Commentary to the International Monsanto Tribunal verdict

Introduction
Daimonie (Facebook) blogs about science and scepticism.

This seems to be how anti-GMO
advocates think it works.
In October 2016, I wrote a post just before the International Monsanto Tribunal. Since then, I also started on my series regarding GMOs in general. But back in October 2016, I concluded:

As expected, the entire Fake Tribunal is a theatre. This is not a scientific conference, nor a court of law. It is quite literally a gathering of people that use pseudo-science to promote "natural" and "organic" alternatives to conventional and contemporary agriculture. Their most important part is in no way their data, their science, or the interests they represent; rather, it is their ability to form incomprehensive gish gallop combined with fear-mongering.
By doing so, they cast doubt on the credibility of science and technology. Worse, they negatively influence public opinion on contemporary agricultural technology that has the potential to greatly improve upon agricultural practises, in ways that benefit health through nutrition (e.g. Golden Rice), reduce pesticide usage (e.g. Bt Crops, Lepidoptera-resistant crops) and improve farm life.
Now, the verdict is here. In this post, I will provide a commentary so that you don't have to watch it. I'll also use their Advisory report. Most of their testimonies have been discussed in my earlier blog post. Amusingly, the questions asked have changed and the sections in this commentary do not line up with those of my earlier blog post.


It starts in a scene of deliberate aesthetics, looking like it isn't heavily sponsored. The introductory statement follows.

Judge Tulkens

Judge Tulkens starts out by trying to seem credible; but it is just a kangaroo court. They're not tasked or required to use anything, even though she claims they do. All of the judges are, apparently, practising legal experts or judges. No official from Monsanto turned up at the kangaroo court, and she points this out for some reason. A lot of references to legal matters follows, clearly an attempt at seeming credible. The tribunal has taken up the form of the court of criminal justice in the Hague.

Here comes the first funny claim. They received the written reports of scientific experts; of course, not of any of the thousands of scientists in the field that agree with GMO safety. Nor any of the precision agriculture Nobel prize winners. Judge Tulkens says the tribunal has no reason to doubt the sincerity or veracity of the testimony before them, which wasn't cross examined nor given under oath. And she points out they can't establish the facts of the allegations. What exactly are you doing? Isn't cross-examination sort of the point? The tribunal is thus trying to inform the media, politicians and public of the consequences of heavy speculation. How useful!

Mr. Orellana: the right to healthy environment

Mr. Orellana is a law lecturer also involved in Center for International Environmental Law. He starts out by tediously rephrasing the right to a healthy environment to its fundamental character.  He also mentions a number of relevant legal cases, verdicts and such. So, the device by which the tribunal can deliver a verdict is clear; now it just needs premises that are met.

Now, to the witnesses. In rapid fire, Mr. Orellana states that the Tribunal heard testimony and documentation on the impact on human health regarding Monsanto's activities. These issues included soils, plants, animal health, biodiversity, farmers and scientific researchers. These affected both indirectly and directly the right to a healthy environment.

As summarised in their report (II Q1.ii Testimonies) it looks like a heavy list. But all of these cases were discussed in my previous blog post, purely based on some research done before they presented their testimony. It is essentially a list of things that happened when glyphosate was nearby, not even necessarily Monsanto's brand. Glyphosate was nearby, so correlation is confused with causation and Monsanto carries the blame.

Mr. Orellana states that Monsanto has engaged in practises that negatively impacted health and environment. That's simply not true; Monsanto is accused of such, but no precedent exist to show any credibility to the accusation. Neither does peer-reviewed research. The premises are not met, and the verdict as presented is false. I'd like to point to the European Food and Safety Authority (EFSA) report:
The substance is unlikely to be genotoxic (i.e. damaging to DNA) or to pose a carcinogenic threat to humans. Glyphosate is not proposed to be classified as carcinogenic under the EU regulation for classification, labelling and packaging of chemical substances. 
 Even worse, Monsanto engages in GMOs. This is mentioned in relation to Roundup Ready products, and only the IARC report classifying glyphosate as 'probable carcinogen' is mentioned. Not the EFSA or WHO reports that followed quickly after, nor the scientific body of literature showing safety. Just the one, heavily criticised, report classifying it as 'probable carcinogen' (which is interpreted as carcinogen).

Any allegation made is cited as if it is true; this is their base system. Any allegation is taken as true, while anything that does not agree with the obvious story they want to tell is blatantly ignored. These 'legal experts and judges' apparently do not feel this compromises their credibility.

Judge Lamm: The right to food

The format is that the Judge first outlines what is under discussion. In this case, it is not only the right to eat but also to feed properly and sufficiently, healthily and permanently and having the possibility to produce. 

A list of testimonies follows, naturally all showing an infringement of this right. As reported to the Tribunal, Monsanto's activities have negatively affected food availability and interfered with the ability to feed.

One of the more amusing claims is that Monsanto's activities deny access to seeds, because they may be too costly. Given that Monsanto does not control any market and that traditional and non-traditional varieties are available, this is simply false. They complain about Monsanto practise forcing farmers to buy their seeds every year, being denied the option of saving seed. Seed saving is hardly modern, as we've been using various cross-bred hybrids and such for a number of years. A field dedicated to harvesting something usually isn't dedicated to forming seeds for the crops of the next year. What is more, it is not Monsanto practise that denies the option but rather national and international patent law which predates GMO seeds and Monsanto. Judge Lamm notes that seed-patents are also an infringement of the right to food, utterly and blatantly denying the reason why patent law exists in the first place; to allow an innovative company to earn money for their investments. Patent law is a form of intellectual property right.

An interesting point is raised. Judge Lamm states that the rise in Organic agricultural practises illustrates that farmers with less or without pesticides and other chemicals is feasible, and that these can deliver the yield to feed the world population. She then claims that this is a sustainable approach to agriculture. The first claim is false, in the sense that the organic yield gap simply means it is less effective (e.g. PLOS One, organic yield averages to 80% of conventional). The second fails in part because the lower yield means the impact per product unit is reduced. If the environmental impact is 90% of that of conventional at 120% of the area, the impact is higher - pure land use is environmental impact as well. And that is on average; I wonder what happens if we take modern practises such as no-till farming.

Judge Lamm: Right to Health

The right to health encompasses both physical and mental aspects. It extends to well-being, such as living arrangements and such. Judge Lamm claims Monsanto not only affected negatively the health of communities but also their mental health. She claims that Monsanto has used PCBS, pesticides and GMOs - "dangerous substances" as she calls them - in their activities.

PCBs are an organic chlorine compound used as coolant fluids, heat transfer fluids and carbon-less copy paper. If you wonder why we haven't heard this one before, it seems to be because it wasn't brought up by any witness. Monsanto and Monsanto spin-offs have been involved in various (real) court cases regarding PCBs, PCB misuse, PCB dumping and so on. While all rather horrid, the real horror is that some part of the company knew of the problems (per internal leaked documents, 2002 [Wiki]). Apparently, these documents were presented to the Tribunal at some point. I'm not sure if this was Monsanto, the agriculture company which exists today, or the former Monsanto Chemical Company. 

Now, we turn to RoundUp. She notes that the combination with RoundUp ready (glyphosate-resistant) crops has led to an increase in glyphosate. What she doesn't mention that this is just about the popularity of this particular herbicide rather than the total amount of pesticide used or its environmental impact. It's well documented that the total amount used is decreased and that glyphosate has less environmental impact than (most) alternatives. The IARC report is mentioned again, but not the EFSA report mentioned earlier nor the Joint FAO/WHO meeting which concluded that it is not necessary to establish an ARfD for glyphosate or its metabolities in view of its low acute toxicity. They do cite an Open letter which states concern over the EFSA decision in 2015. Rather than look at the EFSA decision, they're citing an open letter. Another report is mentioned, this one also not by a scientific body.

They do mention EFSA and EPA reports, but note that some researchers said they disagreed at a later time. This panel notes considerations beyond their ability to judge. They state that while the studies didn't show significant results, a meta-analysis does. With all due respect, Judge Lamm, but can I interest you in a rigorous course of high-school level statistical analysis? This effectively means that yes, they do ignore the EFSA and EPA reports, but refer to the Minutes of a meeting to justify this. In those minutes, the panel concluded that the EPA's review and evaluation chose relevant studies and has a sound, appropriate and acceptable approach. The panel members agreed that based on the evidence presented, there is no association between glyphosate and any solid tumor, leukemia or Hodgkin's lymphoma. This is evidence of clear quote mining. Based on the above, I reject their claim: they do ignore EFSA and EPA reports without justifiable basis. She also states another report does not take into account the risk of exposure; a rather nonsensical point, as this is neither a part of hazard assessment nor particularly relevant as it is extremely low.

Next, they cite 'evidence from released Monsanto documents of an effort to manipulate the science'. Documents from last month can't really be properly addressed as either testimony or documents submitted to the Tribunal. What is more, there is no evidence of such. There is no scientific controversy over glyphosate safety. A singular paper wouldn't even change that.

Regarding GMOs, they claim an absence of consensus. The consensus is there and is very clear. It is underlined by various agencies, such as WHO, EPA and EFSA. There is a public controversy, which doesn't matter, and different countries or unions differ on opinions regarding how strongly the precautionary principle should lead. They point to some concerns such as inadequate design of regulatory frameworks (which are updated frequently) and dependency on company data. The latter is easy to address; do independent studies show the company data is false? So far, they do not (regarding GMOs). There is no lack of transparency and independent researchers are free to test. They also repeat the claim that 'pesticide and herbicide usage per hectare dramatically increased', which is false. The absolute usage and its impact per area has decreased, even though crop land and yield has increased. This is well documented:
 On average, adopters of GE glyphosate-tolerant (GT) soybeans used 28% (0.30 kg/ha) more herbicide than nonadopters, adopters of GT maize used 1.2% (0.03 kg/ha) less herbicide than nonadopters, and adopters of GE insect-resistant (IR) maize used 11.2% (0.013 kg/ha) less insecticide than nonadopters. When pesticides are weighted by the environmental impact quotient, however, we find that (relative to nonadopters) GE adopters used about the same amount of soybean herbicides, 9.8% less of maize herbicides, and 10.4% less of maize insecticides.

Judge Shrybman: Freedom Indispensable for Scientific Research

Judge Shrybman begins by stating that objective and unbiased scientific research gives critical and necessary information about (environmental) risk, a rather nice statement. He also mentions the right to freedom of expression and the right to give and receive information. He states that the need for such has become particularly acute with the election of political leaders with little if any regard with science, fact or reality based advocacy or decision making. Again, this statement is very fine by itself although it seems the Tribunal is not held against this standard. 

He mentions most testimonies were given by scientists, either public or private. A quick look at their advisory report tells us that (for instance) he means Seralini, widely criticised for inadequate and quality-lacking experimental and analytical methods. Also Lovera, who was accused of both incompetency and corruption. I have detailed these testimonies in my earlier blog post. Judge Shrybman then lists a number of accusations, concluding that the accusations must be true. It is honestly beyond me why alleged legal experts are treating accusation and allegation as fact without any cross examination that does not serve their story.

Judge Shrybman details that there is a difference between making claims where legitimate scientific uncertainty exists and taking direct steps to discredit or silence scientists when their findings are not convenient to a particular business model. The latter involves conduct that frustrates the scientific project and that is intended to undermine and silence scientists. This conduct offends and can not be reconciled. I agree, but that begs the question: What are you doing there?

Judge Tulkens: War crimes complicity and Agent Orange

Agent Orange was used as a part of the herbicidal warfare program of the Vietnam War US Military, which had a number of agrochemical companies make (parts of) Agent Orange. So the simple answer is that the US Military is the complicit party, while all manufacturers are informed contributors.

Judge Tulkens starts out with a short summary of Agent Orange and its use in the Vietnam War. A legal action was filed against various of the Agent Orange manufacturers. These decided to pay collectively a fee to these Vietnam veterans. 

Concerning Monsanto's complicity, she claims no evidence was provided and the Tribunal cannot make a definite finding. Nevertheless, they are judged guilty. It is true; the old Monsanto company did manufacture Agent Orange and did know what it was contributing to. Whether that makes a company a war criminal is a different question. It is unlikely any individual working at the company at that time is still working there. How is this even a relevant question? Is a steel manufacturer that knowingly provided steel to an arms company that provided missiles for the US military complicit in their use of that missile against a civilian target? I don't think so, but the sheer weight of the question turns many away from pursuing even such far-removed manufacturers as an employer.

Mr. Orellana: Ecocide

Could past and present practise of Monsanto constitute causing serious damage to the environment significantly affecting the ecosystem services upon which humans rely? He summarises legal precedent for the crime of ecocide, which seems fine.

Surprisingly, the verdict is that Monsanto is guilty. The reasons are the manufacturing of herbicide used by the US military, the large scale use of dangerous agrochemicals in industrial agriculture, the engineering, production, introduction and release of genetically engineered crops, severe contamination of the ecosystem and introduction of PCBs into the environment.

To summarise what we have said about this; the Tribunal accepts all allegations as true. Regarding its role as a manufacturer for herbicides in militant use, the question is whether you condemn all manufacturers or are unjustly targeting Monsanto. Monsanto merely produces agrochemicals, the one under discussion being glyphosate which was found to be safe by numerous agencies across the world. I'm not sure what industrial agriculture must be, as this is a fantasy construct to contrast organic farms and has no real meaning, nor is any such meaning detailed in the report or presentations. Contamination of the ecosystem seems to be bad practise of various parties, not equal to nor limited to Monsanto. Introduction of PCBs is similar to the question of military use herbicides; Monsanto wasn't the only one to do so nor where they the only one to continue after research came up that showed it was not safe.

Conclusion

All allegations are true; we conclude that the alleged kangaroo court is actually a kangaroo court.
There is not too much exciting here; while they cite relevant legal literature as precedent, the devices to condemn Monsanto require premises to be true that have been deconstructed in my earlier blog post and by various agencies, scientists, scientific bodies and others around the world.  As the premises are rejected, the conclusions are discarded. The Kangaroo court has no valid conclusion, other than the organic industry clearly having a majority in donations around this theatre. Actually, we don't know that because they didn't release their sponsors and their contributions.

Luckily, the totally not-involved public is given the word. After a few questions from the public, a man stands up in the back of the audience. This man, dressed in a suite already carries a microphone and, by clear accident and providence, already is standing in front of the cameras (plural). It is Seralini, who had no involvement at all. He outlines his CV in French and then asks about lobbies and claims to be a victim of these lobbies. He then takes about fifteen sentences to ask about the data of papers submitted to regulatory bodies and transparency regarding this matter. He claims nobody was able to get the data which was authorised by the industry and repeats the lie that he proved glyphosate, or rather, Monsanto's brand, was much more toxic. He claims it is not the active ingredient of Round Up, and neglects to mention that both private and public enterprise have tested both glyphosate and Round Up, that there are other producers of glyphosate and that the widespread criticism he faces based on the lack of any solid science in his work. This takes up about half an hour of question time, where the judges mostly underline whatever it is that Seralini said. A few more short questions follow.

The attempt at impressive theatre shown in the Kangaroo court did little that wasn't expected. I can't stress enough that all allegations were accepted without cross examination, and any contribution that did not serve the anti-pesticide, anti-GMO worldview was blatantly ignored. Consider that they used minutes of a meeting, which outlines agreement with findings of EFSA, EPA and WHO, to dismiss those same findings. This because it noted a few scientists 'disagreed' (rather, they pointed out areas where the evidence should be increased). 






































Let's talk about GMOs, part two: What are we using them for?



Introduction

In my first post, I discussed what DNA is and how it relates to phenotypical (outward) change through the central dogma of molecular biology. In the second post I discussed the techniques of genetic engineering, including the novel and prodigal technique called Crispr/Cas9.


We will now look at the current uses of the technology. I  do notwant to include research purposes in this overview, but in the next post because they are very relevant. We'll start with the most important current application, and move down from there towards areas that are not fully developed yet.

Medicine

I think this is our most important current application. We've already covered Insulin when discussing the technique, but there are others. The general name they use for this application is biologics made by recombinant DNA technology. A more common name is bio-pharmaceutical, also known as biologic(al) medical product. In principle, bio-pharmaceutical refers to any pharmaceutical whose manufacturing involves biological sources [wiki]. Wikipedia gives a list of the major kinds of such bio-pharmaceuticals:
An example of a book written on the principles and
applications of biotechnology. [ISBN-13: 978-1555814984]

  • Blood factors
  • Thrombolytic agents
  • Hormones 
  • Interferons
  • Interleukin-based products
  • Vaccines
  • Monoclonal antibodies
  • Additional (e.g. tumour necrosis factor, therapeutic enzymes)

This seems to bode well for the amount of examples we can find. This is not surprising; universities offer both undergraduate and graduate programs that focus on this topic alone. For illustration, I found a book (right) that is purely on the principles and applications of recombinant DNA technology, first written in 1994 and now in its 4th edition (2009). I'll try to give an example of each item in the list.

Hemophilia is caused by the failure to produce certain proteins required for blood clotting. A patient has a reduced or no ability to form blood clots and continues to bleed, even from minor injuries. In the late 1950s, the technique used for treatment of Hemophlia was 'Fresh Frozen Plasma'. In essence, donor blood was centrifuged so that only the plasma (containing trace amounts of clotting factors) was left. This was then administred, in large quantities, to Hemophilia patients. The technique was later refined; the trace amounts were separated. Even so, the amount of donor blood used to make a small amount of clotting factor was tremendous.  An additional issue is that viruses, such as hepatitis viruses and HIV, were transmitted with the plasma-derived clotting factors. Steps were taken to prevent this, but it was still of great concern. In 1984, the factor VIII gene was cloned. Three years later, recombinant human factor VIII entered clinical trials in humans. Factor IX was cloned as well, and both (recombinant) factors entered the market in the 90s [Hematology.org].

Thrombolysis is the breaking down of blood clots using medication. It is almost the opposite of blood clotting factors. It is often used as emergency treatment to dissolve blood clots that form in arteries feeding the heart and brain. These are the main cause of heart attacks and strokes. Another good application is clots in the legs, pelvic area or upper extremities. While these seem innocent, the clots or pieces of it can break off and travel to an artery in the lungs, causing acute pulmonary embolism [webMD]. I found a rather nice resource detailing the modifications made for the production of Tenecteplase, one of the approved medicines for Thrombolysis. It also notes the 'Pharmocodynamics', which explains how they work. In this case, it binds to clots rich in  'fibrin' and cleaves another protein. That protein's two parts degrade the fibrin, which eliminates the blood clot (Thrombus). Chinese hamster ovary cells are the cell line of choice. No, that doesn't mean hamsters are slaughtered for this purpose; they are cells first derived from a chinese hamster, but have mutated to reproduce under laboratory conditions. This is called an Immortalised Cell Line. The chinese hamster line is a common one, often used for this sort of research or application. A plasmid induces the gene for human Tenecteplase and a antiobiotic resistant gene. The cell line is allowed to reproduce and is then purged to find the succesful cells. There is no antibiotic present in the final product [FDA].

The most important human Hormone has already been discussed: Insulin. Another example is the Human Growth Hormone, first extracted from the pituitary glands of cadavers. Recombinant biotechnology made it possible to produce these hormones in bacteria. The recombinant form is called somatropin [Wiki]. Human growth hormone stimulated growth, cell reproduction and regeneration in humans and other animals. Somatropin is used as a prescription drugs to treat children's growth disorders and adult deficiencies. It is also banned from use by competitors in sports as a performance enhancing drug.

Interferons are a group of signalling proteins made and released in response to the presence of several pathogens such as viruses, bacteria, parasites and tumour cells. In a typical scenario, a virus-infected cell will release interferons causing nearby cells to heighten their anti-viral defences. An example is Intron® A ([Merck]. This is another example of plasmid-induced medicine production in E. coli (like Insulin). It is used as a prescription medicine for various types of cancer, sometimes along with chemotherapy. Additionally for chronic hepatitis B and C with stable liver problems. Note that it has serious side effects [FDA], but that these are not related to the technique but to the substance.

Interleukins are another group of signalling proteins, first observed by white blood cells (leukocytes). A number of rare deficiencies of interleukin have been described, featuring autoimmune diseases and immune deficiencies. For example, Interleukin 2 greatly promotes the cell division, and thus amount, of certain T cells. T cells are an elementary part of the immune system. Aldesleukin is an example of recombinant interleukin (2), and is used against cancer, in particular metastatic liver and skin cancer [chemocare].

Recombinant vaccines are a subject so important that Nature, the giant of scientific publishing was my top result on Google. Nature features a dedicated 'subject' on recombinant vaccines. They summarise:
A recombinant vaccine is a vaccine produced through recombinant DNA technology. This involves inserting the DNA encoding an antigen (such as a bacterial surface protein) that stimulates an immune response into bacterial or mammalian cells, expressing the antigen in these cells and then purifying it from them.

A new and exciting form is just entering the research area, which we'll talk about in the next post. For now, I want to explain about 'DNA vaccines' and 'protein subunit vaccines' [genScript], both of which have just left the research area. Before we do that, we need to get a confusing term out of the way. An antigen is a molecule capable of inducing an immune response on the infected/host organism. It is any substance that causes the immune system to produce antibodies against it. Often, antigen and antibody confused.

DNA vaccines insert a purified plasmid, often cultured in E. coli, into the vaccinated individual. These plasmid only produce the antigen, leading the body to recognise and response as if the entire pathogen was there. However, only a antigen is made; it does nothing to you. But as they say, we need to use and boost our immune system. While that is a rather flawed statement, vaccines do exactly that; we use our immune system by giving it a harmless antigen that will teach it what to attack [WHO]. I do want to point out only a few are available for veterinary use and but a singular one for human use [Wiki]

Protein subunit vaccines are another example of the power of recombinant DNA technology. Antigens are produced in E. coli or other cells (e.g. yeast) and purified from there. Injection of these antigens triggers the immune response and memory, so that the recipient becomes immune. The two techniques listed here, and the new one I'll return to, are all techniques that synthesise part of a virus so that the host can be immunised in a carefree manner [Journal of Pharmacy and Pharmacology]. For instance, the protein subunit vaccine for Hepatitis B is composed of synthesised surface proteins only [GELifeSciences].

Monoclonal antibodies bind to specific antigens and induce an immune response. They are a part of possible cancer treatment, with several approved by the FDA for this purpose. They are also used for autoimmune disease because they can target the triggers of these diseases. Of course, you need to make a specific antibody; in the case of cancer, it can even be that you need to tailor the antibody to this specific cancer [Wiki]. It is clear where genetic engineering plays its role, typically by the plasmid method on E. coli [Methods in Molecular Biology]

I think the above gives a fair indication of genetic engineering in medicine. A lot of different applications have been found, and this increases ever more. And you can imagine what the impact of Crispr/Cas9 will be, as it makes the entire process significantly simpler.

Genetically Modified Crops

Ordinary rice (left) versus Golden Rice (right) [[Business Insider].

This is where the public debate rages. I will remind readers that this post is not about the safety of things, but about the current applications of the technology. We will discuss the safety, regulations and ethics behind it in another part of the series. In this section, I want to discuss the currently available technologies. I won't go into the companies or their practises. I also won't discuss the future uses, which is a different post in the series.

A lot of the currently available crops are herbicide resistant. The first technology we want to look at are GM soybeans, whose patent has expired and which are now in the public domain.

The soybean are engineered to be resistant to the herbicide glyphosate. You might have heard of that pesticide; it has accumulated interest when one branch of the World Health Organisation (WHO) classified it as 'probably carcinogenic to humans', much like grapefruit juice or working a night shift [IARC]. That branch was then found to be wrong by other branches of the WHO, but also by other agencies such as the European Food Safety Authority [EFSA]. As one of the most used pesticides [Nature] it understandably receives a lot of criticism from anti-pesticide groups and the organic lobby. While this might seem about safety, it is too recent and relevant not to mention it. We'll discuss this in depth in the safety post.
Acreage in Yield of soybean in the US [USDA].

Why would you want a glyphosate-resistant soybean? Is it so you can spray more? Not really, because glyphosate is expensive. While it is true that a farmer can spray more herbicide in a single application, the amount of applications over a growing season is less. A rather nice report featuring a metric (number representing fairly) of the toxicity of a pesticide and the amount of active ingredient shows that the amount of active ingredient has been reduced since 1996 and the environmental impact has fallen by 20.4% [ BioFortified ]. A more recent version of that report, covering the period of 1996-2013, concludes:

Across all of the countries that have adopted GM Herbicide Tolerant soybeans since 1996, the net impact on herbicide use and the associationed environmental impact has been: 

  • In 2013, a 3.1% increase in the total volume of herbicide active ingredient applied (6.8 million kg) but a 9.3% reduction in the environmental imapct 9measured in terms of the field EIQ/ha load);
  • Since 1996, 0.1% less herbicide active ingredient has been used (2.3 million kg) and the environmental impact applied to the soybean crop has fallen by 14.5%.
However, these seem to be absolute numbers. The chart on the left shows us that since 1996, a 15% increase in the acreage of soybeans in the US is also present. That would indicate a 13% decrease in herbicide active ingredient per acre, which I consider to be a good result. The environmental impact would have lessened by more than 25% per acre. (If we assume the increase in the US is typical.) The report also details the increases in yield for soybeans and many other GM crops.

Most of us feel uncomfortable with the idea of spraying pesticides on our crop. However, we have no such issue with plants that deal with their pests by themselves. Why not make them? These plants would be extremely specific, only dealing with the pests that feed on them. This could reduce insecticide use, pest suppression and conservation of beneficial natural enemies. More than 66 million hectares of Bt crops were planted in 2011 alone, accounting for 67% of corn in the US in 2012 and 79-95% of cotton planted in Australia, China, India and the United states during 2012-2012. In India, for instance, the average Bt cotton farmer realises a pesticide reduction of roughly 40% and a yield increase of 30-40%. Farmers their satisfaction is reflected in a high willingness to pay for Bt seeds [Journal of Agrobiotechnology Management & Economics].

Of course, such uses have drawbacks, particularly insects evolving resistance. Numerous strategies are being implemented and concern to deal with that problem. The primary one is called the refuge strategy, which is that most of the rare resistant pests (mutants) surviving on Bt crops will mate with the relatively abundant susceptible pests from nearby refuges of host plants without Bt toxins. If inheritance of resistance is recessive, the progeny from such mating will die on Bt crops, delaying the evolution of resistance [Nature Biotechnology].
Timeline of the Golden Rice project [IRRI].

There are of course many possible other traits that could be introduced in GM crops. For example, Golden Rice. Vitamin A deficiency is prevalent among the poor whose diets are based mainly on rice or other carbohydrate-rich, micro-nutrient-poor calorie sources. Rice does not contain any pro-vitamin A, which their body could convert to vitamin A. Vitamin A deficiency compromises the immune systems of approximately 40% of children under five in the developing world, being most severe in Southeast Asia and Africa [goldenrice]. What if we could make a simple change, introducing a variant of Rice that does have pro-vitamin A? This is the driver behind the International Rice Research Institute. The project is non-commercial; the idea is to develop the product and give it to those who need it. In June 2016, 123 Nobel Laureates wrote to Greenpeace, the UN and the governments around the world. They stated that organisations opposed to modern plant breeding, with Greenpeace in the lead, have repeatedly denied these facts and opposed biotechnological innovations in agriculture. That they misrepresented the risks, benefits and impacts, supporting the criminal destruction of approved field trials and research projects. They specifically called for these organisations to abandon their campaign against GMOs and Golden Rice in particular [SupportPrecisionAgriculture].

Again, I am not able to give you a complete overview of all the different technologies that are the result of modern plant breeding, as the laureates so beautifully put it. However, I do think this is a nice first look at it. Having covered medicine and crops, we move on to modified Animals.

Animals

I've alluded to genetic engineering as a vibrant and active area of research. For animals, this too is true. We will again skip this topic, because we discuss the use of genetic engineering in research in the next post. Given both the difficulty of genetically modifying image and the strong precautionary principle at work, the number of technologies that we can discuss in this section is limited.

First, we have an anticoagulant antithrombin that is gathered from a genetically modified goat. It is the first medicine produced using genetically engineered animals, producing the same amount of antithrombin in a year as ninety thousand blood donations. It has been approved by the FDA in 2009. A point of concern that is always stronger when we speak of animals is the well-being of the host animal. There are no ill-known effects; it's just a milk goat [Wiki].

A genetically altered zebrafish, called the GloFish for the US brand, is a patented brand of fluorescent zebrafish with bright red, green and orange fluorescent colour. It is only sold as a pet in the United States and Taiwan. Originally, the fish was a part of a research project. Zebrafish are model organisms, a species that is extensively studied to understand particular biological phenomena. Dr. Gong and his colleagues at the National University of Singapore were working on making a zebrafish that would signal pollution by selectively fluorescing in the presence of pollution [Wiki]. The concept of having fluorescent fish that respond to certain chemicals seems to be one that he is still interested in.

AquAdvantage salmon via Nature news.
AquAdvantage salmon is a genetically modified Atlantic salmon that is also triploid. Triploids means that the gametes of these animals have three sets of chromosomes [Wiki], a trait that is not characterised as genetic modification. The sterility arises when only one sex is released; having three sets of chromosomes, their gonad development is impaired. For female triploids, they do not develop at all. Male triploids undergo partial or altered puberty, but cannot reproduce [Sustainable Fish Farming]. Triploid fish have been used in the US state of Washington to stock waterways for angling. AquAdvantage salmon have the trait of directly producing a growth hormone protein, achieving accelerated growth rates. Inducing triploidy reduces the risk of interbreeding with wild-type fish and increases growth by removing the stress of reproduction [Wiki]. The AquAdvantage salmon has been approved for human consumption, and is the first genetically modified animal with that approval [Nature News].

Conclusion

We've now discussed a number of currently available technologies. Surprising to some, most of the current applications lie in medicine, not agriculture. Part of the reason that medicine is not discussed as much is because it is seen as a more complex topics, the details of which people do not want to know. Agriculture and farming in general is quite complex, but more accessible to people their imagination. Even so, people often think that their experience managing a few square metres of vegetables is sufficient data to make claims about agriculture.

Additionally, especially when discussing the genetically modified salmon, we have touched on some safety concerns. In the next post, we will continue speaking of genetic engineering applications, now with a focus on applications in research. That will be a difficult post, largely because research concerns are often quite complex and arcane. However, it will also be interesting because it opens up the Horizon of genetic engineering applications.




Let's talk about GMOs, part one: What techniques are used?

Introduction


In my previous blog post, I discussed what DNA is and how it relates to phenotypical change through to central dogma of molecular biology. In this post, we will discuss techniques currently in use.

The techniques I want to discuss are not only those that have been used in genetic engineering, such as those used commercially to engineer the Bt. crops. I also want to discuss those that are currently in use, particularly Crispr. The latter is a very recent technique that has revolutionised the way genetic engineering works.The goal is not to give an extensive review of techniques, but rather to give readers some insight into common techniques.

Producing Insulin

Synthetic biology doesn't make nice stories. The amount of different tools (enzymes) used and their abbreviated names obfuscate the essence of any story.I want to discuss a few specific tools.

Let's first discuss the bacterium; E. coli. This bacterium can be found in the lower intestine of most warm-blooded organisms and is a common model bacterium in biological research. It has a circular segment of DNA carrying its genetic information; this literally means a circle of DNA.

Typical bacteriophage (wiki).
In 1972, researchers devised how to make recombinant DNA by using two tools (Wiki). The first tool was a restriction enzyme. Restriction enzymes are very specific enzymes that can cut DNA at a specific code (wiki). For instance, the Hind-iii restriction enzyme cuts DNA at the sequence 'AAGCTT' (wiki). The second tool is a DNA ligase (wiki), which is an enzyme that fuses two large pieces of DNA.

But that doesn't put the DNA inside a host cell's genetic material, such as E. coli. What is used for that is a bacteriophage. Bacteriophages are actually bacteria-viruses. As most viruses, they're quite simple - a protein 'vessel' that transports genetic material. A bacteriophage floats around until it comes near a bacterium. It then bonds to the wall of the bacterium, where it breaks down a part of it and empties into the bacterium - the host cell.

As I said, they're fairly simple. Simplicity is not always ideal, but leaves an opening for genetic engineers. Bacteriophages can be 'loaded' with DNA of another origin. In that way, a genetic engineer might make DNA that he or she wants to insert into E. coli - such as the DNA that produces Insulin. The method used by the researchers in 1972 can be combined with PCR (1983) to create plasmids of your choice.  PCR is a technique that thermally separates double-stranded DNA into single-stranded DNA. You then add DNA Polymerase, which is an enzyme that copies DNA. This gives you double stranded DNA again, after which you start the next cycle by thermal separation. This chain of reproduction cycles is called the Polymerase Chain Reaction, or PCR. The following picture give an illustration. The difference with the picture is that when you use to create the DNA 'primer' (short piece) that you want is that the red building block actually extends over the edge of the green one. PCR used for DNA sequencing only adds the nucleotides (=single letters) so that it copies the DNA many times.
PCR reaction.The red primer (and blue complementary chain) will outnumber the green strands in a few cycles. As the added nucleotides (letters) are just molecules, the product of the PCR reaction can truly be called artificial. It's not (e.g.) plant DNA; it is a sequence equal to that present in plants. 

After you have the primer, you use the 1972 method to create a plasmid. Plasmids can be easily migrated into E. coli cells, which then have their own circular DNA floating around and the added plasmids. Some biophages will target the plasmid rather than the E. coli genetic material. Their 'goal' is that they switch their own biophage material with a piece of the E. coli genetic material. They do this by having a 'tail', which matches a part of the host material. Then follows the biophage material - instructions for making biophages - and finally another tail. Sometimes, it messes up - it inserts instructions to make more biophages with the DNA already in place, rather than the DNA of the biophage. The trick is to get the biophage to take the DNA primer we inserted.

You now have 'loaded' phages and 'wild' phages. A common way to make the distinction in the end was to use e.g. Insulin plus an antibiotic resistance gene. If you succeed, you can purge the 'wild' population with antibiotics.

The bacteriophages are added to a solution with E. coli, and they insert their DNA packages into the host cells. However, we want to have the 'insertion DNA' added to the genetic material of the host cell. Adding restriction enzymes, which break the DNA of the E. coli cells would be a good start. Adding a ligase would then randomly incorporate the 'insertion DNA' into the host genetic material. It is also possible to remove the antibiotic resistance gene. For this, you need to put code sequences on both side of the antibiotic code, called FRT regions. By then infecting the E. coli cell with a flippase, a plasmid of baker's yeast, you can remove the antibiotic gene. Flippase will sit between the two FRT sites, and twist - causing a loop. It then connects the two FRT regions - cutting off the remainder, which is antibiotic code in our example.

A lot of phages come with their own enzymes and ligases, which greatly simplifies the process. For E. coli, a common choice is the lambda phage (wiki).That is the basis of phage engineering. To summarise:

  1.  Use synthetic biology (PCR) to create the DNA segment you want to insert. Add the code for resistance to an (old) antibiotic and FRT regions around it. Turn it into a plasmid.
  2. Infect E. coli cells with your plasmid, then load phages. The phages multiply, and some get loaded with material from your plasmid.
  3. Add the phages to a population of E. coli bacteria.
  4. Purge the E. coli so that only those that have the segment remain.
  5. Remove the antibiotic gene.
  6. Feed them, nourish them and farm the Insulin they produce. 

This succeeded in 1978 (wiki), after just six short years (with a different technique to make the primer). This alone is a good indicator of how well these techniques worked. And don't forget the advances in techniques such as DNA sequencing, which allows you to check whether you succeeded in your engineering. For that, you'd add enough food that your genetically-enhanced E. coli multiplied their numbers, then separate part of their population and sequence their DNA.

Improving the quality, quantity or availability of food. 

Genetically engineering E. coli and yeast is all good, but can we do something else with it? Can we change the DNA of a plant so that it can withstand bugs, droughts, colds, rains and heat flares? That is one of many questions one can consider when hearing of genetic engineering.

Let's take one step back. Earlier, I mentioned DNA tails on the phage DNA that had to match the host. Why? The keyword here is affinity. A lot of molecular biology and biochemistry turns into the question of whether it is likely to happen. For something to be likely, we need to know the energy of something not happening versus that of it happening. Technically, we also need the temperature. The required concept is called a Boltzmann factor, and is part of the foundation of the Atomic theory. Before you run off, that's just the theory of there being atoms. Using Boltzmann statistics, we can derive most of the thermodynamic quantities discovered during the industrial age. Boltzmann statistics work well on the smaller scales, too. You can predict the natural shape of a large protein, for instance, based on Boltzmann statistics. If you do that for DNA, you find that something is needed to store our DNA (~2m length) in our cells.

The tails are the complementary code of a region of code on the DNA. Because they are complimentary, they have a very high 'affinity' for that region - the energy of bonding is very, very low. Most principles in nature (most of the physics you were taught in high school!) ultimately boil down to minimisation of energy. So it is for chemistry - affinity means minimal energy, and therefore far more likely.  The higher the affinity, the lower the energy of the combined parts, and the more likely it is for them to combine.

With a gene gun, you can take plant cells and deliver DNA-coated particles to them (wiki) . The DNA-coated particles are often just gold particles covered in plasmids - which we know you can design. This way, plasmids are delivered into a plant cell, a process that is called Transfection. Sometimes, these plasmids are incorporated into the host genetic material. This appears to be a lucky event rather than a targeted one. The same concept is used with plant cells as with E. coli; you use antibiotics or herbicide to kill the cells without the new code. Before you ask, antibiotics do kill other cells; it depends on the antibiotic. Some, for instance, just destroy cell walls - be they plant or otherwise. For a fully developed organism, this is unlikely and dosage is low. For a plant cell near a researcher trying to find the successful transfections (stable transfection), dosage is extremely high. Hence, antibacterial agents work.

Cetus corporation started out with automated breeding methods to select greater amounts of chemical feedstock, antibiotics and vaccine components. It entered the new fields of biotechnology. Later it merged with another company to create Agracetus, which created the first Roundup Ready crops using a gene gun. Five years later, this company was bought by Monsanto, creating the Monsanto Agracetus campus. That's right - Monsanto didn't first create that technology. And this wasn't a single corporation going into the fields of agriculture. The potential was realised, the race was on. To quote from Sir Terry Pratchett's works, it was steam engine time. Health organisations, such as the WHO, were already wondering about policy to evaluate these new, exciting products while not giving in to technological enthusiasm.

There are other methods of editing plants - such as using the Ti Plasmid,  also called the natural genetic engineer. However, the above should give you some insight into how this is done.

Crispr/Cas9

Illustration of Crispr/Cas9. See text for explanation.  Source: Nature
This is the new technique. The novel method that is revolutionising Academia - I can't tell you how often the abbreviation features on posters around the biophysics building (my workplace). The variety and enthusiasm with which people have grabbed onto this new technique since its introduction around 2012 is astonishing. In theoretical physics, a Nobel Prize was just given away for a field founded by a paper that was forgotten and ignored for thirty years or more (topological insulators).  Not so for biologists - they grabbed onto Crispr/Cas and immediately started working with it.

To explain the system, I will use some images from a Nature Biotechnology review. The illustration of Crispr/Cas9 is quite clear. Cas9 is a nuclease, a sort of molecular scissor. Cas9 is very particular; you give it a piece of Guide RNA (remember, the letters match with DNA - the backbone is slightly different). This way, the Cas9 only cuts at that particular code, called the specificity of the nuclease. In general, it has very high specificity, meaning that it only seldom (less than 1%) cuts at the wrong location. In the next image, a slightly more complicated systematic illustration is shown. In this figure, we have naturally occuring (a) Crispr systems that incorporate foreign DNA. This is the natural defence system of numerous bacteria - cutting up infections. In (b) we find the engineering approach, where the researcher provides the Guide RNA and the Cas9 enzyme cleaves (cuts) at that code. In (c) we see the natural guide RNA (top) and the engineered guide RNA (gRNA). As you can see, these are not that different.



After cleaving, it is possible to insert DNA.This is done by hijacking the repair pathways. In that way, the cleavage introduced by Cas9 can be filled up with the code that you wanted to insert. That story is slightly more specific, and I will leave it to the interested reader to search for it. There are two possibilities. The breaks introduced by Cas9 can be repaired by non-homologous end joining (NHEJ) or by homology-directed repair (HDR) pathways. See also the image to the right.
Repair of cleaved DNA with code
supplied by researcher (blue).
Another interesting application of Cas9 is inactivation. There's such a thing as dead Cas9, which has been modified so it can't actually cut. It will still move toward the targeted code, so it can block other molecular machines from sitting there. In this way, sites can be deactivated and the effect of that can be studied.

One other benefit of Crispr/Cas9 is that it can be done in vitro. That's pretty powerful, and can be used for all sorts of things. Let me put down a hopeful hypothesis. Gene-therapy for cancer. Take a sample from the tumor and from healthy tissue, and find code sequences that are different. Use those to target Cas9 to cancer cells, introducing a terminator gene. That's exactly what it sounds - it terminates the cell, by making it mess up its chemistry. Not only do you hit the tumor you sampled from, but also the smaller tumors it seeded. Can this become reality? Honestly, I don't know - my knowledge of the subject matter is broad, not specific. However, I do hope this can become reality!

Conclusion

Genetic engineering turned out to be a set of powerful techniques. The different techniques are different in simplicity of use, of concept and of specificity. But they are quite specific, and we have the technology to compare wild (unedited) DNA to target (edited) DNA to see if we succeeded. And we usually do.

Crispr/Cas9 is mostly making the rounds because of simplicity, broad applicability (many organisms) and because it also has extra options. It's also likely to get a Nobel prize, likely shared between a large number of people. And that's not only the prize for biology, but also that for medicine. And because food issues can lie at the root of conflict (war), it might even be the one for peace.

Yes, the technique has me excited. It's even more specific than the previous techniques, it has a high success rate, a high specificity, a broad applicability and so much more. Honestly, I can't wait to see what this will turn up. And by that I mean not only in commerce, but also in medicine, in agriculture, in helping developing agriculture, in understanding of nature and things I can't even imagine.

We've now discussed what DNA is and how we can change it. In the next post, I'll try to give you an overview of current uses of genetic engineering.