Giving Tuesday - Your donation is most welcome

 Today is Giving Tuesday.  We're making a request to anyone who supports what we are doing (please see the entries in this blog) towards global health and well being.  Please donate to Project GRFT.  We have an increasing need for funding as we move forward with our work.  Any donation large or small is welcome.  As part of Counter Culture Labs we have 501c non-profit status so if you pay taxes in the US, your donation is deductible.   

Here's how to donate.  Go to https://www.counterculturelabs.org/ and scroll down to the donate button.  There is also Venmo on the page.  Please specify that the donation is for Project GRFT.  Then it will be directed to the project, rather than the general CCL fund.  

You can also identify your donation by emailing nenufarmoleculesforlife@gmail.com with your name and the date of your donation.  We'll forward this to our treasurer who will make sure that your donation is channeled to Project GRFT.

Thanks from the Project GRFT crew!

Project GRFT gene gun at Counter Culture labs

 

At Counter Culture labs, we have set up our first gene gun.  It was designed and built by physicist and inventor David Johnson PhD.  In the video he fires the first shot from the gene gun in the new lab environment.  DNA plasmid coated Au microparticles are shot into a rice callus which will then be incubated at 25C.  Compressed Helium gas provides the force to propel the particles into the plant cells.  

project GRFT at CCL

 Project GRFT, after relocating and setting up our gene gun inside CCL is back to shooting DNA coated microparticles into rice calluses.

Two of our volunteers, Kat and Patrick, in the hood and washing the Au microparticles with ethanol.

They are using molecular biology grade ethanol and we do 3 washes

after adding the ethanol -

time to vortex!

2018 article on GRFT/Rice

https://share.america.gov/how-rice-could-ward-off-hiv-infections/

Rice plate

 

Today at Counter Culture Labs

Kat, Patrick and Grier check the plates in the incubator

Every organism has a food they like.  We feed the rice calluses by plating the embryos on Callus Induction Media (CIM) 

They are masses of cells and with care will eventually grow into rice plants 

As they grow we break them apart.

We re-plate the pieces and the pieces grow. 

Callus growth is slow and we are working on monitoring how much growth takes place over time.  

Every organism thrives in a certain temperature range.

The incubator is set at 25C for the calluses.

The calluses on CIM are kept in the dark.  

At later stages different media is used and light is introduced so that photosynthesis can occur and then roots and shoots form.

Another confirmation of the power of GRFT

 "Quite possibly, the most encouraging inhibitor of MERS-CoV is Griffithsin, a 12.7 kDa lectin found in the Griffithsia species (red-green growth). It has three sugar restricting areas that permit it to tie accurately to glycans on CoV protein spikes and forestall viral connection to have cells, with extraordinary strength exhibited in vitro concentrates against MERS-CoV (EC50 of 0.125 M) and various CoV strains (EC50 of 0.00320.33 M) (Millet et al., 2016). Griffithsin additionally seems to have a low foundational poisonousness, with an explicitness list of 303100 against HcoV cells (contrasted with human colorectal adenocarcinoma or fibroblast cell lines) (O’Keefe et al., 2010), demonstrating that it very well may be one of the leading contenders for the animal and clinical preliminaries against SARS-CoV-2 (Mani et al., 2020)."

excerpt from:

In silico investigation and potential therapeutic approaches of natural products for COVID-19: Computer-aided drug design perspective

https://www.frontiersin.org/articles/10.3389/fcimb.2022.929430/full

DNA plasmid under construction

Project GRFT update.  Sreenivas Eadara and the Project GRFT DNA crew in Baltimore are making good progress with the plasmid construct assembly.  

Beautiful bands:

2022 project GRFT ongoing labwork Updates

 

After a rather disastrous Winter with flooding and other problems at CCL, and the closure of one of our other Bay Area labs due to financial limitations, we are recovering. Spring has been a time of growth and progress for project GRFT.  Micropropagation of rice by Hector Vera on agar has got the early problems with contamination under control and cloning is progressing nicely using a progression of media.  We now have plates with happily growing rice calluses that we are continually preparing for transformation by particle bombardment.  Hector is finishing up a lab complete with a gene gun.  UCB students Allison Nakagawara and Maya Douglas joined the project and have been working on preparation of media and buffers, monitoring callus growth and cloning the calluses.  David Alfred Johnson, PhD and David Finn have teamed up to work on the nozzle end of the gene gun. 3d printed parts had ragged edges and were not able to withstand the temperatures in the autoclave.  David Johnson has built two gene guns, one is at CCL and Hector has the other one.  David Finn has located a high-temperature printer to print the parts with clean edges and able to withstand the heat of autoclaving.  In earlier experiments the blast from the gun blew the target plant material completely off the plate.  The solution being tried now is to place the target between to disks of stainless steel fine mesh inserted into the nozzle.  Sreenivas Eadara and the team at Johns Hopkins are working on assembly of the plasmid construct under the guidance of Noam Prywes, PhD (who is doing a research project at UCB to explore rubisco biochemistry and chloroplast transformation technology in plants). 

Photo taken on the 15^th of July 2022 at Counter Culture Labs in Oakland, CA.

From left to right: Allison Nakagawara, Maya Douglas, David Finn, Eddy Spinner, David Johnson, and Hector Vera.

New Clinical Trials of Griffithsin pharmaceutical designed against infection by coronaviruses

This is huge.  

Dr. Kenneth Palmer's group have been working hard to get a Griffithsin based pharmaceutical into clinical trials for some time and now a phase 1b trial is beginning. 

The pharmaceutical is designed to prevent entry into the cells by coronaviruses.

https://clinicaltrials.gov/ct2/show/NCT05437029

https://discoveria.com/studies/NCT05122260

The Pharmaceutical is in the form of a nasal spray which is an effective route to block the coronaviruses that gain access through the respiratory system.

Phase one trials are the first of four phases of clinical trials with human volunteers (They are now accepting volunteers for this study) designed to test the pharmaceutical on healthy individuals to see if any unwanted detrimental effects occur from the drug.  

If and when Q-Griffithsin is approved by the FDA, the method of biomanufacturing will likely involve large scale expression in plants, in this case nicotiana (tobacco).  

Congratulations, Dr. Palmer, on getting clinical trials underway!

Free STEM supplies for educators

 Our friends at Bio-link Depot in Oakland, California are having an event 1 May '22

Registration is required please follow this link:

https://mailchi.mp/dc53af3df94b/update-your-contact-info-save-the-date-for-open-house-8658925?e=d718c48211

https://www.eventbrite.com/e/bio-link-depot-open-house-may-1-tickets-320360004397

First phase 1 clinical study of GRFT for prevention of HIV

 Researchers at Albert Einstein College of Medicine completed phase one clinical study of a griffithsin topical.  This is designed to be applied as a gel to the vagina before sex to inhibit HIV infection.  Phase one trials are done are to see what effect the pharmaceutical has on healthy humans.   The results of this study are very promising,  This is a huge step forward, and we look forward to the next phases of clinical trials.

Here's a link to the paper:

 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0261775 

Meanwhile our group is looking into what the optimal pH of a buffer for adhesion of DNA to W and AU microparticles would be.   We would like to hear from anyone who has done particle bombardment on the subject of what buffers worked best.

important considerations when preparing projectiles

 Basically when coating particles we need to pay careful attention to:

1. pH: This is most important!
2. Timing of incubation
3. Temperature of incubation
4. Salt concentration
5. Size of particles
6. Freshness of Spermidine (0.1M)
7. 2.5 M CaCl2 is filter sterilized and aliquots prepared ready to use.
8. Purity and concentration of DNA
9. Keeping particles suspended in the buffer solution while adding the spermidine and CaCL2
10. We have seen problems with particles sticking to the 1.5 Ml centrifuge tubes. DNA Lo-Bind tubes should work better.