The n-of-1 patient is that one person with a unique genetic mutation that causes an ultra-rare disease, designated as a disease with less than 30 patients in the whole world. The advent of affordable genomic sequencing has identified millions of n-of-1 patients, which is becoming a large and growing population with desperate needs.
Though a great progress is made in identifying n-of-1 patients, and ruling the genetic causes of their diseases; usually, drugs that work in patients with the common mutations, often do not work for these unique patients.
n-Lorem is a foundation created by Dr. Stanley Crooke, the founder, chairman and chief executive officer of Ionis Pharmaceuticals, a global leader in RNA-targeted therapy. This foundation was created to provide individualized treatments for patients with ultra-rare diseases using the technology developed at Ionis Pharmaceuticals.
The mission of n-Lorem is to use the versatility and specificity of antisense technology to kindly offer experimental antisense oligonucleotides (ASO) medicines to treat the n-of-1 patient.
Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded DNA-mimics that can alter the RNA, and consequently protein expression and function. Because they can manipulate the intermediate step between gene and protein translation – at the pre-mRNA level – ASOs can reduce, or restore, or modify protein expression by different mechanisms.
Very simply: DNA gets converted into mRNA. ASOs function in between the two. As such, if the DNA gene has a mutation and codes a dysfunctional mRNA, and consequentially a dysfunctional protein that causes a disease, an ASO can change that. The ASO has the correct complementary code to produce the precise mRNA, and automatically, the precise protein needed to perform the right function. By intervening in the step before protein gets translated, it makes sure that nothing goes wrong and that the mutated gene doesn’t go any further, preventing disease from happening.
Anyone can apply to n-Lorem for a potential treatment. The proposals are approved and prioritized based on certain criteria, such as the severity of the disease, feasibility of developing an ASO treatment for the genetic cause of the disease, degree of potential benefit vs. potential risks, practicality of treatment, availability of physician and institution to treat patient, and other intricacies of the condition.
The unique patient needs to work with a physician that makes the connection to n-Lorem, who will then make an informed decision about whether a patient is appropriate to receive an experimental ASO treatment through a Commission.
It’s outstanding that the FDA reaction to n-Lorem has been very supportive; and, in fact, initial guidance has been put in place in January 2021 to reach more patients in need.
Once regulatory permission has been given, an investigator-led clinical trial is initiated and the patient can receive their custom experimental ASO treatment at no personal cost and will all clinical support.
This cost-free individualized therapy is possible for Ionis Pharmaceuticals because of the inherent efficiency and versatility of the ASO technology. The knowledge of modern ASOs mechanisms and specific possible effectiveness in selected organs, with different possible routes of application, together with integrated safety databases, allows a dive for the treatment of unique patients.
It’s honourable to use science to help the n-of-1 patient and their families.
The gut microbiome is a community of microorganisms that lives in our gastrointestinal tract. It is so far, the most studied microbial community in healthy humans, because of its known role in a range of functions and diseases, like Inflammatory Bowel Disease (IBD)1,2.
To gain perspective on the magnitude of the bacterial presence inside of us, and potential effects on our bodies, the human body expresses 20,000 eukaryotic genes while the gut microbiome expresses 3.3 million prokaryotic genes. This suggests that the genetic contribution of the microbiome to humans may be many hundreds of times greater than the genetic contribution from the human genome.
Most of the microbes in the microbiome do not cause disease. In fact, we need them to perform many important functions that we cannot do ourselves. Microbes digest food to generate nutrients for host cells, synthesize vitamins, help to absorb nutrients and minerals, produce short-chain fatty acids, metabolize drugs, detoxify carcinogens, stimulate renewal of cells in the gut lining, and activate and support the immune system1.
The fermentation by-products acetate, propionate, and butyrate are important for gut health; and, provide energy for epithelial cells, enhance the integrity of the epithelial barrier, and provide immunomodulation and protection against pathogens1.
Current investigations explore resident bacterial gene function, and the potential role it might have in human health and metabolism. Each individual has its own microbiome, and no one common microbe is present in all body sites or all individuals.
Researchers identified the composition of different individual microbiomes, but they also identified the metabolic pathways of the microbial communities found in different body sites (e.g., skin, colon, liver…). What is interesting is that microbial membership diverges greatly between healthy individuals; but, the metabolic pathways of our own microbiomes is very similar, with common ‘housekeeping’ properties that maintain cell function and a functional body site ecosystem3,4.
The interactions between the gut microbiota and our bodies immune system begins at birth4. The microbiota shapes the development of the immune system; and, in turn, the immune system shapes the composition of the microbiota. This cross-talk between the microbes and our bodies is transmitted through a vast array of signaling pathways that involve many different classes of molecules, and extend upon multiple organs such as the gut, liver, muscle, and the brain. This creates axes of metabolic pathways, or highways of chemical communication, between the gut and the different organs in our bodies.
Because the gut microbiome is highly malleable, it can be altered throughout our lifespan by environmental factors, such as diet, stress and medication. What we have seen during the last 60 years, is an increaseincidence of gut dysbiosis, which is an imbalance in the intestinal bacteria that leads to disease.
As such, there is much interest in developing new therapeutic tools for manipulating the composition of the gut microbiota to benefit our health. A better understanding of how variations in this symbiotic relation within us, supraorganisms, will contribute to disease risk and health sustainability; and, will point the way to new therapeutic interventions and disease prevention strategies.
Danone, a leading yogurt multinational food corporation, is developing “precision probiotics”, for example. Researchers at Danone aim to tailor probiotics to an individual’s diet, phenotype, lifestyle, age, gender, genetics and microbiome. The intention it’s to bring to the gut activities or functions that are not provided by our own gut microbiome, or our own genes.
It’s funny that around 1920’s, Isaac Carasso, the creator of Danone, first started selling yogurt in pharmacies, using ferments isolated from the Institute Pasteur, and label it as health-food. It’s like going full circle.
It’s funny that this pandemic can prove all anti-evolutionists wrong.
Nothing like seeing Charles Darwin natural selection right in front of your eyes at the speed of light. Just look at the SARS-CoV-2 viral evolution…
In his book “On the origin of species” written in 1859, Charles Darwin defined natural selection as the “principle by which each slight variation of a trait, if useful, is preserved”. What does this mean (?), it means the individuals best adapted to their environments are more likely to survive and reproduce.
What we are seeing now, is that the mutations of SARS-CoV-2 that better promote spreading, are the ones that are becoming more common among the population, even when derived in different locations. The virus is changing and evolving to spread more rapidly, because natural selection will optimize the level of virulence that maximizes pathogen fitness – expressed as the basic reproductive number (R0)1,2.
On average, comparative data from previous studies tell us that, low-virulence infections have a greater chance of successfully establishing transmission cycles in humans than virus with higher mortality3. As such, as before, the virus actually just wants to spread and not kill.
But, since the environment also affects transmissibility, there are more factors on the equation “when will this madness end” than we would have wished for.
For example, in the evolutionary trade-off between virulence and transmissibility, because intra-host virus replication is needed to allow inter-host transmission, it is almost impossible for natural selection to optimize all traits simultaneously1 and give us some peace.
For example, in the case of the Myxoma virus (MYXV) in rabbits, this evolutionary trade-off leads to an ‘intermediate’ virulence being more advantageous to the virus than a higher virulence1,4. This happens because the rabbit host dies before inter-host transmission, in the case of higher virulence; and, with lower virulence the virus goes absolutely nowhere, because it does not increase virus transmission rates. A similar trade-off model has been proposed to explain the evolution of HIV virulence1,5.
Unfortunately, experimental studies in some viruses have shown that high virulence can promote certain advantages, as in the case of malaria, where a higher virulence was shown to provide the Plasmodium parasites with a competitive advantage within hosts1,6. Or, in the case of the rabbit haemorrhagic disease virus (RHDV), where there is evidence that virulence has increased through time, probably because virus transmission often occurs through flies that feed on animal carcasses, making host death selectively favourable1,7.
Let’s thank the Gods that SARS-CoV-2 is NOT transmissible through flies.
So, current evolutionary theory tells us that it is possible to anticipate the direction of virulence evolution, if the key relationship between virulence and transmissibility, and hence viral fitness, is understood1. Crucial to this is the analysis of the intersection between genomics and evolutionary studies, what is called phylogenomics.
This field of science provides a way to understand virulence evolution, and creates a number of hypotheses that can be tested using appropriate experimental cell assays and bioinformatic tools8,9.
The collaboration of public health and research teams worldwide has now allowed the publication of 620,338 SARS-CoV-2 genomes in GISAID (http://www.gisaid.org/) (as of February 25, 2020)9. At the same time, a dynamic nomenclature system for SARS-CoV-2 has been described to facilitate real-time epidemiology revealing links between global outbreaks that share similar viral genomes10. At the root of the phylogeny are two lineages, A and B; where, A is likely ancestral, as it shares two distinguishing variants with the closest known bat viruses. Further linage designations link new variants to geographically distinct populations, B.1 in the Italian outbreak, then other parts of Europe and the world; and, B.1.1 being the major European lineage which was spread throughout the world. However, many of the major lineages are now present in most countries, and recapitulate the global diversity of SARS-CoV-2, indicating that most local epidemics were seeded by a large number of independent introductions of the virus.
The current evolutionary tree of SARS-CoV-2 shows multiple introductions of different variants across the globe, with introductions from distant locations seeding local epidemics, where infections sometimes went unrecognized for several weeks and allowed wider spread11. The tree topology actually indicates that SARS-CoV-2 viruses have not diverged significantly since the beginning of the pandemic11. These results show that, so far, SARS-CoV-2 has evolved through a non-deterministic, noisy process; and, that random genetic drift has played the dominant role in disseminating unique mutations throughout the world11.
There remains an urgent need for a SARS-CoV-2 vaccine as a primary countermeasure to contain and mitigate the spread; and, the virus’s surface S (Spike) protein continues to be an attractive vaccine target,because it plays a key role in mediating virus entry into the cells, and is known to be immunogenic.
Of course, the virus was only recently identified in the human population with a short time frame relative to the adaptive processes that can take years to occur.
But, the most recent findings show us that the SARS-CoV-2 viruses that are currently circulating, constitute a homogeneous viral population, to which the current vaccines available will be sufficient to mitigate the spread.
Soon, SARS-CoV-2 will become just another viral acquaintance during the winter, like a common cold
1 Geoghegan, J. L. & Holmes, E. C. The phylogenomics of evolving virus virulence. Nature Reviews Genetics19, 756-769, doi:10.1038/s41576-018-0055-5 (2018).
2 Bull, J. J. & Lauring, A. S. Theory and empiricism in virulence evolution. PLoS Pathog10, e1004387, doi:10.1371/journal.ppat.1004387 (2014).
3 Geoghegan, J. L., Senior, A. M., Di Giallonardo, F. & Holmes, E. C. Virological factors that increase the transmissibility of emerging human viruses. Proc Natl Acad Sci U S A113, 4170-4175, doi:10.1073/pnas.1521582113 (2016).
4 Kerr, P. J. et al. Next step in the ongoing arms race between myxoma virus and wild rabbits in Australia is a novel disease phenotype. Proceedings of the National Academy of Sciences114, 9397-9402, doi:10.1073/pnas.1710336114 (2017).
5 Fraser, C., Hollingsworth, T. D., Chapman, R., de Wolf, F. & Hanage, W. P. Variation in HIV-1 set-point viral load: epidemiological analysis and an evolutionary hypothesis. Proc Natl Acad Sci U S A104, 17441-17446, doi:10.1073/pnas.0708559104 (2007).
6 de Roode, J. C. et al. Virulence and competitive ability in genetically diverse malaria infections. Proc Natl Acad Sci U S A102, 7624-7628, doi:10.1073/pnas.0500078102 (2005).
7 Di Giallonardo, F. & Holmes, E. C. Viral biocontrol: grand experiments in disease emergence and evolution. Trends Microbiol23, 83-90, doi:10.1016/j.tim.2014.10.004 (2015).
8 Stern, A. et al. The Evolutionary Pathway to Virulence of an RNA Virus. Cell169, 35-46.e19, doi:10.1016/j.cell.2017.03.013 (2017).
9 Sjaarda, C. P. et al. Phylogenomics reveals viral sources, transmission, and potential superinfection in early-stage COVID-19 patients in Ontario, Canada. Scientific Reports11, 3697, doi:10.1038/s41598-021-83355-1 (2021).
10 da Silva Filipe, A. et al. Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland. Nat Microbiol6, 112-122, doi:10.1038/s41564-020-00838-z (2021).
11 Dearlove, B. et al. A SARS-CoV-2 vaccine candidate would likely match all currently circulating strains. bioRxiv, 2020.2004.2027.064774, doi:10.1101/2020.04.27.064774 (2020).
RNA Viruses, such as the SARS-CoV-2, HIV and Influenza, tend to pick up mutations rather quickly, once they are copied inside the cells of our bodies. This happens because the little cell workers that copy the RNA (called enzymes) are sloppy workers and prone to make errors.
The SARS-CoV-2 virus genetic code has just 30,000 nucleotides blocks of RNA, or letters that can spell at least 29 genes1; and, the most common mutations are single-nucleotide changes between viruses from different people, that have little effect on the overall performance of the virus infection rate, but that allow researchers to track the spread by linking closely-related viruses2.
In fact, sequencing data actually suggests that coronaviruses change more slowly than most other RNA viruses, because they have “proofreading” mechanisms that correct potentially fatal copying mistakes, accumulating only two single-letter mutations per month in its genome — a rate of change about half that of Influenza and one-quarter that of HIV2.
The problem is, that scientists can spot mutations faster than they can make sense of them, or what problems they may cause. Many mutations will have no consequence for the virus’s ability to spread or cause disease, because they do not alter the shape of a protein; whereas those mutations that do change proteins are more likely to harm the virus than improve it2. For example, in the beginning of the pandemic in Singapore, the ∆382 deletion made the infection of the virus milder3. On the contrary, the G614 variant showed a fitness advantage of infection, where individuals had a higher concentration of pseudotyped virions, suggestive of higher upper respiratory tract viral loads, but not with increased disease severity4.
If we think about it, the virus just wants to spread, but not actually kill – because, if it kills, it can no longer spread.
More recently, a new SARS-CoV-2 virus variant showed up in the UK, where multiple spike protein mutations are seen (deletion 69-70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H)5. Changes are also seen in other genomic regions, inclusively in the receptor binding domain (N501Y, “Nelly”).
The unusually high number of spike protein mutations suggests that the variant has not emerged through gradual accumulation of mutations6. Instead, one possible explanation for the emergence of the variant, is prolonged SARS-CoV-2 infection in a single patient, potentially with reduced immunocompetence, similar to what has previously been described7,8. Such prolonged infection in immunocompromised patients can lead to accumulation of immune-escape mutations at a higher rate.
This explanation is also suggested to the current SARS-CoV-2 virus variant found in Brazil (P.1)9, and in South Africa (501Y.V2)10.
Unfortunately, this shows that the emergence of successful variants with similar properties is not so rare6; and, just like the need to develop new influenza vaccines every season, that also might be the case for SARS-CoV-2. The persistence of the pandemic, may enable accumulation of immunologically relevant mutations in the population, even as vaccines are developed.
But, because science tries to be one step ahead, this is exactly what manufacturers are already working on, specifically the ones using mRNA technology that can quickly adapt their platforms to the current mutations seen.
1 Naqvi, A. A. T. et al. Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach. Biochim Biophys Acta Mol Basis Dis1866, 165878-165878, doi:10.1016/j.bbadis.2020.165878 (2020).
3 Young, B. E. et al. Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study. The Lancet396, 603-611, doi:10.1016/S0140-6736(20)31757-8 (2020).
4 Korber, B. et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell182, 812-827.e819, doi:10.1016/j.cell.2020.06.043 (2020).
Moderna uses an efficient method to make large quantities of individual mRNAs with high purity, based on a technique developed in 1984 by D. Melton and colleagues at Harvard University and published in Nucleic Acids Research. In here bacteriophage polymerases were used to transcribe plasmid DNA and make purified RNAs. D. Melton went further and showed that he could transcribe a synthetic mRNA injected into frog oocytes, and get expression of b-globin (Krieg & Melton, Nucleic Acids Research, 1984). With this report, the protocol to make pure mRNAs was settled.
Today, at Moderna, they moved on from this idea to a GMP drug-product inside of a vial in under two months. In the case of the COVID-19 vaccine, they went from a sequence of the virus to having the material ready for a Phase I clinical trial in 45 days.
This automated process starts with back-translation using Moderna’s proprietary computer algorithm, that has been defined over the years to pick the best mRNA sequence for the target in question.
Then, the therapeutic mRNA is engineered to avoid the innate immune sensors that defend against RNA viruses and might destroy the vaccine. These defences come in to main types: Toll-like receptors (TLR) in the endoplasmic reticulum, and RIG-I/MDA-5-like receptors in the cytoplasm.
To avoid TLRs, Moderna places modified nucleotides. This technique is based on a paper by Diebold and colleagues published in Science (2004), where the researchers took macrophages from mice and incubated them with different polymers (polyA, polyC, polyG, polyI, polyU), then checking for an IFN-alpha response. What they have seen was that the only polymer that created the IFN-alpha response was the one that contain Uridine (polyU), which seems to be the only nucleotide that is being recognized by the TLR-driven innate immune response. As such, at Moderna, mRNAs are engineered to replace all U’s with N1-methyl-pseudiuridine without affecting base-pairing, but avoiding TLR recognition. N1-methyl-pseudiuridine is a naturally occurring nucleotide that our bodies recognize as native.
Another problem though, is avoiding the creation of double-stranded RNA (dsRNA) to escape the RIG-I/MDA-5-like receptors. The state-of-the-art used in Moderna to avoid this trap, is HPLC purification to reduce dsRNA content. This is a technique based on Karikó & Weismann (2011, Nucleic Acids Research), that results in very little dsRNA in the end-product, and an avoidance of the innate immune response. The developed technique at Moderna has recently been published in Science Advances (June 2020), where Moderna scientists describe a highly sensitive assay to detect dsRNA and purify therapeutic mRNAs.
(It seems Moderna scientists are now going even further, and have reengineer T7 RNA polymerase not to produce dsRNA at all – although this has not been published yet)
The third step to make a good mRNA-drug, and a successful vaccine, is to find an efficient delivery method. With that in mind, Moderna researchers looked back at work done by Wolff and colleagues in the 90s (Science, 1990, vol.247, 1465-1468), where they were able to directly inject RNA in the mouse skeletal mouse and have protein expression, with no special delivery system involved.
In 2014, researchers at Moderna were able to go further, and measured with a Luciferase system, the in vivo expression of biologically active proteins in a dose-dependent manner, by injecting mice with Moderna’s naked mRNA. Nowadays, Moderna targets different tissues via multiple Routes of Administration with specific delivery vehicles (ROAs: direct injection in the heart or tumours, subcutaneous, intravenous, intramuscular).
Most ROAs require mRNA encapsulation with Lipid nanoparticles (LNPs, 80-100nm). Each encased particle contains 2-6 molecules of mRNA, phospholipid cholesterol, a PEG lipid that keeps the lipid nanoparticles stable avoiding aggregation, and an ionizable lipid that at low pH interacts with the mRNA. These mRNA-LNPs are akin to endogenous lipid transport complexes, slightly bigger than Very-Low Density Lipoproteins (VLDLs), and seen as “friendly bubbly characters” by our bodies.
At Moderna, they empower a rational-structure LNP design by analysing and engineering each individual step of the component mixture, to enhance chemical/physical stability of mRNA-LNPs to allow an optimal biodistribution, cellular uptake, endosomal escape and protein expression. In comparison to the competitor LNPs in the field, which have a 1-2% delivery rate, Moderna’s LNPs can deliver 30% mRNA into cells. To date, Moderna has published two peer-revied articles describing these efforts and their applications in different ROAs (Sabis … Benenato, Mol Ther, 2018; Hassett … Britto, Mol Ther Nuc Acids, 2019).
Finally, there needs to be the right knowledge to engineer the best mRNA sequence for a particular purpose in order to make a good mRNA-drug or vaccine.
So, there needs to be translation initiation fidelity, so that it always starts in the right place and there’s faithful decoding; and, also, the ribosome needs to stop in the right place.
The mRNA needs to have a functional half-life, to function before it gets destroyed.
Also, the therapeutics or vaccine needs to hit the correct cell type, which is achieved by putting “off-logic gates”, like microRNAs target sites in the 3’prime UTR, that cause the mRNA to be degraded in case it hits an undesired cell type.
At Moderna, they have all steps in place for a rapid development of mRNA as medicines:
Efficient methods to make large quantities of individual mRNAs at high purity;
Ability to avoid the innate immune sensors that defend against RNA viruses;
Efficient delivery methods,
Knowledge of how to engineer the best mRNA sequence for a particular purpose.
Besides proprietary algorithm to define the mRNA sequence and unique LNP design; one of the big differences between BioNTech/Pfizer vaccine and Moderna, is that Moderna does not use self-amplifying RNA in their vaccines. According to Dr Melissa Moore, Chief Scientific Officer at Moderna Therapeutics Inc., self-amplifying RNA first starts by building dsRNA which pushes-up the immune response against the vaccine, as such Moderna hasn’t gone down that road to avoid such road-blocks.
The important take-out message is not which vaccine we take.
With a mRNA vaccine, the cell is doing the protein itself, and the antigen-presenting cells are exhibiting it on their surface, which causes an activation of B and T cells.
This protects us and others from developing this devastating disease.
The principle of mRNA therapeutics is to introduce therapeutic messenger (m) RNAs encoding the genetic information for a protein, into a cell of interest. The mRNA structure is designed to increase half-life, translation and protein functionality.
The mRNA synthesis is made by in vitro transcription using a linear DNA template, which provides more than 500 copies of mRNA per template. They promote a transient expression of the encoded protein/antigen, and are degraded into nucleotides, without the formation of toxic metabolites. Since mRNA is highly sensitive, it gets degraded after a short-time, with no risk of genomic integration.
This one process can be used to manufacture essentially any mRNA sequence.
The raw reaction mixture has mRNA and all types of impurities, both process-related (T7 RNA polymerase, remaining building blocks, hydrolyzed DNA…), and product-related impurities (break-off transcripts, side products…), that need to be further purified with high affinity chromatographic methods.
Once purified RNA is obtained, the therapeutic is ready to deliver to the patient.
BioNTech together with their partner Pfizer and FosunPharma, managed to do all this in a record time of 84 days to develop a COVID-19 vaccine.
Their scientists designed multiple antigen variants using the SARS-CoV-2 Spike (S) protein.
Next, they ran preclinical and toxicology evaluation tests in vitro and in vivo (e.g., in vitro expression data, antibody titers in animal models, pseudo-virus neutralization (pVN) assay in animal models), so they could discover the most active antigen and bring it into the clinic. Also, using their Good Manufacturing Practice (GMP) facilities they produced small scale batches for fast entry into the clinic to demonstrate safety, tolerability (i.e., low reactogenicity), and immunogenicity.
Afterwards, they initiated clinical trials, where not one but four vaccine candidates were simultaneously studied, in order identify the safest and most effective candidate for further development. In parallel, there was already preparation of larger manufacturing batches of the candidate with the best output in the clinical studies; and, the identification of collaboration partners to develop and provide the vaccine worldwide (Pfizer, FosunPharma).
BioNTech and Pfizer studied three mRNA types during all Phase I/II studies, to evaluate safety and efficacy. The basis for such studies was that different types of mRNA vaccines lead to different responses.
As such, vaccine prototype (1) was a Uridine mRNA (uRNA) with strong intrinsic adjuvant effect, strong antibody response, that stimulates the production of more CD8+T cells then CD4+T cells.
Vaccine prototype (2) was a Nucleoside-modified mRNA (modRNA), with a moderate intrinsic adjuvant effect, very strong antibody response, lower cytokine induction, that stimulates more CD4+T cells then CD8+T cells.
In the case of vaccine prototype (3), this was a Self-amplifying mRNA (saRNA) with long duration, and a higher likelihood for good efficacy antibody response with lower dosage.
Early and constant interaction with the regulatory agencies (e.g., EMA) has allowed that BNT162b2 vaccine candidate raced to the finish line, being now the leading candidate from BioNTech & Pfizer; and, currently in review for conditional marketing authorization approval.
It is outstanding that if a new variant of the virus is required (e.g., due to a mutated virus sequence), a new GMP batch of a possible vaccine could be available within half of the time (around 42 days).
If there is a positive message out of these dark days, is that Science is there to save us from our doom.
Today’s top selling drugs are mostly biologics, which means proteins (e.g., monoclonal antibodies) being used as medicine. The global therapeutic monoclonal antibody market was valued at approximately US$115.2 billion in 2018, and is expected to generate revenue of $150 billion by the end of 2019, and $300 billion by 20251. Thus, the market for therapeutic antibody drugs has experienced an explosive growth with new drugs being approved for treating various human diseases, including many cancers, autoimmune, metabolic and infectious diseases, which have increasingly fewer adverse effects due to their high specificity.
But, these types of treatments carry their own limitations. Besides of a long period of development, they can only access the extracellular space, the serum. Yet, ~2/3 of the human proteome are intracellular or transmembrane proteins; which means that biologics cannot treat disorders that are caused by these types of proteins.
As such, an alternative mechanism to make these proteins is needed in order to go further in the advancement of therapeutics.
One such mechanism, is to supply the mRNA that encodes that protein. As such, any protein can become a drug. And, since only the coding region varies from mRNA-drug to mRNA-drug; once the delivery vehicle problem is resolved (e.g., using lipid nanoparticles), the development of new mRNA drugs should be quite fast in comparison with other types of medicine.
The other positive aspect of mRNA as therapeutics, is that it can be used in many different types of modalities: prophylactic vaccines, cancer vaccines, intertumoral immune-oncologicals, localized regenerative therapeutics, systemic secreted medicine, and cell surface therapeutics…
Or, another possibility, is the personalization of cancer vaccines, where different patient-specific neoepitopes are sequenced, and mRNA is created that encodes those 20-35 specific tumour-neoepitopes.
Those mRNAs can also encode cytokines, that are able to boost the immune system to attack a tumour, like intertumoral immune-oncologicals therapeutics.
In the field of localized regenerative therapeutics, there is currently a project between Moderna Therapeuticsand AstraZeneca in Phase II clinical trials, where Vascular Endothelial Growth Factor-A (VEGFA) mRNA is injected NAKED, without lipid nanoparticles, directly into the heart muscle of patients with a recent Myocardial Infarction (MI) to help rebuild blood vessels.
Of course, many challenges still exist.
mRNA needs to be rightly sequenced and be made at high purity. It also needs to have the ability to avoid the innate immune sensors, like Toll-like receptors (TLR) in the endoplasmic reticulum, and RIG-I/MDA-5-like receptors in the cytoplasm. What researchers do to avoid the first ones (TLRs) is to use a Pseudouridine, which looks like a Uridine and still base-pairs, but escapes TLR recognition. To avoid the RIG-I/MDA-5-like receptors, researchers need to highly purify the therapeutic mRNA, so that no double-stranded RNA exists (or, reengineer T7 RNA polymerase not to produce dsRNA at all).
The next step on making a good mRNA-drug is to find an efficient delivery method. For that, most therapeutics will need some sort of mRNA encapsulation with Lipid nanoparticles, which are akin to endogenous lipid transport complexes, similar to Very-Low Density Lipoproteins (VLDLs).
Finally, in order to make a good mRNA-drug, there needs to be knowledge on how to engineer the best mRNA sequence for a particular purpose. As such, there needs to be translation initiation fidelity, so that it always starts in the right place and there’s faithful decoding; and, also, the ribosome needs to stop in the right place. There needs to be high translation efficiency, a functional mRNA half-life, and hit the correct cell type, by putting off-logic gates, like microRNAs target sites in the 3’prime UTR and causing the mRNA to be degraded in case it hits an undesired cell type. Also, the mRNAs need to be tailored to the protein type (e.g., secreted, mitochondrial).
So, let’s see what the future brings from mRNA therapeutics…
1 Lu, R.-M. et al. Development of therapeutic antibodies for the treatment of diseases. Journal of Biomedical Science27, 1, doi:10.1186/s12929-019-0592-z (2020).
In humans, anti-tumour immunity is usually very effective, because our T-cells (a type of a white blood cell) can detect the presence of specific cancer neoantigens. These are short chains of aminoacids (peptides) that appear due to tumour-specific mutations, and mark the cancer cell as foreign. They are highly immunogenic, because they are not present in normal tissues; and therefore, evade central thymic tolerance and activate our immune system to destroy them.
Although neoantigens were long-envisioned as optimal targets for an anti-tumour immune response, their efficient discovery and evaluation only became possible recently, with the availability of massive parallel sequencing for detection of all coding mutations within tumours; and, of machine learning approaches to reliably predict those mutated peptides with high-affinity binding of autologous human leukocyte antigen (HLA) molecules.
It has been shown that vaccination with these specific neoantigens can expand the pre-existing neoantigen-specific T-cell populations; and, induce a broader repertoire of new T-cell specificities in cancer patients. This can actually tip the intra-tumoral balance, in favour of an enhanced tumour control.
Research studies now demonstrate the feasibility, safety, and immunogenicity of vaccines that target up to 20 predicted personal tumour neoantigens (e.g., Melanoma Neovax anti-PD11). Vaccine-induced polyfunctional CD4+ and CD8+ T-cells can target unique tumour neoantigens; and, not only that, these T-cells can have persistent memory. This because, studies from the Dana-Farber Cancer Institute and Dr. Catherine Wu’s lab, show that patients have a continuous response 5-7-years later after the initial vaccination, with a second-wave of immune response that shows epitope spreading from the initial epitope. What this means, is that the patient immune system was able to recognize and target tumour cells, even when they try to disguise themselves years later.
Researchers have gone further: at Genocea Biosciences they are developing precision neoantigen selection with their branded ATLAS platform. They can optimize the set of neoantigens for inclusion in the vaccine, by excluding inhibitory antigens (called Inhibigens) that suppress the patient’s immune system, blocking the internal growth of the tumour cells.
1 Ott, P. A. et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature547, 217-221, doi:10.1038/nature22991 (2017).
Theanine was discovered in 1949 by the Japanese researcher Yajiro Sakato1, as a new amide in the water extract of the Japanese green tea Gyokuro (玉 露) – also called “precious dew” due to its dark green colour and high price in Japanese markets, because of its unique characteristic taste of sweetness and “umami”2.
The green tea leaves from specialized varieties of the tea plant Camellia sinensis (Ericales plants) have enormous amounts of theanine, which they absorb from the roots of the plant depending on the nitrogen supply of the soil3, 4. A soil that is rich in nitrogen will promote the biosynthesis of the non-protein aminoacid L-Theanine from Glutamic Acid and Ethylamine (by the enzyme theanine synthetase)2. Since the commercial price of green tea is almost directly proportional to its theanine content, to obtain theanine-rich, good quality green tea leaves, a large amount of nitrogen fertilizer must be supplied to the cultivated plants throughout the growth period (with problematic effects on the environment)2.
Theanine is then transported via the xylem fluid, from the roots to the young bushes leaves. And, because light is necessary to the conversion of theanine into cathechins in the leaves of the plant, when Camellia sinensis bushes are protected from direct sunlight for a couple of weeks just before harvest, they have a high theanineamino acid content2. Even though cathechins are polyphenols with known antioxidant properties, they are also responsible for the astringent flavour of green tea. So, for an optimum taste, cathechins must be balanced with theanines. With less sunlight, there is less photosynthesis and leaf senescence; and, less theanine being converted into catechin, keeping the unique sweet-umami flavor characteristic of Gyokuro green tea.
Theanine is also known as N5-ethyl-L-glutamine due to its structural similarities to L-glutamic acid, which is the most abundant excitatory neurotransmitter in our brains5. Researchers think that L-theanine mechanism of action might be mediated by glutamate receptors, and it might act as a partial agonist for the N-methyl-D-aspartate receptor6.
Theanine has known relaxing and anxiolytic effects, via the induction of the slow alpha-brain waves in the occipital and parietal regions of the human brain7. Plus, it doesn’t have any additive or side effects that are usually associated with conventional sleep inducers.
There is only one IF….
In addition to L-Theanine, Camellia sinensis leaves grown in the shade also have a high level of caffeine8, which decreases slow brain activity and keeps us awake (by increasing beta-wave activity). This because, the buds and young leaves of Camellia plants contain more caffeine than mature leaves8. As such, besides a high level of theanine, Gyokuro or Matcha green tea powder (which goes through the same shade process before harvest), also have high levels of caffeine.
What is interesting, is that this dual effect of L-Theanine and Caffeine in Gyokuro or Matcha green tea powder, seem to have a synergistic effect in decreasing mind wandering and enhancing our attention to target stimuli9, 10. This was shown in a very small randomized clinical trial, that used functional Magnetic Resonance Imaging (fMRI) to scan the brains of subjects, after they ingested L-Theanine and Caffeine supplements while performing a visual task.
So, if we want to focus and stay awake: a cup of Gyokuro or Matcha, will keep our attention sharp as a Japanese sword.
If, on the other hand, we are not feeling very calm, anxiety is setting in, or if sleep is taking too long because the news are only “so-so” at the moment: 200-400mg of L-theanine could help us keep the zen mood, and have a good night sleep11.
1. Sakato Y. Studies on the Chemical Constituents of Tea
Part III. On a New Amide <b>Theanine</b>. Nippon Nōgeikagaku Kaishi. 1950;23:262-267.
2. Ashihara H. Occurrence, biosynthesis and metabolism of theanine (γ-glutamyl-L-ethylamide) in plants: a comprehensive review. Nat Prod Commun. 2015;10:803-10.
3. Ruan J, Haerdter R and Gerendás J. Impact of nitrogen supply on carbon/nitrogen allocation: a case study on amino acids and catechins in green tea [Camellia sinensis (L.) O. Kuntze] plants. Plant Biol (Stuttg). 2010;12:724-34.
4. Huang H, Yao Q, Xia E and Gao L. Metabolomics and Transcriptomics Analyses Reveal Nitrogen Influences on the Accumulation of Flavonoids and Amino Acids in Young Shoots of Tea Plant ( Camellia sinensis L.) Associated with Tea Flavor. J Agric Food Chem. 2018;66:9828-9838.
5. Unno K, Furushima D, Hamamoto S, Iguchi K, Yamada H, Morita A, Horie H and Nakamura Y. Stress-Reducing Function of Matcha Green Tea in Animal Experiments and Clinical Trials. Nutrients. 2018;10:1468.
6. Sebih F, Rousset M, Bellahouel S, Rolland M, de Jesus Ferreira MC, Guiramand J, Cohen-Solal C, Barbanel G, Cens T, Abouazza M, Tassou A, Gratuze M, Meusnier C, Charnet P, Vignes M and Rolland V. Characterization of l-Theanine Excitatory Actions on Hippocampal Neurons: Toward the Generation of Novel N-Methyl-d-aspartate Receptor Modulators Based on Its Backbone. ACS Chem Neurosci. 2017;8:1724-1734.
7. Kobayashi K, Nagato Y, Aoi N, Juneja LR, Kim M, Yamamoto T and Sugimoto S. Effects of L-Theanine on the Release of α-Brain Waves in Human Volunteers. Nippon Nōgeikagaku Kaishi. 1998;72:153-157.
8. Ashihara H and Suzuki T. Distribution and biosynthesis of caffeine in plants. Front Biosci. 2004;9:1864-76.
9. Kahathuduwa CN, Dhanasekara CS, Chin SH, Davis T, Weerasinghe VS, Dassanayake TL and Binks M. l-Theanine and caffeine improve target-specific attention to visual stimuli by decreasing mind wandering: a human functional magnetic resonance imaging study. Nutr Res. 2018;49:67-78.
10. Hidese S, Ogawa S, Ota M, Ishida I, Yasukawa Z, Ozeki M and Kunugi H. Effects of L-Theanine Administration on Stress-Related Symptoms and Cognitive Functions in Healthy Adults: A Randomized Controlled Trial. Nutrients. 2019;11:2362.
11. Williams JL, Everett JM, D’Cunha NM, Sergi D, Georgousopoulou EN, Keegan RJ, McKune AJ, Mellor DD, Anstice N and Naumovski N. The Effects of Green Tea Amino Acid L-Theanine Consumption on the Ability to Manage Stress and Anxiety Levels: a Systematic Review. Plant Foods Hum Nutr. 2020;75:12-23.
A macrophage is a hungry immune cell that engulfs and eats all things that don’t have a good reputation in our body (e.g., cellular debris, pathogens…); and, microglia cells are the resident macrophage population of the Central Nervous System (CNS)1. They function as sentinels of local infection in the brain, backing both innate and adaptive immune responses, and account for 10-15% of all cells found in the brain and spinal cord2.
Microglia cells are also involved in the maintenance of brain homeostasis, contributing to mechanisms that underly learning and memory. They constantly survey their local microenvironment – like patrols – extending their motile processes, or hands/legs, to make a brief contact with neuronal synapses. This continuous synaptic plasticity, throughout our lifetime, is essential to control maladaptive learning and memory, such as addiction3. For example, the number of synapses in the brain regions of the nucleus accumbens, amygdala and dorsomedial striatum increase when we expose our brains to addictive substances (such as alcohol, or opiates); and, decrease upon withdrawal due to the action of microglia cells4. As such, microglia cells help to modify and eliminate synaptic structures when they grow too much, or, are on the way to touch too many other neurons5 – because, neurons tend to be touchy and to enjoy a synaptic orgy.
Whenever a neuron starts to freak out that it has too many synapses and it needs help regulating its neuronal “touchy” behaviour, then the synapse extends a greeting “hand” (filopodia) and “Hi5s” the neighbouring microglia cell, telling her that it needs help remodelling. Once “Hi5ed”, the microglia cell starts nibbling on the synapse6 – cutting all the excess – and, avoiding that that specific neuron gets assigned a bad “sexual” reputation. It’s like behaviour counselling, transforming and remodelling, but neuron-wise and with a microglia cell as the counsellor…
Even though microglia cells are essential and extremely helpful; like everything in life, they can also go haywire, ending up pruning too many synapses, and destroying healthy tissue. An uncontrolled activation of the microglia can be directly toxic to neurons, because they can release inflammatory cytokines (IL-1, TNF-alpha, IL-6, Nitric Oxide, Prostaglandine E2, and Superoxide)7, and lead to excessive pruning of neuronal synapses3.
The most recent research in the pathophysiology of depression and anxiety shows that abnormalities in microglia cells have a central role in the development of these diseases8. For example, a neuroimaging study in depressed patients, revealed that stronger depressive symptoms related with microglial activation in brain regions associated with mood regulation (the prefrontal, anterior cingulate, and insular cortices of the brain)9. Additionally, post-mortem studies of depressed suicide victims showed microglial activation and macrophage accumulation within the anterior cingulate cortex brain region10.
Persistent stress activates a chronic low-inflammatory state in our bodies that enhances our inflammatory response to challenges11. Social stress causes the release of inflammatory monocytes into the circulation8, which end up reaching the Blood Brain Barrier (BBB) and its endothelial cells. This low-systemic inflammation that travels through our vessels, encourages the migration of the brain resident microglia cells to the area of the cerebral vessels. In here, microglia cells make physical contact with endothelial cells of the BBB, and “sense” the inflammatory environment that is present in the blood (aka, inflammatory cytokines activate receptors in the microglia cells). If there is sustained inflammation, then some of the microglia cells can “become neurotic” and start nibbling the end-feet of healthy cells, making the BBB more permeable and, consequently, damaging the protective BBB shield function12. This is turn, leaks inflammatory cytokines from the blood into the brain tissue, further activating more microglia cells, that start cutting synapses from healthy neurons.
What this means is that a persistent low-grade inflammation can trigger microglia activation and change the functional connectivity of healthy neurons in major brain emotional centers13. Because our immune system can interact with the neurocircuitry that is involved in emotion regulation and behaviour, a chronic low-inflammation derived from stress can influence the development of various neuropsychiatric disorders, like depression and anxiety.
But, what can we do to avoid falling in this trap?
Eat well, sleep well, do sports and have a good laugh with friends. All things that inhibit inflammation, and make us feel good.
1. Ginhoux F, Lim S, Hoeffel G, Low D, Huber T. Origin and differentiation of microglia. Frontiers in Cellular Neuroscience. 2013;7
2. Lawson LJ, Perry VH, Gordon S. Turnover of resident microglia in the normal adult mouse brain. Neuroscience. 1992;48:405-415
3. Neniskyte U, Gross CT. Errant gardeners: Glial-cell-dependent synaptic pruning and neurodevelopmental disorders. Nat Rev Neurosci. 2017;18:658-670
4. Spiga S, Talani G, Mulas G, Licheri V, Fois GR, Muggironi G, et al. Hampered long-term depression and thin spine loss in the nucleus accumbens of ethanol-dependent rats. Proc Natl Acad Sci U S A. 2014;111:E3745-3754
5. Tremblay M-È, Lowery RL, Majewska AK. Microglial interactions with synapses are modulated by visual experience. PLOS Biology. 2010;8:e1000527
6. Weinhard L, di Bartolomei G, Bolasco G, Machado P, Schieber NL, Neniskyte U, et al. Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction. Nature Communications. 2018;9:1228
7. Kim YS, Joh TH. Microglia, major player in the brain inflammation: Their roles in the pathogenesis of parkinson’s disease. Exp Mol Med. 2006;38:333-347
8. McKim DB, Weber MD, Niraula A, Sawicki CM, Liu X, Jarrett BL, et al. Microglial recruitment of il-1β-producing monocytes to brain endothelium causes stress-induced anxiety. Mol Psychiatry. 2018;23:1421-1431
9. Setiawan E, Wilson AA, Mizrahi R, Rusjan PM, Miler L, Rajkowska G, et al. Role of translocator protein density, a marker of neuroinflammation, in the brain during major depressive episodes. JAMA Psychiatry. 2015;72:268-275
10. Suzuki H, Ohgidani M, Kuwano N, Chrétien F, Lorin de la Grandmaison G, Onaya M, et al. Suicide and microglia: Recent findings and future perspectives based on human studies. Frontiers in cellular neuroscience. 2019;13:31-31
11. Miller GE, Rohleder N, Cole SW. Chronic interpersonal stress predicts activation of pro- and anti-inflammatory signaling pathways 6 months later. Psychosom Med. 2009;71:57-62
12. Haruwaka K, Ikegami A, Tachibana Y, Ohno N, Konishi H, Hashimoto A, et al. Dual microglia effects on blood brain barrier permeability induced by systemic inflammation. Nature communications. 2019;10:5816-5816
13. Kim J, Yoon S, Lee S, Hong H, Ha E, Joo Y, et al. A double-hit of stress and low-grade inflammation on functional brain network mediates posttraumatic stress symptoms. Nature Communications. 2020;11:1898
Our ability to learn and memorize new things profits from sleep; and, sleep loss leads to cognitive impairment that can only be reversed by closing our eyes and sleeping1. The more time we spend awake, and the further we engage with learning activities, the more our brains will demand for sleep.
The synapse is the structure that allows the neuron (or nerve cell) to pass an electrical or chemical signal to another neuron, or to the end effector cell that produces the action demanded from our brain. Synapses are the foundation of neuronal plasticity, and, in the adult brain, synapses can change their strength and size within minutes or hours in response to a new experience and learning1. Recent research has shown that the need for sleep and synaptic function are strongly linked together.
Sarah B. Noya and her colleagues2 from the Institute of Pharmacology and Toxicology of the University of Zürich (Switzerland) have recently shown that 70% of the synaptic transcripts change during our 24h circadian cycles. The transcripts and proteins related to synaptic signaling, accumulate before the active phase of the bodies and get further cleared out during the day. In the meantime, proteins that are associated with the body metabolism and translation, accumulate in the synapses just before the resting phase or sleeping time. As such, just before we go to bed, the synapses get congested with protein information from our bodies daily function, that needs to be compartmentalized and processed.
We can imagine the synapses as a clerk’s room filling up with boxes and parcels that need to be deliver to the proper address.
But what is interesting, is the result that comes from another study published at the same time, from Franziska Brüning and her team3 at the Institute of Medical Psychology of the Ludwig Maximilian University of Munich (Germany). This research study shows that sleep deprivation abolishes nearly all of the compartmentalization of these accumulating proteins at the synapses (98%); which means that, without a proper shut eye, the synapses get completely clogged with accumulating “protein-parcels” that don’t get removed. When a chemical or electrical information wants to get through the synapses the next day, it can’t because there’s accumulating protein transcripts that haven’t been properly processed, or phosphorylated. The information gets stalled, due to the congestion at the synapses.
So, next time your brain feels fizzle in the morning, and throughout the day; promise yourself (and your brain) to go to bed early, and have a proper night’s rest!
1. Cirelli C and Tononi G. Linking the need to sleep with synaptic function. Science. 2019;366:189-190.
2. Noya SB, Colameo D, Bruning F, Spinnler A, Mircsof D, Opitz L, Mann M, Tyagarajan SK, Robles MS and Brown SA. The forebrain synaptic transcriptome is organized by clocks but its proteome is driven by sleep. Science. 2019;366.
3. Bruning F, Noya SB, Bange T, Koutsouli S, Rudolph JD, Tyagarajan SK, Cox J, Mann M, Brown SA and Robles MS. Sleep-wake cycles drive daily dynamics of synaptic phosphorylation. Science. 2019;366.
According to a recent study by Daghlas and colleagues1, compared to sleeping 6 to 9 h/night, short sleepers have a 20% higher risk of having a heart attack; but, if you are a long sleeper (i.e., sleeping >9h/night), than your chances are even worse, because your risk increases to 34%. Even though the researchers don’t know the underlying cause for such susceptibilities, they claim sleeping too much or too little boosts inflammation in the body, which is associated with the development of heart disease. If you have a genetic predisposition for heart disease, this study found that sleeping between 6-9h, actually reduces your risk of having a heart attack by 18%, which is actually very good news, since not only diet and exercise can help you keep your heart healthy. More and more data, supports the evidence that we should consider sleep to be an adjustable and controllable risk factor for our good heath status2.
Speaking of diet, another study published recently in the Journal of the American Heart Association by Hyunju Kim and his team3, showed that healthy plant‐based diets, which are higher in whole grains, fruits, vegetables, nuts, legumes, tea, and coffee, and lower in animal foods, were associated with a lower risk of cardiovascular disease mortality and all‐cause mortality. Of course, they didn’t explore if the quality of plant foods (either healthy plant foods, or less-healthy plant foods) within the “framework of plant‐based diets” would be associated with cardiovascular disease and all‐cause mortality in the general population.
But, what is intriguing is that, another recent study by Tammy Tong and colleagues4, examined the associations of vegetarianism with risks of ischemic heart disease (i.e., coronary artery disease) and stroke. The results of this study showed that vegetarians had 20% higher rates of total stroke than meat eaters – which was equivalent to 3x more cases of stroke over 10 years; and, the associations for stroke did not soothe after adjustments to other disease risk factors. As the authors of the study say, vegetarian and vegan diets have become increasingly popular in recent years, partly due to perceived health benefits, as well as concerns about the environment and animal welfare; but, what the evidence suggests, is that vegetarians might have different disease risks compared with non-vegetarians. The study group of vegetarians and vegans in this cohort had lower circulating levels of several nutrients (e.g., vitamin B12, vitamin D,essential amino acids,and long chain n-3 polyunsaturated fatty acids), and differences in some of these nutritional factors could contribute to the increased stroke risk. Not only that, but a number of studies in Japan5, 6, showed that individuals with very low intake of animal products, also had an increased incidence and mortality from hemorrhagic and total stroke, implying that some factors connected with animal food intake might be protective for stroke.
Its like Yin and Yang from ancient Chinese philosophy. Rather than opposing, or standing at the sides, our health and life is made of complementary forces that interact to form a dynamic system. It’s all about balance and balancing the sides (and diets).
1. Daghlas I, Dashti HS, Lane J, Aragam KG, Rutter MK, Saxena R and Vetter C. Sleep Duration and Myocardial Infarction. Journal of the American College of Cardiology. 2019;74:1304-1314.
2. Tobaldini E, Fiorelli EM, Solbiati M, Costantino G, Nobili L and Montano N. Short sleep duration and cardiometabolic risk: from pathophysiology to clinical evidence. Nat Rev Cardiol. 2019;16:213-224.
3. Kim H, Caulfield LE, Garcia-Larsen V, Steffen LM, Coresh J and Rebholz CM. Plant-Based Diets Are Associated With a Lower Risk of Incident Cardiovascular Disease, Cardiovascular Disease Mortality, and All-Cause Mortality in a General Population of Middle-Aged Adults. J Am Heart Assoc. 2019;8:e012865.
4. Tong TYN, Appleby PN, Bradbury KE, Perez-Cornago A, Travis RC, Clarke R and Key TJ. Risks of ischaemic heart disease and stroke in meat eaters, fish eaters, and vegetarians over 18 years of follow-up: results from the prospective EPIC-Oxford study. BMJ. 2019;366:l4897.
5. Kinjo Y, Beral V, Akiba S, Key T, Mizuno S, Appleby P, Yamaguchi N, Watanabe S and Doll R. Possible protective effect of milk, meat and fish for cerebrovascular disease mortality in Japan. J Epidemiol. 1999;9:268-74.
6. Sauvaget C, Nagano J, Allen N, Grant EJ and Beral V. Intake of animal products and stroke mortality in the Hiroshima/Nagasaki Life Span Study. Int J Epidemiol. 2003;32:536-43.
The Habenula, is an area of our brains close to the pineal gland, that is involved in pain processing, reproductive behaviour, nutrition, sleep-wake cycles and stress responses, among other things1. A professor I used to know always said the Habenula was the Master of the Brain… and, indeed, recent research has provided evidence that this tiny bundle of nerves is able to produce Dimethyltryptamine (DMT), a psychedelic drug, “cousin” to the famous LySergic acid Diethylamide (LSD).
DMT is internally bio-synthesized by the enzymes Aromatic-L-Amino acid DeCarboxylase (AADC) and Indolethylamine-N-Methyltransferase (INMT). Dean and colleagues2 were able to identify INMT messenger RNA in human tissues, by using a RNAscope in situ assay system; a highly sensitive technique, which proved for the first time a clear-cut identification of DMT and its enzymes in human brain.
Outstanding, was the discovery that there was a significant increase of DMT levels in the rat brain after stimulation of experimental cardiac arrest; showing for the first time, that the brain is capable of synthesizing and releasing DMT under stress.
This ultimately raises the possibility that this phenomenon may also occur in human brains, when we experience situations of extreme stress. The researchers attest that the cardiac arrest-induced increase of DMT may be related to “near-death experiences”, as reported by Timmermann and collegues3. This group recently reported that human subjects given exogenous DMT, experienced “near-death”-like mental states, including the subjective feeling of transcending one’s body and entering an alternative realm, perceiving and communicating with ‘entities’, and themes related to death and dying.
It’s unbelievable that the more we know about how our body and brain functions, the more I realize that our mind is a construction of our organic biological nature.
What we sometimes perceive as a mystical experience is probably just rooted in an organic mechanism that is tricking our minds into a “trip”.
1. Namboodiri VM, Rodriguez-Romaguera J and Stuber GD. The habenula. Curr Biol. 2016;26:R873-R877.
2. Jon G. Dean TL, Sean Huff, Ben Sheler, Steven A. Barker, Rick J. Strassman, Michael M. Wang & Jimo Borjigin Biosynthesis and Extracellular Concentrations of N,N-dimethyltryptamine (DMT) in Mammalian Brain. Scientific Reports. 2019;9.
3. Timmermann C, Roseman L, Williams L, Erritzoe D, Martial C, Cassol H, Laureys S, Nutt D and Carhart-Harris R. DMT Models the Near-Death Experience. Front Psychol. 2018;9:1424.
Cabal, is a term defined in the Merriam-Webster dictionary, as the contrived scheme of a group of persons secretly united in a plot (as to overturn a government, for example).
But, if you talk in terms of Biology, cabals are also a series of synergistic venom peptides essential for the capture of prey. One animal venom can be a complex mixture of 10-200+ short chains of amino acids linked by bonds (peptides), working in a concerted mode to regulate physiological function, with very potent and precise molecular targets1.
For example, cone snails, a small venomous marine mollusk that hunts fish and worms, has ~850 species identified, with each expressing many thousands of unique peptides that selectively target a diverse range of voltage- and ligand-gated ion-channels, transporters and G-protein couple receptors2.
These tiny wonders of nature have the ability to switch between predatory and defensive venom regimes. For example, if they just want to stunt a predator causing a flaccid paralysis, they will produce venom that has high levels of muscle blockers (motor cabal), and that inhibit sodium channels and nicotinic acetylcholine receptors. But, if in the mean time, they change their minds and intend to eat the prey, they use a combination of peptides that cause a rigid paralysis. This lightning-strike cabal has excitatory peptides that inhibit potassium channels and delay inactivation of sodium channels, causing the prey to lie “dead” until it is happily digested in an underwater banquet.
But, how does the cone snail decide whether it is fear or hunger that it’s “feeling” in that moment?
The simple neuronal circuit of the cone snail shifts from a contented state of inertia, to an active motion, stimulated by internal hunger and an appetite stimulus – just like us, slushing from the couch to the fridge looking for our night prey… The hunting activity of the Conus is then organized by a basic set of behavioral transitions. Once the cone snail detects a fish, through sensory signals, it becomes much more active and moves towards the fish extending its rostrum– a massive funnel formed by the muscular walls of the snail sheath; and, a long, thin trunk extends out in the open, where a harpoon-like tooth shoots out to pierce the skin of the fish3 – imagine if we could actually do the same to that bag of cookies that is lying in the shelve right next to the couch.
The active feeding of the cone snail tends to inhibit the avoidance, and the snail changes to a prevention mood once its appetite is satisfied4.
1. Angell Y, Holford M and Moos WH. Building on Success: A Bright Future for Peptide Therapeutics. Protein Pept Lett. 2018;25:1044-1050.
2. Himaya SWA, Mari F and Lewis RJ. Accelerated proteomic visualization of individual predatory venoms of Conus purpurascens reveals separately evolved predation-evoked venom cabals. Sci Rep. 2018;8:330.
3. Olivera BM, Seger J, Horvath MP and Fedosov AE. Prey-Capture Strategies of Fish-Hunting Cone Snails: Behavior, Neurobiology and Evolution. Brain Behav Evol. 2015;86:58-74.
4. Gillette R and Brown JW. The Sea Slug, Pleurobranchaea californica: A Signpost Species in the Evolution of Complex Nervous Systems and Behavior. Integr Comp Biol. 2015;55:1058-69.
In 1958, in the Yale laboratories, A.B. Lerner and colleagues isolated melatonin from the pineal gland of bovines1. They were surprised that after 40 years of research they had finally found the active component that lightened the frog skin color, inhibiting the darkening effect of the Melanocyte Stimulating Hormone (MSH); hence the name, Melatonin1. Disappointingly, the skin lightening properties of melatonin could not be further demonstrated and the project was abandoned2. In the 90’s, melatonin got back on the coolness charts of science, with Reppert and Weaver calling it “Madness” in a Cell article in 1995. During this time, it was discovered its function in regulating the seasonal and circadian rhythms3, the presence of its specific G-coupled receptors in different tissues4; and, its antioxidant properties5. Since then, melatonin has been widely studied and continues to wonder over its broad range of therapeutic effects. From helping on jet-lag relief6, with insomnia7, being an anti-aging agent8, neuro-protective9, and also, improving cardiovascular diseases10: melatonin-madness continues until today.
Melatonin is widely accepted as a nutritional supplement being prescribed for sleep regulation in jetlag and adult sleep disorders; but in 2011 the U.S.A Food and Drug Administration (FDA), issued a warning to a company selling “relaxation brownies”, stating that the synthetic melatonin used in them hasn’t been proved safe as a food additive. Most commercial products are offered at dosages of 1-3mg of melatonin, which causes a spike of melatonin in the blood, reaching much higher levels than those that are naturally produced in the body somewhere between 50 and 200 pg/mL.
But, why use synthetic melatonin when this molecule is present in appetizing plants, nuts, fruits, meats, beverages and other foods11? The levels of melatonin in foods are much lower than those given as a nutritional supplement; but it has been proven that eating such foods drastically increases the circulating melatonin levels in the range of physiological concentrations, which peak at nighttime12, 13.
Maldonado and colleagues have shown that different types of beers are rich in melatonin; and, the more melatonin they have got, the greater is their alcoholic degree14. No wonder some people claim that beer makes them sleepy. But, Molfino15and I have made the same question: if the volunteers were sleepy, was it because of the melatonin or an alcoholic-mediated effect? So far, there’s still no answer to that question.
But, Garcia-Moreno and Maldonado’s group have shown, that Barley, which is malted and grounded in the early brewing process, and Yeast, during the second fermentation, are the largest contributors to the enrichment of beer with melatonin16. From this, we can deduct that not only beer has melatonin, but whatever drink where fermentation occurs, will also be rich in it. Logically, wine was also found to be a rich source of the Madness-molecule. In fact, beer has around 0,09 ng/mL while wine is up to 129,5 ng/mL11. And again, Rodriguez-Naranjo and colleagues showed that melatonin is formed during the alcoholic fermentation, because it is absent in the grapes and musts17.
Lerner ABC, J.D.; Takahashi, Y.; Lee, T.H.; Mori, W. Isolation of melatonin, a pineal factor that lightens melanocytes. J Am Chem Soc. 1958;80:2587.
Jiki Z, Lecour S and Nduhirabandi F. Cardiovascular Benefits of Dietary Melatonin: A Myth or a Reality? Front Physiol. 2018;9:528.
Arendt J. Melatonin and the pineal gland: influence on mammalian seasonal and circadian physiology. Rev Reprod. 1998;3:13-22.
Reppert SM, Godson C, Mahle CD, Weaver DR, Slaugenhaupt SA and Gusella JF. Molecular characterization of a second melatonin receptor expressed in human retina and brain: the Mel1b melatonin receptor. Proc Natl Acad Sci U S A. 1995;92:8734-8.
Hardeland R, Reiter RJ, Poeggeler B and Tan DX. The significance of the metabolism of the neurohormone melatonin: antioxidative protection and formation of bioactive substances. Neurosci Biobehav Rev. 1993;17:347-57.
Sletten TL, Magee M, Murray JM, Gordon CJ, Lovato N, Kennaway DJ, Gwini SM, Bartlett DJ, Lockley SW, Lack LC, Grunstein RR, Rajaratnam SMW and Delayed Sleep on Melatonin Study G. Efficacy of melatonin with behavioural sleep-wake scheduling for delayed sleep-wake phase disorder: A double-blind, randomised clinical trial. PLoS Med. 2018;15:e1002587.
Riemann D, Baglioni C, Bassetti C, Bjorvatn B, Dolenc Groselj L, Ellis JG, Espie CA, Garcia-Borreguero D, Gjerstad M, Goncalves M, Hertenstein E, Jansson-Frojmark M, Jennum PJ, Leger D, Nissen C, Parrino L, Paunio T, Pevernagie D, Verbraecken J, Weess HG, Wichniak A, Zavalko I, Arnardottir ES, Deleanu OC, Strazisar B, Zoetmulder M and Spiegelhalder K. European guideline for the diagnosis and treatment of insomnia. J Sleep Res. 2017;26:675-700.
Day D, Burgess CM and Kircik LH. Assessing the Potential Role for Topical Melatonin in an Antiaging Skin Regimen. J Drugs Dermatol. 2018;17:966-969.
Zhao Z, Lu C, Li T, Wang W, Ye W, Zeng R, Ni L, Lai Z, Wang X and Liu C. The protective effect of melatonin on brain ischemia and reperfusion in rats and humans: in vivo assessment and a randomized controlled trial. J Pineal Res. 2018:e12521.
Liu Y, Li LN, Guo S, Zhao XY, Liu YZ, Liang C, Tu S, Wang D, Li L, Dong JZ, Gao L and Yang HB. Melatonin improves cardiac function in a mouse model of heart failure with preserved ejection fraction. Redox Biol. 2018;18:211-221.
Meng X, Li Y, Li S, Zhou Y, Gan RY, Xu DP and Li HB. Dietary Sources and Bioactivities of Melatonin. Nutrients. 2017;9.
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Since life has emerged on Earth, 3.7. billion years ago, the rising and setting of the Sun has been a constant. Whether it was light or dark, it was day or night. Humans, and all other organisms, have evolved with this imposed biological rhythm; and, human physiology was determined by the light/dark cycle.
Every cell in our bodies exhibits a circadian rhythm, a 24h-cycle synchronized to a light/dark pattern. But, how do cells deep inside in our bodies know when it’s dark outside? The answer to that question is Melatonin (N-acetyl-5-methoxytryptamine), the messenger from our brain to communicate to our cells that it is dark. The master clock of the body, the Suprachiasmatic Nucleus (SCN) receives darkness information from the retina and sends it to the brain. From there it provides an input to the Pineal Gland, to produce melatonin. Melatonin secretion increases in the evening, and is stimulated by darkness; whereas light rapidly suppresses melatonin production.
Melatonin is not unique to humans. It is spread through out the animal kingdom, plants, bacteria, and even unicellular organisms have it. There’s no species that has been identified so far that does not contain Melatonin. This expresses the importance of this molecule to life.