The exchange of small molecules, or metabolites, between organisms and their environment gives rise to the complexity of life on Earth1. One of the examples of such complexity is ourselves, our body and the full array of microorganisms (microbiota) that live on and in us, humans.
Just like us, stony corals (Scleractinia) also live in a symbiosis with associated microorganisms. These include bacteria, archea, fungi, viruses, and single-cellular algae called Symbiodinium.
Under normal conditions, these algal cells provide 90 to 95% of the energy required for stony corals to live happily1. The algae provide lipids, carbohydrates, amino acids, and O2 to Scleractinia. In return, the algae get to recycle nitrogen and other inorganic compounds from the coral, that they can use to fuel their own cell metabolism.
Climate change causes a rise in water temperatures; and, this form of environmental heat stress, disrupts the symbiotic relationship between corals and its algae, which results in coral bleaching.
Researchers have now discovered that heat stress destabilizes this basic nutrient exchange between corals and their algae; and, this turn of events happens long before the bleaching process becomes obvious.
In fact, Rädecker and his colleagues2 at the Red Sea Research Centre, discovered that heat stress causes the coral to need more energy, which swings the coral-algae symbiosis from a nitrogen- to a carbon-limited state. This actually ends up reducing the movement and recycling of carbon, and creates an animosity between these life partners.
The coral starts disliking the algae, because the algae it’s not fully doing its work – recycling carbon. As such, the stony coral starts thinking that sheltering such lazy algae is not beneficial anymore. On top of that, the poor coral needs to catabolically degrade more aminoacids, to compensate for the increased energy demands triggered by the warmer waters. Such demanding “cooking tasks” from the coral side, create fury, and actually increase the release of ammonium; which in turn, promote the algae to grow. So, not only does the algae not clean the building properly, but calls its algal friends and they move in.
Under stress, the coral kicks the algae out. But since this marriage is essential to its life support, the Scleractinia cannot feed itself properly anymore. The stony coral goes hungry, and slowly dies out….
1 Williams, A. et al. Metabolomic shifts associated with heat stress in coral holobionts. Science Advances7, eabd4210, doi:doi:10.1126/sciadv.abd4210 (2021).
2 Rädecker, N. et al. Heat stress destabilizes symbiotic nutrient cycling in corals. Proceedings of the National Academy of Sciences118, e2022653118, doi:10.1073/pnas.2022653118 (2021).
When we mention the word Serotonin (5-hydroxytryptamine, 5-HT), we immediately think of the brain and the Central Nervous System (CNS). People tend to associate serotonin to depression, or mood, or feelings of well-being1.
Although that is correct, truth be told, the majority of the serotonin in the human body is actually produced in the gut. In fact, 95% of total serotonin is manufactured by the Enterochromaffin cells (or, Kulchitsky cells) in the gastro-intestinal tract (GI)2,3. These cells live next to the gut epithelium, that covers the cavity of the GI tract, playing a crucial role in the regulation of bowel movements and secretions. If you think that the gut is almost 9 meters (or 30 feet) long, then that’s a lot of cells producing serotonin.
When in the 50’s, Betty M. Twarog and Irvine H. Page discovered that the brain produced its own serotonin4; then, the gut-made serotonin got reduced to its “Aschenputtel” origins, and relinquished to the favela quarters of the body. As such, brain-derived serotonin always got more attention than its gut-derived counterpart – like a rich vs. poor-cousin type of reputation.
Platelets, also called thrombocytes, are small un-nucleated fragment of cells that, when activated, form blood clots (thrombus) and prevent bleeding.
Platelets do not make serotonin, butcan take it up as they circulate through the gut, and carry it along the blood stream6,7. As such, the serotonin produced in the intestine can be carried all over the body. As the chemical messenger serotonin is, it can influence any other cell, in whatever other location, as long as it has a serotonin receptor on it. As such, peripheral serotonin has now discovered its path back into the limelight, and recent research has strengthened the influence that gut-made serotonin has in other parts of the body, functioning as an intestinal-derived hormone.
Once again, the “Aschenputtel” storycomes into mind, but this time through its “Cinderella” version. Let’s take a look…
For example, gut-derived serotonin can directly regulate the liver and mediate liver regeneration8. In Non-Alcoholic Fatty Liver Disease (NAFLD), a group of conditions that are characterized by excessive fat accumulation in the liver and closely track the global public health problem of obesity, researchers showed that inhibiting gut-derived serotonin synthesis could resolve hepatic fat accumulation8,9.
Peripheral serotonin can also be a negative regulator of bone density, by specifically inhibiting osteoblast formation and leading to osteoporosis10 – a common feature in patients with inflammatory bowel disease (IBD). This happens through the action of a common receptor: the low-density Lipoprotein Receptor-related Protein 5(LRP5), which is expressed in both osteoblasts and enterochromaffin cells11. LRP5 inhibits the expression of an important ingredient for serotonin production (Tryptophan hydroxylase-1, Tph1); as such, when LRP5 is deficient or inactivated due to inflammation or disease, blood levels of serotonin are elevated decreasing osteoblast formation; and, consequently, reducing bone mass1,11.
Epidemiologic data suggests a role of serotonin, or Selective Serotonin-Reuptake Inhibitors (typically used as antidepressants, SSRIs) in the development of venous thrombosis12. In fact, patients with depression were reported to have higher incidences of venous thromboembolism in general13; and, the use of SSRIs is associated with an increased venous thromboembolism risk14. No wonder, serotonin and platelets are “brothers in arms”, ready to block any blood vessel along their way….
Serotonin and its receptors are also present in the immune system, where evidence suggests it contributes to both innate and adaptive responses. There is now clear evidence of a straight communication between the immune system, the gut and the brain via serotonin15,16.
On top of all and because we are not alone, our gut microbiota plays a critical role in regulating our colonic serotonin. Indigenous spore-forming bacteria (Sp) promote serotonin biosynthesis in our enterochromaffin cells, and with that they can significantly modulate GI movements and platelet function – together with many aspects of our physiology17,18. We now know that the microbiota colonizes the GI tract after birth, with a continuous maturation during the first years of life19. Researchers have now showed in animal models that this developing gut microbiota regulates the development of the adult enteric nervous system via intestinal serotonin networks20. What this actually means, is that the actions of our intestinal bugs during the beginning of our life are determinant for the development of our “gut brain”, our second brain. How about that?…
If we ruminate about it, when we “think” with our gut, we are actually listening to our bugs. By directly signalling our cells to produce serotonin and develop a network of neurons as soon as we are born, our gut-bugs are actually finding a way to communicate with us – the host – in the serotonin language.
Now, we just need to understand what are they telling us…
1 Gershon, M. D. 5-Hydroxytryptamine (serotonin) in the gastrointestinal tract. Curr Opin Endocrinol Diabetes Obes20, 14-21, doi:10.1097/MED.0b013e32835bc703 (2013).
2 Bellono, N. W. et al. Enterochromaffin Cells Are Gut Chemosensors that Couple to Sensory Neural Pathways. Cell170, 185-198.e116, doi:10.1016/j.cell.2017.05.034 (2017).
3 Yaghoubfar, R. et al. Modulation of serotonin signaling/metabolism by Akkermansia muciniphila and its extracellular vesicles through the gut-brain axis in mice. Scientific Reports10, 22119, doi:10.1038/s41598-020-79171-8 (2020).
4 Twarog, B. M. & Page, I. H. Serotonin Content of Some Mammalian Tissues and Urine and a Method for Its Determination. American Journal of Physiology-Legacy Content175, 157-161, doi:10.1152/ajplegacy.1922.214.171.124 (1953).
5 Zilla, P. et al. Scanning electron microscopy of circulating platelets reveals new aspects of platelet alteration during cardiopulmonary bypass operations. Tex Heart Inst J14, 13-21 (1987).
6 Morrissey, J. J., Walker, M. N. & Lovenberg, W. The absence of tryptophan hydroxylase activity in blood platelets. Proc Soc Exp Biol Med154, 496-499, doi:10.3181/00379727-154-39702 (1977).
7 Hughes, F. B. & Brodie, B. B. The mechanism of serotonin and catecholamine uptake by platelets. J Pharmacol Exp Ther127, 96-102 (1959).
8 Wang, L. et al. Gut-Derived Serotonin Contributes to the Progression of Non-Alcoholic Steatohepatitis via the Liver HTR2A/PPARγ2 Pathway. Frontiers in Pharmacology11, doi:10.3389/fphar.2020.00553 (2020).
9 Choi, W. et al. Serotonin signals through a gut-liver axis to regulate hepatic steatosis. Nature Communications9, 4824, doi:10.1038/s41467-018-07287-7 (2018).
10 Lavoie, B. et al. Gut-derived serotonin contributes to bone deficits in colitis. Pharmacol Res140, 75-84, doi:10.1016/j.phrs.2018.07.018 (2019).
11 Yadav, V. K. et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell135, 825-837, doi:10.1016/j.cell.2008.09.059 (2008).
12 Rieder, M., Gauchel, N., Bode, C. & Duerschmied, D. Serotonin: a platelet hormone modulating cardiovascular disease. J Thromb Thrombolysis52, 42-47, doi:10.1007/s11239-020-02331-0 (2021).
13 Takeshima, M. et al. Prevalence of Asymptomatic Venous Thromboembolism in Depressive Inpatients. Neuropsychiatr Dis Treat16, 579-587, doi:10.2147/NDT.S243308 (2020).
14 Parkin, L. et al. Antidepressants, Depression, and Venous Thromboembolism Risk: Large Prospective Study of UK Women. J Am Heart Assoc6, doi:10.1161/jaha.116.005316 (2017).
15 Baganz, N. L. & Blakely, R. D. A dialogue between the immune system and brain, spoken in the language of serotonin. ACS Chem Neurosci4, 48-63, doi:10.1021/cn300186b (2013).
16 Jacobson, A., Yang, D., Vella, M. & Chiu, I. M. The intestinal neuro-immune axis: crosstalk between neurons, immune cells, and microbes. Mucosal Immunology14, 555-565, doi:10.1038/s41385-020-00368-1 (2021).
17 Yano, J. M. et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell161, 264-276, doi:10.1016/j.cell.2015.02.047 (2015).
18 Reigstad, C. S. et al. Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells. Faseb j29, 1395-1403, doi:10.1096/fj.14-259598 (2015).
19 Bäckhed, F. et al. Dynamics and Stabilization of the Human Gut Microbiome during the First Year of Life. Cell Host Microbe17, 690-703, doi:10.1016/j.chom.2015.04.004 (2015).
20 De Vadder, F. et al. Gut microbiota regulates maturation of the adult enteric nervous system via enteric serotonin networks. Proc Natl Acad Sci U S A115, 6458-6463, doi:10.1073/pnas.1720017115 (2018).
Precision medicine is an emerging approach to medical care. It takes into account individual variations in genetic make-up, metabolism and other biological and environmental factors, to better determine which treatment and prevention strategies for a particular disease may work better for which groups of people1.
Today, precision medicine is routinely integrated into the care of cancer patients, and has provided substantial increases in cancer free survival2. For example, translational studies investigating signatures within the tumour that promote disease progression, can predict individual responses to standard and targeted chemotherapy regimens2,3.
In the cardiovascular field, even though the prognosis for people with heart failure has improved in recent decades as research studies demonstrate the effectiveness of various medications, precision medicine is still in its infancy.
Some aspects of precision medicine are routinely used by healthcare providers, like the blood level of the biomarker called B-type natriuretic peptide, which is a sensitive indicator of whether heart failure is worsening or if treatments are helping1,4. This biomarker can also help the doctor determine whether shortness of breath symptoms in an individual are due to heart failure, or another medical problem1.
But as it is now, patients diagnosed with heart failure are still offered essentially identical treatments, regardless of whether their disease was caused by coronary artery disease, genetic mutations, or an autoinflammatory processes2.
Also, historically, clinical trial participants have been predominantly white people with particular genetic variants; but, individuals with different racial and ethnic ancestry have different genetic variants, and therefore, may not have the same response to a certain medication or treatment1.
While this “one-size-fits-all’ approach has led to improvements in clinical outcomes in large populations, the individual response rates continue to vary tremendously; and, it is often difficult to distinguish patients who will achieve a favourable response, from those who will experience disease progression, and ultimately succumb to their illness2.
As such, in the cardiovascular field, it is urgent to personalize heart failure care by identifying groups of patients more likely to develop heart failure, and tailoring which medications and other therapies could be most effective for them1. Currently, many individuals are left poorly treated, and there is substantial room for improvement2.
Key studies demonstrating selective efficacy of certain drugs in patients harbouring specific genetic variants, indicate a direction where treatment responses can be predicted using individual genetic information.
Given the recent cost reductions in exome sequencing, for example, this can now be used routinely to identify genetic variants that predict heart failure prognosis, and specific responses to medical and device-based therapies. Such information can further provide critical insights into new disease mechanisms, like Lamin A mutations that display a molecular phenotype that is dramatically distinct from other forms of dilated cardiomyopathy5.
More and more we come to realize that despite a common surface phenotype or symptomology, certain mutations may actually give rise to distinct diseases that need to be appropriately treated.
As such, researchers need to increase clinical trial diversity, so that optimal treatment approaches can be found for each population group. Also, the power of supercomputing should be used to rapidly predict the outcomes of possible new treatments. And, processes for sharing information across large databases shouldbe put in place with guarantees of patient privacy (e.g., cloud-based platforms), so that clinicians/scientists can quickly collaborate and share data internationally.
Moreover, updated health-wearable devices, artificial intelligence and other deep learning technologies strategies will ultimately be employed to develop testable hypotheses from large datasets, and provide precision-personalized approaches to cardiovascular health care.
Let’s hope Precision will be more than a one night stand on Heart’s Tinder list….
5 Cheedipudi, S. M. et al. Genomic Reorganization of Lamin-Associated Domains in Cardiac Myocytes Is Associated With Differential Gene Expression and DNA Methylation in Human Dilated Cardiomyopathy. Circ Res. https://pubmed.ncbi.nlm.nih.gov/30739589/124, 1198-1213, doi:10.1161/circresaha.118.314177 (2019).
A healthy lifestyle is the cornerstone of cardiovascular health.
Lifestyle interventions are already a key component of primary prevention in low-risk cardiovascular disease groups, and serve as an important aide to pharmacotherapy in higher-risk groups.
But according to the new guidelines by the American Heart Association (AHA) and the American College of Cardiology (ACC)1, a first line of therapy for mild to moderate–risk groups are lifestyle-only approaches for a proper blood pressure and blood cholesterol management.
As such, the next time you go to the doctor, you might get an exercise prescription instead of an order to visit the pharmacy.
This is a major change in the idea of health, promoted by not taking a pill, but having a look at lifestyle in order to improve health – and avoid the numerous side-effects that certain medications can have.
An exercise prescription is an individualized physical activity program designed using the Frequency (how often?), Intensity (how hard?), Time (how long?), and Type (what kind?), or the FITT principle developed by the American College of Sports Medicine (ACSM).
Although most health care professionals and patients are aware that physical activity is recommended for good health, the abundance of scientific and lay recommendations for activity can be difficult to distil. As such, framing the exercise prescription by the FITT principle provides clinicians with more structured guidance on how to recommend exercise to their patients.
The updated FITT exercise recommendations for adults with elevated blood pressure are the following:
Frequency: in most, preferably all days of the week due to the transient Blood Pressure lowering effects that last for up to 24 hours after an exercise session;
Intensity: Moderate, any intensity of exercise has been shown to lower Blood Pressure;
Time: >20 to 30 minutes per day to total >90 to >150 minutes per week of continuous or accumulated exercise of any duration;
Type: Emphasize aerobic or resistance exercise alone or combined, due to the recent evidence showing the Blood Pressure lowering effects of exercise do not vary by exercise modality2.
The updated FITT exercise prescription recommendations propose more exercise options in less time, that hopefully will translate to better exercise adherence.
Furthermore, long-term exercise produces changes in the availability of receptors that can control the release of monoamines, like the Serotonin-1A receptor of the Raphe Nuclei4, and Dopamine-2 receptor in the Striatum5.
Regular exercise has antidepressant/anxiolytic properties, and results in dramatic alterations in physiological stress responses.
In addition to antidepressant and anxiolytic properties, the Serotonin system (5-HT) has also been linked to cognitive function; since, a distress of the 5-HT system is associated with cognitive syndromes, such as Alzheimer’s disease6.
So, don’t shy away, and take at least a 20 min quick walk today.
It’s free, and it’s good for you!
1 Gibbs, B. B. et al. Physical Activity as a Critical Component of First-Line Treatment for Elevated Blood Pressure or Cholesterol: Who, What, and How?: A Scientific Statement From the American Heart Association. Hypertension0, HYP.0000000000000196, doi:doi:10.1161/HYP.0000000000000196.
2 Pescatello, L. S. et al. Physical Activity to Prevent and Treat Hypertension: A Systematic Review. Med Sci Sports Exerc51, 1314-1323, doi:10.1249/mss.0000000000001943 (2019).
3 Buhr, T. J. et al. The Influence of Moderate Physical Activity on Brain Monoaminergic Responses to Binge-Patterned Alcohol Ingestion in Female Mice. Front Behav Neurosci15, 639790-639790, doi:10.3389/fnbeh.2021.639790 (2021).
4 Greenwood, B. N. et al. Freewheel running prevents learned helplessness/behavioral depression: role of dorsal raphe serotonergic neurons. J Neurosci23, 2889-2898, doi:10.1523/jneurosci.23-07-02889.2003 (2003).
5 Clark, P. J. et al. Wheel running alters patterns of uncontrollable stress-induced cfos mRNA expression in rat dorsal striatum direct and indirect pathways: A possible role for plasticity in adenosine receptors. Behav Brain Res272, 252-263, doi:10.1016/j.bbr.2014.07.006 (2014).
6 Meltzer, C. C. et al. Serotonin in aging, late-life depression, and Alzheimer’s disease: the emerging role of functional imaging. Neuropsychopharmacology18, 407-430, doi:10.1016/s0893-133x(97)00194-2 (1998).
Dry skin and atopic dermatitis have been associated with changes in the variety of the skin microbiome.
Our skin, as the largest organ in our body, has a huge array of commensal microbes that support a healthy skin barrier. One of those is Staphylococcus epidermidis, one of the most abundant bacterial species of the skin microbiome1.
This chubby mutualistic, Gram-positive, facultative anaerobe constitutes up to 90% of the aerobic resident flora of our skin, and has been associated with a healthy-looking skin2. It does not like to be lonely, and usually appears in pairs or tetrads on the surface of our skin, like a protecting biofilm.
Dry skin, for example, is associated with an increase in microbial diversity along with a decrease in microbial load in comparison to more sebaceous areas of the skin, that are usually populated by lipophilic bacteria such as Cutibacterium acnes – that tend to cause those unwanted teenager-look-a-like pimples that nobody likes…
Lactic Acid is one of the Natural Moisturizing Factors (NMF) of the skin barrier, that is essential to maintain the hydration and a slightly acidic pH of the skin surface (i.e., “acid mantle”)3. Higher lactic acid concentrations and lower skin surface pH are known to increase our epidermal renewal and promote a healthier skin.
New in vitro data suggests that Staphylococcus epidermidis, may be one of the major sources of lactic acid in the skin1.
But only if fed the right way.
It seems that 1% colloidal oat increases Lactic Acid production by this particular bacteria species, making it rely less on simple sugars such as glucose for its metabolism; and, instead use more complex carbohydrates derived from oat.
Oatmeal-containing skin moisturisers significantly changed the metabolism of the Staphylococcus epidermidis, breaking down starch and promoting good gene expression, with an increased DNA and aminoacid synthesis, and an improved ATP metabolism.
How about that?
Bacteria on a diet makes your skin look healthier!
Next time you think about which moisturiser to buy in the drug store: don’t forget to feed your skin microbiome it’s oatmeal!
1 Liu-Walsh, F. et al. Prebiotic Colloidal Oat Supports the Growth of Cutaneous Commensal Bacteria Including S. epidermidis and Enhances the Production of Lactic Acid. Clin Cosmet Investig Dermatol14, 73-82, doi:10.2147/CCID.S253386 (2021).
2 Baviera, G. et al. Microbiota in healthy skin and in atopic eczema. Biomed Res Int2014, 436921, doi:10.1155/2014/436921 (2014).
3 Thueson, D. O., Chan, E. K., Oechsli, L. M. & Hahn, G. S. The roles of pH and concentration in lactic acid-induced stimulation of epidermal turnover. Dermatol Surg24, 641-645, doi:10.1111/j.1524-4725.1998.tb04221.x (1998).
Disrupted sleep is a major feature of Alzheimer’s disease (AD), and it usually appears years before symptoms of cognitive decline emerge.
It seems prolonged wakefulness aggravates the production of amyloid-beta (Aβ) species, which is a major driver of AD progression.
This sleep loss tends to further accelerate AD, with this tendency becoming a vicious cycle of sleepiness and AD advancement.
Unfortunately, the mechanisms by which Aβ affects sleep are still unknown and reason for much research.
Recently, Özcan and team of researchers have shown that in zebrafish, Aβ acutely and reversibly can enhance or suppress sleep in the fish as a function of the length of the oligomer, that is the number of Aβ molecules bounded together.
Genetic disruption analysis has shown that short Aβ oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1). While longer Aβ forms, can actually induce sleep through a pharmacologically tractable Prion Protein (PrP) signalling cascade.
What this data shows, is that Aβ can actually trigger a bi-directional sleep/wake switch in the zebrafish.
And, what this could mean, is that alterations to the brain’s Aβ oligomeric environment, such as during the progression of AD, may lead to disrupt sleep through changes in acute signalling events through similar receptors.
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.