Jerry’s Favs from the Recent 7th Cambridge Symposium

  • DNA Can Function as an Enzyme
  • RNA Polymerase Activity Without Proteins
  • Systemic Brain Delivery of Therapeutic Oligos

The 7th Cambridge Symposium on Nucleic Acids Chemistry and Biology took place September 3-6, 2017 at historic Queens’ College, Cambridge, which was founded in 1448 by Margaret of Anjou (who was then Queen of England by marriage to King Henry VI). Yours truly had the honor of participating in this event and presenting one of TriLink’s posters on the company’s new types of chemically modified mRNA for mRNA therapeutics. As done for other conferences I’ve attended on behalf of TriLink, I wish to share here my personal favorites among the many lectures, which are still fresh in my mind. However, I hasten to emphasize that, while choosing these “favs” is biased a bit by my scientific interests, all the lectures topics are worth looking at later by perusal of the symposium program of nearly forty presentations.

Taken from na-cb.co.uk

By the way, the symposium’s logo artistically depicts a DNA helix under a wooden bridge, better seen in the accompanying picture of the actual bridge over the River Cam at Queens’ College. Built in 1749, it has become known as the Mathematical Bridge for reasons you can read later, and appears to be an arch but is composed entirely of straight timbers. The historical connection between the DNA double helix and Cambridge is that in 1953 Watson & Crick proposed this now famous deoxynucleic acid structure as the molecular basis for genetics, which I’ll comment on again at the end of this post.

Taken from worldachitecture.org

Overview of the Symposium

Mike Gait. Taken from histmodbiomed.org

This symposium is the 7th in a popular conference series going way back to 1981 that brings together nucleic acids scientists across a broad area but with emphasis on chemistry, biochemistry and structure. Michael (Mike) Gait, who is at the Medical Research Council Laboratory of Molecular Biology in Cambridge, originated this series and has been a key organizer for all seven conferences. Participants come from all over the world and include professors, students, and companies—as well as Nobel Laureates (this year Jack Szostak of telomeres fame).

In addition to Mike, the organizing committee included Sir Shankar Balasubramanian, who was recently knighted for his contributions to next-generation sequencing and research on G-quadruplexes, the latter of which I featured here a few years ago. Other committee members were Rick Cosstick, Phil Holliger, and Chris Lowe.

Subject areas this year included:

  • Nucleic acids as therapeutics (including antisense, RNAi, aptamers, immune recognition, cell delivery)
  • RNA and DNA structures and their protein complexes (duplexes, quadruplexes, RNA and DNA enzymes, riboswitches, protein complexes + assemblies)
  • Nucleic acids chemistry applied to cells and cell mechanisms (genomes, evolution, repair, cell manipulation)
  • Nucleic acids as tools, structural assemblies and devices (nanostructures, cages, arrays, supra-molecular chemistry)

Marv Caruthers. Taken from colorado.edu

The showcased and highly prestigious Nucleic Acids Award was presented to Marvin (“Marv”) Caruthers in recognition of his seminal contributions to the synthesis of oligodeoxynucleotides (aka “oligos”) based on the use of phosphoramidite chemistry. This mechanistically elegant chemistry enabled much faster and more efficient coupling for automated synthesis of oligos, which fundamentally transformed all manner of basic and applied research with DNA. His award lecture was titled Synthesis, Biochemistry, and Biology of New DNA Analogues, some of which has been recently published. My previous several posts commenting on Marv, who has been a professor at the University of Colorado in Boulder since 1973, can be read later here.

Jerry’s Favs from the Symposium

To encourage inclusion of unpublished results and other types of “late breaking news” from the lab, the organizers forbade use of Twitter or other real-time social media, blogging, or taking pictures of slides being shown. Consequently, what I can say here is restricted to published papers related to my favs. Keeping this limitation in mind, here are my personal top-three talks that I consider to be tied (i.e., have equivalent scientific importance).

DNA Can Function as an Enzyme!

This lecture by Scott Silverman at the University of Illinois, Urbana-Champaign, dealt with DNA enzymes (aka deoxyribozymes), which were first reported in 1996 by Carmi et al., and are of interest because they expand enzymatic functionality from naturally occurring proteins to synthetic nucleic acids. DNA enzymes can be evolved in vitro starting with random sequences of DNA and applying suitable selection.

In a published account titled Pursuing DNA Catalysts for Protein Modification, Silverman has provided a lengthy and chemically detailed description of his use of in vitro selection to develop DNA catalysts for many different covalent modification reactions of peptide and protein substrates. While interested readers can consult Silverman’s account for various examples, it’s illustrative to consider the molecular design strategy depicted below that was used to evolve a synthetic DNA functional-equivalent of naturally occurring protein kinases that, by definition, carry out protein phosphorylation.

Taken from Silverman Acc Chem Res (2015)

In this case, modular deoxyribozyme design involved a stretch of 40 randomized bases (N = A/G/C/T) having a hairpin loop conjugated to a tyrosine (Tyr)-containing peptide on one end, and an ATP-binding aptamer on the other end. This was intended to experimentally assess whether it would be helpful to provide a predetermined small-molecule binding site in the form of an aptamer, which would cooperate functionally with an initially random catalytic region (N40) from the onset of selection. The selection outcome established that while modular deoxyribozymes that utilize a distinct predefined aptamer domain can indeed be identified, such DNA catalysts do not have any functional advantage relative to nonmodular analogues selected simultaneously for binding and catalysis, at least for this test case of tyrosine kinase activity using an ATP phosphoryl donor.

RNA Polymerase Activity Without Proteins!

By analogy to use of in vitro selection to evolve DNA enzymes from complex pools of random sequences of DNA, complex mixtures of unrelated RNA sequences can also be subjected to in vitro selection to evolve RNA enzymes (aka ribozymes). Indeed, as noted and cited in a talk by co-organizer Philipp Holliger, the emergence of an RNA catalyst capable of self-replication is considered a key transition in the origin of life in the prebiotic “RNA World” first hypothesized by Walter Gilbert in the 1980s. How such self-replicating (replicase) ribozymes emerged from the pools of short RNA oligomers arising from prebiotic chemistry and non-enzymatic replication, however, is unclear.

In a published version of Holliger’s talk addressing this important open question, his laboratory carried out an elegant series of experiments demonstrating that RNA polymerase ribozymes can assemble from catalytic networks of RNA oligomers that are each no longer than 30 nucleotides. Additionally, they found that entropically disfavored assembly reactions are driven by iterative freeze-thaw cycles. Such cooling (to freeze)-warming (to melt) cycles for aqueous solutions of RNA oligo reactants are notionally opposite to heating (to dissociate)-cooling (to hybridize) cycles used for amplification by PCR.

Interested readers can peruse Holliger’s publication for details about these novel findings, but for the purposes of this blog the schematic shown below depicts and describes the mechanism for assembly wherein relatively short RNA oligomers undergo serial ligations and “grow” into a self-replicating RNA polymerase. To me, these results provide an amazing glimpse backward in time to how the RNA World may have evolved!

Assembly of a RNA polymerase ribozyme (RPR 1234) from oligonucleotides devoid of pre-activation. (a) Schematic representation of the assembly trajectory involving (anti-clockwise from top left), ribozyme (blue) cleavage of a short 3′ tail (red) generating a 2′, 3′ cyclic phosphate (>p) (red dot), dissociation of the cleaved tail and strand exchange to cognate substrate (orange) followed by ligation of substrate 5′ OH with >p. (b) Network diagram of RPR 1234 assembly from 4 tailed fragments 1, 2, 3 and 4. Tailed input fragments can ligate to their cognate 5′fragments but must be cleaved (red lines) before ligation to 3′fragments. Taken from Holliger Nat Chem (2015).

Systemic Brain Delivery of Therapeutic Oligos!

Taken from igtrcn.org

A talk by Fazel Shabanpoor titled Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy described work that he had just published with a group of collaborators that included symposium co-organizer Mike Gait, whose lab interests have recently focused on cell-penetrating peptides. This was a fav for me because it had multiple interesting elements: (1.) systemic brain delivery, which is a widely recognized challenge; (2.) “weirdly” structured morpholino oligos, which have backbone structures quite unlike DNA that I’ve commented on here previously; and (3.) splice-switching antisense oligos (SSOs). The latter class of molecules (SSOs) base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Readers interested in SSOs—currently a “hot topic”—can consult a recent comprehensive review, while SSOs for spinal muscular atrophy (SMA) has been featured in previous blog here.

Shabanpoor’s lecture highlighted the fact that development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, Shabanpoor et al. investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching 20-mer phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. They identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript.

Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. It was concluded that this work provides proof of principle for the ability to select new “peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform” for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

Parting Thoughts and The Eagle

I hope that you can now appreciate why these three lectures were my favs from the 7th Cambridge Symposium. Silverman’s conversion of DNA into an enzyme that can phosphorylate a protein is an exciting demonstration of the power of bio-organic chemistry to manipulate DNA to do things it can’t do naturally. Holliger’s demonstration of how RNA polymerase ribozymes may have evolved gives credence to the RNA World hypothesis, and indicates that this supposed prebiotic environment may have provided critical freezing and thawing cycles over many millennia of molecular evolution. In the present biotic world, humans afflicted with neuromuscular and neurodegenerative diseases may benefit from Shabanpoor & Gaits’ new peptide paradigms to enhance CNS delivery and activity of therapeutic oligos.

In conclusion, I should mention that researchers who work with DNA will invariably visit The Eagle when in Cambridge, which is a pub only a short walk from Queens’ College. This pub, which dates back to 1667, is quite famous because it is known with certainty to be the place where Francis Crick interrupted patrons’ lunchtime on February 28, 1953 to announce that he and James Watson had ‘discovered the secret of life’ after they had come up with their proposal for the structure of DNA. Today the pub serves a special ale dubbed “Eagle’s DNA” to commemorate the discovery. Trust me when I say that this ale is mighty tasty, because I enjoyed a pint of it, and while standing in the que for that brew, was inspired to capture this image to share here.

As usual, your comments are welcomed.

Personal photo using a Samsung Galaxy S8

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Sniffing Out Prostate Cancer

  • Current Prostate-Specific Antigen (PSA) Tests Are Less than 25% Accurate
  • Trained “Sniffer” Dogs Detect Prostate Cancer-Specific VOCs in Urine with 98% Accuracy
  • Researchers Hope to Analyze VOCs using Gas Chromatography and Validate Results with Canine Studies to Develop Highly Accurate, Non-Invasive Tests for Prostate Cancer

We’re all familiar with news events involving dogs specifically trained to sniff out fugitives, explosives, or cocaine (among other things). This remarkable canine ability is due to some fascinating olfactory factoids. A dog’s sense of smell is a thousand times more sensitive than that of humans, and a dog has more than 220 million olfactory receptors in its nose, while humans have only 5 million.

Taken from finearts. com

Interestingly—if not amazingly—canine sniffing sensitivity has been investigated as a novel means of detecting cancer by simply smelling urine, or more accurately, smelling volatile organic compounds (VOCs) emitted from a urine sample. The following sections provide synopses of some notable publications dealing with new and improved methods for detection of prostate cancer. While reading this post, keep in mind that the principle here is analogous to humans being able to smell a distinctive VOC in their urine after eating asparagus, which is due to formation of volatile asparagusic acid. But I digress…

Dogs Sniff Out Prostate Cancer

The Problem: Prostate cancer represents the fifth most frequent cancer in the world, and according to current CDC statistics is still the number one killer of men in the US, followed by lung and colon cancers, as shown in the chart below.

Taken from health.am

Prostate-specific antigen (PSA) testing is currently used for detection of prostate cancer. Details of the testing process can be read elsewhere, but I’ll briefly describe the steps of the exemplary assay depicted below. In the first incubation phase, specific autoantibodies (present in the sample, calibrators or controls) bind to the immobilized antigen. In the second incubation phase, the dimethyl acridinium ester (DMAE) conjugate reacts with the coated magnetic particle-autoantibody complexes. Non-bound material is washed away after every incubation step, and chemiluminescence is activated by the addition of “trigger” solutions (hydrogen peroxide and an alkali) resulting in oxidation of the ester to a photo-excited form. Return to a stable state is accompanied by the emission of light, which is measured and expressed in Relative Light Units (RLU). A direct relationship exists between the amount of total PSA in the sample and the RLUs detected.

Taken from en.menarinidiagnostics.fr

Since PSA testing is a great tool for the detection of prostate cancer, there is a strong need for more accurate tests. According to an NIH fact sheet, approximately 75% of men who have prostate biopsies due to elevated PSA levels DO NOT have prostate cancer. In fact, over 1 million unnecessary prostate biopsies will be performed in the US this year alone. Reported costs for this biopsy procedure range from $1,500-$6,000, resulting in billions of wasted dollars each year. Moreover, this huge false positive PSA rate exposes millions of men worldwide to an invasive procedure that has risks including sepsis and death.

Taken from pinterest. com

A Canine Solution? In 1989, Williams & Pembroke provided the first evidence for sniffer dogs that could detect VOCs from melanoma cancer in human urine samples. Fast forwarding to 2014, Taverna et al. reported that the olfactory system of highly trained dogs detects prostate cancer in urine samples. Two 3-year-old female German Shepherd explosion-detection dogs were trained to identify prostate cancer-specific VOCs in urine samples from 362 patients with prostate cancer (low-risk to metastatic) and on 540 healthy controls free of any kind of cancer.

Amazingly, dog 1 sensitivity was 100% and specificity was 98.7%, while for dog 2 sensitivity was 98.6% and specificity was 97.6%. Analysis of the few false-positive cases revealed no consistent pattern in participant demographics or tumor characteristics. It was concluded that “[a] trained canine olfactory system can detect prostate cancer specific VOCs in urine samples with high estimated sensitivity and specificity. Further studies are needed to investigate the potential predictive value of this procedure to identify prostate cancer.” While I did not find any such confirmatory follow-up studies with trained dogs, I did find the following investigations of non-canine alternatives to PSA. Interestingly, one of these studies is based on the knowledge that dogs can accurately detect VOCs in urine and the study plans to validate its results with canine studies.

Taken from chromedia.org

A Chromatographic Solution? As a chemist, its seemed reasonable to me to assume that state-of-the-art separation technology could be applied to VOC analysis to develop a more practical and reproducible replacement of PSA tests. I was gratified to find one such report in 2016 by a British research team that used gas chromatography (GC), depicted below, which is commonly employed for separation and detection of smallish, volatile molecules such as VOCs.

Dubbed “Odoreader,” the GC system was developed by a team led by Chris Probert from the University of Liverpool’s Institute of Translational Medicine and Norman Ratcliffe from the University of the West of England in Bristol. The researchers tested the Odoreader on 155 men presenting to urology clinics, of which 58 were diagnosed with prostate cancer, 24 with bladder cancer and 73 with hematuria or weak urine stream without cancer.

For prostate cancer diagnosis, this GC equipped with an automated data analysis system classified samples with 95% sensitivity and 96% specificity, while for bladder cancer diagnosis, the system had 96% sensitivity and 100% specificity. It was concluded that the results of this pilot study “indicate that the GC system is able to successfully identify patterns that allow classification of urine samples from patients with urological cancers,” adding that “larger cohort studies are planned to investigate the potential of this system.”

Not surprisingly, these very promising results have prompted others to investigate analogous GC methods capable of elucidating the structures of key molecules in the mixture of VOCs associated with prostate cancer. Mangilal Agrawal at Indiana University, together with his postdoc, Amanda Siegel, are doing so by coupling the power of GC to separate molecules and the power of mass spectrometry to identify molecules. Then they plan to validate these biomarkers with canine studies much like the one discussed above. Once validated, they will use the biomarkers to develop a non-invasive ‘strip sensor’ or dipstick test that can be used at doctors’ offices to detect the presence of prostate-specific VOCs. They presented preliminary findings using this GC/MS technology at the 2017 National American Chemical Society Meeting in a 15-minute press release video session with Q&A that I watched with interest.

In conclusion, my hope is that these GC based methods, in combination with continued canine studies, will soon lead to much more accurate strip sensor tests to replace PSA testing. These more accurate tests will benefit millions of men around the world by avoiding unnecessary prostate biopsies, and reduce health care costs.

As usual, your comments are welcomed.

Addendum

Taken from Deng et al.

Aptamers (which I’ve blogged about previously and can be prepared from randomized oligonucleotide libraries from TriLink), are also being extensively investigated as potentially more specific prostate cancer detection tests than antibody-based immunoassays. One recent example reported by Deng et al. is depicted below. Basically, a three-layer core–shell nanostructure consisting of a silver core, a silica spacer, and a fluorescent dye RuBpy-doped outer silica layer was fabricated, and allows metal-enhanced fluorescence (MEF). A target-triggered MEF ‘turn-on’ strategy based on the optimized composite nanoparticles was successfully constructed for quantitative detection of prostate specific antigen (PSA), by using RuBpy as the energy donor and BHQ-2 as the acceptor. The hybridization of the complementary DNA of PSA-aptamer immobilized on the surface of the MEF nanoparticles with PSA-aptamer modified with BHQ-2, brought BHQ-2 in close proximity to RuBpy-doped silica shell and resulted in the decrease of fluorescence. In the presence of target PSA molecules, the BHQ-PSA aptamer is dissociated from the surface of the nanoparticles with the fluorescence switched on.

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Highlights from the 2017 AgBio Innovation Showcase Held by UC Davis

  • An Inconvenient Truth About Unsustainable Global Food Supply
  • Agricultural Biotechnology (AgBio) is Providing Transformative Solutions
  • Highlights from the Inaugural AgBio Innovation Showcase

Taken from the journal.ie

With expected global population to reach 8.3 billion in 2030, it’s clear that excessive exploitation of food resources is no longer sustainable and the problem will simply worsen with environmental problems and effects of climate change. This ominous outlook by food experts is reminiscent of former Vice President Al Gore’s dire vision for global warming in an award winning documentary film in 2016 titled An Inconvenient Truth.

This very real challenge of achieving adequate and sustainable food supplies—globally, not just for developed countries—has been, and continues to be, addressed by nucleic acid-based agricultural biotechnology (aka AgBio). At the forefront of this battle is development of genetically modified foods (aka genetically engineered foods or bioengineered foods), which are foods produced from organisms that have had changes introduced into their DNA using the methods of genetic engineering. Genetic engineering techniques allow for the introduction of new traits, as well as greater control over traits than was possible with previous methods such as selective breeding and mutation breeding.

Taken from intelligencesquaredus.org

Last year, I published a blog about genetically modified organisms (GMOs) in which I made fairly general comments about complex government regulatory issues related to “science vs. semantics” and varying degrees of country/consumer acceptance and rejection. This blog is somewhat of a follow-up to that post, and I will share specifics from the 2017 AgBio Innovation Showcase held by the University of California Davis at its World Food Center, which include GMO and non-GMO technologies. The Center was founded in 2013 as an institute aimed at “bridging agriculture, food science, nutrition, veterinary medicine, public health and policy in new and transformational ways.”

2017 AgBio Innovation Showcase

Taken from agshowcase.com

This inaugural event was held on May 8-9 and featured the most promising AgBio and AgTech startups and research projects. The showcase featured solutions in high-value, nutritious agriculture and food from across the globe. The four major showcase themes were Automation & Robotics, Boosting Nutrition & Sensory Value, Innovation in the Livestock & Dairy Sectors, and Water Management. I’ve selected several highlights that are summarized below. Takeaways from panel discussions about the future of agriculture can be read elsewhere.

Ag Biotech

  • Afingen – This biotech start-up was spun out of Lawrence Berkeley National Laboratory (LBNL) in 2014 and features technology based on proprietary cisgenesis. Cisgenesis involves modification of a recipient plant with a natural gene from a crossable plant. Importantly, cisgenic plants can harbor one or more cisgenes, but they do not contain any transgenes and therefore yield new, improved plant varieties that are classified non-GMO.
  • Taken from whattsupwiththat.com

    Bee Vectoring Technology – How this Canadian company cleverly turns bees into delivery agents that deposit biological products on crops for pest management is best understood by watching this video (details for which may be read in a patent). In brief, powder-form biologics to be delivered are placed in commercially-reared bee. The biologics stick to the bees’ feet and are released when the bee collects pollen from the targeted crop.

    Taken from saipanhydroponics.com

  • MiraculeX – A unique West African plant protein called miraculin (named for its “miracle” ability to transform sour foods into sweet treats), makes it possible to bite into a lemon and taste nothing but sweet lemonade. MiraculeX reportedly inserts the protein’s DNA into the genetic code of ordinary lettuce, which is grown hydroponically and in less than a week is ready to be harvested for processing.
  • Trace Genomics – This startup service in San Francisco provides advice to growers based on analysis of their soil. Growers simply provide a soil sample, from which TraceGenomics extracts DNA from the organisms in the soil and prepares a sequencing library to analyze the soil microbial community. Interpreted results are provided along with information about soil health, nutritional status, and corresponding recommendations for how to improve crop yield and quality.

Diagnostics

  • AstRoNa Biotechnologies – This UC Davis startup aims to commercialize an easy-to-use, hand-held pathogen detection device to quickly monitor food contamination by bacteria, viruses, and fungi. It’s basically “farm-to-table” analysis. The team reportedly developed a disposable test kit to capture and amplify RNA of pathogens, focusing on coli O157:H7. A fully automated handheld instrument is under development and will feature sample multiplexing, quantitative detection, and software to create a traceable record of safety—recording time, location, user, and results in real time.
  • SnapDNA – This startup has an R&D agreement with the US Department of Agriculture to develop rapid, highly specific tests for foodborne pathogens, including Salmonella enterica and human noroviruses (the latter of which is featured in an earlier blog). I was unable to find many details, but a board member states that SnapDNA is “a semiconductor-based bio-chip and multiplexed DNA detection platform.” Adding that “[a]major differentiator of SnapDNA is the specificity to detect DNA at the level of microbial strains in a fast, low cost test, major pain points in current systems.”

Food Science & Animal Health

  • Taken from Wikipedia.org

    Bonumose – This startup in Virginia is scaling up enzymatic production of tagatose (pictured below), which—unlike sucrose and high fructose corn syrup—does not raise blood sugar levels, is low-calorie, and does not cause tooth decay. Beyond not being harmful to health, tagatose provides positive health benefits: it is an effective prebiotic (good for gut health), blocks adsorption of sucrose and starch, is clinically-proven to reduce blood sugar levels in diabetics, contributes to a feeling of satiety, and breaks up dental biofilm. Even better, tagatose is nearly indistinguishable from sucrose in terms of taste and food functionality. And it blends very well with high intensity sweeteners such as stevia. I want some asap!

  • Resilient Biotics – This El Cerrito, California startup utilizes deep sequencing to characterize host genotypes, commensal microbial communities, and pathogen strain variants for microbiome resolution to rapidly identify important genetic elements and key microbial strains that influence states of health and disease. Heuristic search methods can rapidly pinpoint diagnostic biomarkers for pathogen identification and risk prediction. Resilient Biotics is actively developing live biotherapeutics to address major infectious diseases of the respiratory tract in production animal systems.

AgBio Meets CRISPR

As if the 2017 AgBio Innovation Showcase wasn’t stimulating enough, I was thrilled to discover another upcoming meeting that combines two of my favorite topics: agbio and CRISPR. Devoted readers of my blogs will recall numerous past postings on CRISPR for gene editing and other useful manipulations of genomic DNA. My search of Google Scholar indicated no AgBio CRISPR publications to date, but that will likely change, as evidenced by the upcoming conference.

Interested readers can register at the link above and download a detailed agenda and list of confirmed speakers. In doing so, it is apparent that this conference will comprehensively cover the newest topics and the regulatory status related to CRISPR/Cas9 technology.

I look forward to reading about these developments, and posting comments in a future blog titled AgBio Meets CRISPR. If you happen to be attending this conference, please share details about what you learned in the comments section below.

As usual, your comments are welcomed.

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Highly Visible Invisible Food Safety

  • Recent FDA Food Safety Act Focuses on Prevention Rather Than Response
  • PCR Provides a Powerful Approach for Assuring Foodborne Safety
  • Safer Food via Immuno-PCR Commercialized by Invisible Sentinel 

Annual Cost of Foodborne Illness and U.S. FDA Response

Every year contaminated food sickens some 48 million people in the U.S., necessitates 128,000 hospitalizations, and results in 3,000 deaths, according to recent estimates from U.S. Centers for Disease Control and Prevention. Extrapolating these numbers to other developed countries isn’t straightforward, but I used available information to guess that the number of cases of foodborne illness worldwide is roughly 4-5 times greater than the US.

In addition to the toll in human suffering, food contamination that is discovered too late exacts a heavy financial cost on the food industry and the public. A study supported by the Pew Charitable Trusts has estimated that food contamination costs the United States about $152 billion a year after accounting for lost workdays and reduced quality of life, as well as medical expenses.

Continuing outbreaks of foodborne illness have led consumer groups to call for tighter regulation. The result was the FDA Food Safety Modernization Act (FSMA)—the most sweeping reform of U.S. food safety laws in more than 70 years—signed into law by President Obama on January 4, 2011. It aims to ensure the U.S. food supply is safe by shifting the focus from responding to contamination to preventing it. Here’s how.

The U.S. FDA now has a mandate to require food-safety controls. Companies across the food-production and food-distribution network must write outbreak-prevention plans, monitor the performance of their controls, and specify the corrective actions they will take when necessary.

A detailed summary of the FSMA and a host of related information can be read about at this link that includes Guidance for Industry, Final Rules, and Presentations.

PCR Powered Prevention 

In a nutshell, the well-known power of PCR to unambiguously identify and quantify microorganisms lends itself to prevention of foodborne contamination from entering the food supply. Other important attributes of PCR for such prevention are its highly evolved instrumentation and ancillary sample preparation methods, which together provide for fast time-to-results in either centralized labs that receive express-shipped samples, or decentralized (aka point-of-need) facilities at or very near the food source.

Most major commercial suppliers of PCR instrumentation and reagents offer a line of products aimed at food safety per se or quality control for microorganisms that are detrimental to taste, smell, shelf life, and other producer or consumer concerns. An example of such is ETS Labs in California, which uses real-time PCR to detect a full range of wine and juice spoilage organisms to help ensure quality in the wine making industry. This genetic analysis method, which utilizes TriLink’s Hot Start CleanAmp dNTPs under a licensing agreement, detects microbial populations directly in wine or juice. Results are routinely reported within two business days, giving winemakers the ability to address problems before wine defects occur.

Regular readers of this blog will recall that I have touted the advantages of PCR in various contexts, including some aspects of food chain validation and tracing from “farm-to-fork.” However, as indicated above, the present post is focused on preventing foodborne illnesses and showcasing innovators in this space, namely Invisible Sentinel.

Highly-Visible Invisible Sentinel

Truth be told, I can’t recall how I first came across Invisible Sentinel, but I’m glad I did because it’s an interesting story from the perspective of innovation in food safety technology as well as opportunity in an emerging market.

Taken from phillymag .com

Technology-wise, the first thing that caught my attention was the remarkably small size of the PCR read-out device developed by Invisible Sentinel, which is pictured below. Before getting into the specifics of what’s packed inside of this tiny gizmo, I should mention that there are up-front sample prep and PCR thermal cycling steps that must be performed before using the device. These steps have been simplified but involve conventional approaches that can be read about at this link rather than discussed here.

Much more intriguing to me was how PCR amplicons are detected by Invisible Sentinel’s Veriflow® DNA Signature Capturing Technology, which more accurately involves visualization by eye as opposed to fluorescent signal detection commonly used for real-time PCR measurements. Since functional details for this visualization system are not provided on Invisible Sentinel’s website or various YouTube videos, I’ll briefly summarize below what I found in a recently issued Invisible Sentinel patent that describes its version of what is known as Immuno-PCR.

The following schematic, taken from this patent, depicts visualization of two different amplicons derived from 5’-labeled primers: digoxigenin/TAMRA amplicon 20 and FITC/TAMRA amplicon 40 using three antibodies 15, 30, and 50 that are either attached to a membrane, bridge, or bind streptavidin-gold nanoparticles. The latter nanoparticles are visualized by eye, but only if both amplicons form the complex shown. Variations of this scheme can be used for visualization of a single amplicon.

Taken from US Pat. No. 9,347,938

Visualization in this manner is formally analogous to pregnancy strip tests that show two bands for a positive result and only one band for a negative result. Interested readers should consult the aforementioned patent for details regarding how input amplicons undergo lateral flow to ultimately bind to antibody 10 attached to the test membrane shown above.

Exemplary Applications

According to Invisible Sentinel, 114 companies in the U.S. and more than 50 internationally use the technology at more than 250 different sites in 18 countries.

For example, Wawa Inc. (which owns dairy and beverage manufacturing plants as well as 715 convenience stores in six states) has adopted Veriflow®, as has Refresco Gerber Partner for monitoring juice spoilage. WholeVine Products in California, which produces a variety of products from grape seeds and skins, has begun using Veriflow® to make sure its plant equipment and surfaces are pathogen-free.

Although Invisible Sentinel’s website provides a list of currently available tests, I thought it would be useful to provide the following links to several self-explanatory published applications that I found by searching Google Scholar:

Invisible Sentinel Identifies New Market Opportunities for PCR

Invisible Sentinel was started by a pair of entrepreneurs with science backgrounds. Nicholas Siciliano, 37, graduated from Villanova with a degree in chemistry in 2004 and obtained a doctorate in immunology and microbial pathogenesis from Thomas Jefferson University in 2015. In between, he was a biotech consultant and worked as a researcher at the University of Pennsylvania School of Medicine.

Nicholas Siciliano (left) and Benjamin Pascal in their lab. Taken from articles.philly .com

Benjamin Pascal, 35, has a bachelor’s degree in political communication from George Washington University in 2003 and a master’s in business administration from Lehigh University in 2009. He learned biology at the National Institute for Medical Research in London, and then spent several years in R&D at B. Braun Medical Inc.

According to an article in the NY Times, the two wanted to create a diagnostic device that was faster, easier and cheaper to use. They began with $235,000 from friends and family, but the recession made it tough to bring institutional investors onboard. In 2009, they raised another $1.1 million from friends and family, $2 million more in 2011, and raised $7 million at the end of 2013.

Invisible Sentinel’s sales have been on the rise. The company posted revenue of $50,000 in its first year of sales in 2013, $1.1 million in 2014 and more than $4 million in 2015. It has ambitious projections of $30 million in 2018 and $60 million in 2020. The company expected to turn a profit in 2016.

While Invisible Sentinel may have been one of the first to identify the significant market opportunity for food safety monitoring devices, they currently face formidable competition in larger companies such as Romer Labs’ RapidChek®, Bio-Rad Laboratories’ iQ-Check® and DuPont’s Bax® system. Invisible Sentinel is hoping to capture significant market share with its low cost of entry and easy-to-use system. The company can reportedly set up an in-house lab for about $5,000 and train almost anyone to use it in less than a day. Invisible Sentinel kits cost more than others (about $10 per test compared to an industry average of $4 to $8), but the lower capital equipment and lab set up costs are said to greatly offset the higher test costs.

In closing, I hope that you have found this piece on food safety and immune-PCR in the context of Invisible Sentinel to be a nice example of how nucleic acids-based technology is enabling improved food safety.

As usual, you are welcomed to share your comments here.

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Advances in Aptamer Applications – Part 2

  • Top Cited Aptamer Publications Over the Past Three Years
  • Jerry’s Picks for Top 3 Aptamer Publications So Far This Year
  • TriLink Products Cited in Numerous Aptamer Publications

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. My first blog on aptamers was titled Aptamers: Chemistry Bests Mother Nature’s Antibodies. This purposefully provocative claim was intended to emphasize the growing body of evidence that collectively indicates aptamers can perform better than antibodies in many applications.

NMR-derived structures of aptamers binding to either a large protein or small molecule. Taken from genelink .com

Because it has been nearly four years since that boastful blog in 2013, I thought it was time to survey aptamer applications published since then to comment on what has been trending or is otherwise notable. I found more than 1,500 articles in PubMed for 2014 through 2017 (estimate) that have the search term “aptamer” in the title or abstract. Given this huge number of publications, I used Google Scholar citation frequency as a numerical indicator of interest, importance and/or impact for these publications in each year. I also decided to focus on original publications that, by definition, excludes review articles. 

Top 3 Cited Publications in 2014

  1. Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet–aptamer nanoprobe (109 citations)

This Chinese team of researchers led by uber-prolific Weihong Tan, about whom I’ve previously blogged, designed a novel methodology for imaging tumor cells using quenched-fluorescent aptamers. In the presence of target cells, the binding of these “dark” aptamers to cell surface markers weakens the adsorption of aptamers on MnO2 nanosheets causing partial fluorescence recovery (i.e., unquenching), thus illuminating the target cells, as well as facilitating endocytosis into target cells. After endocytosis, reduction of MnO2 nanosheets by glutathione further activates the fluorescence signals and generates large amounts of Mn2+ ions as a contrast agent for magnetic resonance imaging (MRI).

Taken from pubs.rsc.org

  1. A phase II trial of the nucleolin-targeted DNA aptamer AS1411 in metastatic refractory renal cell carcinoma (88 citations)

Taken from mct.aacr.org

The anticancer mechanism of action for DNA aptamer AS1411, which has multiple G-quadruplex moieties that disrupt cancer cell replication following nucleolin-mediated uptake, is depicted below and detailed elsewhere. In this clinical study, it was found that AS1411 appears to have limited activity in patients with metastatic renal cell carcinoma. However, rare, dramatic and durable responses can be observed and toxicity is low. Further studies with AS1411 and other nucleolin-targeted compounds may benefit from efforts to discover predictive biomarkers for response.

  1. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 (61 citations)

Aflatoxin B₁ structure. Taken from wikipedia.org

Aflatoxin B₁ (AFB1) produced by Aspergillus flavus and A. parasiticus is considered the most toxic aflatoxin and it is highly implicated in hepatocellular carcinoma in humans. In this work by Korean researchers, a rapid and simple dipstick assay based on an aptamer has been developed for determination of AFB1 contamination in food. The dipstick assay format was based on a competitive reaction of a biotin-modified aptamer specific to AFB1 between target and Cy5-modified DNA probes. Streptavidin and anti-Cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min.

Top 3 Cited Publications in 2015

  1. Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus (63 citations)

Taken from sciencedirect .com

Staphylococcus aureus (S. aureus) is one of the most important human pathogens and causes numerous illnesses. This report by Iranian researchers describes a sensitive and highly selective dual-aptamer-based sandwich immunosensor for the detection of S. aureus. As depicted below, a biotinylated primary anti-S.aureus aptamer was immobilized on streptavidin coated magnetic beads (MB), which serves as a capture probe. A secondary anti-S.aureus aptamer was conjugated to silver (Ag) nanoparticles such that, in the presence of target bacterium, a sandwich complex is formed on the MB surface and the electrochemical signal of Ag is measured by anodic stripping voltammetry.

  1. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles (59 citations)

Chloramphenicol. Taken from Wikipedia .com

Chloramphenicol (CAP) shown below is a naturally occurring antibiotic that is artificially manufactured for use in veterinary and human medicine. Due to its adverse effects in humans, use of the antibiotic is restricted and, in Europe, ‘zero tolerance’ for CAP in food products has been legislated. In this report by Chinese researchers, detection of CAP uses aptamer-conjugated magnetic nanoparticles for both recognition and concentration, together with upconversion nanoparticles for detection. The method was validated for measurement of CAP in milk vs. a commercially available enzyme-linked immunosorbent assay (ELISA) method.

  1. A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus (48 citations)

Taken from mdpi .com

This work by Chinese investigators describes a novel aptamer/graphene interdigitated gold electrode piezoelectric sensor for detecting S. aureus by binding to the aptamer, which is immobilized on the graphene via the π–π stacking of DNA bases, as depicted below. When S. aureus is present, aptamer dissociates from the graphene and thus leads to change of oscillator frequency of the piezoelectric sensor.

Top 3 Cited Publications in 2016

  1. Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen (38 citations)

This study in the UK uses a thiolated DNA aptamer for prostate specific antigen (PSA) immobilized on the surface of a gold electrode. Controlled electropolymerization of dopamine around the complex served to create an imprint of the complex following removal of PSA. This molecularly imprinted polymer (MIP) cavity was found to act synergistically with the embedded aptamer to provide recognition properties superior to that of aptamer alone. A generalized depiction for producing a MIP is shown below.

Taken from sigmaaldrich .com

  1. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device (34 citations)

Taken from wikipedia.org

Monitoring the periodic diurnal variations in cortisol (aka hydrocortisone, show below) from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. This publication from China reports use of aptamer-functionalized gold nanoparticles pre-bound with electro-active triamcinolone for detection of cortisol based on its competitive binding to the aptamer by monitoring a signal from the displaced triamcinolone using square wave voltammetry at graphene-modified electrodes. The assay was benchmarked vs. ELISA and radioimmunoassays.

  1. Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance (32 citations)

Taken from Liu et al. Biomaterials (2016)

In yet another publication from China, Liu et al. report use of a G-quadruplex nanostructure to target cancer cells by binding with nucleolin, in a manner analogous to that mentioned above. A second component is double-stranded DNA (dsDNA), which is rich in GC base pairs that can be applied for self-assembly with doxorubicin (Dox) for delivery to resistant cancer cells. These nanoparticles were found to effectively inhibit tumor growth with less cardiotoxicity.

Jerry’s Top 3 Publication Picks for 2017-to-Date

Here are my Top 3 “fav” aptamer articles published during the first half of 2017, and my reasons for these aptamer selections—pun intended. Interested readers can consult the original publication for technical details.

  1. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

CRISPR/Cas9 is unquestionably—in my opinion—the hottest topic in nucleic acid-based R&D these days, as I have previously blogged about. Off-target effects of CRISPR/Cas9 can be problematic, so using targeted delivery to cells of interest is an important approach for mitigating this problem. In this study, an aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery with adaptability to other situations. The chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand, and the approach “provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.”

  1. A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva

This report claims the first ever development of a split aptamer that achieves enhanced target-binding affinity through cooperative binding. In this instance, a split cocaine-binding aptamer incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. This system afforded specific, ultra-sensitive, one-step fluorescence detection of cocaine in saliva without signal amplification. This limit of detection meets the standards recommended by the European Union’s Driving under the Influence of Drugs, Alcohol and Medicines program.

  1. Detection of organophosphorus pesticide–Malathion in environmental samples using peptide and aptamer based nanoprobes

Environmental contamination with pesticide residues has necessitated the development of rapid, easy and highly sensitive approaches for the detection of pesticides such as malathion, a toxic organophosphorus pesticide, widely used in agricultural fields. These Indian investigators employed an aptamer, cationic peptide and unmodified gold nanoparticles. The peptide, when linked to the aptamer renders the gold nanoparticles free and therefore, red in color. When the aptamer is associated with malathion, however, the peptide remains available to cause the aggregation of the nanoparticles and turn the suspension blue. The sensitivity was tested in real samples and the results implied the high practicability of the method.

Aptamer Publications in 2014-Present Citing TriLink Products

I was pleasantly surprised to find more than 250 publications on aptamers in Google Scholar citing the use of TriLink products since 2014. This volume of literature is way too large to summarize succinctly, so I decided to do a quick scan to select the following items that provide an indication of the broad diversity of applications partially enabled by TriLink products:

2’-F-UTP. Taken from trilinkbiotech .com

In closing, I should first mention that, while scanning the aptamer/TriLink publications mentioned above, it was evident that the most frequently cited TriLink products were 2’-F-CTP and 2’-F-UTP, which are incorporated into aptamers to impart nuclease resistance, as discussed on a TriLink webpage.

My second and last comment is that, as you may have noticed, there seems to be a high proportion of aptamer publications coming out of China and/or coauthored by Chinese investigators collaborating with researchers in other countries. This despite the fact that Chinese publications in Life Sciences are ~6-times fewer that those from the US, according to reliable statistics. I have no idea why this is so, but thought it’s an intriguing factoid.

As usual, your comments are welcomed.

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Phoenix in Mythology and Sequencing

  • Like a Phoenix, Helicos Sequencing is Being Reborn
  • Direct Genomics in China to Launch the Genocare Clinical Sequencer
  • SeqLL in the USA to Launch Benchtop tSMS Sequencer

A phoenix as depicted by F.J. Bertuch (1747–1822). Taken from Wikipedia.org

In ancient Greek mythology, a phoenix is a bird that is cyclically regenerated or reborn by arising from the ashes of its predecessor, which dies in a show of flames and combustion. In contrast to a phoenix, modern biotech methods generally “die” in utility by being displaced with faster, better, and/or cheaper methods rather than undergoing “rebirth” in the context of a new application. However, a method developed by a company named Helicos (scarily close to Helios associate with a phoenix) may prove to be a rare exception. Perhaps this is destiny, but I digress…

Helicos Sequencing

Successful Sanger sequencing of a human genome in the early 2000s spawned numerous efforts to develop faster, better, and/or cheaper methodology to enable genomic analysis on a routine basis. Among the early contenders there was Helicos BioSciences, which was founded in 2003 by several principals including then—and still—uber-famous Stephen Quake.

Helicos sequencing technology, which is depicted below and outlined elsewhere, was especially attractive because it was “true” single-molecule sequencing (i.e. sample prep did not require prior PCR or other amplification, thus greatly simplifying the workflow). Moreover, the technology uniquely allowed direct RNA sequencing, thus obviating the need to first convert RNA into cDNA.

Main steps for primer(P)-based, single-color (Cy3 dye) Helicos sequencing, in this example using two passes. Taken from Harris et al. Science (2008)

3’-Unblocked reversible terminator. Taken from Chen et al. (2013)

Details for how this sequencing-by-synthesis occurs can read in various proof-of-concept publications. However, it’s worth noting here that the 3’-unblocked reversible terminator nucleotide triphosphate monomers have a cleavable linker attached to a detectable dye. Helicos referred to these as “Virtual Terminator” nucleotides since they are efficiently incorporated by a polymerase yet block incorporation of a second nucleotide on a homopolymer template.

So, with these methodological advantages going for it, why did Helicos file for bankruptcy in 2012? Press coverage at that time stated ‘rough financial sledding and tough competition from rival next-generation sequencing companies.’ In my humble opinion, this lack of commercial success was primarily due to the HeliScope Genetic Analysis System (pictured below) being way too big (think upright freezer-refrigerator), far too expensive ($1,350,000), and its ~35-base reads too short on performance—pun intended.

Two Phoenix-Like Versions of Helicos Sequencing

Fast forwarding about five years from the 2012 bankruptcy filing by Helicos brings us to recent reports of two independent efforts to bring back Helicos sequencing in commercially viable formats and contexts, think Phoenix rising from the ashes.

Jiankui He and the GenoCare sequencer (credit Xinjie Tian). Taken from Cyranoski Nature Biotechnology (2016)

The first of these is led by Jiankui He, Founder/CEO of Direct Genomics in Shenzhen, Guangdong, China, as well as Associate Professor at South University of Science and Technology of China in Shenzhen. He, coincidentally, was a postdoc with Helicos cofounder Stephen Quake, who is reported to lead the scientific advisory board for this new company.

The company’s website homepage states the following:

“Direct Genomics is providing physicians with the first single molecule sequencer built exclusively for the clinic. The technology simplifies genome sequencing by reading individual DNA and RNA molecules directly from patient’s blood or tissue samples, which delivers significant improvement in cost and speed. Together with clinicians, Direct Genomics is making genetics an affordable part of everyday patient care.”

Perusal of scant technical information on the company’s website suggests to me that a smaller sized, TIRF-optics-enabled instrument running Helicos-type sequencing has been developed. A story about Direct Genomics by David Cyranoski in Nature Biotechnology states that $100 “clinical sequencing” is being targeted, with a blood-draw to report turnaround time of 20 hours. A very recent publication I found provides details for resequencing the Escherichia coli genome by the Direct genomics platform named GenoCare.

The company’s website lists the following clinical applications:

  • Non-invasive prenatal testing (NIPT)
  • Tumor diagnosis
  • Early-stage cancer prediction
  • Pre-implantation genetic diagnosis (PGD)

The second Phoenix-like rebirth of Helicos sequencing has been developed by SeqLL, which was co-founded in 2013 by William St. Laurent and Daniel Jones, who previously held various technical positions at Helicos. Statements and a video on SeqLL’s website indicate to me that the sequencing technology is essentially that originally developed and patented by Helicos, which is still trademarked as True Single Molecule Sequencing (tSMS™).

William St. Laurent. /   Daniel Jones. Taken seqll.com

SeqLL has been operating as a tSMS™ service provider, but in October 2016 announced the launch of the tSMS™ System Early Access Program giving researchers access to its new benchtop system “designed to deliver unparalleled quantitative RNA and specialty DNA sequencing results to both academic and industry research partners.” I should add that a big, strong bench is needed given that the physical specs are 30 x 30 x 60 inches and 1,000 pounds! Nevertheless, SeqLL recently announced an SBIG grant for improving its direct RNA sequencing technology, which I think could prove to be a driver for adoption.

In conclusion, I think it’s very interesting to see Helicos sequencing coming back to life, if you will, in not one but two different commercial contexts, both of which will hopefully be successful. This despite current ‘tough competition from rival next-generation sequencing companies,’ as observed in the 2012 bankruptcy story about Helicos mentioned above. First and foremost, among that competition is Oxford Nanopore, which I’ve blogged about previously, and whom offers single-molecule sequencing that seems to me to be faster, better, and cheaper for both DNA and RNA, directly.

As usual, your comments are welcomed.

Postscript

After this blog was written, it was reported in GenomeWeb that Direct Genomics plans to deliver 50 instruments this year to SinoTech Genomics, a startup based in Shanghai that offers both clinical and research sequencing services. Direct Genomics CEO Jiankui He is quoted as saying that ‘SinoTech Genomics [is] committed to ultimately purchasing 700 GenoCare platforms,’ and that Direct Genomics ‘has the capacity of producing around 1,000 GenoCare instruments per year,’ which would be very impressive based on past operational experience with manufacturing Sanger sequencers at ABI.

The piece went on to report that Direct Genomics also ‘aims to launch GenoCare in the US in September.’ Regarding what’s inside the box, so to speak, ABI veteran Bill Efcavitch, who previously served as chief technology officer of Helicos, is quoted as saying that ‘the main difference between the former Helicos technology and the GenoCare platform is in the hardware. It’s completely different engineering.’ He added, however, that it still uses Helicos’ virtual terminator chemistry.

Ocean ‘Dandruff’ DNA to Better Study Marine Biology

  • DNA Barcoding for all Organisms has Numerous Applications
  • DNA Barcodes from Water Samples Greatly Aide Marine Biologists
  • Aquatic Environmental DNA (eDNA) Proves to be Informative ‘Dandruff’

Human DNA identity analysis is now commonplace methodology that’s frequently featured in newspaper stories, TV crime series, or “who dun it” movies. The same principle (i.e. using a characteristic DNA pattern or signature) applies to identification of all animals, birds, insects, and microbes. Actually, DNA barcoding extends to any organism, whether it is alive or has been dead for hundreds of thousands of years (so long as it’s preserved by fossilization).

Taken from gajitz.com

Marine biologists face a serious challenge with accounting for very diverse forms of marine life that exists in a mindboggling huge volume of water. Consequently, it’s not surprising that analysis of water-borne, marine DNA barcodes—as proxies for going to and counting fish—is rapidly trending in utility and importance. Known formally as environmental DNA (eDNA), the aquatic version has been humorously referred to as ocean ‘dandruff’ by Christopher Jerde of the University of Nevada in Reno (which, ironically, is landlocked and distant from any ocean.) But I digress. Before diving further (pun intended) into ocean dandruff, let’s briefly review the background of DNA barcoding.

DNA Barcodes 101

Prof. Paul Herbert. Taken from uoguelph.ca

In 2003, Prof. Paul Herbert and coworkers in the Department of Zoology at the University of Guelph in Canada published a seminal study titled Barcoding animal life: cytochrome c oxidase subunit 1 (CO1) divergences among closely related species that fundamentally changed the field of taxonomy. In a nutshell, Herbert’s team showed it was feasible to classify millions of species based only on DNA sequence of the mitochondrial gene CO1. In the intervening, relatively short amount of time, there have been thousands of publications dealing with applications and extensions of this concept, which is now recognized to be very powerful and promising albeit with some limitations.

Typically, DNA barcodes are identified by sequencing after PCR amplification of one or more specific genetic loci such as CO1. Following proof that a DNA barcode can differentiate the species of interest, single- or multiplex quantitative PCR (qPCR) can be used to enumerate relative amounts of sample from the field.

The advent of high-throughput sequencing technologies applicable to complex mixtures of individually tagged samples then gave rise to “metabarcoding,” about which interested readers can consult many publications for specific topics.

Craig Venter steers his research yacht, Sorcerer II, under the Sydney Harbour Bridge in his quest to collect microbes from the world’s waters. Photo: Dallas Kilponen. Taken from smh.com.au

BTW, among the many pioneering scientific ventures by uber-famous Craig Venter, is his Global Ocean Sampling Expedition aboard his research yacht, Sorcerer II. The expedition is a quest to unlock the secrets of the oceans by sampling, sequencing and metabarcoding DNA of all (or most) microorganisms living in these waters.

Lest you think this was a well-intended but unproductive journey—some say junket—by Venter and coworkers, here’s a link to peruse 16 resultant publications that I found by searching PubMed. To watch and listen to Venter talk about this work, you can click here for an educational and entertaining—as usual with Venter—TED Talk on Sampling the Ocean’s DNA that’s had over 550,000 views!

Ocean ‘Dandruff’

Now that we’ve covered the basics of DNA barcoding and metabarcoding, let’s turn back to ocean dandruff. Dandruff, simply put, is dead skin cells. Using dandruff as an intended witty metaphor for ocean eDNA is a bit misleading as marine eDNA is comprised of a complex mixture of cellular matter from scales, feces, decomposing tissue, etc. of fish and all other present or past sea creatures. Consequently, the design and specificity of primers for PCR is of paramount importance for obtaining—let alone interpreting—DNA barcodes based on fragment size or sequence.

As reported by Miya et al., monitoring the occurrence of fish species-specific eDNA PCR fragments (~70–300 bp) has traditionally used conventional electrophoretic gel separation and detection. More recently, qPCR using fluorogenic probes has been employed owing to the method’s sensitivity, specificity and potential to quantify the target DNA. For example, it has been possible to accurately estimate the biomass of common carp in a natural freshwater lagoon using qPCR of eDNA concentrations and biomass in aquaria and experimental ponds.

Miya et al. also describe the development of a set of PCR primers for metabarcoding mitochondrial DNA of 880 species of fish. They sampled eDNA from four tanks with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing. Out of the 180 marine fish species contained in the four tanks, they detected 168 species (93.3%) distributed across 59 families and 123 genera. That’s quite an impressive accomplishment.

Ocean Dandruff Case Studies

Since there are so many fish-related applications of DNA barcodes, I’ve selected several recent examples that are indicative of the utility of ocean ‘dandruff’—and are quite interesting, in my opinion. The first case in point exemplifies how eDNA can be used to deal with rare and endangered species, which are either very hard to find or can be dangerously distressed by catching to obtain samples.

Green SturgeonBergman et al. report that a decline in abundance of North American Green Sturgeon located in California’s Central Valley has led to its listing as Threatened under the Federal Endangered Species Act in 2006. While visual surveys of spawning by these Green Sturgeon are effective at monitoring fish densities in concentrated pool habitats, results do not scale well—pun intended. By contrast, eDNA provides a relatively quick, inexpensive tool to efficiently identify and monitor Green Sturgeon DNA.

Taken from mthsecology.wikispaces.com

These investigators concluded that follow-on work based on this first-ever eDNA study of Green Sturgeon has the potential to provide better knowledge of the spatial extent of Green Sturgeon spawning that could help identify previously unknown spawning habitats and discover factors influencing habitat usage, guiding future conservation efforts.

Monterey Bay—The second case study, by Port et al., involves taking stock of the marine mammals and fish in Monterey Bay using eDNA and, importantly, comparing the results obtained to those from traditional dive surveys.

In brief, this team of researchers from several universities and the Monterey Bay Aquarium Research Institute found that eDNA assessments picked up almost all the organisms scuba divers spied underwater—plus many more that human eyes missed. Here’s some detail on how they did this.

At each scuba survey location as well as at sites offshore, ~1 gallon of water was sampled several feet above the bottom. Four types of habitats were sampled: sea grass beds, Monterey Bay’s unique “Kelp Forest,” sandy areas and rocky reefs. Onshore, in a “clean” (DNA-free) lab, these water samples were filtered to collect cells containing eDNA for storage at −80 °C until eDNA extraction at a university clean lab. A vertebrate‐specific primer set targeting a small region of the mitochondrial DNA 12S rRNA gene was used for PCR followed by gel purification.

Researchers collecting water in Monterey Bay for eDNA analysis. Courtesy Jesse Port. Taken from mercurynews.com

After quantification, pooled amplicons (each having a sample index sequence) were paired-end sequenced on the Illumina MiSeq platform using a 20% PhiX spike‐in control to improve the quality of low‐diversity samples. The conclusions are worth quoting because—in my opinion—the findings represent a new era in marine biology based on nucleic acid analysis:

“We find spatial concordance between individual species’ eDNA and visual survey trends, and that eDNA is able to distinguish vertebrate community assemblages from habitats separated by as little as ~60 meters. eDNA reliably detected vertebrates with low false‐negative error rates (1/12 taxa) when compared to the surveys, and revealed cryptic species known to occupy the habitats but overlooked by visual methods. This study also presents an explicit accounting of false negatives and positives in metabarcoding data, which illustrate the influence of gene marker selection, replication, contamination, biases impacting eDNA count data and ecology of target species on eDNA detection rates in an open ecosystem.”

Restated more simply, eDNA analysis of the water picked up 11 of the 12 fish and marine mammals that the divers observed, and—importantly—identified 18 additional animals the divers missed! The efficiency and improvement offered by eDNA analysis compared to traditional seek-and-count methods has been echoed in an editorial I found by Hoffmann et al. titled, tongue-in-cheek, Aquatic biodiversity assessment for the lazy.

Invasive Gobies—The third and final case study deals with detection of invasive, non-native fish to assess whether eDNA can provide a better advanced warning system for detecting these unwanted creatures and implementing eradication steps.

Gobies are an invasive fish species that has colonized freshwaters and brackish waters in Europe and North America. One of them, the round goby (Neogobius melanostomus), pictured below, is among the worst invaders in Europe. Current methods to detect the presence of these gobies are labor intense and not very sensitive. Consequently, populations are usually detected only when they have reached high densities and when management or containment efforts are futile.

Taken from animal.memozee.com

To improve monitoring, Swiss and Canadian collaborators developed an assay based on the detection of eDNA in river water, without detecting any native fish species, which is obviously an important assay criterion. The eDNA assay requires less time, equipment, manpower, skills, and financial resources than conventional monitoring methods such as electrofishing, angling or diving. Samples can be taken by novices and the assay can be performed by any molecular biologist on a conventional PCR machine. Therefore, this assay enables environment managers to map invaded areas independently of fishermen’s reports and fish community monitoring.

I could go on and on with examples of utility and the many advantages provided by eDNA for marine biology, but I’m sure you get the picture. I hope that you agree with me that eDNA analysis is a very valuable type of trending nucleic acid-based methodology.

As usual, your thoughts or comments are welcomed.

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You and Your Microbiome – Part 3

  • Top 10 Cited Microbiome Publications are Summarized
  • Welcome to the New World-View of “Holobionts”
  • TriLink Products Cited in Numerous Microbiome Publications

It’s been almost two-and-a-half years since posting Part 2 in this series on microbiomes, which I first began in 2013, and the publication rate keeps accelerating, with about 7,000 articles indexed in PubMed in 2016—way more than the mere 35 in 1996. This vast amount of new microbiome information being published annually led me to use the following search strategy to guide my selection of what’s trending in importance for microbiomes.

Basically, I used Google Scholar to search for publications since 2015 that had the term “microbiome” in the title and, among those items found, used the number of citations as a quantitative indicator of interest, importance, and/or impact. But before summarizing my findings for these Top 10 Most Cited Microbiome articles, here’s what you can read in my previous two postings on microbiomes in case you missed them or want to refresh your memory:

Proportion of cells in the human body. You are comprised of much more than what you think you are! Taken from amnh.org

Meet Your Microbiome: The Other Part of You

  • What’s in your microbiome? Why does it matter?
  • Next-generation sequencing is revealing that you and your bacterial microbiome have a biological relationship.

You and Your Microbiome – Part 2

  • Global obesity epidemic is linked to gut microbiome.
  • Investments in microbiome-based therapies are increasing.

Top 10 Cited Microbiome Publications 

The following articles, which were all published in 2015, are listed in decreasing order of the number of citations in Google Scholar. Titles are linked to original documents for interested readers to consult, and synopses represent my attempt to capture essential findings.

1. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile (369 citations)

C. difficile (From lactobacto.com)

Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens such as Clostridium difficile, which is a major cause of antibiotic-induced diarrhea in hospitalized patients. It was found that Clostridium scindens, a bile acid 7-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration as a probiotic, enhances resistance to C. difficile infection.

2. Dynamics and stabilization of the human gut microbiome during the first year of life (298 citations)

Applying metagenomic sequencing analysis on fecal samples from a large cohort of Swedish infants and their mothers, the gut microbiome during the first year of life was characterized to assess the impact of mode of delivery and feeding. In contrast to vaginally delivered infants, the gut microbiota of infants delivered by C-section showed significantly less resemblance to their mothers. Nutrition had a major impact on early microbiota composition and function, with cessation of breast-feeding, rather than introduction of solid food, being required for maturation into an adult-like microbiota.

Graphical abstract by Bäckhed et al. Cell Host & Microbe (2015)

3. Structure and function of the global ocean microbiome (238 citations)

Taken from Sunagawa et al. Science (2015)

Metagenomic sequencing data from 243 ocean samples from 68 locations across the globe was used to generate an ocean microbial reference gene catalog with >40 million novel sequences from viruses, prokaryotes, and picoeukaryotes. This ocean microbial core community has 73% of its abundance shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

4. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis (200 citations)

Taken from factvsfitness.com

The brain-gut axis is a bidirectional communication system between the central nervous system and the gastrointestinal tract. Serotonin functions as a key neurotransmitter at both terminals of this network. Accumulating evidence points to a critical role for the gut microbiome in regulating normal functioning of this axis. The developing serotonergic system may be vulnerable to differential microbial colonization patterns prior to the emergence of a stable adult-like gut microbiota. At the other extreme of life, the decreased diversity and stability of the gut microbiota may dictate serotonin-related health problems in the elderly. Therapeutic targeting of the gut microbiota might be a viable treatment strategy for serotonin-related brain-gut axis disorders.

5. The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes (184 citations)

Taken from dtc.ucsf.edu

Colonization of the fetal and infant gut microbiome results in dynamic changes in diversity, which can impact disease susceptibility. To examine the relationship between human gut microbiome dynamics throughout infancy and type 1 diabetes (T1D), a cohort of 33 infants genetically predisposed to type 1 diabetes (T1D) was examined to model trajectories of microbial abundances through infancy. A marked drop in diversity was observed in T1D progressors in the time window between seroconversion and T1D diagnosis, accompanied by spikes in inflammation-favoring organisms, gene functions, and serum and stool metabolites. These trends in the human infant gut microbiome thus distinguish T1D progressors from nonprogressors.

6. The microbiome of uncontacted Amerindians (150 citations)

Taken from robertharding.com

Sequencing of fecal, oral, and skin bacterial samples was used to characterize microbiomes and antibiotic resistance genes (resistome) of members of an isolated Yanomami Amerindian village in the Amazon with no documented previous contact with Western people. These Yanomami harbor a microbiome with the highest diversity of bacteria and genetic functions ever reported in a human group. Despite their isolation, presumably for >11,000 years since their ancestors arrived in South America, and no known exposure to antibiotics, they harbor bacteria that carry functional antibiotic resistance (AR) genes, including those that confer resistance to synthetic antibiotics. These results suggest that westernization significantly affects human microbiome diversity and that functional AR genes appear to be a feature of the human microbiome even in the absence of exposure to commercial antibiotics.

7. Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology (136 citations)

Taken from dtc.ucsf.edu

Individuals with obesity and type 2 diabetes differ from lean and healthy individuals in their abundance of certain gut microbial species and microbial gene richness. This study in humans found that, at baseline, A. muciniphila was inversely related to fasting glucose, waist-to-hip ratio and subcutaneous adipocyte diameter. Subjects with higher gene richness and A. muciniphila abundance exhibited the healthiest metabolic status. Individuals with higher baseline A. muciniphila displayed greater improvement in insulin sensitivity markers and other clinical parameters. A. muciniphila is therefore associated with a healthier metabolic status and better clinical outcomes for overweight/obese adults.

8. Host biology in light of the microbiome: ten principles of holobionts and hologenomes (132 citations)

Today, animals and plants are no longer viewed as autonomous entities, but rather as “holobionts“, composed of the host plus all of its symbiotic microbes. The term “holobiont” refers to symbiotic associations throughout a significant portion of an organism’s lifetime, with the prefix holo- derived from the Greek word holos, meaning whole or entire. Holobiont is now generally used to mean every macrobe and its numerous microbial associates, and the term importantly fills the gap in what to call such assemblages. Symbiotic microbes are fundamental to nearly every aspect of host form, function, and fitness, including traits that once seemed intangible to microbiology: behavior, sociality, and the origin of species. Microbiology thus has a central role of in the life sciences, as opposed to a “bit part.”

Taken from researchgate.net

9. The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development (131 citations)

The nasopharynx (NP) is a reservoir for microbes associated with acute
respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked
to asthma development. The NP microbiome examination during the first year of life in a cohort of 234 children led to characterization of viral and bacterial communities, and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns.

10. Insights into the role of the microbiome in obesity and type 2 diabetes (128 citations)

Obesity and type 2 diabetes (T2D) are associated with changes in the composition of the intestinal microbiota, and the obese microbiome seems to be more efficient in harvesting energy from the diet. Lean male donor fecal microbiota transplantation (FMT) in males with metabolic syndrome resulted in a significant improvement in insulin sensitivity and increased intestinal microbial diversity, including a distinct increase in butyrate-producing bacterial strains. Such differences in gut microbiota composition might function as early diagnostic markers for the development of T2D. The rapid development of FMTs provides hope for novel therapies in the future.

TriLink Products Cited in Microbiome Publications

It always amazes me to learn about the many ways TriLink products are used in basic and applied science. When I searched Google Scholar for publications containing “TriLink [and] microbiome” I found 21 items, among which the following were selected to illustrate diversity of these product types and uses:

Takeaway Messages

In summary, several takeaways should now be apparent to you. The first takeaway is that there is continuing explosive growth of microbiome publications in all manner of life-related research, as evidenced by both the introductory PubMed graph and wide spectrum of subjects covered by the Top 10 Cited Publications mentioned above.

The second takeaway is best summarized in publication #8 above, “[t]oday, animals and plants are no longer viewed as autonomous entities, but rather as ‘holobionts’, composed of the host plus all of its symbiotic microbes.” Each of us is indeed inextricably comprised of our human cells and symbiotic microbiota in or on us—like it or not, and for better or worse.

The final takeaway is that TriLink products play a contributing role in elucidating and applying this new world-view of halobionts.

As usual, your comments are welcomed.

Impossible Foods and Other Achievements of Pat O. Brown

  • Brown’s Microarray Publications Started a Revolution in DNA/RNA Analysis
  • Open Access Publishing Was an Unintended Consequence of His Microarray Research
  • Brown’s Passion for Bettering Earth Led to Invention of the Plant-Based Impossible Burger

Impossible Foods is a company founded by Patrick “Pat” O. Brown that wants to transform the global food system by inventing foods we love, without compromise. It’s first commercial product uses 0% meat and 100% plants to recreate everything—i.e. sights, sounds, aromas, textures and flavors—of a big, juicy burger, aptly named the Impossible Burger. “Impossible” because this was not thought to be doable, as many “veggie” burgers have fallen short on appearance, texture and—importantly—taste.

But before I tell you more about the circumstances and science of this game changer of a burger by Brown and company, let me start with his background as related to nucleic acid research, specifically microarrays.

Pat O. Brown and Microarrays

Pat O. Brown. Taken from Wikipedia.org

Brown received his BS, MD, and PhD degrees all from the University of Chicago, where he worked and published with Nicholas R, Cozzarelli on topoisomerases, which are enzymes that participate in the overwinding or underwinding of DNA. Brown did his postdoctoral research with uber-famous Nobel Laureates J. Michael Bishop and Harold Varmus at the University of California, San Francisco.

Brown went on to become a professor at Stanford University and in 1995 was the first to report (along with his colleagues) the use of microarrays for high-throughput analysis of nucleic acid. This seminal article published in venerable Science magazine and titled Quantitative monitoring of gene expression patterns with a complementary DNA microarray has now been cited more than 11,000 times in Google Scholar.

This publication described general methods for attaching cDNA probes for genes of interest onto glass microscope slides using a high-speed arraying machine (aka robotic printing). These were then hybridized to fluorescently labeled cDNA derived from mRNA by reverse transcription with dNTPs including labeled dCTP akin to dye labeled dNTPs offered by TriLink for such applications. Slides were then fluorescently scanned to obtain “spots” having pseudo-color intensities for quantitation relative to a “spike in” reference gene, as shown below.

Taken from Brown & coworkers Science (1995).

This paper triggered the genesis of what would become a highly competitive microarray industry, which I think of as going from “seeing spots to seeing dollars.” Interested readers can find much information about this in a review by pioneering experts during that time. A brief synopsis of this commercialization involving Brown is as follows.

From Microarrays to Open Access

Taken from plos.org

During his time at Stanford, Brown and his coworkers were using microarrays to generate huge amounts of data on gene expression profiling that required detailed analysis of even larger amounts of information previously published in many different journals. Although many of these journals were available online via a subscription, others were not, and almost all strictly forbade downloading and automated analysis. This thwarted Brown and others from compiling databases for anyone to use as needed. In other words, it prevented Open Access—allowing everyone, everywhere to have unrestricted, free access to this information.

Brown mulled over various ways for researchers to share their data, and in a coffee shop discussion with Harold Varmus, who was then Director of the NIH, they agreed on the possibility of a NIH-hosted computer server where scientists could post their work, and where it would be organized in a systematic way. Shortly thereafter in 1999, Varmus posted on the Director’s website a draft proposal for something that was dubbed e-Biomed.

In 2001, Brown helped lead the Public Library of Science (PLOS) initiative to make published scientific research open access and freely available to researchers in the scientific community. PLOS quickly grew in popularity, as have other Open Access journals, and PLOS now publishes roughly 20,000 papers per year. TriLink researchers—including yours truly—are pleased to be PLOS authors in a November 2016 report titled Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation, which has been viewed more than 2,700 times as of June 2017.

Impossible Foods

Taken from ajitvadakayil.blogspot.com

Apparently pioneering Open Access wasn’t enough for Brown and in 2009 he decided to devote his sabbatical to a daunting—if not impossible—challenge: eliminating conventional meat production from animals, which he estimated to be the world’s largest environmental problem, according to a reported interview. Reducing meat consumption, Brown reasoned, would free up vast amounts of land and water, as well as mitigate climate change due to methane emitted by animals (specifically, 8% of the world’s water and 15% of greenhouse gas emissions, according to one report). In addition, there would be elimination of enormous quantities of chemical fertilizers that are harmful to water systems.

Taken from impossiblefoods

‘All you have to do is make a product that the current consumers of meat and dairy prefer to what they’re getting now,’ Brown said and succeeded in raising $3 million in venture capital seed money. His startup company—aptly named Impossible Foods—then raised $108 million, in a whopper of a deal (pun intended) for development of its initial product—a plant-based burger, also aptly named the Impossible Burger.

The Impossible Burger is made from all-natural ingredients such as wheat, coconut oil and potatoes. What makes this burger unlike all other veggie burgers is an ingredient called heme. Heme is commonly associated with hemoglobin—the red pigment in blood—but is also found in other hemoproteins, including those in plants, albeit in low abundance compared to red meat. Therein lies the part of this story I decided to research: how does Impossible Foods obtain large amounts of plant heme having the desired properties for their burger?

Leghemoglobin taken from web.mst.edu

In an Impossible Foods patent by Brown et al., I found soy leghemoglobin identified as one such exemplary heme, which is pictured right. Plant cells within the nodule produce leghemoglobin to serve as an oxygen carrier to the bacteria within the nodule, similar to hemoglobin in blood. This enables the bacteria to obtain enough oxygen for respiration but ensures that the oxygen is in a bound form so that it cannot harm nitrogen fixing enzymes inside the bacteria. Cutting open a nodule reveals the red color typical of leghemoglobin when it binds oxygen, as seen below.

Impossible Foods biomanufacturing facility. Taken from psmag.com

According to the patent, biosynthetic leghemoglobin was expressed and purified using recombinant DNA technology for protein production, and then shown by SDS-PAGE gel and mass spectrometry to be identical to soybean leghemoglobin isoforms purified from soybean root nodules. Given the advanced state-of-the-art of industrial-scale recombinant production, I assume the Impossible Foods processes pictured right can be scaled-up to reduce cost.

If you think that Brown’s burger probably falls short of what you’d want for a meat substitute, think again. After watching an unbiased—I assume—and rather entertaining video of numerous taste testers (including meat eaters and a life-long vegan) give it positive reviews, I set out to sample an Impossible Burger. It was served as two sliders topped with sun-dried tomatoes, cavolo nero, vegan sun-dried tomato mayonnaise on a poppy seed bun served with chickpea panelle. I found the taste and texture nicely meat-like, but the $16 cost a bit tough to swallow—pun intended.

Meat-Substitutes are Ethically Compelling and Becoming Big Business

Readers interested in the ethically compelling case for developing meat substitutes like the Impossible Burger may be interested in a newspaper story about a for-credit curriculum now offered by the University of California, Berkeley. In that article, I was particularly impressed by the extent of other investments in commercialization of meat-substitutes:

  • In direct competition with Impossible Foods, which has raised a total of $183 million, Beyond Meat (which counts both multibillionaire Bill Gates and meats giant Tyson Foods as investors), sells The Beyond Burger™ as well as other meatless products.
  • In 2014, Pinnacle Foods (Vlasic® pickles, Birds Eye® vegetables) bought meatless food producer Gardein for $154 million.
  • Last year, Monde Nissin (instant noodles, etc.) of the Philippines purchased US-based Quorn for $831 million. Quorn’s fungus-derived mycoprotein can be processed to look and taste like chicken nuggets, sausage or patties.
  • Some startups such as Mosa Meat in Holland and Perfect Day in Berkeley are pushing the genetic engineering toward completely biosynthetic “meat” and “milk,” respectively, as recently reported in The Economist.

In conclusion, I think you’ll agree with me that the aforementioned accomplishments of Pat O. Brown give him good reasons to be smiling so broadly in his picture above. I certainly would be.

As usual, your comments are welcomed.

Postscript

After writing this blog, I read about Memphis Meats in San Leandro, California, which—like Mosa Meat and Perfect Day—has been developing cell culture-based technology to produce “meat.” Focusing on chicken, the company is quoted as saying that ‘the taste and texture is similar to that of the real thing, just a bit spongier.’ While this seems promising, it currently costs around $9,000 to produce a pound of Memphis Meats’ poultry, compared to a bit over $3 for a pound of chicken breast. However, the company hopes to reduce costs drastically and to launch a commercial product in 2021. I hope it does, but I think it won’t.

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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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