Oakland University Research Study: Evaluation of Finger-Tip Blood Testing for COVID19 Antibody (IgG & IgM).

Research Volunteers Needed

Evaluation of Finger-Tip Blood Testing for COVID19 Antibody (IgG & IgM).

I am Meron Tarekegn, a medical student at Oakland University William Beaumont School of Medicine, under the direction of Dr. Kenneth Mitton PhD, Associate Professor of Biomedical Sciences, Oakland University. While COVID-19 antibody status can be measured using clinical lab testing services, the current tests require drawing blood from veins in the arm and shipping of blood collection tubes to central labs.

We are conducting a research study to test the feasibility for using a fingertip blood sample to detect if a person has developed antibodies to the COVID-19 coronavirus. This test format will use an indicator system that gives a result in 15 minutes and could potentially be used for on-site testing and by mobile testing teams. To use such an on-site finger-tip blood test, we must first confirm that the test method can detect persons with antibodies to COVID-19, and that the test is reliable. This is a research study to evaluate the testing method itself and will not provide volunteers with any medical diagnosis.

We are recruiting individuals who are over the age of 18 to state whether they have received any COVID-19 coronavirus related testing and whether they have experienced symptoms and their onset. We especially desire to have subjects who:

1) have already been subjected to a COVID-19 antibody test by a clinical lab testing service and have either a positive or negative result.

OR

2) have had a confirmed diagnosis of COVID-19 and have since recovered from the illness and tested negative for the COVID-19 virus OR have been clinically cleared by medical staff (i.e. department of health and/or your primary care physician).

For the testing, volunteers would provide a small finger-tip blood sample. The finger stick procedure is very similar to the way a diabetic checks his or her own blood sugar. Your finger will be stuck once, using a sterile single-use finger-tip lancet (very small with a sharp point). The interviewer will collect drops of your blood from a finger stick, mix the blood sample in a solution to carry out the test, and then load the mixed sample on to the indicator test. The research will take approximately 5-15 minutes of your time.

Your participation in this study is voluntary. The fingertip testing will take place in the Eye Research Institute on the campus of Oakland University.

If you have any questions concerning the research study or wish to consider volunteering, please email our research team members, and mention “Serum Antibody Research” in your subject line or text, to:

igenomes@gmail.com

We will reply with an email to confirm your preferred method of communication (i.e. phone or email) so that we can confirm your eligibility for the research study, review how the test will be done, and how we can schedule your participation should you decide to volunteer.

Oakland University IRB #: 2020-92, Recruitment Script Approved Version 09/24/20

Welcome to 2020: DNA Sequencing for Children with Rare Inherited Retinal Diseases.

Meet Pearl. The new DNA-sequencer in PRRL (Pediatric Retinal Research Laboratory)

UPDATE September 2020: 

Just in time for the 4th of July, on July 3rd 2019, we installed and activated an Illumina iSeq-100 DNA sequencing system. Dr. Mitton, Wendy Dailey, Jennifer Felisky, Naomi Haque, Michael Sun, and Ed Guzman from the ERI all contributed to the setup and eventual ongoing use of this system for high-throughput DNA-sequencing to discover mutations in 8 genes involved in the following rare and inherited conditions:

FEVR (Familial Exudative VitreoRetinopathy), Norrie Disease, and Retinoschisis (RS).

This was done to establish the first DNA-sequencing research service at Oakland University applied to the discovery of genetic disease causing mutations. Our goal is to use these molecular investigations to advance our clinical understanding of these blinding diseases that impacting patients of Associated Retinal Consultants, (Royal Oak Michigan).

Unlike several health care systems in Europe and Canada, DNA sequencing costs are not covered by health insurances in the United States. So for Americans, the access to even DNA-sequencing of genes we already know may be involved in a patient’s disease is simply not available because of the prohibitive costs. As molecular biologists working in vision science, my lab group has developed and then tested a targeted DNA-sequencing panel to analyze many genes related to these rare conditions for only a few hundred dollars per sample. Several developments in technology over the last several years have now made this possible.

  • We use a strategy called “Ampliseq “which is based on PCR (Polymerase Chain Reaction). Using a computational (in silico) design process from Illumina, we designed a large number of PCR primer pairs to copy and amplify all the exon and flanking intronic DNA sequences from 8 genes of interest. These are genes that we either know or think can be involved in these rare pediatric retinal diseases.
  • Each PCR product is about 250 base pairs in length and the total sequence from these 8 genes is over 30,000 base pairs. The primer pairs are mixed in three different pooled combinations (three PCR reactions per patient).
  • Up to 16 patient DNA samples are processed at the same time by PCR amplification at one time. The PCR products are linked to Illumina end sequences, purified, QC tested to make what is called a sequencing library. These are stored frozen until about 40 to 50 samples are ready.
  • The sequencing libraries from 40-50 patients are mixed together and loaded into a processing cartridge that is then loaded into the iSeq100. The single use cartridge contains all the sequencing microfluidic chemistry reagents, and a sequencing flow cell is also plugged into the cartridge.
  • Using a fluorescent diod-array imaging system, 4.5-Million PCR library products, averaging 250 base pairs each in length, are sequenced simultaneously to generate well over 1.2 GigaBases of DNA sequence. The illumina primers contain a unique 4-basepair sequence for each patient’s sequencing library, so the iSeq100 software then sorts all the sequencing reads into separate folders for each patient using this “DNA barcode”.
  • Each Patient’s DNA sequence fragments are then aligned to a reference Human genome sequence and then any differences in the patient’s DNA sequence from the reference genome is reported to us.
  • We use special bioinformatics software in the cloud and some programming tools called “R” to sort these DNA sequence variants and discover changes that would change or impact the normal proteins encoded by the 8 genes of interest.

We have run four iSeq-100 cartridges as of Sept 2020, I am happy to report here that we have successfully sequenced over 90 persons from our Families who have FEVR, Norrie Disease and Retinoschisis. FEVR and Norrie DIsease results in an incomplete retinal vasculature, especially in the periphery of the retina, resulting in a lack of oxygen to the cells. Retinoschisis results in a delicate retina that can suffer layer-separation and retinal detachment due to the functional loss of the Retinoschisis protein, a protein forms an essential structural glue to hold retinal layers together. FEVR, Norrie Disease, and Retinoschisis can all have effects that lead to blindness.

The result of the above process is that we now have the ability to test 8 genes for about $250 per person, and complete the testing in one week starting from a few drops of blood. Previously, only 2 of these genes in a DNA sample could be sequenced for about $4,500, using different service laboratories, taking months. Not only will this make it possible to sequence more genes for more patients, we can also use this new genetic testing research system to provide advanced training to Oakland University’s undergraduate, graduate and medical school students.

While we are in a pandemic in 2020 we have not stopped work on analysis of our sequencing data for patients and we continue to get ready to sequence more persons with these orphan (rare) retinal conditions. Additional undergraduate students and medical students from Oakland University and MSU are getting their feet wet in genetic analysis over the 2020/2021 year, including Zaid Yahya, Tim Page Jr., Amanda Petrelli Cicerone, and Esi Benedict (MSU).

We also help to raise the funds for DNA sequencing so that no Family has to pay for any DNA sequencing we do. To that end we are doing our 2nd annual Walk for Vision 2020, virtual even, to raise funds for the Pediatric Retinal Research Foundation who have supported the start up of our new sequencing research service for these rare diseases. http://prrf.blueskysweet.com/

Repairing and Regeneration, Medicine’s Holy Grail and our Technology Transfer Project

Over the last two decades my natural tendency to pursue research projects closer and closer to future clinical impact has taken me from a very focused study of the mechanisms of regulating gene expression in the retina to understanding retinal diseases that cause most new cases of blindness in the United States each year. Those diseases involve damage to the integrity of the blood-retinal-barrier and even the loss of blood microvessels that nourish the neural retina. As you can see, poking around my website, we work on things like Vascular Endothelial Growth Factor and Wnt (“wint”) signaling to understand what keeps endothelial cells of the retinal blood vessels happy and then to understand how these normal system are perturbed to cause harm.

Along the way, I have worked with Dr. Kim Drenser MD PhD and Dr Michael Trese MD, of Associated Retinal Consultants (Michigan) and the Ophthalmology department in the Beaumont Health System. An atypical Wnt-protein called Norrin has emerged as a protein essential for development of a human baby’s neural retinal vasculature. Long before I came to this project area, real human Families with an inherited condition called Norrie Disease had Sons who were missing a full retinal vasculature, similar to very young premature infants who suffer the same physical problem: a lack of blood supply to nourish an oxygen starved retina. While it was a very rare inherited condition, the eventual discovery of the gene (NDP) and its protein (Norrin) made it clear that this protein is essential to form the initial blood vessels of the neural retina.

Research we have done in animal models, and research in other laboratories, have also revealed that Norrin is also required to establish a strong blood-retinal-barrier which is formed by the endothelial cells that line the interior of blood vessels. But, how can we possibly use this information for a future therapeutic tool? One way would be to make a protein based on human Norrin in large enough and pure enough amounts to be used for both more testing in models and hopefully clinical trial use in human subjects one day. The first step is to come up with a biotechnological solution to make the protein and purify it and to fold it in its required natural 3-D shape so that it has the same activity as Norrin protein made naturally in the retina.

The later project is what I have contributed, using my knowledge and experience designing and producing proteins in bacteria (E. Coli strains used for protein production), to the production of a Norrin activity protein. After demonstrating some preliminary data on this process, I wrote a research plan submitted with a local small business firm Retinal Solutions LLC (Michigan) to obtain NIH STTR (Small business technology transfer) funding to initially iron out the process to make the protein pure, and free of toxic impurities, suitable for testing in the mammalian eye. These begin with a Phase-1 STTR, if one is successful at obtaining the funding. STTR grants require a company, like Retinal Solutions LLC, to work with a non-profit academic partner (that would be me and Oakland University) to develop a technology with the goal of benefiting US citizens with future medical treatments, drugs, and devices.

I am pleased to note that as the Primary Investigator on the project, we obtained funding for the one year Phase-1 STTR from the National Eye Institute (NIH), and we started this developmental research this Fall 2019. In this project, the basic scientist like me, works with my clinical colleagues at Retinal Solutions LLC (Dr. Trese, Dr. Drenser) to identify a clinical need and bring science to bare on the problem. While there are no guarantees of success in any drug development, clear scientific evidence for the genetic necessity of Norrin to make retinal blood vessels simply makes Norrin an excellent candidate as a therapeutic tool, a factor, that would be needed to repair and regenerate the retinal microvasculature and its strong blood-retinal-barrier. Even if Norrin is not the solution on its own, it would likely be one of the essential members required for a therapeutic team. As a protein that is already natural in the neural retina, there is also a much reduced likelihood that there would be any toxicity or ill effects. One this is for certain, just thinking about this as a dream and not trying to see if the dream can come true is not something we wish to do.

So wish us lots of luck as we make Norrin in bacteria, refold it, and put it through our testing in the Eye Research Laboratory and the protein purity characteristics testing by Retinal Solutions LLC. If all goes to plan, then the next future step will be the Phase-II STTR to get this Protein therapeutic closer to future use in human clinical trials. That is the dream, one step at a time.

Ken Mitton

On doing Science well.

As for many biomedical, basic-science research labs, my research flows and changes over time as we make new discoveries that lead us to new questions we form even as we uncover the answers to previous questions. That is the nature of basic science, and it is the way science investigation has always brought the most benefits to people and medicine in particular. While many organizations and countries have attempted to focus research support (funding) into specific diseases, it turns out that the overwhelming majority of high-impact medical discoveries have come from “serendipity”. That is, great useful ideas and tools were discovered to treat diseases simply by exploring how things work.

For example, drugs for controlling high cholesterol were not discovered by deciding to start making drugs for treating high cholesterol. In the course of biochemists investigating how our cells make cholesterol in the first place, chemicals were used to block enzymes to help figure out how cholesterol was made. Some of these chemicals were obviously the idea to become new drugs that could block cholesterol made in the body. Latanoprost, one of the later generation of drugs developed in the ’80s for reducing high intraocular pressure (IOP), was based on the discovery that prostaglandins made by some cells in the eye could increase the aqueous outflow in the eye, and reduce pressure. The basic science was elucidated in animal models. Again, a basic science discovery in the laboratory of physiologist Laszlo Bito at Columbia University was adopted by a Pharma company as the way to make drugs that mimic natural prostaglandins to produce this new class of drugs. As a result, thousands of people around the world have another class of drugs to reduce their intraocular pressure and reduce their risk of vision loss from Glaucoma.

So, you never really know where benefits will arise for biomedicine. That is why many research funding agencies, such as the NIH (USA) and the MRC (UK), understand the importance of funding physiologists and biochemists to explore how things work. In our case, how things work in the eye, and the retina of the eye.

Sincerely,

Ken Mitton

Lab News: Summer’s Start 2019

Current Lab Member and Lab Alumni News: May 2019

Dr Ken Mitton and Wendy Dailey attended ARVO 2019 (May) and presented research on VEGFA isoform regulation of primary human retinal endothelial cells. Also, on Norrin regulation of PLVAP expression in the same cell type. Dr. Mitton co-presented a third abstract with Tom Dzialozsynski, Western University (London Ontario) regarding Gensingoside effects on normalization of serum lipids and slowing of cataract formation in diabetic rats. John Trevithick, PhD, Professor of Biochemistry and Kinesiology (Western University), Tom and Ken’s former supervisor and long time friend and colleague was also on the abstract. Dr Trevithick passed away just over a year ago. The 2019 ARVO meeting was the first one ever outside the United States. Vancouver BC. We wanted to make sure that John Trevithick’s research was presented at this first ever ARVO meeting held in Canada. (Dr. Mitton obtained his PhD under John Trevithick at Western University, London Ontario). Without John Trevithick’s influence, the many many Michiganders trained in biosciences in our lab since 2001 would not have had their opportunities here. Life is connections, and at times a series of wonderful accidents. Peter Chen MD, graduated OUWB school of medicine. The second OUWB medical EMBARK program student in the ERI. Michael Sun has joined our group as the 5th EMBARK program student in the ERI, just completing his M1 year in our OUWB program. Michael will be working with us to help establish patient DNA sequencing for rare inherited retinal vascular diseases in our Pediatric Retinal Research Laboratory.

The 2019 Summer Undergraduate Program in Eye Research has begun, with six Oakland University Undergraduate science students learning about, and contributing to, the NIH and PRRF funded research projects in our group. You can learn more about this program at:

http://www.oakland.edu/ERI

John R. Trevithick, PhD, A Canadian Vision Research Pioneer Leaves Us at Age 82.

It is my sad task to note the passing of John Trevithick, PhD, at age 82, at his home in London Ontario. (2/20/2018)

John is well known to many ARVO (Association for Research in Vision and Ophthalmology) and ISER (International Society for Eye Research) members, since the early 70’s as one of the first researchers who took up the lens as a model for cell differentiation. A pioneer in the explorations of cAMP in N. Crassa, he sought to explore this signaling molecule in mammalian cell differentiation. The lens epithelial to fiber cell transition continues during the entire life of the mammalian lens.

            Along the way John explored cataract mechanisms of course but as a biochemist he always was interested in the fundamental knowledge of cell physiology and biochemistry that could be revealed. His lab was one of the first groups to optimize culture media conditions to permit the study of whole living lenses in tissue culture. This permitted the precise manipulation of external and internal chemistry and was used to correlate research done with diabetic rat models and the RCS model. Interesting effects on the RCS lens posterior opacity caught his attention in this model, where degeneration was occurring in the retina. Along with diabetic cataract models his group explored the contribution of antioxidant biochemistry to degenerative processes in the lens and retina. Some surprising findings, of dramatically slowing cataract formation and other degenerations by supplementing endogenous antioxidants resulted in a couple Nature publications. I joined his lab in 1988 as a PhD student, at the University of Western Ontario.

            John took me, and other students, to many meetings around the world and he spent a lot of care teaching us to repeat work and make sure we published reproducible data. He let us write papers early and we tended to graduate with a few publications already completed. With John, I enjoyed discovering that the diabetic rat lens was iso-osmolar for several weeks by dumping Taurine and free amino acids to perfectly balance the increase in sorbitol. Then this amino acid deficiency led to the loss of GSH, ATP production, ion transport capacity, and finally the loss of fluid regulation. This was a lesson John taught many of us, that if you delve into the biochemistry of even an old model system, you will likely find some new knowledge. Do not assume that a decade of literature on a topic has documented everything important.

            John was Emeritus professor of Biochemistry and had continued also as a professor in Kinesiology, working to explore the effects of exercise on the eye in normal and diabetic rats, working with Tom Dzialoszynski and Earl Nobel. Most recently they spent the last several years on space radiation effects upon the lens. That work, funded by the Canadian Space Agency, involves trips to UBC and the use of their busy cyclotron facility. John, always curious to the very end and finding ways to fund vision research in Canada, which does not have a vision-focused funding category as we have in the USA.

If any members of the vision research community have a memory of John Trevithick they would like to share, please email to me at 

Mitton@oakland.edu

Tom and I are planning to write a short memorial paper about John and his contributions to vision science in Canada and internationally. Many of us owe our thanks to John for the time he devoted to ARVO, ISER, vision science and teaching the next generation of scientists.

A mostly complete list of John Trevithick’s publications in PubMed are found here:

John Trevithick Papers in PubMed

On the pathway to having to teach your own kids: Teacher Shortages in all States as Americans treat teachers so bad.

From twitter @kpmitton:

 #Education in #TrumpVos era. 

Americans treat teachers so bad, soon they will have to teach their own kids! 
#Teachershortage in 50 states. 

Some states are lowering certification requirements so you do not even need a college degree.

#OKLAHOMA’s TEACHER OF THE YEAR leaves state! 

Read more about Student Suicide Investigation; and DeVos Proposes New Student Loan Rules  to make it harder for students to get money back when defrauded by for profit colleges.

  Http://n.pr/2ETJ0Fr

Being A Girl: A Brief Personal History of Violence

A very good read. A very important read.

The Belle Jar

1.

I am six. My babysitter’s son, who is five but a whole head taller than me, likes to show me his penis. He does it when his mother isn’t looking. One time when I tell him not to, he holds me down and puts penis on my arm. I bite his shoulder, hard. He starts crying, pulls up his pants and runs upstairs to tell his mother that I bit him. I’m too embarrassed to tell anyone about the penis part, so they all just think I bit him for no reason.

I get in trouble first at the babysitter’s house, then later at home.

The next time the babysitter’s son tries to show me his penis, I don’t fight back because I don’t want to get in trouble.

One day I tell the babysitter what her son does, she tells me that he’s just a little boy, he doesn’t know…

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