Desperate Things

 

“The mass of men lead lives of quiet desperation. What is called resignation is confirmed desperation. From the desperate city you go into the desperate country, and have to console yourself with the bravery of minks and muskrats. A stereotyped but unconscious despair is concealed even under what are called the games and amusements of mankind. There is no play in them, for this comes after work. But it is a characteristic of wisdom not to do desperate things..” - Henry David Thoreau

Is the modern day life science professional wise? Does s/he go to work each day with a purpose to discover new things or have they found employment that they desperately need? In my 13 year career in Biotechnology I was merely employed. During that time I learned many important things but they were learned on the periphery of my official work. 

Biotechnology is a wonderful thing. What most biotech employees do is not. They can employ the technology but does it result in scientific advancement? They turn on the machines, fill out the paperwork and go home as early as they can. Many of these employees are fully aware that their work is not science. 

Take, for example, an employee at Theranos. At the high point there were nearly 700 people working at Theranos. 2 (CEO and President) went to jail, 2 (laboratory technicians) were whistle blowers. In between were a whole lot of people engaged in "desperate things". 

Thought experiment: You have been working at Theranos for a few months. You are driving to work. The sun is coming up and it is going to be a beautiful day. But you are worried. You have to give a presentation soon and the results you need are just not coming in. You know what needs to go up on that PowerPoint slide. You have a meeting with your technicians. Will they have the data you need to please your superiors? 

The desired outcome IS the only instruction. Those above you have given you instructions on what they need from you. You have then given instructions to your group. They must now go into the laboratory and achieve the desired outcome. The problem is that they are using a machine designed by other people. The machine cannot achieve the desired outcome. They turn on the machine, inject the sample, and await the results. They show up to the meeting with the results. 

As you drive to work you imagine a genius has arrived at Theranos. He knows that the samples are not properly biochemically presented to the machine. He makes the adjustments that only a true biochemist can make.  The results come in easily. Eureka! The machine works! Anyone can now prepare the samples, inject them into the machine and get accurate results. With great pride and joy you will stroll into your presentation with results sure to please all in attendence. You acknowledge and thank your staff. You finish the presentation by laying the ground work for full bonuses and future promotions. 

But you are not the genius who can make everything work. Even worse, you are just smart enough to know that the project was doomed from the start. Why? Bad science. The finger prick sampling of blood introduces factors that destroy accurate testing. You know this. You know your technicians can't overcome this reality. You need the job however. You have kids, a mortgage, a new car. Everyone thinks you are a success. As you drive to work on a beautiful day, you are living a life of quiet desperation. You must throw your technicians under the bus. 

If you read Bad Blood you will remember the story of George Shultzs' grandson. He was a technician at Theranos who was not getting the desired results. His superiors were threatening him to get those results or else. He became a whistle blower by talking to a reporter at  the Wall Street Journal. His superiors, those who drove to work on beautiful days with worried minds, began to suspect the technician would spill the beans. He went to visit his grandfather only to find two Theranos attorneys awaiting his arrival in a room on the second floor. It was an ambush. He needed to sign the nondisclosure contract or face dire consequences. He bravely became a whistle blower rather then live a life of quiet desperation. 

This is of course the extreme. But most scientists face unpleasant consequences when the desired results are not forthcoming. True science will overcome this dilemma eventually. True science works and will provide the path to making things work. True science will only allow for it's proper application. This is known as technology. You can keep trying but only proper application produces useful predictable technology. You must use the scientific method. If not you must live a life of quiet desperation. 

The Value of An Empowered Laboratory Professional

“The trouble with the world is not that people know too little; it's that they know so many things that just aren't so. ”

― Mark Twain

The main reason some believe Sars-Cov-2 was man made is the Furin Cleavage site. It is found in the new virus as four amino acids P R R A. There is a reasonable probability that this could happen in nature. What reduces the probability is that the R's (Arginine) are coded for by the nucleotides CGG. 

In the genetic code there are 6 codons that code for R. In human beings the codon CGG is the most frequently found. 21% of the R codons in the human genome are CGG. In Sars-Cov-2 CGG however it is the least used codon.  Only 3% of R codons in Sars-Cov-2 are coded for by CGG. See the article below: SARS-CoV-2 origin: an affair of codons? 

Occam's Razor posits that the answer with the least amount of assumptions is the most likely to be true. 

It is simply a fact that the natural evolution theory of Sars-Cov-1 to Sara-Cov-2 requires more assumptions than does the man made theory. Considering the improbable Arginine inclusion in the improbable Furin Cleavage site insertion, the lab leak theory is the Occam's Razor choice. 

The logical fallacy Appeal To Authority: The fallacy of appeal to authority makes the argument that if one credible source believes something that it must be true.

When the funding of "gain of function" research became an issue Dr. Fauci needed to clarify what he knew he had been funding. "...gain of function is a very nebulous term...", he stated in a hearing with Rand Paul. In his defense he discussed PC3O which was developed between 2014 and 2016 and officially codified in 2017. Gain of function can be good or bad for us. Whenever we are altering DNA sequences in living organisms however, we run the risk of "a dangerous situation". We must always proceed with extreme caution. 

The question Dr. Fauci now had to answer was whether or not he violated the man made guidelines defining "gain of function". His defense is that the research at The Wuhan Institute of Virology would not reach the criteria of "research that might lead to a dangerous situation". He is most likely correct. He did not sign off on inserting the Furin Cleavage site into a viable version of Sars-CoV-1. The research he funded did involve gain of function but not in a genome that encoded for a dangerous virus. What happened after they approved the grant is unknown hoewever. 

Quantity vs quality is part of the problem. In 2017 2,442,608 science and engineering articles were published. The NIH/NIAID does not have the ability to validate the mountain of work done with their grant money. The NIH has a budget of $45B. 10% of that money is used in its own laboratories. 

Test the tests. Use that $4.5B spent on NIH labs to test our investments. Once the recipients of the grants have submitted their narratives of what took place in their laboratories... CHECK THEIR WORK. It is not sufficient to peer review in carpeted offices. Check in the lab by an empowered laboratory work force. Invest in people who wear white lab coats because they need to keep bad things off of their civilian clothing. Cultivate their careers. Develop specialized laboratory professionals though continuing education. Make that work force a deterrent to faulty thinking from PhDs who find laboratory work beneath them. 

People do not know that Dr. Fauci and most scientists with whom he communicates NEVER set foot in a laboratory. Sars-CoV-2 most likely escaped from The Wuhan Institute of Virology in the body of a laboratory technician. We will never know the truth by combing through grant proposals. The truth lies in the notebooks and databases used by the laboratory staff. 

In the case of Sars-CoV-2 there might have been a clue left in the RNA. Laboratory manipulations leave clues. An experienced laboratory work force would be a powerful tool in identifying manipulations or clues. Currently we have the authority of Dr's Fauci and Collins vs the Furin Cleavage site with CGG Arginine codons. 

SARS-CoV-2 origin: an affair of codons?

Antonio R. Romeu1 and Enric Ollé2
1: Chemist. Professor of Biochemistry and Molecular Biology at the Rovira i Virgili University.

Tarragona. Spain. Corresponding author. Email: antonioramon.romeu@iubilo.urv.cat
2: Veterinarian, Biochemist. Associate Professor of the Department of Biochemistry and

Biotechnology of the Rovira i Virgili University. Tarragona. Spain. Email: enric.olle@urv.cat

Abstract

The furin cleavage site, with an arginine doublet (RR), is one of the clues of the SARS-CoV-2 origin. This furin-RR is encoded by the CGG-CGG sequence. Because arginine can be encoded by six codons, in a previous work we found that in SARS-CoV-2, CGG was the minority arginine codon (3%). Also, analyzing the RR doublet from a large sample of furin cleavage sites of several kinds of viruses, we found that none of them were encoded by CGG-CGG. Here, we come back to the core of the matter, but from the perspective that in the human genome, in contrast, CGG is the majoroty arginine codon (21%). Here, we highlighted that the 6 arginine codons provide genetic markers to a traceability on the RR origin in the furin site, as well as, to weigh the probability of the theories about the origin of the virus.

Key words

SARS-CoV-2 origin, Furin Cleavage Site, Arginine Codon Usage, Bioinformatics.

As it is known (1,2,3) and we have also addressed (4), the origin of SARS-CoV-2 could be reduced to the origin or acquisition of the furin cleavage site in its S protein. This was a gain of function: there had been an insertion in the S gene that had caused the S protein to gain more capacity for human infection. SARS-CoV-2 belongs to the group of Betacoronaviruses, Sarbecoviruses (Lineage B). It was surprising that it was the only Sarbecovirus species with such furin site. At present, still nobody knows how and when it got to the virus. this is a key point in the controversy over the origin of the virus pandemic.

Before going ahead, some basic concepts of biology need to be absolutely clear. Based on the universal genetic code and in the protein sinthesis, when the sequence of a gene is read in frames of three, the combination of the 4 letters taken three by three, results in the existence of 64 triplets or codons. Since there are only 20 protein amino acid, there are more than enough codons to go around, allowing some amino acids to be specified by more than one codon. The arginine (whose symbol is R), can be encoded by any of the 6 triplets: AGG, AGA, CGA, CGC, CGG, CGT.

 

In SARS-CoV-2, the furin site is characterized by the insertion of a 4 amino acid sequence (PRRA), which corresponds to the insertion of 12 nucleotides (3 x 4). In SARS-CoV-2, the RR doublet of the furin site is encoded by the CGG-CGG codons. It is worth bearing in mind that furin cleavage site, with RR doublet, is common in the world of viruses (5) (including Coronaviruses, but excluding Betacoronaviruses). Recombination with other viruses is the most plausible explanation for the acquisition of this site in SARS-CoV-2 (6). However, to analyze the likelihood of such virus recombination, we screened the databases, and analyzing the RR doublet from a large sample of furin cleavage sites of several kinds of viruses. We found that there were no RR doublets encoded by the CGG-CGG codons (4). We observed that AGA triplet was the majority codon involved in these viral RR doublets. In all genetic recombination, there is always a donor and an acceptor of genetic material; and the donor code is passed to the acceptor. If the SARS-CoV-2 has acquired the furin site by recombination with another virus, at the moment, from the information available in the genomic databases, we can't know what it may have been.

With these results, we were interested in determining the arginine codon usage in SARS-CoV-2. Studying the composition of all its proteins, we found (4): AGG (13%), AGA (45%), CGA (5%), CGC (10%), CGG (3%), CGT (24%). The AGA triplet was the majority, and interestingly, CGG was the minority. In the specific case of S protein, of the 42 arginines it has, 20 are encoded by AGA, and only 2 by CGG (4). It was surprising that CGG-CGG codons, were those that encoded the RR doublet of the SARS-CoV-2 furin site.

Since each species has its own codon usage. Regarding the amino acid arginine in Homo sapiens, the frequency of use of triplets is (7): AGG (20%), AGA (20%), CGA (11%), CGC (19%), CGG (21%), CGT (9%). This pattern of the human genome contrats with that of the virus. In the human species, the CGG triplet is the majority (21%) and in the virus it is the minority (3%). It is surprising that in SARS-CoV-2, the furin site RR doublet uses the arginine majority codon of the human genome.

Circumstance like this fuels the great dilemma on conflicting theories about the origin of the virus: natural or laboratory (biotechnological).

The theory that supports a natural origin of SARS-CoV-2, should consider that two independent events have occurred in time: (i) the insertion by mutation or recombination of a 12 nucleotides sequence, encoding the furin site, in a strategic site of the S gene; and (ii) such inserted sequence must contain the majority human arginine codon: CGG. Both events already have a low probability, however, since they are independent and had had to be simultaneous, the probability would be even lower.

The theory that supports a laboratory origin of SARS-CoV-2, should contemplate that the insertion of the furin site in the virus genome had been in a controlled way. Considering the current applications of genetic engineering, genes of certain human proteins are now inserted (cloned) into microorganism genomes suitable for their commercial manufacture. A typical example is the insulin production obtained from the yeast Sacharomyces cerevisae. In these circunstances, human genes are biotechnology optimized with the majority codons of the recipient microorganism. In the case of the SARS-CoV-2 furin cleavage site, it gives the impression that a 12-nucleotide sequence had been cloned into the genome of a given virus originating the SARS-CoV-2. Of course, this cloning sign can only be glimpsed since arginine has 6 triplets in the universal genetic code. In the impossible case that arginine had 2 codons, such as lysine (by the way, lysine is also a positive

amino acid and present in viral furin sites), that traceability does not it would make sense and, there would be no doubt about the natural origin of the virus.

This is the state of the art on the origin of SARS-CoV-2, from the genetic perspective. The issue must also be approached with a forensic genetic mindset. Results based on genetic markers are always expressed in stochastic or probabilistic terms. In genetics, there are no absolute certainties, but there are evidences so highly probable that allow sentencing of guilt or the determination of paternity and/or maternity relationships. Thus, under the umbrella of the sequence analyses, the origin of SARS-CoV-2 cannot be proved as a mathematical theorem. However, between two theories, which have the same consequences, the simplest explanation is usually the most probable: Occam's razor.

Acknowledgements

This work has not been awarded grants by any research-supporting institution.

Competing interest declaration

All authors declare that they have no conflicts of interest.

References

1. Britt Glaunsinger. Coronavirus biology. The second lecture in the COVID-19, SARS-CoV-2 and the Pandemic Series. University of California, Berkeley. 2020. Accessed June 2, 2021. https://www.youtube.com/watch?v=r2mOU2qOCYs.

2. Nicholas Wade. Origin of Covid — Following the Clues. Accessed June 2, 2021. https://nicholaswade.medium.com/origin-of-covid-following-the-clues-6f03564c038.

3.Kristian G Andersen, Andrew Rambaut, W Ian Lipkin, Edward C Holmes, Robert F Garry. The proximal origin of SARS-CoV-2. Nat. Med. 26:450-452, 2020. PMID: 32284615. doi: 10.1038/s41591-020-0820-9.

4. Romeu, A.R.; Ollé, E. SARS-CoV-2 and the Secret of the Furin Site. Preprints 2021, 2021020264 (doi: 10.20944/preprints202102.0264.v1). Accessed June 1, 2021..

5. Elisabeth Braun, Daniel Sauter. Furin-mediated protein processing in infectious diseases and cancer. Clin. Transl. Immunol. E1073, 2019. PMID: 31406574. doi.org/10.1002/cti2.1073.

6. William R Gallaher. A palindromic RNA sequence as a common breakpoint contributor to copy- choice recombination in SARS-COV-2. Arch. Virol. 165:2341-2348, 2020. PMID: 32737584. doi: 10.1007/s00705-020-04750-z.

7. GenScript Codon Usage Frequency Table(chart) Tool. Accessed June 2, 2021. https://www.genscript.com/tools/codon-frequency-table.

By What Authority?

The Jews closed the canon of The Old Testement in 100 AD at the Synod of Jamnia. At that time they decided which were the canonical books of the Hewbrew scripture. The books of The New Testament were decided upon at the Senate of Rome under Pope Damasus in 382 AD. The recipients of the message of the old and new testaments were already in place. The authorities were selected, the message was selected and then canonized in the Bible. 

In science we don't close the books. We leave room for improvements or complete reconstruction. 

We don't talk about "the science" so much here. We talk about the scientific method, which is what Feynmans CCS was all about. "But there is one feature I notice that is generally missing in cargo cult science. That is the idea that we all hope you have learned in studying science in school--we never say explicitly what this is."

The "idea we all hope you have learned" is the scientific method. How do we get to the truth? How many times have we, as individuals, gotten something wrong? We argue and we shout and we point our fingers at the other guy. The bitterness in our online debates demonstrates something the psychologists should be studying. What is road rage? What is cyber bullying about? Why are we so different in different situations? I will suggest that we are different because there is a place in all of our minds where opposition to our thoughts is never encountered. It is a safe space.

It is here, this place inside our minds where we can find the scientific method. The Bible is questioned by Atheists, just not in church while the preacher is giving his sermon. We all must go out into he world where our thoughts are questioned. We must first develop our narrative so we can survive and evolve. 

When we find ourselves alone and we choose to sit and think, it is there where we can explore without fear or interruption. Our minds can roll on freely getting it right and wrong. Recognizing when we are wrong is good. Others publicly recognizing how wrong we are can hurt our feelings. Therefore we must think alone and allow ourselves to make mistakes. We must also thrive to correct our wrong thinking with truths.

Biotechnology has failed to nurture the right people. The industry has evolved selecting those who best cave to authorities like Dr's. Fauci and Collins. When the Covid vaccine was introduced where were the experts asking about the MOA of mRNA vaccines? How could we introduce a paradigm shift to vaccinations when faced with a pandemic? Why was there so little emphasis on treatment? What was our understanding of how viruses pass through the human race? Did we really think we could just hide in our homes while the virus waited for us just outside the front door? There were some left to ask questions, but they too learned the powers of authority and dogma. 

I won't talk about the origins of the pandemic at this time. I will simply say that the pandemic showed us the problem with dogmatic science. Fauci created the NIAID bible during the HIV/AIDS era. Koch's postulates were ignored. Ineffective and harmful solutions, such as AZT, were allowed. Using the same authority (Fauci) and the same NIAID bible, we simply went through the pandemic with minimal resistance to it's destructive path. We learned nothing and we saw what happens when Cargo Cult Science takes center stage. True science will not canonize the Covid 19 book however. We must all go to that safe space in our minds and think about the many aspects of the pandemic. Never stop thinking and asking questions. Just be aware of whom you may be offending and why they take offense. 

 I'll end with another Feynman classic quote that sums up the problem of thinking in a public place, not in that safe place in your mind where the scientific method thrives. 

“I would rather have questions that can't be answered than answers that can't be questioned.”

― Richard Feynman