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. …

San Diego Shines Among Top 10 Biotech Product Innovators in 2014

  • Five of the Top 10 Biotech Products Picked by The Scientist were Developed by San Diego Companies
  • Most of the Innovations Involve Genomics
  • List Includes Several Repeat Winners as well as new Leaders in Cutting-Edge Technology

Recently The Scientist published its annual list of top 10 innovations for 2014. There are several repeat winners this year, including Illumina with two new sequencers and Leica Microsystems with a new 3D superresolution microscope. There are also a number of exciting new products associated with cutting edge technology in fields like human organ models and a Twitter-like site to handle the ever-increasing number of scientific publications. One of the most notable attributes of this year’s list, however, is that half of the award winning companies are based right here in San Diego.

Before zoning in—pun intended—to biotech in the San Diego area, let’s take a quick look at the map below that shows the number of biotech R&D organizations in the U.S. as of August 2014. Probably not surprising to many of you, California is the numerical leader (821), followed by Massachusetts (267), Maryland (173), New York (138), Pennsylvania (134), and New Jersey (110). While these East Coast runners-up in aggregate are a geographical biotech hub numerically comparable the Golden State, the sheer magnitude of California’s biotech industry is unquestionably impressive.

This colorized map highlights regional variations in the number of organizations in the biotech R&D sector. Taken from caliper.com via Bing Images.

This colorized map highlights regional variations in the number of organizations in the biotech R&D sector. Taken from caliper.com via Bing Images.

Money wise by city, San Francisco ($1.15B), Boston ($933M), and San Diego ($387M) were the top three for venture capital investing in 2013, according to data assembled by Thompson Reuters. Job wise, 1.3 million people are directly involved in biosciences, with another 5.8 million workers in related industry sectors, according to recent government statistics for the U.S. biotech industry. That’s 1 out of every ~17 full-time employees in the U.S., which is a heck of lot of biotech brains and brawn!

San Diego’s Biotech Beach is Booming

Over the years, San Diego has become known as Biotech Beach—southern California’s answer to the San Francisco Bay Area moniker of Bay Biotech. While many of us have always found Biotech Beach to be inspiring and stimulating, it’s nice to see that the area is getting some official recognition.

Click here for an interactive version of this map of Biotech Beach taken from by BioSpace.

Click here for an interactive version of this map of Biotech Beach taken from by BioSpace.

As mentioned at the beginning of this post, San Diego is home to 5 of the top 10 product innovations of 2014 reported in The Scientist. You can see in the map above that TriLink BioTechnologies is literally in the middle of Biotech Beach where this innovation occurred—along with loads more of biotech R&D. So, I thought it apropos to briefly mention each of The Scientist’s notable products. With a mental drum roll, here’s the rank-ordered list:

  1. DRAGEN Bio-IT Processor developed by Edico Genome (La Jolla), shrinks the physical bulk of genomic analysis to a chip that could be installed in a server the size of a desktop computer, drastically shrinking the cost of data analysis.
  1. MiSeqDx from Illumina (San Diego) is a breadbox-size gadget that brings next-generation sequencing to clinical labs.
  1. HiSeq X Ten, the newest sequencer from Illumina (San Diego), reaches a long-anticipated milestone: the $1,000 human genome.
  1. IrysChip from BioNano Genomics (San Diego) provides a high-throughput platform for visualizing large-scale genomic structure, with applications for mapping, assembly, and evolutionary analyses.
  1. RainDrop Digital PCR System developed by RainDance Technologies (Billerica, Massachusetts) provides a droplet digital PCR platform with sensitivity and specificity for a wide variety of applications.
  1. TCS SP8 STED 3X by Leica Microsystems (Buffalo Grove, Illinois) is a new generation, super-resolution microscope allowing 3D imaging at several frames per second.
  1. exVive3D Liver from Organovo (San Diego) may eventually obviate the need for animal models in a number of fields, from drug development to environmental toxicology.
  1. HAP1 Knock-Out Cell Lines developed by Haplogen Genomics (Vienna, Austria) uses CRISPR-Cas9 technology to knock out any gene a customer wants to target.
  1. PreciseType Human Erythrocyte Antigen Test from Immucor (Norcross, Georgia) is an array-based test for faster, better blood donor/patient matching critical for transfusions.
  1. Sciencescape offered by Sciencescape (Toronto, Ontario, Canada) uses big data to allow users to rapidly navigate thousands of papers to find, monitor, and share research publications of interest.

In closing, I should note that there’s always been a spirit of innovation in and around San Diego, which is one of the reasons the founders of TriLink were inspired to start their own company here almost 20 years ago. I think I can safely speak for all of us involved in the San Diego biotech community when I say that we are beyond excited to see what will emerge from this area over the next 20 years!

Very cool.

Taken from hayadan.org via Bing Images.

Taken from hayadan.org via Bing Images.

Please take a moment to share your favorite products from 2014 in the comments section below. I look forward to hearing about the innovations that you find particularly intriguing.

Thank You & Happy Holidays!

I’d like to thank all of the loyal readers of my blog. Writing these posts brings me much joy and I’m honored that so many of you enjoy reading them. I hope all of you have a chance to spend the holidays celebrating with your friends and loved ones. I look forward to seeing what trends will develop in the field of nucleic acids in 2015, and I invite you to stay tuned and discuss these exciting advances with me. I’ll be back on January 5 with the first blog post of the year.

Happy New Year!

Broccoli May Reduce Symptoms of Autism

  • Study Reports Remarkable Response to Broccoli Extract
  • Autism Advocacy Group Offers Cautious Optimism
  • Father/Son Cozy Combo for Commercialization Pathway

I’m quite sure that most of you, like me, are very familiar with various ways to improve or preserve your well-being by doing such things as adopting a Mediterranean diet, eating less red meat and more chicken, fish and beans, drinking a glass of red wine, and so on. And like me, you might be somewhat skeptical of the actual benefits but, nevertheless, try to follow some of these recommendations—especially if you enjoy a nice merlot.

A small new study found that a chemical in broccoli sprouts may help alleviate the symptoms of autism. Credit: James Baigrie/Getty Images. Taken from ABC News.

A small new study found that a chemical in broccoli sprouts may help alleviate the symptoms of autism. Credit: James Baigrie/Getty Images. Taken from ABC News.

Having said that, I was tempted to ignore a very recent ABC News story with a headline that read “Broccoli Sprout Extract May Help Curb Autism Symptoms” were it not for two things: firstly, autism is a very common and challenging disorder, and secondly the story referred to a publication in the Proceedings of the National Academy of Sciences (PNAS), which is a very highly regarded scientific journal. So, let’s consider some facts about autism, and then delve into the publication.

Autism Facts

Here are selected facts about autism taken from NIH.gov—my “go to” source of reliable information about all things health related.

What is Autism?

Autism is more accurately referred to as “autism spectrum disorder” (ASD) because it covers a wide range of complex neurodevelopment disorders, characterized by social impairments, communication difficulties, and restricted, repetitive, and stereotyped patterns of behavior. Classical ASD is the most severe form of ASD, while other conditions along the spectrum include a milder form known as Asperger syndrome, as well as childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD-NOS). Although ASD varies significantly in character and severity, it occurs in all ethnic and socioeconomic groups and affects every age group. Experts estimate that 1 out of 68 persons have an ASD. Interestingly, males are four times more likely to have an ASD than females.

Taken from giniesayles.com via Bing Images.

Taken from giniesayles.com via Bing Images.

What are Some Common Signs of Autism?

The hallmark feature of ASD is impaired social interaction. As early as infancy, a baby with ASD may be unresponsive to people or focus intently on one item to the exclusion of others for long periods of time. A child with ASD may appear to develop normally and then withdraw and become indifferent to social engagement.

Many children with an ASD engage in repetitive movements such as rocking and twirling, or in self-abusive behavior such as biting or head-banging. Children with an ASD don’t know how to play interactively with other children. Some speak in a sing-song voice about a narrow range of favorite topics, with little regard for the interests of the person to whom they are speaking.

Children with characteristics of an ASD may have co-occurring conditions, including Fragile X syndrome (which causes mental retardation), epileptic seizures, Tourette syndrome, learning disabilities, and attention deficit disorder.  About 20 to 30 percent of children with an ASD develop epilepsy by the time they reach adulthood.

What Causes Autism?

Given the prevalence of autism, you may find it surprising that scientists aren’t certain about what causes ASD, although there’s agreement on the likelihood that both genetics and environment play a role—nature vs. nurture. Researchers have identified a number of genes associated with the disorder. Studies of people with ASD have found irregularities in several regions of the brain. Other studies suggest that people with ASD have abnormal levels of serotonin or other neurotransmitters in the brain. These abnormalities suggest that ASD could result from the disruption of normal brain development early in fetal development. Where the disruption is caused by defects in genes that control brain growth and regulate how brain cells communicate with each other, possibly due to the influence of environmental factors on gene function.

Do Symptoms of Autism Change Over Time?

Thankfully for many children, symptoms improve with treatment and with age; however, children whose language skills regress early in life—before the age of 3—appear to have a higher than normal risk of developing epilepsy or seizure-like brain activity. During adolescence, some children with an ASD may become depressed or experience behavioral problems, and their treatment may need some modification as they transition to adulthood. It’s also encouraging to know that many people with an ASD are able to work successfully and live independently or within a supportive environment.

What Research is Being Done?

NIH is currently funding and coordinating 11 Autism Centers of Excellence (ACE). The ACEs are investigating early brain development and function, social interactions in infants, rare genetic variants and mutations, associations between autism-related genes and physical traits, possible environmental risk factors, potential new medications and biomarkers.

I’m “big on biomarkers” for all diseases, and was pleased to find a promising press release about Stemina Biomarker Discovery receiving a $2.3 million investment from the Nancy Lurie Marks Family Foundation to support its clinical study of biomarkers in the blood of children with ASD. Using blood samples, Stemina was able to distinguish patients with autism from typically developing children with 81% accuracy, which I think is amazingly good given the complexity of ASD. Moreover, Stemina CEO Elizabeth Donley was quoted as saying “What is exciting about the data we are generating … is that we are beginning to identify metabolic subtypes in comparing one child with ASD to another. This has the potential to revolutionize the way children are diagnosed and treated based on the individual’s metabolism.”

PNAS Publication on Autism and Broccoli

Now that we’ve covered the basics of autism, let’s see how broccoli may help ease ASD symptoms. Full details of this collaborative study are freely available here in a PNAS publication from this past September entitled “Sulforaphane treatment of autism spectrum disorder (ASD).” The investigators outline four premises that led them to test treatment of ADS with sulforaphane, which is an isothiocyanate derived from broccoli—as well as other cruciferous vegetables such as Brussels sprouts or cabbages—and has the remarkably simple molecular structure shown below.

Taken from Wikipedia.

Taken from Wikipedia.

Here’s a short synopsis provided by Dr. Cindy Haines on MedlinePlus, which is a part of NIH that has the stated objective of providing “trusted health information for you.”

Researchers included 44 young men with moderate to severe ASD in a small study involving the chemical sulforaphane. The participants, ages 13 to 17, were randomly assigned to take either a daily dose of sulforaphane extracted from broccoli sprouts or placebo. Investigators and caregivers were not told who was receiving the active substance. Behavior and social interaction were evaluated at the start of the study and then at 4, 10 and 18 weeks. Half of the teenagers underwent a final assessment about a month after treatment ended.

The results:

  • At 18 weeks, 46% of the sulforaphane recipients showed significant improvement in social interaction,
  • 54% in atypical behavior
  • and 42% in verbal communication.

The researchers say improvements were so noticeable that by the end of the treatment period, both staff and family members correctly guessed treatment assignments. Still, they point out the chemical did not work for everyone.

About one third of those taking sulforaphane showed no improvement. They say a larger study including adults and children is needed to confirm any therapeutic benefits.

Comments on this Study by Autism Speaks®

To get independent, informed opinions about this study, I consulted Autism Speaks® and found the following blog comments posted by developmental pediatrician Paul Wang, Autism Speaks® senior vice president for medical research. BTW, I’ve underlined three of his comments that I thought were particularly important:

Today, a lot of parents are talking about adding broccoli sprouts to their kid’s salads and sandwiches. Can this help? Hurt?

The amount of sulforaphane that was administered in the study is many times higher than you can reasonably get through food. Even sulforaphane-rich foods like brussels sprouts, broccoli and broccoli sprouts don’t have enough of the chemical to get you close. So eating these vegetables can’t be expected to improve autism symptoms. Within reason though, eating sulforaphane-rich vegetables is safe and healthy.

What about taking sulforaphane supplements or giving them to a child with autism? Are they safe?

I would caution against starting sulforaphane supplements at this time. First and foremost, this was a very small trial – much too small to assure safety. There was actually a potentially worrisome side effect in the study: Two of the 29 boys and men taking sulforaphane had seizures during the study. Both had a history of seizures in the past, so this could have been a coincidence. However, none of those taking the placebo, or dummy treatment, had seizures during the study.

The study also showed a small increase in liver enzymes in study participants who received sulforaphane. None of these individuals showed any symptoms related to this side effect. However, it poses the possibility that sulforaphane may produce liver inflammation.

It’s important to remember that anything powerful enough to exert biological effects – even beneficial effects – also has the potential to produce unwanted side effects. Just because sulforaphane is found in vegetables doesn’t mean it’s safe. There are many chemicals found in nature that can be toxic. This is particularly true when these chemicals are concentrated into a supplement. Much more study is needed to understand sulforaphane’s actions in the body – for good or bad.

Also, though sulforaphane supplements have been on the market for some time, nutritional supplements don’t go through the kind of rigorous safety testing required for pharmaceutical medicines. So we don’t have good safety data on these products.

No doubt, some people will decide to take sulforaphane supplements based on this study’s findings, regardless of potential safety concerns. How can they select a reputable brand? What would be a safe and reasonable dose?

The brand of supplement used in the study was a patented, pharmaceutical-grade product not available for purchase over the counter. So there’s no way of using the study’s results to gauge the effectiveness or safe doses of the many related health-food products with lesser quality-control during manufacturing.

In the study, the researchers used doses ranging from 50 to 150 µmol daily, depending on the participant’s weight. Their weights ranged from around 120 to 220 pounds.

So if an individual or parent decides to try these supplements – despite safety concerns – I would urge them to work closely with a physician to monitor possible reactions. This monitoring needs to include, but not be limited to, seizures. For example, blood work should probably be done to monitor liver enzyme levels.

Cozy Combo for Commercialization Pathway

I noticed that the conflict of interest statement in this PNAS paper reveals that U.S. patent applications have been filed by three Johns Hopkins University inventors, who include Paul Talalay, one of the corresponding authors, who is also a member of the prestigious National Academy of Sciences. The statement adds that he has divested himself from all potential financial benefits. Furthermore, the sulforaphane is not a commercial product, and has been licensed by Johns Hopkins to Brassica Protection Products LLC, whose CEO, Anthony Talalay, is the son of Paul Talalay.

While inventor Paul Talalay’s apparent largess is laudatory, Hopkins University’s licensing to a company run by his son, Anthony, seems a bit too cozy—in my opinion. On the other hand, publically stating these facts seems to imply that all parties involved are comfortable with legalities.

Anyway, I became interested in learning more about this company, and dug around, so to speak on the internet. The following is some of the backstory on Brassica Protection Products that I gleaned from researching Paul Talalay’s publications and reading a 2013 interview of Anthony Talalay by Adam Stone with bizjournal.com.

The company was founded in 1996 based on Paul Talalay’s long-held belief that certain chemicals in plants might prove to be useful in preventing cancer by inducing enzymes that safely metabolize cancer-causing molecules. Originally the focus was on commercializing broccoli sprouts, which had 20-times more of sulforaphane than mature broccoli, but there were farm-to-market distribution problems, and—perhaps more problematically, I think—people didn’t want to change their eating habits.

Anthony Talalay should be happy about the very promising clinical data reported for a product sold by his company, Brassica Protection Products. Credit: Harry Bosk; taken from bizjournals.com.

Anthony Talalay should be happy about the very promising clinical data reported for a product sold by his company, Brassica Protection Products. Credit: Harry Bosk; taken from bizjournals.com.

After years of disagreement, the company’s board of directors finally convinced Anthony Talalay to abandon this approach and instead commercialize an extract of broccoli sprouts, which is prepared by methods you can read here. That was done three years ago, and business has doubled for each of the past three years.

The company was said to be trying to raise money from family and friends in order to expand operations and sales, which I think ought to be relatively easy now based on the very promising clinical data published in PNAS.

My parting comment is that April 2015 is National Autism Awareness Month, but don’t wait—become engaged now! The Autism Society website offers loads of information and ways to get involved plus advice for living with autism.

As usual, your comments are welcomed.

You and Your Microbiome – Part 2

  • Global Obesity Epidemic is Linked to Gut Microbiome
  • DNA Sequence-Based Microbiomes Accurately Associate with Obesity
  • Blue Agave Margaritas Contain Beneficial Gut Microbes
  • Investments in Microbiome-based Therapies on the Rise, but is there Hype?

Last August, my post entitled Meet Your Microbiome: The Other Part of You dealt with growing recognition that trillions of microbes—mostly bacteria but also fungus—reside in and on each of us, and influence our health status. Moreover, the compositions of these microbiomes change with our diet, what we drink or breath, and who we contact—family, pets, and close friends.

Since then, I’ve collected a string of microbiome articles delving into the implications of this dynamic, symbiotic relationship, and selected some topics that I thought were “blogworthy.” This Part 2, as it were, focuses on overweight/obesity, microbiome therapy, and burgeoning business opportunities.

Overweight/Obesity Epidemic

A recent article in The Wall Street Journal highlights the obesity epidemic that much of the world is facing today. Since 1980, obesity rates have risen by 28% among adults and 47% among children. By 2013 approximately 29% of the world population was said to be overweight or obese. That equates to about 2.1 billion people, the majority of which live in developing countries.

This is a BIG problem—no pun intended—given the very serious consequences of these scary statistics. Policies and programs introduced years ago in countries like the USA have obviously not reversed the continual increase in obesity.

graph

From Wall Street Journal article by Betsy McCay, published May 29, 2014.

Dieting, Exercise and Microbiota

Obesity is considered by the World Health Organization to be preventable by limiting food-derived energy intake from total fats and sugars; increasing consumption of fruits, veggies, etc. and—of course—regular physical activity (60 minutes a day for children and 150 minutes per week for adults).

Some of the roughly 1,000 bacterial species in the human gut help make us fat, while others keep us lean. Centre for Infections/Public Health England/Science Photo Library (taken from Nature).

Some of the roughly 1,000 bacterial species in the human gut help make us fat, while others keep us lean. Centre for Infections/Public Health England/Science Photo Library (taken from Nature).

There could, however, also be links between obesity and bacteria living in our guts, according to Sarah DeWeerdt’s freely accessible article in venerable Nature magazine from which selected snippets are as follows.

Consider what Liping Zhao, a microbiologist at Shanghai Jiao Tong University in China, found after he put a severely obese man on a strict diet. Over the course of 6 months, the man shed ~100 pounds. As he lost weigh, a group of bacteria known as Enterobacter became undetectable in his stool samples, even though they had previously made up 35% of the microbes in his gut.

This dramatic change in human gut microbiota—estimated to be ~1,000 species of bacteria—might be coincident with weight loss, but Zhao and other researchers think otherwise, and believe that these bacteria actually play a key role in regulating body weight.

DeWeerdt quotes Fredrik Bäckhed—a researcher at the University of Gothenburg in Sweden who investigates the gut microbiota using mouse models—as noting that ‘There are a lot of studies in humans, but those are only associations. There are a lot of studies of causation, but those are only in animals.’

How to translate results from studies of lab mice into treatments for humans in the real world is no simple matter, and will likely be the subject of much continued experimentation and hot debate.

The Role of Prebiotics

We’ve all heard about probiotics, but when it comes to studying obesity it’s important to also understand prebiotics. Unlike probiotics which are live organisms, prebiotics are non-living substances (usually carbohydrates) that function as food sources for beneficial bacteria. Research indicates that both pro- and prebiotics are key to controlling obesity. Researchers have shown that in mice prebiotics, particularly oligofructose, can reverse certain gastric problems. DeWeerdt quotes Patrice Cani, a researcher of metabolism and nutrition at the Catholic University of Louvain in Belgium as saying ‘We found that mice fed with oligofructose had an improved gut barrier function.’ ‘The mice that were given prebiotics also had improved metabolic markers, reduced fat mass and reduced inflammation,’ Cani added.

Harvesting a Blue Agave plant in Mexico involves hard work before processing into Agave Tequila and Agave Nectar (taken from inari-hof.de via Bing Images). Blue Agave Margarita is a refreshing source of oligofructose prebiotic (taken from experience-it-island-thyme.blogspot.com via Bing Images).

Harvesting a Blue Agave plant in Mexico involves hard work before processing into Agave Tequila and Agave Nectar (taken from inari-hof.de via Bing Images).
Blue Agave Margarita is a refreshing source of oligofructose prebiotic (taken from experience-it-island-thyme.blogspot.com via Bing Images).

The most widely used prebiotic is oligofructose (aka fructo-oligosaccharides or FOS)—a type of soluble but indigestible carbohydrate fiber found in the Blue Agave plant, as well as fruits and vegetables such as bananas, onions, chicory root, garlic, asparagus, barley, wheat, jicama, leeks and the Jerusalem artichoke. Some grains and cereals, such as wheat, also contain oligofructose. The Jerusalem artichoke—and its relative yacón—together with the Blue Agave plant have been found to have the highest concentrations of oligofructose in cultured plants. Both yacón and Agave nectar are becoming popular substitutes for sugar—BTW, Agave Nectar Margaritas have a 5-Star rating at Food.com!

As the saying goes, “too much of a good thing can be bad.” One report cautions that 15 g or more of FOS can produce side effects such as gas, bloating and general intestinal discomfort, and doses higher than 40 g might cause diarrhea (taken from prebiotin.com via Bing Images).

As the saying goes, “too much of a good thing can be bad.” One report cautions that 15 g or more of FOS can produce side effects such as gas, bloating and general intestinal discomfort, and doses higher than 40 g might cause diarrhea (taken from prebiotin.com via Bing Images).

It’s not clear, however, if the benefits of prebiotics that were seen in mice studies translate to humans. In 2013, Cani and his team conducted a study giving obese women a daily supplement of oligofructose and inulin (a similar substance). After three months, the women ‘showed a slight decrease in fat mass and a reduction in blood levels of an inflammation-promoting molecule. But the results were not really equivalent to the ones we observed in mice,’ Cani told DeWeerdt.

As another caveat regarding studies of prebiotics, consider the results of a study conducted regarding the dietary regimen followed by the subject in his aforementioned obesity study. For 9 weeks, about 100 participants followed a diet that included whole grains, traditional Chinese medicinal foods and prebiotics. The participants all showed ‘improved markers of metabolic health and lower levels of potentially harmful bacteria, including Enterobacter, but they only achieved a modest weight loss of ~6 kg on average.’ Since this average weight loss is almost 10-times less than that achieved by Zhao’s severely obese patient, more factors need to be considered.

Microbiomes Sort “Lean from Obese” with 90% Accuracy!

The advent of fast and cheap DNA sequencing has enabled not only detection and quantification of the type of bacteria in the gut, but also the genes that these microorganisms are expressing, in addition to our own human genes. BTW, researchers in this field generally refer to the collection of bacterial species present in the gut as the microbiota (aka gut flora), and the collection of corresponding expressed genes as the microbiome. In this regard, I find it fascinating—and somewhat difficult to accept—that the ongoing Human Microbiome Project is extending the definition of what constitutes a human to include microbiome, so “it” is technically part of “you”!

The MetaHIT Consortium—a European effort to determine the associations between gut microbes and chronic diseases—sequenced the microbiomes of 169 obese and 123 non-obese individuals. They found that those with fewer bacterial expressed genes tended to have more body fat and other markers of poor metabolic health compared with people with a more diverse microbiome.

The stunning conclusion was that microbial genes are a much better readout of whether you’re likely to be obese or not than human genes are. I repeat—with emphasis—this remarkable conclusion:

…microbial genes are a much better readout of whether you’re likely to be obese or not than human genes…

At the Risk of Oversimplifying this Finding, Genes in your Microbiome Seemingly Influence your Tendency to be Obese or not more so than your own Human Genes!

DeWeerdt reports that other investigators have come to a similar conclusion, and on a quantitative basis claim that microbial genes sort the lean from the obese with 90% accuracy, whereas human genes do this with much less accuracy, i.e. only 58% of the time.

Cause or Effect?

So the really big question is whether these microbial changes are a cause or an effect of the obesity. A step toward deciphering this puzzle has been convincingly addressed by very clever—I think—experimentation wherein the microbiota of an obese mouse is transferred to a microbiota-free mouse.

Jeffrey Gordon, director of the Center for Genome Sciences & Systems Biology at Washington University in St. Louis, Missouri, found back in 2004 that when a microbiota-free mouse is colonized with gut microbes from a normal mouse, it experiences a 60% increase in body fat over the course of 2 weeks—despite eating less food than it did before the transfer.

The simplest interpretation—in my opinion—is that microbes in the gut of these mice increased the ability of mice to store fat. How this occurs biochemically is, of course, not at all clear and will require much more investigation.

BTW, in a variant of this line of research, it has been shown that germ-free mice that receive gut microbes from an obese human donor gain more weight than those that receive them from a lean person. While this might seem surprising, I think it’s actually somewhat expected based on functional biochemical similarity of mouse and human genes that may nevertheless be different genetically, i.e. at the level of DNA sequence.

In Hot Pursuit of Microbiome Therapies

Sarah Reardon reports in Nature magazine that roughly $500 million has been spent on microbiome research since 2008. However, the only major therapy resulting from this sizable investment has been the use of fecal transplants for treating life-threatening gut infections or inflammatory bowel disease—discussed in my earlier blog.

This seeming absence of clinical impact by investment dollars may change due to large pharmaceutical companies viewing this area as a new source of revenue. This past May, Pfizer announced plans to partner with Second Genome, a biotechnology firm in South San Francisco, California, to study the microbiomes of ~900 people comprising a group with metabolic disorders and, of course, a control group.

At virtually the same time, Paris-based Enterome revealed that it had raised $13.8 million in venture capital to develop tests that are intended to diagnose inflammatory and liver diseases based on measuring the composition of gut bacteria. Based on the aforementioned microbiota vs. microbiome issue, I’m somewhat skeptical of this approach by Enterome. Maybe they’ll find otherwise.

Reardon also reports that Joseph Murray—a gastroenterologist at the Mayo Clinic in Rochester, Minnesota—fed gut bacterium Prevotella histicola to transgenic mice with human-like immune systems, and found suppression of inflammation caused by multiple sclerosis and rheumatoid arthritis. He is hoping to develop this into a therapy with biotech firm Miomics in New York.

Similarly, Vedanta Biosciences in Boston, Massachusetts, is conducting preclinical trials of a pill containing microbes that suppress gut inflammation. I checked out Vedanta’s website, and it’s worth visiting to watch a video entitled Telling “Good” from “Bad,” which is about the immune system evolving to differentiate ‘good’ microbes from ‘bad’ microbes, as related to its microbe-based therapeutic approach.

And last June, Second Genome announced a deal with Janssen Pharmaceuticals of Beerse, Belgium, to study the microbial populations of people with ulcerative colitis, in the hope of identifying new drugs and drug targets. Already in the clinic, Microbiome Therapeutics, a biotechnology company in Broomfield, Colorado, is currently conducting trials with two small molecules that select for ‘good’ gut bacteria to help people with diabetes to take up insulin more easily.

From all of the above, there’s no doubt in my mind that microbiomes need to be factored into assessment, modulation, and maintenance of a person’s health, and much R&D is clearly moving in these directions. On the other hand, my sense is that progress will be slower than hoped for, partly because there’s much to be discovered, and partly because people aren’t like mice: we can choose to eat what we wish, indulge our cravings, and skip taking medications.

Chime in with your comments if you think I’m being too pessimistic about this.

BTW, for those of you who do want to change your diet so as to hopefully beneficially change your microbiomes, you can consider engaging with uBiome, which is a small start-up that offers DNA sequence-based microbiome analysis as part of ongoing research involving likeminded persons. An explanatory—and humorous video—can be accessed here.

Hyping the Microbiome?

“Maybe the microbiome is our puppet master” says Carl Zimmer in his NY Times article headlined above (Credit: Jonathan Rosen).

“Maybe the microbiome is our puppet master” says Carl Zimmer in his NY Times article headlined above (Credit: Jonathan Rosen).

Throughout all of the above, you’ll notice that weight-related conclusions derived from legitimate scientific inquiries are properly couched with caveats and the need for further investigations. By contrast, some reports may be pushing the envelope of credulity, as discussed in an engaging but anonymous article in GenomeWeb entitled “Hyping the Microbiome.” One notable quote is by William Hanage, an associate professor of epidemiology at the Harvard School of Public Health, who says that ‘Microbiomics risks being drowned in a tsunami of its own hype.’ He also points to a blog Jonathan Eisen, who gives awards for ‘overselling the microbiome,’ and provides an updated list of links to questionable claims.

Hanage partly blames the media for generating this hype. I agree with this finger pointing, and my personal favorite is this headline and accompanying visual in the NY Times.

Our Microbiome May Be Looking Out for Itself

Postscript

After writing this post, a publication in Nature reported “dynamics and associations” of microbial communities across the human body, based on detailed analysis of data from the Human Microbiome Project. Following is a portion of the abstract that draws three conclusions (see underlines), the first of which I find fascinating:

First, there were strong associations between whether individuals had been breastfed as an infant, their gender, and their level of education with their community types at several body sites. Second, although the specific taxonomic compositions of the oral and gut microbiomes were different, the community types observed at these sites were predictive of each other. Finally, over the course of the sampling period, the community types from sites within the oral cavity were the least stable, whereas those in the vagina and gut were the most stable. Our results demonstrate that even with the considerable intra- and interpersonal variation in the human microbiome, this variation can be partitioned into community types that are predictive of each other and are probably the result of life-history characteristics. Understanding the diversity of community types and the mechanisms that result in an individual having a particular type or changing types, will allow us to use their community types to assess disease risk and to personalize therapies.