Friday, December 27, 2013

Genetically Silenced Apple. Science or beliefs?

 A biotechnology melodrama in Tweets.

Hendrick Goltzins The Fall of Man 

Have you heard of the non-browning  Arctic Apple engineered to market sliced apples and reduce waste? The GMO apple is galaxies away from fruit Adam and Eve tasted in the Garden of Eden.

 It's worth reading some science going into its creation to learn if we feel at ease taking a bite and tasting this modern fruit....dwelling a bit on the tree of knowledge good or evil, science or belief?

If, like me,  you have ever felt like some GMO scientists sound more evangelical than scientific by the time you are done reading this modern  Twitter drama there will be less doubt your feelings are legitimate.

Though it might give you a little headache if you aren't  a scientist, I think it'll be worth your time. Knowledge is like that. Biotechnology science is as fascinating as it gets, even if a little complicated.

 It could be a fun exercise in separating science from illusions of science-beliefs.

If you aren't science minded skip the details. Ask yourself a basic question "am I seeing statements fitting better in the science or religion category"? The fog might lift a just a little.

Science 101: 
Customarily presented in a form of shiny citations to data ( non-scientists might find annoying).
Usually containing raw numbers, science symbols, charts or graphs.
Core tenet:  questioning assumptions.:
 " What is the evidence for my theory/   hypothesis /  belief     ???????"

Religion--belief based.
No data, proof, or evidence required.*
Science citations inevitably absent.
Assertions made requiring zero proof.
* Don't recall the last time they showed numbers or graphs at a religious ceremony.
 It is a good thing, too -can't remember the last spiritual, transcendental graph I've seen, do you? 


According to the New York Times, these apples are neither wanted by consumers nor apple growers. 

I have some questions about testing before taking a bite, don't you? [1] [7]
Were precise profiling techniques used e.g. transcriptomics, proteomics, metabolomics ( the "omics")?
Any feeding trials conducted on laboratory rats?
The answers appear to be "None" & "No".

If approved, the Apple will be one of the very few products designed with silencing technology intended for use as food for people, and it will have evaded proper safety testing.  [9]

Unlike the common herbicide-tolerant and insecticidal GMOs,  the GMO apple was not created by the addition of genes. (+) 

A genetic engineering technique called RNAi or RNA interference was usedDouble-stranded RNA (dsRNA) which binds messenger RNA (mRNA) preventing the production of the browning enzyme; PPO  was introduced.  (*)

So as promised here comes the Tweeter-drama. As the USDA was wrapping up its public comment period on the Arctic apple advocates  took to Twitter, where the following ensued and was  storify'ed  for you.

 Darryl Hannah tweeted a link to her followers encouraging them  to voice their disapproval

 Predictably "scientists"  flocked to "educate the masses".

The website is a  glossy promotional site without a single science citation to be found.

So I pressed the scientist & Co again, and again, and again, and again to post links to science on off-target effects (OTE).
 You see it is very well known that today's silencing technology leads to unpredictable unintended effects causing surprises.
 " When designing therapeutic short-interfering RNAs (siRNAs), off-target effects (OTEs) are usually predicted by computational quantification of messenger RNAs (mRNAs) that contain matches to the siRNA seed sequence in their 3' UTRs ( untranslated regions) [..]

We observed no correlation between the number of computationally predicted OTEs and the actual number of seed-dependent OTEs (P=0.76). On average only 20.5% of actual transcriptional OTEs were seed-dependent (i.e., predicted)." [6]

Sometimes as discovered in a study on bees - scientists silenced genes they didn't  even know existed!  [3]

 A study on insecticidal RNAi reported that dsRNA  engineered to kill insects produced unintended secondary dsRNA with different sequences and different targets than originally planned which did the insects in. [4] A similar process of generation of secondary dsRNA has been described in plants.[8]

Off-target effects causing unpredictable gene silencing are far from conspiratorial. They are discussed by USDA's scientists [5]

 Suzy, the scientist claims  there have been 10yrs of testing which, while true,  is misleading because  they were years of agronomic research and development, rather than safety testing.

  Not a single study on any experimental animal-lab rat or human- exposed to this silenced fruit  is anywhere to find. Not a single "omics" study ( transcriptomics, metabolomics or proteomics) [7]  is cited in spite of multiple requests.

 Thus saying  they are nutritionally equivalent is a leap of faith- not rigorous science.

The tweet below is where it got really interesting!

1. She is unaware that a study recently completed at Rutgers University demonstrating short dsRNA (microRNA)  in the viscera of rabbits fed a therapeutic (antiviral) microRNA engineered tomato.  The scientists filed a patent for plant-based therapeutic applications using this technology.  Thus, her declarative statement that microRNA won't survive digestion is steeped in a brew of ignorance  and is scientifically untrue.

2. It should be clear to readers that absence of PPO ( browning enzyme) and its gene in humans is irrelevant in the context of off-target effects in the plant itself or animals ingesting the silencing gene carrying apples.

3. She appears unaware of a study demonstrating microRNA from bacteria, fungi and even insects has been detected in human plasma. How did they get there? Did she not know or has she not bothered to ask?  [2]. Oh, yeah--Monsanto insists microRNA can't be absorbed, and is still arguing that microRNA are a contaminant to avoid safety testing its crops.

4. But, there is not a single study anywhere in the literature showing rates of degradation of double-stranded RNA at Ph 2!   Never mind in the range of gastric pH's (2-5)  in adult humans.

 So Suzy, the Scientist, can Not back up any of her claims. Her only refuge would be to call me names, which she already tried, but I skipped that part because ad hominem statements starting with " you are...X, Y, ...Z" are so common in these GMO  "conversations/ religious conversions."

 Thus, I pressed the scientists to show me a study on degradation of dsRNA at ph2. Here is what happened.

Dr Suzy the Scientist posts a  link to a study on chemical hydrolysis of ribonucleic acids. Not only is it completely irrelevant to dsRNA, but the hydrolysis reaction takes place after an hour of boiling. How many people do you know who stew their stomach contents at 220F for an hour?

As a brilliant scientist  watching this little drama unfold commented: "Funny how they so adamantly claim these things but cannot show any data."

The stuff sounds pretty indestructible now.  Of course, it isn't, because we have lots of enzymes called pancreatic RNases to digest RNA, Suzy the Scientist evidently forgot about.

OK!  Finally! After days of unscientific nonsense, we arrive where we should have started --at our first science citation. Or rather  a citation to a study on pancreatic ribonucleases (RNases), enzymes that degrade RNA, which, by the way,  have little to do with chemical degradation at pH 2.

First, no data on how fast the dsRNA disappears from the stomach, nor anywhere else. It was bacterial dsRNA, not plant dsRNA.  Nor was this a paper measuring acid stability, but enzymatic stability.

No relevance to how fast the dsRNA disappears and whether or not it is rendered inert during digestion time under physiological conditions.
(Thanks to Dr Heinemann!)

I did eventually get a citation to a second study on degradation of dsRNA in the stomach kindly posted by Dr David Tribe aka GMOpundit.

The trouble is that Dr Tribe's study actually showed  that  dsRNA is stable for extended periods of time relative to time in stomach (10 to >24 hours) and is  not more unstable at pHs that might be relevant to human digestion (5)

Fig 3  at >ph 6.8. dsRNA detectable up to 10 hours at all pHs tested.
Detectable at 24 hours at all except pH 6.8 (and this appears to be an anomaly).
Fig 4 they test to only as low as pH 5, not pH 2  Still present after 24 hours at the LOWEST tested pH of 5!

Curiously enough, Dr Tribe stated "  Simply put, the relevant wheat RNA molecules will be rapidly digested in the gut to harmless, simple nutrients." on his blog discussing silenced wheat.

It turns out there might not have been any science evidence to back up that assumption/ hypothesis/belief.

As far as I can see in this little melodrama, NO studies showing these novel stable RNA molecules that we've never eaten- showing it gets degraded in the human gastro-intestinal tract - were cited at all.

I am of the mindset now that some "scientists" either have never questioned their deeply held assumptions, maybe since high school. Someone way smarter than myself once said:  "assumptions are called ASSumptions because they make ASSes out of us". Or they are willfully selling illusions they know nothing in common with science, and much more in common with religious beliefs.

Unfortunately, the unconscionable denial of risks of RNAi is hindering promising medical research!, which is most definitely very tragic.


1. Securing the safety of genetic modification
Dr Jack A. Heinemann

2. The complex exogenous RNA spectra in human plasma: an interface with human gut biota? 
 PLos One 2012;7(12):e51009. doi: 10.1371/journal.pone.0051009. Epub 2012 Dec 10.

3.  Non-Target Effects of Green Fluorescent Protein (GFP)-Derived Double-Stranded RNA (dsRNA-GFP) Used in Honey Bee RNA Interference (RNAi) Assays

4. Nat Biotechnol 2007 Nov;25(11):1322-6. Epub 2007 Nov 4.
Control of coleopteran insect pests through RNA interference.

5.  RNAi-Based Insecticidal Crops:  Potential Effects on Nontarget Species
Jonathan G. LundGren and Jian J. duan

6. Br.J.Cancer 2013 Feb 5;108(2):450-60. doi: 10.1038/bjc.2012.564. Epub 2013 Jan 8.Lack of correlation between predicted and actual off-target effects of short-interfering RNAstargeting the human papillomavirus type 16 E7 oncogene.

7.  Environment International 37 (2011) 1285–1293
Review Molecular profiling — a tool for addressing emerging gaps in the comparative risk
assessment of GMOs.
Jack A. Heinemann a,b,⁎, Brigitta Kurenbach a,b, David Quist b
a School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand b GenØk – Centre for Biosafety, Tromsø, Norway
Table 1
Expert views on profiling.
Method Positive perspectives on profiling
High throughput sequencing. High-throughput sequencing proved to be a powerful and quantitative method to sample transcriptomes deeply at maximal resolution. In contrast to hybridization, sequencing showed little, if any, background noise and was sensitive enough to detect
widespread transcription in N90% of the genome, including traces of RNAs that were not robustly transcribed or [were] rapidly degraded. (Wilhelm et al., 2008)
Proteomics Therefore, DIGE [DIfference Gel Electrophoresis] is highly appropriate for comparative profiling of knockout, transgenic, or isogenic germplasm as well as defined pharmacological or stress induced responses as recent reports suggest. Another important consideration is that quantitative studies using 2-D gels in general, and 2-D DIGE in particular, can be performed with any plant
species and are not restricted to plants with sequenced genomes. (Thelen and Peck, 2007)
Proteomics Inter-experimental reproducibility is not good using 2-dimensional polyacrylamide gel electrophoresis approaches, however, an excellent differential display technique (DIGE) allows multiple samples to be compared on the same gel and is a powerful tool in biomarker discovery for laboratory exposure studies which is quantifiable. (Van Aggelen et al., 2010)
Metabolomics [Catchpole et al. (2005)] demonstrated the compositional similarity between GM and non-GM potatoes using mass spectrometric fingerprinting as a primary screen, which was then amended by detailed quantitative profiling analyses. This approach (i.e. combining non-targeted and targeted analytical methods) appears feasible even for screening large numbers of transgenic plants, and the application of both supervised and unsupervised data analysis techniques ensures independency from statistical bias. (Rischer and Oksman-Caldentey, 2006)
Metabolomics [The Catchpole et al. (2005) study was] the first report of a large scale metabolomic analysis of field grown GM potato plants, and it highlights the importance of analytical technology and appropriate data analysis for the safety assessment of GM crops. (Colquhoun et al., 2006)
Metabolomics The work reviewed here demonstrates that the tools that are currently available for metabolite profiling are mature and robust enough to facilitate their use in the investigation of biological processes.(Schauer and Fernie, 2006)
Metabolomics In particular, an impressive number of natural foods, spices and beverages have already been the subject of detailed metabolomic-based component analysis, including milk, grapes, tomatoes and tomato juice, rhubarb, beer, celery seeds, coriander as well as many other herbs and spices. These analyses used a combination of NMR, GC–MS, LC–MS and CE techniques
to identify up to 100 different phytochemicals or 200 different carbohydrates in selected fruit, vegetable or beverage samples. (Wishart, 2008)
Mass spectrometry [It is now] possible to choose from a variety of techniques, such as flow injection electrospray ionisation mass spectrometry (FIEMS), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR), which facilitate rapid fingerprinting of crude extracts, [to provide profiles of chemical compositions of GM plants].(Rischer andOksman-Caldentey, 2006)


9. A comparative evaluation of the regulation of GM crops or products containing dsRNA and suggested improvements to risk assessments
  • a School of Biological Sciences, University of Canterbury, Christchurch, New Zealand
  • b Centre for Integrated Research in Biosafety, University of Canterbury, Christchurch, New Zealand
  • c Crop Science Department, Federal University of Santa Catarina, Florianópolis, Brazil
  • d Health and the Environment, School of the Environment, Flinders University, Australia
  • e Institute of Health and Environmental Research, Adelaide, South Australia, Australia
  Open Access


(+) Technically, the Arctic Apple was created by inserting a new piece of DNA. The new piece is designed to be transcribed like any other gene making a single stranded (ss)RNA. The ssRNA has internal complementarity, so once it is made it forms a stem loop structure (see Fig 1C of the paper I link to above). This structure is then a substrate for DICER (or Drosha) and can subsequently enter the RISC complex that directs it to mRNA targets (intended or not). [9]  See cool video here

In theory, it is not necessary to introduce the DNA to get the effect. Animals take up the dsRNA directly and there are plans to make dsRNA-based pesticides that are directly taken up by plants or animals. Ironically, those pesticides may not need regulatory approval because some are arguing that unless there is a DNA step, as in the Apple, it is no longer genetic engineering! Another attempt to subvert safety testing using semantics.

(*)   mRNA which guides production of protein should not be confused with miRNA ( microRNA), which are an example of a short double stranded (ds) silencing RNA.