Hope for SARS-CoV-2 vaccine

There is hope for a vaccine. Serious hope. It will take probably 12 to 18 months until we have it. Maybe a little bit longer. But it looks like we will have it. Just continue your physical distancing, wear masks, be considerate to your fellow humans and it’ll be alright. We’ll bounce back and because we – the people – make up this extraordinary concept of economy, so shall it. After this pandemic will pass, hopefully we will not return to normal; instead, we will emerge a bit wiser, a bit more careful, a bit more compassionate. Meanwhile, throw some grateful words to your nearest scientist, whether or not they work on COVID-19.

REFERENCE: Callaway, E. (28 April 2020). The race for coronavirus vaccines: a graphical guide, Nature, 580: 576-577 (2020). doi: 10.1038/d41586-020-0121-y. FREE FULLTEXT PDF

By Neuronicus, 1 May 2020

Chloroquine-induced psychosis

In the past few days, a new hot subject has gripped the attention of various media and concerned the medical doctors, as if they don’t have enough to deal with: chloroquine. That is because the President of the U.S.A., Donald Trump, endorsed chloroquine as treatment of COVID-19, a “game changer”, despite his very own director of the National Institute of Allergy and Infectious Diseases (NIAID), Dr. Anthony Fauci, very emphatically and vehemently denying that the promise of (hydroxy)chloroquine is beyond anecdotal (see the White House briefing transcript here).

Many medical doctors spoke out urging caution against the drug, particularly against the combination the President endorses: hydroxychloroquine + azithromycin. As I understand it, this combo can be lethal as it can lead to fatal arrhythmia.

As for the (hydroxy)cloroquine’s possibility to help treat COVID-19, the jury is still out. Far out. Meaning that there have been a few interesting observations of the drugs working in a Petri dish (Liu et al. 2020, Wang et al., 2020), but as any pharma company knows, there is a long and perilous way from Petri dishes to pharmacies. To be precise, only 1 in 5000 drugs get from pre-clinical trials to approval and it takes about 12 years for this process to be completed (Kaljevic et al., 2004). The time is so long not because red tape, as some would deplore, but because it takes time to see what it does in humans (Phase 0), what doses are safe and don’t kill you (Phase 1), does it work at all for the intended disease (Phase 2), compare it with other drugs and evaluate the long-term side effects (Phase 3) and, finally, to see the risks and benefits of this drug (Phase 4). While we could probably get rid of Phase 0 and 4 when there is such a pandemic, there is no way I would submit my family to anything that hasn’t passed phases 1, 2, and 3. And those take years. With all the money that a nation-state has, it would still take 18 months to do it semi-properly.

Luckily for all of us, chloroquine is a very old and established anti-malarial medicine, and as such we can safely dispense of Phases 0, 1, and 4, which is fine. So we can start Phase 2 with (hydroxy)chloroquine. And that is exactly what WHO and several others are doing right now. But we don’t have enough data. We haven’t done it yet. So one can hope as much as they want, but that doesn’t make it faster.

Unfortunately – and here we go to the crux of the post -, following the President’s endorsement, many started to hoard chloroquine. Particularly the rich who can afford to “convince” an MD to write them a script for it. In countries where chloroquine is sold without prescription, like Nigeria, where it is used for arthritis, people rushed to clear the pharmacies and some didn’t just stockpiled it, but they took it without reason and without knowing the dosage. And they died. [EDIT, 23 March 2020. If you think that wouldn’t ever happen in the land of the brave, think again, as the first death to irresponsible taking chloroquine just happened in the USA]. In addition, the chloroquine hoarding in US by those who can afford it (is about $200 for 50 pills) lead to lack of supply for those who really need it, like lupus or rheumatology patients.

For those who blindly hoard or take chloroquine without prescription, I have a little morsel of knowledge to impart. Remember I am not an MD; I hold a PhD in neuroscience. So I’ll tell you what my field knows about chloroquine.

Both chloroquine and hydroxychloroquine can cause severe psychosis.

That’s right. More than 7.1 % of people who took chloroquine as prophylaxis or for treatment of malaria developed “mental and neurological manifestations” (Bitta et al.,  2017). “Hydroxychloroquine was associated with the highest prevalence of mental neurological manifestations” (p. 12). The phenomenon is well-reported, actually having its own syndrome name: “chloroquine-induced psychosis”. It was observed more than 50 years ago, in 1962 (Mustakallio et al., 1962). The mechanisms are unclear, with several hypotheses being put forward, like the drugs disrupting the NMDA transmission, calcium homeostasis, vacuole exocytosis or some other mysterious immune or transport-related mechanism. Because the symptoms are so acute, so persistent and so diverse than more than one brain neurotransmitter system must be affected.

Chloroquine-induced psychosis has sudden onset, within 1-2 days of ingestion. The syndrome presents with paranoid ideation, persecutory delusions, hallucinations, fear, confusion, delirium, altered mood, personality changes, irritability, insomnia, suicidal ideation, and violence (Biswas et al., 2014, Mascolo et al., 2018). All these at moderately low or therapeutically recommended doses (Good et al., 1982). One or two pills can be lethal in toddlers (Smith & Klein-Schwartz, 2005). The symptoms persist long after the drug ingestion has stopped (Maxwell et al., 2015).

Still want to take it “just in case”?

P.S. A clarification: the chemical difference between hydroxychloroquine and chloroquine is only one hydroxyl group (OH). Both are antimalarial and both have been tested in vitro for COVID-19. There are slight differences between them in terms of toxicity, safety and even mechanisms, but for the intents of this post I have treated them as one drug, since both produce psychosis.

REFERENCES:

1) Biswas PS, Sen D, & Majumdar R. (2014, Epub 28 Nov 2013). Psychosis following chloroquine ingestion: a 10-year comparative study from a malaria-hyperendemic district of India. General Hospital Psychiatry, 36(2): 181–186. doi: 10.1016/j.genhosppsych.2013.07.012, PMID: 24290896 ARTICLE

2) Bitta MA, Kariuki SM, Mwita C, Gwer S, Mwai L, & Newton CRJC (2 Jun 2017). Antimalarial drugs and the prevalence of mental and neurological manifestations: A systematic review and meta-analysis. Version 2. Wellcome Open Research, 2(13): 1-20. PMCID: PMC5473418, PMID: 28630942, doi: 10.12688/wellcomeopenres.10658.2 ARTICLE|FREE FULLTEXT PDF

4) Good MI & Shader RI. Lethality and behavioral side effects of chloroquine (1982). Journal of Clinical Psychopharmacology, 2(1): 40–47. doi: 10.1097/00004714-198202000-00005, PMID: 7040501. ARTICLE

3) Kraljevic S, Stambrook PJ, & Pavelic K (Sep 2004). Accelerating drug discovery. EMBO Reports, 5(9): 837–842. doi: 10.1038/sj.embor.7400236, PMID: 15470377, PMCID: PMC1299137. ARTICLE| FREE FULLTEXT PDF

4) Mascolo A, Berrino PM, Gareri P, Castagna A, Capuano A, Manzo C, & Berrino L. (Oct 2018, Epub 9 Jun 2018). Neuropsychiatric clinical manifestations in elderly patients treated with hydroxychloroquine: a review article. Inflammopharmacology, 26(5): 1141-1149. doi: 10.1007/s10787-018-0498-5, PMID: 29948492. ARTICLE

5) Maxwell NM, Nevin RL, Stahl S, Block J, Shugarts S, Wu AH, Dominy S, Solano-Blanco MA, Kappelman-Culver S, Lee-Messer C, Maldonado J, & Maxwell AJ (Jun 2015, Epub 9 Apr 2015). Prolonged neuropsychiatric effects following management of chloroquine intoxication with psychotropic polypharmacy. Clinical Case Reports, 3(6): 379-87. doi: 10.1002/ccr3.238, PMID: 26185633. ARTICLE | FREE FULLTEXT PDF

6) Mustakallio KK, Putkonen T, & Pihkanen TA (1962 Dec 29). Chloroquine psychosis? Lancet, 2(7270): 1387-1388. doi: 10.1016/s0140-6736(62)91067-x, PMID: 13936884. ARTICLE

7) Smith ER & Klein-Schwartz WJ (May 2005). Are 1-2 dangerous? Chloroquine and hydroxychloroquine exposure in toddlers. The Journal of Emergency Medicine, 28(4): 437-443. doi: 10.1016/j.jemermed.2004.12.011, PMID: 15837026. ARTICLE

Studies about chloroquine and hydoxychloroquine on SARS-Cov2 in vitro:

• Liu, J., Cao, R., Xu, M., Wang, X., Zhang, H., Li, Y., Hu, Z., Zhong, W., & Wang, M. (18 March 2020). Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro. Cell Discovery, 6 (16), https://doi.org/10.1038/s41421-020-0156-0 ARTICLE | FREE FULLTEXT PDF
• Wang, M., Cao, R., Zhang, H., Yang, X., Liu, J., Xu, M., Shi, Z., Hu, Z., Zhong, W., & Xiao, G. (18 March 2020). Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Research, 30: 269–271. https://doi.org/10.1038/s41422-020-0282-0. ARTICLE | FREE FULLTEXT PDF

First study about chloroquine and hydoxychloroquine on SARS-Cov2 in vivo below. Unfortunately, it has some methodological flaws, see here and here, which hopefully will be corrected once the peer-reviewers will take a closer look at it. UPDATE [31-3-2020]: It seems the article is flawed in more than one way, with serious ethical issues (timeline of treatment doesn’t match the methods reported, patients appear and disappear from data points, graphs different depending on the venue, published in the same journal where the author is editor, no peer-review, no blind, no placebo, controls barely tested, plus, no reputable researcher should announce that they are the genius that cured COVID-19 on a YouTube video, particularly when they get to publish in 24 hours anyway).

Anyway, here it is:

• Gautret P, Lagier J-C, Parola P, Hoang VT, Meddeb L, Mailhe M, Doudier B, Courjon J, Giordanengo V, Esteves Vieira V, Tissot Dupont H,Colson SEP, Chabriere E, La Scola B, Rolain J-M, Brouqui P,  Raoult D. (20 March 2020). Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial. International Journal of Antimicrobial Agents, PII:S0924-8579(20)30099-6, https://doi.org/10.1016/j.ijantimicag.2020.105949. ARTICLE | FREE FULLTEXT PDF

These studies are also not peer reviewed or at the very least not properly peer reviewed. I say that so as to take them with a grain of salt. Not to criticize in the slightest. Because I do commend the speed with which these were done and published given the pandemic. Bravo to all the authors involved (except maybe the last one f it proves to be fraudulent). And also a thumbs up to the journals which made the data freely available in record time. Unfortunately, from these papers to a treatment we still have a long way to go.

By Neuronicus, 22 March 2020

Treatment for lupus

Science has trends, like everything else. Some are longer or shorter lived, depending on how many astonishing discoveries are linked to that given subject. The 2000’s were unquestionably the years of the DNA. Many a grant have been written (and granted) for whole-genome surveys of this and that. Alternative splicing followed. The ’10s saw the rise of various -omics: transcriptomics, metabolomics, proteomics etc. Then everybody and his mamma got on the cart of epigenetics. With a side of immune stuff. Now, move aside epigenetics, here comes the microbiome. And CRISPR.

That is not to say that the not so hip subjects of the bygone years are thoroughly squeezed of knowledge and we throw them aside like some dry dead end and never touch them again. Not at all, not a bit. The trends only mark the momentary believes of the purse holders about which direction the next panaceum universalis will jump from.

Here comes a groundbreaking paper on the gut microbiome. It’s groundbreaking because it comes with a cure for systemic lupus erythematosus (SLE). Possibly autoimmune hepatitis and others autoimmune diseases as well.

An autoimmune disease is a terrible malady that is often incurable and sometimes deadly. It happens when the immune system starts attacking the body. One hypothesis as to why that happens posits that after a particular infection, maybe a particularly nasty one, the immune system doesn’t stop attacking, but now in the absence of an enemy it turns on its own body in genetically susceptible individuals.

Vieira et al. (2018) worked with genetically susceptible mice. And the bombshell comes right there in the first page: after treatment with an oral antibiotic (vancomycin or ampicillin, but not neomycin), mice genetically designed to develop lupus had lower “mortality, lupus-related autoantibodies, and autoimmune manifestations” (p. 1156). Then the researchers took a closer look at the bodies of these mice and observed that 82% of the mice had spleens and livers infected with Enterococcus gallinarum, a gut bacterium that should stay in the gut. But this bacterium is capable of weakening the gut barriers by loosening the tightness of the junctions between gut cells and then migrate to liver, spleen, and lymph nodes. Its high abundance in these places triggers a systemic immune response. Then the authors force-fed some germ-free mice with E. galinarum and saw that the mice developed systemic autoimmune pathology.

As if that’s not enough of a news story, the researchers developed a vaccine against this bacterium. The vaccine is very specific (being made of heat-killed E. gallinarum) and results in reduced levels of serum autoantibodies and prolonged survival rate in the lupus-prone mice.

So people don’t quibble, and rightly so, that those are rodents and humans are not (well, most of them, anyway), the authors looked at the liver biopsies of three humans with SLE and five with autoimmune hepatitis (AIH). They were positive for E. gallinarum, but the controls, i. e. healthy humans, were not. Also, when healthy human liver cells were stimulated with E. gallinarum they displayed autoimmune responses, just like in the murine cells. Finally, you don’t have to undergo a liver biopsy to see if you’re infected with E. gallinarum, just a specific blood test to see if you have increased antibody titers against this bug (or its RNA) as most SLE and AIH patients did.

Needless to say, I am extremely happy with this paper. Who wouldn’t be?! It’s a cure paper! I know, I know, they don’t say that, but what does this sound to you?:

“Administration of oral vancomycin or an intramuscular vaccine against E. gallinarum prevent translocation, Th17/Tfh cell induction, autoantibody production and autoimmune-related mortality (Supplemental, p. 62).”

Call it a very promising cure or a highly effective treatment if you like, but it stares you in the face for what it is as it did the researchers who already patented their stuff and are currently conducting clinical trials.

Most of the paper is in the Supplemental material, not in the 4 pages and a bit in Science. So even if the paper is under the paywall, the Supplementals are not. Be ready for a 71 page worth of 167 MB of data though.

REFERENCE: Manfredo Vieira S, Hiltensperger M, Kumar V, Zegarra-Ruiz D, Dehner C, Khan N, Costa FRC, Tiniakou E, Greiling T, Ruff W, Barbieri A, Kriegel C, Mehta SS, Knight JR, Jain D, Goodman AL, Kriegel MA (9 Mar 2018). Translocation of a gut pathobiont drives autoimmunity in mice and humans.  Science, 359(6380):1156-1161. doi: 10.1126/science.aar7201. PMID: 29590047, DOI: 10.1126/science.aar7201. ARTICLE |  Supplemental Material | Yale press release

By Neuronicus, 8 April 2018

The FIRSTS: mRNA from one cell can travel to another cell and be translated there (2006)

I’m interrupting the series on cognitive biases (unskilled-and-unaware, superiority illusion, and depressive realism) to tell you that I admit it, I’m old. -Ish. Well, ok, I’m not that old. But this following paper made me feel that old. Because it invalidates some stuff I thought I knew about molecular cell biology. Mind totally blown.

It all started with a paper freshly published two days ago and that I’ll cover tomorrow. It’s about what the title says: mRNA can travel between cells packaged nicely in vesicles and once in a target cell can be made into protein there. I’ll explain – briefly! – why this is such a mind-blowing thing.

Fig. 1. Illustration of the central dogma of biology: information transfer between DNA, RNA, and protein. Courtesy of Wikipedia, PD

We’ll start with the central dogma of molecular biology (specialists, please bear with me): the DNA is transcribed into RNA and the RNA is translated into protein (see Fig. 1). It is an oversimplification of the complexity of information flow in a biological system, but it’ll do for our purposes.

DNA needs to be transcribed into RNA because RNA is a much more flexible molecule and thus can do many things. So RNA is the traveling mule between DNA and the place where its information becomes protein, i.e. ribosome. Hence the name mRNA. Just kidding; m stands for messenger RNA (not that I will ever be able to call that ever again: muleRNA is stuck in my brain now).

There are many kinds of RNA: some don’t even get out of the nucleus, some are chopped and re-glued (alternative splicing), some decide which bits of DNA (genes) are to be expressed, some are busy housekeepers and so on. Once an RNA has finished its business it is degraded in many inventive ways. It cannot leave the cell because it cannot cross the cell membrane. And that was that. Or so I’ve been taught.

Exceptions from the above were viruses whose ways of going from cell to cell are very clever. A virus is a stretch of nucleic acids (DNA and/or RNA) and some proteins encapsulated in a blob (capsid). Not a cell!

In the ’90s several groups were looking at some blobs (yes, most stuff in biology can be defined by the all-encompassing and enlightening term of ‘blob’) that cells spew out every now and then. These were termed extracellular vesicles (EV) for obvious reasons. Turned out that many kinds of cells were doing it and on a much more regular basis than previously thought. The contents of these EVs varied quite a bit, based on the type of cells studied. Proteins, mostly, and maybe some cytoplasmic debris. In the ’80s it was thought that this was one way for a cell to get rid of trash. But in 1982, Stegmayr & Ronquist showed that prostate cells release some EVs that result in sperm cell motility increase (Raposo & Stoorvogel, 2013) so, clearly, the EVs were more than trash. Soon it became evident that EVs were another way of cell-to-cell communication. (Note to self: the first time intercellular communication by EVs was demonstrated was in 1982, Stegmayr & Ronquist. Maybe I’ll dig out the paper to cover it sometime).

So. In 2005, Baj-Krzyworzeka et al. (2006) looked at some human cancer cells to see what they spew out and for what purpose. They saw that the cancer cells were transferring some of the tumor proteins packaged in EVs to monocytes. For devious purposes, probably. And then they made to what it looks to me like a serious leap in reasoning: since the EVs contain tumor proteins, why wouldn’t they also contain the mRNA for those proteins? My first answer to that would have been: “because it would be rapidly degraded”. And I would have been wrong. To my credit, if the experiment wouldn’t take up too many resources I still would have done it, especially if I would have some random primers lying around the lab. Luckily for the world, I was not in charge with this particular experiment and Baj-Krzyworzeka et al. (2005) proceeded with a real-time PCR (polymerase chain reaction) which showed them that the EVs released by the tumor cells also contained mRNA.

Now the 1 million dollar, stare-in-your-face question was: is this mRNA functional? Meaning, once delivered to the host cell, would it be translated into protein?

Six months later the group answered it. Ratajcza et al. (2006) used embryonic stem cells as the donor cells and hematopoietic progenitor cells as host cells. First, they found out that if you let the donors spit EVs at the hosts, the hosts are faring much better (better survival, upregulated good genes, phosphorylated MAPK to induce proliferation etc.). Next, they looked at the contents of EVs and found out that they contained proteins and mRNA that promote those good things (Wnt-3 protein, mRNA for transcription factors etc.). Next, to make sure that the host cells don’t show this enrichment all of a sudden out of the goodness of their little pluripotent hearts but is instead due to the mRNA from the donor cells, the authors looked at the expression of one of the transcription factors (Oct-4) in the hosts. They used as host a cell line (SKL) that does not express the pluripotent marker Oct-4. So if the hosts express this protein, it must have come only from outside. Lo and behold, they did. This means that the mRNA carried by the EVs is functional (Fig. 2).

Fig. 2. Cell-to-cell mRNA transfer via extracellular vesicles (EVs). DNA is translated into RNA. A portion of RNA is transcribed into protein and another portion remains untranscribed. Both resultant protein and mRNA can get packaged into a vesicle: either a repackage into a microvesicle (a budding off of the cell membrane that shuttles cargo to and forth, about the size of 100-300nm) or packaged in a newly formed exosome (<100 nm) inside a multivesicular endosome (the yellow circle). The cell releases these vesicles in the intercellular space. The vesicles dock onto the host cell’s membrane and empty their cargo.

What bugs me is that these papers came out in a period where I was doing some heavy reading. How did I miss this?! Probably because they were published in cancer journals, not my field. But this is big enough you’d think others would mention it. (If you’re a recurrent reader of my blog, by now you should be familiarized with my stream-of-consciousness writing and my admittedly sometimes annoying in-parenthesis-meta-cognitions :D). So how did I miss this? How many more great discoveries have I missed? Am I the only one to discover such fundamental gaps in my knowledge? And thus the imposter syndrome takes root.

Just kidding, I don’t have the imposter syndrome. If anything, I got a superiority illusion complex. And I am absolutely sure that many, many scientists read things they consider fundamental to their way of thinking about the world all the time and wonder what other truly great discoveries are out there already that they missed.

Frankly, I should probably be grateful to this blog – and my friend GT who made me do it – because without nosing outside my field in search of material for it I would have probably remained ignorant of this awesome discovery. So, even if this is a decade old discovery for you, for me is one day old and I am a bit giddy about it.

This is a big deal because of the theoretical implications: a cell’s transcriptome (all the mRNA expressed in a cell) varies not only due to its needs, activity, and experiences, but also due to its neighbors’! A cell is, more or less, its transcriptome. Soooo… if we can change that at will, does that means we can change the type or function of the cell too? There are so many questions that such a discovery raises! And possibilities.

This is also a big deal because it opens up not a new therapy, or a new therapy direction, or a new drug class, but a new DELIVERY METHOD, the Holy Grail of Pharmacopeia. You just put your drug in one of these vesicles and let nature take its course. Of course, there are all sorts of roadblocks to overcome, like specificity, toxicity, etc. Looks like some are already conquered as there are several clinical trials out there that take advantage of this mechanism and I bet there will be more.

Stop by tomorrow for a freshly published paper on this mechanism in neurons.

REFERENCES:

1) Baj-Krzyworzeka M, Szatanek R, Weglarczyk K, Baran J, Urbanowicz B, Brański P, Ratajczak MZ, & Zembala M. (Jul. 2006, Epub 9 Nov 2005). Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes. Cancer Immunology, Immunotherapy, 55(7):808-818. PMID: 16283305, DOI: 10.1007/s00262-005-0075-9. ARTICLE

2) Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P, & Ratajczak MZ (May 2006). Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia, 20(5):847-856. PMID: 16453000, DOI: 10.1038/sj.leu.2404132. ARTICLE | FREE FULLTEXT PDF 

Bibliography:

Raposo G & Stoorvogel W. (18 Feb. 2013). Extracellular vesicles: exosomes, microvesicles, and friends. The Journal of Cell Biology, 200(4):373-383. PMID: 23420871, PMCID: PMC3575529, DOI: 10.1083/jcb.201211138. ARTICLE | FREE FULLTEXT PDF

By Neuronicus, 13 January 2018

Arnica and a scientist’s frustrations

When you’re the only scientist in the family you get asked the weirdest things. Actually, I’m not the only one, but the other one is a chemist and he’s mostly asked about astrophysics stuff, so he doesn’t really count, because I am the one who gets asked about rare diseases and medication side-effects and food advice. Never mind that I am a neuroscientist and I have professed repeatedly and quite loudly my minimum knowledge of everything from the neck down, all eyes turn to me when the new arthritis medication or the unexpected side-effects of that heart drug are being brought up. But, curiously, if I dare speak about brain stuff I get the looks that a thing the cat just dragged in gets. I guess everybody is an expert on how the brain works on account of having and using one, apparently. Everybody, but the actual neuroscience expert whose input on brain and behavior is to be tolerated and taken with a grain of salt at best, but whose opinion on stomach distress is of the utmost importance and must be listened to reverentially in utter silence [eyes roll].

So this is the background on which the following question was sprung on me: “Is arnica good for eczema?”. As always, being caught unawares by the sheer diversity of interests and afflictions my family and friends can have, I mumbled something about I don’t know what arnica is and said I will look it up.

This is an account of how I looked it up and what conclusions I arrived to or how a scientist tries to figure out something completely out of his or her field. First thing I did was to go on Wikipedia. Hold your horses, it was not about scientific information but for a first clarification step: is it a chemical, a drug, an insect, a plant maybe? I used to encourage my students to also use Wikipedia when they don’t have a clue what a word/concept/thing is. Kind of like a dictionary or a paper encyclopedia, if you will. To have a starting point. As a matter of fact Wikipedia is an online encyclopedia, right? Anyway, I found out that Arnica is a plant genus out of which one species, Arnica Montana, seems to be popular.

Then I went to the library. Luckily for me, the library can be accessed online from the comfort of my home and in my favorite pajamas in the incarnation of PubMed or Medline as it used to be affectionately called. It is the US National Library of Medicine maintained by the National Institutes of Health, a wonderful repository of scholarly papers (yeah, Google Scholar to PubMed is like the babbling of a two-year old to the Shakespearian sonnets; Google also has an agenda, which you won’t find on PubMed). Useful tip: when you look for a paper that is behind a paywall in Nature or Elsevier Journals or elsewhere, check the PubMed too because very few people seem to know that there is an obscure and incredibly helpful law saying that research paid by the US taxpayers should be available to the US taxpayer. A very sensible law passed only a few years ago that has the delightful effect of having FREE full text access to papers after a certain amount of months from publishing (look for the PMC icon in the upper right corner).

I searched for “arnica” and got almost 400 results. I sorted by “most recent”. The third hit was a review. I skimmed it and seemed to talk a lot about healing in homeopathy, at which point, naturally, I got a gloomy foreboding. But I persevered because one data point does not a trend make. Meaning that you need more than a paper – or a handful – to form an informed opinion. This line of thinking has been rewarded by the hit No. 14 in the search which had an interesting title in the sense that it was the first to hint to a mechanism through which this plant was having some effects. Mechanisms are important, they allow you to differentiate speculation from findings, so I always prefer papers that try to answer a “How?” question as opposed to the other kinds; whys are almost always speculative as they have a whiff of post factum rationalizations, whats are curious observations but, more often than not, a myriad factors can account for them, whens are an interesting hybrid between the whats and the hows – all interesting reads but for different purposes. Here is a hint: you want to publish in Nature or Science? Design an experiment that answers all the questions. Gone are the days when answering one question was enough to publish…

Digressions aside, the paper I am covering today sounds like a mechanism paper. Marzotto et al. (2016) cultured a particular line of human cells in a Petri dish destined to test the healing powers of Arnica montana. The experimental design seems simple enough: the control culture gets nothing and the experimental culture gets Arnica montana. Then, the authors check to see if there are differences in gene expressions between the two groups.

The authors applied different doses of Arnica montana to the cultures to see if the effects are dose-dependant. The doses used were… wait, bear with me, I’m not familiar with the system, it’s not metric. In the Methods, the authors say

“Arnica m. was produced by Boiron Laboratoires (Lyon, France) according to the French Homeopathic pharmacopoeia and provided as a first centesimal dilution (Arnica m. 1c) of the hydroalcoholic extract (Mother Tincture, MT) in 30% ethanol/distilled water”.

Wait, what?! Centesimal… centesimal… wasn’t that the nothing-in-it scale from the pseudoscientific bull called homeopathy? Maybe I’m wrong, maybe there are some other uses for it and becomes clear later:

“Arnica m. 1c was used to prepare the second centesimal dilution (Arnica m. 2c) by adding 50μl of 1c solution to 4.95ml of distilled ultra-pure water. Therefore, 2c corresponds to 10−4 of the MT”.

Holy Mother of God, this is worse than gibberish; this is voluntary misdirection, crap wrapped up in glitter, medieval tinkering sold as state-of-the-art 21st century science. Speaking of state-of-the-art, the authors submit their “doses” to a liquid chromatograph, a thin layer chromatograph, a double-beam spectrophotometer, a nanoparticle tracking analysis (?!) for what purposes I cannot fathom. On, no, I can: to sound science-y. To give credibility for the incredulous. To make money.

At which point I stopped reading the ridiculous nonsense and took a closer look at the authors and got hit with this:

“Competing Interests: The authors have declared that no competing interests exist. This study was funded by Boiron Laboratoires Lyon with a research agreement in partnership with University of Verona. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.”

No competing interests?? The biggest manufacturer of homeopathic crap in the world pays you to see if their product works and you have no competing interest? Maybe no other competing interests. There were some comments and replies to this paper after that, but it is all inconsequential because once you have faulty methods your results are irrelevant. Besides, the comments are from the same University, could be some internal feuding.

PLoS One, what have you done? You’re a peer-reviewed open access journal! What “peers” reviewed this paper and gave their ok for publication? Since when is homeopathy science?! What am I going to find that you publish next? Astrology? For shame… Give me that editor’s job because I am certain I can do better.

To wrap it up and tell you why I am so mad. The homeopathic scale system, that centesimal gibberish, is just that: gibberish. It is impossible to replicate this experiment without the product marketed by Boiron because nobody knows how much of the plant is in the dose, which parts of the plant, what kind of extract, or what concentration. So it’s like me handing you my special potion and telling you it makes warts disappear because it has parsley in it. But I don’t tell you my recipe, how much, if there anything else besides parsley in it, if I used the roots or only the leaves or anything. Now that, my friends, it’s not science, because science is REPLICABLE. Make no mistake: homeopathy is not science. Just like the rest of alternative medicine, homeopathy is a ruthless and dangerous business that is in sore need of lawmakers’ attention, like FDA or USDA. And for those who think this is a small paper, totally harmless, no impact, let me tell you that this paper had over 20,000 views (real science papers get hundreds, maybe thousands).

I would have oh so much more to rant on. But enough. Rant over.

Oh, not yet. Lastly, I checked a few other papers about arnica and my answer to the eczema question is: “It’s possible but no, I don’t think so. I don’t know really, I couldn’t find any serious study about it and I gave up looking after I found a lot of homeopathic red flags”. The answer I will give my family member? “Not the product you have, no. Go to the doctors, the ones with MDs after their name and do what they tell you. In addition, I, the one with a PhD after my name, will tell you this for free because you’re family: rub the contents of this bottle only once a day – no more! – on the affected area and you will start seeing improvements in three days. Do not use elsewhere, it’s quite potent!” Because placebo works and at least my water vial is poison free.

Reference: Marzotto M, Bonafini C, Olioso D, Baruzzi A, Bettinetti L, Di Leva F, Galbiati E, & Bellavite P (10 Nov 2016). Arnica montana Stimulates Extracellular Matrix Gene Expression in a Macrophage Cell Line Differentiated to Wound-Healing Phenotype. PLoS One, 11(11):e0166340. PMID: 27832158, PMCID: PMC5104438, DOI: 10.1371/journal.pone.0166340. ABSTRACT | FREE FULLTEXT PDF 

By Neuronicus, 10 June 2017

EDIT [7 December 2022]: Look at that! There is a warning after the DOI that appeared on my website through no involvement of my own saying the paper has been retracted roughly 2 years after publication. Good! The official reason for retraction? They used nothing to find nothing, their “findings” are just “standard noise”. Told you so! PubPeer link here.

This is what PLoS One is saying about it (my highlights):

The PLOS ONE Editors have discussed the study design and results reported in this article with experts in RNA-seq analysis, statistical analysis and members of our Editorial Board. Based on our assessment and the advice received, and in light of the above concerns, we have determined that the results presented in this article do not provide sufficient support for claims about effects of Arnica m. on gene expression. Hence, we are retracting this article due to concerns about the study design and about the validity and reliability of the reported conclusions. We regret that these issues were not fully addressed during the article’s pre-publication peer review.
In addition to the above, the PLOS ONE Editors hereby notify readers that the Competing Interests statement was incorrect for this article and should have explicitly stated that Boiron Laboratories, a company that provided funding support for this study, markets homeopathic products including various dilutions of Arnica m.
MM, CB, DO, EG, and PB did not agree with retraction. AB, LB, and FDL did not respond.

Drink before sleep

Among the many humorous sayings, puns, and jokes that one inevitably encounters on any social medium account, one that was popular this year was about the similarity between putting a 2 year old to bed and putting your drunk friend to bed, which went like this: they both sing to themselves, request water, mumble and blabber incoherently, do some weird yoga posses, cry, hiccup, and then they pass out. The joke manages to steal a smile only if someone has been through both situations, otherwise it looses its appeal.

Being exposed to both situations, I thought that while the water request from the drunk friend is a response to the dehydrating effects of alcohol, the water request from the toddler is probably nothing more than a delaying tactic to postpone bedtime. Whether there may or may not be some truth to my assumption in the case of the toddler, here is a paper to show that there is definitely more to the water request than meets the eye.

Generally, thirst is generated by the hypothalamus when its neurons and neurons from organum vasculosum lamina terminalis (OVLT) in the brainstem sense that the blood is either too viscous (hypovolaemia) or too salty (hyperosmolality), both phenomena indicating a need for water. Ingesting water would bring these indices to homeostatic values.

More than a decade ago, researchers observed that rodents get a good gulp of water just before going to sleep. This surge was not motivated by thirst because the mice were not feverish, were not hungry and they did not have a too viscous or a too salty blood. So why do it then? If the rodents are restricted from drinking the water they get dehydrated, so obviously the behavior has function. But is not motivated by thirst, at least not the way we know it. Huh… The authors call this “anticipatory thirst”, because it keeps the animal from becoming dehydrated later on.

Since the behavior occurs with regularity, maybe the neurons that control circadian rhythms have something to do with it. So Gizowski et al. (2016) took a closer look at  the activity of clock neurons from the suprachiasmatic nucleus (SCN), a well known hypothalamic nucleus heavily involved in circadian rhythms. The authors did a lot of work on SCN and OVLT neurons: fluorescent labeling, c-fos expression, anatomical tracing, optogenetics, genetic knockouts, pharmacological manipulations, electrophysiological recordings, and behavioral experiments. All these to come to this conclusion:

SCN neurons release vasopressin and that excites the OVLT neurons via V1a receptors. This is necessary and sufficient to make the animal drink the water, even if it’s not thirsty.

That’s a lot of techniques used in a lot of experiments for only three authors. Ten years ago, you needed only one, maybe two techniques to prove the same point. Either there have been a lot of students and technicians who did not get credit (there isn’t even an Acknowledgements section. EDIT: yes, there is, see the comments below or, if they’re missing, the P.S.) or these three authors are experts in all these techniques. In this day and age, I wouldn’t be surprised by either option. No wonder small universities have difficulty publishing in Big Name journals; they don’t have the resources to compete. And without publishing, no tenure… And without tenure, less research… And thus shall the gap widen.

Musings about workload aside, this is a great paper, shedding light on yet another mysterious behavior and elucidating the mechanism behind it. There’s still work to be done though, like answering how accurate is the SCN in predicting bedtime to activate the drinking behavior. Does it take its cues from light only? Does ambient temperature play a role and so on. This line of work can help people that work in shifts to prevent certain health problems. Their SCN is out of rhythm and that can influence deleteriously the activity of a whole slew of organs.

Summary of the doi: 10.1038/nature19756 findings. 1) The light is a cue for suprachiasmatic nulceus (SCN) that bedtime is near. 2) The SCN vasopressin neurons that project to organum vasculosum lamina terminalis (OVLT) are activated. 3) The OVLT generates the anticipatory thirst. 4) The animal drinks fluids.

Reference: Gizowski C, Zaelzer C, & Bourque CW (28 Sep 2016). Clock-driven vasopressin neurotransmission mediates anticipatory thirst prior to sleep. Nature, 537(7622): 685-688. PMID: 27680940. DOI: 10.1038/nature19756. ARTICLE

By Neuronicus, 5 October 2016

EDIT (12 Oct 2016): P.S. The blog comments are automatically deleted after a period of time. In case of this post that would be a pity because I have been fortunate to receive comments from at least one of the authors of the paper, the PI, Dr. Charles Bourque and, presumably under pseudonym, but I don’t know that for sure, also the first author, Claire Gizowski. So I will include here, in a post scriptum, the main idea of their comments. Here is an excerpt from Dr. Bourque’s comment:

“Let me state for the record that Claire accomplished pretty much ALL of the work in this paper (there is a description of who did what at the end of the paper). More importantly, there were no “unthanked” undergraduates, volunteers or other parties that contributed to this work.”

My hat, Ms. Gizowski. It is tipped. To you. Congratulations! With such an impressive work I am sure I will hear about you again and that pretty soon I will blog about Dr. Gizowski.

Painful Pain Paper

There has been much hype over the new paper published in the latest Nature issue which claims to have discovered an opioid analgesic that doesn’t have most of the side effects of morphine. If the claim holds, the authors may have found the Holy Grail of pain research chased by too many for too long (besides being worth billions of dollars to its discoverers).

The drug, called PZM21, was discovered using structure-based drug design. This means that instead of taking a drug that works, say morphine, and then tweaking its molecular structure in various ways and see if the resultant drugs work, you take the target of the drug, say mu-opioid receptors, and design a drug that fits in that slot. The search and design are done initially with sophisticated software and there are many millions of virtual candidates. So it takes a lot of work and ingenuity to select but a few drugs that will be synthesized and tested in live animals.

Manglik et al. (2016) did just that and they came up with PZM21 which, compared to morphine, is:

1) selective for the mu-opioid receptors (i.e. it doesn’t bind to anything else)
2) produces no respiratory depression (maybe a touch on the opposite side)
3) doesn’t affect locomotion
4) produces less constipation
5) produces long-lasting affective analgesia
6) and has less addictive liability

The Holy Grail, right? Weeell, I have some serious issues with number 5 and, to some extent, number 6 on this list.

Normally, I wouldn’t dissect a paper so thoroughly because, if there is one thing I learned by the end of GradSchool and PostDoc, is that there is no perfect paper out there. Consequently, anyone with scientific training can find issues with absolutely anything published. I once challenged someone to bring me any loved and cherished paper and I would tear it apart; it’s much easier to criticize than to come up with solutions. Probably that’s why everybody hates Reviewer No. 2…

But, for extraordinary claims, you need extraordinary evidence. And the evidence simply does not support the 5 and maybe 6 above.

Let’s start with pain. The authors used 3 tests: hotplate (drop a mouse on a hot plate for 10 sec and see what it does), tail-flick (give an electric shock to the tail and see how fast the mouse flicks its tail) and formalin (inject an inflammatory painful substance in the mouse paw and see what the animal does). They used 3 doses of PZM21 in the hotplate test (10, 20, and 40 mg/Kg), 2 doses in the tail-flick test (10 and 20), and 1 dose in the formalin test (20). Why? If you start with a dose-response in a test and want to convince me it works in the other tests, then do a dose-response for those too, so I have something to compare. These tests have been extensively used in pain research and the standard drug used is morphine. Therefore, the literature is clear on how different doses of morphine work in these tests. I need your dose-responses for your new drug to be able to see how it measures up, since you claim it is “more efficacious than morphine”. If you don’t want to convince me there is a dose-response effect, that’s fine too, I’ll frown a little, but it’s your choice. However, then choose a dose and stick with it! Otherwise I cannot compare the behaviors across tests, rendering one or the other test meaningless. If you’re wondering, they used only one dose of morphine in all the tests, except the hotplate, where they used two.

Another thing also related to doses. The authors found something really odd: PZM21 works (meaning produces analgesia) in the hotplate, but not the tail-flick tests. This is truly amazing because no opiate I know of can make such a clear-cut distinction between those two tests. Buuuuut, and here is a big ‘BUT” they did not test their highest dose (40mg/kg) in the tail-flick test! Why? I’ll tell you how, because I am oh sooo familiar with this argument. It goes like this:

Reviewer: Why didn’t you use the same doses in all your 3 pain tests?

Author: The middle and highest doses have similar effects in the hotplate test, ok? So it doesn’t matter which one of these doses I’ll use in the tail-flick test.

Reviewer: Yeah, right, but, you have no proof that the effects of the two doses are indistinguishable because you don’t report any stats on them! Besides, even so, that argument applies only when a) you have ceiling effects (not the case here, your morphine hit it, at any rate) and b) the drug has the expected effects on both tests and thus you have some logical rationale behind it. Which is not the case here, again: your point is that the drug DOESN’T produce analgesia in the tail-flick test and yet you don’t wanna try its HIGHEST dose… REJECT AND RESUBMIT! Awesome drug discovery, by the way!

So how come the paper passed the reviewers?! Perhaps the fact that two of the reviewers are long term publishing co-authors from the same University had something to do with it, you know, same views predisposes them to the same biases and so on… But can you do that? I mean, have reviewers for Nature from the same department for the same paper?

Alrighty then… let’s move on to the stats. Or rather not. Because there aren’t any for the hotplate or tail-flick! Now, I know all about the “freedom from the tyranny of p” movement (that is: report only the means, standard errors of mean, and confidence intervals and let the reader judge the data) and about the fact that the average scientist today needs to know 100-fold more stats that his predecessors 20 years ago (although some biologists and chemists seem to be excused from this, things either turn color or not, either are there or not etc.) or about the fact that you cannot get away with only one experiment published these days, but you need a lot of them so you have to do a lot of corrections to your stats so you don’t fall into the Type 1 error. I know all about that, but just like the case with the doses, choose one way or another and stick to it. Because there are ANOVAs ran for the formalin test, the respiration, constipation, locomotion, and conditioned place preference tests, but none for the hotplate or tailflick! I am also aware that to be published in Science or Nature you have to strip your work and wordings to the bare minimum because the insane wordcount limits, but you have free rein in the Supplementals. And I combed through those and there are no stats there either. Nor are there any power analyses… So, what’s going on here? Remember, the authors didn’t test the highest dose on the tail-flick test because – presumably – the highest and intermediary doses have indistinguishable effects, but where is the stats to prove it?

And now the thing that really really bothered me: the claim that PZM21 takes away the affective dimension of pain but not the sensory. Pain is a complex experience that, depending on your favourite pain researcher, has at least 2 dimensions: the sensory (also called ‘reflexive’ because it is the immediate response to the noxious stimulation that makes you retract by reflex the limb from whatever produces the tissue damage) and the affective (also called ‘motivational’ because it makes the pain unpleasant and motivates you to get away from whatever caused it and seek alleviation and recovery). The first aspect of pain, the sensory, is relatively easy to measure, since you look at the limb withdrawal (or tail, in the case of animals with prolonged spinal column). By contrast, the affective aspect is very hard to measure. In humans, you can ask them how unpleasant it is (and even those reports are unreliable), but how do you do it with animals? Well, you go back to humans and see what they do. Humans scream “Ouch!” or swear when they get hurt (so you can measure vocalizations in animals) or humans avoid places in which they got hurt because they remember the unpleasant pain (so you do a test called Conditioned Place Avoidance for animals, although if you got a drug that shows positive results in this test, like morphine, you don’t know if you blocked the memory of unpleasantness or the feeling of unpleasantness itself, but that’s a different can of worms). The authors did not use any of these tests, yet they claim that PZM21 takes away the unpleasantness of pain, i.e. is an affective analgesic!

What they did was this: they looked at the behaviors the animal did on the hotplate and divided them in two categories: reflexive (the lifting of the paw) and affective (the licking of the paw and the jumping). Now, there are several issues with this dichotomy, I’m not even going to go there; I’ll just say that there are prominent pain researchers that will scream from the top of their lungs that the so-called affective behaviors from the hotplate test cannot be indexes of pain affect, because the pain affect requires forebrain structures and yet these behaviors persist in the decerebrated rodent, including the jumping. Anyway, leaving the theoretical debate about what those behaviors they measured really mean aside, there still is the problem of the jumpers: namely, the authors excluded from the analysis the mice who tried to jump out of the hotplate test in the evaluation of the potency of PZM21, but then they left them in when comparing the two types of analgesia because it’s a sign of escaping, an emotionally-valenced behavior! Isn’t this the same test?! Seriously? Why are you using two different groups of mice and leaving the impression that is only one? And oh, yeah, they used only the middle dose for the affective evaluation, when they used all three doses for potency…. And I’m not even gonna ask why they used the highest dose in the formalin test… but only for the normal mice, the knockouts in the same test got the middle dose! So we’re back comparing pears with apples again!

Next (and last, I promise, this rant is way too long already), the non-addictive claim. The authors used the Conditioned Place Paradigm, an old and reliable method to test drug likeability. The idea is that you have a box with 2 chambers, X and Y. Give the animal saline in chamber X and let it stay there for some time. Next day, you give the animal the drug and confine it in chamber Y. Do this a few times and on the test day you let the animal explore both chambers. If it stays more in chamber Y then it liked the drug, much like humans behave by seeking a place in which they felt good and avoiding places in which they felt bad. All well and good, only that is standard practice in this test to counter-balance the days and the chambers! I don’t know about the chambers, because they don’t say, but the days were not counterbalanced. I know, it’s a petty little thing for me to bring that up, but remember the saying about extraordinary claims… so I expect flawless methods. I would have also liked to see a way more convincing test for addictive liability like self-administration, but that will be done later, if the drug holds, I hope. Thankfully, unlike the affective analgesia claims, the authors have been more restrained in their verbiage about addiction, much to their credit (and I have a nasty suspicion as to why).

I do sincerely think the drug shows decent promise as a painkiller. Kudos for discovering it! But, seriously, fellows, the behavioral portion of the paper could use some improvements.

Ok, rant over.

EDIT (Aug 25, 2016): I forgot to mention something, and that is the competing financial interests declared for this paper: some of its authors already filed a provisional patent for PZM21 or are already founders or consultants for Epiodyne (a company that that wants to develop novel analgesics). Normally, that wouldn’t worry me unduly, people are allowed to make a buck from their discoveries (although is billions in this case and we can get into that capitalism-old debate whether is moral to make billions on the suffering of other people, but that’s a different story). Anyway, combine the financial interests with the poor behavioral tests and you get a very shoddy thing indeed.

Reference: Manglik A, Lin H, Aryal DK, McCorvy JD, Dengler D, Corder G, Levit A, Kling RC, Bernat V, Hübner H, Huang XP, Sassano MF, Giguère PM, Löber S, Da Duan, Scherrer G, Kobilka BK, Gmeiner P, Roth BL, & Shoichet BK (Epub 17 Aug 2016). Structure-based discovery of opioid analgesics with reduced side effects. Nature, 1-6. PMID: 27533032, DOI: 10.1038/nature19112. ARTICLE 

By Neuronicus, 21 August 2016

Inhaling a bitter tasting solution may help with asthma (don’t try this at home, yet)

Asthma is an inflammatory disease of the lungs’ airways. The airway smooth muscle (ASM) expresses a large number of G protein-coupled receptors (GPCRs). The GPCRs are proteins bound to the cell membrane that sense what happens outside the cell and thus signal the cell to engage in appropriate responses. There are many, many types of GPCRs (in the upper hundreds) all over the body. Furthermore, alternative splicing (that is reshuffling parts of the gene that codes for a protein in such a way that you can get several different proteins from the same gene) may produce new types.

In an a effort to characterize the GPCRs in the ASM in the hope of finding an asthma pharmacological target, Einstein et al. (2008) found many more types of these receptors than previously thought, produced mainly by alternative splicing. In a subsequent study, the same group found out that some of these GPCRs are the same GPCRs that are expressed by your tongue in order to taste bitterness (Desphande et al., 2010)! The researchers were not expecting this.

Moreover, the bitter receptors (called TAS2Rs) in the lungs are fully functional, that is they respond to bitter substances like quinine. The response is, surprisingly, that of relaxation of the airways. It’s surprising because the role of bitter receptors in the tongue is to signal avoidance of bitter foods, because they usually contain toxins. So Desphande et al. (2010) (and anyone else in their shoes) would have expected a similar role for the bitter receptors in the lungs: that is, upon smelling something bitter the airways would close to prevent further poisoning. The data proved this expectation to be wrong.

The work so far has been done in isolated human cells. If quinine relaxes the ASM in an Petri dish, would it do so also when the ASM is still attached to its owner? So the researchers gave some bitter inhalants to some mice who had asthma and this treatment DECREASED the airway obstruction in a dose-dependent manner.

Asthma hits 300 million people worldwide and more than a quarter million die of it per year. So this research sparks great hopes for a new treatment direction.

References:

1. Einstein R, Jordan H, Zhou W, Brenner M, Moses EG, & Liggett SB (1 Apr 2008, Epub 24 Mar 2008). Alternative splicing of the G protein-coupled receptor superfamily in human airway smooth muscle diversifies the complement of receptors. Proceedings of the National Academy of Sciences of the United States of America, 105(13):5230-5. doi: 10.1073/pnas.0801319105. Article | FREE FULLTEXT PDF

2. Deshpande DA, Wang WC, McIlmoyle EL, Robinett KS, Schillinger RM, An SS, Sham JS, & Liggett SB. (Nov 2010, Epub 24 Oct 2010). Bitter taste receptors on airway smooth muscle bronchodilate by localized calcium signaling and reverse obstruction. Nature Medicine, 16(11):1299-304. doi: 10.1038/nm.2237. Article | FREE FULLTEXT PDF 

By Neuronicus, 10 March 2016

The Firsts: Anandamide (1992)

Cannabis, the plant whose psychoactive tetrahydrocannabinol (THC) binds to the same receptors in the brain as anandamide.

A rare tragedy took place in France a few days ago when a Phase I clinical trial for a new drug destined to improve mood and alleviate pain has resulted in one person dead and five other hospitalized. Phase I means that the drug successfully passed all animal tests and was being tried for the first time in humans to test its safety (efficacy and potency are tested in phase II and III, respectively).

The trial has been suspended and an investigation is on the way. So far, it appears that both the manufacturer (Bial) and the testing company (Biotrial) have followed all the guidelines and regulations. The running hypothesis is that the drug (BIA 10-2474) is acting on an unexpected target. What does that mean?

BIA 10-2474 is a FAAH inhibitor (fatty acid amide hydrolase). This enzyme breaks down anandamide, which is an endocannabinoid. In other words, is a neurotransmitter in the brain that binds to the same receptors as THC, the main active component of marijuana. So, if you give someone BIA 10-2474, the result would be an increase in the availability of anandamide, presumably with anxiolytic and analgesic effects (yes, similar to smoking weed).

There are other FAAH inhibitors out there that had been previously tried in humans and they were never marketed not because they were unsafe, but because they were ineffective in producing the desired results, i.e. less pain and/or anxiety.

So we don’t know yet why BIA 10-2474 killed people, but the bet is that in addition to FAAH, it also binds to some other protein. Why they didn’t discover this in animal trials, is a mystery; perhaps the unknown protein is unique to humans? By the looks of the drug’s structure, I think is computer generated, meaning is composed of a bunch of functional groups that someone put together in the hopes that it would fit neatly on the target binding site; but so many functional groups thrown in together might bind unexpectedly to other places than the intended. More on the story in Nature.

Anyway, that was the very long intro to today’s featured paper: the discovery of anandamide. Which happened very recently, in 1992, by the Mechoulam group at the Hebrew University of Jerusalem, Israel. Anandamide is the first endocannabinoid to be isolated. Mechoulam’s postodcs, William Devane and Lumir Hanus, used mass spectroscopy and NMR (nuclear magnetic resonance, MRI is an application of the same principles) to identify and isolate the molecule in a pig brain. And then they named it, fittingly, the “amide of bliss”…

Of note, members of the same Mechoulam group identified two more of the six known endocannabinoids. The three pages paper is highly technical, but I am assured (by a chemist) that is an easy-peasy read for any organic chemist.

Reference: Devane WA, Hanus L, Breuer A, Pertwee RG, Stevenson LA, Griffin G, Gibson D, Mandelbaum A, Etinger A, & Mechoulam R (18 Dec 1992). Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science, 258(5090):1946-9. PMID: 1470919, DOI: 10.1126/science.1470919.  Article | Research Gate Full Text

By Neuronicus, 18 January 2016

Yeast can make morphine

Opiates like morphine and heroin can be made at home by anybody with a home beer-brewing kit and the right strain of yeast. In 2015, two published papers and a Ph.D. dissertation described the relatively easy way to convince yeast to make morphine from sugar (the links are provided in the Reference paper). That is the bad news.

The good news is that scientists have been policing themselves (well, most of them, anyway) long before regulations are put in place to deal with technological advancements by, for example, limiting access to the laboratory, keeping things under lock and key, publishing incomplete data, and generally being very careful with what they’re doing.

Complementing this behavior, an article published by Oye et al. (2015) outlines other measures that can be put in place so that this new piece of knowledge doesn’t increase the accessibility to opiates, thereby increasing the number of addicts, which is estimated to more than 16 million people worldwide. For example, researchers can make the morphine-producing yeast dependent on unusual nutrients or engineer the existing strain to produce less-marketable varieties of opiates or prohibit the access to made-to-order DNA sequences for this type of yeast and so on.

You may very well ask “Why did the scientists made this kind of yeast anyway?”. Because some medicines are either very expensive or laborious to produce by the pharmaceutical companies, the researchers have sought a method to make these drugs more easily and cheaply by engineering bacteria, fungi, or plants to produce them for us. Insulin is a good example of an expensive and hard-to-get-by drug that we managed to engineer yeast strains to produce it for us. And opiates are still the best analgesics out there.

Reference: Oye KA, Lawson JC, & Bubela T (21 May 2015). Drugs: Regulate ‘home-brew’ opiates. Nature, 521(7552):281-3. doi: 10.1038/521281a. Article | FREE Fulltext PDF

By Neuronicus, 2 January 2016

Tryptophan-rich foods and happiness

The paper I feature today is not an experimental study, but an editorial written as a short review (5 pages). A not very good one, I’m afraid.

Neurochemical imbalances are to be found in virtual any brain disorder. Probably the most known is the serotonin depletion associated to depression, which is the main reason why SSRIs (selective serotonin reuptake inhibitors) are so widely prescribed for the disorder. With the caveats that serotonin is but one player, that it has many receptors involved in different aspects of the disease and “depression” is an umbrella term for a host of behaviors, this editorial focuses on non-pharmacological ways to address the depletion of serotonin. Noble goal, poor execution.

In a nutshell, Young (2007) argues that there are 4 ways to increase serotonin availability in the brain:
1) effortful focusing on positive things, either via psychotherapy, talk, social interactions, mediation or just mental exercises to consciously improve mood. I’m sure that the thought of trying to focus on the positive thoughts never crossed the minds of depressed people! Of course that this is how healthy people regulate their moods, everybody is sad or suffers loss at some point in their life and a lot of people snap out of it by engaging in those suggested behaviors, but the trouble with depression is that it persists despite efforts to be positive. The author should know that crying “Cheer up!” to a depressed person never works, but chances are they would feel even more alienated because they’ve tried that already!
2) exposure to bright light (3000 lux). No contention here. Light therapy is successful in treating seasonal depression. We should all get more light.
3) exercise. It’s unclear which kind, aerobic or to fatigue, but probably either would work.
4) eating tryptophan-rich foods (like meat, cheeses or eggs). Why tryptophan? Because the brain can make serotonin out of tryptophan, but serotonin itself is too big of a molecule to enter the brain (i.e. doesn’t cross the brain blood barrier). But the author admits that “although purified tryptophan increases brain serotonin, foods containing tryptophan do not” (p. 396) soooo,… then eating tryptophan-rich foods will NOT increase the serotonin. But then he goes on saying that drinking milk or eating nixtamalized corn increases serotonin (verbatim: “Acute ingestion of alpha-lactalbumin by humans can improve mood and cognition in some circumstances, presumably owing to increased serotonin” and “Breeding corn with a higher tryptophan content was shown in the 1980s to prevent pellagra; presumably, it also raised brain serotonin” p. 396-397). Utterly confusing and self-contradictory.

I also want to make a big note here:
a) there is no reliable evidence that eating tryptophan-rich foods increases the brain serotonin. Otherwise, instead of paying for Prozac, you would buy a huge bottle of tryptophan pills from the nearest dietary supplements store. Which brings me to my second point:
b) why don’t we give tryptophan supplements instead of SSRIs? Tryptophan is sold in USA as a dietary supplement which I think is a tremendously dangerous thing to allow (in most EU countries is considered a drug, so you can’t buy it from the shoddy dietary supplements stores). Because its efficacy in depression is inconclusive at best, i.e. most studies did not find significant improvements, while others showed improvement only in a subpopulation of depression sufferers. But it can induce nausea, sleepiness, confusion, depression, and even dementia symptoms and death. And interacts badly with other drugs or even with carbohydrate-rich foods, like pizza or pasta.

This is definitely not among the best papers I have read. It has many speculations supported by un-replicated studies. Or, when such studies are sparse, the reasoning relies on evolutionary speculations elevated to the rank of causal explanations (e.g. we spend so much time indoors, therefore depression is on the rise; conversely, our ancestors spent more time outside, therefore they were happier). Although I agree with Young that we should invest more research into non-pharmacological ways to improve brain dysfunctions, we need to do so in a more pragmatical manner that just telling people to think positive. Ok, rant over.

Reference: Young SN (Nov 2007). How to increase serotonin in the human brain without drugs. Journal of Psychiatry and Neuroscience, 32(6):394-399. PMID:18043762, PMCID:PMC2077351. Article | FREE FULLTEXT PDF

By Neuronicus, 3 December 2015

Putative mechanism for decreased spermatogenesis following SSRI

The SSRIs (selective serotonin reuptake inhibitors) are the most commonly prescribed antidepressants around the world. Whether is Prozac, Zoloft or Celexa, chances are that 1 in 4 Americans (or 1 in 10, depending on the study) will be making a decision during their lifetime to start an antidepressant course or not. And yet adherence to treatment is significantly low, as many people get off the SSRI due to their side effects, one of the main complains being sexual dysfunction in the form of low libido and pleasure.

Now a new study finds a mechanism for an even more worrisome effect of citalopram, (Celexa), an SSRI: the reduction of spermatogenesis. Prasad et al. (2015) used male zebrafish as a model and exposed them to citalopram in 3 different doses for 2- or 4-weeks period. They found out that the expression in the brain of the serotonin-related genes (trp2 and sert) and gonadotropin genes (lhb, sdhb, gnrh2, and gnrh3) were differently affected depending on the dose and durations of treatment. In the testes, the “long-term medium- and high-dose citalopram treatments displayed a drastic decrease in the developmental stages of spermatogenesis as well as in the matured sperm cell count” (p. 5). The authors also looked at how the neurons are organized and they found out that the serotonin fibers are associated with the fibers of the neurons that release gonadotropin-releasing hormone 3 (GnRH3) in preoptic area, a brain region in the hypothalamus heavily involved in sexual and parental behavior in both humans and fish.

Shortly put, in the brain, the citalopram affects gene expression profiles and fiber density of the serotonin neurons, which in turn decreases the production of GnRH3, which may account for the sexual dysfunctions that follow citalopram. In the testes, citalopram may act directly by binding to the local serotonin receptors and decrease spermatogenesis.

Reference: Prasad P, Ogawa S, & Parhar IS. (Oct 2015, Epub 8 Jul 2015). Serotonin Reuptake Inhibitor Citalopram Inhibits GnRH Synthesis and Spermatogenesis in the Male Zebrafish. Biololy of Reproduction. 93(4):102, 1-10. doi: 10.1095/biolreprod.115.129965. Article | FREE FULLTEXT PDF

By Neuronicus, 11 November 2015

Fat & afraid or slim & brave (Leptin and anxiety in ventral tegmental area)

A comparison of a mouse unable to produce leptin thus resulting in obesity (left) and a normal mouse (right). Courtesy of Wikipedia. License: PD

Leptin is a small molecule produced mostly by the adipose tissue, whose absence is the cause of morbid obesity in the genetically engineered ob/ob mice. Here is a paper that gives us another reason to love this hormone.

Liu, Guo, & Lu (2015) build upon their previous work of investigating the leptin action(s) in the ventral tegmental area of the brain (VTA), a region that houses dopamine neurons and widely implicated in pleasure and drug addiction (among other things). They did a series of very straightforward experiments in which the either infused leptin directly into the mouse VTA or deleted the leptin receptors in this region (by using a virus in genetically engineered mice). Then they tested the mice on three different anxiety tests.

The results: leptin decreases anxiety; absence of leptin receptors increases anxiety. Simple and to the point. And also makes sense, given that leptin receptors are mostly located on the VTA neurons that project to the central amygdala, a region involved in fear and anxiety (curiously, the authors cite the amygdala papers, but do not comment on the leptin-VTA-dopamine-amygdala connection). For the specialists, I would say that they are a little liberal with their VTA hit assessment (they are mostly targeting the posterior VTA) and their GFP (green fluorescent protein) is sparsely expressed.

Reference: Liu J, Guo M, & Lu XY (Epub ahead of print 5 Oct 2015). Leptin/LepRb in the Ventral Tegmental Area Mediates Anxiety-Related Behaviors. International Journal of Neuropsychopharmacology, 1–11. doi:10.1093/ijnp/pyv115. Article | FREE PDF

By Neuronicus, 28 October 2015

Orgasm-inducing mushrooms? Not quite

Claims that there is an orgasm-inducing mushroom in Hawaii may not be entirely accurate. Author and licensing of the above drawing unknown.

A few weeks ago, the social media has bombarded us with the eye-catching news that there is a mushroom in Hawaii whose smell induces spontaneous orgasms in women, but not men, who found its smell repugnant.

Except that it appears there is no such mushroom. Turns out the 14 year old paper is written by the president of a Hawaiian company that sells organic medicinal mushrooms. Not only written, but funded, as well. This is enough to damn the credibility of any study (that’s why scientists must declare competing interest when submitting a paper). But it also seems that the study has major fundamental flaws, like not having a single objective measure (of the quantity of spores, for example), is done under non-controlled environmental conditions (the participants seem to have known what was expected from them), there have been no replications, etc. Actually, it should have been suspicious to me from the start that nothing happened in the following 14 years; you would think that such claims would have been replicated, or at least the mushroom identified. But, as they say, hindsight is 20-20. Here is some nice little reporting exposing the business in Huffington Post and ScienceAlert.

I am not blaming the science media outlets on this one too much, like IFL Science or NBC affiliate, as I thought of covering this study myself, should I have been able to get my hands on the full text of the paper. In all honesty, who wouldn’t want to read that paper, especially since the abstract speculated on the mushroom’s spores having hormone-like chemicals that mimic the human neurotransmitters released during sexual encounters? But I (and others) have searched in vain for the full text and the most parsimonious explanation is that it was buried or withdrawn.

The trite but true message is: even the science media (including this one) is prone to mistakes. Interested in something? Go to the source and read the whole paper yourself, even the small print (like the one with competing interests), and only then make an opinion. That’s why I always post the links to the original article.

Reference: Holliday, J.C. & Soule, N. (2001). Spontaneous Female Orgasms Triggered by Smell of a Newly Found Tropical Dictyphora Species. International Journal of Medicinal Mushrooms, 3: 162-167. Abstract | Debunking in The Journal of Wild Mushrooming | Debunking in Discover Magazine

By Neuronicus, 17 October 2015