Posted as an aid to your understanding.

mRNA Display Design of Fibronectin-based Intrabodies That Detect and Inhibit Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein*

Hsiang-I. Liao‡,1,
C. Anders Olson§,1,
Seungmin Hwang‡,
Hongyu Deng‡,
Elaine Wong‡,
Ralph S. Baric¶,
Richard W. Roberts‖ and
Ren Sun‡,**,2

+
Author Affiliations
From the ‡Department of Molecular and Medical Pharmacology and
the **California Nano System Institute, UCLA, Los Angeles, California 90095,
§Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125,
the ¶Department of Epidemiology, University of North Carolina, Chapel Hill, North Carolina 27599, and
the ‖Department of Chemistry, Chemical Engineering, and Biology, University of Southern California, Los Angeles, California 90089-1211
↵2To whom correspondence should be addressed: 650 Charles E. Young Dr. South, CHS 23-120, UCLA, Los Angeles, CA 90095. Fax: 310-825-6267; E-mail: rsun@mednet.ucla.edu.

↵1 Both authors contributed equally to this work.

From http://www.jbc.org/content/284/26/17512.full

…The ability to detect and inhibit protein function is central to molecular and cellular biology research. To date, phage display and monoclonal antibody production have been the most common routes to design reagents for protein detection and inhibition, antibodies and antibody-like reagents that serve as high affinity, high specificity molecular recognition tools (1). Totally in vitro selection methods using alternative scaffolds are becoming more common to produce affinity reagents with improved and expanded functionality (2, 3). For example, ribosome display and mRNA display enable creating 1–100 trillion-member peptide and protein libraries that surpass immunological and phage display diversities by 3–5 orders of magnitude (4).

Antibodies or antibody-like molecules are important because they can serve as diagnostics, probes for studying proteins in vivo, and potential therapeutics (or surrogate ligands for therapeutic design/screening). Regarding biology, antibodies used inside living cells, denoted “intrabodies,” are appealing because they provide an alternative to genetic knock-outs, dominant negative mutations, and RNA interference strategies, enabling targeting proteins in a domain-, conformation-, and modification-specific fashion as well as identifying hot spots for protein interaction (5, 6). For example, green fluorescent protein-labeled intrabodies can act as molecular beacons to determine real time, live cell localization of endogenous target proteins rather than non-native expression of green fluorescent protein target fusions (7).

Although antibodies often demonstrate laudable affinity and selectivity, these proteins are likely to be suboptimal as a general approach to create intracellular reagents. Most notably, antibodies contain disulfide bonds that are likely to be reduced in the cytosol, thus impeding their proper folding and function (8). To overcome the paucity of functional intrabodies generated by in vitro selection methods, in vivo screens may be employed at the expense of combinatorial diversity (9). On the other hand, it has been demonstrated that intracellular antibodies can generate aggresomes, which may inhibit the ubiquitin-mediated degradation pathway and promote apoptosis (10–12).

Ideally, intrabodies would be as follows: 1) easy to produce in a broad variety of cells; 2) stable; 3) specific; 4) high affinity; 5) highly selective; 6) functional in intracellular environments; and 7) noninterfering with normal cellular processes. Recently, ribosome display has been used to generate protein affinity reagents based on ankyrin domains (DARPins), which detect and inhibit kinase or proteinase function in vivo (13, 14). Although this scaffold is powerful, it is structurally very different from antibodies as it utilizes a discontinuous binding surface rather than the continuous surface generated by the CDR loops in antibody VH and VL domains.

Our approach here has been to use mRNA display to design disulfide-free antibody-like proteins that can be used to create general protein targeting tools. To do this, we used a protein library based on the 10th fibronectin type III domain of human fibronectin (10Fn3)3 (15, 16). The 10Fn3 domain was developed as an antibody mimetic by Koide et al. (16) because of the following: 1) it is topologically analogous to the immunoglobulin VH domain; 2) it is exceptionally stable; 3) it presents a continuous protein interaction surface; and 4) it expresses well in both eukaryotic and bacterial cells (16). We recently described construction and characterization of a 3 × 1013 member 10Fn3 library (15) and validated this library by developing proteins and fluorescence resonance energy transfer sensors that recognize IκBα in a phosphoserine-specific fashion (17). There the selected 10Fn3 functioned in vivo, blocking proteasome-mediated degradation of full-length IκBα efficiently.

Here we have targeted the severe acute respiratory syndrome (SARS-CoV) nucleocapsid protein (N). SARS-CoV is a unique member of the Coronaviridae family with only 20% sequence identity to the closest homolog (18). There is a need for reagents and methods that can be used to detect new infectious entities as they arise. Indeed, the recent SARS epidemic was unexpected, reaching an 8% fatality rate despite the fact that coronaviruses typically are involved in ∼30% of common cold infections. N protein is 422 amino acids long, phosphorylated, and composed of two structured domains linked by a nonstructured domain. The N-terminal domain (NTD) is a putative RNA binding domain, and the C-terminal domain (CTD) mediates self-association (Fig. 1A) (19, 20). The unstructured middle domain interacts with the membrane (M) protein, anchoring M protein to the viral core. The two structured domains act in concert to bind genomic RNA, oligomerize, and form the final packaged ribonucleoprotein complex.

We chose N as our target for several reasons. First, the N protein is the most abundant protein produced by SARS virus. Second, N plays multiple roles in vivo, including binding/packaging the viral genomic RNA, mediating interactions with the viral membrane (via the M protein), acting in genome replication, and exerting control over host cell processes (21, 22). Finally, no therapeutic reagents currently target N protein; therefore, new inhibitory N-directed ligands represent an important potential new route for developing anti-SARS drugs.

After six rounds of selection, we were able to generate molecules that detect SARS N protein in vitro and modulate its SARS replication in vivo in a domain-specific manner. The selection yielded six high affinity molecules that recognize the CTD and two molecules that require the NTD for binding. We confirmed the interaction between the selected 10Fn3 proteins and N protein both in vitro and in vivo by pulldown, co-immunoprecipitation, and immunofluorescence microscopy. Seven of the 10Fn3-based intrabodies inhibit replication, ranging from 11- to 5900-fold, recognizing at least two nonoverlapping epitopes/hot spots in a synergistic manner. These molecules represent new tools for detecting SARS virus, assessing N function in living cells, and identifying regions of N critical for virus proliferation.

Abstract follows…

On January 21, 2020 https://www.nationalreview.com/2020/01/how-far-left-will-democrats-push-virginia/?amp asked, “How Far Left Will Democrats Push Virginia?”

The article’s first paragraph outlines the Democrat’s planned mandates to further limit Virginian’s R2KBA; the rest of the story is deserving of an unfolding only Paul Harvey can deliver; Click the National Review link for the sense of democrat direction. The Paul Harvey link provides the context.

The democrat focus on social policy is targeting religious adoption agencies protections and encouraging elementary and secondary school students who see themselves as transgender to use the locker rooms and showers they prefer; democrats are amending themselves into a position where they deny our Creator (again) as they attempt to assume the role of supreme ruler-state, transforming the unsuspecting formerly free, if they are successful, to the no status subjugated.

Like one of their leaders, Paul Begala, so quaintly put it… Stroke of the pen. Law of the Land. Kinda cool.

ARTICLE I

BILL OF RIGHTS

Section 18. Personal Reproductive Liberty.

That an individual’s right to personal reproductive autonomy is central to the enjoyment of life and liberty and shall not be denied or infringed upon unless justified by a compelling interest of the Commonwealth and achieved by the least restrictive means.

Yes, I am aware Begala was expressing his delight in the power of the Executive Order. The question is, when was the last time you recognized any democrat politician’s expression of respect for the Rule of Law when they found themselves bound to it not to their benefit?

The National Review points out this year’s short legislative period gives the Virginia democrats only until February 11 to pass their efforts into law. But we are talking ‘fundamental change’ here and you have to understand that Virginia’s fundamental change is a concept coming from waaaay far above the Virginia democrat criminal brain.

Crossing your fingers and hoping they won’t beat the clock is not going to help. Virginia citizens must continue to apply pressure to resist the chains of slavery successfully.

We will all get our turn to resist. The criminal elite wants America’s guns. And more.

December 25, 2019 https://www.bucksafa11.org/2019/12/25/when-you-get-down-to-it/

December 20. 2019 https://www.bucksafa11.org/2019/12/20/somebody-has-to-ask/

December 5, 2019 https://www.bucksafa11.org/2019/12/05/red-flags-and-target-marking/

September13, 2019 https://www.bucksafa11.org/2019/09/13/lest-we-forget/

September 21, 2019 https://www.bucksafa11.org/2019/09/21/somehow-missed-this-story/

September 10, 2019 https://www.bucksafa11.org/2019/09/10/another-bipartisan-assault-scheduled/

October 13, 2018 https://www.bucksafa11.org/2018/10/13/pa-democrat-governor-signs-feel-good-republican-anti-rkba-law/

And from http://voluntarysociety.org/conditioning/misc/peoplespottage.html

…Americans now are of three kinds, namely: those who are very unhappy about what has happened in one lifetime to their world — to its morals, principles and ways of thinking —and have intuitions of a dire sequel; those who only now begin to read the signs and are seized with premonitions of disaster; and three, those who like it.

It is impossible to say what proportion any one of these three divisions bears to the total. It is impossible, furthermore, at any moment of time to say what the people want or don’t want. They probably do not know. And what they say may be so like writing on the sand that a tide not of their making will wipe it out. This is riddle.

Suppose a true image of the present world had been presented to them in 1900, the future as in a crystal ball, together with the question, “Do you want it?”

No one can imagine that they would have said yes — that they could have been tempted by the comforts, the gadgets, the automobiles and all the fabulous satisfactions of mid-century existence, to accept the coils of octopean government, the dim-out of the individual, the atomic bomb, a life of sickening fear, the nightmare of extinction. Their answer would have been no, terrifically. You feel very sure of that, do you? You would have said no yourself?

Then how do you account for the fact that everything that has happened to change their world from what it was to what it is has taken place with their consent? More accurately, first it happened and then they consented.

They did not vote for getting into World War I. They voted against it. The slogan that elected President Wilson in 1916 was: “He kept us out of war.” Then in a little while we were in it and supporting it fanatically.

They did not vote for the New Deal. They voted against it. That is to say, they elected Mr. Roosevelt on a platform that promised less government, a balanced Federal budget, and sound money. Nevertheless, when it came, they embraced the New Deal, with all its extensions of government authority, its deficit spending and its debasement of the currency.

They did not vote for getting into World War II. So far as they could they voted against it. Annotating in 1941 the 1939 volume of his Public Papers, Mr. Roosevelt wrote: “There can be no question that the people of the United States in 1939 were determined to remain neutral in fact and deed.” They believed him when he said, during the 1940 campaign, “again and again and again” that their sons would never be sent to fight in foreign wars. So he was elected a third time on his pledge to keep the country out of war.

Immediately afterward, in March 1941, came Lend-Lease. By any previous interpretation of international law, Lend-Lease was an act of war — the government of one country giving arms, ammunition, and naval vessels to a belligerent nation. Not long after that, actual shooting began in the Atlantic, but for a while its meaning was disguised. Our navy was escorting cargo trains of Lend-Lease goods across the Atlantic, under pretense of patrolling the waters, and German submarines were trying to sink the cargo vessels; the trouble was that when the protecting U. S. Navy vessels appeared the Germans would shoot only in self-defense, because Hitler did not want to attack, whereas what Mr. Roosevelt needed to release him from his antiwar pledges was an attack.

That went on until, in October, 1941, Admiral Stark, Chief of Naval Operations, sent a message to all fleet commanders saying: “Whether the country knows it or not we are at war.” And still there had been no attack that would release Mr. Roosevelt and unite the country for war. After a cabinet meeting on November 25, 1941, Henry L. Stimson, the Secretary of War, writing in his diary, defined the problem that had been discussed that day. It was how to “maneuver” the Japanese “into the position of firing the first shot.”

Pearl Harbor solved the problem. But in fact we had already been in the war for at least nine months.
They never voted for the Welfare State, with its distortions of the public debt, its basic socialism, its endless vista of confiscatory taxation, its compulsions and its police-like meddling with their private lives. Certainly they never voted for it in the way the English voted for socialism. Yet step by step they accepted it and liked it.

They did not vote for the United Nations, nor for putting the United Nations flag above American troops in foreign countries, nor for the North Atlantic Pact, which may involve us in war automatically and thus voids the Constitutional safeguard which says that only the Congress can declare war. A report entitled “Powers of the President to send Armed Forces Outside of the United States,” signed by the chairman of the Foreign Relations Committee of the Senate, says: “The use of the Congressional power to declare war has fallen into abeyance, because wars are no longer declared in advance .”

And to all of this the people have consented, not beforehand but afterward.

They have never voted on a foreign policy that steered the ship from the American main at the top of the world to the international shoals of extreme danger.

Whereas in 1945 the American word was law in the world and the Chief of Staff could report to the President that “the security of the United States now is in our own hands,” five years later the government was telling the people they would have to fight for survival against the aggressor for whom we had swapped Hitler; and that we could save ourselves only with the aid of subsidized allies in Europe. Never having voted for it, having had in fact nothing to say about it, people nevertheless accepted it as if it had been inevitable in the pattern of American destiny.

They never voted for whittling away the restraints imposed by the Constitution on the power of executive government. They were deeply alarmed when, in a letter to the chairman of a House Committee, President Roosevelt asked why the Constitution should be permitted to stand in the way of a desirable law; and their feeling for the sanctity of the Constitution was so strong that when Mr. Roosevelt proposed to enlarge the Supreme Court in order to pack it with New Deal minds he was defeated by a spontaneous protest of extraordinary intensity.

Nevertheless, since then the mind of the Supreme Court has changed. What Mr. Roosevelt had been unable to do by onslaught was done by death and old age. As conservative judges fell out, their seats were filled by men whose sympathies inclined to the Welfare State. By a series of reinterpretations of the Constitution, the reformed Supreme Court has so relaxed the austerities of the supreme law as to give government a new freedom.

In this process it has cast itself in a social role.

Formerly its business was to say what the law was, according to the Constitution; if people did not like the law they could change it, only provided they changed it in a lawful manner by amending the Constitution. Now the Supreme Court undertakes to say what is justice, what is public welfare, what is good for the people and to make suitable inflections of the Constitution. Thus law is made subordinate to the discretions and judgments of men, whereas the cornerstone of freedom was that the government should be a government of law, not of men.

They did not vote to debase the dollar. Everything that has happened to money was done to it by government, beginning with the deceptive separation of people from their own gold, then a confiscation of the gold, then making it a crime for a private citizen to own gold, together with a law forbidding contracts to be made in any kind of money but irredeemable paper currency, and finally the dishonorable repudiation of the promissory words engraved on its bonds.

All of this with an air of leave-these-things-to-the-wisdom-of-government, as if people could not understand the mysteries of money. That was absurd. The controlling facts about money are not mysterious. By contrast, in 1896, there was a very grave monetary question to be settled. It was silver versus gold; or inflation versus sound money. It was taken to the people, and the people, not the government decided it. The people voted for sound money.

Enough of this history if it serves to indicate that in our time, actually in a few years, a momentous change has taken place in the relationship between government and people. It is commonplace to say that people have lost control of government. It is a thing too vast, too complex, too pervasive in all the transactions of life to be comprehended by the individual citizen. Indeed, as the Hoover Commission was able to show, the government no longer comprehends itself.

While the number of those who administer, or assist to administer, executive government has increased fivefold, and while the expenditures of Federal government have increased twenty times in twenty years, the power of the individual to resist the advance of its authority has not increased at all. In fact it has diminished. Even organized pressure groups, such as farmers and union labor, no longer resist. They ride it and use their influence to gain freer access to the illusory benefits that now flow in all directions from Washington.

Those who remember what the American world was like in the preceding generation do not need the record. The change it indicates is known to them by feeling…

Update follows.

On this day that the intensity of the attacks on America is ramped up by the Democrats and their Republican collaborators in a time branded by one of their taskmasters as the end of an era, a message of resistance.

from https://gunwatch.blogspot.com/2020/01/how-virginia-sheriffs-can-enforce-their.html Second Amendment Sanctuaries.

A Townhall.com article reported today that “Congressman Ro Khanna (D-CA) claimed Tuesday there were no embassy protests or deaths of American contractors under the Obama administration.

Now, everybody knows that’s a lie but reading that article could not have been timed better. It nudged me into looking at Obama era posts where I found a good ol’ ‘smoking gun’ reference. Ah, those were the days.

The link to frontpage does not work. Use http://shoebat.com/2014/05/03/distribution-list-smoking-gun-benghazi-email-included-muslim-brotherhood-agent/ if you want to get the pertinents.

The Benghazi Mayekar/Alhassani/Obama connection was one notable embassy protest that comes to mind. Samir Mayekar moved on to https://www.chicagobusiness.com/government/citys-economic-development-czar-takes-tricky-balancing-act and Mehdi K. Alhassani of https://twitter.com/mehdi3344?lang=en fame, who… a few hours before the Benghazi attack, Alhassani met in the White House with Samir Mayekar, a George Soros ‘fellow’ for an unscheduled visit…

Here’s some Sharia financing information you might find interesting.

http://gulfbusiness.com/2014/03/bahrain-eyes-external-sharia-audits-islamic-banks/#.VGqdnyx0z-g

February 2011, out of concern that foreign commercial banks that previously submitted to sharia law were mixing Islamic banking activities and conventional banking activities Qatar ordered non-islamic banks close their Islamic finance divisions in Qatar by the end of the year

http://www.businessinsider.com/qatar-banks-sharia-law-2011-2

Six months prior to that announcement, the Qatar Central Bank issued new rules that effectively gave conventional banks that opened sharia compliant windows a choice: Full submission to sharia banking methods or be limited to conventional banking activities.

To appreciate the sharia arm twisting behind the new rules, one only has to understand “The estimated 15-20% annual growth rate of Islamic services has encouraged conventional banks to open sharia-compliant windows in recent years and the new rules will therefore have a significant impact on their growth. Islamic finance has been an important driver in attracting new customers. Islamic assets among the country’s banks grew at an average of 54.3% between 2003 and 2010, compared to 37.8% in conventional assets over the same period, according to MEED.”

That quote is from http://www.oxfordbusinessgroup.com/news/qatar-new-rules-islamic-banking

Major corporations that consider themselves sharia compliant in one fashion or another, in other words corporations not yet faced with the choice of a short knife elimination from a perceived expanding market or a full commitment to sharia compliance include: Bank of America, Barclays, Bloomberg, Century21, J.P. Morgan Chase and even Coca Cola.

The longer but probably incomplete list can be read at http://www.shariahfinancewatch.org/blog/shariah-financial-institutions/

Palantir and Sharia compliant Financial corporations… put them together and what do you get?

Anything for the cause. I put this together back when the Obama crowd was getting pushy about armed citizenry.

A well-regulated militia, being necessary to the security of a Free State, the right of the people to keep and bear Arms, shall not be infringed.

1) Do you believe the County Sheriff is the highest ranking LEO in your county?

O yes O no

2) Do you believe the County Sheriff owes primary allegiance to the State/Federal Government?

O yes O no

3) Do you believe it is acceptable for the County Sheriff to avoid publicly declaring an allegiance to the Second Amendment?

O yes O no

4) Do you believe the concept of holding politicians feet to the fire improves their character and level of respect for constituents?

O yes O no

5) Do you think voting a non-performing, Constitutional disrespecting politician out of office would improve your and your family’s life and well-being?

O yes O no

6) Do you believe the Second Amendment empowers you to join or start a militia group?

O yes O no

7) Do you think a County militia is relevant in the Twenty-first Century?

O yes O no

8) If you said yes to question #7, do you think there is any difference between a Sheriff’s posse and a militia?

O yes O no

9) If you said no to question #8, would you be willing to join a Sheriff’s posse to resist State/Federal gun registration or collection?

O yes O no

10) Do you think Sheriff would ever, if the need arose, call for a posse to resist any egregious State/Federal action contrary to our Constitution?

O yes 0 hell yes O no O Hell no

Having once again come across an article where the democrats accused the White House of the democrat party’s favorite activity, playing games in a most dangerous fashion, I thought it was time to look into the somehow empowered democrats propensity of using their political energy weapons in the most domestic of fashions while they express horror at any actions their opposition party takes that appear to defend US from foreign enemies the democrats helped birth.

My effort paid off by providing an unexpected introduction to Nawal El- Saadawi and I consider it a blessing when I encounter a truth delivered like the arrival of the eye of a category five hurricane.

Nancy Pelosi’s hammer should be so powerful.

Other weekend reading provided enlightenment from a different direction by Burton L Visotzky who explained the emergence of “rabbinic Judaism”, a form of Judaism that emerged from the rubble of the Herodian Temple and in his words was “shaped and defined by rabbis” using vocal transmissions of what the rabbis called their “Oral Torah” that lead to a Judaism that replaced the Israelite Temple religion.

If one thought about it one might see the similarity in the way our Constitution took a mortal hit from the Patriot Act after the takedown of the World Trade Center and the ready adaptation of rabbinic Judaism after the Romans destroyed their temple in 70 CE; as Rabbi Visotzky put it, “In short, what is now called “Judaism” was invented in the matrix of Roman culture.”

What’s more of a concern for US is the recent proclamation about special status being provided Jews and the daily news media accounts of people arrested for verbally assaulting Jewish people.

You see, when the rabbis were developing the oral tradition they were also giving their own interpretation of Scripture, an effort intended to replace the Temple as the source of authority. While modern rabbis retain that authority they also are assuming the mantle the recent status of special provides and that adds a whiff of the infallibility the Roman/Jesuit pope is in the process of casting away.

While some may celebrate the likely end of the Roman Church’s reign, one important thought to remember is the Church foundation was always presented as based on the teachings of Jesus who made clear the difference between right and wrong and the origins and the consequences of each.

Rabbinical Judaism presents moral leadership as equivocating virtue without value. Regularly. An Outback society of no rules just rights. And that, my friend, just leads to a walk to the locker room