WATER Sulfate’s Critical Role for Maintaining Exclusion Zone Water: Dietary Factors Leading to Deficiencies Seneff S1*, Nigh G2 1 Computer Science and Artificial Intelligence Laboratory, MIT Cambridge MA 02139, USA; [email protected] 2 Naturopathic Oncology, Immersion Health, Portland, OR 97214, USA; [email protected] * Correspondence: Stephanie Seneff [email protected] Keywords: Structured water; sulfate; exclusion zone; cobalamin; eNOS; glyphosate; metformin; vegan diet Received: June 21, 2019; Revised: September 30, 2019; Accepted: October 22, 2019; Published: December 18, 2019; Available Online: December 18, 2019 10.14294/WATER.2019.5

Abstract Introduction Biological water exists in at least two dis- Water is essential for life, but the signifi- tinct forms: bulk and interfacial. While the cance of water’s unique properties that former possesses widely understood prop- make it essential is an under-represented erties, little has been written about the for- topic in the research literature. A small mation, maintenance, and functional role group of dedicated researchers have devot- of interfacial water in living systems. In this ed their lives to understanding the unique paper we equate interfacial water with Ex- properties of water. Gerald Pollack PhD, clusion Zone (EZ) water described by Pol- and Professor at the University of Wash- lack and propose that it is the sulfate mol- ington, is perhaps the best-known member ecule that plays a fundamental role in pro- of this group, due in part to his remark- viding the interfacial negative charge that able skill in translating dense science into builds and maintains the EZ in biological terms that are comprehensible to the non- systems. We further propose novel roles for expert. He has succeeded in popularizing endothelial (eNOS), the notion of EZ water, which is a “fourth erythrocytes, and cobalamin in sulfate pro- phase” of water (beyond solid, liquid, and duction and ongoing regulation. Two exog- gas) characterized by a regularized crystal- enous agents, the diabetes drug metformin line hexamer structure, yielding a gel that and the herbicide glyphosate, and one life- excludes solutes and also extrudes protons style factor, vegetarianism/veganism, can (Pollack and Clegg, 2008). As a result, the contribute to reduced sulfate production gel itself is negatively charged, and a volt- and subsequent loss of EZ water. A set of age drop is created at the interface with compensatory changes in the body, often neighboring “unstructured,” or bulk, liquid normally considered to be discrete patholo- water. This voltage drop can be as high as gies, serve to reestablish adequate sulfate -200 mV between EZ water and the adja- supply in the face of these and other det- cent unstructured water. The charge sepa- rimental impacts on sulfate production. ration that is induced forms a battery that Finally, we review additional pathologies can supply energy to the tissues (Hwang associated with cobalamin deficiency and et al. 2018). This gel phase of water is the suggest that they, too, can be linked to the dominant phase present in the human restoration of sulfate metabolism. body.

WATER 11, 22-42, December 18, 2019 22 WATER Highly hydrophilic synthetic materials “liquid ice.” The recognition of this unique such as Nafion and ceramic powder can structuring of water grew as new technolo- be used to simulate biological membranes. gies allowed for closer investigations. In Water exposed to these materials separate 1971, Damadian (1971) first published his into a heterogeneous structures with sharp findings, obtained using NMR, that it was boundaries dividing EZ water from un- evident that “cancerous tissue has a lower structured water. The EZ water has a nega- degree of organization and less water struc- tive electric potential, whereas ordinary ture than normal tissue.” Later imaging low-density water has a positive polarity. techniques such as MRI and CT confirmed In an experimental study of water exposed the same loss of water structure regarding to ceramic powder at a 3% concentration, tumors and other pathological tissue (Un- the zeta potential (ZP) was measured to be ger et al., 1988). Iwama et al. (1992) re- -37.7 mV, and the voltage drop reached a ported a significant difference in spin-lat- maximum at -80 mV (Hwang et al. 2018). tice cross-relaxation times between control This is a model for the arrangement of water water protons vs. protons in water of peri- molecules in the vasculature, where hydro- tumoral edema, noting that loss of water philic substances bound to the endothelial structure may be an early event in edema membrane create EZ water that interfaces formation. with unstructured water in the blood, and this creates a battery that supplies energy Vink and Duling (2000) noted a 0.4-0.5μm to the endothelial cells. A similar process thick gelled layer of water lining the endo- takes place surrounding all the cells in the thelium of all healthy vessels. This layer tissues. The charge separation and battery was noted to regulate solute penetration effect are an important aspect of water in based upon molecular size and charge. The the support of life, and we will be reviewing authors had coincidentally also referred the evidence behind these assertions. to this layer as an “apparent endothelial exclusion zone.” Pizzitutti et al. (2007) discussed the “anomalous behavior” of hy- Evidence for the Unique Structuring dration water surrounding proteins. Ap- parently unfamiliar with the research into of Water Within Living Systems biological EZ water, the authors observed The recognition of water as holding a that “Water around biomolecules slows unique position in the processes of life down with respect to pure water,” and “[t] dates back hundreds of years, at least to the he origin of such behavior remains elusive.” 16th century, when Paracelsus proclaimed It is highly likely that they were witnessing that water is “the matrix of the world and of the altered properties of water forming the all its creatures.” (Jolande, 1988) With the EZ layer around the proteins under inves- advent of modern technologies, however, tigation. that has allowed for a much more detailed Studying collagen, Melacini et al. (2000) understanding of the role water is playing noted the importance of water in stabilizing in the maintenance of the living state and its triple helix crystalline structure. They its relationship to health and disease. We found that water within the collagen helix briefly review, here, some representative studies in a large body of literature. forms a “semi-clathrate-like structure that surrounds and interconnects triple helices Szent-Gyorgyi (1956) noted that the cy- in the crystal lattice.” Water within the ma- toplasm of our cells contains little or no trix of bone is likewise highly structured, a “random water,” but instead contains wa- structuring that has been found associated ter that is highly constrained, much like a not only with the organic macromolecules

WATER 11, 22-42, December 18, 2019 23 WATER such as collagen and proteoglycans, but specific ways in which sulfate, associated with the mineral surfaces as well. Water, with various macromolecules, contributes in fact, seems to play a foundational role in to this water structuring and to general ho- orienting mineral nanoparticles into paral- meostasis of the organism. We show that lel arrangements within the bone matrix, sulfate plays a role in maintaining the EZ providing this orientation even in the ab- water lining blood vessels and in maintain- sence of organic molecules (Duer and Veis, ing the proper viscosity of the circulating 2013). blood. An excellent 2007 review by Le Bihan, Glycosaminoglycans (2007) catalogues the wide-ranging evi- dence to date that biological water has both As described in more detail below, we hy- unique structure(s) and physical proper- pothesize that water is maintained in this gel form through the special assistance of ties. The following year, Ball (2008) ex- sulfate molecules that are anchored to the panded that review to cover the active role extracellular matrix of most cells in the water plays in a wide range of cellular and body. Thus, sulfate takes on a dual role: physiological functions. Most critically, it as a vital structural component of the ma- has become evident that most biological trix, and as the dominant water-structuring water, both intra- and extracellular, is high- component within that matrix. The sulfate ly structured and does not exist in the bulk molecules are complexed with long amino- state. Pathology, in turn, is associated with sugar chains to form various biologically the loss of water structure. Most recently interesting molecules such as heparan sul- Sharma et al. (2018) found that several fate, chondroitin sulfate, keratan sulfate nutrients and medications had the effect of and dermatan sulfate. These chains, col- extending (thickening) the EZ layer in an in lectively, are called glycosaminoglycans vitro model, while the herbicide glyphosate (GAGs), and are either attached to mem- (the active ingredient in Roundup®) was brane-bound proteins or attached to free- found to narrow the EZ layer. The authors floating proteins bound to hyaluronan, note the documented role of the EZ in sta- a very large polysaccharide that sprawls bilizing protein folding, and propose that across the extracellular matrix space and the positive or negative health impact of also helps to maintain the gel (Seneff et al., various nutrients or toxicants may be due 2015). These sulfated amino-sugar chains to their effect on biological EZ water. are also attached to lipid molecules, such as Maintenance of EZ water is important both ceramide, to form the sulfonated lipid, sul- intracellularly and extracellularly. A review fatide (Takahashi and Suzuki, 2012). by Kerch (2018) differentiates between Chaotropes and Kosmotropes the “tightly bound water” associated with healthy cells and tissue in a wide range of Contrary to most of the extracellular tissue conditions, and the “loosely bound water” water, the water in the circulating blood associated with an equally wide range of must remain in the liquid phase. Small diseases and with aging. ions can be characterized by their effects on water structuring/destructuring along a scale called the Hofmeister scale, where EZ Water and Sulfate ions that tend to structure water (i.e., form a gel) are called “kosmotropes,” represent- Given this critical role of EZ water for ing one extreme, and ions that destructure maintenance of normal cellular physiology water (maintaining a liquid phase) are and organismal health, we turn now to the called “chaotropes.” (Russo, 2008) Sulfate

WATER 11, 22-42, December 18, 2019 24 WATER is among the strongest of the biological this is considered to be an inactive state. kosmotropes, and this is what makes it so Calcium entry and subsequent calmodulin important in maintaining the gelled water binding and phosphorylation cause eNOS in the extracellular matrix of most cells, to leave the membrane, and become acti- where the relative sulfate concentration is vated to produce NO (Takahashi and Men- much higher than in the blood. Its presence delsohn, 2003). in the bloodstream will also have a gelling effect, thereby increasing blood viscosity. Much has been written about a dysfunc- tional “uncoupled” state of eNOS where it By contrast, nitrate is a chaotrope, and it produces superoxide rather than NO (Zwei- will therefore act to decrease blood viscos- er et al., 2011, Duan and Kwan, 2011, Land- ity and maintain adequate flow. Both free messer et al., 2003). This is very danger- nitrate and free sulfate are present in sub- ous because the superoxide reacts with NO stantial amounts in the blood, and are high- to form peroxynitrite (ONOO−) a highly ly regulated and maintained in a constant damaging oxidizing agent. It has been pro- balance. One effect of this tight regulation posed that eNOS is a moonlighting is to keep the blood viscosity within a nar- (Seneff et al., 2015, Seneff et al., 2012), row range, the high shear rate measure- that synthesizes not only NO but also sulfur ment spanning between 14.28 and 19.40 dioxide (SO2), and that it switches between (208 Sec-1) from critically low to critically these two under exquisite external control, high, respectively (Nwose, 2010). Free sul- mediated by the viscosity of the flowing fate levels in the blood are maintained usu- blood (Melkumyants et al., 1989) and com- ally at the level of 0.3 to 0.4 micromolar municated through electromagnetic signal- (Morris and Levy, 1983). ing. This is in fact the reason for its complex control mechanisms. We believe evidence for this dual role of eNOS is compelling, Endothelial Nitric Oxide Synthase and that it is further supported by evidence we present here. Endothelial nitric oxide synthase (eNOS) is is one of three isoforms of an enzyme that In this model, eNOS synthesis of SO2 catalyzed by sunlight. Infrared light ex- synthesizes nitric oxide (NO) from arginine, pands the EZ water (Chai et al., 2009), UV the other two being neuronal NOS (nNOS) light mobilizes electrons within the EZ wa- and inducible NOS (iNOS) (Knowles and ter, and flavins bound to eNOS respond to Moncada, 1994, Griffith and Stuehr, 1995). blue light by emitting electrons needed to The isoform, eNOS, is highly expressed in oxidize the sulfur atom (Seneff et al., 2015, endothelial cells lining the vascular wall; Seneff et al, 2012). Light, including infra- hence its name. The enzyme has a very red, provides the energy source for the for- complex requirement for cofactors, and a mation of EZ water (Hwang et al. 2018). very complex regulatory mechanism (Zwei- er et al., 2011, Zou et al., 2002). The co- While hypothetical, this model not only ex- factors include heme, plains several anomalies associated with (BH4), nicotinamide adenine dinucleotide eNOS within red blood cells (RBCs), it also phosphate (NADPH) and the nucleotides accounts for the tight feedback regulation flavin adenine dinucleotide (FAD) and fla- between these two cardinal viscosity regu- vin mononucleotide (FMN). It also houses -2 - lators, SO4 and NO3 . A consequence is a zinc atom in a cavity formed between two that eNOS dysfunction, caused by environ- molecules of the eNOS dimer. eNOS can be mental toxins and resulting in the release of found bound to caveolin in caveolae formed superoxide, leads not only to local damage, in the membrane of the endothelial cell, and particularly in the endothelial wall, but also

WATER 11, 22-42, December 18, 2019 25 WATER to impairment of the SO4-2 / NO3- balance. were present in the RBC extracellular GAGs were sulfated. Sulfate is estimated to con- EVSP, NO and Viscosity Regulation tribute around 17% of the negative charge Flowing blood induces an electromagnet- in typical sulfated oligosaccharides (Roux ic field called the electrokinetic vascular et al., 1988). We predict that it contributes streaming potential (EVSP), which acts as a much larger percentage in RBCs, because a signal to stimulate endothelial cells to re- the GAGs in RBCs are highly sulfated. lease NO (Trivedi et al., 2013). This field A study comparing diabetic patients to can increase the response of the endothelial normal controls revealed that the RBCs of wall to ATP stimulation by up to a factor of diabetics had reduced levels of sialic acid, seven. The magnitude of the signal depends which was associated with increased risk on the zeta potential (ZP), which in turn re- of aggregation (rouleaux formation) and flects the amount of negative charge carried adherence to the vascular endothelial wall. on the surface of the RBCs, establishing a The authors proposed that reduced nega- hydration shell. There is evidence that EZ tive charge on the RBC membrane led to water extends beyond this hydration shell these defects (Rogers et al., 1992). It has for several micrometers (Yoo et al., 2011). been demonstrated that RBCs lose their The EVSP also carries a frequency compo- negative charge and their sialic acid as they nent synchronized to the heartbeat. The re- age, and this may be an important marker sponse intensity (production of NO) rises of senescence (Syed and Rizvi, 2013). with an increase in the heart rate. As previ- eNOS in RBCs ously stated, nitrate, an oxidation of NO, is a chaotrope that destructures wa- Red blood cells express eNOS in relatively ter, reducing the viscosity of the blood and high concentrations, and this has presented improving flow rates. Increased flow is ben- a puzzle to researchers, because nitric oxide eficial for rapid distribution of oxygen and would be expected to interfere with hemo- nutrients to over-exercised muscle cells as- globin’s ability to carry oxygen, like car- sociated with intense exertion, which also bon monoxide does (Mehta et al., 2000). sharply increases the heart rate. Furthermore, RBCs have poor support for transport of the arginine across The normal ZP of RBCs is -15.7 millivolts their membranes. They produce high lev- (Fernandes et al., 2011). The source of the els of arginase, which swiftly clears any ar- negative charge in the RBC membrane has ginine that enters the cell, thus apparently been little studied, but it is logical that sul- rendering their eNOS useless (Kang et al., fate is an important contributor, since it is 2000). In addition, the eNOS of RBCs is a negatively charged molecule and a promi- mostly concentrated near the plasma mem- nent component of the cell glycocalyx. brane (Kleinbongard et al., 2006). In other Erythrocyte membranes contain both sialic cells, as mentioned previously, membrane- acid (Syed and Rizvi, 2013) and sulfated bound eNOS is considered inactive with re- GAGs (Srikanth et al., 2012), and these are spect to NO (Chen et al., 2012). probably covalently linked, forming com- plex sugar structures similar to those de- A considerably more plausible model, as scribed in Roux et al., (1988). Srikanth et we have suggested, is that RBCs use their al. (2012) demonstrated that RBCs contain membrane-bound eNOS to synthesize sul- both chondroitin sulfate and dermatan sul- fur dioxide (SO2) rather than NO (Seneff fate, two of the sulfated GAGs that populate et al., 2015, Seneff et al, 2012). Just as NO the extracellular matrix of most other cells. spontaneously oxidizes to nitrite and then In this study, most of the disaccharides that to nitrate, SO2 would also be spontaneously

WATER 11, 22-42, December 18, 2019 26 WATER oxidized to sulfite and then enzymatically plausible that excess serum cholesterol oxidized to sulfate by sulfite oxidase. Keep- would induce synthesis of SO2 to support ing in mind that sulfate and nitrate are at sulfate synthesis and its conjugation with opposite poles of the Hofmeister series, cholesterol via a sulfoconjugase enzyme to with sulfate being a kosmotrope and nitrate form CS. This would be particularly impor- being a chaotrope, it could be intelligent tant for membrane-bound eNOS in RBCs, design in biology to have a single enzyme because these cells normally carry signifi- switch between sulfate and nitrate synthe- cant amounts of CS in their membrane. sis under exquisite control of signals de- This helps maintain their biconcave shape rived from blood parameters to maintain (Przybylska et al., 1998) and also contrib- blood viscosity within narrow limits. This utes significantly to the maintenance of an model places the RBC at the center of sys- extended hydration layer that is dependent temic blood viscosity homeostasis though upon CS content (Faure et al., 1996). regulation of EZ water in the circulatory system and beyond. Within the three-dimensional compart- ment of the cytoplasm of RBCs, water is Cholesterol and Cholesterol Sulfate predominantly in the bulk state. Only 10% is interfacial, associated with the hydra- Many sterols are sulfated in transit, and tion layer around hemoglobin molecules cholesterol sulfate (CS) is among the most (Stadler et al., 2008). At the two-dimen- common sulfated sterols present in the sional RBC membrane, though, associated blood plasma (Strott and Higashi, 2003). water is gelled, or, as we now understand it, Sulfation makes cholesterol amphiphilic, EZ water. As noted by Schneider & Schnei- rendering it unnecessary to package it up der in their seminal study of RBC mem- inside lipid carrier molecules such as high brane water content, the relatively high hy- density liporprotein (HDL) and low-density drations at higher humidities may reflect a lipoprotein (LDL). CS is produced in large gel-type swelling or capillarity of the mem- amounts in the skin and released into the brane matrix (Schneider et al., 1972). This membranes of HDL particles, which likely membrane-associated EZ water is depen- helps distribute it to the tissues. CS’s rate of dent upon the CS content of the membrane. inter-membrane exchange is approximate- ly ten times faster than that for cholesterol, Individuals with a genetic defect in the ste- allowing it to more readily enter liposomal roid sulfatase gene leading to ichthyosis membranes (Rodriguez et al., 1995). The have been found to have extremely elevat- sulfated cholesterol molecules would pro- ed levels of CS in the membranes of their vide a negatively charged field around the RBCs as well as in the plasma (Bergner and HDL particle, as well as creating an EZ wa- Shapiro, 1981). Their plasma CS levels were ter shell that can protect the particle from measured to be on average 3,300 micro- oxidation and glycation damage. grams/100 ml as compared to under 350 micrograms/100 ml in normal adults. Their High serum cholesterol (e.g., high LDL par- RBCs were even more extreme, containing ticles) is a central pathogenic factor in en- on average 7,500 micrograms/100ml com- dothelial dysfunction, causing a reduction pared to less than 300 micrograms/100 ml in the release of NO, and this effect is likely in normal adults. This represents a 30-fold mediated through upregulation of caveolin increase in the membrane CS content. expression (Feron et al., 1999). The eNOS bound to caveolin at the plasma membrane Collectively, these findings suggest to us is inactive with respect to NO synthesis. two important, previously unknown roles However, if membrane-bound eNOS can that RBCs normally play in physiology. synthesize SO2 instead, then it becomes First, through their sheer volume they con-

WATER 11, 22-42, December 18, 2019 27 WATER tribute to the overall viscosity of the blood membrane-bound eNOS molecules. through this “entrapment” of local water - into an EZ layer at the RBC surface. Sec- H2S and bisulfide (HS ) are both trans- ond, they play a central role in the distri- ported very rapidly across the erythrocyte bution of cholesterol to the tissues. In this membrane (Jennings, 2013). RBCs typi- regard, the removal of the sulfate ligand cally carry significant levels of glutathione. is an essential part of the process. We hy- Oxidized glutathione reacts with H2S in the pothesize that, under normal conditions, following simple reaction: CS is only housed temporarily in the RBC GSSG + H2S → GSSH + GSH membrane. Once released, it decomposes into cholesterol and sulfate, and these two This reaction is actually protective against nutrients are supplied to the endothelial acute poisoning by excessive H2S exposure, cells lining the capillaries and to the tissues as it has been shown that pretreatment of and organs. Sulfate anions can be attached mice with GSSG protects them from an oth- to the extracellular GAGs to reinforce their erwise lethal dose of sodium sulfide (Smith sulfate supplies. and Abbanat, 1996). However, it also sug- gests that RBCs may be able to exploit en- In this model, high serum cholesterol is po- dogenously produced hydrogen sulfide gas tentially an indicator of cholesterol sulfate to both reduce oxidized glutathione and deficiency. Since sulfation renders the mol- synthesize sulfite using eNOS enzymati- ecule water soluble, impaired sulfation ne- cally, catalyzed by sunlight. The two highly cessitates that cholesterol be carried within conserved cysteine residues of eNOS be- a lipid particle in an esterified form. At the come glutathionylated as a modification same time, the sulfate anion, secured in a that enables binding to caveolae and turns lipid membrane and bound to cholesterol, off NO synthesis (Duan and Kwan, 2011). is prevented from acting as a kosmotrope to gel the circulating blood and disrupt flow. Neutrophils exposed to hydrogen sulfide gas under oxidative conditions produce sul- Garlic is universally recognized as a nutri- fite, and they are absolutely dependent on tionally important food that is protective NADPH oxidase to do so (Mitsuhashi et al., against cardiovascular disease, atheroscle- 2005). Neutrophils also are known to ex- rosis, hyperlipidemia, thrombosis, hyper- press eNOS (deFrutos et al., 2001), so their tension and diabetes (Banerjee and Maulik, synthesis of sulfite could also be catalyzed 2002). It is believed that much of its benefit by eNOS in collaboration with NADPH derives from the fact that it is an excellent oxidase, as has been proposed for RBCs. source of sulfane sulfur (diallyl sulfide, di- This suggests to us that inflammation, e.g., allyl disulfide and diallyl trisulfide) (Iciek et within the rheumatoid joint, which induces al. 2016). RBCs extract sulfur from garlic both the expression of reactive oxygen spe- and release gas, which then induces vasore- cies and the infiltration of neutrophils into laxation acting as a gasotransmitter (Bena- the artery wall, may be a process that is vides et al., 2007). Glucose processing needed to renew sulfate supplies to the vas- through the pentose phosphate pathway culature (Wright et al., 2010). This is yet in RBCs restores NADPH to the reduced another way the body provides the sulfate state following its participation in redox necessary to maintain water structure both reactions in the cell. We suggest here that locally and globally. among these redox reactions is NADPH’s involvement in the mobilization of elec- trons to catalyze the synthesis of SO2/sul- fite from hydrogen sulfide (H2S) by RBC

WATER 11, 22-42, December 18, 2019 28 WATER Cobalamin as a Regulatory Agent ter structuring throughout the body. In this section, we first propose that cobala- Environmental Factors Leading to min (vitamin B12) has a catalytic role in the Cobalamin Deficiency synthesis of sulfate by eNOS. We then dis- Cobalamin is a crucial vitamin for health, cuss environmental factors that we believe are disrupting the supply of cobalamin to even though it only catalyzes a few reac- the body, mainly glyphosate, metformin tions. Cobalamin deficiency is widespread, and a strict vegan diet. Finally, we show with about 20% of the US population being how the metabolic effects of the loss of co- at or below the critical symptomatic thresh- balamin orchestrate a well-choreographed old of 200pmol/L (Allen, 2008). This is systemic response involving multiple or- due in part to the complex process involved gans, which is aimed at maintaining sulfate in cobalamin absorption, which often gets supplies to the vasculature at the expense disrupted. Plants neither require nor pro- of the health of the organs. duce cobalamin, so a vegan diet, becoming increasingly popular in the United States, is Cobalamin’s Critical Role in Sulfate likely to be a risk factor for cobalamin defi- Synthesis ciency (Antony, 2003). The manifestations of cobalamin deficiency are complex, but Cobalamin is an essential B vitamin with extremely interesting. They reflect the in- several critical roles in the body. Howev- tricacies of biological control mechanisms er, we argue here that there is yet another whereby seemingly diverse symptoms or- role that has been to date overlooked by chestrate a higher-level plan that works a the research community. Cobalamin is ac- “work-around” solution. This is necessi- tive in two known forms: adenosylcobala- tated when the mechanisms that typically min and methylcobalamin. However, it is synthesize sulfate from sulfane sulfur and recognized that cobalamin can also con- hydrogen sulfide are derailed. jugate with glutathione. In fact, studies predict that aquacobalamin rapidly reacts Vitamin B12 is a large molecule that is with reduced glutathione, irreversibly, to sensitive to degradation in acidic environ- form glutathionylcobalamin (Xia et al., ments. These features make it very difficult 2004). Early studies that investigated this to deliver cobalamin to the tissues. The reaction found that the eventual reaction process of cobalamin transport through product was sulfitocobalamin, through an the gut and past the gut lining is complex. unspecified subsequent reaction sequence It begins with binding of dietary cobalamin (Pezacka et al., 1990, Farquharson and Ad- to the protein haptocorrin, secreted by the ams, 1977). Intriguingly, the structure of the salivary glands in the oral cavity. Binding to eNOS molecule is such that there is a large haptocorrin protects B12 from degradation cavity where the bulky cobalamin molecule in the acidic environment of the stomach. should fit perfectly (Wheatley, 2007). Thus, Pancreatic proteases digest the haptocor- the observations from these published pa- rin in the upper intestine. Parietal cells in pers can be pieced together to propose that the stomach produce intrinsic factor, which eNOS binds to glutathionylcobalamin. This enters the upper intestines and then binds complex secures a sulfane-sulfur atom, to the cobalamin following its release from likely from an ambient hydrogen sulfide haptocorrin. Without intrinsic factor, only molecule, which then gets oxidized to SO2/ 1% of the cobalamin will be absorbed. sulfite via the superoxide that is released from eNOS when it is in its uncoupled state. Metformin is a very popular drug widely In this way, cobalamin becomes a central used to help control blood sugar levels in player in the sulfate balance and, thus, wa- patients suffering from Type II diabetes. It

WATER 11, 22-42, December 18, 2019 29 WATER has become clear that a side effect of long- pared to a neutral pH of 7.0. This is an im- term metformin therapy is cobalamin defi- portant point when considering glyphosate ciency (Kibirige and Mwebaze, 2013). Stud- exposure in the stomach lumen, where the ies have shown that metformin is effective pH is extremely low (1.5 to 3.5). Given this, at reducing blood sugar even when it is ad- one can predict that the parietal cells in the ministered in a delayed-action formulation stomach will be especially susceptible to di- that stays in the gut and never even reaches etary glyphosate exposure. One of the im- the liver, much less the general circulation portant roles of parietal cells is to release (Song, 2016). Part of metformin’s effects intrinsic factor, which is essential for cobal- on blood sugar is likely due to disruption of amin transport across the gut wall (Festen, cobalamin absorption in the gut, with co- 1991). balamin deficiency leading to suppressed Furthermore, cobalamin depends upon co- hepatic release of glucose from glucagon balt as a . A study of cows exposed stores (Ebara et al., 2001, Ebara et al., to glyphosate examined blood levels of a 2008). suite of minerals, and found alarmingly low Glypohosate is the active ingredient in the levels of just two minerals: cobalt and man- pervasive herbicide, Roundup®. It is the ganese. Both were at levels well below the most used herbicide in the world: its us- minimum of the normal range in all cows at age rate has increased dramatically over eight different farms (Krüger et al., 2013). the past two decades, in step with the dra- The cobalt atom in cobalamin is housed in matic growth in GMO Roundup-Ready the center of a corrin ring, which forms the crops such as corn, soy, canola and sugar core of the cobalamin molecule. Corrin is beets (Benbrook, 2016, Swanson et al., assembled from four molecules of pyrrole, 2014). Glyphosate’s manufacturer, Mon- and pyrrole synthesis has been shown to be santo, has long maintained that glyphosate disrupted by glyphosate. Glyphosate blocks is a very safe herbicide with little risk to hu- the enzyme δ-aminolevulinic acid synthase, man health from exposure. Glyphosate has the first step in the synthesis of the pyrrole however been found to be present as a con- ring (Kitchen et al., 1981). It likely does taminant in many common foods, and in- this through substrate inhibition, acting as dependent researchers are finding evidence an analogue of glycine. that it is considerably more toxic than the regulators have been led to believe (Séralini In addition, eNOS itself is sensitive to et al., 2014, Bonfanti et al., 2018, Santovito glyphosate toxicity. It contains a heme et al., 2018). group, which, like the corrin ring, is com- posed of pyrrole units whose synthesis The most common cause of vitamin B12 would be suppressed by glyphosate. Fur- deficiency in developed countries is im- thermore, eNOS is an orphan member of paired absorption due to a loss of gastric the cytochrome P450 (CYP) family of en- intrinsic factor. Surprisingly, glyphosate is zymes (Gorren and Mayer, 2007): a study able to diffuse passively across lipid mem- of rats showed that liver CYP branes, i.e., across cell plasma membranes, were severely suppressed by glyphosate as demonstrated in an elegant set of ex- (Hietanen et al., 1983). RBCs express the periments conducted by B.N. (Ehrl et al., L-type amino acid transport protein LAT1 2018). The ability of glyphosate to cross (Mann Dosier et al., 2017), which has been cell membranes depends critically on the shown to actively take up glyphosate into acidity of the medium. Glyphosate’s mem- cells (Xu et al., 2016). Glyphosate circu- brane permeability increases by an order of lating in the serum would be taken up by magnitude at pH 4.1 (mildly acidic), com- RBCs along glycine transport channels,

WATER 11, 22-42, December 18, 2019 30 WATER and RBCs have a major need for glycine (Carbonero et al., 2012); as a precursor to heme synthesis for their hemoglobin as well as for their eNOS and • The cumulative production of H2S other heme-dependent enzymes. Finally, (Jung and Jeong, 2014) disrupts the eNOS depends on both zinc and iron (in gut barrier (Medani et al., 2010, Ijssen- heme) as catalysts, and glyphosate has nagger et al., 2016), and H2S produced been shown to chelate these two cations, in the gut diffuses into circulation to act as well as cobalt, making them unavailable as substrate for eNOS to generate SO2. (Mertens et al., 2018). Sulfite oxidase, the This is transformed as previously de- enzyme that converts sulfite to sulfate, can scribed to biologically active sulfate, also be expected to be impaired by glypho- thus restoring systemic sulfate stores; sate due to potential chelation of its cofac- • Taurine, stored in the brain and heart, tor molybdenum, as well as a dependency is released in the context of cobalamin on heme whose synthesis is suppressed by deficiency and consequent NMDA re- glyphosate, as previously described. ceptor activation (Menéndez et al., Consequences of Cobalamin Defi- 1993); ciency • Circulating taurine can bind to bile salts If, as we hypothesize here, eNOS’s synthesis in the liver, after which it can be con- of sulfate depends critically on cobalamin, verted to sulfate in the digestive tract then cobalamin deficiency can be predict- with assistance from the gut microbes ed to lead to sulfate deficiency over time. (Carbonero et al., 2012). What is interesting is that the other enzy- Cobalamin deficiency leads to peripheral matic reactions that cobalamin catalyzes, nerve injury and subsequent Wallerian de- such as methylmalonyl CoA mutase and generation of peripheral nerves, whereby methionine synthase, involve critical path- the ends of axons distal to the site of injury ways which, when impaired due to cobala- begin to disintegrate. This involves upregu- min deficiency, initiate major adjustments lation of cystathionine beta synthase (CBS) in the way the body manages to obtain ad- and increased production of H2S, contrib- equate sulfate. These combined effects of uting to the H2S pool for vascular oxida- cobalamin and sulfate deficiency initiate a tion. Further, myelin degradation products, broad range of compensatory changes in including cysteine and sulfatide, may then the body, many of which are highly symp- be acted upon by CBS, cystathionine γ‐ tomatic and, we suggest, incorrectly con- (CSE) and cysteine dioxygenase (CDO) to sidered to be unrelated pathologies. These generate additional H2S (Jung and Jeong, changes are, in brief: 2014). • Sulfur-fixing bacteria proliferate, pre- All of these adaptations are problematic dominantly in the large bowel, but also in terms of health, because they can lead occasionally in the small bowel; to several pathologies such as; dysbiosis • Dietary sulfur, sulfomucins, sulfated and small intestinal bacteria overgrowth hormones, and other compounds are (SIBO) (Banik et al., 2016), and inflamma- preferentially converted by these bac- tory bowel diseases such as Crohn’s (Arijs et al., 2013) and ulcerative colitis (Ijssen- teria into H2S through well-established pathways (Carbonero et al., 2012); nagger et al., 2016, Zhu et al., 2016, Coutin- ho et al., 2017). In addition, there may be • These same bacteria also harvest sul- systemic symptoms of high H2S, including fate from bile salts, converting it to H2S hypotension (Betowski, 2004) and brady-

WATER 11, 22-42, December 18, 2019 31 WATER cardia (Liu et al., 2011); concentration and tial for the synthesis of methionine from memory impairment (Rosenegger et al., homocysteine. When this pathway is dis- 2004, Tvedt et al., 1991, Eto et al., 2002); rupted, methylation capacity is severely rheumatic conditions (Muniraj et al., 2017), impaired, and homocysteine accumulates severe neuropathy, dementia, and arterial to unhealthy levels. Since DNA synthesis damage inducing thrombosis and cardio- depends on methyl groups, cobalamin de- vascular disease, among other possible is- ficiency causes a reduced proliferation rate sues. Further, the oxidative conditions cre- of nascent RBCs, resulting in macrocytic ated by uncoupled eNOS for conversion of anemia. At the same time, homocysteine H2S to SO2 also increase the risk of throm- induces an inflammatory cascade in the ar- bosis and cardiovascular disease, neurolog- tery wall, which causes the release of super- ical disorders, and many other conditions oxide to support the oxidation of the sulfur associated with excess oxidative stress. in homocysteine to sulfate, as discussed in a paper by McCully published in 2011 (Mc- Within this model, these pathologies are Cully, 2011). Thus, homocysteine can par- unfortunate side effects of our bodies’ at- tially compensate for the loss of eNOS-de- tempts to generate sulfate, compensate for rived sulfate, but at a severe cost, as inflam- low cobalamin, and ultimately maintain ad- mation drives cardiovascular disease (Golia equate EZ water within tissue and the blood et al., 2014). stream. Maintenance of EZ water can only happen when sulfate is in sufficient supply. Vitamin B12 deficiency leads to increased cholesterol uptake and increased choles- Since sulfate deficiency impairs the ZP of terol synthesis in adipocytes. Through the RBCs, another adaptive biological compen- mechanism of epigenetics, genes for pro- sation is to reduce the number of RBCs in teins involved in cholesterol synthesis and circulation. Macrophages need adequate uptake are upregulated as a consequence of sulfate to clear the DNA in the nuclei that hypomethylation of their promoter regions RBCs release as they mature (Bratosin et (Adaikalakoteswari et al., 2015). These al., 1998). The RBC population as a whole authors wrote: “Our clinical observations needs to be reduced in order to protect from show strong associations of vitamin B12 possible agglutination due to insufficient deficiency with BMI, triglycerides, and to- negative charge and EZ water as a conse- tal cholesterol, and our in vitro studies in quence of cholesterol sulfate deficiency in adipocytes show that vitamin B12-deficient their membranes, as described in section 5 conditions induce de novo cholesterol bio- above. synthesis.” (Adaikalakoteswari et al., 2015, Regulatory Mechanisms to Maintain p. 10). Epicardial adipose tissue (EAT) ac- Vascular Health at the Expense of counts for 20% of the total weight of the the Heart and Liver heart, and obesity is associated with an in- crease in the amount of EAT (Golia et al., With these concepts in mind, it is now pos- 2014). EAT is infiltrated with macrophages sible to examine the diverse consequences that penetrate the tissue in response to in- of cobalamin deficiency as it applies to both flammatory signals. In patients with cardio- EZ water formation and related patholo- vascular disease, EAT produces higher lev- gies, schematized in Figure 1. Throughout els of atherogenic and inflammatory cyto- this discussion it will be important to keep kines (Baker et al., 2006). We hypothesize in mind the fact that anything compromis- that, as proposed in Seneff et al. (2015), the ing sulfate production will ultimately have increased cholesterol storage in fat cells ac- a negative impact on EZ water formation cumulating within cardiovascular arteries throughout the body. Cobalamin is essen- provides a ready supply of cholesterol wait-

WATER 11, 22-42, December 18, 2019 32 WATER ing to become cholesterol sulfate once sul- of a vegan diet, which is generally depleted fate becomes available. in both nutrients. A study on rats noted that cobalamin deficiency led to an increase Glycine is a source of methyl groups via the in plasma glycine and serine levels (Ebara glycine cleavage system (Kikuchi, 1973). At et al., 2001): this could be explained by a least for pigs, it appears that cobalamin de- suppression of the glycine cleavage system, ficiency suppresses the ability to use methyl which would also explain the results in the groups derived from glycine to synthesize pig study above. The excess bioavailabil- choline, and that this leads to severe fatty ity of glycine due to impaired methylation liver disease under conditions of dietary pathways could be advantageous for heme choline deficiency (Johnson et al., 1955). synthesis, since glycine is a substrate for Choline deficiency due to insufficient di- pyrrole ring synthesis. The heme is needed etary sources along with impaired cobala- for the enzymes eNOS and sulfite oxidase min absorption could be a contributory fac- involved in synthesizing sulfate from H2S. tor in the epidemic we are seeing today in non-alcoholic fatty liver disease (NAFLD) Another curious aspect of cobalamin defi- (Corbin and Zeisel, 2012). NAFLD is the ciency is that it impairs the adenylyl cyclase most common liver condition worldwide, system in the liver, leading to a reduction affecting nearly one in three people in the in the synthesis of glucose from glycogen industrialized world (Smith and Abbanat, stores in response to hormonal stimuli (Eb- 1996). Deficiencies in choline and cobala- ara et al., 2008). It may also be a practical min could be especially relevant in the case response, because the tissues are less able

Figure 1: Schematic of metabolic pathways disturbed by B12 deficiency, that lead to alternative approaches to maintaining sulfate supplies to the tissues, associated with disease. Both glyphosate and metformin suppress B12 bioavailability.

WATER 11, 22-42, December 18, 2019 33 WATER to take up glucose and store it in the extra- neled into the citric acid cycle (see Figure cellular matrix GAGs because of the sulfate 1), providing an alternative source of fuel, deficiency problem. This makes glucose given that propionate is no longer metabo- more dangerous as a glycating agent. At the lized via the citric acid cycle. In response same time, the lipid particles suspended in to cobalamin deficiency, stored taurine in the blood, as well as the RBCs and platelets, the brain can be mobilized as a resource to are more susceptible to glycation damage boost sulfate levels, systemically. from glucose because they do not have an adequate shield of gelled water surround- Discussions (Seneff et al. 2013) indicate ing their membranes due to insufficient that ammonia has a very high pKa and is membrane-bound sulfate. therefore one of the few bioavailable mol- ecules that can substitute for taurine as a Stealing Taurine from the Brain for buffer to support the high pH of the mi- Sulfate Synthesis by Gut Microbes tochondrial matrix (Hansen et al., 2010). This allows astrocytes and neurons to re- Cobalamin also catalyzes an important lease their stored taurine (Albrecht et al., reaction that converts malonate into suc- cinate, feeding into the citric acid cycle to 1994), which can then be transported to produce ATP. A precursor to malonate is the gut microbes conjugated to bile acids. propionate, a short chain fatty acid pro- While taurine is a very stable molecule that duced by the gut microbes in the process of human cells are unable to further metabo- metabolizing branched chain amino acids, lize, the gut microbes are able to oxidize fatty acids, cholesterol and sugars via fer- taurine to sulfate, thus supplying sulfate mentation (Banerjee et al., 1999). A con- to the host. The enzyme in Escherichia coli sequence of this blockage is that both pro- that oxidizes taurine to sulfite, taurine di- pionate and methylmalonate accumulate , depends upon alpha-keto gluta- to high levels. Both are problematic in in- rate as a reducing agent (Van der Ploeg et teresting ways. Propionate interferes with al., 2001, Eichhorn et al., 1997). Thus, the the detoxification of ammonia in the liver. increased bioavailability of glutamate due Ammonia detoxification to urea involves to suppression of the urea cycle consequen- a pathway that begins with N-acetylgluta- tial to cobalamin deficiency facilitates tau- mate as substrate. rine oxidation to sulfite by E. coli. NMDA activation induces the release of taurine Acetyl-CoA is a substrate for N-acetylgluta- from neurons (Menéndez et al., 1993). An mate synthase, and propionyl-CoA acts as a in vitro study on rat hippocampal slices re- competitive inhibitor of this enzyme (Rabi- vealed that glyphosate induces neurotoxic- er et al., 1979, Rabier et al., 1986). Conse- ity by a mechanism that involves activation quently, both ammonia and glutamate ac- of NMDA receptors and subsequent calci- cumulate to toxic levels. Both have adverse um uptake and increased release of gluta- effects on the brain, leading to a chronic mate, a neurotoxin, into the synaptic cleft low-grade encephalopathy and neurotoxic- (Cattani et al., 2014). Decreases in gluta- ity. Related to this, rats fed a diet that was thione content and increases in lipoperoxi- high in fermentable carbohydrates exhib- dation were indicators of oxidative stress. ited behaviors characteristic of anxiety and aggression that were associated with high A transcriptome analysis from 2013 of levels of propionate in the caecum. This Escherichia coli protein expression in re- was a consequence of fermentation by the sponse to glyphosate exposure revealed gut microbes (Hanstock et al., 2004). The that many proteins involved in taurine excess glutamate that is spared from pro- transport were down-regulated (Lu et al., cessing through the urea cycle can be chan- 2013). Taurine transporter subunits tauA,

WATER 11, 22-42, December 18, 2019 34 WATER tauB, and tauC, panC (a taurine transport and easily attacked by immune cells (Brais- system permease protein), ompF (an ATP- sant et al., 2013, Kristensen et al., 1993). binding component of the taurine trans- The myelin is likely more accessible to im- port system), and ompT (a periplasmic mune attack because of a destructuring of protein of the taurine transport system) the surrounding water due to insufficient were all sharply downregulated by E. coli sulfate in the glycoproteins and glycolipids in response to glyphosate exposure. OmpF that surround the fatty sheath. Erosion of was downregulated by a factor of 11, and it the myelin sheath may be the reason for an was among the most downregulated of all association between cobalamin deficiency the downregulated proteins. This strongly and Alzheimer’s disease (McCaddon et al., suggests that glyphosate disrupts the me- 2002). The major protein in the myelin tabolism of taurine into inorganic sulfate sheath is called myelin proteolipid protein by microbes. peptide, and it is rich in cysteine residues, a potential source for sulfate. But, perhaps Taurine-conjugated bile acids (taurocho- even more convenient as a direct source of late) have been identified as a risk factor sulfate is the sulfatide, a sulfated glycolipid for colon cancer, and as a potential expla- normally present in high amounts in the nation for the link between a diet high in myelin sheath. Sulfatide levels in the brains animal-based foods and colon cancer (Rid- of Alzheimer’s patients plummet very early lan et al., 2016). This was attributed to the in the disease process (Han et al., 2002). conversion of sulfur-containing amino ac- These authors found an astonishing 93% ids to H2S by sulfur-reducing bacteria such depletion of sulfatide already in the very as Desulfovibrio and Bilophila wadswor- early stages of Alzheimer’s disease. A study thia. In particular, B. wadsworthia are no- comparing Alzheimer’s patients to controls table for their ability to process taurine into showed that the Alzheimer’s patients had H2S (Carbonero et al., 2012). A diet high significantly elevated levels of methylmalo- in milk and milk fat led to over-represen- nic acid (p = 0.0097), which was associated tation of B. wadsworthia in the mouse gut, with cobalamin deficiency (Kristensen et likely due to an overabundance of taurine- a., 1993). We hypothesize that the myelin conjugated bile acids (Devkota et al., 2012). sheath is attacked in order to retrieve sul- Excessive levels of H2S corrode the gut lin- fate from the sulfatide to help restore sul- ing and induce an inflammatory response fate levels in the circulation. leading to DNA damage, while the microbi- ally metabolized bile acids cause degrada- In addition to dementia, cobalamin defi- tion of tumor-suppressing proteins such as ciency is also linked to multiple other neu- P53 (Qiao et al., 2001). It is here hypoth- rological diseases, including myelopathy, esized that these and other H2S-producing peripheral neuropathy and optic neuropa- microbes flourish in an environment where thy (Saperstein and Barohn, 2002). Elevat- common commensals such as E. coli are un- ed serum methylmalonic acid and homo- able to process taurine and other reduced- cysteine are diagnostic features. It is likely sulfur sources into sulfate. that these conditions are a consequence of a stripping of the sulfatide and sulfur-en- Destruction of the Myelin Sheath riched proteins in the myelin sheath of pe- Propionate and malonate (as methylmalo- ripheral neurons. nic acid), as well as ammonia, all of which are elevated in response to cobalamin defi- ciency, are major disruptors of the myelin sheath surrounding nerve fibers. They cause the myelin to become much more fragile

WATER 11, 22-42, December 18, 2019 35 WATER Conclusion Discussion with Reviewers In this paper we have proposed that the 1. In your opinion what agents will sulfate molecule plays a unique role within be suggested in the nearest future to biology as the source of the negative charge increase the exclusion zone size and needed for the formation and maintenance health of biological tissues? of EZ water in living system. We have de- scribed unique roles for eNOS and cobala- If we are correct that the maintenance of min within the system of ongoing sulfate health is dependent upon the proper uti- production and regulation. It is our asser- lization of sulfur and its metabolites, then tion that several diverse and seemingly un- those compounds that play a role in sulfur related pathological processes might have a metabolism in general, and hydrogen sul- common origin as compensatory process- fide metabolism in particular, would be ex- es to reestablish sulfate sufficiency for EZ pected to have uniquely beneficial roles in water maintenance. We also find that pa- health maintenance. Many examples sup- thologies traditionally associated with co- port this idea. balamin deficiency can be best understood Coenzyme Q10 is an essential component through the impact that that deficiency has of mitochondrial electron transfer. Much on sulfate metabolism, and the compensa- research has shown its beneficial role in en- tory changes that this causes. hancing energy production. A lesser known While our proposed roles for eNOS and co- role of CoQ10, though, is as an essential balamin in this model are unique, we be- cofactor in the activity of sulfite: quinone lieve the circumstantial evidence presented reductase (SQR), where it facilitates con- - here is compelling and warrants further version of H2S to SO3 , thus feeding into the -2 research. If our model of EZ water produc- SO4 pool via the SUOX enzyme. tion and maintenance through the inter- There are several other nutrients that play face of water and sulfated molecules is cor- -2 a lesser-known role in H2S and SO4 me- rect, it has wide implications in health and tabolism and distribution around the body, medicine. Further, if we are correct that including vitamin D, , curcumin, glyphosate has a broad range of detrimen- resveratrol, and others. These are, perhaps tal impacts on sulfate production, it adds to not coincidentally, among the nutrients the growing body of evidence that its wide- with the strongest health-supporting evi- spread use requires renewed scrutiny and dence. While their role in sulfur metabo- regulation. lism is virtually never mentioned in articles Science progresses to a large extent by spec- on their health benefits, we propose that it ulative hypotheses that, through further in- may be as significant as their antioxidant vestigation, transform into established fact. and other properties. It is our hope that these ideas might provide In this respect, it is our prediction that a foundation for understanding the forma- agents that might be suggested in the fu- tion and maintenance of interfacial water ture for increasing the size of the exclusion in the body, and eventually progress to a zone will be agents that contribute to the set of therapeutic strategies for addressing -2 H2S/SO4 cycle in the body we have de- the wide-ranging pathologies that might be scribed in this paper. They will do so in a related to its loss. way that ultimately facilitates production and/or distribution and/or utilization of -2 SO4 and its critical role in supplying the electronegative charge to the hydrophilic

WATER 11, 22-42, December 18, 2019 36 WATER surfaces within the body. Abbreviations 2. In your opinion what novel physi- Cytochrome P450 CYP cal methods will be suggested in the nearest future to increase the exclu- Electrokinetic Vascular sion zone size and health of biologi- Streaming Potential EVSP cal tissues? Endothelial Nitric Oxide Synthase eNOS To our knowledge, the only physical ther- Epicardial adipose tissue EAT apy thus far shown to increase the size of the exclusion zone is the application of in- Exclusion Zone EZ frared light, with Pollack reporting that IR of a wavelength 3000nm having the great- Flavin Adenine Dinucleotide FAD est impact on EZ extension. The scope of Flavin Mononucleotide FMN the electromagnetic spectrum is astonish- ing, encompassing 20 orders of magnitude. Glycosaminoglycans GAGs Given the increasing number of health ben- efits discovered for very specific frequen- High Density Lipoprotein HDL cies along that spectrum, it is our predic- Inducible Nitric Oxide Synthase iNOS tion that electromagnetic medicine will be a growing field of interest. The full EM Low Density Lipoprotein LDL spectrum is essentially a virtually untapped Nicotinamide Adenine ocean of potential specific frequencies that Dinucleotide Phosphate NADPH might have EZ-extending properties. Nitric Oxide NO Non-alcoholic Fatty Liver Disease NAFLD Author Contributions Peroxynitrite ONOO- Both authors contributed significantly to all aspects of the paper. Small Intestinal Bacterial Overgrowth SIBO Sulfur Dioxide SO Funding 2 Tetrahydrobiopterin BH4 This research was funded in part by Quanta Computers, Taiwan under the auspices of Zeta Potential ZP the Qmulus project.

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