Refining Antioxidant (ROS & RNS) Therapy in Autism - Selenium and Molybdenum

Today’s post is about further refining antioxidant therapy.

As we saw in a recent post, oxidative and nitrosative stress is a very common feature of autism and is treatable with OTC products.

The cheapest antioxidant, N-acetylcysteine (NAC), looks to be the best one, but there are numerous others with exotic names and equally exotic prices.

Today we just look at selenium and molybdenum.  Selenium was on my to-do list for a long time because it affects some key enzymes call GP_X (glutathione peroxodases).

Molybdenum was enthusiastically recommended in a recent comment and this blog has previously touched on Molybdenum Cofactor Sulfurase (MOCOS).

Rather surprisingly, there is a commercial product that contains NAC, Selenium and Molybdenum. 

Selenium and GP_X (glutathione peroxodases)

There are eight different glutathione peroxodases, but GPx1, GPx2, GPx3, and GPx4 are all made from selenium.

GP_X speeds up the antioxidant reactions that involve glutathione (GSH).

In autism we know that both GSH and GP_X are lacking.

We know how to make more GSH, just take some NAC.  But what about the catalyst GP_X? 

There may be an equally easy way to increase that. 

Selenium and Thyroid  Enzymes

Selenium is also part of the three deiodinase enzymes D1, D2 and D3.

The active thyroid hormone is called T3, but most of what is circulating in your body is the inactive pro-hormone form called T4.

Deiodinase 1 (D1)  both activates T₄ to produce T₃ and inactivates T₄. Besides its increased function in producing extrathyroid T₃, its function is less well understood than D2 or D3.

Deiodinase 2 (D2), located in the ER membrane, converts T₄ into T₃ and is a major source of the cytoplasmic T₃ pool.  It looks like some people with autism may lack D2 in their brain.

Deiodinase 3 (D3) prevents T₄ activation and inactivates T₃. It looks like some people with autism have too much D3 in their brain.

D2 and D3 are important in homeostatic regulation in maintaining T₃ levels at the plasma and cellular levels.

·        In hyperthyroidism D2 is down regulated and D3 is upregulated to clear extra T₃

·        in hypothyroidism D2 is upregulated and D3 is downregulated to increase cytoplasmic T₃ levels

Serum T3 levels remain fairly constant in healthy individuals, but D2 and D3 can regulate tissue specific intracellular levels of T3 to maintain homeostasis since T3 and T4 levels may vary by organ.  

It appears that some people with autism may have central hyperthyroidism, meaning in their brain.

Regular readers may recall this post:-

Central Hypothyroidism or Low Brain D2 Levels in Autism

The major source of the biologically active hormone T3 in the brain is the local intra-brain conversion of T4 to T3, while a small fraction comes from circulating T3. 

As evidence derived from in vitro studies suggests, in response to oxidative stress D3 increases while D2 decreases (Lamirand et al., 2008; Freitas et al., 2010).  As we know in the autistic brain we have a lot of oxidative stress.

Furthermore, in ASD, the lower intra-brain T3 levels occur in the

Absence of a systemic T3 deficiency (Davis et al., 2008), most likely due to the increased activity of D3.

So in some autistic brains we have too much D3 which is inactivating T3 and preventing T4 being converted to T3.

Reduced D2 is reducing the conversion of T4 to T3. 

We would therefore want to increase D2 in some autism.

This can be achieved by:-

·        Reducing oxidative stress, which we are already sold on. 

·        We can also potentially upregulate the gene that produces D2 using some food-based genetic therapy. Kaempferol (KPF) appears to work and may explain why broccoli sprout powder makes some people go hyper and some others cannot sleep  

The Small Polyphenolic Molecule Kaempferol Increases Cellular Energy Expenditure and Thyroid Hormone Activation

The cAMP-responsive gene for type 2 iodothyronine deiodinase (D2), an intracellular enzyme that activates thyroid hormone (T3) for the nucleus, is approximately threefold upregulated by KPF

·        Perhaps low levels of selenium differentially affect the synthesis of D1, D2 and D3?

  

Where does selenium come from? 

We know from Chauham/James that selenium levels are reduced in autism, but we also know that selenium levels vary widely by geography.  

You get selenium from your diet and the level of selenium in the soil varies widely.  It is widely held that most healthy people should have plenty selenium in their diet. 

In the following paper there is an analysis of Selenium status in Europe and the Middle East.

Since we have plenty of Polish readers I have included the chart with the Polish data (on the left).  It shows that Polish people may be a little deficient in selenium.

You can see the level of selenium in Poland is below that needed to optimise plasma GPx activity.

So if you already have reduced GPx activity, because of autism, and you really need to make the most of your limited glutathione (GSH) because you have oxidative/nitrosative stress, then a little extra selenium could be just what the doctor should have ordered.

  

A Review of Dietary Selenium Intake and Selenium Status in Europe and the Middle East

Se is an essential non-metal trace element [3] that is required for selenocysteine synthesis and is essential for the production of selenoproteins [4]. Selenoproteins are primarily either structural or enzymatic [2], acting as catalysts for the activation of thyroid hormone and as antioxidants, such as glutathione peroxidases (GPxs) [5]. GPx activity is commonly used as a marker for Se sufficiency in the body [6], where serum or plasma Se concentrations are believed to achieve maximum GPx expression at 90–100 μg/L (90.01 μg/L as proposed by Duffield and colleagues [7] and 98.7 μg/L according to Alfthan et al. [8]). However, plasma selenoprotein P (SEPP1) concentration is a more suitable marker than plasma GPx activity [9]. Prospective studies provide some evidence that adequate Se status may reduce the risk of some cancers, while elevated risk of type 2 diabetes and some cancers occurs when the Se concentration exceeds 120 μg/L [10]. Higher Se status has been linked to enhanced immune competence with better outcomes for cancer, viral infections, including HIV progression to AIDS, male infertility, pregnancy, cardiovascular disease, mood disorders [2] and, possibly, bone health [11–14].

  

Selenium and NAC for Rats with TBI

Perhaps not surprisingly, selenium and NAC have been found beneficial for Rats unfortunate enough to have sufferred a traumatic brain injury (TBI).

 N-Acetylcysteine and Selenium Modulate Oxidative Stress, Antioxidant Vitamin and Cytokine Values in Traumatic Brain Injury-Induced Rats

It has been suggested that oxidative stress plays an important role in the pathophysiology of traumatic brain injury (TBI). N-acetylcysteine (NAC) and selenium (Se) display neuroprotective activities mediated at least in part by their antioxidant and anti-inflammatory properties although there is no report on oxidative stress, antioxidant vitamin, interleukin-1 beta (IL)-1β and IL-4 levels in brain and blood of TBI-induced rats. We investigated effects of NAC and Se administration on physical injury-induced brain toxicity in rats. Thirty-six male Sprague–Dawley rats were equally divided into four groups. First and second groups were used as control and TBI groups, respectively. NAC and Se were administrated to rats constituting third and forth groups at 1, 24, 48 and 72 h after TBI induction, respectively. At the end of 72 h, plasma, erythrocytes and brain cortex samples were taken. TBI resulted in significant increase in brain cortex, erythrocytes and plasma lipid peroxidation, total oxidant status (TOS) in brain cortex, and plasma IL-1β values although brain cortex vitamin A, β-carotene, vitamin C, vitamin E, reduced glutathione (GSH) and total antioxidant status (TAS) values, and plasma vitamin E concentrations, plasma IL-4 level and brain cortex and erythrocyte glutathione peroxidase (GSH-Px) activities decreased by TBI. The lipid peroxidation and IL-1β values were decreased by NAC and Se treatments. Plasma IL-4, brain cortex GSH, TAS, vitamin C and vitamin E values were increased by NAC and Se treatments although the brain cortex vitamin A and erythrocyte GSH-Px values were increased through NAC only. In conclusion, NAC and Se caused protective effects on the TBI-induced oxidative brain injury and interleukin production by inhibiting free radical production, regulation of cytokine-dependent processes and supporting antioxidant redox system.

  

Dietary selenium stabilizes glutathione peroxidasemRNA in rat liver

  

And now to Molybdenum 

Molybdenum (Mo) is a trace dietary element necessary for human survival.

Low soil concentration of molybdenum in a geographical band from northern China to Iran results in a general dietary molybdenum deficiency, and is associated with increased rates of esophageal cancer.  Compared to the United States, which has a greater supply of molybdenum in the soil, people living in those areas have about 16 times greater risk for esophageal cancer.
So you would not want to have molybdenum deficiency.

Four Molybdenum-dependent enzymes are known, all of them include molybdenum cofactor (Moco) in their active site: sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and mitochondrial amidoxime reductase.

Moco cannot be taken up as a nutrient, and thus it requires to made in your body from molybdenum.

If your body cannot make enough Moco you may develop what is called molybdenum cofactor deficiency, which would ultimately kill you. It is ultra rare.

Symptoms include early seizures, low blood levels of uric acid, and high levels of sulphite, xanthine, and uric acid in urine.


When caused by a mutation in the MOCS1 gene it is called the type A variant.

Molybdenum cofactor deficiency may indeed be extremely rare, but MOCS1 is a known autism gene.  Perhaps there exists partial molybdenum cofactor deficiency, which is not rare at all?

Source:-  Identification of candidate intergenic risk loci in autism spectrum disorder

MOCOS (Molybdenum cofactor sulfurase)


Molybdenum cofactor sulfurase is an enzyme that in humans is encoded by the MOCOS gene.

MOCOS sulfurates the molybdenum cofactor of xanthine dehydrogenase (XDH) and aldehyde oxidase (AOX1), which is required for their enzymatic activities.

MOCOS is downregulated in autism and is suggested to induce increased oxidative-stress sensitivity, which would not be good.

So it looks like we need a clever way to upregulate MOCOS.

You need adequate molybdenum cofactor (Moco), for which you do need adequate molybdenum.

You need the genes MOCS1 and MOCOS to be correctly expressed.

SIRT1 activation, which is a future therapy for Alzheimer’s, is suggested to increase MOCOS, as may NRF2.

Sirtuin-activating compounds (STAC) are chemical compounds having an effect on sirtuins, a group of enzymes that use NAD+ to remove acetyl groups from proteins. They are molecules able to prevent aging related diseases like Alzheimer's, diabetes, and obesity.  There is quite a long list that includes ranges from polyphenols such as resveratrol, the flavonols fisetin, and quercetin also butein, piceatannol, isoliquiritigenin,

Fisetin is found in strawberries, cucumbers and supplements.  In normal animals, fisetin can improve memory; it also can have an effect on animals prone to Alzheimer's.

Modulation of p25 and inflammatory pathways by fisetin maintains cognitive function in Alzheimer's disease transgenic mice

Here is the excellent French paper on MOCOS:-

Olfactory stem cells reveal MOCOS as a new player in autism spectrum disorders

With an onset under the age of 3 years, autism spectrum disorders (ASDs) are now understood as diseases arising from pre- and/or early postnatal brain developmental anomalies and/or early brain insults. To unveil the molecular mechanisms taking place during the misshaping of the developing brain, we chose to study cells that are representative of the very early stages of ontogenesis, namely stem cells. Here we report on MOlybdenum COfactor Sulfurase (MOCOS), an enzyme involved in purine metabolism, as a newly identified player in ASD. We found in adult nasal olfactory stem cells of 11 adults with ASD that MOCOS is downregulated in most of them when compared with 11 age- and gender-matched control adults without any neuropsychiatric disorders. Genetic approaches using in vivo and in vitro engineered models converge to indicate that altered expression of MOCOS results in neurotransmission and synaptic defects. Furthermore, we found that MOCOS misexpression induces increased oxidative-stress sensitivity. Our results demonstrate that altered MOCOS expression is likely to have an impact on neurodevelopment and neurotransmission, and may explain comorbid conditions, including gastrointestinal disorders. We anticipate our discovery to be a fresh starting point for the study on the roles of MOCOS in brain development and its functional implications in ASD clinical symptoms. Moreover, our study suggests the possible development of new diagnostic tests based on MOCOS expression, and paves the way for drug screening targeting MOCOS and/or the purine metabolism to ultimately develop novel treatments in ASD.  

Lately, a diminished seric expression of glutathione, glutathione peroxidase, methionine and cysteine has been highlighted in a meta-analysis from 29 studies on ASD subjects.⁴⁵ Along this line, purines and purine-associated enzymes are recognized markers of oxidative stress. ROS are generated during the production of uric acid, catalyzed by xanthine oxidase and XDH.⁴⁶ Conversely, uric acid is nowadays recognized as a protective factor acting as a ROS scavenger.^{47, 48} Interestingly, allopurinol, a xanthine oxidase inhibitor, was found efficient in reducing symptoms, especially epileptic seizures, in ASD patients displaying high levels of uric acid.⁴⁹ However, in our cohort, only 3 out of 10 patients exhibited an abnormal uric acid secretion. It can therefore be postulated that still unknown other MOCOS-associated mechanisms may have a role in the unbalanced stress response observed in ASD OSCs.

Identifying and manipulating downstream effectors of MOCOS will be the next critical step to better understand its mechanisms of action. In parallel, we plan to ascertain some of its upstream regulators. For example, bioinformatic analyses revealed that the promoter region of MOCOS includes conserved binding sites for transcription factors such as GATA3 and NRF2. In addition, other putative interactors, such as the NAD-dependent deacetylase sirtuin-1 (SIRT1), may have a regulatory role on MOCOS expression. Interestingly, these three genes have been associated with ASD, fragile X syndrome, epilepsy and/or oxidative stress.^{54, 55, 56, 57} In conclusion, our study opens an unexplored new avenue for the study of MOCOS in ASD, and could set bases for the development of new diagnostic tools as well as the search of new therapeutics.

Conclusion

It looks like a little extra selenium may be in order to increase those GPx enzymes that are need to speed up aspects of the antioxidant activity of GSH.

When it comes to molybdenum, things get much more complex. You certainly do not want to be deficient in molybdenum and you do not want Molybdenum cofactor deficiency; you also do not want molybdenum cofactor Sulfurase (MOCOS) mis-expression.

It is fair to say that quite likely there is a problem related to molybdenum that affects oxidative stress in autism; but it is not yet clear what to do about it.  I rather doubt the solution is as simple as just a little extra molybdenum, but it is easy to try.

On the plus side, we see that if you have autism, epilepsy and high uric acid you are likely to benefit from allopurinol, which also seems to help in COPD.

There is nothing new about allopurinol possibly be effective in some autism, as from this 25 year old book, Diagnosis and Treatment of Autism.

Again we see that activating NRF2 looks a good idea, that applies to both autism and COPD.

One thing to note is that NRF2 activators are good for cancer prevention, but if you have a cancer you want NRF2 inhibitors.

NRF2 activators include sulforaphane (SFN), R-alphalipoic acid (ALA), resveratrol and curcumin.  SFN is by far the most potent.  Resveratrol and curcumin have a problem with bioavailability.

Why not Cocoa Flavanols for Autism?

  

Judging by my blog statistics, lots of people are interested in broccoli (Sulforaphane) to treat autism.  Thanks to the patents held by Johns Hopkins, you can expect to hear much more about Sulforaphane in the coming years.

Meanwhile, Columbia University and Mars, the chocolate people, have released a study showing that “flavanoids” in cocoa can do wonders for memory loss in older people.  In effect, they can restore memory in 60 years olds to where it was 20 or 30 years earlier.

If you take a step back and look at what is known by science about oxidative stress and antioxidants, all will become much clearer.

Oxidative Stress Pioneers

In an earlier post we met Paul Talalay, a German-American, who worked at Johns Hopkins.  He specializes in foods that protect you from cancer.  He is Mr Broccoli. 

It turns out that perhaps the real pioneer in this field is a 100% German, called Helmut Sies, who also studies foods that act as antioxidants and nutrients that provide protection from cancer.  We have his very detailed diagram below, that explains the relationship between many of the factors involved in oxidative stress.  I wish I had found it earlier.  I added the six outer boxes.

If you want to read clever studies about this subject, just include Helmut Sies in your search; for example “selenium Helmut Sies”.

Redox Pioneer: Professor Helmut Sies

On this graphic you will see GSH (Glutathione).  When you take NAC (N-acetylcysteine) you directly raise the level of GSH.  When eat broccoli you activate Nrf2, which is a Redox switch, just under the traffic light in the graphic.

When you eat certain flavonoids, like Cocoa, or carotenoids like lycopene (found in tomatoes), you again promote the anti-oxidative free radical scavenger effect.  Look in the blue boxes under diet.

Not on the diagram, we also have flavonolignans which are natural phenols composed of a part flavonoid and a part lignan. As pointed out in a comment in the last post by Seth Bittker, one interesting  flavonolignan is Silibinin, which has anti-oxidant and chemoprotective effects

Note the presence of (Coenzyme) Q10 in the yellow box.  This is part of the mitochondrial cocktail suggested by Dr Kelley from Johns Hopkins for regressive autism.  Q10 is depleted by statins.

Glutathione peroxidases, in the yellow box, are also very interesting.  These are selenium-containing enzymes.  GPx (x goes from 1 to 8)  catalyze the reduction of H₂O₂ and organic hydroperoxides to harmless products. This function helps to maintain membrane integrity and to reduce further oxidative damage to molecules such as lipids and lipoproteins with the associated increased risk of conditions such as atherosclerosis.  It appears GP₁ may be defective in autism and this is contributes to increased oxidative stress.  This area has been well studied due to its impact on heart disease.  You appear to be able to counter the lack of GPx with yeast-bound selenium, other forms of selenium do not work, due to a lack of bioavailability. A post will appear just on Selenium.

There are several other potent (exogenous) antioxidants that we have come across:-

• Alpha lipoic acid also known as ALA or Tioctic acid (found  in Dr Kelley’s cocktail)
•   L-Carnosine (studied by Dr Chez )
•  Vitamin C (suggested by many, including Dr Kelley)

Another day, another anti-oxidant

In human health, two well used anti-oxidant drugs are Alpha lipoic Acid (ALA,  also known as Tioctic acid) and N-acetyl cysteine (NAC).  They share many similar effects.

•       Potent antioxidant
•       Increase insulin sensitivity
•       Improve memory in those with mild cognitive          impairment
•       May lower blood pressure
•       Improve behavior in autism

NAC is widely used to treat Chronic obstructive pulmonary disease (COPD) and ALA is used to treat diabetic neuropathy. Perhaps they could be interchanged

·        NAC has a chemoprotective effect
·        ALA has been shown to induce cell cycle arrest in  human breast cancers      cells

Back to Cocoa Flavanols and Mars

This flurry of activity was driven by a well publicized study done at Columbia University Medical Center (CUMC), using a high cocoa flavanol concentration drink provided by Mars.

Dietary cocoa flavanols reverse age-related memory decline

   

In the CUMC study, 37 healthy volunteers, ages 50 to 69, were randomized to receive either a high-flavanol diet (900 mg of flavanols a day) or a low-flavanol diet (10 mg of flavanols a day) for three months. Brain imaging and memory tests were administered to each participant before and after the study. The brain imaging measured blood volume in the dentate gyrus, a measure of metabolism, and the memory test involved a 20-minute pattern-recognition exercise designed to evaluate a type of memory controlled by the dentate gyrus.
The high-flavanol group also performed significantly better on the memory test. “If a participant had the memory of a typical 60-year-old at the beginning of the study, after three months that person on average had the memory of a typical 30- or 40-year-old,” said Dr. Small. He cautioned, however, that the findings need to be replicated in a larger study—which he and his team plan to do.

This is very impressive.  But how do the other anti-oxidants compare?

Well, without funding from Mars, researchers only managed the money to test ALA and NAC on mice; but as you might expect, the result was similar.

The antioxidants alpha-lipoic acid and N-acetylcysteine reverse memory impairment and brain oxidative stress in aged SAMP8 mice

Chronic administration of either LA or NAC improved cognition of 12-month-old SAMP8 mice in both the T-maze footshock avoidance paradigm and the lever press appetitive task without inducing non-specific effects on motor activity, motivation to avoid shock, or body weight. These effects probably occurred directly within the brain, as NAC crossed the blood-brain barrier and accumulated in the brain. Furthermore, treatment of 12-month-old SAMP8 mice with LA reversed all three indexes of oxidative stress. These results support the hypothesis that oxidative stress can lead to cognitive dysfunction and provide evidence for a therapeutic role for antioxidants.

Cocoa Flavanols are good for your heart

This is also good news, but it does seem that antioxidants are generally very good for your heart.

First cocoa.

In this study blood pressure, glucose, insulin and cholesterol were all markedly affected for the better by the cocoa as was cognitive function.

This is great;  but it is what Helmut Sies has been telling the world for many years.

Benefits in Cognitive Function, Blood Pressure, and Insulin Resistance Through Cocoa Flavanol Consumption in Elderly Subjects With Mild Cognitive Impairment

The Cocoa, Cognition, and Aging (CoCoA) Study

Abstract—Flavanol consumption is favorably associated with cognitive function. We tested the hypothesis that dietary flavanols might improve cognitive function in subjects with mild cognitive impairment. We conducted a double-blind, parallel arm study in 90 elderly individuals with mild cognitive impairment randomized to consume once daily for 8 weeks a drink containing _990 mg (high flavanols), _520 mg (intermediate flavanols), or _45 mg (low flavanols) of cocoa flavanols per day. Cognitive function was assessed by Mini Mental State Examination, Trail Making Test A and B, and verbal fluency test. At the end of the follow-up period, Mini Mental State Examination was similar in the 3 treatment groups (P_0.13). The time required to complete Trail Making Test A and Trail Making Test B was significantly (P_0.05) lower in subjects assigned to high flavanols (38.10_10.94 and 104.10_28.73 seconds, respectively) and intermediate flavanols (40.20_11.35 and 115.97_28.35 seconds, respectively) in comparison with those assigned to low flavanols (52.60_17.97 and 139.23_43.02 seconds, respectively). Similarly, verbal fluency test score was significantly (P_0.05) better in subjects assigned to high flavanols in comparison with those assigned to low flavanols (27.50_6.75 versus 22.30_8.09 words per 60 seconds). Insulin resistance, blood pressure, and lipid peroxidation also decreased among subjects in the high-flavanol and intermediate-flavanol groups. Changes of insulin resistance explained _40% of composite z score variability through the study period (partial r2_0.4013; P_0.0001). To the best of our knowledge, this is the first dietary intervention study demonstrating that the regular consumption of cocoa flavanols might be effective in improving cognitive function in elderly subjects with mild cognitive impairment. This effect appears mediated in part by an improvement in insulin sensitivity.

There are more cocoa studies:-

Enhancing dentate gyrus function with dietary flavanols improves cognition in older adults  The effect of acute consumption of a flavonol-rich cocoa drink on cerebral vasomotor reactivity in African Americans

Mars Cocoa Science website

Cocoa Flavanols as a therapy for Autism

Based on the work of Helmut Sies and the trials funded by Mars, it is pretty obvious that 1,000mg of cocoa flavanols a day would very likely have a marked effect on someone with autism, assuming that is they were not already taking NAC, ALA, Carnosine, Broccoli, Sulforaphane or Selenium.  500 mg should also have an effect.

Choice of antioxidant

The question is what is the ultimate treatment for oxidative stress in autism?

I guess this will depend on exactly what type of autism you have (regressive or not), to what extent you have a mitochondrial dysfunction and whether you have any genetic dysfunction related to oxidative stress.

What works best in Billy, may be suboptimal in Charlie, but still much better than nothing at all.

It looks to me that NAC and ALA will likely be the most potent antioxidants.

If you live in the US, you can buy cocoa flavanols in standardized doses from Mars.  One capsule = 125mg of cocoa flavanols.   I have to add that I am far more inclined to believe Mars, than those supplement companies out there.  You can buy tablets saying they contain 50 mcg of Selenium, but what do they really contain? 

You can also buy “high flavanol” raw (non-alkalized) cocoa powder in big bags.  This lighter brown cocoa has lost far less of the flavonoids in the processing process.  In theory, a 5g teaspoon of the very best one will contain (on a good day) 415 mg of flavavols.

Mars are only supplying their CocoaVia products in North America, so if you want to try cocoa flavanols you have a few options:-

·        8.5 teaspoons of standard raw cocoa  (content will vary widely)

                or

·        1.2 teaspoons of “Chococru” upmarket raw cocoa

                or

·        4 capsules of CocoaVia from Mars  

Each of the above should give you 500mg of cocoa flavanols, which would look like a good starting point.  As with NAC, the studies show that the benefit increases the more you take, but the extra benefit drops off.

If somebody in the US tries CocoaVia, do let us know the result.

Not surprising, Mars tell us on the label that the product is not intended for children.  I do not suppose they ever thought of it being an autism therapy either.

I do like the idea of the redox switch, Nrf2, which Sulforaphane is known to activate.  I also like the idea of the enzyme GP₁ that acts as catalyst in the oxidation/reduction process.

The science is around 20 years old and nobody has yet figured it all out;  they probably will not conclusively do so in the next 20 years either.

Food for thought!