Broccoli May Reduce Symptoms of Autism

  • Study Reports Remarkable Response to Broccoli Extract
  • Autism Advocacy Group Offers Cautious Optimism
  • Father/Son Cozy Combo for Commercialization Pathway

I’m quite sure that most of you, like me, are very familiar with various ways to improve or preserve your well-being by doing such things as adopting a Mediterranean diet, eating less red meat and more chicken, fish and beans, drinking a glass of red wine, and so on. And like me, you might be somewhat skeptical of the actual benefits but, nevertheless, try to follow some of these recommendations—especially if you enjoy a nice merlot.

A small new study found that a chemical in broccoli sprouts may help alleviate the symptoms of autism. Credit: James Baigrie/Getty Images. Taken from ABC News.

A small new study found that a chemical in broccoli sprouts may help alleviate the symptoms of autism. Credit: James Baigrie/Getty Images. Taken from ABC News.

Having said that, I was tempted to ignore a very recent ABC News story with a headline that read “Broccoli Sprout Extract May Help Curb Autism Symptoms” were it not for two things: firstly, autism is a very common and challenging disorder, and secondly the story referred to a publication in the Proceedings of the National Academy of Sciences (PNAS), which is a very highly regarded scientific journal. So, let’s consider some facts about autism, and then delve into the publication.

Autism Facts

Here are selected facts about autism taken from NIH.gov—my “go to” source of reliable information about all things health related.

What is Autism?

Autism is more accurately referred to as “autism spectrum disorder” (ASD) because it covers a wide range of complex neurodevelopment disorders, characterized by social impairments, communication difficulties, and restricted, repetitive, and stereotyped patterns of behavior. Classical ASD is the most severe form of ASD, while other conditions along the spectrum include a milder form known as Asperger syndrome, as well as childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD-NOS). Although ASD varies significantly in character and severity, it occurs in all ethnic and socioeconomic groups and affects every age group. Experts estimate that 1 out of 68 persons have an ASD. Interestingly, males are four times more likely to have an ASD than females.

Taken from giniesayles.com via Bing Images.

Taken from giniesayles.com via Bing Images.

What are Some Common Signs of Autism?

The hallmark feature of ASD is impaired social interaction. As early as infancy, a baby with ASD may be unresponsive to people or focus intently on one item to the exclusion of others for long periods of time. A child with ASD may appear to develop normally and then withdraw and become indifferent to social engagement.

Many children with an ASD engage in repetitive movements such as rocking and twirling, or in self-abusive behavior such as biting or head-banging. Children with an ASD don’t know how to play interactively with other children. Some speak in a sing-song voice about a narrow range of favorite topics, with little regard for the interests of the person to whom they are speaking.

Children with characteristics of an ASD may have co-occurring conditions, including Fragile X syndrome (which causes mental retardation), epileptic seizures, Tourette syndrome, learning disabilities, and attention deficit disorder.  About 20 to 30 percent of children with an ASD develop epilepsy by the time they reach adulthood.

What Causes Autism?

Given the prevalence of autism, you may find it surprising that scientists aren’t certain about what causes ASD, although there’s agreement on the likelihood that both genetics and environment play a role—nature vs. nurture. Researchers have identified a number of genes associated with the disorder. Studies of people with ASD have found irregularities in several regions of the brain. Other studies suggest that people with ASD have abnormal levels of serotonin or other neurotransmitters in the brain. These abnormalities suggest that ASD could result from the disruption of normal brain development early in fetal development. Where the disruption is caused by defects in genes that control brain growth and regulate how brain cells communicate with each other, possibly due to the influence of environmental factors on gene function.

Do Symptoms of Autism Change Over Time?

Thankfully for many children, symptoms improve with treatment and with age; however, children whose language skills regress early in life—before the age of 3—appear to have a higher than normal risk of developing epilepsy or seizure-like brain activity. During adolescence, some children with an ASD may become depressed or experience behavioral problems, and their treatment may need some modification as they transition to adulthood. It’s also encouraging to know that many people with an ASD are able to work successfully and live independently or within a supportive environment.

What Research is Being Done?

NIH is currently funding and coordinating 11 Autism Centers of Excellence (ACE). The ACEs are investigating early brain development and function, social interactions in infants, rare genetic variants and mutations, associations between autism-related genes and physical traits, possible environmental risk factors, potential new medications and biomarkers.

I’m “big on biomarkers” for all diseases, and was pleased to find a promising press release about Stemina Biomarker Discovery receiving a $2.3 million investment from the Nancy Lurie Marks Family Foundation to support its clinical study of biomarkers in the blood of children with ASD. Using blood samples, Stemina was able to distinguish patients with autism from typically developing children with 81% accuracy, which I think is amazingly good given the complexity of ASD. Moreover, Stemina CEO Elizabeth Donley was quoted as saying “What is exciting about the data we are generating … is that we are beginning to identify metabolic subtypes in comparing one child with ASD to another. This has the potential to revolutionize the way children are diagnosed and treated based on the individual’s metabolism.”

PNAS Publication on Autism and Broccoli

Now that we’ve covered the basics of autism, let’s see how broccoli may help ease ASD symptoms. Full details of this collaborative study are freely available here in a PNAS publication from this past September entitled “Sulforaphane treatment of autism spectrum disorder (ASD).” The investigators outline four premises that led them to test treatment of ADS with sulforaphane, which is an isothiocyanate derived from broccoli—as well as other cruciferous vegetables such as Brussels sprouts or cabbages—and has the remarkably simple molecular structure shown below.

Taken from Wikipedia.

Taken from Wikipedia.

Here’s a short synopsis provided by Dr. Cindy Haines on MedlinePlus, which is a part of NIH that has the stated objective of providing “trusted health information for you.”

Researchers included 44 young men with moderate to severe ASD in a small study involving the chemical sulforaphane. The participants, ages 13 to 17, were randomly assigned to take either a daily dose of sulforaphane extracted from broccoli sprouts or placebo. Investigators and caregivers were not told who was receiving the active substance. Behavior and social interaction were evaluated at the start of the study and then at 4, 10 and 18 weeks. Half of the teenagers underwent a final assessment about a month after treatment ended.

The results:

  • At 18 weeks, 46% of the sulforaphane recipients showed significant improvement in social interaction,
  • 54% in atypical behavior
  • and 42% in verbal communication.

The researchers say improvements were so noticeable that by the end of the treatment period, both staff and family members correctly guessed treatment assignments. Still, they point out the chemical did not work for everyone.

About one third of those taking sulforaphane showed no improvement. They say a larger study including adults and children is needed to confirm any therapeutic benefits.

Comments on this Study by Autism Speaks®

To get independent, informed opinions about this study, I consulted Autism Speaks® and found the following blog comments posted by developmental pediatrician Paul Wang, Autism Speaks® senior vice president for medical research. BTW, I’ve underlined three of his comments that I thought were particularly important:

Today, a lot of parents are talking about adding broccoli sprouts to their kid’s salads and sandwiches. Can this help? Hurt?

The amount of sulforaphane that was administered in the study is many times higher than you can reasonably get through food. Even sulforaphane-rich foods like brussels sprouts, broccoli and broccoli sprouts don’t have enough of the chemical to get you close. So eating these vegetables can’t be expected to improve autism symptoms. Within reason though, eating sulforaphane-rich vegetables is safe and healthy.

What about taking sulforaphane supplements or giving them to a child with autism? Are they safe?

I would caution against starting sulforaphane supplements at this time. First and foremost, this was a very small trial – much too small to assure safety. There was actually a potentially worrisome side effect in the study: Two of the 29 boys and men taking sulforaphane had seizures during the study. Both had a history of seizures in the past, so this could have been a coincidence. However, none of those taking the placebo, or dummy treatment, had seizures during the study.

The study also showed a small increase in liver enzymes in study participants who received sulforaphane. None of these individuals showed any symptoms related to this side effect. However, it poses the possibility that sulforaphane may produce liver inflammation.

It’s important to remember that anything powerful enough to exert biological effects – even beneficial effects – also has the potential to produce unwanted side effects. Just because sulforaphane is found in vegetables doesn’t mean it’s safe. There are many chemicals found in nature that can be toxic. This is particularly true when these chemicals are concentrated into a supplement. Much more study is needed to understand sulforaphane’s actions in the body – for good or bad.

Also, though sulforaphane supplements have been on the market for some time, nutritional supplements don’t go through the kind of rigorous safety testing required for pharmaceutical medicines. So we don’t have good safety data on these products.

No doubt, some people will decide to take sulforaphane supplements based on this study’s findings, regardless of potential safety concerns. How can they select a reputable brand? What would be a safe and reasonable dose?

The brand of supplement used in the study was a patented, pharmaceutical-grade product not available for purchase over the counter. So there’s no way of using the study’s results to gauge the effectiveness or safe doses of the many related health-food products with lesser quality-control during manufacturing.

In the study, the researchers used doses ranging from 50 to 150 µmol daily, depending on the participant’s weight. Their weights ranged from around 120 to 220 pounds.

So if an individual or parent decides to try these supplements – despite safety concerns – I would urge them to work closely with a physician to monitor possible reactions. This monitoring needs to include, but not be limited to, seizures. For example, blood work should probably be done to monitor liver enzyme levels.

Cozy Combo for Commercialization Pathway

I noticed that the conflict of interest statement in this PNAS paper reveals that U.S. patent applications have been filed by three Johns Hopkins University inventors, who include Paul Talalay, one of the corresponding authors, who is also a member of the prestigious National Academy of Sciences. The statement adds that he has divested himself from all potential financial benefits. Furthermore, the sulforaphane is not a commercial product, and has been licensed by Johns Hopkins to Brassica Protection Products LLC, whose CEO, Anthony Talalay, is the son of Paul Talalay.

While inventor Paul Talalay’s apparent largess is laudatory, Hopkins University’s licensing to a company run by his son, Anthony, seems a bit too cozy—in my opinion. On the other hand, publically stating these facts seems to imply that all parties involved are comfortable with legalities.

Anyway, I became interested in learning more about this company, and dug around, so to speak on the internet. The following is some of the backstory on Brassica Protection Products that I gleaned from researching Paul Talalay’s publications and reading a 2013 interview of Anthony Talalay by Adam Stone with bizjournal.com.

The company was founded in 1996 based on Paul Talalay’s long-held belief that certain chemicals in plants might prove to be useful in preventing cancer by inducing enzymes that safely metabolize cancer-causing molecules. Originally the focus was on commercializing broccoli sprouts, which had 20-times more of sulforaphane than mature broccoli, but there were farm-to-market distribution problems, and—perhaps more problematically, I think—people didn’t want to change their eating habits.

Anthony Talalay should be happy about the very promising clinical data reported for a product sold by his company, Brassica Protection Products. Credit: Harry Bosk; taken from bizjournals.com.

Anthony Talalay should be happy about the very promising clinical data reported for a product sold by his company, Brassica Protection Products. Credit: Harry Bosk; taken from bizjournals.com.

After years of disagreement, the company’s board of directors finally convinced Anthony Talalay to abandon this approach and instead commercialize an extract of broccoli sprouts, which is prepared by methods you can read here. That was done three years ago, and business has doubled for each of the past three years.

The company was said to be trying to raise money from family and friends in order to expand operations and sales, which I think ought to be relatively easy now based on the very promising clinical data published in PNAS.

My parting comment is that April 2015 is National Autism Awareness Month, but don’t wait—become engaged now! The Autism Society website offers loads of information and ways to get involved plus advice for living with autism.

As usual, your comments are welcomed.

You and Your Microbiome – Part 2

  • Global Obesity Epidemic is Linked to Gut Microbiome
  • DNA Sequence-Based Microbiomes Accurately Associate with Obesity
  • Blue Agave Margaritas Contain Beneficial Gut Microbes
  • Investments in Microbiome-based Therapies on the Rise, but is there Hype?

Last August, my post entitled Meet Your Microbiome: The Other Part of You dealt with growing recognition that trillions of microbes—mostly bacteria but also fungus—reside in and on each of us, and influence our health status. Moreover, the compositions of these microbiomes change with our diet, what we drink or breath, and who we contact—family, pets, and close friends.

Since then, I’ve collected a string of microbiome articles delving into the implications of this dynamic, symbiotic relationship, and selected some topics that I thought were “blogworthy.” This Part 2, as it were, focuses on overweight/obesity, microbiome therapy, and burgeoning business opportunities.

Overweight/Obesity Epidemic

A recent article in The Wall Street Journal highlights the obesity epidemic that much of the world is facing today. Since 1980, obesity rates have risen by 28% among adults and 47% among children. By 2013 approximately 29% of the world population was said to be overweight or obese. That equates to about 2.1 billion people, the majority of which live in developing countries.

This is a BIG problem—no pun intended—given the very serious consequences of these scary statistics. Policies and programs introduced years ago in countries like the USA have obviously not reversed the continual increase in obesity.

graph

From Wall Street Journal article by Betsy McCay, published May 29, 2014.

Dieting, Exercise and Microbiota

Obesity is considered by the World Health Organization to be preventable by limiting food-derived energy intake from total fats and sugars; increasing consumption of fruits, veggies, etc. and—of course—regular physical activity (60 minutes a day for children and 150 minutes per week for adults).

Some of the roughly 1,000 bacterial species in the human gut help make us fat, while others keep us lean. Centre for Infections/Public Health England/Science Photo Library (taken from Nature).

Some of the roughly 1,000 bacterial species in the human gut help make us fat, while others keep us lean. Centre for Infections/Public Health England/Science Photo Library (taken from Nature).

There could, however, also be links between obesity and bacteria living in our guts, according to Sarah DeWeerdt’s freely accessible article in venerable Nature magazine from which selected snippets are as follows.

Consider what Liping Zhao, a microbiologist at Shanghai Jiao Tong University in China, found after he put a severely obese man on a strict diet. Over the course of 6 months, the man shed ~100 pounds. As he lost weigh, a group of bacteria known as Enterobacter became undetectable in his stool samples, even though they had previously made up 35% of the microbes in his gut.

This dramatic change in human gut microbiota—estimated to be ~1,000 species of bacteria—might be coincident with weight loss, but Zhao and other researchers think otherwise, and believe that these bacteria actually play a key role in regulating body weight.

DeWeerdt quotes Fredrik Bäckhed—a researcher at the University of Gothenburg in Sweden who investigates the gut microbiota using mouse models—as noting that ‘There are a lot of studies in humans, but those are only associations. There are a lot of studies of causation, but those are only in animals.’

How to translate results from studies of lab mice into treatments for humans in the real world is no simple matter, and will likely be the subject of much continued experimentation and hot debate.

The Role of Prebiotics

We’ve all heard about probiotics, but when it comes to studying obesity it’s important to also understand prebiotics. Unlike probiotics which are live organisms, prebiotics are non-living substances (usually carbohydrates) that function as food sources for beneficial bacteria. Research indicates that both pro- and prebiotics are key to controlling obesity. Researchers have shown that in mice prebiotics, particularly oligofructose, can reverse certain gastric problems. DeWeerdt quotes Patrice Cani, a researcher of metabolism and nutrition at the Catholic University of Louvain in Belgium as saying ‘We found that mice fed with oligofructose had an improved gut barrier function.’ ‘The mice that were given prebiotics also had improved metabolic markers, reduced fat mass and reduced inflammation,’ Cani added.

Harvesting a Blue Agave plant in Mexico involves hard work before processing into Agave Tequila and Agave Nectar (taken from inari-hof.de via Bing Images). Blue Agave Margarita is a refreshing source of oligofructose prebiotic (taken from experience-it-island-thyme.blogspot.com via Bing Images).

Harvesting a Blue Agave plant in Mexico involves hard work before processing into Agave Tequila and Agave Nectar (taken from inari-hof.de via Bing Images).
Blue Agave Margarita is a refreshing source of oligofructose prebiotic (taken from experience-it-island-thyme.blogspot.com via Bing Images).

The most widely used prebiotic is oligofructose (aka fructo-oligosaccharides or FOS)—a type of soluble but indigestible carbohydrate fiber found in the Blue Agave plant, as well as fruits and vegetables such as bananas, onions, chicory root, garlic, asparagus, barley, wheat, jicama, leeks and the Jerusalem artichoke. Some grains and cereals, such as wheat, also contain oligofructose. The Jerusalem artichoke—and its relative yacón—together with the Blue Agave plant have been found to have the highest concentrations of oligofructose in cultured plants. Both yacón and Agave nectar are becoming popular substitutes for sugar—BTW, Agave Nectar Margaritas have a 5-Star rating at Food.com!

As the saying goes, “too much of a good thing can be bad.” One report cautions that 15 g or more of FOS can produce side effects such as gas, bloating and general intestinal discomfort, and doses higher than 40 g might cause diarrhea (taken from prebiotin.com via Bing Images).

As the saying goes, “too much of a good thing can be bad.” One report cautions that 15 g or more of FOS can produce side effects such as gas, bloating and general intestinal discomfort, and doses higher than 40 g might cause diarrhea (taken from prebiotin.com via Bing Images).

It’s not clear, however, if the benefits of prebiotics that were seen in mice studies translate to humans. In 2013, Cani and his team conducted a study giving obese women a daily supplement of oligofructose and inulin (a similar substance). After three months, the women ‘showed a slight decrease in fat mass and a reduction in blood levels of an inflammation-promoting molecule. But the results were not really equivalent to the ones we observed in mice,’ Cani told DeWeerdt.

As another caveat regarding studies of prebiotics, consider the results of a study conducted regarding the dietary regimen followed by the subject in his aforementioned obesity study. For 9 weeks, about 100 participants followed a diet that included whole grains, traditional Chinese medicinal foods and prebiotics. The participants all showed ‘improved markers of metabolic health and lower levels of potentially harmful bacteria, including Enterobacter, but they only achieved a modest weight loss of ~6 kg on average.’ Since this average weight loss is almost 10-times less than that achieved by Zhao’s severely obese patient, more factors need to be considered.

Microbiomes Sort “Lean from Obese” with 90% Accuracy!

The advent of fast and cheap DNA sequencing has enabled not only detection and quantification of the type of bacteria in the gut, but also the genes that these microorganisms are expressing, in addition to our own human genes. BTW, researchers in this field generally refer to the collection of bacterial species present in the gut as the microbiota (aka gut flora), and the collection of corresponding expressed genes as the microbiome. In this regard, I find it fascinating—and somewhat difficult to accept—that the ongoing Human Microbiome Project is extending the definition of what constitutes a human to include microbiome, so “it” is technically part of “you”!

The MetaHIT Consortium—a European effort to determine the associations between gut microbes and chronic diseases—sequenced the microbiomes of 169 obese and 123 non-obese individuals. They found that those with fewer bacterial expressed genes tended to have more body fat and other markers of poor metabolic health compared with people with a more diverse microbiome.

The stunning conclusion was that microbial genes are a much better readout of whether you’re likely to be obese or not than human genes are. I repeat—with emphasis—this remarkable conclusion:

…microbial genes are a much better readout of whether you’re likely to be obese or not than human genes…

At the Risk of Oversimplifying this Finding, Genes in your Microbiome Seemingly Influence your Tendency to be Obese or not more so than your own Human Genes!

DeWeerdt reports that other investigators have come to a similar conclusion, and on a quantitative basis claim that microbial genes sort the lean from the obese with 90% accuracy, whereas human genes do this with much less accuracy, i.e. only 58% of the time.

Cause or Effect?

So the really big question is whether these microbial changes are a cause or an effect of the obesity. A step toward deciphering this puzzle has been convincingly addressed by very clever—I think—experimentation wherein the microbiota of an obese mouse is transferred to a microbiota-free mouse.

Jeffrey Gordon, director of the Center for Genome Sciences & Systems Biology at Washington University in St. Louis, Missouri, found back in 2004 that when a microbiota-free mouse is colonized with gut microbes from a normal mouse, it experiences a 60% increase in body fat over the course of 2 weeks—despite eating less food than it did before the transfer.

The simplest interpretation—in my opinion—is that microbes in the gut of these mice increased the ability of mice to store fat. How this occurs biochemically is, of course, not at all clear and will require much more investigation.

BTW, in a variant of this line of research, it has been shown that germ-free mice that receive gut microbes from an obese human donor gain more weight than those that receive them from a lean person. While this might seem surprising, I think it’s actually somewhat expected based on functional biochemical similarity of mouse and human genes that may nevertheless be different genetically, i.e. at the level of DNA sequence.

In Hot Pursuit of Microbiome Therapies

Sarah Reardon reports in Nature magazine that roughly $500 million has been spent on microbiome research since 2008. However, the only major therapy resulting from this sizable investment has been the use of fecal transplants for treating life-threatening gut infections or inflammatory bowel disease—discussed in my earlier blog.

This seeming absence of clinical impact by investment dollars may change due to large pharmaceutical companies viewing this area as a new source of revenue. This past May, Pfizer announced plans to partner with Second Genome, a biotechnology firm in South San Francisco, California, to study the microbiomes of ~900 people comprising a group with metabolic disorders and, of course, a control group.

At virtually the same time, Paris-based Enterome revealed that it had raised $13.8 million in venture capital to develop tests that are intended to diagnose inflammatory and liver diseases based on measuring the composition of gut bacteria. Based on the aforementioned microbiota vs. microbiome issue, I’m somewhat skeptical of this approach by Enterome. Maybe they’ll find otherwise.

Reardon also reports that Joseph Murray—a gastroenterologist at the Mayo Clinic in Rochester, Minnesota—fed gut bacterium Prevotella histicola to transgenic mice with human-like immune systems, and found suppression of inflammation caused by multiple sclerosis and rheumatoid arthritis. He is hoping to develop this into a therapy with biotech firm Miomics in New York.

Similarly, Vedanta Biosciences in Boston, Massachusetts, is conducting preclinical trials of a pill containing microbes that suppress gut inflammation. I checked out Vedanta’s website, and it’s worth visiting to watch a video entitled Telling “Good” from “Bad,” which is about the immune system evolving to differentiate ‘good’ microbes from ‘bad’ microbes, as related to its microbe-based therapeutic approach.

And last June, Second Genome announced a deal with Janssen Pharmaceuticals of Beerse, Belgium, to study the microbial populations of people with ulcerative colitis, in the hope of identifying new drugs and drug targets. Already in the clinic, Microbiome Therapeutics, a biotechnology company in Broomfield, Colorado, is currently conducting trials with two small molecules that select for ‘good’ gut bacteria to help people with diabetes to take up insulin more easily.

From all of the above, there’s no doubt in my mind that microbiomes need to be factored into assessment, modulation, and maintenance of a person’s health, and much R&D is clearly moving in these directions. On the other hand, my sense is that progress will be slower than hoped for, partly because there’s much to be discovered, and partly because people aren’t like mice: we can choose to eat what we wish, indulge our cravings, and skip taking medications.

Chime in with your comments if you think I’m being too pessimistic about this.

BTW, for those of you who do want to change your diet so as to hopefully beneficially change your microbiomes, you can consider engaging with uBiome, which is a small start-up that offers DNA sequence-based microbiome analysis as part of ongoing research involving likeminded persons. An explanatory—and humorous video—can be accessed here.

Hyping the Microbiome?

“Maybe the microbiome is our puppet master” says Carl Zimmer in his NY Times article headlined above (Credit: Jonathan Rosen).

“Maybe the microbiome is our puppet master” says Carl Zimmer in his NY Times article headlined above (Credit: Jonathan Rosen).

Throughout all of the above, you’ll notice that weight-related conclusions derived from legitimate scientific inquiries are properly couched with caveats and the need for further investigations. By contrast, some reports may be pushing the envelope of credulity, as discussed in an engaging but anonymous article in GenomeWeb entitled “Hyping the Microbiome.” One notable quote is by William Hanage, an associate professor of epidemiology at the Harvard School of Public Health, who says that ‘Microbiomics risks being drowned in a tsunami of its own hype.’ He also points to a blog Jonathan Eisen, who gives awards for ‘overselling the microbiome,’ and provides an updated list of links to questionable claims.

Hanage partly blames the media for generating this hype. I agree with this finger pointing, and my personal favorite is this headline and accompanying visual in the NY Times.

Our Microbiome May Be Looking Out for Itself

Postscript

After writing this post, a publication in Nature reported “dynamics and associations” of microbial communities across the human body, based on detailed analysis of data from the Human Microbiome Project. Following is a portion of the abstract that draws three conclusions (see underlines), the first of which I find fascinating:

First, there were strong associations between whether individuals had been breastfed as an infant, their gender, and their level of education with their community types at several body sites. Second, although the specific taxonomic compositions of the oral and gut microbiomes were different, the community types observed at these sites were predictive of each other. Finally, over the course of the sampling period, the community types from sites within the oral cavity were the least stable, whereas those in the vagina and gut were the most stable. Our results demonstrate that even with the considerable intra- and interpersonal variation in the human microbiome, this variation can be partitioned into community types that are predictive of each other and are probably the result of life-history characteristics. Understanding the diversity of community types and the mechanisms that result in an individual having a particular type or changing types, will allow us to use their community types to assess disease risk and to personalize therapies.    

DIY Chromosomes

  • This DIY (do-it-yourself) is Actually DIT (do-it-together)!
  • A Global Network of Undergraduates are Collectively Assembling Designer Chromosomes for Yeast
  • The Inspirational Jolt for this Harnessing of “Student Power” Came in a Caffeinated Coffee Conversation

At a conference held over a decade ago, visionary geneticist Ronald Davis suggested that researchers should synthesize artificial yeast chromosomes and insert them in a living cell. Many of the attendees, including Jef Boeke, didn’t think much of the idea. According to a News Focus article in Science by Elizabeth Pennisi, Davis made the bold prediction that artificial yeast would be the next significant milestone in the then-emerging field of synthetic biology. Boeke didn’t share Davis’s vision, mostly due to the seeming insurmountable task of designing and synthesizing a 12.5-million-base genome comprising 16 chromosomes. According to Pennisi, as Boeke listened to Davis’s talk, he says, ‘I remember thinking “Why on Earth would you want to do that?’“

However, Boeke’s opinion about his own rhetorical question completely flip flopped, and has led to an inspiring “collective can do” attitude among scientists in this field. Led by Boeke, hundreds of researchers around the global are now working together as a gigantic team to successfully achieve a goal befitting a “grand challenge,” which would otherwise not be possible.

Here’s the interesting story of what has happened and how it came about.

Boeke (a geneticist who recently moved from Johns Hopkins University to New York University Langone Medical Center) and his colleagues have just finished the first complete synthetic yeast chromosome. They are well on their way to putting together several more—thanks to technological advances in manufacturing DNA oligonucleotides and—importantly—a global army of collaborators who are—amazingly to me—mainly undergraduate students!

computo

It’s only a cartoon, but definitely not a joke…“designer chromosomes” are now a reality (credit: Dave Simonds, taken from economist.com).

This post, which borrows from Pennisi’s article, briefly touches on only a bit of the truly impressive science—which can be read in detail in the original publication by Boeke and his army—and instead emphasizes the enthusiastic undergraduate student participation, along with an equally interesting—I think—odyssey as to how this project progressed to where it is now.

Why Build Completely Customized Yeast?

Good question. For centuries, Saccharomyces cerevisiae (S. cerevisiae), also know as brewer’s, baker’s, or budding yeast, has played a pivotal role in our food and drinks, from baked goods to beer and wine. It’s also a “workhorse” for cellular and molecular biologists. In 1996, it became the first eukaryote to have its genome fully sequenced. Since then yeast geneticists have studied it every which way—facilitated in part by being able to incorporate foreign DNA through a process called homologous recombination. There is even a comprehensive website dedicated to all things yeast.

In contrast, the “ultimate modification” of yeast is designed completely in silico (i.e. using computer programs), and assembled in the lab piece by piece using chemically synthesized DNA oligonucleotides. Each of the ~6,000 genes can thus be pondered with regard to being kept, modified or removed. The same applies to other genetic elements. Moreover, exogenous genes (i.e. not found in natural yeast) or other exogenous functional genetic elements can be inserted to obtain “custom content” genetic variants intended for specific applications, such as production of commercially valuable products.

Caffeinated Coffee Conversation Jolts Inspiration

Why tinker with yeast when we could ‘synthesize the whole thing.’ Jef Boeke, New York University (credit: Jef Boeke/New York University; taken from sciencemag.org).

Why tinker with yeast when we could ‘synthesize the whole thing.’ Jef Boeke, New York University (credit: Jef Boeke/New York University; taken from sciencemag.org).

I’m sure it was more than the caffeine, but its jolt probably helped a little based on Pennisi’s story about Boeke’s “aha moment.” The following anecdote is taken from Pennisi’s article. Boeke traces his change of heart about the synthetic yeast project to 2006, while over coffee, his Johns Hopkins colleague Srinivasan Chandrasegaran was trying to convince him to make a large number of zinc-finger nucleases. These DNA-modifying enzymes are Chandrasegaran’s specialty, and a toolkit full of them would make the yeast genome easier to manipulate. Boeke wasn’t that interested and, almost as a joke, suggested a more drastic way to take control of the yeast genome: synthesize the whole thing. To his surprise, Chandrasegaran jumped on the idea, and the pair brought Joel Bader, a computer scientist at Johns Hopkins, into the discussion.

Despite Boeke’s dismissal of Davis’s proposal just 2 years earlier, the trio at Hopkins quickly concluded that making an artificial yeast genome would be worthwhile if it could be a testbed for learning about the genome itself. They decided to start with the 90,000-base “R” arm of chromosome 9—the shortest in the yeast’s genome—and spent a year arguing about how the synthetic sequence should differ from the natural one. The trio considered just including the genes they wanted, ‘but we quickly realized it would be very risky to eliminate whole bunches of genes’ without really knowing in advance what the effect of that loss on the whole system would be,’ Boeke recalls.

So the team used the natural sequence of the chromosome 9 arm as the basic building block, and incorporated DNA that would enable them to ‘induce changes at will’. Basically, when activated by a chemical added to cells, it kicks off a chromosomal version of ‘musical chairs.’ By using this process, they were able to generate new yeast strains with different properties. Over time, they have been able to add stability to the yeast genome, helping to ensure integrity of the strain. Readers interested in technical aspects of how they did this—and other genetic edits such as removal destabilizing transposons and non-coding RNA—are referred to the original publication.

From Buy It to DIT—More Inspiration

After months of work and with the help of Bader, Boeke designed a version of the chromosome 9 arm using a software program. He then contracted with a biotech company to synthesize the chromosomal arm. The company was having difficulty synthesizing such a long piece of DNA and they didn’t hear back regarding the project for almost a year. During that time, Boeke began to wonder if he could speed things up himself. He came up the idea of offering a course dedicated to building a synthetic yeast genome. In 2007, the course was offered for the first time as a summer school class, which to me is reminiscent of the innovative strategy used by Mark Twain’s fictional character, Tom Sawyer, to achieve painting a seemingly endless and impossibly high fence. In this case, it’s building a big genome by making it educational for many student workers. sawyer

When tasked with painting a fence in front of his house, Tom Sawyer convinced others that doing so is more fun than sitting in the shade watching other people paint. As other children congregate to watch Tom Sawyer paint, he extols the fun of painting, and eventually has the other children clamoring to get to paint the fence (taken from postcrossing.com via Bing Images).

The course proved to be extremely popular and 6 years later it’s one of the most in-demand classes that Hopkins offers. Every section is packed —even on Friday nights! ‘We were overwhelmed with interest’ from biology, engineering, computer science, public health, and other majors,’ said Boeke, who is working with his colleagues to keep the course going at Hopkins despite his move to NYU.

Student-Power Drives Yeast Project Assembly

Genome builder, Katrina Caravelli (credit: Marty Taylor/Johns Hopkins University; taken from sciencemag.org).

Genome builder, Katrina Caravelli (credit: Marty Taylor/Johns Hopkins University; taken from sciencemag.org).

As a former professor and still active researcher, I have nothing but high praise for Boeke’s Tom Sawyer-like innovation to harness interest and enthusiasm of students through an educational course that achieves a truly grand challenge: a totally synthetic designer-yeast genome. More importantly, here’s what Pennisi reported about a couple of representative undergraduate student genome-builders, James Chuang and Katrina Caravelli, whose “Build A Genome” coursework consisted of sequences of DNA comprised of just four letters: A, C, G, and T in DNA:

For students, the course offers intensive training in synthetic biology and the thrill of taking part in frontline research. ‘I was really fascinated by the potential and by my ability to have an impact,’ recalled Caravelli, who signed up for the course in 2008 and is now Boeke’s senior lab coordinator at New York University. During the semester-long course, students learned basic molecular biology procedures such as performing PCR and cloning DNA in bacteria.

Each student committed to completing 10,000 DNA bases during the course. ‘My building blocks went to chromosome 8,’ Chuang, now a graduate student in biomedical engineering at Boston University, proudly told Pennisi. After being supplied with the DNA sequence he needed to synthesize, he started out with 16 DNA oligonucleotides, each about 75 bases long, ordered from a commercial DNA synthesis company. The ends of some pieces overlapped, so when he mixed the pieces together with enzymes, they self-assembled into 750-base spans dubbed building blocks. After making sure the building blocks had the correct sequence, he put them into bacteria to generate many copies of the newly assembled DNA. Now, Johns Hopkins graduate student Jingchuan Luo is putting those bigger DNA sections into yeast and making “minichunks” about 3,000 bases long. If the yeast stays healthy, then she adds the next chunk in line to it, and so on. Her goal: to make a yeast strain with a totally new chromosome 8.

Caravelli recalls that her own bacteria sometimes wouldn’t grow with the yeast segment in their midst. She enjoyed figuring out why. ‘The trial-and-error process challenges you to think properly like a scientist.’

Boeke’s course has proved such a success that three other U.S. universities are hosting or will soon host their own. Because it’s now cheaper to buy 750-base segments than to have students make them, current students start with such DNA blocks and turn them into 3000-base spans, and, ultimately, 10,000-base chunks. Class productivity has soared. Each student’s target is now 30,000 to 50,000 bases.

The students have high hopes for their work. ‘I want to see synthetic biology do really useful things for society,’ Chuang told Pennisi. ‘The synthetic yeast genome provides a template for doing [that].’

synthesis

Initial assembly steps carried out by undergraduate students (taken from sciencemag.org).

Global Momentum and Sc2.0

After the students in Boeke’s course completed chromosome 9, they moved on to chromosome 3. It took 49 students over 18 months to successfully assemble the 272,871 bases. Their work was detailed in a publication in Science in March of this year. The publication showed that ‘yeast carrying the shortened, modified chromosome grew normally and looked little different from their natural counterparts under almost all of the growing conditions tested.’

You can watch and listen to Jef Boeke talk about this achievement in a 2 minute video.

As they neared completion of chromosome 3, the team continued work on other chromosomes as word was spreading about the novel work coming out of Hopkins. Ying-Jin Yuan of Tianjin University in China heard about the project and was eager to participate. He convinced Huanming Yang from BGI to get involved and Yang helped organize the first synthetic yeast genome meeting, held in Beijing in 2012. Yuan didn’t want to stop there. He forged an international collaboration with Boeke and through his own 2012 “Build-A-Genome” course, he enlisted the help of 60 Chinese students and assembled all the bases needed for chromosome 5.

Popularity of the project continued to grow in other labs around the world. Tom Ellis, a synthetic biologist at Imperial College London, had attended Yang’s meeting in Beijing and was also intrigued by Boeke’s work. He helped organize a second synthetic yeast meeting in July 2013. Soon after, the British government committed £1 million to the project.

The momentum has continued. These like-minded yeast chromosome-builders have created a great website for their project that is called Sc2.0. This site is definitely worth visiting, especially to read the FAQ and learn more about the international Team members. I’ve included a great visual from the website that depicts where in the world each of the 16 chromosomes is being built.

xvi

The goal of the yeast genome project is to complete each of the yeast’s chromosomes within 2 years. This will be done by various partners around the global. Once the individual chromosomes are complete, Boeke will lead a team through the largest challenge of all—putting all of the chromosomes together into one organism. ‘It’s a little surprising that we can have this grand idea with so many moving parts and multiple organizations making different chromosomes,’ Boeke’s senior lab coordinator, Katrina Caravelli told Pennisi. But so far, ‘it’s working well.’

The Vision for Custom Yeast

Many yeast biologists are waiting in anticipation for the synthetic yeast genome project to be completed as they are eager to begin testing it themselves. Aside from studying how induced mutations influence function of genes, the way will be open for various applications, such as a new model for human diseases. Boeke, for example, told Pennisi that he ‘would like to install in yeast all of the genes of the molecular pathway that, in humans, is defective in Lesch-Nyhan syndrome, a rare disorder characterized by gout and kidney and neurological problems, including self-mutilating behavior. By modeling the molecular defect in yeast, he hopes to figure out how the mutation affects eukaryotic cells in general.’

On the other side of the equation, Kirsten Benjamin, a synthetic biologist at Amyris Inc., is looking to study what’s wrong with the synthetic strain. ‘We’re going to find a bunch of ways where it doesn’t work,’ she says. This, she says, ‘will allow us as a scientific community to discover all these unappreciated phenomena.’

While the commercial applications for synthetic genomes are still to be determined due to uncertainty around financial feasibility, it’s not the long term viability that Boeke is after. ‘In a way, you can put it as a kind of milestone, like the first human genome was a milestone for genomics,’ he says. ‘When we finish it, we can really plant a flag in it.’

My opinions on this are that the cost for synthesizing DNA oligonucleotides has continually decreased, while innovative methods for up-scaled mass production and stitching together oligos have continually improved. In concert, these will nullify the aforementioned ‘too costly’ argument and enable custom synthesized genomes.

What do you think?

As always, your comments are welcomed.

Children at Risk from Deadly Respiratory Virus EV-D68

  • Frightening Statistics From CDC
  • CDC Updated U.S. Map of Outbreak & Advice of What to be Aware
  • CDC Develops Rapid Real-Time RT-PCR Test for Detection
  • Some Speculate on Linking Outbreak to the “Southern Border Invasion”

Ebola virus is dominating news reports lately, and perhaps rightly so considering the worldwide impact. Turning our attention, however, to actual incidents of infection and death in the U.S., enterovirus (EV) D68 poses a much greater threat and warrants our attention—especially if you or your friends have young children.

On September 24, Eli Waller’s parents were worried that their 4 year-old son had pink eye and kept him home from school so that he wouldn’t infect other children. He seemed otherwise healthy. What happened next was shocking.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

‘He was asymptomatic and fine, and the next morning he had passed,’ said Jeffrey Plunkett, the township’s health officer. ‘The onset was very rapid and very sudden,’ quoted the NY Times.

A week later the Centers for Disease Control and Prevention (CDC) confirmed that Eli had been infected with EV-D68.

EV-D68 was seen as early as August of this year as hospitals in Missouri and Illinois reported increased visits from children with respiratory illness. Soon, the virus was identified in 43 states and detected in 594 patients, 5 of which died.

After reading this very sad—if not frightening—story, I decided to research EV-D68 for this “hot topic” blog, which I’m dedicating to little Eli Waller.

EV-D68 Facts in the U.S. for 2014

Following are facts and related information I’ve selected from the CDC website for EV-D68 that is well worth consulting if you or your friends have any young children.

  • The United States is currently experiencing a nationwide outbreak of EV-D68 associated with severe respiratory illness. From mid-August to October 30, 2014, CDC or state public health laboratories have confirmed a total of 1105 people in 47 states and the District of Columbia with respiratory illness caused by EV-D68.
Activity of enterovirus-D68-like illness in reporting states. Taken from CDC website for EV-D68.

Activity of enterovirus-D68-like illness in reporting states. Taken from CDC website for EV-D68.

  • Every year, various species of EV and rhinovirus (RV) cause millions of respiratory illnesses in children—see my Postscript below for details. This year, EV-D68 has been the most common type of enterovirus identified, leading to increases in illnesses among children and affecting those with asthma most severely.
  • CDC has prioritized testing of specimens from children with severe respiratory illness. There are likely many children affected with milder forms of illness. Of the more than 1,400 specimens tested by the CDC lab, about half have tested positive for EV-D68. About one third have tested positive for an enterovirus or rhinovirus other than EV-D68.
  • As of October 22, EV-D68 has been detected in specimens from seven patients who died and had samples submitted for testing. Investigations are ongoing; CDC reviews and updates available data every Thursday.

Lakeia Lockwood, mother of D’Mari Lockwood receiving treatment, said he was “Struggling to breathe, coughing. [Doctors] said the airways were so tight they actually, in Gary, said I almost lost him.” Taken from wgntv.com via Bing Images.

Lakeia Lockwood, mother of D’Mari Lockwood receiving treatment, said he was “Struggling to breathe, coughing. [Doctors] said the airways were so tight they actually, in Gary, said I almost lost him.” Taken from wgntv.com via Bing Images.

What CDC Is Doing about EV-D68

According to a CDC website for EV-D68, the following are now operative:

  • CDC is continuing to collect information from states and assess the situation to better understand EV-D68, the illness caused by this virus, and how widespread EV-D68 infections may be within each state and the populations affected.
  • CDC is helping states with diagnostic and molecular typing for EV-D68. CDC has obtained one complete genomic sequence and six nearly complete genomic sequences from viruses representing the three known strains of EV-D68 that are causing infection at this time.
  • Comparison of these sequences to sequences from previous years shows they are genetically related to strains of EV-D68 that were detected in previous years in the United States, Europe, and Asia. CDC has submitted the sequences to GenBank to make them available to the scientific community for further testing and analysis.
cold

Researchers at The Genome Institute at Washington University have recently sequenced the genome of EV-D68, which is similar to the germ that causes the common cold, rhinovirus, shown above. This picture, taken from a Bioscience Technology interview with senior author Gregory Storch, who is quoted as saying that ‘Having the DNA sequence of this virus enables additional research. It can be used to create better diagnostic tests. It also may help us understand why this epidemic seems to be producing severe and unusual disease, and why it’s spreading more extensively than in the past.’

CDC Developed a New, Rapid Test for EV-D68 Detection

In an important breakthrough that once again demonstrates the “power of PCR,” CDC has developed a new PCR-based test to quickly detect EV-D68, enabling CDC to process over 2000 specimens. As a result, the number of confirmed EV-D68 cases increased substantially in the past few weeks as CDC began using the test on October 14. About 40% of the specimens have tested positive for EV-D68.

Because EV-D68 is an RNA virus, CDC’s new lab test utilizes reverse transcription polymerase chain reaction (RT-PCR), which offers fast, “real-time” detection. More information about RT-PCR in general is available at TriLink’s webpage for CleanAmp™ One-Step RT-PCR 2X Master Mix.

M. Steve Oberste, chief of the polio and picornavirus laboratory branch at CDC, recently told PCR Insider that until the new CDC test was released on October 14, the agency was detecting the virus using a 10 year-old semi-nested PCR protocol from J. Clin. Microbiol followed by sequencing. CDC’s new test is specific for the VP1 gene of EV-D68, and ‘is a straightforward TaqMan® rRT-PCR test’, Oberste said. The sample prep step remains standard RNA or total nucleic acid extraction.

The lab is currently working on publishing the new method, as well as putting the basic protocol on the CDC website. ‘That way, states and clinical labs can adopt it as [a Laboratory Developed Test (LTD)],’ Oberste said.

Is CDC Hiding EV-D68 Link to Illegal Alien Kids?

At the risk of my being accused of posting politically incorrect comments, the above question is the headline of an online editorial in Investor’s Business Daily on October 17 that, within five days—when I wrote this blog—prompted over 500 comments and more that 7,500 tweets.

I won’t comment on the editorial’s assertion of CDC’s ‘bungling…about the Ebola outbreak’ but I am of the opinion—at least for now—that CDC is probably not ‘hiding’ anything about EV-68. On the other hand, I strongly agree with the editorial’s comment that ‘dispersal of …unaccompanied minors, throughout the U.S. without proper medical screening is an appalling dereliction of duty by…an administration sworn to protect the health and safety of American citizens.’

Let’s hope that the EV-D68 outbreak—regardless of its origin–ends soon, without further loss of children like little Eli Waller.

Postscript

For readers interested in details of microbiology, here is selected information taken from Blomqvist et al. published in J. Clin. Microbiol. (2002).

The family Picornaviridae contains two large and important genera of common human pathogens, Enterovirus and Rhinovirus. In structural and genetic properties, these two genera are very much alike. However, rhinoviruses, which multiply mainly in the nasal epithelium, differ from enteroviruses, infectious agents of the alimentary tract, by their sensitivity to acid and by their growth at a lower optimal temperature. The genus Enterovirus contains 64 serotypes pathogenic to humans, which have been distinguished by the neutralizing antibodies against them. There may still be uncharacterized serotypes, as some clinical enterovirus isolates are not typeable by existing antisera and show genetic segregation indicative of an independent serotype. Nucleotide analysis of the RNA genomes of different human enterovirus (HEV) serotypes has provided new insight into the classification of enteroviruses, resulting in the division of these viruses into four main genetic clusters, designated HEV species A to D. Poliovirus serotypes 1 to 3 are genetically related to HEV-C but are classified as a species of their own. The genus Rhinovirus contains 102 serotypes, which are numbered from 1 to 100. Serotype 1 contains two subtypes, 1A and 1B. More recently, a strain referred to as the Hanks strain has been proposed to represent a new serotype.

Pseudouridine: 2014 Modified Nucleobase of the Year

  • A Minor Modified Nucleobase Playing a Major Role in RNA Therapeutics
  • Nucleobase is “Acrobatically” Formed in RNA by an Enzyme
  • Its Original Identification is Linked to the Atomic Bomb! 

The Molecule of the Year was started in 1989 by Science magazine and in 1996 was changed to ‘Breakthrough of the Year’. After a brief hiatus, the Molecule of the Year designation was revived in 2002 by the International Society for Molecular and Cell Biology and Biotechnology Protocols and Research. If you peruse the list of these lauded molecules, you’ll find that DNA has received this coveted award twice: “PCR and DNA polymerase” (1989) and “DNA repair enzyme” (1994).

While the Molecule of the Year is fascinating and well deserved, I think we should show some love for individual nucleobases given that this blog focuses on “what’s trending in nucleic acid research”—and since TriLink scientists are “The Modified Nucleic Acid Experts”. So, I thought it would be apropos (and fun) to start an annual post on Modified Nucleobase of the Year.

After mulling over which modified nucleobase merits this accolade for 2014, I decided on pseudouridine (aka 5-ribosyluracil)—an unusual isomer of uridine—for several reasons that are given below, followed by some history of its discovery that I found to be quite interesting.

pseudo-u-1st

Pseudouridine-Modified RNA Therapeutics

An earlier post entitled “Modified mRNA Mania” commented on relatively recent research demonstrating an exciting new paradigm in therapeutics based upon in vivo delivery of modified mRNAs to express target proteins. These proteins encoded by modified mRNAs have been applied to vaccines, cellular reprogramming, and other therapeutic modalities. In that post, I go on to indicate how such proof-of-concept publications have quickly spawned several new start-ups and mega-million dollar investments by Big Pharma companies. Although it’s still early days, the future for modified mRNA therapeutics is very promising.

Pseudouridine-5′-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors. Pseudo-UTP, along with 5-methylcytidine-5′-triphosphate (5-methyl-CTP) has shown innate immune suppression in cell culture and in vivo, while actually enhancing translation.

For example, Warren et al. determined an efficient means of reprogramming multiple human cell types using modified mRNA that can express the four primary reprogramming proteins. These cells are referred to as induced pluripotent stem cells (iPSCs). Warren et al. found that modified mRNA substituted with Pseudo-UTP, 5-Methyl-CTP and ARCA effectively evaded the cell’s innate immune response, a crucial component in their success. Reduced toxicity due to substitution with Pseudo-UTP, 5-Methyl-CTP was critical since it allowed repeated transfection with modified mRNA over several weeks.

Readers interested in these approaches can access two posters here by TriLink scientists and collaborators.

“Acrobatic” Enzymatic Synthesis of Pseudouridine from Uridine

From the structures of uridine and pseudouridine shown above, you’ll notice that the red-colored nitrogen (N) and a double-bonded carbon in uridine have “switched places” in pseudouridine. Whether and how nature does this switch via one or more enzymes are intriguing questions that have been extensively investigated for many years.

The non-controversial answer to the first question about “whether” this switch occurs naturally is that nature has indeed evolved an enzyme for isomerizing uridine to pseudouridine that is named—appropriately—pseudouridine synthase. The second question about “how” is less clear, if not controversial, to some. Here’s why:

Pseudouridine synthase achieves this “acrobatic” positional switch on uridine that is already incorporated in RNA, as opposed to during biosynthesis of monomeric uridine prior to RNA synthesis. Since only a small number of uridines in RNA are converted to pseudouridine, understanding which ones are isomerized has been investigated. Readers interested in this can consult a recent report by Sibert & Patton entitled “Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements.”

The controversial aspect of “how” deals not with sequence context but mechanistic details, which Santi and coworkers in 1999 proposed as follows, based on elegant substitution of hydrogen (H) with fluorine (F) at the C-5 ring position—BTW, my apologies for the organic chemistry “arrow pushing” but it’s a necessary evil to put up with to show the “how”. 

onthe

On the other hand, subsequent work by Spedaliere et al. has led to questioning whether a different mechanism is operative. This is explored in detail  in a publication entitled The pseudouridine synthases: revisiting a mechanism that seemed settled.” Readers interested in this alternative view are encouraged to read that publication.

Pseudouridine-Modified Oligonucleotides

I hope this brief overview has spurred you to learn about the amazing things you can do with pseudouridine. Companies like TriLink offer custom chemical synthesis of pseudouridine-modified oligonucleotides that can be used for a wide variety of physicochemical, biochemical, or biological studies. Among biological applications, RNA interference (RNAi) using short, double-stranded RNA (aka siRNA) is especially important and widely employed. RNAi has been expertly reviewed by Beal & Burrows et al. with regard to incorporation of modified bases to probe and enhance RNAi.

Highlighted in the Beal & Burows review are comprehensive investigations by Nawrot and coworkers, who incorporated pseudouridine, as well as other modified nucleobases, at various positions to evaluate thermodynamic stability and gene silencing activity. As detailed in those results, certain locations for pseudouridine provided potent gene-silencing activity. I think you’ll see that these publications and reviews support my claim that pseudouridine is truly deserving of Modified Nucleobase of the Year status.

Fractionating Fractions and Chasing Spots

While I believe I’ve provided significant justification for my obesssion with pseudouridine, I understand that some of you may not be convinced. If the current range of potential uses and pivotal roles of this facscinating nucleobase haven’t been compelling enough, perhaps I’ll make you a convert when you learn more about this nucleobase’s origin.

Several groups of investigators in the 1950s were engaged in identifying minor constituents of RNA by developing then state-of-the-art chromatographic methods to separate hydrolyzed RNA into its components. Those of you who may not be familiar with column chromatography or paper chromatography simply need to know that eluted (aka separated) fractions are collected and further fractionated, or paper spots are cut out, followed by analyses to determine purity and identity. Sounds easy, but it’s not—especially in the 1950s.

Column chromatography in the 1950s (taken from Wikipedia)

Column chromatography in the 1950s (taken from Wikipedia)

What these early investigators actually did for “fractionating fractions and chasing spots” of minor RNA constituents is way too complicated to get into here. So, I’ve taken the liberty to focus on the following work of Waldo E. Cohn, who has the distinction of being the first to definitively identify the very unusual structure of pseudouridine, and is associated with its naming.

Waldo E. Cohn:  From the Atomic Bomb to Pseudouridine

In researching Cohn’s work leading up to identifying and naming pseudouridine, I learned about how his earlier involvement with the ultra-secret Manhattan Project to develop the first atomic bomb led to pioneering investigations of radioactive 32P biodistribution in rats! Here’s a brief excerpt on that taken from Simoni et al., which I’ll tie back to Cohn’s pseudouridine story.

Dr. Ralph Overman uses a probe counter to check the shoes of Dr. Waldo E. Cohn for radioactive contamination on June 14, 1946 in Oak Ridge, Tennessee (AP Photo/Clinton Labs; taken from snippits-and-slappits.blogspot.com via Bing Images).

Dr. Ralph Overman uses a probe counter to check the shoes of Dr. Waldo E. Cohn for radioactive contamination on June 14, 1946 in Oak Ridge, Tennessee (AP Photo/Clinton Labs; taken from snippits-and-slappits.blogspot.com via Bing Images).

By the mid-1930s, the cyclotron had been invented and developed at the University of California at Berkeley by Ernest O. Lawrence. With it came the capability of creating artificial radioactive isotopes. One of the first radioactive isotopes produced was 32P, which was immediately put to use in seeking solutions to biological problems.

Waldo E. Cohn was a graduate student at University of California Berkeley working with his thesis advisor Prof. David M. Greenberg. The 32P was prepared in the cyclotron of the Radiation Laboratory at Berkeley by bombardment of red phosphorus with deuterons. After purification and oxidation to H3PO4, the radioactive material was administered to rats, and the distribution of radioactivity in various tissues was followed with time, which you can read about here and is considered a “JBC Classic”.

Cohn’s experiences with radioactive isotopes as a graduate student shaped his career. After receiving his Ph.D. degree in Biochemistry from Berkeley in 1938, and a postdoc at Harvard, he was recruited to the Metallurgical Laboratory of the University of Chicago in 1942 as a member of the Manhattan Project. His role was to study the biological effects of fission products on biological systems. Cohn moved to the Oak Ridge National Laboratory (ORNL) where he became Senior Chemist and Group Leader of the Oak Ridge Biology Division, a position that he held until his retirement in 1975.

Cohn was involved in the formation of the Isotope Distribution Committee, which was assigned the responsibility of making isotopes available to qualified researchers. In 1946, Cohn and Paul Aebersold published a landmark paper in Science that established the administration of isotope distribution and preparation of the catalog of isotopes that were available for research. It was one of the first post-war efforts toward the “peaceful use” of atomic energy. Cohn became well known for his efforts to establish and enforce standardized biochemical nomenclature. From 1965 to 1976, he was Director of the Office of Biochemical Nomenclature of the National Academy of Sciences.

Now back to pseudouridine!

The Right Scientist at the Right Place

Cohn’s experience with radioisotopes coupled with his expertise in nucleic acid-related chemistry and the unique resources offered by ORNL added up—in my opinion—to him being the right scientist at the right place to doggedly pursue identification of minor constituents of RNA.

Here are actual excerpts from his key publications on this hunt.

note note2

In concluding this post, I’d like to add one more historical “factoid” that I thought was interesting. I tracked down the identity of “Dr. A Michelson.” He is actually A. M. Michelson who coauthored with über-famous Sir Alexander Todd the first synthesis of ATP published in Nature in 1948. My thanks to Drs. Cohn and Michelson for their respective roles in giving “birth” to pseudouridine, which in turn has enabled development of new molecular tools for nucleic acid-based applications.

As we look at the historical significance as well as modern applications of pseudouridine, I hope I’ve convinced you of the amazing work this nucleobase is capable of. Let me know if you support my nomination for Modified Nucleobase of the Year, or share your nominations for other nucleobases below.

Reflections on Advances in Medicinal Oligonucleotides

  • Oligos Are Not “Magic Bullets”
  • Oligos Have, Nevertheless, Enabled New Drug Paradigms
  • Oligos Continue to Attract Significant Corporate Investments

Reflections

The 10th Annual Meeting of the Oligonucelotide Therapeutics Society (OTS) is in full swing today in San Diego, CA where it began on Oct 12 and concludes on Oct 15. Having worked on antisense oligos since the early days (~30 years ago) participating in this meeting led me to several thoughts that I wish to share with you in this post.

First of all, contrary to many sceptics in those early days, the concept of using synthetic oligonucleotides as an entirely new class of medicinal agents has not only survived but also greatly expanded in terms of the biological target/mechanism of action and types of oligo constructs used—each with a seemingly endless array of chemical modifications to evaluate. In approximate chronological order of discovery, these targets and the types of oligos they have now come to include are listed below coincidentally, most of these are represented in the 2014 OTS agenda.

Target
mRNA
dsDNA
transcription factors
immunostimulation
proteins
splice junctions
RNA interference
Oligo Type
antisense
triplex
dsDNA decoys
CpG
aptamers
blockers
siRNA

 

 

 

 

 

Secondly, while it has been possible for oligo chemists to design and synthesize a plethora of modified oligos to achieve optimized nuclease stability, binding to target, etc., efficient delivery has remained the single most challenging problem to deal with. In talks on medicinal oligos, this situation is oftentimes eluded to as something to the effect of “there are only three remaining problems to solve: delivery, delivery, and delivery.”

Lastly, contrary to early hopes of being Dr. Ehrlich’s “magic bullets” (see caption below), oligos didn’t quite prove to be the new paradigm for a speedy concept-to-clinic solution. As all of us in the oligo world know, oligo-based therapeutics have encountered long and costly R&D timelines and clinical development paths typical of all other classes of therapeutic compounds.

magic

Dr. Ehrlich’s Magic Bullet is a 1940 biographical film starring Edward G. Robinson, based on the true story of the German doctor and scientist Dr. Paul Ehrlich (14 March 1854 – 20 August 1915), who worked in the fields of hematology, immunology, and chemotherapy. His laboratory discovered arsphenamine, a synthetic arsenic-containing compound that provided the first effective medicinal treatment for syphilis, thereby initiating and also naming the concept of chemotherapy. Ehrlich popularized the concept of a “magic bullet”. In 1908, he received the Nobel Prize in Physiology or Medicine for his contributions to immunology.

Notable Advances

Given the aforementioned breadth of therapeutic targets being pursued with oligos, selecting notable advances for comment here is necessarily limited to only a few, and is obviously highly subjective. Having said that—and with apologies for not highlighting other important advances—here are my favorites.

Ebola Drug – As reported in a Scientific American story, Thomas W. Geisbert, now at the University of Texas Medical Branch at Galveston, and his many collaborators have devised a highly promising treatment that has saved the lives of six monkeys infected with Ebola virus—the causative agent of the current Ebola disease outbreak recently commented on in a previous post. One of Geisbert’s collaborators, Ian MacLachlan of Burnaby, British Columbia–based Tekmira Pharmaceuticals, and his team have received a $140-million grant from the U.S. Department of Defense to develop the therapy further.

Working together, the scientists engineered an siRNA to prevent the Ebola virus from making a particular protein, without which it cannot replicate itself. “If you knock out that one, in theory you knock out everything,” Geisbert says. The researchers also designed another siRNA to thwart manufacture of a second protein that the virus uses to weaken an infected individual’s immune system. There is no danger of the siRNAs interfering with typical cellular duties because the targeted viral proteins do not exist in the cells of humans or other mammals.

MacLachlan and his colleagues encapsulated the lab-made siRNAs in Tekmira’s proprietary lipid nanoparticle (LNP) formulation to enable vascular delivery and transport across cellular membranes.

Simplified cartoon of Tekmira’s lipid nanoparticles (LNPs) formulations of siRNA (taken from an interview with Ian MacLachlan in GEN via Bing Images).

Simplified cartoon of Tekmira’s lipid nanoparticles (LNPs) formulations of siRNA (taken from an interview with Ian MacLachlan in GEN via Bing Images).

Then they injected the preparation into several rhesus macaques, which had been infected with Ebola virus less than an hour earlier. In one study, two of three monkeys given a total of four doses of the treatment in the first week after exposure survived. In a second study designed to test the effectiveness of a higher dose, all four monkeys that received seven siRNA injections lived. Tests revealed that the treated monkeys had far fewer virus molecules in their blood than is typical for an infected animal. The macaques tolerated the siRNA injections well, and those that survived were still healthy 30 days later.

The study was a “milestone,” says Gary Kobinger of the University of Manitoba, who is working on a different Ebola treatment based on antibodies. He believes Geisbert and his team “are leading the effort toward clinical development.”

Exon Skipping – This novel strategy is used to restore the reading frame within a gene. Exons are the sections of DNA that contain the instruction set for generating a protein; they are interspersed with non-coding regions called introns. The introns are later removed before the protein is made, leaving only the coding exon regions.

Splicing naturally occurs in pre-mRNA when introns are being removed to form mature-mRNA that consists solely of exons. The mechanism behind exon skipping is a mutation specific antisense oligonucleotide (AON). The AON binds to the mutated exon, so that when the gene is then translated from the mature mRNA, it is “skipped” over, thus restoring the disrupted reading frame. This allows for the generation of an internally deleted, but largely functional protein.

Shown below is a schematic diagram of how this applies to the AON named Drisapersen, developed by Prosensa and currently in late stage development for treating Duchenne Muscular Dystrophy (DMD) patients. Drisapersen induces exon 51 skipping in the dystrophin gene and is intended for ~13% of all DMA patients. On June 3, 2014, Prosensa announced that the United States Food and Drug Administration (FDA) outlined a regulatory path forward for drisapersen, under an accelerated approval pathway, based upon existing data.

Drisapersen binds to exon 51 in pre-mRNA leading to an in-frame mRNA transcript that produces a shortened but functional protein like that in Becker muscular dystrophy (BMD), which is a less form of muscular dystrophy.

Drisapersen binds to exon 51 in pre-mRNA leading to an in-frame mRNA transcript that produces a shortened but functional protein like that in Becker muscular dystrophy (BMD), which is a less severe form of Duchenne muscular dystrophy.

Notable Corporate Investments

Santaris Pharma – According to an Aug 4, 2014 press release, Roche announced that it has agreed to acquire Santaris Pharma based near Copenhagen, Denmark. Santaris Pharma has pioneered its proprietary Locked Nucleic Acid (LNA) platform that has contributed to an emerging era of RNA-targeting therapeutics.

Roche plans to maintain Santaris Pharma’s operations in Denmark, where the existing site will be renamed Roche Innovation Center Copenhagen. Under the terms of the agreement, Roche will make an upfront cash payment of $250 million to Santaris Pharma shareholders and make additional contingent payments of up to $200 million based on the achievement of certain predetermined milestones.

Isis Pharmaceuticals – As detailed in multiple press reports here, Isis Pharmaceuticals has announced that it has earned three $1 million payments for advancement of each of three programs with GlaxoSmithKline (GSK), a $10 million milestone payment from Biogen-Idec, and a $15 million milestone payment from AstraZeneca.

Isarna Therapeutics – It was announced earlier this year that Isarna Therapeutics GmbH raised $7.6 million from its current investor groups to further its TGF-ß therapeutics using oligos.

In closing, my reflections turned to thinking that, while it has taken more time and money than originally envisaged by early proponents of medicinal oligos, the pipelines are full and investment is strong.

All of us in the oligo therapeutics arena are greatly interested in hearing about what you are doing with medicinal oligos, so please feel free to share in the comments section below. Also, if you’re at OTS stop by the TriLink booth or look for me during the talks, I’d love to meet you face-to-face.

#OligoMeeting2014

October is Down Syndrome Awareness Month

Down syndrome remains the most commonly diagnosed chromosomal condition with approximately 6,000 afflicted babies born in the U.S. each year. This means that Down syndrome occurs in about 1 out of every 700 babies.

In recognition of Down Syndrome Awareness Month, this post provides information about Down syndrome taken from the U.S. Centers for Disease Control (CDC) and Prevention website. It also provides a personal story about John Nguyen, a coworker at TriLink, whose first born son, Jordan, has Down syndrome. I’d like to first introduce the Nguyen’s story and then continue on with an overview of Down syndrome causes, risk factors and diagnosis.

The Nguyen Family

When John and his wife, Jessica heard, “Your child may have Down syndrome,” they, like most parents who receive this news, were saddened and confused. What did this mean? Will he or she be healthy? What do we do next? So many questions and emotions shook up their world in a way they never expected.

baby boyWhen they welcomed Jordan into the world, his condition was still unknown and his heart needed to be monitored so they were only able to hold him for a few seconds before he was whisked away. But that’s all they needed to know they were a family and they were in this together. Jordan spent 10 days in the NICU. In addition to having Down syndrome, he was also diagnosed with a congenital heart condition called Ebstein’s anomaly.

Jessica said, “Although it was difficult in the beginning, we quickly realized how lucky we were and how amazing he is. His heart condition remains stable. Throughout this journey we have remained positive and refuse to let his diagnoses damper or put a limit to what Jordan can accomplish and achieve… The route to parenting we thought we were going to take was detoured and I consider it a blessing. We now get to appreciate things we never previously considered. We have met people we never would have met otherwise. And now know a love so deep that we never could have imagined. Love not only for and from our son but also from the love and support of our family, friends, and community. Those questions and emotions in the beginning really shook up our world….but in the best way possible.”

boyJohn and Jessica actively fundraise for the Down Syndrome Association of San Diego, whose mission is to enhance the quality of life for people with Down syndrome and their families through education, social and support programs. They participate in the Buddy Walk® each year.

TriLink is donating $5* to the Nguyen’s Buddy Walk® team for each share this post receives. Help us spread awareness about this common and often misunderstood condition.

Types of Down Syndrome  

Down syndrome, which was first characterized by English physician John Langdon Down in 1862, is a condition in which a person has an extra chromosome. Typically, a baby is born with 46 chromosomes—23 from both mother and father. Babies with Down syndrome have an extra copy of one of these chromosomes, chromosome 21. A medical term for having an extra copy of a chromosome is ‘trisomy.’ Down syndrome is also referred to as Trisomy 21. This extra copy changes how the baby’s body and brain develop, which can cause both mental and physical challenges for the baby.

Taken from http://theoneinathousand.blogspot.com/2012/10/october-is-down-syndrome-awareness-month.html

Taken from http://theoneinathousand.blogspot.com/2012/10/october-is-down-syndrome-awareness-month.html

There are three types of Down syndrome. People often can’t tell the difference between each type without looking at the chromosomes because the physical features and behaviors are similar.

  • Trisomy 21: ~95% of people with Down syndrome have Trisomy 21, wherein each cell in the body has 3 separate copies of chromosome 21 instead of the usual 2 copies.
  • Translocation Down syndrome: This type accounts for ~3% of people with Down syndrome. This occurs when an extra part or a whole extra chromosome 21 is present, but it is attached or “trans-located” to a different chromosome rather than being a separate chromosome 21. 
  • Mosaic Down syndrome: This type affects ~2% of the people with Down syndrome, wherein some of their cells have 3 copies of chromosome 21, but other cells have the typical two copies of chromosome 21.

Causes and Risk Factors

Researchers know that Down syndrome is caused by an extra chromosome, but no one knows for sure why Down syndrome occurs or how many different factors play a role. One factor that increases the risk for having a baby with Down syndrome is the mother’s age. Women who are 35 years or older when they become pregnant are more likely to have a pregnancy affected by Down syndrome than women who become pregnant at a younger age. However, the majority of babies with Down syndrome are born to mothers less than 35 years old, because there are many more births among younger women.

Diagnosis

Doctors can diagnose Down syndrome during pregnancy or after the baby is born. Some families want to know during pregnancy whether their baby has Down syndrome. Diagnosis of Down syndrome during pregnancy can allow parents and families to prepare for their baby’s special needs. 

Screening for Down syndrome

Screening tests often include a combination of a blood test, which measures the amount of various substances in the mother’s blood (e.g., MS-AFP, Triple Screen, Quad-screen), and an ultrasound, which creates a picture of the baby. During an ultrasound, one of the things the technician looks at is the fluid behind the baby’s neck. Extra fluid in this region could indicate a genetic problem. These screening tests can help determine the baby’s risk of Down syndrome. Rarely, screening tests can give an abnormal result even when there is nothing wrong with the baby. Sometimes, the test results are normal and yet they miss a problem that does exist.

A relatively new test used since 2010 to detect certain chromosome problems including Down syndrome, screens for small pieces of the developing baby’s DNA that are circulating in the mother’s blood. This test is recommended for women who are more likely to have a pregnancy affected by Down syndrome, especially mothers over the age of 35. The test is typically completed during the first 3 months of pregnancy, and it is becoming more widely available from Sequenom, Natera, and other companies.

Following a positive screening test, diagnostic tests are usually performed in order to confirm a Down syndrome diagnosis. Types of diagnostic tests include:

  • Chorionic villus sampling (CVS)—examines material from the placenta
  • Amniocentesis—examines the amniotic fluid (the fluid from the sac surrounding the baby)
  • Percutaneous umbilical blood sampling (PUBS)—examines blood from the umbilical cord

These tests look for changes in the chromosomes that would indicate a Down syndrome diagnosis.

What to expect after birth

Most pregnancies involving Down syndrome are uncomplicated and proceed normally. Once a baby with down syndrome is born, however, he/she should receive an echocardiogram (an ultrasound picture of the heart) because almost half of all babies born with Down syndrome have a heart defect. The doctor will usually order tests of the baby’s blood to confirm whether there is an extra chromosome (chromosome 21) in the baby’s cells and make an official diagnosis.

Living with Down syndrome

Down syndrome is a lifelong condition. Services early in life will often help babies with Down syndrome improve their physical and intellectual abilities. Most of these services focus on helping children with Down syndrome develop to their full potential and include speech, occupational, and physical therapy. These services are offered through early intervention programs in each state. Children with Down syndrome may also need extra help or attention in school, although many children are included in regular classes.

In addition to state-run programs, families of Down syndrome children often find support and resources by connecting with one another and joining national organizations such as National Down Syndrome Society and National Association for Down Syndrome. Several campaigns including Buddy Walk® and Down Syndrome Awareness Month have taken shape over the past few decades to raise awareness of and acceptance for those with Down syndrome. It is important for everyone to understand that people with Down syndrome can lead productive and fulfilling lives well into adulthood. They hold jobs, live independently and contribute to their communities.

Share this blog and raise money for Down syndrome research

In support of Down Syndrome Awareness Month, I hope you’ll take a moment to find out more about Down syndrome, or better yet to support a fundraising or awareness effort in your community. I personally invite you to share this blog to help raise money* for Down syndrome.

Thanks to all of our loyal readers, we reached our initial goal and raised $1,500 for the Buddy Walk® in less than 8 hours! But we’re not stopping there, we’ll donate an additional $1* for every new share we receive.

* up to $2,500.

Hot Topic in Oligonucleotide Therapeutics: Nanomedicine

  • What is it?
  • Why is it Hot?
  • How do Nucleic Acids Fit in?

The upcoming Oligonucleotide Therapeutics Society (OTS) annual meeting on Oct 12-15 in San Diego has an agenda with a number of presentations that could each qualify as a hot topic to highlight in this blog post. While mulling over which to pick, the talk by Prof. Weihong Tan—an extraordinarily prolific researcher at the University of Florida—entitled DNA-based molecular medicine and nanomedicine triggered my decision to comment on nanomedicine because it’s at the nexus of multiple scientific disciplines and various aspects of medicine.

What is Nanomedicine?

Nanomedicine, like almost everything these days, has a wiki site; however, it simply defines nanomedicine as “the medical application of nanotechnology,” which begs questions of what’s nanotechnology and what are some of these applications? So, I dug deeper and found—to my surprise—that there is no widely agreed upon definition of nanomedicine!

Instead, several related but different definitions of nanomedicine have been adopted by groups of expert researchers that actually practice nanomedicine. There’s a scholarly editorial in the International Journal of Nanomedicine (IJM) that addresses this curious circumstance. At the risk of “losing you” by being pedantic, here are several snippets that were my key takeaways from this article:

  • “Although defining a term such as nanomedicine may sound simple, by comparing several main funding agencies from around the world, one quickly realizes that a uniform international definition of nanomedicine does not currently exist. This is typical of a new field, but can be problematic to those trying to understand the field, make significant contributions to it, and especially in how the public views nanomedicine.”
  • “[In NIH’s 2006] Roadmap for Medical Research in Nanomedicine, [it] is defined as ‘an offshoot of nanotechnology [that] refers to highly specific medical interventions at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve’.”
  • “[N]anomedicine emerged from nanotechnology which is generally defined by the creation and use of materials at the level of molecules and atoms (sometimes specifically less than 100 nm, other times this dimension is more diffuse and confusing).”

The last point resonated with me, and prompted a search for a picture that puts nanometer dimensions into perspective relative to familiar objects. The scale shown below nicely achieves that and highlights the 10-100 nm range applicable to representative nanodevices used in nanomedicine.

Nanometer dimensions of common objects (taken from piterest.com via Bing Images).

Nanometer dimensions of common objects (taken from piterest.com via Bing Images).

In closing this section, it’s worth pointing to two authoritative sources of up-to-date information on nanomedicine. First is the NIH website dedicated to nanomedicine, and second is the American Society for Nanomedicine (ASN) website. BTW, the current president of ASN is Georgetown University Prof. Esther Chang, whose cell-targeted nanoparticle delivery investigations were featured here earlier this year in a post entitled Nanomedicine: Using ‘Tiny Little FedEx Trucks’ to Target Breast Cancer Tumors.

Why is Nanomedicine Hot?

Before offering my personal perspectives on why nanomedicine is hot, perhaps I should first convince you that it is hot. For instance, entering a general search of PubMed using the word nanomedicine found over 8,000 publications since 1999, with the number of nanomedicine-related publications growing exponentially since 2006—coinciding with the aforementioned NIH Roadmap’s funding. These articles reached an impressive rate of ~4 publications per day in 2012.

This intense interest in nanomedicine over that short period is underscored by a similar search of Google Scholar that found over 97,000 items, while that for Google Patents found nearly 24,000 items.

As for why nanomedicine is hot, let me first echo what I said in the introduction above, i.e., nanomedicine is at the nexus of multiple scientific disciplines and various aspects of medicine. Given this circumstance, nanomedicine as an application is exceptionally diverse and, hence, powerful. This potential power, which can enable major advances having medical significance, is emphasized in the concluding statement taken from the IJM editorial mentioned above:

“IJN takes a firm stance…and emphasizes nanomedicine research in which significantly changed medical events are elucidated only by concentrating on nanoscale events. In this respect, our attempt to separate nanomedicine from other traditional medical research fields is a focus on significantly changed medically related events that result by concentrating solely on the nanoscale.”

How do Nucleic Acids Fit in?

I’m neither clairvoyant nor privy to Prof. Tan’s upcoming lecture at OTS, but I’ve used the illustration below to point out a prime example of the kind of application his talk will likely refer to: tumor-specific, nanoparticle-encapsulated, drug delivery enabled by nucleic acid aptamers. This therapeutic application of aptamers—and its “chemical cousin” wherein imaging agents are delivered—can be read about in two earlier posts from October and November of last year. In brief, nucleic acids provide 3D-structure for aptamers to allow specific bonding to “molecular markers” on target cells or tissue.

Targeted nanoparticles for imaging and therapy (taken from medicalnewstoday.com via Bing Images).

Targeted nanoparticles for imaging and therapy (taken from medicalnewstoday.com via Bing Images).

More futuristic applications of DNA in nanomedicine are predicated on predictable “self-assembly” of nano-scale structures via base pairing to achieve a functional state. This remarkably powerful attribute is unique to nucleic acids. This is elaborated on in the following abstract of a review by Chhabra et al. entitled DNA Self-assembly for Nanomedicine:

“Self-assembling DNA nanostructures based on rationally designed DNA branch junction molecules have recently led to the construction of patterned supramolecular structures with increased complexities. An intrinsic value of DNA tiles and patterns lies in their utility as molecular pegboard for deterministic positioning of molecules or particles with accurate distance and architectural control. This review will discuss the state-of-art developments in self-assembled DNA nanostructural system. Biomedical aspects of information guided DNA nanostructures will also be summarized. We illustrate both the use of simple DNA artworks for sensing, computation, drug delivery and the application of more complex DNA architectures as scaffolds for the construction of protein and nanoparticle arrays.” 

The Future is Now!

You may be thinking that achieving self-assembling DNA nanostructures for practicing nanomedicine is decades away and, even if doable then, it will be limited to only a handful of experts. Parabon NanoLabs, however, is proving that may not be true.

Parabon NanoLabs, with the support of an NSF Technology Enhancement for Commercial Partnerships grant, is using a simple “drag-and-drop” computer interface and DNA self-assembly techniques, to develop a new automated method of drug development that could reduce the time required to create and test medications.

“We can now ‘print,’ molecule by molecule, exactly the compound that we want,” says Steven Armentrout, the principal investigator on the NSF grants and co-developer of Parabon’s technology, according to an online interview. “What differentiates our nanotechnology from others is our ability to rapidly, and precisely, specify the placement of every atom in a compound that we design.”

The Parabon Essemblix Drug Development Platform combines computer-aided design (CAD) software with nanoscale fabrication technology, developed in partnership with Janssen Research & Development, LLC, part of the Janssen Pharmaceutical Companies of Johnson & Johnson.

Scientists can use the CAD software to design molecular pieces with specific, functional components for development of a new drug-delivery agent, as depicted below. The software then optimizes the design using a cloud supercomputing platform that uses proprietary algorithms to search for specific sets of DNA sequences that can self-assemble those components.

Parabon Essemblix process (credit: Parabon NanoLabs)

Parabon Essemblix process (credit: Parabon NanoLabs)

With the resulting sequences, the scientists chemically synthesize trillions of identical copies of the designed molecules. The process, from conception to production, can be performed in weeks, or even days — much faster than traditional drug discovery techniques that rely on trial and error for screening potentially useful compounds, the company says.

Parabon R&D scientists also claim that in vivo experiments, funded by an NSF SBIR award, have validated this approach to faster, more targeted rational drug design, which—as the saying goes—is a good thing.

Frankly, I’m amazed and truly excited about this advance in nanomedicine made possible by oligo base-pairing rules! I also look forward to hearing Prof. Tan’s talk at OTS and finding out more about the latest developments in nanotechnology. If you’re attending OTS, please look for me at the talks as I’d really like to meet you!

As always, your comments are welcomed.

Practical Applications of DNA Testing

  • Italy is Keeping Cities Beautiful by DNA Testing Dog-Droppings
  • Fines Issued to Owners who Don’t Pick Up
  • Smart Idea or Story-plot for an Italian Comic Opera?

As you know, I like to explore all things related to nucleic acids. In this post, I take a little respite from scientific analysis and instead explore an interesting real-life application for DNA testing. As summer comes to an end and we’ve all returned from our recent holidays, this post provides a tie-in to Naples, which is both a great vacation destination and a place where dog-droppings are a controversial topic. Sound intriguing? Read on.

Naples’ historic city center, which is listed by UNESCO as a World Heritage Site, is the largest in Europe, covering 4,200 acres and enclosing 27 centuries (!) of history. This historic city is leading the way in employing modern technology to keep its streets clean. Naples is the first metropolitan city to use “CSI-style” DNA forensics to rid its sidewalks of dog poop and impose stiff fines to those who let Fido leave his DNA behind. It’s a story that strangely juxtaposes quaint European old-city charm with an interesting mix of science and societal topics to evoke a wide spectrum of opinions.

By the way, in researching Naples, I learned that it has long been a major cultural center with a global sphere of influence, particularly during the Renaissance and Enlightenment eras. In the immediate vicinity of Naples are numerous culturally and historically significant sites, including the Palace of Caserta and the Roman ruins of Pompeii and Herculaneum. Naples is synonymous with pizza, which originated in the city as Neapolitan flatbread and migrated to America with the Italians. Neapolitan music has furthermore been highly influential, credited with the invention of the romantic guitar and the mandolin, as well as notable contributions to opera.

Good Idea or Waste of a City’s Resources?

Despite its glorious past, the city is not without it’s slew of current problems. Tommaso Sodano, the vice mayor of Naples, recently acknowledged in an interview that the city is facing many challenges related to huge debt, unfunded service agencies, wide-spread organized crime and general filth related to illegal dumps and dog droppings…yes, poop.

The city administration is positioning Naples ‘at the cutting edge of dog-waste eradication’. The initiative takes DNA samples of dogs to create a database used to identify and fine owners who leave their dog’s poop on city streets.

“I know some people find it funny,” Mr. Sodano is quoted as saying, smiling, “that with all the problems the city has, we would focus on dog poop. I know that.”

Via Toledo is Naples’s principal shopping street and a “must do” for tourists (taken from Wikipedia)

Via Toledo is Naples’s principal shopping street and a “must do” for tourists (taken from Wikipedia)

While it may sound a bit humerous at first, it’s an issue that is wide-spread and difficult to control as there are some 80,000 dogs in Naples. While other cities have tried everything from mailing dog poop back to its owner to publicly shaming offenders, Naples is pursuing a more modern approach. Blood samples will be taken from every dog in the city and used to create a database matched with contact information for the dog’s owner. ‘When an offending pile is discovered, it will be scraped up and subjected to DNA testing. If a match is made in the database, the owner will face a fine of up to 500 euros, or about $685.’ The program is in its infancy, but officials say it’s already making an impact on Via Toledo and surrounding areas where it’s being piloted.

Veterinary workers in Naples drew blood from Fiona, a pit bull, for the DNA database. Credit Gianni Cipriano for the NY Times.

Veterinary workers in Naples drew blood from Fiona, a pit bull, for the DNA database. Credit Gianni Cipriano for the NY Times.

Many residents and officials are skeptical that the program will work given the city’s existing challenges with basic services like garbage collection and sewage. Others disagree with the program’s expenditures in light of the city’s mounting debt. Sodano remains committed to the program saying “The main goal is respect for the rules.” He’s quick to add that other mounting issues shouldn’t prevent city administration from keeping Naples beautiful. “Governing Naples,” he said, “certainly requires a sparkle of madness.”

What’s the DNA-Based ID Used?

I tried, unsuccessfully, to find out what DNA-based identity testing is being used in Naples. In researching this, however, I did find the abstract of a relatively recent publication that suggests to me that Naples may be analyzing mitochondrial DNA (mtDNA), analogous to TriLink’s forensic products (mitoPrimers™) for human identity testing using PCR-sequencing.

According to this abstract, the researchers sequenced the entire ∼16 kb canine mtDNA genome of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. They then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region sequence—identical in all dogs—for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. They identified a total of 71 informative SNPs that they concluded are “useful forensic tools to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).”

Even though we don’t know the exact details of the testing being used in Naples, I wrote this post because I find the practical applications of DNA-based testing to be fascinating. If you know of other real-life applications, please share them in the comments section below.

ALS Ice Bucket Challenge: What You May Not Know but Should

  • The Story of How this Challenge Began
  • How Baseball and the “Big Bang” are Connected to this Challenge
  • How a Dearth of Genetic Understanding Led to Crowdfunding ALS Research
  • The First Drugs May be on the Horizon

How It Began

For those very few of you who have for some reason been disconnected from mainstream and social media, the ALS Ice Bucket Challenge involves dumping a bucket of ice water on someone’s head to promote awareness of the disease amyotrophic lateral sclerosis (ALS) and encourage donations—typically $100—to research ALS. The challenge dares nominated participants to be recorded having a bucket of ice water poured on their heads, and challenging others to do the same. A common stipulation is that nominated people have 24 hours to comply or forfeit by way of a charitable financial donation.

Those of you already familiar with the ALS Bucket Challenge may, however, not know that it was first issued by golfer Chris Kennedy to his cousin Jeanette Senerchia of Pelham, New York, whose husband, Anthony, has had ALS for 11 years.

Anthony and Jeanette Senerchia of Pelham and their daughter Taya, 6, watch as local politicians participate in the ice bucket challenge, July 25, 2014 outside Pelham (NY) Town Hall. Photo: Tania Savayan; taken from lohud.com via Bing Images.

Anthony and Jeanette Senerchia of Pelham and their daughter Taya, 6, watch as local politicians participate in the ice bucket challenge, July 25, 2014 outside Pelham (NY) Town Hall. Photo: Tania Savayan; taken from lohud.com via Bing Images.

According to a local newspaper story, Jeanette Senerchia, 40, said “I was going to donate,” but they were really relentless with their texts counting down the time.” When she finally poured icy water on herself, having her 6-year-old daughter Taya record it, she posted the video on Facebook and “it went crazy,” she said. In a little more than a week, 1,000 people across the country, in Canada, Europe and Japan had joined in.

Soon the challenge circled back to Pelham, where Town Supervisor Pete DiPaola took a dousing, along with Town Councilman Timothy Case and Court Clerk Fran Ardisi. State Assemblywoman Amy Paulin and Westchester County Legislator Jim Maisano also got in on the act.

Girl Scouts from Troop 1662 did the honors in front of Town Hall, pouring the water from giant, 5 gallon Home Depot buckets baring the words “Let’s DO this.”

Photo taken from lohud.com.

Photo taken from lohud.com.

In drought-stricken regions a cool—pun intended—variant has been proposed as seen in this video wherein San Luis Obispo, California photographer Brittany App—yes, it’s really her last name—has a compromise: instead of dumping a bucket of ice water on one’s head, she is challenging people to live on only 5 gallons of water for a day.

BTW, lest we get caught up in all the fun associated the ALS Ice Bucket Challenge, keep in mind that ALS currently has no cure and affects an estimated 350,000 individuals around the globe, killing more than 100,000 annually. The disease can impact anyone, anywhere, regardless of age, ethnicity, or socioeconomic background.

Famous Persons with ALS

While I will echo the last sentences again, it’s apparent that society in general has a fascination—for lack of a better word—in knowing which famous persons have reached a certain age, or married, or passed away, or become afflicted with a disease. In the case of ALS, a website lists a number of such persons among which the most famous are American baseball great Lou Gehrig and British theoretical physicist Stephen Hawking. Captions for their photos below provide further information about each of these two “professional polar opposites” who nevertheless have ALS in common.

lou

Henry Louis “Lou” Gehrig (June 19, 1903 – June 2, 1941), born Ludwig Heinrich Gehrig, was an American baseball player in the 1920s and 1930s, and set several Major League records and was popularly called the “The Iron Horse” for his durability. His record for most career grand slam home runs (23) still stands today. At the midpoint of the 1938 season, Gehrig’s performance began to diminish. After six days of extensive testing at Mayo Clinic in Rochester, Minnesota, the diagnosis of ALS was confirmed on June 19, Gehrig’s 36th birthday. ALS is commonly referred to as “Lou Gehrig’s disease” in the U.S. Photo taken from exequy.wordpress.com via Bing Images.

 

 

hawking

Professor Stephen Hawking—born Jan 8, 1942 in Oxford, England—has conducted work concerning the basic laws that govern the universe itself. Along with Roger Penrose, he has shown that Einstein’s General Theory of Relativity implied space and time would have a beginning in the, “Big Bang,” and end in “black holes.” In regards to the disability Stephen experiences, he has some things to say: “I am quite often asked: How do you feel about having ALS? The answer is, not a lot. I try to lead as normal a life as possible, and not think about my condition, or regret the things it prevents me from doing, which are not that many.” Photo taken from picfind.bloguez.com via Bing Images.

 

 

 

ALS Genetics in Brief

Given that this blog deals with “all things nucleic acids,” it’s apropos to mention a bit about the genetics, but quickly add that they are quite complex, as can be read about here.

There is a known hereditary factor in familial ALS. A defect on chromosome 21, which codes for superoxide dismutase, is associated with ~20% of familial cases of ALS, or about 2% of ALS cases overall. This enzyme is a powerful antioxidant that protects the body from damage caused by superoxide, a toxic free radical generated in the mitochondria. Free radicals are highly reactive molecules produced by cells during normal metabolism. Free radicals can accumulate and cause damage to DNA and proteins within cells.

This mutation has over 100 different genotypes. The most common ALS-causing mutation is a mutant SOD1 gene, seen in North American patients; this is characterized by an exceptionally rapid progression from onset to death. The most common mutation found in Scandinavian countries, D90A-SOD1, is more slowly progressive than typical ALS and patients with this form of the disorder survive for an average of 11 years.

In 2011, a genetic abnormality known as a hexanucleotide repeat was found in a region called C9orf72, which is associated with ALS combined with frontotemporal dementia ALS-FTD, and accounts for some 6% of cases of ALS among white Europeans. The gene is also found in people of Filipino descent.

I found it rather surprising that, where no family history of the disease is present—i.e., in a whopping ~90% of cases—there is no known cause for ALS! How stressful it must be to be afflicted with a disease having no known cause and no drug. Consequently, it’s easy to understand the importance of raising more research funding through the ALS Ice Bucket Challenge, and other money raising endeavors such as the following.

Crowdfunded ALS— Project MinE

I’ve previously commented here on the rapidly growing popularity of crowdfunding as a new, socio-web-based mechanism for “reaching out” to obtain money for conducting scientific research—including ALS. Project MinE is an independent, large-scale, whole-genome research project that has been initiated by two Dutch individuals with ALS and started on World ALS day (June 21) last year. These individuals provide their personal views on Project MinE at their Treeway website, which provides several modes of “communal contributing” that include jumping into Amsterdam canals (!!) as well as the now “classic” Ice Bucket Challenge.

Project MinE is a research project aimed at systematically interrogating the human genome for common and rare genetic variation in ALS—aka genetic “data mining.” The project will involve obtaining donated-DNA sequence information for 15,000 ALS samples and 20,000 healthy controls to obtain a large number of single-nucleotide polymorphisms (SNPs). This and additional sequencing will be performed on a sample size large enough to reliably analyze whole genome sequencing data outside of a family context.

The long-term benefit of the approach taken for project MinE is a catalogue of many non-ALS whole genomes that can be used to investigate other human diseases, including Diabetes Mellitus, some types of cancer, and other neurological disorders. Project MinE is the largest genetic study worldwide for ALS and was started in the second quarter of 2013. Complete information—and details on how to donate may be found at the Project MinE website, which I encourage you to visit.

Harvard Team Reports Possible ALS Drug Target

I thought it would be best to end this post on a positive note—specifically a possible drug target for ALS that is the subject of an online account by Cynthia Fox in Drug Discovery & Developement. Snippets of this exciting story are as follows.

A Harvard University team reported they may have found an ALS therapy—or two! When they blocked a gene for prostanoid receptor DP1 in ALS brain glia cells in a dish, neurons made from human embryonic stem (ES) cells were “completely protected” from death.

When they created ALS mice with that same gene deleted, the mice lived 6.7 percent longer.

This helps validate the idea that neurons made from human stem cells—in a dish—can be drug screens, team leader Kevin Eggan told a press conference. The 6.7 percent survival increase may rise even more, he said, if/when their DP1 antagonist is given with a drug his team earlier found has anti-ALS properties. And as both drugs are FDA-approved for other indications, clinical trials could move fast.

“We think this is a significant advance—both in terms of the use of stem cells for understanding disease, and with respect to understanding the degenerative processes of ALS and how we might inhibit them,” said Eggan.

Let’s all hope that this all proves to be true, and soon!

As always, your comments are welcome.