplification were determined by qPCR as described previously for HHV-5 , poliovirus , adenovirus , and influenzavirus (CDC protocol of real-time RT-PCR for swine influenza A H1N1, 28 April 2009). For HHV-4, real-time PCR was performed as described previously , but with modified primers (CTTCTCAGTCCAGCGCGTTT and CAGTGGTCCCCCTCCCTAGA) and a modified probe (FAM-CGTAAGCCAGACAGCAGCCAATTGTCAG-TAMRA). All reactions were performed on a ViiA7 Real-Time PCR System (Life Technologies/Thermo Fisher Scientific, Waltham, MA) with the TaqMan RNA-to-Ct 1-Step Kit. One-microliter template (out of 25-μl extraction eluate or 50-μl amplification reaction, respectively) was used with 10-μl master mix, 0.25 μM of each primer, 0.125 μM probe in a total volume of 20 μl. Each sample was tested in duplicates with the following cycling conditions: 30 min at 48 °C, 10 min at 95 °C, 50 cycles of 15 s at 95 °C, and 1 min at 60 °C.
For experiments using different ratios of virus input, virus stocks were quantified by digital PCR using the QuantStudio 3D Digital PCR System (Life Technologies/Thermo Fisher Scientific), which allows for absolute quantification without the need of a standard. Reactions were performed in a final volume of 15 μl with 7.5 μl 2× QuantStudio 3D Digital PCR Master Mix, 0.25 μM of each primer and 0.125-μM probe (same primer and probes as used in the qPCR assays described above). The template was diluted as necessary for optimal digital PCR readout. For RNA virus quantification, the reaction additionally contained 0.4 μl 40× TaqMan RT Enzyme Mix and an initial reverse transcription step for 15 min at 48 °C followed by 10 min at 96 °C. Cycling conditions were 50 cycles of 2 min at 60 °C, 30 s at 98 °C, and 60 °C for 2 min. After read-out on the QuantStudio 3D Digital PCR instrument, the raw data was imported into QuantStudio 3D AnalysisSuite Cloud Software (version 3.0.2.2) to calculate absolute copy numbers.
Human clinical samples were obtained from healthy blood donors (Zurich blood donor service, Schlieren, Switzerland) or from diagnostic samples (tested negative for poliovirus, HHV-4, HHV-5, influenzavirus A, and adenovirus) and stored at −20 °C. Samples were centrifuged at 2000 rpm for 10 min (Heraeus Multifuge X3 R, Thermo Fisher Scientific), spiked with viruses to achieve quantitative PCR threshold cycles (ct values) of 22–25, which are considered as high positive samples in diagnostic tests for most viruses. Filtration was done using a 0.45-μm PES filter (TPP, Trasadingen, Switzerland). Freeze-thaw cycles, if applicable, were performed by freezing the samples at −20 °C.
Nuclease treatment with DNase and RNase was performed as previously described  with reagent volumes scaled up for 1000 μl of clinical sample. Briefly, a nuclease mix containing 120 μl DNase (0.92 mg/ml, Roche, Basel, Switzerland), 10 μl RNaseA (0.77 mg/ml, Qiagen, Hilden, Germany), 130 μl 10× nuclease buffer (400 mM Tris-HCl, 100 mM NaCl, 60 mM MgCl2, 10 mM CaCl2; pH 7.9), 30 μl PBS, and 10-μl water was added. The reaction was incubated for 1 h at 37 °C in a thermoshaker at 1400 rpm. Samples were treated with protease (0.71 mg/ml, Qiagen) for 30 min at 37 °C to remove nuclease activity.
QIAamp Viral RNA Mini Kit (Qiagen), PureLink Viral RNA/DNA Mini Kit (Life Technologies/Thermo Fisher Scientific), and NucliSENS EasyMAG system (BioMérieux, Craponne, France) were used according to the manufacturer's instructions. Two input volumes of spiked plasma were tested, 500 and 1000 μl, and eluted into 25 μl to achieve a high sample concentration. Large starting volumes were loaded into the extraction columns in multiple steps according to the manufacturer's instructions, if necessary.
In a first protocol for unbiased nucleic acid amplification , we processed RNA and DNA viruses combined in a single reaction, called here the "combined protocol." We changed our protocol to include separate amplification steps for RNA and DNA and replace T7 polymerase with DNA Polymerase I Large Klenow Fragment (NEB Biolabs, Ipswich, MA), called the "separate protocol" [9, 42]. For a direct comparison, Klenow fragment was used for both workflows.
For the RNA workflow, cDNA was generated by reverse transcription with a primer containing a random octamer linked to an anchor sequence ATCGTCGTCGTAGGCTGCTCNNNNNNNN [16, 36, 37]. Five microliters of eluate was used as template in a total volume of 20 μl, with 5 μM of random primer, 1 mM of dNTPs, 1× first strand buffer, 20 mM DTT, and 20 U/μl of SuperScript III (Invitrogen/Life Technologies). The template and random primers were heated at 65 °C for 5 min, followed by reverse transcription at 42 °C for 60 min and inactivation at 96 °C for 5 min. Prior to second strand synthesis, cDNA was denatured at 94 °C for 2 min and cooled down to 10 °C for 5 min. The second strand was synthesized with 5 U/μl DNA Polymerase I (Klenow) in 10× NEB buffer in a final volume of 10 μl, at 37 °C for 30 min followed by an enzyme inactivation step at 75 °C for 20 min. An additional step of second strand synthesis used in the initial, combined protocol was omitted.
The DNA workflow started at the denaturation step at 94 °C for 2 min and was performed with the same random primer as used in the RNA workflow prior to second strand synthesis. Second strand synthesis was performed using the same conditions as described for the RNA workflow. Further amplification with the anchor primer and AmpliTaq Gold (Thermo Fisher Scientific) was performed as previously described , but separately for the RNA and DNA workflow.
The quality and size of the anchor PCR products were assessed by capillary gel electrophoresis (Fragment Analyzer, Advanced Analytical, Ames, IA). PCR products were quantified with PicoGreen (Invitrogen/Thermo Fisher Scientific) and diluted to 0.2 ng/μl. DNA and RNA preparations were pooled in equal concentrations for constructing sequencing libraries with the NexteraXT protocol (Illumina, San Diego, CA). Individual samples were dual indexed during the library preparation and pooled for sequencing. Libraries were sequenced on a MiSeq (Illumina) for 1 × 150 cycles with version 3 reagents and the "FASTQ only" workflow. Samples were demultiplexed using MiSeq Reporter v2.4.60. Raw sequencing reads are available from the Zenodo repository (10.5281/zenodo.814807). Reads were processed with a dedicated bioinformatic pipeline "VirMet" version 0.3.3 developed in our laboratory (https://github.com/ozagordi/VirMet/releases/tag/v0.3.3) . Briefly, reads were quality-filtered by removing low quality bases (average PHRED score below 20), reads shorter than 75 bp and reads with low entropy (i.e., consisting mainly of repeats). Read passing quality filters were cleaned from non-viral reads by aligning with STAR [48, 49] against, in this order, human, bacterial, bovine, and canine genomes. Reads not matching any of the above genomes were aligned with BLAST  against an in-house viral database that contains approximately 46,000 different virus sequences. For each sequencing read that passed the quality filter, the BLAST hit with lowest e value was reported, given the identity was higher than 75%. Reads which did not match genomes used in the cleaning step and did not match viral genomes included in the database were reported as of unknown origin.
Optimization of the protocol was assessed by comparing the virus amplicon concentrations after random amplification by qPCR, by evaluating the fractions of reads of different taxonomic categories after sequencing, and by counting the absolute number of reads and the fraction of the total quality filtered reads for each individual spiked virus. All sequencing experiments were performed in duplicates or triplicates. Statistical analysis was done in R version 3.3.2 using linear models . Coverage plots for viruses used for spiking were generated by mapping virus reads reported by our VirMet pipeline with smalt (http://www.sanger.ac.uk/science/tools/smalt-0, default thresholds) against the following reference genomes: Human adenovirus 7 (GenBank AY495969.1), Human poliovirus 1 Mahoney (V01149.1), and HHV-5 strain AD169 (X17403.1).
First, three different methods of total nucleic acid extraction were tested: QIAamp Viral RNA Mini Kit, PureLink Viral RNA/DNA Mini Kit, and the automated NucliSENS EasyMAG system. Plasma from healthy donors was spiked with different viruses (adenovirus, poliovirus, HHV-4, influenzavirus A) and extracted, and the concentration in the eluate determined by qPCR. EasyMAG extraction was most efficient for both RNA viruses, while virus concentrations for DNA viruses were similar to PureLink extraction. The Qiagen extraction kit led to the lowest recovery of viral genomes for all tested viruses (Additional file 1: Figure S1 and Table S2). Thus, the EasyMAG system was selected as standard extraction method for all further experiments.
In order to assess the effect of sample preparation on the sensitivity of metagenomic sequencing, we spiked plasma from healthy donors with both RNA and DNA viruses (poliovirus, adenovirus, and HHV-4). All viruses were spiked at a qPCR threshold cycle (ct value) in the range of 22–25. Different orders of sample processing corresponding to different pre-analytical situations were then tested using the virus-spiked plasma: the condition "same day" extraction comprised filtration, extraction, and short-term storage before further processing at −20 °C; "pre-processed" comprised filtration, storage at −80 °C, and later extraction; "archived" samples comprised storage at −20 °C, filtration, and extraction. In all three conditions, each sample was processed filtered as well as non-filtered.
In all conditions, the fraction of virus reads significantly increased as a result of filtration. Same day extraction with filtration and extraction followed by freezing showed the highest enrichment of virus reads (Fig. 1a).
Considering individual viruses used for spiking, for all three viruses, significantly more reads were reported for filtered samples, both in numbers and in the fraction of total reads passing quality filtering (Fig. 1b, c; Additional file 1: Table S3). Conditions pre-processed and archived (including a freezing step before extraction) proved better for DNA viruses (adenovirus and HHV-4) than "same day extraction". Of note, in all conditions, the highest number of spiked virus reads was reported for poliovirus and the lowest number for HHV-4 (Fig. 1b, c), although the ct values of the input for each virus were similar. For all further samples, we chose pre-processed as standard method.
Next, we tested the effect of nuclease treatment, which takes advantage of the presence of a stable virus capsid that protects the viral genome from digestion, in metagenomic sequencing of plasma spiked with two RNA viruses (poliovirus and influenzavirus A) and two DNA viruses (adenovirus and HHV-4). Spiked plasma incubated at 4 °C without reaction buffer and nucleases served as a control.
In samples that were treated with nucleases, we observed an increase in the fraction of reads passing quality filtering (Fig. 2a, Additional file 1: Table S4). This is a result of the digestion of human DNA containing a lot of repetitive (low entropy) sequences and therefore fewer reads to be removed in quality filtering.
With nuclease treatment, reads of viruses increased significantly, while human background reads decreased (Fig. 2b). Freeze/thaw cycles did not improve virus enrichment by nuclease treatment (Fig. 2b).
Considering sequencing reads from the individual viruses, nuclease treatment enriched all viruses, except the large, enveloped DNA virus HHV-5 (Fig. 2c, Additional file 1: Table S5). Incubation alone, without the enriching effect of nuclease treatment, resulted in a reduced recovery of the RNA viruses influenza and polio.
In order to optimize unbiased amplification of nucleic acids to represent the entire virus population in a sample, we divided the workflow to process RNA and DNA in two separate reactions and changed the enzyme for second strand synthesis from T7 DNA polymerase to Klenow Large Fragment . For comparison, sample volumes were kept identical for both workflows. Using qPCR after random PCR amplification, higher amplicon concentrations were obtained for DNA viruses processed in the separate DNA workflow, when compared to the combined protocol (data not shown). After sequencing, the fraction of virus reads was enriched in the separate protocol for DNA when compared to the combined reaction as well (Fig. 3a). Most importantly, higher numbers of sequencing reads were obtained for all viruses in the separate workflows compared to the combined workflow, especially also for HHV-4 and influenzavirus (Fig. 3b, Additional file 1: Table S6).
As shown before , sample preparation can influence the coverage of viral genomes. We therefore aligned virus reads reported by the combined and separate workflow for each virus to its reference genome. Reads were uniformly distributed along the reference genomes in both workflows (Additional file 1: Figure S2).
We further tested if a higher input volume into the reverse transcription reaction and the downstream anchor PCR would increase virus recovery. Using the separate protocol, input strategy 1 used 5 μl of the extract into the reverse transcription reaction and 3 μl of the reverse transcription reaction into the anchor PCR; input strategy 2 used double the amount (10 and 6 μl, respectively). Viral amplicon concentrations after the anchor PCR were higher for input strategy 2 (Additional file 1: Figure S3A). However, the fraction of virus reads and reads assigned to each spiked virus were at similar levels for both input strategies (Additional file 1: Figure S3B and S3C, Additional file 1: Table S7).
Our final workflow is depicted in Fig. 4.
After establishing a workflow for unbiased, metagenomic sequencing using spiked plasma samples, we used our new protocol for testing a highly multiplexed viral pathogen reagent (11/242-001, National Institute for Biological Standards and Control, South Mimms, UK) containing 25 different human viruses from different virus families  (Table 1). We processed and sequenced 1000 μl of the reagent. As our bioinformatic pipeline VirMet determines the taxonomic origin of each individual read, we summed up reads at the species level and noted the most commonly reported strain. Of the 25 viruses expected in the reagent, we detected 17 and 15 different viruses in two replicates, respectively, with high numbers of reads (Table 1). Previous studies have sequenced the same viral pathogen reagent [30, 52]. We randomly subsampled our raw reads to match the numbers analyzed in these studies. Using 2,000,000 reads, we still detected 17 viruses in one of the replicates. Seven of the eight viruses we did not detect (Coronavirus 229E, Norovirus GI and GII, Influenzavirus A and B, Human parainfluenza virus 3) were missed by 15 to 79% of other methods as well . Comparing our results to similar workflows (filtration, nuclease treatment, random amplification, NexteraXT, MiSeq) as, for instance, the sample preparation methods N1-N4 presented in Li et al. , our numbers of reads were higher as reported and we identified more virus species (Table 1, 150,000 reads). As previously shown, additional non-targeted viruses are present in the reagent and were detected by us as well (e.g., Bovine viral diarrhea virus, Bocavirus, Enterovirus; Table 1).
In order to confirm that our protocol detects viruses in a reproducible and quantitative manner, we sequenced plasma samples spiked with different ratios and concentrations of an RNA virus (poliovirus or influenzavirus) and a DNA virus (adenovirus). First, we spiked a concentration of 50,000 copies/μl of both viruses; we then spiked ten times more and ten times less of one virus while keeping the other virus constant at 50,000 copies/μl, and vice versa for the other virus.
After extraction, viral amplicons were quantified in the eluate and the ratio of spiked viruses was perfectly maintained (Additional file 1: Figure S4). After random amplification and sequencing with the separate workflow, we calculated the ratio of the number of reads of the two viruses, as the number of total reads in each sample varied and the viruses are amplified differentially. The ratio of the virus reads in a sample correlated well with the difference in ct values in the initial sample, showing that our method amplifies and detects both viruses in a reproducible and quantitative manner (Fig. 5).
In order to test if our method established with plasma also works for other clinical samples, we spiked the same volume of plasma, urine, throat swab, and two different stool suspensions with the same amount of viruses. After sequencing, the fraction of virus reads in urine and throat swab was similar as for plasma (Fig. 6, Additional file 1: Table S8). For stool samples, however, the amount of unknown background reads was substantially higher and virus reads, if detected, were mostly bacteriophages. For the individual viruses that were spiked, the number and fraction of virus reads were significantly decreased in both stool samples. A more stringent virus enrichment protocol is therefore needed to sequence stool samples to achieve the same sensitivity [53, 54].
In this study, we developed and validated a sample preparation protocol for unbiased amplification and high-throughput metagenomic sequencing of viruses in routine diagnostic use. In an unbiased metagenomic approach, prior knowledge of the virus sequence is not required. In principle, it can therefore detect any virus. However, sample processing needs to be optimized for recovery and amplification of viral genomes that might be present only in very small amounts in clinical samples. We optimized such a protocol using plasma samples spiked with different classes of viruses.
First, we tested filtration, extraction, and nuclease digestion procedures in order to find optimal conditions for virus enrichment and reduction of unwanted, non-viral reads. Filtration proved to be indispensable, as the number of virus reads after sequencing significantly increased. Additionally, sample pre-processing including a freeze thaw cycle showed the best enrichment (Fig. 1), and for best integration into the daily laboratory workflow, we chose pre-processed for further experiments. Two of the extraction methods tested here have been compared before with a higher scoring for the QIAamp Viral RNA Mini Kit compared to the PureLink Viral RNA/DNA Mini Kit . Other studies reported limited coverage of the genome using a magnetic bead-based method similar in principle to the EasyMAG . However, we did not observe this as we obtained good coverage of the entire genome (Additional file 1: Figure S2).
Nuclease treatment, which takes advantage of the stable virus capsid that shields the viral genome from digestion, significantly reduced the fraction of human reads and increased the amount of reads passing the quality filter in our analysis pipeline. This is a result of the digestion of human DNA containing high numbers of repetitive (low entropy) sequences and important for maximizing the amount of quality reads to increase sensitivity. Nuclease treatment enriched for all viruses, except HHV-5, which might, probably due to its size or envelope, not be as stable as smaller viruses. While the combination of low-speed centrifugation, filtration, and nuclease-treatment also showed the greatest increase in the proportion of viral sequences in other studies  and filtration and DNase treatment lead to dramatic improvements , some studies only found minor differences among the methods with or without filtration and nuclease digestion . The reason for these discrepancies might be that some studies compare pre-sequencing nucleic acid concentrations while others compare sequencing reads. Enrichment methods in general decrease the absolute concentration; however, in our hands, the overall gain of nuclease treatment to increase the ratio of virus over host prevails.
Poliovirus reads were recovered in highest numbers in our experiments, although the amount of spiked viruses was adjusted to similar levels (ct values). This might be due to the fact that poliovirus has the smallest virion and genome size among the spiked viruses, what might have facilitated its efficient enrichment and amplification. In contrast, HHV-4 virus is a large virus in respect to virion and genome size and often only single reads could be detected. It was suggested that filtration might actually have an adverse effect on large virus genomes such as herpesvirus and mimivirus . In other studies of virus profiling in clinical specimens, lower numbers of HHV-4 reads as expected were also reported . However, experiments using HHV-5, which belongs to the same virus family as HHV-4, resulted in many more virus reads. Therefore, it is conceivable that other factors than virus and genome size are influencing the number of recovered viral genomes.
Separating the random amplification into two separate workflows for RNA and DNA was more advantageous for the DNA viruses than for the RNA viruses. This could be either a result of concomitant amplification of DNA viruses in the RNA workflow or an indication that DNA genome amplification was negatively affected during the reverse transcription and second strand synthesis in the previous combined workflow. Yet, for both the combined and separate workflow, reads were uniformly distributed across the reference genomes and we did not observe differences.
In order to validate our method, we sequenced a multiplexed viral pathogen reagent that was expected to contain 25 viruses across different families, genome types, and sizes . At most, we identified 17 different viruses (Table 1). We did not detect any reads for eight viruses supposed to be in the reagent, all of them single-stranded RNA viruses (Coronavirus 229E, Norovirus GI and GII, Influenzavirus A and B, Metapneumovirus, Human Parainfluenzavirus 3). However, most of these viruses were present at very low concentration in the viral pathogen reagent (undetectable by qPCR after mixing of the reagent) and were also frequently not detected by other groups that probed this reagent. In the study by Mee et al. , a wide range of different sample volumes and enrichment, amplification, and sequencing methods were used. Stochastic effects in detection of very low abundant viruses and differences in analysis thresholds could have played a role as well. Comparing our results to more similar workflows (filtration, nuclease treatment, random amplification, NexteraXT, MiSeq), our numbers of reads were higher than reported and we identified more virus species . Therefore, we think that our method, which might still have potential for improved sensitivity, shows no bias against a certain virus family or genome type (e.g., ssRNA).
To assess how robust our sample preparation is, we correlated different ratios of spiked viruses with the number of corresponding sequencing reads. In both virus combinations and experiments performed, there was a good correlation between the ratio of spiked viruses and the ratio of the resulting reads (Fig. 4). Direct correlations of viral copies with percentages of sequencing reads have been suggested [18, 54]. However, a direct quantification of input copy numbers based on sequencing reads seems not applicable. Viruses have different properties such as the presence of an envelope, types of genomes, and virion sizes, and certain virus or genome types are amplified preferentially as we have seen with Poliovirus in our experiments. Adding to the complexity, different composition of the genetic background in clinical samples will strongly influence the yield of virus reads. Nevertheless, the correlations showed that the ratio of sequencing reads is preserved over different input ratios and concentrations, signifying that the amplification method itself preserves the relative contribution of different viruses in a sample.
Finally, spiking the same amount of viruses in different samples showed that the protocol presented here could be applied not only for plasma, but also for other clinical samples such as urine and swabs. A more stringent virus enrichment protocol is needed to sequence stool samples or biopsies, as those contain a lot of bacterial or human background reads, respectively [53, 54].
A metagenomic virus sequencing protocol, as presented here, allows diagnostic laboratories to potentially identify any virus present in clinical samples with a single analysis. Such an approach reduces the time and cost spent today on multiple tests performed for each distinct virus and allows detecting rare or novel viruses not accounted for in routine test panels. Characterization of the virome and its alterations in specific disease settings might help to better understand and manage infectious diseases. Finally, our analysis highlights the need for validation methods and standards for metagenomic sequencing approaches in clinical diagnostics.
A correction to this article is available online at https://doi.org/10.1186/s40168-017-0351-x.
We thank Edward Mee from the NIBSC (South Mimms, Hertfordshire, UK) for providing us with the multiplexed viral pathogen reagent. We thank the IMV Diagnostics team, Merle Schanz, and Silke Stertz for providing virus stocks.
Funding was provided by the Clinical Research Priority Program "Viral Infectious Diseases" of the University of Zurich. The funding body did not have any role in the design of the study, in the collection, analysis, and interpretation of data, and in writing the manuscript.
Raw sequencing data are available on the Zenodo repository (10.5281/zenodo.814807). The analysis pipeline "VirMet" is available on github (https://github.com/ozagordi/VirMet/releases/tag/v0.3.3).
DWL, AT, and MH conceived and designed the study. DWL, FDG, VK, and SS performed experiments. DWL, OZ, JB, KJM, and MH analyzed the data. DWL and MH wrote the manuscript. All authors read, commented on, and approved the final manuscript.
Plasma was obtained from anonymous blood donations of healthy individuals at the Zurich Blood Transfusion Service (http://www.zhbsd.ch). Other clinical samples were obtained from anonymized leftovers used for routine diagnostic tests. Research using anonymized biological material is not restricted by the Swiss Federal Act on Research involving Human Beings.
KJM has received travel grants and honoraria from Gilead Sciences, Merck Sharp & Dohme, Bristol-Myers Squibb, and ViiV. The University of Zurich received research grants from Gilead Sciences, Merck Sharp & Dohme, and Roche for studies that KJM served as principal investigator and advisory board honoraria from Gilead Sciences. The other authors declare that they have no competing interests.