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Nothing illustrates that change is the essence of life quite like the butterfly.  How does this cloudless sulphur caterpillar, eating the toxic blooms of a cassia bush, become a butterfly? How did it know the cassia toxin would protect it from predators ensuring a next generation? How did it select the bright colors that warn birds that it has a toxin and they should stay away? And how does it transform into the butterfly for the next phase of its life?

All information for life is carried in genes encoded by DNA. The DNA is a hard code passed from generation to generation from parents to their offspring. If you look closely at two caterpillars you may note differences in size or color.  These differences are phenotypic expressions of genes.  Phenotype is controlled by modifying molecules that are applied to DNA and can be changed by diet and environment.  Amazingly, DNA modifiers can be inherited from prior generations.  A well-studied transgenerational DNA modifier is the effect of starvation on the genome.  Epigenetics is the field of science discovering and understanding DNA modifications and an important branch of this science is an ability to treat disease.

The manipulation of gene expression and silencing can be purposeful and directed. A particularly useful analogy is that DNA is the organisms operating system, OS, while epigenetics refers to apps that can be applied to the OS.  The apps give profound plasticity to increasing or decreasing expression of genes. Manipulating gene expression is a natural phenomenon and could be exploited as a step to personized medicine.

An organism’s genotype confers the code for everything required to survive. At conception, the stem cell has unlimited potential, the cell receives signals from its local environment and begins a one-way path to its destiny. DNA modifications are tags that are added onto the DNA by the surroundings that up or down regulate, or even silence, genes.  Daughter cells from the differentiated cells inherit the DNA modifications.  The collective modifications are the resulting phenotype of the organism. Epigenetics is about gene expression or gene silencing, understanding the apps. There are two periods of resetting DNA, at conception, when epigenic tags from the parent are erased and at six to eight weeks of gestation (in humans) when tags are again reset.

The epigenetic clock is a term to describe when on or off signals are expressed. Epigeneticists also realized there is epigenetic memory passed from the mother to the offspring because some tags are not erased. How is the DNA controlled by an epigenetic tag? DNA is packaged in the cell by wrapping around proteins called histones.  Mechanisms of histone control are acetylation and methylation.  Acetylation is the modification that relaxes DNA histone binding by an enzyme, histone acetylase.  Relaxing the binding of a section of DNA makes the gene available for reading and therefore expressed.

This image shows the DNA helix (orange tube) physically packaged in the cell, wrapped around histone proteins (green balls).  The position of acetylation of histone lysine residues by histone acetylase (HAT) are illustrated by the gray strings. You can click this image to read the discussion in Nature and the source of the image.  When a gene is expressed, a section of the DNA is unwrapped from its histone, read by enzymes that write/edit/erase the RNA that will code for protein synthesis.  The tightness of DNA wrapping on a histone is determined by modifications on the DNA.  A histone deacetylase (HDAC) will prevent unwrapping and inhibit gene expression because it will remove the acetyl group from histones lysine residues.  An enzyme that inhibits histone deacetylase results in gene expression, this is a technique that is being employed to create drugs to treat Alzheimer’s disease. You can imagine how this system can regulate the amount of a protein that is produced.

Methylation is a modification that make genes inactive. Methylation is inherited to daughter cells.  That means gene silencing can be inherited from cell to cell. An adaptation of genes to overcome silencing is a change in location in a chromosome. Methyl donor compounds, and those found in the diet, are also treatment opportunities to overcome disease by silencing genes because increasing DNA methylation is a mechanism that inactivates genes. The drug budesonide, which is DNA methyltransferase activator, results in increased methylation of DNA and ultimately decrease in the size of some tumors that are under the control of tumor suppressor genes. The research suggests that modification of DNA methylation either by hypo- or hypermethylation is effective in different types of tumors.

Genes can gain/lose function.  If a gene is moved to a different location in the chromosome or there is a mutation in the genetic code function can change.  Some bits of DNA are known to be particularly sensitive to moving location and some areas of DNA code are hyperexcitable and can be particularly prone to mutation. In disease, a tumor suppressor gene can be turned off and result in cancer. It would be possible to impact epigenetic tags to increase the expression of a tumor suppressor gene and stop the tumors growth.

In addition to acetylation and methylation, some RNA molecules are regulatory.  Regulatory RNA’s can neutralize genes or block messenger RNA. This process isn’t all or nothing and gives fine tuning to gene expression.

Transgenerational epigenetic inheritance is recognized and is the transmission of epigenome or epigenetic markers from one generation to the next. This adaptation doesn’t affect the structure of DNA but provides a unique survival mechanism allowing organisms to adapt to changing conditions. The effect of starvation has been studied,  decreased nutrition was shown to affect subsequent generations through modifications that are inherited from the mother.

The cloudless sulphur larvae becomes a butterfly because the epigenetic tags on the DNA are uniquely choreographed. The larval genes are silenced after sufficient food intake and the next stage genes are turned on, giving wings for flight.

There are many interesting ideas that come to mind, one is the unmet need for biomarkers and treating metabolic diseases.

Disease pathologies arise from dysregulation of the immune system, oxidative stress, abnormal axonal transport, and other metabolic malfunctions. Discovering epigenetic controls of the pathologies in metabolic dysregulation may lead to targeted treatments and hope for patients.

Right click on this link to open an active PDF in a new window.  Click on the pictures in the PDF and you will see more epigenetics information, including exciting You Tube Ted talks.

lsu-2265-labComplicated diseases require complicated answers.  Neurodegenerative diseases are a broad class of maladies that don’t have a simple answers.  Some 18 years ago it was suggested to the EPM society president that a “virtual” project should be launched.  The vision included several labs, each taking a part in an aspect of EPM, sharing reagents, protocols, and data- to quickly advance a good diagnostic. With every new EPM society president the idea was awakened and alas, quickly put to bed.

Our laboratory investigates disease pathologies involved in protozoal encephalomyelitis (EPM)  and amyotrophic lateral sclerosis (ALS).  Over the years we developed a panel of biomarkers and determined treatment responses in the EPM-suspect horse and determined the effects of drugs on various  biomarkers.  How much faster this would have been in a Virtual Laboratory!  I’m pleased to report virtual sharing is springing up everywhere.  For example, the Chan Zuckerberg Initiative funds collaborative pilot projects addressing key challenges in the biology of neurodegenerative diseases.

Neurodegenerative Disease Research Inc, es.t April 21, 2020, funds a Virtual ALS Laboratory.  So far, there are 14 primary researchers and the group is growing.  Biomarkers are sorely needed and that is one focus of the group.  Ideally, biomarkers change over time when given an effective treatment.  A useful biomarker in murine, equine, and human ALS is neurofilament light (NfL).  Neurofilaments are  prognosticators of axon damage, the level of NfL changes quickly with an effective treatment. We use NfL levels found in horses that have demyelinating polyneuropathies to detect axon damage.  The marker is useful to determine the cellular response to an effective therapy.

The NDR Virtual lab uses patient driven research, transformative technology, imaging, single cell biology, neuroscience, genomics, stem cells, biochemistry, computational biology, bioengineering, and  immunology.  Laboratories with a track record and an idea are funded to move their project along while incorporating experiments designed to cross-over with another laboratory.   The Virtual lab is far more effective that the sum of the parts. Data from the  initial funding is coming in and we hope to have some really great news for the ALS community soon.

raccoon dogIs anyone untouched by the Covid19 pandemic?  Some of our critical research projects are on hold, mostly due to no PPE for personnel to handle study lab animals and also travel restrictions.  We are fully functional at our lab.  We are using any extra time to evaluate data and share our findings with veterinarians and horse owners through Zoom meetings.  Check out the Facebook meeting posts to find the links and join us in our Zoom room.  Until then, here are some facts that help us focus on what is important. The information is up to date as of mid-April.

The SARS-CoV-2 strain of a novel Coronavirus appeared in Asia in 2019 and is known as Covid2019…Covid19 for short.  SARS is an acronym for severe acute respiratory syndrome caused by a betacoronavirus that is transmitted by contact with fomites.  Fomites are infectious materials transmitted by contact with respiratory droplets or body fluids.  Unfortunately for us this virus is transmitted by airborne particles.  Symptoms include fever, headache, body aches, dry cough, hypoxia (lack of oxygen), and usually pneumonia. For you molecular biologists,  SARS-CoV are enveloped, positive-sense, single-stranded RNA virus that infect the epithelial cells within the lungs. SARS-CoV-1 binds the ACE2 (angiotensin-converting enzyme 2) receptor and infects humans, bats, and palm civets. We learned about SARS-CoV-1 in 2003.  There are seven coronaviruses that infect humans. In the 2003 SARS outbreak 9% of patients with confirmed cases died, the hardest hit population was over 60 and over 50% of these people died.

The evolution of  SARS-CoV is interesting.  There were two different strains of SARS-CoV isolated in China in 2003, indicating there were separate species crossing events.  The virus came from wild animals sold as food in a market in Guangdong, China and it was isolated from asymptomatic masked palm civets.  This virus was able to infect humans, raccoon dogs, ferret badgers, and domestic cats. The virus could not be maintained in tissue culture and it did not infect bats until the virus was altered in 2008 in a laboratory to contain a human receptor binding domain. That indicated to researchers that bats could be asymptomatic and serve as a natural reservoir for the virus. And a note for Julie, the raccoon dog, also known as the mangut, tanuki or neoguri, is a canid indigenous to East Asia. It is the only canid species that can climb trees.

The recent ancestor for all coronavirus existed in 8000 BCE. Some say the virus existed 55 million years ago. They coevolved with birds and bats. Bats are the reservoir for alpha and betacoronavirus  while birds serve the same role for the gamma and deltacoronavirus. The global range allowed evolution and dissemination of this virus family.

The path to human infection is from leaf-nose bats to horseshoe bats, civets, and finally to humans. Bovine coronavirus evolved from rodents and crossed species to equids. It was in the 1890’s when bovine coronavirus jumped to people, and the likely cause of the “flu” pandemic that same year.

Of interest to us is that human corona virus (OC43) causes respiratory infections and is suspected of playing a role in neurological diseases. Mouse hepatitis virus (MHV) is a coronavirus that causes epidemic murine illness that has a high mortality.  Prior to the discovery of SARS-CoV, MHV had been the best-studied coronavirus both in vivo (in the animal) and in vitro (in tissue culture) as well as at the molecular level. Some strains of MHV cause a progressive demyelinating encephalitis in mice which has been used as a murine model for multiple sclerosis.

Human coronaviruses vary in risk factors and disease severity.  Some (MERS-CoV) are deadly to more than 30% of those infected and others just cause an irritating, common cold.The human coronavirus discovered in 2003, SARS-CoV-1 has a unique pathogenesis because it causes both upper and lower respiratory track infections.

Six species of human coronaviruses are known.  One species is subdivided into two different strains, making seven strains of human coronaviruses altogether. Four of these strains continually circulate in the human population and produce the generally mild symptoms of the common cold: OC43, HKU1, HCoV-229E, NL63. These viruses cause about 15% of commons colds (the majority of colds are infections caused by rhinoviruses).

Four coronavirus strains have a seasonal incidence occurring in the winter in temperate climates.  There is no preference towards a particular season in the tropics.  Three strains (two species) produce symptoms that are potentially severe; all three of these are β-CoV strains: MERS-CoV, SARS-CoV-1, SARS-CoV-2. The two SARS-CoV strains have occurred in the last 17 years, both from Chinese wet markets.

How does all this affect us? The Center for Disease Control has updated its guidelines for essential workers that have been exposed to people infected with SARS-CoV-2. Workers that do not feel sick are able to return to work so long as they take their temperature before leaving the workplace, wear a face mask at all times, and practice social distancing.

The department of Homeland Security and Health and Human Services outlines how removing shelter-in-place type restrictions after 30 days would lead to a second, very high peak in the number of cases and deaths.  The number of expected deaths is 300,000. The spike in deaths was projected to occur abut 150 days after lifting the stay-at-home restrictions. We are staying put so you can expect more Zoom meetings.

Its concerning that United States hospitals are seeing a shortage of antibiotics, antivirals, and sedatives required by patients on ventilators.  Increased demand and effects of the pandemic has halted production of some drugs. The University of Minnesota has analyzed the supply chain and identified 156 drugs that could go into shortage in the next 90 days, but they have not released the list.

The numerous studies and information on chloroquine on the outcome of COVID-19 patients are so far inconclusive or halted due to a high number of complications. A vaccine trial, called the Solidarity Vaccine Trial will evaluate multiple candidate vaccines at the same time, against a placebo. The expectation is the researchers will have results in 3-6 months due to high enrollment and an adaptive design. We will be in line for that vaccine when it is ready.

Pathogenes laboratory wishes you and your families well.  We will continue to be here to help you with your horses by testing sera, developing new avenues of research and answering your questions about neurodegenerative diseases. Give us a call or join us through Zoom meetings.

It will soon be our 20th Anniversary at Pathogenes!  We’ve paved new ground and published most of our work because I grew up in the “publish or perish” era.  Now days science is all about technology transfer, it’s “patent or perish”! You may have participated in some things we invented that didn’t get off the ground, looking at down-regulation of molecules in response to S. neurona infection. I never cease to be amazed that this assay predicted the degree of illness a horse would have in a few months. It was expensive and cumbersome to run and ultimately discarded.  How about our horse-side antibody assay?  Who could predict that would strike out?  In a mere 15 minutes you knew if antibodies were present in serum or CSF.
I loved that one. Here is what it looked like:Dipstick assay

We took some side trips, identifying a cell that could support the replication of millions of PRRS virus in vitro, we grew 7 species of Eimeria in cell cultures resulting in a vaccine that would eliminate all those hen houses, and developed monoclonal antibodies, one  against S. suis and provided a local University with a much needed pig-herd vaccine. We collaborated with folks in Germany (looking at pigeon sarcocystosis), Norway (an acquired polyneuritis in Norwegian horses) and Canada. Sarcocystosis and Toxoplasmosis in dogs and cats didn’t escape our interest. But our heart belongs to people dealing with neuromuscular diseases in horses. We remember Amy and Ty from years ago, their picture shows the bond we have with our horses.

We have a paper, in press, that blazes a new trail for others to follow.  The paper explains the rationale for detecting S. fayeri toxin in horses, if you want the punch line--we detect cysts in live horses! We aren’t giving up our quest for new treatments or determining how disease progresses in horses, mouse or man. Horses have always led the field in neuromuscular research, spinal nerves were an important source of myelin for research in Myasthenia Gravis and Guillain Barre syndrome.  As most of you know, we got our ideas and direction from the work done in the 1980’s.  Wish we could say it was our idea and our hard work, but really, we  just pieced together a logical story from published literature.

Our current  directions are identifying the inherited genes that predict late onset ataxia in horses and defining the expressed protein environment in response to specific treatments given to animals with neurologic disease.  We are interested in the molecular targets of drugs in the nervous system and why they act for weeks after a dose.  We are surprisingly close to figuring that one out! You'll need special tubes to participate in these studies, contact us to find out if you can be a part of this research.

What stands out in our work is our ability to give clinicians information about their case and how it compares to hundreds of similar cases across the country.  We document all the information derived from our assays and connect the data to the feedback we get from you.  It’s time to catch up and let us know about your horse.  Even if our last contact was years and years ago, you are still in our system and gentle on our minds. Why, we may even have a picture of your horse if you sent one! We’d like to hear from you.  Here are some useful links:

To give us your update please use this form:  https://docs.google.com/forms/d/e/1FAIpQLSc_4HNIEtaCYI4qgt8X99OHypTSOhaJYv0iXO4kXJZ0hqIUPA/viewform?usp=sf_link

A veterinarian can give us information about a horse with neurological disease: https://docs.google.com/forms/d/e/1FAIpQLSeLU8t3ROPE9nrPjhSuReg4C2ZpmsCrYPyzmMQ8yuwVgMfong/viewform?usp=sf_link

A horse owner can give us information about a horse with neurological disease: https://docs.google.com/forms/d/e/1FAIpQLScB0jNG9dHpnNc2AhOIn8KpNVxMFc4QZ2YvSIJHyrh86hZAFQ/viewform?usp=sf_link

If your horse was treated we’d like a post-treatment update: https://docs.google.com/forms/d/e/1FAIpQLSevDKLdGqXFdr_JrH2W8h_4xbgBYJAxXH4Ydt-_vKUjlffy5A/viewform?usp=sf_link

If you suspect polyneuritis equi this form is appropriate:https://docs.google.com/forms/d/e/1FAIpQLScv4wQlg1pW13VPCuZHaG3yGsxQDnF5C2FrHkjgCH7mrT6Swg/viewform?usp=sf_link

We have been a bit quiet lately and that’s because a friend is very sick.  He has amyotrophic lateral sclerosis (ALS). In order to help him, the Pathogenes team needed to catch up on the particulars of this most horrible disease.  There is no cure for ALS and quite frankly, no useful treatments.  It has taken us a few months to read stacks of papers and gather a team of experts. And form a plan.  Our plan isn’t simple and it isn’t easy.  It’s complicated and can change.  As we formulate and fine tune our approach to stopping the progression of ALS our experts review our work for scientific accuracy and feasibility. We don’t mind that no one has heard of what we propose.  We will test our hypothesis and march forward.

You can find a lot of information on ALS on the Web and we won’t repeat it here.  You can check out our ALS tab for some links we found useful. It is worth pointing out that there is familial ALS (fALS) in which genetics plays a big part.  Only 10-15% of patients with ALS (PALS)…these folks are acronym heavy…get fALS.  Most people get spontaneous ALS (sALS), close to 90%.  There are different camps and controversies concerning the pathways involved in disease and how to approach reversing the progress of motor neuron (MN) death, but an overriding theme of those that investigate ALS is the compassion and sharing of information.  Basically, ALS is associated with an enzyme mutation (superoxide dismutase, SOD) and/or other mutations that cause MN death.  Motor neurons make muscles work. You need motor neurons to breathe.

What stirred us to take a break from our bench work and communicate with our EPM-centric following is learning that dogs get spontaneous ALS!   Dog-ALS is associated with the enzyme superoxide dismutase, the mutation is in SOD1.  The onset of dog disease is late in life.  That means there are ALS cases in people, dogs, and genetically engineered mice.  Horsey people realize there are unknown causes of spontaneous neuromuscular disease in horses that cause them to be wobbly.  These horses can progress until they can’t get around and are euthanized.  In some cases there is no diagnosis and no treatment.  We reviewed two of these diseases.

Equine Motor Neuron Disease (EMD) is an acquired neurodegenerative disease in horses affecting the lower motor neurons of adult horses. The disease is characterized by the onset of abnormal nerve function and muscle wasting resulting from the deterioration of motor neurons and myopathy. Horses from 15 months to 25 years old can get EMD. EMD is considered to be a multifactorial disease, however a dietary deficiency in vitamin E is considered to be a major predisposing factor in its development. This is largely related to when horses have a decreased antioxidant capacity leading  to accumulation of free radials and that results in oxidative damage to the ventral motor neuron cells. Could a decreased function of horse-SOD be a factor?

No one knows what causes Equine degenerative myeloencephalopathy (EDM) that is a diffuse, degenerative disease which primarily causes damage to the horse’s spinal cord. EDM is considered to be an advanced form of neuron-axon dystrophy. EDM may have a genetic basis. Horses can develop EDM and equine motor neuron disease (EMD) at the same time and in association with an underlying vitamin E deficiency. Horses with EDM show clinical signs of a general proprioceptive  ataxia-“I don’t know where my feet are” and an abnormal base-wide stance while at rest. Horses will usually start to show signs of EDM when they are 6 to 12 months old. Horses with mild cases of EDM may present as performance-related problems. At first the condition produces subtle signs, being nothing more than  "clumsy" but ataxia slowly progresses as clinical signs are usually slow and insidious. Ataxia signs will become more apparent and worsen over time. Paralysis and spastic muscular movements will become more evident, until late stages where the horse is unable to get up from laying down without assistance. The only way to get a definite diagnosis that a horse has EDM is by conducting post-mortem examination shortly after death.

We’d like to test horses for EMD and EDM for antibody against neurofilaments.  It’s a serum test.   Once we have some results we will share them with everyone.  If we can demonstrate that horses also have a form of ALS, and why shouldn’t they?, we can start looking at treatments in this species.  If you have a horse with a diagnosis of EMD or EDM send us serum and we will test it.  Be sure and have a firm diagnosis.  Not just a “This is on the differential” or “It’s nothing I’ve seen before!”. Testing is expensive and we’re proposing to pay for it.  We need to know and have proof that several diseases have been ruled out.  You can email us for the form to send in a sample for this specific testing until we put a submission form up on our site. We want late cases as well as cases that are early.

The funny thing is that the deeper we delve into ALS and our approach to treatment, some paths seem to converge. As the ALS community gets closer to understanding the pathophysiology of disease we are finding common roads.  All roads seem to be leading to Rome after all.

At some later date when we have some good news for your ALS friends we will ask you to share what we find.  Until then, we are going back to work.

Jim