Preparing particles - theory

 

What is happening when we coat either Au or W particles?   

Adsorption

Adsorption is where a layer of molecules cling to a surface.

1.       There is the adsorbate (i.e. DNA plasmid)

2.       There is the adsorbent (i.e. W)

What attracts the adsorbate and the adsorbent to each other so that they are held together in Adsorption?

DNA has a – charge.  W has + charge.  Under the right conditions, a bond is formed.  If the DNA is in solution with the W and the bond forms, the DNA will precipitate and adsorption occurs.  The DNA is not present in the solution anymore, it is now coating the W microparticle.

That’s very beautiful and simple to understand but as may be expected the devil’s in the details. 

In this post we hit you up for a donation

As you may be aware, today has been designated as Giving Tuesday.  Though I don't much relish begging or soliciting donations, it needs to be done.   

Please note that project GRFT has non-profit status through Counter Culture Labs.  

We depend on your donations as well as the long hours of hard work put in by a small group of volunteers to keep moving forward.  

Our goal is to produce, store and make available transgenic rice containing Griffithsin (GRFT) worldwide for research purposes.   By producing the rice locally, it solves certain supply chain problems that we are seeing a lot of now.  It empowers people living without much resources to obtain this valuable material by simply farming rice.   The GRFT can be purified from the rice quickly and simply using chromatography methods not too different from making drip coffee.    

I'm sure that everyone who reads this wants there to be an end to the global pandemic we have been experiencing.  That takes a global solution.  

We are fortunate in the US to have access to advanced vaccines.  Yet in many developing countries, the vaccination rate is very small, around 2% in some areas.  GRFT is not meant to substitute for vaccination and we would encourage everyone that hasn't yet got vaccinated with access to vaccines to roll up your sleeves and get vaxxed!  

GRFT has not so far been approved as a pharmaceutical.  It needs to go through clinical trials.

Research has shown GRFT to be a most potent antiviral, inhibiting cellular infection by coronaviruses as well as other viruses.

What we are doing in our project is developing the biomanufacturing of this non-toxic antiviral polypeptide by expressing it in rice.   This can be done in any location where climate permits, or in greenhouses.  There's no need for expensive industrial laboratories with bioreactors with this method.   We can keep the cost to a minimum supporting the 21st century wisdom that states: medicine for people who need it rather than for profits.

Right now we have lab expenses such buying gold nanoparticles to be coated with DNA and shot into rice calluses to enter the cells and transform the rice to express GRFT.

If you, dear reader, are in agreement with this and our efforts towards ending pandemics, present and future, please help, here's a link to contribute:

https://www.counterculturelabs.org/

Please put into the special instructions box:

GRFT

Let's work together to put stop viral outbreaks from becoming pandemics!

Thanks!

preparing rice seed to grow embryos and calluses

 Here's Hector in the lab as he prepares rice seed to grow on media plates

https://youtu.be/Qdc9-8sgymg

note: the husked seeds were washed with bleach to sterilize them

Hector's work on project GRFT

Introducing Mr. Hector Vera who is one of our talented, dedicated and hard working volunteers at project GRFT.  Hector speaks from outside a laboratory in Oakland, California where our gene guns are now being built and tested.  

Open Insulin - changing the paradigm

 The older sibling of Project GRFT is Open Insulin. They recently made a quality introductory video about their work which may be viewed here    We share the goals of  medicine for people, not profit, and valuing open source technology.   

Project GRFT in the lab and in the fields - a plan

 

People have asked the question, “If Griffithsin is such a potent anti-viral, why is it not being manufactured?  Why is it not generally available and being used to stop viral outbreaks such as Covid-19?”  It’s a very good question.  There are a few efforts being made to biomanufacture griffithsin and at least one to get it into clinical trials.  In March, 2020, I couldn’t find anything to indicate that it was in any pharma company pipeline. I decided that if no one is working on this, I would put my shoulder to the wheel and do work on its development.  First I reached out to researchers around the world who had worked with griffithsin.  I reached out to my friends in the biohacking community.  There were a few people who responded at first and we started a group of friends to take on a rather enormous project with a goal of making griffithsin available to the world. 

The arts of molecular biology and biotechnology demonstrate that genes from one organism may be placed into another organism which will read the sequence information and from it make a peptide or polypeptide in the cells.  There is a large and thriving industry based on this.  We were searching for the best method for biohackers with limited resources to use to produce griffithsin on an epic scale.  Most of our experience with protein expression had been with bacteria so far.  E-coli is widely used for this because it grows rapidly and is relatively inexpensive and easy to work with.  Bacteria has certain drawbacks, however.  For example bacteria is a prokaryote, lacking organelles and the protein folding may not work well in eukaryotes such as animals.  On the other end of things, much pharmaceutical Biomanufacturing uses animal cells for expression.  The folding then is correct for animal or human use.  The drawback with animal cells is that they are difficult to work with, grow slowly, and are extremely subject to contamination by any number of organisms.  Stringent laboratory procedures are needed to ensure sterility during transfection and the growth cycle.  This works well for pharmaceutical companies with the resources to carry it out, however Biomanufacturing using animal cells is very expensive to set up and maintain. 

Some of the scientists who responded to our inquiries used plants to express griffithsin with good results.  Dr. Yavar Vafaee, Ph.D. had published his research: “Heterologous production of recombinant anti-HIV microbicide griffithsin in transgenic lettuce and tobacco lines” and was very kind in advising and getting me started in the exciting new direction of plant molecular biology.  We also were fortunate to find Dr. Evangelia Vamvaka, Ph.D. who had been working in Dr. Jennifer Doudna’s lab at UCB.  She had previously done a great deal of research with griffithsin and published papers such as “Rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against HIV”.  After reading her papers and speaking with her, we decided that her method was the one that would work the best to meet our biomanufacturing goals as biohackers with limited resources.  Expression in rice has a number of advantages, such as rice being non-toxic and generally regarded as safe, and when harvested and dried, it can be stored for long periods of time without trouble. Dr. Vamvaka’s research shows that the folding of the protein is correct and that the antiviral properties are present as expressed in rice.  

Our group, Project GRFT formed, started meeting and working.   Our goal is to grow transgenic rice containing the polypeptide griffithsin and to make this rice available worldwide for research.        To be continued....

Griffithsin vs Coronaviruses

 

Griffithsin vs Coronaviruses

 

On the 18^th of March 2021, there are 120,915,219 confirmed cases of SARS-CoV-2  with 2,674,078 confirmed deaths reported (WHO) 

UPDATE:  As of the 22nd of  November 2021, there are now 258,172,735 confirmed cases of  SARS-CoV-2   and  5,158,642  confirmed  deaths recorded (Johns Hopkins Coronavirus  Resource  Center)

Please note that SARS is an acronym for severe acute respiratory syndrome.  

The SARS-CoV-2 viral infection may cause extreme damage to the respiratory system but also affects other organs of the body.

Coronaviruses are single stranded RNA viruses.  They are enveloped viruses.  The RNA is enclosed inside a protective spherical coat of protein.  These relatively large virions (virus particles) and have around 74 mushroom shaped protrusions, the spikes.  This gives the virion a look that some say resembles a crown, which is the origin of the name coronavirus. Some of the glycoproteins are structural, such as the famous spike protein, and others are glycosylated non-structural proteins.  These envelope and spike glycoproteins play a role in the infection of cells by attaching to a host cell and acting as a doorway for endocytosis or the taking in of the now naked RNA by the receptors of the cell (the envelope does not enter the cell). Once inside the host cell, in the cytoplasm, the RNA can instruct the ribosomes to replicate more of the virus.

Remembering that GRFT is a lectin, with the ability to bind carbohydrates, we can now start to see how the antiviral action of GRFT is to attach to the glycoproteins, such as the spike protein and block the viral entry into the host cell. 

3 deadly coronaviruses, SARS, MERS, and Covid-19

 

SARS coronavirus first appeared in humans in 2002 with 8096 reported cases.

MERS (Middle East respiratory syndrome) had an outbreak in 2012 with 2260 reported cases.

Covid-19 first appeared in 2019

SARS and MERS show a higher percentage of fatalities than Covid-19 with MERS being the most deadly.

Covid-19 is more easily transmissible than SARS or MERS. 

SARS, MERS and Covid-19 were infecting animals, probably bats, at first and then zoonotic transmission occurred, the jump from animal to human.  Besides bats, with MERS, camels were a reservoir of the virus and humans were infected from camels (Please don’t touch a sick camel, or at least wash your hands after).

Once a coronavirus made the jump, human to human transmission spread it quickly. 

 

Is it probable that GRFT can prevent infection by coronaviruses?

We had been doing research related to Griffithsin for a few years and in March 2020 when the covid-19 pandemic was sweeping across the world, we searched through our library of papers and found one, published in the March 2010 issue of Journal of Virology:

Broad-Spectrum In Vitro Activity and In Vivo Efficacy of the Antiviral Protein Griffithsin against Emerging Viruses of the Family Coronaviridae. (O'Keefe BR, Giomarelli B, Barnard DL, Shenoy SR, Chan PK, McMahon JB, Palmer KE, Barnett BW, Meyerholz DK, Wohlford-Lenane CL, McCray PB Jr. Broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family Coronaviridae. J Virol. 2010 Mar;84(5):2511-21. doi: 10.1128/JVI.02322-09. Epub 2009 Dec 23. Erratum in: J Virol. 2010 May;84(10):5456. PMID: 20032190; PMCID: PMC2820936)

This excellent paper shows the results of many experiments.  4 strains of SARS were tested individually in vitro and griffithsin showed remarkable antiviral action, preventing the viral cytopathic cell death from the viral infection.  GRFT bound to the Coronavirus spike protein. Experiments were also successfully performed on other coronaviruses that don’t infect humans but are found in animals.   GRFT was also shown to be of very low toxicity to the host cells.

In vivo experiments were performed on mouse models using a mouse-adapted SARS virus.

Control groups of mice got nothing at all (sham) or only GRFT (control) but the important groups observed were the mice getting SARS virus alone and the group getting both SARS and doses of GRFT.  The griffithsin was applied into the tiny mouse nostrils, because that is a good place to administer it when the virus attacks the respiratory system.  Picture for a moment, scientists in lab coats pipetting into mouse nostrils!  This may have caused the mice some discomfort but it also saved them from dying from SARS.  The mouse group that got only the virus sickened, losing weight, and then 70% of the group died, while 30% recovered. 

The group of lab mice that got the SARS virus and also doses of GRFT up their tiny noses didn’t lose weight and had 100% survival. 

Important points made that stand out:

“Due to the proven threat from

SARS-CoV infections and the possibility of future zoonotic

transmission of coronaviruses, efforts have been initiated to

identify agents that could either reduce infection or suppress

the deleterious cytokine response to SARS-CoV infection”

 

“The broad range of Coronaviridae species sensitive to GRFT is a significant attribute for this antiviral protein, as this group of viruses appears to be capable of continuing zoonotic evolution and transfer to human hosts”

 

10 years later, a novel Coronavirus made the leap from animal to human hosts, there was an outbreak, and the world was unprepared to stop it. As we all know it became a raging global pandemic.

 

In 2016, an article was published in the journal Antiviral Research about in vitro studies done using GRFT to stop MERS from infecting cells:

 

Middle East respiratory syndrome coronavirus infection is inhibited by griffithsin.

Jean K. Millet, Karin Seron, Rachael N. Labitt, Adeline Danneels, Kenneth E. Palmer, Gary R. Whittaker, Jean Dubuisson, Sandrine Belouzard

 

Again, GRFT was shown to bind to the spike protein and prevent infection, this time by the MERS coronavirus.

 

Again, scientists urged more development of griffithsin:

“In conclusion, griffithsin has a low cytotoxicity, likely interacts with any coronavirus spike proteins because of their highly glycosylated nature and is able to hamper coronavirus spike protein functions. Griffithsin should be considered as an interesting drug candidate to develop for the treatment and/or prevention of current but also future emerging coronavirus infections.”

 

3 years later, in Wuhan, China, a novel coronavirus started infecting humans and as humans we were unprepared and defenseless.

 

In a 2020 letter to the editor of Virilogica Sinica, we find confirmation that the entry inhibiting antiviral GRFT also works on the SARS Cov-2

 

Griffithsin with A Broad-Spectrum Antiviral Activity by Binding Glycans in Viral Glycoprotein Exhibits Strong Synergistic Effect in Combination with A Pan-Coronavirus Fusion Inhibitor Targeting SARS-CoV-2 Spike S2 Subunit

Yanxing Cai, Wei Xu, Chenjian Gu, Xia Cai, Di Qu, Lu Lu, Youhua Xie & Shibo Jiang

 

One interesting development reported in this letter regards the pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike, EK1 working synergistically with GRFT. 

 

We could easily see from the research that has been done that GRFT has the potential to prevent a coronavirus outbreak from becoming a pandemic.  It may be too late to do anything about the present one that we are facing.  Vaccines have been developed, tested, produced and deployed in one year which is a huge accomplishment.   It’s not a matter of if but a matter of when the next coronavirus makes the leap from animal to human.  We were not prepared for the covid-19 onslaught.  A small but dedicated group of scientists and biohackers, decided to start to prepare for the next outbreak without delay. 

                                                   

To be continued….

Project GRFT: The story so far

In this and the posts to follow, we will introduce project GRFT, what this project is doing, and why it's important.  GRFT = Griffithsin.  The first part of our story starts here. 

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Seaweed

In the 1800’s a widow, Mrs. Amelia Griffiths (1768-1858), roamed the seacoasts of Devon, Cornwall and Dorset with her wicker basket in hand, passionately collecting and cataloging the marine algae commonly known as seaweed.  In honor of Mrs. Griffiths and her contributions to the science of phycology, the Swedish marine botanist Carl Adolph Agardh named a genus of red marine algae “Griffithsia”. 

Griffithsia setacea from Griffiths’ books. © 2014 Royal Albert Memorial Museum & Art Gallery, Exeter City Council

Griffithsia are eukaryotic organisms of the Rhodophylum (Rhodo, from the ancient Greek, Rhodon, Rose and phylum which in the taxonomy stack is below kingdom and above class). Griffithsia are found in the oceans of the world. They dance gracefully in Neptune’s realm off the Coast of New Zealand. 

Viruses

In the late 1800’s, tobacco plantations in the Ukraine and Bessarabia (now Moldova) were, like many other plantations worldwide, infected with a disease that caused spots on the leaves causing a mottled appearance or mosaic pattern.  This was called mosaic disease because the patterns sometimes resembled mosaics.  The disease could and did destroy entire tobacco crops.  Scientists in Russia and Europe started an inquiry into the cause of the disease.  Was it a mold or a bacteria?  Was it something else?  Conducting experiments, they found that the agent which infected the plants passed through filters with a small pore size that would have stopped bacterial cells.  It could not be seen with the microscopes in use at the time.  It would not grow on prepared media on which bacteria or fungi would thrive.  So this was something as yet undiscovered.  Dmitry I. Ivanovsky and Martinus W. Beijerinck started the groundwork for what would become Virology, the study of viruses. 

What is a virus?  First we should look at what a virus is not.  Animal, vegetable, or mineral it is not.  Fungus or bacteria it is not.  Viruses can't be considered cells or independently living organisms. Perhaps viruses are best imagined as being exquisite alien machines running a program to inject nucleotides into  living cells causing the cellular mechanism to produce more of the virus.  Viruses are made of nucleotides often dressed up in a protein coat or capsid. 

Not all viruses cause health problems.  Some we find useful for a wide variety of applications including Biomanufacturing by the expression of of recombinant protein in organisms.  Viruses considered pathogenic to humans only number about 100 - 200 varieties. These disease causing viruses, however, represent a major health concern and are a significant cause of mortality and suffering worldwide.

Prevention of viral outbreaks

 

Prevention of the spread of pathogenic viruses may be accomplished in several ways, for example: 

1.        Physical distancing, physical barriers.

These keep the virus particles, known as virions, away from cells by methods such as masking and wearing other PPE, also by air filtration.

 

2.       Vaccines

Vaccination stimulates the immune response with the body producing antibodies against the particular virus.   Vaccination usually only requires 1-3 doses of the vaccine and may provide immunization for a lifetime.

 

 

Antivirals

Antivirals prevent the virus from hijacking cells in the host and replicating.  The antiviral works typically by blocking entry into the cell by the virus.  Antivirals need to be taken whenever there is danger of the transmission of a virus.  Antivirals generally work against pathogenic viruses when administered before or at the first stages of infection.  

Antiviral lectins

A lectin may be defined as a carbohydrate binding protein. 

Lectins are found in everywhere in nature including in the food we eat. 

An example of an antiviral lectin is BanLec or banana lectin.  Found in the bananas Musa acuminata and Musa balbisiana, BanLec has been shown to be effective against HIV. 

Other examples of effective antiviral lectins found in nature are cyanovirin-N, scytovirin, and microvirin.   

Griffithsin

   Of all the known antiviral lectins found in nature, perhaps griffithsin, the red seaweed protein, is the shining star. Besides being free of significant toxicity, griffithsin is the most powerful of any of the lectins at inhibiting enveloped viruses from entering cells.  

Griffithsin, or GRFT is a 121 amino acid, 12.7 kDa protein.

Wild type GRFT:

Please take a look at position 31 above.   In wild GRFT, this is a non-standard amino acid and in recombinant GRFT this is generally replaced by substituting alanine (the red A).

The yellow arrows designate the secondary B-sheet structures of the protein.

GRFT is a dimer and has 3 triangular prismatic blades in the Beta sheets.  These separately have been shown to have some antiviral properties, however together in griffithsin’s domain swapped dimer they are more powerful, just as two hands can grab and hold with more strength than one hand.  This dimeric structure has six carbohydrate binding sites that work together.  

An interactive 3D model of the GRFT protein structure may be found here, scroll down to "Structure" and play with it.

 

Future episodes to follow!

work in progress

Thanh's gene gun rapid fire magazine design for our hardware working group.

 

Please help support the project:

(new link)

https://charity.gofundme.com/o/en/campaign/help-us-build-a-gene-gun