Monday, January 2, 2017

What are isogenic lines and why should they be used to study GE traits?

There has been quite a lot of talk about the latest paper from Seralini's group that claims that there are substantial metabolome differences between genetically engineered corn and non-GE corn. The paper was published in an online journal run by the Nature group (and not in Nature as some websites are claiming). At first glance, this paper seems to detail some results that are seriously concerning. However, when one examines the methodologies used, several glaring issues emerge that challenge the conclusions reached from the results presented. Many others have addressed several of the methodological problems with this study, but I'd like to focus on the corn lines that they used and the claims that they were isogenic as the entire experiment hinges on using the correct lines. 

To start with, I need to explain what an isogenic line is as most people (even other scientists outside of the plant sciences) do not know what this is. When discussing isogenic lines, it's not a single line but at least two lines. Genetically, these lines differ by only a few genes but are identical beyond that. Achieving this is nearly impossible, so researchers will use near-isogenic lines (genetically these are at least 99% identical). Generally speaking, near-isogenic lines (NIL) are not available for purchase and must be generated. To do this, a donor plant with the gene of interest (in this case, the gene would be a GE trait like glyphosate resistance or Bt production) is crossed with what is called the recurrent parent (see figure below). Using genetic markers (in a process called marker assisted selection), progeny with the appropriate genes are then back crossed against the recurrent parent and the trait selected for until the progeny are 99% genetically identical to the recurrent parent. It takes time and effort to generate an NIL like this, and there simply are no shortcuts to getting there.



Figure caption: In plant breeding, selected individuals are crossed to introduce or combine desired trait characteristics into new offspring; this necessitates numerous generations of backcrossing to establish the desired trait characteristics fully. Each successive backcross increases the genetic similarity of the new offspring to the recurrent parent, e.g. 75% similar at BC1 through to 99.2% by BC6. These numbers are based on how much of the recurrent parent genome can be theoretically regained at each step; however slight variations can occur. Marker-assisted methodologies that utilize DNA markers to enable selection of plant individuals that contain the greatest number of favorable alleles can reduce the number of generations required to get close to 99% similarity as adopted in the generation of the inbred variants of this study. From Harrigan et al., 2016 via PMC. DOI: 10.1007/s11306-016-1017-6

So what do NILs have to do with this new study and why do they matter? 
In order for the authors to clearly demonstrate that the differences seen are due to the GE trait (NK603, resistance to glyphosate), they need to use lines that have nearly identical genetic backgrounds. This is because it is well known that different lines have different transcript expression patterns, metabolomes, flavors, etc. For example, this paper by Wen et al. looked at the differences in the kernel metabolomes from different corn lines. This is not unexpected as different lines can have different phenotypes. One only has to look at a seed catalog to see the variation that is available for crops such as tomatoes or apples and yet these are the same species. Because of this, a NIL is needed to demonstrate that observed differences are due to the gene of interest and not the normal variation that is seen between lines.


In this study, the authors state that they are using isogenic lines in several places, but in the materials and methods they state they used the "closest" isogenic lines. For example, they state in the abstract that they used isogenic lines (see figure below).


In the materials and methods they state this: 


These are not isogenic or near-isogenic lines. The DKC stands for DEKALB seeds, which is owned by Monsanto, and the numbers are the identifiers used by the company to denote the line. These numbers are akin to a catalog number used to sell seed. The lines offered one year may not be the same ones offered the next. It's also important to note that these numbers do not denote lineages, which is a carefully guarded trade secret for seed companies. The authors did not provide any information on the lineage of the two lines (DKC 2678 or DKC 2675 [which was also labeled as DKC 2575 in some places in the manuscript]) and just because they have similar numbers, that does not mean that they are genetically related. To help illustrate this, I found a DEKALB catalog from 2012. On page 5, there is a line, DKC 27-55 that has VT Double PRO technology (a stacked Bt trait). On page 6, there is a line called DKC 27-45 that has a single Bt trait (YieldGard Corn Borer)and RoundUp Ready 2. DKC 27-55 was new for 2012 and DKC 27-45 was on the market for awhile and was a recommended line for growing silage (for more on what silage is, see this video from the Peterson Farm Brothers harvesting silage for their cattle). Although these two lines have similar DKC numbers, they have different traits and very different uses. Beyond just picking two lines with similar catalog numbers, there is another issue with their choice of lines; they used hybrids. 

With hybrids, two unrelated lines are crossed and the resulting progeny has higher yield through a process called hybrid vigor. It's very common for corn to be hybridized and it's something to be mindful of for a study looking at genetic effects as this can introduce a source of genetic variation if the same parent is not used in the hybridization process. If the lines did not share the same hybridization parent, then they would not be isogenic even if the original two lines were isogenic (for which no evidence is provided that they are). They ordered seeds that were already hybridized and provided no information on which parent was used to make the hybrid. Since there is no guarantee that the two hybrids share the same genes from the same hybridization parent, these cannot be considered isogenic. To do so is bad science.

The choice of lines used in this study introduces several major sources of variation that make it impossible to account for. Because the lines are not isogenic (or near-isogenic) and there is no information on if they were hybridized to the same parent line, it is impossible to say if the observed differences are due to the transgenic trait or due the fact that lines with differing genetics were used. The risk of misinterpreting the results based on this would be far too great and this type of experiment is too expensive to waste money like that. This is one of the reasons why researchers will generate their own NILs. Other issues with the study include the poor plot design that lacks randomization (or any other standard design for an experiment like this), lack of replications (different blocks within the field, other locations, repeated growing seasons, etc.) and the lack of information about these lines. No pedigree is offered and these lines are no longer on the market (I couldn't find any information on these lines online), so we can't say for sure how likely it is that they are closely related or not. This is bad science and this paper never should have made it through the peer review process with these and other major methodological issues intact (not to mention all of the grammatical and typographic errors).

This type of experiment really required the use of NILs that the researchers made themselves by crossing and backcrossing. Luckily such an experiment was published prior to the submission of this paper. Harrigan et al. (2016) examined this same issue. However, the experimental design for this study was superior in every way. The Harrigan study generated NILs for the same trait that the Seralini paper did (NK603); however, they generated four lines each that were transgenic or not as well as the recurrent parent (as a control). These lines were hybridized with two different female testers and planted in randomized complete block design with three replicate blocks in three separate locations (Illinois, 
Minnesota, and Nebraska). The metabolomes were then measured by GC-MS. They found few differences between the lines but that genetic variation between the lines accounted for more differences than the GE trait, for which no trait-specific effect was found.

As I stated above, there are numerous other issues with the methodology used in the Seralini paper that are not limited to just the use of improper lines. Generating NILs is time and labor intensive, but if you are truly trying to answer this type of question it is simply something that must be done. You cannot take shortcuts with experiments like this and you cannot assume that just because two lines have similar numbers in a seed catalog that they are related. Shortcuts in science lead to bad results that are a waste of time and money. 

Thursday, December 29, 2016

What has Andy Wakefield done for science?

With the resurgence of Andy Wakefield through his "documentary" Vaxxed, his prior work has come back into focus. It's clear that his work as a researcher contains many errors of omission and commission, but why is the scientific community certain that something nefarious happened in his prior work? There is of course the excellent work from Brian Deer showing how the paper published in The Lancet was fraught with fraud and undisclosed conflicts of interest (being paid nearly a half a million pounds to show that the MMR is dangerous is kind of a conflict of interest). That investigation led to Andy losing his medical licence in the UK. However, much of the focus has been on the 1998 Lancet paper and not as much on his other work. I'll discuss one of his other papers here as it is incredibly clear that at best, inept data analysis and sample handling was done.   

In 2002, Andy was an author on a paper entitled "Potential viral pathogenic mechanism for new variant inflammatory bowel disease" published in Molecular Pathology (the PubMed Central deposit can be found here). The paper uses qPCR detection of the Measles virus using the Taqman chemistry (see image below). 


This is how Taqman qPCR works.

The results of the paper look normal to the lay person; however, if a researcher who is familiar with qPCR looks at the raw data from the paper, an enormous problem is evident. I rarely share youtube videos; however, this video from C0nc0rdance explains qPCR and why the issue is such a huge deal.


For those who don't want to watch the video, the problem is simple. qPCR is done using a thermocycler that uses a laser to detect a fluorescent target and is linked to a computer. The software on the computer automatically does calculations for determining what is positive and what is not. One of the calculations that is done is the threshold for determining what is considered a positive reaction and what is not. In the 2002 study, the threshold was manually lowered so that samples that should have been negative came up as positive. These samples were all after cycle 30 which means that it was a small quantity of material being detected. Additional analysis of the raw data by Dr. Bustin (an expert on qPCR) found that these were false positives due to contamination. So we have two major issues to contend with here. First the samples were contaminated (qPCR is sensitive to contamination so extra care has to be taken to prevent this). Second, the data was improperly analyzed and the Ct threshold improperly lowered so that these samples became positive. At best, this is poorly conducted science that should be retracted. At worst, if the threshold was knowingly altered to generate positives, this would be scientific misconduct. It's not likely that the contamination was intentional as it was in low quantities (why spike a sample with an amount that would look like contamination rather than spike with an amount that would clearly be positive?) and some of the researchers involved in this study published a subsequent study that found no measles virus when the experiment was replicated (three independent labs did the tests on each of the samples), so the adjusted analysis was probably done in ignorance rather than malice.

But this paper (and several others) did have a positive impact on the scientific community. It lead to the creation of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE). These standards dictate what the minimum amount of information needs to be included with a paper using qPCR data. This includes information on how the samples were extracted, how the test was validated, what controls were used with the test, and how the data were analyzed. Many of the top journals have adopted these guidelines and many researchers follow them even if it isn't required for publication (it's just good science). 
Information needed to fulfill the MIQE guidelines.
So even though Andy Wakefield is at best an incompetent researcher (and a con artist at worst), he has done something good for science. As a result of his work (and the work of others), the standards for qPCR reporting have been increased so that the results are standardized across disciplines and publications. It does bring up an interesting question, how many of Wakefield's previous studies are faulty and what should be done about them. In this case, a correction should have been submitted, at the very least, when it became clear that the data analysis was not done properly and that the resulting conclusions are suspect. 

However, one thing is clear. Andy Wakefield is not the virtuous crusader for the truth that has been unfairly attacked by the forces of evil. Despite the narrative that he tells, his work speaks for itself. It is full of errors and improprieties that make it all suspect. His actions are what led to him being shunned by the scientific community. Andy shouldn't be blaming anyone but Andy. 

Saturday, December 3, 2016

Genetic modification of Influenza A virus reveal a novel vaccine production strategy

Researchers have developed a new type of vaccine. By genetically modifying the genome of Influenza A virus to require a non-standard amino acid (Ne-2-azidoethyloxycarbonyl-L-lysine which has been used in synthetic biology previously) that some microbes use. What makes this amino acid unique is its codon (three base RNA sequence that signals what the amino acid should be). In most organisms, the codon used (UAG) is actually what is known as a stop codon (it stops production of the protein chain), so this means that any gene that contains that codon will stop prematurely unless grown in an organism with that non-standard amino acid. A lot of work went into finding places in the viral protein that the non-standard amino acid could be inserted without interfering with the function of the protein. Once several sites were identified, the researchers grew the virus in kidney cells that had been genetically modified to use the non-standard amino acid. The end result was genetically stable progeny viruses that required the non-standard amino acid.

When the altered influenza virus was injected into mice, it did not replicate and the mice developed a strong immune response to the virus. Researchers were able to safely inject the mice with a dose of the modified virus that was about 100,000 times the dose of the wild-type virus that would kill half of the mice injected (LD50). The modified virus also interfered with the wild-type influenza virus in co-infections of the two.  

The implications for this development could be astounding. With this, researchers could develop a vaccine that uses a fully infectious virus that has been modified to need this amino acid to replicate. However, caution is warranted at this point. A lot of work still remains to develop this into a functional vaccine. It must go through further trials to see if it works in primates and then there are all the human clinical trials that are needed. This work is promising and certainly should be pursued further.


This negative stained transmission electron microscopic (TEM) image shows recreated 1918 influenza virions that were collected from supernatants of 1918-infected Madin-Darby Canine Kidney (MDCK) cells cultures 18 hours after infection. Photo credit: Cynthia Goldsmith courtesy of the CDC.

Friday, December 2, 2016

Poinsettias and pathogens



Poinsettias are a very interesting plant. Originally, this plant grows as either a small shrub or a tree in its native range (Mexico). However, the little potted poinsettias that can be bought at the store never grow close to that size. There is a good reason for this: the potted poinsettias are infected with a pathogen known as a phytoplasma. Phytoplasmas are small bacteria that lack a cell wall and are limited to the phloem tissue within plants. They are spread by vegetative propagation, grafting and by sap-sucking insects that feed in the phloem. Normally phytoplasmas are associated with severe disease in plants by altering the structure of plants, such as causing the petals of flowers to develop into leaves instead of petals. One such disease is aster yellows which infects over 300 different plant species in 38 families (see picture below).
Aster yellows on the Purple coneflower (Echinacea purpura). Via wikipedia
But what does this have to do with poinsettias? It turns out that the phytoplasma that infects the potted poinsettias also causes stunting, but doesn't cause severe distortion of the leaves. Not only is the plant stunted, but it produces additional branches where normally the plant has a single branch. The trait has been used since the 1920's to sell poinsettias during November/December in North America. This trait was found to be transferable by grafting but could be lost when the plant was subjected to heat treatment or tissue culture techniques. It was thought that another pathogen that is common in poinsettias was the cause of the stunting, Poinsettia mosaic virus, but it wasn't until the 1990's that a phytoplasma was shown to be the causal agent of the stunting. Previous work found that the virus wasn't completely associated with this trait as plants without the virus developed stunting and free branching. This article contains further information on the history of poinsettias and the work to determine why commercial poinsettias are stunted and free branching. This article discusses how to care for poinsettias.

A poinsettia tree in Mexico. Via Petal Passion

A potted poinsettia. Via pixabay.


The phytoplasma infecting poinsettias is a perfect example of how not all pathogenic organisms are bad and that they might be of benefit. This is certainly the case in the ornamental industry as it is the basis of the entire ornamental poinsettia industry.

Wednesday, November 2, 2016

Does Zika damage the testicles? Maybe

A lab mouse. Picture via Pixabay

A new study is causing quite the stir. Researchers used a mouse-adapted African strain of Zika to examine if the virus damages the testicles, as Zika has been detected in the sperm for up to 10 weeks post infection. In this latest study, severe damage and significantly smaller testicles were observed in infected mice. By day 21 the damage was much more extensive. Starting at day 14, sperm count and sperm motility dropped and by day 42, the differences were large. Overall, the fertility of infected mice dropped from around 80% to roughly 25% and the number of viable fetuses from the sperm of infected males dropped from 25 (from uninfected sperm) to 10. The researchers also tested this with mice infected with Dengue and an isolate of the Asian strain of Zika, but Dengue didn't damage the testes and the Asian strain caused less damage. The researchers hypothesize that the difference seen between the African and Asian strains is due to the Asian strain not being adapted to mice whereas the African strain was (it replicated less efficiently than the African strain in the mouse model).

Mouse testicles either infected with Zika or not, after 7 (a), 14 (b) or 21 days (c) post infection. Adapted from Govero et al., 2016; doi: 10.1038/nature20556

This study, although valuable, does have some limitations. First, a mouse-adapted strain was used which could result in mutations that make the virus different than the ones circulating in humans. Second, the work was done in a mouse model and sometimes research in mice doesn't translate to humans. However, this was just a preliminary study and something of concern was noted. These findings more than justify further work to examine if Zika causes damage to human testicles and if it can reduce fertility. 

Saturday, October 29, 2016

Zika causes birth defects (another case of Moms Across America ignoring science for ideology)

A friend of mine (Iida from Thoughtscapism <FB; website> sent me an interesting story that seems to be gaining steam in the anti-GMO world: Zika doesn't cause birth defects. Before I get started on debunking their claims, it's important to give a proper background on what Moms Across America truly represents and why they are not a valid source of information on anything related to science.

Moms Across America started off as an organization that was opposed to genetically engineered crops (colloquially known as GMOs). They rabidly oppose agrochemicals (glyphosate is a favorite target of theirs) and are well known for outrageous claims that they found glyphosate in X substance. They have claimed to find it in breastmilk, wine, granola bars and more recently vaccines. The group has recently branched out as being opposed to vaccines (although in all fairness, Zen said this was her personal belief and not necessarily the views of the organization), they have acted as a lobby wing and advertiser for organic food, and it now appears to be anti-science in general with their latest post. Researchers have taken the time to look at the methods used in the case of their breastmilk-glyphosate study and even tried to replicate it using proper testing protocols. They have also faced backlash for their attacks on breastfeeding (apparently to help market non-GMO formula, or rather that is what some suggest) and for parent-shaming those who do not "care" enough to feed their kids organic foods. In short, the positions of this group tend to be opposed to science and favor bad science over well designed and accurate studies and science. 

So what does this have to do with Zika and birth defects? In Zen's latest anti-science article she makes a series of claims (sourced from one Ian Trottier) that have no basis in science. She blames the "overblown" hysteria around Zika for allowing $1.9 billion in taxpayer dollars to go to Big Pharma for vaccines and Big Chemical for pesticides. Naturally, in her mind both vaccines and pesticides are far more dangerous than a virus, despite all the evidence to the contrary. First, I'll discuss her "evidence" and the four points that she tries to use to prove that Zika isn't a danger and then I'll discuss what the actual scientific evidence is for Zika causing birth defects (as she wrongly claims that there is no science to back up the idea that Zika causes birth defects). 

In the article, Zen tries to link to several "sources" to claim that Zika doesn't cause birth defects. The first link claims to have all sorts of evidence from science journals that Zika does not cause birth defects. This is what she says about the source: "The website sources articles from the New England Journal of Medicine, Lancet, PubMed, EPA, CDC and others. Additionally, site developer, Ian Hamilton Trottier, has conversed with experts at Lancaster University in the UK and the London School of Hygiene and Tropical Medicine. That includes experts based in the US." However, when you click the link you get the following:


Zen's first source that is "full" of scientific journal articles that "demonstrate" that Zika does not cause birth defects. 
Later in the article, she tries to cite MiamiCAN.org as a source. However, when you click on that link, it is for a domain that is for sale:


Zen's second attempt at linking a source for her claims.

The third source does list the four claims that Zen mentions in her article, but lacks any sources to back up the assertions. 


The fourth time seemed to be the charm as when MiamiCAN.com was linked, you get a website that has some sources. However, every source on that page deals with naled and does not back up the assertions that there is no credible scientific evidence that Zika causes birth defects. Furthermore, Zen claims that Dr. Michael Diamond "is essentially quoted as saying there is nothing that suggests detection of ZIKA as being the actual cause of MICROCEPHALY in the subjects he's studied. Dr Diamond recently published a review that links ZIKA to being present in tear ducts."
Dr. Diamond is indeed a flavivirus expert (the genus that Zika virus belongs to) and has published extensively on Zika (and other viruses and immunology in general). Since I cannot find the quote that Zen is alluding to and the fact that Dr. Diamond's work has been instrumental in confirming the link between Zika and birth defects, one is forced to assume that this quote is from the early days of the Zika outbreak in Brazil, when researchers did not know what we know now about Zika causing birth defects. This is a cherry picked quote that attempts to use an appeal to authority to confirm Zen's ideology despite the fact that the source has clearly demonstrated the opposite of what she is claiming.

The four points of the article basically boil down to "we don't know" and pesticides. She tries to link the use of the pesticides to both Monsanto and the Nazi bombing of France in a clear attempt to poison the well. Pesticides that have been ruled out as being involved with microcephaly, like 
pyriproxyfen, get mentioned as a possible cause. I won't get into that here as I've addressed pyripoxyfen before. It's sort of a rehashing of the claims that Mike Adams made about Zika that I also debunked previously, except Zen mentions paraoxonase as to why naled is really causing the birth defects attributed to Zika. However, her assertion that children have lower levels of paraoxonase in their liver does not automatically mean that the levels of this enzyme are the reason for birth defects. It ignores the fact that the mother's liver will help protect the growing baby. A study did find that lower maternal levels of one of the enzymes in this class did lead to a smaller head circumference, but it was nowhere near the level of reduction seen in babies born with congenital Zika disorder. I don't have the space to get further into the claims surrounding this enzyme as it is complicated.

But what about the evidence that supports the link between Zika infection during pregnancy and birth defects? How solid is the science on this? I'll outline just some of the key findings that support that Zika virus causes birth defects. The body of scientific literature is extensive on this topic and one only has to look to find it.


1) The Zika virus crosses the placental barrier
2) The complete genome of Zika has been recovered from the amniotic fluid and brain tissue of an infected fetus. 
3) Zika virus has been shown to disrupt brain organoid formation and deplete neural progenitor cells in vitro.
4) Case studies have shown a link between congenital Zika infection and birth defects.  
5) Zika virus infection caused birth defects in a mouse model. Specifically the Asian strain is more damaging than the African strain. 
6) A case-control study has demonstrated a clear link between congenital Zika virus infection and birth defects. 

Some of this work has been published since May 2016 when this article summarizing the evidence for congenital Zika virus infection and birth defects was published. This article concluded that the available evidence had met the criteria for a causal link to be established. Since then, the evidence supporting the link has only gotten stronger and at this point attempts to deny the link shows a clear lack of understanding of science. This infographic I made summarizes the evidence for Zika being a TORCH pathogen (TORCH pathogens cause birth defects).


The Zika virus is a TORCH pathogen that causes birth defects.

This article from Zen is a clear cut case of her ignoring the science that clearly demonstrates that the Zika virus causes birth defects. Although to be fair, it's entirely possible that she just doesn't understand the science. Based on her past history, that cannot be entirely discounted; however, not understanding science isn't an argument against it. The real moral here is that people need to be careful what they use as sources. Moms Across America, much like Natural News or Joseph Mercola, have clearly demonstrated that they are not a valid source when it comes to science. They cherry pick quotes and ignore all of the scientific evidence that contradicts their ideology. Moms Across America really shouldn't be used as a source for anything. 

Saturday, October 22, 2016

Sexual transmission of a bee virus


A queen honeybee. Via wiggledanceforme.


When sexually transmitted infections are discussed, one might think of human viruses, such as HIV or herpes. However, other organisms can become infected with viruses from sex. A new study describes how one of the more debilitating bee viruses, Deformed Wing Virus (DWV), can be sexually transmitted from infected male drones to otherwise healthy queens after mating. As the name might suggest, DWV severely deforms the wings of infected bees (see picture below) and impairs their cognitive function, specifically impairing the learning behavior and memory retention. Because of this, it wasn't known if infected drones would be successful at mating or not.

Carniolan honey bee with Deformed wing virus. Photo credit, Xolani90, via Wikipedia. 

In order to test this, the researchers took extensive steps to ensure that queen honeybees were free of viruses. They came from the same virus-tested colony as larvae and were then reared in nurse colonies that lacked a queen. That colony was screened for the varroa mite (300 bees were examined and none had the mite) and common bee viruses tested for in 60 worker bees. These include DWV, Black Queen Cell Virus, Sacbrood Virus, Chronic Bee Paralysis Virus and the ABPV complex made up of Acute Bee Paralysis Virus, Kashmir Bee Virus and Israeli Acute Paralysis Virus. A total of 30 queens met this criteria and were used in the study. They were then allowed to mate with DWV-infected male drones for at least 7 min; nine did not mate for the full time leaving 21 queens that fulfilled the requirements. If the drone's endophallus was still present in the queen after mating, then it was removed and tested for DWV; three of these had high levels of DWV. They found that seven of the queens developed high levels of systemic DWV infection; one of these queens died shortly after ovipositing (laying eggs) due to the infection. A further 15 queens had medium levels of DWV that varied in titer by tissue and eight queens had low levels of DWV.

This study answers an important question: can DWV-infected drones pass the virus on to queens by mating? The answer is a clear yes. Part of the reason why this study is important is because DWV is one of the major viruses associated with colony collapse disorder. Complicating matters, previous work has demonstrated that the varroa mite prefers drone brood (the varroa mite transmits all the viruses listed in this post to bees). 
Another issue is that if a queen dies, then the colony will be lost. Up to 25% of lost colonies are the result of loss of the queen. This new discovery could have implications for managing honeybees and lead to the development of strategies to mitigate these losses.