Cell-Free DNA in Urine Developed for Early Detection of Liver Cancer

  • Exponential Increase in Publications on Cell-Free Biomarkers
  • Short Fragments of DNA in Urine Are Cancer Biomarkers
  • Rising Rate of Liver Cancer Prompts Earlier Detection by Urine Testing

Based on the large number of publications I found in PubMed, it is safe to say that investigations of “cell-free DNA biomarkers” have been on an upward trend in the field of nucleic acids. There has been an exponential increase in these publications during recent years, as shown in my chart below.

In my opinion, these articles revealed several broad technical categories such as diseases of interest, sources of samples (i.e. blood, feces, urine, tissue), methods of DNA isolation, and analytical methodologies. Importantly, these mostly academic or sometimes research-use-only applications are now transitioning towards becoming FDA-approved nucleic acid-based tests for in vitro diagnostics. An authoritative, up-to-date FDA list of these tests can be viewed here.

Many types of malignancies are included on this list, but despite its prevalence, liver cancer is not among them. Consequently, recent news about a promising candidate nucleic acid-based test for liver cancer using urine, which is arguably the most common and simplest of specimens to collect, prompted me to do some further research for this blog.

Urine and blood have become particularly interesting due to ease of access and safety compared to traditional, invasive biopsies. I have previously blogged about these non-invasive sample types in a post titled Sniffing Out Prostate Cancerin urine, and another post titled Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics.

Short Fragments of DNA in Urine Are Cancer Biomarkers

Although circulating DNA in the blood of cancer patients was detected and reported on four decades ago, such DNA was not investigated as a means of providing possible biomarkers until recent years, as reviewed by Anker et al. Urine is collected body fluid that has been filtered through the kidneys by nephrons, which are the microscopic structural and functional units of the kidney. Kidneys remove waste from the blood and return the cleaned blood back to the body. In 2000, Botezafu et al. reported that small amounts of cell-free circulating DNA in the blood can pass the kidney barrier into urine, and tumor-specific sequences can be detected in DNA isolated from urine.

In 2004, urine was first described as a useful source of circulating DNA for molecular diagnosis and prognosis. This landmark publication by a large multi-institutional team is titled Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. The corresponding author, Ying-Hsiu Su, is now with JBS Science (JBSS), a small startup company that followed up this initial observation with a publication in 2017 by Hann et al. titled Detection of urine DNA markers for monitoring recurrent hepatocellular carcinoma. A synopsis of this paper is featured later on in this blog, preceded by a short background on hepatocellular carcinoma (HCC).

HCC on the Rise and the Need for Early Detection

Credit: Kateryna Kon

According to a recent review by Ghouri et al., an epidemic of HCC, the most common malignancy of the liver, has spread beyond the predominance in Eastern Asian, and has been increasing in the Northern hemisphere, especially in the U.S. and Western Europe. It occurs more commonly in males in the fourth and fifth decades of life. Ghouri et al. add that, among all cancers, HCC is one of the fastest growing causes of death in the U.S. and poses a significant economic burden on healthcare. Chronic liver disease due to hepatitis B virus (HBV) or hepatitis C virus (HCV) and alcohol accounts for the majority of HCC cases. HCC is reportedly the fourth leading cause of cancer death worldwide.

According to Ghouri et al., the global burden of cancer in 2012 was an all-time high of 14 million cases, and that number is expected to grow to 22 million over the next two decades. Liver cancers have the seventh highest age-adjusted incidence rate in the world, with 0.8 million cases diagnosed for the year 2012.

Hann et al. note that HCC constitutes 70–85% of all types of liver cancer. The high mortality rate of HCC (wherein 85% of patients die within 5 years) is mainly due to late detection and a high recurrence rate. Rates of recurrence range from 15% for liver transplantation to nearly 100% for surgery or ablation. Recurrence is most common within 2 years.

Importantly, Hann et al. go on to provide the following statement in regards to the need for early detection of HCC:

MRI scan of the abdomen, liver cancer.

“Early detection of recurrent HCC has been difficult with the currently available diagnostic methods and serial imaging. Notably, there are no specific guidelines addressing how HCC recurrence should be monitored. Magnetic resonance imaging (MRI)/computed tomography (CT) imaging is the gold standard for diagnosis, although it is expensive and has limited utility in the detection of small tumors (< 2 cm), tumors in the presence of previously treated lesions (especially from local ablation), cirrhosis, obesity, and dysplastic nodules. Thus, there is an urgent unmet medical need to have a sensitive test for monitoring HCC recurrence.”

Early Detection of HCC Proof-of-Concept

Hann et al. then demonstrate the proof-of-concept (aka feasibility) of using urine for early detection of recurrent HCC by detecting three known HCC-associated DNA modifications: TP53 249T mutation (shortened TP53m), aberrant promoter methylation of glutathione S-transferase pi 1 (mGSTP1), and Ras association domain family 1 isoform A (mRASSF1A). These genes are then compared to the MRI imaging in a small (n = 10) blinded prospective study. These three DNA markers were chosen because of the reported availability of sensitive, cell-free DNA PCR assays for each of them:

  • TP53 in complex with DNA.

    Mutations in the TP53 gene are associated with approximately 50% of human cancers, as featured in a previous blog. A clamp-mediated PCR assay, followed by melting curve analysis, was available to detect TP53m. In this assay, the clamp suppressed 107copies of wild-type templates and permitted detection of TP53mtemplate, with a sensitivity of 0.1% (1:1000) of the mutant/wild-type ratio, assessed by a reconstituted standard.

  • For mGSTP1detection, an available methylation-specific PCR assay for the 5′-end region of the GSTP1 promoter targeted specific CpG sites with a forward primer, a reverse primer and a fluorogenic probe. Using this assay, mGSTP1 was detected with an assay sensitivity of 10 copies per reaction.
  • Quantitative methylation-specific PCR was similarly available for detection of

Interested readers can consult the aforementioned 2004 publication by Su et al. for extensive methodological details on how the low-molecular weight DNA in urine was isolated, as well as more information on the relationship of this DNA to circulating cell-free DNA and HCC tumor DNA.

Rather than try to paraphrase the conclusions of Hann et al., here is a quote of what they report:

“This study demonstrates the potential applicability of using urine DNA markers in combination with serum [alpha fetoprotein] AFP for the early detection of HCC recurrence in a small 10-case study. HCC recurrence is known to be the major factor for poor prognosis. In this small 10-case study, MRI identified recurrence in 5 out of 10 patients (cases 1–5). Encouragingly, for all 4 recurrent patients that remain in the study (cases 1–4), urine DNA markers were found to be elevated in urine samples as early as 9 months before MRI confirmation.

Although this is a small longitudinal 10 patient study, the potential of these urine DNA markers for management of HCC recurrence and important characteristics of HCC recurrence is demonstrated.”

“Bridge Award” by the National Cancer Institute for Commercialization

At the beginning of this blog, I made a passing reference to coming across recent news about a promising urine-based test for liver cancer. That news was actually a press release titled JBS Science, Inc. awarded $3 million bridge award by National Cancer Institute [NCI] to commercialize a urine test for liver cancer screening. While this press release led me to do the background research for the technical aspects described this blog, it also led me to discover that additional steps for commercialization will be carried out with funding by an NCI “Bridge Award,” which was new to me as a funding mechanism.

Credit: Lightspring. Modified by Jerry Zon

Since many of my readers are either in academia or small businesses, and seek support from the NIH, I thought it would be useful to look into the Bridge Award program. Briefly, the NCI’s Bridge Award is a specific type of Small Business Innovation Research (SBIR) funding to help small businesses bridge the funding gap between the end of an SBIR award and the subsequent round of financing needed to advance R&D toward a commercial product, as is visually depicted here.

Under the terms of the Bridge Award, SBIR-funded companies can apply for an additional $3M to cover both preclinical andclinical development costs. NCI Bridgeawardees are expected to obtain matchingfunds from investors, such as drug ordevice manufacturers, foundations, universities,or angel investors, for this 3-year award.

Since the Bridge Award must be matched by third-party investors, the NCI is providing a significant amount of “seed capital” and a not insignificant imprimatur, if you will, that together facilitate investments by third-parties.

The NCI provides a link to its SBIR Bridge Award “success stories,” which I found included numerous small companies covering various areas of cancer therapies, diagnostics, and cancer imaging technologies.

As usual, your comments are welcomed.

Addendum

After writing this blog, I decided to “deep dive” into databases for any information related to the novelty of a urine test for early detection of liver cancer, as it occurred to me that urine ought to be an obvious biofluid to look at for biomarkers.

I eventually found a lengthy review published in 2017 by Sengupta and Parikh titled Biomarker development for hepatocellular carcinoma early detection: current and future perspectives. I highly recommend this review to interested readers because it covers all types of biomarkers (i.e., proteins, DNA, RNA, and metabolites). Importantly, it discusses requisite phases of biomarker discovery that are necessary before a biomarker is appropriate for use in clinical practice.

As for the novelty of testing urine, a word search for urine in this comprehensive review by Sengupta and Parikh confirmed that the now NCI Bridge Award commercialized DNA-based urine test for early detection of HCC is indeed the first of its kind.

Recent Outbreaks of Acute Flaccid Myelitis (AFM) in Children Are Cause for Concern

  • AFM Is a Polio-Like Disease Confirmed in 24 States Across the U.S.
  • An Enterovirus Is Associated with AFM
  • AFM Has No Cure, but a Preventative Vaccine Appears Possible

About a month ago, a tweet about an AFM outbreak in children in Pittsburgh caught my attention. Because I had never heard of AFM, I searched scientific literature, learning that the disease is somewhat similar to polio, and has sporadically occurred in clusters around the world for quite a few years. One of the causative agents of AFM seems to be an enterovirus that mutates into various genotypes and phenotypes, based on information gathered from genome sequencing, PCR-based analyses, and clinical data. Now, combatting AFM and avoiding potentially major outbreaks are issues of growing concern.

This blog on AFM is my attempt—as a non-expert in virology—to distill complicated science and epidemiology into commentary that fits into what’s trending in nucleic acids research. Having said that, I’ll now start with a brief synopsis of AFM as a disease and its presumptive causative agent, “enterovirus D68” (EV-D68), and I’ll finish with some information on a possible protective vaccine against this debilitating illness.

Basic Facts About AFM

In general, my go-to sources for authoritative, up-to-date information on diseases are the National Institutes of Health (NIH) website, and the Centers for Disease Control (CDC) and Prevention website. Each web page is thoroughly referenced, with convenient links to primary sources. Here is a brief synopsis of information that I found on AFM by searching “Acute Flaccid Myelitis” on the NIH and CDC sites.

Child sleeping using an assisted-breathing device. iStock Credit: Juanmonino

Symptoms: AFM is a rare disease that affects the spinal cord, specifically the area of the spinal cord called gray matter. This causes the muscles and reflexes in the body to become weak. Symptoms of AFM include sudden (acute) weakness in the arm(s) or leg(s), along with loss of muscle tone (flaccid), and decreased or absent reflexes. In some cases, AFM can cause facial weakness, drooping of the eyelids, and difficulty swallowing, speaking, or moving the eyes. The most severe symptom of AFM is respiratory failure, which can happen when the muscles involved with breathing become weak. This can require urgent support and utilization of an assisted-breathing device, like the one pictured above.

Interested readers can access more information by perusing recent videos on AFM via YouTube. Most of these videos discuss a spike or outbreak of AFM in a particular state, and refer to AFM as a “polio-like” illness, an oversimplification that is widely used in mainstream media reports. Be forewarned that several posts among these AFM videos offer comments that I would politely describe as unscientific, if not plain bizarre.

Testing muscle response to nerve impulses using EMG. Credit: Romaset

Diagnosis: Most cases of AFM occur in children. Unfortunately for patients and parents, AFM can be difficult to diagnose because the symptoms are similar to other neurological diseases, such as Guillain-Barre syndrome (GBS), acute disseminated encephalomyelitis (ADEM), and transverse myelitis. Diagnosis may include an MRI of the spine, testing of the cerebral spinal fluid (CSF), tests checking nerve speed (nerve conduction velocity; NCV), and muscle response to nerve messages (electromyography; EMG). According to the CDC, as of October 31st 2018, there are 191 reported cases under investigation in 24 states across the U.S., of which 72 have been confirmed to be AFM cases.

Treatment: There is no specific treatment for AFM, but a neurologist specialized in treating brain and spinal cord illnesses may recommend certain interventions on a case-by-case basis. For example, neurologists may recommend physical therapy to help with arm or leg weakness caused by AFM. However, the extent of recovery varies – although some patients may make a full recovery, most have continued muscle weakness. The long-term outcomes of people with AFM remain unknown.

Causes: I was quite surprised at the CDC website’s statement that “AFM or similar neurologic conditions may have a variety of possible causes such as viruses, environmental toxins, and genetic disorders. Oftentimes, despite extensive lab tests, the cause of a patient’s AFM is not identified.” Given the sophistication of modern medicine and molecular diagnostics, I was expecting more definitive information. This in turn prompted me to research the reported virus-associated causes of AMF, which you can read about below.

EV-68 Is a Causative Agent for AMF

Enterovirus

Enteroviruses are a genus of positive-sense single-stranded RNA viruses, and they are named based on their transmission-route through the intestine (enteric means intestinal). A simplified depiction of the basic structural elements of an enterovirus is shown here. Classification of viruses can be inherently complicated, and it is not a topic that can be easily discussed due to the evolution of naming conventions, which sometimes appear to be a seemingly incomprehensible mix of Latin or Greek nomenclature, letters, and numbers.

Human enteroviruses are currently classified into 12 species: enteroviruses A through J (excluding letter I), and rhinoviruses A through C. Enteroviruses isolated relatively recently are named with a system of consecutive numbers. For example, EV-D68 belongs to enterovirus D. EV-D68 has a genome that contains a single open-reading frame, coding for a poly-protein (P1), the precursor of four viral capsid proteins, VP1, VP2, VP3, and VP4, and seven non-structural proteins, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. VP1 and VP3 are the major antigenic epitopes.

EV-D68 was first isolated in 1962 in California, from children with pneumonia and bronchiolitis. According to a June 2018 publication by Sun et al., there have been only 26 cases of documented EV-D68 respiratory disease in the U.`S. from 1970 to 2005. However, the upsurge of EV-D68 cases in the past few years showed clusters of infections in Europe, the Americas, Asia, Oceania, and Africa. In particular, more than 1,000 cases, including 14 deaths, were reported during an epidemic of EV-D68 infection in 2014 in the U. S., resulting in strong public attention toward this virus, as well as intensified research efforts on how to combat it.

A year later in 2015, Huang et al. reported carrying out a metagenomic shotgun sequencing protocol on clinical samples and negative controls, the results of which allowed for the assembly of 20 EV-D68 genomes: 6 complete, and 14 near-complete. A comparative genomic analysis revealed that EV-D68 strains circulating in the 2014 outbreak were significantly different from prior ones, and that they actually belong to a new clade. Importantly, two functional mutations in EV-D68 may alter its protease cleavage efficiency, thus leading to increased rate of viral replication and transmission.

In 2015, Zhuge et al. reported on the diagnostic utility of an EV-D68-specific real-time reverse transcription-PCR (RT-PCR) developed by the CDC. Nasopharyngeal swab specimens from patients testing positive for rhinovirus or enterovirus were assessed using this EV-D68 RT-PCR, and the data were compared to results from partial sequencing analysis of the EV genome. The EV-D68 RT-PCR data showed diagnostic sensitivity and specificity of 98.6% and 97.5%, respectively. It was concluded that EV-D68 RT-PCR is a reliable assay for detection of EV-D68 in clinical samples, and it has the “potential to be used as a tool for rapid diagnosis and outbreak investigation of EV-D68-associated infections in clinical and public health laboratories.”

EV-D68 Is Spread in Multiple Ways

Although a number of whole-genome sequencing studies on EV-68 have now been reported in the context of comparative genomics, my inner “alarm bell” was triggered by a Lednicky et al. report. The researchers collected EV-D68 from classroom air using a filter-based air sampling method, and from environmental surfaces by swab sampling. Then, they amplified and sequenced the complete genome of the enterovirus. Here is a brief description of what they found and concluded:

EV-D68 was detected in 4-of-6 air sampler filters, and in 12-of-16 desk tops in a university classroom. cDNA synthesis was performed with avian myeloblastosis virus (AMV) reverse transcriptase and random hexamers on the viral nucleic acids extracted from filters or swabs, and PCR was performed using a panel of respiratory virus primers. Quantitative RT-PCR tests executed after the virus was identified indicated 400 to 5,000 genomic equivalents of EV-D68/m3 in the air samples. Viral RNA from the air sample with the highest concentration of virus was used for sequencing.

The researchers concluded that, “[a]s with our findings, high levels of airborne enteroviruses [have previously been] detected in a pediatric clinic, and this may be a common finding in indoor settings with enterovirus-infected individuals. Our work also suggests that young adults can produce airborne EV-D68 and raises the question of whether airborne transmission is important for spreading the virus.”

According to an article that I found in a journal on hygiene, EV-D68 can be found in bodily fluids such as saliva, mucous, sputum, and feces. It is transmitted through direct contact, including shaking hands, touching contaminated objects or surfaces, changing diapers of an infected person, or drinking water containing the virus. Following infection, the virus can be shed in stool for several weeks, and in the respiratory tract for up to 3 weeks. Shedding of EV-D68 can occur even in the absence of symptoms.

EV-D68 Vaccine Status

Vaccinating a baby.

The Benjamin Franklin axiom that “an ounce of prevention is worth a pound of cure” is as true today as it was when Franklin said it, especially when referring to modern vaccines. I researched the status of vaccines to protect against AMF, and found the following report by Dai et al.

These researchers describe the development of a virus-like particle (VLP)-based experimental EV-D68 vaccine. They found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1, and that they were visualized as spherical particles of ∼30 nm in diameter. Immunization of mice with EV-D68 VLPs resulted in production of serum antibodies that displayed potent serotype-specific neutralizing activities against EV-D68 virus in vitro.

Importantly, passive transfer of anti-VLP sera completely protected neonatal recipient mice from lethal EV-D68 infection. Moreover, maternal immunization with these VLPs provided full protection against lethal EV-D68 challenge in suckling mice. According to Dai et al., “these results demonstrate that the recombinant EV-D68 VLP is a promising vaccine candidate against EV-D68 infection.”

Hispid Cotton Rat (Sigmodon Hispidus).

Along similar lines, Patel et al. have found that cotton rats are permissive to EV-D68 infection without virus adaptation. Three different strains of EV-D68 studied all showed the ability to produce neutralizing antibody upon intranasal infection or intramuscular immunization. Patel et al. concluded that “our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.”

An EV-D68 mRNA Vaccine Seems Feasible

In 2017, a high-visibility Nature publication by Pardi et al. demonstrated that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA–LNP), encoding the pre-membrane and envelope glycoproteins of Zika Virus (ZIKV), elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 μg of nucleoside-modified ZIKV mRNA–LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 μg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination.

In my opinion, this landmark proof-of-concept study, which I’m pleased to say used modified mRNA comprised of 1-methylpseudouridine-5′-triphosphate obtained from TriLink, should be readily extended to EV-D68. The work of Dai et al. mentioned above, together with the information gained from genomic sequencing of EV-D68, provide the conceptual basis for synthesis of modified mRNA encoding one or more antigenic proteins as potential vaccines. These candidates could then be tested in one or both of the animal models mentioned above.

Unfortunately, drug and vaccine companies tend to be reluctant to invest in research targeting rare diseases. Development of a vaccine against AFM requires adequate financial resources. One can only hope that such resources become available, perhaps through a charitable foundation, the NIH, or maybe even crowdfunding, artistically depicted here.

The NIH has taken the lead on placebo-controlled clinical trials for a ZIKV vaccine; however, as detailed in a September 14, 2018 Science news article, a steep drop in Zika cases has undermined this and other planned related trials. This has led researchers to consider trials in which subjects are deliberately exposed to ZIKV, but studies like these would be faced with serious ethical and safety issues. Given the rarity of AFM cases, clinical evaluation of the efficacy of a candidate vaccine against AFM would encounter similar issues.

As usual, your comments are welcomed.

Footnote

After writing this blog, I became motivated to do a literature search for treatment of AFM. I found a 2017 publication by Tyler and coworkers titled Evaluating Treatment Efficacy in a Mouse Model of Enterovirus D68-Associated Paralytic Myelitis. This study evaluated 3 widely used empirical therapies for their ability to reduce the severity of paralysis in a mouse model of EV-D68 infection: (1) human intravenous immunoglobulin (hIVIG), (2) fluoxetine, and (3) dexamethasone.

Antibody binding to a virus.

Importantly, hIVIG, which was shown to contain neutralizing antibodies for EV-D68, reduced paralysis in infected mice and decreased spinal cord viral loads. Fluoxetine had no effect on motor impairment or viral loads. Dexamethasone treatment worsened motor impairment, increased mortality, and increased viral loads.

The following concluding statement from this 2017 publication is especially promising: “Results in this model of EV-D68-associated AFM provide a rational basis for selecting empirical therapy in humans.” In my opinion, of the 3 therapies investigated in this mouse model, treating patients with readily available hIVG seems to be the strongest course of action.

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Spotlight on TriLink Product Applications

  • Nearly 500 Publications in 2017 Cite Use of TriLink Products
  • Jerry Spotlights 20 Citing Oligos, Nucleotides, mRNA and Aptamers
  • 10 of These 20 Spotlighted Items Show Global Reach of TriLink Products

While thinking about possible topics to blog about, it occurred to me that researching recent publications on the applications of TriLink products would likely lead to many options. Using Google Scholar to do just that, I was given nearly 500 items, which is indeed plenty. However, choosing which to feature was neither an easy nor objective task. Having said that, and with sincere apologies to publications not spotlighted here, my “faves” and comments are given below, listed arbitrarily (not ranked) in four product categories: oligonucleotides, nucleotides, mRNA, and aptamers.

Taken from depositphotos.com

For convenience, each publication title can be clicked on to access the original article. Links to the cited TriLink products are also provided, alongside links to other adjunct information. Several trending “hot topics” and previous blogs are also noted.

Oligonucleotides

Taken from researchgate.net

Nucleotides

8-oxo-dGTP; taken from TriLink BioTechnologies // dPTP; taken from TriLink BioTechnologies

mRNA

Modified mRNA for new therapeutic approaches continues to be an amazingly hot area of R&D, which I have previous dubbed “modified mRNA mania” in a previous blog. Interested readers can peruse this link to ~300 items found in my Google Scholar search for TriLink and mRNA publications in 2017.

pseudo-UTP; taken from TriLink Biotechnologies // 2-thio-UTP; taken from TriLink Biotechnologies

Aptamers

2’-F-dCTP; taken from TriLink BioTechnologies // 2’-F-dUTP; taken from TriLink BioTechnologies

Global Reach

A pleasantly surprising aspect of the selected-product search results given above is the worldwide distribution of researchers using TriLink products. This global reach, if you will, is evident from the following countries outside of the USA, which I made point of mentioning:

The Netherlands, India, Austria, Switzerland, Turkey, Germany, Italy, Belgium, Republic of Korea, and Denmark.

All of the publications listed above were selected solely on the type of TriLink product used. Given the relatively small “sample size” of these selected publications, which are only 20-of-500, finding investigators in 10 countries outside of the USA is a compelling testimonial for the TriLink global reach.

World Science Day

Truth be told, when I was searching for a fitting image to visually convey the concept of “global science,” I came across the fact that the United Nations Educational, Scientific, and Cultural Organization (UNESCO) has designated November 10 as World Science Day, with an emphasis on peace and development. The stated intention is to highlight “the important role of science in society and the need to engage the wider public in debates on emerging scientific issues. It also underlines the importance and relevance of science in our daily lives.”

Taken from monitor.co.ug

According to UNESCO, “[t]he theme for 2018 is ‘Science, a Human Right’, in celebration of the 70th anniversary of the Universal Declaration of Human Rights (art. 27), and of the Recommendation on Science and Scientific Researchers. Recalling that everyone has a right to participate in and benefit from science, it will serve to spark a global discussion on ways to improve access to science and to the benefits of science for sustainable development.”

To me, this is a long-term objective which is indeed critical for betterment of future generations.

As usual, your comments are welcomed.

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FDA Approves First-of-a-Kind Test for Profiling Cancer Genes

  • Memorial Sloan Kettering Develops a Next-Generation Sequencing (NGS) Assay for 468 Genes
  • FoundationOne Does the Same for a 324 Gene Assay
  • Some View These Approvals as Tearing Down Conceptual Walls Between System Biology and Clinical Practices

Taken from jamanetwork.com (Credit: C. Lynm)

This blog’s title, which recently flashed around the globe as headline news, deserves to be echoed here, as it signals the achievement of a major milestone in nucleic acid-based diagnostics. Ever since the emergence of powerful PCR and sequencing methods for DNA analysis, there have been thousands of publications dealing with discovery and validation of genes associated with the genesis or signature of various types of cancer. A great deal of literature has also appeared regarding possible use of DNA analysis for cancer diagnostics.

Despite these continuing advances, the transition from research findings to doctors using DNA diagnostics has been hindered by the relatively long and costly process of gaining approval by regulatory authorities. In the U.S., the regulatory authority is the Food and Drug Administration (FDA). Readers interested in regulatory aspects can use this link to access FDA guidance for genetic testing in general. The focus herein is to provide a brief overview of what underlies the headline news echoed in the title of this blog.

Mutational Landscape of Metastatic Cancer

Molecular pathology of cancer has historically relied upon low-throughput approaches to interrogate a single allele in a single sample, such as those listed at this FDA website. By contrast, massively parallel “next generation” sequencing (NGS) has enabled a dramatic expansion in the content and throughput of diagnostic testing. However, the complexity of clinical NGS testing has prevented laboratories from achieving large-scale implementation, which is needed in order to maximize the benefits of tumor genomic profiling for large populations of patients. In addition, the clinical utility of mutation profiling requires evaluation of how molecular results are influencing therapeutic decisions in different clinical contexts.

To address this issue, the Memorial Sloan Kettering (MSK) Cancer Center developed a targeted tumor sequencing test, MSK-IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), to detect gene mutations and other critical genetic aberrations in both rare and common cancers. This test therefore detects all protein-coding mutations, copy number alterations, selected promoter mutations, and structural rearrangements in 341 (and more recently, 468) cancer-associated genes, as detailed by Zehir et al. This large team of investigators prospectively sequenced tumors from more than 10,000 cancer patients, who presented with a vast array of solid tumor types.

Taken Zehir et al. Nature Medicine (2017)

The DNA sample prep and analysis methodology described by Zehir et al should be appreciated as a tour de force of integrated automated sample handling systems. The method operates in conjunction with barcoded adapters, PCR, multiplexed DNA capture by biotinylated hybridization probes, and paired-end 100-base pair NGS reads – all to a mean depth of coverage of 718X.

Breakthrough FDA Approvals

On November 15th 2017, the FDA announced approval of MSK-IMPACT as a tumor profiling assay, i.e. an in vitro diagnostic (IVD) test, which can identify a higher number of genetic cancer mutations (biomarkers) than any test previously reviewed by the agency. The IMPACT test works by comparing a tumor tissue sample to a “normal” sample of tissue or cells from the same patient. This serves to detect genetic alterations that might help guide treatment options.

While the test is intended to provide information on cancer biomarkers, its results are not conclusive for choosing a corresponding treatment according to the FDA, which added that the assay is >99% accurate and capable of detecting a mutation at a frequency of ~5%. Detection of certain molecular changes (microsatellite instability) using the IMPACT test was concordant >92% of the time across multiple cancer types in 175 cases when compared to traditional methods of detection. Importantly, the Centers for Medicare & Medicaid Services (CMS) cover the cost of this test.

Shortly thereafter, on November 30th, the FDA announced approval of FoundationOne CDx (F1CDx) as an NGS-based IVD test that can detect genetic mutations in 324 genes and two genomic signatures in any solid tumor type. This test will also be covered by CMS. Additionally, based on individual test results, the new diagnostic can identify which patients with any of 5 tumor types may benefit from 15 different FDA-approved targeted treatment options. Its results provide patients and health care professionals all of this information in one test report, avoiding duplicative biopsies.

Tearing Down the Walls

A very recently published commentary by Allegretti et al. argues that, besides the many practice-changing implications, MSK-IMPACT and F1CDx approval by the FDA “tears down the conceptual walls dividing system biology from clinical practice, diagnosis from research, prevention from therapy, cancer genetics from cancer genomics, and computational biology from empirical therapy assignment.”

These authors further opine that that MSK-IMPACT and F1CDx have moral and ethical implications. For the first time, the FDA implies—and, the authors posit, some may say FDA plainly endorses the view—that “each patient at an advanced cancer stage has the right to have her/his cancer genome deciphered at the highest possible level of complexity compatible with current knowledge and technology, linking molecular information to state-of-the-art systemic therapies, as they become available.”

Therefore, according to Allegretti et al., extended NGS testing is becoming the standard of care in oncology. In the near future, “NGS profiling will likely be requested at progressively earlier stages, leading to a change in the engagement rules. No longer will the oncologist request a single assay for a single therapeutic option, e.g. BRAF or RAS mutational status for specific pathway blockade in specific cancers. On the contrary, it is implicit in [these] FDA approvals that the entire mutation catalogue will have to be made available to the medical team as soon as possible after diagnosis, and much before any specific therapy becomes applicable.” Pictorially, they view the new paradigm as follows:

Taken from Allegretti et al. J Exp Clin Cancer Res (2018)

I hope you will forgive my extensive quoting from Allegretti et al., but I believe it’s important to accurately convey the importance of their argument, given the profound impact of this type of thinking. Only time will tell the extent to which change become reality, as these innovations must deal with the hurdle of difficult dynamics between cancer care, cost containment, and societal conscience.

As always, your comments are welcome.

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The Scientist’s Top 10 Innovations in 2017—and Jerry’s Top Pick

  • Multiplexed Assays Are Trending
  • Miniaturization is Big—Pun Intended
  • Jerry’s Top Pick is an Amazing 4 Inch Cube

Taken from the-scientist.com

Welcome to my first blog of the New Year, 2018! My New Year’s resolution is to “double down” by giving my best effort to provide interesting and informative content about what’s trending in nucleic acid research. Having said that, and in keeping with tradition, this first blog of the year pairs my comments on the Top 10 Innovations in 2017, as reported by the The Scientist, with my personal “fav” for the best new product launched last year.

So, with an imaginary loud flourish of trumpets, read on to learn about The Scientist’s 10 winners that I’ve listed from 10th to 1st place—to build your interest—after which I comment on my personal fav.

Top 10 Innovations in 2017 Reported by The Scientist

10. TrueCut Cas9 Protein v2 from Thermo Fisher Scientific is a next-generation CRISPR-Cas9 protein engineered to deliver maximum editing efficiency across a range of genes and standard cell lines, as well as stem cells, T cells, and primary cells.

With regard to this product, I think it’s worth mentioning that TriLink offers mRNA-encoded Cas9, as an alternative to delivering Cas9 protein per se. Also, my several past blogs on CRISPR can be accessed here.

9. TSQ Altis Triple Stage Mass Spectrometer from Thermo Fisher Scientific robustly and reliably quantitates most analyte types, even in complex samples such as plasma and tissue, thus enabling wide applicability, including forensic toxicology and clinical research.

8. Chromium from 10x Genomics for profiling single-cell gene expression, enables deep profiling of complex cell populations, is provided as a complete droplet-based system of reagents, barcodes, hardware and software for sample prep prior to high-throughput sequencing.

7. Edit-R crRNA Library—Human Genome from Dharmacon provides users with an arrayed library of synthetic crRNA guides in a “one-well-per-gene” format, with four distinct guides per gene for redundancy to improve statistical power.

6. HiBiT Protein Tagging System from Promega is a new detection system for quantifying proteins in or on a cell of interest, using a small and easily integrated 11-amino-acid tag (HiBiT) that interacts with a complementary large 156-amino-acid component leading to bioluminescence.

5. SR-X Ultra-Sensitive Biomarker Detection System from Quanterix offers more than 80 different assays to test samples (e.g. blood, serum, cerebral spinal fluid, single-cell lysates) for cytokines or other markers of neurodegeneration or neuroinflammation, and more.

4. Blaze from Intabio is a system for detecting and identifying protein isoforms that aims to save pharma companies time in lab prep work for QC of biologics manufacturing. Launch will be “within the next few months” and “pricing is still yet to be set,” according to The Scientist.

3. QGel Assay Kit for Organoids from QGel provides fully synthetic extracellular matrix to reproducibly grow research organoids, which are miniaturized and simplified version of an organ produced in vitro.

2. i-STAT Alinity from Abbott is a handheld, cartridge-based blood-testing device for user-friendly point-of-care assays on a blood sample of just several drops—including glucose levels and hematocrit—with results directly delivered to a patient’s medical record for clinicians within 2-10minutes. Alinity is available in about 50 countries, but Abbott is waiting for a few more assays to be cleared by the US Food and Drug Administration before selling it stateside.

Regarding this product, I should mention that my several past blogs on point-of-care can be accessed here.

1. IsoCode Chip from IsoCode is new single-cell technology allowing researchers to characterize cells based on the proteins they secrete—as many as 42 different cytokines, chemokines, and other types of molecules. IsoCode chips contain thousands of long microchambers that house only single cells. Within each microchamber, 15 spatially separated slots contain up to three different antibodies targeting specific secreted proteins; upon binding, each antibody fluoresces in a different color to distinguish the proteins. This provides the ability to simultaneously profile thousands of individual T cells or immune cells at one time.

Taken from The Scientist Dec 2017

IsoCode chips come in 10 different panels, ranging from 24 to 42 antibodies per panel, at a cost of $500–$600. The automated IsoLight imaging and workflow platform can be purchased starting at $200,000. But the IsoCode chips can also be paired with other fluorescence microscopy systems.

Jerry’s Fav for the Best Innovation in 2017

My personal pick for this honor goes to the world’s first on-site Legionella DNA test to prevent Legionnaires’ disease, which was released this past November by the Canadian company Spartan Bioscience. According to a press release, it is the first on-site DNA test for Legionella bacteria and it can detect and quantify Legionella in only 45 minutes, compared to 10-14 days for off-site sample analysis using traditional culturing methodology. The system pictured here consists of a coffee-cup-sized portable DNA analyzer called the Spartan Cube, which employs a single-use disposable test cartridge.

Taken from ctvnews.ca

This innovative product stood out for me because it brings together the following topics that I have separately blogged about:

  • Reoccurring outbreaks of potentially fatal Legionnaire’s disease, such as that which recently hit New York City, and also shut down Disneyland.
  • Decentralized analytical testing for more rapid “sample-to-answer” applications on-site, i.e. in the field—wherever that might be—or at point-of-care in hospitals, clinics, and elsewhere.
  • Miniaturization and simplification of qPCR using cleverly engineered devices, such as the Spartan Cube, in conjunction with single-use disposable test cartridges.

Legionella is a common environmental bacterium that can infect the cooling towers of Heating, Ventilation, and Air Conditioning (HVAC) systems of large buildings. Infected cooling towers release aerosolized water droplets contaminated with Legionella into the surrounding air. Globally, there are hundreds of thousands of office-building towers, hospitals, hotels, shopping malls, and other large buildings at risk for infection by Legionella. Weekly testing with the Spartan system can rapidly detect Legionella bacterial growth early, and thus allow cleaning and decontamination of the cooling tower before Legionella reaches dangerous levels to human health.

In addition, and also importantly, traditional culture test methods can underestimate the Legionella concentration on site. The Centers for Disease Control and Prevention (CDC) found that Legionella culture can underestimate actual Legionella levels by a factor of 10 or more. Culture incorrectly reported that water samples were negative for Legionella an average of 11.5 percent of the time when in fact they were positive.

Paul Lem. Provided by Paul Lem

According to Paul Lem, M.D., CEO of Spartan Bioscience, who I contacted about cost to customers, “the price for the Cube and Legionella tests is $5-10K/building/year, depending on the building. It’s a subscription model.”

Regarding my further inquiry about testimonials to date, Dr. Lem provided a copy of a November 26, 2017 article reported in The Globe and Mail which quotes him as saying that “several property managers are testing the device at close to 100 properties, including BGIS and Ottawa’s KRP Properties, owned by tech entrepreneur [Sir] Terry Matthews.”

The article also states that “the market could be worth billions of dollars globally, encompassing office buildings, malls, hospitals, schools, theme parks, spas and so on. ‘Nobody really knows because the market doesn’t really exist yet,’” said Lem.

For the sake of increased public safety toward exposure to Legionella, let’s all hope that this application of the amazing Spartan Cube is indeed very successful. And, moreover, that 2018 is a great year for other nucleic acids-based innovations, many of which I look forward to blogging about here.

As usual, your comments are welcomed.

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Legionnaires’ Disease Outbreak in New York

  • First Identified as a New Pathogen 40 Years Ago, Legionella Persists
  • Legionella’s Life Cycle Involves “Biological Sanctuaries”
  • qPCR Proven to Outperform Antibody-Based Detection of Legionella

When I read about an outbreak of Legionnaires’ disease (LD) in New York City, baseball legend Yogi Berra’s famous quote, “It’s déjà vu all over again” immediately came to mind, along with the irony of Berra playing for the New York Yankees. So, if you’re much younger than me, you’ll likely not know why “It’s déjà vu all over again” and you may wonder who Berra was. You can read about him later elsewhere, but for now you should read on to learn about Legionnaires disease and why déjà vu is apropos.

History of LD

Notable positive events during 1976 in the United States included our Bicentennial Celebration, unveiling by NASA of the first space shuttle (the Enterprise), establishment of Apple Computer Company by Steve Jobs and Steve Wozniak, and Silly Love Songs by Paul McCartney and Wings ascending to #1 on the charts. While many of these events were the beginning of fabulous things to come, one proved to be the beginning of something catastrophic. American Legionnaires who gathered in Philadelphia, Pennsylvania for the Bicentennial were struck with a mysterious epidemic of fatal respiratory disease.

Taken from networks.org

Sadly, 182 members of the Pennsylvania American Legion were affected, and 29 individuals died after they returned from the convention in Philadelphia. The epidemiological and microbiological studies continued for months before scientists began to understand what had happened. Much of the basic framework of our knowledge of Legionnaires disease, as the epidemic came to be known, was developed by a team from the CDC and the Pennsylvania Department of Health, as detailed elsewhere.

Taken from case1study.wikispaces.com

The cause of the disease remained a mystery until 1977 when an investigative team led by J. E. McDade and C. C. Shepard (of the Leprosy and Rickettsia Branch, Virology Division, Bureau of Laboratories, CDC) reported on the isolation of a Gram-negative bacillus found in patient samples. As often done for naming pathogens after sources, the genus of this rod-shaped bacterium was aptly named Legionella. Legionella includes the species L. pneumophila, which caused the pneumonia-like illness medically named legionellosis, but commonly referred to as LD.

2017 LD Outbreak Hits New York City—Again

In June of this year, forty years after the first characterization of Legionella, it’s lethal infectivity reoccurred in an outbreak in the Upper East Side of the Manhattan borough of New York City, leaving one person dead and six other people sickened. According to a newspaper account, this outbreak occurred within 11 days, and may have been triggered by contacting contaminated water as has happened in other cases.

While this incident affected relatively few people compared to other previous outbreaks, including one in the Bronx borough of New York City in 2015 that killed 15 people and sickened more than 70, it’s a scary reminder of the persistence of Legionella in the environment. In this regard, it has been reported that 200 to 400 cases of the illness are recorded each year in New York, despite the monitoring of 6,000 water systems wherein Legionella can flourish in warm conditions. This environmental factor provides a segue into what genomic sequencing has revealed about Legionella.

Genomics-Based Insights on Legionella

The bacterial pathogen L. pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause severe pneumonia associated with Legionnaires disease. As detailed elsewhere, recent advances in genome sequencing has had a major impact on understanding of the pathogenesis, evolution and genomic diversity of Legionella.

A lipopolysaccharide cell wall and several outer membrane proteins are essential virulence factors. Central to the pathogenesis of L. pneumophila is its Type IV secretion system, which translocates over 270 effector proteins into the host cell, thus allowing this bacterium to manipulate host cell functions to its advantage and assures intracellular survival and replication.

Within aquatic media, as depicted below, Legionella exist as part of biofilms, which provide a protective environment—or biological sanctuary, if you will—wherein the bacteria exhibit marked increase in resistance to biocidal compounds and chlorination. Aside from the resultant difficulty of purging water systems to be free of Legionella, these bacteria can invade and multiply within protozoa (which are ubiquitous and include amoeba), thus providing yet another biological sanctuary. Protozoa are present in all aquatic or moist environments, and can be found in even the most inhospitable parts of the biosphere, thus providing further protection to Legionella.

Taken from Comas Nature Genetics (2016)

The actual infectious particle is not known but may include excreted legionellae-filled vesicles, intact legionellae-filled amoebae or free legionellae that have lysed their host cell. Transmission to humans occurs via mechanical means, such as air-conditioning units, taps and showerheads, as well as others listed by the World Health Organization (WHO).

Infection in humans occurs by inhalation of the infectious particle and establishment of infection in the lungs. After ingestion by macrophages, L. pneumophila have been found to inhibit acidification and maturation of its phagosome. Following a 6–10 hour lag period, the bacteria replicate for 10–14 hours until macrophage lysis releases dozens of L. pneumophila progeny.

It’s worth noting that, according to WHO, there is no direct human-to-human transmission of Legionella, which in my opinion is why incidence of LD remains relatively low.

Gardening Can Be Bad for Your Health—No Joke.

Unfortunately, there are other ways of contacting LD besides ingestion of tainted water. At the risk of sounding flippant, gardening can be seriously bad for your health because of contracting LD by breathing in aerosolized Legionella from contaminated soil. This is especially true in New Zealand, which has the highest incidence of LD in the world, according to a recent publication, with L. longbeachae being the most clinically relevant species. This infectious agent is predominantly found in soil and composted plant material. Most cases occur over spring and summer, and the people at greatest risk are those involved in gardening activities.

Taken from lawrieco.com.au

Some agricultural experts advocate smelling soil to assess its quality, stating that “[t]he smell of a soil can often reveal its state of health, sweet or offensive or plain bland,” and adding “the smell does not actually come from the dirt itself, but from soil microbes that inhabit a healthy soil environment. Sweet smelling soil has good levels of organic carbon which is vital to supporting the world of billions of beneficial bacteria and fungi in every cup of healthy soil.”

These soil sniffing experts, however, fail to consider the presence of pathogenic organisms including L. longbeachae. I, for one, will carefully avoid purposefully smelling any soil when gardening, and will instead be sure to wear a good mask capable of filtering out aerosolized Legionella, as you should too!

Nucleic Acid-Based Detection of Legionella

Rapid and effective diagnosis of LD is extremely important so that timely and appropriate therapy can be provided, thereby lowering the morbidity and mortality rates and reducing the health and economic costs associated with this disease. Surprisingly, diagnosis is reportedly established solely by time-consuming microbiological tests. Luckily, it looks like testing procedures could soon change for the better, thanks to PCR and NGS.

Taken from corisbio .com

Earlier this year, Christovam et al. assessed the accuracy of various detection tests in patients suspected of being infected with Legionella and in patients with laboratory-confirmed LD. Investigators analyzed urinary Legionella antigen detection, direct fluorescent antibody (DFA) staining, serological testing and PCR vs. culture analysis (the reference standard). The sensitivity and specificity for PCR were 83 % and 90 %, respectively, whereas DFA sensitivity and specificity were 67 % and 100 %, respectively. Moreover, PCR had high sensitivity and specificity for early diagnosis of LD.

Taken from letsfixit.co.uk

While the study results reported by Christovan seem promising, less definitive results have been reported. Krøjgaard et al., who compared culture and qPCR assays for the detection of Legionella in 84 samples from shower hoses and taps in a residential area before and after two decontaminations. Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc. was said to be “dubious.” However, the rapid detection of high concentrations of Legionella by qPCR was said to be valuable as an indicator of risk, although it may be false positive compared to culture results. Detection of a low number of bacteria by qPCR was said to be a strong indication for the absence of risk.

Not surprisingly, the advent of powerful next-generation sequencing (NGS) is emerging as a better method for genus-specific, sensitive and quantitative determination of Legionella. In 2017, Pereira et al. reported findings from a study using NGS to differentiate 20 pathogenic strains of Legionella in fresh water systems. A genome standard and a mock community consisting of six different Legionella species demonstrated that the reported NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. Comparison of quantification by real-time PCR showed consistency with the NGS data, thus indicating that NGS “provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method.”

Concluding Comments

Aside from providing a brief introduction and update on LD, my additional intent was to alert readers—without undue alarm—to the myriad circumstances in which Legionella can infect humans. According to the aforementioned list provided by WHO, the most common form of transmission of Legionella is inhalation of contaminated aerosols produced in conjunction with water sprays, jets or mists. Infection can also occur by aspiration of contaminated water or ice, particularly in susceptible hospital patients.

Researching transmission of Legionella in Google Scholar led me to find additional information (see links below) that you may find useful or interesting.

Thankfully, as I’ve said before, Legionella is not transmitted human-to-human. The scary aspect of Legionella, however, is that it’s continually mutating, which raises the specter of emergence of a strain that can spread within a human population. Let’s hope that this doesn’t happen and/or that modified mRNA vaccines can be quickly produced to combat that possibility.

As usual, your comments are welcomed.

Addendum

After finishing this blog, there was a Reuters news report on October 9, 2017 that Michigan’s top medical official, Dr. Eden Wells, will be charged with involuntary manslaughter for her role in the city of Flint’s water crisis, which was linked to an outbreak of LD that caused at least 12 deaths. Dr. Eden Wells would become the sixth current or former official to face involuntary manslaughter charges related to this crisis, which principally involved lead contamination in the city’s water supply.

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Sniffing Out Prostate Cancer

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

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

Taken from finearts. com

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

Dogs Sniff Out Prostate Cancer

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

Taken from health.am

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

Taken from en.menarinidiagnostics.fr

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

Taken from pinterest. com

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

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

Taken from chromedia.org

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

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

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

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

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

As usual, your comments are welcomed.

Addendum

Taken from Deng et al.

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

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Highlights from the 2017 AgBio Innovation Showcase Held by UC Davis

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

Taken from the journal.ie

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

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

Taken from intelligencesquaredus.org

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

2017 AgBio Innovation Showcase

Taken from agshowcase.com

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

Ag Biotech

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

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

    Taken from saipanhydroponics.com

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

Diagnostics

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

Food Science & Animal Health

  • Taken from Wikipedia.org

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

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

AgBio Meets CRISPR

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

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

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

As usual, your comments are welcomed.

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Advances in Aptamer Applications – Part 2

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

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

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

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

Top 3 Cited Publications in 2014

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

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

Taken from pubs.rsc.org

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

Taken from mct.aacr.org

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

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

Aflatoxin B₁ structure. Taken from wikipedia.org

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

Top 3 Cited Publications in 2015

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

Taken from sciencedirect .com

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

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

Chloramphenicol. Taken from Wikipedia .com

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

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

Taken from mdpi .com

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

Top 3 Cited Publications in 2016

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

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

Taken from sigmaaldrich .com

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

Taken from wikipedia.org

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

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

Taken from Liu et al. Biomaterials (2016)

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

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

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

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

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

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

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

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

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

Aptamer Publications in 2014-Present Citing TriLink Products

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

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

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

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

As usual, your comments are welcomed.

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Studying Telomeres in Space

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

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

Taken from keywordsuggest.com

What are telomeres?

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

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

Taken from weeklyglobalresearch.wordpress.com

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

Taken from 2014hs.igem.org

‘Houston, we have a problem’

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

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

Taken from energeticnutrition.com

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

Twins and telomeres

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

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

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

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

Studying telomeres in space by PCR

Taken from geeky-gadgets.com

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

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

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

Julian Rubenfein. Taken from nydailynews.com

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

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

Your thoughts or comments here are welcomed.

Postscript

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

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

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

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