Resonant Neuromelanin – Cell Immune Sovereignty Key –—
By Edward DeVere II
- World wellness at viral speed, safety and affordability may start with the first reseller of the level 3 lab validated Covid over in minutes, essential oils and the melanin master activation formula. By 12/31, a Columbus, Ohio veterinarian and paramedic, and a New Mexico doctor of nutripathy with 30 years of wellness work with individuals, governments and military, may be the first to bring both products to major US markets.A Sedona, Arizona new health center may make it a 3 three some; with leaders of 3 locales seeking to openly test and increase health options for those who are free to choose them.All product inquiries should first go to the article 314 author, text preferred,(936) 718-2747.
Daily zoominars on all related topics may be arranged at beyond viral speed and effectiveness.Chaga and shilajit, master sources for new melanin super immunity communities may be a part of the symbiotic viral acceleration, to meet and exceed the earth’s unprecedented loss of super health prevention and treatment of any health crisis. this may be accomplished by 1/7/2021 for a true inauguration of collaboration restoration as never before.
- Existence is both fractal and linear – this document highly reflects and is not light reading, in one sense. Please validate 9 of over 100 references and resist snap evaluations for the great potential of any truly patient, objective reader.The most diplomatic compliment, and or option to multiple global vaccinations may be the safe,affordable,melanin millennium activation,made enmasse ,in most communities with humanphotosynthesis.com standards,which,in concert with this millennium manifesto shows immunity restoration of all biomes may outspeed climate devastation.See worldometers.info for real time confirmed cases by country. The content you validate in this overview may empower virtually any one who’s consistent with new action, proving they dare to care.NASA’s 2014 statement that melanin versions are required for all life in all galaxies is a word to the melanin informed, that, amplifying melanin vitality in all people and environments ,for immunity and the higher emotional and intelligence is likely the single most essential, effective regeneration campaign to compliment all nature and evidence based technological and educational innovations.The Italian national agency for new technologies made melanin 1 billion times more conductive for industrial use. The great reset fourth industrial revolution,blending bio and digital systems will be greater opportunities for humanity; only if there’s a matching fund for amplifying a super natural paradigm like melanin activation in the population and the planetary eco body itself.See the dark secret of life at brunonic.com, and see if there is a more universal platform science for true enmasse human and planetary evolution?We may make nature’s most malleable molecule billions of times better for humanity’s commonwealth of all cells, the greatest pool of super potential in this galaxy awaiting turn key activation.Wei Cao,post doctoral ,Chicago north western polymer chemist and the Nathan Gianneschi’s group of multinational university life quality visionaries,are non synthetically,amplifying, as never before, cellular defense and repair.
- By Kristopher Benke
Authors: Wei Cao, Alex J. Mantanona, Haochuan Mao, Naneki C. McCallum, Yang Jiao, Claudia Battistella, Valeria Caponetti, Nanzhi Zang, Matthew P. Thompson, Marco Montalti, J. Fraser Stoddart, Michael R. Wasielewski, Jeffrey D. Rinehart, Nathan C. Gianneschi
Journal: Chemistry of Materials
Melanin is a biological component that we are keenly aware of because of its visibility in the skin and hair. We know its presence can influence an individual’s likelihood of participating in science, either as a scientist or subject. Divorced from its societal implications, the chemical itself displays a fascinating array of properties and functions in the body which come as a consequence of its unique structure.
Each melanin pigment is a polymer-like molecule made up of subunits of the neurotransmitter dopamine and other similar molecules like L-DOPA, the dopamine precursor that is used to treat Parkinson’s disease (Figure 1). Melanin has a high amount of disorder, meaning no two melanin molecules are exactly the same. They may differ in how many subunits they contain, how they are connected, and how they are stacked. Additionally, there is not just one type of melanin. Instead, melanin is a larger category of pigments which consists of two main types: eumelanins, which are the source of black and brown color in the body, and pheomelanins, which give red hair its color. There’s even a variety present in the brain known as neuromelanin that offers protection to neurons –and which is diminished in individuals with Parkinson’s disease.
A key function of melanin is its ability to protect bodily tissue from damage by radicals, which are highly reactive and often short-lived molecules that contain unpaired electrons. Electrons most frequently exist in pairs, in the body and otherwise, but when they don’t, they can have drastic effects, both good and bad. Therefore, the body continuously tries to eliminate unintentional –or even intentionally formed –radicals before they can cause cellular damage.
A common form of radicals in the body are reactive oxygen species (ROS) which include peroxides and superoxides (O2–). The presence of both molecules can be curbed by protectant molecules like melanin and antioxidants. Unintentionally formed “external” radicals come from a number of sources, including ionizing radiation like sunlight and x-rays, as well as air pollutants and cigarette smoking. The body can also purposefully create “internal” radicals; for example, hydrogen peroxide is used for metabolic processes in the mitochondria and nitric oxide in the regulation of blood pressure.
Yet despite the damaging effect that radicals can have, melanin itself contains radicals. In fact, melanin production relies on the generation of radicals to form bonds, and when you shine ultraviolet and visible light on melanin it produces more radicals! This is not as haphazard as it seems because the radicals inside melanin are well-contained and act as traps for the outside radicals; when they combine, both are rendered harmless.
Researchers have sought to enhance the protective properties of melanin by making a synthetic melanin with increased radical content. Conveniently, unenhanced synthetic melanins already exist in the form of nanoparticles (Figure 2A) and are made with the same building blocks as natural melanins, including dopamine. So, Cao et. al. decided to modify a proportion of the building blocks in the nanoparticles so that they produced more radicals while still reacting to form larger nano-sized structures, resulting in so-called “radical NPs” (Figure 2B). The building blocks were modified to contain a well-known radical-containing molecule called TEMPO, which in other applications is used to detect radical-containing molecules.
The increased presence of radicals was detected by magnetometry, a technique used to measure the magnetic field produced by unpaired electrons. This increase in radical content was confirmed using electron paramagnetic resonance, or EPR spectroscopy. EPR spectroscopy is a specialized technique used to detect unpaired electrons and the molecular environments they exist in, and in fact, melanin was one of the first molecules in which radicals were detected using this technique.
Next, the researchers demonstrated the effect that the radical NPs could have in human skin. When the radical NPs were mixed with human skin cells, they were taken up by the cells and accumulated at the nucleus, where our radiation-sensitive DNA is stored (Figure 3A). This was a promising first sign that the radical NPs were acting like natural melanin, so next they tested if their presence would protect the cells from ionizing radiation. Cao et al. introduced a dye into the cells that fluoresced when exposed to ROS and irradiated the cells with ionizing X-ray light. Looking at cells under a microscope, they found that the fluorescence that appeared in the unprotected cells was absent in the radical NP treated cells (Figure 3B).
Enhanced melanin such as that found in these radical NPs synthesized by Cao et al. has the potential to act as a radioprotective material. Such materials are useful to protect from occupational hazards present at nuclear power plants and in the healthcare industry. This method may even eliminate the need for heavy lead jackets used during medical X-ray scans to avoid DNA-damaging ionizing radiation. Finally, the melanin-like design of these radical NPs makes them biocompatible, meaning they could be used to minimize the negative impacts of cancer treatment by protecting the non-cancerous parts of the body.
Like the quanta9.com tech nuclear quantum physicists, nano altering and value adding ,and super charging melanin as a real world wellness,nano resonant neuro melanin reality and premise.
To change the source ,course and direction of our world,changed as never before by variations of one invisible organism,which revealing to all humans what they don’t know ,must learn about and over come, now ,or loose all, they have ever valued.
Independent virologists, admit there are millions of far more infectious and contagious ,lethal pandemic infectious organisms ,some already validated in India and Brazil,like the nipah and hendra viruses.see Deborah Mackenzie Herculean decades of virology and pandemic.science based research.while most don’t know enough to deny the enclosed data .many mores dream of myriad new injections at any cost .literally.
What remedies will you choose? Before you and your world community is rushed into compliance with science that you don’t comprehend or control and its effects are daily more likely to be out of control of even the increasingly competing and confusing providers.
See the YouTube, “The World’s Most Powerful X-ray laser just gave rise to a molecular black hole”.That million orthodox molecular wave may easily be March 4th by that very date. The most essential catalyst for earth’s viral restoration is the viral awakening of whole system’s awareness of each human. In that light, what’s your solution for the tidal reversal of earth and human devastation?
Melanin partnerships can fluorish with any person or enterprise such as eesystem.com, myclar8ty.com, avisamyco.com, consciousvitality.com,birchboys.com, parsiherbs.com, nadonnainternational.com, dmvoils.com and others, too numerous to mention ,whose values reflect melanin,as a potential aid in all ventures ,that prioritize amplifying nature’s venture,which the universal melanin molecule may be the most adoptable,adaptable example.
For those who prove ready to do the new,melanin activation may amplify any phase of their reality;with receptivity and accountability.
Drink more clean water and get 15 minutes of sunlight daily, and enjoy healthy music; if only through a window. Melanin millennium activation, explained in this ebook blog, continually optimizes all three.
If any reader will scroll through this treatise; it’s promised they will find higher ground and useful content.
What’s your warp speed return to normal global plan? Besides the one with billions of people and dollars; ready to adopt it?
The new melanin for a super human community,clinical medical science foundation,to compliment or exceed any treatment and prevention of pandemic causing viruses ,Bacteria etc,centers around the brilliant science teams of Dr.Arturo Solis Herrea,in Mexico, Kishalay Paria, Hee Ho park in South Korea, Zheng Wang at the D.C. naval research lab, and Henry H. Lee, Boston Wyss Institute.
fungalmelani.com is one of the few ,mass producing,high grade,Therapeutic melanin,sustainably sourced,for its greatest single application;natural medicine for a new and improved mankind,in these times.
Microbally made melanin could be made in any locale, regardless of climate;dozens of vertical market enhancements,from large scale water treatment,food products and stronger materials for construction,vehicles and clean power,biofarmceuticals and more.
$10 billion funded ,project warp speed is an all nation all military agreement to vaccinate the world population with new innoculations for the new viruses which may spread far faster and with more devastation than virus 1, which ,revamped your world as you know it ,only since 12/19/2020.billions are added monthly to this runaway new virus new vaccine viral pandemonium.
$1 billion for proven, natural compounds ,to compliment,match and or exceed conventional thought ,warp speed projects are the first ,true hedge fund ,for a stable future for humanity,increasingly on the endangered species list.
Those who fund them,may be the strategic managers of a new humanity far more immune to divisiveness and empowered to enjoy the life quality only given to an awakened ,United,global civilization with a new health and wealth,that is out of this world.
The warped project has 3 million Pfizer doses Fda approved and distribution ready and as 12/18 ,40 million in addition with the emergency approved mRNA Moderna,another genetically altering injection,you are assured is safe and effective,without full disclosure of the safe and effective studies in the lab and in real world settings.
The continuing new viruses and vaccines seems to be the greatest mega trend in all of humanity’s endeavors.if it’s not the only hope;those who have options must step up or fade in the wake of industrialized synthetic viral automation of a synthesized,near irreversible,earth,including all her inhabitants.
Pfizer’s former,chief science officer,Mike Yeadon and many medical doctors called for a suspension of all vaccines on 12/1/2020 by the European medical association.
Moderna and many mRNA vaccine pose ,these formulas will keep triggering antibody production from the body,not more side effects.
Virologist,Dr.Ian Jones at UK University of Reading and Duke University’s ,immunologist Dr.Stephanie Langel ,call for second opinions .Among thousands of similar professionals who have not created an actual option like Puerto Rico’s senior oncologist has mentioned throughout this document; making Puerto Rico the island of hope for a moon shot landing of super immunity and many other frontiers of a new lease on life and it’s very purpose for all 8 billion people.
Orlando Bravo,Puerto Rico’s first billionaire,dedicated to ending social,educational and financial inequity in his country of origin,is an ideal candidate to seed round the greatest inequality;the virally dwindling,lack of scaleable health and freedom for Puerto Rico and all countries.as a corporate turn-around artist,his greatest work of art can be the turn around of earth to a clean,bill of health.
12 people of this capital,ethic and visionary ability.may transcend what no government coalition may remedy as realistic windows to a better world ,close faster in all countries.
40 million of America’s 220 million population ,risk residential eviction from failure to pay rent ,after the cdc eviction moratorium expires in 12/31/2020,says the ,nonpartisan,nonprofit,Aspen Institute.the us census bureau reports 83 million find it difficult to meet food,rent ,mortgage and car payments.
Similar viral collapse of any semblance of equity or financial health in any demographic,is a reality for every country since only 12/19/2019.
If there’s an option to project warp speed ,the existence of the world as we’ve known it as humans may depend on its unicorns and more proof in Puerto Rico and a dozen other ,daring islands of success,outside Puerto Rico.
$1-12 billion from 1 to 12 private capital sources ,by March to July could build the foundation for a new year for a completely new world.
If a quorum of the 30 billionaires ,who’ve moved to Puerto Rico since the unique tax breaks were authorized,the recovery of Puerto Rico and all nations from the greatest storm,ever to reach landfall on planet earth,could actually win the discovery of a new world that ancient and modern,colonial,conquistadors would envy.
Nano super immunity now for the human community also needs a project warp speed team.the new atom and eve,victory oils and melanin millennium magic,need god fathers and mothers for a garden of eden gone global and ever more viral.
Investment proposals are available for wise and kind ,Capital sources as of 12/18/20 .starting at $20,000 and by 1/08,ranging from 1 billion and beyond.warp speed plan 2 ,is now here.
Collectively, this army of melanin covid over for billions-research team has validated the unique,universal ,critical ,affordable and mass scaleable,melanin production for the likely regeneration of human immunity to thwart covid and many new emerging infectious agents and go beyond a return to normalcy to a new world of super wellness,easily achieved,compared to complications of storage,training,tracking and testing;much of which may literally be obsolete.Flooding all cells with bioactive oxygen and hydrogen sounds innocuous until people all cellulary informed as to how primordial a regenerative dynamic that truly is–the biologically erudite-see it after reading anyone of the melanin immuno therapeutic studies of which there are dozens from over 10 countries.
Though these new era melanin civilization regeneration pioneers aren’t formally working together;one true investor in a unified melanin ecosystem will bring potential Olympic gold standards of daily living to the majority of the world’s population as never before.
The four pillars of melanin millennium science mastery mentioned in the previous paragraph,with their senior colleagues could easily deserve 12 Nobel prizes.
- My super life science partners proposed to develop a resonant melanin to super charge all cell-to-cell regenerative data in all human cells. As world leaders rush to synthetically immunize & 5g-300g, and genetically modify all 8 billion people by 2023; the new neuromelanin activation in each person may be the most primordial humane family plan to compliment all other best natural practices, products and policies whatever happens.The superneuroresonantmelanin has never been seen on this planet as a natural mass produced hue manna. It’s the opposite of conventional vaccine geoengineering.
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- The creative process for the end product synergizes the integrated use of scalar fusion hyperinfusion from a noted self-taught bioscalar physicist- eesystem.com, a new three-way scalar precursor holoscience, from ra-key.com (the source of the open sourced 432 universal therapeutics spectrum).On 12/7, England will be the first western country to start its mass injection campaign. Russia started their release roll out months ago before even completing late stage credit clinical trials.China started two months after the 12/19/2019 Covid release, and ispromoting their vaccines in competition with the warp speed vaccine in 100 of the 194 countries. Canada has stockpiled more vaccines per capita than any other country and promotes vaccine use with no clinical trials in it’s joint venture with Sino Biologics in China.China’s silk Road $8 trillion joint venture with 100 countries and counting may be the single largest mass vaccination trend. China’s Sino Vac wants to vaccinate every human and animal on earth for all diseases.Global health freedom prosperity options satellite to cell are exponentially expanding via quantumgeneration.io and qchange.ai.Quantacbd.com’s applied quantum physics process, in FDA trials for HIV prevention and treatment, the electron atom spin, in any specific atom, to match the receptor site of any human cell.They alone may exponentially reinvent resonant nano neuromelanin, essential oils, any vitamin, mineral, element, photon etc., and every phase of medicine, agriculture, water treatment, and reforestation.Our new world era of health and safety is inner standing and mimicking how all minute infectious agents and our intrinsic sovereign immune cellular secrets work and making them non active or super active in resonance with their higher natures ,on the sub atomic,atomic and molecular level by non synthetically,altering the the naturally occurring compounds like plants,essential oils,melanin minerals,vitamins,etc, on those invisible levels, on a long term basis.The following white paper that shows how and the resulting new era health wealth benefits for world wellness, now gone viral ,is one of 12 examples of true super health science for all human and planetary systems that a true dedicated reader will find by persistence and patience with the style and evidence of this living and, in flux. Non perfect,nano resonant article 314. Share this warp speed solutions series,or any part:and help it go viral in an ever expanding, populist,grassroots,groundswell spiral.
Page Authors:Arthur Mikaelian, PhDBeverly Hills, CA, USJon Kavulic Science Writer Boise, ID, USJosh Sinolinding Medolife CorporationR & D Lab.Los Angeles, CA, US
ABSTRACT Accelerated by broad legalization,the CBD market in the United States is exploding at an unprecedented rate. Quanta (headquartered in Los Angeles, California) originally entered the CBD product industry using first of a kind Mikaelian Resonance Technology or more commonly known in the CBD industry “Quanta’s patented polarization technology.”This technology is used to enhance the effectiveness and consistency of all Quanta products. For decades scientists have attempted to produce stable polarization of molecules and failed. This Polarization technology has achieved just that and is backed by multiple scientific tests confirming its efficacy. As such, Polarization is a historic technological breakthrough, which will revolutionize the way the world processes nutrients, medicines and active ingredients. Quanta possess an exclusive right to utilize the polarization in the preparation of its CBD (and other cannabinoid)products, offering it a unique competitive advantage in an emerging market.WHITE PAPER CBD in the Quantum Era –Polarization and its effects on anti-inflammatory abilities of cannabidiol (CBD)Quantum biology refers to applications of quantum mechanics and theoretical chemistry to biological objects and problems. Many biological processes involve the conversion of energy into forms that are usable for chemical transformations, and are quantum mechanical in nature.
CHALLENGES Despite the modern Gold Rush that is the wildly expanding CBD market, many challenges do exist, though these challenges can also be seen as opportunities. The mainstream American CBD market is so new no definitive CBD brands have been established as far as the public is concerned.Many brands have been shown to have problems with product quality and have produced significantly inconsistent results for the user. The key challenge is how can an emerging brand gain user loyalty and define itself in the market as separate from the pack.SOLUTIONS Quanta has addressed this problem through its exclusive license of the polarization technology. This allows for a unique marketing strategy and a product that produces superior, consistent results in a manner, which provides a cost benefit to the company. These results are backed by scientific data,resulting in a new form evidence-based alternative solution.
THE AMERICAN CBD MARKET CBD has absolutely exploded into mainstream America. The Brightfield Group (a marketing research group specializing in the CBD industry) has estimated that the American CBD market will reach a stunning $22 BILLION a year by 2022. CBD products are already available in national chains such as Kroger, CVS, Walgreens and Neiman Marcus.QUANTAINC.Quanta (a Nevada corporation) is a Los Angeles, California producer of CBD-products. Founded in 2016by Eric Rice.It currently produces a variety of CBD products such as muscle rubs and vapes but is currently in the process of branching out into other cannabinoid products including those consisting of CBN and CBG.Quanta has a unique competitive advantage in the CBD market as all its products are processed using a form of “polarization” via Mikaelian Resonance Technology. Quanta has exclusive rights to this technology for cannabinoid processing.QUANTA’S SCIENTIFIC STUDY ON THE ANTI-INFLAMMATORY EFFECTS OF POLARIZED CBD Background Inflammation is a rapid immune system response to injury and has been documented as early as the 1st century. The World Health Organization recognizes chronic inflammatory diseases as the leading cause of death in the world.Within the numerous inflammation pathways is a protein that has been the focus of many anti-inflammatory drugs since its discovery: Cyclooxygenase, isoform 2 (COX-2). COX-2, also known as Prostaglandin endoperoxide H synthase 2 (PGHS2). This makes the COX enzymes an attractive target for non-steroidal anti-inflammatory drugs (NSAIDs), though COX-2 may be a preferred target to avoid inhibiting COX-1’s gastroprotective products.Cannabidiol (CBD) has a multitude of potential medicinal uses as an anticonvulsant, sedative and antiemetic, but a topic of intense study has been (CBD) and (CBDA)’s role as an anti-inflammatory drug .
Materials and MethodsCBD was supplied in the form of an isolate powder. Purity was checked to be around 99.585% via UPLC-PDA by DB Labs (Las Vegas, USA). Recombinant COX-2 (human) enzyme was sourced from Cayman Chemical with their commercially available Fluorescent Inhibitor Screening Kit (Cayman Chemical Company, MI). CBD was prepared in ethanol prior to the assay. COX-2 activity was measured by the oxidation of 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) to resorufin as a byproduct of the reduction of PGG2 to PGH2. The assay was performed on a black 96 well plate in triplicate with fluorescence measured with emission/excitation at 485/535 nm.Figure 2.Bar graph showing the activity of COX-2 enzymes exposed to each sample. The negative control uses 100% ethanol to show uninhibited activity. Nonpolarized CBD showed a slight decrease in COX-2 activity, while High-Intensity Polarized (B) CBD and Medium-Intensity Polarized (C) CBD showed a marked reduction in anti-inflammatory activity.Figure 3. Bar graph showing the COX-2 inhibition effect of each sample in relation to the inhibition effect of the Negative Control sample (non-CBD). The negative control uses 100% Ethanol, which displayed NO inhibition in COX-2 activity. Nonpolarized CBD seemed to increase COX-2 inhibition, while High-Intensity Polarized (B) CBD showed drastic inhibition of COX-2 activity. Medium-Intensity Polarized (C) CBD showed a slight inhibition of COX-2 activity.Annabelle Manalo, PhDDr. Annabelle Manalois a CBD expert who has been repeatedly featured in Forbes Magazine and has spoken before Congress regarding the medical potential of CBD.In a laboratory experiment, reviewed and endorsed by Dr. Manalo, polarized CBD utilizing Mikaelian Resonance Technology (MRT) was tested in comparison to non-polarized CBD and a negative control in their inhibition of the COX-2 enzyme—a standard marker for inflammation.“COX 2 Inhibition is a key attraction to anti-inflammatory mechanisms today. The understanding of the role that cannabinoids play on COX 2 Inhibition, specifically CBD and its variations, is controversial. The data presented here lends promise to the importance of the stabilization of the CBD molecule whether by delivery or in this case, polarization. Furthermore, the possibility that CBD may cause an upregulation of COX2 while polarized CBD inhibits COX2 with such significance is a striking result and absolutely worth solidifying. Does the polarization of the CBD molecule mimic the function of CBDA? More studies should follow in order to prove such a novel claim.”-Dr. Annabelle Manalo (May 2019)
Results; The negative control showed no inhibition of COX-2 activity; non-polarized CBD showed significant inhibition of COX-2. However, heavily polarized CBD processed with MRT was shown to be 572.81% MORE EFFECTIVE in inhibiting the activity of COX-2 than non-processed CBD.In summary, though non-processed CBD showed effectiveness in inhibiting inflammation, CBD processed usingMRT was remarkably more potent, greatly inhibiting the activity of COX-2.In a brief exploratory study, polarized ibuprofenalso showspromising results.The study is currently ongoing.SUMMARY AND FUTURE OUTLOOKDue to the exclusivity Quanta possesses to access Polarizationfor all cannabinoids, Quanta is exceedingly well-positionedto become a dominant brand due to its marketing and product quality advantages. In addition, MRT’s cost benefit advantage should lead to enhanced profitability for the company.For the future Quanta is looking to expand into other product lines such as nootropics, anti-aging, skin care and agriculture. It should be noted, the studies done on the effectiveness of Polarizationwere using the fifth generation of the invention. The inventor, Dr. Arthur Mikaelian, is presently finalizing the completion of the seventh generation of the technology which he has optimistically described as a “giant leap forward” in the efficiency of the innovation.These are certainly historically exciting times for the CBD industry, Quanta, and the patented polarization technology.EXTENDED INFORMATION: MIKAELIAN RESONANCE TECHNOLOGY Mikaelian Resonance Technology (Polarization)appliesnon-invasive, quantum biotechnology that is capable of significantly increasing the potencyof bioactive natural organic or synthetic products, in some cases from 200% to 500%, as well as increasing their absorption and bioavailability.Dr. Arthur Mikaelian, the inventor of Polarization,received a full US Patentin 2012for his polarization technology. It is a profound scientific breakthrough that excites the electron state of specific molecules so that they more readily engage in biochemical activity. Polarizationdoes this by specifically altering the spin of the electron field and pushing these electrons into a higher, outer orbit. In doing so, these MRT-processed molecules have a greater zone in which they can “latch onto” other molecules making them more reactive, intensifying their biochemical activity manyfold.Scientists have experimented for years with the excited electron state, but have repeatedlyfailed in achieving any consistent stabilization of the excited electron state, sometimes with the results only lasting seconds. MRT is a game-changing breakthrough in that the fifth-generation technology has produced commercially viable stability. This will allow manufacturers to reliably warehouse
Their product without fear of losing potency,so that they can nimbly respond to supply-chain needs.Polarizationwill revolutionize nutritional, supplemental, and pharmaceutical processing and manufacturing. Polarizationis inexpensive to implement in the manufacturing process and allows Quanta to use smaller amounts ofactive ingredients,while having stronger results for the consumer. This is a significant cost benefit to the company, as well as allowing for unique promotional claims using evidence-based results.Polarizationis going to change theway we do things and the way that products are formulated. In any industry if a business has an exclusivelicense to utilize Polarization, they will have a unique competitive advantage within that industry.This is the future.EXTENDED INFORMATION: THE AMERICAN CBD MARKETCBD (cannabidiol) is a marijuana-derived substance which in contrast to THC (tetrahydrocannabinol) does not have intoxicating effects when consumed. CBD products have seen explosive growth in recent years with truly impressive growth predicted for at least the next five years.In 2018 the American CBD market hit $600 million, a 200% growth over the previous year. This was before the Farm Bill was passed on December 20thof that same year.The Farm Bill has legalized the production of hemp with a THC level of 0.3% or less, but placed no limitation upon its CBD content. As a result, we are expecting an explosion of legal CBD production in the United States, as farmers across the country have embraced theindustry.As quoted in CBS News and Rolling Stone Magazine, The Brightfield Group (a marketing research group specializing in the CBD industry) has estimated that the American CBD market will reach a stunning $22 BILLION a year by 2022 with CBD products beingmade available in national chains such as CVS, Walgreens and Neiman Marcus.0 5 10 15 20 25 2018 2022 $ Billions American CBD Market $22 billion by 2022 American CBD Market Figure 1. As quoted in CBS News and Rolling Stone Magazine, The BrightfieldGroup (a marketing research group specializing in the CBD industry) has estimated that the American CBD market will reach a stunning $22 BILLION a year by 2022 with CBD products being made available in national chains such as CVS, Walgreens and Neiman Marcus.
EXTENDED INFORMATION: VARIOUS STUDIES SHOWING THE POLARIZATIONTECHNOLOGY’S EFFECTIVENESSQuanta Inc. does not want to limit its activity to cannabinoids only. It would like to expand its product lines into nootropics, anti-aging, skin care and agriculture markets.Agriculture Study The followingresearch was aimed to establish effects of the Polarization Technologyon Auxin plant growth hormone.This work investigated the exogenous effect of Mikaelian Resonance frequencies to Ryegrass seeds germination,where specific targeted Electromagnetic photon emission was directed to Ryegrass seeds prior to seeding.Stem Cell StudyThe aim of the belowstudy was to assess the migratory capability of mononucleated pluripotent stem cells (MNCs) to target human hepatic sinusoidal endothelial cells (HHSEC, Adult) by integration of DNA conjugated gold nanoparticles(AuNP).0% 20% 40% 60% 80% 100% Growth (%) Summary of Study Comparing Growth of Non-polarized vs (-) Polarized Ryegrass Seeds Control (-) Polarized Figure 4.MRT was used to target auxin plant growth hormone with ryegrass seeds. The polarized seeds germinated 400% more quickly than non-polarized seeds and reached full maturity 260% faster than non-polarized seeds.
Study of the Effects of Scorpion Venom on a Pancreatic Ductal Adenocarcinoma Cell Line
The study has validated an in vitromodel, where in the various venom concentrations treated with polarization and conjugated with gold nanoparticles (AuNP), we observed a >95% apoptosis in the human pancreatic ductal adenocarcinoma (HPDAC) cell line.0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 MNC Samples Live MNC Count Exp. #2 – MNCs Attached to Liver Cells MNC conjugated with (+ pol) AuNP 24 hrs. MNC conjugated with (+ pol) AuNP 2 hrs. MNC conjugated with (+ pol) AgNP 2 hrs. MNC Control 0% 20% 40% 60% 80% 100% Venom Concentrations % SIA Specific Induced Apoptosis (SIA) in Human Pancreatic Cancer Line 1.4 μg/ml 2.8 μg/ml 4.2 μg/ml 5.6 μg/ml 5.6 μg/ml & AuNP (+ pol) 5.6 μg/ml & AuNP (- pol) Figure 5.Stem cells conjugated with gold nanoparticles polarized with MRT bound with liver tissue 37% more efficiently than stem cells not conjugated with polarized gold nanoparticles.Figure 6.In one study a polarized solution of scorpion venom and gold nanoparticles killed OVER 95%of cultured pancreatic cancer cells without exhibiting any damage to healthy cells.
Geneticist Ronald Crystal, studied the progenitors of world popular mRNA vaccines, 20 years ago, with Russian MD, pulmonary immunologist Alexander Chuchalin. Dr. Chuchalin is Russia’s top doctor and respiratory expert, who left the Russian ministry health council after president Putin fast tracked what he claims is the first covid vaccine, Sputnik V, after August 11, 2019 when Putin claimed he gave it to his own daughter.
Rushing vaccines to public use without long-term trials and transparency as her side effects and worse, is literally trying to outpace the rate of infection, and leading physicians in over 100 countries say it’s a pandemic, in and of itself.
Global vaccination with no exceptions is endorsed by all 194 country leaders in GAVI (Global Alliance for Vaccines and Immunizations), and in America, Project Warp Speed. Currently, two billion doses of covid-19 have been ensured by GAVI. 2 billions doses for 94 countries will be distributed in 2021 by first quarter.
Australia’s medical doctors association president, Omar Khorshid, agrees with Russia’s Dr.Chuchalin, and Sandra Frihoffer, American MD and trainer of MDs, former 4 year CNN medical correspondent and adviser to the CDC and FDA.Professor Rachel Martin,is UC Irvine’s lead exobiology investigator predicting protease properties on extremeophile organisms on land, sea, air in earth and elsewhere.
Computational molecular modeling finds how molecules and microbes stay healthy or not. Sound relevant?
All things all relevant;depending on the ethics and bandwidth of the student:these days;few on earth may say they know enough to bring earth’s populations the life quality all seek:knowingly or not.
Protease are critical to all life and all viral function.melanin activation is pivotal in mitigating the furin protease pivotal to the survive and the thrive covid and colleagues,auto pilot design.
Patient readers will find a peer reviewed study from Korea on melanin and before and after covid patient x rays recovery with hee ho park’s version of the nano resonant melanin we independently foresaw in article 314’s title.
RNA from each cells nucleus instructs the ribosome 3-d printer for protein production .the corona RNA mimics human RNA,with out its 3 functional proteases .covid etc..is over now.
Martin’s lab is repurposed to find the stealth ,covid 3 main protease mutantions so people can collectively out smart all viral agents in all form ,and learn and master the secrets of human cell super sovereign wellness.get this cell phone provider and charge all your 1.6 super computer cells with this data plan.
If readers will send 313 or parts to all the references in this cellular common sense we may have a global sovereign cellular independence ,not another declaration with an intrinsic expiration.
If eesystem.com is the richest source of life wave particle,field generation;human photosynthesis.com is the the world’s most bio active receptor sites that may defend,amend and ascend every cell in every human and animal as well as the most activating formula to automate and enrich the continuation of that process.
Melanin activation of higher states of awareness and compassion is not a pill spectator sport and requires an intention to improve regardless of past or present and innately compliments all collaborative,life positive views.
Present policies of all national governments is accelerating toward less choices and more synthetic outer and inner environments.
The path of least resistance for a dialogue between all divided parties and individuals may be irreversibly elected via 1 million to 100 million people freely choosing the melanin activation party and open ended partnerships.
The hope for 12/16/2020, FDA Puerto Rico authorization of herd scale trials of unique essential oils in public schools and among the medical doctors themselves, to stop and prevent Covid, and other such infectious agents, may be the most pivotal doorway to human health wealth freedom of choice; allowing similar innovations to naturally, safely, and quickly immunize all nations for an unheralded unification, not extinction devastation. The hope for trial will be conducted by Dr. Marcial Vega, oncologist and professor of three medical schools.
The covid alter ego, second wave is here; the certificate of vital individual’s defense.
Makes super computing analysis of covid over remedies 147 trillion times a second.intrucept.com says their vast library of cell immunity factors may add 5,000 different immunity improvers to any covid and other global health crises.
On 12/16 we spoke to Jerome Baudry,Hewlett-Packard partner at Huntsville,Alabama,whose supercomputers found 53 natural compounds from indigenous traditions that may add to new molecular and sub atomic super human immunity and longevity for billions in way that far exceeds,yet may work with project warp speed,agreed by all presidents to vaccinate the world population multiple times by 2023.
Baudry’s international coalition super cloud computer has found 300 naturally occurring compounds that may have high performance covid etc relevance to supplement any vaccine and or create a new era of nature’s vaccines.this warrants mega media attention and is one of one thousand indigenousi digital global normalcy that very few world influencers are evaluating.
These compounds find viral receptor sites ,neutralize them and even help human cells be stronger,longer in multiple ways;surpassing survival and building paths to humanity thriving.
Covid’s 2 protein commandeer human cells to virally mass assemble its covid proteins needed to do a corporate take over of all human cells.
The 3 cellular vote thieves formula of covid are the the ,mpro and the spike protein that attaches to human cells-the crown like ace2,seeks to start the invasion on the cell surface.
Baudry’s warp speed cell wellness army,found an average of 30 natural compounds that intercept and neutralise all 3 key entry ways designed by covid-19 and potentially, any non benign virus ,of any origin.
The Texas based super computer called sentinelldoes 147 trillion calculations per second .outlearning any virus and person ;excepting humans will to learn new era,quantum leap all cells wellness.hew
Lett packard’s sentinel can learn in weeks what took up to 10 years to learn
Fast track to a thriving humanity anyone?
Dr.Veneetha Menon,assistant computer science is a partner of Dr.Baudry, Jim Steele is a project contact.
Dr.Gary Zank,also at UAH and his quest for extra terrestrial life will find the cosmic melanin over covid an option that’s simultaneously here and out if this world.Zank and the Hubble space telescope team may find interstellar melanin is the hidden hand in heath for every living cell,Star and solar system and the omnipresent and eternal molecule that needs human ethnics to do its best work for life quality in any systems,living or inanimate,anywhere and the the chip in the cosmic computer.
Dr. Zank ,Dr. Gang li and Dr.Juanxiang Hu,seek to learn where life prospers in any environment;now that’s your mission and that of all people;as it’s always been.
The melanin molecule may be the original,eternal,life thriving molecular best friend for all collaborative life forms.mimic it ;learn,amplify,share ,thrive and revive earth’s life while you still can.
In the new pandemics,who can afford not to get a 2nd opinion. There’s a 1000 different covid and more therapeutics with ctap,found at N.C. Biotech center. Can you afford not to do your own research;new options may be a 1000 times safer and effective than the sources you currently use.
- Rate your covid relevant I.Q. In 5 minutes:300 I.Q- your FDA approved clinicals trials to prevent and treat covid etc non synthetically, start by 1/20/2020, you won’t be bought by vested interests and have an executive team in place by 12/15/2020.200 I.Q.-200 you inform 3 billion people by 1/20/2020 that any action plan similar to the one described in the preceding sentence is a reality in a documentary, like plandemic that had 8 million to 2 billion viewers in 90 days, took $2000 to make and was seeded by the book,plague of corruption. 180% -you read 314 document A-Z, checking all references, realize 20-180 IQ may be true and resolve to take action so 100 people per month,simply do the same.179 I.Q. and below;you motivate a risk taker and Manhattan moon shot maker like Andy Ruiz,jr.,who has a minimum,1 million action oriented fans to read 314 -a-z by 1/1/2020. Prepareforchange.net, update to cv-19 is a 360 on vaccination, legal, freedom of choice options, and updates on all aspects of covid over.
CAN PURE-LIGHT® HELP PREVENT THE SPREAD OF CORONAVIRUS?
The Pure-Light® Super-Oxygen® light bulbs and surface coating technology… have repeatedly shown the ability to kill 99.9% of harmful viruses and bacteria that it has been tested against within hours on surfaces and in the air within 8-10 ft of the bulb and a coated surface when exposed to a light source, with the PLT bulbs actually being the light source. (see lab reports and research). The Super-Oxygen®technology has been shown to kill SARS viruses, which Coronovirus is a typeof. And so the answer is a very probable YES, though it has not been specifically tested against the Coronavirus Covid-19.
WHAT MAKES PURE-LIGHT® SUPER OXYGEN® TECHNOLOGY SO IMPORTANT?
ANSWER: CONTINUAL PROTECTION NO MATTER THE CURRENT DISEASE “OUTBREAK”
Every year there are seasonal cold, flu and disease outbreaks. And of course there is the ever present problems with Toxic Mold, E-Coli, Salmonella, MRSA, and a dozen other deadly and harmful pathogens. Because of the way that the Pure-Light Super-Oxygen technology works, these negative super oxygen molecules are attracted to these pathogens and literally dissolves them…no matter what new version of pathogen it is. This is a naturally occurring process that nature has been using for millions of years outdoors and in our own bodies that Pure-Light has harnessed and made affordable to help protect our families and the planet from the some of the deadly contaminants there are in the world.
A $25 charcoal mask is available via buyactivatedxmcharcoal.com. Activated charcoal supplements may purge heavy metals etc. in some vaccines.
Acute respiratory distress syndrome
Novel coronavirus disease 2019
Type I interferon
Severe acute respiratory syndrome coronavirus 2
- Vitamin C:
- Vitamin D:
Vitamin- D receptor
Tumour necrosis factor
Major histocompatibility complex (MHC) class 1
Transmembrane protease serine 2 precursor
How Bipolar Ionization Works to Clean the Air of Pollutants
Airborne particles are charged by the ions causing them to cluster and be caught in filters
As they divide to reproduce, bacteria and virus cells bond with oxygen ions and are destroyed
Odorous gases and aerosols oxidize on contact with oxygen ions and are neutralized
Oxygen ions cause a chemical reaction with VOCs breaking down their molecular structure
See Bipolar Ionization in Action
Watch Plasma Air prevent mold growth over 6 days in a controlled laboratory experiment.
Watch Plasma Air remove smoke in 60 seconds in a controlled laboratory experiment.
How Plasma Air Compares to Reactive Air Purification Methods
Plasma Air’s bipolar ionization technology is a superior solution because it proactively treats the air in the occupied space at the source of contamination. Traditional end-of-pipe solutions utilize a reactive “pass-through” or “filtered” approach.
Plasma Air Particle Filtration HEPA / Fine Grain Filters1 Carbon Filters2 Ultraviolet3 Biofilters Chemical Scrubber PCO4 Description Bipolar Ionization Pre-filters, bag filters, pre-treated filters, fiberglass filters for larger airborne particles Captures fine airborne particles Absorbs and filters chemicals Ultraviolet light kills germs and remove airborne particles Treats exhausted air through biological media by reacting with contaminants Treats exhausted air through chemical media by reacting with contaminants Filtered media is coated with material that reacts with chemicals Energy savings Up to 30% None None None None None None None Pressure drop Low Low High Medium None Very high High Medium Particle size Small Large(> 5um) Small(< 0.01um) N/A N/A N/A N/A Large Treats air in room Yes No No No No No No No Treats Make-up / Supply air Yes Yes Yes Yes Yes No No Yes Treats return air Yes Yes Yes Rarely Yes No No Yes Treats exhaust air Yes Rarely No Yes Rarely Yes Yes Rarely Capital costs Low Low Medium High Medium Very High Very High High O&M Costs Low Low Low to medium High Low Very High Very High High Energy Costs Low Low High High High High High Medium Disposal Costs None Low Depends on contamination High Medium High High High Success rates5 99% Low 0-99.7% Low Low Low Low Low1) HEPA filters add significant pressure drop resulting in larger horsepower motors and increased fan energy. 2) Carbon has high first cost, additional space requirements in the air handling units and prohibitive media replacement costs. 3) At high levels, UV can create noxious gases and is mutagenic. Bacteria kill rates are negligible with no effectiveness in the occupied space. 4) PCO can produce formaldehyde. 5) Success Rate based on independent tests on airborne pathogens
What Are Oxygen Ions?
Ions are molecules or atoms that contain an electrical charge and exist in nature in various sizes. Small ions only last between 30 and 300 seconds before losing their charge, but are extremely active.
Small ion densities range from 900 to 1,100 negative ions and 1,000 to 1,200 positive ions per cubic centimeter (ions/cm3) in pristine natural environments. At sea level ion density is typically around 500 negative and 600 positive ions/cm3. In cities and inside buildings ion levels drop by 80% to 95% and can be barely detectable in small spaces.
As ion density decreases, so does the air quality. By increasing the quantity of both positively and negatively charged small oxygen ions, air quality is improved. This is the basis of Plasma Air’s bipolar ionization technology.
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Details of mass production at low costs could make Artuo’s melanin immunity formula vastly more available and affordable.
Production of Melanin Pigment by Fungi and Its Biotechnological Applications
By Sandra R. Pombeiro-Sponchiado, Gabriela S. Sousa, Jazmina C. R. Andrade, Helen F. Lisboa and Rita C. R. Gonçalves
Submitted: June 1st 2016Reviewed: December 28th 2016Published: March 1st 2017
Production of the microbial pigments is one of the emerging fields of research due to a growing interest of the industry for safer products, easily degradable and eco-friendly. Fungi constitute a valuable source of pigments because they are capable of producing high yields of the substance in the cheap culture medium, making the bioprocess economically viable on the industrial scale. Some fungal species produce a dark-brown pigment, known as melanin, by oxidative polymerization of phenolic compounds, such as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol or 1,8-dihydroxynaphthalene (DHN) or 3,4-dihydroxyphenylalanine (DOPA). This pigment has been reported to act as “fungal armor” due to its ability to protect fungi from adverse conditions, neutralizing oxidants generated in response to stress. Apart from the scavenging activity, melanin exhibits other biological activities, including thermoregulatory, radio- and photoprotective, antimicrobial, antiviral, cytotoxic, anti-inflammatory, and immunomodulatory. Studies have shown that the media composition and cultivation conditions affect the pigment production in fungi and the manipulation of these parameters can result in an increase in pigment yield for large-scale pigment production. This chapter presents a comprehensive discussion of the research on fungal melanin, including the recently discovered biological activities and the potential use of this pigment for various biotechnological applications in the fields of biomedicine, dermocosmetics, materials science, and nanotechnology.
- biological activity
- industrial applications
chapter and author info
Considering the harmful effects of synthetic dyes on human health and to the environment, developmental process for obtaining pigments from natural sources has become significant worldwide. Microbial pigments have gained attention owing to a growing interest of the industry in safer products, easily degradable, eco-friendly and do not cause harmful effects. The pigment production from microorganisms is considered more advantageous because it is a more efficient and cost-effective process than chemical synthesis of pigments. Microorganisms are also more feasible sources of pigments in comparison to pigments extracted from plants and animals because they do not have seasonal constraints, do not compete for limited farming land with actual foods, and can be produced easily in the cheap culture medium with high yields [1–6]. Besides, the microorganisms produce an extraordinary range of pigments that include several chemical classes such as carotenoids, melanins, flavins, phenazines, quinones, monascins, violacein, or indigo, as shown in Table 1.
Pigment Microorganism Indigoidine (blue-green) Streptomyces aureofaciens CCM 323, Corynebacterium insidiosum Carotenoid (orange) Gemmatimonas aurantiaca T-27 Melanin (black-brown) Kluyveromyces marxianus, Streptomyces chibanensis, Cryptococcus neoformans, Aspergillus sp., Wangiella dermatitidis, Sporothrix schenckii, and Burkholderia cepacia Prodigiosin (red) Serratia marcescens, Rugamonas rubra, Streptoverticillium rsubrireticuli, Serratia rubidaea, Vibrio psychroerythrus, Alteromonas rubra, and Vibrio gazogenes Zeaxanthin (yellow) Staphylococcus aureus, Vibrio psychroerythrus, Streptomyces sp., and Hahella chejuensis Canthaxanthin (orange) Monascus roseus, Bradyrhizobium sp. Xanthomonadin (yellow) Xanthomonas oryzae Astaxanthin (red) Phaffia rhodozyma, Haematococcus pluvialis Violacein (purple) Janthinobacterium lividum Anthraquinone (red) Paecilomyces farinosus Halorhodopsin and rhodopsin (pink Halobacterium halobium Rosy pink Lamprocystis roseopersicina Violet/purple Thiocystis violacea, Thiodictyon elegans Rosy peach Thiocapsa roseopersicina Orange brown Allochromatium vinosum Pink/purple violet Allochromatium warmingii
Among microbial species, fungi represent an economically significant source of these compounds because they can act as microbial cell factories producing high yields of metabolites with great diverse chemical structures combined with ease of large-scale cultivation [7–9].
As shown in Table 1, some fungal species produce a dark-brown pigment, known as melanin. In general, this pigment is located in the outermost layer of the cell wall associated with chitin (referred as cell wall-bound melanin), but in some fungi, melanin can also be found outside the fungal cell, usually in the form of granules in culture fluids .
Fungal melanins are negatively charged, hydrophobic pigments of high molecular weight formed by oxidative polymerization from phenolic and/or indolic compounds, such as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol or 1,8-dihydroxynaphthalene (DHN) or 3,4-dihydroxyphenylalanine (DOPA). Most Ascomycota fungi synthesize DHN-melanin from the polyketide synthase pathway, whereas few species are able to produce melanin through L-DOPA, in a pathway that resembles mammalian melanin biosynthesis [11–13].
The melanin pigment is not essential for fungal development, but it has been reported to act as “fungal armor” due to its ability to protect the microorganisms from harmful environmental conditions. In vitro studies have shown that melanized fungi are more resistant to UV light-induced and oxidant-mediated damages, temperature extremes, hydrolytic enzymes, heavy metal toxicity, and antimicrobial drugs than those nonmelanized [10, 14–17]. Recent studies have shown that in industrial and roadside areas, there is an increase in the proportion of dark melanin-containing fungi, as Cladosporium and Alternaria, which were more resistant to contamination by heavy metals and unsaturated hydrocarbons. Radionuclide contamination also led to a change in fungal communities, with an increased proportion of melanized fungi. For example, melanized fungal species as Cladosporium spp., Alternaria alternata, Aureobasidium pullulans, and Hormoconis resinae were found to colonize the walls of the damaged reactor at Chernobyl where they are exposed to a high constant radiation field [18, 19].
The presence of melanin in the cell wall is also correlated with enhanced virulence of parasitic fungi, as Paracoccidioides brasiliensis, Sporothrix schenckii, and Exophiala (Wangiella) dermatitidis [17, 20, 21]. This pigment protects the conidia against digestion by proteases and hydrolases secreted by competitive microorganisms or against bactericidal and fungicidal proteins of animal origin, such as defensins, magainins, or protegrins . This effect was observed for Cryptococcus neoformans, whose in vitro melanization has been associated with resistance against host effector cells, oxidants, microbicidal peptides, and amphotericin B [23–25], and in Wangiella (Exophiala) dermatitidis, when the polyketide synthase gene WdPKS1 associated on melanin production was disrupted, this strain has become more susceptible to voriconazole and amphotericin B . Others studies suggest that melanin contributes to fungal pathogenesis because this pigment alters the host defense response mechanisms, decreases phagocytosis, and reduces the toxicity of microbicidal peptides, reactive oxygen species, and antifungal drugs as well as to play a significant role in fungal cell wall mechanical strength [27, 28].
Although the molecular structure of fungal melanin remains enigmatic, significant progress has been made in understanding particular aspects of its macro- and microstructure. These advances allow to elucidate the molecular mechanisms of the various biological functions of melanin . Studies have shown that the effect of melanin enhancing the survival of fungi under adverse conditions can be mainly due to its powerful free radical scavenger properties, acting as a “sponge” for other free radicals generated by the fungus in response to environmental stress [20, 29, 30]. Apart from this scavenging ability, melanin exhibits other biological activities, including thermoregulatory, photoprotective, antimicrobial, antiviral, cytotoxic, anti-inflammatory, radioprotective, and immunomodulatory [13, 17, 18, 31–34].
Since melanin has characteristics of functional materials and bioorganic, a growing number of researchers see this pigment with great interest, taking advantage of their properties for numerous biotechnological applications in cosmetics, pharmaceutical, electronic, and food processing industries [12, 19, 35].
The purpose of this chapter involves a comprehensive discussion of the research on fungal melanin, including the recently discovered biological activities and the potential use of this pigment for several biotechnological applications. Additionally, we discussed the ways to explore the metabolic potential of the pigment-producing fungi by manipulation of cultivation conditions to improve performance of the process, increasing yields, and reducing cost, for large-scale production.
2. Factors influencing the melanin production
Microbial pigment production is now one of the emerging fields of research due to its potential for various industrial applications, as foodstuff, cosmetics, pharmaceutical, and textile manufacturing processes. However, it is known that for the success of microbial fermentation processes, it is necessary to choose the correct productive culture strain and to determine the appropriate cultivation conditions [4, 8, 36].
An ideal pigment-producing microorganism should be capable of using a wide range of C and N sources; must be tolerant to pH, temperature, and minerals concentration; and must give reasonable pigments yield. The nontoxic and nonpathogenic natures, coupled with easy separation from cell biomass, are also preferred qualities. The potential of using filamentous fungi as pigment sources is due to their extraordinary metabolic versatility because they can be cultivated over a wide range of temperatures (10–50°C), pH (2–11), salinity (0–34%), and water activity (0.6–1) and under oligotrophic or nutrient-rich conditions. They can grow in different culture systems (submerged and solid), and fermentation protocols have been established for large-scale industrial processes. In addition, these organisms can be genetically modified to increase productivity and quality of the produced pigments [37, 38].
In order to improve performance and reduce the cost of pigments produced by microbial fermentation, it is essential to identify the nutritional and physical factors that have a greater influence on the cell growth and metabolite biosynthesis [4, 6, 39, 40].
Several studies have shown that the composition of the growth medium, nature and concentration of carbon and nitrogen sources, minerals, vitamins, temperature, pH, the presence of oxygen and aeration, light, stress, and irradiation, among others, affect the growth and pigment production in fungi and that the manipulation of the culture conditions can result in enhanced pigment production [41–47].
Experimental evidences indicate that the growth temperature influences the performance of the pigment production process, but this effect depends on the type of organism. Pseudomonas requires 35–36°C for its growth and pigment production, while in Monascus purpureus, maximum pigment production was observed at 30°C with a reduction of the yield at 37°C . Another study in Monascus sp. J101 reported that the yield of pigment at 25°C was ten times higher than at 30°C, probably due to long growing (120 hours) and lower viscosity of the broth at 25°C compared to 30°C . Studies developed in our laboratory, using a melanin-overproducing mutant (MEL1) from Aspergillus nidulans fungus, showed that the higher production of pigment occurred at incubation temperature of 28°C compared to 37°C .
Researches support that the pH of the medium also affects the growth of fungi and type of pigment produced. In species of Monascus, the pH influences the yield and quality of the produced pigment, with the highest red pigment excretion and production at alkaline pH [51, 52]. Studies on wood-inhabiting fungi indicate that pH of the substrate potentially plays an important role in fungal melanin formation. Fungi Trametes versicolor and Xylaria polymorpha tested on wood substrates produced maximum pigmentation at the pH range 4.5–5.0, except for Scytalidium cuboideum, which produce maximum intensity of red pigment at pH 6 and blue pigment at pH 8 . In our study with the hypermelanized mutant (MEL1) from A. nidulans, we observed an increase in the production of pigment when the initial pH of the culture was at 6.8 compared to pH 8.0 . Metabolically, the effects of pH and temperature on fungal pigment production is associated with changes in protein activity, so that the culture conditions may control certain activities such as cell growth, production of primary and secondary metabolites, fermentation, and oxidation processes of the cell .
The influence of light on intra- and extracellular pigment production was studied in five pigment-producing fungi: M. purpureus, Isaria farinosa, Emericella nidulans, Fusarium verticillioides, and Penicillium purpurogenum . These authors concluded that the cultivation in the total absence of light increased biomass and production of extracellular and intracellular pigments in all fungi. The fungi grown under red light have no effect, and green or yellow light resulted in worsening effect in all the fungi, thus postulating the existence of photoreceptors responsive to dark and light in all the fungi. In a similar study,  noted that the production of pigment by Monascus species also was favored when the fungus was grown in the dark.
Some studies report that the pigment synthesis requires proper aeration probably related to the oxygen dependency of some enzymatic reactions responsible for the production of pigment. In Monascus ruber, it was observed that the highest levels of pigments production were obtained at an aeration rate of 0.05 L min−1, which appeared to be clearly sufficient for providing the fungus with oxygen and removing carbon dioxide . In our studies, it was noted that no melanin pigment production takes place during stationary cultivation of hypermelanized mutant (MEL1) from A. nidulans, indicating that the formation of this pigment involves the oxidative polymerization of the precursors .
Carbon and nitrogen are necessary for cellular metabolism, and these sources are related to the formation of biomass, the type produced pigment, and the yield of the desired substance. These nutrients may regulate the expression of genes of interest and activate important metabolic pathways for the production of pigments [45, 58, 59]. In general, glucose, an excellent carbon source for growth, interferes with the formation of many secondary metabolites, including pigments. For example, the pigment production by Penicillium sp. was evaluated in the presence of 10 different carbon sources, and the maximum mycelial growth was obtained with fructose, whereas the maximum pigment production was obtained with soluble starch . This result shows that the increased biomass does not necessarily result in increased pigment production because pigments produced by fungi are secondary metabolites whose production usually occurs at the late growth phase (idiophase) of these microorganisms . The pigment production capability of fungal species belonging to the genera Penicillium, Aspergillus, Epicoccum, Lecanicillium, and Fusarium was evaluated in different culture media, and the results showed that the complex media, as potato dextrose (PD) and malt extract (ME), favored increased pigment production . According to the authors, these media contain nutrients that can regulate the expression of genes of interest and activate metabolic pathways important for the production of pigments.
Studies have demonstrated that the promoting or repressing effect of a nitrogen source on pigment production is strain dependent. It has been reported that various types of peptone, used as a nitrogen source, are able to promote an increase in the production of pigments in many species of fungi [55, 59, 62, 63]. However, M. purpureus was not able to grow in media containing peptone, and a maximum yield of the pigment was achieved when the media were supplemented with yeast extract (1%) and monosodium glutamate (5%) as nitrogen source . In M. ruber, the use of glutamic acid as a nitrogen source showed promising results, either as stimulating the accumulation of extracellular pigments or contributing to increase the efficiency of the pigment production process . The production of high amounts of extracellular melanin by the fungus Gliocephalotrichum simplex was obtained in cultures supplemented with tyrosine (2.5%) and peptone (1%) .
The optimization of medium composition is an important strategy to increase pigment production because some sources of carbon and nitrogen can be more easily assimilated and promote higher yields of the desired product. During the optimization experiments to enhance the production of melanin by Auricularia auricula, it was observed that soluble starch, tyrosine, peptone, CaCO3, and K2HPO4 had positive effects, while glucose, (NH4)2SO4, MgSO4, CuSO4, and FeSO4 negatively impacted melanin production . In other study with A. auricula, it was observed that yeast extract, tyrosine, and lactose have significant effects on pigment production and the optimization of medium resulted in 2.14-fold higher melanin concentration than that of the unoptimized medium .
Since the substrates for the production of pigment strongly influence the cost of the bioprocess, there is a need to select cheap and efficient substrates to make the process economically viable on the industrial scale. Large amounts of agro-industrial residues generated from diverse economic activities have attracted strong industry interest on the utilization of these residues as inexpensive substrates to support the growth of microorganisms in bioprocesses. This strategy may represent an added value to the industry and also helps in solving pollution problems, reducing or preventing their disposal in the environment [1, 66, 67].
Various studies have reported the successful utilization of agro-industrial residues for the production of fungal pigments. The use of corn cob powder as a substrate for production of pigments by M. purpureus resulted in greater pigment production  than other substrates, as jackfruit seed , corn steep liquor , and grape waste . In the black yeast Hortaea werneckii, it was observed that rice bran acts as the cheapest source for increased production of melanin by than wheat bran and coconut cake . Wheat bran extract, L-tyrosine, and CuSO4 represent the best combination of medium components to obtain the maximum melanin yield from the fungus A. auricula in submerged culture . A study conducted in our laboratory evaluated the use of corn steep liquor, sugarcane bagasse, and molasses as nutritional source on pigment production by melanin-overproducing mutant (MEL1) from A. nidulans. We observed that, in the presence of 0.2% corn steep liquor, an increase in the pigment production occurred, while a high yield of biomass was obtained at a concentration of 2%. The supplementation of medium with molasses and sugar cane bagasse hydrolysate did not have a positive effect on pigment production but promoted an increase in the fungal growth. These results indicate that corn steep liquor contains substances that stimulate the synthesis of pigment and it represents a low-cost fermentation medium for large-scale production of the pigment melanin by MEL1 mutant for future industrial applications .
3. Pathways of melanin biosynthesis
Various techniques, including electron paramagnetic resonance , X-ray diffraction , infrared, ultraviolet and visible spectroscopy , and nuclear magnetic resonance , have been used to elucidate the melanin structure from different organisms. These studies have shown that fungi can produce different types of melanins by oxidative polymerization of phenolic or indolic compounds [11, 27].
Melanin in cell walls of Basidiomycotina is derived from phenolic precursors, as glutaminyl-3,4-dihydroxybenzene (GDHB) or catechol. In the parasitic fungus Ustilago maydis, polymerization of catechol dimers with the formation of fibrils of melanin was shown . The precursor of melanin in Agaricus bisporus and other Basidiomycetes is a metabolite of the shikimic acid pathway-γ-glutaminyl-4-hydroxybenzene oxidized under the action of peroxidase and/or phenolase into γ-glutaminyl-3,4-benzoquinone, followed by its polymerization . C. neoformans, a pathogenic basidiomycetous yeast, is known to synthesize DOPA-melanin when o-diphenolic compounds, such as 3,4-dihydroxyphenylalanine, are present in the culture medium. This fungus may use a wide array of substrates, such as D- and L-dopamine , homogentisic acid , catecholamines, and other phenolic compounds , maximizing its ability to produce melanin. Polymerization of exogenous substrates in this fungus occurs under the action of laccase . However, it is important to emphasize that different properties are observed for melanins derived from different substrates. Comparison of the catecholamines L-dopa, methyldopa, epinephrine, and norepinephrine shows differences in term of color, yield, and thickness of the cell wall melanin layer. It was also observed that the pigments vary in the strength of the stable free radical signal detectable by EPR [13, 83].
In the Ascomycota fungi, melanin pigment is generally synthesized from the pentaketide pathway in which 1,8-dihydroxynaphthalene (DHN) is the immediate precursor of the polymer, as described by Bell and Wheeler  based on genetic and biochemical evidence obtained from Verticillium dahliae and W. dermatitidis [84, 85]. Figure 1 shows a general model for fungal dihydroxynaphthalene (DHN)-melanin biosynthesis. In this pathway, the polyketide synthase (PKS) converts malonyl-CoA to 1,3,6,8-tetrahydroxynaphthalene (1,3,6,8-THN), which undergoes several reduction and dehydration reactions to produce scytalone, 1,3,8-trihydroxynaphthalene (THN), and vermelone. A further dehydration step leads to the intermediate 1,8-dihydroxynaphthalene (DHN), which is polymerized to DHN-melanin, possibly by a laccase enzyme [10, 13, 27].
However, some species of this class, including Cladosporium resinae, Epicoccum nigrum, Hendersonula toruloidea, Eurotium echinulatum, Humicola grisea, and Hypoxylon archeri, do not produce this type of pigment [11, 28, 86–88]. In the genus Aspergillus, DHN-melanin has not been identified in some members, as A. nidulans and A. niger. Bull  identified dopachrome (indole 5,6-quinone 2-carboxylic acid) and melanochrome (indole 5,6-quinone), which are intermediates in the DOPA-melanin pathway, in A. nidulans mutants defective in the production of melanin. Other studies confirmed the indolic nature of the melanin produced by A. nidulans [11, 90]. In A. nidulans strains, one tyrosinase was identified as the enzyme responsible for the production of melanin pigment, based on its substrate specificity (DOPA substrate) and susceptibility to inhibitors [91, 92]. In a recent study, our group characterized the pigment produced by A. nidulans mutants as DOPA-melanin according to the results obtained with specific inhibitors of DHN- and DOPA-melanin pathways .
The production of DOPA-melanin has also been investigated in other fungi such as Neurospora crassa , Podospora anserina , A. nidulans , A. oryzae, and C. neoformans . A biosynthesis pathway for fungal DOPA-melanin, proposed by , is shown in Figure 2, which strongly resembles the pathway found in mammalian cells, though some of the details may differ.
In this pathway, there are two possible starting molecules, L-dopa and tyrosine. If L-dopa is the precursor molecule, it is oxidized to dopaquinone by laccase. If tyrosine is the precursor, it is first converted to L-dopa and then dopaquinone. The same enzyme, tyrosinase, carries out both steps. Dopaquinone, a highly reactive intermediate, forms leucodopachrome, which is then oxidized to dopachrome. Hydroxylation (and decarboxylation) yields dihydroxyindoles, which can polymerize spontaneously to form DOPA-melanin [10, 27, 97].
Some fungi have more than one biosynthetic pathway of melanins. For example, Aspergillus fumigatus synthesizes DHN-melanin  and also produces a second type of melanin, piomelanins, from homogentisic acid by the tyrosine degradation pathway that protects the cell wall of hyphae from ROS, and gray-green DHN-melanins determine the structural integrity of the cell wall of conidia and their adhesive properties . In Agaricus bisporus, melanins are formed from DOPA by tyrosinase and from γ-glutaminyl-4-hydroxybenzene by peroxidase and phenolase .
The extracellular fungal melanin, which is found in culture fluids usually in the form of granules, can be formed from some culture components, which are autoxidized or are oxidized by phenoloxidases released from the fungus during autolysis [10, 11, 27].
4. Biological activities of melanin
Despite the difference in their origins, melanin pigments have a number of common characteristics that allow them to fulfill their protective function. Several biological functions of melanins are closely associated to their chemical composition and structure. The presence of unpaired electrons in the melanin structure is responsible for various properties, including antioxidant, semiconductor, optical, electronic, and radio- and photoprotective .
The effect of melanin enhancing the survival of fungi under adverse conditions is mainly due to its function as an extracellular redox buffer, which can neutralize oxidants generated by the fungus in response to environmental stress . It has been reported that melanin contributes for virulence of C. neoformans, protecting the pathogen against free radicals generated immunologically . In W. dermatitidis and A. alternata, melanin confers resistance to oxidants permanganate and hypochlorite, representing a key role in pathogenesis of infections caused by these fungi . Studies have shown that melanin of zoopathogenic and phytopathogenic fungi is essential for their parasitizing, due to its antioxidant properties .
Melanin pigment extracted from several fungal species has shown the ability to scavenge free radicals (reactive nitrogen and oxygen species), becoming a potential natural antioxidant. Melanins produced by Exophiala pisciphila and Aspergillus bridgeri ICTF-201 exhibited a significant DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity comparable with that of synthetic melanin, indicating its antioxidant potential [102, 103]. Melanin produced by Schizophyllum commune showed high free radical scavenging activity in a dose-dependent manner, when the melanin concentration was increased from 10 to 50 μg, the scavenging activity was also increased from 87% to 96%, similar to those obtained using ascorbic acid (standard compound used to measure free radical scavenging activity) . Melanin pigment of Fonsecaea pedrosoi has antioxidant potential by reducing Fe(III) to Fe(II), ensuring the balance of its redox chemical microenvironment and minimizing the effect of oxidation of fundamental structures on fungal growth . Similar results were also observed for melanin from Ophiocordyceps sinensis, which proved to be an effective DPPH radical scavenger and a strong ferrous iron chelator . The chelating power of fungal melanin can be explained by various functional groups present in the structure of this pigment, which provide an array of multiple nonequivalent binding sites for metal ions [14, 22].
It has been reported that substances acting as antioxidants protect cells from ROS-mediated DNA damage, which can result in mutation and subsequent carcinogenesis. The excess free radicals may attack cellular constituents, as the cell membrane, nucleic acid, protein, enzymes, and other biomolecules, by peroxidation, resulting in the severe damage of cell functions and subsequent serious deleterious effects on the organism . It has been reported that melanin protects melanocytes and keratinocytes from the induction of DNA strand broken by hydrogen peroxide, indicating that this pigment also has an important antioxidant role in the skin . Studies in our laboratory showed that melanin extracted from hyperpigment-productive mutant (MEL1) of A. nidulans has the ability to scavenge the biological oxidants, as HOCl, and may be a promising material in cosmetic formulations to protect the skin against possible oxidative damage .
There is experimental evidence that fungal melanin may also act as an anti-aging drug, due to its action in reducing the generation of free radicals, clearing away the free radicals produced in excess, and enhancing the activities of antioxidant enzymes. Studies have shown that one of the major causes of aging is the surplus free radicals produced during the oxidative metabolism in the human body . It was demonstrated that the melanin produced by fungus Lachnum singerianum YM296 significantly inhibited the formation of lipid peroxidation products and slowed down the aging process, elevating the levels of superoxide dismutase, glutathione peroxidase, and catalase and decreasing the level of malondialdehyde in mice liver and brain homogenate and serum, suggesting that this pigment could be used as a new anti-aging drug .
Researches have also shown that some fungal melanin exhibits immunomodulatory activity through the inhibition of pro-inflammatory cytokine production in T lymphocytes and monocytes, as well as fibroblasts and endothelial cells [12, 110, 111]. During an inflammatory response, cells of the innate and acquired immune systems release a variety of mediators, such as nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukins (IL), and the reactive nitrogen and oxygen species, which are implicated in the pathogenesis of a number of inflammatory diseases .  reported that treatment of macrophages activated in vitro with melanin from the fungus F. pedrosoi inhibited the production of nitric oxide and Th1 cytokines. The study performed by  showed that the expression of inducible nitric oxide synthase gene decreased and lower levels of cytokines, such as IL-12 and TNF-α, were observed when activated macrophages were incubated with melanized cells of the Fonsecaea monophora fungus. Our studies demonstrated that melanin extracted from a highly melanized mutant (MEL1) of A. nidulans inhibited NO production in LPS-stimulated macrophages, with a maximum response of 82% inhibition, and also showed a dose-dependent inhibitory effect on TNF-α production, reaching an inhibition of 51.86% at a melanin concentration of 100 μg/mL. These results suggest that melanin from A. nidulans has potential as an anti-inflammatory agent and may be used in the future for development of new drugs with therapeutic utility .
Some studies have proposed that fungal melanin exhibits anti-radiation activity in vivo and in vivo and then could be explored as a probable radioprotector [16, 115]. Since melanin has a stable free radical population, it is thought that the radioprotective properties of this pigment result from a combination of physical shielding and quenching of cytotoxic free radicals generated by radiation .  showed that Lachnum extracellular melanin (LEM404) had strong anti-ultraviolet radiation activity because the survival rates of Escherichia coli, Staphylococcus aureus, and Saccharomyces cerevisiae under UV radiation were significantly increased after in vitro addition of LEM404. Compared with the control groups, the antioxidant defense systems, such as superoxide dismutase and glutathione peroxidase activities, were improved significantly in mice of experiment groups, and the reactive oxygen species detected by malondialdehyde content were decreased significantly. These results confirmed that fungal melanin could be used as component of photoprotective creams mainly for its free radical scavenging rather than its light absorption properties. The probable mechanisms of radioprotection by melanin appear to be modulated in pro-survival pathways, immune system, and prevention of oxidative stress. It was reported that melanin isolated from the fungus G. simplex reduced the radiation-induced overproduction of pro-inflammatory cytokines (IL-6 and TNF-α), which might help in the recovery from radiation injury by preventing the aggravation of inflammation and oxidative stress . This study confirmed the possible use of melanin-coated nanoparticles for protecting against radiotoxicity during radioimmunotherapy .
Recent studies have demonstrated that, in addition to the ability of transferring electrons arising under the action of radiation, melanin also possesses ionic conductivity due to its ability to transform any type of radiation energy not only into heat but also use it for the maintenance of redox processes in cells . It was assumed that melanin pigments, participating in redox reactions, are able to perceive the energy of radiation (UV, visible light, and radiation) and convert it into useful reducing power for metabolic processes. This hypothesis is supported by the discovery of melanized fungi in soils contaminated by radioactive nuclides and areas around the damaged Chernobyl nuclear reactors, which not only survive high radiation levels but also have enhanced growth upon exposure [16, 19, 119, 120]. Owing to its semiconductor property, melanin becomes a promising material for organic bioelectronic devices like transistors, sensors, and batteries .
Fungal melanins also exhibit growth inhibitory effect against various microorganisms. The extracellular melanin isolated from S. commune showed significant antibacterial activity against E. coli, Proteus sp., Klebsiella pneumonia, and Pseudomonas fluorescens and antifungal activity against dermatophytic fungi, Trichophyton simii, and T. rubrum . The A. auricula melanin displayed inhibitory activity on biofilm formation of the three bacterial strains, E. coli K-12, Pseudomonas aeruginosa PAO1, and P. fluorescens P-3, and there was a proportional reduction in biofilm biomass with the increase in pigment concentration. Confocal laser scanning microscopy (CLSM) analyses showed that the three strains formed thick and compact biofilms when grown in the absence of pigment, but the presence of A. auricula melanin resulted in thinner and looser cell aggregations on surfaces instead of normal biofilm architecture. This study suggested that A. auricular melanin inhibits quorum-sensing (QS)-regulated biofilm formation in all strains tested without interfering with their growth . Silver nanoparticles incorporated Yarrowia lipolytica melanin exhibited antimicrobial activity against the pathogen Salmonella paratyphi, and they were also effective at disrupting biofilms on polystyrene as well as glass surfaces . These nanoparticles displayed excellent antifungal properties toward an Aspergillus sp. isolated from a wall surface, suggesting the application of these nanoparticles as effective paint additives. The melanin-silver nanostructures with broad-spectrum antimicrobial activity against food pathogens also have potential applicability in food processing and food packaging industries .
The anti-cell proliferation effect of fungal melanin in tumoral cell lines has already been demonstrated.  reported that the extracellular melanin produced by the fungus S. commune was effective against human epidermoid larynx carcinoma cell line (HEP-2) in a concentration-dependent manner, indicating its potential application in cancer chemoprevention and chemotherapy.
The evaluation of the effect of fungal melanin on non-tumor cells is also interesting because it may serve as alternative to acute in vivo toxicity testing, avoiding the indiscriminate use of animals. The melanin produced by A. bridgeri was evaluated in vitro cytotoxicity assay using cell lines TE 355.Sk derived from normal human skin fibroblasts and HEK-293 derived from human embryonic kidney cells, and no cytotoxicity was observed against the two cell lines . In our studies, the toxicity of the melanin from A. nidulans was also evaluated due to its potential practical application as antioxidant and anti-inflammatory agent. The results showed that the viability of mouse macrophages remained greater than 90% when these cells were treated with a high melanin concentration (100 μg/mL), indicating that this pigment has low cytotoxicity . We also showed that the toxicity of A. nidulans melanin on mouse fibroblast McCoy cell line, after metabolic activation with hepatic S9 microsomal fraction, was much lesser (CI50 = 413.4 ± 3.1 μg/mL) than known cytotoxic agents such as cyclophosphamide (CI50 = 15 ± 1.2 μg/mL). In this study, we demonstrated that this melanin pigment did not induce gene mutations in different strains of Salmonella typhimurium used in the Ames assay. Based on these results, we suggest that the melanin produced by A. nidulans does not cause significant damage to the cellular components and might be used in the future for development of new therapeutic drugs .
5. Biotechnological applications of melanin
With the current knowledge about physical and chemical properties and the broad spectrum of biological activities, fungal melanins have attracted growing interest for their potential use in the fields of biomedicine, dermocosmetics, nanotechnology, and materials science.
5.1. Bioelectronic applications
In recent years, the electronics industry has been driven to develop materials and components that are cheaper and more environmentally friendly. As melanin has characteristics of functional materials and bioorganic, a growing number of researchers in the fields of materials science and organic electronics see the melanin with great interest, taking advantage of their properties for applications in organic electronic devices. Melanins present interesting optoelectronic properties, such as high optical absorption in the UV-Vis range, good transmission electronic, and ionic conductivity appreciably, pointing this biomaterial as a promising active component in organic electronic devices with low environmental impact [118, 121, 125–127].
Among the physical properties of melanin, the electrical conductivity is one of the most interesting to investigate in the perspective of technological application. The electrical conductivity properties of this biopolymer are similar to those of amorphous semiconductor solids, and then it can be considered an organic semiconductor, which is largely available and biocompatible and, consequently, cheaper and easier to process with respect to inorganic semiconductors, as silicon germanium. In particular, it can be considered a promising material for sensors and photovoltaic devices, due to broadband spectral absorbance and charge transport properties .
The technical literature describes the integration of organic semiconducting polymers as melanin in silicon electronic devices in view of the possibility of achieving multifunctional systems that combine electrical and optical properties of semiconductors, the structural versatility and mechanical characteristics of materials, and processing polymeric . The production of devices based on thin film melanin exhibited electrical conductivity comparable to that of amorphous silicon . In this study, melanin films showed excellent thermal stability and adhere well to glass substrates and silicon, indicating the possibility of using this technique for the production of films from synthetic melanin. Other groups have published various device architectures with applications such as memory (metal-insulator-semiconductor geometries) , batteries , and biomimetic interfaces .
Deposition of homogeneous melanin layers for optoelectronics application is an issue of considerable technological relevance. Synthetic melanin thin films deposited by spray-coating presented features ascribed to an amorphous semiconducting material . They also showed that further improvement of conductivity together with an increased absorption in the NIR region, by doping the synthetic melanin macromolecule, could make this material a good candidate for optical sensing applications. It has been reported that the iron-melanin coating markedly enhances the catalytic activity of the gold nanoparticles (AuNPs) for both the hydrogen peroxide electroreduction and hydrogen evolution reaction . This strategy may be used to improve nanomaterials with potential applications as efficient catalysts and electrocatalysts. Studies have shown that synthetic melanin-like nanoparticles complexed with paramagnetic Fe3+ ions have potential as a highly efficient and nontoxic contrast agent for magnetic resonance imaging instead of Gd3+-based contrast agents, which can cause nephrotoxicity .
The optical and electronic properties of melanin have attracted the attention of researchers for the production of continuous thin films from conventional synthetic melanin, which have been used for a number of different device configurations, including chemi-sensors, next-generation solar cells, and a range of other detectors [126, 130, 134]. Potential also exists to use melanin films as an effective radiation sensitizer that could greatly improve the spectral range and efficiency of superconducting transition-edge bolometers .
The metal chelation properties of melanin offer interesting possibilities for melanin-based metal ion sensing. A piezoelectric sensor system capable of real-time detection of metal ions was constructed by cross-linking melanin onto the gold electrode of quartz crystal microbalance (QCM) and showed high sensitivity and selectivity to metal ions particularly for Hg(II) .
Melanin has many other interesting properties, such as ultraviolet absorption, which has been utilized to prepare optical lenses or filters. Studies have shown that it is possible to use melanin as an ultraviolet, visible and near-infrared absorbing pigment in opthalmic devices, protective eyewear, windows, packaging material, umbrellas, canopies, and other similar media suitable for providing protection from radiation [139, 140]. The incorporation of the melanin in solid plastic films of polyvinyl alcohol (PVA-melanin film system) to be used in conjunction with other plastics to make laminated sheets or lenses, including sunglasses, ski goggles, ophthalmic prescription lenses, helmets, windows, light filters for artificial lighting, and other light filters that protect people from potentially damaging UV and high-energy visible light has also been reported .
5.2. Medical applications
Despite its high biocompatibility, the use of melanin as a novel biomaterial in pharmaceutical and biomedical applications reported in literature is still scarce. A study performed with melanin nanoparticles as biocompatible drug nanocarriers, using metronidazole (antibiotic drug), showed that melanin could be a very interesting nanocarrier drug release device because it strongly responds to pH, being a very interesting feature for the treatment of intestine and colon diseases, which would greatly benefit with pH targeting . Another study showed that systemic melanin-covered nanoparticle (MN) administration reduced hematologic toxicity in mice treated with radiation and that these structures provide efficient protection to bone marrow against radiotoxicity during radioimmunotherapy and in some cases external beam radiation therapy, permitting the administration to tumors of significantly higher doses .
Melanin has also been used to treat various types of malignant cancer tumors, disorders of the immune system including AIDS, diseases of blood origin and disorders due to the disturbances in cell homeostasis, and complex and hardly curable mental disorders (schizophrenia, epilepsy) involving nervous and other regulatory systems. A study on the use of melanin for the treatment of Parkinson’s disease, an amelioration in the monkeys’ overall functional ability and secondary motor manifestations by the administration of an effective amount of melanin in monkeys treated with MPTP (1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine), a toxin that causes a neurodegenerative disease, was observed. This study demonstrated that toxin-induced Parkinson’s disease could be prevented in the melanin-treated animals because the administered melanin causes chelation or scavenging of toxins, such as MPTP, thus preventing a neurodegenerative disease, such as Parkinson’s disease. The results of this study also showed that melanin administration to aid the recovery of neurons in a mammal having neuron injury suggests that melanin can be used to treat Alzheimer’s disease .
Owing to their ability to increase the permeability of the blood-brain barrier, the melanin is also useful as carriers for other therapeutic agents, which must reach brain tissue to produce their therapeutic responses . Two examples of such therapeutic agents that will cross the blood-brain barrier when linked to melanin are boron and nerve growth factor. According to the same authors, the melanin is also an effective vehicle for the transport of boron to cancerous sites in the body, mainly when the cancerous cells to be treated are located in the brain, because this pigment binds boron very strongly. The melanin can also function as a carrier for nerve growth factor due to the ability to get nerve growth factor across the blood-brain barrier, and this is the major advantage over conventional therapy.
In recent years, efforts have been focused on investigating the potential use of this pigment as active material in tissue repair engineering. Bettinger et al.  reported that thin films of melanin were found to enhance Schwann cell growth and neurite extension in rat pheochromocytoma cells (PC12 cells) compared to collagen films in vitro. Melanin films also induced an inflammation response that was comparable to silicone implants in vivo, and the implants were significantly resorbed after 8 weeks. These results showed that melanin thin films have great potential in the reconstruction of tissues, being biodegradable, and possess inflammatory response comparable to silicone. Another study of the biocompatibility of melanin thin films demonstrated that the melanin film effectively supports the growth of undifferentiated stem cells and their differentiation into neuronal precursors and neurons . They related that high-quality melanin thin films display appealing features, such as reversible conductivity by controlled hydration—dehydration steps—excellent biocompatibility with stem cells, and water-resistant adhesion, for bioelectronic applications, e.g., in organic electrochemical transistors (OECTs), which can translate cellular activity into electrical signals [125, 147]. It has also been reported that melanin thin films possess highly desirable physical and biological properties that make them ideal for organic bioelectronic devices .
In cosmetic industry, there are great interests in the melanin, especially to protect against the noxious effects of UV radiation by incorporation in skin photoprotection formulations [35, 148]. The protective action of melanin is related to its high efficiency to absorb and scatter photons, particularly the higher-energy photons from the UV and blue part of the solar spectrum. Very likely, melanin photoprotection is also due to its ability to quench excited states of certain molecules and scavenge ROS that may be generated in pigmented cells . Development of methods for producing melanin soluble in aqueous cosmetic buffers at physiological pH and temperature may make possible the use of this pigment as ingredients of face and hand creams, lotions, antiaging ointments, or foundation makeups, acting as a screen and antioxidant for the protection against photoinduced skin damages . Other dermocosmetic applications of melanins include the use of the pigment for hair dyeing and the development of novel strategies for hair recoloration .
Since melanin has a stable free radical population, it is thought that the radioprotective properties of this pigment result from a combination of physical shielding and quenching of cytotoxic free radicals generated by radiation . Some studies suggest the possible use of melanin-coated nanoparticles in medicine, mainly for protecting patients against the harmful effects of gamma rays during radioimmunotherapy [34, 151]. Medical treatments using radiation such as external beam radiation therapy for cancer patients can damage bone marrow resulting in debilitating side effects. In experimental models, melanin can successfully shield bone marrow from such side effects. Mice treated with melanin-coated nanoparticles have higher white blood cell and platelet counts than control mice after radiation treatment . It has been reported the use of melanin, a biopolymer with good biocompatibility and biodegradability, intrinsic photo-acoustic properties, binding ability to drugs, and chelating property to radioactive metal ions, as an efficient endogenous nanosystem for imaging-guided chemotherapy . According to the authors, melanin nanoparticles could successfully enter into the tumor and act as an efficient drug-delivery system, thereby greatly increasing the safe utility of the drugs for tumor treatment and significantly lowering the dosage used and its side effects.
A valuable biotechnological approach to the melanin-mediated synthesis of silver nanostructures with broad-spectrum antimicrobial activity has been developed. Silver nanostructures synthesized with melanin derived from Y. lipolytica displayed excellent antifungal activity against an Aspergillus sp. isolated from a wall surface, indicating its potential application as effective paint additives . The melanin-mediated nanostructures with broad-spectrum antimicrobial activity against food pathogens may be considered suitable for many practical food packaging applications because they can effectively inhibit the growth of pathogens and increase the shelf life of packed food products .
5.3. Environmental applications
The chemical structure of melanin presents many oxygen-containing groups, including carboxyl, phenolic and alcoholic hydroxyl, carbonyl, and methoxy groups, which have the ability to bind to a broad spectrum of substances . In literature, studies have confirmed that fungal melanin acts as metal chelators, enhancing the biomass-metal interaction and consequently its biosorption capacity . Study conducted by  showed that a melanin-rich strain of the fungus Cladosporium cladosporioides biosorbed 2.5- to 4-fold more Ni, Cu, Zn, Cd, and Pb ion than non-melanic Penicillium digitatum. These authors also studied the culture of C. cladosporioides in different growth times and found that a culture grown for two days is not pigmented and has only 34% of Cd adsorption rate that obtained for pigmented biomass after 4 days of growth . Another study reported that melanized fungus Armillaria adsorb high concentrations of cations from the surrounding environment; some ions (Al, Zn, Fe, Cu, and Pb) were 50–100 times more concentrated on rhizomorphs than in soil . The results obtained in our laboratory using a melanin-overproducing mutant (MEL1) from A. nidulans fungus [31, 93] showed that biosorption capacity for neodymium and lanthanum varied with stage of growth of this mutant; the biomass obtained after 72 hours of growth exhibited a 75% increase compared to the biomass of 48 hours. This result is related to melanin production during growth of the MEL1 mutant, since the biomass 48 hours is slightly pigmented, while the 72 hours biomass is dark due to the increased production of pigment . Therefore, the pigmented biomass of the MEL1 mutant may be considered as a promising biosorbents for removal/recovery of the rare earth elements from wastewater due to the presence of the melanin increase significantly metal complexing capacity, improving the efficiency of biosorption process .
Some melanized fungi have shown to be good candidates for bioremediation of contaminated sites, due to the ability of fungal melanin to bind to heavy metals and radionuclides in contaminated sites. Experimental evidence shows that the accumulation of 90Sr by conidia or mycelium by a range of microfungal species is greater in pigmented than in unpigmented species .  In a study on the uptake efficiency of the radiocesium (137Cs) and radiocobalt (60Co) in melanized and nonmelanized fungi, it was observed that 60% of both radionuclides were uptaken by melanin of A. alternata and Aspergillus pulverulents. These results can be explained by melanin or other natural pigments present in the cell wall of these fungi that can act as the radiation receptor and/or as an energy transporter for metabolism. Other studies have demonstrated the potential application of the melanized fungi for the removal of radionuclides and heavy metals from aqueous solutions, providing an alternative means to affect cleanup of industrial effluent [16, 120, 160–164]. It has been reported that fungal melanin arranged in nanoparticles protects against extremely high levels of ionizing radiation and suggests that the protective efficacy of this pigment is a function of its chemical structure, the presence of stable free radical, and spatial arrangement . According to the authors, these nanoshells have the potential use for environmental bioremediation, for example, to prevent the spread of radioactive contamination to ground water because the melanin is expected to encapsulate the radioactive particles and thereby reduce their spread. In this way, melanin nanoshells may be used to contain radiation from radioactive waste and biomedical radioactive materials.
Melanin possesses physicochemical properties and biological activities that make it a suitable biomaterial for a wide range of applications in cosmetic, pharmaceutical, electronic, and food processing industries. In addition, this pigment has a considerable interest biotechnological because it can be produced on a large scale with low cost, making its use for future practical applications economically advantageous. However, it is necessary to expand the knowledge about the structure-property-function relationships for the development of melanin-based technology. In the context, we hope that the information in this book will be useful and will encourage a greater number of researches on fungal melanin, which might be useful to deploy innovative and sustainable solutions for human health and the environment.
DNase-coated melanin-like nanospheres for treating sepsis in severe COVID
Coronaviruses are a group of RNA viruses that cause disease in mammals and birds. In humans and birds, they cause respiratory infections that can range from mild to fatal. Mild illnesses in humans include some cases of the common cold. In contrast, more deadly strains can cause SARS, MERS, and now COVID-19 disease. The most recent type of coronavirus, Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), falls under the beta coronavirus category, along with the previous SARS-CoV-1 and MERS-CoV. There have been no significant developments in the treatment approach for this beta-type CoV, making it imperative to design and develop a unique therapeutic pathway to minimize the transmission of SARS-CoV-2 and the effects of COVID-19.
COVID-19 disease caused by the SARS-CoV-2 virus causes a wide range of illnesses ranging from mild respiratory diseases, severe progressive pneumonia and acute respiratory distress syndrome, elevation of cytokines, multiple organ failure to death. Neutrophils, which make up about 70% of the white blood cells in our bodies, are higher in the severe progression of COVID-19 disease. The abnormal activation of neutrophils triggers the release of extracellular neutrophil traps (NETs) and extracellular DNA, resulting in multiple organ failure and septic shock.
Because severe COVID-19 is very difficult to treat, researchers at the Korea Research Institute of Biosciences and Biotechnology (KRIBB) suggested that DNase-1 could be used to dissolve extracellular neutrophil traps (NETs), thereby halting further progression becomes COVID-19. Since DNase has a short half-life in blood plasma, Wonha Lee and colleagues suggested applying DNase to the surface of the nanoparticles in order to stabilize and maintain DNase activity. The researchers used polydopamine to bind DNase to the nanoparticle surface. The research is published in the journal Advanced science.
Physicochemical characterization of pMNSs. a) Production of DNase-I pMNSs. b) Size distribution of bMNSs, pMNSs and DNase-I pMNSs. c) Zeta potentials of bMNSs, pMNSs and DNase-I pMNSs. d) Scanning electron microscope (SEM) images of bMNSs, pMNSs and DNase-I-pMNSs (scale: 500 nm). e) Migration profile of pure DNA after digestion with free DNase I, pMNS and bMNSs as well as various amounts of DNase I pMNSs. pMNSs, PEG-coated melanin-like nanospheres; bMNSs, naked bio-inspired melanin-like nanospheres.
Development of the novel treatment strategy for COVID-19
First, the researchers analyzed the computed tomography of severely infected COVID-19 patients whose lungs were severely damaged. They found that the patients had higher neutrophil counts and lower lymphocyte counts, another type of white blood cell that is responsible for fighting infection. They also compared the extracellular DNA, DNAse and Citrullinated Histone H3 in healthy and COVID-19 infected patients. Lee found that extracellular DNA and citrullinated histone H3 levels increased in severely infected individuals, while DNase I levels decreased in infected individuals.
The scientists then synthesized the melanin-like nanoparticles using the oxidation of dopamine. The surface of the melanin-like nanoparticles was immobilized with DNAse-I and polyethylene glycol (PEG). The visualization of synthesized and coated nanoparticles under the scanning electron microscope showed that the morphology of the nanoparticles was spherical and had a uniform size distribution. The researchers evaluated the degradability of DNAse-I and PEGylated nanoparticles and found that DNA was degraded at a nanosphere concentration of more than 1 microgram. They also evaluated the DNAse I binding efficiency and stability over time on the nanoparticles by BCA assay and treatment with FBS with PBS and PBS media only. As the concentration of DNAse-I increased, so did the binding efficiency, and the DNASe-I coated nanospheres were only stable in PBS media for up to 72 hours.
Effect of DNAse-I-PEGylated melanin-like nanoparticles on COVID-19 patients
Severely affected COVID-19 patients suffer from a decrease in DNAse-I in their system, requiring the use of DNAse-I-PEGylated melanin-like nanoparticles. The group of scientists from the Korea Institute of Biosciences and Biotechnology treated the plasma of COVID-19 patients and surprisingly found that both free DNAse and DNAse-coated nanoparticles reduced extracellular DNA levels. In addition, they found that factors such as myeloperoxidase, neutrophil elastase, which are responsible for extracellular neutrophil traps and consequently increase the severity of COVID-19, were alleviated to a greater extent when treated with DNAse-I PEGylated melanin-like nanospheres. More than half of the hospitalized COVID-19 patients had sepsis, which was caused by uncontrollable inflammatory reactions. The inflammation is characterized by increased levels of cytokines. They quantified the immune factors responsible for inflammation after treatment with DNase-I and DNAse-I-coated nanoparticles, and found that the factors decreased significantly, which promised to use these DNAse-coated nanoparticles as a potential treatment approach can be.
Effect of nanoparticles on the Cecal Ligation and Perforation (CLP) treated septic mouse model
The researchers further validated the synthesized DNase-I-PEGylated nanoparticles in vivo using a CLP mouse model. To create a CLP model, the opening of the large intestine, known as the appendix, is cut open and holes are made to trigger inflammation that mimics human sepsis. The KRIBBS scientists administered DNase-I melanin = similar nanospheres to the CLP mouse model and found that the number of neutrophils decreased dramatically. The scientists also realized a 40% survival rate and a reduction in cytokine levels in the CLP models when nanoparticles were administered, confirming that these nanoparticles can be used as a possible treatment strategy.
The researchers showed that extracellular DNA, one of the factors responsible for the progression of COVID-19, can act as a likely target for the treatment of SARS-CoV-2 virus-induced sepsis. They also found that the delivery of DNase-I PEGylated melanin-like nanospheres suppressed extracellular DNA expression, which in turn slows the progression of sepsis in severe COVID-19 patients. They say injecting these nanoparticles early can potentially stunt the progression of COVID-19 disease. Although the efficacy of bioinspired nanospheres has been verified in both in vitro testing of blood samples and in vivo testing of the septic mouse model, Lee recommends validating the nanoparticles under appropriate in vivo sepsis-induced animal models prior to clinical studies COVID-19 carried out in human patients. However, the PEGylated melanin-like nanospheres could be a promising treatment approach for SARS-CoV-2 sepsis.
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Bioinspired DNase‐I‐Coated Melanin‐Like Nanospheres for Modulation of Infection‐Associated NETosis Dysregulation
The current outbreak of the beta‐coronavirus (beta‐Cov) severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) began in December 2019. No specific antiviral treatments or vaccines are currently available. A recent study has reported that coronavirus disease 2019 (COVID‐19), the disease caused by SARS‐CoV‐2 infection, is associated with neutrophil‐specific plasma membrane rupture, and release excessive neutrophil extracellular traps (NETs) and extracellular DNAs (eDNAs). This mechanism involves the activation of NETosis, a neutrophil‐specific programmed cell death, which is believed to play a crucial role in COVID‐19 pathogenesis. Further progression of the disease can cause uncontrolled inflammation, leading to the initiation of cytokine storms, acute respiratory distress syndrome (ARDS), and sepsis. Herein, it is reported that DNase‐I‐coated melanin‐like nanospheres (DNase‐I pMNSs) mitigate sepsis‐associated NETosis dysregulation, thereby preventing further progression of the disease. Recombinant DNase‐I and poly(ethylene glycol) (PEG) are used as coatings to promote the lengthy circulation and dissolution of NET structure. The data indicate that the application of bioinspired DNase‐I pMNSs reduce neutrophil counts and NETosis‐related factors in the plasma of SARS‐CoV‐2 sepsis patients, alleviates systemic inflammation, and attenuates mortality in a septic mouse model. Altogether, the findings suggest that these nanoparticles have potential applications in the treatment of SARS‐CoV‐2‐related illnesses and other beta‐CoV‐related diseases.
In recent decades, there have been recurrence of large‐scale epidemics attributed to coronaviruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV), Middle East respiratory syndrome coronavirus (MERS‐CoV), and most recently, SARS‐CoV‐2, which was discovered in patients with severe pneumonia in December, 2019.[1–3] CoVs are classified into four categories, that is, alpha‐CoV, beta‐CoV, gamma‐CoV, and delta‐CoV. Interestingly, SARS‐CoV, MERS‐CoV, and SARS‐CoV‐2 are beta‐CoV lineage viruses. Currently, there are no specific antiviral treatments or vaccines available for these beta‐CoVs; therefore, it is crucial to develop novel treatment strategies to address the rapid spread of this virus.
The full spectrum of coronavirus disease 2019 (COVID‐19)—the disease associated with SARS‐CoV‐2 infection—ranges from mild respiratory tract illness to severe progressive pneumonia, acute respiratory distress syndrome (ARDS), serum pro‐inflammatory cytokine elevation (cytokine storm), multiple organ failure (MOF), and death. SARS‐CoV‐2 infections are associated with both uncontrolled inflammatory innate immune responses and impaired adaptive immune responses, leading to local and systemic tissue damage. The late stage of the disease is difficult to treat, as the radical immune reactions propagate toward cytokine storms, acute respiratory distress syndrome (ARDS), and septic shock, leading to increased patient mortality.[5, 6] The underlying mechanism as well as initiators that trigger and propagate the cytokine storm, subsequently leading to the development of severe COVID‐19 are unknown. A recent study has proposed that neutrophilia might be linked to poor outcomes in patients with severe COVID‐19, and it is believed to play a critical role in COVID‐19 pathogenesis. NETosis, a special type of neutrophil‐specific programmed cell death, is a process in which reticular structures consisting of chromatin and granular proteins are formed.[8, 9] It has been suggested that excessive amount of NETosis, which lead to a hyperinflammatory response, is associated with organ damage and MOF. Hence, it is postulated that aberrant activation of neutrophils followed by release of neutrophil extracellular traps (NETs) and extracellular DNAs (eDNAs) in the peripheral blood may contribute to organ damage and mortality in sepsis‐related diseases. It is predicted that similar outcomes may arise in COVID‐19 patients.
Inspired by our observations and based on a recently published report, we suggest that the dissolution of a basic constituent of NET structure—DNA—using DNase‐I may be an appropriate strategy for preventing NET‐related pathogenesis in SARS‐CoV‐2 patients. Previously, it had been demonstrated that the delivery of a recombinant DNase‐I by inhalation led to the dissolution of NETs in the airways of cystic fibrosis (CF) patients, that resulted in clear mucus and ameliorated symptoms. In addition, the use of an actin‐resistant DNase in CF patients in Phase I and II clinical trials showed promising results (NCT02605590, NCT02722122). When tested using animal models, delivery of DNase‐I through the airways resulted in increased survival,[14–16] leading us to believe that DNase‐I may help dissolve NETs and prevent further progression to ARDS and sepsis in severe COVID‐19 patients. However, exogenously administered recombinant DNase‐I showed only a modest effect, presumably due to the short half‐life of DNase‐I in the blood plasma. Previous studies have reported that conjugation of DNase‐I onto the surface of nanoparticles enhances the stability and preserves the activity of DNase‐I in blood plasma.[17, 18]
In this study, we report for the first time, demonstration of DNase‐I‐coated melanin‐like nanospheres (DNase‐I pMNSs) for modulation of sepsis‐associated NETosis dysregulation. Polydopamine‐based DNase‐I MNSs have a structure similar to that of squid ink and skin melanin.[19–22] DNase‐I was readily attached to the nanoparticle surface through the excellent adhesive properties of polydopamine.[23, 24] Intravenous injection of bioinspired DNase‐I MNSs alleviated systemic inflammation and attenuated mortality in a septic mouse model. We also show that exogenously administered DNase‐I MNSs exhibit a suppressive effect against neutrophil activities, eDNA level, and cytokine storm. Thus, our data support further investigation of DNase‐I MNSs as potential tools to treat SARS‐CoV‐2‐mediated illnesses.
2.1 Increased Neutrophil and Reduced Lymphocyte Counts in Severe COVID‐19 Patients
Computed tomography (CT) was performed, and blood samples from COVID‐19 patients were analyzed on their admission to the hospital (Figure S1, Supporting Information, and Table 1). CT imaging revealed that patients with severe clinical manifestations had severe lung tissue damage, indicating an increase in the severity of septic symptoms (Figure S1, Supporting Information). As reported previously, severe clinical manifestations such as ARDS and sepsis, were mostly observed in elderly patients (mean age 72.2 ± 13.1) who showed increased neutrophil counts and decreased lymphocyte counts when compared with the less severe group (Table 1).[25, 26] The later symptom lymphopenia is widely recognized to be associated with the severity of diseases.
All patients (n = 60) Mild patients (n = 40) Severe patients (n = 20) p‐Value Characteristics Age [y] 33.7 ± 16.4 55.4 ± 17.1 72.2 ± 13.1 0.014 ARDS 11 (18.3) 0 (0) 11 (55) <0.001 Sepsis 13 (21.7) 0 (0) 13 (65) <0.001 Discharged 40 40 (100) 0 (0) <0.001 Died 16 0 (0) 16 (80%) <0.001 Complete blood count White blood cell count, × 109L−1 [normal range 4–10] 6.5 ± 3.4 5.9 ± 3.6 9.1 ± 2.6 0.005 Neutrophil count, × 109 L−1[normal range 3–7] 4.6 ± 3.4 4.1 ± 3.2 7.7 ± 3.3 <0.001 Lymphocyte count, × 109 L−1[normal range 1.5–4] 1.4 ± 0.7 1.5 ± 0.7 0.8 ± 0.3 <0.001 Hemoglobin, g dL−1 [normal range 12–17] 13.0 ± 1.6 13.5 ± 1.7 13.0 ± 1.6 0.253 Platelets, × 109 L−1 [normal range 140–400] 237.3 ± 104.9 245.0 ± 107.9 186.7 ± 64.9 0.061
2.2 Increased Levels of NETosis Markers in Severe COVID‐19 Patients
Levels of extracellular DNA (eDNA), DNase‐I and citrullinated histone H3 (Cit‐His H3) were measured in 20 healthy control volunteers (normal group), and 60 blood samples from COVID‐19 patients, including mild patients and severe patients. The median serum eDNA level in the normal group was 0.41 (0.29–0.53) µg mL−1. In comparison, the level of eDNA was slightly increased to 0.85 (0.58–1.12 µg mL−1) in the 40 mild COVID‐19 patients and drastically increased to 2.83 (2.46–3.20) µg mL−1 in the 20 severe COVID‐19 patients (Table 2). The median serum Cit‐His H3 levels in the blood samples showed much more drastic differences. In the normal group, the Cit‐His H3 median level was slightly increased from 0.05 (0.04–0.06) µg mL−1 to 0.30 (0.02–0.62) µg mL−1 in mild COVID‐19 patients, and significantly increased to 17.74 (14.83–20.65) µg mL−1in severe COVID‐19 patients. A slightly different effect was observed for DNase‐I, that is, in the normal group, the median DNase‐I level was slightly increased from 2.11 (1.73–2.49) µg mL−1 to 3.11 (2.04–4.18) µg mL−1 in the mild COVID‐19 patients, but decreased to 0.97 (0.67–1.27) µg mL−1 in severe COVID‐19 patients.
Normal patients (n = 20) Mild patients (n = 40) Severe patients (n = 20) NETosis eDNA [mg mL−1] 0.41 ± 0.12 0.85 ± 0.27 2.83 ± 0.37*** DNase‐I [mg mL−1] 2.11 ± 0.38 3.11 ± 1.07 0.97 ± 0.30*** Cit‐His H3 [mg mL−1] 0.05 ± 0.01 0.30 ± 0.32 17.74 ± 2.91***
2.3 Preparation and Characterization of DNase‐I pMNSs
The bare bioinspired melanin‐like nanospheres (bMNSs) were surface‐modified with DNase‐I to improve the stability and thus activity of DNase‐I (Figure 1a).The melanin‐like nanoparticles were synthesized through oxidation of dopamine based on the protocol published in our previous paper. Then, DNase‐I and PEG were immobilized on the surface of nanoparticles in a one‐pot process. The particle sizes of bMNSs, pMNSs, and DNase‐I pMNSs were similar, approximately 170 nm (Figure 1b). The surface charge of bMNSs was measured to be −12.4 mV, which neutralized to −0.15 mV when the PEG coating was introduced to generate pMNSs (Figure 1c). The surface of the DNase‐I pMNSs was negatively charged (−10.9 mV) due to coating with DNase‐I. Data also confirmed the spherical appearance and relatively monodispersed size of the nanoformulations (Figure 1d). The DNA degradation capability of DNase‐I pMNSs was evaluated using agarose gel electrophoresis (Figure 1e). Free DNase‐I (1 U) completely degraded 1 µg DNA. pMNSs and bMNSs did not degrade DNA due to the absence of DNase‐I on their surface. However, DNase‐I pMNSs degraded DNA at a concentration > 1.0 µg of nanospheres, suggesting that the effect was due to the activity of coated DNase‐I on pMNSs. To evaluate the amount of DNase‐I that can be bound to pMNSs, nanoparticles were prepared with various ratios of pMNSs and DNase‐I. The binding contents and binding efficiency of DNase‐I on pMNSs were measured through BCA assay (Table S1, Supporting Information). As the feed amount of DNase‐I increased, the actual amount of DNase‐I binding to the nanoparticles increased, but the binding efficiency of DNase‐I was limited to less than 83%. However, the DNase‐I pMNSs3 containing ≈45% of DNase‐I showed excellent stability and DNA degradation properties, thus DNase‐I pMNSs3 was selected and used in further experiments. Then, to analyze the binding stability of nanoparticles and DNase‐I, the activity of DNase‐I over time in PBS containing 10% FBS or PBS only media, was tested. When DNase activity was tested after incubation of long‐acting DNase‐I in media containing 10% FBS for 72 h (Figure S2(a), Supporting Information), the activity of DNase‐I pMNSs was maintained for up to 36 h, but the activity decreased after 48 h. However, in PBS only media (Figure S2(b), Supporting Information), the activity of DNase‐I pMNSs was maintained for up to 72 h.
2.4 Exogenous DNase‐I pMNSs Alleviated NETosis Factors in the Plasma and Neutrophils of Severe COVID‐19 Patients
After observing an increase in the levels of NETosis‐related factors, we hypothesized the necessity to compensate for the loss of endogenous DNase‐I in severe COVID‐19 patients. We therefore validated the effects of DNase‐I pMNSs on reduction of the neutrophils in NETosis. To demonstrate the effects of DNase‐I on DNA degradation, we treated the plasma of severe COVID‐19 patients with either free DNase‐I or DNase‐I pMNSs. The results showed that both forms of DNase‐I significantly reduced the eDNA levels (Figure 2a), and that exposure of DNase‐I to the plasma of severe COVID‐19 patients increased the activity of DNase‐I (Figure 2b). We also observed markedly reduced NET levels, MPO activity, and NE levels in neutrophils of severe COVID‐19 patients upon treatment with DNase‐I pMNSs (Figure 2c–e).
As shown in Table 1, over 50% of the severe COVID‐19 patients were diagnosed with ARDS and sepsis on admission to the hospital. Typically, sepsis is accompanied by a cytokine storm, a deadly uncontrollable systemic inflammatory response resulting from the burst of vast amounts of pro‐inflammatory cytokines by immune effector cells. Therefore, we evaluated the effects of DNase‐I pMNSs on NF‐κB activation and cytokine secretion from neutrophils. The results showed that the activity of NF‐κB and secretion of cytokines IL‐1β, IL‐6, IFN‐γ, and TNF‐α were slightly reduced upon treatment with free DNase‐I, and were further drastically reduced upon treatment with DNase‐I pMNSs (Figure 2f–j).
2.5 DNase‐I pMNSs Alleviated NETosis Factors in a Septic Mouse Model
After confirming reduction in neutrophil counts and neutrophil‐related NETosis factors in severe COVID‐19 patients upon treatment with DNase‐I MNSs in vitro, we further validated the effects of DNase‐I pMNSs in vivo. Based on a recent report that neutrophil activity is a valid target for preventing or reducing sepsis and enhancing survival, we tested the exogenous administration of the DNase‐I pMNSs in an in vivo setting: cecal ligation and perforation (CLP)‐treated septic mouse model (Figure 3a). CLP mouse model is the most frequently used model to investigate the complex molecular mechanisms of sepsis. Sepsis is induced via polymicrobial infectious focus within the abdominal cavity, followed by bacterial translocation into the blood compartment, which triggers a systemic inflammatory response. Similar phenotypes are observed in the CLP model, and severe COVID‐19 patients; NETosis, cytokine release syndrome (CRS), and multiple organ failure (MOF) syndrome can be induced in the CLP model. Hence, the CLP model can be used to represent the SARS‐CoV‐2‐induced severe COVID‐19 patients. The CLP model was considered an appropriate in vivo model for the current setting due to the absence of available alternatives. To confirm the anti‐NETosis effects of DNase‐I pMNSs, CLP‐operated mice were intravenously injected with phosphate‐buffered saline (PBS; control group), PEG‐Nano, free DNase‐I (100 units), or DNase‐I pMNSs (100 units) at 12 h and 24 h post‐CLP‐operation. We measured the DNase‐I activity 24 h after drug administration (48 h post‐CLP‐operation); Colón et al. measured the activity after 18 h, Yoshikawa et al. measured the activity after 24 and 48 h NET, and Czaikoski et al. measured the activity at 48 h. Previously, it was reported that neutrophil activity increases after CLP surgery as a function of time. To effectively suppress the rapidly increasing NET, the drug was administered at 12 and 24 h and the effect was confirmed 24 h after drug administration (48 h post‐CLP‐operation). All of the CLP‐operated mice died within 90 h of CLP induction when PBS, PEG‐Nano, or free DNase‐I were administered. This demonstrated the high mortality rate of the CLP‐operated sepsis model. Previously, the pharmacodynamics of recombinant human DNase‐I in the serum was analyzed, indicating a short half‐life, and a lack of in vivo effect of free DNase‐I. Interestingly, DNase‐I pMNSs demonstrated a 40% survival rate for the CLP‐operated septic mice for over 132 h, leading to the full recovery of these mice. We then confirmed the effect of DNase‐I pMNSs on the lungs, and we found a significant reduction in the morphological changes caused by CLP, including pulmonary edema, hemorrhage, alveolar collapse, and inflammatory cell infiltration (Figure 3b).
To further evaluate the efficacy of DNase‐I pMNS treatment in a therapeutic setting, we evaluated the levels of neutrophils, NETs, eDNA, DNase activity, NE activity, and MPO activity in the intraperitoneal cavity of CLP‐operated mice (Figure 3c–h). In agreement with the effects observed in the plasma and neutrophils of severe COVID‐19 patients (Figure 2), similar results were observed in which DNase‐I pMNSs induced a drastic reduction in the levels of peritoneal neutrophils, NETs, eDNA, NE activity, and MPO activity compared to those in the control group (PBS treatment) and free DNase‐I group. It was also clearly demonstrated that DNase activity increased dramatically in DNase‐I pMNSs group, compared to the control group (PBS treatment).
Next, we evaluated the effect of the DNase‐I pMNSs on the regulation of inflammatory responses in the CLP‐operated sepsis model. As mentioned before, systemic inflammation associated with sepsis often causes MOF syndrome, and it has been reported that the major organ targets of MOF syndrome are the kidney and liver. CLP treatment significantly increased the serum levels of hepatic injury markers alanine transferase (ALT) and aspartate transaminase (AST) (Figure S3a and b, Supporting Information), renal injury markers blood urea nitrogen (BUN) and creatinine (Figure S3c–d, Supporting Information), tissue injury marker lactate dehydrogenase (LDH) (Figure S3e, Supporting Information), and pneumonia and sepsis marker C‐reactive protein (CRP) (Figure S3f, Supporting Information). Treatment with DNase‐I pMNSs led to apparent reductions in the serum levels of all these markers.
We also evaluated the absolute number of neutrophils, leukocytes, and total white blood cells (WBCs) in the blood of the CLP‐operated sepsis model (Figure S4a–c, Supporting Information). Similar to the effect shown in the intraperitoneal cavity of CLP‐operated sepsis model (Figure 3c), DNase‐I pMNSs caused a drastic reduction in the number of neutrophils and the total number of WBCs in the blood relative to those observed in the control group (PBS treatment) (Figure S4a and c, Supporting Information). However, compared to the control group (PBS treatment), no difference was observed in the number of leukocytes in the blood (Figure S4b, Supporting Information). Cytokine array analysis on lung tissue in the CLP‐operated mouse model revealed the upregulation of inflammatory cytokines (Figure 4). The upregulated cytokines included IL‐6, IL‐8, IL‐1β, CCL2, etc. The high level of cytokines observed in the lung tissue in the CLP‐operated mouse model supports the sudden rise in the systemic inflammatory response, including the triggering of the “cytokine storm” after SARS‐CoV‐2 infection. However, upon treatment with DNase‐I pMNSs, the level of cytokines such as IL‐6, IL‐8, IL‐1β, and CCL2, was significantly reduced in the lung tissue samples. As SARS‐CoV‐2 causes damage to vascular endothelial cells and induces severe lung damage, we evaluated whether DNase‐I can protect vascular barrier disruption by observing the transendothelial permeability in the septic mouse model (Figure S5, Supporting Information). As a result, free DNase‐I was only effective initially and the effect disappeared after 72 h upon administration, but in the group administered with DNase‐I pMNSs, the survival rate was maintained even after 72 h. The observed features in the blood of animal models support the improved outcomes after the DNase‐I pMNSs administration. To verify whether the ability of the nanoparticle‐bound is DNase‐I concentration dependent, we generated various concentrations of DNase‐I pMNSs, and observed concentration‐dependent survival rate in vivo (i.e., DNase‐I as variable and nanoparticles as constant; 25, 50, and 100 units) (Figure S6, Supporting Information).
NETosis, a neutrophil‐specific oxidative explosion‐dependent process, leads to the release of neutrophil extracellular traps (NETs). NETosis is a special type of programmed cell death in which reticular structures consisting of a DNA backbone and a number of functional proteins are formed. Initially, the neutrophil elastase (NE) degrades the core histone protein and the linker H1 histone protein, resulting in neutrophil nuclei losing their characteristic shape. Then, a combination of nuclear rupture and granular membranes leads to chromatin decondensation and the release of NETs into the extracellular space, enhanced by myeloperoxidase (MPO). These NETs correspond to extracellular filaments of uncondensed chromatin coated by numerous granular proteins. During this process, Cit‐His H3 is thought to be involved in the formation of NETs. NETs have been linked to severe infections such as sepsis, in which the release of chromatin with neutrophil granular proteins serves as an additional defense of the innate immune system against circulating microbes, including bacteria, fungi, protozoa, and viruses. Recently, Zuo et al. verified that sera from patients with COVID‐19 have elevated levels of cell‐free DNA (eDNA), myeloperoxidase‐DNA (MPO‐DNA), and citrullinated histone H3 (cit‐His H3), and demonstrated that the latter two (MPO and cit‐His H3) are specific markers of NETs. In patients with severe COVID‐19, lymphopenia has been observed as a collective and representative characteristic, accompanied by an increased ratio of neutrophils as well as an increase in the absolute neutrophil count.[25, 26] In addition, a recent study has proposed that NETosis is perhaps linked to poor outcomes in patients with COVID‐19 and is believed to play a crucial role in COVID‐19 pathogenesis. It is postulated that aberrant activation of neutrophils, an increase in the release of NETs, and eDNAs in peripheral blood may contribute to organ damage and mortality in COVID‐19 patients.
A recently published report and in our own independent work have demonstrated elevated levels of NETosis markers, such as eDNA, MPO and NE.[10, 37, 38] These are characteristic features of critically ill patients with COVID‐19.Hence, we hypothesized that DNase‐I could be used to ameliorate the dissolution of the main constituent of NET structure, DNA, and thus prevent NET‐related pathogenesis, including progression toward ARDS and sepsis in severe COVID‐19 patients. However, it is quite challenging to treat patients with high levels of NETosis, and it is linked with high mortality and morbidity rates. Therefore, a novel NETosis‐targeting agent able to tackle and reduce NETosis in the blood is essential to prevent further progression toward sepsis. Currently, there are no FDA‐approved drugs to treat severe COVID‐19. Existing antivirals and antimalarials that are being used in clinical trials for the treatment of SARS‐CoV‐2 have shown therapeutic efficacy in only patients with mild conditions and not in severe COVID‐19 patients with ARDS or sepsis. Remdesivir has shown some promising results in mild COVID‐19 patients; however, it is still an investigational antiviral drug and is not currently FDA‐approved to treat or prevent any diseases, including COVID‐19. This highlights the urgent need for an alternative means of treating severe COVID‐19 patients by effectively suppressing the inflammation‐mediated MOF syndrome.
Although degrading the NET structure could serve as a promising strategy to suppress NET‐related complications in COVID‐19 patients, it has been reported that the half‐life of DNase‐I in the blood is very short. Previously, a polydopamine‐coated polymeric nanoparticle was proposed as a long‐acting DNase‐I formulation.[17, 18] In this study we report, we demonstrated DNase‐I‐coated melanin‐like nanospheres (DNase‐I pMNSs) for the amelioration of sepsis‐associated NETosis (Figure 1). The DNase‐I pMNSs possess a structure similar to that of squid ink and skin melanin.[19–22] As melanin nanoparticles using polydopamine are processed in water without using organic solvents, the synthesis of melanin nanoparticles is environmentally friendly. In addition, the excellent adhesion properties of polydopamine enable the effective immobilization of the biomolecules on the surface of nanoparticles, thereby enabling various bioapplications. The interaction between the surface of melanin nanoparticles and DNase‐I is Michael addition or Schiff base reaction between the catechol group of melanin and the amine group of DNase‐I.[40, 41] We observed that the DNase‐I pMNSs maintained their activity in a biologically relevant solution (media containing FBS) for 36 h (Figure S2, Supporting Information). This is thought to be because the DNase‐I attached on the nanoparticle surface could leech off or exchange in the presence of FBS. Nonetheless, it is assumed that the 36 h‐stability of the nanoparticles is sufficient even under the biological settings (during circulation in the blood containing human serum albumin) after injection.
We also performed a comparative study using blood samples from severe COVID‐19 patients and demonstrated the suppression of NETosis factors such as eDNA, NET, MPO, NE, and cytokine levels (Figure 2). Through the administration of exogenous DNase‐I pMNSs in the CLP‐operated septic mouse model, we demonstrated that DNase‐I pMNSs effectively reduce sepsis‐associated NETosis factors (Figures 3 and 4). We also showed that administration of the DNase‐I pMNSs significantly reduces NETosis factors such as eDNA, NET, MPO, and NE in the CLP‐operated septic mice (Figure 3). These observations are also consistent with cytokine array analysis in lung tissue from the CLP‐operated mouse model, which revealed that DNase‐I pMNS treatment lead to a reduction in the level of sepsis‐related pro‐inflammatory cytokines, such as IL‐6, IL‐8, IL‐1β, and CCL2 (Figure 4). In particular, intravenous injection of DNase‐I pMNSs alleviated the systemic inflammation and attenuated mortality in a septic mouse model, resulting in a 40% survival rate for the CLP‐operated septic mice at 132 h post‐induction (Figure 3a), demonstrating that the mice had fully recovered. The free DNase‐I is effective at the beginning of administration (Figure 3f), but in the group administered with DNase‐I pMNSs, it was confirmed that the effect lasted until 72 h and improved the survival rate (Figure 3a). The free DNase‐I is effective up to about 24 h after administration, and the DNase‐I‐bound nanoparticles (DNase‐I pMNSs) acted longer and improved the mortality (Figure 3a).
These results indicate that early administration of the DNase‐I pMNSs during the early phase of sepsis may slow progression toward ARDS and sepsis, thereby potentially treating the symptoms in severe COVID‐19 patients. Our data support further investigation of DNase‐I pMNSs for the treatment of severe COVID‐19‐mediated illnesses. Although the suggestion to use DNase‐I pMNSs as a potential treatment for severe COVID‐19 is based entirely on in vitro blood samples derived from severe COVID‐19 patients and in vivo studies using the CLP‐operated septic mouse model as a surrogate for SARS‐CoV‐2 infection, our results demonstrate that NETosis factor, cytokine release, and lung damage were significantly alleviated after treatment. In addition, sepsis‐associated inflammatory response including, NF‐κB activation were inhibited upon the administration of DNase‐I pMNSs. It is worth noting that the efficacy of DNase‐I pMNSs needs to be further validated in an appropriate in vivo setting using an ARDS‐ or sepsis‐inducing infection animal model before moving forward to clinical trials on patients with COVID‐19.
Based on our data from severe COVID‐19 patients, we demonstrated that eDNA, a NETosis factor, is a potential target for the treatment of SARS‐CoV‐2‐induced sepsis. We suggest therapeutic delivery of a DNase‐I pMNSs for the suppression of eDNA, thereby alleviating the progression of sepsis in severe COVID‐19 patients. We confirmed the effectiveness of the DNase‐I pMNSs in blood samples collected from actual COVID‐19 patients as well as in a CLP‐operated septic mouse model. DNase‐I pMNSs is a promising bioinspired vehicle and demonstrates a potential new strategy for SARS‐CoV‐2‐induced sepsis therapy. There are currently no other reports that target the NETosis factor for the treatment of severe COVID‐19 patients. Recently, DNase‐I was delivered to dissolve NETs in patients with CF, and our findings implicate DNase‐I pMNSs as a suitable material for clinical translation and for preventing the progression of sepsis in severe COVID‐19 patients. Our findings indicate the possibility of using NETosis factors as diagnostic targets in severe COVID‐19 patients.
5 Experimental Section
Dopamine hydrochloride and poly(ethylene glycol) (PEG, four arm‐amine termini, HCl salt) were obtained from Sigma (PA, USA) and JenKem (TX, USA), respectively). NaOH solution (1 N) and Tris buffer (10 mM, pH 8.5) were purchased from Daejung (Suwon, Korea) and Biosesang (Seoul, Korea), respectively. Recombinant DNase‐I was purchased from Roche (Basel, Switzerland). Pierce BCA Protein assay kit was from Thermo Scientific (MA, USA). Dulbecco’s phosphate‐buffered saline (PBS), and fetal bovine serum (FBS) were from Welgene (Gyeongsan, Korea) and Gibco (CA, USA), respectively.
At Yeungnam University Medical Center, whole blood samples were collected from patients after they were diagnosed with the SARS‐CoV‐2 infection at a public health center in Daegu, Republic of Korea. Patients with COVID‐19 sepsis were defined using criteria provided by the Sepsis Consensus Conference Committee. The human study protocol was approved by the Institutional Review Board of Yeungnam University Hospital at Daegu in Korea (YUH 2020‐03‐057, 2020‐05‐031‐001).
Neutrophil Counts of Patients with COVID‐19
Complete blood counts were analyzed in venous blood samples within 24 h of the admission of patients in the hospital. Neutrophil count was analyzed using a Sysmex XE‐2100 Automated Hematology System (TOA Medical Electronics, Kobe, Japan).
NET Enzyme‐Linked Immunosorbent Assay (ELISA)
NETs were generated from freshly isolated neutrophils (1 × 105 cells) by stimulating the cells with phorbol‐myristate acetate (25 nM) (PMA, Sigma‐Aldrich, MO, USA) or control media (RPMI 1640 supplemented with glutamine, penicillin, and streptomycin) and analyzed using a fluorometric technique as previously described. NET production was measured as arbitrary fluorescent units (AFUs).
Plasma samples were analyzed to quantify the release of granule matrix proteins upon degranulation in peripheral blood mononuclear cells (PBMCs) of SARS‐CoV‐2‐infected patients and mice, using a human MPO ELISA kit (BMS2038INST, Invitrogen) and mouse myeloperoxidase ELISA kit (MBS700747, MyBioSource), respectively.
Cit‐His H3 ELISA
Cit‐His H3 concentration in cell culture media or SARS‐CoV‐2 patient sera was determined using a human citrullinated histone H3 ELISA Kit (MBS7254090, MyBioSource).
Quantification of Plasma Extracellular DNA (eDNA)
To quantify eDNA from the plasma of SARS‐CoV‐2‐infected patients or mouse plasma, plasma samples were centrifuged at 16800 × g for 10 min and DNA was extracted using a Qiagen QIAamp DNA Mini Blood Mini Kit according to the manufacturer’s protocols (Qiagen, Valencia, CA, USA) The purified eDNA was quantified using a NanoDrop spectrophotometer.
ELISA for DNase‐I Activity
Plasma samples were diluted 1:50 and analyzed using digestion buffer spiked with double‐stranded DNA (1 µg mL−1). Samples were stained with PicoGreen (Invitrogen) according to the manufacturer’s protocol. After incubation at 37 °C for 5 h, reduction in PicoGreen staining (fluorescence emission, Em) was then measured using a fluorometer.
Preparation of DNase‐I pMNSs
Bare melanin‐like nanospheres (bMNSs) were synthesized by dopamine hydrochloride, as presented in our previous report (Figure 1). Dopamine hydrochloride (10 mg) was dissolved in deionized water (DW, 50 mL). Then, NaOH solution (50 µL, 1 N) was added to the dopamine hydrochloride solution. The reaction was stirred for 24 h, and the color gradually changed to black as the reaction proceeded. For purification, the prepared bMNSs were collected via centrifugation at 17 000 rpm (27237 × g‐force) for 20 min and washed with deionized water (DW) three times. To prepare DNase‐I pMNSs, surface engineering of bMNSs with DNase‐I was performed according to our previous reports.[17, 18] The resulting bMNSs (10 mg) were re‐suspended in Tris buffer (5 mL, 10 mM, pH 8.5) containing DNase‐I (2, 5, 10, or 20 mg) and poly(ethylene glycol) (10 mg, PEG, four arm‐amine termini, HCL salt), and stirred at 4 °C for 3 h. The prepared DNase‐I pMNSs were purified in the same manner as above and washed with DW multiple times. We also prepared PEG‐coated MNSs (pMNSs) as a control, using the same method as for DNase‐I pMNSs, but without DNase‐I addition.
Characterization of MNS
The particle size and surface charge of nanospheres were measured by dynamic light scattering (DLS: Malvern Instruments, Southborough, Massachusetts). The morphology of the nanospheres was observed by field emission scanning electron microscopy (FE‐SEM: JEM‐7500F, Akishima, Japan). The enzymatic activity of DNase‐I pMNSs was confirmed via degradation of highly polymerized salmon sperm DNA (Sigma‐Aldrich, PA, USA) by gel electrophoresis. For this, bMNSs, pMNSs, or DNase‐I pMNSs were incubated with 1 µg of salmon sperm DNA for 10 min at 37 °C, followed by gel electrophoresis.
For density gradient isolation of PBMCs, Percoll (pH 8.5–9.5; Sigma‐Aldrich, UK) was used as previously described. The PBMCs (95% purity and 97% viable according to trypan blue exclusion) were resuspended in RPMI 1640 media (Sigma‐Aldrich). For discontinuous density gradient centrifugation of neutrophils, 1‐step Polymorphs (Axis‐Shield, Oslo, Norway) were used. To further increase the purity, the neutrophil population was purified using CD45 antibody‐conjugated magnetic beads and magnetic‐activated cell sorting (MACS). Trypan blue dye exclusion showed that the general viability of the neutrophils was >95%. To analyze the effect of DNase‐I on inhibition of cytokine production, neutrophils were isolated from COVID‐19 patients and treated with DNase‐I MNSs. DNase‐I MNSs were also administrated to CLP‐operated septic mice. Supernatants were used for cytokine assay using ELISA, and cell lysates were used for analysis of NF‐kB activity.
Nuclear extracts were prepared, and TransAM assays were performed as previously described. The activity of individual NF‐κB subunits was determined via ELISA using NF‐κB Family Transcription Factor Assay Kit (43296; Active Motif, Carlsbad, CA, USA). Briefly, nuclear extracts (2 µg) were placed into wells of NF‐κB consensus oligonucleotide‐coated 96‐well plates. Plates were incubated with NF‐κB primary antibody, and then binding was detected using HRP‐conjugated secondary antibody included with the kit. For analysis, the optical density (OD) at 450 nm was measured using a Tecan Spark microplate reader (Tecan, Austria GmbH, Austria).
Levels of inflammatory cytokines IL‐1b, IL‐6, IFN‐g, and TNF‐α in the supernatant were measured after treating PBMCs with pMNSs, Free‐DNase‐I, and pMNSs‐DNase‐I (100 unit) using human ELISA kits (Quantikine ELISA, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocols.
Animal experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University College of Medicine (IACUC No. No. SKKU IACUC2020‐06‐29‐2). Six‐ to seven‐week‐old C57BL/6 male mice (18–20 g) were obtained from Orient Bio (Seongnam, Korea). Mice were used after a 12‐day acclimatization period. Five animals per cage were housed under controlled temperature at 20–25 °C and humidity of 40–45% with a 12:12 h light/dark cycle. Mice were fed a normal rodent pellet diet and supplied with water ad libitum.
Cecal Ligation and Puncture
The CLP‐operated septic mouse model was prepared as previously described.Briefly, a 2‐cm midline incision was made to expose the cecum and adjoining intestine. The cecum was then ligated tightly using a 3.0‐silk suture 5.0 mm from the cecal tip, punctured with a 22‐gauge needle, and then gently squeezed to extrude feces from the perforation site. The cecum was then returned to the peritoneal cavity, and the laparotomy site was sutured using 4.0‐silk. For sham operations, the cecum of animals was surgically exposed, but not ligated or punctured, and then returned to the abdominal cavity.
In Vivo Neutrophil Migration Assays
To assess neutrophil migration, CLP‐operated mice were treated with pMNSs, free DNase‐I, and pMNSs‐DNase‐I (100 units) 6 h after CLP surgery. Mice were then euthanized, and the peritoneal cavities were washed with 5 mL of normal saline. Neutrophils were counted using an auto hematology analyzer (Mindray, BC‐5000 Vet). Results are expressed as neutrophils × 106 per peritoneal cavity.
RNA from lung tissue was extracted using an RNeasy mini‐kit (Qiagen Venlo, Netherlands) according to the manufacturer’s protocols. RNA‐seq libraries were prepared using the TruSeq RNA Sample Prep kit v2 (Illumina) according to the manufacturer’s protocols. RNA‐seq libraries were pair‐end sequenced on an Illumina Hi‐seq 3000/4000 SBS kit v3 (MACROGEN Inc.). All RNA‐seq data were mapped using the Tophat package against Affymetrix Human Gene 2.0 ST arrays (902136). Remaining mRNA was used for qPCR analysis. Fold‐change was determined using the R package limma, and P‐values were Benjamini‐Hochberg (BH) adjusted. The array results are available in the Gene Expression Omnibus (GEO) database of NCBI (Accession code: GSE101126).
Hematoxylin and Eosin (H&E) Staining and Histopathological Examination
Male C57BL/6 mice were CLP‐operated and then intravenously administered pMNSs, free DNase‐I, or DNase‐I pMNSs (100 units) at 12 h or 24 h after CLP (n = 5). At 72 h post‐CLP operation, mice were euthanized. Lung specimens were removed from mice for the analysis of phenotypic changes. H&E staining was performed using a standard protocol.
Cytokine Levels in the Plasma of Septic Mice
Fresh serum was used for analysis of AST, ALT, BUN, creatinine, and LDH levels using biochemical kits (MyBioSource). Values were measured using an ELISA plate reader (Tecan, Austria GmbH, Austria).
All the in vitro and in vivo data were analyzed via two‐tailed unpaired t‐test using the Graphpad prism 7 software, the prepared sample sizes were n ≥ 3, and the statistical significance was set at P <0.05. A more detailed information for each experiment is provided in the Figure legend. All data normalization processes were carried out according to the manufacturer’s protocol. Data transformation and evaluation of outliers were not used in our study.
H.H.P., W.P and Y.Y.L contributed equally to this work. This work was supported by the following grants: the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2019R1C1C1006300, 2019R1A4A1028700, 2018R1C1B6001120, 2020R1A4A3078645, and 2020R1A4A4079817). This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2019M3C9A6091949). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (2018M3A7B4071204) This work was also supported by the BK21 FOUR Project.
Conflict of Interest
The authors declare no conflict of interest.
The 2 charts show how super immune mycology derived melanin may be for all people:
Birch Boys Fungal Melanin, Our Patent Pending Novel Extraction Process
by Maya Duncan-WhiteAugust 30, 20204 Comments
What Makes Chaga the King of Herbs?
If you’ve ever harvested Chaga, somewhere along your journey you may have wondered about its tough, dark, seemingly charred surface. Some have even made the mistake of scraping it away, assuming it has no value… Until they are told the truth:
“The black is the best part“
August 25, 2020 marks an important milestone for Birch Boys, Inc., with the filing of our first patent! Over the last six months, we have been perfecting the process of isolating Melanin from several fungal species, including our champion fungus, Chaga (Inonotus obliquus).
What is Melanin?
Melanin is a class of pigments produced by plants, animals, and fungi to protect cells from damage and promote survival. Scientific research on melanin is exploding with new innovations in the fields of nanotechnology, biomedicine, and materials science. It is being investigated for use in biomedical devices as a biocompatible and biodegradable component. For the same reasons, melanin is also perfectly poised to be a skincare solution with antioxidant properties and UV radiation absorption. Previously, commercially available melanin was limited to synthetic or squid ink derived melanins, but Birch Boys Inc. is changing that with our sustainably produced natural fungal melanins.
Spores of Innovation
Garrett Kopp (founder and President of Birch Boys, Inc.) was faced with a quandary when he stumbled into some research on melanin. Knowing it was a major constituent of Chaga, he realized Birch Boys was in a unique position to produce melanin. Research on melanin and its applications is exploding, but much of it is focused on using synthetic or squid ink-derived melanins, and both of these have obvious drawbacks. Namely, synthetic melanins are subject to post-synthetic transformations that can cause degradation of the product. This raises quality and safety concerns, especially if it is synthesized using toxic or harmful chemicals. With squid ink, the process involves living animals, so there’s ethical as well as safety and sustainability concerns. So, in a culture that is finally pushing for natural and ethical products, where is the natural and ethical melanin? It’s right here.
Garrett contacted me, Maya Duncan-White, a friend from college, and conveniently a Chemist. Garrett and I were both a part of the Clarkson School, an early entrance college program at Clarkson University. We skipped our senior year of high school in order to start college. While unorthodox at the time (my high school even tried to convince me I wasn’t allowed to do this), it gave us the opportunity to get a head start on education and careers, with far better resources and opportunities than we would have had in high school. Garrett poured his heart into his business, and I poured mine into science and research.
Function of Melanin in the Epidermis
Melanin is a general term for a group of pigments produced by plants, animals, and fungi. In humans, we are most familiar with eumelanin and pheomelanin. Everyone has eumelanin in their skin, but skin tone is dictated by the amount of melanin concentrated in the skin cells. Pheomelanin is an orange-toned melanin that gives red hair and freckles their color. Melanin serves a similar purpose for fungi, providing protection from harmful UV rays and acting as an antioxidant and free-radical scavenger. The high melanin content in Chaga is responsible for its unprecedented ORAC score (Oxygen Radical Absorbance Capacity). The ORAC score is a quantified measure of antioxidants, and to give some context, Chaga has 1,300 times as many antioxidants as blueberries! So, when you drink Chaga tea, you are drinking soluble melanin and experiencing its benefits.
Many studies have shown that melanins support the survival of fungi in many different ways in addition to antioxidant activity, including resistance to infection, increased ability to survive in extreme environments (temperature, radiation, and improved mechanical strength of cell walls. In fact, species of melanized fungi have been found thriving in the nuclear reactors of Chernobyl!
Abundant Applications for Fungal Melanin
Fungal melanins are the obvious choice for a natural and sustainably produced melanin for use in research and in skin care products. Current research initiatives have identified melanin as a choice biocompatible, biodegradable material to be used to advance the fields of biomedicine and biomedical engineering, materials science, and nanotechnology. Melanins have been used in the design of drug delivery systems, coatings on implanted devices, and even in edible batteries! Melanin also shows promise for use in water filtration systems, with the ability to chelate and effectively filter heavy metals from contaminated water. This feels appropriate since mushrooms have always been nature’s cleaners, with the incredible ability to break down toxic waste like oil spills and even cigarette butts. So with this, I say: The future is melanin. The future is fungi.
Thanks to Our Customers During the Coronavirus Pandemic
Lastly, Garrett and I want to thank everyone who has been alongside us on this journey: Our loyal customers, our wholesalers, and our local harvesters. Your support gave us the opportunity we needed to pursue this; YOU fueled the successful outcome of our discovery and we are so thankful. We ask for your continued participation and feedback as we explore new markets and customers for fungal melanin. If you know anyone who may need wholesale melanin, whether they are academic researchers, skin care companies, or someone with an innovative idea, feel free to send them our way and email us at email@example.com and firstname.lastname@example.org.
Birch Boys is a wonderful and unique company because we value our relationships with our customers and listen to them. So please, we would love to hear from you! Thank you so much for reading.
You can look forward to four new incoming blogs over the month of September, NATIONAL MUSHROOM MONTH! Up next is the healing history of the Adirondacks. Stay tuned, and thank you for celebrating with us!
If you are interested in purchasing fungal melanin or wouldlike to request a quote, submit an inquiry here.
Comparing Functional Fungi for Healing Benefits
by Garrett KoppSeptember 24, 20202 Comments
Highlights of Healing Mushrooms
A common misconception is that a single mushroom extract is good for a single benefit. Companies use this misconception for marketing and to distinguish their products from one another, but it could not be farther from the truth. If you read our blogs and look at our product labels, you will find that almost all of our mushroom products share some key benefits. While it is true that each mushroom differs in its specific composition of mycochemicals, most offer shared health benefits:
- Immunomodulatory Activity
- High Antioxidant Content
- Anti-Inflammatory Activity
There is no mutual exclusivity in medicinal mushrooms. A single mycochemical may have many benefits, and a single benefit can be attributed to many mycochemicals. These niche mycological compounds work together synergistically to support nearly every aspect of your health.
Major Bioactive Components:
Chances are you have heard of beta-glucans, but might not have ever heard how they work. Let’s change that! Beta-glucans stimulate the immune system because our immune cells recognize them as invasive. Our immune system, in turn, is stimulated. It gets to work producing more immune cells and working harder to protect our body. It’s like having a nice, strong coffee before a productive day… but for our immune system.
Antioxidants: Phenolic Compounds
Antioxidants and antioxidant supplements are everywhere now. So what sets mushrooms apart? In addition to being a 100% natural source, mushrooms are an extremely potent source of a plethora of antioxidant phenolic compounds. Not one, not two, but countless different phenolics work together to prevent cellular and DNA damage due to oxidative stress. In order to achieve this, antioxidants scavenge and extinguish free-radicals in our bodies. Free radicals are highly reactive chemical species that cause disease and cancer by damaging our cellular membranes and DNA. Antioxidants support our cellular and DNA health and have anti-cancer, anti-inflammatory, and immune-boosting activity.
Chaga, in particular, is the breadwinner when it comes to antioxidants. Chaga contains a high amount of melanin, which is a potent phenolic antioxidant. We always hear about berries being the “best” source of antioxidants, but Chaga has 1,300 times the antioxidant concentration of blueberries. Yes, that is a lot.
Triterpenoids (or triterpenes) are a class of compounds with immunomodulatory activity, including antiviral, antimicrobial, and anticancer activity. Reishi is a potent source of triterpenoids. There is a reason Reishi has been lauded as the key to vitality and longevity for thousands of years in traditional chinese medicine. You can even use your taste buds as evidence of the presence of triterpenoids in Reishi as they are responsible for the bitter flavor. Ganoderic acid, lucidenic acid, and ganoderiols are unique triterpenoids found only in Reishi. These compounds have been researched for their anti-inflammatory, immunomodulatory, antiviral, and hepatoprotective (hepato: liver) properties.
While Reishi is widely known for its high concentration of medicinal triterpenoids, many other medicinal mushrooms also contain unique triterpenoids. Lion’s Mane, a popular nootropic mushroom, contains two such compounds: Hericenones and Erinacines. Found only in Lion’s Mane mushrooms, hericenones and erinacines are small molecules with the ability to stimulate synthesis of Nerve Growth Factors (NGF) in the brain and promote the growth and survival of your brain’s neurons. I can’t fully express in words how incredible this is! Triterpenoids also possess immunomodulatory and anti-inflammatory activities. For me, Lion’s Mane is the one mushroom I never forget to include in my daily health regimen (Wink!).
All of the mycochemical groups listed down the left column encompass a broad subgroup of chemicals, which I have listed more specifically and notated in the Extra Science Footnotes below!
The Role of Solubility Properties in Extract Formulation
We get asked a lot why we use water and ethanol for our extracts: Why not just water? Why do we combine them? Why ethanol? The shortest answer: Using both water and ethanol allows us to extract the highest amount of beneficial compounds.
All chemicals follow a set of solubility rules: the structure of a chemical determines which solvents (e.g. water, alcohol, oil, etc.) it is soluble in. Every chemical has a “preference” for a particular type of solvent. Luckily, many mycochemicals prefer water, but some do not, so we use ethanol to coax the rest of the mycochemicals into our extract. Dual extracts will offer a higher potency and a broader range of benefits and compounds than a simple water extract will. Anyone who tells you that alcohol extracts are useless or undesired has not done their research. Beneficial phytosterols, glycosides, and are all readily solubilized by ethanol!
Another question we get a lot: Why is my tincture/extract cloudy? Is this bad? Answer: No, it is perfectly normal! A cloudy tincture means that there are particulates in the liquid. This is a result of those pesky solubility rules. When we combine our water and ethanol extracts, somethings may precipitate out of solution. The precipitate causes the tincture to appear cloudy. Just make sure to shake up your extract bottle before use!
Reishi Mushroom – History and Benefits of “The Mushroom of Immortality”
by Garrett KoppJune 24, 20202 Comments
A Guide to Reishi Mushrooms
Reishi mushroom, Ganoderma lucidum or Ganoderma tsugae, is also known as “lingzhi,” which translates to “spiritual/miraculous/sacred/effective” “mushroom” in Chinese. It has been used in traditional Chinese medicine for over 2,000 years and is venerated as the “mushroom of immortality”.
History of Reishi Mushroom
The first textual mentions of reishi date back to the Han dynasty, over 2,000 years ago, when Chinese healers discovered medicinal properties. Ancient Chinese scripts documented the mushroom as an “elixir of immortality”. In addition to these texts, reishi has also shown up in ancient artworks related to Taoism. Going back even further, ancient carvings, paintings, and furnitures featuring reishi have been discovered. One of the first texts to document the medicinal value of herbs was Shen Nong Ben Cao Jing, written during the Chinese Han dynasty. Within the book, botanical, zoological, and mineral substances are recorded. Reishi is featured and described as a mushroom with therapeutic properties, anti-aging effects, tonifying effects, strengthening cardiac function, enhancing vital energy, and increasing memory capability. In 1596, the Bencao Gangmu, a compendium of medicinal material, was published listing 6 subspecies of reishi for different medicinal benefits.
Wild reishi remains a rare commodity, and before people began cultivating and growing it, access was mostly limited to Chinese nobility. As the years went by, it became a staple for many people across all of Asia. Reishi mushroom remains a commonly used traditional treatment for many ailments across modern-day Asia, and its use in western cultures is starting to grow dramatically as well.
Reishi Mushroom Benefits
Reishi is being increasingly used across the world to assist with general health and wellness. As many as 279 bioactive compounds have been isolated from reishi as of 2015. It is used to boost the immune system, provide stress relief, assist with sleep, lower high blood pressure and high cholesterol, provide antioxidants, and fight cancers. Accordingly, research has been conducted to establish these properties. I will discuss this impressive research in the following sections.
1. Immune System Support
Your immune system functions to fight off illnesses by identifying and seeking out potentially harmful organisms, foreign material, and mutated cells. As you age, your immune system reduces the production of immune cells and causes slower and less effective responses. This can lead to an increase in illnesses, inflammation, and cancer risk.
Reishi contains three major immunomodulating substances: triterpenoids, proteins, and polysaccharides like beta-d-glucans. A 2002 study states that these increase mitogenicity (cell reproduction) and activation of immune cells, thus supporting an effective immune response and contributing to reishi’s anti-inflammatory and anticancer properties. A 2018 study also related the immunomodulatory activity of reishi to its potent anticancer properties due to its ability to regulate the expression levels of immune cytokines in blood serum. A 2002 study found that several polysaccharides, among them beta-d-glucan, and gylans isolated from G. lucidum also exhibited immunostimulating activity. The isolated compounds increased proliferation of lymphocytes in vitro, that is, rapidly increased the number of white blood cells.
Allergies are also caused by a fault in the immune system. The cumbersome symptoms of seasonal allergies are caused by your body releasing a flood of histamines when you come in contact with a harmless substance, like pollen, that your immune system has misidentified as a foriegn invader that must be destroyed. Reishi has been shown to regulate release of histamines, which may help alleviate or lessen unpleasant symptoms. A 2006 article argues for reishi to be taken as “immunonutrition” in order to treat histamine-mediated allergic responses.
It is important to note that Anaphylaxis is different from mild to severe allergies. It is the result of a serious and sometimes deadly sensitivity to something, most commonly peanuts, shellfish, and bees. Reishi should not be used to treat or prevent Anaphylaxis. In cases of Anaphylaxis, an epipen and a trip to the hospital is usually necessary.
2. Stress Relief
Reishi is classified as an adaptogen. Adaptogens help the body maintain homeostasis by stabilizing physiological processes related to stress. Adaptogens support the neurological, endocrine, and immune systems and allow a more positive physical response to stress.
3. Anticancer Activity
The most impressive medicinal aspect of reishi is its potential as an anticancer agent. Reishi contains beta-glucan polysaccharides, a well-researched immunostimulant and anticancer agent. In a 2006 study, ganoderic acid (reishi is the only known natural source of ganoderic acid) was isolated from reishi and found to be cytotoxic to various human carcinoma cell lines and significantly less toxic to healthy cells. The researchers conducted a mouse study and found that the growth of human solid tumors was suppressed after treatment with ganoderic acid. They also showed that the proliferation (rapid growth) of highly metastatic lung cancer cell lines was inhibited by induced cell death and arrest of the cell cycle. The study concluded that reishi was a potentially useful chemotherapeutic agent. In a 2003 study, G. lucidum was shown to suppress cell adhesion and migration of highly invasive breast and prostate cancer cells and researchers concluded that it could reduce tumor invasiveness. In a 2018 publication, a mouse model was used to establish the antitumor activity of G. lucidum extract. They found that the extract inhibited tumor growth without causing liver or kidney toxicity nor bone marrow suppression. Immune cytokines are associated with the survival, growth, and progression of cancers, and in this study, the reishi extract was found to modulate the levels of immune cytokines in blood serum, which increased antitumor immune system activity.
4. Cardiovascular Health
Reishi contains triterpenoids, a compound reported to possess antihypertensive and hypocholesterolemic activity, meaning it can lower blood pressure and cholesterol levels, respectively. In a 2018 study, researchers investigated whether reishi may be able to treat hypertension without incurring the negative side effects of many current hypertension drugs. Aqueous reishi extract was administered to rats with hypertension and after 7 weeks of treatment, blood pressure was reduced to levels comparable to treatment with losartan (a commonly prescribed medication for high blood pressure treatment). However, reishi has no known negative side effects. The study also found that after reishi treatment, cerebral blood flow was increased and neurotransmitter ratios had shifted toward more excitatory as opposed to inhibitory. This means that reishi could also be a potential nootropic and support healthy brain function and stimulation.
5. Antioxidant Activity
Antioxidants are compounds that prevent cell damage or cell death from highly reactive molecules called free radicals. Free radicals cause damage to DNA and are linked to aging, cancer, and other diseases. In a 1999 study, terpenes and polysaccharides were isolated from reishi and tested for their antioxidative properties. All samples were shown to have antioxidant activity, with terpene extracts the highest comparatively. The terpene extract was further studied and found to contain ganoderic acids and lucidenic acid as the most major components.
Birch Boys and Reishi: The Harvesting Process
Here at Birch Boys, we only use 100% wild-harvested reishi mushrooms. We harvest our reishi from hemlock trees in the Adirondack Mountains of New York state. All of our mushrooms are sustainably harvested.
Technically speaking, Red Reishi is a name that can refer to two subspecies of mushrooms, either Ganoderma tsugae or Ganoderma lucidum. G. tsugae is more often found in colder regions, such as the Northeastern United States. G. lucidum prefers warmer climates, such as Asia, South America, and Australia.
That begs the question, which one is better? In short, it depends on the quality of its habitat, among other environmental factors. Despite being recognized as two separate species, comparative analysis suggests that the bioactive components within the two mushrooms fall somewhere between ‘extremely comparable’ and ‘virtually identical’. The informed consensus is that you should worry less about the type of reishi you’re getting and more about where you’re getting it from.
Most Reishi products contain G. lucidum, as it has become highly commercialized. G. lucidum is most often sourced in bulk from Chinese farms that grow their reishi artificially on spawn-logs or from bags of mulch or wood-pulp substrate.
At Birch Boys, we do things differently to offer you the most natural and healthy products. We harvest the North American variant of reishi, G. tsugae, which is commonly referred to as the “Hemlock Bracket” because it grows on fallen hemlock trees. We work with loggers, foresters, and outdoor professionals to sustainably harvest reishi from its natural habitat. Our harvesting domain spans 220,000 acres of North American forest where we have exclusive mushroom foraging rights.
Quick Tip: If you want to forage for reishi on your own, be sure to practice sustainable harvesting techniques. To do this, make sure that you have waited until after the mushroom has dropped its spores prior to harvesting. You’ll know the spores have dropped by observing the dust of rusty-brown spore powder beneath the mushroom. See the image below for an example.
While we know there is so much more we can learn about reishi, one thing is for sure… this “mushroom of immortality” has one of the longest established histories of medicinal use of any mushroom in the world. Reishi has managed to maintain its permanence in Chinese medicine as well as catching on in the western world, where it is reaching new heights through scientific studies.
The Best Reishi Products
Bao, Xing-Feng, et al. “Structural Features of Immunologically Active Polysaccharides from Ganoderma Lucidum.” Phytochemistry, Pergamon, 17 Jan. 2002, www.sciencedirect.com/science/article/abs/pii/S0031942201004502.
Boh, Bojana, et al. “Ganoderma Lucidum and Its Pharmaceutically Active Compounds.” Biotechnology Annual Review, Elsevier, 15 Sept. 2007, www.sciencedirect.com/science/article/pii/S1387265607130106.
Gao, Yihuai, and Shufeng Zhou. “The Immunomodulating Effects of Ganoderma Lucidum (Curt.: Fr.) P. Karst. (Ling Zhi, Reishi Mushroom) (Aphyllophoromycetideae).” International Journal of Medicinal Mushrooms, Begel House Inc., 2002, www.dl.begellhouse.com/journals/708ae68d64b17c52,2ea43b2e1c72ed1c,0444eb934f7280cf.html.
Powell, Martin. “The Use of Ganoderma Lucidum (Reishi) in the Management of Histamine-Mediated Allergic Responses.” Gale Academic Onefile, The Townsend Letter Group, May 2006, go.gale.com/ps/anonymous?id=GALE%7CA145341877&sid=googleScholar&v=2.1&it=r&linkaccess=fulltext&issn=&p=AONE&sw=w.
Shevelev, Oleg B, et al. “Hypotensive and Neurometabolic Effects of Intragastric Reishi (Ganoderma Lucidum) Administration in Hypertensive ISIAH Rat Strain.” Phytomedicine : International Journal of Phytotherapy and Phytopharmacology, U.S. National Library of Medicine, 1 Mar. 2018, www.ncbi.nlm.nih.gov/pubmed/29519314.
Siwulski, Marek, et al. “Ganoderma Lucidum (Curt.: Fr.) Karst. – Health-Promoting Properties. A Review.” Sciendo, Sciendo, 1 Sept. 2015, content.sciendo.com/view/journals/hepo/61/3/article-p105.xml.
Sliva, Daniel. “Ganoderma Lucidum(Reishi) in Cancer Treatment – Daniel Sliva, 2003.” SAGE Journals, 2003, journals.sagepub.com/doi/abs/10.1177/1534735403259066.
Tang, Wen, et al. “Ganoderic Acid T from Ganoderma Lucidum Mycelia Induces Mitochondria Mediated Apoptosis in Lung Cancer Cells.” Life Sciences, Pergamon, 6 Sept. 2006, www.sciencedirect.com/science/article/abs/pii/S0024320506006801.
Zhao, Ruolin, et al. “The Effect of Ganoderma Lucidum Extract on Immunological Function and Identify Its Anti-Tumor Immunostimulatory Activity Based on the Biological Network.” Nature News, Nature Publishing Group, 23 Aug. 2018, www.nature.com/articles/s41598-018-30881-0.
hu, Min, et al. “Triterpene Antioxidants from Ganoderma Lucidum.” Wiley Online Library, John Wiley & Sons, Ltd, 8 Sept. 1999, onlinelibrary.wiley.com/doi/abs/10.1002/(SICI)1099-1573(199909)13:6%3C529::AID-PTR481%3E3.0.CO;2-X.
Disclaimer: The statements on this website have not been evaluated by the U.S. Food and Drug Administration. These products are not intended to treat, fight, cure, or prevent any disease, illness, or ailment. The information presented herein is not a substitute for a consultation with a licensed physician.
We read with great interest the review article by Aparna et al. in the recent issue of your journal. I would like to commend the authors for their endeavor to highlight the under-recognized public health problem and suggesting potential solutions for the same, but at the same time have the following comments to offer, explanation to which will benefit the readership of the journal.
- The diagnostic cutoffs of the levels of serum vitamin D given in Table 1 are based on the Endocrine Society Clinical Practice Guideline (ESCPG) 2011 which are relatively old. A recently published review of guidelines suggests that all international guidelines except ESCPG agreed upon the cutoff of >20 ng/ml as sufficient, 12–20 ng/ml as insufficient, and <12 as deficient. The same cutoffs have been endorsed by Indian Academy of Pediatrics. Statistically, also, the cutoff of 20 ng/ml is more appropriate as it coincides with the level that would cover the needs of 97.5% of the population. Defining a standard cutoff is very necessary as increasing the cutoff will greatly affect the prevalence rate of insufficiency and will increase the treatment rate. Also, the cutoff for toxicity is much lower than given by authors
- As highlighted by authors that the 25(OH)D rather than 1,25-dihydroxyvitamin D levels should be measured as it greatly depends upon parathyroid hormone concentrations. The same has been endorsed by the American Academy of Nutrition as well as other prominent societies. The other more important reasons for recommending 25(OH)D levels are the very short half-life of 1,25-dihydroxy vitamin D (4 h), little or no relationship of serum levels of 1,25-dihydroxyvitamin D to vitamin D stores, and practical challenge to measure with accuracy due to its picomolar concentrations and lipophilic nature. The measurement of 1,25-dihydroxyvitamin D levels should be reserved for specific conditions like chronic kidney disease, etc. Recently, there is a surge in the request of 1,25-dihydroxyvitamin D levels; hence, it is very important to educate the physicians about these limitations
- Authors should highlight the role of vitamin D in the pathogenesis of recurrent wheeze as well as asthma. Recent Cochrane review of high-quality studies showed that vitamin D reduced the risk of asthma exacerbation in children as well as adults, hence reduced emergency visits as well as hospitalization. Also, it has been seen that prolonged vitamin D deficiency may lead to stunting and vitamin D supplementation may be an intervention to prevent and mitigate childhood stunting
- Apart from those highlighted by authors, few other reasons for vitamin d Deficiency pandemic in India are wrong cooking practices, the high prevalence of lactose intolerance, increased intake of coffee and tea, and lack of adequate supplementation/fortification.
As highlighted by authors, we physicians must understand ourselves as well as educate the common peoples and the policy makers that sunlight alone is not sufficient, the adequate supplementation, as well as fortification of staple foods, is the key to halt this pandemic.
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What coalitions will mirror the global scope and effect, with true and clinically proven health options, the magnitude of A.C.T. access to Covid-19 tools’ four horseman? 1- As of 11/19/2020, leaders of 189 countries of the world 194 agreed to vaccinate 1/2 billion of their collective global constituencies by March and 2 billion by 2022, with by then, up to 300 wonderful, different synthetic compounds to choose from with full disclosure of every ingredient of every vaccine. 2- diagnostics testing 9 million by May 2021. 3- testing Covid treatments. 4- Global automated remote and point of sales, mark, monitor and monetized technocratic bio tech economy and education. One universal principle is that all will have access, regardless of locale, income or education.Hee Ho (Daniel) Park, PhD water engineering, Kang Won, National University (One of 10 core national universities with a 270 legacy test in South Korea) is the lead developer of the nano melanin vitamin D, Covid etc., treatment prevention trial, 13 member, interdisciplinary team is His 41 research works won 256 citations. His site is covid19.elsevierpure.com. See the involvement of Tae Wook Han and Dook. kribb.re.kr is a part of The global wellness melanin life science next step in human revolution, as is the national agency, ncov.mohwgo.kr, with Covid stats and 351 articles.
Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses
Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8–35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.
Citation: Carpentier AF, Geinguenaud F, Tran T, Sejalon F, Martin A, Motte L, et al. (2017) Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses. PLoS ONE 12(7): e0181403. https://doi.org/10.1371/journal.pone.0181403
Editor: Stephen J. Turner, Monash University, AUSTRALIA
Received: December 30, 2016; Accepted: July 2, 2017; Published: July 17, 2017
Copyright: © 2017 Carpentier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Funding support for this study was provided by: Oligocyte (non-profit association); ADNA (non-profit association). The authors received no other specific funding for this work.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: The AP/HP (Assistance Publique de Hopitaux de Paris) filed a provisional patent application (“Immunostimulatory Compositions”, PCT/EP2016/078794, filed November the 25th, 2015) on this vaccine approach. AF Carpentier & C Banissi are listed as inventors. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
The recent developments in the ability to identify genetic mutations that can be recognized by the immune system (neo-epitopes), along with the success of immune checkpoint inhibitors, have renewed the interest for cancer vaccines [1,2,3]. Antibody responses, commonly induced with modern vaccines; are not helpful where intra-cellular antigens are involved, which is usually the case in cancer. Immunity in cancer is mainly mediated by cytotoxic T- lymphocytes (CTLs) [4,5]. CTLs recognize short peptidic epitopes (eight to ten amino acid residues) that result mainly from degraded intracellular proteins, and that are displayed by major histocompatibility complex (MHC) class I molecules on the surfaces of target cells and/or antigen presenting cells (APCs). However, CTL responses are much more difficult to trigger in humans than antibody responses , as the induction of CTL responses requires the extracellular antigen (usually presented by MHC class II molecules) to be redirected to the MHC class I pathway in a process called cross-presentation . To achieve cross-presentation, and subsequently the induction of CTL responses, several formulations have been developed, including viral vectors, water-in-oil emulsions, dendritic cell purifications, DNA vaccines, and combinations with immune adjuvants such as Toll-like receptors (TLRs) or toxins [4, 5, 7–11]. However, these approaches face barriers to clinical development, such as poor stability, high manufacturing costs, and inadequate immune responses in humans [12,13]. Another limitation is seen with subunit vaccines which use a small part of an antigen. Indeed, peptides are typically poorly immunogenic, and both adjuvants and high antigenic doses are therefore usually needed to elicit an effective immune response [12,13].
Melanin, synthetized within the melanosomes of epidermal melanocytes, is widely found in mammalian skin and plays a major role in the protection of skin cells against mutagenic ultraviolet (UV) light rays . Interestingly, melanocytes exhibit phagocytic functions, and phagosomes are transported from the cell surface to the melanosomes, supporting the idea that melanosomes, that contain many lysosomal enzymes, are part of the lysosomal degradation pathway [15–17]. Further studies have shown that melanocytes may contribute to the phagocytosis of invading pathogens and can act as antigen presenting cells [15,18]. We therefore investigated whether melanin itself could act as an adjuvant for immune responses against specific antigens.
Here, we show that the attachment of synthetic melanin to small peptidic epitopes can be used in vaccine formulations to efficiently trigger immune responses. Moreover, the addition of synthetic melanin to the vaccine formulation enables a dramatic reduction in antigen dose compared with the doses required for other conventional vaccine approaches.
Preparation and characterization of synthetic melanin and peptide vaccine formulations
The oxidation of L-Dopa in alkaline conditions is an established process for obtaining synthetic melanin . Thus, to prepare the vaccine formulations, a solution of L-Dopa was mixed with a solution of peptide corresponding to the human gp100 epitope  (weight ratio L-Dopa: epitope of 1:4), and the mixture was then oxidized at pH 8.5 in aerated conditions (Fig 1a). Under these conditions, the colorless L-Dopa solution turned black, a process that we monitored using UV spectroscopy (Fig 1b). The kinetics of L-Dopa oxidation, as assessed by the 350/280 nm ratio, were similar with or without peptide (Fig 1c). When filtered through a 10-kDa filter, a black material was retained in the upper chamber, and could be easily resuspended in a saline solution. Transmission electronic microscopy (TEM) images showed nanoparticles with sizes mainly between 10 and 20 nm (Fig 1d). When the reaction was monitored via SDS-PAGE, the free peptide disappeared within 18 h (Fig 1e). This suggests that melanin bound to gp100 (forming gp100-melanin). The binding of the peptide to gp100 was confirmed by FTIR spectroscopy. The amide II’ band at 1455 cm-1, which is characteristic of peptides  was observed with the gp100-melanin spectrum but not with the control melanin spectrum (Fig 1f).
Distribution of melanin in draining lymph nodes
The induction of antigen-specific immunity relies on the direct interaction of DCs with naive T-cells that occur in the T-cell zone of lymph nodes. To assess the distribution of the vaccine formulations in vivo, mice were injected subcutaneously with [gp100-melanin + CpG-28] or saline, and sacrificed on days 2 or 7 (n = 3/group). To avoid any bias caused by natural melanin, these experiments were carried out in BALB/c mice, which are naturally devoid of melanin. Black pigmentation of the draining inguinal lymph nodes was macroscopically visible on day 2 post-injection in gp100-melanin-injected animals (Fig 2a & 2b). Fontana-Masson staining confirmed numerous melanin-laden macrophages in the sinuses (Fig 2c) and, to a lesser extent, in the paracortical areas, which is a T-cell zone (Fig 2d). The pattern of melanin distribution was similar at days 2 and 7 post-injection, and in mice injected with gp100-melanin (without CpG-28). No melanin was observed in mice that received saline only. These results show that the vaccine formulation effectively reached the draining lymph nodes in vivo.
Immunization with low-dose gp100-melanin triggers CTL responses
We then assessed the ability of gp100-melanin to trigger an immune response in mice. Free gp100 peptide and gp100-melanin and were used as vaccine preparations alone or mixed with the TLR9 agonist CpG-28 (CpG) . When combined with CpG, gp100-melanin, but not gp100, induced a significant number of IFNγ-secreting lymphocytes (p<0.001) (Fig 3a). Similar data were obtained when a TLR3 agonist was used (S1 Fig). If the gp100 epitope was added in the vaccine formulation after L-Dopa had been oxidized instead of before, no significant CTL response was observed (p<0.01) (Fig 3a). The minimal weight ratio of L-Dopa:epitope required to induce significant immunity was 1:1, with the best response observed at a ratio of 4:1 (Fig 3b). The minimal dose of the gp100 epitope required to induce CTLs was 0.5 μg (p<0.01, when compared to the lowest concentration tested) (Fig 3c). At both 10 and 50μg of the gp100 epitope, our vaccine formulation compared favorably (p<0.01) with the combination of incomplete Freund’s adjuvant (IFA) and a TLR9 agonist, a combination that is commonly used to trigger CTL responses [22,23] (Fig 3c). After two 2 initial immunizations with gp100-melanin + CpG, the specific CTL response progressively diminished over a period of months and was still seen after 4 months (S2 Fig).
Immunization against other MHC class I epitopes also triggers CTL responses
We then checked that the efficacy of our vaccine combining synthetic melanin and CpG was not limited to the gp100 epitope and can be generalized to other peptides containing MHC class I epitopes. We investigated the ability of this formulation to trigger CTL responses with a self-epitope derived from the murine Ephrin-A2 protein (EphA2) and with a long synthetic peptide (30 amino acids) containing the classic ovalbumin SIINFEKL epitope (pOVA30). The peptides were incubated for 18 h with L-Dopa to generate EphA2-melanin and pOVA30-melanin and then combined with CpG for immunizations. These epitopes alone were not immunogenic or were poorly immunogenic, whereas immunization with either EphA2-melanin or pOVA30-melanin induced the production of a significant number of IFNγ-secreting lymphocytes (p<0.001 and p<0.01 for EphA2 and pOVA30, respectively) (Fig 3d). Taken together with the results described above, this suggests combination with melanin successfully increased the CTL-based immune response to vaccination against various peptidic epitopes.
Immunization with the melanin-adjuvant vaccine formulation induces effector memory CD8+ T-cells
To characterize the CTL phenotype, mice were immunized subcutaneously with either [pOVA30 + CpG] or [pOVA30-melanin + CpG] on days 0 and 14 and sacrificed on day 21. The percentage of SIINFEKL-specific T cells (dextramer+ T-cells) within the CD8+ lymphocyte population was significantly enhanced in mice given [pOVA30-melanin + CpG] compared with mice given [pOVA30 + CpG] (mean ± SD: 1.42 ± 0.13% vs 0.1 ± 0.04%, respectively; p = 0.05) (data not shown). Analysis of these dextramer+ CD8+ cells using multiparametric flow cytometry showed a phenotype of effector memory CD8+ T-cells, with low CD62L expression and high T-bet and granzyme expression in the [pOVA30-melanin + CpG] group (Fig 4).
Subcutaneous injections of pOVA30-melanin protect against established syngenic tumors
We next investigated whether these CD8+ T-cells were functional in vivo. Ovalbumin-transfected cells (E.G7-OVA) were injected subcutaneously into C57BL/6 mice, and the mice were immunized on days 4 and 18 with [melanin + CpG-28], [pOVA30-melanin + CpG-28], [pOVA30 + CpG-28], or [pOVA30-melanin]. All of the mice developed measurable tumors. A significant decrease in the tumor growth compared with that in the control groups was observed only after immunization with [pOVA30-melanin + CpG-28] (p<0.001) (Fig 5a). Complete tumor regression occurred in 2/10 mice. Depletion of CD8+ cells by monoclonal antibodies abrogated this anti-tumor effect (Fig 5b). These results suggest that the vaccine elicited an immune response that allowed a tumor growth inhibition, which was mediated by CD8+ T-cells.
Immunization with melanin formulation without any TLR agonist
We finally assessed the ability of our formulation to trigger an immune response without any TLR agonist. The synthetic peptide pOVA35, that contains both a MHC class II (ISQAVHAAHAEINEAGC) and a MHC class I (SIINFEKL) ovalbumine epitopes, was incubated for 18 h with L-Dopa to generate pOVA35-melanin. Mice were immunized subcutaneously with either free pOVA35 or pOVA35-melanin on days 0 and 14 (without CpG), and the immune response was assessed on day 21. The pOVA35 peptide alone was not immunogenic, whereas immunization with pOVA35-melanin induced the production of a significant number of IFNγ-secreting lymphocytes against the CD4 epitope (p<0.01) (Fig 6). The presence of the MHC-class II epitope allowed the mice immunized with pOVA35-melanin, but not those immunized with pOVA35 only, to mount a significant CTL response against the MHC-class I epitope (p<0.01) (Fig 6).
Protection against UV radiation and radical scavenging are the most acknowledged functions of melanin in mammals . Here, we showed that melanin, when synthesized in the presence of antigens, may play a role in antigen-specific immunity. Indeed, epitope-containing peptides associated with synthetic melanin triggered specific immune responses, with effector memory CD8+ lymphocytes expressing granzyme. These immune responses were functional, because immunization against tumor antigens elicited an anti-tumor effect in vivo. Furthermore, induction of CD8+ lymphocytes with a short MHC class I epitope can be easily obtained either by embedding a MHC class II epitope within the peptides, or by adding a TLR agonist to the vaccine formulation, thus providing the “second signal” for cross-presentation [4,24].
The mechanism underlying the efficacy of our approach most likely involves an antigen carrier effect provided by melanin. Indeed, no immune response was observed if peptides were added after L-Dopa had been oxidized. This observation does not support an immunostimulatory effect of melanin itself on immune cells but instead indicates that a close association between the peptides and synthetic melanin is required. Cross-linking reactivity is a well-known property of catechol moieties and L-DOPA after oxidation [25,26], and the association between melanin and our peptides was indeed documented by FITR spectroscopy. After subcutaneous injection of our peptide-melanin nanoparticles, melanin was found in the draining lymph nodes. Such a migration within lymph nodes, in which T-lymphocytes are primed, is commonly observed with nanoparticles ≤200nm . In humans, melanin deposits are commonly described in cases of dermatopathic lymphadenopathy, a lymph node pathology secondary to skin disease . Interestingly, melanin deposits in our model were mainly seen in the sinuses and in the paracortex, which is a T-cell zone, and were still detected within lymph nodes 7 days after injection. It might be hypothesized that this slow clearance of melanin contributes to the efficacy of our vaccines by enabling a slow antigen release.
Cross-presentation of antigens by nanoparticulate formulations is a well-described process in immunology . As synthetic melanin aggregates into nanoparticles , its ability to enhance immune responses to combined to antigens thus appears logical. Whether this phenomenon spontaneously occurs with natural melanin in living animals to protect against infections is unclear. In invertebrates, melanin plays a role in immune defense by encapsulating invading pathogens, a process known as melanization. However, such a process does not trigger specific immunity and does not appear to occur in vertebrates . Yet, several facts support the idea that immunity and pigmentation are functionally linked in mammals. Numerous studies have suggested that darker-skinned individuals may be less susceptible to bacterial and fungal skin diseases . In addition, in mice with melanocytosis, melanin granules in the skin are continuously captured and transported to regional lymph nodes by Langerhans cells [32,33]. Finally, normal melanocytes display antigen-presenting functions [15,18]. However, the role of melanin itself in these immune processes has never been addressed. Our results showing that melanin can act as an antigen carrier shed new light on the putative mechanism involved in the above described observations.
The vaccine formulation presented in this study has three major advantages in the field of subunit vaccines. First, our approach is a simple and efficient way to trigger robust CTL responses, whereas most other vaccine technologies fail to do so . Second, very short MHC class I epitope can be used if a MHC class II epitope is embedded within the peptides, or if a TLR agonist is added to the vaccine formulation. Finally, very low doses of antigens are required. For example, 50 μg of the poorly immunogenic gp100 epitope was insufficient to trigger a CTL response when administered with the classic combination of IFA plus a TLR9 agonist . By contrast, as little as 0.5 μg of the epitope was sufficient to trigger a significant CD8+ immune response when using our formulation. The advantages of our melanin-nanoparticle vaccine over other nanoparticle-vaccines such as liposomes or natural polymers are under study. However, the superior efficacy of our formulation when compared to the combination of IFA and a TLR9 agonist, which is one of the best adjuvant known to trigger CTL responses [22,23], suggests that our approach deserves further developments.
In conclusion, the conjugation of synthetic melanin to short peptides represents a very simple means of triggering T-cell response. This approach should be particularly useful for immunizations against cancer, for which immunizations against neo-epitopes are currently under intense investigations [1,2].
Materials and methods
Peptides and synthesis of melanin
The KVPRNQDWL (gp100), FSHHNIIRL (EphA2), SIINFEKL, SQAVHAAHAEINEAGR, SMLVLLPKKVSGLKQLESIINFEKLTKWTS (pOVA30) and SLKISQAVHAAHAEINEAGRLRGSIINFEKLTKWR (pOVA35) endotoxin free peptides were purchased from Genosphere Biotechnologies (Paris, France). These peptides correspond to, or contain, H-2b epitopes (underlined) of the human glycoprotein 100 , the mouse Ephrin A2  and the ovalbumin protein , respectively. The pOVA35 peptide also contains a mouse MHC class II epitope (bold) . Stock solutions of peptides (10 mg/ml) and L-Dopa (2.2 mg/ml) (Sigma-Aldrich, Saint-Quentin-Fallavier, France) were prepared in Milli-Q purified water. For the preparation of synthetic melanin, peptides (10 μg/mouse, unless specified) were mixed with L-Dopa (100 μg/mouse, unless specified). The solutions were then incubated for 18 h at pH 8.5 under vigorous agitation (Eppendorf Thermomixer, 1000 rpm, 20°C) to ensure continuous oxygenation of the solution.
UV visible spectra were obtained using a JASCO V630 spectrophotometer (JASCO, Lisses, France). The solution of polymerizing L-Dopa was diluted 1/20, and spectra were recorded using 1cm path length quartz cuvette after different incubation times. Particle sizes were determined by transmission electron microscopy (Tecnai 12, ImagoSeine platform, France). Samples were prepared by depositing a drop of the above solution on carbon-coated copper grids placed on a filter paper. Fourier transform infrared (FTIR) spectra were recorded on a Tensor 27 spectrophotometer (Bruker, Karlsruhe, Germany) at a resolution of 1 cm-1, and the data treatment was performed using the Opus program. Because of interfering vibrations of H2O at 1645 cm-1, spectra were recorded in D2O. Deuteration experiments were performed by drying the samples and dissolving them in 1.5 μl D2O solution (>99.8% purity, Euriso-Top; CEA, Saclay, France). Solutions were deposited between two ZnSe windows.
Tricine-SDS-PAGE analysis was performed according to the protocol published by Schägger . Briefly, samples were mixed with 5x sample buffer (containing glycerol, SDS, β-mercapto-ethanol and bromophenol blue) and heated for 4 min at 95°C. Aliquots containing 2 μg of peptide in a final volume of 12.5 μl were loaded on an electrophoresis gel using 4%, 10% and 16% acrylamide/Bis (29:1) for the stacking, spacer and resolving gel, respectively. Following electrophoresis, the gels were stained with Coomassie Brilliant Blue R-250 and imaged with the ChemiDoc XRS+ system (Bio-Rad Laboratories, Marnes la Coquette, France).
Mice and immunization protocols
Female C57BL/6, or BALB/c mice (Janvier Labs, Le Genest-Saint-Isle, France) were 5–6 weeks old at the initiation of the experiment and were kept under specific-pathogen-free conditions. All animal experiments were approved by the ethics committee of Paris Descartes University (Project APAFIS #5337 N° 2016021517305775) and performed in accordance with European Union guidelines for animal experiments. Peptides (10 μg/mouse, unless specified) mixed with L-Dopa (100 μg/mouse, unless specified) were incubated for 18 h under the above described conditions. When specified, the phosphorothioate oligonucleotide CpG-28 (5’-TAAACGTTATAACGTTATGACGTCAT) (Oligovax, Paris, France), a B-type CpG-ODN [9, 38], or polyinosinic:polycytidylic acid (poly I:C), a TLR3 agonist (Sigma-Aldrich, Saint-Quentin-Fallavier, France) were added to the vaccine formulations (10 μg/mouse) just before the immunizations. Mice were immunized subcutaneously in the flank (100 μl/injection). As controls, some mice were injected with peptides + 50 μg of CpG-28 emulsified in incomplete Freund’s adjuvant (Sigma-Aldrich, Saint-Quentin-Fallavier, France). Mice were euthanized on the indicated day by cervical dislocation. Spleens and inguinal lymph nodes were surgically removed under aseptic conditions.
The inguinal lymph nodes were surgically resected, fixed in 4% neutral buffered formalin for 24 h and embedded in paraffin. Five-micron-thick sections were then cut and stained with Fontana-Masson stain. The slides were reviewed by a pathologist (A.M.) using an Olympus BX51 light microscope, and images were obtained using a SPOT Insight Digital Camera (Diagnostic Instruments).
Epitope-specific IFNγ production by splenocytes was determined as previously described . Briefly, single-cell suspensions of splenocytes (5 x 105 cells/well) were stimulated at 37°C in 5% CO2 for 21 h with 10 μg/ml of the epitopes KVPRNQDWL for human gp100, FSHHNIIRL for murine EphA2, SIINFEKL or SQAVHAAHAEINEAGR for ovalbumin (Ovalbumine MHC class I and class-II epitopes, respectively). When spots indicating IFNγ production appeared, they were counted using a Cellular Technology Ltd system and analyzed using ImmunoSpot 5.0.3 software (Cellular Technology Ltd., Ohio, USA). The results are presented as the mean of triplicate wells and the number of IFNγ spot-forming cells (SFCs) per 5 x 105 cells.
Multiparametric flow cytometry and antibodies
Single-cell suspensions of splenocytes (1 x 106 cells/well) were first stained for 45 min at 4°C with H-2Kb (SIINFEKL) dextramers conjugated to phycoerythrin (PE) (Immudex, Copenhagen, Denmark). Cells were then incubated with Pacific Blue™anti-mouse CD3 (Biolegend, San Diego, CA, US), anti-mouse CD8a APC-eFluor®780 (eBioscience, San Diego, CA, US) and anti-mouse CD62L (L-Selectin) Alexa Fluor® 700 (eBioscience) antibodies for 30 min at 4°C. After fixation and permeabilization using Fix/Perm buffer (eBioscience), cells were intracellularly stained with anti-human/mouse T-bet PE-Cyanine7 (eBioscience) and Alexa Fluor® 647 anti-human/mouse Granzyme B (Biolegend) antibodies for 1 h at 4°C. Flow cytometry analysis was performed using a BD LSRII flow cytometer and BD FACSDIVA software (Becton Dickinson, NJ, US). The results were analyzed using FLOWJO software.
Tumor models and in vivo CD8+ T-cell depletion
Under anesthesia with 2.5% isoflurane, C57BL/6 mice were injected subcutaneously with 5 x 104 cells stably transfected with a plasmid expressing chicken ovalbumin (E.G7-OVA) (American Type Culture Collection, ATCC, USA). Mice were then immunized subcutaneously on days 4 and 18 after tumor graft. Tumors were measured every 3–4 days using calipers, and the tumor volumes were calculated using the following formula: π/6 x length x width2. Mice were sacrificed when tumors reached a maximal diameter of 20 mm. In the same tumor model, CD8+ T cells were depleted in vivo as follows: 100 μg of anti-CD8 mAbs (rat IgG2b mAb, clone YTS 169.4 from Proteogenix, France) per mouse or isotype control mAbs were injected intraperitoneally two days before therapeutic vaccination and then once per week until the end of the experiment.
For statistical analysis, continuous variables are presented as the mean for normally distributed variables and as the median for non-parametric variables. Associations between non-normally distributed variables were calculated using the Mann–Whitney test and between normally distributed variables by the paired Student t-test. Differences in tumor size among the various groups were determined using the ANOVA repeated-measures test. All reported P-values were based on two-sided tests at a significance level of 0.05. The Bonferonni’s correction was applied in case of multiple tests within the same experiment. All statistical analyses were performed using Statview 5.0 software®.
S1 Fig. CTL response after subcutaneous immunizations in C57BL/6 mice with gp100 + poly I:C or gp100-melanin + poly I:C (poly I:C = Polyinosinic:polycytidylic acid; 10μg/mouse).
Mice were sacrificed 8 days after immunizations. (n = 8 mice/group with pooled data from 2 different experiments). SFC: spot-forming cells; Bars = median. *p<0.001.
S2 Fig. Evolution of the CTL response after 2 subcutaneous immunizations (day 0 and day 14) with gp100-melanin + CpG.
Each point represents an individual mouse (n = 8 mice/group with pooled data from 2 different experiments of 4 mice each). SFC: spot-forming cells; Bars = median. * p < 0.01; **p<0.001, when compared to splenocytes stimulated with control epitopes.
- 1. Yadav M, Jhunjhunwala S, Phung QT, Lupardus P, Tanguay J, Bumbaca S, et al. Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature. 2014;515: 572–576. pmid:25428506
- 2. Verdegaal EM, Verdegaal EM, de Miranda NF, Visser M, Harryvan T, van Buuren MM, et al. Neoantigen landscape dynamics during human melanoma-T cell interactions. Nature. 2016;536: 91–95. pmid:27350335
- 3. Camisaschi C, Vallacchi V, Vergani E, Tazzari M, Ferro S, Tuccitto A, et al. Targeting Immune Regulatory Networks to Counteract Immune Suppression in Cancer. Vaccines. 2016;4: 4. pii: E38. pmid:27827921
- 4. Reed SG, Orr MT, Fox CB. Key roles of adjuvants in modern vaccines. Nat Med. 2013;19: 1597–1608. pmid:24309663
- 5. Foged C, Hansen J, Agger EM. License to kill: Formulation requirements for optimal priming of CD8(+) CTL responses with particulate vaccine delivery systems. Eur J Pharm Sci. 2012;45: 482–491. pmid:21888971
- 6. Jensen PE. Recent advances in antigen processing and presentation. Nat Immunol. 2007;8: 1041–1048. pmid:17878914
- 7. Simsova M, Sebo P, Leclerc C. The adenylate cyclase toxin from Bordetella pertussis—a novel promising vehicle for antigen delivery to dendritic cells. Int J Med Microbiol. 2004;293: 571–576. pmid:15149033
- 8. Forde GM. Rapid-response vaccines—does DNA offer a solution? Nat Biotechnol. 2005;23: 1059–1062. pmid:16151391
- 9. Maubant S, Banissi C, Beck S, Chauvat A, Carpentier AF. Adjuvant properties of Cytosine-phosphate-guanosine oligodeoxynucleotide in combination with various polycations in an ovalbumin-vaccine model. Nucleic Acid Ther. 2011;21: 231–240. pmid:21787231
- 10. Sandoval F, Terme M, Nizard M, Badoual C, Bureau MF, Freyburger L, et al. Mucosal imprinting of vaccine-induced CD8+ T cells is crucial to inhibit the growth of mucosal tumors. Sci Transl Med. 2013;5: 172ra20. pmid:23408053
- 11. Maisonneuve C, Bertholet S, Philpott DJ, De Gregorio E. Unleashing the potential of NOD- and Toll-like agonists as vaccine adjuvants. Proc Natl Acad Sci U S A. 2014;111: 12294–12299. pmid:25136133
- 12. Lewis JJ. Therapeutic cancer vaccines, using unique antigens. Proc Natl Acad Sci USA. 2004;101 Suppl 2: 14653–14666.
- 13. Goldman B, DeFrancesco L. The cancer vaccine roller coaster. Nat Biotechnol. 2009;27: 129–139. pmid:19204689
- 14. Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mammalian skin and its hormonal regulation. Physiol Rev. 2004;84: 1155–1228. pmid:15383650
- 15. Le Poole IC, Mutis T, van den Wijngaard RM, Westerhof W, Ottenhoff T, de Vries RR, et al. A novel, antigen-presenting function of melanocytes and its possible relationship to hypopigmentary disorders. J Immunol. 1993;151: 7284–7292. pmid:8258725
- 16. Diment S, Eidelman M, Rodriguez GM, Orlow SJ. Lysosomal hydrolases are present in melanosomes and are elevated in melanizing cells. J Biol Chem. 1995;270: 4213–4215. pmid:7876179
- 17. Schraermeyer U. Transport of endocytosed material into melanin granules in cultured choroidal melanocytes of cattle—new insights into the relationship of melanosomes with lysosomes. Pigment Cell Res. 1995;8: 209–214. pmid:8610072
- 18. Gasque P & Jaffar-Bandjee MC. The immunology and inflammatory responses of human melanocytes in infectious diseases. J Infect. 2015;71: 413–421. pmid:26092350
- 19. Bridelli MG. Self-assembly of melanin studied by laser light scattering. Biophys Chem. 1998;73: 227–239. pmid:17029729
- 20. Van Stipdonk MJ, Badia-Martinez D, Sluijter M, Offringa R, van Hall T, Achour A. Design of agonistic altered peptides for the robust induction of CTL directed towards H-2Db in complex with the melanoma-associated epitope gp100. Cancer Res. 2013;69: 7784–7792.
- 21. Baenziger JE, Méthot N. Fourier transform infrared and hydrogen/deuterium exchange reveal an exchange-resistant core of alpha-helical peptide hydrogens in the nicotinic acetylcholine receptor. J Biol Chem. 1995;270: 29129–29137. pmid:7493938
- 22. Speiser DE, Liénard D, Rufer N, Rubio-Godoy V, Rimoldi D, Lejeune F, et al. Rapid and strong human CD8+ T cell responses to vaccination with peptide., IFA., and CpG oligodeoxynucleotide 7909. J Clin Invest. 2005;115: 739–746. pmid:15696196
- 23. De Titta A, Ballester M, Julier Z, Nembrini C, Jeanbart L, van der Vlies AJ, et al. Nanoparticle conjugation of CpG enhances adjuvancy for cellular immunity and memory recall at low dose. Proc Natl Acad Sci USA. 2013;111: 19902–19907.
- 24. Azmi F, Ahmad Fuaad AA, Skwarczynski M, Toth I. Recent progress in adjuvant discovery for peptide-based subunit vaccines. Hum Vaccin Immunother. 2014;10: 778–796. pmid:24300669
- 25. Waite JH. Reverse engineering of bioadhesion in marine mussels. Ann N Y Acad Sci. 1999;875: 301–309. pmid:10415577
- 26. Lee H, Scherer NF, Messersmith PB. Single-molecule mechanics of mussel adhesion. Proc Natl Acad Sci U S A. 2006;103: 12999–13003. pmid:16920796
- 27. Manolova V, Flace A, Bauer M, Schwarz K, Saudan P, Bachmann MF. Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol. 2008;38: 1404–1413. pmid:18389478
- 28. Burke JS, Sheibani K, Rappaport H. Dermatopathic lymphadenopathy. An immunophenotypic comparison of cases associated and unassociated with mycosis fungoides. Am J Pathol. 1986;123: 256–263. pmid:3486598
- 29. Song C, Noh YW, Lim YT. Polymer nanoparticles for cross-presentation of exogenous antigens and enhanced cytotoxic T-lymphocyte immune response. Int J Nanomedicine. 2016;11: 3753–3764. pmid:27540289
- 30. Viljakainen L. Evolutionary genetics of insect innate immunity. Brief Funct Genomics. 2015;14: 407–412. pmid:25750410
- 31. Mackintosh JA. The Antimicrobial Properties of Melanocytes, Melanosomes and Melanin and the Evolution of Black Skin. J Theor Biol. 2001;211: 101–113. pmid:11419954
- 32. Hemmi H, Yoshino M, Yamazaki H, Naito M, Iyoda T, Omatsu Y, et al. Skin antigens in the steady state are trafficked to regional lymph nodes by transforming growth factor-beta1-dependent cells. Int Immunol. 2001;13: 695–704. pmid:11312257
- 33. Yoshino M, Yamazaki H, Shultz LD, Hayashi S. Constant rate of steady-state self-antigen trafficking from skin to regional lymph nodes. Int Immunol. 2006;18: 1541–1548. pmid:16966493
- 34. Yamaguchi S, Tatsumi T, Takehara T, Sasakawa A, Yamamoto M, Kohga K, et al. EphA2-derived peptide vaccine with amphiphilic poly(gamma-glutamic acid) nanoparticles elicits an anti-tumor effect against mouse liver tumor. Cancer Immunol. Immunother. 2010;59: 759–767. pmid:19943047
- 35. Ahonen CL, Doxsee CL, McGurran SM, Riter TR, Wade WF, Barth RJ, et al. Combined TLR and CD40 triggering induces potent CD8+ T cell expansion with variable dependence on type I IFN. J Exp Med. 2004;199: 775–784. pmid:15007094
- 36. Kroeger DR, Rudulier CD, Bretscher PA. Antigen presenting B cells facilitate CD4 T cell cooperation resulting in enhanced generation of effector and memory CD4 T cells. PLoS One. 2013; 8: e77346. pmid:24155947
- 37. Schägger H. Tricine-SDS-PAGE. Nat Protoc. 2006;1: 16–22. pmid:17406207
- 38. Carpentier A, Metellus P, Ursu R, Zohar S, Lafitte F, Barrié M, et al. Intracerebral administration of CpG oligonucleotide for patients with recurrent glioblastoma, a phase II study. Neuro Oncol. 2010;12: 401–408. pmid:20308317
Recent advances in melanin-like nanomaterials in biomedical applications: a mini review
Biomaterials Research 23, Article number: 24 (2019) Cite this article
Melanins are a group of biopigments in microorganisms that generate a wide range of colorants. Due to their multifunctionality, including ultraviolet protection, radical scavenging, and photothermal conversion, in addition to their intrinsic biocompatibility, natural melanins and synthetic melanin-like nanomaterials have been suggested as novel nano-bio platforms in biomedical applications.
Recent approaches in the synthesis of melanin-like nanomaterials and their biomedical applications have briefly been reviewed. Melanin-like nanomaterials have been suggested as endogenous chromophores for photoacoustic imaging and radical scavengers for the treatment of inflammatory diseases. The photothermal conversion ability of these materials under near-infrared irradiation allows hyperthermia-mediated cancer treatments, and their intrinsic fluorescence can be an indicator in biosensing applications. Furthermore, catechol-rich melanin and melanin-like nanomaterials possess a versatile affinity for various functional organic and inorganic additives, allowing the design of multifunctional hybrid nanomaterials that expand their range of applications in bioimaging, therapy, theranostics, and biosensing.
Melanin-like natural and synthetic nanomaterials have emerged; however, the under-elucidated chemical structures of these materials are still a major obstacle to the construction of novel nanomaterials through bottom-up approaches and tuning the material properties at the molecular level. Further advancements in melanin-based medical applications can be achieved with the incorporation of next-generation chemical and molecular analytical tools.
Melanin refers to a group of biopigments in microorganisms, plants and animals that generates a wide range of colors from yellow to red (known as pheomelanin) and brown to black (called eumelanin). Melanin determines hair, eye, and skin color in humans and acts as a protective shield by adsorbing harmful ultraviolet (UV) light from solar radiation and scavenging reactive oxygen species (ROS) to prevent DNA damage to nearby cells . Adsorbed solar energy can be dissipated as heat, contributing to the thermoregulation of animals, which is especially important for cold-blooded animals . In the brain, neuromelanin is produced in some populations of catecholaminergic neurons with uncertain circumstances, with attention due to its hypothetical relationship to neurodegenerative disorders such as Parkinson’s disease [3, 4]. Melanin also plays a major role in the innate immune system of insects against bacterial and fungal pathogens . Its multifunctionality and intrinsic biocompatibility have inspired scientists to develop novel nanomaterials by mimicking its chemical structure and material properties.
Among various precursors, dopamine (DA) has been a major precursor in the in vitro synthesis of melanin-like nanomaterials because it can be nonenzymatically crosslinked to 5,6-dihydroxyindole (DHI) under relatively moderate conditions, such as mildly basic pH (~ 8.5) and/or in the presence of dissolved oxygen in aqueous reaction solutions (details will be discussed in chapter 2.1). In addition to melanin-like properties, the versatile adhesive properties of DA-derived melanin (also called polydopamine, pDA) has allowed limitless applications in biomedical and environmental fields since it was first reported in 2007 . pDA was first synthesized as a material-independent surface coating material that mimics the chemical composition of mussel adhesive proteins, and it has also been suggested as a synthetic model of natural melanin, in particular neuromelanin in the brain, to elucidate the unexplored mechanisms of natural melanin in nature . pDA possesses various functional groups, such as protonated and/or unprotonated amine groups, catechols, and dihydroxyindole rings, that participate in multimodal molecular interactions on the surface of various types of materials constructing multifunctional coatings and adsorbing a bunch of molecules and ions as adsorbents (details will be discussed in chapter 2.2).
Here, we briefly review recent advances in natural melanin and melanin-like nanomaterials, such as pDA, that have been established for biomedical applications. Their synthesis and fabrication into nanoparticles, hollow nano/microcapsules, and core@shell structures with a controlled size and shape will be summarized in chapter 2, and their emerging applications in bioimaging, theranostics, sensors, and drug delivery systems will be introduced in chapter 3.
Synthesis and characterization of melanin-based multifunctional nanomaterials
Synthesis of artificial melanin
Melanogenesis in nature is initiated through the activation of tyrosinase, laccase and other polyphenol oxidases in response to external stimuli such as chemical stresses (metal ions, ROS, oxidizing agents), UV exposure, and pathogenic attack (particularly in insects) . These enzymes convert the precursors (tyrosine, DOPA, and DA) into major intermediates, DHI and/or 5,6-dihydroxyindole-2-carboxylic acid (DHICA), that can be further polymerized and assembled into melanin granules. Inspired by biosynthesis in nature, the synthesis of melanin-like nanomaterials has been achieved with natural precursors as well as their synthetic analogs crosslinked via enzymes in vitro. F. Li et al. reported that pDA nanoparticles synthesized by laccase were more stable in strongly acidic and alkaline solutions than the chemically synthesized nanoparticles . A. Lampel et al. systematically demonstrated the relationship between the structural variation of precursors and the resulting melanin-like material properties by tuning the amino acid sequence of self-assembled tripeptide crystalline precursors containing phenylalanine, aspartic acid, and tyrosine residues . Enzymatic polymerization via tyrosinase not only formed covalent crosslinking between tyrosine residues but also altered noncovalent interactions in preordered crystalline tripeptides, resulting in a wide range of disordered melanin-like pigments synthesized. Surface-immobilized enzymes have allowed for the site-specific deposition of melanin-like nanoparticles in proximity to enzymes; however, the synthesis was self-terminated due to the uncontrolled adhesion of the synthesized particles to enzymes .
Chemical oxidation processes can be the ultimate alternatives to the enzymatic synthesis of melanin-like nanomaterials that allows for cost-effective mass production with reproducibility. Melanin-like pDA nanoparticles have been synthesized with tunable sizes ranging from a few nanometers to hundreds of nanometers in aqueous solution at mildly basic pH values by the addition of different ratios of sodium hydroxide (NaOH) or ammonium hydroxide (NH4OH) to DA. During these processes, X. Wang reported that radicals could further control the growth of the particles by either quenching the radical intermediates of pDA or accelerating seed formation . Particle size was also affected by buffers; N. F. D. Vecchia et al. reported that the particle size of pDA increased with increasing initial DA concentration in phosphate or bicarbonate buffers, but inhibition of particle growth was observed in Tris buffer . The authors suggested that the covalent attachment of Tris molecules to the surface of the growing pDA surface prevented particle growth. X. Jiang et al. confirmed the effect of the water-alcohol co-solvent system on pDA synthesis via an ammonia solution and established an empirical formula that predicts the size of the resulting particles in mixed solvents . Chemical oxidants such as KMnO4, NaIO4, and CuSO4, and organic bases such as piperidine, have also been used for the synthesis of melanin-like particles and coatings in acidic pH conditions or in organic solvents [15,16,17]. Finally, temperature is another factor that can control the size of the growing particles .
Supramolecular assembly through noncovalent interactions has been suggested to be critical in regulating the shape and properties of melanin-like nanomaterials in addition to covalent crosslinking . Theoretical and experimental studies have suggested various types of noncovalent interactions, such as hydrogen bonding, van der Waals interactions, and π-π stacking between oligomeric intermediates . In 2012, Hong et al. experimentally isolated a trimer composed of DA and DHI assembled through unknown noncovalent interactions, suggesting that the building blocks for noncovalent assembly could be sufficiently low as oligomers, not polymers . They subsequently reported experimental evidence in 2018 that cation-π interactions can be the major driving force for the molecular assembly of oligomeric intermediates into granules . In addition to experimental studies, computational methods suggested that large molecular weight oligomers are less likely to form, and tetramers can be notably more stable than the other forms, implying that the major building blocks for noncovalent assembly can be quite repetitive, although the resulting granules possess diversity in their material properties .
Synthesis of melanin-like hybrid nanomaterials
During both the enzymatic and chemical syntheses of melanin-like nanomaterials, additives can be simultaneously incorporated via either covalent crosslinking to growing oligomers or physically entrapped in particles through noncovalent interactions to tune their material properties (as summarized in Table 1). For example, fluorescent pDA nanoparticles were obtained in the presence of glutathione (GSH) as an additive to a DA precursor with potential applications in biosensing . Ferric ion-complexed pDA nanoparticles were synthesized in one-pot and further annealed at 650 °C for 3 h, resulting in carbonized pDA embedded with 3–5 nm sized Fe3O4 as an organic/inorganic hybrid nanocatalyst . A recent study by D. Wang et al. showed that DA precursors participated in constructing a metal-organic framework (MOF) with Mn2+ and organic linkers and further polymerized as a part of a MOF crystal, showing great potential for photothermal therapy (PTT) . Hong et al. reported that cations such as the quaternary ammonium cation and potassium ions showed a remarkable affinity to building blocks of pDA via cation-π interactions, generating superhydrophilicity in one-pot synthesized coatings . Targeting moieties as well as therapeutic agents could also be incorporated into growing melanin-like nanoparticles, with potential in bioimaging and theranostics  (details are shown in chapter 3.3).
As-prepared melanin-like nanoparticles can be chemically reactive core templates for multifunctional core@shell nanostructures. Metal ions can chelate to the surface of nanoparticles for bioimaging, such as in photoacoustic imaging (PAI)  and magnetic resonance imaging (MRI) [40, 41]. Silver ions could be adsorbed and simultaneously reduced on the surface of artificial melanin, allowing for the green synthesis of reductant-free organic/inorganic hybrid nanoparticles with antibacterial activity . Nucleophiles such as thiols and amines are commonly used moieties in immobilizing organic molecules on the surface of as-prepared melanin-like nanoparticles without the additional chemical reagents . Thiolated polyethylene glycol could be used for the surface PEGylation of melanin-like nanoparticles to enhance the biocompatibility and stability in vivo [28, 35]. Targeting moieties such as RGD, folic acid, and triphenylphosphonium (TPP) could be immobilized on the surface of nanoparticles with anticancer drugs such as doxorubicin (DOX) and camptothecin (CPT) for tumor-targeting therapeutic delivery [29, 52,53,54,55] (details are shown in chapter 3.2). Interestingly, tumor cell lysates adsorbed on pDA nanoparticles acted as an immune booster for vaccine development in colorectal cancer .
Melanin-like nanomaterials, and in particular pDA, could be coated on the surface of any kind of template/core material to construct the reverse type of aforementioned core@shell nanostructures. pDA coating on the surface of inorganic nanoparticles enhances colloidal stability and prevents agglomerization and corrosion of inorganic nanoparticles [36, 37, 57,58,59]. In addition, it has been reported that pDA coating is not only biocompatible but also attenuates the intrinsic toxicity of inorganic materials such as quantum dots in vivo . Melanin-like coating can also provide an adhesive nano-thin layer between the core and outer materials, expanding further design of theranostic nanomaterials with multifunctionality [61,62,63,64]. Moreover, by further modification of these processes, hollow melanin nanocapsules could be synthesized by dissolving sacrificial core materials of as-prepared core@melanin hybrid nanoparticles .
Biocompatibility and biostability of melanin-like multifunctional nanomaterials
As a great candidate for biomedical applications, one of the strengths of melanin-like nanomaterials is their biocompatibility and long-term stability. They are not only well-dispersed in aqueous buffered medium, but also remained stable when stored in those solution without notable physical changes for up to at least several weeks [27, 59]. Coating of them to inorganic nanoparticles improve the water dispersibility and colloidal stability without altering thickness of the modification for long period of time , allowing a variety of both in vitro and in vivo biomedical applications.
Dopamine causes oxidative stress to cells and is cytotoxic, but it has been experimentally proven that melanin-like nanomaterials like pDA possesses good biocompatibility. In vitro cytotoxicity has been evaluated in many different types of mammalian cell lines including HeLa, 4 T1, HepG2, and NIH3T3; the cell viability was retained greater than 90% after incubating with those nanoparticles for up to several days [27, 35, 59, 65]. Hong et al. reported that there is still non-negligible amount of free dopamine that physically entrapped in synthesized pDA, but it is rarely released from pDA due to strong non-covalent interactions and therefore does not cause severe cytotoxicity . In vivo studies have confirmed that the biocompatibility of those nanomaterials contribute to attenuate the intrinsic cytotoxicity of conventional biomaterials. Hong et al. reported that pDA coating significantly reduced the leukocyte population alternation caused by conventional CdSe core and ZnS-capped nanocrystals in blood after tail vein injection to mice . In addition, it was reported to suppress the macrophage adhesion after 4 days and the foreign body giant cells (FBGCs) formation after 14 days from subcutaneous implantation of biodegradable poly (L-lactic acid) film in rats . Similarly, Liu et al. investigated the long-term biocompatibility as well as ultra-stability of pDA coating in vivo; pDA-coated gold nanoparticles were mainly uptaken by the Kupffer cells in the liver, the organ with the highest accumulation, while they were uptaken by a variety of cells in the spleen, the organ with the second highest accumulation. They were intact within those cells for at least six weeks without causing notable histological toxicity in mice .
Anti-inflammatory and photothermal therapy
Melanin-like nanoparticles, and in particular pDA, have been suggested as therapeutics in the treatment of anti-inflammatory diseases because of the abundant phenolic groups that serve as radical scavengers. Zhao et al. demonstrated that 80 nm-sized pDA nanoparticles efficiently scavenged either H2O2− or lipopolysaccharide (LPS)-induced cellular ROS in vitro, and further study in murine acute peritonitis models confirmed the successful suppression of in vivo inflammation . Similarly, Bao and his colleagues validated the antioxidant capacity of 160 nm-sized pDA nanoparticles and provided potential applications in periodontal disease treatment to relieve oxidative stress without any side effects .
Abundant phenolic groups in melanin-like nanoparticles not only scavenge radical species but also quench photosensitizers such as chlorin e6 (Ce6). Photosensitizers generate ROS, including singlet oxygen species, to kill nearby cells upon light exposure in photodynamic therapy (PDT). The current drawback of conventional photosensitizers is their undesirable photoactivation . To overcome this, Han and his colleagues developed complexed nanoparticles consisting of pDA and a photosensitizer conjugated to hyaluronic acid . Since the synthesized nanoparticles release the photosensitizer from the quencher pDA only in response to the degradation of hyaluronic acid by the tumor-localized intracellular enzymes (e.g., hyaluronidase), tumor-specific photoactivation is allowed . Similarly, Z. Dong et al. reported a calcium carbonate-pDA composite hollow nanoparticle with the loaded photosensitizer Ce6 that is released from pDA and photoactivated only in an acidic tumor environment .
Natural melanin and melanin-like synthetic nanoparticles possess broadband adsorption from the UV to near-infrared (NIR) range . Therefore, they can provide efficient photothermal conversion upon NIR irradiation, which can be used in hyperthermia-mediated cancer treatments, also called PTT. The major advantage of PTT is the spatial control of light exposure for target-specific treatment, but the cytotoxicity of currently used photosensitizers and the limited penetration of the light source into the skin are obstacles in the widening of its application . Recent studies have suggested that melanin-like nanoparticles can be the ultimate photosensitizers used in PTT because of their biocompatibility and biodegradability in addition to their strong NIR-reactive photothermal conversion efficiency. Liu et al. reported that melanin-like nanoparticles showed a photothermal conversion efficiency of 40% upon NIR irradiation with an 808 nm laser and did not cause long-term toxicity in rats in the absence of light exposure . Furthermore, the synergistic effects of PTT and chemotherapy could be achieved by synthesizing drug-loaded pDA nanoparticles: the acidic nature of the tumor environment triggers the release of anticancer drugs such as DOX and 7-ethyl-10-hydroxycamptothecin (SN38), and the simultaneous hypothermia caused by light-excited pDA could successfully suppress tumor growth in a tumor xenograft model .
Currently, nanotechnology for drug delivery has received great attention because it allows for personalized healthcare by improving the targeting ability and drug loading efficiency to improve the therapeutic efficacy and reduce the side effects . Therefore, a variety of nano-carriers with different sizes, structures and surface properties have been designed for smart drug delivery to date . Among them, researchers have witnessed that melanin-like nanoparticles have many advantages in terms of their drug loading efficiency due to the strong π-π interactions between the aromatic rings on drugs and melanin in addition to their biocompatibility and biodegradability . Furthermore, a variety of molecules could be physically adsorbed or chemically immobilized on artificial melanin to achieve multifunctionality. Table 1 summarizes the use of melanin-like nanomaterials in smart drug delivery systems.
Melanin-like nanomaterials can act as a core template, a coating or an adhesive layer between the core and the outer materials. Ho and his colleague compared the drug-loading efficiency and release profile of a model anti-cancer drug, CPT, adsorbed on the surface of as-prepared pDA nanoparticles in a size ranging from 75 nm to 400 nm, adjusted synthetically by pH . As the size of the particles that were synthesized at a pH of approximately 7.5–8 increased, the drug loading efficiency also increased due to an advanced interior volume for the drugs of up to 11.81 μg per 1 mg of particles. In contrast to the loading efficiency, however, the drug release was much faster from the smaller particles, which were synthesized at pH 9 . Cui and his colleagues developed a pH-dependent drug releasing platform by the synthesis of pDA hollow capsules decorated with DOX via a pH-cleavable linker . Over 85% of the DOX showed a sustained released up to 12 h at pH 5, mimicking the tumor environment, whereas only 20% of DOX were released at neutral pH . Targeting moieties could also be incorporated on the surface of artificial melanin templates with therapeutics for targeted drug delivery. W.-Q. Li et al. co-immobilized TPP with DOX on the surface of nanoparticles as the targeting moiety for mitochondria . The developed nanoparticles can overcome drug resistance in long-term chemotherapy . In addition, a gene delivery system was established by immobilization of amine-rich polymers such as polyethylenimine (PEI) on the surface of templated pDA NPs followed by complexation with plasmid DNA . In this system, the toxic nature of PEI is suppressed by the conversion of the primary amine groups to secondary and tertiary amines during covalent conjugation with pDA .
Artificial melanin can also be an outer coating of nanomaterials to control drug release profiles and enhance biocompatibility and stability. A nano-thin coating layer of pDA on as-prepared insulin particles enabled pH-responsive insulin release . Sustained release of insulin was obtained up to 40 h at pH 7.4, while only 29% was released at pH 5.4 . W. Cheng et al. also employed pDA coating on DOX-loaded mesoporous silica nanoparticles as a pH-sensitive gatekeeper . They further immobilized folic acid on the surface-coated pDA for targeted cancer therapy . S. Li et al. reported that pDA coating with casein could stabilize the zein-resveratrol nanocomplex against environmental stresses, including pH, salinity, storage, redispersion, and UV irradiation, and enhance the antioxidant activity . Finally, core-shell nanostructures have been synthesized by using artificial melanin-like pDA as an adhesive layer. M. Oroujeni et al. adopted pDA to immobilize the drug carrier 6-thio-β-cyclodextrin on the surface of magnetic nanoparticles, and hydrophobic diclofenac (DCF) was subsequently inserted into the surface-immobilized 6-thio-β-cyclodextrin . Oligonucleotide-based aptamers and molecular probes have also been covalently immobilized on pDA-coated multifunctional nanoparticles via Michael addition reactions for potential applications in biosensors and nanomedicine .
Bioimaging and theranostics
Photoacoustic (PA) imaging is a noninvasive, nonionizing, and low-cost imaging method based on converting irradiated light energy absorbed by target molecules into thermal energy [28, 36]. Melanin and its derivatives, including pDA, have been suggested as endogenous chromophores that can produce PA signals in vivo . Zhang et al. synthesized a melanin-loaded nano-liposome for PA imaging- and MR imaging-guided photothermal cancer therapy, and the longitudinal relaxivity, r1, of the synthesized nano-liposomes was 0.25 mM− 1 s− 1 at 7 T . Ju et al. reported that pH-induced aggregation of melanin nanoparticles resulted in increased PA signal strength by generating overlapping thermal fields between aggregated nanoparticles . In addition, various metal ions and reduced metals, targeting moieties, and therapeutic agents are allowed to be integrated with melanin-like nanoparticles for multimodal imaging and theranostics. Cationic poly-L-lysine-coated melanin nanoparticles could be used for anionic glycosaminoglycans (GAGs)-targeted PA imaging for the early diagnosis of articular cartilage degeneration in osteoarthritis . Repenko et al. reported that melanin-coated gold nanomaterials with various shapes could be used as gastrointestinal imaging probes for PA imaging with excellent dispersibility, minimal cytotoxicity, and augmented PA responses .
In addition to PA imaging, organic/inorganic hybrid nanomaterials composed of melanin-like nanomaterials and inorganic metal ions have emerged for MR imaging-based theranostics. High loading efficiency of various metal ions on melanin-like nanomaterials can be achieved for improved contrast, and further incorporation of poly (ethylene glycol) (PEG) increases the circulation time in vivo. Ju and his colleagues reported that the longitudinal relaxivity, r1, of Fe3+-loaded pDA nanoparticles was 17 mM− 1 s− 1 at 3 T, whereas that of the commercially available Gd-DOTA under the same conditions was 7.1 mM− 1 s− 1. Similarly, Gd3+-loaded 13 nm sized natural melanin was synthesized for MRI with high stability in vivo and a high r1 value . Various metal ions, such as Fe3+, Cu2+ and Mn2+, could be loaded onto pDA nanoparticles for not only bioimaging but also simultaneous photothermal cancer therapy [39,40,41]; further co-immobilization of photosensitizers and therapeutic agents allows for multimodal theranostic applications. Wang et al. synthesized PEGylated-polydopamine (PDA) nanoparticles with the photosensitizer IR820 in addition to Fe3+ for MR imaging-guided combined photothermal and PDT . pDA nanoparticles loaded with ICG, DOX, and Mn2+ could be used for MR imaging-guided combined chemotherapy and PTT in vivo . Lin and his colleagues designed novel melanin-coated Fe3O4 nanoparticles with surface-adsorbed 64Cu isotopes for multimodal imaging-guided cancer phototherapy . The high brightness observed on T1-weighted images, the high affinity for metal ions, strong absorbance in the NIR region, and biocompatibility of natural melanin contributed to the MR imaging, PA imaging and photothermal imaging/therapy, and 64Cu was employed for additional PET imaging .
UV irradiation induces the autofluorescence of both natural melanin and synthetic melanin-like nanomaterials . Therefore, the in-situ synthesis of these fluorescent nanomaterials could be an indicator in biosensing applications. Kong et al. developed a GSH sensing platform based on the MnO2-induced synthesis of fluorescent polydopamine nanoparticles (FPNPs) as an indicator in the absence of GSH, because GSH inhibits FPNP synthesis by reducing MnO2 to Mn2+ . Similarly, they also detected ascorbic acid by using CoOOH as an oxidant to synthesize FPNPs instead of MnO2 because CoOOH is reduced to CO2+ in the presence of ascorbic acid and loses its activity to synthesize FPNPs .
Furthermore, chemical modification and/or partial degradation could enhance the intrinsic fluorescence intensity of as-prepared melanin-like nanoparticles. Gu and his colleagues synthesized tens of nanometer-sized FPNPs via ethylenediamine-induced partial degradation of as-prepared pDA nanoparticles . The synthesized FPNPs were photostable and biocompatible, so they could be applied to the bioimaging of neuromast hair cells in the lateral line of zebrafish . Similarly, Lin’s group obtained FPNPs by using hydroxyl radicals instead of ethylenediamine because hydroxyl radicals can induce the partial degradation of as-prepared pDA nanoparticles by introducing hydroxyl groups onto the particles while reducing the π-π interactions . FPNPs were used for the detection of Fe3+ via electron-transfer-induced fluorescence quenching after the chelation of Fe3+ to the catechol groups of the FPNPs . Interestingly, Yin et al. reported that further chemical reduction could enhance the fluorescence intensity of the as-prepared FPNPs; they first synthesized FPNPs by sodium periodate-induced oxidation and then treated the FPNPs with sodium borohydride for the chemical reduction that tunes the surface of the as-prepared FPNPs to become full of hydroxyl groups . The quantum yield of these FPNPs was 5.1%, which is the highest rank among the reported FPNPs so far .
In addition to the intrinsic fluorescent properties of melanin-like nanomaterials, these nanomaterials can be applied in biosensing applications as fluorescence quenchers. Wang et al. synthesized mesoporous pDA nanoparticles and applied them as quenchers for fluorophore-conjugated single-strand DNA (ssDNA) probes to detect the downregulated let-7a and upregulated miRNA-21 in different types of cancer cells . ssDNA probes were adsorbed on the surface of pDA nanoparticles through noncovalent bonds, such as π-π stacking and electrostatic attraction, and then released from the surface by complexation with their complementary miRNAs, resulting in the recovery of their fluorescence . Interestingly, mesoporous pDA nanoparticles successfully protected surface-bound ssDNA probes from cleavage by DNase I, which was used to target miRNA recycling for signal amplification . Ma’s group synthesized a novel upconversion@pDA core@shell nanoparticle with surface-complexed Cy3-labeled aptamer probes for the detection of cytochrome C (Cyt C) in living cells. The fluorescence of the probes was quenched at the surface of the nanoparticles and then recovered by simultaneous dissociation and binding to Cyt C in the cytosol immediately after cellular uptake . In addition, the steady upconversion luminescent signals from the core of the synthesized nanoparticles served for not only intracellular imaging but also as an internal standard for the quantitative measurement of Cyt C from Cy3-labeled aptamers .
UV shielding in dermatology
Melanin protects nearby cells in skin against sunlight by absorbing harmful ultraviolet (UV) rays and scavenging free radicals that cause DNA damage. Thus, synthetic melanin-like nanomaterials can be emerging as biocompatible sunscreens and next-generation therapeutics for people with melanin deficiency, such as albinism and vitiligo, which increases the risk of skin cancer . Huang et al. demonstrated that melanin-like nanoparticles with a diameter of about 200 nm can be endocytosed, undergo perinuclear aggregation, and form a supranuclear cap in human epidermal keratinocytes, similar to natural melanosomes occurring in human skin . Accumulated melanin-like nanoparticles reduced reactive oxygen species (ROS) and prevented DNA damage in cells under UV irradiation . C. Wang et al. suggested polydopamine-encapsulated polymeric gels as bioinspired sunscreens with superior UV shielding properties, nonphototoxicity, and nonirritating nature . Similarly, Y. Wang et al. compared the UV shielding properties of polydopamine nanoparticles with natural melanin granules encapsulated in polymer matrix, indicating that decrease in the size of nanoparticles lead to increased UV-shielding and visible light transparency properties with reduced light scattering . However, most of synthetic melanin-like nanomaterials up to date are brown-to-black due to the broad range absorbance from UV to near-IR range, which is a major obstacle in cosmetic applications. In addition to UV, absorbance manipulation in visible range of those materials can be the next-step to be achieved.
Melanin is a biopigments group in nature that possesses multifunctionality including ultraviolet protection, radical scavenging, and photothermal conversion. Its multifunctionality as well as intrinsic biocompatibility have inspired researchers to design novel melanin-like nanomaterials. Various enzymatic and chemical oxidation processes from natural precursors and their synthetic analogues have been reported, and additives can be easily incorporated in situ or form core-shell structures that expand the achievable scope of multifunctionality. Synthesized melanin-like nanomaterials have been emerging in various biomedical applications including therapy, bioimaging, and biosensing. In addition, the electrical conductivity and ion transport of melanin in nature have opened the very recent applications in soft bioelectronics. However, their bottom-up design/fabrication and tuning material properties at the molecular level are still limited because the chemical structure of these materials has not been fully elucidated thus far. Understanding the heterogeneity of the chemical structure and molecular weight distribution of oligomeric/polymeric building blocks and the driving force for nanoscale assembly via next-generation chemical and molecular-level analytical tools will be the next step forward to advance melanin-based biomedical applications in the future.
Availability of data and materials
- Cyt C:
Foreign body giant cells
Fluorescent polydopamine nanoparticle
Magnetic resonance imaging
Poly (ethylene glycol)
Reactive oxygen species
Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 2007;21(4):976–94.
Goodman G, Bercovich D. Melanin directly converts light for vertebrate metabolic use: heuristic thoughts on birds, Icarus and dark human skin. Med Hypotheses. 2008;71(2):190–202.
Haining RL, Achat-Mendes C. Neuromelanin, one of the most overlooked molecules in modern medicine, is not a spectator. Neural Regen Res. 2017;12(3):372–5.
Fedorow H, Tribl F, Halliday G, Gerlach M, Riederer P, Double KL. Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson’s disease. Prog Neurobiol. 2005;75(2):109–24.
Kan H, Kim CH, Kwon HM, Park JW, Roh KB, Lee H, et al. Molecular control of phenoloxidase-induced melanin synthesis in an insect. J Biol Chem. 2008;283(37):25316–23.
Lee H, Dellatore SM, Miller WM, Messersmith PB. Mussel-inspired surface chemistry for multifunctional coatings. Science. 2007;318(5849):426–30.
Huang YR, Li YW, Hu ZY, Yue XJ, Proetto MT, Jones Y, et al. Mimicking melanosomes: polydopamine nanoparticles as artificial microparasols. ACS Cent Sci. 2017;3(6):564–9.
Pillaiyar T, Namasivayam V, Manickam M, Jung SH. Inhibitors of Melanogenesis: an updated review. J Med Chem. 2018;61(17):7395–418.
Li F, Yu YY, Wang Q, Yuan JG, Wang P, Fan XR. Polymerization of dopamine catalyzed by laccase: comparison of enzymatic and conventional methods. Enzyme Microb Tech. 2018;119:58–64.
Lampel A, McPhee SA, Park HA, Scott GG, Humagain S, Hekstra DR, et al. Polymeric peptide pigments with sequence-encoded properties. Science. 2017;356(6342):1064–8.
Strube OI, Bungeler A, Bremser W. Enzyme-mediated in situ synthesis and deposition of nonaggregated melanin protoparticles. Macromol Mater Eng. 2016;301(7):801–4.
Wang XH, Chen Z, Yang P, Hu JF, Wang Z, Li YW. Size control synthesis of melanin-like polydopamine nanoparticles by tuning radicals. Polym Chem. 2019;10(30):4194–200.
Della Vecchia NF, Luchini A, Napolitano A, D’Errico G, Vitiello G, Szekely N, et al. Tris buffer modulates polydopamine growth, aggregation, and paramagnetic properties. Langmuir. 2014;30(32):9811–8.
Jiang XL, Wang YL, Li MG. Selecting water-alcohol mixed solvent for synthesis of polydopamine nano-spheres using solubility parameter. Sci Rep. 2014;4:6070.
Kim DJ, Ju KY, Lee JK. The synthetic melanin nanoparticles having an excellent binding capacity of heavy metal ions. B Korean Chem Soc. 2012;33(11):3788–92.
Ponzio F, Barthes J, Bour J, Michel M, Bertani P, Hemmerle J, et al. Oxidant control of polydopamine surface chemistry in acids: a mechanism-based entry to superhydrophilic-superoleophobic coatings. Chem Mater. 2016;28(13):4697–705.
You I, Jeon H, Lee K, Do M, Seo YC, Lee HA, et al. Polydopamine coating in organic solvent for material-independent immobilization of water-insoluble molecules and avoidance of substrate hydrolysis. J Ind Eng Chem. 2017;46:379–85.
Ju KY, Lee Y, Lee S, Park SB, Lee JK. Bioinspired polymerization of dopamine to generate melanin-like nanoparticles having an excellent free-radical-scavenging property. Biomacromolecules. 2011;12(3):625–32.
d’Ischia M, Napolitano A, Pezzella A, Meredith P, Sarna T. Chemical and structural diversity in eumelanins: unexplored bio-optoelectronic materials. Angew Chem Int Ed. 2009;48(22):3914–21.
Kaxiras E, Tsolakidis A, Zonios G, Meng S. Structural model of eumelanin. Phys Rev Lett. 2006;97(21):218102.
Hong S, Na YS, Choi S, Song IT, Kim WY, Lee H. Non-covalent self-assembly and covalent polymerization co-contribute to polydopamine formation. Adv Funct Mater. 2012;22(22):4711–7.
Hong S, Wang Y, Park SY, Lee H. Progressive fuzzy cation-π assembly of biological catecholamines. Sci Adv. 2018;4(9):eaat7457.
Chen CT, Martin-Martinez FJ, Jung GS, Buehler MJ. Polydopamine and eumelanin molecular structures investigated with ab initio calculations. Chem Sci. 2017;8(2):1631–41.
Zhao H, Zeng ZD, Liu L, Chen JW, Zhou HT, Huang LL, et al. Polydopamine nanoparticles for the treatment of acute inflammation-induced injury. Nanoscale. 2018;10(15):6981–91.
Bao XF, Zhao JH, Sun J, Hu M, Yang XR. Polydopamine nanoparticles as efficient scavengers for reactive oxygen species in periodontal disease. ACS Nano. 2018;12(9):8882–92.
Dong ZL, Feng LZ, Hao Y, Chen MC, Gao M, Chao Y, et al. Synthesis of hollow biomineralized CaCO3-polydopamine nanoparticles for multimodal imaging-guided cancer photodynamic therapy with reduced skin photosensitivity. J Am Chem Soc. 2018;140(6):2165–78.
Liu YL, Ai KL, Liu JH, Deng M, He YY, Lu LH. Dopamine-melanin colloidal nanospheres: an efficient near-infrared photothermal therapeutic agent for in vivo cancer therapy. Adv Mater. 2013;25(9):1353–9.
Wang XY, Zhang JS, Wang YT, Wang CP, Xiao JR, Zhang Q, et al. Multi-responsive photothermal-chemotherapy with drug-loaded melanin-like nanoparticles for synergetic tumor ablation. Biomaterials. 2016;81:114–24.
Ho CC, Ding SJ. The pH-controlled nanoparticles size of polydopamine for anti-cancer drug delivery. J Mater Sci Mater Med. 2013;24(10):2381–90.
Li WQ, Wang ZG, Hao SJ, He HZ, Wan Y, Zhu CD, et al. Mitochondria-targeting polydopamine nanoparticles to deliver doxorubicin for overcoming drug resistance. ACS Appl Mater Interfaces. 2017;9(20):16794–803.
Priyam A, Nagar P, Sharma AK, Kumar P. Mussel-inspired polydopamine-polyethylenimine conjugated nanoparticles as efficient gene delivery vectors for mammalian cells. Colloid Surface B. 2018;161:403–12.
Cui JW, Yan Y, Such GK, Liang K, Ochs CJ, Postma A, et al. Immobilization and intracellular delivery of an anticancer drug using mussel-inspired polydopamine capsules. Biomacromolecules. 2012;13(8):2225–8.
Li H, Jia Y, Feng XY, Li JB. Facile fabrication of robust polydopamine microcapsules for insulin delivery. J Colloid Interface Sci. 2017;487:12–9.
Cheng W, Nie JP, Xu L, Liang CY, Peng Y, Liu G, et al. pH-sensitive delivery vehicle based on folic acid-conjugated polydopamine-modified mesoporous silica nanoparticles for targeted cancer therapy. ACS Appl Mater Interfaces. 2017;9(22):18462–73.
Ju KY, Kang J, Pyo J, Lim J, Chang JH, Lee JK. pH-induced aggregated melanin nanoparticles for photoacoustic signal amplification. Nanoscale. 2016;8(30):14448–56.
Repenko T, Rix A, Nedilko A, Rose J, Hermann A, Vinokur R, et al. Strong photoacoustic signal enhancement by coating gold nanoparticles with melanin for biomedical imaging. Adv Funct Mater. 2018;28(7):1705607.
Lin J, Wang M, Hu H, Yang XY, Wen B, Wang ZT, et al. Multimodal-imaging-guided cancer phototherapy by versatile biomimetic theranostics with UV and gamma-irradiation protection. Adv Mater. 2016;28(17):3273–9.
Chen L, Ji Y, Hu XM, Cui C, Liu H, Tang YF, et al. Cationic poly-L-lysine-encapsulated melanin nanoparticles as efficient photoacoustic agents targeting to glycosaminoglycans for the early diagnosis of articular cartilage degeneration in osteoarthritis. Nanoscale. 2018;10(28):13471–84.
Ge R, Lin M, Li X, Liu SW, Wang WJ, Li SY, et al. Cu2+-loaded polydopamine nanoparticles for magnetic resonance imaging-guided pH- and near-infrared-light-stimulated thermochemotherapy. ACS Appl Mater Interfaces. 2017;9(23):19706–16.
Miao ZH, Wang H, Yang H, Li ZL, Zhen L, Xu CY. Intrinsically Mn2+-chelated polydopamine nanoparticles for simultaneous magnetic resonance imaging and photothermal ablation of cancer cells. ACS Appl Mater Interfaces. 2015;7(31):16946–52.
Liu FY, He XX, Zhang JP, Chen HD, Zhang HM, Wang ZX. Controllable synthesis of polydopamine nanoparticles in microemulsions with pH-activatable properties for cancer detection and treatment. J Mater Chem B. 2015;3(33):6731–9.
Hong SH, Sun Y, Tang C, Cheng K, Zhang RP, Fan QL, et al. Chelator-free and biocompatible melanin nanoplatform with facile loading gadolinium and copper-64 for bioimaging. Bioconjug Chem. 2017;28(7):1925–30.
Gu GE, Park CS, Cho HJ, Ha TH, Bae J, Kwon OS, et al. Fluorescent polydopamine nanoparticles as a probe for zebrafish sensory hair cells targeted in vivo imaging. Sci Rep. 2018;8:4393.
Yin HG, Zhang KL, Wang LJ, Zhou K, Zeng J, Gao D, et al. Redox modulation of polydopamine surface chemistry: a facile strategy to enhance the intrinsic fluorescence of polydopamine nanoparticles for sensitive and selective detection of Fe3+. Nanoscale. 2018;10(37):18064–73.
Wang ZQ, Zhang JX, Chen F, Cai KY. Fluorescent miRNA analysis enhanced by mesopore effects of polydopamine nanoquenchers. Analyst. 2017;142(15):2796–804.
Ma L, Liu FY, Lei Z, Wang ZX. A novel upconversion@polydopamine core@shell nanoparticle based aptameric biosensor for biosensing and imaging of cytochrome c inside living cells. Biosens Bioelectron. 2017;87:638–45.
Chen MH, Wen Q, Gu FL, Gao JW, Zhang CC, Wang QM. Mussel chemistry assembly of a novel biosensing nanoplatform based on polydopamine fluorescent dot and its photophysical features. Chem Eng J. 2018;342:331–8.
Ang JM, Du YH, Tay BY, Zhao CY, Kong JH, Stubbs LP, et al. One-pot synthesis of Fe (III)-Polydopamine complex nanospheres: morphological evolution, mechanism, and application of the carbonized hybrid nanospheres in catalysis and Zn-air battery. Langmuir. 2016;32(36):9265–75.
Wang DD, Wu HH, Zhou JJ, Xu PP, Wang CL, Shi RH, et al. In situ one-pot synthesis of MOF-polydopamine hybrid nanogels with enhanced photothermal effect for targeted cancer therapy. Adv Sci. 2018;5(6):1800287.
Gao Y, Wu X, Zhou L, Su Y, Dong CM. A sweet polydopamine nanoplatform for synergistic combination of targeted chemo-photothermal therapy. Macromol Rapid Commun. 2015;36(10):916–22.
Luo HY, Gu CW, Zheng WH, Dai F, Wang XL, Zheng Z. Facile synthesis of novel size-controlled antibacterial hybrid spheres using silver nanoparticles loaded with poly-dopamine spheres. RSC Adv. 2015;5(18):13470–7.
Fan QL, Cheng K, Hu X, Ma XW, Zhang RP, Yang M, et al. Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging. J Am Chem Soc. 2014;136(43):15185–94.
Li YY, Jiang CH, Zhang DW, Wang Y, Ren XY, Ai KL, et al. Targeted polydopamine nanoparticles enable photoacoustic imaging guided chemo-photothermal synergistic therapy of tumor. Acta Biomater. 2017;47:124–34.
Jiao L, Xu Z, Du W, Li H, Yin M. Fast preparation of polydopamine nanoparticles catalyzed by Fe2+/H2O2 for visible sensitive smartphone-enabled cytosensing. ACS Appl Mater Interfaces. 2017;9(34):28339–45.
Dong ZL, Gong H, Gao M, Zhu WW, Sun XQ, Feng LZ, et al. Polydopamine nanoparticles as a versatile molecular loading platform to enable imaging-guided cancer combination therapy. Theranostics. 2016;6(7):1031–42.
Wang XL, Wang N, Yang Y, Wang XX, Liang JY, Tian XX, et al. Polydopamine nanoparticles carrying tumor cell lysate as a potential vaccine for colorectal cancer immunotherapy. Biomater Sci Uk. 2019;7(7):3062–75.
Yavuz E, Tokalioglu S, Patat S. Core-shell Fe3O4 polydopamine nanoparticles as sorbent for magnetic dispersive solid-phase extraction of copper from food samples. Food Chem. 2018;263:232–9.
Cheng YX, Zhang SP, Kang N, Huang JP, Lv XL, Wen K, et al. Polydopamine-coated manganese carbonate nanoparticles for amplified magnetic resonance imaging-guided photothermal therapy. ACS Appl Mater Interfaces. 2017;9(22):19296–306.
Liu XS, Cao JM, Li H, Li JY, Jin Q, Ren KF, et al. Mussel-inspired polydopamine: a biocompatible and ultrastable coating for nanoparticles in vivo. ACS Nano. 2013;7(10):9384–95.
Hong S, Kim KY, Wook HJ, Park SY, Lee KD, Lee DY, et al. Attenuation of the in vivo toxicity of biomaterials by polydopamine surface modification. Nanomedicine Uk. 2011;6(5):793–801.
Tao W, Zeng XW, Wu J, Zhu X, Yu XH, Zhang XD, et al. Polydopamine-based surface modification of novel nanoparticle-aptamer bioconjugates for in vivo breast cancer targeting and enhanced therapeutic effects. Theranostics. 2016;6(4):470–84.
Wang CX, Zhou JJ, Wang P, He WS, Duan HW. Robust nanoparticle-DNA conjugates based on mussel-inspired polydopamine coating for cell imaging and tailored self-assembly. Bioconjug Chem. 2016;27(3):815–23.
Zhu DW, Tao W, Zhang HL, Liu G, Wang T, Zhang LH, et al. Docetaxel (DTX)-loaded polydopamine-modified TPGS-PLA nanoparticles as a targeted drug delivery system fore the treatment of liver cancer. Acta Biomater. 2016;30:144–54.
Oroujeni M, Kaboudin B, Xia W, Jonsson P, Ossipov DA. Conjugation of cyclodextrin to magnetic Fe3O4 nanoparticles via polydopamine coating for drug delivery. Prog Org Coat. 2018;114:154–61.
Nieto C, Vega MA, Marcelo G, Valle EM. Polydopamine nanoparticles kill cancer cells. RSC Adv. 2018;8(63):36201.
Han J, Park W, Park SJ, Na K. Photosensitizer-conjugated hyaluronic acid-shielded polydopamine nanoparticles for targeted photomediated tumor therapy. ACS Appl Mater Interfaces. 2016;8(12):7739–47.
Tran ML, Powell BJ, Meredith P. Chemical and structural disorder in eumelanins: a possible explanation for broadband absorbance. Biophys J. 2006;90(3):743–52.
Zou LL, Wang H, He B, Zeng LJ, Tan T, Cao HQ, et al. Current approaches of photothermal therapy in treating cancer metastasis with nanotherapeutics. Theranostics. 2016;6(6):762–72.
Din FU, Aman W, Ullah I, Qureshi OS, Mustapha O, Shafique S, et al. Effective use of nanocarriers as drug delivery systems for the treatment of selected tumors. Int J Nanomedicine. 2017;12:7291–309.
Liu FY, He XX, Lei Z, Liu L, Zhang JP, You HP, et al. Facile preparation of doxorubicin-loaded upconversion@polydopamine nanoplatforms for simultaneous in vivo multimodality imaging and chemophotothermal synergistic therapy. Adv Healthc Mater. 2015;4(4):559–68.
Li SM, Li ZX, Pang JF, Chen J, Wang HD, Xie QL, et al. Polydopamine-mediated carrier with stabilizing and self-antioxidative properties for polyphenol delivery systems. Ind Eng Chem Res. 2018;57(2):590–9.
Zhang L, Sheng DL, Wang D, Yao YZ, Yang K, Wang ZG, et al. Bioinspired multifunctional melanin-based nanoliposome for photoacoustic/magnetic resonance imaging-guided efficient photothermal ablation of cancer. Theranostics. 2018;8(6):1591–606.
Ju KY, Lee JW, Im GH, Lee S, Pyo J, Park SB, et al. Bio-inspired, melanin-like nanoparticles as a highly efficient contrast agent for T1-weighted magnetic resonance imaging. Biomacromolecules. 2013;14(10):3491–7.
Wang JJ, Guo Y, Hu J, Li WC, Kang YJ, Cao Y, et al. Development of multifunctional polydopamine nanoparticles as a theranostic nanoplatform against cancer cells. Langmuir. 2018;34(32):9516–24.
Elleder M, Borovansky J. Autofluorescence of melanins induced by ultraviolet radiation and near ultraviolet light. A histochemical and biochemical study. Histochem J. 2001;33(5):273–81.
Kong XJ, Wu S, Chen TT, Yu RQ, Chu X. MnO2-induced synthesis of fluorescent polydopamine nanoparticles for reduced glutathione sensing in human whole blood. Nanoscale. 2016;8(34):15604–10.
Zhao YY, Li L, Yu RQ, Chen TT, Chu X. CoOOH-induced synthesis of fluorescent polydopamine nanoparticles for the detection of ascorbic acid. Anal Methods. 2017;9(37):5518–24.
Lin JH, Yu CJ, Yang YC, Tseng WL. Formation of fluorescent polydopamine dots from hydroxyl radical-induced degradation of polydopamine nanoparticles. Phys Chem Chem Phys. 2015;17(23):15124–30.
Wang CP, Wang D, Dai TJ, Xu P, Wu P, Zou Y, et al. Skin pigmentation-inspired polydopamine sunscreens. Adv Funct Mater. 2018;28(33):1802127.
Wang Y, Wang XF, Li T, Ma PM, Zhang SW, Du ML, et al. Effects of melanin on optical behavior of polymer: from natural pigment to materials applications. ACS Appl Mater Interfaces. 2018;10(15):13100–6.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1D1A1B07045249 to S.H.) and the Cooperative Research Program for Agriculture Science and Accounts of Chemical Research Technology Development (Project PJ01323201 to S.H.) from the Rural Development Administration of the Republic of Korea.
Jihyo Park and Haeram Moon contributed equally to this work.
Department of Emerging Materials Science, DGIST, Daegu, 42988, South Korea
Jihyo Park, Haeram Moon & Seonki Hong
The J. P, H. M, and S. H wrote and improved the final manuscript. All authors read and approved the final manuscript.
Correspondence to Seonki Hong.
Ethics approval and consent to participate
Mycotech private Ltd in Goa, India may make medical grade melanin from over 1,000 different microbes as may other labs like the naval research lab in Bethesda, Maryland, Korea, Italy, and Japan. Melanin, one of nature’s primal key stones may be made for billions, inexpensively and with the highest enviornmental standards to meet the greatest challenge ever to all people.
Melanocytes and melanin represent a first line of innate immunity against Candida albicans
Melanocytes are dendritic cells located in the skin and mucosae that synthesize melanin. Some infections induce hypo- or hyperpigmentation, which is associated with the activation of Toll-like receptors (TLRs), especially TLR4. Candida albicansis an opportunist pathogen that can switch between blastoconidia and hyphae forms; the latter is associated with invasion. Our objectives in this study were to ascertain whether C. albicans induces pigmentation in melanocytes and whether this process is dependent on TLR activation, as well as relating this with the antifungal activity of melanin as a first line of innate immunity against fungal infections. Normal human melanocytes were stimulated with C. albicanssupernatants or with crude extracts of the blastoconidia or hyphae forms, and pigmentation and TLR2/TLR4 expression were measured. Expression of the melanosomal antigens Melan-A and gp100 was examined for any correlation with increased melanin levels or antifungal activity in melanocyte lysates. Melanosomal antigens were induced earlier than cell pigmentation, and hyphae induced stronger melanization than blastoconidia. Notably, when melanocytes were stimulated with crude extracts of C. albicans, the cell surface expression of TLR2/TLR4 began at 48 h post-stimulation and peaked at 72 h. At this time, blastoconidia induced both TLR2 and TLR4 expression, whereas hyphae only induced TLR4 expression. Taken together, these results suggest that melanocytes play a key role in innate immune responses against C. albicans infections by recognizing pathogenic forms of C. albicans via TLR4, resulting in increased melanin content and inhibition of infection.melanocytes, melanin, antifungal activity, Candida, Toll-like receptors
Melanocytes, dendritic cells of neuroectodermal origin, are a major component of the epidermis. Although they are located in the basilar layer, they extend their dendrites to make contact with other epidermal cells such as keratinocytes and Langerhans cells [1,2]. Melanocytes synthesize a pigment called melanin, and their main role is to protect the skin against ultraviolet (UV) radiation by transferring melanin (synthesized in specialist organelles called melanosomes) to keratinocytes [1–3]. Melan-A (formerly MART-1) and gp100 (or Pmel-17) are antigens involved in melanosome maturation. Melan-A, the concentration of which correlates with melanin content, is necessary for gp100 function. As a result, it plays an important role in regulating pigmentation .
The presence of melanocytes in organs other than the skin (and therefore not involved in protection against UV radiation) raises the question of whether their sole role is to be an UV scavenger. Comparative and developmental biological studies of melanization suggest a different scenario, that is, melanization is a very important component of the innate immune response . In invertebrates, melanocytes modulate melanin production during infection; in humans, inflammation often leads to hypo- or hyperpigmentation [3,5]. Melanin (specifically eumelanin, or black melanin) has antimicrobial properties [1,5,6]. In addition, the activation of Toll-like receptors (TLRs) 1, 2, 4, 5, and 7, particularly TLR4, is associated with an increase in melanocyte pigmentation [6,7].
Candida albicans is an opportunistic pathogen that colonizes the skin and mucosae. An important virulence mechanism of C. albicans is its ability to transform from the yeast (blastoconidia) form into a hyphae, a process associated with tissue invasion . TLR2 and TLR4 are involved in the differential recognition of blastoconidia and hyphae [9,10]. Candidiasis is responsible for more than 50% of all systemic fungal infections . Although a recent study explored the role of nonprofessional cells such as epithelial cells in innate immune responses to C. albicans , few studies have examined the participation of melanocytes. Early findings suggest that C. albicans negatively regulates melanogenesis at the transcriptional level .
Here, we present evidence that TLR4-mediated innate immune responses induce the melanization of pathogenic forms of C. albicans, resulting in increased melanin content and reduced infectivity.
Materials and methods
TLR ligands, C. albicans supernatants, and crude extracts
Lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Staphylococcus aureus (both from Sigma-Aldrich, St. Louis, MO, USA) were used at 100 ng/ml and 10 μg/ml, respectively. Candida albicans (American Type Culture Collection [ATCC] 14053, Manassas, VA, USA) was grown for 24 h at 37°C in Sabouraud glucose broth (BD-Difco, Sparks, MD, USA; 30 g/L). Cells were pelleted by centrifugation at 4000 g for 10 min at 4°C. The supernatant was then filtered through a 10-KDa membrane filter (Amicon-Ultra-0.5 mL, Merck-Millipore Corporation, Billerica, MA, USA) and heated at 100°C for 20 min. Crude extracts of C. albicans were prepared by resuspending C. albicans (1 ml of 0.5 McFarland inoculum) in 49 ml of yeast extract peptone dextrose (YPD) medium from BD-Difco (Sparks, MD, USA) (to obtain 100% blastoconidia) or synthetic Lee’s medium broth (to obtain 100% hyphae)  and grown for 3 h at 30°C and 37°C, respectively. Cell pellets were obtained by centrifugation at 4000 g for 10 min, washed twice in phosphate-buffered saline (PBS) from Life Technologies Corporation (Carlsbad, CA, USA), suspended in 1 ml of PBS, and then lysed by agitation with glass beads for 30 min. Finally, the cells were heated to 100°C for 1 h followed by centrifugation at 16c000 g for 10 min at 4°C. The soluble fraction was used in all subsequent assays adjusting proteins at a final 200 μg/ml, measured in a VITROS 5600 analyzer (Ortho Clinical Diagnostics, Rochester, NY, USA).
Measurement of melanin content
Normal human melanocytes (between passages 4 and 8; Invitrogen) were plated at a density of 1–5 × 106 cells per well in Medium 254 containing human melanocyte growth supplement (GIBCO) and then stimulated with filtered (0.22 μm) and heated C. albicans supernatant for 24, 48, or 72 h. Filtered Sabouraud glucose broth was used as a vehicle and LTA and LPS (at 100 ng/ml and 10 μg/ml, respectively) were used as positive controls. After incubation, the cells were trypsinized, counted, and spun down. The pellets were resuspended in sodium hydroxide for 90 min at 37°C according to a protocol described by Ahn et al. (7). Finally, the cell lysates were loaded into a microplate reader (Lab-Tec, Quinta Normal, Santiago, Chile) and the optical density was measured at 485 nm. To calculate the melanin concentration in the samples, a standard curve was prepared using known concentrations of synthetic melanin (Sigma-Aldrich, St. Louis, MO, USA). To assess differences between blastoconidia and hyphae, melanocytes were stimulated with crude extracts of both forms. In this case, PBS was used as a vehicle.
Melanosome antigens and measurement of TLR2 and TLR4 expression
Melanocytes (1–5 × 106 cells/well) were stimulated with crude extracts of blastoconidia or hyphae for 0, 24, 48, 72, or 96 h. Cells were then collected and analyzed by flow cytometry. Briefly, cells were detached by scraping and centrifuged at 3000 g for 5 min. The supernatants were discarded and the cells were resuspended in wash solution (1% PBS/3% fetal bovine serum [FBS], GIBCO, Life Technologies Corporation, Carlsbad, CA, USA). To examine the expression of TLR2 and TLR4 genes, total RNA was extracted with TRIzol Reagent (Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer’s instructions. TLR2 and TLR4 mRNA expression was measured using Taqman primers and probes (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA), in a StepOne thermal cycler (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer’s recommendations. Cells were incubated with mouse anti-human Melan-A, mouse anti-human gp100 (Abcam, Cambridge, MA, USA), or mouse anti-human TLR2 and TLR4 (e-Bioscience, San Diego, CA, USA) and analyzed by flow cytometry (FACSCalibur flow cytometer; BD Biosciences, San José, CA, USA).
Antibodies mediated blocking of TLRs
To probe the participation of TLR2 and/or TLR4 in melanization, melanocytes at a density of 1–5 × 106 cells per well were stimulated with crude extracts of blastoconidia and hyphae in the absence or presence of anti-TLR antibodies. Anti-human CD284 (TLR4) and anti-human CD282 (E-biosciences, San José, CA, USA) were used in this experiment. Anti-mouse immunoglobulin G (IgG), IgG2a kappa (E-biosciences, San José, CA, USA), was used as the isotype control. Briefly, control and stimulated cells were incubated without and with the antibodies at a dilution of 1:1000 in PBS/3% FBS for 2 h at 37°C. Then, cells were washed in PBS and trypsinized, counted, and spun down. The pellets were resuspended in sodium hydroxide for 90 min at 37°C, as described previously. Optical density of melanin was measured at 485 nm.
Antifungal activity of melanin
Antifungal susceptibility testing was performed using the microdilution broth method published in the European Committee for Antimicrobial Susceptibility Testing 7.1 definitive document, with some modifications . Briefly, synthetic melanin was dissolved in dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO, USA) to obtain a stock solution. This solution was diluted and then added to culture medium to yield a final concentration of 2500 μg/ml and 1% of DMSO for testing C. albicans (ATCC strain 14053).
Antifungal activity of melanocyte lysates
Nonstimulated and Candida-stimulated melanocytes (crude extracts) were grown for 72 h at a density of 1–5 × 106 cells per well, trypsinized, washed twice in PBS, and resuspended in PBS containing 10% DMSO. Next, melanocytes were lysed by agitation with glass beads. Five hundred microliters of the melanocyte lysates were mixed with 10 μl C. albicans (104 colony-forming units/m) and incubated at 37°C for 30 min. The yeast was then plated onto Sabouraud agar and incubated for 18–24 h at 37°C.
The differences between groups were analyzed using the Student t test, analysis of variance, and post hoc test. The results were considered statistically significant at a P value <0.05.
C. albicans induces melanocyte melanization
To obtain an in vitro melanization pattern, normal human melanocytes were stimulated with two TLR ligands, LTA or LPS, and the results compared with those obtained for melanocytes stimulated with C. albicans supernatant. Stimulation with LPS led to greater increases in melanocyte melanin content than stimulation with LTA, particularly at 72 h (Fig. 1A). Since YPD medium could stimulate pigmentation by itself, it could have been a factor in the observation that the C. albicans supernatant was more stimulatory. The following experiments were performed using crude extracts of C. albicans blastoconidia and hyphae that were previously washed in PBS and suspended in the same buffer. Consistent results were obtained with the crude extracts of blastoconidia and hyphae at 72 h, although the hyphae crude extract induced stronger melanization (similar to that induced by LPS; Fig. 1B). The melanin concentration in the melanocytes was calculated by reference to a standard curve (see Materials and methods section).
Candida albicans melanocyte melanization. (A) The melanin content of melanocytes increases over time (24, 48, and 72 h post-stimulation) with vehicle (yeast extract peptone dextrose broth), C. albicans supernatant, lipoteichoic acid (LTA), or lipopolysaccharide (LPS). (B) Crude blastoconidia or hyphae extracts of C. albicans with vehicle (phosphate-buffered saline) at 72 h post-stimulation. (C) Optical microscopy images of melanization in melanocytes stimulated with blastoconidia and hyphae extracts of C. albicans at 24 and 72 h post-stimulation. This figure shows an increase in size and hyperpigmentation in melanocytes stimulated with hyphae extracts (10×). Results are representative of three independent experiments. *, P < 0.05; **, P < 0.01. Gray triangle indicates an increase in the melanin content. This Figure is reproduced in color in the online version of Medical Mycology.
Crude extracts of C. albicans induce the expression of melanosome markers
To track the genesis of melanin induction caused by C. albicans, we examined the expression of major melanosome markers over time. When melanocytes were stimulated with crude extracts of C. albicans, expression of Melan-A and gp100 was induced at 24 h. This expression decreased at 48 h and 72 h as the melanin content of the melanocytes increased (Figs. 1A, 2A). Hyphae induced higher expression of gp100 than blastoconidia (Fig. 2B).
Candida albicans induces the expression of early melanosomal antigens (Melan-A and gp100) in melanocytes at 24, 48, and 72 h post-stimulation. (A) FACS plots showing the time course of antigen expression. x-axis: fluorescence level; y-axis: events number. (B) Fold of induction of Melan-A and gp100 relative to nonstimulated melanocytes. B, blastoconidia; H, hyphae. Gray triangles indicate a decrease in melanosomal antigens at 72 h of stimulation. This Figure is reproduced in color in the online version of Medical Mycology.
C. albicans induces TLR expression
At first glance, it appeared as if the C. albicans (both the blastoconidia and hyphae forms) induced differential expression of TLRs and melanin production by melanocytes. When melanocytes were stimulated with crude extracts of C. albicans at 24 h, we found that blastoconidia induced greater expression of TLR2 mRNA than hyphae (Fig. 3A), hyphae induced greater expression of TLR4 mRNA than blastoconidia (Fig. 3B), and the expression of TLR2 mRNA was higher than that of TLR4 mRNA. At the protein level, 20% of melanocytes constitutively expressed TLR2, which increased upon stimulation by blastoconidia (Fig. 3C), but was not time dependent. On the other hand, TLR2 expression did not increase upon stimulation by hyphae extracts (Figs. 3C and 3D). Nonstimulated melanocytes did not express TLR4, although a higher percentage of cells expressed TLR4 upon stimulation with blastoconidia than with hyphae (Figs. 3Eand 3F ). Minimal levels of TLR4 expression were observed upon stimulation with hyphae; however, this increased over time, and maximal levels were attained after 96 h of stimulation (Fig. 3E and 3F).
Melanocyte expression of Toll-like receptors (TLRs) 2 and 4 is induced by stimulation with Candida albicans crude extracts (blastoconidia and hyphae). (A) TLR2 induction at the mRNA level. (B) TLR4 induction at the mRNA level. (C) TLR2 induction at the protein level (assessed by FACS). (D) Fold induction of TLR2 relative to control (assessed by FACS). (E) TLR4 induction at the protein level (assessed by FACS). (F) Fold induction of TLR4 compared to nonstimulated melanocytes (assessed by FACS). (G) Melanization reduction by antibody-mediated blocking of TLT4. Control, nonstimulated melanocytes; OD, optical density; GADPH, glyceraldehyde 3-phosphate dehydrogenase.
Anti-TLR4 antibodies block the melanization in hyphae extract–stimulated melanocytes
To study a possible role of TLRs in melanization induced by blastoconidia and hyphae extracts of C. albicans, cells were incubated with specific anti-TLR2 and anti-TLR4 antibodies. Only TLR4 was blocked post-stimulation with hyphae extracts but not with blastoconidia extracts (Fig. 3G).
Antifungal activity of melanin and melanocyte lysates
Synthetic melanin (at 0.03 mg/ml) inhibited C. albicans growth by 50% (Fig. 4A, B) and was fungicidal at concentrations >0.06 mg/ml (Fig. 4C). Melanocyte lysates inhibited the growth of C. albicans, with stimulated cells being more inhibitory than nonstimulated cells (Fig. 4D).
Both synthetic melanin and melanocyte lysates inhibit the growth of Candida albicans. (A) Graphic representation of C. albicans inhibition by synthetic melanin (assessed by spectrophotometry), expressed in growth percentage (%). (B) Visual inhibition of C. albicans growth by synthetic melanin in a broth microdilution test. (C) Fungicidal activity of synthetic melanin assessed onto culture plate. (D) Inhibitory activity of melanocyte lysate on C. albicans onto culture plate. Results are representative of three independent experiments. *, P < 0.05; **, P < 0.01. The C. albicans inocula contained 104 ufc/ml C. albicans. This Figure is reproduced in color in the online version of Medical Mycology.
These results suggest that melanocytes are not only melanin factories but they apparently play a key role in the innate immune response against C. albicansinfections by recognizing both commensal and pathogenic forms of C. albicansand reacting by regulating their melanin content. This process inhibits the growth of C. albicans, particularly when melanocytes are stimulated by the hyphae form. Consistent with this, an earlier study of the etiology of depigmentation in response to some fungal infections suggested that C. albicans negatively regulates melanogenesis at the transcription level . Following the publication of epidemiological data that showed that persons with darker skin have increased resistance to Trichophyton mentagrophytes and C. albicans infections , we examined the hypothesis that pathogenic forms of C. albicans induce melanocyte pigmentation as a first line of innate immunity against fungal infections.
Melanin synthesis by mammalian melanocytes is regulated at the genetic, biochemical, and environmental levels . Melanocytes induce the expression of early melanosomal antigens, which are related to melanosome maturation and the regulation of melanization [4,16]. Here, we showed that Melan-A and gp100 were induced after 24 h of stimulation by C. albicans extracts, suggesting that they play a role in melanin synthesis. Whereas the expression of Melan-A decreased at 48 h post-stimulation, that of gp100 decreased at 72 h. This hierarchical expression pattern suggests that Melan-A is required for gp100 function . Melanosomal antigen expression disappeared after 72 h of stimulation, whereas the level of pigmentation peaked at this time, probably due to the cessation of early-phase melanosome maturation.
An interesting finding was that melanocytes induced the expression of TLR2 and TLR4 upon stimulation by
C. albicans and that this expression was different after exposure to blastoconidia and hyphae (the pathogenic form). Thus, while blastoconidia induced higher expression of TLR2 mRNA, hyphae induced higher expression of TLR4 mRNA (Fig. 3A, B). Previous reports describe differential TLR responses in other cell types in response to both forms of C. albicans [9,17]. At the protein level, TLR2 (but not TLR4) was constitutively expressed in 20% of melanocytes (Fig. 3A), but TLR2 expression increased upon stimulation with C. albicans blastoconidia but not upon stimulation with hyphae. This increase was consistent over time (Fig. 3C and D). TLR4 induction was lower than that of TLR2, and blastoconidia-induced TLR4 expression was stronger than that caused by hyphae. Unlike that of TLR2, TLR4 expression increased over time, reaching a peak at 72 h (blastoconidia) or 96 h (hyphae). This behavior reflects the melanogenesis, suggesting a role for TLR4 in this process [6,7].
To test the direct participation of TLRs, melanocytes were incubated with anti-TLR2 and anti-TLR4 antibodies, resulting in a reduction of melanization in hyphae extract–stimulated melanocytes in the presence of anti-TLR4 antibodies. This finding corroborates a role of TLR4 in melanogenesis induced by the pathogenic form of C. albicans. Although blastoconidia extracts induce melanization, hypha extracts induce a stronger pigmentation and additionally cause changes in the melanocytes morphology, which increases their size and melanin content (Fig. 1C). This could be related to TLR4 induction by hyphae; however, other receptors could be involved in melanogenesis; therefore, more studies are needed to dissect the specific mechanism.
Melanin not only protects against UV radiation, it also has antimicrobial properties. An important biological role of melanocytes, melanosomes, and melanin is to inhibit the growth of bacteria, fungi, and parasites, which may explain the presence of melanocytes at different sites within vertebrates [1,5] and the immunomodulatory properties of melanin from different sources . In this study we demonstrated that synthetic melanin inhibited or killed C. albicans as effectively as melanocyte lysates, especially after C. albicans stimulation. This could be explained, at least in part, by the higher melanin content of stimulated cells. In addition, we noted that the minimal inhibitory concentration of melanin for C. albicans (0.03 mg/ml) was equivalent to the physiological concentration in hyphae extract–stimulated melanocytes (Fig. 1B), supporting the idea of a defensive role of melanization in C. albicans infections. While the mechanism underlying the inhibitory/cytotoxic properties of melanin is not fully understood, previous studies suggest that reactive quinone intermediates produced during melanin biosynthesis per se and/or hydrogen peroxide have antifungal properties . Furthermore, melanin absorbs organic and inorganic compounds by acting as a “cationite.” For example, in the eye, melanin is able to bind bacteria-derived toxins such as botulinum A [1,5]. Thus, melanin could adsorb different compounds within the cell wall of C. albicans or directly disrupt the cell membrane. Futures studies will focus on gaining a better understanding of these mechanisms.
This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT), project 11110160 and Programa de Mejoramiento de la Calidad y Equidad de la Educación-Universidad de Chile (MECESUP-UCH 0717), project.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.
Role of Vitamin D in Athletes and Their Performance: Current Concepts and New TrendsMirian de la Puente Yagüe,1 Luis Collado Yurrita,2,*Maria J. Ciudad Cabañas,2 and Marioa A. Cuadrado Cenzual2
Over the past decade, interest in research in relation to vitamin D (VITD) has been growing exponentially, partly due to the increased prevalence of its deficiency in the population and the association between the deficiency of VITD and a wide range of diseases [1,2,3]. The importance and versatility of vitamin D in the organism is becoming increasingly evident. VITD plays an active role in immune function, protein synthesis, muscle function, cardiovascular function, inflammatory response, cell growth and musculoskeletal regulation [2,3,4,5].
A priori, athletes might seem to have sufficient levels of VITD. However, the latest research shows that this assumption is wrong. In the last decade, the scientific community has conducted studies on VITD levels in various groups of athletes including runners, basketball players, jockeys, gymnasts and even dancers, showing that these levels in athletes are comparable to those of the general population. However, recent publications show that these levels will considerably depend on geographical location, and on the type of sport, whether it is indoor or outdoor, etc. A line of special interest is the influence of VITD deficiency on athlete morbidity [8,9,10,11,12]. The deficiency of this vitamin is generally widespread in the athletic population with an increase in morbidities associated with it, and the appearance of osteomalacia and osteoporosis [10,11,12]. Given the high prevalence of its deficiency and its negative potential on morbidity, the possible determination of VITD levels in athletes is considered part of the screening routine [7,10].
In relation to VITD supplementation in athletes with deficiency, several studies have shown that this increases muscle strength. Higher serum levels of vitamin D are associated with reduced injury rates and better sports performance. It is important to correctly identify people with vitamin D deficiency who need supplements to help optimize their performance and prevent future injuries [1,10,11].
Finally, it seems that there is a paradoxical relationship between ethnicity and VITD concentration. As an example, white-skinned subjects generally have lower levels of VITD but higher bone mineral density and decreased risk of fracture [6,12].
This review was prepared by searching available medical and scientific literature from PubMed, EMBASE and Cochrane Library. Nutrition, endocrinology, biochemistry, orthopedics, sports and toxicology journals, among others, were analyzed as well as by reviewing several books, conference proceedings, government publications.
2. Synthesis and Metabolism of Vitamin D
On the one hand, VITD is a micronutrient, since its deficiency can be treated by supplementation, and it is also a prohormone, seeing that its precursors are transformed into active metabolites. It comes in two biologically inactive forms, cholecalciferol (vitamin D3) and ergocalciferol (vitamin D2) [2,4,13].
Vitamin D is mostly synthesized in the skin. Cholecalciferol, or vitamin D3, is the primary source of endogenous VITD and is formed through the interaction of ultraviolet B (UVB) radiation after sun exposure with 7-dehydrocholesterol, which is stored inside the plasma membrane of every skin cell. Ergocalciferol, or vitamin D2, represents a small percentage and has its origin in exogenous dietary intake [14,15,16]. Vitamin D is difficult to obtain through diet because very few foodstuffs contain the vitamin naturally, the exceptions being the liver of fatty fish, mushrooms and eggs, among others. Supplementation or fortification with vitamin D2 and D3, such as milk and other dairy products, cereals, etc., currently implies an exogenous supply [14,16].
The VITD obtained from sun exposure, food or supplementation is biologically inert and must undergo two hydroxylations in the organism to become active, the first being performed in the liver by the CYP2R1 enzyme where it is converted to 25-hydroxyvitamin D3 (calcidiol). The second being performed in the kidney and other tissues by the CYP27B1 enzyme to form 1.25-dihydroxyvitamin D3 (calcitriol) that is the biologically active form. The active metabolite of vitamin D is transported through the bloodstream by the binding protein vitamin D (BPD), reaching numerous skeletal and extraskeletal target organs. In fact, the CYP27B1 enzyme is present in many target cells in the body to allow local synthesis of calcitriol. In addition, vitamin D receptors (VDR) are present in most tissues [15,16,17,18].
3. Vitamin D Mechanism of Action
The functions of VITD are performed in the body via two pathways through endocrine and autocrine mechanisms [19,20,21,22,23]. The endocrine mechanism is the most studied and works by increasing intestinal calcium absorption and osteoclastic activity. Vitamin D is essential in bone growth, density and remodeling [13,18,21,22,23]. When vitamin D levels decrease below normal limits, PTH increases bone resorption to meet the body’s demands for calcium. This means that low levels of VITD lead to an increase in bone turnover with an added risk of bone injury such as stress fractures, which are very common in athletes.
The second mechanism of action of vitamin D involves an autocrine pathway. Although it is not so well known, this pathway is essential since it hosts many of the organism’s key metabolic processes, such as signaling processes, expression and genetic response, hormone protein synthesis, immune/inflammatory response, turnover and cell synthesis. Without VITD, the ability to effectively respond to physiological and pathological symptoms would be totally altered [19,20,21,22]. This vitamin works as a modulator of up to 2000 genes involved in cell growth, immune function and protein synthesis [15,21,23].
The autocrine pathway seems to be the most important in relation to the action of vitamin D on skeletal muscle function. Targets for the VITD receptor have been identified in almost every body tissue. VDR regulates expression in hundreds of genes that perform essential bodily functions. The discovery of VDR in muscle suggests the importance of the role of VITD in muscle tissue [19,20,21,22,23].
At present, the existing theory is that an adequate concentration of vitamin D in the blood is necessary to optimize the function of genomics . This role as a genetic modulator explains how vitamin D can affect a variety of physiological functions, such as bone health, muscle function, inflammation and immunity, all important for health, training and performance [19,21].
4. Prevalence of Deficiency and Insufficiency of Vitamin D in Athletes
Not only is it estimated that 1 billion people in the world currently have VITD deficiency, but the progressive increase in its prevalence worldwide is also worrying [5,9,24,25,26,27,28]. Most articles evidence that VITD deficiency is widespread across the world and at prevalence rates that meet the criteria of a pandemic (definition of a pandemic: “an epidemic occurring worldwide, or over a very wide area, crossing international boundaries and usually affecting a large number of people”) [5,14,25]. However other authors questioned this sentence . VITD deficiency is a frequent finding among the American population, so much so that 36% to 57% of adults are deficient [27,28,29]. This deficiency is also common in Europe mostly countries in Northern European latitudes (>35° N) such as the UK, Ireland, Denmark, France, Germany, etc. [5,25,29]. A similar prevalence has been found even in areas where there is greater sun exposure, such as Australia, the USA and Saudi Arabia [8,27,28,29]. In Canada, 30–50% of children and adults have VITD deficiency. Similar data has been found in other countries, see Africa, New Zealand, Brazil, etc., evidencing a high risk of VITD deficiency in both adults and children [24,25,27,29].
The main factors for VITD deficiency are cultural and environmental influences. The major cause for the VITD deficiency pandemic is the lack of awareness of the population that sun exposure is the main source of vitamin D. In relation to food sources of VITD, it is difficult to obtain vitamin D through the diet because very few foods naturally contain the vitamin, exceptions being the liver of fatty fish such as salmon, sardines, herring and red meat. Actually, diet source includes fortified foods such as milk, fat spreads and cereals. Due to the critical role played by exposure to sunlight and, in particular, to ultraviolet radiation in the synthesis of VITD, any factor that alters this mechanism will contribute to VITD deficiency, such as the decrease of UVB radiation reaching the earth’s surface, the use of sunscreens, melanin that diminishes the effectiveness of sun in producing VITD, polluting atmospheric particles, latitude, weather, lifestyles, etc. [8,9,12,19,22,24,25,29]. In addition, numerous endogenous factors can alter the production of vitamin D and can induce its deficiency, such as its altered metabolism, malabsorption or insufficient intake in one’s diet [24,25,29].
Current strategies in Public Health include dietary supplementation with VITD and education of young children and adolescents. Such initiatives have an important effect on the decrease in the prevalence of developmental problems such as rickets and stunting [2,3,5,8,9]. Other strategies support no need to perform screening everyone for the VITD status. It is more cost-effective to increase food fortification with VITD . However, symptoms of VITD deficiency in adults, osteoporosis, osteomalacia and immune deficiencies are ignored in most cases. Patients with VITD deficiency have musculoskeletal pains that are often misdiagnosed as fibromyalgia, chronic fatigue syndrome and myositis, among others [30,31,32,33,34,35,36,37].
In relation to athletes, VITD deficiency within the global athletic population also follows the same patterns [7,10,12,30,31,32,33,34,35,36,37]. When VITD levels in professional athletes are analyzed, we observe that they are all affected in a similar way. The different studies present the following results: among basketball professionals, 32% of the athletes were found to be deficient and 47% had VITD insufficiency. Among National American Football League players, 26% had VITD deficiency and 42% to 80% showed levels of insufficiency. Of Liverpool’s professional football players, 36% showed deficiency or insufficiency [30,31,32,33,34,35,36].
Furthermore, multiple studies have shown that dark-skinned athletes have a higher risk of suffering from secondary alterations due to VITD deficiency [26,28,34,35]. One study showed that athletes with high concentrations of melanin in their skin need up to 10 times longer exposure to ultraviolet (UVB) radiation to generate the same reserves of VITD as light-skinned athletes. A study by Mehran et al. on professional hockey players in which vitamin D deficiency was 0% and insufficiency only appeared as 13% should be noted. The authors attributed this low frequency to race, since 96.2% of the players were Caucasians [34,37].
In relation to the degree of solar exposure and athleticism, the distance to the equator, season and weather will dictate the source of solar VITD. The production of VITD from the solar source will obviously be influenced by hours of sunshine, pollution, sun block, skin pigment, age, etc. During the summer months and/or countries with more hours of sunshine, UVB radiation from the sun can be absorbed in sufficient amounts to synthesize VITD [4,6,19,32]. However, during the winter months, the angle of the sun prevents UVB radiation from reaching latitudes above 35–37 degrees. When analyzing levels of VITD in athletes, it should be taken into account that these can vary according to the season, place of training, type of sport and skin color [4,6,30,35].
According to some authors, the levels of vitamin D are generally lower in the winter months [30,31,33,34]. However, suboptimal vitamin D levels occur even in sunny countries near the equator when the sun is avoided or the skin is protected. Despite all the factors mentioned above, a high prevalence of vitamin D deficiency has been documented in athletes in both outdoor and indoor sports [3,6,25,35,36,37]. A recent meta-analysis that groups together 23 studies composed of 2313 athletes found that 56% had insufficient levels of vitamin D . Koundourakis et al. [11,12] showed that professional Greek football players who trained at a latitude of 35.9° did not have insufficient levels of vitamin D. Almost identical levels were reported in players of the National Football League, in elite gymnasts in Australia and in young Hawaiian skaters and a variety of other athletes around the world. These findings were observed regardless of sun exposure. In a recent study conducted in Israel at a favorable latitude (31.8° N) for sunshine, 73% of athletes were deficient in vitamin D [1,3,6,25,35,37].
Finally, in relation to dietary recommendations, studies find that athletes do not come close to meeting these in most countries. One study found that only 5% of college athletes met the US Recommended Dietary Allowance (RDA) .
5. Assessment of Vitamin D Status Determination of 25-OHVITD
Status of levels of insufficiency or deficiency of VITD can be defined by using an indicator to determine the blood level of total 25-hydroxy vitamin D (25-OHVITD). This indicator is currently considered as the most qualified to show the body store of vitamin D [38,39,40].
The measurement of blood levels of 25-OHVITD will show us the cutaneous production of VITD that is obtained from food and supplements. It should be noted that the plasma average life is approximately 15–20 days and is recognized as a biomarker of exposure. However, it is controversial whether blood levels of 25-OHVITD could be considered as a biomarker of effect (e.g., relationship with the state of health, etc.) since serum 25-OHVITD levels do not indicate the amount of VITD stored in body tissues [40,41,42].
Unlike 25-OHVITD, the determination of 1.25 dihydroxy vitamin D (1.25 (OH)2 VITD) is generally not a good indicator of VITD levels, since it has a very short average life (barely 15 h) and its serum concentrations are closely regulated by PTH calcium and phosphate. In fact, levels of 1.25 (OH)2 VITD do not drop significantly until a severe deficiency of VITD occurs [38,39].
The latest research is aimed at being able to measure the unbound fraction of 25-OHVITD, or rather, the fraction of VITD that is not bound to plasma proteins and that exerts biological activity. The unbound form can pass through the cell membrane and, therefore, carry out its function [42,43,44].
After several years of research, a new method was developed in 2017 that allows the concentration of unbound 25-OHVITD to be measured. This method measures the concentration of the unbound fraction, based on an enzyme-linked immunosorbent immunoassay (ELISA). The separation of unbound and bound forms, as well as the capture of the former, is achieved through the use of a monoclonal antibody (anti-25-OHD), disrupting as little as possible the balance between both forms . Following the appearance of this method, new investigations are emerging, although the usefulness of measuring unbound 25-OHVITD has yet to be established in normal clinical practice.
6. Levels and Classification of Vitamin D Levels
In the past decade, there has been an exponential increase in the prevalence of deficiency in the population, and in some studies, it is claimed that we are facing an emerging epidemic situation in relation to low blood levels of 25-OHVITD. From the above, it can be clearly deduced that it is extremely important to adequately define the status of deficiency and insufficiency and optimal levels of vitamin D in the population. The definition of VITD levels for its classification has traditionally been very controversial. At present, it is suggested that its establishment should be based on levels and on clinical and disease risk markers [5,9,45,46,47,48]. Some authors propose that the clinical ranges of vitamin D need to be based on the association of 25-OHVITD deficiency, osteomalacia, rickets and the approximate concentration at which PTH rises sharply. On the other hand, it is proposed that the limit for insufficiency should be the concentration at which the PTH plateau and calcium absorption are maximized [5,9,45,48].
There are studies which suggest that a value of 25-OHVITD >30 ng/mL should be considered as acceptable for maintaining bone health and reducing the risk of fracture in healthy young people and adults, while others suggest that necessary levels should be set at >40 ng/mL [3,8,25,45]. On a more conservative basis, the US Institute of Medicine (IOM) states that concentrations of ≥20 ng/mL (50 nmol/L) should meet the needs of 97.5% of the population [5,9,14,29]. The IOM also establishes an inappropriate level of VITD when levels are between 12 and 20 ng/mL (30 and 50 mmol/L), and finally, people are at risk of VITD deficiency when their levels are below 12 ng/mL (30 nmol/L). Serum concentrations above 125 nmol/L (>50 ng/mL) are associated with potential adverse effects, and finally, levels above 150 ng/mL should be considered as toxic . Unfortunately, there are currently no precise thresholds to classify the condition of athletes although Close et al. argue that those athletes with serum levels of 25-OHVITD below 12 ng/mL should be considered for supplementation, in accordance with IOM guidelines  (Table 1).
Serum 25-Ohvitd Concentrations (nmol/L) Health Status (ng/mL) vitamin D Status < 30 < 12 Associated with VITD deficiency, leading to rickets in infants and children and osteomalacia in adults Severely Deficient 30 to 50 12 to 20 Generally considered inadequate for one and overall health in healthy individuals Deficient/
50 to 125 20 to 50 Generally considered adequate for bone and overall health in healthy individuals Adequate > 125 > 50 Emerging evidence links potential adverse effects to such high levels, particularly >150 nmol/L (>60 ng/mL) Inadequate/
7. Role of Vitamin D and Its Relationship with the Condition of the Athletes
7.1. Effect of Vitamin D on Calcium Homeostasis and Bone Balance
Traditionally, it has been assumed that the main function of vitamin D was the maintenance of calcium homeostasis and serum phosphate. An adequate amount of vitamin D and calcium is required for the development, growth and integrity of bones. Currently, VITD has been shown to influence bone health by activating the expression of genes that improve intestinal absorption and renal reabsorption of calcium (in association with an increase in PTH) and bone turnover . Vitamin D also contributes to the mobilization of calcium of the bone by means of osteoclastogenesis that results from the activation of several genes, including the activator of the K-ligand nuclear factor receptor (RANKL) and the RANKL system [3,5,49,50,51].
On the other hand, VITD is closely related to the parathyroid hormone (PTH). Together, these hormones closely regulate the concentration of calcium in the serum. Chronic VITD deficiency leads to secondary hyperparathyroidism. This combination of vitamin D deficiency and an elevated PTH level can cause excessive mobilization of calcium from the bone to maintain circulating calcium levels at the expense of bone mineral density [51,52,53,54,55].
Furthermore, research suggests that the concentration of VITD in the blood is associated with bone mineral density (BMD) and/or mineral content in the hip and lumbar vertebrae of women throughout their lives [51,53]. Current literature shows inconsistent associations between bone mineral density (BMD) and vitamin D levels [51,52], particularly in racial minorities and athletic populations. The load stimulus to which the musculoskeletal system is subjected through a high-intensity dynamic sports activity is believed to compensate for 25-OHVITD deficiency and prevent poor bone health in athletes. However, Hamilton et al. demonstrated that BMD and the level of 25 [OH] D were not statistically linked in a study conducted in male athletes from the Middle East, suggesting that genetic polymorphism in path 25 [OH] D/1.25 [OH] D can explain some of these differences. While it is considered that athletes should have “sufficient” vitamin D concentrations to optimize bone mineral density (BMD), the exact value to “optimize” bone health is still unclear .
Finally, vitamin D also increases the activity of the insulin-like growth factor 1 (IGF-1) through induction of its receptor expression, which has a crucial effect on bone formation both in vitro and in vivo [55,56].
7.2. Effect of Vitamin D on Fractures
A particularly relevant section refers to the action of VITD in stress fractures, which are frequently observed in athletes, which represent from 0.7% to 20% of all clinical injuries in sports medicine. Although it has already been stated that vitamin D levels can affect BMD, there is less knowledge of the role of vitamin D in fracture healing and there is no scientific evidence on it. A review found that vitamin D reduces, increases or has no effect in the soft callus formation phase during the fracture healing process . Other studies find conflicting results regarding the effect of vitamin D in the callus mineralization phase . However, a recent investigation found lower serum levels of 25-OHVITD in patients with delayed fracture consolidation, while other studies found no differences between patients with diaphyseal fractures and those who presented delayed healing .
7.3. The role of Vitamin D in the Skeletal Muscle
Vitamin D has been shown to be a powerful modulator of skeletal muscle physiology [57,58,59,60]. Vitamin D influences it by activating the expression of genes that influence muscle growth and differentiation, particularly in fast-twitch fibers (type II) [60,61,62]. In addition, enlarged interfibrillar spaces and infiltration of fat, fibrosis and glycogen in muscular dystrophies are shown in muscle biopsies of individuals with VITD deficiency . Biopsies of 12 patients with VITD deficiency, before and after treatment with the vitamin, found atrophy of type 2 muscle fibers before treatment and significant improvement after it .
It should be noted here that both genomic and non-genomic effects of VITD are crucial for muscle performance. In fact, vitamin D affects calcium and phosphate transport by muscles through cell membranes, phospholipid metabolism and muscle cell proliferation and differentiation .
The VDR exerts its effects in two pathways:
The first, the genomic pathway (slow or nuclear), through which the transcription and translation of the target genes are modified. This finding suggests that vitamin D promotes muscle cell proliferation and differentiation [15,20,60,61,62].
The second mechanism is the non-transcriptional signaling pathway associated with the membrane (rapid, non-genomic or membrane), in which the receptor for 1.25-OHVITD is located. It has been shown that this mechanism enhances the interaction between myosin and actin in the sarcomere, making the force of muscle contraction stronger [11,15,20,57,63] (Figure 2).
In relation to physical exercise and its impact on athletes, it is argued that the low level of VITD could directly affect muscle strength and performance. Studies in young people and in elderly who are non-athletes found that low VITD levels were negatively associated with muscle strength markers [51,58]. For athletes with VITD deficiency, supplementation with the vitamin probably improves certain parameters of muscle performance [32,60]. In injured athletes, insufficient VITD also seems to delay rehabilitation and recovery after orthopedic surgery [49,63].
From a clinical point of view, a potential association between VITD and muscle function is also suggested, since myopathy was strongly associated with severe VITD deficiency . Barker et al. found that 93% of the patients who presented common clinical symptoms of non-specific musculoskeletal pain had VITD deficiency.
To conclude, it is suggested that vitamin D is beneficial for people as it increases the synthesis of muscle proteins, the concentration of adenosine triphosphate (ATP), strength, jump height, jumping speed and power, as well as the capacity to perform aerobic and anaerobic exercise. Physical performance could be significantly improved and/or preserved with adequate levels of vitamin D. Vitamin D also prevents muscle degeneration and reverses myalgia .
7.4. Effects of Vitamin D on Lung Function
Vitamin D insufficiency has been associated with impaired lung function, asthma and chronic obstructive pulmonary disease (COPD). On the other hand, vitamin D deficiency resulted in deficits in lung volume and correlated with multiple indices of compromised lung function and increased airway reactivity . These actions of VITD favor alveolar structural integrity, pulmonary compliance, vital capacity and oxygen exchange [64,65,66,67].
Among the population of athletes, exercise performance and aerobic capacity (VO2max) depend on all these lung functions above. Adequate VO2max levels are needed in all sports activities. However, the results found for different authors in the athletic population in relation to VITD deficiency and athletes are inconclusive [64,65,66,67]
7.5. Vitamin D and Cardiovascular Function
First, we must bear in mind that the regular practice of intense physical exercise is associated with several structural and cardiac electrophysiological adaptations that improve diastolic filling and facilitate a sustained increase in cardiac output, which is essential for athletic performance. The vast majority of athletes show relatively slight structural and electrical changes, which are considered within the conventional definition of normal limits. Such cardiac adaptations are collectively known as “Athlete’s Heart” and are often reflected in the ECG and imaging studies .
Numerous factors influence the adaptations of athlete hearts, including sports modality, duration and intensity of training, age, ethnicity, gender, anthropometry and substance abuse to improve performance .
A small proportion of athletes develop pronounced changes that overlap with phenotypic expressions of heart disease involved in sudden cardiac death associated with exercise (SCD). In these circumstances, distinguishing between physiological adaptation and cardiac pathology is challenging, but a misdiagnosis can have serious consequences. Emerging studies suggest that ethnicity is a major determinant of cardiovascular adaptation to exercise, which should always be considered during the evaluation of an athlete. It is a well-established fact that ethnicity is one of the factors that influence the manifestations of an athlete’s heart [67,68,69].
It is also recognized that many professional athletes have vitamin D deficiency and, currently, no study has examined the association between vitamin D levels and the cardiac structure and function in healthy athletes. One thing to bear in mind is that recent research has detected an association between VITD and sudden cardiac death in athletes, finding a strong relationship between severe VITD deficiency and sudden cardiac death [67,68,69,70].
8. Mechanism of Action of Vitamin D in Cardiovascular Function
Vitamin D receptors (VDR) are present throughout the heart and vascular system, specifically located in myocytes and cardiac fibroblasts [1,9,20]. The activated form of VITD, 1-2OH VITD, participates in the structural remodeling of cardiac muscle and vascular tissue and activate myocyte contractility [57,59,61,63].
There is scientific evidence that vitamin D deficiency has long-term cardiovascular adverse effects. Vitamin D deficiency negatively affects cardiac contractility, vascular tone, cardiac collagen content and cardiac tissue maturation. This is mainly because VITD deficiency causes an increase in PTH levels that can lead to left ventricular hypertrophy. This hypertrophy can alter the filling capacity of the ventricle and ejection fraction leading to possible hypoxia of muscle tissue and a decrease in athletic performance [68,69,70]. It has also been evinced that in patients who presented a severe VITD deficiency, supplementation treatment resulted in an improvement in cardiac muscle function .
At the vascular level, there are vitamin D receptors in the vascular wall, so this vitamin is believed to affect vascular physiology and its pathophysiology [71,72,73,74]. Vitamin D insufficiency is related to increased arterial stiffness and endothelial dysfunction in blood vessels and promotes atherogenesis . Severe vitamin D deficiency causes an alteration in the adaptive immune response favoring vascular dysfunction, insulin resistance and arteriosclerosis [67,68]. These factors are critical for aerobic and anaerobic exercise performance and resistance capacity . Additionally, low serum levels of vitamin D can cause pathological myocardial hypertrophy, increased blood pressure and endothelial dysfunction. This confluence of alterations supports the assumption that inadequate levels of vitamin D could negatively influence cardiorespiratory capacity, influencing the supply of oxygen and nutrients to the exercising muscle.
Recent data has shown a high prevalence of vitamin D deficiency among ethnicities, particularly among Arab athletes. Vitamin D deficiency is associated with hypertension, myocardial infarction and stroke, as well as other diseases related to cardiovascular diseases. To date, the association between vitamin D levels, ethnicity and cardiovascular function in athletic populations has not been studied .
8.1. Action of Vitamin D in the Immune System
Different studies have proved that vitamin D affects innate and adaptive immunity through its VDR action [71,72,73]. Vitamin D affects both T and B cells. In resting conditions, the expression of VDRs shows low activity in both T and B cells, but in infectious diseases, they increase their activity, which suggests a crucial role in adaptive immunity .
Vitamin D can reduce inflammation by its inhibitory effect on proinflammatory cytokines such as interleukin-6, which converts monocytes into macrophages and produces more inflammatory cytokines [11,15,20]. Interleukin-6 can be early increased in intense training [71,72] and it is believed to be related to the appearance of muscle damage during training. On the other hand, it has been shown that vitamin D reduces the production of other proinflammatory cytokines such as interferon, interleukin-2 and tumor necrosis factor-6 [37,38,39,40]. Low levels of VITD in the general population and in athletes (especially after intense exercise) result in an increase in IL6 and TNFα. Therefore, vitamin D improves this inflammatory response [71,72,73].
By endorsing the above, the insufficiency of vitamin D in athletes is associated with a higher frequency of diseases, including common colds, influenza and gastroenteritis. In athletes, the incidence of respiratory diseases is higher (especially at the elite level), suggesting that low levels of vitamin D may favor the vulnerability of these professionals to upper respiratory tract infections, while individuals with higher levels of vitamin D show a lower propensity to them [65,66,73].
8.2. Effects of Vitamin D on the Nervous System
Vitamin D affects the central and peripheral nervous systems. Vitamin D receptors are present throughout the brain, including the primary motor cortex, which is the region that coordinates movement [9,11,74].
In turn, vitamin D also affects neuronal differentiation, maturation and growth. It also exerts direct neuroprotective effects through the synthesis of proteins that play a vital role in neural activity, including transmission. The GABAergic function is the main “brake” in the brain that affects muscle relaxation through corticospinal neurons [12,20,74,75]. The effects of vitamin D on GABAergic tone and on serotonin and dopamine are crucial for muscle coordination and for avoiding central fatigue, a condition associated with the synaptic concentration of several neurotransmitters. A high proportion of serotonin and dopamine affects exercise performance due to its effect on the general feeling of tiredness and perceptions of effort [12,15,17,76]. Another mechanism, through which vitamin D affects the brain and sports performance, may involve nociceptors, or rather, the sensory nerve cell that responds to noxious stimuli by sending signals to the spinal cord and brain. Nociceptors are full of VDR and 1α-hydroxylase. When these receptors transfer pain signals to the brain, an inhibitory physical response takes place. The relevance of this mechanism and physical activity/vitamin D is based on recent findings in animal studies that indicate that vitamin D depletion could result in hyperinnervation and nociceptive hypersensitivity in deep muscle tissue and loss of balance without affecting muscle strength or cutaneous nociceptive response [12,21,75]. Based on this finding, we could speculate that nociceptive hyperinnervation and hypersensitivity in deep muscle tissue could cause a false appearance of myalgia during physical activity that could reduce performance in individuals with vitamin D deficiency.
9. Supplementation with Vitamin D in Athletes
Despite the special attention given to the diet of athletes, we must bear in mind that some micronutrient deficiencies may appear. It is generally believed that if athletes follow a balanced diet, they will not require supplements [3,8,9,77,78,79,80]. However, this idea may be too simplistic. First, determining dietary fitness in athletes can be challenging.
The micronutrient requirements of these professionals may vary depending on the duration, intensity and type of training [30,32,35,80,81,82]. Secondly, for some micronutrients, especially vitamin D, there may not be ample food sources. The importance of this issue lies in the fact that an athlete’s micronutrient status can affect their physical performance . On the other hand, VITD activity is related to the adequate presence of other nutritional factors and it is very important to know the status of other nutrients, like magnesium. Magnesium plays an important role in bone mineralization due in part to its positive influence in the synthesis of active VITD. New research evidence that magnesium implementation can potentiate the effectiveness of VITD activity .
VITD deficiency leads to an increased risk of morbidity that could negatively influence athletic performance and significantly shorten the lifespan of professional athletes. Although some researchers have reported the improved effect of VITD supplementation on physical performance, the issue remains controversial [83,84,85,86,87,88,89,90,91,92].
Suboptimal VITD levels appear both in athletes who mainly train indoors, and at higher latitudes, and in those who train outdoors at lower latitudes [53,81,86]. We must remember that one of the factors that has the greatest influence on vitamin D levels is exposure to sunlight. Anything that limits the amount or quality of sun exposure, can compromise vitamin D levels [78,79,80].
Few published studies categorically state that vitamin D supplementation benefits neuromuscular and aerobic performance. In a recent randomized placebo-controlled trial, the effect of vitamin D (5000 IU per day over a period of eight weeks) on speed times and vertical jumps in a cohort of athletes was evaluated. The group that received vitamin D supplements recorded a substantial increase in vertical jump heights from the beginning to the end of the study period, while no change was observed in the placebo-controlled group .
Wyon et al. [90,91] found an improvement in neuromuscular performance in elite ballet dancers in a study of oral vitamin D3 supplementation. A significant increase in isometric strength (18.7%) and vertical jump (7.1%) was observed. The intervention group showed a significant decrease in the number of injuries with respect to the control group. However, other studies were unable to document any benefits following vitamin D supplementation in athletes with adequate or moderately deficient levels of vitamin D prior to supplementation. Close et al. examined the effects of vitamin D3 supplementation on serum concentrations of 25 [OH] D and on various exercise performance rates in athletes. At the start of the study, 57% of the participants were found to have VITD deficiency. However, despite the increase observed in serum levels of vitamin D, none of the groups showed an improvement in exercise performance compared to control ones [31,32]. Carswell et al.  in 967 young healthy military recruits found there was no influence of status VITD on muscular strength. Although supplementation restored VITD sufficiency, the beneficial effects on exercise performance remain unclear. However, they found a fairly positive association between VITD status and endurance performance.
10. Supplementation with the Appropriate Dose of Vitamin D
It seems that vitamin D supplementation in the general population is important for preventing and avoiding its deficiency. However, there is a lot of controversy regarding the appropriate supplement doses. In athletes, it is even more controversial.
Carlberg et al. suggest that a threshold level of VITD is not enough to asseverate the needs of VITD in individuals. The efficiency of the molecular response to VITD is critical for establishing the appropriate dose of VITD in each individual. This researcher’s evidence that VITD supplementation and his dose is related to the “personal vitamin D response Index” [88,91].
As stated above, the Institute of Medicine (IOM) concluded, in the 2011 consensus statement, that 25 (OH) D levels of 20 ng/mL (50 nmol/L) meet the needs of at least 97.5% of the (North American) population at all stages of life .
The Recommended Dietary Allowance (RDA) to meet the requirements of the IOM of vitamin D for the US and Canada is 600 IU for children and adults under 70 years of age and 800 IU for those over 70 years old. Although the US recommendation is higher than the one established in other countries, many VITD experts believe that these recommendations were established for bone health maintenance, but may not be sufficient to maintain non-skeletal benefits, as well as the optimal health and performance of athletes [3,48,52,77,86].
The Endocrinology Society estimated that 600–800 IU were not sufficient to ensure adequate levels and raised the recommended intake to 1500–2200 IU/day for individuals who do not have adequate sun exposure to maintain adequate vitamin D levels [3,5,8]. In relation to athletes, there is no evidence to suggest that their requirements are different from those of the general population.
A randomized controlled clinical trial in 70 athlete subjects was randomly divided into two groups, VITD supplementation and control. They found that weekly uptake of 50.000 IU VITD improved only certain athlete performance tests, and they conclude that the optimum dosage for athletes needs further studies .
Additionally, possible intoxications due to inadequate vitamin D supplementation should be taken into account. Vitamin D toxicity may be the result of the intake of excessive amounts of supplements of this vitamin. No cases of vitamin D toxicity have been reported from sunlight or regular food intake. The symptoms of vitamin D toxicity are produced by the resulting hypercalcemia which can lead to anorexia, frequent urination, excessive thirst, nausea, vomiting and, in severe cases, altered mental status and kidney failure. Many cases of vitamin D intoxication are the result of improperly manufactured supplements . Some athletes and coaches live in the belief that “if a little is good, more is better”, which is a dangerous misconception. It is very important that the supplementation is carried out by professionals with knowledge on the subject and who are aware that, although VITD intoxication is very rare, it can occur. The most frequent cases are due to unintentional consumption of extremely high doses, and in many cases due to industrial error [3,5,8,83].
The purpose of our review was to investigate the relevance of vitamin D in athletic performance. We review recent advances in this field and novel insights about vitamin D supplementation in athletes.
Low vitamin D status could negatively impact the health and training efficiency of athletes. Research to date suggests that certain athletes are at risk for suboptimal vitamin D status, which may increase risks for stress fractures, acute illness, and suboptimal muscle function.
The emerging evidence about vitamin D and athletic performance suggests the need to determine vitamin D concentration in athletes but further research is necessary to characterize the true vitamin D status by simply measuring free vitamin D rather than total 25-OHVITD.
In relation to the prevention of vitamin D deficiency, we must be aware that sun exposure is the main source. Unfortunately, there is evidence concerned about the possibility that sun exposure, if uncontrolled, may promote skin cancer. On the other hand, we must also take account nutrition in athletes and vitamin D. A personalized nutrition plan should develop. Sufficiency of essential minerals and micronutrients, like magnesium, are critical to enhancing activation of vitamin D.
One interesting theory is based on individual molecular response to vitamin D. Athletes with personalized supplementation of vitamin D, will contribute to obtain optimized clinical benefits. Future studies could determine the optimal VITD threshold and determine supplementation recommendations.
Although previous studies seem to suggest that VITD supplementation in athletes may have a beneficial effect on athletic performance, these results cannot be generalized. Unnecessary supplementation with high doses of vitamin D might be a relatively common practice, without proven benefit, and even with the risk of harm.
Future research is needed, focusing on double-blinded supplementation and optimal VITD levels in athletes and to investigate VITD potentially positive influence on exercise performance and the benefits of VITD supplementation on athletic performance.
M.d.l.P.Y.: Writing—original draft preparation, formal analysis; investigation, resources, L.C.Y.: Writing—review and editing, M.J.C.C.: validation, M.A.C.C. Conceptualization and methodology; All authors have read and agreed to the published version of the manuscript.
This research received no external funding.
Conflicts of Interest
The authors declare no conflict of interest.
References1. Todd J.J., Pourshahidi L.K., McSorley E.M., Madigan S.M., Magee P.J. Vitamin D: Recent advances and implications for athletes. Sport Med. 2015;45:213–229. doi: 10.1007/s40279-014-0266-7. [PubMed] [CrossRef] [Google Scholar]2. Zhang R., Naughton D.P. Vitamin D in health and disease: Current perspectives. Nutr. J. 2010;9:65–71. doi: 10.1186/1475-2891-9-65. [PMC free article] [PubMed] [CrossRef] [Google Scholar]3. Holick M.F., Binkley N.C., Bischoff-Ferrari H.A., Gordon C.M., Hanley D.A., Heaney R.P., Murad M.H., Weaver C.M. Guidelines for preventing and treating vitamin D deficiency and insufficiency revisited. J. Clin. Endocrinol. Metab. 2012;97:1153–1158. doi: 10.1210/jc.2011-2601. [PubMed] [CrossRef] [Google Scholar]4. Owens D.J., Allison R., Close G.L. Vitamin D and the Athlete: Current Perspective and new challenges. Sports Med. 2018;48(Suppl. 1):S3–S16. doi: 10.1007/s40279-017-0841-9.[PMC free article] [PubMed] [CrossRef] [Google Scholar]5. Cashman K.D., Dowling K.G., Škrabáková Z., Gonzalez-Gross M., Valtueña J., De Henauw S., Moreno L., Damsgaard C.T., Michaelsen K.F., Mølgaard C., et al. Vitamin D deficiency in Europe:pandemic? Am. J. Clin. Nutr. 2016;103:1033–1044. doi: 10.3945/ajcn.115.120873. [PMC free article] [PubMed] [CrossRef] [Google Scholar]6. Halliday T.M., Peterson N.J., Thomas J.J., Kleppinger K., Hollis B.W., Larson-Meyer D.E. Vitamin D status relative to diet, lifestyle, injury, and illness in college athletes. Med. Sci. Sports Exerc. 2011;43:335–343. doi: 10.1249/MSS.0b013e3181eb9d4d.[PubMed] [CrossRef] [Google Scholar]7. Larson-Meyer D.E., Willis K.S. Vitamin D and athletes. Curr. Sports Med. Rep. 2010;9:220–226. doi: 10.1249/JSR.0b013e3181e7dd45. [PubMed] [CrossRef] [Google Scholar]8. Hossein-nezhad A., Holick M.F. Vitamin D for health: A global perspective. Mayo Clin. Proc. 2013;88:720–755. doi: 10.1016/j.mayocp.2013.05.011. [PMC free article] [PubMed] [CrossRef] [Google Scholar]9. Larson-Meyer D.E. The importance of Vitamin D for Athletes. Sports Sci. Exch. 2015;28:1–6. [Google Scholar]10. Koundourakis N.E., Androulakis N.E., Malliaraki N., Tsatsanis C., Venihaki M., Margioris A.N. Discrepancy between exercise performance, body composition, and sex steroid response after a six-week detraining period in professional soccer players. PLoS ONE. 2014;9:e87803. doi: 10.1371/journal.pone.0087803.[PMC free article] [PubMed] [CrossRef] [Google Scholar]11. Koundourakis N.E., Avgoustinaki P.D., Malliaraki N., Margioris A.N. Muscular effects of vitamin D in young athletes and non-athletes and in the elderly. Hormones. 2016;15:471–488. doi: 10.14310/horm.2002.1705. [PubMed] [CrossRef] [Google Scholar]12. Bikle D.D. Vitamin D Metabolism, Mechanism of Action, and Clinical Applications. Chem. Biol. 2014;20:319–329.[PMC free article] [PubMed] [Google Scholar]13. Last J. A Dictionary of Epidemiology. 4th ed. Oxford University Press; Oxford, UK: 2001. [Google Scholar]14. Plum L., De Luca H. The Functional Metabolism and Molecular Biology of Vitamin D Action. Clin. Rev. Bone Miner. Metab. 2009;7:20–41. doi: 10.1007/s12018-009-9040-z.[CrossRef] [Google Scholar]15. Norman A.W. From vitamin D to hormone D: Fundamentals of the vitamin D endocrine system essential for good health. Am. J. Clin. Nutr. 2008;88:491S–499S. doi: 10.1093/ajcn/88.2.491S.[PubMed] [CrossRef] [Google Scholar]16. Verstuyf A., Carmeliet G., Bouillon R., Mathieu C. Vitamin D: A pleiotropic hormone. Kidney Int. 2010;78:140–145. doi: 10.1038/ki.2010.17. [PubMed] [CrossRef] [Google Scholar]17. Lips P. Vitamin D physiology. Prog. Biophys Mol. Biol. 2006;92:4–8. doi: 10.1016/j.pbiomolbio.2006.02.016. [PubMed] [CrossRef] [Google Scholar]18. Holick M.F., Garabedian M. Vitamin D: Photobiology, metabolism, mechanism of action, and clinical applications. In: Favus M.J., editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. 6th ed. American Society for Bone and Mineral Research; Washington, DC, USA: 2006. pp. 129–137. [Google Scholar]19. Wang Y., Zhu J., De Luca H.F. Where is the vitamin D receptor? Arch. Biochem. Biophys. 2012;523:123–133. doi: 10.1016/j.abb.2012.04.001. [PubMed] [CrossRef] [Google Scholar]20. Holick M.F. Vitamin D: Extraskeletal health. Endocrinol. Metab. Clin. N. Am. 2010;39:381–400. doi: 10.1016/j.ecl.2010.02.016. [PubMed] [CrossRef] [Google Scholar]21. Christakos S., Ajibade D.V., Dhawan P., Fechner A.J., Mady L.J. Vitamin D: Metabolism. Endocrinol. Metab. Clin. N. Am. 2010;39:243–253. doi: 10.1016/j.ecl.2010.02.002.[PMC free article] [PubMed] [CrossRef] [Google Scholar]22. Morris H.A., Anderson P.H. Autocrine and paracrine actions of vitamin d. Clin. Biochem. Rev. 2010;31:129–138.[PMC free article] [PubMed] [Google Scholar]23. Forrest K.Y., Stuhldreher W.L. Prevalence and correlates of vitamin D deficiency in US adults. Nutr. Res. 2011;31:48–54. doi: 10.1016/j.nutres.2010.12.001. [PubMed] [CrossRef] [Google Scholar]24. Ovesen L., Andersen R., Jakobsen J. Geographical differences in vitamin D status, with particular reference to European countries. Proc. Nutr. Soc. 2003;62:813–821. doi: 10.1079/PNS2003297. [PubMed] [CrossRef] [Google Scholar]25. Angeline M.E., Gee A.O., Shindle M., Warren R.F., Rodeo S.A. The effects of vitamin D deficiency in athletes. Am. J. Sports Med. 2013;41:461–464. doi: 10.1177/0363546513475787. [PubMed] [CrossRef] [Google Scholar]26. Ginde A.A., Liu M.C., Camargo C.A., Jr. Demographic differences and trends of vitamin D insufficiency in the US population, 1988–2004. Arch. Intern. Med. 2009;23:626–632. doi: 10.1001/archinternmed.2008.604. [PMC free article] [PubMed] [CrossRef] [Google Scholar]27. Manson A.E., Brannon P.M., Rosen C.J., Taylor C.L. Vitamin D Deficiency-Is there Realy a Pandemic? N. Engl. J. Med. 2016;375:1817–1820. doi: 10.1056/NEJMp1608005. [PubMed] [CrossRef] [Google Scholar]28. Holick M.F. The Vitamin D deficiency pandemic: Aproaches for diagnosis, treatment and prevention. Rev. Endocr. Metab. Disord. 2017;18:153–165. doi: 10.1007/s11154-017-9424-1.[PubMed] [CrossRef] [Google Scholar]29. Farrokhyar F., Tabasinejad R., Dao D., Peterson D., Ayeni O.R., Hadioonzadeh R., Bhandari M. Prevalence of vitamin D inadequacy in athletes: A systematic-review and meta-analysis. Sports Med. 2015;45:365–378. doi: 10.1007/s40279-014-0267-6.[PubMed] [CrossRef] [Google Scholar]30. Morton J.P., Iqbal Z., Drust B., Burgess D., Close G.L., Brukner P.D. Seasonal variation in vitamin D status in professional soccer players of the English Premier League. Appl. Physiol. Nutr. Metab. 2012;37:798–802. doi: 10.1139/h2012-037. [PubMed] [CrossRef] [Google Scholar]31. Close G.L., Russell J., Cobley J.N., Owens D.J., Wilson G., Gregson W., Fraser W.D., Morton J.P. Assessment of vitamin D concentration in non-supplemented professional athletes and healthy adults during the winter months in the UK: Implications for skeletal muscle function. J. Sports Sci. 2013;31:344–353. doi: 10.1080/02640414.2012.733822. [PubMed] [CrossRef] [Google Scholar]32. Rankinen T.S., Lyytikainen E., Vanninen I., Penttila R., Uusitupa M. Nutritional status of the Finnish elite ski jumpers. Med. Sci. Sports Exerc. 1998;30:1592–1597. doi: 10.1097/00005768-199811000-00006. [PubMed] [CrossRef] [Google Scholar]33. Farrokhyar F., Sivakumar G., Savage K., Koziarz A., Jamshidi S., Olufemi R., Ayeni D.P., Bhandari M. Prevalence of Vitamin D inadequacy in athletes: Effects of Vitamin D supplementation on serum 15-OH Hydroxyvitamin D concentration and physical performance in athletes.A systematic review and meta-analysis of Randomized Controlled Trials. Sports Med. 2017;47:2323–2339. doi: 10.1007/s40279-017-0749-4. [PubMed] [CrossRef] [Google Scholar]34. Mehran N., Schulz B.M., Neri B.R., Robertson W.J., Limpisvasti O. Prevalence of vitamin D insufficiency in professional hockey players. Orthop. J. Sports Med. 2016;4:1–4. doi: 10.1177/2325967116677512. [PMC free article] [PubMed] [CrossRef] [Google Scholar]35. Maroon J.C., Mathyssek C.M., Bost J.W., Amos A., Winkelman R., Yates A.P., Duca M.A., Norwig J.A. Vitamin D profile in National Football League players. Am. J. Sports Med. 2015;43:1241–1245. doi: 10.1177/0363546514567297. [PubMed] [CrossRef] [Google Scholar]36. Dubnov-Raz G., Livne N., Raz R., Rogel D., Constantini W. Vitamin D concentrations and physical performance in competitive adolescent swimmers. Pediatr. Exerc. Sci. 2014;26:64–70. doi: 10.1123/pes.2013-0034. [PubMed] [CrossRef] [Google Scholar]37. Carter G.D. 25-hydroxyvitamin D assays: The quest for accuracy. Clin. Chem. 2009;55:1300–1302. doi: 10.1373/clinchem.2009.125906. [PubMed] [CrossRef] [Google Scholar]38. Heureux N. Vitamin D testing-where are we and what is on the horizon? Adv. Clin. Chem. 2017;78:59–101. [PubMed] [Google Scholar]39. Bikle D., Bouillon R., Thadhani R., Schoenmakers I. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D sta- tus? J. Steroid Biochem. Mol. Biol. 2017;173:105–116. doi: 10.1016/j.jsbmb.2017.01.007. [PubMed] [CrossRef] [Google Scholar]40. Chun R.F., Peercy B.E., Orwoll E.S., Nielson C.M., Adams J.S., Hewison M. Vitamin D and DBP: The free hormone hypothesis revisited. J. Steroid Biochem. Mol. Biol. 2014;144:132–137. doi: 10.1016/j.jsbmb.2013.09.012. [PMC free article][PubMed] [CrossRef] [Google Scholar]41. Nielson C.M., Jones K.S., Bouillon R. Osteoporotic Fractures in Men (MrOS) Research Group. Role of assay type in determining free 25-hydro- vitamin D levels in diverse populations. N. Engl. J. Med. 2016;374:1695. doi: 10.1056/NEJMc1513502.[PMC free article] [PubMed] [CrossRef] [Google Scholar]42. Johnsen M.S., Grimnes G., Figenschau Y., Torjesen P.A., Almås B., Jorde R. Serum free and bio-available 25-hydroxyvitamin D correlate better with bone density than serum total 25-hydroxyvitamin D. Scand. J Clin. Lab. Investig. 2014;74:77–83. doi: 10.3109/00365513.2013.869701. [PubMed] [CrossRef] [Google Scholar]43. Heureux N., Lindhout E., Swinkels L. A direct assay for measuring free 25- hydroxyvitamin D. JAOAC Int. 2017;100:1318–1322. doi: 10.5740/jaoacint.17-0084. [PubMed] [CrossRef] [Google Scholar]44. Gallagher J.C., Sai A.J. Vitamin D insufficiency, deficiency, and bone health. J. Clin. Endocrinol. Metab. 2010;95:2630–2633. doi: 10.1210/jc.2010-0918. [PMC free article] [PubMed] [CrossRef] [Google Scholar]45. Hollis B.W. Circulating 25-hydroxyvitamin D levels indicative of vitamin D sufficiency: Implications for establishing a new effective dietary intake recommendation for vitamin D. J. Nutr. 2005;135:317–322. doi: 10.1093/jn/135.2.317. [PubMed] [CrossRef] [Google Scholar]46. Institute of Medicine. Food and Nutrition Board . Dietary Reference Intakes for Calcium and Vitamin D. National Academy Press; Washington, DC, USA: 2010. [Google Scholar]47. Holick M.F. Vitamin D. In: Shils M.E., Shike M., Ross A.C., Caballero B., Cousins R.J., editors. Modern Nutrition in Health and Disease. 10th ed. Lippincott Williams & Wilkins; Philadelphia, PA, USA: 2006. [Google Scholar]48. Wacker M., Holick M.F. VitaminD—Effects on skeletal and extraskeletal health and the need for supplementation. Nutrients. 2013;5:111–148. doi: 10.3390/nu5010111. [PMC free article][PubMed] [CrossRef] [Google Scholar]49. Kopeć A., Solarz K., Majda F., Słowińska-Lisowska M., Mędraś M. An evaluation of the levels of vitamin D and bone turnover markers after the summer and winter periods in polish professional soccer players. J. Hum. Kinet. 2013;38:135–140. doi: 10.2478/hukin-2013-0053. [PMC free article] [PubMed] [CrossRef] [Google Scholar]50. Bischoff-Ferrari H.A., Dietrich T., Orav E.J., Dawson-Hughes B. Positive association between 25-hydroxy vitamin D levels and bone mineral density: A population-based study of younger and older adults. Am. J. Med. 2004;116:634–639. doi: 10.1016/j.amjmed.2003.12.029. [PubMed] [CrossRef] [Google Scholar]51. Maimoun L., Manetta J., Couret I., Dupuy A.M., Mariano-Goulart D., Micallef J.P., Peruchon E., Rossi M. The intensity level of physical exercise and the bone metabolism response. Int. J. Sports Med. 2006;27:105–111. doi: 10.1055/s-2005-837621.[PubMed] [CrossRef] [Google Scholar]52. Hamilton B., Grantham J., Racinais S., Chalabi H. Vitamin D deficiency is endemic in Middle Eastern sportsmen. Public Health Nutr. 2010;13:1528–1534. doi: 10.1017/S136898000999320X.[PubMed] [CrossRef] [Google Scholar]53. Burgi A.A., Gorham E.D., Garland C.F., Mohr S.B., Garland F.C., Zeng K., Thompson K., Lappe J.M. High serum 25-hydroxyvitamin D is associated with a low incidence of stress fractures. J. Bone Miner. Res. 2011;26:2371–2377. doi: 10.1002/jbmr.451. [PubMed] [CrossRef] [Google Scholar]54. Li Y.C. Vitamin D, renin, and blood pressure. In: Ho-Lick M.F., editor. Vitamin D Physiology, Molecular Biology, and Clinical Applications. 2nd ed. Humana Press; Boston, MA, USA: 2010. pp. 937–953. [Google Scholar]55. Iwamoto J., Sato Y., Takeda T., Matsumoto H. Analysis of stress fractures in athletes based on our clinical experience. World J. Orthop. 2011;2:7–12. doi: 10.5312/wjo.v2.i1.7.[PMC free article] [PubMed] [CrossRef] [Google Scholar]Retracted56. Koundourakis N.E., Androulakis N.E., Malliaraki N., Margioris A.N. Vitamin D and exercise performance in professional soccer players. PLoS ONE. 2014;9:e101659. doi: 10.1371/journal.pone.0101659. [PMC free article] [PubMed] [CrossRef] [Google Scholar]57. Cannell J.J., Hollis B.W., Sorenson M.B., Taft T.N., Anderson J.J. Athletic performance and vitamin D. Med. Sci. Sports Exerc. 2009;41:1102–1110. doi: 10.1249/MSS.0b013e3181930c2b.[PubMed] [CrossRef] [Google Scholar]58. Girgis C.M., Clifton-Bligh R.J., Hamrick M.W., Holick M.F., Gunton J.E. The roles of vitamin D in skeletal muscle: Form, function, and metabolism. Endocr. Rev. 2013;34:33–83. doi: 10.1210/er.2012-1012. [PubMed] [CrossRef] [Google Scholar]59. Girgis C.M., Mokbel N., Cha K.M., Houweling P.J., Abboud M., Fraser D.R., Mason R.S., Clifton-Bligh R.J., Gunton J.E. The vitamin D receptor (VDR) is expressed in skeletal muscle of male mice and modulates 25-hydroxyvitamin D (25OHD) uptake in myofibers. Endocrinology. 2014;155:3227–3237. doi: 10.1210/en.2014-1016. [PMC free article] [PubMed] [CrossRef] [Google Scholar]60. Sato Y., Iwamoto J., Kanoko T., Satoh K. Low-dose vitamin D prevents muscular atrophy and reduces falls and hip fractures in women after stroke: A randomized controlled trial. Cerebrovasc. Dis. 2005;20:187–192. doi: 10.1159/000087203. [PubMed] [CrossRef] [Google Scholar]61. Oh J.H., Kim S.H., Kim J.H., Shin Y.H., Yoon J.P., Oh C.H. The level of vitamin D in the serum correlates with fatty degeneration of the muscles of the rotator cuff. J. Bone Joint Surg. Br. 2009;12:1587–1593. doi: 10.1302/0301-620X.91B12.22481.[PubMed] [CrossRef] [Google Scholar]62. Barker T., Henriksen V.T., Martins T.B., Hill H.R., Kjeldsberg C.R., Schneider E.D., Dixon B.M., Weaver L.K. Higher serum 25-hydroxyvitamin D concentrations associate with a faster recovery of skeletal muscle strength after muscular injury. Nutrients. 2013;4:1253–1275. doi: 10.3390/nu5041253. [PMC free article][PubMed] [CrossRef] [Google Scholar]63. Peter N., Black M.B., Scragg R. Relationship between serum 25-Hydroxyvitamin D and pulmonary function in the Third National Health and Nutrition Examination Survey. Chest. 2005;128:3792–3798. [PubMed] [Google Scholar]64. Laaksi I., Ruohola J.P., Tuohimaa P., Auvinen A., Haataja R., Pihlajamaki H., Ylikomi T. An association of serum vitamin D concentrations < 40 nmol/L with acute respiratory tract infection in young Finnish men. Am. J. Clin. Nutr. 2007;86:714–717.[PubMed] [Google Scholar]65. He C.S., Handzlik M., Fraser W.D., Muhamad A.S., Preston H., Richardson A., Gleeson M. Influence of vitamin D status on respiratory infection incidence and immune function during 4 months of winter training in endurance sport athletes. Exerc. Immunol. Rev. 2013;19:86–101. [PubMed] [Google Scholar]66. Ksiazek A., Zagrodna A., Dziubek W., Pietraszewski B., Ochmann B., Slowinska-Lisowska M. 25(OH)D3. Levels Relative to Muscle Strength and Maximum Oxygen Uptake in Athletes. J. Hum. Kinet. 2016;50:71–779. doi: 10.1515/hukin-2015-0144.[PMC free article] [PubMed] [CrossRef] [Google Scholar]67. McGreevy C., Williams D. New insights about vitamin D and cardiovascular disease: A narrative review. Ann. Intern. Med. 2011;155:820–826. doi: 10.7326/0003-4819-155-12-201112200-00004. [PubMed] [CrossRef] [Google Scholar]68. Judd S., Tangpricha V. Vitamin D deficiency and risk of cardiovascular disease. Circulation. 2008;117:503. doi: 10.1097/MAJ.0b013e3181aaee91. [PMC free article] [PubMed] [CrossRef] [Google Scholar]69. Lavie C.J., Dinicolantonio J.J., Milani R.V., O’Keefe J.H. Vitamin D and cardiovascular health. Circulation. 2013;128:2404–2406. doi: 10.1161/CIRCULATIONAHA.113.002902. [PubMed] [CrossRef] [Google Scholar]70. Willis K.S., Smith D.T., Broughton K.S. Larson-Meyer DE.Vitamin D status and biomarkers of inflammation in runners. Open Access J. Sports Med. 2012;3:35. [PMC free article][PubMed] [Google Scholar]71. Barker T., Martins T.B., Hill H.R., Kjeldsberg C.R., Dixon B.M., Schneider E.D., Henriksen V.T., Weaver L.K. Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans. Cytokine. 2014;65:134–137. doi: 10.1016/j.cyto.2013.12.004. [PubMed] [CrossRef] [Google Scholar]72. Gombart A.F., Borregaard N., Koeffler H.P. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1,25-dihydroxyvitamin D3. FASEB J. 2005;19:1067–1077. doi: 10.1096/fj.04-3284com. [PubMed] [CrossRef] [Google Scholar]73. Wrzosek M., Lukaszkiewicz J., Wrzosek M., Jakubczyk A., Matsumoto H., Piątkiewicz P., Radziwoń-Zaleska M., Wojnar M., Nowicka G. Vitamin D and the central nervous system. Pharmacol. Rep. 2013;65:271–278. doi: 10.1016/S1734-1140(13)71003-X.[PubMed] [CrossRef] [Google Scholar]74. Mpandzou G., Aït Ben Haddou E., Regragui W., Benomar A., Yahyaoui M. Vitamin D deficiency and its role in neurological conditions: A review. Rev. Neurol. 2016;172:109–122. doi: 10.1016/j.neurol.2015.11.005. [PubMed] [CrossRef] [Google Scholar]75. De Luca G.C., Kimball S.M., Kolasinski J., Ramagopalan S.V., Ebers G.C. Review:the role of vitamin D in nervous system health and disease. Neuropathol. Appl. Neurobiol. 2013;39:458–484. doi: 10.1111/nan.12020. [PubMed] [CrossRef] [Google Scholar]76. Heaney R.P., Holick M.F. Why the IOM recommendations for vitamin D are deficient. J. Bone Miner. Res. 2011;26:455–457. doi: 10.1002/jbmr.328. [PubMed] [CrossRef] [Google Scholar]77. Uwitonze A.M., Razzaque M. Role of Magnesium in Vitamin D activation and function. J. Am. Osteopath. Assoc. 2018;118:181–189. doi: 10.7556/jaoa.2018.037. [PubMed] [CrossRef] [Google Scholar]78. Razzaque M.S. Magnesium: Are we consuming enough? Nutrients. 2018;10:1863. doi: 10.3390/nu10121863.[PMC free article] [PubMed] [CrossRef] [Google Scholar]79. Wyon M.A., Koutedakis Y., Wolman R., Nevill A.M., Allen N. The influence of winter vitamin D supplementation on muscle function and injury occurrence in elite ballet dancers: A controlled study. J. Sci. Med. Sport. 2014;17:8–12. doi: 10.1016/j.jsams.2013.03.007. [PubMed] [CrossRef] [Google Scholar]80. Peeling P., Fulton S.K., Binnie M., Goodman C. Training environment and Vitamin D status in athletes. Int. J. Sports Med. 2013;34:248–252. doi: 10.1055/s-0032-1321894. [PubMed] [CrossRef] [Google Scholar]81. Constantini N.W., Arieli R., Chodick G., Dubnov-Raz G. High prevalence of vitamin D insufficiency in athletes and dancers. Clin. J. Sport Med. 2010;20:368–371. doi: 10.1097/JSM.0b013e3181f207f2. [PubMed] [CrossRef] [Google Scholar]82. Jones G. Pharmacokinetics of vitamin D toxicity. Am. J. Clin. Nutr. 2008;88:582S–586S. doi: 10.1093/ajcn/88.2.582S. [PubMed] [CrossRef] [Google Scholar]83. Bescos Garcia R., Rodriguez Guisado F.A. Low levels of vitamin D in professional basketball players after wintertime: Relationship with dietary intake of vitamin D and calcium. Nutr. Hosp. 2011;26:945–951. [PubMed] [Google Scholar]84. Carsmell A.T., Oliver S.J., Wentz L.M., Kashi D.S., Roberts R., Tang J.C., Izard R.M., Jackson S., Allan D., Rhodes L.E., et al. Influence of Vitamin D supplementation by sunlight or oral D3 on exercise performance. Med. Sci. Sports Exerc. 2018;50:2555–2564. doi: 10.1249/MSS.0000000000001721. [PMC free article][PubMed] [CrossRef] [Google Scholar]85. Holick M.F., Binkley N.C., Bischoff-Ferrari H.A., Gordon C.M., Hanley D.A., Heaney R.P., Murad M.H., Weaver C.M. Evaluation, treatment, and prevention of vitamin D deficiency: An endocrine society clinical practice guideline. J. Clin. Endocrinol. Metab. 2011;96:1911–1930. doi: 10.1210/jc.2011-0385. [PubMed] [CrossRef] [Google Scholar]86. Alimoradi K., Nikppyeh B., Ravasi A.A., Zahedirar N., Kalayo S.A., Neyestani T.R. Efficacy of Vitamin D in physical performance of Iranian elite Athletes. Int. J. Prev. Med. 2019;10:100–108. [PMC free article] [PubMed] [Google Scholar]87. Carlberg C. Nutrigenomics of Vitamin D. Nutrients. 2019;11:676. doi: 10.3390/nu11030676. [PMC free article][PubMed] [CrossRef] [Google Scholar]88. Minisola S., Ferrone F., Danese V., Cechetti V., Pepe J., Cipriani C., Colangelo L. Controversies Surrounding Vitamin D: Focus on Supplementation and Cancer. Int. J. Environ. Res. Public Health. 2019;16:189. doi: 10.3390/ijerph16020189.[PMC free article] [PubMed] [CrossRef] [Google Scholar]89. Plitz S., Marz W., Cashman K.D., Kiely M.E., Whiting S.J., Holick M.F., Grant W.B., Pludowski P., Hiligsman M., Trummer C., et al. Rationale and plan for Vitamin D, food fortification: A review and guidance. Front. Endocrinol. 2018;9:373–379.[Google Scholar]90. Bolland M.J., Grey A., Avenell A. Effects of Vitamin D supplementation on musculoskeletal health. A systematic review, meta-analysis, and trial sequential analysis. Lancet Diabetes Endocrinol. 2018;6:847–858. [PubMed] [Google Scholar]91. Carlberg C., Haq A. The concept of the personal Vitamin D reponse index. J. Steroid Biochem. Mol. Biol. 2018;175:12–17. doi: 10.1016/j.jsbmb.2016.12.011. [PubMed] [CrossRef] [Google Scholar]
92. Wyon M.A., Wolman R., Kolokythas N., Sheriff K., Galloway S., Mattiussi A. The effect of Vitamin D supplementation in elite adolescent dancers on muscle function and injury incidence. A randomized double-Blind study. Int. J. Sports. Physiol. 2018;12:1–15. [PubMed] [Google Scholar]
Melanin nanoparticles hinder tumours with new theranostics approach
Nanoparticles loaded with skin pigment helps scientists diagnose and treat tumours simultaneously with therapy/diagnostics: ‘theranostics’.
It is sprayed, in synthetic form, onto NASA spaceships to protect them from radiation. It has also featured in recent research into how its absorptive properties have allowed fungi to adapt and thrive inside the radioactive exclusion zone of the Chernobyl disaster. Now the dark skin pigment Melanin, which protects our body from the sun’s damaging rays by absorbing light and dissipating it as heat, could become an essential tool in the diagnosis and treatment of cancer.
Longevity.Technology: The strength, in our view, of the theranostics approach is that it isn’t presented (at this stage) as an option for curing tumours. The research team are, instead, presenting a highly viable method to find tumours and slow them, making them a widely applicable Longevity option for buying time against many different types of cancer, including the ones for which we don’t currently have a cure.
The TRL score for this Longevity.Technology domain is currently set at: ‘Late proof of concept demonstrated in real life conditions.’
The TRL score for the technology addressed in this article is: ‘Technology has completed initial trials and demonstrates preliminary safety data.’
This revelation comes from the research of scientists from The Technical University of Munich (TUM) and Helmholtz Zentrum München. Writing in Nature Communications , they show how they created melanin-loaded nanoparticles (derived from cell membranes) that helped not just to image tumours in mice, but also to slow their growth.
The team were able to create both of these useful effects at once because of melanin’s ability, rare amongst biological polymers, to produce heat when excited. Their method goes like this: first they inject the melanin (transported in biological nano-containers called outer membrane vesicles or OMVs) directly into the tumour before heating it with pulses of an infrared laser. The heat from the infrared causes the tissue to expand slightly, before cooling and contracting when the beam is switched off, releasing pressure waves of ultrasound as it shrinks back to its original size. As tumours expand and contract at different rates to healthy tissue, the specific ultrasound signature they give off can be used to locate them in the body.
So far so standard photoacoustic imaging: a technique that is new but far from unique to medical research. What makes their contribution exciting is what happened to the melanin during this procedure: when exposed directly to the beam, it absorbed the infrared and dissipated it as heat, causing the temperature of the tumour tissue to rise from 37°C up to a maximum of 56°C. This dramatic temperature shift not only killed some of the tumorous cells, but also provoked a bodily immune response to the inflammation, weakening the cancerous tissue further.
This contrasts strongly with control tumours without the melanin injection, which only reached a maximum temperature of 39°C, and grew at a significantly faster rate than those with. And what’s more, the images produced by the treatment delivered were very sharp, and in high-contrast with the surrounding healthy tissue. What’s more, the OMV transporters are biocompatible, biodegradable and can be easily and inexpensively produced in bacteria, even in large volumes.
Professor Vasilis Ntziachristos, the leader of this research, places his team’s new method among the first of many breakthrough therapies in a new field. “Our melanin nanoparticles fit into the new medical field of theranostics – where therapy and diagnostics are combined. This makes them a highly interesting option for use in clinical practice,” he says . They are now developing their melanin-OMV combo for use in further clinical trials.
Researchers investigating possible link between vitamin D deficiency and COVID-19
For months, many of us have stayed home on the advice of health experts, quarantining indoors to help slow the spread of the coronavirus. Now some researchers are investigating the possibility that spending a little more time in the sun could actually help prevent COVID-19.
That’s because sunshine is a key ingredient for our bodies to make vitamin D.
After seeing a correlation between severe COVID-19 patients and vitamin D deficiency, the researchers hypothesized that the vitamin, absorbed through sunlight by the skin, could play a role in helping the body’s immune system fight the novel coronavirus.
An earlier observational study of 499 patients by the University of Chicago found that patients with untreated vitamin D deficiencies were almost twice as likely to test positive for COVID-19 compared to those with enough vitamin D.
More research is needed still, and it’s also unclear whether low vitamin D levels are simply an effect of severe disease, rather than a cause.
“The correlation is not causation. So we need to actually do a clinical trial,” said Arizona State University researcher James Adams.
The question at the heart of these trials is, does vitamin D deficiency cause severe COVID-19 symptoms, or is vitamin D deficiency just another symptom of COVID-19?
Adams’s theory is that rather than treating COVID-19 symptoms after the fact, vitamin D could play a role in preventing serious infections —resulting in milder infections or perhaps no infection at all. To test this, he is running a preliminary study of vitamin D in COVID-19 patients in conjunction with the Southwest College of Naturopathic Medicine.
In the phase one clinical trial, researchers will administer supplemental vitamin D for two weeks to participants who have tested positive for COVID-19 but have not yet developed severe symptoms. All participants in the study must also show a vitamin D deficiency in initial screenings.
After two weeks, researchers will measure participants’ vitamin D levels and measure any changes in the severity of COVID-19 symptoms. The study will continue to track symptoms at four weeks and six weeks after initial treatment.
In addition to measuring vitamin D, the study will also measure other cells that are important for overall health, such as vitamin C.
Low levels can go unnoticed
Vitamin D deficiencies are more common than one might think, according to naturopathic doctor Sarah Trahan, who works at the college and is collaborating with Adams on the study.
“Generally anyone we test, if they’re not supplementing, they’re deficient,” she said.
Many of these deficiencies tend to go unnoticed, according to Lise Alschuler, a naturopathic doctor at the University of Arizona’s Andrew Weil Center for Integrative Medicine.
While deficiencies become more obvious when patients have less than 20 nanograms of vitamin D per milliliter of blood, she said many of her patients have between 20 and 30 nanograms per mL, which is less than the 40-50 nanograms per mL that she considers ideal.
“So they’re not going to get rickets, but they’re going to have insufficient vitamin D for optimal health,” Alschuler said. “And that’s where a lot of people fall, even in Arizona, surprisingly, because we have so much sun.”
Screening for vitamin D deficiency is not done as frequently by physicians as Alschuler would hope, but it’s something she is working to raise more awareness about.
Deficiencies in vitamin D tend to be more prevalent among those who are older, obese or who have darker colored skin. So far, people in these categories have also been linked with disproportionate rates of severe COVID-19 and COVID-19 related deaths.
“These are all three major risk factors with COVID-19, and they all relate to vitamin D,” Adams said.
Older people tend to make less vitamin D, whereas the melanin in darker skin blocks more UV rays from the sun, which are needed to make vitamin D, according to Trahan.
According to a CDC study published in 2006, 21% of non-Hispanic white people are at risk of having inadequate levels of vitamin D, versus 73% of Black people and 42% of Hispanic people.
The higher rate of deficiency among overweight people is thought to be because vitamin D dissolves in fat, so more vitamin D is being absorbed by fat cells and not going into the bloodstream.
Vitamin D deficiencies may not be to blame for the disproportionate impact of COVID-19 on these populations. There could be other factors at play such as socioeconomic status, genetics or increased presence of other diseases.
Vitamin supplements are one option
Still, Adams thinks it’s worth investigating, especially since administering normal doses of vitamin D is extremely safe and relatively cheap.
“Let’s try to make the immune system as healthy as possible,” he said. “If someone has a weakened immune system because of vitamin D deficiency, then other treatments like vaccines may not work as well.”
Rather than being the main treatment to prevent or cure COVID-19, Adams sees vitamin D as a potential supplemental treatment in addition to drugs or vaccines, especially since it is thought to play a role in immune system health.
While vitamin D has no serious risks when taken at the right levels, Trahan cautioned against taking too much supplemental vitamin D. Taking too much could increase the risk for kidney damage, kidney stones or other complications from too much calcium buildup in the blood.
To get a normal level naturally, Trahan said people with lighter skin can go outside in the middle of the day for about 15 minutes without sunscreen on, since sunscreen blocks vitamin D absorption. For those with darker skin, she said it may take about twice as long to get normal levels from the sun.
Because unprotected sun exposure could increase the risk for skin cancer, taking supplements as an alternative is typically fine if a person takes less than 4,000 international units of vitamin D daily, according to Martin Hewison, a professor at the University of Birmingham in the U.K. who has studied the vitamin for years.
“There has never been a single example of any toxic side effects in anybody taking less than 4,000 international units per day,” he said.
Understanding the role of vitamin D
While vitamin D is often thought of as important for calcium absorption and bone health, its exact function in the immune system is not fully understood and needs to be further researched.
Many cells in the body have vitamin D receptors, which are areas on the cell that vitamin D can bind with.
“Those receptors, they’re like a basket, and when it catches a vitamin D ball, basically the receptor activates and it creates actions within the cell,” Alschuler said.
One effect is that vitamin D seems to help initiate an inflammatory response in cells, but also regulate inflammation to keep it in check, according to Alschuler. In particular, vitamin D may be able to suppress inflammatory signals called cytokines. Targeting inflammatory cytokines is important because an over-release of these signals causes an immune system overreaction, known as cytokine storms, which have been blamed for many COVID-19 deaths and damaging side effects.
But vitamin D also interacts with other key cells in the immune system, according to Hewison.
“It’s a good jack of all trades,” Hewison said. “It helps in all the places where you’d want it to…it’s not just a one-trick pony.”
People with lower levels of vitamin D seem less able to make a protein called cathelicidin, which helps destroy the outside membrane or envelope of bacterias and viruses.
“It just punches holes in the membranes of the bacteria and kills them,” Hewison explained.
Vitamin D also helps promote what is essentially an immune system housekeeping process called autophagy, where the body removes damaged or failing cells and recycles them to create newer, healthier cells, Hewison said. This process can help eliminate invading organisms from the body, he said.
One study Hewison worked on suggested that vitamin D could control iron levels inside a cell, which help give the cell energy but can be taken advantage of by viruses that enter the cell. In the case of a viral infection, he said the vitamin appears to help iron leave the cell so that it does not get hijacked for use by the virus.
‘We know its potential’
While these findings are intriguing, Hewison said there has not been much success with using vitamin D for infection treatment.
“This is more important for long term preventative measures,” he said. “Once you’ve got the disease…does it work really as well as we think? I think the simple answer is no.”
He says this is because once an infection gets into a body’s system, the bacteria or virus will try to evade immune system responses promoted by vitamin D.
He added that there is no clear-cut evidence that vitamin D supplements would prevent COVID-19, but that further studies were needed.
“We know its potential. The real issue is going to be getting some good studies that answer a key question,” he said. “Is vitamin D deficiency a causative factor in these sorts of diseases or are the diseases actually contributing to vitamin D deficiency?”
As someone who has been taking 2,000 international units of vitamin D daily for years and hasn’t had a cold or cough since, Hewison is certainly a believer in the potential preventative power of vitamin D, but some in the scientific community are more skeptical, making obtaining funding for further studies a challenge.
However, he thinks that in the case of this pandemic, it’s worth investigating any potential benefits, especially one this cheap and plentiful.
“What have you got to lose?” he said.
Further study is needed
Rather than wait for grant funding, which can take months, researchers at ASU are fundraising money for their study, which has enrolled 40 participants. Adams said they hope to get funding to enroll an additional 60 participants so that they can get better statistical results.
Since the study is not giving placebos to any participants, it’s possible that any recorded benefits could just be the result of a bias called the placebo effect. In other words, improvements could result from a belief in the treatment, rather than as a result of the treatment itself. Without a placebo, there is no control baseline to measure the treatment against.
To better analyze the data, Adams said researchers will compare it to historical data of COVID-19 symptom development.
Another potential issue with this study is that participants must be able to speak English. This could lead to an overrepresentation of white participants.
However, Adams said the study is open to all races and ethnicities and that approximately 40% of the participants enrolled so far are minorities, and many of them are Hispanic. The reason for the English requirement, he said, is that including more languages in the study would require hiring additional staff and translators.
“We would have to have all aspects of the process done by someone who is fluent in that language,” he said. “It would be so much more expensive.”
The research team hopes to complete the study in another month or two, and if it goes well, Adams hopes to do a placebo-controlled study of 10,000 participants. He plans to submit a proposal to the National Institutes of Health to secure more funding for the next stage of study.
Instead of enrolling people who have tested positive for COVID-19, he said the phase 2 study would enroll those who haven’t yet tested positive for COVID-19. The study would compare participants who received a placebo with those who received vitamin D to see if the latter group has lower rates of COVID-19 after a six month period.
While he hopes these studies will show a positive impact in lowering the rate of COVID-19, Adams is not marketing vitamin D as a cure to stop the pandemic and believes that if it proves effective, it will be just one piece of the puzzle.
“We hope that we can reduce their severity of symptoms,” Adams said. “We’re not recommending this as a treatment now, we’re saying this is a treatment we think is worth investigating.”
Wine & chocolate stop Covid? Study finds certain foods can kill the coronavirus1 Dec, 2020 12:15
A range of foods and beverages, including dark chocolate, green tea, and muscadine grapes could be a secret weapon in the fight against Covid-19. New research has revealed they contain compounds that can kill the virus.
Scientists studied what happened when the virus that causes Covid-19 – SARS-CoV-2 – came up against chemical compounds found in plants known for their anti-inflammatory and antioxidant properties.
Fascinatingly, they discovered that compounds from green tea, two varieties of muscadine grapes, cacao powder, and dark chocolate were able to bind to a particular enzyme, or protease, in the virus and stop it reproducing.
The researchers, from North Carolina State University, carried out lab studies and computer simulations to examine how the so-called main protease (Mpro) in the coronavirus reacted to the plant compounds.
“Mpro in SARS-CoV-2 is required for the virus to replicate and assemble itself. If we can inhibit or deactivate this protease, the virus will die,” the study’s corresponding author, DeYu Xie, explained.
The investigations discovered that the chemical compounds found in the foods and drinks fill up a ‘pocket’ in the Mpro, rendering it useless, effectively stopping the virus from replicating.
“Green tea has five tested chemical compounds that bind to different sites in the pocket on Mpro, essentially overwhelming it to inhibit its function,” Xie said.
“Muscadine grapes contain these inhibitory chemicals in their skins and seeds. Plants use these compounds to protect themselves, so it is not surprising that plant leaves and skins contain these beneficial compounds.”AFRICAN AMERICAN LONGEVITY ADVANTAGE: MYTH OR REALITY? A RACIAL COMPARISON OF SUPERCENTENARIAN DATA
by ROBERT YOUNG
Under the Direction of Frank J. Whittington ABSTRACT
Demographic researchers have identified a crossover pattern between the mortality rates of the Caucasian-American and African-American oldest-old (80+) populations for over a century. Debate has centered on whether the crossover effect is due to age misreporting or the heterogeneity hypothesis or if it continues beyond age 99. This thesis addresses these issues by using new data from the SSA‘s study of supercentenarians. The study identified 355 persons aged 110 or older whose ages could be verified, creating the first reliable American dataset for this population group. Analysis of the data has indicated that mortality rates at ages 110-115 were significantly lower for African-American supercentenarians than for their Caucasian- American counterparts, and that the African-American proportion of the population increased steadily with age. The results of this analysis show that the crossover phenomenon is multicausal and cannot be fully accounted for by age misreporting, suggesting a need to consider genetic and environmental impacts on racial variations in maximum human longevity.
Vaccine side effects, contaminants, genetic factors, expense, availability, etc. may be obsolete after the melanin Manhattan west haven a project fast tracks it’s trial for safety and efficacy. It’s non adversarial, regarding any medical, political, military, media educational agency concerning any covid plan.
The world may have more than enough synthetic approaches to prevention and treatment of covid, it’s next wave, and who knows how many versions. The non synthetic collaboration invitation doesn’t ask anyone to bet their future on any covid over plan, much less, that of the planet.Avisamyco in CA also mass produces high grade melanin.Covid relevant biopharma medicinal products are grown with pharmaceutical grade potency and purity in organisms like microbes, fungi, and enzymes, aren’t necessarily drugs per se, and may be regulated as such.in history‘s greatest sci-fi novel series, Dune, the melange spice that gifted all humans superhuman wellness was the most valued commodity in all galaxies, as well as the chloridian super immunity, non synthetic bio chemical that created the elite Nobel class of Jedi. Science faction is the New Testament true news to trump all other world developments.Whatever the chosen name, soma, living water, etc. all ancient and modern super human civilizations hold that the melanin molecule is the dark matter bridge, to all visible and invisible worlds.To make the resident super melanin real for billions of people; we need global planners who act locally, with scientists, political, military, educational and celebrity influencers for a generation of world collaboration and unification.
1/20/2020 is the inauguration of melanin global wellness melanin activation now- text (936) 718-2647 for global events venue and ground swell viral speed benefits. We are developing the melanin WHO dashboard.Study Suggests Coronavirus Disease is Mutating2020-11-06 15:56:00Sara Karlovitch, Assistant Editor
Severe acute respiratory syndrome coronavirus 2, the virus that causes coronavirus disease 2019 (COVID-19), is accumulating genetic mutations that may have made it more contagious, according to a study published in mBIO.
The COVID-19 pandemic has caused more than 1.19 million deaths and there have been over 46.4 million confirmed cases worldwide, according to the World Health Organization. Mutations to the virus are a combination of natural drift, which are genetic mutations that don’t help or hurt the virus, and pressure from the human immune system, according to the study.
The study included more than 5000 patients with COVID-19 in Houston, Texas. Investigators found that a mutation known as D614G, which is located in the spike protein that pries open cells for viral entry, may have made the virus more contagious.
According to the study, during the initial wave of the pandemic, 71% of novel coronaviruses identified in patients in Houston had this mutation. During the second wave of the outbreak, that number had jumped to 99.9% prevalence.
“The virus continues to mutate as it rips through the world,” said study co-author Ilya Finkelstein, PhD, associate professor of molecular biosciences at The University of Texas at Austin, in the press release. “Real-time surveillance efforts like our study will ensure that global vaccines and therapeutics are always one step ahead.”
A total of 285 mutations have been identified across thousands of infections, though most do not affect disease severity, according to the study. Ongoing studies are surveilling a third wave of patients to determine how the virus is adapting to neutralizing antibodies produced by the immune system.
What do we know about the novel coronavirus’s 29 proteins?
These biomolecules could hold clues to why the virus is so infectious and to how to stop itApril 1, 2020Credit: NIAID-RML
Scientists across the globe are gunning to understand the novel coronavirus, called SARS-CoV-2, and what makes it so contagious and deadly.
Several members of the coronavirus family infect humans: four cause the common cold and two—SARS-CoV and MERS-CoV—have triggered dangerous epidemics. The novel coronavirus’s closest kin is SARS-CoV, which jumped species from bats to civets to humans to cause the severe acute respiratory syndrome (SARS) epidemic of 2002–2003. That SARS outbreak infected over 8,000 people. The current coronavirus pandemic has infected more than 880,000 people and killed more than 44,000 as of April 1, according to data from Johns Hopkins University.
“From a molecular perspective, figuring out why the virus is so much more transmissible than past viruses is where we should be looking right now,” says Robert Kirchdoerfer, a structural biologist at the University of Wisconsin–Madison who studies how coronaviruses fuse with host cells.
While people infected with the early 2000s SARS virus showed severe symptoms almost right away, people infected with SARS-CoV-2 can spread the virus before they show symptoms or when they are just mildly sick. A study published in Nature reports that patients shed virus most efficiently in the first week of illness, when their symptoms are mild (2020, DOI: 10.1038/s41586-020-2196-x). The US Centers for Disease Control and Prevention now estimates that 25% of people infected with the virus show no symptoms.
“That gives it an advantage to spread,” says Melanie Ott, a virologist at the University of California, San Francisco, and a member of the Quantitative Biosciences Institute’s COVID consortium. There must be something in the virus’s biological makeup that facilitates this silent transmission and that makes its effect on its human hosts so variable. But researchers don’t yet know what it is.
The RNA genome of SARS-CoV-2 has 29,811 nucleotides, encoding for 29 proteins, though one may not get expressed. Studying these different components of the virus, as well as how they interact with our cells, is already yielding some clues, but much remains to be learned, Ott says.Coronavirus proteinsCredit: Adapted from Nature (DOI: 10.1038/s41433-020-0790-7) and bioRxiv (DOI: 10.1101/2020.03.12.988865)/C&EN/Shutterstock
Coronaviruses are named for the crown of protein spikes covering their outer membrane surface. Early work on the novel coronavirus has focused on these spike proteins—also called S proteins—because they are the keys that the virus uses to enter host cells. In both SARS-CoV and SARS-CoV-2, the S protein binds to a receptor called angiotensin converting enzyme 2 (ACE2) to hack its way into host cells.
At the amino acid level, the spike proteins on SARS-CoV-2 are about 80% identical to those on SARS-CoV. “The residues which interact with ACE2 are conserved, compared to [the first] SARS, but the residues in between are different, and there are also some insertions,” says Rolf Hilgenfeld, a structural biologist who studies coronaviruses at the University of Lübeck.
Studies so far suggest that the new virus’s spike proteins bind to ACE2 significantly more strongly than those of SARS-CoV. “That is probably one of the reasons it spreads more easily and is more infectious,” Hilgenfeld says.From a molecular perspective, figuring out why the virus is so much more transmissible than past viruses is where we should be looking right now.Robert Kirchdoerfer, structural biologist, University of Wisconsin–Madison
A non-peer-reviewed preprint posted on medRxiv on March 27 reports that ACE2 is expressed especially strongly in the lungs of people with lung diseases (2020, DOI: 10.1101/2020.03.21.20040261). That observation may partly explain how these underlying conditions make people more susceptible to infection by the novel coronavirus.
Stronger binding with ACE2 isn’t the only clue to the virus’s power. S proteins are made up of two segments, S1 and S2, that must be cleaved at two sites to expose a peptide that initiates fusion with ACE2 on a host cell. A mutation in the SARS-CoV-2 S protein allows an enzyme called furin, which is made by many types of human cells, to do the first cut. This ability to get human enzymes to do its work primes the virus for fusing with ACE2—providing another possible explanation for this virus’s infectiousness, says Carolyn Machamer, a cell biologist at Johns Hopkins University who studies the basic biology of coronaviruses. “It can get that first clip before it even comes into contact with a receptor.”
Researchers are working to identify the best way to interfere with the S protein’s interaction with ACE2. ACE2 is present in many organs throughout the body and interfering with it may have side effects, so researchers want to avoid hitting the receptor and are instead developing antibodies or peptides that bind and disable specific segments of the S protein.
The other 28
Of the 29 SARS-CoV-2 proteins, four make up the virus’s actual structure, including the S protein. One group of the other 25 coronavirus proteins regulates how the virus assembles copies of itself and how it sneaks past the host immune system. These so-called nonstructural proteins are expressed as two huge polyproteins that are then cleaved into 16 smaller proteins. An enzyme called the main protease, which performs 11 of those cleavages, is also a highly promising drug target. Hilgenfeld and his colleagues recently reported the structure of the main protease and identified an inhibitor that can block it.
Many of the virus’s nonstructural proteins are still poorly understood. “I think we are just going to have to go brute force and probably piece through the genome and study a lot of these proteins individually,” says Anthony Fehr, a biologist at the University of Kansas. These studies will help scientists both understand the underlying biochemistry that gives SARS-CoV-2 its nasty kick and identify other ways to fight the virus.
Fehr’s lab works on a nonstructural protein called NSP3, a component of which blocks the host’s efforts to fight off the virus. In particular, this protein shuts down host enzymes called PARPs, which prevent viruses from replicating, and interferes with cellular calls for the release of virus-fighting immune proteins called interferons. This protein could be a drug target, he says. “The virus would be dead in the water if it didn’t have a way to counter the interferon response.”
The third group of proteins in the novel coronavirus are accessory proteins. Coronaviruses don’t need these proteins to replicate in a test tube, but they do need the molecules to counteract the host’s innate immune system. However, accessory proteins are the least-well understood. “I’m interested in how the differences in these small proteins affect pathogenesis,” says Susan Weiss, a microbiologist who studies coronaviruses at the University of Pennsylvania. Her lab has studied accessory proteins in other coronaviruses; they plan to determine how mutations in these genes affect SARS-CoV-2’s ability to counteract host immune response and replicate.
Structural biology continues to be a key method for studying the new virus’s proteins. The differences between SARS-CoV-2 and the earlier SARS-CoV, “must have their basis in protein structure, but it’s quite difficult to correlate the 3-D structures—if you even have the 3-D structures—with differences in function,” Hilgenfeld says.
However, researchers have other tools to investigate SARS-CoV-2, says Susan Daniel, a biomolecular engineer at Cornell University who studies how coronaviruses enter cells. For example, electron spin resonance can provide information about how peptides involved in viral fusion get buried in the membranes of host cells; circular dichroism spectroscopy can provide insight about how alpha-helical structures within viral proteins change under specific conditions; isothermal titration calorimetry can hint at how viral peptides interact with various ions, which can give clues about their conformations; and nuclear magnetic resonance can provide important structural information not found in crystal structures.
Meanwhile, other labs are rushing to explore how differences in immune system responses might affect how severely ill an infected person may get. For example, Ott’s lab is beginning to infect organoids, which are models of lungs and other organs made of complex cell cultures, with the virus to watch how infections proceed. “The scientific community has come together in an unprecedented way,” Ott says. “Everybody basically has shifted their attention to the virus and is looking for ways to contribute meaningfully.”
This story was updated on April 13, 2020, to correct a statistic about the 2002–03 SARS epidemic. That virus didn’t kill more than 8,000 people worldwide; it infected that many. It killed over 700.
Could the Induction of Trained Immunity by β-Glucan Serve as a Defense Against COVID-19?
As the SARS-CoV-2 virus wreaks havoc on the populations, health care infrastructures and economies of nations around the world, finding ways to protect health care workers and bolster immune responses in the general population while we await an effective vaccine will be the difference between life and death for many people. Recent studies show that innate immune populations may possess a form of memory, termed Trained Immunity (TRIM), where innate immune cells undergo metabolic, mitochondrial, and epigenetic reprogramming following exposure to an initial stimulus that results in a memory phenotype of enhanced immune responses when exposed to a secondary, heterologous, stimulus. Throughout the literature, it has been shown that the induction of TRIM using such inducers as the BCG vaccine and β-glucan can provide protection through altered immune responses against a range of viral infections. Here we hypothesize a potential role for β-glucan in decreasing worldwide morbidity and mortality due to COVID-19, and posit several ideas as to how TRIM may actually shape the observed epidemiological phenomena related to COVID-19. We also evaluate the potential effects of β-glucan in relation to the immune dysregulation and cytokine storm observed in COVID-19. Ultimately, we hypothesize that the use of oral β-glucan in a prophylactic setting could be an effective way to boost immune responses and abrogate symptoms in COVID-19, though clinical trials are necessary to confirm the efficacy of this treatment and to further examine differential effects of β-glucan’s from various sources.Keywords: COVID-19, SARS-CoV-2, trained immunity, β-glucan, innate immunity
Throughout evolution, the majority of cellular life (~97%) has existed without a canonical adaptive immune system capable of generating memory responses (1). In fact, until the appearance of jawed fish 500 million years ago, features of adaptive immunity did not exist (2). Despite this, plants, protists, invertebrates and lower animals certainly had a prescient need to protect themselves from recurrent infections. As such, it is known that in these organisms, the innate immune system evolved ways of programming memory-like features in order to non-specifically prevent infection of common pathogens. This protection in plants is known as Systemic Acquired Resistance (SAR), which is responsible for the observation that following inoculation with attenuated organisms, plants benefit from subsequent protection against a myriad of different infectious agents such as fungal, viral and bacterial pathogens (3). While of course the engagement and activation of adaptive immune responses in humans to protect against sinister infectious agents such as the SARS-CoV-2 virus is important, in seeking ways to quickly protect human life, we stand to learn a great deal from our evolutionary immunological origins in memory-like innate immune responses.
The formal principle of TRIM in humans has been recognized for almost a century, where the first recognized study of TRIM came from Sweden in 1934 and showed that infants given the Bacille Calmette-Guérin (BCG) vaccine against Mycobacterium tuberculosis (TB) had an increased survival rate compared to unvaccinated infants, which could not only be attributed to being immune to TB (4). In the late 90s several studies came out that explored the protective effects of β-Glucan, BCG and other vaccines against non-specific secondary pathogens that further supported the concept of TRIM (5–10). More recently, a 2017 study in Denmark showed that early administration of BCG was associated with a reduced mortality rate of 38% within the neonatal period (11). Though the BCG vaccine has gained the most general attention as a known inducer of TRIM, there are several other compounds that also act as potent initiators of TRIM. One such inducer is β-glucan, which is a naturally occurring polysaccharide found in the cell wall of yeast, bacteria and fungi. Like the BCG vaccine, β-glucan is known to induce a phenotype of TRIM, though the mechanism of action is known to be different from BCG.
Following exposure to β-glucan, innate immune cells undergo epigenetic reprogramming that results in cellular activation, augmented cytokine production, and changes in metabolic function that include increased aerobic glycolysis in addition to dose-dependent changes in oxidative phosphorylation (12, 13). Alterations in histone methylation and acetylation are important epigenetic alterations that occur which are responsible for the positive regulation of gene expression. When these “trained” cells then come into contact with heterologous secondary stimuli they are programmed to produce a more robust immune response (14, 15). Accordingly, studies have shown that following treatment with β-glucan, mice were more resistant to bacterial infections such as Staphylococcus aureus (16) and parasitic infections such as Leishmania braziliensis (17). Importantly, β-glucans of various sources have also been widely shown to have significant anti-viral effects, and have been shown to decrease the severity of both upper and lower respiratory tract viral infections (18–24). We posit that these anti-viral effects could likely be due to the induction of TRIM, though more definitive research is needed to determine whether the general immune stimulatory effects of β-glucans or the induction of TRIM is directly responsible.
As of June 24, 2020, 9.4 million people have been diagnosed with a confirmed case of COVID-19, hundreds of thousands of people have been hospitalized, and over 481,000 people have died worldwide. COVID-19 has presented the modern world with a challenge that global health-care infrastructures have not seen in over a century since the 1918 Spanish influenza pandemic. Though there are several promising vaccine candidates on the horizon, it cannot be expected that a vaccine against SARS-CoV-2 will bring any proximate relief, which indicates that in the interim, it is necessary to focus on effective and easily deployed therapeutics to increase immunity against SARS-CoV-2. Accordingly, several studies have been quickly initiated to investigate whether the induction of TRIM, through the administration of the BCG vaccine, can help protect against COVID-19. On March 30, 2020, the BRACE trial was initiated in Australia, which aimed to give the BCG vaccine to up to 4,170 healthcare workers in order to determine if BCG vaccination can reduce the incidence and severity of COVID-19 during the 2020 pandemic. Due to the excitement and promise of this trial, on May 3, 2020, the Bill and Melinda Gates Foundation gave a 10-million-dollar grant to expand this trial to 10,000 healthcare workers. In support of this study, one epidemiological investigation by Miller et al., has shown a correlation between the universal BCG vaccination policy and reduced morbidity and mortality due to COVID-19 (25).
While the excitement regarding the use of BCG as a prophylactic treatment for COVID-19 is warranted, considering that β-glucan can be administered orally, has an extremely high safety profile, does not require a person to access healthcare to receive the treatment, and is known to act similarly to the BCG vaccine in terms of augmenting innate immune responses, there is a strong argument to be made in favor of the use of β-glucan to prophylactically treat against COVID-19 as well. Here-in, we will highlight the known anti-viral impacts of β-glucan, review the known mechanisms of β-glucan-induced TRIM that could lead to protection against COVID-19, and present our personal view about the immune response to SARS-CoV-2 in the scope of TRIM. Additionally, though there is strong evidence to support the use of β-glucan as an anti-viral agent, COVID-19 has presented with a unique clinical course that involves the development of cytokine storm and thromboembolic events which often lead to mortality. As such, it is also important to consider that the immunostimulatory effects of β-glucan could be detrimental to the subset of patients who do develop cytokine storm and hyperinflammation, and so further research and understanding of the anti-viral mechanisms of β-glucan are needed before conclusions are made, which will also be discussed.
Natural Compound β-Glucan
β-Glucans are a heterogenous group of polysaccharides found abundantly in the cell walls of yeast, bacteria and fungi. They are made of glucose molecules linked together by (1–3), (1–4) or (1–6) β-glycosidic bonds, with varying branching structures coming off of the linear backbone. Despite the rich diversity of glucan structures, only β-glucans that consist of a β(1, 3) linked D-glucose backbone with β(1, 6) branching side chains are classified as biological response modifiers, and are known to have immunogenic properties (26, 27). The majority of these immunogenic β-glucans are purified from fungus and yeast. Importantly, unlike other natural products, β-glucans preserve their bioactivity even after oral digestion (28). In the human diet, β-glucans are abundantly found where oat, barley, wheats, yeasts, and certain mushrooms are rich sources of β-glucan. One cooked cup of oatmeal can have up to 2 mg of β-glucan, however for reference, therapeutic oral doses of β-glucan can contain up to 500 mg (29). Orally administered β-glucan is thought to mediate immunogenicity through receptor-mediated interactions with M cells which translocate luminal immunogens into Peyer’s patches, which then interact with resident macrophages and dendritic cells (DCs) (30). Another mechanism is through direct interaction of β-glucan and DCs in Peyer’s patches whose projections may extend through the apical epithelial cells and into the intestinal lumen (31, 32). Once β-glucans reach gastrointestinal macrophages, they will travel through the bloodstream or lymph system to target the bone marrow, spleen and lymph nodes (33).
There have been several routes of administration studied regarding β-glucan that include oral, intra-muscular (IM), intra-venous (IV), intra-nasal (IN), and intra-peritoneal (IP) administration. A particular challenge to research on β-glucan is the relative diversity of route of administration, which can lead to very different effects. While in animal studies IM and IP administration are relatively simple, in a human population these routes could be considered too invasive. For this reason, the majority of human studies conducted using β-glucan have used oral β-glucan. As discussed above, oral administration β-glucan is shown to exert immunogenic properties, however it is likely that the systemic administration of β-glucan through either IV or IM routes would result in more pronounced effects. Weighing the immuno-stimulatory function vs. the ease and safety of administration is certainly important, however in this context further studies are needed to determine the best approach (34).
Known Anti-Viral Properties of β-Glucan
Antiviral Properties of β-Glucan in Animal Studies
Along with the long list of anti-pathogenic bacterial properties, β-glucan has also shown promising anti-viral properties (19–21, 35). With regards to relevance to COVID-19, β-glucan has shown marked efficacy in abating viruses that impact the upper and lower respiratory tracts and those that culminate in a viral pneumonia. For example, one study showed that the administration oral β-glucan to pigs 3 days prior to infection with swine influenza virus (SIV) decreased the severity of microscopic lung lesions induced by SIV and decreased the detectable SIV nucleic acid present within the lungs days 5, 7, and 10 post-inoculation. Interferon gamma (IFN-γ) and nitric oxide (NO) levels were significantly increased in the bronchoalveolar lavage fluid from the β-glucan treated pigs (20). Enhanced anti-influenza properties have also been observed in mice that have been administered β-glucan, where Vetvicka et al., showed that a 2-week regimen of oral of β-glucan resulted in decreased mortality due to influenza infection. The suppression of phagocytosis is a well-known feature of influenza infections, which significantly contributes to disease pathogenesis, and importantly, this study showed that β-glucan increased the phagocytic capacity of neutrophils (36). β-Glucan was also shown to increase the production of IL-1β, TNF-α, and IFN-γ in peripheral blood, and potentiated the antibody response to influenza infection as compared to controls. Viral titers were shown to be significantly reduced after day 1 post-infection, with viral levels shown to be specifically lowered in heart tissues (19). In agreement with these studies, reports show that in addition to enhanced cytokine functions, a potential mechanism of increased protection from upper and lower respiratory viral infections could be due to increased number, phagocytic capacity and lysosomal enzymatic activity of alveolar macrophages (AMs) (37). These changes to the function and number of AMs may play a very important role in effective viral clearance within the lungs. A study conducted by Medina-Gall et al. that used zebrafish intraperitoneally injected with β-glucan and then subsequently challenged with spring viremia of carp virus (SVCV), a deadly virus that causes significant mortality in carp populations, supported this. Here they showed that β-glucan treated fish exhibited a significant increase in survival at 14 days post-treatment (23, 38, 39).
Antiviral Properties of β-Glucan in Human Studies
Human studies confirm these findings in animals, where yeast (1, 3)-(1–6) β-glucan was shown to decrease the severity of physical symptoms of upper respiratory tract infections (URTI) (24). This study was also shown to decrease the systolic and diastolic blood pressure of participants receiving β-glucan. This may have specific implications for the use of β-glucan in the setting of COVID-19, as patients with the most severe symptoms requiring intensive care unit (ICU) treatment were shown to have significantly increased blood-pressure compared to those not needing ICU care (40). Another study using β-glucan from the Pleurotus ostreatus mushroom significantly reduced the incidence of lower respiratory tract infections and the frequency of the flu and flu-like disease in children (18). A study in older adults age 50-70 who received a β-glucan supplement for 90 days exemplified the protective effects of β-glucan in this high-risk group. Here the number of days that a patient experienced symptoms of a URTI was decreased. The blood from treated individuals also showed increased IFN-γ (35). Finally, in two double-blind, randomized, placebo-controlled studies, orally administered yeast-derived β-glucan was shown to significantly reduce the number of common cold episodes by 25% and led to a milder progression of severe common cold episodes (41, 42). Though of course the symptoms and outcomes of COVID-19 are known to be far more severe than a “common cold” there is evidence here that the administration of β-glucan could lead to a decrease in the severity and an improvement of outcomes, especially in the most vulnerable populations.
It must be noted that in these animal and human studies, β-glucan is shown to impact the immune response which likely benefits anti-viral responses, but it not examined whether these effects are a result of TRIM or a result of β-glucan directly stimulating immune cells which leads to better viral control. Moving forward, studies using β-glucan in viral settings should seek to make this important distinction. This is especially important because if a TRIM-mediated mechanism is at play, the use of β-glucan as a prophylactic would be the indicated clinical course, however if it is due to direct immuno-stimulatory effects, β-glucan could be used as a therapeutic.
Trained Innate Immunity (TRIM)
What Is TRIM?
While β-glucan itself causes direct stimulation of immune responses, β-glucan has also been known to act as a training agent which results in amplified immune responses when these trained cells are exposed to a secondary, heterologous, stimulus. Evolutionarily speaking, living multicellular organisms have long been fighting off fungal and bacterial pathogens, and so overtime, it makes sense that organisms lacking adaptive responses would devise a way to protect themselves against these repeated infections. That anti-fungal and bacterial TRIM was likely retained in higher vertebrates, resulting in the TRIM observed following administration of β-glucan or other elements that resemble fungal and bacterial antigens.
Animal studies using β-glucan support the paradigm of TRIM, where exposure to β-glucan followed by a secondary infection with Staphylococcus aureus results in protection against the pathogen (5). As Netea et al. points out in his excellent recent review article on TIRIM, models of TRIM using various training agents have shown protection against a host of relevant lethal pathogens such as Streptococcus pneumonia, Toxoplasma gondii, Escherichia coli, and rotavirus (43–46). Further, the various examples of the BCG vaccine and β-glucan affording protection against secondary infections, such as Candida albicans, in a macrophage specific manner, ultimately leads to the idea that the exposure of innate immune cells, specifically myeloid cells, to specific training stimuli results in a non-specific immune protection (7, 9, 17, 47, 48).
Human studies further support the idea that the induction of non-specific immunity following exposure to an unrelated primary pathogen is driven by innate immune cells. For example, the presence of a latent herpesvirus infection has been shown to protect from future infections against Listeria monocytogenes and Yersinia pestis in a macrophage dependent manner (46, 49). This data holistically points to the concept that by stimulating the immune response with one pathogen, it is possible to fortify it against infection by another. With this understanding, it is possible to take advantage of such immune responses by using a stimulant, such as β-glucan, that does not actually make an individual sick, but does have the benefit of generating primed immune cells that will respond to a host of lethal infections.
The Mechanisms of TRIM
Innate immune memory primarily involves macrophages and monocytes, though DCs, and Natural Killer cells (NKs) have also been shown to be involved in TI (14, 50, 51). It has been observed that the effects of TRIM can last for weeks to months, which led to the question of whether cells in the periphery were themselves trained, or whether the administration of a training agent such as β-glucan could impact the bone marrow (BM) which may lead to a more lasting TRIM phenotype. Further, considering that many of the cells known to be involved in TRIM are terminally differentiated, and thus unable to pass their phenotype on to their progeny, it was hypothesized that HSCs may be impacted. Accordingly, it was shown that the administration of intraperitoneal β-glucan treatment results in a biased expansion of Lin-Sca1+cKit+ (LSKs) and Multipotent Myeloid Progenitor 3 (MPP3) HSCs in the BM which are skewed toward the myeloid lineage through GM-CSF and IL-1 (52). In mice treated with β-glucan, the induction of a systemic inflammation using LPS resulted in increased responsiveness and cytokine production from these cells that was seen to last for up to 1 month (53). This education and alteration of HSCs in the BM is responsible for the generation of “central” memory which creates a repertoire of innate cells possessing innate immune memory features, which then migrate to peripheral tissues to generate peripheral memory (46, 54).
Epigenetic Regulation of TRIM Relating to Antiviral Responses
While the molecular mechanisms of TRIM are still being elucidated, data suggests that epigenetic, metabolic, and mitochondrial alterations each play an integral role. In addition to the described pathways of Dectin-1 activation leading to increased cytokine release, activation of the Dectin-1 receptor by β-glucan also causes important changes to the epigenetic status of immune gene promoters. An example of the epigenetic priming induced by β-glucan is that upon Dectin-1 activation, nuclear factor of activated T-cells (NFAT-1) is dephosphorylated, which results in its translocation through the nuclear membrane. NFAT-1 mediates β-glucan-driven epigenetic training by upregulating immune gene-priming long non-coding RNAs (IP-incRNAs) which culminates in increased levels of trimethylation of histone H3 at lysine 4 (H3K4me3) at promoter sites (14, 55). High levels of H3K4me3 are associated with robust levels of gene expression, and so this epigenetic effect results in more vigorous cytokine production upon re-stimulation of β-glucan-primed immune cells (56). Such epigenetic modifications driven by β-glucan result in inflammatory genes that are ideally positioned to be rapidly activated by secondary infections or stimuli, such as a virus.
The anti-viral effects of epigenetic reprogramming due to the induction of TRIM have already been supported in the context of training the immune response with the BCG vaccine, and so it is likely that β-glucan works in the same way. In a study by Arts et al. it was shown that the BCG vaccine protected from experimental viral infection through the induction of genome-wide epigenetic reprogramming and the upregulation of IL-1β (57). An important note about this experiment is that while the authors used the BCG vaccine to induce TRIM, β-glucan driven TRIM also shows epigenetic regulations that lead to an increased production of IL-1β, indicating that it is likely β-glucan administration would have shown similar effects (58, 59). Additionally, in this experiment, an attenuated strain of the yellow fever virus vaccine was used. Yellow fever is a member of the Flavivirus genus, which are a group of single stranded positive sense RNA viruses. Considering that coronaviruses are also positive sense RNA viruses, there is reason to believe that these findings support the idea that β-glucan could be an effective prophylactic for COVID-19.
Metabolic Regulation of TRIM Relating to Antiviral Responses
Metabolic changes are also a prominent feature of β-glucan induced TRIM, as vital energy metabolites regulate chromatin-modifying epigenetic enzymes, methylation, histone modification, and the position of the nucleosome by acting as substrates and co-factors. Consequently, the energy state of a cell and the metabolic programs that are initiated as a result of β-glucan stimulation tightly modulate the transcription of immunogenic genes (60). The metabolic switch from oxidative phosphorylation toward aerobic glycolysis is a key feature of TRIM, which has been shown to be mediated through the AKT/mTOR/HIF1α pathway (61). Other notable metabolic features of TRIM are a decrease in itaconate, a product of the decarboxylation of cis-aconitate, and increased fumarate and mevalonate accumulation through upregulation of the TCA cycle following stimulation with LPS. β-glucan signaling notably inhibits the LPS mediated upregulation of immune-responsive gene-1 (IRG-1), the enzyme that is responsible for itaconate generation, and stimulates the activity of succinate dehydrogenase, leading to increased fumarate production (62). This is critically important as itaconate is known to induce immune tolerance and anti-inflammatory properties in human monocytes (63, 64).
With regards to the impact of this on anti-viral protection, there is evidence that high levels of itaconate and its derivatives inhibit key Type-I interferon production during viral infection (65, 66). Relating this to SARS-CoV-2 infection, there is current research that suggests that SARS-CoV-2 demonstrates significant sensitivity to Type-I interferon signaling (67). There is also evidence that the ability of SARS-CoV-2 to downregulate type I IFN responses is tightly associated with disease severity, and SARS-CoV-2 has been shown suppress type I IFNs in response to viral infection (68, 69). Indeed, it has been shown that stimulation of DCs with fungal β-glucan stimulates IFN-β production, which in turn activates CD8+ T-cells and leads to their increased proliferation, and secretion of IFN-γ and Granzyme-B (70). Thus, for these reasons, using β-glucan to metabolically upregulate Type I IFN responses may lead to better overall viral control.
Discussion of β-Glucan and Trim in the Scope of COVID-19
The Viral Pathogenesis of SARS-CoV-2
The SARS-CoV-2 virus is known to bind to the angiotensin-converting enzyme-2 (ACE-2) expressed on various tissues including the heart, kidney, bladder, and especially the lung. In the lungs, SARS-CoV-2 binds to ACE 2 expressed on type II alveolar cells to gain entry to the cells (71, 72). Type II alveolar cells themselves will respond to viral infections through the recognition of pathogen associated molecular patterns (PAMPs), which for a ssRNA virus such as SARS-CoV-2, will likely be genomic viral ssRNA or dsRNA. While SARS-CoV-2 is a positive-sense single stranded virus, dsRNA is an obligate intermediate of positive-stranded RNA viruses, which will accumulate during replication cycles and work as a cytosolic PAMP (73). These PAMPs will be recognized through TLR3 or TLR7 endosomal RNA receptors and the cytosolic RNA sensors RIG-I and MDA5. This signaling causes activation and nuclear translocation of the transcription factors NF-κB and IRF3 which cause type I IFN anti-viral responses that are capable of suppressing early stage viral replication (69, 74). It is thought that the epithelial cells are the main source of anti-viral responses in the first 24–48 h of infection, however in order to mount a sustained immune response, it is necessary that these viral signals are carried over into innate immune cells which can then translate these into adaptive immune responses.
There are several mechanisms that are likely responsible for robust macrophage responses to SARS-CoV-2. First, Type II alveolar cells will secrete a host of inflammatory cytokines in response to viral infection such as IL-1β, IL-6, TNF-α, CXCL10, and CCL2 that will act to recruit other inflammatory cells to help abate the viral infection (74). Alveolar macrophages in the lung have also been shown to express ACE 2, which may indicate that they too are susceptible to infection with SARS-CoV-2 and upon being infected will not only present viral epitopes on MHC I and MHC II for CD8+ and CD4+ recognition, but will also activate anti-viral IFN type I signaling (75, 76). It is also probable that viral infection of type II pneumocytes results in their eventual apoptosis, which leads to subsequent phagocytosis of these cells by macrophages, resulting in another important mechanism of antigen uptake (77). Further relaying the vitally important role of innate immune cells in responses to SARS-CoV-2, one recent study used single cell RNA sequencing to identify novel receptors of SARS-CoV-2 to understand which immune cells come into contact with SARS-CoV-2 infected cells. This study indicated that macrophages most frequently communicate with the targets of SARS-CoV-2 through chemokines and phagocytic signaling (78). Such studies indicate that the ability of innate immune cells to survive infection with SARS-CoV-2 and maintain the capacity to educate adaptive responses is vital for successful protection.
Innate Immune Responses in COVID-19
Information about the nature of the SARS-CoV-2 virus and the related immune responses are still emerging, and many aspects of the viral pathogenesis are still unknown. Interestingly, there seems to be a dynamic role for immune responses, where a lack of competent Th1 adaptive immune responses and decreased CD4+ and CD8+ T-cells, resulting in lymphopenia, have been observed in some patients with the most severe disease, while at the same time, overly robust immune responses leading to cytokine storm are also being observed in the most severe cases (79–81). An interesting hypothesis to explain this could be that innate immune responses are critical in early stages, however their most important role is actually in their ability to swiftly and energetically activate Th1 type adaptive responses. When macrophages and DCs fail to galvanize and educate T-cell and B-cell activation, they continue to aberrantly secrete cytokines such as IL-6 and TNFα in efforts to control viral infection, however this results in cascading inflammation, eventually resulting in cytokine storm. This hypothesis would be consistent with observed clinical data of increased IL-6 and TNFα in patients with the most severe responses (82, 83). Our hypothesis is strongly supported by work from Zhao et al., who showed that in mice infected with SARS-CoV, severe disease was correlated with slow kinetics of viral clearance and delayed activation and transit of respiratory DCs to the draining lymph nodes, leading to deficient virus-specific T-cell responses. They also showed that an inhibitory subset of alveolar macrophages prevented the development of immune responses, which could be reversed by giving a treatment, poly I:C, that stimulates TLR3 activation and leads to cellular activation of AMs and DCs (84). While this research relates to SARS-CoV and not SARS-CoV-2, the viruses are known to share a relatively high degree of sequence homology, so there is reason to believe that similar mechanisms are at play between the two viruses due to their similar viral structure (85).
A recent publication by Zhang et al. utilized bronchoalveolar lavage fluid (BALF) from healthy controls and patients with both mild and severe COVID-19 to perform single-cell RNA sequencing. In their model, they identified four groups of human macrophage subsets in the lung and tracked how these changed in COVID-19. Interestingly, they found that AMs, defined by transcriptomics and expression of FABP4, were significantly decreased in COVID-19 infection as compared to healthy controls, and more significantly depleted in severe infections as compared to mild ones. This indicates that the function and presence of AMs are specifically impacted due to SARS-CoV-2, and that their presence likely plays a critical role in protecting against the progression of symptoms (86). Yao et al. have shown that AMs can be targeted for training, and other studies have shown that following β-glucan treatment, AMs in the lung show enhanced IL-1 production and phagocytic properties (37, 87). Though the ability of β-glucan to specifically induce TRIM in alveolar macrophages has not been shown, β-glucan has been shown to enhance cellular activity, cytokine production and phagocytosis in alveolar macrophages, indicating that TRIM may be involved (88).
Taking this into consideration, we pose that in addition to the general immunological benefits of β-glucan, the mechanism of SARS-CoV-2 and related immune responses highlights a very relevant and specific role for β-glucan, as it is known to impact innate immune cells in such a way that they not only are more effective at fighting initial infections, but that they are also better at activating adaptive immune responses. As a result of TRIM induced by β-glucan, we hypothesize that macrophages and DCs would have increased phagocytic capacity, which could not only lead to better viral control, but also to better processing and presentation of viral particles on MHCs (26). Trained macrophages could also elicit enhanced NK cell and neutrophil function. It is also known that β-glucan polarizes tolerogenic M2 macrophages to an M1 phenotype, which would result in increased activation and cytokine secretion, and increased propagation of Th1 T-cell responses (89). Adding to this enhanced activation, it has also been shown that autocrine type I IFN signaling in DCs stimulated with fungal β-glucan promotes antigen presentation to CD8+ T-cells, which in the context of the paper written by Zhao et al., could be an extremely important way to boost immune responses against SARS-CoV-2 (70). There is also evidence that β-glucan treated and trained DCs are more efficient at supporting B-cell responses and the production of neutralizing antibodies, which further helps to transition the early innate immune response toward a long-lasting, hyper specific adaptive response (90). We ultimately theorize that the activation of macrophages, DCs, NK cell and neutrophils due to TRIM induced by β-glucan may result in more effective initial responses to infection, enhanced T and B-cell responses against SARS-CoV-2, and an overall decrease in the duration and severity of symptoms in COVID-19.
As previously mentioned, while the induction of robust innate immune responses should generally benefit anti-viral processes, COVID-19 has posed a specific challenge to clinicians due to the development of a hyperinflammatory state marked by increased serum levels of inflammatory chemokines and cytokines, that is a major cause of disease severity and death (40, 79, 91, 92). Like other corona viruses, SARS-CoV-2 has been shown to result in respiratory failure due to local hyperinflammation and ARDS, which has been linked to Macrophage Activation Syndrome (MAS) (93–95). Patients with severe disease have been shown to have increased levels of IL-6, TNFα, MCP1, MIP1A, and IP10, which is also correlated with endothelial dysfunction and increased levels of D-dimer (96). Contrastingly, patients with moderate disease that experience mild symptoms and quickly recover from infection are known to show only modest increases in serum cytokines (97). Taking all of this information together, it is likely that as postulated above, rapid and efficacious initial immune responses are essential for control of viremia, however when these mechanisms fail, dysregulated immune responses prevail resulting in hyper-inflammation and rapid decompensation. For this reason, using an immunostimulant such as β-glucan in later stages of disease could be inappropriate, and could further exacerbate disease. In this setting, therapeutics that quell the immune response such as inhibitors of IL-6 and TNFα would be most appropriate and have shown some degree of clinical promise (98, 99).
Taking this together, we postulate that β-glucan would be best used in the prophylactic setting, where it could utilize processes of TRIM to prime innate immune cells and help to fortify the initial immune responses in the general population to prevent potential SRAS-CoV-2 infection. It could also contribute to a decrease in symptoms in mild and moderate patients. It cannot be ruled out however, that pre-treatment with β-glucan could further exacerbate the already severe hyperinflammation that develops in some patients. Therefore, clinical trials are needed to determine the safety profile and the efficacy of β-glucan in the prophylactic anti-viral setting.
Exploring the Age Demographics of COVID-19 in Relation to TRIM
Another interesting facet of COVID-19 is that age bears a strong negative association with disease severity, where children, especially those under 18, do contract COVID-19 but see relatively few immediate serious adverse effects (100, 101). Though children rarely develop ARDS due to COVID-19, recent reports suggest that COVID-19 is related to the development of a Kawasaki disease-like syndrome in the pediatric population. There are several theories that have been posed to explain why older adults have the highest mortality rate. There are two potential theories that we could like to explore here. The first, is that as stated above, the ability of innate immune cells to educate adaptive immune responses is the critical synapse in mounting viral protection against SARS-CoV-2, and when this fails, innate immune responses prevail, resulting in hyperinflammation, and cytokine storm. Around age 20, the thymus begins to erode, resulting in a decreased production of naïve T-cells, and an increased relative ratio of more differentiated T-cell subsets. CD8+ T-cells specifically are seen to decline drastically with age due to this thymic loss (102, 103). Incidentally, the rate of CD8+ T-cell decline is also more pronounced in men, which could possibly be why men seem to experience worse outcomes due to COVID-19 (104, 105). It can thus be hypothesized that the ability of the innate immune system to educate adaptive immune responses, and the following generation of CD8+ T-cells specific for SARS-CoV-2 and the production of neutralizing antibodies by B-cells is significantly reduced in adults, and potentially specifically male adults. While the use of β-glucan would not replenish naïve CD8+ T-cells, as discussed above it can aid in the ability of innate cells to uptake antigen and reinforce the potency of presentation to T-cells, which could help improve outcomes for the most at risk.
A second hypothesis as to why children are relatively unscathed during this pandemic relates to the induction of TRIM due to routine vaccination schedules in children, which usually last until age 18. While the BCG vaccination is best associated with the induction of TRIM, there is evidence that childhood immunizations can lead to heterologous non-specific immunological effects, which is likely due to the induction of TRIM (106). As children in the United States do not receive the BCG vaccination, other required vaccines would have to be responsible for these effects. Fittingly, cohort studies of the measles, diphtheria-tetanus, and diphtheria-tetanus-pertussis vaccination are correlated with increased non-specific immunogenicity (107). This, of course, relates to the earlier mentioned findings that in countries where individuals routinely receive the BCG vaccine, there are observed lower mortality rates due to COVID-19. This data is certainly preliminary, however supports the idea that the induction of systemic TRIM can help protect against COVID-19 (108). It will be important to closely monitor the results of the aforementioned clinical trials to see if this correlation holds and can be supported more than just circumstantially. Even more, while the BCG vaccine is extremely useful in preventing TB and even in treating bladder cancer, there can be serious adverse effects which include, but are not limited to, the formation of an injection site abscess, lymphadenitis, severe local reactions, and even death (109–111). Though death due to BCG vaccination is rare, it is shown to be associated with an immunocompromised status (111). As immunocompromised patients are a high-risk group in regard to COVID-19, this indicates that the BCG vaccine could not be used to protect these patients who desperately need to be protected. For these reasons, there lies a strong argument that use of natural compound β-glucan to induce TRIM and to reinforce innate immune responses in a prophylactic setting could be an effective therapeutic, that would carry a relatively lower cost and increased safety profile compared to other interventions such as the BCG vaccination, especially in the at-risk populations.
Understanding the exact mechanism of the immune response to SARS-CoV-2 will surely guide therapeutic and preventative interventions moving forward. It will also be critically important to understand why some patients develop a hyperinflammatory syndrome as this will shape prevention and treatment strategies. As we work to understand these mechanisms, incipient data is showing that innate immune responses in COVID-19 are essential in mounting a successful immune response and when this process fails, hyperinflammation occurs. β-Glucan has been shown to possess a range of anti-viral properties, and we submit that its role as an inducer of TRIM could possibly aid immune responses against SARS-CoV-2 and could help to prevent severe clinical courses. While we await the development of an effective vaccine, we will need to focus on preventative and therapeutic options that can be safety and quickly implemented to bolster immune responses.
We hypothesize that the use of oral β-glucan in the prophylactic setting may be an efficient, low-cost and safe way to help support this immune response, however clinical research and trials are needed to confirm the safety and efficacy of this treatment, and determine which sources and specific doses of β-glucan may be most effective in this context. Further while oral β-glucan would be the safest route of administration and does show important physiological effects, the method of β-glucan administration must also be further studied. In this regard, we pose that research on this topic is important, and the development of clinical trials to answer these questions are necessary in order to evaluate this potentially important treatment. Additionally, given the development of hyperinflammatory responses in severe COVID-19 patients, exclusion criterion should be considered and implemented. Finally, as we seek to understand the anti-viral mechanisms of β-glucan, it is important to make the distinction between general immunostimulatory effects and effects due to the induction of TRIM. Understanding whether TRIM processes are responsible for anti-viral responses will surely give further insight into other potential anti-viral strategies, as the novel SARS-CoV-2 is not the first, nor will it be the last time the human population must deal with a viral pandemic.
Data Availability Statement
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.
AG contributed to the conceptualization, planning, and writing of the manuscript. JY contributed to the conceptualization, planning, direction, and editing of the manuscript. All authors contributed to the article and approved the submitted version.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
A New Farming Technique Using Drastically Less Water Is Catching On
But not everyone is on board.
Filipino melon grower Denis Miguel was intrigued to hear of a young Indian farmer who in 2011 had broken the world record for growing rice by using an unconventional method of cultivation that needed only half as much water and one-tenth as many seeds but resulted in spectacular yield increases.
Miguel, from Isabela province, had never grown rice before, but he teamed up with a local rice farmer to try out the system. Last year, he reaped the equivalent of 10.8 tons of rice per hectare, or four times as much rice as the farmer usually grew on that land.
He was astonished. “A harvest of 10.8 tons per hectare on a rain-fed farm which used to produce only 2.5 tons was a great success. I was a newbie rice farmer. It was my first attempt at rice farming. It was an eye opener to all the people who were witness,” he said in an email.
Miguel is not alone. Reports from China, India, Southeast Asia and Africa suggest that average yield increases of 20 to 50 percent are regularly being achieved by farmers adopting the “system of rice intensification” (SRI), which aims to stimulate the root system of plants rather than trying to increase yields in the conventional way by using improved seeds and synthetic fertilizers.
Rice is the major staple crop of nearly half the world and is primarily grown by small farmers. Seedlings are traditionally planted in large clumps in flooded fields. One kilogram of rice typically requires about 660 gallons of water.
SRI, in contrast, involves the careful spacing of fewer but younger plants, keeping the topsoil around the plants well-aerated by weeding, using manure and avoiding flooding.
What was a grassroots movement spreading slowly by word of mouth since it was developed by French priest Henri de Laulanié in Madagascar in 1983 is now growing fast as regional governments in China and India join anti-poverty groups like Oxfam to back the method.
According to the SRI International Network and Resources Center at Cornell University, an estimated 10 million rice farmers in 60 countries have adopted SRI.
“It has the potential to reduce the amount of water, money and labor that farmers in developing countries need to spend. Time and again, farmers have seen improvements in yield, profitability and resilience,” says Norman Uphoff, professor of international agriculture at Cornell.
The idea of using less to gain more is seen as an important innovation for adapting farming to climate change and a way to increase yields at a time when human populations are growing fast but traditional plant breeding and genetically modified techniques have failed to increase yields more than a few percentage points, says Uphoff.
In Bihar, one of India’s poorest states, more than 335,000 hectares of rice are grown using SRI methods. Scientist Anil Kumar Verma from the rural nongovernmental organization