DNA Day 2017

  • There are Now Millions of DNA-Related Publications
  • Some of the Top 5 Cited Papers on DNA Will Surprise You
  • You Probably Won’t Guess Top 5 Most Frequently Cited

Deciding what to post here in recognition of DNA Day 2017 was just as challenging as it has been in past years, primarily because there’s so many different perspectives from which to choose. After much mulling, and several abandoned approaches, I settled on featuring DNA publications that have received the most citations, as an objective metric—not just my subjective opinions about topics I think are significant or otherwise interesting.

Before getting to the numbers of DNA-related papers and some of the most cited papers, here’s a quick recap of what was posted here in the past, starting with the inaugural blog four years ago:

2013—60th Anniversary of the Discovery of DNA’s Double Helix Structure

2014—My Top 3 “Likes” for DNA Day

2015—Celebrating Click Chemistry in Honor of DNA Day

2016—DNA Dreams Do Come True!

Explosive Growth of DNA Publications

Regular readers of my blogs will know that I frequently use the NIH PubMed database of scientific articles to find publications by searching keywords, phrases, or authors. A convenient feature of these searches is providing “results per year” that can be exported into Excel for various purposes. Some preliminary searches indicated that DNA-related articles can be indexed by either DNA or PCR, or cloning, or other terms among which sequencing was notable. The majority, however, were indexed as either DNA or PCR, which together gave nearly 1.7 million items—an astounding number. This number is even much greater since PubMed excludes some important chemistry journals, as well as patents.

Diving deeper into these numbers, I thought it helpful to look at the publication volumes and rates for DNA, sequencing DNA, and PCR through 2015 starting from 1953, 1977, and 1986, respectively. These respective dates correspond to seminar publications by Watson & Crick, Maxam & Gilbert, and Mullis & coworkers. The results shown in the following graph attest to my often stated “power of PCR” as premier method in nucleic acid research, which we’ll see again below in another numerical context.

Top 5 Cited Papers

During my perusal of the above literature in PubMed generally related to DNA, I thought it would be interesting to find, and share here, which specific papers have the distinction of being most frequently cited. Citations are not available in PubMed, but are compiled in Google Scholar, which led me to these Top 5 that are listed from first to fifth.

Frederick Sanger (1918-2013) Taken from newscientist.com

  1. DNA sequencing with chain-terminating inhibitors

Frederick Sanger, the eponymous father of the “Sanger sequencing” method published in 1977, received the 1980 Nobel Prize in chemistry for this contribution. He also received the 1958 Nobel Prize in chemistry for sequencing insulin, and is the only person to win two Nobel Prizes in chemistry. Uber-famous DNA expert Craig Venter is quoted as saying that ‘Fred Sanger was one of the most important scientists of the 20th century,’ [who] ‘twice changed the direction of the scientific world.’

  1. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method

Kenneth J. Livak, PhD
Taken from archive.sciencewatch.com

The most commonly used method to analyze data from real-time, quantitative PCR (RT-qPCR) experiments is relative quantification, which relates the PCR signal of the transcript of interest to that of a control sample such as an

untreated control. The derivation, assumptions, and applications of this method were published in 2001 by Livak & Schmittgen. I overlapped with Ken Livak at Applied Biosystems, which pioneered commercilaization of RT-qPCR reagents and instrumentation at the time. He is currently Senior Scientific Fellow at Fluidigm Corp.

Sir Edwin M. Southern Taken from ogt.co.uk

3. Detection of specific sequences among DNA fragments separated by gel electrophoresis

Sir Edwin Mellor Southern, FRS, the eponymous father of “Southern blotting” DNA fragments from agarose gels to cellulose nitrate filters published in 1975, is a Lasker Award-winning molecular biologist, Emeritus Professor of Biochemistry at the University of Oxford and a fellow of Trinity College. He is also Founder and Chief Scientific Advisor of Oxford Gene Technology.

  1. Prof. Bert Vogelstein, MD
    Taken from hhmi.org

    A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity

This paper by Feinberg & Vogelstein published in 1983 describes how to conveniently radiolabel DNA restriction endonuclease fragments to high specific activity using the large fragment of DNA polymerase I and random oligonucleotides as primers. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments. His group pioneered the idea that somatic mutations represent uniquely specific biomarkers for cancer patients, leading to the first FDA-approved DNA mutation-based screening tests, and now “liquid biopsies” that evaluate blood samples to obtain information about underlying tumors and their responses to therapy (an area that I’ve touted in previous blogs). A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 109 dpm/μg of DNA can be obtained using relatively small amounts of precursor. These “oligolabeled” DNA fragments serve as efficient probes in filter hybridization experiments.

  1. Kary B. Mullis, PHD
    Taken from TED.com

    Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

In 1988, Kary B. Mullis and coworkers (then at Cetus Corp.) published in venerable Science a method using oligonucleotide primers and thermostable DNA polymerase from Thermus aquaticus to amplify genomic DNA segments up to 2000 base pairs to detect a target DNA molecule present only once in a sample of 105 cells. Since that time, polymerase chain reaction (PCR)-related technology has evolved to now routinely enable a variety of single-cell analyses of DNA or RNA. Dr. Mullis received the 1993 Nobel Prize in chemistry for his 1983 invention of PCR, which his website says ‘is hailed as one of the monumental scientific techniques of the twentieth century.’

Top 5 Papers by Citation Frequency

While writing the above section, it occurred to me that ranking these five publications by total number of citations-to-date in Google Scholar doesn’t account for differences in the number of years between the year of publication and now. I did the math to calculate the average citation frequency per year, and here’s the totally surprising—to me—result: relative gene expression methodology published by Livak & Schmittgen is by far the most frequently cited of the Top 5, according to this way of ranking:

  1. 2001, relative gene expression, Cited by 69560 = 4,637 avg. citations per year
  2. 1977, Sanger sequencing, Cited by 32662 = 1,701
  3. 1975, Southern blotting, Cited by 21201 = 796
  4. 1988, PCR, Cited by 18785 = 671
  5. 1983, oligolabeled DNA, Cited by 21200 = 642

I should point out that, as transformative methods such as these gradually become widely recognized as “standard procedures,” researchers tend to feel it unnecessary to include a reference to the orignal publication. Consequenly, citation frequency decreases with time even though cummulative usage increases. In other words, 25 years from now average citations per year for relative gene expression will have likely decreased, and be surpassed by a new “method of the decade,” so to speak.

Prediction for the Future

This line of reasoning leads me to close with some speculation about what DNA-related technique might emerge as the next “method of the decade” that tops the above ranking by citation frequency.

My guess is that it will be Multiplex genome engineering using CRISPR/Cas systems by Zhang & coworkers that has been cited by 4145 at the time I’m writing this piece, only four years from its publication in venerable Science in 2013. Some of my blogs have already commented on various aspects of CRISPR/Cas9, which is among genome editing tools offered by TriLink.

As usual, your comments are welcomed.

Autism Awareness Month – April 2017

  • Sequencing for Diagnosis of Autism Holds Promise
  • Several Genetic-Risk Testing Procedures are Available
  • More Than 40 Autism Publications Using TriLink Products

The first National Autism Awareness Month was declared by the Autism Society in April 1970 with the aim of educating the public about autism. Autism is a complex mental condition and developmental disability, characterized by difficulties in the way a person communicates and interacts with other people. Autism can be present from birth or form during early childhood, typically within the first three years. Autism is a lifelong developmental disability with no single known cause.

The puzzle pattern of this ribbon reflects the complexity of autism, while the colors and shapes represent the diversity of people and families living with this spectrum of disorders. Taken from drdiane.com

People with autism are classed as having Autism Spectrum Disorder (ASD) and the terms autism and ASD are often used interchangeably. The term “spectrum” refers to the wide range of symptoms, skills, and levels of disability in functioning that can occur in people with ASD, which includes Asperger syndrome. Some children and adults with ASD are fully able to perform all activities of daily living while others require substantial support to perform basic activities. ASD occurs in every racial and ethnic group, and across all socioeconomic levels. However, boys are significantly more likely to develop ASD than girls. The latest analysis from the U. S. Centers for Disease Control and Prevention (CDC) estimates that 1 in 68 children has ASD.

Taken from myaspergerschild.com

According to the CDC, diagnosing ASD can be difficult, since there is no medical test, like a blood test, to diagnose the disorders. Doctors look at the child’s behavior and development to make a diagnosis.” More details from the CDC are provided at this link.

Notwithstanding this current difficulty for diagnosis of ASD, research has led to continuing progress toward possible blood tests for ASD, which is the focus of this blog and supplements an earlier posting here on treating autism with a broccoli nutraceutical.

ASD and Exome Sequencing

My Google Scholar search for “autism and sequencing” led to a mindboggling list of more than 47,000 items! When ordered by relevance rather than date of publication, two publications were each cited ~1,000-times following “back-to-back” appearance in venerable Nature magazine in 2012. This computes to a combined average of ~400 citations per year, or a fraction more than one citation per day on average, which to me signals significant attention by the ASD research community and thus worth commenting on herein.

Sanders et al., in the first of these two widely cited studies, carried out exome sequencing in 238 families wherein each pair of parents was unaffected by ASD but had a child who was affected (aka proband), and in 200 of these families there was an unaffected sibling. This study design feature is important in view of the widely held idea that complex personality traits are derived by a combination of “nature and nurture,” i.e. genetics inherited from parents and that which is learned or otherwise acquired by familial and all external events.

Before synopsizing what was found, I should note that germline single-base mutations spontaneously arise during mitosis in every generation, and are termed de novo single nucleotide variants (SNVs). Identifying SNVs remained refractory to analysis at the whole genome or exon level until the advent of next-generation sequencing (NGS) technologies.

Sanders et al. found that the total number of non-synonymous (i.e. changes in the amino acid sequence of proteins) de novo SNVs—particularly highly disruptive nonsense and splice-site de novo mutations—are associated with ASD. They concluded that their results “substantially clarify the genomic architecture of ASD, demonstrate significant association of three genes—SCN2A, KATNAL2 and CHD8—and predict that approximately 25–50 additional ASD-risk genes will be identified as sequencing [more] families is completed.”

Neale et al., in the second widely cited study, likewise conducted exome sequencing but on only 175 ASD probands and their parents. Nevertheless, they found that the proteins encoded by genes that harbored de novo non-synonymous or nonsense mutations showed a higher degree of connectivity among themselves and with previous ASD genes as indexed by protein-protein interaction screens. They concluded that their results “support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold,” but did acknowledge the strong evidence reported by Sanders et al. for individual genes as risk factors.

ASD Genetic-Risk Testing

The American Academy of Pediatrics (AAP) in 2013 issued a statement on ethical and policy issues for genetic screening of children for ASD that was prompted in part due to then recent progress by IntegraGen—a small French genomics company—on development of a gene test that uses a cheek swab to screen infants and toddlers for 65 genetic markers associated with autism. Highlights of the AAP’s statement include:

  • Genetic screening can be particularly useful for diagnosing older babies and children with developmental disorders such as autism.
  • Genetic screening should be made available for all newborns. However, parents should have the right to refuse screening after being informed of the benefits and risks.
  • The decision to offer testing or screening should be based primarily on the best interest of the child.

Taken from autismspeaks.org

By way of an update, I’m pleased to add that in a 2015 press release by IntegraGen it was announced that its ARISk® Test became the first test marketed in the U. S. to assess the risk of autism spectrum disorder in children. Among the following IntegraGen statements about the ARISk® Test, I think it’s most important to note the caveats I’ve bolded for emphasis:

  • The test does not confirm or rule out a diagnosis of ASD for the child tested.
  • The test is intended to be used together with a clinical evaluation and other developmental screening tools.
  • Intended for children with early signs of developmental delay or ASD and in children who have older siblings previously diagnosed with an autism spectrum disorder.
  • A genetic score, based on the total number of genetic markers associated with autism identified, is used to estimate the child’s risk of developing ASD.
  • Intended for use for children 48 months and younger. The ARISk® Test is not available for prenatal testing.

Taken from integragen.com

More recently, Courtagen—cofounded by my former Life Technologies colleague Kevin McKernan (coinventor of SOLiD® NGS)—has commercialized its sequencing analyses for ASD and other neurodevelopmental conditions. According to a Courtagen posting, “[i]n the absence of a known single-gene disorder, ASD likely involves a complex combination of both genetic and environmental factors that influence early brain development. Multi-gene panels, such as Courtagen’s devSEEK® panels, provide clinicians with information on a number of genes commonly associated with ASD and autistic features. Clinicians can then use information from multi-gene panels to tailor treatments that meet the patient’s unique genotype and symptoms.”

Some interesting—to me—logistical and operational information about devSEEK® (237 genes) is as follows:

  • Turn-around time for results is 4-6 weeks.
  • DNA for sequencing is extracted from a single saliva sample. No blood draw or muscle biopsy required; however, blood and muscle tissue are accepted.
  • Courtagen works with patients, physicians, and insurance carriers to pre-approve each test. Courtagen will bill the insurance company and is willing to handle an appeal process as needed.
  • A secure physician online portal is available for ordering genetic tests and accessing patient reports when completed. Genetic counselors are available to address questions regarding Courtagen test results.

ASD Research and TriLink

While mulling over how to conclude this Autism Awareness Month blog featuring genetic testing for ASD, I wondered about TriLink’s role in advancing autism research by virtue of its various nucleic acid-related products being used for autism investigations. I was pleased and proud to find more than 40 items by searching Google Scholar for articles with the words “autism and TriLink.”

Perusal of these items revealed that the most cited (450-times) report was a 2012 publication in highly regarded Cell titled MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System, which used TriLink 5-methyl-2′-deoxycytidine-5′-triphosphate (5m-dCTP). Given the apparent significance of this publication, I won’t try to give a short, simplified synopsis but rather quote the following part of the authors’ summary:

“We report that 5hmC [5-hydroxymethylcytosine] is enriched in active genes and that, surprisingly, strong depletion of 5mC [5-methylcytosine] is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.”

I also noted a 2016 Cutting-Edge Review in Arteriosclerosis, Thrombosis, and Vascular Biology titled A CRISPR Path to Engineering New Genetic Mouse Models. These investigators utilized TriLink Cas9 mRNA for gene editing analogous to that reported by others for CRISPR/Cas9-mediated knockout of the autism gene CHD8 (see above). This led to transcriptomic profiling showing that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.

In conclusion, I must say that I learned much new information about autism while researching this blog, which I hope you found informative as well as interesting. If so, I have achieved my goal of either increasing or reaffirming your awareness of autism, and the availability of genetic risk-assessment tests.

As usual, your comments here are welcomed.

Postscript

Recently, a team of academic researchers in Arizona made headlines with their publication in Microbiome reporting ties between autism symptoms and the composition and diversity of a person’s gut microbes, aka “gut microbiome,” about which I’ve commented on in several previous blogs.

The participants, who were 18 children with ASD (ages 7–16 years), underwent a 10-week treatment program involving antibiotics, a bowel cleanse, and daily fecal microbial transplants over 8 weeks. Remarkably, the new therapy seemed to provide some long-term benefits, including an 80% improvement of gastrointestinal symptoms associated with ASDs and roughly a 20% – 25% improvement in autism behaviors, including improved social skills and better sleep habits.

Click here for a simplified, educational video on this work by the principal investigator, Prof. James B. Adams at Arizona State University.

I should emphasize that this is a very small study, and much more research will be needed to verify and firmly establish possible benefits and risks. Interested readers should contact Prof. Adams regarding any questions they might have.

Finding Frankfurter Fraud Featuring Famously Familiar PCR

  • While Thousands of PCR-based Tests for Food Authentication Exist, Commercial Adoption is Lax
  • PCR Tests for Halal Frankfurter Products Reported by Malaysian Team
  • PCR-enabled Next Generation Sequencing of USA Frankfurters Exposes Extensive Mislabeling and Adulteration

Regular readers of this blog will know that (a) I favor alliterations, (b) frequently feature PCR-based topics, and (c) I am fond of food facts involving nucleic acids, all of which are found in the title of this posting. While writing style and food are both a matter of taste, so to speak, it’s almost impossible to comment on nucleic acids without involving PCR, in one way or another, as PCR is—in my opinion—the most widely used and important method in molecular biology.

Having said this, and knowing that this summer alone Americans will likely consume an estimated 7 billion (!) hot dogs, (a.k.a. frankfurters or wieners), I thought it apropos to now feature finding frankfurter fraud by PCR. But before getting to that, I thought it’s worth commenting first on a frankfurter vs. wiener vs. hot dog and other “meaty” definitions to get us “linked” up—please pardon the puns, another of my penchants.

Frankfurter, Wiener, Hot Dog Lexicon

Frankfurter sausages and sauerkraut. Taken from tripadvisor.com

While it’s a fact that a resident of Frankfurt, Germany is properly called a Frankfurter, one of Frankfurt’s pork sausage specialties is also called a frankfurter—but spelled with lower case f—and is short for Frankfurter Würstchen, which go back to the 13th century. By the same token, wiener refers to a pork and beef sausage specialty introduced in the 18th century in Vienna, Austria—a city called Wien in German, and hence the word wiener.

Frankfurters and wieners look very similar, with the main culinary distinction being absence of serving with the bun, which by contrast is characteristic of a hot dog. Readers interested in the origin of the hot dog’s name and bun usage will learn at this link—pun intended—that there are various and widely different claims for this name and usage.

Hot dogs in buns. Taken from clowns4kids.com

Dog Factory, a short film by uber-famous Thomas Edison in 1904 poked fun at what went into hot dogs. Taken from wikipedia.org

In one such claim, the term dog is said to have been linked—there I go again—to sausages made from dog meat, as popularized in an old spoof by Thomas Edison (!) pictured below. This issue of what meat(s) hot dogs and similar sausages contain now segues into finding frankfurter fraud using PCR.

Finding Food Fraud by PCR

Before getting to the meat of this matter (oops!) involving frankfurters, I thought it would be informative to provide some larger perspective on finding food fraud, generally, utilizing the power of PCR for specific detection of nucleic acids that are characteristic of a given species of any food whether it be meat, fish, vegetable, etc. My search of Google Scholar for articles with all of the terms “food, fraud, and PCR” gave ~3,900 items. Here are some selected samplings, the first of which was taken from several I found that used TriLink products—hooray!

  • US Food & Drug Administration researchers employed species-specific primers from TriLink for multiplex real-time PCR analysis of salmon and trout species in a range of 80 commercial products in North America. 4 instances (5%) of fraud were found.
  • By contrast, a whopping 40% of commercial pet food products tested by PCR for the presence of eight meat species (bovine, caprine, ovine, chicken, goose, turkey, porcine, and equine) were found to be potentially mislabeled, according to a study by academic researchers.
  • Saffron produced from dried stigma of Crocus sativus is considered to be the most expensive food spice in the world, as ~200,000 (!) flowers must be carefully hand-picked (!) to produce only 1 kg of spice. Combating saffron fraud with PCR has already led to ~150 publications (!).
  • PCR-based food authentication to screen for possible allergens or GMOs is important for prevention of potentially life-threatening food contamination or alleviating consumer perceptions—or perhaps misperceptions, as I’ve commented on previously.

Finding Frankfurter Fraud by PCR

As for finding frankfurter fraud by PCR, the aforementioned ~3,900 items from my Google Search of food, fraud, and PCR was sub-searched for frankfurter, which led to only 3 reports titled as follows:

The third item, which is by a team of Malaysian researchers and is the most recent, offers some interesting introductory perspectives on strict religious, cultural, or geographical restrictions over the consumption of certain meats in the context of commercial frankfurters.

For example, pork is totally unacceptable to Malaysia’s large Muslim population, as well as Jewish and certain Christian denominations. On the other hand, Egyptians prefer buffalo because of their cultural preferences, while some Indians and Europeans avoid beef because of religious requirements and the fear of bovine spongiform encephalopathy (aka “Mad Cow Disease”—click here for an FDA update), respectively.

In this Malaysian study, 100% beef, buffalo, or pork frankfurters were prepared as models, and then fully heat-processed in the laboratory to simulate conventional manufacturing procedures. Additional beef, buffalo, or pork frankfurter models were deliberately contaminated by “spiking” in 1%, 0.5%, or 0.1% of buffalo and pork, beef and pork, and beef and buffalo meat, respectively. PCR was then performed using species-specific primer pairs for two genes (cytochrome b and NADH dehydrogenase subunit 5) for cross-validation. Twenty different halal-branded (i.e. pork-free) “beef” frankfurters from Malaysian markets were tested. While no pork was detected in any of the tested “beef” frankfurters, they were all beef- and buffalo-positive, thus revealing that all of the investigated Malaysian commercial “beef” products were buffalo-adulterated.

Halal Certification by PCR

The above mentioned concern for non-pork halal-assurance piqued my interest as to the extent of PCR usage for halal-related certification, and my subsequent Google Scholar search for “halal, certification, PCR” gave nearly 350 items. This relatively large number of reports signals quite widespread adoption of PCR. I encourage those interested to peruse these items later, but will mention here that the following article is the most cited—close to 100 citations as of February 2017—Identification of pork derivatives in food products by species-specific polymerase chain reaction (PCR) for halal verification

In closing, I think it’s worth noting in the context of halal certification—and increasingly popular “democratization” of technology, about which I’ve previously offered comments—a French start-up company now offers an antibody-based “dip stick” kit (Halal Test) for anyone to use for pork-free halal-assurance at home or even when eating out. Amazing.

Taken with copy write permission from French duo launch HalalTest: ‘We want to democratize analysis’ by Rachel Arthur+Rachel ARTHUR, 05-Nov-2014

Hot Dog! — There’s Now an NGS Food Authentication Service Company

Frankly—pun-to-be intended—I don’t know how “hot dog!” became an exclamatory phrase for good news. It applies, however, to the fact that Clear Labs Inc. (a 2014 start-up in Palo Alto, California) has proven that several issues of concern for consumers of hot dogs can be successfully addressed by PCR/NGS methods.

In a Clear Labs’ poster abstract for the 2016 International Association of Food Protection meeting, results were reported for a study of 345 hot dog products sold by national brands to compare product label information and ingredient lists with the results of NGS analyses. Following DNA extraction, universally accepted regions for animals, plants, and bacteria were PCR-amplified for NGS, which revealed that ~15% of these products had ingredient substitution, unexpected ingredients, or hygienic issues. In addition, 10% of all products labelled as vegetarian contained detectable levels of meat DNA. Vegetarian products also accounted for 67% of hygienic issues, such as human DNA.

At the risk of overly generalizing these findings, I believe that they probably reflect very widespread issues in the food industry, which we as consumers are virtually helpless to deal with, and can only hope that US FDA regulations begin to mandate PCR-based food certification.

Having said this, I didn’t want to end with a “downer.” Therefore, I’ll conclude with some hopefully fun facts about hot dogs, which—if you’re wondering—can be made from beef, pork, turkey, chicken, or a combination (but must be labeled as such, according to FDA information worth reading later).

Fun Frankfurter (aka Hot Dog) Facts

Just for fun, I calculated that the 7 billion frankfurters to-be-consumed by Americans this summer would stretch 56,818 miles (assuming each one were 6 inches long), which would wrap around Earth’s equator 2.3 times if placed end-to-end!

Some other fancinating frankfurter feats:

  • Taken from wikipedia.org

    The world’s longest hot dog was 197 feet and was prepared by Shizuoka Meat Producers and the All-Japan Bread Association for the latter’s 50th anniversary celebration in 2006 at the Akasaka Prince Hotel:

  • The world’s most expensive hot dog is the “California Capitol City Dawg”, served at Capitol Dawg in Sacramento, California and cost $145.49. Proceeds from the sale of each 18-inch long, 3-pound super “Dawg” are donated to the Shriners Hospitals for Children.
  • The annual Nathan’s Hot Dog Eating Contest is held on Independence Day at Nathan’s Famous original, and best-known restaurant in Coney Island, a neighborhood of Brooklyn, New York City. The current champion is Joey “Jaws” Chestnut, who ate a stomach-busting 70 (!) hot dogs and buns in only 10 minutes (!) at the 2016 championship.

Taken from elitedaily.com

As usual, your comments here are welcomed.

Evolving Polymerases to Do the Impossible

  • Polymerases Aren’t What They Used to Be! 
  • Scripps Team Evolves Polymerases That Read and Write With 2’-O-Methyl Ribonucleotides
  • Key Reagents for Romesberg’s “Molecular Moonshots” Are Supplied by TriLink BioTechnologies

Long-time devotees of these posts will likely remember a blog several years ago about Prof. Floyd Romesberg at the Department of Chemistry, The Scripps Research Institute who achieved a seemingly impossible feat. Namely, designing a new pair of complementary bases such that DNA replicating in E. coli would be comprised of six bases, thereby creating a six-base genetic code that is expanded from Nature’s four-base code.

Floyd E. Romesberg. Taken from utsandiego.com

More recently, Romesberg has cleverly outfoxed Nature once again, this time by evolving nucleic acid polymerases into mutant polymerases that can do what heretofore seemed impossible. He and his research team’s publication (Chen et al.) is a tour de force of experimental methodology that is not easily read, and is even harder to simply summarize in a short space like this blog. Consequently, I’ll first tell you what was accomplished, then give a short synopsis of principal new methodology, and close by commenting on the significance of this fascinating work.

Doing the Impossible

Romesberg’s lab successfully achieved what I think of as “multiple molecular moonshots,” wherein a Taq polymerase (which normally reads and writes DNA during PCR), was evolved by novel selection (SELEX) methods into mutant polymerases that are able to transcribe DNA into 2’-O-methyl (2’-OMe) RNA, and reverse transcribe 2’-OMe RNA into DNA for PCR/sequencing.

As depicted below, this was exemplified using a 60-mer DNA template and 18-mer 2’-OMe RNA primer to produce a fully-modified 48-mer 2’-OMe RNA by means of an evolved mPol and all four A, G, C and U 2’-OMe NTPs, which I’m proud to say were bought from TriLink BioTechnologies! This type of molecular evolution of a polymerase has no precedent.

DNA template   5’ ————————————- 3’

RNA primer                                 ←←← 3’ xxxxxx 5’

mPol ↓ 2’-OMe NTPs

Determining the fidelity of this seemingly impossible molecular transformation was addressed by achieving a feat of comparable impossibility! As depicted below, the aforementioned 48-mer 2’-OMe RNA product was hybridized to a DNA primer for reverse transcription into a 48-mer complementary DNA (cDNA) strand, using an evolved mPol, together with all four A, G, C and T unmodified dNTPS, which were also purchased from TriLink. This unprecedented conversion of 2’-OMe RNA into cDNA was followed by conventional PCR/sequencing, the results of which demonstrated relatively high fidelity.

2’-OMe template   5’ xxxxxxxxxxxxxxxxxxxxxxxxxxx 3’

DNA primer                                           ←←← 3’ —— 5’

mPol ↓ dNTPs

cDNA                        3’ ————————————– 5’

How They Did It

In the selection cycle shown below, (1) phage-display libraries were used to expose individual polymerases (Pol) on E. coli. cells in proximity to chemically attached primer/template complexes of interest, which are mixed with natural or modified triphosphates including biotin (green; B)-labelled UTP to extend the primer. (2) Phage that display active mutant polymerases (mPols) are isolated with streptavidin (SA) beads. After washing to remove nonspecific binders, phage cleaved from the beads are used to re-infect E. coli. (3) Heat-treated lysates of E. coli that express the recovered mPols are next subjected to plate-based screening using 96-well plates coated with primer/template complex and extension buffer that contained natural or modified triphosphates and B-UTP, incorporation of which is chromogenically detected. (4) Mutants that give rise to the most activity are selected for individual gel-based analysis, from which (5) promising candidates are selected for further diversification (e.g., by gene shuffling, as depicted) and then subjected to additional rounds of evolution.

Taken from Chen et al. Nature Chemistry (2017)

What is the Significance

In a previous blog, I’ve commented on increasing interest in the utility of aptamers, which are oligonucleotides that can specifically bind small molecules or motifs in proteins, and thus be used to build electronic sensors or studied as potential therapeutic agents rivaling antibodies. Therapeutic aptamers, like antisense oligonucleotides, require incorporation of chemical modifications to impart stability toward nucleases in blood or cellular targets.

Burmeister et al. have previously reported methods for mPol transcription of a DNA template into a fully modified, nuclease-resistant 23-mer 2’-OMe RNA aptamer—also using TriLink’s 2’-OMe NTPs! However, they encountered considerable experimental difficulties in generating this therapeutically promising 23-mer against vascular endothelial growth factor. These technical issues have now been surmounted by the mPol-evolution approaches in the present work by Romesberg’s team, which enabled improved access to longer 2’-OMe RNA aptamers with reasonable efficiency and fidelity.

Moreover, the present study is the first to evolve an mPol for reverse transcription of fully modified 2’-OMe RNA into DNA, which can then be amplified by PCR and/or sequenced, thereby opening the door for a variety of new analytical methods. Most importantly, the molecular mechanism by which these remarkable mPol activities was evolved, namely, the stabilization of an interaction between the “thumb and fingers domains,” may be general and thus useful for the optimization of other Pols. In that case, we can look forward to further advances in evolving other Pols to do the impossible—hopefully using modified nucleotide triphosphates from TriLink!

As usual, your comments are welcome.

CRISPR-Mediated Interference (CRISPRi) of Long Non-Coding RNA (lncRNA)

  • More Methodology from CRISPR Mania
  • lncRNA Function Blocked by CRISPRi
  • Mysteries of lncRNA Can Now be Deciphered by CRISPRi

This blog is about yet another example of a powerful new methodology spawned by intense scientific interest in using CRISPR-related technologies. This near mania for all things CRISPR is reflected by there being ~5,000 (!) publications already in PubMed only ~5 years after seminal papers appeared.

I chose the present blog topic because it involves use of CRISPR for genome-wide identification of functional long non-coding RNA (lncRNA) in human cells. In an earlier blog about lncRNA, which are now recognized to be regulators of gene expression encoded by what was originally defined as “junk” DNA, it was pointed out that it is inherently difficult to experimentally identify such regulation by lncRNA. Thanks to CRISPR this task is now much less daunting as you’ll learn below, following a couple of introductory sections to set the stage.

Repurposing CRISPR/Cas9 Using “Dead” Cas9

Qi et al. very cleverly—at least to me—recognized that the CRISPR/Cas9 system could be repurposed as an RNA-guided platform for sequence-specific control of gene expression by finding a catalytically inactive mutant Cas9 protein that lacked exonuclease (i.e. cutting) activity of wild-type Cas9, and instead blocked transcription by RNA polymerase (RNAP), as depicted below. These researchers coined the overall process as “CRISPR interference” (CRISPRi) and loosely referred to such a mutant Cas9 as “dead” Cas9 (dCas9).

Taken from Qi et al.

Interested readers are encouraged to consult this publication by Qi et al. to fully appreciate the extensive amount of work that went into translating the above concept into practice, and supporting the proposed mechanism of action. In my opinion, it’s a tour de force example of applying hypothesis-driven, state-of-the art molecular biology to devise a new method—in this case specifically blocking transcription of a DNA region using CRISPRi in conjunction with target-specific short guide RNA (sgRNA).

Adding Functionality to Down-Regulate Transcription

Just as organic chemists can design and synthesize small molecules having desired functional properties, molecular biologists can design and produce complex macromolecules having desired functional elements. The latter is nicely exemplified by Gilbert et el., who demonstrated that fusion of dCas9 to transcription factor effector domains having repressive regulatory functions enables efficient transcriptional repression in human (or yeast) cells via sgRNA that target genes of interest.

Taken from Liu et al. (2017)

As depicted below, Gilbert et al. used dCas9 fused to the Krüppel associated box (KRAB) domain, which is a transcriptional repression domain, and Green Fluorescent Protein (GFP) as a reporter gene targeted by sgRNA. They employed RNA-sequencing to quantify the transcriptome of GFP-positive HEK293 cells expressing dCas9-KRAB or a negative control construct. It was shown that CRISPRi is highly specific, as GFP was the only gene that was significantly suppressed by GFP-targeting sgRNA. Averaged data from two independent biological replicates indicated that no gene other than GFP changes by >1.5-fold.

Genome-Scale CRISPRi to Identify Human lncRNA

According to Liu et al., it has not been possible to predict which lncRNA loci are functional or what function they perform. Consequently, there is a need for large-scale, systematic approaches to interrogating the functional contribution of lncRNA loci. This sizeable team of collaborators from various institutions in the San Francisco Bay area, therefore, developed a genome-scale screening platform using CRISPRi with dCas9-KRAB and a library of sgRNA.

Taken from Liu et al. (2017)

As depicted below for the overall approach, they first designed a CRISPRi Non-Coding Library, which targets 16,401 lncRNA genes each with 10 sgRNAs per transcription start site. The required 170,262 sgRNAs were not synthesized chemically, but rather produced intracellularly by first using array-based sgDNA synthesis followed by clonal (i.e. individual sgDNA sequence) incorporation into lentivirus, which in turn were transfected into seven types of cells for screening. More detail on such lentiviral libraries is given in a Footnote at the end of this blog.

As indicated pictorially above, they applied this pooled screening approach to identify lncRNA genes that modify robust cell growth for induced pluripotent stem cells (iPSC) and six well-known, transformed human cell lines (K562, U87, etc.). This led to identification of 499 lncRNA loci that modified cell growth upon CRISPRi targeting.

Interestingly—at least to me—372 (~75%) and 299 (~60%) of these 499 growth-modifying lncRNA loci were distal to a protein coding gene (PCG) or mapped enhancer, respectively. The diagram below, taken from a review by Vance & Ponting, depicts “distal” effects of lncRNA away from PCG between two chromosomes (chr). What “triggers” transcription of the lncRNA from chr A and how it “finds” its cognate PCG on chr B are open and indeed intriguing questions.

Taken from Vance & Ponting (2014)

In addition to these high percentages of distal effects, Liu et al. found the following surprising results with regard to cell-type specificity of lncRNA function:

“Remarkably, 89% of the lncRNA gene hits modified growth in just one of the cell lines tested, and no hits were common to all seven cell lines. Although nearly all of the hit genes were expressed in the cell line in which they exhibited a growth phenotype, expression alone was insufficient to explain the cell type specificity of their function.”

“[Thus,] in contrast to recent studies that found that essential protein-coding genes typically are required across a broad range of cell types, we show that lncRNA function is highly cell type-specific, a finding that has important implications for their involvement in both normal biology and disease.”

Following are some of the major unanswered questions about lncRNA posed in a review I recommend reading for more background on lncRNA:

  • How does the manner in which lncRNAs are transcribed, processed, and regulated differ from that of other RNAs?
  • Are lncRNAs evolutionarily conserved, both in terms of their primary sequences and secondary structures?
  • Are all lncRNAs functional? Which ones have detectable biological functions in cells or in the whole organism?
  • Does the pervasive transcription that generates the lncRNA transcripts play a regulatory role distinct from the steady-state accumulation of the lncRNAs?
  • Can lncRNAs be exploited for clinical applications and therapeutics?

After reading this review, I thought to myself that there are many open questions about lncRNA but no comprehensive answers yet deciphered. When I then checked Google Scholar for items with both “deciphered” and “lncRNA” as terms, I found there were over 1,800 such items. Evidently, there are quite a few authors who, like me, view unknown functions of lncRNA as a cipher. I suspect that much of the now mysterious lncRNA function will eventually be deciphered thanks, in part, to the power of CRISPRi.

Your comments are welcomed.

Footnote

Readers interested in lentiviral sgRNA library construction and use for screening target cells can find general information at this website from which the following self-explanatory schematic provides a high-level overview of the workflow.

Taken from cellecta.com

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

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

Honey! I shrunk the lab! 

Taken from gene-quantification.de

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

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

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

Lab-in-a-Box 

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

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

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

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

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

The Bento Lab. Taken from Bento Lab

Lab-on-a-Robot

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

Taken from Berg et al. (2008)

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

Lab-on-a-Drone

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

Victor Ugaz. Taken from tamu.edu

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

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

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

Taken from Ugaz & coworkers (2016)

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

Taken from Ugaz & coworkers (2016)

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

Taken from Ugaz & coworkers (2016)

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

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

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

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

As always, your comments here are welcomed.

Nanopore Sequencing by Synthesis (Seq-by-Syn)

  • Yet Another Notable Achievement Involving George Church, ‘The Most Interesting Scientist in the World’ 
  • Team of 30 Coauthors Reports Seq-by-Syn with DNA Polymerase-Nanopore Protein Construct on an Integrated Chip
  • Challenging Improvements Needed for Commercial Reality

Prof. George M. Church. Taken from evolutionnews.org

Devotees of my blog will know that I’m prone to word play such as calling myself a “huge” fan of “tiny” nanopores for DNA sequencing, about which I’ve previously opined. They will also recall that I’m an admitted scientific admirer of George Church, who I think is The Most Interesting Scientist in the World.

Having said this, it’s not surprising that I closely follow what’s trending in nanopore sequencing, and also make an attempt to read all of Church’s papers as they get published because they are almost invariably quite interesting, involve “big ideas,” and in some new way are very educational, at least for me. Following are my comments about a recently published paper on nanopore sequencing in venerable Proceedings of the National Academy of Sciences of the United States of America (aka PNAS) wherein Church is the designated corresponding author.

Backstory

The seminal origins and early history of nanopore sequencing have been recently chronicled and criticized—then clarified—in Nature Biotech in several “To the Editor” items, which collectively provide enlightening insights into who did what when, so to speak. Those of us who are ‘Nanoporati’—a clever term tweeted by Nick Lowman—should definitely read those Nature Biotech items. For now, however, I’ll set the stage, as it were, by echoing a bit of what I’ve posted in the past for nanopores.

Patented but prophetic (i.e. no data) methods for nanopore sequencing DNA is actually a relatively old (~20 year) idea posited by Church and other creative visionaries. On the other hand, nanopore sequencing was first reduced to practice commercially not too long ago by Oxford Nanopore Technologies (ONT). Many years of delay between concept and commercialization was due to the need for gradual evolution of lots of “nanopore-ology” and sequencing biochemistry, as well as developing highly sophisticated electronics and complex algorithms for data analysis.

Nanopore Sequencing-by-Scanning (Seq-by-Scan)

Taken from rsc.org

As depicted below, and as can be best seen in a video, ONT’s commercially available MinION Seq-by-Scan system essentially involves threading a strand of DNA through a protein-based nanopore and converting resultant ionic current fluctuations into nucleotide base sequence.

While there are issues with base-calling accuracy, the remarkably small and readily portable MinION provides fast, real-time sequencing results for a wide variety of applications. These included unique or otherwise compelling Point-of-Care analyses, such as pathogen surveillance, which has been achieved in remote geographical locations and even in outer space aboard the International Space Station, as I’ve previously posted.

Nanopore Seq-by-Syn

In contrast to DNA Seq-by-Scan using a nanopore, which is challenged by pore-based differentiation of similarly sized A, G, C, and T bases, DNA Seq-by-Syn has no such limitation as it uses the DNA as a template for base-by-base (i.e. stepwise) detection of enzymatic synthesis of complementary DNA. Various Seq-by-Syn methods and challenges have been discussed elsewhere, and currently available commercial systems include those from Illumina and PacBio. The former employs nucleotides that are reversible terminators equipped with cleavable fluorescent “tags” on each base. The latter detects fluorescently labeled tags on polyphosphates released upon nucleotide incorporation.

The presently featured DNA Seq-by-Syn publication by Stranges et al., which builds upon two earlier reports cited therein, differs from the above approaches by using nanopore-based detection of mass tags rather than fluorescent tags. In principle, mass tags could afford higher accuracy compared to DNA Seq-by-Scan. However, as will now be explained, achieving improved accuracy is far easier said than done.

The general approach taken to demonstrate proof-of-concept for mass-tagged nanopore DNA Seq-by-Syn is depicted below in simplified cartoon form, but involves a true tour de force—in my opinion—of three key technologies. The first is design and synthesis of the nucleotides with appropriate mass tags, which involves very sophisticated chemistry that is best appreciated by reading detailed, extensive supporting information (SI) for Stranges et al. and SI for an earlier publication by Fuller et al. In a nutshell, these nucleotides have 5’-hexaphosphates linked to relatively large mass tags comprised of complex oligonucleotide structures.

Taken from Stranges et al. PNAS 2016

The second area of technical innovation involves attachment of a single molecule of ϕ29 DNA polymerase to each α-hemolysin (αHL) nanopore in such a manner as to retain its enzyme activity and be positioned such that every released mass tag transits through (i.e., is “captured” by) the nanopore leading to base identification by its current signature. As depicted below in two related representations, each of these heteroheptameric pores is comprised of one modified αHL subunit to which a peptidyl SpyTag moiety is attached, and six unmodified αHL subunits. This allows attachment of one ϕ29 DNA molecule modified with a cognate peptidyl SpyCatcher moiety at a predetermined, time-average distance from the pore.

Taken from Stranges et al. PNAS 2016.

The third key area of innovation deals with insertion of the enzyme-pore conjugate into a lipid bilayer residing on a silanized array (aka chip) of 256 Ag/AgCl electrodes such that there is one functional pore per electrode. Interested readers are encouraged to consult the publication for details, as well as check out related fabrication and methods patents that I found by searching Google Scholar.

Representative Results

The first image shown above depicts what base tag-specific detection would ideally look like if each of the four different bases would have a characteristic current-blockage intensity and persistence. In addition, all pores would ideally function similarly. Not surprisingly, given the stochastic nature of single-molecule systems in general, Stranges et al. found less than ideal behavior.

For example, out of 70 single pores obtained, 25 captured two or more tags, whereas only six of those pores showed detectable captures of all four tagged nucleotides. Data obtained for the pore with the most transitions between tag capture levels (i.e. the best results) is shown below, while results for the other five are given in the SI.

Taken from Stranges et al. PNAS 2016

To quote the authors:

“All four characteristic current levels for the tags and transitions between them can be readily distinguished…Homopolymer sequences in the template, and repeated, high-frequency tag capture events of the same nucleotide in the raw sequencing reads were considered a single base for sequence alignment. We recognized 12 clear sequence transitions in a 20-s period. Out of the 12 base transitions observed in the data, 85% match the template strand, showing that this method can produce results that closely align to the template sequence.” 

Interested readers need to consult and carefully read the SI for Stranges et al. regarding the interpretation of the “repeated, high frequency capture events,” such as that exhibited by C in the above current vs. time plot.

All of the above snippets in aggregate suggest to me that, while this huge amount of work has made progress toward one approach to Seq-by-Syn, many improvements will need to be made before achieving a robust system to successfully compete in the commercial sector.

Authorship, Affiliations, and Acknowledgments

The relatively large team of 30 coauthors listed for Stranges et al. include the following numbers of investigators and affiliations: 1 at Arizona State Univ., 4 at Harvard, 11 at Columbia University, and 14 at Genia Technologies, which is a Santa Clara, CA company that was acquired by Roche in 2014, and is part of Roche Sequencing.

Acknowledgments in Stranges et al. refer to support by Genia and NIH Grant R01 HG007415, which I found was awarded to coauthors George M. Church (Harvard), Jingyue Ju (Columbia), and James J. Russo (Columbia). The end of the abstract of this grant reads as follows:

“The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques. We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system. These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine.”

The conflict of interest statement in Stranges et al. indicates that the technology described therein (called “Nanopore SBS”) has been exclusively licensed by Genia, and that specified coauthors are entitled to royalties through this license. In addition, Church is a member of the Scientific Advisory Board of Genia.

Parting Comments

Long gone are the days when government-funded academic researchers thumbed their noses, if you will, at commercial development. Nowadays almost all academics parlay their government grants into university patents that get licensed to companies, usually with some type of corporate involvement of said academics.

I hasten to add that I’m not implying that NIH-funded academic research being a “seed” for corporate profitability is negative—especially in view of its Small Business Innovative Research (SBIR) program—but rather view it as a paradigm shift for the better, as it allows academic creativity to be harnessed into applications that can hopefully greatly benefit society.

In conclusion, and coming back to George Church, who I highlighted in the introduction to this blog, I must say that he might very well be the academic researcher with the longest list of technology transfer, advisory roles, and founded companies—13 to date—according to a public list that is truly mind boggling, at least to me.

As usual, your comments are welcomed.

Postscript

After writing this blog, Roche announced on December 15, 2016 that “it has officially notified Pacific Bioscience (PacBio) of its intention to terminate its [2013] agreement and efforts to develop a sequencing instrument for use in the clinical research and clinical market using their Single Molecule, Real-Time (SMRT®) technology,” about which I have commented previously. The announcement went on to say Roche would instead focus on internal development efforts” and “actively pursue multiple technologies and commercial strategies.” A GenomeWeb headline was more specific:  “Roche Will Focus on Genia’s Nanopore Technology for Dx Market After Ending Deal With PacBio.”

On December 30, 2016 it was reported that the University of California (UC) filed a patent suit against the Chief Technology Officer (CTO) at Genia, and Genia Technologies, claiming the CTO produced key inventions during his time at UC that he later assigned to Genia, but which should have automatically been assigned to UC. Stay tuned…

Top 10 Innovations 2016

  • Sequencing with Sequel, but with a Shocking Surprise
  • Endonuclease CRISPR-Cas9 Makes the Cut Twice—Pun Intended
  • My Predicted 2016 Innovation Winners Made Lots of News

Welcome to my first blog of the New Year, 2017! My New Year’s resolution is to continue to do my best in providing interesting and informative content about what’s trending in nucleic acid research. As in the past, this first blog of the year comments on the Top 10 Innovations in 2016 that were selected by a panel of judges and published last month in The Scientist. Also like the past, you can peruse TriLink’s top products by clicking here.

So, with an imaginary loud flourish of trumpets, read on to learn about the 10 winners, starting with 1st place.

  1. Milo from ProteinSimple enables single-cell Western blotting in a benchtop instrument that allows researchers to search for specific proteins in about 1,000 single cells simultaneously. A titered cell-suspension is pipetted on a 1-by-3-inch glass microscope slide covered by a 30-micron gel-layer dotted with 6,400 microwells. Some wells will remain empty, but ~1,000 will collect individual cells for antibody-based protein analysis, following lysis and other steps, all done automatically. Indeed simple!
  2. ExVive Human Kidney Tissue from Organovo is a replica of the kidney proximal tubule created using 3D bioprinting, which is like uber-trending 3D printing with plastic, but instead uses tiny aggregates of cells. This novel product offers drug developers a reliable means of testing for renal toxicity that is more predictive than traditional cell culture, and avoids animal testing.
  3. Sequel System from Pacific Biosystems is not neither small (see below) nor inexpensive ($350,000), but is nevertheless a third the size and weight—and half the cost—of PacBio’s original long-read, single molecule, real-time (SMRT) sequencer, PacBio RS II, about which I’ve favorably blogged several times in the past. Moreover, Sequel has seven-times the throughput of PacBio RS II.

Taken from fiercebiotech.com

Development of what would become Sequel was announced by PacBio in 2013 as part of a potentially $75M deal with Roche Diagnostics aimed at DNA sequencing-based products for clinical diagnostics. Surprisingly—if not shockingly—in December 2016, after Sequel was launched, Roche stated it was terminating this deal with PacBio in order to focus on its internal development efforts.

At this time, I can only speculate that Roche’s internal efforts include single-molecule DNA sequencing using nanopore technology developed by Genia Technologies, Inc., which Roche acquired in 2014 for $125M—plus even greater contingent payments. By remarkable coincidence, I had been drafting a blog about Genia from a purely technical perspective, but will now update that for posting on January 24th as a sequel to Sequel—pun intended.

  1. Lumos from Axion Biosystems is a 48-well light-delivery device allowing researchers to incorporate cutting-edge optical assay techniques, such as optogenetics, into their in vitro research. Lumos delivers user-specified intensity and duration of light with up to four wavelengths simultaneously for assay flexibility.
  2. LentiArray CRISPR Libraries from Thermo Fisher Scientific make applying lentivirus-encoded CRISPR for gene editing more accessible to researchers. Given the continuing explosive-like interest in DNA endonuclease CRISPR-Cas9, which in 2015 also made the Top 10 cut—pun intended—I was expecting to find this endonuclease system among the 2016 Top 10, too. What surprised me, however, was seeing this system split into two parts—pun intended—i.e. CRISPR per se and separately as Cas9, which you’ll find below.
  3. nCounter Vantage 3D Panels from NanoString utilize an automated microscope that counts color-coded barcodes conjugated to target molecules that are mRNAs, DNAs, proteins, and even phosphorylation status of proteins—all at the same time, which I think is quite a technical achievement. The nCounter analysis system ranges from $149,000 to $280,000, and the nCounter Vantage 3D Panels run from $275 per sample and upward.

Taken from nanostring.com

  1. ZipChip from 908 Devices is a cleverly designed microfluidic chip that radically speeds up sample prep for mass spectrometry, requires only a few microliters of sample volumes, and broadens the range of materials that a mass spectrometer can handle. In less than 3 minutes per sample, ZipChip can process cell growth media, cell lysates, blood, plasma, or urine. The 1-by-3-inch chip is in a small box, less than a foot long, which mounts directly onto a mass spec. The device costs $30,000 and an auto sampler adds another $20,000 to the price.
  2. HAP1 Cells from Horizon have earned a spot in The Scientist Top 10 Innovations for the third year in a row—this time as Turbo GFP Tagged HAP1 Cells, which were selected for their ability to tag proteins of interest with green fluorescent protein (GFP) without requiring that the gene be overexpressed. Turbo GFP cells, which are custom-made using CRISPR-Cas9 gene-editing technology, cost $3,400 and take about 16-18 weeks to develop.

Roger Y. Tsien (February 1, 1952 – August 24, 2016).
Taken from wikipedia.org

As a sad side note, Roger Y. Tsien, who was awarded the 2008 Nobel Prize in Chemistry for his discovery and development of GFP, in collaboration with Osamu Shimomura and Martin Chalfie, passed away in 2016 at the age of 64.

  1. Prime sCMOS Camera from Photometrics is a 4.2-megapixel camera that has a built-in algorithm to reduce what is called “shot noise”—the variation inherent in measurements taken using light microscopes—without having to acquire many extra images and then average across them, or increase the light intensity, which can damage samples. The Prime sCMOS camera costs $15,950.
  2. GeneArt Platinum Cas9 Nuclease from Thermo Fisher Scientific is a recombinant Streptococcus pyogenes Cas9 protein purified from E. coli that contains a nuclear localization signal that aids in delivery to target-cell nuclei, where Cas9 works in conjunction with CRISPR. I should add that CRISPR-based gene editing is alternatively achieved by transfection of Cas9 mRNA, which is offered by TriLink, and used as described in a recent exemplary publication by a team of international collaborators.

Revisiting Jerry’s Predictions for 2016 Top 10 Innovations

Devotees of my blog may recall the following predictions I offered in January 2016 as winning innovations-to-be:

  • Direct Genomics in China will resurrect and morph Helicos single-molecule sequencing into a diagnostics instrument.
  • GnuBIO—acquired by Bio-Rad—will offer its long-delayed next-generation sequencing system.

While none of these were selected by The Scientist, they did make news in various ways:

  • Oxford Nanopore’s VolTRAX system for automated sample has very recently been launched. I should also note that its tiny MinION sequencer was rocket-launched—literally—to the International Space Station (ISS) for evaluation in rapid identification of pathogens that might infect astronauts on the ISS, as I’ve commented on in a previous blog. Launch puns intended.
  • Direct Genomics recently announced its GenoCare Analyzer, the world’s first single-molecule genome sequencer that is being engineered exclusively for the clinic, and promises to improve the cost, speed, and quality of clinical genome sequencing by directly reading a patient’s original DNA or RNA molecules without prior amplification.
  • GnuBio now offers a fully integrated sequencing platform which allows users to simply load genomic DNA onto cartridge, place the cartridge into the GnuBIO sequencer, and then press “run.” Within hours, results can be exported directly from the instrument with real-time informatics onboard.

Although my picks weren’t among those in The Scientist list for 2016, I take satisfaction in believing that choosing winning biotechnology products is like art appreciation or judging beauty, both of which are in the eye of the beholder, who can disagree on what their eyes behold.

As usual, your comments are welcomed.

Nanobombs for Light-Activated MicroRNA Drug Delivery

  • Nanoparticles Designed to “Explode” Upon Irradiation with Light
  • Light Used Can Penetrate Skin and Tissue
  • Nanoparticle “Cargo” of MicroRNA Released Locally, Where Needed 

Every once in a while the title of a publication grabs my attention so much that I just have to read it, and that’s what happened when I read this one A Near-Infrared Laser-Activated “Nanobomb” for Breaking the Barriers to MicroRNA Delivery.

Taken from ajamtafireworks.com

My interest was primarily piqued by the word ‘nanobomb,’ which I had never heard of even though I consider myself quite well-read scientifically. Moreover, microRNA (miRNA) and drug delivery are both currently trending hot topics. I couldn’t find an eye catchy image of a nanobomb other than fireworks by this name sold in India, which are oddly touted as being “pollution free”! But I digress…

Delivery of miRNA and other types of nucleic acid-based drugs continues to be perhaps the biggest challenge facing successful clinical development of this broad new class of drugs. In that regard, an expert review titled Nucleic acid delivery: the missing pieces of the puzzle?, written by my good friend and long-time collaborator Prof. Frank Szoka (UCSF), is a “must read” for those of you who are interested in further details and perspectives by an expert.

Prof. Xiaoming He. Taken from bme.osu.edu

Another “hook” in the nanobomb article’s title was its use of lasers, which are also trending in biomedical or biotechnological applications. So, all of this taken together, led me to request a pdf from the corresponding coauthor, Prof. Xiaoming He at Ohio State University, who promptly complied. Since it’s a pay-to-read publication, I’ve tried my best to convey the essence of this work in what follows.

Symphony of Science

Like a symphony, which is an elaborate musical composition for full orchestra, I learned that Prof. His nanobombs for laser-induced drug delivery are also quite elaborate and composed of a full array of scientific principles. To my surprise, a Google Scholar search of the term “nanobomb” gave a large number—more than one hundred—of items that included a wide variety of ways to use nano-size, i.e. sub-micron materials and light to somehow “blow up” or otherwise kill diseased cells, and thus fall under the umbrella-term photodynamic therapy.

Carbon nanotubes (~100 nm diameter) seen through an electron microscope. Taken from item.fraunhofer.de

For example, there’s a report in 2005 coauthored by Eric Wickstrom—who coincidentally is a friend of mine from early antisense oligo days—on the first application of single-wall carbon nanotubes (SWCNT) as potent therapeutic nanobombs for killing breast cancer cells in vitro. This is accomplished by adding water molecules into SWCNTs, which then get adsorbed on target cells for 800-nm (red) laser light irradiation to vaporize the entrained water.

A more elaborate rendition of nanobomb drug delivery was published in 2013 by Lu et al., who reported that intracellular gold nanoparticles under laser irradiation generate “nanobubbles” that can kill cells. To formulate a targeting gold nanoparticle as a cancer cell-specific “nanobomb,” a 31-nt DNA aptamer was conjugated to the gold surface. While biological results from this approach have yet to be published, there’s a good tutorial on the properties and applications of gold nanoparticles that are commercially available in discrete sizes, as seen here:

Taken from simaaldrich.com

Symphonic Variations of Science

This section heading extending my musical metaphor for nanobomb drug delivery is meant to convey the fact that the study by Prof. He and his coworkers featured herein is a symphony-like variation of photodynamic therapy composed of many parts. This work stands out—in my opinion—as being a remarkably comprehensive proof-of-concept involving lots of in vitro data and, more importantly, compelling in vivo animal results. I think you’ll “tune in”—musical pun intended—on this as I now summarize the complex scientific symphony by He and coworkers pictured below.

(A) Three agents are encapsulated inside nanoparticles: microRNA-34a (miR-34a) anti-mRNA drug for gene therapy of prostate cancer stem cells, indocyanine green (ICG) for absorbing laser light, and ammonium bicarbonate for gas generation under heating. (B) Laser light causes the nanoparticles to expand, penetrating the cancer cell’s endosomal/lysosomal barrier (green circles), blowing up cancer cells (yellow), and releasing miR-34a to inhibit protein CD44, which is crucial for cancer stem cell survival. Credit: Hai Wang et al./Advanced Materials. Taken from kurzweilai.net

What’s the Nanobomb?

The top part of the above depiction labeled (A) doesn’t do justice to the elegant methodology used to prepare the water-in-oil-in-water (W-in-O-in-W) double-emulsion nanobomb, so interested readers will have to consult the publication by He and coworkers for details. However, as shown in A, the nanobomb’s active components are indocyanine green (ICG), ammonium bicarbonate (NH4HCO3) and microRNA-34a (miR-34a), the latter of which I found is a single-stranded 22-nt RNA rather than the misleading drawn double-stranded species. Chitosan’s amino groups form a polyplex with miRs like miR-34a.

I also found many references to miR-34a as an anticancer agent, and in this instance it serves to negatively regulate CD44 overexpression, which is associated with survival and growth of prostate cancer stems cells (CSCs) as previously studied by a number of other groups.

Indocyanine green (ICG). Taken from Wikipedia

Having said this, the real novelties of this nanobomb are—in my opinion—its “ignition” and “explosive” materials, namely ICG and NH4HCO3, respectively. Some among you may recognize from the name or structure of ICG that it’s a “chemical cousin,” so to speak, of cyanine-3 and cyanine-5 dyes popular for labeling, which BTW are now available from TriLink as NHS esters upon inquiry.

The ICG dye is used to absorb laser light and convert the light’s energy into heat, which then causes NH4HCO3 to very rapidly generate CO2 and NH3 gases and thus release miR-34a (as depicted in part B of the above cartoon from He and coworkers). This “release” aspect deserves the following further comments.

Why’s a Nanobomb Needed?

This rhetorical question relates to the “D-word,” i.e. delivery of miR (or other classes of oligo agents) into a cell’s cytoplasmic compartment where it is intended to block target mRNA expression after its release from the endosome/lysosome compartment, as depicted in B above. The following electron micrograph shows what these sub-compartments look like.

Electron micrograph of endosomes in human HeLa cells. Early endosomes (E), 5 minutes after internalization; late endosomes/ multivesicular bodies (M) and lysosomes (L) are visible. Bar, 500 nm. Taken from Wikipedia.com

As depicted by He and coworkers, laser light via ICG is used to “ignite” or trigger “explosive” release of miR-34a from these compartments, rather than rely on natural phenomenon or use of pH-sensitive triggering mechanisms. For those of you who are not familiar with the endosome/lysosome compartment, details can be found in the aforementioned review by Prof. Frank Szoka, which was coauthored by Juliane Nguyen.

Is the Nanobomb Effective In Vivo?

Saline (control); HLPP = formulation; miR-34a (R) unformulated plus laser light (L); HLPP with ICG (I) and NH4HCO3 (A) plus L; HLPP with A and R plus L; HLPP with I and A and R plus L. Taken from He and coworkers.

Prof. He and his coworkers carried out extensive controlled experiments in vitro and in vivo to assess efficacy (as well as biodistribution and safety) of their nanobombs, and to me the most compelling data are shown below. Briefly, mice capable of harboring tumors derived from subcutaneously placed human prostate CSCs were treated with various formulations or control, and afterwards the extent of tumor growth was accessed in various ways including by tumor weight. It’s obvious in (C), shown below, that the only statistically significant (*) effect was obtained by the nanobomb formulation.

But is This Nanobomb Practical?

Here’s what He and coworkers state with regard to the scope and practicality of their nanobomb approach:

“Although the tissue penetration of the NIR [near infrared laser] is limited to less than ~1 cm and multiple polymers are needed for preparing the nanosystem, NIR could be delivered into deep tissue using minimally invasive approaches [e.g. endoscopy] and the preparation of the nanosystem using the double-emulsion method is quite straightforward. Therefore, the present study demonstrates the great potential of the NIR laser-activated “nanobomb” for microRNA delivery to achieve augmented cancer therapy.”

Time will tell what aspects of this approach will ultimately be reduced to practice in the clinic and/or lead to an approved photodynamic therapy for cancer. However, in the meantime, I’m betting that it will also “ignite” others to explore variations on this symphony of science.

As usual, your comments are welcome.

Postscript

After this blog was written, Jalani et al. published a paper titled Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery. This work is conceptually related—in my opinion—to the above nanobombs in that low energy red-light-absorbing nanoparticles emit higher energy UV light (i.e. photon upconversion) to simulataneously image and release drugs specially absorbed on these nanoparticles. This NIR light penetration, UV imaging, and drug release are said therein to be effective down to a depth of ~2 cm of tissue, which is twice that reported above by He and coworkers.

Frightening Fungus Among Us

  • Clinical Alert for Candida auris (C. auris) Issued by CDC
  • US Concerned About C. auris Misidentification and Drug Resistance
  • Sequencing C. auris DNA in Clinical Samples is Preferred for Identification
Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

When I was a kid and didn’t know better, there was a supposedly funny rhyme that “there’s fungus among us.” While this saying is thankfully passé nowadays, the growing number of infections by a formerly obscure but deadly fungus is frightening. This so-called “superbug” is an antibiotic-resistant fungus called Candida auris (C. auris) that’s worth knowing about, and is the fungal focus of this blog.

First, Some Fungus Facts

Fungi are so distinct from plants and animals that they were allotted a biological ‘kingdom’ of their own in classification of life on earth, although that was only relatively recently, i.e. 1969. There are 99,000 know fungi, which exist in a wide diversity of sizes, shapes and complexity that extends from relatively simple unicellular microorganisms, such as yeasts and molds, to much more complex multicellular fungi, such as mushrooms and truffles.

It was previously thought that genomes of all fungi are derived from the genome of the model fungus Saccharomyces cerevisae, which has been used in winemaking, baking and brewing since ancient times. However, genome sequencing of more than 170 fungal species has revealed that, while the genome size of S. cerevisae is only ~12 Mb, seven species of fungus have genome sizes larger than 100 Mb. This is attributed to various evolutionary pressure-factors generating transposable elements, short sequence repeats, microsatellites, and genome duplication, and noncoding DNA.

Fungal cell walls are made up of intertwined fibers mostly comprised of long chains of chitosan, the same tough compound found in the exoskeletons of animals such as spiders, beetles and lobsters. The chitin in fungal cells is entangled with glucans and other wall components, such as proteins, forming a mass that protects the cell membrane behind it—and posing a formidable barrier against antifungal drugs.

Taken from Wikipedia.org

Taken from Wikipedia.org

In researching whether there are any nucleic acid drugs against fungi, I found one early patent by Isis (now Ionis) Pharmaceuticals for use of antisense phosphorothioate-modified oligonucleotides for the treatment of Candida infections, but virtually no other reports. I suspect that will change in the future as pathogenic fungi and other disease-causing microbes become more resistant to conventional drugs.

Fungal infections of the skin are very common and include athlete’s foot, jock itch, ringworm, and yeast infections. While these can usually be readily treated, infections caused by pathogenic fungi have reportedly risen drastically over the past few decades. Moreover, with the increase in the number of immunocompromised (burn, organ transplant, chemotherapy, HIV) patients, fungal infections have led to alarming mortality rates due to ever increasing phenomenon of multidrug resistance.

Segue to a Serious Situation

Emergence of drug-resistant fungi is, in part, the segue to the serious story of the present blog. The other part being incorrect identification of a certain fungus as being a common candida yeast, which is not only scary but seemingly inexcusable in today’s era of highly accurate PCR-based assays to accurately identify microorganisms. Here’s the situation in a nutshell.

  1. auris infection, which is associated with high mortality and is often resistant to multiple antifungal drugs, was first described in 2009 in Japan but has since been reported in countries throughout the world. Unlike many Candida infections, C auris is a hospital-acquired infection that is contracted from the environment or staff of a healthcare facility, and it can spread very quickly.

To determine whether C. auris is present in the United States and to prepare for the possibility of transmission, the Centers for Disease Control (CDC) and Prevention issued a clinical alert in June 2016 requesting that C. auris cases be reported.

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

This official alarm bell, if you will, was triggered by the following facts:

  • Many isolates are resistant to all three major classes of antifungal medications, a feature not found in other clinically relevant Candida
  • auris identification requires specialized methods such as a MALDI-TOF mass spectrometry or sequencing the 28s ribosomal DNA, as pictured below.
  • Using common methods, auris is often misidentified as other yeasts, which could lead to inappropriate treatments.

The CDC subsequently found that seven cases were identified in Illinois, Maryland, New York and New Jersey. Five of seven isolates were either misidentified initially as C. haemulonii or not identified beyond being Candida. Five of seven isolates were resistant to fluconazole; one of these isolates was resistant to amphotericin B, and another isolate was resistant to echinocandins. While no isolate was resistant to all three classes of antifungal medications, emergence of a new strain of C. auris that is would pose a serious public health issue.

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Based on currently available information, the CDC concluded that these cases of C. auris were acquired in the U.S., and several findings suggest that transmission occurred:

  • First, whole-genome sequencing results demonstrate that isolates from patients admitted to the same hospital in New Jersey were nearly identical, as were isolates from patients admitted to the same Illinois hospital.
  • Second, patients were colonized with auris on their skin and other body sites weeks to months after their initial infection, which could present opportunities for contamination of the health care environment.
  • Third, auris was isolated from samples taken from multiple surfaces in one patient’s health care environment, which further suggests that spread within health care settings is possible.

A related Fox News story adds that C. auris was found on a patient’s mattress, bedside table, bed rail, chair, and windowsill. Yikes!

While the above situation in the U.S. might not seem particularly worrisome to you, the potential for emergence of more infectious C. auris strains with higher lethality should be of concern. That has already reportedly occurred in several Asian countries and South Africa. Obviously, deployment of the best available methods for pathogen identification can, in principle, lessen the likelihood of the emergence and/or spread of C. auris in the U.S. and other countries.

Case for Point-of-Care C. auris Nanopore Sequencing?

Taken from extremtech.com 

Taken from extremtech.com

Regular readers of my previous blogs know that I’m an enthusiastic fan of the Oxford Nanopore Technologies minION sequencer, which is proving to be quite useful for characterizing pathogens in very remote regions on Earth—and even on the International Space Station to diagnose astronaut infections! Notwithstanding various current limitations for minION sequencing of microbes, it seems to me that it would be relatively straightforward to generate minION data for many available samples of pathogenic fungi and genetically related microbes to assess the feasibility using minION for faster, cheaper, better unambiguous identification of C. auris minION in centralized or Point-of-Care applications.

Taken from rnaseq.com

Taken from rnaseq.com

If you think this suggestion is farfetched, think again, after checking out these 2016 publications using minION:

The 51.4-Mb genome sequence of Calonectria pseudonaviculata for fungal plant pathogen diagnosis was obtain using minION.

The first report of the ~54 Mb eukaryotic genome sequence of Rhizoctonia solani, an important pathogenic fungal species of maize, was derived using minION.

Sequence data is generated in ~3.5 hours, and bacteria, viruses and fungi present in the sample of marijuana are classified to subspecies and strain level in a quantitative manner, without prior knowledge of the sample composition.

CDC on C. auris Status and FAQs

In the interest of concluding this blog with the most up-to-date and authoritative information, I consulted the CDC website and found statements and replies to FAQs that are well worth reading at this link.

As a scientist, my overriding question concerns the lack of adoption of improved microbiological methods by hospitals and clinics. The above noted misidentifications of C. auris infections resulting from use of flawed lab analyses seems unacceptable. Although I don’t know all the facts or statistics to generalize, I suspect that there are other incorrect lab analyses due to use of outdated methods. On the other hand, I’m hopeful that, with the FDA’s widely touted Strategic Plan for Moving Regulatory Science into the 21st Century, the section entitled Ensure FDA Readiness to Evaluate Innovative Emerging Technologies—think nanopore sequencing—becomes actionable, sooner rather than later.

Changing established—dare I say entrenched—clinical lab tests is not simple or easy, but if it doesn’t begin it won’t happen, about which I’m quite certain. I can only wonder why development of infectious disease analytical methods and treatments seem to require a crisis. Sadly, I think it boils down to the complexities and socio-political dynamics of who pays.

Frankly, it’s my personal opinion that maybe it’s time Thomas Jefferson’s philosophy about hammering guns into plows is directed to health care.

Postscript

After writing this blog, I learned that T2 Biosystems has received FDA approval to market in the U.S. the first direct blood test for detection of five yeast pathogens that cause bloodstream infections: Candida albicans and/or Candida tropicalis, Candida parapsilosis, Candida glabrata and/or Candida krusei.

Yeast bloodstream infections are a type of fungal infection that can lead to severe complications and even death if not treated rapidly. Traditional methods of detecting yeast pathogens in the bloodstream can require up to six days, and even more time to identify the specific type of yeast present. The T2Candida Panel and T2Dx Instrument (T2Candida) can identify these five common yeast pathogens from a single blood specimen within 3-5 hours.

T2Candida incorporates technologies that break the yeast cells apart, releasing the DNA for PCR amplification for detection by greatly simplified, miniaturized nuclear magnetic resonance (NMR) technology, as can be seen in this video.

In my opinion, this fascinating new technology is another example of what could be rapidly deployed toward detecting C. auris.