Me and My Money…a child’s story of diabetes

M&M Cover 300 small

Copyrighted in 2012, Me & My Money…a child’s story of diabetes, took two years to write…

From birthing thoughts, jotting ideas, typing a manuscript, carrying a daily journal, contacting an illustrator,  building the manuscript, proofreading and editing again and again and again and…you get the idea…this dream came to fruition.  It had do be done.  There is no other book like it — as far as my research goes — that reflects, mimics, or encourages diabetic children, T1Ds.  The book is now available to the general public in paperback form.  Celebrate!!

Submitting to a marketable world with a targeted audience, networking, promoting, speaking, through the main character, Kali with her dog, Money, brings attention to the increase of diabetes in animals – up 300% this past decade!

Through the wonderful insightfulness of illustrator Amy Pichly-Meyer who is responsible for the clarity of expression with the cover design, along with all 37 colored  illustrations, this book was built for you.  I am thankful for her friendship and abilities.

Briefly, the main character, Kali, is presently a fourth grader.  She was diagnosed with diabetes at the age of two.  Her rapid growth with blooming and wizened independence results from learning how to take care of herself.  Wayne, a best friend, knows her very well and together they embark on adventures through Wayne’s hobby, their separate and shared responsibilities, mutual friends, and more.  But Kali cannot forget her special pack!  She must carry this with her everyday, everywhere.

This book, along with the recent publication of Me & My Money Too…a child’s story with diabetes, are tools to understanding this disease.  Minute-by-minute self-care is introduced and forthcoming in this illustrated, easy-to-read chapter book, 104 pages long, appropriate for 8 years olds and available at in paperback edition as well as Kindle e-readers.   Local bookstores carry copies as well as local libraries with requests.  Please enjoy the following Chapter excerpts…

Chapter 1, Book One

Due to keeping up with my chores, I occasionally have some change in my pocket.  That’s because my parents give me a weekly allowance that I can spend when I want.

“Make sure you spend wisely,’ Dad says.  I’m not quite sure what “spend wisely” means, but I think I do okay with what I have.  For instance, one of my friends from school, Wayne, asked me to go to the hobby store one Saturday afternoon.  Because it is school summer vacation time, we could go almost any time, depending on our parents.  However, Saturday’s are best.

Wayne and I go way back, to kindergarten.  He is the only red-head kid in our whole grammar school, never mind our fourth grade.  He’s a full blown carrot top with thick hair in wavy curls.  This makes him real easy to spot in a crowd.  The girls in our class like Wayne more than the boys do.  I think this is because he has good manners and is polite.  It is either that or his clear sky blue eyes that sparkle.  Even his eyelashes are orange, matching his hair.  Most girls I know like blue eyes.  I never thought about it much.  My eyes are dark brown to match my light brown and blonde streaky hair.

Wayne is a pale looking kid.  Most of the time, the only color on Wayne’s face is because of his tannish-brown colored freckles.  I swear, if he didn’t have freckles, he would be white as a ghost!  I tease him about that, pretending that I can’t see him sometimes.  We laugh.  He blushes all red and pink.  It’s funny to see.  Wayne is shorter than I am — for now.  He teases me about my long brown hair — so flat, so straight and thin.  “There’s nothing to your hair,” he tells me as he quickly flicks it in the air.  “You don’t even have to brush it.  Your hair just hangs there off your fat head,” he often tells me as if I need to be reminded. 

I think Wayne is jealous of my straight hair.  Brushing his hair is a struggle because it is very thick.  Mine is a lot easier because it is stick straight.  He hates when his mother brushes his hair because “she always pulls it out of my head,” he told me one time.  “And you have a chubby little pug-nose.  Not like my pointy one at all,” he compares.  “But you get a tan in the summer.  I don’t tan, I burn.  It’s awful,” he explains.  That is true.

One summer that I remember, his mother covered his face and arms with white suntan lotion so  he wouldn’t get burned while playing with me in my back yard.  He looked so funny.  That stuff made him smell like the beach.  I laughed at him.  He didn’t like that.  And after a while, the suntan lotion disappeared.  But we stayed friends.  He wears that stuff all the time.  Sure, I use it too, but when I forget to rub it on my skin, I don’t get too worried.

“I guess you look like your father,” I told him because I think he does.  “I look like my mother,” I said.  And that’s okay with me because my mother is beautiful.  My Dad’s okay looking — for a Dad.  Sometimes he has a moustache.  Mom told him to shave it off because it made her sneeze when they kiss.  Eeeuu!  Sneezing and kissing, yuck!

“Kali,” my mom called up the stairs.  “Wayne is on the phone for you.”

“Okay.  Thanks.  I’ll get it up here,” I told her.

“Hi Wayne.  Wassup?”

“Hey.  Can you come to the train store with me today or what?  I’m looking for a certain model train engine and I think the Viking Hobby Store will have it.”

Well, I have no particular interest in model trains, so I knew I wouldn’t buy anything but I would go along with him anyway.  This is a wise decision.  After all, he is my very best friend.  Very best friends do things for each other whether we need to or like to or not.  It was Saturday morning anyway.  Cleaning my room could wait until I got back.  I was thinking, planning.

“Mom, Dad, is it okay with you if I go to the hoppy store with Wayne today?  He thinks he found the perfect train engine for his set.  He wants me to go and help him check it out.”

“I don’t mind.  Is your room picked up?” Mom asked.

“Not all the way yet, Mom,” I replied.  “I can finish when I get back.  All I have to do is fold my clothes and put them away.  A load of my socks and some t-shirts are still in the dryer.”

“Well, all right.  Did you ask your father?” Mom wanted to know.  “And don’t forget to take a snack and some juice boxes with you.”

“I know.  I already have a pack of peanut butter nabs in my sack with some water.  I don’t know where Dad is.  I thought he was in the kitchen with you.  Maybe he’s in the garage.”

“Okay,” continued my mother.  “If you don’t see your father on your way out, I’ll tell him.  If you do, please remind him that we’re going shopping today.  Oh, are you and Wayne taking your bikes or walking?”

“I think we’ll walk.”  And out the back door I went.

Chapter 2

Wayne lives four houses away from me.  I took the short-cut over to his house through a wildflower field.  I often come here to pick my mother some flowers.  I like to pick flowers for her.  There are all kinds of wildflowers in this field: yellow ones, pink ones, purple ones with yellow centers.  I don’t know the names of these flowers, I just know they are pretty and Mom likes them.

After I met Wayne on the front porch of his house, we proceeded to walk to this particular ‘train store’ as he calls it.  It is really a hobby store that has lots of other things of interest besides trains.  I’ve been in there with one of my older cousins.  She was looking for some particular, special types of paint brushes made with camel’s hair.  I thought that was cool — paint brushes made out of a camel’s hair!  There are small glider planes in this store with hand-held motor devices; there are different kinds of wood burners and wood carving tools; there are even lots of different colored rubber fish bait things — lures.  I liked those because they are shiny, squishy, and they squiggle.  Things that would attract a fish — go figure!  Wayne had been going on about a certain model train engine he saw in here a while ago.  It took him weeks and weeks to save up his allowance money to buy it.

“Hey, before we walk too far, is your insulin pump filled up?” Wayne asked.  “I hope it doesn’t beep like crazy while we are out.  I would hate to have to leave early so you could fix it.”

“Yes, it’s fine.  I filled it up with insulin yesterday morning and changed the needle site.  We are good to go!”  I planned this on purpose.  I knew he would ask.  I am diabetic.  Wayne knows.  Among many other things, I do take care of myself and my diabetes.  I have to test my blood sugar levels many times each day.  Diabetes is a disease that causes me not to digest food properly.  Because of that, I wear an insulin pump.  I used to have to take insulin shots before I got the pump.  Having the pump is much easier.  An insulin pump squirts insulin into my body, automatically, a tiny bit at a time.  A “squirt” is maybe the size of a pencil tip or the size of a period at the end of a sentence.  My parents had to give me insulin shots before I learned to give them myself.  I still keep syringes handy, in case the pump breaks down for whatever reason.  This is called having “Plan B” which is important. 

Read more of Kali’s young life with diabetes in “Me & My Money…a child’s story of diabetes.”  Available at

Pray, Pray, Pray

“Why Catholics Say the Rosary”

“The Rosary as we know it was developed in the Middle Ages.  It evolved out of an ancient practice in which people who could not read Scripture recited 150 Our Fathers for the 150 Psalms.  Strings of beads, called ‘pater-nosters’ were used to keep track of the prayers.

“Over time, this practice merged with the Scriptures greeting to Mary (‘Hail Mary, full of grace…’) and with meditations on the lives of Christ and Mary.

“In groups or in a family, one person can say the first half of each prayer, while others respond with the second half.

“The Rosary is, at its heart, a biblical prayer, which is why Scripture verses are often used for meditation with each mystery.”

“How to Pray the Rosary”

  1. “Begin with the Sign of the Cross.  Pray the Apostles’ Creed while holding the crucifix (part of a set of Rosary beads.)
  2. Pray an Our Father.
  3. Pray three Hail Marys.
  4. Pray the Glory Be, announce the first mystery, than pray an Our Father.
  5. Pray ten Hail Marys (called a decade).
  6. Pray the Glory Be and the Fatima Prayer.
  7. Before each decade, announce the next mystery, followed by an Our Father.  For each set of ten Hail Marys (or beads on the Rosary string), pray ten Hail Marys while meditating on the mystery.
  8. After the five decades are completed, pray the Hail, Holy Queen and the concluding Rosary Prayer.”

In these 21st century days of strife and hardship, of neglect and forgotten promises, prayers are consoling to the human mind and soul.

When you need to know more, please let me know by leaving a comment.

Thank you…A. K. Buckroth

You’re Invited….

Come Walk With Me!  October 20th and/or October 7th.

Do you like to walk?  Would you like to walk with me and others, many of whom you know?  Many with whom you’ll become fascinated?  Acquainted?  Well, everybody’s got a story, right?

The following are Upcoming, 2018, Community Events, Family Friendly with a Wonderful Cause! (google this link for all the informational details) has all the details you need to get involved.  As I say, “Four miles ain’t nothin’.”  Join me.  Be a part, a peice.


Know a diabetic? Love a diabetic? care for a Diabetic?    October 7, 2018     AND/OR  October 20, 2018

Through “Andrea’s Angels,” the Juvenile Diabetes Research Foundation is making fundraising climes toward a cure for diabetes.  Thorough details can be found in the links above.  “What’s a cuppla more miles?”  lol.


Andrea K. Roth (aka: A. K. Buckroth)

Traveling with Diabetes: Tips from JDRF (Juvenile Diabetes Research Foundation)



Summer Travel Checklist

Planning a trip before summer ends?
Double check our packing list to make sure you have everything you need to manage your type 1 diabetes (T1D) wherever you go!

A travel letter from your doctor with written approval to travel with insulin and syringes
Insulin pens
Test strips
Ketone test strips
Back-up snacks for quick carbs
Emergency glucagon
Equipment including blood glucose meters, CGMs and insulin pumps

You’ll find this packing list and more helpful ways to make the most of your summer—while keeping T1D in check—in JDRF’s Summer Guide.



This blog regards the down side of complaining by award-winning author and publisher, Steve Liddick.  I you are unfamiliar with Steve’s works, they can be found here:

I specifically posted this in mind of all the T1Ds around the world.  You have been heard.  This “reflection” is proof.  Now, where was I?  Oh….



by Steve Liddick

“You know how people say, “I don’t like to complain, but . . .?” Well, I don’t like to complain, Not just because it doesn’t do a bit of good to moan about this and that. No, I try to avoid complaining around other people because they don’t want to hear it. They have their own problems and don’t need to take on mine.

“You want to kill a conversation, just start complaining about something—anything—and watch as people drift away from you. The more often a discouraging word is heard, the more cloudy the skies are all day.

“Complaining takes many forms. Say you’re at a four-way stop intersection and some clodhopper pulls out in front you when it’s not his turn. You lay on your horn, make all kinds of specialty hand gestures, and scream nasty bits about the offender’s maternal parentage.

“Who do the other two drivers at the intersection get upset with? Not the aforementioned clodhopper who cut you off. They stare white-hot daggers at the guy making the fuss. Nobody likes a complainer?

“When people greet you with “Hi, how you doin’?” you are expected to say, “Great, how’re you?” Start unloading woes on them and watch as their eyes glaze over and they remember meetings they’re late for.

“I am here to tell you that, while nobody wants to hear your beefs, it is injurious to one’s health to keep it all bottled up. Something has got to give. An outlet must be found for the steam that is building, threatening to explode your head.

“Mental health professionals are of no use at all. In fact, when they say, “and how do you feel about that?” you know they’re not really listening and that just ticks me off that much more.

I can’t even get it out of my system by yelling at my wife. She knows where the frying pans are and I have to sleep sometime.”

Diabetes Research “Is Complicated”


T1D Science Explained: Beta Cells

We’re excited to share with you the exciting research and progress being made on beta cell replacement therapies and what that means in our pursuit of a cure for type 1 diabetes (T1D).

In our pancreas, we find beta cells: a type of cell located in the pancreatic islets that produce insulin. Unfortunately, in people with T1D, these cells are attacked and often destroyed by the body’s own immune system.

Scientists do not know why exactly this misguided attack happens, but recent studies are uncovering promising findings: that beta cells may become stressed before the onset of T1D and potentially trigger it and that we are born with a finite number of beta cells that die off in those with T1D. Some researchers are working on finding the cause of the immune attacks while others are focused on how to protect and replace beta cells. Restoring beta cell function could mean insulin independence—in essence, a cure. Below is a summary of how far your support has allowed us to progress in beta cell replacement therapies.

Islet Transplantation

Previous islet cell transplantations have showed a lot of benefits for those with T1D. Clinical trial patients have seen improvements in glycemic control and some islet recipients remained independent from insulin for up to five years. While these transplants are not possible for widespread use—there are a limited number of available transplants and a need for strong immunosuppressive drugs to protect the donor cells from the continued immune attacks—this research was a big step forward in T1D research and brought us closer to the next step: encapsulation.


Encapsulation aims to take the benefits of islet transplantation while eliminating the need for immunosuppressive drugs. By adding a protective barrier to beta cells before they are implanted, we can shield them from immune system attacks while still allowing insulin and glucose to flow through the cells and the pancreas. Researchers are getting close. A recent partnership between Eli Lilly and Sigilon Therapeutics—based on JDRF-funded work—is testing a cell encapsulation strategy that would make beta cells essentially unrecognizable to the immune system and free to function as they are meant to, while Novo Nordisk has recently announced its commitment to bring beta cell replacement closer to clinical trials.

Gene Modification

Other studies are exploring how to protect cells from the immune system attack from another angle. JDRF-funded research is seeking the answer to a vital question: How can the beta cell protect itself from the immune attack, without the need for a capsule or coating to shield it? Based on exciting progress made in genome editing technologies, T1D researchers are looking to edit the DNA sequences in the beta cell responsible for attracting immune cells. This technology could eliminate, or lessen, the need for encapsulation altogether by altering beta cells to alleviate the stress that triggers the attack and to protect themselves from autoimmune attacks.


Beta cell replacement therapies have incredible potential to reduce and even eliminate the dependence of those with T1D on insulin. The research is complex, but we want to make sure you’re kept in the loop as the research unfolds because we know it will have tremendous impact for everyone connected to T1D.


Find out more.
Support T1D research

Viruses May Cause T1D and Other Autoimmune Diseases

Posted in Diabetes Research News

“Viruses are the cause of many health conditions and affect the body in different ways. As scientists learn more about these viruses, they can develop targeted strategies for preventing and treating them. A major breakthrough was recently discovered involving a very common virus known as the Epstein-Barr Virus or EBV.

“EBV is most commonly known for causing mononucleosis (mono) or the “kissing disease” since it is often transmitted via saliva. By age 20, more than 90 percent of the population in developed countries will be infected by the disease. This rate spikes in under-developed countries with more than 90 percent of the population being affected by age 2. There is no cure for the virus – it remains in the body for life, though may not have a noticeable impact.

“However, researchers have found that the effect it can have at a cellular level may be more significant than previously realized. Scientists from the Cincinnati Children’s Hospital’s Center for Autoimmune Genomics and Etiology have published a study potentially linking EBV to seven diseases, including T1D. One of the Diabetes Research Connection’s own Scientific Review Committee members, Matthias Von Herrath, was an author on an article cited by the study in its research.

“Typically, the body responds to viruses by increasing the production of antibodies by B cells. These antibodies then attack and destroy the virus. However, with EBV, the virus actually takes over the B cells and re-programs them using transcription factors. This alters the way that B cells respond and can change their basic function, which may increase the risk of developing other diseases. The scientists have narrowed it down to one factor in particular – the EBNA2 protein.

“Transcription factors associated with this protein attach to and change sections of person’s genetic code. Depending on where they attach, it could contribute to different diseases including T1D, lupus, multiple sclerosis, rheumatoid arthritis, celiac disease, and more. Identifying what is happening on a cellular level could help researchers to develop more targeted treatment options and potential cures for these diseases. The study also opens doors for more in-depth research regarding how transcription factors may affect other gene variants and diseases.

“These findings are very encouraging in better understanding some of the underlying factors that may contribute to T1D. More research is necessary to explore each disease in particular and the potential impact from EBV and the EBNA2 protein. The Diabetes Research Connection is excited to see where these discoveries may lead moving forward and how it could change the future of T1D treatment. The DRC provides funding to early-career scientists pursuing novel research studies on type 1 diabetes to improve prevention strategies, treatment options, and management techniques as well as potentially find a cure. Learn more about current projects and provide support by visiting”

Happily copied and pasted for you by A. K. Buckroth, #buckroth.

By-Products of Diabetes by E. Ashley Skinner

…Copy & Pasted from Facebook on May 11, 2018….

“I can’t say this will be my last post for a while, but maybe the last of many…. love U all! Certainly, in the most difficult moments of life U realize who R true friends, or ppl who really do appreciate U. Unfortunately, some friends will click “like”, but they don’t really take time to read your status if they see it’s long. I have decided to publish this message to make ppl aware of the horrific byproducts of type 1 diabetes. This disease ruins your neural, cardiovascular❤️, & renal systems & can cause blindness….& the drug, Insulin, that is prescribed, doesn’t fix the true problem of the disease & could also be the very thing that kills U. (The true problem of type 1 diabetes is the Immune system attacking things it shouldn’t.)  Now I’m looking 👀for those ppl who’ll have the time🕛to read this post ’til the end. Type 1 Diabetes is very invasive & destructive to our bodies, even after treatment w/Insulin, & medications sometimes for resistance to insulin, or if you’re a type 2. Your body’s still struggling w/itself trying to regulate foreign invaders & knowing what to keep, & what to kill, & the spiking & dropping of your blood sugars doesn’t ever stop.🛑. Plz, in honor of a family member, or friend who has died💀, or who continue to fight diabetes, of any type, copy & paste (not share) on your page. So I’m gonna find out who reads📖my posts. Please write “done” in the comments.”

Copyright 2018.

Just lettin’ you know…again.  Author A. K. Buckroth

“Shameless Hussy Looks Back” by June Comarsh Gillam

My friend, June Gillam, caretaker and long-time spouse of a diabetic mentioned herein, wrote this poem with great love for him.  Her shared and devotional intentions are purposely written for all to learn.

After he wheeled his chair in, all independent like
from his morning shower just the other day
and transferred smoothly onto the king-sized bed,
I finished toweling him off and rubbed it dry,
fluffing up the curly golden short hairs it nested in,
resplendent in its pinky glow.

“No gray hairs down here!” I marveled,
bending over my mate of 29 years
to rub my face lightly back and forth
on his sweet soft sausage,
rolling it around
beneath my lips.

Back when we were new –
He in his black leather boots, and
I in my shameless hussy moans –
He would have sprung up tall and hard so fast
I couldn’t have finished the toweling off
before he put to use that splendid package,
with his bull balls slung low,
and banging gently on my bum.

I would cling to his skin
and ride him up the steep sides
of the mountain, onto the high plateau,
leaving my mind behind
in the merciful death of thoughts,
simply flying his horse of many colors
across the vast and lasting mesa.

After each of his blood sugar dramas
like a five-way coronary bypass,
two carotid artery surgeries,
claudication of the legs
and losing a toe here and there
he resumed his relentless and leather-booted interest.
Although requiring some modifications, some assistance
from those little blue pills and from those brown
packages that arrived in the mail with various humming
and buzzing adult toys he wanted to try out.
“I’ll never quit,” he vowed and indeed
each time he rose again, triumphant.

My libido shriveled
as spontaneous flights across the highland mesa
faded into movies in my memory,
and it turned into an unexpected duty to search
to find my shameless hussy’s eager heart.

“I am still interested,” he says today,
but that soft pink sausage resting like a newborn’s
tells a different story.

“Did you think I would give up?”

“I had lately come to hope exactly that
this year since his near death, two-month stay
in ICU with kidney failure and dialysis,
open wound dressings, and half year of physical therapy
since the amputation of his left leg, above the knee.

“I sent away for a book on sex for the disabled,”
he says, “but it hasn’t arrived yet.”

One day soon, the mail will come –
I’m digging deep into the great divide of time
to find my shameless hussy long-departed self”

Come back, come back, come back!

I cry out to her.

–  I can’t do this all alone!


Needles be Gone for Type One Diabetes Patients (…Hurrah!)

As a T1D for too long, this article concerning research for a cure of this slow killer, known as diabetes, is exciting – to me.  I hope you find it exciting as well. 

“Project Researcher: Yo Suzuki, Ph.D. – J. Craig Venter Institute

“Project Description

“Success will bring more NIH funding and could eliminate needles! Insulin delivery using infusion pumps can be effective for treatment of T1D, but it does not completely protect T1D patients from the long-term effects of the disease or enable a normal non-diabetic lifestyle. Diabetes research has focused on using insulin-producing cells isolated from cadavers or made from stem cells in T1D patients. While some of the research has yielded promising results, nothing yet has radically changed general approaches to treat patients.

“Our approach is to give bacterial cells that naturally live in our body the ability to function like our insulin-producing cells, to synthesize insulin when blood glucose levels are high to maintain proper glucose levels in T1D patients. In the proposed project, we will establish the feasibility and safety of this approach by making bacterial strains with this function and testing them in mouse systems. If successful, our research will form the basis for a bacterial treatment that can circumvent the struggle of injected insulin therapy and the issues regarding the rejection of transplanted human cells.

“Project Details

“An important ingredient of our study is a set of bacteria recently discovered to naturally reside deep within the human skin, in a layer previously thought to have only human cells. This layer of the skin contains blood vessels and is, therefore, suitable for implanted cells to monitor blood glucose levels and release insulin for systemic distribution. These bacteria do not cause problems in our body. Unlike transplants, they do not trigger host immune response. Unlike insulin pumps, they can enter this deep layer of the skin without puncturing the skin!

“Advances in biology now enable making changes to DNA, the genetic material, in these bacteria. Our team at the J. Craig Venter Institute is at the forefront of these advances to produce safe and beneficial bacteria. To make the deep-skin bacteria function like insulin cells, we will introduce a gene into them for making a version of insulin. Bacteria cannot make native insulin by themselves, but there is a type that can be made in bacteria and is as effective as native insulin. We will also introduce a DNA piece containing three genes for making a glucose sensor to control insulin production in the bacteria.

“It is critical that insulin-producing bacteria do not infect healthy individuals. Therefore, we will install a mechanism to prevent the bacterial cells from spreading beyond the designated host. We will then paint the mouse skin with our bacteria and see if blood glucose levels drop.

“This critical proof-of-concept experiment will tell us if this skin bacteria-based approach has promise and deserves continued support. If we are successful, we will have experimental data that will attract additional funding from the NIH. Establishing a system that can be tested in actual T1D patients will involve many rounds of experimentation and improvement. For example, our future bacterial cells will have more sophisticated safety features. However, tools needed for this approach are already available in basic form. Moreover, the eventual product may be superior to any other products under development. Therefore, we strongly feel that the work needs to be started now toward determining the viability of this approach. Your support could help revolutionize T1D therapy.


“Hello, my name is Yo Suzuki. I am an Assistant Professor at the J. Craig Venter Institute. Our institute is known for its expertise in reading and writing genomes, blueprints for life. When writing genomes, our focus is to design and build beneficial microbes. I have been engineering microbes for 11 years, and finally, a connection is made between my skills and the opportunity to contribute to curing type I diabetes. The goal of this project is to create a bacterial strain that can respond to glucose and produce potent insulin analogs in a mouse. Our long-term goal will be to develop an engineered bacterial strain as a surrogate for beta cells in T1D patients. If we are successful, we will have a microbial treatment that circumvents the struggle of injected insulin therapy and problems with a transplant approach. Our project is the first and critical step toward this long-term goal. Our approach is innovative, but many tools to enable this approach are already available, including harmless bacteria that enter the skin in a non-invasive manner and live in a layer of skin appropriate for glucose sensing and insulin administration. Our tools to control these bacteria will only improve. Therefore, we have to start the work now to test the viability of this promising approach. Your support is greatly needed and appreciated.


“Update on 4-11-18  (6 Month Progress Report)

“Executive Summary

“The purpose of this project is to test to see if skin bacteria that respond to glucose levels and produce insulin can be engineered and introduced into laboratory animals to reduce their blood glucose levels, as a precursor to establishing engineered skin bacteria as painless substitutes for glucose sensors and insulin pumps. During the first six months, we made rapid progress and established genetic tools for engineering skin bacteria. This accomplishment sets the stage for the second phase of the project where expression modules for insulin genes and regulatory mechanisms for sensing glucose will be developed.

“Productivity: The work performed is summarized under the aims proposed for the project.

“Aim 1. Establish genetic engineering tools in selected deep skin bacteria.

“Completed: We collected six Gram-positive and three Gram-negative strains of bacteria isolated from skin samples. We determined the minimal inhibitory concentrations for nine antibiotics commonly used for genetic engineering. This test revealed that there are many strain-antibiotic combinations that can be used in our study. The Gram-positive strain Staphylococcus epidermidis ATCC12228 was the only strain tested among the nine strains for the capacity to re-enter the skin (unpublished result, R. Gallo). Therefore, we focused our resources on this strain. Gram-negative bacteria have been noted to be more proficient at proper folding of secreted proteins derived from other organisms, although there are examples where Gram-positive cells were used for making and secreting insulin. We decided to keep the Gram-negative strains as backup strains and continue to acquire genetic engineering tools for them. This marks the attainment of an initially proposed milestone (selection of strains).

“We previously developed a genetic engineering approach where synthetic DNA fragments loaded with an enzyme called transposase in vitro is introduced into bacteria. Transposase facilitates the integration of DNA fragments into the genome. Because this approach is effective in a wide variety of organisms, we tested it in the S. epidermidis strain with limited tools. To cost-effectively perform this procedure, we purified transposase and confirmed the activity of the purified enzyme. We then used the enzyme and succeeded in introducing a DNA fragment only containing a puromycin resistance gene into the S. epidermidis strain. The positive result was obtained when the DNA sample was pretreated with the lysate of the organism. The idea was that methylases in the lysate generated the methylation pattern on DNA found in the native organism so that the incoming DNA was accepted as its own DNA.

“Aim 2. Implement a biocontainment measure.

“In progress: Before we engineer the capability to express and secrete insulin in the S. epidermidis strain, a mechanism for biocontainment needs to be implemented, as an organism capable of expressing human insulin would be a hazard to laboratory workers, should they become infected. We are attempting to use CRISPR genome editing to knock out the thyA gene needed to make thymidine, an ingredient for DNA, to make our bacterial cells dependent on thymidine supplied from outside and keep the cells within designated culture tubes.

“Aim 3. Express SCIs in deep skin bacteria.

“In progress: Recent studies have resulted in single-chain insulin analogs (SCIs) that match native insulin in potency (Hua et al., 2008, J. Biol. Chem. 283:14703-14716). In beta cells, proinsulin is folded and cleaved to produce biologically active insulin, which consists of two peptides that are linked by disulfide bonds. Because native bacteria cannot cleave proinsulin, SCIs are essential for the strategy of cell-intrinsic and self-sufficient production of active insulin within bacteria. We are currently designing expression constructs incorporating the published SCI-57 design (Hua et al., 2008).

“Aim 4. Evaluate the capacity of bacterially produced SCIs to stimulate glucose uptake in adipocytes.

“Not started. We plan to test whether the bacterially produced SCIs are biologically active by treating mouse adipocytes with the bacteria in a glucose uptake assay. We expect this work to be started as soon as the SCI-producing strains are made. We have culture cell expertise needed to prepare the adipocytes.

“Aim 5. Evaluate the ability of SCIs to reduce blood glucose in mice after application of the engineered bacteria to the mouse skin.

“Not started. We plan to determine if the SCI-producing bacteria can colonize the skin of a mouse to result in a reduction of blood glucose levels in the mouse. We will prepare for this work when the SCI- producing bacteria are made.

“Aim 6. Implement a glucose-mediated regulation of SCI production.

“Not started. For effective blood glucose control, it is critical that SCI production be adjusted based on glucose concentration. We initially proposed to use a glucose sensor in Gram-negative bacteria, but we decided to focus on a Gram-positive strain. We will start researching mechanisms available in Gram-positive bacteria to enable a smooth transition to this phase of research.

“Synergistic Activity: The Diabetes Research Connection grant enables the critical first step toward a microbial treatment for diabetes that circumvents the struggle of injected insulin therapy. The preliminary data obtained in this project were incorporated into a grant pre-proposal submitted by J. Glass (JCVI) to the Larry L. Hillblom Foundation. We have identified a suitable NIH grant mechanism (PAR-18-434) to further develop our approach. We also attended the 2017 Chemical and Biological Defense Science and Technology Conference in Long Beach, California sponsored by Defense Threat Reduction Agency. Skin biology is an important aspect of their portfolio, and we received useful feedback on our research from experts.

Update on 2-06-18

“Last time I told you that I purified an enzyme called transposase to be used for facilitating a process called transformation, to put engineered DNA constructs into cells from outside. It turned out that the transformation step was still not easy for skin bacteria, but by communicating with scientists from Australia and multiple universities in the U. S. working on related bacteria of the Staphylococcus species, I got better at the process. I started getting colonies, or dots in a Petri dish each originating from a single transformed cell. Tests confirmed that these colonies had the DNA material I introduced into the cells. With this process being established, the next step for me will be to knock out a gene (thyA gene) needed to make an ingredient for DNA, to make our bacterial cells dependent on the ingredient (thymidine) supplied from outside, so that we can keep the cells at designated sites like a culture flask (as opposed to my skin). Also, the time is right for making a DNA construct for expressing insulin to be introduced into the skin bacteria. Designing DNA constructs is what I love the most. I am grateful that your support as it enables us to proceed to this phase of the project.

“Update on 11-22-17

“Toward my aim, one goal of establishing genetic engineering tools for skin bacteria, I wanted to test the approach of introducing synthetic DNA fragments loaded with transposase in vitro.  Transposase facilitates the integration of DNA fragments into the genome.  I believe that this is a widely applicable approach that is suitable for our project with multiple target organisms to engineer.  For optimizing this process, I would need quite a bit of transposase, but this is an expensive reagent if you buy it.  Just 10 µl of it costs $500, and you can use all 10 µl in one experiment.  Therefore, I decided to make my own preparation of transposase.  I received a DNA construct containing a transposase gene that can be expressed in E. coli from a research group in Sweden.  I introduced this construct into an E. coli strain suitable for recombinant protein production.  I induced transposon production, lysed the cells, and purified the protein from the lysate using an affinity chromatography column.  I confirmed the activity of the purified transposase using a standard assay in our laboratory.  I succeeded in this process and obtained an amount of transposase worth $93,000.  Encouraged by this result, I am now tackling establishing transformation protocols for skin bacteria.”

Thank you, Dr. Suzuki.  Readers, what do you think about this?  I’d like to hear from you.

Just sayin’…. #buckroth