Spartan Cube—The World’s Smallest Molecular Diagnostic Device

  • Records Are Meant to be Broken—Including Those for PCR Diagnostics
  • Spartan Bioscience Claims It’s Cube is World’s Smallest Mol Dx Device 
  • The Cube was Launched at the Recent AACC Clinical Lab Expo 

Prologue

[22]-annulene. Taken from Wikipedia.com

[22]-annulene. Taken from Wikipedia.com

In the spirit of the recent Olympic games, and as the saying goes—records are meant to be broken. When I was in grammar school, a big buzz in sports was who would be the first to break the 4-minute mile; answer: Roger Bannister in 3 minutes and 59.4 seconds in 1954—now 17 sec faster. In my college craze days, it was how many persons can fit into a Volkswagen Beetle; answer: I couldn’t find the first feat, but the current record is 20 crammed into an old style Beetle in 2010. Then during my graduate organic chemistry studies, there was interest in synthesizing increasingly larger annulenes—completely conjugated CnHn monocyclic hydrocarbons akin to benzene (n=6); answer: [22]-annulene with n=22 (see below) synthesized by F. Sondheimer. But I digress…

Our collective fascination with records—and beating them—also applies to all sorts of instruments for health-related sciences, such as the most powerful MRI imaging systems (currently from GE) or longest-DNA-sequencing system (currently from PacBio). Due to the seemingly endless utility of PCR, there is a continual stream of claims for the fastest PCR system (currently from BJS Biotechnologies) or—more to point herein—smallest PCR system.

To wit, regular readers of my blog will recall an April 2016 post titled World’s Smallest Real-Time PCR Device, which referred to a hand-held system reported by Ahrberg et al. for real-time, quantitative PCR (qPCR). That system is pictured below next to the original system commercialized by ABI in the 1990s that weighed 350 pounds and was 7 feet long!

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

It seems that PCR records fall as easily as those in the Olympics. Not even a year later, Ahrberg’s claim is being challenged by Canadian company Spartan Bioscience, which recently introduced its Cube device at the 2016 AACC Annual Scientific Meeting & Clinical Lab Expo. Following are some technical details that I thought were worth sharing.

Taken from businessinsider.com

Taken from businessinsider.com

Cube Facts

Given its amazingly small size of only 4 x 4 x 4 inches, there apparently has been some remarkable engineering achievements to be able to squeeze-in what’s needed for the rapid heating and cooling required for PCR thermal cycling. Ditto for the optics required to enable fluorescence detection. Like other relatively small devices intended for emerging Point-of-Care (POC) applications in a doctor’s office or clinic, there is wireless connectivity to a laptop that serves as the user interface for operation and data analysis, as well as a power source for the Cube via a USB cable.

You’re likely wondering by now how much the Cube will sell for. Unfortunately, I was not able to obtain a list price from Spartan’s CEO at this time, so we’ll all have to wait and see.  I’ll post the answer as a comment to this blog as soon as I find out the price.

Inside the Cube—Perhaps 

I actually don’t know exactly what’s inside the Cube, but some possibilities of what might be are as follows. I’ve based these educated guesses on a Spartan Bioscience patent (US pat. no. 8,945,880) by Paul Lem and others entitled Thermal cycling by positioning relative to fixed-temperature heat source. As depicted below, a hot block provides a heat source at a fixed temperature to thermally cycle PCR reaction vessels that can be precisely moved by a micrometer to and from the hot block in a repeated manner.

Taken from US patent no. 8,945,880

Taken from US patent no. 8,945,880

As for how fluorescence might be measured to monitor each PCR reaction in real-time, one possibility is depicted below. Basically, each reaction tube is proximate to excitation light from an LED, and has a slit at the bottom for emitted light that is collected and processed into a typical real-time PCR curve.

Taken from US patent no. 8,945,880

Taken from US patent no. 8,945,880

Before the Cube

Prior to launching the Cube, Spartan Bioscience has been selling an FDA-Cleared in vitro diagnostic product called Spartan RX, which is also relatively compact, and carries out fully automated—“cheek swab-to-result”—PCR analysis of certain Cytochrome P450 2C19 (CYP2C19) genotypes. Roughly 1-in-3 people carry CYP2C19 mutations that can impair metabolism of a wide variety of commonly used drugs. Consequently, these PCR-based results are a valuable aid to clinicians in determining strategies for therapeutics that are metabolized by the Cytochrome P450 2C19.

I was favorably impressed by the fact that this CYP2C19 assay qualifies for reimbursement from Medicare and most insurers, according to the company’s website, which adds that there is an ongoing 6,000-patient clinical trial entitled Tailored Antiplatelet Initiation to Lessen Outcomes due to Clopidogrel Resistance after Percutaneous Coronary Interventions (TAILOR PCI).

The Spartan RX cheek swab POC results have been recently compared to centralized genotyping with a TaqMan® allelic discrimination assay (Life Technologies) using qPCR and with the GenID® reverse dot-blot hybridization assay (Autoimmun Diagnostika GmbH). Published results indicate excellent agreement, and led to the following conclusions by the authors: “Compared to both laboratory-based genotyping assays, the POC assay is accurate and reliable, provides rapid results, can process single samples, is portable and more operator-friendly, however the tests are more expensive.”

I look forward to finding out more about the Cube and the PCR results it can obtain. I’ll post more information on my blog as it becomes available. As usual, your comments are welcomed.

Postscript

To me, there’s something visually intriguing about a cube, perhaps because it’s one of the so-called Platonic Solids, which have been known since antiquity and studied extensively by the ancient Greeks.

Taken from acs.org

Taken from acs.org

Platonic solids such as the cube have also fascinated chemists, as evidenced by there being a substantial amount of published literature on the synthesis and physical properties of platonic solids. For example, cubane (C8H8) is a synthetic hydrocarbon molecule that consists of eight carbon atoms arranged at the corners of a cube, with one hydrogen atom attached to each carbon atom. A solid crystalline substance, cubane was first synthesized in 1964 by Philip Eaton and Thomas Cole. Prior to this work, researchers believed that cubic carbon-based molecules would be too unstable to exist.

Taken from bitrebels.com

Taken from bitrebels.com

On the fun side, the cube was morphed—so to speak—into what became an amazingly popular game, or should I say, object of competition. Rubik’s Cube is a 3-D combination puzzle invented in 1974 by Hungarian sculptor and professor of architecture Ernő Rubik. If you’re wondering about the world’s record for solving this puzzle, it’s currently a mind-boggling 4.9 sec, according to a list (with video links) of this and past records that appear to have been broken regularly, just as I opined at the beginning of this blog. But I digress…yet again.

Virtual Reality for Graphene Nanopores and Space Station Sequencing 

  • Simulated Sequencing Takes Virtual Reality Way Beyond Games  
  • In Silico Simulations Suggest Possible 99.99% Accuracy for Graphene Nanopores 
  • minION Nanopore Sequencer is Sent to the International Space Station

Prelude

oculusvr

Taken from oculusvr.com

This blog is mostly about an international team of researchers who are using Virtual Reality (VR)—in the form of computational modeling—to simulate a new approach to DNA sequencing using nanopores made out of graphene. While VR is a hot trend in all sorts of so-called immersion media, such as those offered by Oculus (that was acquired by Facebook for $2 billion in 2014), computation-based VR has been used by scientists for simulating molecular interactions for a relatively long time. However, extending molecular simulations to complex (aka many-atom) systems like nanopores and DNA has had to wait for bigger, faster, cheaper computing.

In this blog, I’ll also discuss the recent launch of a commercially available nanopore sequencer for the first ever DNA sequencing in space using a self-landing rocket operated by Space X (co-founded by uber-famous multi-billionaire entrepreneur Elon Musk). It’s hard for me to even imagine what seemingly incongruent mix of topics could be more intriguing than these. As the now trendy saying goes, you can’t make this stuff up. But I digress…

Lift off! Taken from am1070theanswer.com                     Self-landing! Taken from indianexpress .com

Lift off! Taken from am1070theanswer.com    –  Self-landing! Taken from    indianexpress.com

Nanopore Sequencing 

baseFrom an earlier blog you’ll know that I’m a huge fan of tiny nanopores for sequencing, which is a 20+ year old concept, as depicted below from a seminal patent wherein DNA was envisaged as moving through a pore-in-lipid bilayer leading to base-dependent transient blockage of ionic current from which sequence is determined.

Taken from nature.com

Taken from nature.com

After two decades, this prophetic concept of nanopore sequencing has recently been realized, and commercialized by Oxford Nanopore Technologies (ONT) using “bionanopore” technology. Comparing the images above and below, you’ll see that bionanopores are, in many respects, quite similar to the first described nanopores, wherein a pore-forming protein, α-hemolysin (gray), is embedded in a lipid bilayer (blue). On the other hand, there is an attached DNA-processive enzyme, 29 DNA polymerase (brown), that feeds in the single strand of DNA for sequencing; details may be found elsewhere.

An alternative strategy for nanopore sequencing is to replace this type of bionanopore (composed of biological macromolecules) with a pore constructed of non-biological materials, notably silicon-based semiconductors that enable electrical signal generation and data processing. This would-be evolution of nanopore sequencing from biological constructs to various types of solid-state materials can be read about elsewhere.

Whither Goest Graphene?

It seems the next step in the progression of nanopore technology is those made of graphene—the trivial name for a very special form of carbon that was long known but exceedingly difficult to make. In fact, the process is so difficult that Andre Geim and Kostya Novoselov at The University of Manchester were awarded the 2010 Nobel Prize in Physics for their work enabling the production and characterization of graphene.

Geim and Novoselov. Taken from rsc.org 

Geim and Novoselov. Taken from rsc.org

Graphene is a two-dimensional array or “sheet” of carbon atoms that is usually depicted by the ball-and-stick model (pictured at the left below) as a one-atom-thick sheet of otherwise infinite dimensions. Since nothing is infinite in the real world, sheets of graphene have edges to which hydrogen is bonded, but for simplicity is ignored. This carbon-carbon bonding with carbon-hydrogen edges is akin to that in polycyclic aromatic hydrocarbons familiar to readers who are chemists.

Taken from 3dprint.com  

Taken from 3dprint.com

Taken from Bayley (2010) in nature.com

Taken from Bayley (2010) in nature.com

Because of graphene’s unique electrical properties and single-atom-thin structure, the basic idea is that a nanometer-size hole in graphene might be made—somehow—to allow DNA and ions to pass through and thus generate electrical signals—somehow—that are accurately deciphered—somehow—into DNA sequence. Oh, and let’s not forget that this sequence information must differentiate—somehow—3’->5’ from 5’->3’ directional pass through. All these “somehows” are meant to indicate that it’s far easier to imagine the concept of graphene nanopore sequencing, as fancifully shown below, than to actually do it.

Taken from Mechant et al. Nano Letters (2010)

Taken from Mechant et al. Nano Letters (2010)

The most daunting practical problem deals with how to “drill” tiny holes in graphene. One approach has been to use controlled electron-beam exposure in a transmission electron microscope. Initial demonstration of this approach was published in 2010 by Merchant et al. in Nano Letters in a paper titled DNA Translocation through Graphene Nanopores, from which the schematic left is taken.

In this device, a few-atoms-layer piece of graphene (1-5 nm thick) having an ~10 nm hole is suspended over a 1 μm diameter hole in a 40 nm thick silicon nitride (SiN) membrane suspended over an ~50 × 50 μm2 aperture in a silicon chip coated with a 5 μm silicon oxide (SiO2) layer in such a way that a bias voltage (VB) is applied between the reservoirs to drive DNA through the nanopore. Although DNA could be detected, the graphene pore size was too big to allow sequence detection.

Taken from Chang et al. Nano Letters (2010)

Taken from Chang et al. Nano Letters (2010)

Similar studies by Schneider et al. were also reported in Nano Letters in 2010, which appears to be a watershed year for this journal inasmuch as another noteworthy nano-detection scheme for DNA was described therein by Chang et al.—but with an important new feature. Namely, using gold electrodes (in yellow, below) separated by only 2 nm and conjugated to dC, a derivative of dG (blue balls) apparently was able to H-bond (magenta) to dC—based on dC-dG complementarity and detected as electron tunneling signals. This transient, base pair-specific H-bonding is what has now been further investigated by others albeit in the following form of Virtual Reality.

Virtual Reality Nanopore Sequencing

In contrast to the above “real” experiments, others have simulated reality using mathematical calculations based on theoretical chemistry, which is Virtual Reality that has physical significance well beyond simply playing games. Mathematical modeling or computation simulations are phrases generally used to describe these so-called in silico “experiments” that serve as indications of what could be done, in theory, if this Virtual Reality is actually translatable to the real world. But I digress…

An international team of investigators in the U.S., Germany, and Netherlands has recently reported studies titled Nucleobase-functionalized graphene nanoribbons for accurate high-speed DNA sequencing. Although this article is a dreaded “pay-to-read” article, there is a brief news piece about it at the website for the U.S. National Institute of Standards and Technology (NIST) where some of this work was conducted.

Taken from nist.gov

Taken from nist.gov

As is evident from the schematic shown below, these investigators borrowed from the aforementioned types of publications to imagine a graphene nanopore having its internal edges functionalized with nucleobase moieties that could potentially H-bond with DNA bases in a sequence specific manner—à la Chang et al. Under appropriate conditions, this could provide the basis for sequencing via measurement of induced current fluctuations.

More specifically, they imagined a sheet (aka ribbon) of graphene 4.5 x 5.5 nm with several nucleobase moieties attached to a 2.5 nm nanopore. In animated simulations (which are linked at the NIST website), you can watch how this sensing device would perform at room temperature in water with attached cytosine H-bonding to detect G in DNA.

When you watch this simulation, you’ll immediately notice how “wiggly” DNA is due to random motions of its constituent groups and atoms. You’ll also see detection of each translocating (i.e. passing G) as an increasing signal being recorded in real-time. While time as a parameter in this simulation is real, the simulation itself is not real, but rather virtual reality based on state-of-the art theoretical calculations by a computer.

With that caveat in mind, the performance was said to be 90% accurate (due to missed bases rather than wrongly detecting a base) at a rate of 66 million bases per seconds, which to me is mindboggling ultra-fast. Moreover, if this device could be fabricated as four sequentially-located graphene pores each functionalized with either C, G, A, or T, the researchers estimate that “proofreading” would increase accuracy to 99.99%, as required for sequencing the human genome.

Virtual reality is, well, not reality. And sometimes dreams and reality go in opposite directions. However, if indeed the above imaginary device and simulations were to become reality—nanopore sequencing would indeed be advanced dramatically from today’s performance.

ONT’s minION Sequencer in Space

In transitioning back to reality, it’s almost unbelievable to me that ONT’s minION nanopore sequencer—which I’ve blogged about before—was sent to the International Space Station (ISS) in April 2016 to carry out the first ever sequencing of DNA in space. If that wasn’t enough “buzz”, then the fact that this was achieved by uber-famous Elon Musk’s Space X company made it way more so, along with much ado in successfully landing the rocket’s first-stage on a relatively tiny platform in the ocean. This is all amazing stuff.  And to think that not so long ago, we were thinking how great it would be if self-landing rockets were really possible, not just a fun concept in video games and sci-fi movies!  Maybe virtual reality isn’t that far from becoming reality, but I digress….

The first aim of putting the minION nanopore into space is to demonstrate the feasibility of nanopore sequencing in microgravity. That being done would then allow use of the minION to rapidly sequence astronaut samples in the ISS to diagnose, for example, an infectious disease or other health issue.

Interested readers can peruse elsewhere much more about this historic milestone, as well as watch and listen to a short (but very exciting) video titled Space Station Live: Big DNA Science in a Small Package. The video was posted on Twitter on July 21st and features the minION device (aka The Biomolecular Sequencer).

NASA minION flight hardware for the ISS experiments packaged for shipment to the ISS. Credit: NASA/Sarah Castro. Taken from spaceref.com

NASA minION flight hardware for the ISS experiments packaged for shipment to the ISS. Credit: NASA/Sarah Castro. Taken from spaceref.com

I look forward to learning the results of the minIONs research in space. I hope that it’s “mission accomplished!” and of great use in years to come. BTW, if you’re a “space buff” like me, you can watch and listen to ISS-Mission Control live streaming 24-7 at this website.

As usual, your thoughts about this blog are welcomed as comments.

DIY CRISPR Kit – Door to Democratization or Disaster?

  • Gene Editing with CRISPR is All the “Buzz”
  • Low-Cost CRISPR Kit Being Sold to DIY “Biohackers”
  • What is the Balance Between Democratization and Preventing Disaster?

The dictionary definition of democratization is the transition to a more democratic political regime. Since democracy emphasizes the role of individuals in society, democratization is generally perceived to be good. This political concept of democratization is being increasingly morphed, if you will, to describe the transition of science and technology from trained specialists in traditional labs to any individual, anywhere—including someone’s kitchen table.

Taken from oocities.org

Taken from oocities.org

Lest you get the impression I’m an elitist, and not in favor of fostering better understanding—and appreciation—of science by non-scientists everywhere, I definitely am not. I want the value of science to be widely appreciated. Even if I weren’t of that opinion, democratization of science and technology is already evident in this exemplary cartoon indicating how DNA is now familiar to virtually everyone. But I digress…

Taken from diy-bio.com

From diy-bio.com

It is evident that molecular biology has also undergone democratization based on emergence of so-called “do it yourself” (DIY) advocates of biology (DIY-BIO), which on the surface seems like a good thing. But, as I’ll expand upon below, DIY-BIO has morphed in a way which has elevated concerns that a well-intentioned DIY aficionado anywhere can now access genetically powerful CRISPR reagents that might inadvertently unleash a harmful home-made organism.

CRISPR Basics

First off, I should note that gene editing by CRISPR—thankfully short for “clustered regularly-interspaced short palindromic repeats”—actually involves another component named Cas9—short for CRISPR associated protein 9. Cas9 is an enzyme that recognizes single guide RNA (sgRNA) hybridized to one strand of specifically targeted DNA via the 5’end of sgRNA, as depicted in green in the mechanism below. The remaining sgRNA has a double-stranded “stem” (black, red) and loop (purple) internal structure, and a 3’ end with several stem-loop structures (red).

Taken from jeantet.ch

Taken from jeantet.ch

The scissors indicate Cas9 cutting both strands of DNA, which thus allows for insertion of so-called donor DNA and, consequently, enabling a variety of genetic manipulations in plants, bacteria, human or animal cells. Chemically synthesized sgRNA that target any gene of interest can be readily designed for purchase, along with Cas9 in the form of biosynthetic Cas9 mRNA encoding this necessary protein component.

CRISPR’s importance as an emerging, useful tool for gene editing is evident from the number of publications in PubMed that have approximately doubled each year since the seminal to give an estimated 2,500 publications indexed to CRISPR as a search term. Unfortunately (but perhaps not surprisingly given the billion-dollar implications), there is an ongoing dispute over inventorship involving the Broad Institute (see Feng Zhang patent), the University of California, and the University of Vienna.

Biohacker Promotes DIY CRISPR Kit

Josiah Zayner (Taken from http://www.ifyoudontknownowyaknow.com)

Josiah Zayner (Taken from http://www.ifyoudontknownowyaknow.com)

As mentioned in the introduction, self-proclaimed “biohackers” who are avid fans and practitioners of DIY molecular biology, have been busily “doing their thing” for some time now without much cautionary publicity. That’s changing, however, as a result of the advent of CRISPR together with relatively easy access to its sgRNA and Cas9 reagents. One case in point involves Josiah Zayner, who has a PhD from the Department of Biochemistry and Molecular Biophysics at the University of Chicago and now lives in the San Francisco Bay Area.

Zayner’s online biographical sketch states that he is “very active in Biohacking and DIY Science and run[s] an online Biohacking supply store The ODIN.” By visiting the website for The ODIN, which reportedly raised $65,000 by crowdfunding online via Indiegogo, you’ll find various items for conducting molecular biology experiments, along with an “about” page stating that “smaller groups of people, small labs or even DIY Scientists on their own can do amazing things if they have access to resources that are normally only available to large heavily funded labs and companies.”

While this seems all fine and good is some ways, the item offered by The ODIN that has led to controversy is the first-ever DIY kit for CRISPR. This, according to an article in The Mercury News, “raises the specter—deeply troubling to some experts—of a day when dangerous gene editing is conducted far from the eyes of government regulators, posing risk to the environment or human health”.

The article goes on to quote one expert who said The ODIN kit is sold for manipulating yeast and could never be used to alter human genes, while another expert cautioned that the kit can teach basic principles to do so with appropriate modifications. Another problem is inadvertent conversion of yeast into a harmful microorganism that might be accidentally spread.

Taken from mercurynews.com

Taken from mercurynews.com

While I share these concerns, it will be virtually impossible to prevent individuals or small groups intent on nefarious activities using CRISPR technology. On the other hand, I have to admit that I would be very concerned if I were living next door or otherwise nearby Josiah if he is indeed practicing what he’s preaching, so to speak, using CRISPR in his kitchen as pictured right.

CRISPRized Plants, Too

If you think that DIY is a passing fad with few devotees, think again. Aside from the main DIY-BIO website that you can peruse, a recent online article in Fusion talks about a couple of DIY enthusiasts doing things that make the hairs on my neck stand up, as the saying goes. For instance, David Ishee, a 30-year-old Mississippi resident who never attended college, does at-home experiments in his shed using online kits for growing plants, but will now use CRISPR to carry out gene editing.

Ishee reportedly will use software like DeskGen that advertises its “on-demand CRISPR libraries” for gene editing, and is quoted as saying “That gives me a lot of new options. Up until now, all the genetic edits I’ve made have been limited to plasmids and unguided genomic insertions. That limits the kinds of cells I can work with and the types of work I can do.”

So what will Ishee do? The answer is that nobody but he knows. If his genetically edited plants grow and seeds get carried by the wind, they could someday end up in your backyard. What then? Who knows? Could be creepy.

Possibly harmful, irreversible consequences of completely democratized CRISPR are completely unknown. Therein lies the essence of the problem that has many experts quite concerned, as reported in Fusion. I share that concern.

Parting Shot

In closing this brief story about DIY synthetic biology using CRISPR, I must say that I wish journalists writing for newspapers and other media would stick to news that is factual and not interpreted for commentary that is flat out wrong or intentionally provocative. My case in point is the following big font, bold letters headline:

“Finally, your chance to play God!”

This was used by time.com to recycle the aforementioned piece by The Mercury News. Shame on time.com for this misleading and totally wrong exclamation. But I digress…

I would greatly appreciate knowing your thoughts about DIY CRISPR by sharing them here as comments.

Zon on Zon’s Zebrafish

  • Leonard Zon Uses Zebrafish to “Fish” for Candidate Drug Compounds
  • Two Candidate Drugs are in Clinical Trials for Cancer Treatments
  • Zon Interviews L. Zon
Leonard I. Zon, M.D. Taken from pediatrics.mc.vanderbilt.edu.

Leonard I. Zon, M.D. Taken from pediatrics.mc.vanderbilt.edu.

The first time I was asked if I was related to the scientist Leonard Zon, I honestly had to reply that I didn’t know, and out of curiosity later looked up his publications, which were quite numerous for a then newish investigator. His current biosketch expertise includes pioneering research in the new fields of stem cell biology and cancer genetics. Dr. Leonard I. Zon is the Grousbeck Professor of Pediatric Medicine at Harvard Medical School, an Investigator with the Howard Hughes Medical Institute, and Director of the Stem Cell Program at Children’s Hospital Boston—that’s impressive!

I found Leonard Zon’s unusual zebrafish-based research and accomplishments therefrom definitely blogworthy, and the coincidences of both our surnames and involvement in science are kind of an unusual “double-doppelgänger,” if you will. In any case, it’s always a surprise to meet your double, even if only in name and profession.

A few regular readers of my blog will likely smile and think that Zon on Zon’s Zebrafish is yet another instance of my penchant for alliteration. However, reading the following snippets about Leonard Zon’s clever—dare I say zany—use of zebrafish for his research will illustrate why they are unusual, interesting and commercially viable.

On the other hand, I must admit that I too smiled at the thought of this unique opportunity to post an interview of one Zon by another Zon, which reminded me a bit of Zappa Plays Zappa. But I digress…

Zebrafish as a Model for Organogenesis

Leonard Zon currently has nearly 250 research publications listed in PubMed, which is a large number by any measure, but that’s even more impressive when you take into account that his first was in 2002—only 14 years ago. That translates to an average of about 3 publications every 2 months each year!

Zon’s inaugural publication in 2002 was an in-depth review in venerable Science in which the abstract presciently reads in part as follows:

Organs are specialized tissues used for enhanced physiology and environmental adaptation. The cells of the embryo are genetically programmed to establish organ form and function through conserved developmental modules. The zebrafish is a powerful model system that is poised to contribute to our basic understanding of vertebrate organogenesis. This review develops the theme of modules and illustrates how zebrafish have been particularly useful for understanding heart and blood formation.

As will be elaborated below, Zon’s most recent publication in 2016—also in Science—has extended the zebrafish model to now include melanoma. If you’re asking yourself, why use zebrafish, the answer is partly due to convenience derived from the unique features and accelerated life cycle of zebrafish.

Zebrafish life cycle. Taken from en.wikipedia.com.

Zebrafish life cycle. Taken from en.wikipedia.com.

Seen right, these advantages include its small size, easy care, and rapid generation time. In addition—and very importantly—the embryos and growing zebrafish are transparent, allowing for continuous observation of developing organs under the light microscope.

Mutagenesis screens allow examination of defects in early organogenesis and late organ function. These many advantages of investigating zebrafish—and Zon’s huge facility comprising thousands of tanks—are nicely explained and shown in a video, which I found well worth viewing. The video also includes Zon’s specially bred, virtually transparent species of zebrafish named—humorously—Casper, after Casper the Friendly Ghost. Pictures below are (a) the transparent Casper zebrafish; (b) the non-transparent wild-type zebrafish; and transparent Casper the Friendly Ghost, which brings back my childhood memories. But again I digress…let’s get back to science!

(a) Casper, (b) wild type zebrafish. Taken from nature.com. Transparent Casper the Friendly Ghost. Taken from enchantedamerica.wordpress.com

(a) Casper, (b) wild type zebrafish. Taken from nature.com. Transparent Casper the Friendly Ghost. Taken from enchantedamerica.wordpress.com

Aided by the availability of DNA sequence information for the zebrafish genome, researchers have published ~2,000 (!) reports dealing with antisense gene “knockdown” using phosphorodiamidate oligonucleotides. By numerical coincidence, the first such report appeared in 2000, and was prophetically entitled Effective targeted gene ‘knockdown’ in zebrafish.

Taken from igtrcn.org.

Taken from igtrcn.org.

As shown below, these rather unusual oligonucleotides—dubbed “morpholinos” by resemblance of the 6-membered ring to morpholine—can be injected directly into zebrafish embryos. Interested readers can consult a detailed “how to” guide on use of morpholinos in zebrafish.

Going forward, however, I expect that uber-hot CRSPR/Cas9 gene editing will be widely adopted, based on the titles of these two pioneering publications in 2016:

Stem Cells and Beyond

Now that we know a bit about Zon’s zebrafish and how to knockdown or edit a gene for functional genomics (aka “gene functionation”), let’s zero in on what Zon studies and how he does that. In a nutshell, the overarching science in Zon’s lab deals with stem cells, which are undifferentiated cells that can differentiate into specialized cells, as well as divide to produce more stem cells, as depicted below.

Taken from nas-sites.org.

Taken from nas-sites.org.

According to Zon’s website, the hematopoietic system that forms various types of blood cells is an excellent model for understanding tissue stem cells. This conceptual relationship is very important because it provides insight to cell differentiation regulation, and involvement in aging, disease, and oncogenesis. In addition, better understandings of the regulation of hematopoietic stem cell biology and lineage differentiation improves diagnosis and treatment of human hematopoietic disorders (aka “blood cancers”) and bone marrow transplantation therapies.

Differentiation of different blood cells from hematopoietic stem cell to mature cells. Taken from wikipwedia.com.

Differentiation of different blood cells from hematopoietic stem cell to mature cells. Taken from wikipwedia.com.

From Zebrafish to Clinic

Scientific theory is nice, but success is best, and Leonard Zon’s theory of using zebrafish to manipulate human stem cells for discovering therapies seems indeed to be headed for success, according to an article in the Harvard Gazette.

Zon and others at the Harvard Stem Cell Institute (HSCI) have published initial results of a Phase Ib safety study wherein 12 adult patients undergoing umbilical cord blood transplantation received two umbilical cord blood units, one untreated and the other treated with the small molecule 16,16-dimethyl prostaglandin E2 (dmPGE2). This molecule had been found in Zon’s zebrafish screen, which I’ll outline below.

Fate Therapeutics, a San Diego-based biopharmaceutical company of which Zon is a co-founder, sponsored the investigational new drug (IND) application, under which the aforementioned clinical program was conducted, thus translating his research findings from the laboratory—dare I say tank—into the clinic.

Zon’s Zebrafish Yield New Approaches to Treat Muscular Dystrophies

According to NIH’s National Institute of Neurological Disorders and Stroke website, muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles that control movement. It adds that there is no specific treatment to stop or reverse any form of MD. Consequently, MD is a compelling target for new drug discovery, and Zon’s zebrafish are being used for such discovery in the following way.

Each zebrafish produces about 200 (!) eggs per week, so Zon’s lab collects and deposits a single egg into each well of a multi-well plate for individual but parallelized treatment with individual chemicals to screen for effects on differentiation, thus screening many compounds in a speedy manner.

Zon and coworkers concluded that “these studies reveal functionally conserved pathways regulating myogenesis across species [zebrafish, mouse, and human] and identify chemical compounds that…differentiate human iPSCs into engraftable muscle.”

Let’s hope that human clinical trials using this novel therapeutic approach enabled by Zon’s zebrafish soon prove successful for treatment of MD.

Zon’s Zebrafish Also Enable Elucidation of Melanoma Development

According to a fact page by the American Cancer Society, skin cancer is the most common of all cancers. About 3.5 million cases of basal and squamous cell skin cancer are diagnosed in this country each year. Melanoma, a more dangerous type of skin cancer, will account for more than 73,000 cases of skin cancer in 2015.

Zon and a large group of 18 coworkers have recently reported in venerable Science work that has been heralded in a New York Times article. This study is very comprehensive and involves lots of “heavy duty” molecular and cellular biology, which you can read in detail in the aforementioned linked article. Snippets of the key findings are as follows.

  • Benign melanocytic skin cells carry oncogenic BRAF-V600E mutations and can be considered a “cancerized” field of melanocytes, but they rarely convert to melanoma.
  • In an effort to define events that initiate cancer, they used a melanoma model in the zebrafish in which the human BRAF-V600E oncogene is driven by the melanocyte-specific mitfa
  • When bred into a p53 mutant background, these fish develop melanoma tumors over the course of many months.
  • The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and is specifically reexpressed only in melanoma tumors, making it an ideal candidate for tracking melanoma from initiation onward.
  • As show below, they developed a crestin:EGFP reporter that recapitulates the embryonic neural crest expression pattern of crestin and its expression in melanoma tumors.
  • They show through live imaging of transgenic zebrafish crestin reporters that within a cancerized field (BRAFV600E-mutant; p53-deficient), a single melanocyte reactivates the NCP state, and this establishes that a fate change occurs at melanoma initiation in this model.
Taken from Zon and coworkers Science 2016.

Taken from Zon and coworkers Science 2016.

Zon Interviews L. Zon

Dr. Zon and some of his 4,000 zebrafish tanks. Taken from bizjournals.com.

Dr. Zon and some of his 4,000 zebrafish tanks. Taken from bizjournals.com.

After researching Leonard Zon’s aforementioned unique—and promising—use of zebrafish to advance basic science and discover new therapies, I contacted him by email to “interview” him, regarding several points. My questions (JZ) and his answers (LZ) are as follows.

JZ: Aside from the cord blood clinical trial mentioned in the Harvard Gazette, are any other human clinical trials being carried out based on your zebrafish findings?

LZ: We have had two chemicals discovered in zebrafish, and ultimately went to a clinical trial. The first was a di-methyl form of PGE2 for cord blood transplantation for leukemia. The second was leflunomide, an arthritis drug that paused transcription in neural crest cells and is being evaluated for metastatic melanoma.

JZ: Is Fate Therapeutics your only startup company?

LZ: I started Scholar Rock about 3 years ago. This company is targeting the TGF-B family of ligands. Has about 25 employees, and is in Cambridge. I am about to start a third company.

JZ: Is your zebrafish method for screening chemicals patented?

LZ: We have patents on several screening methods, but in general we patent the chemicals we find.

JZ: Zebrafish offer many reported advantages for your kind of research, but what is a primary disadvantage?

LZ: The major disadvantage of the zebrafish is that the system occasionally lacks definition. For instance, in the blood system, we have one monoclonal antibody against one epitope. We really need to create reagents for the field that brings it in line with other systems such as mice and humans.

JZ: How many tanks and zebrafish are maintained in your two labs?

LZ: We have 4000 tanks and about 300,000 fish.

JZ: Did you name your transparent Casper zebrafish after Casper the Friendly Ghost?

LZ: Absolutely.

In closing, I should add that the huge amount of information on zebrafish as a model organism for human disease and drug discovery from many labs has been centralized and organized in a database that is available through The Zebrafish Information Network (ZFIN) for researchers to share at the ZFIN Community Wiki.

I hope that you found this blog interesting, and I welcome your comments.

Joe Zon with some of his famous guitars at NAMM Show 2015. Taken from ilan.me.

Joe Zon with some of his famous guitars at NAMM Show 2015. Taken from ilan.me.

Postscript

Truth be told, compared to being asked if I’m related to Leonard Zon, I’m more frequently asked if I’m related to Zon guitars, which apparently are quite well known, and are produced in Redwood City, CA by Joe Zon, who is pictured below. My reply to that frequent question is that I don’t know if I’m related, but will someday look into that, as well as whether I’m distantly related to Leonard Zon.

Death of DNA Dogma?

  • Current Genetic Dogma is DNA → RNA → Protein
  • Two Research Teams Independently Implicate Sperm Short RNA Can Transmit Paternal Genetics
  • More Research Needed to Elaborate the New Dogma

The Central Dogma of all life on Earth is currently understood to be DNA encoding RNA that in turn encodes protein. That genetic inheritance is transferred as DNA was first posited by uber-famous Francis Crick, who coined the term Central Dogma. While dogmatic principles, by definition, should have no exceptions, a few species of viruses can be considered to be exceptional cases in this regard.

The Central Dogma. Taken from biology.tutorvista.com

The Central Dogma. Taken from biology.tutorvista.com

That said, there is now quite a scientific buzz—if not shudder by some—over reports implicating RNA molecules as direct (i.e. non-DNA) agents for mammalian inheritance. My instantaneous mental responses to these surprising—if not shocking—revelations was first, “Wow, who would have thunk?” and then, “I’ve got to share this news in a blog.” So here it is.

Surprising Science in Sperm

Human sperm. Taken from leavingbio.net

Human sperm. Taken from leavingbio.net

While most of us are probably at least passingly familiar with textbook descriptions of the basic structure of sperm and its functional role in reproductive molecular biology, more detailed information on its nucleic acid content is less known. Consequently, shown below is a depiction of the basic structural components of a sperm, DNA content, and primary functions for doing its job, so to speak, in fertilization of an egg.

By way of background, here’s information that I thought was worth sharing. My Google Scholar search results for nucleic acid content of sperm included a very impressive technological accomplishment reported by uber-famous professor/entrepreneur Stephen Quake and co-workers in 2012 on microfluidic separation methods for the first ever genome-wide single-cell DNA sequencing of human sperm. Contrary to what one might intuitively expect, 91 genomes of sperm from a single individual were not identical. Since DNA from only one sperm and one egg combine during fertilization, the exact paternal DNA genotypes in the resultant offspring involves “pot luck,” so to speak.

Regarding RNA, my Google Scholar search led to a paper in 2011 by Krawetz et al. on the first ever report of deep-sequencing of short (18-30 bases) RNA (sRNA) in human sperm (for which TriLink offers a high-performance CleanTag™ kit for sRNA library prep as detailed on this poster). Krawetz et al. found microRNA (miRNA) (≈7%), piwi-interacting RNA (piRNA) (≈17%), and repeat-associated sRNA (≈65%). A minor subset of sRNA within the transcription start site/promoter fraction (≈11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. However, reproductive roles for this molecular menagerie (what I tongue-in-cheek call these various sRNAs) remain speculative.

Fast forwarding to present time leads us to the two “wow” publications in venerable Science that triggered this blog:

While you’ll need to read these publications for details, they collectively raise the following controversial question vis-à-vis the Central Dogma for strictly DNA-based inheritance.

Are You Inheriting More Than Genes from Your Father?

Yes, is the surprising—if not bombshell—answer to this question, which I borrowed from Mitch Leslie’s Science editorial headline. If this conclusion is supported by further studies, it forces a fundamental revision of reproductive molecular cell biology. That’s a very big deal, so to speak, with ramifications not to be under appreciated.

Using sRNA library preparation methods analogous to TriLink CleanTag™ for Illumina deep-sequencing, the USA-Canadian team analyzed sperm from male mice fed a low-protein diet, progeny of which showed elevated activity of genes involved in cholesterol and lipid metabolism. They found that >80% of sRNA were fragments from several kinds of transfer RNAs (tRNAs). Most notably, 5′ fragments of tRNA-Gly-CCC, -TCC, and -GCC shown below all exhibited an approximately 2- to 3-fold increase in low-protein sperm.

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

Arrows indicate ~30- to 34-nt 5′ tRFs. Taken Upasna Sharma et al. Science (2016)

To understand when, where, and how these tRNA fragments were formed, as well as unravel functional significance, the researchers describe an experimental tour de force—in my opinion. This included antisense modified-oligonucleotide “knock-out” of these tRNA fragments, as well as “knock-in” injection of <40-nt sRNA populations purified from control and low-protein sperm into control zygotes.

The researchers concluded that the sperm acquired most of these fragments while passing through the epididymis, a duct from the testicle where the cells mature. Functionally, they also link tRNA fragments to regulation of endogenous retro-elements active in the preimplantation embryo.

In the second study, the China-USA team also found tRNA fragments by deep-sequencing of sRNA. After feeding male mice either a high-fat or low-fat diet, the scientists injected the animals’ sperm into unfertilized eggs, and then measured metabolic performance of the offspring, which ate a normal diet. Progeny of fat-eating fathers remained lean; however, they showed two abnormalities often found in their dads and in humans who are obese or diabetic—abnormal absorption of glucose and insensitivity to insulin.

Like the first study, these researchers also did “knock-in” experiments wherein they inserted the tRNA fragments into eggs fertilized with other sperm. Fragments that came from fathers that ate the high-fat diet resulted in offspring that also showed impaired glucose absorption.

Take Home Messages

At the risk of over simplifying or over generalizing, the aforementioned two studies of sRNA in sperm provide compelling—and stunning—evidence for how tRNA fragments in sperm are responsible for inheritance independent of sperm DNA sequences. So much for dogma.

With regard to specifics, researchers now need to investigate how permanent these changes are, and how quickly they can be reversed by changing diet.

The flip-side of a bad diet adversely influencing offspring is to investigate if and how a good diet imparts better health to offspring.

Please share your thoughts about these reports, conclusion, and implications by commenting here.

Postscript

If you enjoy hip hop music—or just want to chuckle—this YouTube video for the Central Dogma song will get your head bobbing in sync with the music, lead you to smile, and give you a cool visual display of the central dogma.

Nucleic Acid-Based Circulating Biomarkers for Cancer Diagnostics Become Reality

  • Circulating Tumor Cell Blood Tests Approved by FDA
  • Circulating DNA Stool Test Approved for Colorectal Screening to Avoid Colonoscopy
  • Circulating mRNA Urine Test Approved for use to Reduce the Total Number of Unnecessary Prostate Biopsies

Backstory

Taken from sysmex-inostics.com 

Taken from sysmex-inostics.com

According to the NIH National Cancer Institute website, ~1.6 million persons in the U.S. alone will be diagnosed with cancer this year. A very important key to survival is early detection. To enable significantly earlier diagnosis compared to manifestation of clinical symptoms, researchers have been focusing on finding DNA or RNA biomarkers that are circulating in blood, which is readily available and relatively noninvasive compared to traditional biopsies.

exosomesSome of the basic processes underlying this paradigm-shift in cancer diagnostics are depicted in the simplified cartoon wherein tumor cells, or components thereof, pass into the bloodstream. This leads to circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) to investigate and differentiate from their normal counterparts as sources of potential biomarkers.

That task is much easier said than done because of the need to sort through all of the normal components in blood, as well as deal with circulating cells and DNA derived from apoptosis (aka programed cell death) and necrosis that are normal ongoing “background” to contend with. In addition to CTCs and ctDNA, there is active cellular excretion of small (30-100 nm) exosome particles as depicted in the following graphic. Consequently, gene-encoding mRNAs, gene-regulating micro RNAs (miRNA), and potentially other exosomal components, can serve as diagnostic biomarkers.

Snapshots of Recent Commercial Diagnostic Products

My search of PubMed for publications indexed to “circulating biomarkers” AND “cancer” led to ~9,000 items, the vast majority of which have appeared during the past decade at an accelerating annual rate.  In fact, there were ~1,000 publications in 2014 alone—that’s roughly 3 such publications every day! Those interested in perusing this mountain of information later can use this link, as my intention here is to comment on resultant commercial diagnostic products, each of which provides all-important early diagnosis using a simple blood test, or urine or stool.

CTCs

In one of my blogs last year, I asserted that liquid biopsies were (metaphorically) clinically valuable “liquid gold” in a modern day Gold Rush. My evidence for the “rush” was a then recent review in Clinical Chemistry stating 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.” In that vein—double pun intended—who’s struck it rich, so to speak, commercially?

Taken from journal.frontiresin.org

Taken from journal.frontiresin.org

The answer is Veridex, which developed the CELLSEARCH® CTC Test that has the added distinction of being the first FDA-approved in vitro diagnostic (IVD) test for capturing and counting CTCs to determine the prognosis of patients (in this case for metastatic breast, colorectal or prostate cancer). This test utilizes magnetic capture of cancer-specific antibodies as depicted below.  Veridex was subsequently acquired by Jansen Diagnostics, which now offers a complete system for CELLSEARCH® CTC Test comprising sample collection, sample preparation, and sample analysis using unique immuno-magnetic and fluorescence imaging technology.

In addition, a Swiss molecular diagnostics company, Novigenix, offers its blood tests for early detection of cancer. Colox®, its lead product, is designed to significantly reduce mortality from colorectal cancer through early detection and follow-up colonoscopy. Novigenix’s technology is based on predictive gene expression profiles of circulating blood cells and tumor-derived protein markers.

Taken from Soper and coworkers in Chem. Commun. (2015).

Taken from Soper and coworkers in Chem. Commun. (2015).

Although not yet a diagnostic device, Prof. Steven Soper at UNC-Chapel Hill and a team of coworkers have recently published methods whereby captured CTCs can be enzymatically released for further analysis. This release procedure (depicted right) features use of an oligonucleotide linker containing uracil (U) that is cleaved by USER™, which consists of a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.

ctDNA Biomarkers for Colon Cancer Screening

That ctDNA can provide promising biomarkers for noninvasive assessment of cancer has been successfully translated into a commercial product by Trovagene, which tests for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagnostic value of ctDNA.

In addition, Cologuard® (developed by Exact Sciences in Madison, WI) was approved by the FDA as the first stool-based colorectal screening test that detects red blood cells and DNA mutations that may indicate colon cancer or precursors to cancer. Its commercials are frequently seen on TV. Given the inconvenient colon-cleansing required of patients prior to the also unpleasant invasiveness of colonoscopy, it’s not surprising that more and more persons are opting to use this new test.

In fact, Exact Sciences recently reported that during the first quarter of 2016, the company completed approximately 40,000 Cologuard® tests, an increase of more than 260% compared to approximately 11,000 tests completed in the same quarter of 2015. The cumulative number of physicians ordering Cologuard® since launch expanded to more than 32,000. Finding a doctor is relatively easy, as I found out when I located a gastrointestinal (GI) specialist near me who was also in my network—yeh!

Given the high incidence rate of colon cancer, and the traditionally recommended screening process, it was necessary for Exact Sciences to obtain compelling data in a large clinical study. An FDA announcement stated that the safety and effectiveness of Cologuard® was established in a clinical trial that screened 10,023 subjects. The trial compared the performance of Cologuard® to the fecal immunochemical test (FIT), a commonly used non-invasive screening test that detects blood in the stool. Cologuard® accurately detected cancers and advanced adenomas more often than the FIT test.

Other ctDNA Biomarkers

PlasmaSelect-R™ offered by Personal Genomics Diagnostics, which is a service company founded by experts at Johns Hopkins University, analyzes ctDNA in blood for genetic alterations in cancer based on a targeted panel of 63 well-characterized cancer genes. Cell-free DNA is extracted from plasma using proprietary methods for low-abundance sample DNA, and processed using a proprietary capture process for high-coverage next-generation sequencing to allow tumor specific mutations, amplifications, and translocations to be identified with a high sensitivity (allele fractions as low as 0.10%) and specificity. The company states that its “services further the understanding of cancer and facilitate the development of new diagnostics and therapeutics through our pioneering research approaches and novel technologies.” 

In June 2016, Roche announced that the FDA approved the cobas® EGFR Mutation Test v2 for use with plasma samples, as a companion diagnostic for the non-small cell lung cancer (NSCLC) therapy, Tarceva®. It’s important to recognize that this is the first FDA approval of a liquid biopsy test as an aid in clinical decisions, and makes it the only companion diagnostic that is FDA-approved for the detection of the epidermal growth factor receptor (EGFR) gene in tumor DNA derived from plasma (or tumor tissue). NSCLC patients who have EGFR exon 19 deletions or L858R mutations are candidates for the EGFR-targeted therapy Tarceva® (erlotinib) in first-line treatment.

Circulating RNA and miRNA

The discoveries in 1999-2000 of tumor-derived RNA in the blood of cancer patients sparked a new field for studying gene expression noninvasively using quantitative reverse transcription-PCR (qRT-PCR) and then next-generation sequencing. The existence of circulating RNA was surprising because ribonucleases are present in blood. However, mechanisms that protect circulating RNA reportedly include complexation to lipids, proteins, lipoproteins, or nucleosomes, and protection within apoptotic bodies or other vesicular structures.

Cleverly named Molecular Stethoscope is a newish startup co-founded by uber-famous Drs. Stephen Quake and Eric Topol. The company has leveraged Quake’s finding that genome-wide analysis of circulating RNA shows tissue-specific signatures from all of the major organs can be monitored in blood, and Topol’s finding that such signatures can be used to predict imminent occurrence of a heart attack. Coronary artery disease, neurodegenerative diseases, and autoimmune/inflammatory diseases are the company’s current objectives. I’m guessing, however, that cancer might be added or licensed.

My search of the literature indicates that there are far more publications on circulating miRNA, presumably due to its greater abundance resulting from its small size and/or binding to miRNA-related proteins. The biogenesis of miRNA is depicted below.

Taken from nature.com

Taken from nature.com

A review and prospectus for circulating miRNA applied to cancer has been recently published by Bertoli et al. in an article entitled MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer. From my search of this emerging field, some exemplary commercial endeavors are as follows.

The first blood-based cancer diagnostic to exploit exosomes became commercially available in the U.S. in January 2016 via launch of ExoDx Prostate(IntelliScore) by Cambridge, MA-based Exosome Diagnostics. As reported by a large team of medical experts in JAMA Oncology, qRT-PCR was used to compare the urine exosome 3-gene expression with biopsy outcomes in patients with a range of low-to-high prostate-specific antigen (PSA) levels (2 to20 ng/mL).

Taken from nature.com

Taken from nature.com

The investigators concluded that this qRT-PCR assay using urine was associated with improved identification of patients with higher-grade prostate cancer among men with elevated PSA levels and could reduce the total number of unnecessary biopsies from the ~1M total annual biopsies. The complications that have been associated with unnecessary biopsy and overtreatment range from erectile dysfunction and incontinence, to infections, sepsis and serious cardiovascular events.

At the other end of the commercial spectrum, so to speak, startup Miroculus aims to aid in the early diagnosis of cancer by making a low-cost, open-source, decentralized diagnostic they called Miriam pictured below. Their goal is for untrained workers in clinics around the world to be able to use Miriam to screen for cancer.

Taken from miroculus.com

Taken from miroculus.com

Miriam made its—or more gender specific—her public debut at the TEDGlobal conference in Rio De Janeiro in 2014 with TED curator Chris Anderson calling it ‘one of the most thrilling demos in TED history’, according to Miroculus. To see and hear why this opinion is accurate, and how Miriam will work in concert with a smartphone camera and cloud interface, I urge you to check out the ~11 minute TEDGlobal presentation at this link, which also gives a short, layperson introduction to miRNA biomarkers in blood for cancer.

Oh, One More Thing

Taken from graymatters.com

Taken from graymatters.com

Although this post focuses on nucleic acids, it’s worth noting that protein biomarkers in blood are also being investigated. In view of increased awareness and media attention about concussion injuries in the National Football League (NFL), a timely example of protein biomarkers for diagnosis of chronic traumatic encephalopathy (CTE)—which heretofore has not been possible by any test—is in development.

Currently the only way to diagnose CTE is through a post-mortem autopsy, but Aethlon Medical Inc. intends to change that with the diagnostic test being developed by its subsidiary Exosome Sciences. The test being studied is designed to identify an abnormal protein called tau that builds up in brain tissue as a result of repetitive head trauma. CTE researchers believe that they have developed a means of measuring plasma exosomal tau. Researchers thought that exosomes had potential as a means of identifying CTE because they cross the blood-brain barrier and can provide a unique method of measuring certain aspects of the contents of brain cells through a blood test.

Exosome Science was able to use its diagnostic blood test in 78 NFL players with histories of concussions, as well as in a control group made up of 16 athletes involved in non-contact sports. The subjects are all part of a much larger NIH-funded project called DETECT, which is focused on developing a variety of biomarkers for CTE and involves researchers at Boston University School of Medicine and the University of Washington.

Look for a future post here about DETECT involving nucleic acid biomarkers.

As always, your comments are welcomed.

Dietary Intake of Plant miRNA in Humans is Exciting but Controversial

  • Chinese Team Claims Dietary Rice miRNA can Regulate Gene Expression in Humans
  • Collaboration by miRagen Therapeutics and Monsanto Reports Inability to Detect Bioavailability of Dietary Rice miRNA
  • City of Hope Investigators Claim First Evidence for Plant miRNA having Anticancer Activity

Prelude

food

Taken from whale.to

You’re probably familiar with the adage “you are what you eat,” the origins of which I found attributable to 17th century Europeans. As scientists, we can intuitively understand this concept by thinking about our dietary food as biochemical inputs, and our body content as biochemical outputs. Further intuition suggests that this input/output cuts both ways, so to speak, as stated here by noted healthy food advocate, Ann Wigmore.

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Curiously Circular RNA

  • Circular RNA (circRNA) Formation Serendipitously Discovered in 1991  
  • Next-Generation Sequencing Reveals circRNA to be Ubiquitous
  • circRNA can Function as MicroRNA ‘Sponges’ to Regulate Gene Expression

There’s something seductively simple—and curious—about circles, which are unique in having no beginning or end, unlike most other things. On a less philosophical plane, thinking about circles conjures up incongruent memories of delicious doughnuts and geometric definitions from my youthful days going to the neighborhood bakery and diligently taking notes in my high school geometry class, respectively.

donuts

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Genes in Space

  • High School Student’s PCR Experiments Launched to Space Station
  • Program Evaluates Epigenetics Linked to Astronaut’s Altered Immunity
  • Amplyus Has Big Plans for Its Tiny, Low-Cost PCR Device

In my 2013 blog post on the 30th anniversary of the invention of Nobel Prize-winning PCR by Kary Mullis, I ventured to say that PCR of DNA or RNA was the most widely used—and enabling—method for all life sciences on planet Earth. This accolade can now be expanded to extraterrestrial space in view of PCR experiments to be carried out in the International Space Station (ISS) following the April 8th launch from Kennedy Space Center in Florida aboard NASA’s Cargo Resupply Services flight (CRS-8).

Taken from earthkam.org

Taken from earthkam.org

What makes this “out of this world” milestone for PCR even more exciting is that it’s the result of competition among students in high school—yes, high school—to conceive and design PCR-based studies relevant to living in space. Following is a brief synopsis of the program, the winning high school student, and a small startup company with big plans for its low-cost miniPCR™ device.
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National DNA Day 2016 – DNA Dreams Do Come True!

  • Khorana’s Dream of Synthesizing a Gene from Hand-Made Oligos
  • Caruther’s Dream of Automating Oligo Synthesis
  • Venter’s Dream of Fully Automating Gene Synthesis
  • Who’s Dreaming About What’s Next?

DNA Day ImageThis blog acknowledging National DNA Day on April 25th deals with dreams of various sorts, but mainly with gene synthesis, which was only a dream in the 1950s and is now achievable in a way few dreamed possible even a few years ago.

Before I get to DNA gene-dreams that did come true, I want to briefly mention two other dream-like anniversaries. First is the fact that my blog is now beginning its 4th year—yeh!—after its inaugural posting in April 2013 to celebrate 60 years since Watson & Crick’s famous publication of DNA’s helix structure as the fundamental basis for genetic material. Second is this year being TriLink’s 20th anniversary—yeh!—as a leading provider of modified nucleic acids, which co-founders Rick Hogrefe and Terry Beck likely view as their business dream come true. But I digress…

The First Dreamer and Doer Continue reading