[Sciences/Maladies Orphelines] Resume de la conference GLUT1 a Washington, DC

La semaine dernière a eu lieu la conférence sur le déficit GLUT1 2019, qui s’est tenue au Hilton Crystal City. Il était bien situé près de l’aéroport national Ronald Reagan (DCA) facilitant l’accès à la conférence et idéalement situé au 2ème étage de l’hôtel, avec différentes salles de bal utilisées. Avant d’entrer dans les détails, je dois révéler que j’ai assisté à la moitié de la première journée et surtout à des sessions que j’ai jugées pertinentes en tant que scientifique. Si quelqu’un d’autre veut rédiger un résumé sur l’autre session, n’hésitez pas à me contacter, je n’hésiterai pas a l’ajouter à ce post.

Séance du mercredi après-midi:
La première session à laquelle j’ai assisté était une séance plénière de recherche animée par le Dr Juan Pascual (UT Southwestern), qui nous a donné quelques informations sur l’essai clinique en cours des NIH en cours concernant l’essai clinique de triheptanoine. Nutrigenix menait un essai clinique parallèle et, après l’arrivée des premiers résultats, a décidé d’arrêter cet essai. Les premiers résultats du Dr Pascual ont montré une réduction des crises convulsives chez environ 7 patients sur 12 traités par TH. Le traitement a été défini sur une période de temps (si je me souviens bien, il était de 6 mois) et arrêté. Certains patients ont continué à s’améliorer, d’autres ont montré une diminution de l’amélioration. Une caractéristique intéressante était le rapport sur les modifications de l’EEG suggérant une possible interaction entre les circuits excitateurs et les circuits inhibiteurs.

La deuxième session a été animée par le Dr McKenzie Cervenka (Université John Hopkins), qui a interrogé des patients adultes. Une caractéristique intéressante est que tous les enfants initialement diagnostiqués avec GLUT1DS dans les années 1990 sont désormais tous des adultes et que nous apprenons à connaître la maladie à mesure qu’ils vieillissent. Une observation intéressante est que nous avons une surabondance de GLUT1DS chez les adultes. En particulier, un examen des antécédents médicaux suggère que les symptômes bénins signalés dans leur enfance peuvent indiquer une infection à GLUT1DS, mais sont passés inaperçus en raison du manque de diagnostic ou de symptômes cliniques appropriés.
Chez l’adulte, il semble être qualifié d’encéphalopathie / déficience cognitive légère et chronique accompagnée de crises peu fréquentes. Une spasticité et une ataxie variable ont été rapportées, telles que la dyskinésie paroxystique induite par l’exercice. Bien que le niveau recommandé de cétose chez les enfants soit d’environ 5mmol/L, ce niveau ne semble pas être atteint chez les patients adultes (probablement plus autour de 3mmol/L).
Il n’y a aucun signe de différence de sexe ou de genre en ce qui concerne l’apparition (50%), les symptômes les plus courants sont: ataxie (63%), difficulté cognitive (66%) et difficultés d’élocution.
Environ 82% ont signalé certains déclencheurs (excitation, stress ou anticipation, chaleur, faim, fatigue….) Et 67% ont signalé des modifications des symptômes à la puberté.
61% des patients interrogés suivent le régime cétogène (52% du traitement classique et 33% du régime Atkins modifié). Environ 41% des personnes qui suivaient le régime céto n’avaient eu aucune crise pendant leur enfance.
Grâce à la prise de conscience récente, l’âge des patients sans crise se rajeunit.
Environ 46% des patients prenaient des médicaments antiépileptiques, notamment de l’acétazolamide, du lévétiracétam ou de la lamotrigine.
Notamment, 91% des répondants ont trouvé que l’activité physique réduisait les symptômes, 100% étaient capables de faire des activités quotidiennes de base et 36% étaient capables de conduire. Notamment, 19% ont fondé une famille et ont des enfants atteints de GLUT1DS. Cela a amené quelques discussions sur la manière dont les patientes enceintes devraient traiter le KD et sur le besoin urgent de mener davantage d’études pour évaluer l’effet du KD sur la grossesse. Il y a des spéculations selon lesquelles un tel KD pourrait être protecteur chez ces patients et le fœtus.
Une mise à jour récente des directives a été publiée récemment dans Epilepsia et est disponible en accès libre ici: https://onlinelibrary.wiley.com/doi/10.1002/epi4.12225

Les autres recommandations étaient de suivre la ration calcium / créatinine dans l’urine, d’augmenter l’hydratation et de prendre en compte le risque de carence en carnitine induit par l’utilisation de plusieurs antiépileptiques. Les niveaux de carnitine doivent être surveillés 1 et 6 mois après le début de la MK.

Enfin, une présentation d’un scientifique de Sanofi sur la thérapie génique s’explique en particulier par le succès récent et l’approbation par la FDA de deux thérapies géniques des maladies RPE65 et SMA.

Le reste de la journée a été consacré à une session d’affiche, assez succincte (moins de 10 affiches), mais qui a été une expérience enrichissante de permettre à des patients et à un gardien d’interroger mes connaissances scientifiques et de les expliquer. Ce fut une expérience difficile mais enrichissante, ainsi que des données alignées sur d’autres études.

Séance du jeudi matin:
Il y avait une série de différents panels principalement axés sur les patients et sur le régime cétogène.
Le premier orateur était le Dr Eric Kossoff (Université John Hopkins), soulignant les périodes intéressantes pour le régime céto. Il existe plus de 3000 publications à ce jour et 7 essais contrôlés randomisés. Il devient assez intéressant de demander à l’American Epilepsy Society d’organiser une session satellite (ou une session au sein de la session plénière) sur le régime céto en général.
Il semblerait que le régime cétogène chez la souris modifie le microbiote intestinal.

une étude récente citée dans Cell: https://www.cell.com/cell/retrieve/pii/S0092867418305208?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS00967230308%3
Les auteurs ont signalé une diminution de deux types de bactéries (Akkermansia muciniphilia et Parabacteroides spp) chez des patients atteints d’épilepsie réfractaire. Le régime céto a pu être modifié 4 jours chez la souris. Fait intéressant, la restauration de ces bactéries a restauré la protection contre les convulsions. Il n’y a pas encore d’études cliniques à l’appui de ces affirmations.
Il n’y a pas non plus de raison de restreindre les liquides ou les calories.
Tous les régimes semblent être valides, vous choisissez celui que vous pouvez coller. Cependant, il est recommandé d’adhérer au KD pendant <2 ans et à la version Atkins modifiée pendant> 12 ans. Environ 80% des patients ont présenté une réduction des crises épileptiques supérieure à 90% sur le KD et le MAD et 64% n’ont plus besoin d’anticonvulsivants. Le régime céto semble très efficace pour la cognition et la dyskinésie.
Bien que l’arrêt du KD chez les patients atteints d’épilepsie puisse être envisagé environ 2 ans après l’absence de crises épileptiques, le consensus est de continuer chez les patients sous GLUT1DS.
Le régime céto est un véritable engouement depuis deux ans, avec de bonnes informations et beaucoup de mauvaises informations. Il a été recommandé d’utiliser précautionneusement certains produits étiquetés comme compatibles céto.
Une autre discussion a porté sur l’effet à long terme du régime cétogène. Les données actuelles suggèrent que, bien qu’une élévation du cholestérol total et des LDL aient été observés à 3 mois, une normalisation sous la forme d’une diminution semble se produire. Plus d’études longitudinales sont actuellement en cours.
Il n’y a pas de risque décisif de malformations congénitales chez les patients suivant le KD.
Enfin une information rapide sur l’huile de CBD. Certains patients ont admis l’utilisation de l’huile de CBD en tant qu’adjuvants et n’ont trouvé aucun bénéfice supplémentaire. La restriction de l’huile de CBD dans le cadre d’essais cliniques reste a être documente.

Séance du jeudi après-midi:
J’ai moins de notes prises parce que c’était généralement général, mais il y a eu quelques faits saillants de la recherche, y compris ceux du Dr. Umrao Momani (Université de Columbia) en éliminant sélectivement SLC2A1. Il semble que le gène knockdown chez les souris de la petite enfance (P2) et de la petite enfance (P28) ait eu une plus mauvaise performance motrice par rapport à la désactivation au stade avancé. Cela a également été rapporté par une aggravation des crises et une diminution de la densité capillaire dans le cerveau de ces deux groupes.
Le Dr DeVivo a également signalé les problèmes de patients classés comme des patients du type GLUT1DS en raison de la présence de symptômes rencontrés chez les patients GLUT1DS, mais ne montrant aucune mutation connue dans SLC2A1 ni aucune mutation connue des gènes SLC2A3 (GLUT3).

Voici quelques-unes des notes prises lors de la conférence G1D. Rendez-vous à San Diego en 2021!

Advertisements

[Sciences/Rare Diseases] Summary of the GLUT1 Deficiency Conference 2019 – Washington, DC

Last week occurred the GLUT1 Deficiency Conference 2019 that was hosted in the Hilton Crystal City. It was nicely located near the Ronald Reagan National airport (DCA) making it easy access to the conference and conveniently located in the 2ndfloor of the hotel, with different ballrooms used. Before I go into details, I have to disclose that I have attended half of the first day and attended mostly sessions that I considered relevant as a scientist. If anyone else wants to write a summary on the other session, please feel free to contact me, I will add to this post.

Wednesday Afternoon Session:

The first session I have attended was a research plenary session hosted by Dr. Juan Pascual (UT Southwestern) giving us some update on the current NIH clinical trial ongoing when it comes to the Triheptanoine Clinical Trial. Nutrigenix was running a parallel clinical trial and after initial results came in decided to discontinue such trial.

Dr. Pascual initial results reported a reduction in seizures in about 7 patients out of 12 on the TH. The treatment was defined in a time period (if I remember well it was 6 months), and stopped. Some patients continued to improve, some showed a drop in the improvement. An interesting feature was the report of changes in the EEG suggesting a possible interplay between excitatory circuits and inhibitory circuits.

The second session was provided by Dr. McKenzie Cervenka (John Hopkins Univeristy) that has been surveying patients that are adults. An interestin feature is that all children initially diagnosed with GLUT1DS in the 1990s are now all adults and we are learning about the disease as they age.

An interesting observation is that we have a glut of GLUT1DS occuring in adults, in particular a review of medical history suggest that mild symptoms reported in their childhood maybe indicative of GLUT1DS but went under the radar due to the lack of proper diagnosis or clinical symptoms.
In adults, it seems to characterize as a mild and chronic encephalopathy/cognitive impairment with infrequent seizures. Some varying spasticity and ataxia were reported, as the paroxysmal exercise-induced dyskinesia.

Although the recommended level of ketosis in children is about 5mmol/L, such level seems not reachable in adult patients.
There is no signs of sex or gender differences when it comes to occurrence (50%), the most common symptoms are: ataxia (63%), cognitive difficulty (66%), and speech difficulties.
About 82% reported some triggers (excitation, stress or anticipation, warm, hunger, fatigue….), and 67% reported changes in the symptoms when reaching puberty.

61% of the patients surveyed are on the ketogenic diet (with 52% on the classic one and 33% on the modified Atkins Diet). About 41% on the keto diet were seizure-free in childhood.
Thanks to the recent awareness, the age of seizure-free patients is getting younger.
About 46% of the patients were on AEDs, in particular acetazolamide, levetiracetam or lamotrigine.
Notably, 91% of respondent found physical activity reduced symptoms seizures,100% were capable of basic daily activities and 36% able to drive. Notably, 19% started families and have children with GLUT1DS.  This brought some discussion about how pregnant patients should handle the KD and the urgent need for more studies to assess the effect of KD on pregnancy. There is some speculation that such KD maybe protective in these patients and the fetus.
A recent update in the guidelines were published recently in Epilepsia and available as open-access here: https://onlinelibrary.wiley.com/doi/10.1002/epi4.12225

Other recommendations were to follow calcium/creatinine ration in urine, increase hydration, and consider the risk of carnitine deficiency induced by use of multiple AEDs. Carnitine levels should be monitored 1 and 6 months after initiation of KD.

Finally, a presentation from a Sanofi scientist about gene therapy, this is in particular in light of recent success and FDA approval of two gene therapies for RPE65 and SMA diseases.

The rest of the day was a poster session, that was pretty succinct (less than 10 posters) but really was an enjoying experience to have patients and caretaker asking about my science and explaining them. It was some challenging but enriching experience, as well as some data aligning with other studies.

Thursday morning session:

There was a series of different panels mostly driven for patients and about the ketogenic diet. The first speaker was Dr. Eric Kossoff (John Hopkins University) highlighting the interesting times for the keto diet. There is over 3000 publications as of today, and 7 randomized controlled trials. It becomes interesting enough to have the American Epilepsy Society to have a satellite session (or a session within the plenary session) about the keto diet in general. There are some indication that the ketogenic diet in mice is altering the gut microbiota, based on a recent study cited in Cell (https://www.cell.com/cell/retrieve/pii/S0092867418305208?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867418305208%3Fshowall%3Dtrue)

The authors reported a decrease in two types of bacteria (Akkermansia muciniphilia and Parabacteroides spp) in patients with refractory epilepsy. The keto diet was able to alter 4 days in mice. Interestingly, restoring these bacteria restored seizure protection. There is yet any clinical studies to support these claims. There are also no reasons to fluid or calorie restriction.

All diets seems to be valid, you choose which one can stick. However, it is recommended to adhere to KD for <2 years and to the modified Atkins for >12 years. About 80% of patients have over >90% seizure reduction on KD and MAD and 64% no longer required anticonvulsants. The keto diet seems highly effective for cognition and dyskinesia.

Although the discontinuation of KD in epilepsy patients can be considered about 2 years after seizure-free, the consensus is to keep on in GLUT1DS patients.

The keto diet is really entered a craze in the last two years, with some good information and also a lot of bad information. It has been recommended to operate precaution on some products labeled as keto-friendly.

Another discussion was on the long-term effect of ketogenic diet. The current data suggest that although an elevation was observed for total cholesterol and LDL were reported at 3 months, a normalization as a decrease seems to occur. More longitudinal studies are currently performed.

There is not conclusive risk of birth defects on patients following the KD.
Finally a quick information on CBD oil. Some patients admitted the use of CBD oil as adjuvants, found no additional benefits of it. Restriction of CBD oil restrains it the use of clinical trials.

 

Thursday afternoon session:

I have lesser notes taken because it was mostly general but there were some research highlights including from Dr. Umrao Momani (Columbia University) by selectively knocking-down of SLC2A1. It seems that knockdown the gene in infancy (P2) and early years (P28) mice fared worse on motor function compared to late stage silencing. This was also reported by worsened seizures and decrease capillary density in the brain of these two groups. Dr. DeVivo also reported about the issues of patients that are classified as GLUT1DS-like patients due to the presence of symptoms as encountered in GLUT1DS patients but showing no mutations known in SLC2A1 and also no known mutations in SLC2A3 genes.

These are some of the notes taken at the G1D conference, see you in San Diego in 2021!

 

[Sciences/Neurosciences] Propionic Acid Induces Gliosis and Neuro-inflammation through Modulation of PTEN/AKT Pathway in Autism Spectrum Disorder (Abdelli et al., Sci. Rep. 2019)

Once a wise man said: “Be always wary of scientific studies trumpeted by mainstream news outlets as groundbreaking. Once the smoke settles down, the study in question is rarely groundbreaking, but rather limited with a lot of caveats”. If I have to summarize this study, that would be within the lines of the wise man. Despite what news outlets have been selling, this is a study that has its own merits, but its methodological limitations and caveats outweighs the novelty and significance. Especially considering the publication occurred in Scientific Reports, the response of Nature Publishing Group to the open-access model, this is an extra layer of concern as Scientific Reports prestige and quality has suffered major setbacks in the last few years due to papers retracted for blatant scientific misconducts that should have been spotted by reviewers.

About the authors: We have three authors. The first author seems to be a postdoc, as she apparently graduated from the same program from another lab. The second author maybe an undergrad, although a faculty with the same family name is listed. Finally the senior author is a faculty with an expertise (based on the publication record) on gastrointestinal (GI) tract physiology and pathophysiology. However, none of them seems to have a history of publication in any field of neurosciences. This is an important point, because it explains a lot of methodological flaws that anyone with neural stem cell biology (and brain development) could pick easily.

1. Introduction and hypothesis: The authors are basically using the rationale of changes in metabolomics observed in ASD patients and reported by several studies. In these studies, there are some indications that certain patients on the spectrum (especially those qualified as severely disabled) display an impaired GI function, in particular something we could qualify as something similar to inflammatory bowel diseases. There are studies suggesting that such GI condition is associated in a changes in the gut microbiota, yet with a fairly low resolution (we are able to document changes in a family of bacteria, but not able to pinpoint to the level of Genus species yet). In particular, there is a study that was recently published in Cell Stem Cells (very high impact factor journal) that highlighted changes in mice behavior and gene expression profile of several genes associated to autism following the fecal transplant from patient on the spectrum considered severe (https://www.cell.com/cell/fulltext/S0092-8674(19)30502-1).

In this study, the authors speculate that certain metabolites biosynthesized and/or bio transformed by these class of bacteria are contribution to the symptoms. In particular, the authors consider acetate (AC, CH3-COOH) , propionate (PPA, CH3-CH2-COOH) and butyrate (BA, CH3-CH2-CH2-COOH) as potential culprits, citing studies showing an elevated levels of these small chain fatty acids (SCFA) in fecal cultures of ASD patients compared to control patients. The authors also cite two rare genetic diseases such as neonatal propionic acidemia (PA) and propionyl coA carboxylase (PCC). PA will be very useful to us, because it will help us set what we would consider a pathological level of propionic acid (PPA) in blood. Yet comes the most speculative, and I would say the “jumping the shark” moment of the paper. The authors assume that since processed foods are rich in PPA, such amount of PPA can lead to the development of ASD in the fetal brain during pregnancy. That’s a lot of speculations with little or no layers of evidence. We have here the authors trying to make a statement four to five steps too far from the existing literature for several reasons (and also lakcing the literature backing them up) that be identified as the following:

1.   Where is the literature providing evidence that these SCFA cross the GI tract, at which extent (bioavailability studies)?
2.   What are the levels of PPA (and other SCFA) in the plasma/serum levels of neurotypic patients versus patients on the spectrum? For patients suffering from PA, I have found an old study referring to serum PPA as high as 0.337-1.35mM, with a normal level about 0.00337mM (https://www.jpeds.com/article/S0022-3476(81)80004-2/pdf)
3.   How much of PPA can cross the blood-brain barrier? This is an important question to answer. We can try to build on the analogy of the SCFA to ketone bodies (acetoacetate, beta-hydroxybutyrate) that are formed when someone is fasting or forced into a ketogenic diet. Considering that patients suffering from GLUT1 Deficiency Syndrome are showing improvement when put on a ketogenic diet (with BHB levels around 2-3mM), we can speculate these compounds can cross the BBB readily.
4.   How much of PPA can cross the placental barrier? I don’t have any clue either.

These talking points are important because it will determine if the experimental design of a study is sound or deeply flawed, which eventually will set the quality of the paper and the robustness of the conclusion made. That’s something I should mention by now, is that both the authors and the news outlets have been very fond of superlatives and trying to sell that paper at much higher level that it is meant to. Not only it is scientifically inadequate to make extraordinary conclusions without highly robust data to support them, but also it is nowadays dangerous to do so as such studies will be used by scammers, charlatans and other snakeoil salesmen to promote their supplements claimed to “cure autism” and use these type of studies to claim their products is supported by science.

2. Materials and methods: The authors used neural stem cells (NSCs) derived from fetal tissues (obtained from Life Technologies/Thermofisher) and maintained in a classical medium formulation aimed to maintain these NSCs in their pluripotency stage. The cells were passaged no more than three times, according to the author. This is important, as NSCs/NPCs passaging over time will coax them towards the astrocytes lineage compared to neurons (in terms of development, neurons appear earlier and mature earlier than astrocytes).
Now there is something intriguing. The authors claimed they looked at PPA and BA at concentrations ranging from 0.1mM (that would be physiological), 0.5, 1 and 2mM concentration. Considering such treatment would reproduce the fetal brain, we have to factor in what is the amount capable to cross the three barriers: GI barrier of the mother, the placental barrier and finally the BBB. However, the authors never showed what happened to these concentrations except the 2mM which is limit deadly (remember? This is the one that is detected in newborns having the rare genetic mutation). Are the authors trying to model the effect of PPA to model such diseases or are they assuming that the amount of PPA in processed found will be high enough to put the pregnant women into a severe metabolic acidosis? I don’t know but that like a red flag.
The differentiation of these NSCs into neurons/astrocytes were left to occur in a fairly random fashion, as the authors use the same medium used for NSCs maintenance but without growth factors. I think this is an important issue here as we may have a significant variability in terms of yield between passages in particular when it comes to neurons/astrocytes ratio (personal communication with Clive Svendsen).
Otherwise, nothing else really fancy and classical techniques found in any neurosciences studies: Immunocytochemistry, neurite outgrowth, qPCR…

3. Results:
3.1. Figure 1: I am a bit perplexed from what I see and from what I get when it comes to quantifications. The first issue I have is the lack of scale bar. A scale bar tells you how many pixels equals to a length. For example, a 512×512 pixel image may indicate you that 100 pixels equals to 50 micrometers. Here you have to trust that the experimenter did not fudge the data, crop the pictures and really show you a 10x magnification.
For simplicity, I will focus on the Day 10. My concern is about the health of the neurospheres in some of the groups, in particular the BHB-treated group. You can see in controls; we have nice rosette-shaped neurospheres with a dark core reflecting a dense. In contrast, look at the BHB treated group. These neurospheres are small, frail and lack the morphology observed in control. I wonder if BHB at this concentration is showing signs of toxicity? If yes, the authors were not concerned at all by this issue. And this is something concerning. If BHB is neurotoxic, so how can we make a conclusion to BHB as inhibitor. There are ways to show the viability of these neurospheres: Hoechst staining, propidium iodide staining, Fluorojade staining……..Because of this important issue, I will not consider the BHB treatment as valid.

Slide1

If you compare the data shown in Fig.1B and 1C compared to the quantification made in Fig.1A (and shown in the bottom), you can see we have a certain discrepancy here. I would skip the issue in the y-axis labelling (the correct symbol for micrometers is µ (mu) not n(nu)), but compare the 10 days timepoint to the data we actually obtain from the Fig.1A. In scientific publications, you have to be sure that what your representative blot/micrograph picture shows matches your quantitative analysis. In other words, what I see in the micrograph pictures in Fig.1A should be reflected in Fig.1B and 1C.
Then explain me why the differences in diameter reported is not as different between my quantitation (using ImageJ internal functions) is different from the one displayed. How do the authors justify the use of SEM instead of SD, except for making the graph look nicer (you can see the data actually suggest a much more variability that likely undermine the statistical difference)? How does the authors explain the 3x difference in neurosphere counts between me and them? Did they crop the pictures? If so they should have accounted for, and highlights the importance of having a scale bar in micrographs pictures and normalizing such data to a surface area (e.g. pixels2, µm2, mm2….)

3.2. Figure2: These are immunofluorescence pictures of plated neurospheres allowed to differentiate by their own on Matrigel-coated plates (Cultrex). The pictures are okay, although not very convincing for some and certainly not suitable for quantification. The use of flow cytometer is definitively a go-to when it comes to assessing cell populations.

Slide2

We are also here having a mixed results and missing important cell markers. First, the authors should have performed a nestin staining, as nestin is one of the markers present in NSCs/NPCs. Second, the use of GFAP as astrocytes marker has to be taken with a big grain of salt. I am not sure experts in the field would have let this fly with just one marker. GFAP can be expressed by NSCs and NPCs. Showing at least two markers per cell lineages (NeuN/bIII tubulin for neurons, S100B/GLAST1 for astrocytes) would have been much more convincing. bIII tubulin antibody (in particular the one used in this paper) is known to have a very strong non-specific staining. A good bIII tubulin would show nice neurites. I have attached a picture of an iPSC line developed by Sigma-Aldrich. You can use it to compare so you can see what a good bIII tubulin and a good GFAP staining should look like in NPC-derived astrocytes. Here we have just some blobs (that indicates a possible non-specific immunoreactivity) that dangerously overlap with GFAP. Technically, you cannot have a neuron that express both GFAP or bIII tubulin. It is either or but not both. R&D Systems has a nice interactive map that shows you the different cell markers expressed by the neural lineage as its differentiate into neurons and astrocytes here: https://www.rndsystems.com/pathways/neural-stem-cell-differentiation-pathways-lineage-specific-markers

What I am supposed to do with that?

3.3. Figure 3: The authors looked at both GFAP and bIII tubulin at mRNA levels (PCR) and protein levels (by ELISA). I would have personally put the PCR data first, followed by the ELISA data. The PCR data was normalized to GAPDH and the DeltaDeltaCt method was used, which is good, the authors also have represented the apparent bIII tubulin or GFAP/GAPDH ratio, which is good.  However, I am more skeptical on the ELISA data. The reason why? The data is represented as micrograms of protein/microliters of cell extract. I am skeptical why the authors did not run a Western-blot analysis for these two housekeeping genes, since you expect a lot of proteins being expressed. The authors also forgot to mention if they have diluted the samples or just added the crude extract at is. This is important because you can easily blunt the accuracy of your ELISA. LSBio is honestly a cheapskate when it comes to showing a standard curve, unlike more established ELISA kits manufacturer such as R&D Systems or Abcam, that will show you their standard curves and tell you the coefficient of variation in them. The maximum concentration of the standard curve is 1000pg/mL or 1ng/mL, with a detection range of 15.63-1000pg/mL and a sensitivity of 9.38pg/mL. The authors reported concentrations for GFAP was 0.8-3 pg/ml according to their graphs. Something is wrong here, and we have at least 2 reviewers that completely miss that. Are the authors telling me that they were able to detect GFAP and bIII tubulin below the sensitivity level (9.38 and 313pg/mL respectively)? Give me a break! I would also have advised the authors to normalize their concentrations into something meaningful like mg of proteins. It is easy to take a fraction of the cell lysate and measure the total protein concentration by BCA. I ask my students whenever they use an ELISA for quantifying a cellular protein to normalize their amount detected (pg/mL) to a total protein concentration (mg/mL) which allows us to normalize the data. Failure to do this normalization is like showing a Western-blot without a proper loading control (e.g. actin, GAPDH….).

3.4. Figure 4: In this figure, the authors are trying to show the expression of GPR41 (aka free fatty acid receptor 3 or FFAR3) in those cells. Honestly, this is my breaking point of tolerance. First, the authors underwent some cherry-picking of the data, showing you only the PPA treatment in astrocytes (where is the BA treatment? Where are the BHB treatments?) and only the BA treatment in neurons (where is PPA? Where are BHB?).
I am also very skeptical that what the authors call astrocytes are really astrocytes looking. What we see in Fig.4A looks very similar to 4B: very thin cytoplasmic projections looking like neurites. Only neurons form neurites in cultures. Astrocytes have more a flat-shaped feature, sometimes a bit fusiform like shape. Again, the GPR41 protein expression is really up when you have tons of PPA given (mM and more). How come this went through peer-review unabated and have at least 2 reviewers did not notice this gross conundrum in the data?

4. Rest of the figures and conclusion: I can go further with this paper. It was looking very interesting and promising, but the lack of expertise from the authors quickly percolated into loose and inconclusive data. This is the kind of paper you wish the authors would seek feedback from across the street, from some faculty with a neuroscience background and give them an honest feedback to make this paper good and scientifically sound. What we have indeed is a half-baked study, served as the next big thing since sliced bread. Not only the data is far from convincing of the claims made my authors (I would probably accept as a possible model for modelling PA or PCC, but this paper IS ABSOLUTELY NOT SHOWING THAT PPA IS CAUSING AUTISM for several reasons below:

1.   It does not account for the PPA levels found in normal persons, even less provide a study showing PPA levels in people eating processed foods (if such dietary habit even lead to such outcome).
2.   It does not consider that in order to be valid the authors have to show that you have a 100% bioavailability of PPA across the GI barrier, the placental barrier and the BBB, which are not reported or cited by the authors in any credible form.
3.   It does not account that the levels used as so ridiculously high that a pregnant mother would deal with a possibly deadly metabolic acidosis.
4.   It also ignored that BHB was showing signs of neurotoxicity.
5.   There is a worrisome pattern of data cherry-picking, with groups popping in and out intermittently, sometimes even in a complacent manner. This is a no-no and an unacceptable behavior that has no place in any respected peer-reviewed journals. Why did the reviewers overlooked that issue?
6.   There are several inconsistencies in the data, especially whether the axis labels are botched or if the authors really provided measurements that were nornally impossible to reach (below detection limit).

This paper should at least had a “major revision” to fill the gaps. Yet, it went through at least 2 reviewers and none of them were able to see the obvious methodological flaws. As a reviewer for Sci Rep on a seldom basis, I am very concerned about the quality of review provided by the journal in the recent years, especially in light of series of retraction. Conclusions? The news outlets have been trying to sell an overhyped paper that does not live much under scrutiny. This is just “same old, same old” when it comes to journalistic reporting on science (trying to fudge it as groundbreaking), but also opens a dangerous precedent. I will bet that within 12 months, there will be some quack doctors and snake oilsalesmen that will claim they can cure autism by selling you supplements aimed to reduce the PPA or by selling you a dietary fad book, claiming it will cure your child autism by dietary restriction. I guess the keto diet will soon join the casein-free/gluten-free diet as outdated and have another fad being served as dietary torture to children on the spectrum.

[Computer/PC] Someones trash is someones else treasure. Rescuing a 2002/2003 dream PC from the dumpster.

“Someone’s trash is someone’s treasure”. That’s a kind of sentence I like. Not a hoarder, but I am an avid (and budgeted) retro gaming enthusiast with a knack for old computers.
You can say that I grew right as the home computer and video games market grew. I was born with a 2600 joystick in my hand (well to be honest, I was 3 years old when my uncle brought an Atari 2600 home and got to touch the iconic joystick), and early on developed the interest to computer and gaming. I still remember my first contact with a computer (a Thomson MO-5 in my elementary school, with the optical pen and the “turtle” sitting in the room, never to be used unfortunately). Over the years, some computer hit home, given by someone or God knows found somewhere, but never worked (I remember that we had an Alice 90 at some point, and a Yashica YC-64 MSX-based computer. But both lacked their cables and therefore were rendered unusable).
I still have a fond memory of my first computer that actually worked, an Amstrad CPC6128. It came with all the bells and whistle as a super-duper bundle pack: the Amstrad 6128 keyboard, the dedicated color monitor and TV tuner base (taking place right under the monitor, with a SCART and RF input and small speakers no less!), a 15-games pack (including Rambo, Galivan, Cauldron, Sorcery…..) and a computer desk!
The Amstrad CPC6128 unfortunately did not last more than a year, thanks to the infamous 3″ floppy drive, quickly replaced by an Atari 520ST. This one lasted for over 5 years before dying…..from a defective floppy drive (and also by having the Joystick/Mouse port desoldered). That was the beginning of the hiatus for me and computers. I have been playing on my friends computers then, but it took me until 1997 to finally close the hiatus and get back to PC. I bought my first PC back in 1997, through the help of my big brother friend. Because he was in Paris, he could get used components very cheap and mounted my first PC (a Pentium 120 with 16MB RAM mounted on a generic Taiwanese Intel 430FX chipset, an S3 Trio, 1.2GB WD Caviar, a 12x CD-Rom drive, an ESS Audiodrive and a VGA screen that could only do 640×480@60Hz by default). This was my first PC and since then I have mounting and building my own, gathering pieces and buying stuff on the cheap, one component at the time.
To be honest, it has been over 15 years I dropped fixing computers for the fun as I switched to a Mac as I was starting grad school. Since, I have been mostly using Macs and almost never had to put my hands inside a PC Tower.
But about a year ago, I got somehow the flame of PC building on again. This was maybe triggered by watching Youtube videos on various channels including Linus Tech Tips, Lazy Game Reviews, the 8-Bit Guy, Jesus Metal Rocks or Nostalgia Nerd. Let’s say seeing computer builds, especially seeing some old PCs being restored back to their glory ticked me.
A couple of months ago, as I was dumping the trash in the dumpster lining the backalley.  One of the neighbor apparently was doing some cleaning in his garage/man cave as they closed on selling their house and likely prepared their move. There was not one, but three computer and lots of computer stuff that were dumped in the dumpster. Most of it was junk (old cables and adapters, incomplete PC games….). One computer was a Dell Pentium III but DOA. However, there were two Antec cases in that dumpster. One was just an empty case (Big and grey tower), but the second one was almost complete PC. All it needed was a hard drive, a mouse/keyboard and a screen. I took my luck of “finders keepers” and rescued it, as well with some cables (80-pins IDE cables, yum! Pretty useful if you are modding OG Xboxes) and kept it in the garage until I had time to spend effort on it (lets say April/May were crazy months on my professional side, sucking my free time over the weekend).
Low and behold, this computer was almost a time capsule of my college years, sometimes between 2002-2004 period. The PC was an AthlonXP 1700+, mounted on an ASUS A7N8X Deluxe (no less!) built around an NVIDIA nForce2 and harboring an on-baord SATA RAID (that was the early days of SATA), 1GB DDR RAM (2×256 and 1x512MB), 2 Ethernet connectors (yikes!), and 6 USB connectors (including 4 USB2.0).
Accompanying this beefy beast is a BFG Geforce 6600 card (and my first time encountering a graphic card with the need of a Molex connector. I remember seeing them on the 3DFX Voodoo 4 but never had a graphic card needing additional wattage), two optical drives (a DVD-burner and a CD-Burner). The whole stuff was mounted on an Antec case. Back in the days, Antec cases were fairly high-end and pricy cases, much more than the generic Taiwanese case. But you have to give them credit, the design in it is pretty good, mostly screwless and really accessible to easy change compounds in it. This was probably the beast during my college years. My last PC I have before switching to an AthlonXP 1800+ with 256MB RAM, a Geforce 2 GTS (a Leadtek if I remember well), and an 40/100GB WD Caviar IDE drives (I had one for the OS, the other for my personal data).
After unmounting it piece by piece, giving it the TLC it greatly needed (the dust of West Texas can be very unforgiving for electronics), I remounted it and rewired the cables (Antec has nicely designed a backspace behind the drive bays, allowing to use it as a corridor to pass cable throughs),
IMG_0240

Now the beast was restored in all its glory. Cleaned up, rewired to improve the air flow as much as I could. I also took the opportunity to replace the 1700+ with a 2400+ processor bought on eBay for a couple of bucks, some Arctic Silver 5 thermal paste (because the thermal paste must have been long gone) between the CPU and the cooling fan, replaced the DDR RAM with 2x512MB ones (that I had from stocks gutted on computers) to enjoy the Dual Channel feature of the mainboard (you can identify them by the blue color, however it does not allow you to utilize the third memory bank), added a Seagate 320GB SATA drive lying around.

Considering the case is from 2002 (QC stamped on the bottom) the design is pretty cool, with an aeration funnel located just over the CPU ventilation. The removal of 5.25″ drives is pretty sleek and effortless, without the need of screws. These are things we could laugh about in 2019, with all these fancy cases, but back 20 years ago, these were options simply non-existent on the average beige PCs.
The only issue I have is the front USB ports that give me hell to work.  They simply don’t recognize anything plugged in. I gave some TLC to the front plastic and cleaned the small USB controller that provides the front connectors and lead to the mainboard via a special pin connector. Also tweaked the jumper setting the voltage on the USB connector (after reading the manual). Low and behold, it worked! It recognized a flash drive I had some drivers in it. That is great, because it would provide me easy access to plug in USB controllers like a generic Gamestop-compatible Xbox360 wired controller.
For the display, I also went on the cheap and acquired a Dell E197FP monitor on Facebook marketplace for $10. I wished I could find one monitor with the USB extensions, DVI connector and underneath speaker unit (that is really handy when you want to keep a compact computer), but this one will make the job. The nice thing is its old 4:3 tipping a 1280×1024 resolution on a 19″. That would be the same of my good old IIyama 17″ screen I had back then, and most importantly matching the resolution size you want for emulation, especially arcade cabinets using MAME.
For the choice of the OS, I was divided between Windows XP or putting Windows Vista in it since I have found a copy of Windows Vista Ultimate Edition in a thrift store a couple of years ago for a buck. The advantage of Vista over XP is possibly the chance to have a better chance with the EOL. The concern was of course the performance and CPU overload (Vista came in 2006, about 5 years after this generation of machines. That would be trying to install Windows XP on a Pentium II. Yes you can, but the CPU will remain a bottleneck for performance). Vista gave it a 3.2 overall score, which seems okay. But trying to stick best to the era primed over and led me to erase-reinstall Windows XP on that machine.
The biggest issue I have is dealing with the SATA driver. The chipset in it is a Silicon Image 3112 RAID. Usually, that would come in the form of a driver you would insert in the 3.5″ floppy drive during XP installation. But I have no floppy drive for this puppy and don’t have really the need of one (I am not planning to do early MS-DOS emulation on that one, as the mainboard has no ISA slot for an Soundblaster, and the PCI Soundblaster emulation is not great. Honestly, I am better with DOSBox for that purposes).
I have found a XP ISO image on the Net that was already patched with SP3 (very important, because getting hand on the last service pack for XP is not easy indeed) and also slipstreamed with a bunch of SATA drivers. Although the first phase went well (XP detected the drive, formatted it and copied the first batch of files), it would irremediably fail on the first reboot with the BSOD 0x000007B error. This one is commonly encountered on the Web with XP/Vista/7, seems to be the issue on how the OS deals with SATA interface (in some machines, you can ask the BIOS to treat SATA as ATA instead of AHCI. Unfortunately, not on mine. In addition, the absence of USB support for booting external drives put my USB floppy disk out of the game). The solution I found to this problem was to match a SATA driver that was  more recent to the latest BIOS (the mainboard got the latest official BIOS update dated from 09/2004). Using nLite, I created a new ISO that was inclusive of the latest XP driver I found (Silicon Image is long gone and I had to search on the Internet to find a driver) and booted into it flawlessly.
Finally! I was able to get best of both worlds! Windows XP and SATA Hard Drive. The machine is running great so far, although with fairly noisy fans running at full-speed all the time (despite having it plugged to benefit the Q-Fan feature of ASUS motherboard). It is almost like having a hair dryer, a stark contrast of the Dell Optiplex 7010 I have revamped earlier this year (these are pretty good potato PCs that for less than $200 gives you a decent Windows 10 PC, that my son use to play Fortnite on it).
As for now, I just finished to install the OS and its patches, as well as the drivers for it (Nvidia supported this Geforce for almost 10 years, with the latest WHQL driver dated from 2013). My main goal is to recreate somehow the experience I would have with this machine like 16 years ago. I must have some archived ISOs of Office 2003 somewhere, I installed Winamp to recreate my MP3 experience, PowerDVD for the DVD player (I am looking for options to region-hack the DVD-Rom drive (I have a ton of Region 2 DVDs that I brought from France when I migrated in the US), and emulators for anything 8 to 16-bits (the SEGA Genesis/SNES was the best we could come for emulation, with only a burgeoning attempt to emulate the SEGA Saturn/Sony Playstation or Nintendo 64 then).
One of the niceties is that GOG.com allows you to have some executable files of the games you purchase on their website, allowing me to have them run on Windows XP. In the same time, that I hate about Steam. Steam dropped XP support last year. That means you cannot play games on your machine anymore, Steam does not seem to allow you to have an offline copy. So much for buying Doom 3 for a couple of bucks yesterday, but only compatible on XP/Vista machines (to play on Windows 7 and better, you have to buy the BFG edition which is a remastered version designed for newer machines).
That would allow me to run basically games I have been playing from 1994 up to 2004 into this machine. I also have a bunch of educational and game CDs that I bought for the kids back in the days through garage sales, all concealed into a Napster CD sleeve (yep, apparently Napster licensed its name to some CD sleeves).
Ideally, that gives me an excuse to roam into Goodwills and other thrift stores and find some old games running mostly on XP. Ideally, I would like to find games from that era (or a bit earlier), as well as a PS2 keyboard (seems they are rare to find nowadays) to plug that puppy in. I also need to find a small table, ideally a small computer desk just for it. I remember IKEA having a very nice and compact computer desk (if I remember, its name was MIKAEL) that was discrete, yet well conceived for desktop PCs.
For some people, it was an obsolete machine that was taking the dust, but for me it is a nice time capsule that was uncovered, like a old and rusty oil lamp that has something magical about it. Someones trash is someones else treasure.

[Metal/Gothic] Lacuna Coil – In A Reverie (20th anniversary album)

Today marks the 20th anniversary of the release of the first album by the Italian gothic metal band Lacuna Coil “In A Reverie”. My introduction to Lacuna Coil occurred few years later than this album via their album “Unleashed Memories” that a friend lended me over, as I was deep into “The Gathering” back in that days (college years).
Until then, the Italian metal scene was pretty obscure for me, with the Italian music scene mostly represented by some pop artists such as Eros Ramazzotti or Laura Pausini that were spinning on the French airwaves, and in a more sarcastic manner their predominance of the Eurodance.
Lacuna Coil band line up changed a lot over the last 20 years with the exception of the core of the band: Cristina Scabbia and Andrea Ferro. Both made me a very powerful singing duet that is until now pretty unique in the gothic metal scene: both sing clean, no “Beauty and The Beast” duet here. That what makes them unique, as well as their artistic style that has this unique “Lacuna Coil” flavor.
“In A Reverie” is a 9-track album covering about 43 minutes, with two distinct covers: the original cover in which both Cristina and Andrea are posing on their natural clothes, the second one (that I like more) printed for the re-issue of this album. Overall, the album is pretty homogenous, very consistent between tracks, so I will not talk much about it tracks by tracks but rather give my overall feelings in general.
However, there is one track that comes out of the ordinary, that really sounds my favorite. “To Myself I turned”. If you have heard their first EP named “Lacuna Coil”, the whole song is a revamped version of “Falling”, in a better and louder version. It was already a nugget in the EP and Lacuna Coil forged it into a song that is in my opinion the signature of the band.
The album is still spinning on my playlist, 20 years later still holds up decently. What I really liked about the songs were the melancholically lyrics that were just right for me, in a period of time that was emotionally and mentally tough, a period of transition between leaving your teenage years and growing up as an adult. Lacuna Coil was part of that transition and helped cope with emotions that I could not express, that I could not catharsize to other. Since that album, the band has been experiencing a constant change in their line-up, being experiencing various styles (mostly going into a more alternative/mainstream form of metal) but still remains a staple band, one of the few flagship of the Italian metal scene in my library.
Seeing them on tour is next in my bucket list, and I guess my best bet is to see them in Europe, as the band rarely comes abroad and tour in the US (or if they do, in a very few select dates).

[Sciences] Restoration of brain circulation and cellular functions hours post-mortem (Vrselja et al., Nature 2019)

You may have heard about that groundbreaking story last on “pig brains being revived” sounding almost like a scenario of a zombie movie. Let’s say science journalism love to use superlatives and sensationalistic headlines to grab few more clicks and views.
As usual, my skepticism was to first look at the paper and see how the claims hold on. The publication behind that “pig zombie paper” is the study from Vrselja and colleagues published in Nature last week and available here: https://www.nature.com/articles/s41586-019-1099-1
So what is about this story? First it is published in Nature, a top-tier peer-review journal. Second it is a huge paper, coming from Yale University. The paper was initially submitted on February 22, 2018 and got accepted March 1st. You can say a bit more than a year and that suggest that this paper likely went at least two rounds of review and probably more than three peer-reviews (three were named as well as other anonymous). One of the peer-reviewer was Pr. Constantino Iadeccola (Cornell University, NY), a “rock star” in the field of cerebral blood flow (which nicely match for the paper).
Overall, it is a very good paper with some reservations on the greater impact that I will explain later.
To understand the paper, you need to understand first that as until now we consider the brain highly dependent on continuous cerebral perfusion with blood flow to survive. The brain is highly dependent on oxygen and glucose (at least 20% of our daily uptake is taken by this tissue that only represent less than 2% of the human body weight).
We assume that if you stop flowing the brain with blood (e.g. cardiac arrest), you will die within minutes from massive and irreversible brain damage. The whole idea of this paper was: “what if we could maintain a blood flow for 24 hours, can we maintain some neurological function?”. In particular, the authors have developed a kind of artificial blood, cell-free, called BEx. It contains a hemoglobin carrier called Hemopure(R), glucose/pyruvate, as well as a cocktail of neuroprotective agents, antibiotics and some echogenic agents (to measure blood flow). The caveat is that as a control the authors used a simple saline solution without glucose and pyruvate (see supplementary tables below). Considering the importance of glucose for the brain tissue, and the absence of glycogen storage in that tissue, I would argue that this is a non-negligible flaw in the experimental design, giving a serious advantage to the BEx and maybe even overestimating the BEx activity.
Screen Shot 2019-04-17 at 2.06.26 PM

Nethertheless, let’s continue the discussion. Pig brains were not obtained from pigs euthanized for the sole purpose of the experiments, but rather obtained as waste from the slaughterhouse. Thats ethically much more acceptable, even 3R-friendly (as it valorize animal tissues considered as waste) and much more easy for obtaining an IACUC approval. About 300 post-mortem brains were used, I guess mostly for the development and optimization of the technique. The sample size (N) appears to be 32 pigs/group, which is very good for statistical power of analysis.
The surgical procedure (to connect these brain to the system) was about 10 minutes of warm anoxia, which would probably represent a severe cardiac arrest in which CPR is not performed immediately. They exposed these brain to either 1 hour or 10 hours post-mortem interval (PMI) without flow, with control perfusate or with BEx. Note that the perfusion to occur happened about 3 hours since the initial brain flush, the surgical preparation appears indeed tidious, but reproduce a pulsate blood flow similar to what would happen in animals. They also cooled down the brain to 25ºC, which is known that cooler temperature improve the chance of reducing brain damage (the common sense is that drowning in an hypothermic environment (frozen lake) increase your chance of resuscitation compared to drawing in a normothermic environment (swimming pool). The experiment lasted 10 hours for all groups, except the 1 hour PMI group.
The first results shown demonstrated the presence of a functional flow inside the brain tissue, and some vascular reactivity, using nimodipine infusion (a Ca channel blocker commonly used to reduce blood flow) and showing changes in blood flows. In other words, there is a proof of principle that it works.
The second result looked at changes in cerebral edema as a crude estimation of the blood-brain barrier (BBB) function. The control perfusate showed an increased water content, which is not surprising as some of our in-house data (and other studies) suggesting that the BBB function has a greater dependence to glucose than to oxygen when it comes to maintaining the barrier integrity. Since the CP is glucose-free, that is not surprising. The BEx group of course fare better (same level than 1 hour PMI group) but wonder how it would have fared if the CP contained the same amount of glucose and pyruvate. My personal thought is why did not the authors performed a gadolinium imaging of these two brains? They provide some T1 scans, which are nice but confirming changes in the BBB leakiness using gadolinium as contrasting agent would have been better.
Figure 3 show us a series of tissue staining of these brain, in particular from the hippocampal region. As expected, the Nissl staining worsened in the PMI, the presence of CP partially improved the situation and the BEx was similar to the 1 hour PMI and has the lowest cell death (as imaged by active caspase-3). When you look between CP and BEx, the difference is not that dramatic and makes me wonder that if we had the right CP formulation (with same glucose/lactate content), we would unlikely have a difference between CP and BEx, suggesting that perfusion with a saline solution oxygenated and with the correct amount of glucose can do as well than a more complex one.
Figure 4 show that the perfusion with BEx help to maintain astrocytes and microglial cells alive and functional (as measured by the secretion of pro-inflammatory cytokines following treatment with LPS).
Figure 5 show that there are some functional neurons present in BEx, capable to show electrophysiological activity. Small activity (not enough to be detected by EEG) but measurable by patch-clamp analysis.
Overall, it is a nice paper, considering it got published in Nature. There are some interesting stuff, but there are also some questionable limitations and caveats that I would have pointed as a reviewer and expected reviewers from Nature to have pointed it before letting it accepted. It shows that no matter what, never blindly trust a paper even if published in Nature.
The idea is very interesting and can help us better understand the post-mortem brain. It also raises the importance of CPR or any procedure aimed to keep a steady flow in the brain after injury or cardiac arrest and maybe worth considering it.

Anti vaccine activists are angry about a new study…and they didn’t even bother to read it

Left Brain Right Brain

This is a big piece of what the “vaccines-cause-autism” idea is built on. Really bad analyses. Another study shows up showing that vaccines don’t cause autism. People immediately jump to give talking points to their community: “ignore this study! In fact, it’s just another conspiracy to defraud you!!!!!”*

But they know from history, their community doesn’t check. They don’t test whether the talking points hold up. Either that, or they really are this bad at simple math.

One reason I slowed down a lot on writing debunks of the “vaccines cause autism” community is that it’s very repetitive. It only takes a few minutes to see where they make their mistakes. I admit, I have the training and the time to do that checking, but it leaves me wondering. As in, it’s hard to not ask: “are they really this bad at science? Are they so biased they don’t know…

View original post 1,457 more words