Studying Telomeres in Space

  • Telomeres are DNA Biomarkers for Your Biological “Age”
  • Telomere Shortening Due to Stress was Expected During Spaceflight, but Exactly the Opposite has Been Found
  • Raising New Questions to Answer

After you read this blog about studying telomeres in space, I think you will agree with my opinion that scientific advances can sometimes occur amazingly fast. Telomeres (which are peculiar DNA structures that I’ll explain below) went from esoteric Nobel Prize subject matter in 2009 to the focus of spaceflight science in just six short years. Now, telomeres are being investigated by PCR on the International Space Station (ISS)! With a wink and a nod to Star Trek, this is indeed “warp speed” progress!

Taken from keywordsuggest.com

What are telomeres?

A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighboring chromosomes. For vertebrates like us, the repetitive sequence of nucleotides in telomeres is TTAGGG, with the reverse complementary DNA strand being AATCCC, as depicted below. This sequence of TTAGGG is repeated ~2,500 times in humans.

During chromosome replication, the enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, causing the end of the chromosome to be shortened in each replication. The telomeres are thus disposable “buffers” at the ends of chromosomes which are truncated during cell division, as depicted below.

Taken from weeklyglobalresearch.wordpress.com

Telomers, however, are replenished by an enzyme named telomerase. This peculiar enzyme has an embedded RNA template and incorporates DNA nucleotides, as depicted below, and is therefore a special kind of reverse transcriptase. In people, it has been found that telomeres shorten with age in all replicating somatic cells that have been examined. In fact, average telomere length declines from about 11 kilobases at birth to less than 4 kilobases in old age, with the average rate of decline being greater in men than in women. Thus, telomere length can serve as a biomarker of a cell’s biological (versus chronological) “age” or potential for further cell division.

Taken from 2014hs.igem.org

‘Houston, we have a problem’

This now famous phrase, which was used in the past tense by the crew of the Apollo 13 moon flight to report a major technical problem back to their Houston base, echoed in mind when I learned that space flight might lead to telomere shortening. Yikes! This molecular-level change could indeed be a serious problem, and was first suggested by findings from laboratory microgravity simulations reported in 2008 by Chinese researchers. Since it was known that space flight leads to bone loss, they cultured bone stem cells (BSCs) under simulated microgravity in a rotary cell culture system.

This led to significantly decreased activity of telomerase. It was postulated that reduced bone formation in space flight may partly be due to the altered potential differentiation of BSCs associated with telomerase activity, which plays a key role in regulating the lifespan of cell proliferation and differentiation. Additionally, telomerase activation or telomerase replacement may act as a potential countermeasure for microgravity-induced bone loss.

Taken from energeticnutrition.com

If you’re thinking that these “potential countermeasures” are fanciful, you’d better think again. I recently came across a patent that was published last year on methods and compositions for increasing telomerase activity in cells, including pharmaceutical formulations. Moreover, there are now various commercially available supplements claiming to promote telomerase activity, such as that picture below. I hasten to add that I do not advocate use of any such supplement, and that interested readers should consult their primary care physician or certified nutritionist.

Twins and telomeres

Although identical twins are almost the same genetically, differences in environment, diet and other outside factors can affect their health in different ways. Consequently, identical twins have been enrolled in various studies that require deciphering effects due to “nature vs. nurture” (i.e. intrinsic genetics vs. external factors). Part of the Twins Study supported by NASA was aimed at examining the effects of space travel on one of a pair of twins: astronaut Scott Kelly, who stayed on the ISS for one year, while his twin brother, Mark, remained on Earth. In brief, Prof. Susan Bailey at Colorado State University is exploring differences between the twins’ telomeres to determine if telomeres respond differently to spaceflight and then how such changes relate to the various medical endpoints studied by other Twins Study investigators.

Scott Kelly (left) and his identical twin brother Mark in 2015 prior to Scott’s one-year mission to the ISS. Taken from space.com

Preliminary research results for this part of the NASA Twins Study (reported at NASA’s annual Investigators’ Workshop earlier this year) were a quite surprising because they were opposite of what was expected, thus raising more questions than providing answers. It had been theorized that exposure to microgravity and stress during prolonged spaceflight would shorten telomeres, but instead Bailey’s team found telomeres in Scott’s white blood cells increased in length while in space! This finding was rationalized as being due to increased exercise and reduced caloric intake during the space mission. Upon his return to Earth, however, these telomeres began to shorten again.

This is yet another example of a biomedical phenomenon being far more complex that first theorized, and one that becomes less understood as more and more data are obtained. We’ll all have to patiently stay tuned for how this telomeres-in-space story evolves. The good news, as I’ll explore in the next and final section of this blog, is that there are exciting plans to use PCR to measure telomere length extraterrestrially! This is very “far out” science—pun intended.

Studying telomeres in space by PCR

Taken from geeky-gadgets.com

The aforementioned Twins Study involved taking blood samples from an astronaut during spaceflight for lab analysis upon return to Earth. To obtain much more data, and to do so in real time while in space, NASA launched the Genes in Space-2 mission in April 2017. The goal is to determine whether astronauts aboard the ISS can analyze telomeres by PCR reactions in a small thermal cycling device (miniPCR system) and thus measure and monitor telomere changes during spaceflight.

In addition to testing the miniPCR system, the Genes in Space-2 mission has a secondary goal to test the feasibility of techniques used to measure telomere length. Currently, Single Telomere Length Analysis (STELA) is the only suitable technique for use on the ISS due to technical requirements. The Genes in Space-2 mission will also be testing the feasibility of a loop-mediated isothermal amplification (LAMP) colorimetric assay for detection of amplification aboard the ISS. Please stay tuned for updates on the outcome of these very important feasibility experiments.

Scheme for STELA procedure. Taken from Xing et al. (2009)

Julian Rubenfein. Taken from nydailynews.com

As a side note, the Genes in Space competition for 2017 selected this experiment on telomere amplification in microgravity from 375 submissions by nearly 850 students in grades 7 to 12 from across the US. This telomere experiment was proposed by 17-year old Julian Rubinfien from Stuyvesant High School in New York City, who is pictured below. I encourage you to read this interesting, although lengthy interview about his background and the experimental rationale. What’s even more interesting is this short video of Julian at the launch and his comments—very impressive!

I strongly encourage you to read more about all the award-winning experiments in this exciting round of competition among young, highly motivated, advanced students, who I’m sure will be successful in whatever they do in the future.

Your thoughts or comments here are welcomed.

Postscript

Profs. Elizabeth Blackburn and Carol Greiner—who received a Nobel Prize in 2009 for seminal work on telomeres—co-founded Telome Health Inc. (THI) in 2010 to leverage the predictive power of telomere-length assays to help assess health status, disease and mortality risk, and response to specific therapies. THI subsequently announced TeloTest™ as a diagnostic test that measures average telomere length by qPCR. TeloTest™ was the first saliva-based telomere test available on the market, and is currently offered by a company named TeloYears.

The clinical utility of testing telomere length in a saliva-based test was recently reported from an independent, large clinical study sponsored jointly by Kaiser Permanente, University of California, San Francisco (UCSF), and National Institutes of Health. In the study, the average telomere length of 100,000 Kaiser patients was measured and analyzed relative to other health domains and clinical outcomes.

My recently obtained TeloTest™ results from TeloYears indicated that my biological age is 4 years older than my chronological age. Naturally, I was hoping to learn that my telomere-based age would be less than my actual age. Alas, the results are what they are, so I’ll be following diet, exercise, sleep, and stress-management recommendations you can read about at TeloYears Learning Center.

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CRISPR-C2c2 Update: Powerful New Diagnostic Method Using CRISPR

  • CRISPR Craze Continues Led in Part by Wunderkind Feng Zhang
  • Nonspecific CRISPR-C2c2 “Collateral” Cutting Channeled into Diagnostics
  • Turning Biochemical “Lemons to Lemonade”

This blog post is about a new and powerful diagnostic approach based on CRISPR, which I’ll get to below. But first I’d like to point out several reasons why this is an especially interesting development:

  • Taken from spyhollywood.com

    Any new method using CRISPR is more “sizzle” for this “super-hot” technology

  • Feng Zhang, the 32-year-old author of this publication, is regarded as a wunderkind
  • My past blog post on CRISPR-C2c2 “collateral” cutting noted possibilities for turning “lemons into lemonade”
  • Such “lemonade” has emerged as a diagnostic cleverly acronymized as SHERLOCK

So, without further ado, here’s a brief recap of CRISPR-C2c2 as an intro to SHERLOCK (think Holmes), followed by why this acronym is apropos for a diagnostic method that magnifies detection, in this case Zika virus, about which I’ve previously commented. 

What’s CRISPR-C2c2?

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (CRISPR-Cas) adaptive immune systems in microbes contain endonucleases that can be leveraged for CRISPR-based gene editing using targeted CRISPR RNAs (crRNAs), as I’ve outlined in a previous blog post. While such Cas enzymes target DNA, others target RNA and function as RNA-guided RNases, which was reported by Feng Zhang and collaborators in Science last year in an article titled C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.

My blog post about that C2c2 publication provides more information, but the main point related to the present blog post about diagnostics is that after recognition of its RNA target, activated C2c2 engages in nonspecific (aka “collateral”) cleavage of nearby non-targeted RNAs. This post-cleavage non-specificity can be transformed, if you will, from something “bad” to something “good” in terms of mechanisms, which I described as being akin to converting ‘lemons into lemonade’. You’ll now see how SHERLOCK is such ‘lemonade’ concocted by Zhang and coworkers.

What is SHERLOCK?

Taken from US 20140199245 A1

Before answering this question, and not to confuse you, I need to first mention that the nuclease originally called C2c2 was renamed Cas13a to be more systematic, and will be referred to herein as such. Having said that, Cas13a can be reprogrammed with crRNAs to provide a platform for specific RNA sensing. Molecular recognition of an intended RNA target by crRNA in vitro serves as a sequence-specific “trigger” to activate Cas13a’s nonspecific, “collateral” cleavage of labeled RNA reporters. Cleavage of these fluorescently labeled, internally quenched RNAs leads to unquenching and fluorescent light emission for detection, as depicted below for a generic RNase.

Now that you have a sense of how CRISPR-Cas13a can generate a signal, you’re probably asking ‘What is SHERLOCK?’ It’s an acronym coined by a large team lead by CRISPR genome-editing pioneer Feng Zhang and bioengineer James Collins, both from the Broad Institute. SHERLOCK stands for High-sensitivity Enzymatic Reporter UnLOCKing, which is a recently published new method for sequence-specific detection of DNA or RNA in any sample of interest. This general utility represents a major advance in CRISPR-based diagnostics.

Feng Zhang. Taken from be.mit.edu // James Collins. Taken from achetron.com

As depicted below, sample prep workflows allow for input of either double-stranded DNA (dsDNA) or RNA, which are first amplified by recombinase polymerase amplification (RPA) directly or as cDNA, after reverse transcription (RT-RPA), respectively. In either case, further amplification utilizes T7 transcription into RNA (blue = target RNA; yellow = non-target RNA). Following cleavage of target RNA by Cas13a-crRNA, a commercially available “cleavage reporter” (i.e. internally quenched) RNA undergoes “collateral” cleavage to generate a reporter signal (i.e. fluorescence; pictured below) for real-time detection of the target.

Taken from Feng Zhang, Collins & coworkers Science (2017)

An advantage of RPA is that it is performed isothermally, which allows simplification of the detection device by eliminating the need for a thermal cycler, thus lending to incorporation into small, portable point-of-care (POC) systems (about which I previously commented several times). The abovementioned RPA/T7 sample prep methodology was shown to have attomolar (aM, 10−18 mol/L) sensitivity in model systems, and was next investigated for sensitivity and specificity of virus detection, as well as simplification of reagent format.

Zhang, Collins & coworkers constructed lentiviruses harboring genome fragments of either Zika virus (ZIKV) or the related flavivirus Dengue (DENV), and showed that SHERLOCK detected viral particles down to 2 aM could discriminate between ZIKV and DENV. To explore the potential for POC use of SHERLOCK with paper-spotting and lyophilization (aka freeze-drying), they first demonstrated that Cas13a-crRNA complexes lyophilized and subsequently rehydrated could detect 20 femtomolar (fM, 10−15 mol/L) non-amplified RNA, and that target detection was also possible on glass fiber paper.

The other components of SHERLOCK, namely the RPA reagents and T7 polymerase, were already known to be amenable to freeze-drying and storage at ambient temperatures. In combination, freeze-drying and paper-spotting the Cas13a detection reaction resulted in comparable levels of sensitive detection of RNA as aqueous reactions. Although paper-spotting and lyophilization slightly reduced the absolute signal of the readout, SHERLOCK could readily detect mock ZIKV virus at concentrations as low as 20 aM. Most importantly, SHERLOCK detected ZIKV in clinical isolates (ZIKV RNA extracted from patient serum or urine samples and reverse transcribed into cDNA) could be detected at concentrations down to 1.25 × 103 copies/mL (2.1 aM), as verified by qPCR.

Concluding Comments

If you reflect upon the schematic for SHERLOCK, you’ll note that the input can be either DNA or RNA, which get amplified to produce many copies of RNA that serve as substrates for cleavage by Cas13a-crRNA, thus inducing collateral cleavage of reporter RNA to produce a detectable signal. Lest you think SHERLOCK is too costly to be practical, its developers provide a detailed cost accounting that estimates $0.61 per test, which you’re welcome to compare with your cost of conventional qPCR. I’m quite sure that will lead you to concur with me that $0.61 per test is relatively inexpensive.

If you’re questioning whether SHERLOCK is generally applicable, I urge you to read Zhang, Collins & coworkers in its entirety to learn more about SHERLOCK’s proven ability to detect and distinguish (1) various bacterial pathogens, (2) single-base cancer mutations in cell-free DNA, and (3) health-related single-nucleotide polymorphisms (SNPs) benchmarked against 23andMe genotyping data as the gold standard of these SNPs.

In an article in Science about SHERLOCK, Harvard University’s George Church (who I’ve proclaimed is The Most Interesting Scientist in the World and is the co-founded of a CRISPR therapeutics company), sums up his reaction in one word: ‘Wow.’ I agree!

The article concludes with Collins saying the Broad is now ‘aggressively exploring’ how to commercialize SHERLOCK and may launch a startup company. But before a diagnostic comes to market, it must pass muster at regulatory agencies such as the U.S. Food and Drug Administration. I’m betting is does.

I welcome your sharing any thoughts or comments about this new CRISPR-based diagnostic method.

Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from gene-quantification.de

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.

Lab-in-a-Box 

In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab

Lab-on-a-Robot

Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.

Lab-on-a-Drone

A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from tamu.edu

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Update on Zika Virus Detection by RT-PCR

  • Various RT-PCR Assays for Zika Virus Have now Received Emergency Use Authorization (EUA) from the FDA
  • Quest Diagnostics’ Assay Approved by FDA for General Use as a Zika Test
  • Troubled Theranos Touts New “miniLab” for Zika EUA
  • Vaccine Development Progressing Albeit Relatively Slowly
Aedes aegypti mosquito. Taken from wcvb.com

Aedes aegypti mosquito. Taken from wcvb.com

In January 2016, I posted a blog about the then emerging public awareness of Zika virus (ZIKV), which is spread by the bite of infected mosquitos—primarily the Aedes aegypti mosquito. Sadly, ZIKV can be passed from a ZIKV-infected pregnant woman to her fetus leading to development of a brain defect (microcephaly) and/or other malformities. Moreover, ZIKV is now associated with sexual transmission and blood transfusion. This is scary news.

This update was prompted by the additional fact that ZIKV is on the rise and there is still no vaccine, according to the Centers for Disease Control and Prevention (CDC)—my “go to” source for loads of authoritative information about this infectious disease. Weekly updated ZIKV-infection “case counts” in US States and US Territories were given as ~1,200 and ~6,500, respectively on Aug 10th, and increasing to ~3,400 and ~20,000 on Sep 21st —only 6 weeks later!

As I pointed out in my previous blog on ZIKV, absent an anti-ZIKV vaccine, there is considerable interest in mosquito abatement as well as early detection, notably by reverse-transcription PCR (RT-PCR) of this RNA Flavivirus. Now that Zika infection by mosquitos in Florida has been found, leading to several CDC warnings, I thought it would be both apropos and technically interesting to provide the following update on ZIKV structure and RT-PCR.

ZIKV Genome and Structure

As of last month, my PubMed search of “Zika [Title/Abstract] AND RT-PCR [Anywhere]” gave 55 publications. Incidentally, there are now a number of Zika genome sequencing publications, such as this lead reference. It’s worth noting that ZIKV genome sequencing enables monitoring the potential evolution of new genomic variants that might foil existing RT-PCR assays and guide the selection of new primers for RT-PCR.

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

It’s also worth pointing out that Zika’s overall molecular capsid structure is enveloped, spherical, and has a diameter of ~40 nm in diameter, as depicted below in schematic form, and pictured in the accompanying electron microscopic image. The surface proteins are arranged in an icosahedral-like symmetry.

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

ZIKV RT-PCR

In the interest of giving explicit credit to various investigative groups who have developed RT-PCR assays for ZIKV, here are links and short snippets I’ve selected from publications, in chronological order, found in the aforementioned PubMed search. Interested readers are encouraged to check out the original publication for details.

The first report of an RT-PCR assay for ZIKV appears to be by Faye et al. in 2008 at the Institut Pasteur de Dakar in Senegal, who targeted the envelope protein coding region and tested ZIKV isolates previously collected over a 40-year period from various African countries and hosts. The assay’s detection limit and repeatability were respectively 7.7pfu/reaction and 100% in serum; none of 19 other Flaviviruses tested were detected. Faye et al. in 2013 extended this work to include quantitative RT-PCR detection of ZIKV and evaluation with samples from field-caught mosquitoes.

Kinetics of ZIKV detection in urine compared to serum from 6 patients was described by Gourinat et al. in 2015 at the Institut Pasteur, Noumea, New Caledonia using RT-PCR primers and probes previously reported by others. Urine samples were positive for ZIKV more than 10 days after onset of disease, which was a notably longer period than 2-3 days for serum samples. These researchers concluded that urine samples are useful for diagnosis of ZIKV infections, and are preferred to serum wherein virus titer diminishes more rapidly.

Musso et al. in 2015 working in Tahiti, French Polynesia with 1,067 samples collected from 855 patients presenting symptoms of Zika fever found that analysis of saliva samples increased the rate of detection of ZIKV at the acute phase of the disease compared to serum samples. They noted that saliva was of particular interest when blood was difficult to collect, especially for children and neonates.

Most recently, Xu et al. in 2016 in China reported the development of a SYBR Green (intercalator dye)-based qRT-PCR assay for detection of ZIKV. Although their results indicate that the assay is specific, it’s important to note that SYBR-type detection can be subject to nonspecific artifacts, for which TriLink’s proprietary CleanAmp™ Primers can be investigated to potentially ameliorate such problems, as discussed in this downloadable pdf publication by TriLink researchers.

ZIKV In Vitro Diagnostic Assays

As detailed elsewhere, the Secretary of Health and Human Services (HHS) earlier this year determined that “there is a significant potential for a public health emergency that has a significant potential to affect national security or the health and security of United States citizens living abroad and that involves Zika virus.” The Secretary of HHS further declared that “circumstances exist justifying the authorization of the emergency use of in vitro diagnostics for detection of Zika virus…”.

The following RNA-based assays and suppliers are currently listed by the FDA for this Emergency Use Authorization (EUA):

  • xMAP® MultiFLEX™ Zika RNA Assay (Luminex Corporation)
  • VERSANT® Zika RNA 1.0 Assay (kPCR) Kit (Siemens Healthcare Diagnostics Inc.)
  • Zika Virus Real-time RT-PCR Test (Viracor-IBT Laboratories, Inc.)
  • Aptima® Zika Virus Assay (Hologic, Inc.)
  • RealStar® Zika Virus RT-PCR Kit U.S. (Altona Diagnostics)
  • Zika Virus RNA Qualitative Real-Time RT-PCR (Focus Diagnostics)
  • Trioplex Real-time RT-PCR Assay (CDC)

Among these, two are particularly notable—in my opinion. Trioplex Real-time RT-PCR Assay is a multiplexed laboratory test developed by the CDC to simultaneously detect ZIKA, dengue virus, and chikungunya virus RNA, each of which can be transmitted primarily by Aedes aegypti mosquitos to cause infections with similar symptoms. Consequently, it is useful to have a single test that can detect each of these viruses in the same sample. Full details for the Trioplex assay are provided in a 40-page downloadable pdf from the CDC at this link.

In brief, this CDC-developed test uses virus-specific primer pairs and fluorogenic hydrolysis probes each differentially dual-labeled with fluorescent reporter and quencher dyes for in vitro detection of complementary DNA (cDNA). The detection follows reverse-transcription of RNA isolated from clinical specimens including serum, cerebral spinal fluid, urine, and amniotic fluid. It’s worth pointing out that I subsequently found a publication in 2016 by several collaborating academic groups regarding an analogous triplex RT-PCR assay for the same three viruses.

The other notable assay is Zika Virus RNA Qualitative Real-Time RT-PCR developed by Focus Diagnostics, which in April 2016 was the first of the aforementioned ZIKV tests to receive authorization by the FDA for use by qualified labs to detect ZIKV RNA in blood samples of those meeting CDC clinical criteria or of people who may have lived in or traveled to an affected location or had other exposure to the virus. Quest Diagnostics, the parent company of Focus Diagnostics, announced it would make the test broadly available to physicians, including those in Puerto Rico in May 2016.

ZIKV EUA Sought by Theranos for Its new “miniLab”

Theranos, which has been in the news regarding troubles over its stealthy proprietary system for finger-stick blood tests, appears to be pivoting its strategic plans. It announced at the August 2016 American Association of Clinical Chemistry Meeting its R&D for a new, fully automated miniLab system, including analytical and method comparison results of its ZIKV nucleic acid-amplification-based assay.

According to its press release, the company collected finger-stick samples from subjects, including people in the ZIKV-infested Dominican Republic, and shipped those to Palo Alto, California to run on the miniLab. Although I was unable to obtain these particular results, I did find the following figure at the TechCrunch website showing functional components of the miniLab along with an article by Sarah Buhr that’s worth a quick read, in my opinion.

Taken from techcruch.com

Taken from techcruch.com

According to the aforementioned Theranos press release, the company has submitted assay validation data for this Zika assay to the FDA for an EUA. The company also states that it is unaware of any currently available capillary (i.e. finger-stick) test for ZIKV.

I’ll stay tuned for future general information about the miniLab, as well as information specifically related to ZIKV. If I hear of anything, I’ll add as a comment here or in a new post with technical details about its nucleic acid assays.

ZIKV Vaccine Status

I hope this blog has convinced you that RT-PCR of ZIKV has provided improved molecular diagnostics. I’m guessing, that like me, you find it unfortunate that there isn’t a proven anti-ZIKA vaccine as of yet. This is especially frustrating given the fact that Zika disease has been known for more than 50 years, and that it is evidently on the rise globally, including in the USA according to regularly updated CDC statistics.

In February 2016, the Obama administration requested $1.9 billion in funding for the NIH to develop a ZIKV vaccine. The US Congress continues to be deadlocked by partisan politics despite the fact that Florida state and local officials are scrambling to contain the ZIKV outbreak in Miami Beach. This outbreak poses a serious threat to the health of residents, as well to visitors who drive the region’s $24 billion-a-year tourism industry.

Nevertheless, some progress has been reported for US studies in monkeys, and US-based Inovio says it’s received FDA approval to begin studies in humans. Outside the US, Sanofi Pasteur in France is said to be poised for initiating its trials in humans, and French biotech company Valneva is reported to have succeeded in generating a ‘highly purified inactivated vaccine candidate’ using the same technical approach it used for its encephalitis vaccine that is marketed in the United States and Europe.

Lagging—dare I say “glacially slow”—action against ZIKV by the World Health Organization is quite disappointing to me, and is reminiscent of what I’ve commented on in an earlier blog concerning this bureaucracy’s ineffective response to the Ebola virus. If I had my druthers, TriLink’s previously announced engagement by Battelle in development of Ebola mRNA vaccine would somehow materialize for a Zika mRNA vaccine. In this regard, GlaxoSmithKline is reported to be preparing research studies alongside the NIH’s Vaccine Research Center to test a self-amplifying mRNA vaccine technology for Zika. Interested readers can check out this link to a fascinating PNAS publication by Geall et al. on biosynthetic self-amplifying mRNA vaccines delivered in lipid nanoparticles.

As always, your comments are welcomed.

Postscript

Taken from Fleming et al. (2016) ACS Infectious Diseases

Taken from Fleming et al. (2016) ACS Infectious Diseases

One of my previous posts featured recent elucidation of biologically functional G-quadraplexes in living cells. Consequently, it’s apropos to mention here that Fleming et al. at the University of Utah have just now published the first analysis of potential G-quadruplex sequences (PQS) in the RNA genome of ZIKV. As depicted in this artistic cartoon, several PQS were found, with the most stable located near the end of the 3’ untranslated region (3′-UTR). Importantly, these investigators propose a rationale for screening G-quadruplex-binding compounds as a completely new class of anti-ZIKV drug candidates. In my opinion, this is a great example of how basic biochemical research can lead to new strategies for much needed antiviral drugs.

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.

Point-of-Care PCR 2.0

  • Ubiquitome Quickens Pace of POC Apps for Its Freedom4
  • Cepheid Unveils its POC Diagnostics System
  • Hopkins Crew Brews “Coffee Mug-Sized” Gizmo for Fully Automated Chlamydia Testing
Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Regular readers of this blog will recall a recent byline exclaiming “Honey I Shrunk the qPCR Machine”, which spotlighted the unveiling of startup company Ubiquitome’s first point-of-care (POC) product—Freedom4—developed in New Zealand. Up until then, this far away—for me—exotic island country brought to mind folks fondly nicknamed Kiwi—after the native flightless bird, not Chinese fruit. Mightily impressed by this tiny but powerful qPCR device, I vowed to thereafter keep an eye on these Kiwis’ democratized POC apps enabled by its nifty handheld 4-sample high-performance qPCR device.

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Pseudouridine Biomarker for Breast Cancer

As you are probably aware, October is National Breast Cancer Awareness month. Everyone from the NFL to Yoplait yogurt seems to be engaged in campaigns for fundraising and awareness. I think it’s great to see the extensive community support for this worthy cause. Since breast cancer awareness is top of mind this month, I thought I’d follow up my latest blog about pseurdouridine with a ‘mini blog’ highlighting some interesting research involving a pseudouridine biomarker for breast cancer.

I’d also like to mention that TriLink is participating in Breast Cancer Awareness month. For every order placed in October, TriLink will dontate $5 to Susan G Komen to support the upcoming 3-Day Walk being held November 20-22 in San Diego.

3day

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RNA Vaccines: Safer and Better Than Pathogens

  • Polio Virus Accidentally Discharged by GlaxoSmithKline  
  • Anthrax Accidentally Shipped to Numerous Labs
  • New RNA Vaccines are Viewed as Much Safer and Better

Polio Virus Accidental Discharge

Polio virus is pretty but highly pathogenic. Taken from dr.marahimi.com.

Polio virus is pretty but highly pathogenic. Taken from dr.marahimi.com.

The genesis of this blog about safer, better vaccines using RNA instead of pathogenic organisms was an incident that received very little attention in the US but was big news in Europe and—frankly—scared the bejeezus out of me. London-based GlaxoSmithKline (GSK) acknowledged in a report that its vaccine production personnel in Belgium dumped 12 gallons of liquid contaminated with poliovirus into the local sewage treatment plant and subsequently into the Lasne River. Yikes!

A spokesman is quoted as saying that there was “an accidental discharge of cleaning solution that had been used to sanitize equipment used in the manufacture of polio vaccine.” However, chances of anyone developing polio from being exposed to the water were said to be very low, “given the high level of dilution and high level of public vaccination”—vaccination against polio is compulsory in Belgium. That may be true, but the issue is increasing safety, as you’ll see from what follows here.

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Gene-Expression Biomarkers Can Detect Depression

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

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

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

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

  • Depression is a very common illness. The Centers for Disease Control and Prevention estimates that 1-in-10 US adults suffer from depression, which reportedly costs close to $50B annual in lost productivity in the work place. Globally, more than 350 million people of all ages are afflicted with depression, according to recent statistics from the World Health Organization (WHO). By the year 2020, WHO estimates that depression will be the second leading cause of “lost years of healthy life”, following heart disease. Incidentally, as seen from the map below, depression rates around the world vary significantly among countries.
  • Depression is diagnosed based on the patients’ self-report of their symptoms and the evaluation of one or more structured psychiatric interviews with the patient by a psychiatrist, psychologist or primary care physician. The absence of direct, non-subjective measures of depression can lead to relatively lengthy time-to-treatment, non-reporting, or—sorry to say—fraudulent claims and/or treatments based solely on what is said as opposed to what is objectively measured.

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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.”

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