[Sciences/BBB] Acute Necrotizing Encephalitis (of childhood), a blood-brain barrier perspective.

This is a blog post following a request by a page follower on my Facebook account to provide an “layman” perspective on acute necrotizing encephalitis (ANE), also referred as acute necrotizing encephalitis of childhood (ANEC). This is a very short and surely incomplete summary but it should be a great starter to give the current perspective of this condition through the lens of the blood-brain barrier.

It is a condition that was firstly discovered by Mizuguchi and colleagues in 1995 firstly described in infants and toddlers (http://jnnp.bmj.com/content/jnnp/58/5/555.full.pdf). It was firstly described in patients from Asian origin (Japan). It was initially described to occur during the winter period, in particular with region that had experienced an influenza A outbreak. The main clinical feature of the disease marked by the presence in the magnetic resonance imaging (MRI) of increased water content inside the brain, mostly associated with edema (brain swelling). This increased water content can only be explained by the opening of the blood-brain barrier.

Water diffusion between the blood and the brain is tightly regulated by the blood-brain barrier (BBB). The BBB provides two kinds of barrier: a physical barrier (by the presence of tight junctions) and a chemical barrier (by the presence of solute carriers and drug efflux pumps). The case of water as a molecule (H2O) is very interesting. Water is a very small molecule (the molecular weight is 18g/mol or also 18 Daltons) but also a very polarized molecule. Hydrogens and the oxygen atoms forming H2O are not completely neutral, hydrogen carries a tiny positive-charge and oxygen carries two tiny-negative charges (we refer in chemistry as electronegative charges). Think about having a tiny magnet. In the opposite, cell membranes are made of phospholipids. As their name say, they are lipids by definition or what we commonly call them as “fatty acids”. Lipids have a distinct composition, they are mostly formed by carbons and hydrogens. Carbon is not much a magnet atom, it neither likes to carry positive charges nor negative charges. This is why lipids are commonly referred as apolar molecules. Now, polar and apolar molecules behave like water and oil mixed together: they simply do not mix and will sequestrate themselves, usually forming a oil droplet surrounded by water. Water entrance inside the brain is believed to occur mostly via paracellular route, as depicted in the picture below (source: http://www.nature.com/nrn/journal/v7/n1/full/nrn1824.html?foxtrotcallback=true).

main-qimg-a138eec8624c78aa6379a71b994b20ac-c

Tight junctions are very tights, letting water fall through the cracks only in a tiny amount. Imagine having a very good rooftop that only let water fall through one drop every hour. The problem with the opening of the BBB following various factor is the massive entrance of water. Think about having a hole in your rooftop and facing a tropical storm shower outside: you are facing now a massive and unregulated entrance of water inside the brain, leading to a brain swelling.
In peripheral tissue, edema (swelling) formation can naturally expand, resulting in a swollen tissue. The problem with the brain is its anatomical structure: it is encased inside a rigid shell (skull) that has no exit route for the penetrating water. This results in an increased pressure inside the brain (we usually referring as increase in intracranial pressure or ICP). This increased pressure induce a mechanical stress, crushing brain cells via mechanical stress and ultimately neuronal cell death. Such swelling appears to occur in specific brain regions, with a primary lesion site in the gray matter (neurons), with persistent deposition of hemosiderin and white matter (axon fibers) cysts during and after the recovery phase. Until now, we don’t exactly know what cause such disease, but appears as following a viral infection including flu (influenza A and B, swine flu (H1N1), parainfluenza virus), varicella, measles, rubella and various herpesviruses (HHV-6, HHV-7) (https://www.hindawi.com/journals/mi/2015/792578/#B1), although the presence of such viral agents (detection by polymerase chain reaction) in spinal tap as well as post-mortem signs of brain inflammation remains anecdotal.

Interestingly, it seems that patients suffering from ANE undergo a very severe immune response commonly referred as “cytokine storm”, as several studies noted an increase in inflammatory markers (in particular interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha (TNF-alpha) making this phenomenon the most prevalent hypothesis.

Immune cells communicate to each other via a common language called “cytokines”. Cytokines are like a “RED ALERT” system, they signal some breach in security or incoming danger.
Brain microvascular endothelial cells (BMECs) lining the blood side of the BBB can also understand the “cytokine” language and understand such signal as “RED ALERT – OPEN THE BBB SIGNAL” as depicted in the picture below (source: https://www.researchgate.net/profile/Nicolas_Weiss/):

Now where are these cytokines coming from and how they are triggered? It is a very good question. This is where the viral infection comes in. I will not details much about the immune response to viruses, but you can ask @TheMadVirologist for any questions related to viruses. For this I will use a figure that resume the immune response to viruses (source: https://www.researchgate.net/profile/Francoise_Stoll-Keller/).

figure-1-function-of-dendritic-cells-in-the-immune-response-to-virusesfollowing-the

Upon infection, infected cells will display viral particles on the cell surface and will also secrete a protein called “interferon-gamma”. This is a sort of cellular “SOS Danger” to the immune system. Natural killer cells, dendritic cells and macrophages may start the early response, also known as “innate immunity” to contain the viral infection. In addition, free circulating viruses can be spotted by B cells through their array of surface antibodies and trigger what we refer to as “acquired immunity”. Viral infection will trigger an immune response and we can think that maybe an overactive immune system may exaggerate the danger resulting into the “cytokine storm”. Another hypothesis is that such cytokine storm maybe triggered by natural killer (NK) cells.

This hypothesis is further supported by the presence of a higher count of natural killer leukocytes in ANE patients during the recovery phase. Natural killer (NK) cells are immune cells normally targeting cancer cells and cells infected by viruses.  This “cytokine storm” maybe the causative agent of the blood-brain barrier disruption (BBB) by different mechanisms (source: http://stroke.ahajournals.org/content/strokeaha/42/11/3323/).

but appears to occurs via an matrix-metalloproteinase (MMPs) dependent pathway. Under the stimulation of such cytokines, brain endothelial cells and astrocytes may increase the production and releases of MMPs locally. These MMPs act as little scissors that can chop the extracellular matrix supporting brain endothelial cells and astrocytes end-feet processes. In addition, these MMPs can also chop tight junction proteins that are involved in tight junction (TJ) complexes. These TJs are very important as they provide the barrier limiting the diffusion of water and solutes between the blood and the brain.

In addition to the cytokine storm hypothesis, it seems that other factors maybe involved in the pathophysiology of the disease. Until now, Ran binding protein 2 (RANBP2) (http://www.cell.com/ajhg/fulltext/S0002-9297(08)00630-7). RANBP2 is a protein involved in the nuclear pore complex, yet the relevance of this mutation at the blood-brain barrier remains unknown. In neurons, it is associated with cellular structures different from the cell nucleus, in particular it is associated with mitochondria (power house of cells) and microtubules.

Another protein of interest associated is EphB2, a receptor for ephrins (https://www.ncbi.nlm.nih.gov/pubmed/?term=ephb2+blood-brain+barrier). Ephrins play an important role in brain wiring during development (axon guidance) but also play a role in the formation of the vascular tree.

The function of EphB2 and ephrins at the blood-brain barrier remains unclear. However, a recent study identified the expression of EphB2 at the cell surface of endothelial cells including primary human non-BBB (HUVECs) and BBB (HBMECs) endothelial cells. Furthermore, a case report from a patient suffering from systemic lupus erythromatous (SLE), an autoimmune disorder, presenting the case of ANE showed the presence of antibodies in the serum capable to bind selectively to EphB2.

Yet, at this point we don’t know if this antibody binding is enough to trigger the BBB disruption or it requires the recruitment of immune cells to trigger such disruption.

 

 

 

 

 

 

 

 

 

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[Videogames] Fallout – 20th Anniversary

Today marks the 20th anniversary of Fallout! Happy Fallout Day! Man, this is one of the rare game you come back to it, despite being a very tough game (it was that time when you had to save manually), especially straight from the start (yep, that means dying from the first five minutes from a rat bite (yep, a ridiculous rat rat bite that eats you 4HP) and by its game mechanics involving missed hits (when 50% chance of hit is 50% chance of miss, with the odds resetting at each action), weapons malfunctions (loosing a turn because your 10mm pistol jammed) or worse from a critical hit (with your brain and lungs splattered all over the place).
The game was also an amazing experience for those that have dreamt a game taking place in a post-nuclear wasteland (especially when you have been bottle-fed on Mad Max 2, Fist of the North Star and a fistful of Italian Sci-Fi B-movies). Shock and awe right after being introduced by the Interplay logo “for gamers by gamers”.

The use of “Maybe” by Ink Spots was setting the mood, the idea of dystopian chronology that will become the “Pre-War” civilization in a sequence straight out of a Ray Bradbury short story. We only discover that we have been watching a dead civilization and get narrated its fall by Ron Perlman legendary “war…..war never changes” with the music of Mark Morgan in the background.
Here we are, a vault dweller from Vault 13 asked to go out and find a water chip for the whole vault community by the Overseer. It sends you out with a “goodbye and good luck” out of your vault with nowhere to get back inside with only a rusty machete, a 10mm pistol (with scarce amount of ammos) and a pajama as your only environmental suit. Here is the deal: you have 150 days to find a solution for the water chip. Nobody knows where to find one but maybe the nearby sister Vault, Vault 15, may have one. This is it. There is no linear progression, you do whatever you want and can wander anywhere you want straight out the Vault 13 cave. This freedom was very novel.
Another novelty what the character design and carefully choosing traits. You are assigned a number of points through a SPECIAL (strength, perception, endurance,  intelligence, agility and luck) character crafting system associated with skills. Each of them matter on their own as it will dictate your ability to interact with the environment.
In addition, you have the karma system in which your good and bad deeds are buildup and known from the NPCs. You may enter a community as neutral but your interactions will leave a reputation of either an idolized person or a vilified person that can get you shot at first sight.
It is so immersive and engaging that you rarely play the game the same way twice unless you are doing it on purpose. Add also the remarkable sound work by Mark Morgan that makes the atmosphere unique. If you have not played it yet, RUN! Grab a free copy on Steam on install it and play! It is such an immersive game that it is in my Top 10 favorites of all times.

 

[SciFi/TV] Star Trek: The Next Generation 30th Anniversary

Today we celebrate the 30th anniversary of Star Trek: The Next Generation (aka TNG), aired on the US television with the two-part episode “Encounter at Farpoint”. It was the second installment of the Star Trek franchise on the TV, a bit less than 20 years after the last episode (but many syndications through the 70s and 80s made Trek being a staple in the pop culture), a year after the release of “Star Trek IV: The Voyage Home” and in my opinion one of the most ambitious TV Sci-Fi project from the 80s.
The show itself takes place in the 24th Century, almost a century later after “The Original Series” took place, with a new crew and the “Enterprise-D”, a brand-new class of starship.

It is interesting to get back to it, considering its anniversary coincides with the beginning of “Star Trek: Discovery” last week on the CBS All-Access, trying to cash in the fanbase as Games of Thrones do through a subscription fee (honestly, it is outrageous considering the rest of the world have it included inside their Netflix package). It is also interesting how many fans were outraged on the new crew of the USS Enterprise. Apparently there are some fans so extreme they even do not recognize Captain Jean-Luc Picard (Patrick Stewart) as canon. But back then, there was no Internet so not much amplification chamber for trolls.

What I can say that for most (I say most) 170 episodes, it was such an engaging ride spanned through 7 seasons. What I think made it a success?

First, the design of the ship. The ship is wonderful, the computer system (voiced by Majel Barett, Gene Roddenberry’s second wife) and interface (the LCARS) was such a savvy and eye-candy UI interface (remember, the Apple Macintosh just came in few years before and Windows was not the most gorgeous GUI), the bridge is a spartan but yet well designed and spacious, clearly designed for exploration and diplomatic missions.

Second, it was the crew. You can have the nicest ship, yet have a crew you dont feel attached to. Thats how I felt for example with the NX-01 Enterprise crew (ST:Enterprise). Captain Jean-Luc Picard (acted by Patrick Stewart) was in my opinion the best Captain of the Trek franchise. Picard maintained the hierarchy intact but also knew to listen to his senior officers. Picard was the man putting diplomacy first before setting the phaser on maximum charge and most of all Picard had the best quotes ever in episodes highly charged with philosophical meanings. There are so many episodes I can cite where Picard was completely awesome showing his remarkable acting.
For instance “The Inner Light” in which Picard has his brain held captive by a interstellar probe making him revive the last years of a scholar in a planet slowly dying from its star becoming a supernova. Try not to cry on this one. There is also the episode “The Measure of A Man” in which Picard defends Data (always referred as “Mr. Data”) in court as a scientist proclaim that Data is dispensable machine, even if it has a conscience. You have also the famous “Best of Both Worlds (part I and II)” in which the Federation gets first-hand contact with the Borg and get assimilated as Locutus of Borg, leading the massacre of Wolf 359. You have “Darmok and Jalad at Tanagra” in which Picard and the Captain of a new alien race are beamed down on a planet, such alien race using a metaphoric language in their communication. Another one is “Sarek” and “Unification (part I and II)” in which Picard decides to mind-meld with Sarek suffering from a sort of Vulcan form of dementia, holding his condition the time of his diplomatic mission and Picard travel to Romulus to find Spock.  Want some more awesome Picard-centric episode? “The Drumhead”, in which a sabotage on the Enterprise becomes the spark in a chain of reactions resulting in a witch hunt and with one of the most memorable quote from Picard “With the first link, the chain is forged….”. Or “Chain of Command (part 1 and 2)” in which Picard captured and tortured by the Cardassians (playing a saber rattling dance with the Federation via the intermediate of Captain Jellico) is standing up to his oppressor by yelling him “there are four lights!”. Or “Sins of Father” in which Picard stands by his Lt. Commander Worf in front of the Klingon High Council, serving as his mentor in front of the accusations of treasons of his late father. You know what, just check out the video below:
https://youtu.be/smdqe2eluEI

As a first officer, we had Commander William “Will” Riker (played by Johnathan Frakes). He was the man to get beamed down with the away team, he was the womanizer but yet sharing this ambiguous and odd relationship with Deanna Troi (I will come back later to it). You know it is interesting how the “Riker’s beard” made a huge effect on him, by Season 2 you have to admit that such beard was making Riker a pretty handsome man. He was harboring this side of James Kirk, boldly going where no man went before, but was also capable of the most calculated and stern decision that you would expect from a first officer. If I have to pick an Riker-centric episode, I would definitely recommend the episode “Frame of Mind”. Another episode is “The Outcast” in which Riker falls in love with an androgynous alien, discussing about the place of transgenders in society and their acceptance, thirty years before “Caitlin Jenner” and the infamous “bathroom bills”.

We had also Lt. Commander Data (played by Brent Spiner), a unique (well there was two copies, Lore and B-4) android created by Dr. Noonian Soong (the descendant of Arik Soong, the scientist that created the “Augments”). Data was in my opinion one of the best representation of the diversity in the TNG universe. He was unique and very puzzling for any human. I felt Data was showing an allegory of a Aspie: friendly, socially awkward that has trouble to identify social cues, very savvy and sometimes tell much more than expected from him. But Data sometimes showed how much humanity he had. For instance, this “Pen Pal” episode in which he violates the Prime Directive to save a girl he had sympathized through communication channel from a catastrophic geologic event. The other one that really shed a tear was “The Offspring” in which Data creates a child android, giving this child android the choice of its own gender identity (Hello! 1987!). The android decides to become a girl and named “Lal”. Data becomes a father, enjoying fatherhood only to discover that Lal suffers from a fatal electronic malfunction that needs her decommissioning. Try not to shed a tear as a parent, if you learn your child has a terminal disease. But maybe the best acting from Brent Spiner was playing the role of “Lore”, Data’s “older brother” that was dismantled by Soong because of his mercurial and psychopathic traits. Seeing Brent Spiner playing both the naive and friendly Data and the manipulative, deceptive and murderer (he summoned and lured the crystalline entity on Omicron Theta resulting in the killing of any lifeforms on the planet and even tried to do the same to the Enterprise crew). Brent Spiner remarklable acting was again to be seen on Star Trek:Enterprise in the episodes he played Dr. Arik Soong.

We also had Lt. Commander Worf Rozhenko (acted by Michael Dorn), the only Klingon from the fleet taking place after the Khitomer accords that settled the feud of the Klingon Empire with the Federation of planets. Worf grew up amongst humans torn between his Klingon heritage and growing amongst the Federation.  There are so many good episodes with Worf, especially the ones involving his Klingon heritage and his struggle to get accepted amongst his compatriots. For instance, you have this episode in which the Enterprise rescues a Klingon vessel containing three outlaws, putting Worf at odds between his loyalty for the Federation and the loyalty for the Klingons. There is the “Reunion” episode that set Worf into a character of a Greek mythology that will haunt him even in Deep Space Nine.

We had Lt. Commander Geordi LaForge (acted by LeVar Burton), a engineer born with blindness but equipped with a particular set of visor. Despite’s being congenitally blind, Geordi showed to be one of the most talented engineer in the Federation, saving the Enterprise from many situations. Geordi also showed some pride in his disability, in the episode “The Masterpiece Society” in which he faces the astounishment of a society in which eugenism is considered as a virtue and disability a failure that does not have a place.

We also had Counsellor Deanna Troi (played by Marina Sirtis) that played the role of a Betazed, capable to read others mind and emotion. She was the ship counsellor and also part of the dilemma of serving in the same ship than Riker, her former “Imzadi”. Deanna Troi role was at the beginning though to be defined but as the series goes, she showed to be instrumental to the series. My favorite episode with Troi? “Face of The Enemy” in which she is posing as a Roman agent of the dreaded Tal’Shiar, playing diametrically the behavior that is associated with her by playing a ruthless and fearless Romulan agent. We also had Dr. Beverly Crusher (played by Gates McFarren) that played the doctor and confident to Jean-Luc Picard due to their close relationship (Dr. Crusher’s husband was Picard best friend) and of course Wesley Crusher (played by William Wheaton). Wesley was honestly a pain in the neck during the first two seasons, leading to the climatic “Shut up Wesley!” quote.

Another niceties of the Enterprise-D is the holodeck, allowing to recreate a virtual environment that is too real to be true. Imagine breaking the fourth wall and entering inside a movie. Some of the best episodes were Holodeck-centered episodes. For example, the episode in which Picard see himself re-enacting Dixon Hill, giving this “roman noir” episodes straight outta the Prohibition era, the episodes in which Data plays Sherlock Holmes with Geordi as his buddy and by mistake giving life to Moriarty.

But there are so many episodes that really raises questions on the society as we know it and address questions that very few series ever bring on the TV: gender identity (there are even in the season 1 episode a few times you can see men wearing sort robe uniforms), the impact of interfering on a “primitive society” (the episode “Who watch the watchers” is another good one to watch), the issue of being born with a disability and societies in which eugenism is considered as the norm, the challenge of becoming a senior in a society in which becoming a senior is considered inadequate and is marked by your death at your 60th birthday (the episode “Half A Life”) or getting brought into a world in which money and material possession so dear to us has no more meanings (the episode “The Neutral Zone”). There is an episode that question to which extent can we allow us to develop warfare against our biggest enemy (“I Borg” and the virus to contaminate the Borg Collective).

There are also some single gems that does not fit the Trek narrative but are damn good. For example “Yesterday’s Enterprise” in which the Khitomers accords never happened resulting in an all-out battle against the Klingon for over 30 years. There are the episodes with Q, this malicious superpower entity presented in the pilot as the judge setting a trial on the Enterprise crew for the crimes mankind was held responsible. Some of them are plenty funny, some of them are deadly costly to the Enterprise such as “Q Who” in which Q introduce Picard to the Borg.

 

If you want to watch TNG but not interested to buy the whole collection, you have two choices: BBC America usually play them on the weekends and Netflix gives you access to everything Trek (except the movies). I am just warning you that the first two seasons are pretty campy as Roddenberry hired most of the old writers from TOS and feels most of a time some sort of reheated dish. But by the mid-season 2 and the injection of a new generation of writers, the show takes up and becomes very fascinating.

 

 

[Neurosciences/Junk Sciences] Autopsy of a flawed study of aluminum and brain inflammation (Li et al., J Inorg Biochem 2017)

Note: This is a special blog post coauthored by The Mad Virologist and The Blood-Brain Barrier Scientist (this article will be co-published on both our blogs). Another post has already been published on this paper, but we wanted to take a deeper look at everything that is wrong with this paper.

[UPDATE2] The study in question got retracted according to RetractionWatch:
http://retractionwatch.com/2017/10/09/journal-retract-paper-called-anti-vaccine-pseudoscience/

[UPDATE] I would strongly recommend the reader to look at the comments on Pubpeer about this paper. It is terrifying to think how it percolated through peer-review.
https://pubpeer.com/publications/4AEB7C8F30015079E2611157CF8983#undefined

A recent paper by ophthalmologist Chris Shaw was published and immediately touted as being proof positive that the aluminum adjuvants found in some vaccines are responsible for causing autism. Before we get into the paper, I have a few choice things to say about Chris Shaw. Despite not being an immunologist, Shaw has ventured into studying how vaccines and vaccine adjuvants cause neurological disorders such as autism. Shaw made headlines in 2016 when a paper he co-authored that claimed to show a link between the HPV vaccine and neurological disorders was retracted after being accepted by the journal Vaccine. It turns out that the statistics used in the paper were completely inappropriate and there were undisclosed conflicts of interests for some of the authors, including Shaw.These issues should have prevented the paper from being accepted in the first place, but mistakes do happen and science tends  to be self correcting. More surprising is that Shaw claimed that he didn’t know why the paper was retracted and that the science was of the highest quality. Shaw’s previous work has also been described by the WHO as deeply flawed and rejected by that body. This isn’t being brought up to dismiss the paper out of hand but to help illustrate why Shaw’s work is deserving of additional scrutiny. Hopefully by the end of this post, the logic behind the need for additional scrutiny of anything Shaw publishes is abundantly clear. We’ll begin by examining the methods used by Shaw’s research group and point out some of the issues.

Background for experimental design flaws: PK and species issues

One problem that is recurrent with Shaw is his “vaccination schedule” tries to consider rodents, such as mice and rats, as humans in miniature. It is wrong to assume that rodent and human primate species are alike, they’re not and there are notable physiological differences between rodents and non-rodents. For example, there are a couple of studies by Terasaki and colleagues (http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07208.x/abstract) that have shown differences in the expression of solute carriers and drug transporters at the blood-brain barrier. We cannot exclude that such differences may bias the outcome observed in his studies, but this bias applies intrinsically to any in vivo studies based on a rodent model.
There is also the issue of brain development and mapping the vaccination schedule and the brain maturation. In this study (as well in the previous ones), Shaw and colleagues consider that applying vaccines from post-natal day (PND) 3 to 12 is representative of a human infant vaccine schedule. There is some differences in the literature, as previous studies from Clancy and colleagues mapped the PND12 to the 7th gestational months in humans (https://blogs.cornell.edu/bfinlay/files/2015/06/ClancyNeurosci01-17kkli7.pdf), some more recent publications map PND21 to 6th month post natal in humans, making the PND12 around the 3rd month infancy following full-term birth (http://www.sciencedirect.com/science/article/pii/S2352154615001096). You can easily appreciate that by following Shaw flawed experimental design, the total amount of Al administered during a 2 year period has been indeed administered within 90 days of birth, whereas the vaccination schedule according to the CDC does not start before the 2nd month of infancy if we exclude the two injections of Hepatitis B vaccines at birth and after the first month respectively (https://www.cdc.gov/vaccines/schedules/hcp/imz/child-adolescent.html).

In addition to a flaw in the experimental design, we cannot exclude some differences in the pharmacokinetic profile of Al adjuvants between mice and humans. The data available is fairly limited but a recent study from Kim and colleagues (https://www.ncbi.nlm.nih.gov/pubmed/26437923) failed to show a significant brain uptake of Al compared to controls following the single oral administration of different Al oxide nanoparticles at a concentration of 10mg/kg. Furthermore, the approximation of Shaw in terms of total burden of Al from vaccines (550 microg/kg) is not an accurate metric as we have a dynamic process involving absorption, distribution and elimination to occur simultaneously. A daily burden of Al from vaccines is a much more reliable parameter to consider. Yokel and McNamara (https://www.ncbi.nlm.nih.gov/pubmed/11322172) established it about 1.4-8 microg/day for based on 20 injections spanning over a 6-year period in a 20kgs individual.
If we consider Shaw calculation, then the total burden at age 6 would be 1650 microg/kg or 33’000 microg for a 20kgs 6-year old child. That’s about 15 microg/day of daily Al burden from vaccines, a value that is 2 to 10 folds higher than applied to humans. It makes therefore very difficult to compare apples to oranges, as Shaw experimental paradigm is flawed and not representative of a clinical scenario.

Selection of genes to measure:

Selecting which genes to measure is a crucial step in a study like this. If care is not given to ensure that the correct genes are selected, then the study will be a wasted effort. Shaw stated in the paper that they selected genes that were previously published. However, not all of the genes that they measured came from this paper. Only 14 of the genes were from this paper (KLK1, NFKBIB, NFKBIE, SFTPB, C2, CCL2, CEBPB, IFNG, LTB, MMP9, TNFα, SELE, SERPINE1, and STAT4). This leaves 17 genes the were measured but not found in the paper. Two of these can be explained. One gene, ACHE, was mentioned as having been selected because of other work, so it is sourced. The second gene, is the internal control gene beta-actin. This is a housekeeping gene that is often used as an internal control to provide a relative expression from. This leaves 15 genes unaccounted for. We suspect that these genes were selected because they are involved in the innate immune response, but no reason is stated in the paper.

The way these genes were selected is problematic. Because half of the genes seemed to be selected for uncited reasons, this study is what is known in science as a “fishing expedition.” There’s nothing inherently wrong with this type of research and indeed it can lead to new discoveries that expand our understanding of the natural world (this study that increased the number of sequenced viral genomes by nearly tenfold is a good example of this). But what fishing expeditions can show is limited. These types of studies can lead to other studies but they do not show causality. Shaw is claiming causality with his fishing expedition here.

There is also the problem that they used old literature to select their gene targets when much more recent research has been done. By happenstance, they did measure some of these same genes in their study. However, their results do not match has has been measured in children that have been diagnosed with autism. For example, RANTES was shown to be decreased in children with autism. In Shaw’s work there was no statistical difference in RANTES expression between mice given the aluminum treatment and those receiving saline. Likewise, MIP1alpha  was shown to be decreased in developmentally delayed children but was shown to be increased in the aluminum treated mice. This was also the case for ILIb which was found to be elevated in children with moderate autism yet there was no statistical difference between the mice receiving the aluminum treatment and those receiving saline. In fact IL-4 was the only gene to follow an expression pattern similar to what was found in children with severe autism (elevated in both cases). However, there is something odd with the gel in this case. This was the image for figure 4 that was included in the online version of the paper (we have not altered the image in any way). Look closely at the top right panel at the IL-4 samples and the IL-6 samples. You’ll notice that the bands for the control and the aluminum treated mice have different color backgrounds (We enlarged the image to help highlight this but did not adjust the contrast). If these came from the same gel, there would not be a shift in color like this where the treated bands have a lighter color encircling them. The only way this could happen is if the gel was assembled in photoshop. The differences could be real; however, since this image was modified we do not know for sure and this is scientific misconduct. Papers get retracted for this all the time and people have lost their degrees for doing this in their dissertations. These gel results cannot be trusted and the paper hinges on them. The Western blots and issues with them will be discussed below.

22016544_10102969159317918_1868648690_n

The unaltered figure 4.

22052798_10102969159312928_192591888_n

A close up of the panel with the regions in question highlighted.

Semi-quantitative RT-PCR:

In order to quantify the gene expression levels of the genes that Shaw’s group selected, they used an older technique called semi-quantitative RT-PCR. This technique uses the exponential increase in PCR products in order to show differences between expression of a gene under different conditions. There’s nothing wrong with the technique provided one understands what the limitations are. Let’s say you have a large number of genes that you want to measure expression of, but you aren’t sure which genes are going to be responsive and you have limited funds. Semi-quantitative RT-PCR is a good method to screen for specific genes to be examined further by more precise techniques, such as Real-Time RT-PCR, but it’s not appropriate to use this technique and then make statements about precise quantification. Where semi-quantitative RT-PCR excels is with genes that are normally not expressed but can be expressed after some sort of stimulus, such as terpene biosynthesis genes that are induced by insect feeding.

To put it bluntly, semi-quantitative RT-PCR was not used properly in the paper by Shaw. The way that it was used implied that it would be quantitative when the technique is not that precise. Without verification by another method, ideally Real-Time PCR which can determine what the exact abundance of a given target is, these results should be taken with a grain of salt. This would still be the case if there weren’t irregularities in the gel images. With those irregularities, this is absolutely essential and should have prevented this paper from being accepted.

Western-blots and data manipulationPCR and Western-blots data: the owl is not what it seems
As The Mad Virologist mentioned, the semi-quantitative PCR is an old-fashioned RNA quantitation method, with the use of Real-Time quantitative PCR (that quantifies the amplification product at each cycle, using a fluorescent dye as an indicator) is a much more accepted method nowadays (see his section for more details). For Western-blots, the semi-quantitative approach is more accepted but it is important to show data that are consistent between what you show (qualitative) from what you count (quantitative). In Western-blot analysis, we measure the relative darkness of a protein band (the black lines that you see in papers) between treatments and controls. Because you cannot exclude some errors due to the amount of protein loading, we also measure the band intensity for proteins that are very abundant, usually referred as housekeeping proteins (because they play essential functions in cells). In this case, beta-actin (named ACT in the paper was used).
Once you normalize to beta-actin, you can compare the effect of a treatment by comparing the relative band intensity ratios. In both cases (semi-quantitative PCR and Western-blots), “what you see is what you measure” or you have to show a “representative Western-blot” alongside a quantitative data to demonstrate that your quantification matches with band densities. The common practice is the use of image acquisition software like ImageJ to determine band density. Showing Western-blot is nice, but not foolproof. Indeed, Western-blots data (with fluorescence images) is amongst the most common method by which some researchers can manipulate or even falsify data but also the most common type of data that spark a paper retraction. Someone notice something fuzzy on a Western-blot data, creating some questioning reaching to the editors and asking access to the full dataset (usually the X-ray film or the original full scan of the blot). Often, the author will use the excuse “the dog ate the flash drive” or “the hard drive containing the data crashed” if they cannot provide such data.
There are some methods to spot some image manipulation on Western-Blots and include playing with the brightness/contrast, requesting the presence of quantitative data in addition of a representative blot, samples must be coming from a same gel (you cannot use a cookie-cutter and build-your-own perfect gel). There is an excellent article that describe the pitfalls and cases of bad Western-blot data representation if not image manipulation. (https://www.elsevier.com/editors-update/story/publishing-ethics/the-art-of-detecting-data-and-image-manipulationThere are, at this time, different issues raised both in the Western-blots pictures and their subsequent analysis raising the reliability of the data presented in this study.

In this post, we have used the full-resolution pictures provided by the journal website (http://www.sciencedirect.com/science/article/pii/S0162013417300417), opened just pictures in ImageJ to convert such pictures into 8-bit format, invert the lookup tables (LUT) and adjusted the brightness and contrast. We have exported such pictures in Powerpoint to ease the annotation and comments. We recommend the reader to judge by himself/herself and download the full-resolution images as well.

The first concern is by looking at Figure 1C. First, this is the original Fig.1.

1-s2.0-S0162013417300417-gr1_lrg

Then, this is the close-up analysis for Fig.1C

Slide1

There are several issues. First there are some bands that appears as band splicings, in which the author create a custom blots by assembling different bands from different gels. This is a no-no in Western-blots: all bands showed in a blot should come from the same gel. This is why Western-blot is a torture for graduates students and postdocs, you need to show your best blot with all bands showing the same behavior for your quantitative analysis.
Second, the presence of a rectangular grey piece that was added on the top of control 3 TNF band. This is a possible data manipulation and fraud, as you are voluntary masking a band and hiding it. Thats a big red flag on the paper. The third issue of Fig.1C is the consistent feeling of seeing bands either cropped on a grey rectangle or what I call a “Photoshop brushing” in which you brush off using the brush function area of the gel you consider not looking good enough. You can clearly see it with actin as we have a clear line between the blurred blot and a sharp and uniform grey in the bottom half of the blot, compared to the wavy top of the blot. This a grey area that I am not familiar with Western-blot but this is a no-no for any immunofluorescence picture. Any image manipulation that goes beyond the brightness/contrast adjustment and involves alteration of the acquired picture is considered as data manipulation. If you analyze the data upon correcting for the inconsistency of Figure 1C, the graph looks much more different and failed to show any differences between Al-treated and control, when you restrict yourself in over-normalizing it and plot straight the protein/actin band density ratios.

What is also concerning and surprising is the conclusion from the authors that males, not females, showing an inflammatory response. Of course, the authors failed to show the same outcomes from female animals and expect us to trust them on this. The problem is that such conclusion is in direct contradiction with the literature. There is a solid literature supporting the presence of a sexual dimorphism in terms of inflammatory response, in particular in terms of neuroinflammation and autoimmune disorders such as multiple sclerosis (https://www.ncbi.nlm.nih.gov/pubmed/28647490; https://www.ncbi.nlm.nih.gov/pubmed/27870415). There is also a growing call to the scientific community to provide results for both sexes (males and females alike). Although Shaw reports the study was performed in both males and females, he gives us this explanation at the end of section 3.1: Taken together, a number of changes indicative of the activation of the immune-mediated NF-κB pathway were observed in both male and female mice brains as a result of Al-injection, although females seemed to be less susceptible than males as fewer genes were found altered in female brains.

Yet the interesting part comes when Shaw try to compare ikB phosphorylation between males and females following Al injection (Fig.3C). When you analyze the data, you are raising concerns very rapidly. First, we have a possible case of cookie-cutter band in which you just paste a band that seems nice enough in a blank space. This is a very suspicious activity as you can make up data as easy as this. Second, there is again this “Photoshopping brushing/erasing” taking place in that figure, in which I suspect a case of fraudulent activity. As you can see in female, it is as if someone tried to mask some bands that should not have been here. Remember when he said that males but not females showed an inflammatory response? Is it trying to dissimulate data that contradict his claims?

image

Again, lets bring up Figure 3 at its full resolution.
1-s2.0-S0162013417300417-gr3_lrg

Finally, the same issues are persistent and even more obvious in Fig.5A. Again, we have a mixture of different Western-blots image manipulations including bands splicing, Photoshop brushing, cookie-cutter bands……

First, the unedited picture:
1-s2.0-S0162013417300417-gr5_lrg

And below the close up of Fig.5A

Slide3

These are some serious concerns that raise the credbility of this study and can only be addressed by providing a full-resolution (300dpi) of the original blots (X-ray films or the original picture file generated by the gel acquisition camera).  There has been a lot of chatter on PubPeer discussing this paper and many duplicated bands and other irregularities have been identified by the users there. If anyone is unsure of how accurate the results are, we strongly suggest looking at what has been identified on PubPeer as it suggests that the results are not entirely accurate and until the original gels and Western blots have been provided, it looks like the results were manufactured in Photoshop.

 

Statistics:
Long time followers know that I tend to go right to the statistics that are used in papers to see if what they are claiming is reasonable or not. Poor use of statistics has been the downfall of many scientists, even if they are making honest mistakes. It’s a common problem that scientists have to be wary of. One easy solution is to consult with a statistician before submitting a paper for publication. These experts can help point out if the statistical tests that were run are the correct or not. The Shaw paper could have benefited from this expertise. They used a Student’s T Test for all of their statistics comparing the control to the aluminum treated. This is problematic for a couple of reasons. These aren’t independent tests and the data likely does not have a normal distribution, so a T Test isn’t appropriate. Better statistical tests would have been either Hotelling’s T-squared distribution or Tukey’s HSD.  Another issue is how the authors used standard error (SE) instead of standard deviation (SD). To understand why this matters, it helps to understand what the SE and what the SD measure and what these statistics show. The SD measures the variation in samples and how far the measurements are from the mean of the measurements. A smaller SD means that there is low variability in the measurements. The SE measures the likelihood that a measurement varies from the mean of the measurements within a population. Both the SE and SD can be used; however, using the SE is not always appropriate, especially if you are trying to use it as a descriptive statistic (in other words if you are trying to summarize data). Simply put, the SE is an estimation and only shows the variation between the sample mean and the population mean. If you are trying to show descriptive statistics, then you need to use the SD. The misuse of SE when the SD needs to be shown is a common mistake in many research publications. In fact, this is what the GraphPad manual has to say about when to use the SD and when to use the SE:

If you want to create persuasive propaganda:
If your goal is to emphasize small and unimportant differences in your data, show your error bars as SEM,  and hope that your readers think they are SD. If our goal is to cover-up large differences, show the error bars as the standard deviations for the groups, and hope that your readers think they are a standard errors.” This approach was advocated by Steve Simon in his excellent weblog. Of course he meant it as a joke. If you don’t understand the joke, review  the differences between SD and SEM.” The bottom line is that there is an appropriate time to use the SE but not when you are trying to summarize data.

Another issue is the number of animals used in the study. A consensus in published study is to provide a minimal number of animals (usually n=8) needed to achieve statistical significance but also maintain to a minimum to ensure proper welfare and humane consideration for lab animals. In this study, such number is half (n=5). Also the authors are bringing some confusion by blurring the lines between biological replicates (n=5) and technical replicates (n=3). By definition, biological replicates are different organisms that are measured and are essential for statistical analysis as these replicates are independent from each other. Technical replicates are dependent on each other as they come from the same biological samples and are repeated measurements. By considering the latter as statistical relevant, you are biasing yourself to consider a fluke as a biological phenomenon.

 

Conclusions:
Based on the methods that were used in this paper, Shaw et al. went too far in declaring that aluminum adjuvants cause autism. But there are six other key points that limit what conclusions can be drawn from this paper:
1) They selected genes based on old literature and ignored newer publications.
2) The method for PCR quantification is imprecise and cannot be used as an absolute quantification of expression of the selected genes.
3) They used inappropriate statistical tests that are more prone to giving significant results which is possibly why they were selected.
4) Their dosing regime for the mice makes assumptions on the development of mice that are not correct.
5) They gave the mice far more aluminum sooner than the vaccine schedule exposes children to.
6) There are irregularities in both the semi-quantitative RT-PCR and Western blot data that strongly suggests that these images were fabricated. This is probably the most damning thing about the paper. If the data were manipulated and images fabricated, then the paper needs to be retracted and UBC needs to do an investigation into research misconduct by the Shaw lab.

Maybe there’s a benign explanation for the irregularities that we’ve observed, but until these concerns are addressed this paper cannot be trusted.

[Metal/Melodic Death] Arch Enemy – Rise of the Tyrant (10th Anniversary Review)

Today marks the 10th anniversary of the release of Arch Enemy “Rise of The Tyrant”, the seventh album of the band. It is a 49 minutes, 11 tracks packed with fury and rage, in my opinion one of the best AE album from Angela Gossow-era.
We start with the ballistic “Blood On Your Hands” setting the tone: brutal and full of rage, one of my favorite tracks of the album by its fast-paced drums and guitars, Angie roars and Michael’s melodic riffs. We do not have any second of rest as we move with “The Last Enemy”, keeping on the insane speed of “Blood On Your Hands”. We are not yet done with the 10-minutes mark that we have this huge burst of adrenaline, priming you to enter the mosh pit. “I Will Live Again” slows down the pace into a more classical AE, allowing us to better savor on Michael’s melodic riffs and focus on Angie’s lyrics. This classical interlude is only transient as we move to the fourth track “In This Shallow Grave”. Combining fast and slow tempos, with Michael neighing his guitar like a horseman of the Apocalypse, Angie dark lyrics are really hitting deep inside, as some quotes from the lyrics “I have no soul, I have no blood. Genocide! Who is your master in this shallow grave? Alone, in this shallow grave….”. “Revolution Begins” appears in my opinion something you would have heard from “Anthems of Rebellion”, setting aside the brutal riffs and drums of the first tracks to allow the exposure of the lyrics. “Rise of The Tyrant”, the leading track of the album starts with an excerpt of the movie “Caligula” in which Caligula declare himself as a God, following by the brutal riffs and Angie growls. We are at 30 minutes and man this album is damn great in keeping us engaged and beefed up. “The Day You Died” keeps on the classical AE, so we are in good old melodic death metal. “Intermezzo Liberte” is this kind of interlude I love with AE, allowing us to enjoy Michael Amott’s remarkable guitar play. “Night Fall Fast” keep on the straight A line. “The Great Darkness” is maybe the weakest in my opinion but still pretty decent. “Vultures”, the last track of the album, perfectly an almost flawless album. Ten years later, “Rise of The Tyrant” hold up very well in my AE discography alongside with “Doomsday Machine” (my two favorites of AE Angela Gossow-era). If you have been entering Arch Enemy and have to pick an album with Angie fronting the band, pick this one.

[Videogames/Retrogaming] NPR 09/18/2017 – Looking For Analog: Old Button-Mashing Arcades Come Back For A New Generation : NPR

Very interesting article from NPR. I got into the emulation about 20 years ago, using MAME and my old 14.4K modem. Yep, that was my college years.
I still remember that now extinct emulator called Replay+ that was capable of emulating Data East’s “Robocop”. It was such an amazing experience to enter the world of emulation and see the technical prowess of some programmers involved in the MAME projects and emulators, those that spend hours dumping EEPROMs from PCB arcade cabinets, but also seeing people tinkering arcade cabinets like Arcade@Home or even some attempting to rescue old PCBs and arcade cabinets from the fate of slow decay in abandoned warehouse (you have to Google that rescue operation of a abandoned cruise ship somewhere in the UK). I am still preciously keep my own little collection (Atari and SEGA, because SEGA did what Nintendon’t).
The arrival of the Raspberry Pi with frontend like RetroPie, as well as custom cabinets like bar cabinets (illustrated below), brings this glowing and warm feel that all these EEPROMs have an after-life as dumped ROMs and enjoy a second life amongst the retrogaming community.
There is still a legal grey zone about the legality of ROMs but it is also a formidable opportunity to ensure a proper archiving and storage of such precious creations. It is also becoming a run against the clock as prototypes and unfinished games bytes slowly decay from the aggression of time (floppies and hard drives getting de-magnetized, dumping machines dying from wear, dephasing of CRTs in favor of flat panels…..). Yet, the popularity is more than ever (Wreck-It Ralph anyone?). Just look how Nintendo creates a shark frenzy with his classic NES and SNES emulation consoles (don’t let me talk about ATGames, they have been doing terrible jobs in providing decent SEGA retro consoles).
Glad NPR brought this article about retrogaming and ignite the same passion for retrogaming. Who remembers “Silver Spoons” arcade cabinets inside the game room? This is not anymore a dream, almost anyone can have their own arcade cabinet at home. Hmmmm, I should definitely rethink about getting my hands with a hammer and some nails, a soldering gun and a saw….

 

Source: Looking For Analog: Old Button-Mashing Arcades Come Back For A New Generation : NPR

[Metal/Melodic Death] Arch Enemy – Will to Power (90%)

Finally, after a week in waiting….got my Arch Enemy pre-order by mail including a tee and a digital version of the album (including a poster and three stickers, yikes!).
So how does Arch Enemy’s latest album hold? How does Jeff Loomis brings in the band? Interesting to do this review a week ahead on the 10th anniversary of their album “Rise of The Tyrant”.
Well you have the choice: the short or the long comment. The short one? A damn good AE album! The long one? Gonna have to read it through. This is an album made of 13 tracks totaling 54 minutes.
We got introduced by “Set Flame To The Night”, an instrumental opener that for AE fans reminds us the intro of “Khaos Legion” album. “The Race” brings on the big guns and set the tone of the album: its gonna loud, fast and furious. You know something from the good old times when Angie was fronting, that stuff that raises your adrenaline serum levels and gives you to jump into a mosh.
“Blood In The Water” slows down a bit, bringing the classical Michael Amott melodics riffs backed by Jeff Loomis, some classic AE style as I love it :). The “World Is Yours” marks this blend of the old and the new (brought with “War Eternal”). The tunes are catchy, certainly a great one as flagship song for promotion but I am not much impressed. That can be a great primer to the band if you have not listened to the band before. Listen by yourself and judge:

The fifth track, “The Eagle Flies Alone” is slower in pace but it is like the melodic power ballad with Michael and Jeff bringing on the riffs. Thats what I like about AE, powerful melodic riffs with fast-paced brutality. AFE!

“Reason To Believe” is the one posing as a power ballad, imagine stretching the Michael’s interlude into half a song with Alissa bringing on her experience on clean voices from the Agonist on that one, distilled inside this song. Turn on your lighters on that one. Oh yeah!
The seventh track “Murder Scene” brings back the brutal AE, good stuff but not too impressive. That compared to the eight track, “First Day In Hell”, starting like “We Are A Goodless Entity” only to bring it the heavy, hammering tough lyrics.
“Saturnine”, the the 9th track is this very nice instrumental interlude that Michael used to serve to each album. Short but sweet gothic one. “Dreams of Retribution” starts with Michael melodics a la Yngwie Malmsteen only to bring us the power melodic riffs surfing on some tunes we grew accustomed in “War Eternal”. Damn good track! “My Shadow And I” is nice but yeah not really talking to me much. “A Fight I Must Win” is a nice one to listen, straight outta AE playbook. The last song is “City Baby Attacked By Rats” is a very nice cover of the original punk song from GBH. I am not into punk music, but the cover is pretty rad. The original below.

So here we are at the end of the album. What can I say? I got introduced to AE via “War Eternal” and went into their discography retrospectively. Paradoxically, listening to “War Eternal” sounded one of the weakest link of their album. But, there is a big but here. I consider “War Eternal” as a transition and may explain this weakness. “Will To Power” brings the good old AE, Alissa settled into the continuation of Angie but in the same type pushing the band to foray into new avenues like “Reason to Believe”, there is also this idea of reaching out by Michael, maybe as his experience in pushing his Spiritual Beggars project, with the cover of “City Baby…”.
Arch Enemy brings on the big guns, followed by the engaging melodic riffs, bringing you in this state of frenzy prime for some memorable moments in the mosh! Thats some AFE album you handle here!