[Sciences/Stem Cells] Case report of a successful treatment of a junctional epidermolysis bullosa (Hirsch et al., Nature 2017)

This is a very groundbreaking case report here on a patient suffering from junctional epidermolysis bullosa (JEB). This is a genetic condition marked by mutation in the LAMB2 gene, a gene encoding for laminin beta-2 chain of the laminin complex. Laminins are part of the extracellular matrix and play an important role in cell adhesion such as skin cells. Imagine a concrete slab by which your house sits firmly.
In patients suffering from JEB, this slab is wobbly and crackle under the pressure due to some issues in the concrete composition. These patients end up having a very fragile skin that rapidly detach and have a low life expectancy due to high risk of infection and important handicap. Until now, no treatment has been successful, including skin grafts.
In this case report, the authors have taken skin stem cells from this patient and have corrected the mutation by inserting a correct gene copy of LAMB2 via retrovirus. Such stem cells were maintained and expanded into Petri dishes to form skin graft islands. Such islands where then implanted to the patients and successfully grafted in. Apparently the patient has been able to recover and live an almost normal life.
Another “Yes!” moment for science! If you are interested to read more about it, you can find it here:
https://www.nature.com/articles/nature24487

 

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[Metal/Death] Cab Ride Home

I have a strong urge of listening something brutal this morning, in particular something from a band named “Cab Ride Home”. The video is a bit garbage (the audio is horrible!) but it is some good death. I let you with this video. Oh, did I mention that a certain Danica Roem is on the vocals? :p
https://youtu.be/aHvbLXyzank

[Metal/Symphonic] Nightwish – Angels Fall First (20th Anniversary)

Today marks the 20th of the release of the Finnish Symphonic Metal band Nightwish “Angels Fall First”. It is interesting to think that for some reason Tuomas does not consider it as their first album. Sure, it had a limited release, mostly in Finland, but I still consider as their first album.
It is interesting to listen back to this album in its retrospection. It is an 11-track album, lasting a 67 minutes. Coming back from that day, the first thing that comes into the attention is the use of keyboards elements wrapping the guitar riffs. But foremost, it is Tarja that brings on the attention. In my knowledge (and I maybe wrong on this one), this is the first instance of the presence of a trained of a professionally trained mezzo-soprano. Sure, femal-fronted metal bands were already here including in gothic metal bands like The Gathering.
It starts with “The Elvenpath” setting the tone, with excerpt quotes from the 1978 cartoon movie “The Lord of The Rings”. The second track, “Beauty & The Beast” somehow sounds like more Nightwish as we know it today, in which Tuomas keyboards lead the charge. A 10:22 track of some awesomeness in the blend of the instrumental symphonic elements with the guitars melodics played by Empuu. Thats it, the prototypical symphonic metal track that surely inspired many to also experiment the same in their studio. You can take anyone in a blind test, ask him or her the band behind the song and get “Nightwish” cited most of the time. I discovered this album much later than the release, but I can imagine the reaction folks had back then. “The Carpenter”, third track of the album is more in line with gothic metal elements, sounding more familiar. It is also the first track of the album featuring Tuomas in the vocals. I guess there is a reason Tuomas hates this album and also why he does not sing in the other albums :p. It’s okay but yeah kind of a let down from “Beauty & The Beast”. Just listen here.
https://youtu.be/Z4A5HwMOfW8

“Astral Romance” brings up the album to a higher level, that is another favorite in my playlist, some good old Nightwish here (I somehow prefer these kind of outdated tunes from their most recent albums). “Angel Fall First”, the fifth track, is somehow introducing us to the powerful keyboard ballads that Tuomas will produce later in his career. Again, Tarja’s voice is magnificent even in her beginning career. “Tutankhamen” brings the metal kick back in, making it very enjoyable. Again, I could serve it into a blind playlist and someone would swear this track is part of their later album. “Nymphomaniac Fantasia” is a nice one too, but maybe a bit “cheap” in my opinion. “Know Why The Nightingale” is another good one from the album.

“Lappi (Lappland)” is the only Finnish song but oh boy, I can guarantee a lots of headbangers rushed to a Suomi dictionary to understand the lyrics. But who cares? You don’t need to understand it to feel the beautiful in this song. Again, it hints us about the whiz inside Tuomas in handling the keyboards, much more at ease than singing. Some damn good old Nightwish!

The album concludes with “Return To The Sea” inspired by gothic elements and finally “Once Upon A Troubadour”, that is honestly not much my favorite. Blame it on Tuomas or not, I don’t know just not that fond.

[Metal/Doom] Hallatar – No Stars Upon The Bridge (95%)

Hallatar, a side project band former by Amorphis vocalist Tomi Joutsen, Swallow The Sun guitarist Juha Raivio and ex-HIM drummer Gas Lipstick released the tribute album “No Stars Upon The Bridge” to late metal singer Aleah Starbridge, band member of “Trees of Eternity” and partner in life to Juha.
What I can tell that after a year that we lost Aleah to cancer, the pain and sorrow is still present in the metal scene. Hallatar, through this album, is playing into a much more darker and black tone that any of the members usually play in their respective bands. These bands are already playing some fine doom, already heavy in dark and despair. Imagine achieving the most concentrate black tone you can from sadness, sorrow and pain. This is what this album is basically giving us: a very painful, sad and heart wrenching requiem to Aleah, using her own poems put into lyrics and offering into a majestic album that distillate some of the best spirits of all three bands into a masterpiece of modern doom metal.
The album is a 9-track album, a 40 minutes requiem that will shake your from deep in it, reviving some of these painful souvenirs of the lost ones. I am warning you, this is not for the fainted of heart. It is very depressing but so liberating and cathartic.
We start with “Mirrors” with the vocals of Tomi and the signature guitar tones of Juha (that gives this trademark to Swallow and Trees). Slow, doom and gloom, damn good funeral doom introductory track.

The interlude comes from hearing Aleah’s voice reciting her poem “Raven’s Song” (Track 2) and followed by “Melt”. Straight into the playbook from Swallow the Sun, Tomi’s clean voices allows us to fully appreciate the lyrics, accompanying us into the processing funeral march. “My Mistake”, the fourth track song, features Heike Langhans (Draconian) as guest lyrics and keep on into our funeral processing march. “Pieces”, the following track, provides an interlude with its solitary piano with Aleah’s poem haunting us. “Severed Eyes”, is in my opinion this kind of track that is made to heal and remember our dearest love ones that we lost by playing on the dry guitars and the simplicity. “The Maze” is a damn good funeral doom track, an amazing track by its ability to wrench your inner mind and rediscover these dark corners that often get buried into our lives. “Spiral Gate” is the last interlude of Aleah, leaving us with her last goodbye poem and nicely transitioning to the last track “Dreams Burn Down”, the concluding track of the album. Majestic piece of work, like a last farewell to the casket before the burial with Aleah haunting voice. You realize that as the casket is brought down and the first pile of dirt covers it that this is over. That the nightmare is not a nightmare anymore, it is the reality of life and we cannot reverse anything to it. It is time to give our farewell do our dearest love and remember it into our hearts and mind.
What I can say is rarely such an album, fruit of a collaboration of some of the finest doom/gothic Suomi (Finnish) metal bands, can shake someone inner psyche and let it tears the emotion apart. Yes, it is sad and depressing. Yes, it is dark and heart wrenching. But it is also a cathartic album allowing us to anyone listening to it to make the last grievance and keep in our mind the loss one of the most beautiful feminine metal voice we lost in the person of Aleah Starbridge. Rest in peace nightingale :(.

[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.

 

 

 

 

 

 

 

 

 

[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.

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The unaltered figure 4.

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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.

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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.
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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:
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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.