Gene-Expression Biomarkers Can Detect Depression

  • First-ever Lab Test for Depression Found Using RT-PCR
  • FDA Approval as Diagnostic Possible by Early 2016
  • Huge Potential Market as 1-in-10 US Adults Suffer from Depression

While it’s normal for everyone to occasionally feel blue or sad, prolonged bouts of depression that interfere with normal life are indicative of a serious mental health issue. While there are numerous forms and differing severity of depressive disorders, as described at a National Institute of Mental Health (NIMH) website, only two factual aspects of this illness really stand out in my opinion:

Depression is a very common illness. The Centers for Disease Control and Prevention estimates that 1-in-10 US adults suffer from depression, which reportedly costs close to $50B annual in lost productivity in the work place. Globally, more than 350 million people of all ages are afflicted with depression, according to recent statistics from the World Health Organization (WHO). By the year 2020, WHO estimates that depression will be the second leading cause of “lost years of healthy life”, following heart disease. Incidentally, as seen from the map below, depression rates around the world vary significantly among countries.

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

  • Depression is diagnosed based on the patients’ self-report of their symptoms and the evaluation of one or more structured psychiatric interviews with the patient by a psychiatrist, psychologist or primary care physician. The absence of direct, non-subjective measures of depression can lead to relatively lengthy time-to-treatment, non-reporting, or—sorry to say—fraudulent claims and/or treatments based solely on what is said as opposed to what is objectively measured.
Bi-polar Depression © Mena Purdy oil on canvas.

Bi-polar Depression © Mena Purdy oil on canvas.

Fortunately, as you’ll read below, easily measured gene expression-based biomarkers present in conventional blood-draw samples have been recently found to differentiate depressed from normal subjects. Not surprisingly, the academic inventor has filed a patent that—if successfully commercialized—will significantly add to the “biomarker bonanza” that I’ve commented on previously.

Brief Background on Biomarkers for Depression

The search for biomarkers for depression is definitely not new and, in a review of biomarkers for psychiatric diseases, has been traced back more than a century ago to the work of Aloysius Alzheimer in 1901. As a psychiatrist and a neuropathologist, Alzheimer worked with a 51-year-old patient suffering from dementia. Over the next 5 years, he observed her behavior. When she died, he studied her brain, paying particular attention to the senile plaques and neurofibrillary tangles that were present. Alzheimer ultimately discovered the connection between these “biomarkers” and the symptoms the disease that now bears his name.

In recent years, the search for psychiatry-relevant biomarkers of MDD, schizophrenia, bipolar disease, and other important psychiatric/neuropsychiatric disorders has intensified. Searching PubMed Central provided ~1,500 references, from which I selected the following recent examples to give you a flavor of the diversity of technologies being used. These are mostly used in relation to MDD, which is expected to become the most prevalent and costly disease worldwide by 2030. A very depressing thought (bad pun intended)…

  • Gas chromatography/mass spectrometry to identify the differences in global plasma metabolites to screen for diagnostic biomarker panels from the plasma of MDD patients vs. non-depressed (ND) control subjects (Ding et al.)
  • Nuclear magnetic resonance (NMR) spectroscopy to profile urine samples from first-episode drug-naïve MDD subjects vs. ND controls (Zheng et al.)
  • Multiplexed immunoassay based technology to screen plasma samples from MDD patients, schizophrenic patients, and normal control individuals for 79 proteins, including a series of cytokines, chemokines and neurotrophins previously suggested to be involved in the pathophysiology of depression and schizophrenia (Domenici et al.)

Genome-Wide mRNA Expression Profiling

A review by Schmidt et al. emphasizes the large number of “key growth factors, cytokines, hormones, and metabolic markers that could be included in an initial multi-analyte biomarker panel of MDD.” With so many genes to consider, it was only a matter of time before genome-wide microarray (aka “gene chip”) profiling would be applied to discover a panel of candidate genes. However, the way this was done seems very interesting to me.

Magnetic Resonance Imaging (MRI) images of hippocampus (yellow) and amygdala (blue), which are symmetrically located in both lobes of the brain (upper right); taken from Butterworth et al. via Bing Images. Read here about these brain regions as related to depression.

Magnetic Resonance Imaging (MRI) images of hippocampus (yellow) and amygdala (blue), which are symmetrically located in both lobes of the brain (upper right); taken from Butterworth et al. via Bing Images. Read here about these brain regions as related to depression.

A team led by professor Eva E. Redei at the Department of Psychiatry and Behavioral Sciences, Northwestern University in Chicago used established “endogenous depression and chronic stress models,” as detailed here, in both male and female rats in various rat species to simulate human genetic diversity. Brain regions were dissected immediately after decapitation, and individual samples of hippocampus and amygdala (see picture right) were homogenized for RNA isolation and then RT-PCR. Resultant cDNA was hybridized onto GeneChip® Rat Genome 230 2.0 Arrays (Affymetrix), each of which are comprised of >31,000 probe sets that analyze >30,000 transcripts and variants from >28,000 well-substantiated rat genes.

Statistical analyses were used to determine panels of genes that serve as candidate biomarkers; however, it was then necessary to investigate whether these rat-derived candidates correlate with human depressive illness. The results of this scientifically big leap, if you will, from artificially stressed rats to actual human patients with MDD were published by Redei and coworkers late last year in Translational Psychiatry. Basically, a panel of 20 genes was studied by RT-PCR of blood samples from 32 MDD patients (age range 23–83)—before (i.e. baseline) and after 18 weeks of cognitive behavioral therapy (CBT)—and 32 non-depressed (ND) control individuals.

Salient findings are as follows:

  • The abundance of nine mRNA transcripts in blood differed significantly between groups at baseline, suggesting that this panel of blood biomarkers could be diagnostic of a clinical population, i.e. identifies MDD.
  • The blood test also predicts who will benefit from CBT based on the behavior of some of the markers. This will provide the opportunity for more effective, individualized therapy for people with depression.
  • The test showed the biological effects of CBT, the first measurable, blood-based evidence of the therapy’s success.
Eva E. Redei in an interview by Chicago Tonight (taken from Bing Images).

Eva E. Redei in an interview by Chicago Tonight (taken from Bing Images).

By watching a short video of professor Redei in a posted statement by Northwestern University, you can listen to her comments about these findings, and get a sense of her strong convictions that energized this long, 16-year pursuit of blood biomarkers for depression. She is quoted therein as saying “[t]his clearly indicates that you can have a blood-based laboratory test for depression, providing a scientific diagnosis in the same way someone is diagnosed with high blood pressure or high cholesterol.”

While the above quote is very positive, accepted use of these blood biomarkers for MDD will require much more work. In fact, the published report by Redei et al. has the following concluding statement:

“Future studies are aimed at validating these results in a larger patient population and determining the selectivity of these panels in patient population that include other psychiatric illnesses.”

To get more specifics about this statement, I contacted professor Redei by email and asked the following questions to which her replies are given:

JZ: What is the approximate size of a “larger patient population” for MDD biomarker validation that you refer to?

ER: We would like to enroll 100 well-characterized patients with major depression and 100 controls (no depression or other psychiatric illnesses).

JZ: If such a study began patient enrollment at the beginning of 2015, and the results were positive, in roughly what time-frame could FDA approval as a diagnostic be expected if all went well?

ER: As you know, FDA approval process and time is difficult to predict. However, as it is an in vitro non-invasive diagnostic, it would receive expedited review. I believe that the end of 2016 is a possibility.

JZ: Specifically, what other psychiatric illnesses

ER: Bipolar disease is the number one on the list.

Hopefully, this larger clinical study will begin soon and provide results warranting expedited review and approval by the FDA. My guess is that a commercial entity aiming to market MDD biomarkers as a diagnostic product will be the sponsor of this future clinical study. That entity could be either a startup or existing firm, with patent licensing from Northwestern University. Like many academic and research institute investigators seeking patent protection—and commercial profits—professor Redei has filed a patent application related to the aforementioned blood biomarkers for depressive diseases. If a patent is issued, and if the larger, definitive clinical trial indeed validates the diagnostic utility of these biomarkers, the intellectual property would almost certainly have huge commercial value.

Postscript

Fight Depression with Physical Exercise!

If you are feeling depressed, and don’t exercise regularly, you should definitely go to the gym or start jogging or take long walks as a means of combatting the “blues”—as well as build-up muscles and burn-off calories. Many societies through the ages have adopted physical exercise in one form or another as a means of ensuring physical and mental well-being, although scientific evidence to support the latter has been somewhat controversial. Some researchers have concluded that there is evidence to suggest that exercise may be a neglected intervention in mental health care. Others have concluded that there is still a long way to go in clinical randomized control trials to prove the point.

Now, a team led by investigators at the Karolinska Institute in Stockholm has published the first strong evidence—in my opinion—for a molecular biological pathway underlying exercise and resistance to depression. In a nutshell, comparisons of “muscle mice” with normal mice revealed activation of skeletal muscle expression of enzymes that convert kynurenine aminotransferases (KAT) that convert kynurenine into kynurenic acid, a metabolite unable to cross the blood-brain barrier. Reducing plasma kynurenine protects the brain from stress-induced changes associated with depression. Importantly, elevated KAT was also found for a group of human volunteers following a 3-week training program.

‘The big thing is to show that by changing something in the periphery of your body, in muscle, you can affect function in the brain. That was the big novelty of the paper,’ said one of the lead investigators in a BioTechniques interview.

Origin of “Feeling Blue”

When I used the expression “feel blue” to begin this post about biomarkers for depression, it occurred to me that I had no clue about the origin of this commonly used phrase. Since I couldn’t find it in the Online Etymology Dictionary, I resorted to searching the web, and found an interesting discussion-thread debating various origins. Looking into that, I found this interesting—to me—origin of the phrase “feeling blue” at English Language & Usage citing Origins of Navy Terminology:

If you are sad and describe yourself as “feeling blue,” you are using a phrase coined from a custom among many old deepwater sailing ships. If the ship lost the captain or any of the officers during its voyage, she would fly blue flags and have a blue band painted along her entire hull when returning to home port.

The clipper ship Blue Jacket, an 1854 extreme clipper in the Liverpool and Australia trades—named after the blue jackets, a traditional name for sailors in the US and British navies—was lost at sea in 1869 when flax it carried caught fire, according to nine survivors who managed to save 15,000 pounds sterling of gold (taken from vickeryart.com via Bing Images).

The clipper ship Blue Jacket, an 1854 extreme clipper in the Liverpool and Australia trades—named after the blue jackets, a traditional name for sailors in the US and British navies—was lost at sea in 1869 when flax it carried caught fire, according to nine survivors who managed to save 15,000 pounds sterling of gold (taken from vickeryart.com via Bing Images).

As always, your comments are welcomed.

Cheers!

The Most Interesting Scientist in the World: George M. Church

  • Mind Boggling Breadth and Significance of Scientific Publications
  • Serial Entrepreneur and Science Advisor to Many Companies
  • Radical Advocate of Total Openness for Personal Genomics

While seeing for the umpteenth time a Dos Equis beer commercial featuring The Most Interesting Man in the World, I was suddenly inspired to write a blog about The Most Interesting Scientist in the World. After scrolling and polling my memory to decide who that would be, it was an easy decision to pick George M. Church, professor of genetics at Harvard. As I’ll briefly highlight herein, Prof. Church’s contributions continually span a mind boggling spectrum of science that cuts across academic theory, ground breaking “how to” methods, serial entrepreneurship, and—perhaps most importantly—radical openness for personal genomics.

George M. Church and The Most Interesting Man in the World: ‘I don’t always read science, but when I do it’s by George M. Church.’ (taken from Bing Images)

Scientific Superman

Most scientists—including me—would be very happy with being recognized as having made a significant contribution in one area of research. So, what is super impressive to me about George Church are the number, breadth and continuity of his contributions that are truly significant—in my opinion. This is based on a quick scan of his nearly 300 publications to date, from which I selected a dozen that are indicative of this remarkable achievement.  The titles of which are given here, in chronological order, along with a brief comment:

Openness of Personal Genomics

For the past 15 years or so, continuing advances in DNA sequencing pioneered by Church—and many others—have enabled the ability to now sequence the genome of an individual at a cost that is affordable within health care systems in developed countries. While this new era of “personal genomics”is only at its dawn, Church’s vision of the need for complete “openness” is a radical departure from traditional confidentiality of medical information.

This view of the future was actually embodied nearly ten years ago by his founding of the Personal Genome Project (PGP), which is dedicated to creating public genome, health, and trait data. PGP’s stated philosophical and operational positions are that “sharing data is critical to scientific progress, but has been hampered by traditional research practices—our approach is to invite willing participants to publicly share their personal data for the greater good.”

The best leaders do so from the front, so it’s not surprising that Church describes himself as “guinea pig #1” of the PGP, enrollment for which is an online process that you can access here to read about the study guide and consent forms. To get a sense of what this complete openness involves, here’s a link to access each of the approximately 4,000 Participant Profiles, with Church being first on the list as PGP#1, participant ID hu43860C. Clicking his ID link lets you peruse all sorts of medical information such as conditions and medications. BTW, his conditions include narcolepsy, which not to make lite of could partly be due to his super active brain!

I really like the “Nobody is Average” statement and image shown below because they succinctly convey the essence of why personal genomics is important: individual genomes in a population are each different and are therefore best understood as such, rather than an ensemble average.

Nobody is average but what to do about it? That is the challenge of individualized disease prevention based on genomics. Taken from one of a series of must-read postsin blogs.cdc.gov via Bing Images.

Nobody is average but what to do about it? That is the challenge of individualized disease prevention based on genomics. Taken from one of a series of must-read posts in blogs.cdc.gov via Bing Images.

Consequently, the PGP is a critically important feasibility assessment of how an individual’s genome and health information are dealt with. The PGP is still work-in-progress, with benefits and insights to-be-determined at some time in the future. Reading a recently published interim report on “lessons learned” so far gave me the impression that specific, actionable medical benefits have been obtained in relatively few instances. On the other hand, much was favorably said about the “ongoing participatory learning experience this project represents for both its participants and researchers”—sort of kumbaya for personal genomics.

In any event, we’ll have to stay tuned for future reports to see how things pan out for the PGP and its allied efforts, such as the Encyclopedia of DNA Elements (ENCODE) project and the National Institute of Standards and Technology (NIST) Genome in a Bottle (GIAB) consortium. While much has already been published about ENCODE, GIBA is relatively new. It’s a public-private-academic consortium initiated by NIST to develop the technical infrastructure (reference standards, reference methods, and reference data) to enable translation of whole human genome sequencing to clinical practice. The consortium holds biannual public workshops in January at Stanford University and in August at NIST in Gaithersburg, MD.

Serial Entrepreneur and Indefatigable Science Advisor

I sometimes think of George Church as the Duracell Bunny-equivalent for founding genomics-based start-up companies because he keeps doing and doing it while others stop. So too for his being a Science Advisor, because I keep coming across new companies leveraging his sage advice.

In researching these aspects of Church’s professional involvements, I came across a Bloomberg Business Week synopsis that is well worth reading, although it will make you very tired even thinking about how he manages to do all of this over and above his many purely academic interests. Although I won’t list the more than 20 advisory positions, which you can peruse on your own, his 12 startup companies are grouped as follows.

  • Medical diagnostics: Knome, Alacris, AbVitro, and Pathogenica
  • Synthetic biology/therapeutics: Codon Devices, Joule, Gen9, Editas Medicine, Egenesis, enEvolv, and WarpDrive
  • Renewable chemicals, fuels and other products: REG Life Sciences

Among these, I think that Editas Medicine is an especially notable start up to watch going forward because it’s based on currently “white hot” CRISPR/Cas9 and TALENs technologies, which enable unprecedented genome editing, as outlined in a TriLink tutorial. Editas Medicine co-founders are world leaders in this genome editing: George Church (Harvard), Feng Zhang (MIT), Jennifer A. Doudna (UC Berkeley), J. Keith Joung (Harvard), and David R. Liu (Harvard).

According to a 2013 press release, Editas Medicine secured $43 million financing from several well-known venture capital investment firms. It also states that CRISPR/Cas9 and TALENs “have made it possible to modify, in a targeted way, almost any gene in the human body with the ability to directly turn on, turn off, or edit disease-causing genes.” It adds that “Editas’ mission is to translate its genome editing technology into a novel class of human therapeutics that enable precise and corrective molecular modification to treat the underlying cause of a broad range of disease at the genetic level.”

At the risk of throwing cold water on these red hot technologies for genome editing, it will likely take quite a while—and definitely much, much more money—to achieve the first FDA-approved clinical example of a genome-edited therapeutic. Hopefully, I will be proven wrong.

BTW, even if genome editing falls short of enabling new therapeutics, researchers in academia and start ups like Editas will have learned much more about the intricacies of molecular biology, and likely reveal yet another new way to achieve that goal. I’ve always believed that taking risks and learning from mistakes are key to scientific advances, which I why I’m ending this post the following thought.

Taken from quotespictures.com via Bing Images.

Taken from quotespictures.com via Bing Images.

What do you think will happen?  Which of Church’s projects are most interesting to you?  As usual, your comments are welcomed.

We’re Celebrating Click Chemistry In Honor of National DNA Day

  • The Verbification of Click Chemistry 
  • Old Chemistry Morphs into New Applications for DNA and RNA  
  • Amazingly, Phosphorus in DNA and RNA is not Needed for Function 

This post comes only two days after National DNA Day 2015 on April 25th so it’s apropos to feature DNA, but I’d also like to give a nod to the lesser recognized RNA, without which DNA would be akin to music notes in search of a melody.  If you’re a regular reader of this blog, you know my stance on this subject and so I digress…

So-called “Click Chemistry” is trending so “hot” that it has led to a phenomenon known as verbification, which is when a noun becomes a verb by virtue of popularity and linguistic convenience. So, just as Google has become to google for virtually everyone, Click has become to click for synthetic chemists and biotechnologists. Whether or not you’re already familiar with Clicking, I hope to provide herein some interesting snippets about Click, its growing ubiquity, and how it has enabled synthesis of a completely novel, non-phosphorous linkage in DNA that nevertheless functions flawlessly in vivo—a stunning feat never before achieved that has intriguing implications about life. More on that later, but first some snippets about Click.

What is Clicking?

Rolf Huisgen (b1920) in 2004 (taken from Bing Images)

Rolf Huisgen (b1920) in 2004 (taken from Bing Images)

The essential basis of “Click Chemistry” goes back to the early 1960s when Rolf Huisgen at the University of Munich published a series of papers demonstrating reactions of azides with alkynes to give cyclic triazoles, as shown below. In chemistry parlance, these were examples of a “1,3-dipolar cycloaddition,” and were soon eponymously named the “Huisgen reaction,” which I can still recall from chemistry lectures during my graduate studies way back when, giving testimony to both my age and—thankfully—long-term memory that still functioning, but I digress (again)…

structure

K. (Karl) Barry Sharpless (b1941; taken from Bing Images)

K. (Karl) Barry Sharpless (b1941; taken from Bing Images)

Fast forward 40 years to the early 2000s when K. Barry Sharpless at The Scripps Research Institute in San Diego introduced the term “Click Chemistry” to describe an important variant of the Huisgen reaction wherein copper ion is used to catalyze the rate of reaction making it possible to use mild conditions to chemically join, i.e. conjugate, a wide variety of azides and alkynes.

In my opinion, the growing ubiquity of Clicking is evident from the number of publications that I found in SciFinder® by searching for the term “Click Chemistry” which gave over 11,300 items. That computes to an astounding 870 publications per year or 2-3 publications per day! Of these, there were nearly 700 patents, which attests to significant potential commercial value.

Aside from these impressive numbers, Clicking is mostly used for labeling DNA and RNA, as exemplified below.

Beyond Labeling to Life

To me, the most exciting applications of Clicking have been published by Tom Brown, who I have enjoyed knowing personally for many years, and who has recently moved his research team from the University of Southampton to the University of Oxford, UK, where he is Professor of Nucleic Acid Chemistry in the Department of Chemistry.

Although his published work on Clicking first appear only about 7 years ago, Tom and his team have since then reported an amazing 44 Click-related publications. Notable among these are use of ring strain-promoted strategies, which obviate the need for potentially problematic copper ion, and Click ligation of RNA to synthesize ribozymes that are not accessible by enzymatic ligation.

Absolutely unexpectedly—in my opinion—Brown’s team has discovered that Clicking together 3’-alkyne and 5’-azido DNA fragments to obtain triazole linkages of type depicted below in blue are faithfully transcribed in vivo despite the absence of a natural, negatively charged phosphodiester moiety! This completely contradicts our collective conceptual dogma of the evolutionarily-derived sensitivity of DNA polymerases toward DNA template structure.

dna

I’m particularly fascinated by these findings because they further exemplify non-classical forms of DNA/RNA that nevertheless are capable of one or more of the basic life processes, namely, replication, transcription and translation, which I’ve commented on previously. Whereas these earlier blogs highlighted either replication of modified DNA bases or translation of modified RNA bases, the following work by Brown’s team demonstrates transcription of non-phosphorus triazole linkages in DNA.

In one example, a triazole mimic of a DNA linkage was Clicked into two locations within a 300-mer fragment of DNA that was then shown to undergo errorless PCR amplification using various polymerases and normal dNTPs. They next investigated the biocompatibility of triazole-linked DNA within the cellular machinery of E. coli by insertion into a plasmid having ß-lactamase (BLA), as depicted below in Fig. 5 taken from their publication. The plasmid grew and functioned with essentially the same efficiency as the wild-type plasmid, and a control experiment proved that DNA repair processes did not excise and replace these triazole linkages with wild-type linkages.

fig5

Biocompatibility of these Clicked triazole-DNA linkages with transcription in a human cell line was subsequently demonstrated by Brown’s team using a Click-linked gene encoding the fluorescent protein mCherry. After cell growth and monitoring mCherry fluorescence (i.e. protein function), mRNA was isolated and reverse transcribed into mCherry cDNA that was shown by sequencing to be error-free. The authors noted that “this is the first example of a non-natural DNA linker being functional in a eukaryotic cell.”

In closing this section, I think it’s fair to say that the above mentioned findings by Brown’s team support the possibility of Click-based chemical evolution of life forms having non-phosphorus DNA or RNA linkages either in the past or the future, along the lines I’ve commented on previously in Back to the Future with RNA and XNA. It’s also worth musing over the fact that Friedrich Miescher initially discovered DNA based on it containing a large proportion of phosphorus, while Tom Brown discovered a new form of DNA that functions without phosphorus! Bizarre.

Intrepid Serial Entrepreneur

Prof. Tom Brown © Univeristy of Southampton

Prof. Tom Brown © Univeristy of Southampton

In addition to all of the aforementioned creative contributions to Click chemistry of DNA and RNA, Tom Brown’s energy and entrepreneurial talent has been recognized by being awarded the 2014 Chemistry World Entrepreneur of the Year, based on having started three successful biotech companies.

In a lengthy story by Sarah Houlton, Brown talks about how tough times for getting grants in the 1990s led him to do a deal with the Wellcome Trust to transform his Trust-funded DNA synthesis core lab into a commercial venture managed by his wife, Dorcas, a PhD biologist. This new company called Oswel—coming from oligonucleotide synthesis and Wellcome—became profitable and was eventually acquired by Eurogentec.

The Browns decided to try to repeat this performance by using their personal money to start ATDBio in Southampton as an oligonucleotide supply company in 2005, and used its profits to support R&D that continues today in a second facility in Oxford.

At about the same time ATDBio started up, Brown decided to be the sole investor in University of Southampton virologist Rob Powell’s PCR-based diagnostics start-up named Primer Design, which now has about 30 employees in Southampton, and sells kits worldwide for a whole host of applications.

I congratulated Tom on the occasion of his receiving this award, and asked him what’s next? He replied that “[t]he companies are doing well and realistically my current focus is more than ever on academic research—starting new initiatives in Oxford on diagnostic and therapeutic aspects of nucleic acid chemistry, with a long-term hope that my research will lead on to important applications.”

I strongly suspect new and important applications will indeed be found by Tom.

What do you think about DNA and RNA Clicking?

As always, your comments are welcomed.

DNA’s Forgotten Discoverer: Swiss Scientist Friedrich Miescher 

  • Discovered in 1869 in Pus Cells from Bandages of Crimean War Soldiers
  • Miescher Named this New Matter Nuclein and Intuited that it Played a Fundamental Role in Heredity
  • This put the “N” in DNA—Deoxynucleic Acid
  • Children now Isolate DNA from Fruits & Vegetables in Elementary School 

Truth be told, what led me to writing this post was suddenly realizing one day that, although the vast majority of my professional career involves nucleic acids—and DNA in particular—I did not know anything about the discovery of DNA or its naming. My follow-on thoughts were that this was somewhat embarrassing for a blogger focused on nucleic acids, and should be remedied by some homework! This is also good timing since my mind is currently aflutter with all things DNA in anticipation of National DNA Day coming up on April 25. In the event that you recall my past commentary about the bias toward DNA, yes I am still supporting a National RNA Day to balance the ranks, but I digress…

Friedrich Miescher as young man (taken from cienciaytecnicasuabia.blogspot.com via Bing Images)

Friedrich Miescher as young man (via Bing Images)

In doing my so-called homework, I learned about Swiss scientist Friedrich Miescher’s life story and circumstances surrounding his discovery in the late 1860s of new matter that he named nuclein, which eventually became incorporated into the term nucleic acid. Those circumstances, including Miescher’s unusual source of nuclein, were quite interesting to me so I thought they’d be worth sharing in this post, which draws upon a lengthy article by Ralf Dahm, who has written extensively about Miescher, and has a website worth visiting.

Background

Friedrich Miescher, who was born in Switzerland and graduated from Basel University’s Medical School, moved to Tübingen, Germany in 1868 to be trained as a scientist, specifically to study the chemical constituents of cells. He had been inspired to do so by his uncle, Wilhelm His (1831-1904), an eminent physician and professor in Basel, who discovered neuroblasts and coined the term dendrite.

It was His’ conviction that the key to fundamental questions in biology lay in the chemistry of cells and tissues, and Miescher—who had little interest in becoming a physician—was easily persuaded by his uncle to study these questions.

In his first semester at Tübingen, Miescher worked with Adolf Strecker (1822-1871), a leading organic chemist at the time and renown for being the first person to synthesize an amino acid (alanine) in a reaction known today as the Strecker synthesis.

Tübingen castle where Miescher worked in Hoppe-Seyler’s laboratory (pictured below) dates back to 1078. Image taken from tübingen.de via Google Images. 

Tübingen castle where Miescher worked in Hoppe-Seyler’s laboratory (pictured below) dates back to 1078. Image taken from tübingen.de via Google Images.

But Miescher was not so interested in organic chemistry as such, and was instead keen to apply his newly acquired knowledge to explore the chemistry of cells and tissues. So, in autumn 1868, Miescher joined the laboratory of biochemist Felix Hoppe-Seyler (1825-1895), who was one of the pioneers of what was then referred to as physiological chemistry—a new field aiming to unravel the biochemistry of life. Hoppe-Seyler did seminal work on the properties of proteins—notably hemoglobin, which he named—and introduced the term proteid that later became protein.

(taken from Dahm)

Photograph of Hoppe-Seyler’s laboratory in Tübingen castle taken in 1879 where Miescher discovered DNA ten years earlier. The large distillation apparatus provided distilled water. Prior to being made into a laboratory, the room was used as the laundry for the castle. (taken from Dahm)

The Discovery of DNA

I’m fascinated by reading about the discovery of DNA by Miescher in his own words:

“I had set myself the task of elucidating the constitution of lymphoid cells. I was captivated by the thought of tracking down the basic prerequisites of cellular life on the simplest and most independent form of animal cell.” 

He tried to isolate the cells from lymph glands, but found that gave small quantities of low purity, so he switched to leukocytes (aka white blood cells) as his model system, partly because they have relatively large nuclei that he wanted to study. The other reason was availability: this cell type was readily available to him from the local surgical clinic in the form of pus from fresh surgical bandages. According to a book about Miescher, these were in plentiful supply from hospitalization of wounded soldiers from a war in Crimea—which, unfortunately, happens to again be a war-like region currently in the news and referred to as the “Ukraine crisis.”

Once again in Miescher’s own words:

Leukocyte clusters in pus stained with Toluidine (taken from agora.crosemont.qc.ca via Bing Images).

Leukocyte clusters in pus stained with Toluidine (taken from agora.crosemont.qc.ca via Bing Images).

“I was faced with the task of determining, as completely as possible, the chemical building blocks whose diversity and arrangement determines the structure of the cell. For this purpose pus is one of the best materials.”

To begin, Miescher had to develop methods to wash leukocytes and separate the protoplasm from nuclei in order to analyze their constituents. He tested various salt solutions, always checking the outcome of his trials under a microscope—“a very time-consuming task,” Miescher wrote.

After he had an alkaline solution those constituents and then neutralized it, he wrote:

“…I could obtain precipitates that could not be dissolved either in water, acetic acid, very dilute hydrochloric acid, or in solutions of sodium chloride, and which thus could not belong to any of the hitherto known proteins.” 

Unknowingly, Miescher had for the first time, obtained a crude precipitate of DNA that he called nuclein.

He eventually developed a protocol for isolating nuclein in sufficient quantity and purity, and then set about determining its elemental composition. Elemental analysis was one of the few methods available then to characterize novel substances.

Miescher wrote:

“I have tried to detect the essential peculiarities in [nuclein’s] elementary composition, as best the sparse material at my disposal would allow me to.” 

In addition to revealing carbon, hydrogen, oxygen, and nitrogen, he measured a large proportion of organic phosphorus—not inorganic phosphate—in ratios relative to the other elements that differentiated nuclein from any known protein.

Following up on his discovery of nuclein in leukocytes, Miescher examined other tissues and cell types, such as liver, testes, kidney, and yeast cells, and found nuclein there too. Self-confidently, he speculated that, upon further investigation,

“…[the] entire family of such phosphorus-containing substances, which differ slightly from one another, will reveal itself, and that this family of nuclein bodies will prove tantamount in importance to proteins.”

However, there’s an interesting twist—pun intended—in this story of DNA’s discovery!

Troubles Publishing!

Miescher completed his discovery and initial characterization of nuclein in autumn 1869, and began writing up his first-ever scientific publication, finishing the manuscript on December 23, 1869.

However, Hoppe-Seyler was skeptical of Miescher’s data and wanted to repeat the experiments for himself before consenting to their publication. This caution by Hoppe-Seyler was the result of an earlier controversy in his lab regarding a phosphorus-containing compound unrelated to nuclein.

In early 1871, Miescher’s manuscript entitled—as translated to English from German—“On the Chemical Composition of Pus Cells” was included as the first paper in an issue of a journal published by Hoppe-Seyler. It also had a paper on nuclein by another student of Hoppe-Seyler, and an article by Hoppe-Seyler in which he confirmed Miescher’s findings on nuclein, including its unusually high phosphorus content.

Miescher’s Opinion of Nuclein

Miescher was confident about the importance of his discovery. He stated that the new substance he had discovered would prove to be of equal importance to proteins. Concluding his publication he wrote:

“This is how far I have come based on the material at my disposal. It is obvious that elementary analyses apart, a number of simple and obvious experiments are missing, which would likely give essential information on the relationship between nuclein and the other hitherto known groups [of molecules]. I myself will, as soon as possible, report further news. However, I believe that the given results, albeit fragmentary, are significant enough to invite others, in particular chemists, to further investigate the matter. Knowledge of the relationship between nuclear substances, proteins and other closest conversion products will gradually help to lift the veil which still utterly conceals inner processes of cell growth.”

Concluding Comments

Notable subsequent contributions to collectively “lift that veil” include:

  • Proportions of A, G, C, and T bases in DNA between species by Erwin Chargaff and coworkers in the late 1940s and early 1950s
  • In this same timeframe, Oswald T. Avery et al. and Hershey & Chase experiments proving that DNA, not protein, is the carrier of genetic information
  • The double-helix structure of DNA revealed by Watson & Crick in 1953 as the basis for coding
  • Discovery of messenger RNA (mRNA) by Sydney Brenner, et al. in the 1960s

For those of you who would like to know much more about the life and scientific contributions of Friedrich Miescher, I suggest Kathleen Tracy’s book entitled Friedrich Miescher & the Story of Nucleic Acid (Uncharted, Unexplored, and Unexplained), which I found to be an enjoyable and educational read.

While Watson & Crick may be names most associated with DNA, a German biotech company is determined that the world never forgets Friedrich Miescher. Tübingen-based CureVac, which develops RNA vaccines and therapies (see TriLink’s mRNA/longRNA products), plans to restore the University of Tübingen lab where Miescher made his discovery, according to a report in Nature. That space is now a computer room.

CureVac’s CEO, Ingmar Hoerr, is quoted as saying that Miescher “is one of the most important guys in science, and not many people are aware of it.”

After reading this post, you can now appreciate the basis for that opinion.

Oh, by the way, the principles of DNA isolation is now taught in grammer school, and is a favorite for science fairs, such as that pictured below. If Friedrich Miescher could see this now, what would he say?

As usual, your comments are welcomed.

Visitors of all ages at Georg-August-University Göttingen in Germany are intrigued by demontration of how to extract DNA from tomato (taken from karlovsky.net via Bing Images).

Visitors of all ages at Georg-August-University Göttingen in Germany are intrigued by demontration of how to extract DNA from tomato (taken from karlovsky.net via Bing Images).

Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics

  • Invasive Needles and Scalpels Seen as Passé
  • Noninvasive Sampling Advocates Focusing on Circulating Tumor Cells (CTCs) 
  • New Companies are Pursuing the Liquid Biopsy “Gold Rush”

Biopsy Basics

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

As defined in Wikipedia, a biopsy is ‘a medical test commonly performed by a surgeon or an interventional radiologist involving sampling of cells or tissues for examination.’ Biopsies can be excisional (removal of a lump or area), incisional (removal of only a sample of tissue), or a needle aspiration (tissue or fluid removal). Despite the value of these traditional types of biopsies, they are more or less invasive, lack applicability in certain instances, and require accurately “going to the source” of concern, as pictured to the right, for ultrasound-guided breast cancer biopsy. Better methodology is highly desirable and is the topic of this post. By the way, if you want to peruse a lengthy list of scary risks associated with various type of common invasive biopsies, click here to see what I found in Google Scholar by searching “incidence of complications from biopsies.”

Benefits of Liquid Biopsy

Unlike traditional biopsies that invasively “go to the source” of concern, noninvasive liquid biopsies take advantage of relatively recent recognition that some diseases, such as cancer, discharge. Loosely speaking this means cells and/or other characteristic analytes can be readily sampled in blood, urine or even saliva, which are each effectively the “source going to the sample.” This directional flip-flop, if you will, provides liquid biopsies with several important advantages: non-invasiveness that is inherently safer, much wider applicability, and—some argue—better diagnostics. The latter point is expanded upon in an expert review by Tsujiura et al. as follows:

“Surgical and/or biopsy…approaches cannot always be performed because of their invasive characteristics and may fail to reflect current tumor dynamics and drug sensitivities, which may change during the therapeutic process. Therefore, the importance of developing a non-invasive biomarker with the ability to monitor real-time tumor dynamics should be emphasized. This concept, so called ‘liquid biopsy’, would provide an ideal therapeutic strategy for an individual cancer patient and would facilitate the development of ‘tailor-made’ cancer management programs. In the blood of cancer patients, the presence and potent utilities of circulating tumor cells (CTCs) and cell-free nucleic acids (cfNAs) such as DNA, mRNA and microRNA have been recognized, and their clinical relevance is attracting considerable attention.”

In my opinion, the importance of the aforementioned “ability to monitor real-time tumor dynamics” will become increasingly evident as next-generation sequencing costs continue to decrease, thus enabling a patient’s liquid biopsy samples to be analyzed over more time-points, during which viruses or cancer cells can mutate. Following is a real-life story of the latter situation.

Liquid Biopsies for Cancer: A Case Study

Lynn Lewis had her cancer analyzed an easier way: a simple blood test. Credit Michael Nagle for the NY Times.

Lynn Lewis had her cancer analyzed an easier way: a simple blood test. Credit Michael Nagle for the NY Times.

Every personalized medical story has a face and place, and it was Lynn Lewis of Brooklyn who caught my attention in an article by Andrew Pollack in last year’s NY Times. Ms. Lewis had her non-responsive cancer analyzed by means of liquid biopsies, in the form of blood samples, to take advantage of new tests offered by one of many new start-up companies developing safer tests with more actionable information. In a nutshell, here’s Lynn’s story:

Lynn, who at the time of the story was 54 and working as a self-employed lawyer, had already been battling metastatic breast cancer for seven years. In June of 2013, she had a liquid biopsy test of tumor DNA fragments in her blood developed by Guardant Health—then a relatively new start-up company in Redwood City, CA that was investigating use of next-generation sequencing to look at a panel of genes associated with various cancers. The test found a genetic mutation that suggested her disease would be treatable by the Novartis drug Afinitor.

Liquid biopsy blood tests in a lab at Guardant Health. Credit Jim Wilson/the NY Times.

Liquid biopsy blood tests in a lab at Guardant Health. Credit Jim Wilson/the NY Times.

Puzzlingly, Ms. Lewis had been previously treated with Afinitor and it had stopped working. Still, doctors decided to re-administer the drug to her, and again it didn’t work. A second test several months later found additional mutations but did not help determine the best course of treatment.

A third liquid biopsy, however, indicated significant levels of the protein Her2, which drives tumor growth, in some of Ms. Lewis’s cancer cells. The increase in Her2 may not have been detected previously because liquid biopsies sample only a tiny bit of the tumor, which is typically heterogeneous. While tumor heterogeneity is—in my opinion—an inherently confounding aspect of cancer, regardless of how biopsies are obtained, Ms. Lewis was successfully switched to Herceptin and Tykerb, drugs that specifically target Her2-positive cancers.

Lynn’s case to me is less an example of cancer treatment being “trial and error” and more an illustration of the importance of liquid biopsies as a safe, repeatable means of sampling inherently heterogeneous and dynamically changing cancers to obtain new data in real-time.

As a side note, I should add that liquid biopsies can also provide a valuable “sneak preview” of cancer before detection by traditional means. For example, a study published in The New England Journal of Medicine showed that in some cases a liquid biopsy could detect the worsening of breast cancer five months before it could be “seen” by conventional—and BTW potentially harmful—CT scans. This could allow an ineffective therapy to be abandoned earlier in favor of an alternative drug.

So, what’s not to like about liquid biopsies? Pollack claims that, despite all the potential of liquid biopsies, developers of such tests for the most part have not yet rigorously established their accuracy and—more importantly—actionable clinical utility. In other words, does blood testing really help patients? In my opinion, this is the biggest problem facing routine adoption of these biopsies. While clinical investigators can fairly quickly use newly developed assays to examine liquid biopsies “for research purposes only,” widespread sales for diagnostics requires FDA approval that is usually a very slow process. This is especially true for DNA/RNA-based assays that have to account for genotypic (i.e. sequence) differences between patients who are nevertheless grouped together as having the “same” cancer. Hence the emergence of the new era of so-called “N of 1” personalized medicine.

The New Gold Rush

If you agree with me that discovering clinical utility of, and extracting commercial value from liquid biopsies are metaphorically a new Gold Rush, then liquid biopsies are effectively “liquid gold.” As proof that indeed a “rush” for discovery is on, a recent review in Clinical Chemistry states that “the detection and molecular characterization of CTCs are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker.”

While “N of 1” cases like that of Ms. Lewis suggest utility of Guardant’s tests, Pollack’s article asserts that lack of much more and equally compelling evidence for clinical utility has hindered the acceptance of CellSearch®, the first test to detect and count CTCs. Sold by Janssen Diagnostics, a subsidiary of Johnson & Johnson, it was first approved by the Food and Drug Administration in 2004.

Common sense would lead one to accept that having a large number of tumor cells in the blood is cause for worry because of increased probability that the cancer will progress. But it is not always clear what to do with that information, i.e. what is actionable from a clinical perspective? Pollack quotes Dr. Scott Kopetz, an associate professor at the M.D. Anderson Cancer Center in Houston as saying that it’s not useful “to tell someone you have a high chance of a cancer coming back but we don’t know what to do.” Still, some developers of these diagnostic tests claim that detection of these tumor cells could at least provide evidence of ineffective treatments, saving the patient from suffering unnecessary side effects of drug therapies that aren’t working.

Helmy Eltoukh (left), CEO of Guardant Health, with AmirAli Talasaz, President and CTO, in the lab. Credit Jim Wilson/the NY Times.

Helmy Eltoukh (left), CEO of Guardant Health, with AmirAli Talasaz, President and CTO, in the lab. Credit Jim Wilson/the NY Times.

On the other hand, I found a recent study of women diagnosed with breast cancer by traditional mammography, who were also analyzed for CTCs in blood, the latter of which had limited utility. Notwithstanding low diagnostic yield of CTCs such as in this study, there are a growing number of companies working with CTCs, some of which will likely provide actionable data with clinical utility, but you’ll have to stay tuned for those:

Alere                                                     Cynvenio Biosystems

ApoCell                                                 Epic Sciences

Biocept                                                 Fluxion Biosciences

Clearbridge Biomedics                          Rarecells

Creatv MicroTech                                  ScreenCell

SRI International

But some experts say the momentum is shifting to the newer approach of looking for DNA fragments not inside cells but rather released DNA that enters the bloodstream when cancer cells die, and are phenomenologically referred to as circulating tumor DNA (ctDNA). That ctDNA can provide promising biomarkers for noninvasive assessment of cancer, has already been successfully translated into commercial products by Trovagene, which test for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagostic value of ctDNA.

Importantly, assessment of tumor burden was recently demonstrated for non-small-cell lung cancer by collaborators at Stanford University, who reported an ultrasensitive next-generation sequencing method in Nature Medicine showing that levels of ctDNA were highly correlated with tumor volume and distinguished between residual disease and treatment-related imaging changes. Moreover, measurement of ctDNA levels allowed for earlier response assessment relative to radiographic approaches.

Notwithstanding the relative diagnostic value of “inside v. outside” DNA, various investors are placing their bets, so to speak. Guardant initially raised $10 million from Sequoia Capital, the venture capital firm known for backing Apple, Google and, most recently, WhatsApp. In February, the company raised $50 million for a current total of $100 million, which is obviously a big investment in the potential value of tiny analytes. Other start-ups focusing on ctDNA are Boreal Genomics, and Inostics, which was acquired by Sysmex, a Japanese diagnostics company.

Exosomes

In addition to CTC and ctDNA as sources of potentially actionable diagnostic information, small (30-100nm) exosome vesicles are also being extensively investigated. Interested readers can find extensive details for this approach in Michael D. O’Neill’s BioQuick News summary of the fourth annual meeting of the American Society for Exosomes and Microvesicles.

Here, I’ll simply exemplify this approach by pointing to two cases, the first being the team of Qiagen and Exosome Diagnostics Qiagen that is developing kits to capture and process RNA and DNA from biofluid exosomes and other types of microvesicles. The goal is to provide sample prep technology applicable to patient blood, urine, or cerebrospinal fluid for qPCR or next-generation sequencing.

The second example is a communication by a consortium of Japanese academic, government, and corporate investigators published in 2014 in venerable Nature magazine entitled Ultra-sensitive liquid biopsy of circulating extracellular vesicles involving colorectal cancer. Interested readers will find lots of detail in this report; however, the important “punchline” is that cancer markers were detected in samples with early stage colorectal cancer that are invasive. On the other hand, the authors state that further studies are needed to know whether the reported method reduces colorectal cancer mortality as a screening test.

Tool Providers, Too

“Miner with Pick, Shovel and Pan” ca. 1850. Daguerreotype from the collection of Matthew R. Isenburg (taken from georgetowndivide.wordpress.com via Bing Images).

“Miner with Pick, Shovel and Pan” ca. 1850. Daguerreotype from the collection of Matthew R. Isenburg (taken from georgetowndivide. wordpress.com via Bing Images).

While all of the aforementioned companies seeking “gold” from liquid biopsies have “staked their claims,” they all need tools with which to “dig.” Like selling picks and axes during the Gold Rush of yesteryear, modern day biotechnology tool providers such as Illumina, Bio-Rad Laboratories, and RainDance Technologies—to mention just a few—each hope their tools will be used to test for tumor DNA.

“Looking at the Sun and Trying to See the Stars”

Technically, analysis of ctDNA is like the proverbial finding “a needle in the haystack” because it is present in very small amounts compared to other cellular components that contain DNA—not to mention the slew of other non-DNA components.

Because of its metaphorical impact, my favorite quote in Pollack’s article regarding liquid biopsies is that of Nitin Sood, CEO of Boreal Genomics, who said “it’s like looking at the sun and trying to see the stars.” Mistakes can easily be made. And fewer genes can typically be analyzed from a liquid biopsy than from a conventional tumor biopsy.

The biggest payoff, so to speak, would come if liquid biopsies could detect cancer in seemingly healthy people, when it is still curable. A recent study by researchers at Johns Hopkins University found that tumor DNA could be detected in the blood of about half the 223 patients with localized cancers that they tested. However, since it was already known that those patients had cancer, the study did not demonstrate that a blood test could detect cancer earlier than other methods.

Ms. Lewis, the breast cancer patient discussed at the beginning of this post, told Pollack that she did not fully understand the results of her tests, but she was sharing them publicly in hopes that they provide some insight to scientists. We should note that her tests where performed as part of a research project and she was not billed for them.

Unfortunately, performing and analyzing such tests may not be up to the doctors as the FDA’s view of approving diagnostic tests ultimately comes into play. Since convincing the FDA of clinical utility is a long and expensive process, it’s understandable that there is growing public support for doctors using unapproved tests of patient samples “for research purposes only.” On the other hand, not all doctors—and I suspect very few—want to operate in a research mode, and all are likely more than concerned about possible lawsuits from disgruntled patients—or their surviving spouse or other relative.

As always, your comments are welcomed.

Postscript

Liquid biopsies as an emerging trend in applied nucleic acids analysis is moving very quickly. Here are three noteworthy items that I came across after completing this post.

(1.) HealthDay News reported that, based on analysis of methylated DNA—see TriLink Epigenetic Reagents—in 10 breast cancer-specific genes in CTCs, a new test is able to detect with more than 90 percent accuracy recurrent breast cancer in women.

Dr. Joanne Mortimer, director of women’s cancer programs at the City of Hope Comprehensive Cancer Center, in Duarte, California is quoted as saying: “There is reason to be optimistic, and to study this further.”

However, Mortimer, who was not involved with the new research, said the study only included a small number of patients—55 healthy women and 57 who had breast cancer that had spread—and more study is needed.

“This is incredibly needed,” she added, as other tests on the market aren’t very reliable.

According to Mortimer, the worst part of being treated for early stage breast cancer for patients is the period after treatment is over. “Then they live with this uncertainty. They may be cured, they may not. And only time will tell.”

(2.) Written in Blood is the title of another recent article worth reading was written by Ed Yong and published in Nature magazine as a News Feature that is freely (yeh!) accessible here. The byline summarizes the gist of this report:

“DNA circulating in the bloodstream could guide cancer treatment — if researchers can work out how best to use it.”

(3.) A conference on Biofluid Biopsies & Companion Diagnostics on October 27-28 had a jam-packed agenda on all manner of discovery and commercialization involving CTCs and ctDNA, as well as exosomes. I presented this downloadable poster on TriLink CleanAmp™ PCR and RT-PCT products. Check out the speakers and presentations by clicking here.

(4.) Coincidentally, I’ve just returned from Circulating Biomarkers World Congress 2015 held in Boston, MA where I presented a poster on Improved Small RNA Library Preparation Workflow for Next-Generation Sequencing that allows automation of processing liquid biopsy samples to find low levels of miRNA biomarkers.

Drug Developer’s Dream-Come-True May Be Patient’s Nightmare

  • HCV ‘Miracle’ Cure’s $84,000 Price Tag Causes Significant Controversy
  • 2014 Sales of the Drug Register Over $10 Billion
  • Competition is Rising, but Prices aren’t Getting Lower
  • Hepatitis C Vaccination Seems Elusive

Having been involved in drug discovery and development for many years, I can assure you Gilead’s new drug, Sovaldi™, is truly a drug developer’s dream come true. Solvaldi™ (sofosbuvir) is a drug used to protect against hepatitis C virus (HCV). Compared to other antiviral or anticancer agents, this nucleotide-analog prodrug, pictured below, produces an extraordinarily high cure rate of well over 90% in HCV-infected patients after only a 12-week (84-day) course of treatment that involves simply taking only one pill per day. That’s amazing!

Stereochemical structure of sofosbuvir (taken from positivelyaware.com via Bing Images).

Stereochemical structure of sofosbuvir (taken from positivelyaware.com via Bing Images).

This miracle-like drug, however, comes at a seemingly very high price: a single pill costs $1,000—adding up to a total treatment cost of $84,000 per patient. The estimated cost of manufacturing this pill is only $68-$136, so there’s been quite a bit of media coverage devoted to discussing why the cost to consumers is so high, how this effects drug payment systems, and strong concerns about the viability of treatment in underdeveloped countries where HCV infection is relatively high. This seeming price-gouging is also causing a budget dilemma for whether or not to treat U.S. prisoners who have rampant rates of HCV infection. Before getting to these prickly issues, let’s start at the beginning with the discovery of this “drug developer’s dream-come-true.”

Pharmasset’s Discovery and Gilead’s Acquisition

Pharmasset was founded in 1998 by Profs. Raymond Schinazi and Dennis Liotta at Emory University in Atlanta, Georgia, where its laboratories were set up nearby. Through the sometimes scarily “small world of biotech,” I came to know about this start-up company in the early days because a very good friend of mine (nucleic acids expert, Prof. Wojciech J. Stec) was the company’s Vice President for Chemistry from 2001-2003. In retrospect, Prof. Stec and his team obviously made some great contributions with their R&D programs focused on the development of an oral drug for the treatment of HCV, enabling Pharmasset to go public in 2007.

This viral target was technically challenging because of different HCV genotypes that each have to be combated. On the other hand, there was a compelling medical need since then available FDA approved treatments by interferons or the nucleotide analog, ribavirin, were largely palliative. These treatments fell far short of providing a cure, which is obviously the desired outcome of the ideal drug—certainly from the patient’s point of view.

Pharmasset’s molecular design strategy and synthetic chemistry for an anti-HCV drug candidate eventually led to a very impressive publication in J. Med. Chem. in 2010. The publication was entitled Discovery of a β-d-2-Deoxy-2-α-fluoro-2-β-C-methyluridine Nucleotide Prodrug (PSI-7977) for the Treatment of Hepatitis C Virus, which you should peruse if interested in details. At that time, PSI-7977 was already in Phase 2 clinical trials, and was known to function as a prodrug that undergoes “unmasking” in vivo to ultimately generate the triphosphate form of the nucleotide analog. This triphosphate is the active drug moiety by virtue of its interference with HCV RNA replication required during the life cycle of this virus depicted below.

Simplified representation of the HCV life cycle (taken from Wikipedia with acknowledgement Graham Colm as original uploader).

Simplified representation of the HCV life cycle (taken from Wikipedia with acknowledgement Graham Colm as original uploader).

Concurrently, an R&D team in Foster City, California—about 2,100 miles from Atlanta—was also busily engaged in the pursuit of nucleotide analog prodrugs having structures and mechanism of action quite similar to PSI-7977. That team was from Gilead, a relatively new Big Pharma company that was already profiting handsomely from its very successful anti-HIV drug, Viread®. They too reported in J. Med. Chem. a seemingly equally promising report entitled Discovery of the First C-Nucleoside HCV Polymerase Inhibitor (GS-6620) with Demonstrated Antiviral Response in HCV Infected Patients.

In 2011, just one year after Pharmasset’s aforementioned publication, Gilead stunned the drug development world by announcing its intention to acquire PSI-7977 from Pharmasset for a whopping $11 billion. This was a huge bet to place on a molecule that had not yet been studied in definitive Phase 3 trials in a significant number of patients. My personal opinion is that potential risks of serious side effects that would sideline the drug were offset by the extraordinary efficacy of PSI-7977 after a very short treatment period compared to long-term drug exposure during existing chronic treatments. In any case, this huge bet evidenced to me even bigger “corporate chutzpah” by Gilead.

More specifically, a Bloomberg Business story referred to Pharmasset’s data on PSI-7977 showing that 40 patients who received the therapy were responsive after only 12 weeks. There were no significant adverse events, and most importantly about half the patients had been followed up to 24 weeks, and they were all cured. Although there were no significant adverse events, the drug was said to be tested in combination with ribavirin in patients with HCV genotypes 2 and 3, while genotype 1 is most common and hardest to treat.

Concerns about higher cure rate and genotype-related efficacy have been subsequently swept away by a continuous string of impressive FDA approvals, and the narrative has quickly switched to cost of treatment.

Hepatitis C can be cured globally, but at what cost?

This is the rhetorical and quite knotty question raised by Hill & Cooke in a July 2014 article with the same title published in venerable Science magazine. It has been followed by many other articles expressing collective concern about how the world—literally—deals with the cost of Sovaldi™ for the globally staggering number of patients infected with HCV.

These authors state that worldwide, an estimated 185 million people have been infected with HCV, and that untreated, this can lead to cirrhosis, liver failure, and liver cancer. HCV-related fatalities were said to be as high as 500,000 deaths per year, which represents more deaths than tuberculosis or malaria. Clearly, there is an urgent need to treat this mass of humanity with Sovaldi™, which is now known to cure well over 90% of people with HCV in only 12 weeks. But with the $84,000 cost of treatment, which has raised serious issues in developed (aka “rich”) countries such the U.S., it is impossible to afford in undeveloped (aka “poor”) countries. This “who-can-pay-that-much” dilemma also involves the “why-pay-so-much” question given that Sovaldi™’s estimated manufacturing cost is said by Hill & Cooke to be in the range of only $68-136!

©Saharrr/Fotolia.com

©Saharrr/Fotolia.com

In it’s first full year of sales, Sovaldi™ rocketed to the second highest selling drug of 2014, according to a recent report in GEN. Questions about this high price tag, wider access to this miraculous—in my opinion—drug, and cheaper alternatives continue to swirl around. Gilead argues their drug is not just a treatment for hepatitis C but an actual cure, and the high price tag is worth it, especially when compared to the potential costs of treating liver failure. These cost accounting calculations and comparisons of treatments are far too complex for dredging through here, but I do appreciate the point being made by Gilead, as well as the fact that it’s a for-profit company that has to factor in all of its expenses in bringing Sovaldi™ to market, including the acquisition of Pharmasset, and Gilead’s expenses for failed in-house anti-HCV R&D.

Gilead’s Approach to Treatment Access in Developing Countries

Regarding access by relatively “poor” countries, following is Gilead’s stated position, and then my synopsis of what the company has done in the case of India, by way of example.

“Gilead makes it a priority to increase access to its medicines for people who can benefit from them, regardless of where they live or their economic means. In developing countries, Gilead’s treatment access strategies include tiered pricing, voluntary generic licensing (often in advance of U.S./EU regulatory approval), negotiation with national governments, regional business partnerships, product registration, medical education and partnerships with non-profit organizations. This approach has been successfully applied to Gilead’s humanitarian program in HIV over the past ten years, with six million patients now receiving Gilead-based HIV medicines in developing countries.”

In Sept 2014, Gilead demonstrated its corporate largess by executing non-exclusive licensing agreements with seven generic drug manufacturers located in India to produce sofosbuvir for distribution in 91 developing countries. Importantly, these countries reportedly account for more than 100 million persons infected with HCV, or 54% of all such persons worldwide.

To me, there are three genuinely laudable features of these licensing agreements: (1.) Gilead will provide complete documentation for manufacturing to enable the Indian companies to produce sofosbuvir at-scale as soon as possible; (2.) licensees set their own prices for the drug and pay Gilead only a royalty on sales to cover ongoing sofosbuvir-related expenses; and (3.) there are no restrictions on use of sofosbuvir in various combination therapies.

Competition and Discounting by Abbvie

Not surprisingly, many other companies have new HCV treatments in clinical development, among which Abbvie—spun-out of Abbott Laboratories in 2011—has won recent FDA approval for Viekira Pak™ (a so-called “cocktail” of three drugs) to treat HCV genotype 1. It’s also not surprising that Abbvie has priced this drug at $83,319, essentially the same price as Sovaldi™. However, to compete with Gilead, Abbvie negotiated a discount with Express Scripts—the largest U.S. pharmacy benefit manager. According to a press release, details of the discount are unavailable but are believed to be in the range of Sovaldi™ prices in Western European countries: $51,373 in France and $66,000 in Germany—don’t ask me the rationale for those lower prices vs. $84,000 in the U.S.

Hepatitis C: The Next 25 Years

Michael Houghton (taken from mmi.med.ualberta.ca via Bing Images).

Michael Houghton (taken from mmi.med.ualberta.ca via Bing Images).

That’s the intriguing, future-looking title of a recent article in Antiviral Res. by Michael Houghton. I had the pleasure of working with Michael years ago during a collaboration between Lynx Therapeutics and Chiron to investigate an antisense oligonucleotide for inhibiting HCV—a virus that Michael’s lab discovered at Chiron after nearly 6 years of intensive investigations between 1982 and 1988. For discovery of HCV, Michael was awarded the Lasker Award in 2000—awardees of which frequently go on to a Nobel Prize.

Rather than doing an injustice to Michael’s sage wisdom by poorly paraphrasing, following is the verbatim Abstract of his aforementioned published opinion:

Excellent progress has been made in the field of hepatitis C since the discovery of the causative virus in 1989. Screening tests have been produced to protect the blood supply, along with diagnostics to aid therapeutic management, as well as the recent approval of highly effective small-molecule drugs targeting the viral life cycle, which, given in combination, can now cure the vast majority of patients. Future urgent priorities include facilitating the accessibility of these drugs to all of the world’s estimated 170 million carriers before liver cancer and other end-stage liver diseases occur, as well as producing a vaccine to protect individuals at high risk of infection, such as intravenous drug users. Effective control of this viral infection is now clearly in sight.

Inspired by Michael’s urging for a prophylactic vaccine to prevent infection, I went off to research that topic, but came away with the impression that current prospects are not encouraging. This is partly because there already have been numerous investigations, but like HIV, there are currently dim glimmers of hope for a preventative vaccine. This disappointing situation for HCV has been expertly summarized in a recent lengthy publication I found by Verstrepen et al. Aside from running out of new HCV vaccine design strategies to explore, these authors note with alarm that the scientific world has to deal with “the near disappearance of the most relevant animal model for HCV”—the chimpanzee. They say that “public concerns about research with non-human primates, chimpanzees in particular, has eventually led to stop the use of apes for HCV research in Europe, and a significant reduction of the number of animals used in the United States.”

Although it’s not easy, maybe it’s time for society to rethink its priorities.

As always, your comments are welcomed.

Postscriptong>

After this post was completed, The Wall Street Journal published an interview of Arvind Goyal, a medical director for the state of Illinois, by Ed Silverman entitled How One State Decides Who Gets $84,000 Drug that’s well worth reading. One noteworthy snippet is this Q&A, which revealed the high—to me—proportion of patients who have addiction problems, and the state’s triaging philosophy:

WSJ: Let’s talk about the restrictions. One mentions patients can’t have been abusing drugs or treated for alcohol or illicit drugs for 12 months prior to requesting Sovaldi. But during that time, those folks who go untreated can transmit the virus to others.

Dr. Goyal: Remember that one-third of the population for which we were approving Sovaldi take drugs or alcohol, and nobody ever studied if Sovaldi could be safe or effective for such people. But the disease can be dormant and not show symptoms or signs for up to 30 years after getting in the system. Why is it so bad to tell them they should be sober and take the drug in a dependable fashion? If someone is using a street drug such as heroin I can’t be sure they are compliant taking Sovaldi. It’s a total waste.

Top Picks from Tri-Con 2015

  • “Honey I Shrunk the qPCR Machine” Tops Presentations
  • High School Student Wins Popular Vote for Best Poster
  • BioFire Defense FilmArray is More Interesting Exhibitor
  • Extra Bonus: Swimming with the Sharks

The 22nd International Molecular Medicine Tri-Conference—better known as Tri-Con—took place on Feb 15-20 in San Francisco, where I and 3,000+ other attendees from over 40 countries took part in a jam-packed agenda. In this blog I’ll briefly share my top 3 picks—and an “extra bonus”—but first some insights into the challenges involved in navigating a large conference like this.

The first challenge was scoping out four simultaneously occurring “channels”—diagnostics, clinical, informatics, and cancer—to select as many interesting items as possible from all the presentations (500), panel discussions (30), posters (150), and free “lunch-nars.” The new Tri-Con’15 app with a word and name-searchable agenda (including abstracts) made this easier than previous years. I was even able to put selected items into a calendar/to-do list with 15-min reminder alarms—very slick and convenient. Every big conference should have an app like this!

The second challenge came once I was physically onsite. It took a bit of effort to navigate from one room to another in the huge, multi-room Moscone Center without GPS guidance. I was also struggling to make it to the talks and events on time without getting hijacked by bumping into friends—which happened a lot.

The third and final challenge had to do with posters. Given all of the other exciting options during the conference, I really had to focus to stay on-task and make sure I was present at my poster at the specified times, yet alone try to get around to the other posters of interest. This was definitely not easy, since my poster entitled Pushing the Limits of PCR, qPCR and RT-PCR Using CleanAmp™ Hot Start dNTPs attracted a steady stream of interested visitors. But that’s a great challenge to have, so I can’t complain too much.

Anyway, and without further ado, here are my top 3 picks from the conference. There was an impressive amount of material to choose from and it wasn’t easy to narrow it down, but I did finally decided to limit myself to one presentation, one poster, and one exhibit. Oh, and then I decided to sneak in an extra bonus, all of which I hope you find to be as interesting as I did. 

According to Kiwis, Size Does Matter

My personal photo of Dr. Jo-Ann Stanton holding a Freedom4 qPCR instrument that slides open to access four sample tubes.

My personal photo of Dr. Jo-Ann Stanton holding a Freedom4 qPCR instrument that slides open to access four sample tubes.

My pick for the top presentation, which was actually also a poster and exhibit all-in-one, features the amazing achievement of a group of New Zealanders—that’s right, Kiwis!—at the University of Otago. The team, led by Dr. Jo-Ann Stanton (shown right), recently introduced a first-of-a-kind handheld device for performing real-time or quantitative PCR (qPCR) in the field. This remarkable palm-size instrument—which makes me immediately think “Honey, I shrunk the qPCR machine!”—manages to squeeze a plastic four-well sample (10uL-40uL) strip into a thermal cycling block along with an optical system and still only measure a mere 4 x 8 inches. Not only is it’s size impressive, but the instrument will reportedly perform conventional 40-cycle qPCR with SYBR green or FAM in about 50 minutes. To top it off, results are said to be comparable to “gold standard” laboratory systems. The device is being commercialized as the Freedom4 device by a New Zealand-based start-up company named Ubiquitome, which values this little cutie at $25,000 according to a Feb 25, 2015 press release.

In her talk entitled, A Handheld qPCR Device for Use in the Field, Dr. Stanton said the Freedom4 was tested using World Health Organization and International Accreditation New Zealand assays for E. coli O157, influenza, adenovirus, enterovirus, norovirus, and astrovirus. She added that, in side-by-side “benchmarking” tests with clinical samples using much larger laboratory-based instruments—namely the Roche LightCycler® and Stratagene (now Agilent) Mx3000P systems—the Freedom4 was comparable to, and in one case better than, in-laboratory technology. These tests included measures for sensitivity, precision, and inter-assay variability.

What’s really cool with this thermal cycler—pun intended—is that the user interacts with the device via either a tethered connection to a laptop computer or wirelessly to a smart phone. It also runs on batteries!

Dr. Stanton concluded by saying that, with the correct assay panel, this new technology can be easily carried in a backpack, together with portable sample prep kits, to determine the disease-causing species or reveal antibiotic resistance, all in real time either “cow-side” or in the remote clinic. She also envisages forensic applications.

Teenage Attendee is the Overwhelming Favorite for Top Poster

In addition to the great scheduling feature mentioned above, another cool thing about the Tri-Con’15 app was allowing attendees to vote for the best poster. The winner—who certainly got my vote—was S. Pranav of Monta Vista High School, whose poster was entitled Integrative Network Analysis of Epigenetic and Genomic Data for Colorectal Cancer. Remarkably, it was submitted by a high school-level attendee. Although I was unable to catch him/her for a picture and quote, here is my shortened synopsis of the conference abstract:

The Cancer Genome Atlas Project (taken from bg.upf.edu).

The Cancer Genome Atlas Project (taken from bg.upf.edu).

  • In this work we propose an integrative framework to identify epigenetic and genomic drivers as well as deregulated pathways and crosstalk in colorectal cancer using a Bayesian network model derived from The Cancer Genome Atlas (TCGA) data.
  • We first define a set of seed genes implicated for colorectal cancer in literature and enrich them using prior knowledge of pathways connectivity. We derive a unified Boolean signaling network of all major KEGG signaling pathways and use a novel metric based on node degree and pathway coverage for feature selection enrichment.
  • We found that DNA methylation is a major factor in promoting colorectal carcinogenesis. In particular hypo-methylation of anti-apoptosis genes BCL2, BCL2L1 and BCL2L11 and hyper-methylation of pro-apoptosis genes BAK1, BAX and BID seem to throw apoptosis machinery into disarray in colorectal tumors. Other hypo-methylated oncogenes include AKT1, PIK3CA, BRAF, KRAS and APC.
  • The constructed Bayesian network also points to new epigenetic and genetic pathways, which could give further insight into molecular basis of colorectal cancer.

I’m sure you’ll agree with me and the other attendee voters that this is a VERY impressive study for a high school student, who will undoubtedly go on to make further significant scientific contributions.

New Company with Old Roots Steals Spotlight at Exhibits

Being somewhat of a technophile, making this pick was a toughie because of all the really cool stuff that was exhibited. After taking a few deep breaths and letting my mind wander over what I saw, my pick went to BioFire Defense. Here’s why.

Aside from having an interesting founder/instrumentation/company evolutionary history that dates back to Carl Wittwer and Idaho Technology in the 1990s and merging with bioMérieux in 2014, BioFire Defense is a fascinating company that, according to their website, ‘is committed to making the world a healthier and safer place.’ The company provides integrated products and systems to the biodefense and first responder community. These products and systems are relatively new and certainly very important market segments with rapid growth potential. The very slick, key enabling-technology—in my opinion—is the FilmArray system pictured below.

BioFire FilmAssay system (taken from microbiology.publish.csiro.au)

BioFire FilmAssay system (taken from microbiology.publish.csiro.au)

The FilmArray module shown right in a simplified cartoon is actually an amazingly clever and technically sophisticated fluidic system that is about as turnkey of an instrument as I’ve ever seen. A minimally instructed operator need only add the sample, for example blood, in one port, and then add buffer in another port before pushing the start button. From then on, automated cell lysis, DNA/RNA extraction, and PCR lead to the final yes/no result, all without any user intervention or data analysis by a trained expert.

In addition to this outstanding instrument, the company’s assays are quite notable. Earlier this month, BioFire’s FilmArray® Ebola test was awarded the Frost & Sullivan’s 2014 Global New Product Innovation Award. Given the fervent need for innovation in the field of biodefense, I believe BioFire is a company to keep on eye on.

Bonus Highlights

The inaugural Swimming with the Sharks sessions, which were a take on the TV series Shark Tank that I happen to like, involved twelve start-up companies seeking funding via 5-minute value-proposition “pitches” given to a panel of industry leaders (aka judges). Criteria for judging were Clinical Utility, Investor Readiness, and Healthcare Impact.

The top place winner would receive recognition as the “2015 Tri-Con Most Promising Company”, and services valued up to $15,000 from Sales Performance International (SPI) for personalized consulting. The second place winner would receive $5,000 in SPI services.

The twelve competing companies were:

Magnetic Insight
HeatSeq
Beacon Biomedical
Nanovega
Solano Pharmaceuticals
oncgnostics
DxUpClose
Fluoresentric
FibroTx
Molecular Assemblies
CrackerBio
Veramarx

First place went to Fluoresentric, pitched by William Olsen, who promised investors a radically “faster, better, cheaper” version of PCR named Extreme Chain Reaction (XCR™), which would operate as a plug-in to a smart phone that provides power and allows both acquisition and processing. Cost of parts for this apparently remarkable gizmo were said to be less than $200. Some data was shown, but not in detail. How this “faster and better” variant of PCR is actually achieved or differs from PCR were not described. So—in my opinion—the X in XCR™ represents a mysterious X-factor to be revealed in the future.

Second place went to Molecular Assemblies, pitched by Curt Becker, Founder/CEO, who promised investors a totally new method for making DNA. This method uses commercially available, naturally occurring terminal deoxynucleotide transferase (TdT) together with proprietary, synthetic reversible-terminator A, G, C and T monomers to assemble long fragments of high quality DNA. Becker claimed this is “the way nature makes DNA” and is thus differentiated from conventional phosphoramidite chemistry. The method is further said to be a “cost-effective sustainable approach that uses nature’s enzymes to replace the chemicals—thus Introducing Eco-Genes™—a new generation for ‘writing’ genomes and the next big leap in genomics.”

My personal photo of the twelve startup company representatives taken after their Swimming with the Sharks.

My personal photo of the twelve startup company representatives taken after their Swimming with the Sharks.

Wow, all great stuff at Tri-Con 2015!

As usual, your comments are welcomed.

2014 BioGENEius Challenge Top Honor Announced

  • Premier Competition for High School Students Culminates at BIO International Convention
  • Winner, Emily Wang, Developed New Fluorescent Proteins to Improve Biosensing
  • Her Grandmother’s Battle with Cancer Inspired this Award Winning Research

I don’t know about your science achievements while in high school, but mine were limited to getting up early on Saturdays to go to Biology Club to dissect worm, starfish and cat specimens to study anatomy—and trying not to pass out from noxious formaldehyde preservative! Thus, I am constantly amazed by the level of complexity and maturity that I see in young science students today, and I always look forward to seeing who will win the annual BioGENEius Challenge.

The BioGENEius Challenge is the premier competition for high school students that recognizes outstanding research in biotechnology. The Challenge is organized by the Biotechnology Institute, a U.S. based nonprofit organization dedicated to biotechnology education. Its mission is to engage, excite and educate the public, particularly students and teachers, about biotechnology and its immense potential for solving human health, food and environmental problems.

Emily Wang proudly showing her International 2014 BioGENEius Challenge award winning fluorescent proteins. Photo credit: Emily Wang.

Emily Wang proudly showing her International 2014 BioGENEius Challenge award winning fluorescent proteins. Photo credit: Emily Wang.

This past June, Emily Wang—a graduating senior at Gunn High School in Palo Alto, California—was named the winner of the 2014 International BioGENEius Challenge. A panel of judges found that Emily’s research in developing fluorescent proteins to improve biosensing helped her stand out from the 14 other finalists from across the U.S. and Canada competing for the Challenge’s top prize, a $7,500 cash award.

Emily’s win was announced during a luncheon at the hugely popular 2014 BIO International Convention, and was keynoted by even more famous Sir Richard Branson. The International BioGENEius Challenge is one of the few international competitions to host participants at a leading industry conference, thereby providing students with unparalleled access to companies, scientists and innovators in biotechnology.

Emily was not only evaluated on the quality of her research, but also on her research poster presentation and responses to questions testing her scientific knowledge. Moreover, each student’s research was reviewed for the potential commercial and practical applications of their project.

Computer artwork of the molecular structure of green fluorescent protein (GFP). Some central atoms are represented as spheres. The molecule has a cylindrical structure formed from beta sheets (ribbons). GFP is found in the Pacific jellyfish (Aequorea victoria). It fluoresces green when blue light is shone on it, as depicted here (taken from fineartamerica.com via Bing Images).

Computer artwork of the molecular structure of green fluorescent protein (GFP). Some central atoms are represented as spheres. The molecule has a cylindrical structure formed from beta sheets (ribbons). GFP is found in the Pacific jellyfish (Aequorea victoria). It fluoresces green when blue light is shone on it, as depicted here (taken from fineartamerica.com via Bing Images).

Emily’s project, titled, “Illuminating Disease Pathways: Developing Bright Fluorescent Proteins to Improve FRET Biosensing,” seeks to help visualize disease pathways, including cancer metastases.

Before I get into the details of Emily’s project, I should pause to mention the second, third, and fourth place winners, who won $5,000, $2,500 and $1,000, respectively. Those winners were:

  • Logan Collins, Fairview High School, Boulder, CO
  • Neil Davey, Montgomery Blair High School, Silver Spring, MD
  • Nathan Han, Boston Latin School, Boston, MA

Emily’s Project for the International 2014 BioGENEius

With the help of Gayle Kansagor of the Biotechnology Institute, I contacted Emily to get more specific information about her award winning research project for this blog post, and inquire about the availability of the fluorescent proteins that she developed. Emily kindly provided me with the following first-person account.

Layman’s Description

I aimed to create a tool to visualize diseases at the molecular level. I developed Clover3, a bright green fluorescent protein, and mRuby3, a bright red fluorescent protein. Clover3-mRuby3 can be used to image neurons to investigate Alzheimer’s disease, detect and track cancer growth, and observe biological activities with greater clarity than before.

Abstract

The discovery and development of fluorescent proteins, recognized by the 2008 Nobel Prize in Chemistry, enabled a revolution in biological microscopy and sensing. Biosensors employing fluorescence resonance energy transfer (FRET) between fluorescent proteins are powerful tools to non-invasively report biochemical events within living cells. The development of new FRET sensors, however, remains difficult, often due to low FRET dynamic range, which worsens detection of subtle or transient cellular responses. I engineered a new green fluorescent protein Clover3, which confers increased FRET dynamic range onto biosensors and shows improved quantum yield. I also developed a new red fluorescent protein mRuby3, which improves extinction coefficient. Clover3-mRuby3 may benefit a variety of biomedical applications, including imaging of neural structures to investigate Alzheimer’s disease, detecting and tracking of cancer metastases, and monitoring signaling pathways to elucidate disease mechanisms. Clover3-mRuby3 may be used to visualize biological activities with greater clarity than before, which may advance current understanding of illnesses to create new therapies and medicine to improve human health.

Motivation

When I was little, my paternal grandmother passed away due to Stage IV brain cancer. The late detection of cancer filled me with indignation, and I longed to understand how cancer furtively grew and spread. I wanted to create a biological imaging tool, a bright fluorescent protein, to help researchers visualize cancer activities at the molecular level. After reading countless medical papers about fluorescent proteins, I became convinced that the future of cancer research lies in creating new imaging tools from fluorescent proteins. I learned that the innovative field of fluorescent protein engineering had potential to revolutionize cancer research. I also learned, however, that current fluorescent protein technology still had limitations, such as poor photostability and low fluorescence resonance energy transfer (FRET). I wanted to improve current fluorescent proteins and develop a new one that could serve as a valuable tool for scientists to understand disease pathways. With my interest in fluorescent proteins, I contacted Professor Michael Lin at Stanford University’s Department of Bioengineering, which was how I started my current project.

Publication Potential for and Availability of Emily’s Proteins

I asked Emily about the availability of the new fluorescent proteins for research use and she said that one of the earlier proteins, Clover2 can be found on AddGene. Not having heard about AddGene, I visited its website and learned that Addgene is a non-profit plasmid repository which is “dedicated to helping scientists around the world share high-quality plasmids.” Moreover, it has been amazingly effective—in my opinion—at achieving this goal: over the past ten years Addgene has shipped over 350,000 individual plasmids to 5,000 different research institutions. If you’re interested in knowing which of these are the “Top 10” plasmid technologies that have been distributed, you can consult this list.

When I asked Emily about whether this work was being published, she said that it was going to be. However, as of writing this post, I couldn’t find a citation in PubMed, so we’ll have to wait for that publication. I also inquired about a patent application, and was told by her that this was in the process of being dealt with.

Concluding Comments

If you’re wondering, as I was, where Emily has gone for further education, I connected with her on LinkedIn, and learned that she’s a student at Harvard University—undoubtedly studying hard and looking forward to doing more great research.

Emily’s research is a prime example of how sophisticated high school science has become, partly due to increased awareness of the importance of biotechnology in all aspects of modern society.

BTW, in contrast to Emily’s DNA-based plasmid delivery of encoded biosensor proteins, I find it interesting and exciting that modified mRNA is now being used as an alternative approach that provides various advantages. TriLink has recognized the importance of this new methodology, and now offers modified mRNAs encoding a wide variety of reporter proteins that include several popular “colors” of fluorescent proteins. You can find out more about these and other mRNA products on the TriLink website.

As always, your comments are welcomed.

Postscript

As mentioned in Emily’s abstract above, the importance of green fluorescent proteins (GFPs) was recognized by the 2008 Nobel Prize in chemistry, which was jointly awarded to Osamu Shimomura of Boston University and Marine Biological Laboratory, Martin Chalfie of Columbia University, Roger Y. Tsien at the Department of Chemistry and Biochemistry, University of California, San Diego.

teachers

The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins derived from GFP and dsRed. Taken from Wikipedia.

The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins derived from GFP and dsRed. Taken from Wikipedia.

Because of the transformative utility of GFPs and modified analogs having different colors as labels, there are many versions with different excitation and emission properties to suite specific requirements. I couldn’t resist including this cute illustration of spectral diversity provided by the Tsien Laboratory in sunny San Diego.

Back to the Future with RNA and XNA

  • The Prebiotic ‘RNA World’ Predates our DNA-Based Genetics  
  • What About Other Life-Forming Nucleic Acid (XNA)? 
  • New Research Discovers Synthetic XNA Providing Possible Clues 
Taken from blackfilm.com via Bing Images.

Taken from blackfilm.com via Bing Images.

Hopefully, I’ve piqued your interest by hijacking the award-winning film title Back to the Future as the lead-in for this blog. Now let me briefly outline why it’s apropos for coupling with RNA and XNA—i.e. molecules like RNA and DNA but curiously different, as you’ll see herein.

My metaphorical connection between Back to the Future and nucleic acids comes from currently captivating theories about RNA preceding DNA in molecular evolution of life, and—more intriguingly—whether XNA played a primordial role back then, or might in the future.

Said another way, evolution of all forms of life must logically derive from molecular evolution—in my opinion. Consequently, current terrestrial life-encoding genetic molecules of DNA and RNA must have a past and future. But what molecules did they arise from eons ago, and what molecules will they possibly become eons from now?

While we don’t have Doc’s time machine to go back eons to learn what was—or travel eons forward to learn what will be—some clever scientists in the recent past, and many more now, have been investigating how we, like Marty, can conjure compelling theories of what might be foreseen by looking back in time, in a molecular sense.

Ancient, Prebiotic Genetics

As previously commented on here, researchers have been interested in molecular evolution since 1924 when Soviet biologist Alexander Oparin proposed a theory of the origin of life on Earth through gradual chemical changes in carbon and other key atoms in the “primordial soup” of evolving matter. Following Watson & Crick’s discovery of the genetic code in double-stranded DNA in the 1950s, and Sydney Brenner’s elucidation of the existence of mRNA in the 1960s, increasing attention has been directed to how these two particular classes of nucleic acid molecules became the principal basis of all living organisms today.

Taken from disinfo.com via Bing Images.

Taken from disinfo.com via Bing Images.

The short version of this fascinating topic, which can be read about in detail in a lengthy review by Gerald Joyce, proposes that there was an initial evolution of what Walter Gilbert called ‘the RNA World” in his 1986 Nature publication with that provocative title. This prebiotic RNA-centric stage of molecular evolution on Earth is supported by data showing that folded, 3-dimensional RNA structures can have catalytic functions (ribozymes). Moreover, this functionality includes RNA replication using molecular “building blocks” that may also arise from ribozyme-mediated metabolism.

According to Joyce, RNA is capable of performing all of the reactions of protein synthesis; however, this “crowning achievement” of the RNA world “also began its demise.” Thus, evolution of proteins began to provide protein enzymes to increase molecular diversity and—importantly—lead to DNA building blocks for creating DNA-based genomes that were more stable and complex than RNA.

Timeline of events in the early history of life on Earth, with approximate dates in billions of years before the present. Taken from Joyce in Nature.

Timeline of events in the early history of life on Earth, with approximate dates in billions of years before the present. Taken from Joyce in Nature.

What are the Xs in XNAs?

While the aforementioned sequence of molecular evolution pictured above is widely held to be a compelling theory, puzzling questions remain as to how and why RNA and DNA specifically evolved from a complex, molecularly “cluttered”—according to Joyce—cauldron of prebiotic chemicals that presumably included all sorts of nucleic acid-like molecules.

One theoretical viewpoint is that there is a fine balance of dynamic forces between molecular entities which are unstable enough to be reactive—thus being able to form new, larger conjugates—yet stable enough to persist—thus serving either as templates for replication or enzymes to create protein enzymes. Simply put, very special chemical properties of RNA, DNA, and proteins enabled these molecules to become the “big winners” here on Earth as life evolved.

If this is the case, are there “losers” that nevertheless have genetic potential under conditions different from those presently existent on Earth? Maybe under conditions that will exist eons from now, when terrestrial and climatic conditions change drastically. Or perhaps these “losers” could thrive today if present on other so-called “Goldilocks planets” having conditions favorable for some form of life.

All of this leads to what Taylor et al. have stated succinctly in their recent Nature publication that has received much attention:

Catalysis by nucleic acids (and by biopolymers in general) requires as a minimum the presence of chemically functional groups and a framework for their precise arrangement. Synthetic genetic polymers (XNAs) with backbones based on congeners of the canonical ribofuranose share with RNA and DNA a capacity for heredity, evolution and the ability to fold into defined three-dimensional structures, forming ligands (aptamers). 

Remarkably, they have discovered that XNAs (aka Xeno nucleic acids)—exemplified by the structures shown below—can support the evolution of enzymes (XNAzymes), which is considered a key event in the origin of life, pre-dating the appearance of protein enzymes.

structures

XNAzymes comprised of repeating units of ANA, FNA, HNA, or CENA were found by screening corresponding pools of random-sequence oligomers to find specific sequences that exhibited RNA endonuclease (“cutting”) or RNA ligase (“joining”) activities. They also discovered a FNAzyme that ligates FANA oligomers.

These discoveries, which were made using TriLink’s triphosphates of ANA and triphosphorylated RNA, led Taylor et al. to offer the following conclusion:

Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on Earth and elsewhere in the Universe.

In my opinion, this purposefully vague, scientifically couched statement implies that these stunning discoveries with XNA support the possibility of non-DNA/non-RNA–based forms of life on Earth under different “boundary conditions” that may exist in the future, or on other planets—now.

Regarding the latter, Benner et al. have reviewed the principles of organic chemistry and concluded that life—defined as a chemical system capable of Darwinian evolution—may exist in a wide range of environments. These include non-aqueous solvent systems at low temperatures, or even supercritical dihydrogen-helium mixtures that exist in the Universe. They noted that the only absolute requirements may be a thermodynamic disequilibrium and temperatures consistent with chemical bonding.

Incidentally, if you’re curious (as I am) about the availability of fluorine in the early Universe for possible incorporation into FNA, I found a publication on calculations that support the presence of HF and F shortly after the Big Bang.

After researching and thinking about all of the aforementioned science, my mind conjured up Star Trek-like visions of traveling at warp speed “to boldly go where no man has gone before” in search for XNA-based life. Alas, I was born too soon for that.

Human, Klingon, Cardassian and Romulan representatives meet their primeval ancestor in the Alpha Quadrant. Taken from Wikipedia “The Chase” (Star Trek: The Next Generation; Season 6, Episode 20).

Human, Klingon, Cardassian and Romulan representatives meet their primeval ancestor in the Alpha Quadrant. Taken from Wikipedia “The Chase” (Star Trek: The Next Generation; Season 6, Episode 20).

On the other hand, I believe that intergalactic planetary exploration will indeed happen in the future. I’ve always enjoyed reading “The Chase” (Star Trek: The Next Generation; Season 6, Episode 20), which involves Starship Enterprise Captain Picard (Patrick Stewart) and crew discovering puzzling “number blocks” that they ultimately deduce to be fragments of “compatible DNA strands” (think XNAs) recovered by others from different worlds all over the galaxy. The crew eventually believe that they have discovered an “embedded genetic pattern that is constant throughout many different species.” They speculate that this was left by an early race that pre-dates all other known civilizations, and would ultimately explain why so many races are humanoid.

Needless to say, I also enjoyed researching and composing this blog, which I hope you found interesting.

As always, your comments are welcomed.

Better Brewing and Biotechnology

  • After 8,000 Years of Brewing We may Finally be Getting Better Beer!
  • Brewing Craft Beers: it’s All in the Genes
  • An Old Industry Adopts New Ways

Beer Statistics

Later in this post I’ll get to nucleic acids, but let’s start with some brewing statistics that I think you’ll find impressive regardless of whether you drink beer, sip wine, imbibe spirits, or even abstain from alcohol.

Beer is very popular, based on worldwide statistics for beer consumption that speak volumes—pun intended. From one report, I estimated that this year about 200 billion liters of beer will be drunk—no pun intended. Using the real-time Population Clock (which, BTW, is almost scary to watch), I reckon that there are about 5 billion not-too-young-or-old potential beer drinkers on the planet, so that’s 40 liters of beer this year for each of these folks.

Another report I found states that, based on total volume, China consumes about 50 billion liters of beer each year, out-drinking its nearest rival (the United States) by more than 2x! The 24 billion liters consumed by the US, surpasses 3rd place Brazil who consumes 14 billion liters annually. Russia and Germany round out the top five at 9 billion liters each.

If you’re wondering about consumption on a per capita basis, a rank-ordered list of the top 50 countries is available here, with Czech Republic 1st at a whopping ~150 liters/person and India 50th at only ~2 liters per person. Notably, the US is 14th at ~77 liters per person.

Brewing Basics

A 16th-century brewery. The Brewer, designed and engraved in the Sixteenth Century, by J. Amman (taken from Wikipedia).

A 16th-century brewery. The Brewer, designed and engraved in the Sixteenth Century, by J. Amman (taken from Wikipedia).

Clearly, beer is thoroughly enjoyed around the world, so let’s explore the brewing process in a bit more detail. Brewing is simply the production of beer via the breakdown of starch and fermentation of sugar, which, according to Wikipedia, has been going on since around 6,000 BC. Archaeological evidence suggests most emerging civilizations starting with ancient Egypt and Mesopotamia (which is now Iraq) brewed beer in some fashion.

Fast forward about 7,500 years to 1487 AD, when Albert IV, Duke of Bavaria promulgated the Reinheitsgebot (German for “purity order”), sometimes called the “German Beer Purity Law,” specifying three ingredients—water, malt and hops—for the brewing of beer. Since this time-period precedes microbiology, yeast—the necessary fourth ingredient—was not explicitly specified but was fortuitously present in one or more of the other three ingredients.

Hop cone in a hop yard in Hallertau, Germany (taken from Wikipedia).

Hop cone in a hop yard in Hallertau, Germany (taken from Wikipedia).

I know you are familiar with the first ingredient, water, so let’s skip to the good stuff. Malts are derived from cereal grains—primarily barley. When fermented, malt develops enzymes such as proteases, which produce sugars to feed the yeast during the brewing process. Hops are the female flower clusters, or seed cones, of the hop vine Humulus lupulus, and are used as flavor and preservatives in beer. Hops had been used for medicinal and food flavoring purposes since Roman times. By the 7th century, Carolingian monasteries (in what is now Germany) were using hops to make beer, though it wasn’t until the 13th century that widespread cultivation of hops for use in beer was recorded.

While we’re about to discuss yeast and it’s connection to nucleic acid analysis, it’s worth mentioning that hop-derived flavors have led to a number of contemporary genetic analyses. If you’re a fanatical “hop head” beer lover and/or interested in nucleic acid-based applications related to hops, you may find this publication by the Hopsteiner company to be interesting.  This study represents the use of SNPs as molecular markers to more efficiently track favorable traits (e.g., flavor and aroma) during cultivation of new hops.

It’s all in the Yeast!

Although hops are a major contributor to the flavor and aroma of beer, these enjoyable properties also come from the byproduct of yeast growth during the fermentation process. These byproducts form carbon dioxide and, some would say most importantly, alcohol—without which beer would have far less popularity.

Saccharomyces cerevisiae are rather uninteresting looking microorganisms that have exceptionally important utility (taken from Wikipedia).

Saccharomyces cerevisiae are rather uninteresting looking microorganisms that have exceptionally important utility (taken from Wikipedia).

The dominant types of yeast used to make beer are Saccharomyces cerevisiae, known as ale yeast, and Saccharomyces uvarum, known as lager yeast. Also popular are Brettanomyces, which ferments lambics—a type of Belgian beer—and Torulaspora delbrueckii, which ferments Bavarian weissbier. Before the role of yeast in fermentation was understood, fermentation involved wild or airborne yeasts, and a few styles such as lambics mentioned above still use this method today. Emil Christian Hansen, a Danish biochemist employed by the Carlsberg Laboratory, developed pure yeast cultures which were introduced into the Carlsberg brewery in 1883, and pure yeast strains are now the main fermenting source used worldwide.

Since there are thousands of possible Saccharomyces cerevisiae available for brewing beer and they all look similar, it is critical to identify the “good” yeasts and to reject the undesirable, “bad” yeasts. This is where modern nucleic acid-based analysis comes into play, providing tools for characterizing genes responsible for desirable properties of beer.

The Genes of Craft Beer

Clearly yeast is critical to the flavor and aroma of beer. These seemingly simple organisms have been studied extensively, but the connection between the genetic makeup of the yeast and the brewing properties that result from it are not well understood. Recently, two research teams set out to change this and hopefully determine what in the yeast’s genetic code is responsible for the flavor profile they produce.

Genetic mapping of yeasts could lead to custom brews. Sandy Huffaker for The New York Times.

Genetic mapping of yeasts could lead to custom brews. Sandy Huffaker for The New York Times.

The two labs participating in this project are White Labs, a Southern California yeast distributor, and a Belgium lab formed by a collaboration between the Flanders Institute for Biotechnology and the University of Leuven. The researchers involved in this project have sequenced more than 240 strains of yeast from around the world. They will use this sequencing data and the information from over 2,000 batches of beer to try to generate new yeast strands that exhibit particular flavors and properties. Dr. Kevin Verstrepen, director of the Belgian lab involved in this study, says “in a few years we might be drinking beers that are far different and more interesting than those that currently exist.”

Click here to read more about this project in an article reported by William Herkewitz of The New York Times.

The Beer Industry Comes into the 21st Century

The project discussed above represents the remarkable pace of advancement in sequencing as well as a paradigm shift within the beer industry itself. We can all remember how long and costly the Human Genome Project was (almost 10 years and $3 billion, if you lost track). Just over a decade later, a complete yeast genome can be sequenced in a matter of days for a few thousand dollars. But what’s possible through science is meaningless if brewers refuse to adopt the technology. As stated by Randy W. Schekman, a yeast geneticist at the University of California, Berkeley, “until recently, the brewing industry has been remarkably resistant to using the techniques of genetics and molecular biology to improve their brewing strains. It’s long overdue that someone has actually delved into the molecular basis between the differences in brewing strains.

Nice work, if you can get it: Pete Slosberg, the founder of Pete’s Wicked Ale, sampled beer at the lab. Credit Sandy Huffaker for The New York Times.

Nice work, if you can get it: Pete Slosberg, the founder of Pete’s Wicked Ale, sampled beer at the lab. Credit Sandy Huffaker for The New York Times.

I agree—8,000 years is definitely a long time! I look forward to seeing—and hopefully tasting—the results of this nucleic acid-based analysis coming soon to a brewery near me. As usual, your comments are welcomed.

Cheers! Santé! Prost! Salute! Nazdrowie!

Postscript

Intrigued by the role of yeast in brewing, I did a Google Scholar search of “flavor compounds” (all the words) combined with “brewers yeast” (exact phrase) for publications since 2010. Here’s a sampling of found snippets that seemed quite interesting to me:

Yeast: the soul of beer’s aroma—a review of flavour-active esters and higher alcohols produced by the brewing yeast

EJ Pires, JA Teixeira, T Brányik, AA Vicente – Applied microbiology and …, 2014 – Springer… Delvaux FR (2009) Impact of pitching rate on yeast fermentation performance and beer flavour. …T, Ferreira IM (2013) Evaluation of brewer’s spent yeast to produce flavor enhancer nucleotides …HP (1978) The isolation and identification of new staling related compounds form beer …

Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers’ yeast

CT Duong, L Strack, M Futschik, Y Katou, Y Nakao… – Metabolic …, 2011 – Elsevier… in optimizing their yeast strains, particularly with regard to beer stability, the development of novel flavors and economics of … Diacetyl has a butter-like flavor and is particularly undesirable in lager beers. … The latter compound is an intermediate of the valine biosynthetic pathway. …

Monitoring of the production of flavour compounds by analysis of the gene transcription involved in higher alcohol and ester formation by the brewer’s yeast …

Y He, J Dong, H Yin, P Chen, H Lin… – Journal of the Institute …, 2014 – Wiley Online Library… the understanding of gene-regulating mechanisms and biosynthetic pathways of aroma-active compounds during yeast … JP, Winderickx, J., Thevelein, JM, Pretorius, IS, and Delvaux, FR (2003) Flavor-active esters … Part I: Flavour interaction between principal volatiles, Tech. …