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Would it be possible to create a gel, holding viruses, that could alter the human genome when rubbed in?

Would it be possible to create a gel, holding viruses, that could alter the human genome when rubbed in?



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I was wondering if it could be possible to engineer a virus, that when it comes into contact with human hair follicles, it could alter someones natural hair color. Naturally blue hair anyone?

Doubtless there are loads of problems, but i was hoping they could be identified and potentially worked around.


Bio-Genetic Weapon Catastrophe: Virus spread by CIA with nano-Uav as Cyber-Dragonfly Eye

From the pandemic to a world catastrophe from China to Iran to Italy for a dramatic toll of more than 4thousands deaths, 5 times the 813 of SARS in 2003. Meanwhile, the suspicion that was spread by the CIA with a Cyber ​​Insect or other type of nano-drone, as claimed by intelligence sources.

Although CoronaVirus appears absolutely lethal only to older people or those with a weak immune system, its spread is growing exponentially due to the ease of contagion between human beings (saliva, breath, hands) and its resistance of indefinite duration on surfaces and objects. It is true that with a splash of ethanol or another domestic disinfectant the virus can be killed but what is worrying is its easy return.

In fact, he was able to kill both the Chinese hero doctor, Li Wenliang, the ophthalmologist who first raised the alarm in China last December (this is the reason for the code number Covid-19), but also the director of the Wuhan hospital, Li Zhiming.

The first was only 33 years old, the second 51, both of whom were obviously very exposed to this violent respiratory disease but both were also equipped with every necessary medical tool available so far to combat it. About a thousand doctors are infected in China and among them, there are already 6 deaths confirming that prolonged contact with infected people can be fatal even for individuals with good antibodies, adequate protections, and efficient treatments.

Dr. Liu Zhiming, director of Wuhan hospital, who died in recent days

All this highlights the power of the virus which, despite having a mortality rate now around 2% and therefore lower than the respiratory tract syndromes SARS (7%), has a contagious rapidity in some tragically high and, above all, unstoppable areas. It is a viral strain of the large CoronaVirus family: that of simple “common cold”, the upper respiratory tract disorder including cold, cough, maldigola, which, however, can degenerate into pneumonia so severe as to paralyze the lungs thus blocking the blood oxygenation.

The International Committee on Taxonomy of Viruses (ICTV) has already renamed it Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) given its dangerousness already attested in a detailed article published by some Chinese scientists in the February issue of “Science China Life Sciences ”which highlights biochemical anomalies such as to induce the Chinese Communist Party to consider it without a shadow of a doubt a biological weapon.

According to other American experts, it is a war instrument that got out of hand in Wuhan, Chinese second and independent analysts were built and spread by some enemy of Beijing. Among the number one suspects are the United States that all over the world, especially in the countries bordering Russia, China, and the Middle East, have at least 25 laboratories for the production of biogenetic weapons, that is, calibrated on the genome of some ethnic groups. And some 007 confirm that it is a widespread epidemic with nano-drones …

In addition to having started studies on the genetic manipulation of insects such as viral vehicles as defense or attack tools (officially against pests in agriculture), the US has already created DragonflEye, the light-guided cyber-dragonfly for reconnaissance flights, targeted pollination and delivery of “payload”. We will see later in detail the Pentagon partner laboratories that developed the project, started by the CIA in 1970 with the first Insecthoper.

This was the first prototype of “nano-drone” which has also evolved today in the Black Hornet 3, the 10 cm UAV helicopter already in use by the American army and other NATO countries, whose kit can fit in a military backpack. Or a tourist visiting a foreign country…

CHINA AND IRAN: MORE DEATHS IN US ENEMY COUNTRIES

The increase in the number of deaths and infected with COVID-19 (where “CO” stands for corona, “VI” for virus, “D” for disease and 󈬃” indicates the year in which it occurred), as called by the Director-General of the World Health Organization Tedros Adhanom Ghebreyesus on February 11, 2020, it is so dizzying that now no health institution or media in the world is able to provide a real-time update, therefore, the data we are reporting will already be exceeded half now after publication.

If we examine the current number of dead people, we see that the second most affected country is another historic enemy of the United States, Iran, at the center of recent military clashes over the killing of General Qasem Soleimani, commander of the Quds Forces, of the Iranian Pasdaran, in an operation by the Pentagon and the Central Intelligence Agency, the missile attack by the Islamic Revolutionary Guards of Tehran at two American bases in Iraq with 109 soldiers seriously injured for brain damage, and the plane crash of the CIA Flying Command in Afghanistan, still shrouded in mystery …

The main outbreak of infection and deaths in Iran would be in Qom, the place of the main mosque where the Shiites hoisted the red flag of revenge for the death of Soleimani, where it is the most important central, as highlighted by Gospa News in a previous report. nuclear uranium enrichment that resumed working at full capacity after the exit of Washington from the Joint Comprehensive Plan of Action agreement, acronym JCPOA, and even more after the killing of the senior officer of the Pasdaran (another name of the Revolutionary Guards IRGC). An underground bunker that cannot be attacked with missiles but not with a virus…

BIOGENETIC WEAPON’S HIGH RISK FOR HUMANITY

Given the enormity of the contagion, for an independent information body, it is now not only lawful but also a duty to report all the sources that describe SARS-CoV-2 as a biological weapon.

These curious coincidences about its geographic spread in China, a rival of the USA on the commercial level for the battle over tariffs, overcome by an agreement on a few days before the outbreak of the epidemic, and Iran, antagonist on the military and energy front, have been reported to justify the credible validity of the affirmation of a source with important relations with Russia and contacts with the intelligence of various foreign countries which obviously we take care to keep anonymous.

CoronaVirus is a biological weapon spread by a CIA agent in China with nano-drones. The US already has the vaccine» an international subject told us. Disturbing words to be taken with caution at least as much as the official press releases of NATO countries now accustomed for years to declaring lies for the alleged good of the West.

Few words for a huge pandemic that increasingly takes on the contours of a criminal military attack day by day and which confirms the alarm launched a few months ago by the United Kingdom highlighted in the previous Gospa News report on the suspicion – now almost certain – that epidemic was caused by a biogenetic weapon.

The Center for the Study of Existential Risk (CSER) of the University of Cambridge in an August report highlighted the dangers of technology in the war field because «a bio-weapon could be built to target a specific ethnic group based on its genomic profile »Defining this eventuality« extremely harmful and potentially unstoppable.

AMERICAN EXPERTS: COVID-19 IS A BIO-WEAPON

In the same previous reportage, we also reported on the first world expert who said he believed CoronaVirus to be a biological weapon.

Francis Boyle is a professor of international law at the University of Illinois College of Law. He drafted United States national legislation to implement the Biological Weapons Convention, known as the 1989 Combat Terrorism Weapons Act, which was unanimously approved by both chambers of the American Congress and promulgated by President George W. Bush. In an interview with the American website Geopolitics and Empire, he said this war instrument got out of hand at Wuhan’s biosafety level 4 (BSL-4) laboratory.

To believe the bio-weapons theory there is Jeff Brown, China expert, founder with other journalists and international authors of the Bioweapon Truth Commission, (www.bioweapontruth.com), an independent organization that has created the largest archive of documentation, films and audio publications on the United States and the West. In fact, Brown began to study the phenomenon since the US biological weapons attacks on China during the Korean War (1950-1953) and on Japan’s infamous “Unit 731”.

His story is well presented in an article in the British newspaper The Guardian:

Formed in the mid-1930s in Harbin, north-eastern China, Unit 731 conducted lethal experiments on an estimated 3,000 prisoners, who were mostly Chinese and Korean. According to historical accounts, male and female prisoners, named “logs” by their torturers, were subjected to vivisection without anesthesia after they had been deliberately infected with diseases such as typhus and cholera. Some had limbs amputated or organs removed.

«As Japan headed towards defeat in the summer of 1945, the unit’s leader, Lt Gen Shiro Ishii, forbade researchers from discussing their work and ordered the demolition of the unit’s Harbin headquarters. At the end of the war, US authorities secretly granted unit officials immunity from prosecution in return for access to their research. Several former Unit 731 officials went on to have successful careers in medicine, academia and business» reads in post.

This is why Brown, author of China Rising website, himself today, in an interview with Kevin Barrett, international terrorism expert, and host of the radio broadcast Truth Jihad Radio, accuses the US of being the manipulators of the epidemic:: «I’m in contact with people every day in China. And they are on a war footing. They are mobilizing hundreds of millions of people. They’ve mobilized the army.

They have quarantined like 50 million people and hundreds of millions of people are now for the next two weeks going to be working out of their homes. I mean, that’s why the death rate is so low and the number of incidences is relatively low, even though just like you say, the United States has the motive. They have the modus operandi and they have the means to do it.

The interview, published in full on the Veterans Today website, run by veteran Marine officers from Vietnam and military intelligence experts and former CIA agents, was released a few days ago before the news of the 6 dead in Iran came. This fact, however, does not contradict Brown’s reasoning but simply extends it to another ethnic group, the Islamic one of Tehran.

Map of the Pentagon’s Defense Threat Reduction Agency (DTRA) bio-laboratories in the project unveiled by the journalist Dilyana Gaytandzhieva

The existence of specific genetic bioengineering studies tested on insects and bats (among the first suspects as an animal carrying the infection) revealed the same Darpa (Defense Advanced Research Projects Agency), the arms research agency of the Pentagon which officially opened the biology sector in 2014.

To confirm that in at least 25 laboratories controlled by the US Department of Defense there were experiments in the development of biochemical weapons with interactions between viruses and insects was the Bulgarian journalist Dilyana Gaytandzhieva in her investigation into the activities of another even more specific Pentagon entity: Defense Threat Reduction Agency (DTRA).

As posted by Gospa News, the reporter, the first in the world to discover the conventional weapons delivered by the CIA to ISIS terrorists before the Turkish SETA dossier revealed all the jihadist formations supplied with TOW missiles by the American counterintelligence agency, discovered the existence of research in the field of bio-genetics in reference to the Chinese and Russian genomes.

PROTEIN “MODELED” FOR BECOMING AGGRESSIVE

Well, the suspicions of biogenetic manipulation of Gaytandzhieva, a member of Brown’s Bioweapon Truth Commission mentioned above, are now partially confirmed in scientific research disclosed by a video by some exponents of the Chinese Communist Party (CPP) that the Sino-English dual language site GNews (close to communication expert Stephen Bannon, former coordinator of Donald Trump’s winning election campaign) translated from the original language.

«On January 21st, three researchers from the Chinese Academy of Sciences published a joint paper in the “Science China Life Sciences” journal, written in English. They revealed the truth about the novel coronavirus, so please watch this news closely» reads.

There were three researchers who found this information — Pei Hao, a researcher from Institute Pasteur of Shanghai and the Chinese Academy of Sciences, Wu Zhong, a researcher from National Engineering Research Center for the Emergence Drugs, Academy of Military Medical Sciences, and Xuan Li, a researcher from Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences.

The researchers were shocked when they compared the SARS S-protein with Wuhan CoV’s S-protein and found that it doesn’t alter the structural composition of the virus even though four amino acids were replaced.

This research points to an important discovery that the Wuhan CoV S-protein supports strong interaction with human ACE2 molecules, which can lead to infecting the epithelial cells in the upper respiratory tract. This discovery also told us that Wuhan CoV is highly contagious by design. This research has laid a theoretical foundation for China to develop control, test, and intervention of Wuhan CoV in a scientific way. After reading the research report, people are outraged in disbelief. The key point of all these findings is that “four of the important S-protein have been replaced in Wuhan Coronavirus”.

First, the purpose is to disguise Wuhan CoV as SARS to make it more difficult for medical researchers and doctors to differentiate it from SARS. This misleads them into taking SARS-like prevention measures, which can result in the delay of treatment and increased spread of the virus. Secondly, it is so contagious that it spreads rapidly by design – further Chinese Communist Party blamed – Can this type of crime through biotechnology against humanity being committed by bats or bamboo rats? Ten thousand years of evolution cannot produce this type of precise altering of four important S-proteins! The fact that the Wuhan virus was created in a lab under human influence Is indisputable. This is inhumane to the extreme».

In 2010, China won the battle against the SARS virus. In 2015, the PLA hospital won battles against the Ebola virus in Africa. This time, our enemy picked Chinese New Year for a new viral outbreak. We are facing a new challenge. All medical research and epidemic prevention personnel in our country have entered a war-ready state. All the people in the country are on high alert and are combat-ready. The Chinese people will win this war added CCP.

The researchers in the synthetic document called ” Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission” published in the scientific journal as “letter to the editor” are more cautious but highlight the same concerns.

The scientific article on Science China – click to read the document in English

According to the crystal structure of SARS-CoV S-protein RBD domain complexed with its receptor ACE2 (PDB code: 2AJF), the 3-D complex structure of the Wuhan CoV S protein binding to human ACE2 was modeled with structural superimposition and molecular rigid docking – biochemical doctors wrote – So to our surprise, despite replacing four out of five important interface amino acid residues, the Wuhan CoV S-protein was found to have a significant binding affinity to human ACE2. Looking more closely, the replacing residues at positions 442, 472, 479, and 487 in the Wuhan CoV S-protein did not alter the structural conformation.

In summary, our analysis showed that the Wuhan CoV shared with the SARS/SARS-like coronaviruses a common ancestor that resembles the bat coronavirus HKU9-1. Our work points to the important discovery that the RBD domain of the Wuhan CoV S-protein supports strong interaction with human ACE2 molecules despite its sequence diversity with SARS-CoV S-protein. Thus the Wuhan CoV poses a significant public health risk for human transmission via the Sprotein–ACE2 binding pathway.

“FIVE EYES” VACCINE SUDDENLY READY…

American researchers try to dismantle the hypothesis of a laboratory virus in the discussion with various profiles on the specialized Virologica.org site where the debate is focused on the genome and its animal origin (the genetic profile is close to 99% to that of a virus grown in the Malaysian pangolin). One of these scientists, at the end of his digression in which he categorically excludes human manipulation with quirky biochemical arguments, thanks Wellcome Trust for the contribution.

This shows an evident relationship with one of the main financiers on the experiments on the CoronaVirus family before the epidemic. As reported in the previous article, in fact, this foundation was born after the transformation of the American pharmaceutical holding Wellcome taken over by the Glaxo SmithKline group, the notorious GSK, leader in the production of vaccines in the world but also repeatedly investigated and convicted of countless criminal violations on the subject of corruption and health laws, already formally charged with contributing to the search for a vaccine together with the Australian University of Queensland (see previous article).

According to the last news, in record time, the Australian vaccine would have already passed the first tests and its experimentation on animal guinea pigs would have already begun. It must not be forgotten that the intelligence of Australia SIS is part of the international group of secret services of the so-called “Five Eyes” together with the USA, the United Kingdom, Canada, and New Zealand.

Therefore the patent of a new vaccine by the Australians with the help of the British big pharma GSK which is a partner of the Darpa agency of the Pentagon in multiple projects is certainly another reason for suspicion …

Wellcome Trust is instead among the financiers of the Pirbright Institute depositary of patent no. 10130701 in the USA for a ConoraVirus research on avian chickens. The name of this British research center is mentioned in the context of the radio conversation between American journalists Kevin Barrett and Jeff Brown.

«Well, you know, I think people need to understand that the Ebola virus is owned by, patented by the U.S. military. The Zika virus is patented by the Rockefeller Foundation. I just learned that the coronavirus, our favorite friend these last four weeks, is patented by the Pirbright Institute, which is funded by Bill Gates».

Pirbright Institute has obviously immediately denied any involvement with the “human” type of the virus by reiterating that the studies were only on chickens. But it is also clear that if he had really enlarged the field of research he would be careful not to declare it in these days of world emergency.

SERIOUS DAMAGES IN CHINA EVEN FOR SWINE INFLUENCE

Brown himself confirms that he had received information on an estimated projection of 65 million deaths worldwide from an intelligence informer who presented himself with the curious nickname of Uriah Reep.

«Uriah Heep and I talked about how they were obviously attacked with a swine flu virus, African swine flu, which leapfrogged from the area of Georgia, with the Richard Lugar Bioweapons Lab right to China. And then it leapfrogged all over the country, wiped out 40 percent of the country’s swineherd, in the largest swine industry in the world. And then they also did the same thing with the avian flu. When you track the way these two viruses, you know, spread across China like wildfire. I mean, just like on a highspeed train, there’s no way that that could have happened naturally, through the normal movement of livestock and poultry».

In fact, due to the severity of the human pandemic, the swine epidemic has completely gone into the background. This puts pressure on the Chinese financial system due to inflation induced by rising pork prices. The GNews media highlights a very suspicious circumstance: China’s African swine flu came from imported Russian porks as China attempted to stop buying American products to hurt the American farmers.

On Feb 1, 2020, the most influential Chinese military website www.xilu.com published an article to acknowledge that the Wuhan coronavirus is manmade accusing the U.S. of creating such bioweapon against china» Gnews reported that then quoted in full some sentences taken from the Chinese portal.

It is possible that our enemies want to destroy our productivity. It is the so-called unrestricted warfare affecting all aspects of our economy. The CIA can infiltrate China through propaganda warfare and provoke “color revolution” in Hong Kong, why can’t it initiate a biochemical attack in China through manipulation?.

Since its birth in July 2018, Gospa News has elaborated multiple investigative reports on the attempted coup in Venezuela, when in 2019 the country was brought to its knees by continuous blackouts caused by electromagnetic sabotages that left the USA which, in addition to destroying various power plants, killed several people in the hospitals left without light.

We wrote about the civil war in Ukraine caused by the orange revolution that culminated in the massacre in Kiev’s Maidan square caused by Georgian mercenary snipers, on the conflict in Syria, where the White Helmets financed by London and Washington was accused of serious crimes including that of children killed with chlorine in Douma to stage a fake chemical attack by the Damascus army, and on American drone attacks on the Houthi separatists in Yemen which caused thousands of civilian casualties.

Therefore, at this moment, it is easier for me to believe the accusations of the Beijing Communists rather than the NATO silences or denials that come from the White House.

SARS-CoV-2 has all the characteristics to be considered a biogenetic weapon designed specifically to target some ethnic groups, certainly the Chinese but probably also the Iranian Muslims in light of the latest deaths, and it has manifested itself as a perfect viral storm: right in the Chinese New Year period when Asians scattered all over the world return home to celebrate with relatives, and a few weeks before the election consultations for the renewal of the Parliament of Tehran (this weekend), another moment of great movements and contacts between the population. What has happened elsewhere may be the so-called “collateral damage” of any military mission.

According to our source close to foreign intelligence, the virus was spread in China (and probably elsewhere) by a CIA agents equipped with “nano-drones” that are easy to hide during transport and to be used without giving attention to the transport of contagion material in capsules or vitro to be spread or detonated with electromagnetic discharges at the right time.

The technology developed by the Pentagon’s DARPA agency and put at the service of the US Army or the National Clandestine Service, the CIA’s killer operations office, is among the most sophisticated in the world as reported in the previous report on biochemical weapons. Among the possible means available to the American military for the spread of mass weapons are two brand new micro-engineering jewels.

The first goes beyond any human imagination for those who are not in the sector and starts from a rudimentary project.

CYBER-DRAGONFLY AND THE NANO-DRONE

CIA created a nano-sized drone of their own back in the 1970s. At a time when the government required a miniature listening device, they invented a mechanical bumblebee. Their initial design was too difficult to control, so they ditched the idea and invented the Insectothopter, a mini dragonfly drone reads on Globaldroneuav’s website, a Chinese company specializing in the supply of spare parts for the Unmanned Aerial Vehicles and administered by the retired Beijing army colonel Qin Ren-ping, as CEO.

Insectothopter experienced problems with flight control whenever crosswinds were apparent, so it was unused on the field and is now stored at the CIA Museum.

dragonfly, the genetically modified cyber-insect-spy, piloted with light

According to Popular Mechanics, DragonflEye is a genetically modified dragonfly with light-sensitive “steering neurons” implanted in its spinal cord. Insects fitted with a custom backpack filled with sensors are capable of being controlled, but it’s still in development. Flashes of light are used to make the insects fly or move» reads on the same website.

It now appears that the project overcame the first hurdle after some of the genetically modified dragonflies took their first flight. The idea was developed by researchers from the Charles Drak Stark Laboratory and the Howard Hughes HHMI Medical Institute, which specializes in biomedical research and partners in various DARPA projects. In the summer of 2017, their first positive tests had great visibility in the media and on YouTube also with the interview with the researchers.

This system pushes the boundaries of energy harvesting, motion sensing, algorithms, miniaturization and optogenetics, all in a system small enough for an insect to wear J. Wheeler, a biomedical engineer at Draper and Howard Hughes Medical Institute and principal investigator of the technology said in a press release, according to Us TomoNews.

The cyborg dragonflies could be turned into tiny surveillance systems. Other applications of this technology may include guided pollination, payload delivery, and precision medicine and diagnostics is specified.

For two years now nothing has been known about DragonflEye and its possible use in the military field given the consolidated collaboration between the Hughes Institute and the Pentagon. However, it is clear that if a dragonfly can be genetically modified and remotely controlled for pollination in the same way, it can be easily piloted for the spread of a virus.

If this appears to be the most futuristic solution for all its implications, obviously to the detriment of the poor insect completely distorted with suffering that no one will ever know, there is a much less science fiction and more pragmatic alternative already widely used by various NATO armies. It is the nano-UAV Black Hornet produced by the Norwegian Prox Dynamics and already ruthless by the British Armed Forces in Afghanistan since 2012 as a surveillance and reconnaissance drone.

Adopted also by the US Marines, Australian Army and the special departments of the German Army Bundeswehr and the Norwegian Forsvaret, it is the smallest remote-controlled flying device existing since it measures only 10 cm in length with a rotor diameter of 12 cm.

Black Hornet 3 nano-drone that can fly even in GPS-denied areas

The Scandinavian company was then acquired by the American corporation FLIR Systems, Inc., a leader in the design and production of infrared cameras and one of the main suppliers of the US Defense Department. In June 2018, the updated Black Hornet 3 version was presented which to the personal reconnaissance system (PRS) of the original nano-drone added the possibility of navigating in GPS-denied environments, allowing the fighter to maintain awareness situational, threat detection and surveillance wherever the mission takes him.

Having the ability to fly without a GPS connection can, therefore, escape detection of the radar in view of its microscopic size. It is in the fingers of the hand of a soldier, it has an empty weight of 18 g, a cruising speed of 36 km / h, an autonomy of 25 minutes (but in case of problems or insufficient battery it returns to the base autonomously) and a range of 1,600 meters. It is piloted through a monitor connected to the camera installed on the aircraft. The control kit with two devices weighs less than 1kg and can fit in the backpack of a military man as in that of any tourist visiting China…

With these references, we have therefore shown how the spread of a biological weapon via nano-drones supplied by international intelligence sources is perfectly conceivable and practicable. Who could have implemented it, how, where and when mysteries obviously remain.

“Dark Prince” was certainly aware of these mysteries, Michael d’Andrea, commander of the Central Intelligence Agency’s special operations in the Middle East, who disappeared on the Bombardier / Northrop Grumman E-11A spy-plane crashed – or downed – in Afghanistan a few days. before the virus turned out to be a tremendous epidemic in China and many countries around the world. The 007 American bosses suddenly disappeared along with all his secrets …

© 2020 – Fabio Giuseppe Carlo Carisio – no reproduction without authorization – versione originale in italiano


Starting from nothing

When Chinese health authorities were first confronted with the outbreak, it had a disturbing familiarity. They had already dealt with a similar set of symptoms during the SARS outbreak in the early 2000s and had seen the spread of MERS a decade later. Thanks to these and related viruses, we already had a detailed description of the structure of the typical coronavirus genome as early as 2005. That knowledge would undoubtedly prove essential for the first step in developing a rapid diagnostic test: characterization of the genome of the new virus, 2019-nCoV.

Because we know what the average coronavirus looks like, we have been able to identify areas that don't change much over the evolution of new members of this family of viruses. And that allows us to obtain sequences of its genome without first isolating the virus.

The first challenge of sequencing a coronavirus genome is that it's made of RNA rather than DNA. Most of our tools for working with nucleic acids are specific to DNA. Fortunately, we've discovered an enzyme called "reverse transcriptase" that takes RNA and makes a DNA copy of it—transcription is the copying of DNA into RNA this enzyme does the opposite, hence the name. (Reverse transcriptase was first identified in other RNA viruses that need to be copied into DNA as part of infection.) Using reverse transcriptase, researchers were able to make DNA copies of parts of 2019-nCoV as a first step to studying its genome.

But reverse transcription of samples from infected individuals would simply create a mess of DNA fragments from everything present: the patient's own cells, harmless bacteria, and so on. Fortunately, DNA sequencing and analysis techniques have become so advanced that it's now possible to just sequence the whole mess, irrelevant stuff and all, and let computers sort out what's present. Software is able to take what we know about the average coronavirus genome and identify all of the fragments of sequence that look like they came from a coronavirus. Other software can determine how all these fragments overlap and then stitch them together, producing a near-complete coronavirus genome.

At this point, Chinese health authorities recognized that the virus involved in these infections was new, and they rapidly published the virus's genome sequence so that other health organizations could be prepared.


Laser and LED Lights - GHK-Cu Treatments

Miller TR, Wagner JD, Baack BR, Eisbach KJ, Effects of topical copper tripeptide complex on CO2 laser-resurfaced skin. Arch Facial Plast Surg. 2006 Jul-Aug8(4):252-9.

Cangul IT, Gul NY, Topal A, Yilmaz R, Evaluation of the effects of topical tripeptide-copper complex and zinc oxide on open-wound healing in rabbits. Vet Dermatol. 2006 Dec17(6):417-23.

Huang PJ, Huang YC, Su MF, Yang TY, Huang JR, Jiang CP, In vitro observations on the influence of copper peptide aids for the LED photoirradiation of fibroblast collagen synthesis. Photomed Laser Surg. 2007 Jun25(3):183-90.

Stimulation of Hair Growth

Publications on SRCPs and Hair Growth

Methods for the design and testing of copper-peptide complexes with hair growth properties are described.

A wide variety of GHK-Cu analogs were described that increase hair follicle size and increase hair growth in mice and rats.

Study Result Reference
Use of GHK-Cu analogs for hair follicle enlargement and stimulation of hair growth US Patent 5,120,831 New metal peptide complexes and derivatives used for stimulating growth of hair in warm-blooded animals, especially humans. Pickart US Patent 5,177,061 Compositions for stimulating hair growth containing cupric complexes of peptide derivatives including. glycyl-l-histidyl-l-lysine n-octyl ester. Pickart US Patent 5,214,032 New glycyl-histidyl-lysyl copper compounds used in stimulating hair growth. Pickart US 5,550,183 Metal-peptide compositions and methods for stimulating hair growth. Pickart
Stimulation of hair growth in mice GHK analogs with hydrophobic residues were tested and found to stimulate hair growth in rats. The hair follicle stimulating properties of peptide copper complexes. Results in C3H mice. Fors, Pickart and Uno Ann N Y Acad Sci 1991 26642:468-9
Stimulation of hair growth in mice and rats The details of hair stimulation by copper peptides was studied by 1) phototrichogram, 2) folliculogram (micro morphometric analysis), and 3) the rate of DNA synthesis in the follicular cells. The effects were essentially a stimulation of the follicular cell proliferation, resulting in an enlargement of the anagen follicles from vellus to terminal type (therapy) or a maintenance of the piebald terminal follicles (prevention). A SRCP (PC1020) had the effect of follicular enlargement on the back skin of fuzzy rats, covering the vellus follicles. Chemical agents and peptides affect hair growth. Uno and Kurata (University of Wisconsin, Madison, USA) J Invest Dermatol 1993 101(1 Suppl):143S-147S
Minimizing hair loss after cancer chemotherapy Hairloss protection by peptide-copper complex in animal models of chemotherapy-induced alopecia. Awa and Nogimori Journal Of Dermatological Science, Vol: 10, 1995, 99-104
Human hair growth Stimulation of hair growth in humans with GHK-Cu analogs Phototrichogram Analysis of Hair Follicle Stimulation: A pilot clinical study with a peptide-copper complex. Patt, Duncan and Kalis (University of Reims, France) Dermatological Research Techniques, (CRC Press), pp-217-226, 1996
Hair growth in rats Stimulation of hair growth in rats Quantitative Assessment of Peptide-Copper Complex Induced Hair Follicle Stimulation Using the Fuzzy Rat, Uno, Packard, Patt (University of Wisconsin) Dermatological Research Techniques, (CRC Press), pp-227-239, 1996
Hair growth in rats Stimulation of hair growth in rats with GHK-Cu analogs Evaluation of Telogen Hair Follicle Stimulation Using an In Vivo Model: Results with Peptide Copper Complexes. Timpe, Dumwiddie, Patt (Procyte Corp.) Dermatological Research Techniques, (CRC Press), pp-241-254, 1996
Human study of hair growth with GHK-Cu analog Compared GHK-CU analog in Tricomin with 2% minoxidil. Tricomin 2.5% increased hair count by 97 non-vellus hairs while 2% minoxidil increased count by 73 non-vellus hair after 3 months (non-vellus hair count) Procyte Corp. press release 1997
Breakdown resistant, long acting copper-peptides used for stimulation of hair growth Tested copper complexed with protein peptones for hair growth effects in mice. Copper-peptide mixture produced more hair growth in mice than GHK-Cu analogs Pickart US Patent 5,554,375 Tissue protective and regenerative compositions.
Review Skin remodeling and hair growth Pickart L, Effect of copper peptides on hair growth and condition, Body Language Dermatology 2004, Number 7, pages 20-22
Review Skin remodeling and hair growth Pickart L, Skin remodeling copper peptides for improving hair growth, Cosmetics & Medicine (Russia) 2004, Number 3, pages 14-29
Study of human follicles in organ culture AHK-Cu increased follicular cell growth while decreasing progrmmed cell death (apoptosis) Pyo HK, Yoo HG, Won CH, Lee SH, Kang YJ, Eun HC, Cho KH, Kim KH, The effect of tripeptide-copper complex on human hair growth, Arch Pharm Res 2007, vol 7, 834-839.

GHK-Cu Analogs and Stimulation of Hair Growth

Certain analogs of GHK-Cu have the property of enlarging hair follicles and stimulating hair growth. These analogs have more fat-like character than GHK-Cu. This increase in fat-like properties is obtained by either chemically synthesizing fatty acids into the GHK molecule or attaching amino acid residues such as alanine, phenylalanine or leucine to the basic GHK structure.

These analogs originally arose in an attempt to create GHK-Cu analogs which would be retained in body tissues for longer periods of time. However, it was noted that - while such analogs were superior wound healing agents - they also markedly increased hair growth around the periphery of experimental wounds in mice.

These hair stimulating analogs were created by Drs. Steven Lovejoy, Loren Pickart and Boris Weinstein. Skin repair and hair growth enhancement effects are closely linked. New skin appears to arise from the hair follicle. Certain products based on Iamin can be used to both repair skin, increase hair follicle size, and stimulate hair growth. As a person ages, our hair follicles get smaller, producing thinner hair shafts. A major cause of hair follicle miniaturization appears to be due to the development of striking changes in capillaries surrounding the hair follicles. Comprehensive surveys of the male scalp from birth to senescence find very significant changes in the structure of the blood vessels of the scalp. The number of the blood capillary loops supplying the hair follicle is greatly diminished. The inadequate subepidermal circulation that can develop as males age does not provide a rich nutrition for the follicle. Strong hair growth requires a large flow of nutrients such as such as vitamins, minerals and amino acids so that the follicle can actively synthesize new hair.

Blood flow impairments to the follicle, and their reversal, may explain why the administration of copper peptides (such as Tricomin) to the scalp increases hair growth and increase the size of hair shafts. It has long been known that certain copper-peptide complexes strongly stimulate angiogenesis or new blood vessel formation. The increase in hair follicle size and the rate of hair growth caused by the administration of copper-peptides may be due to their causing blood flow changes that provide adequate nutrients to the follicle, producing faster growing hair with thicker hair shafts. Copper ion complexed with certain peptides has both skin repair and hair growth enhancement effects. Examples of this are Tricomin and GraftCyte which are based on Pickart's earlier work (from ProCyte Corporation).

More follicular cell growth and less programmed cell death (apoptosis)

During aging in men and women, there is a progressive decrease in hair follicle size. This produces thinner hair and in time stops new hair growth.

Pyo et al (refererence above) propose, based on studies of human hair follicles, that the actions of copper peptides increase cell growth in cultured hair follicles while decreasing programmed cell death or apoptosis. Copper pepides also decrease the Bax protein which increases apoptosis. Their studies use Ala-His-Lys-copper, as close analog of GHK, that I had found to stimulate hair growth many years ago.

So, the copper peptides may function by slowing the rate of programmed cell death in human hair follicles that ultimately stops human hair growth.

The skin of the mouse to the left was shaved, then treated in three spots with copper peptides. The result is a much more rapid hair growth (the three circular patches of hair) in the three spots treated with copper peptides. While human hair growth will not respond nearly as dramatically as in mice, skin health and hair follicle function are closely interrelated. New skin appears to arise from the hair follicle. As a person ages, our hair follicles get smaller, producing thinner hair shafts. The blood circulation that supplies nutrients and oxygen to the hair follicle send fewer blood vessels to the hair follicle, thus inhibiting the vital flow of nutrients to the hair follicle. Copper-peptide complexes improve skin health and a more healthy skin increases the blood vessel network to the hair follicles resulting in larger follicles that grow hair faster with thicker hair shafts.



In the microscopic images to the left, the magnifications are identical. The top photo is mouse skin untreated with copper-peptides. The bottom photo is mouse skin treated with copper-peptides. Note the larger hair follicles (the elongated purple columns) in the lower photo, the increased content of subcutaneous fat in the skin (the white material in the center of the skin), and the increased thickness of the skin. When we are young, we have a layer of fat under the skin (part of "baby fat") which is greatly reduced as we age. Hair researchers have noted the accumulation of this fat around healthy follicles that are vigorously growing hair, and its relative lack around dormant follicles, have postulated that these cells serve a supportive function for the hair follicle. It must be emphasized that effects in humans on hair follicle health are not as dramatic.

New Hair Follicle Formation?

At times, SRCPs can apparently induce a proliferation of hair follicles, although this phenomena is difficult to reproduce on a consistent basis. The photograph on the top is a microscopic field of mouse hair follicles in an animal treated only with saline. The photograph on the bottom is a similar area of mouse skin treated with copper-peptides and which has a much higher density of hair follicles. Individual experiments on hair follicle multiplication are consistent, that is, the effect is actual when it occurs, but repeated results are difficult to obtain. The variability may be due to different timing in the hair growth cycle or slight changes in the type of, or formulation of, the copper-peptide preparations. Such experiments strongly suggest that, under certain circumstances, new hair follicle formation can be induced in adult animals.

Reducing Hair Loss After Chemotherapy / Reducing Hair Loss During Chemotherapy

The hair loss caused by chemotherapy drugs used for cancer treatment can be minimized with copper peptides. Awa and Nogimori found that application of copper peptides minimize hair loss after chemotherapy and accelerated new hair growth in rats.

Rats were pretreated with SRCPs then exposed to chemotherapeutic drugs. This reduced hair loss.

If the rats were first given chemotherapeutic drugs, the treated with SRCPs later, the SRCPs speeded hair regrowth.

T. Awa, K. Nogimori and R. Trachey, Hairloss protection by peptide-copper complex in animal models of chemotherapy-induced alopecia. J. Derm. Sci. 10, 99-104 (1995)

GHK, Copper, Regeneration, and Stem Cells

The primary cause of aging is a decline in organ function over time. Up until we reach age 20, tissue and organs are maintained in a fully functional and healthy state. But as we age, repair slows and our organs fail to fulfill their biological role. Adult stem cells in organs create new cells for repair and the key protein in activating and supporting stem cell function appears to be protein P63. Without adequate P63, skin ages rapidly as do other tissue of the body.

GHK-Copper increases protein P63 in addition to all of GHK-Copper's other protective and repair functions. This is the final link in understanding GHK-Copper role in the human body as given in the graphic below.

Recent studies revealed that older animals have as many adult stem cells in their bodies as young animals. However, these stem cells are not differentiated into the types of cells needed to rejuvenate older tissue.

From my previous studies, we know the amount of GHK-Copper needed to activate strong systemic healing of skin throughout the body in mice, rats, and pigs. This should be the same as that needed to increase P63 and activate epithelial stem cells since wound healing proceeds via stem cell actions. GHK-Copper must also activate other types of adult stem cells since it has strong healing actions on on the stomach, intestinal linings and bone tissue. Approximately, 75 milligrams of GHK-Copper injected three times a week should be sufficient to activate human stem cells and improve the function of organs in older individuals. It is possible that GHK-Copper might be effective as an oral supplement enclosed in special liposomes that are taken up by the lymphatic system to avoid possible breakdown by intestinal enzymes.

The 75 milligram level of GHK-Copper should be very safe and is 280-fold below expected negative actions of the molecule caused by its blood pressure lowering actions.

In cell culture studies, GHK-copper increases differentiation of embryonic stem cells. However, in an organ culture system, GHK-copper "activated" adult stem cells which then produced more keratinocytes.

GHK tested for stopping stem cell differentiation

GHK-Cu tested for stimulating stem cell differentiation

GHK reduced the clonogenic potential of stem cells by 78%

GHK-Cu increased cell copper by 2162% above the control value and caused stem cell differentiation

Methods of controlling proliferation and differentiation of stem and progenitor cells, United States Patent: 6,962,698, Peled, Tony, Fibach, Eitan, Treves Avi, Gamida Cell Ltd. (Jerusalem, IL) and Hadasit Medical Research Services and Development, Ltd. (Jerusalem, IL)

GHK-copper activated adult stem cells by increasing integrins, P63, and PCNA. This increased keratinocyte proliferation.

Arch Dermatol Res. 2009 Apr301(4):301-6. Copper-GHK increases integrin expression and p63 positivity by keratinocytes. Kang YA, Choi HR, Na JI, Huh CH, Kim MJ, Youn SW, Kim KH, Park KC. Department of Dermatology, Seoul National University College of Medicine, Yeongeon-dong, Jongno-gu, Seoul, Republic of Korea.

Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin.

Cell Stem Cell. 2009 Jul 25(1):64-75. TAp63 prevents premature aging by promoting adult stem cell maintenance. Su X, Paris M, Gi YJ, Tsai KY, Cho MS, Lin YL, Biernaskie JA, Sinha S, Prives C, Pevny LH, Miller FD, Flores ER. Department of Molecular and Cellular Oncology, Graduate School of Biomedical Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.

The cellular mechanisms that regulate the maintenance of adult tissue stem cells are still largely unknown. We show here that the p53 family member, TAp63, is essential for maintenance of epidermal and dermal precursors and that, in its absence, these precursors senesce and skin ages prematurely. Specifically, we have developed a TAp63 conditional knockout mouse and used it to ablate TAp63 in the germline (TAp63(-/-)) or in K14-expressing cells in the basal layer of the epidermis (TAp63(fl/fl)K14cre+). TAp63(-/-) mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis. These phenotypes are likely due to loss of TAp63 in dermal and epidermal precursors since both cell types show defective proliferation, early senescence, and genomic instability. These data indicate that TAp63 serves to maintain adult skin stem cells by regulating cellular senescence and genomic stability, thereby preventing premature tissue aging.

Cellular senescence is a distinctive form of cell cycle arrest that has been suggested to modulate the processes of tumor suppression and aging. Though a detailed understanding of the cellular machinery regulating this process is emerging, a more thorough understanding of the key players linking senescence to organismal aging is needed. The recent discovery that loss of the p53-related protein p63 induces cellular senescence and causes features of accelerated aging provides further evidence that cellular senescence is intimately linked with organismal aging, and identifies p63 as a key regulator of both of these processes.

The p53 family member p63 comprises multiple isoforms and is critical for stratified epithelial development. In this issue of Cell Stem Cell, by generating isoform-specific knockout mice, Su et al. (2009) reveal pivotal roles for TAp63 in the maintenance of dermal and epidermal precursors, genomic stability, and organismal longevity

The distinguishing feature of adult stem cells is their extraordinary capacity to divide prior to the onset of senescence. While stratified epithelia such as skin, prostate, and breast are highly regenerative and account disproportionately for human cancers, genes essential for the proliferative capacity of their stem cells remain unknown. Here we analyze p63, a gene whose deletion in mice results in the catastrophic loss of all stratified epithelia. We demonstrate that p63 is strongly expressed in epithelial cells with high clonogenic and proliferative capacity and that stem cells lacking p63 undergo a premature proliferative rundown. Additionally, we show that p63 is dispensable for both the commitment and differentiation of these stem cells during tissue morphogenesis. Together, these data identify p63 as a key, lineage-specific determinant of the proliferative capacity in stem cells of stratified epithelia.

Wound Healing and Skin Repair Studies Using SRCPs

The healing of surgical wound in 10 control rats and 10 GHK-Cu treated rats was investigated.

The application of GHK-Cu markedly stimulated wound closure

Effect of locally injected medications on healing of pad wounds in dogs. Swaim SF, Vaughn DM, Kincaid SA, Morrison NE, Murray SS, Woodhead MA, Hoffman C.E, Wright JC, Kammerman JR, College of Veterinary Medicine, Auburn University, AL, USA, Am J Vet Res 1996 Vol. 57, 394-9

Evaluation of multipeptide copper complex medications on open wound healing in dogs. Swaim, Bradley, Spano, McGuire and Hoffman (College of Veterinary Medicine, Auburn University, AL, USA) J Amer Ani Hos Assoc. 29, 519-525, 1993

Healing is very impaired in immune suppressed patients and wound contraction is impaired - Rats were immune suppressed with cortisone injections then the effect of GHK-Cu determined.

In immune suppressed rats, collagen synthesis was 23% of that in normal rats. GHK-Cu more than tripled collagen synthesis in these rats, raising it to 77% of normal and restored normal healing. GHK-Cu restored normal wound contraction in suppressed rats

Tested GHK-Cu cream on healing of skin ulcers.

GHK-Cu increased ulcer re-epithelialization

Enhanced healing of ulcers in patients with diabetes by topical treatment of glycyl-l-histidyl-l-lysine Mulder GD, Patt L, Sanders L, Rosenstock J, Altman MI, Hanley ME, Duncan GW, Wound Rep Reg 2:259-269, 1994

Tested GHK-Cu on the healing of linear incision wounds.

GHK-Cu increased wound tensile strength by 74% and neovascularity by 69%.

Tested 2nd generation SRCPs on repair of human nickel allergy injured skin.

Placebo-controlled double-blinded study found an accelerated the recovery of skin after injury and plus potent anti-inflammatory action.

Tested 2nd generation SRCPs on repair human tape stripped damaged skin.

Placebo-controlled double-blinded study found an accelerated the rate of skin repair

Tested 2nd generation SRCPs on repair of human 24-hour detergent damaged skin.

Placebo-controlled double-blinded study found accelerated the rate of skin repair

Tested 2nd generation SRCPs on repair of human acetone damaged skin.

Placebo-controlled double-blinded study found an accelerated the rate of skin repair

Cangul IT, Gul NY, Topal A, Yilmaz R, Evaluation of the effects of topical tripeptide-copper complex and zinc oxide on open-wound healing in rabbits. Vet Dermatol. 2006 Dec17(6):417-23.

Arul V, Kartha R, Jayakumar R, A therapeutic approach for diabetic wound healing using biotinylated GHK incorporated collagen matrices, Life Sci. 2007 Jan 280(4):275-84.

Founding ProCyte for Wound Healing Product Development

In 1985, Barbara Weinstein and I founded ProCyte Corporation to develop GHK-Cu for clinical wound healing uses. William Weinstein, her son and a lawyer, secured the patent rights to the technology for the company. Since GHK-Cu had direct actions on cells important in healing, I named the company ProCyte - Latin words meaning - "for the cell". Dr. John Majnarich gave us laboratory space and supplies.

Since then, GHK-Cu has had numerous successes in experimental wound healing in animal models. However, in human clinical studies it has had a number of limited successes and failed to prove effectiveness in 3rd phase in two clinical trials for drug approval. However, this pattern of successes and failures is similar to other wound growth factors in clinical trials. To date (2005), no successful would healing factor has established itself in the market.

The most successful wound healing agent, Platelet-Derived Growth Factor or PDGF, is sold by Johnson & Johnson as Regranex. But it only improves the closure of diabetic ulcers by about 20%.

Proper dose ranging studies of GHK-Cu in various formulations have never been adequately performed. I designed the first pilot clinical trial in France in 1987-1988 that used a 4% concentration of GHK-Cu in a cream with minimal preservatives since many common preservatives inhibit skin repair. Because of the low preservative level, this cream required refrigeration. This study of 60 patients with diabetic and venous stasis ulcers gave evidence of rapid healing. This apparently successful formulation was never used in later clinical studies, which failed to achieve therapeutic goals in FDA trials for clinical uses.


Bernard Kalis (Universite de Reims) who, along with his colleagues, performed the first successful tests of GHK on the healing of human wounds.


A following study in 1990 designed by Schering Plough Pharmaceutical used a similar cream but with an addition of 1% benzoyl alcohol as additional preservative. In retrospect, the addition of benzoyl alcohol was a mistake since it is now known that benzoyl alcohol inhibits the function of wound fibroblasts about 100-fold more than it inhibits bacteria growth. The GHK-Cu cream improved the healing of wounds caused by Moh's Surgery. However, it did not accelerate the healing of venous stasis ulcers, which may be a poor model for testing wound healing formulations since other growth factors such as PDGF, TGF, FGF, and EGF have failed to demonstrate statistical healing of venous stasis ulcers.

Nonetheless, following this other small human studies gave evidence of wound healing. A well controlled study published in 1993 of 120 diabetic patients in a single medical center found evidence of accelerated healing of diabetic ulcers. A larger double-blinded study in 1995 failed to demonstrate improved closure but did find GHK-Cu to improve re-epithelialization of the ulcers.

This study used 505 patients in 33 medical centers with an average of 17 patients per medical center. Given the variance in treatment procedures at various medical centers, this may have increased the statistical variance and degraded the data. The GHK-Cu concentrations used (2.0% and 0.5%) appear too low for effective healing. Also, benzoyl alcohol, that was known that time to inhibit wound fibroblasts, was inexplicably still used as a preservative.

Because of rapid breakdown of GHK-Cu, dosages should be in the 4% to 10% range and it is best to apply the GHK-Cu in a non-ionic cream that slowly releases the GHK-Cu into the wounded tissue.

Example - Healing of Pre-Ulcer Dermatitis

One of the best uses of SRCPs would be the healing of pre-ulcer dermatitis before the damaged skin develops open sores. The photographs at left are an example of healing "at-risk" skin with pre-ulcer dermatitis. In the top photo, the patient has two open skin ulcers visible in left top and bottom of the photo. On the right of the photo, there are reddish fissures developing into skin ulcers. In the bottom photo, the application of a copper peptide cream to the periphery of the skin ulcers has healed the fissured skin and prevented further ulcer development. It should be emphasized that this particular copper peptide cream was designed specifically for "at-risk" skin in the stage of pre-ulcer dermatitis to help prevent further skin breakdown. It was not approved for the treatment of open skin ulcers.


Example - Healing of Diabetic Skin Ulcers

Persons with diabetes often have slow and inadequate skin repair. The skin complications of diabetes lead to skin that is dry, tends to crack, and is slow to heal. Leg sore and foot sores are the leading cause of lower leg amputations and presently approximately 10% of diabetic patients require an amputation during their lifetime. The primary reason for amputation is infections associated with the development of broken and ulcerated skin. Persons with diabetes are at 15 times the average risk of experiencing limb amputations.

SRCP creams often produce a rapid improvement in skin health and help prevent the development of cracks and fissures in the skin which may turn into skin ulcers. Rapid healing of broken and cracked skin, before an infection sets in, is very important.

Often skin damage in diabetic patients is relatively easy to heal. The best way is to pre-wash affected skin with 3% hydrogen peroxide followed by the application of creams containing SRCPs. In the photograph to left, a women with diabetes had six skin ulcers on her foot and physicians were recommending immediate amputation. Instead she tried the hydrogen peroxide washes followed by application of the copper peptide to the periphery of the skin ulcers. The photographs to the left are either side of the foot before this procedure and those to the right are after 28 days of hydrogen peroxide and the copper peptide cream. After 28 days all the skin ulcers were healed. The ulcers never re-occurred and she only used additional product as needed when her skin became excessively irritated and cracked.

For cracked skin, use a light coating of a copper peptide once daily as needed. For skin ulcers, put the copper peptide around the edge of the ulcers. This aids skin healing from the outer edge of the ulcers. NOTE: Copper peptides are not FDA-approved for use within the ulcer area.

Some clinicians recommend against the use of hydrogen peroxide on damaged skin and state that it increases skin damage. However, my reviews of the last 70 years of medical literature have found many reports of improved skin healing after washes of hydrogen peroxide at low concentrations (1% to 10%). However, skin damage is occasionally observed at higher concentration of hydrogen peroxide. So any use of hydrogen peroxide on damaged skin should use concentrations of 1% to 3% and no higher.


GHK-Cu Clinical Dosage Levels and Pharmacokinetics of Rapid Breakdown In Vivo

While GHK and GHK-Cu biological actions start at 10exp (-12) M and peak at about 10exp (-8) M, clinical studies have used far higher dosages. There may be explanations for this divergence. When GHK-Cu containing creams are applied to wounds or intact skin, the uptake levels are very low - ranging from 0.05 to 0.15%. Alternatively, when GHK-Cu is injected intradermally or into a wound margin. GHK is rapidly cleared from the area with an over 95% clearance in 1 minute. In mice, it has an approximate 20 minute half-life when injected intraperitoneally. It is quickly broken down into free glycine and lysyl-histidine, which is quickly excreted. (Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization, Endo T Miyagi M Ujiie A (Kissei Pharmaceutical Co., Ltd., Minamiazumi, Nagano, Japan) J Chromatogr B Biomed Sci Appl 1997 Apr 25692(1):37-42)

Enhancement of Systemic Wound Healing with GHK-Cu

GHK-Cu can be used to enhance systemic wound repair throughout an animal. In 2.5 kilogram rabbits, H. Paul Ehrlich found that the injection of 1 milligram of GHK-Cu (formulated at a ratio of 2 molecules peptide to one molecule copper) into muscle tissue markedly stimulated healing in areas distant from the the injection site and increased the concentration of circulating wound macrophages. If this technique were used in humans, the injection of a very small amount of GHK-Cu (approximately 30 milligrams) before a surgical operation would accelerate the tissue repair. The amount of copper contained in 30 milligrams of GHK-Cu is about the daily RDA.

Such a technique would be of great value in difficult operations such as hip transplantation surgery in the elderly and in operations in immune suppressed patients. Experiments with immune suppressed rats by H. Paul Ehrlich have found that GHK-Cu normalized wound repair in such situations.

Skin and Hair Transplantation

GHK-Cu improved the transplants of skin grafts in pigs, mice, and humans. For pig skin grafts, the best results were obtained by using GHK-Cu dissolved in a small amount of DMSO (dimethylsulfoxide). Normally in skin grafts, most of the grafted skin dies off, and new skin grows outward from the surviving core of the skin graft. However, the addition of DMSO often so improved the graft "take" that the final graft "take" area exceeded the actual size of the skin graft.

Study Result Reference
US Patents describing methods using GHK-Cu stimulating wound healing of wound and methods for enhancing wound healing through an animal US Patent 4,665,054 New glycyl-L-histidyl-L-lysine copper derivatives of improved resistance to proteolytic enzymes and better fat solubility for use in inhibiting thromboxane production and enhancing wound healing. Pickart US Patent 4,760,051 Compositions containing glycyl-1-histidyl-1-lysine copper(II) enhance the wound healing process without evoking an antigenic response Pickart US Patent 4,810,693 Copper glycyl-L-histidyl-L-lysine complexes enhance the healing of wounds and sores. Pickart US Patent 4,877,770 New glycyl-histidyl-lysine ester copper complex compounds with anti-inflammatory and superoxide dismutase activity useful for enhancing wound healing
Pickart US Patent 4,937,230 Method for healing wounds in horses using a copper complex of Glycyl-L-Histidyl-L-lysine or derivatives on the affected area. Pickart US Patent 5,164,367 Compositions for accelerating wound healing in mammals containing cupric salt or complexes with amino acid or peptide
Wound healing in rats Acceleration of wound healing using glycyl-histidyl-lysine copper (II) Downey, Larrabee, Voci and Pickart (Virginia Mason Research Center) Surg. Forum 573-575, 1985
Healing of rat wounds Chemoattraction of capillary endothelial cells Anti-oxidant actions GHK-Cu was tested on the healing of rat wounds, capillary cell chemoattraction, and superoxide dismutase-like activity. GHK-Cu accelerated the healing of rat wounds and acted as a chemoattractant for capillary endothelial cells at 10exp (-12) M. GHK-Cu also possesses significant superoxide dismutase-like activity. Gly-l-his-l-lys copper(II) - A human growth factor with superoxide dismutase-like and wound healing activities Pickart, Downey, Lovejoy and Weinstein (University of Washington) In: Superoxide and Superoxide Dismutase (Elsevier, 1986) pp.555-558
Wound healing and skin transplantation in mice GHK-Cu was tested by injection for improving the healing of surgical wounds and the take of skin grafts GHK-Cu accelerated the healing of surgical wound and improve the take of skin grafts Iamin: A human growth factor with multiple wound healing properties Pickart In: Biology of Copper Complexes Plenum Press 1987, pp.273-282
Healing of wounds in mice GHK-Cu was tested for improving the closure of surgical wounds GHK-Cu accelerated the healing of surgical wounds in mice Biological activity of human plasma copper-binding growth factor glycyl-L-histidyl-L-lysine. Pickart and Lovejoy Methods Enzymol 1987147:314-28
Wound healing in pigs Periphery of wound injected with 50 micrograms of GHK-Cu in 1% DMSO. Control wounds received 1% DMSO only. By 17 days post wounding, GHK-Cu accelerated healing and induced a "star" pattern of strong wound contraction. Pickart, 1984, Unpublished
Wound healing in pigs The healing of punch biopsies wound in pigs was determined GHK-Cu markedly stimulated wound healing and collagen synthesis. The effect is highly localized to the immediate skin area that is treated Counts, D, Hill E, Turner-Beatty M, Grotewiel M, Fosha-Thomas S, Pickart L. Effect of lamin on full thickness wound healing. Fed Am Soc Exp Biol 1992 A1636
Paw healing in dogs Tested GHK-Cu on paw healing in dogs. Tripeptide-copper complex improved the healing of pad wounds in 12 mature English Pointers. Collagen production was significantly greater in treated pads. Healing was best with light bandaging. Wet bandages nullified the tripeptide-Cu effect.
Dog surgical wounds Effect of GHK-Cu and GHKF-Cu on wound healing of surgical wounds in dogs. GHK-Cu and GHKF-Cu increased wound closure and contraction and markedly increase the production of granulation tissue.
Human - Refractory Venous Stasis Ulcers and Diabetic Ulcers Effect of GHK-Cu non-ionic cream with minimal preservative was tested on 60 patients GHK-Cu cream accelerated wound re-epithelialization Effects of the tripeptide glycyl-l-histidyl-l-lysine copper(II) on healing. Clinical and biochemical correlations. Aupaix, Maquart, Salagnac, Pickart, Gillery, Borel and Kalis J Invest Derm 94:390, 1990
Human - Acute Surgical Wounds Tested GHK-Cu cream with benzoyl alcohol as preservative on healing after Moh's surgery. GHK-Cu increased wound healing and skin re-epithelialization Fish S, Katz I, Hien NR, Briden ME, Johnson JA, Patt, L, Evaluation of glycyl-1-histidyl-1-lysine copper complex in acute wound healing. Wounds 1991, 3:171-177
Healing in immune suppressed rats Stimulation of skin healing in immunosuppressed rats H. P. Ehrlich (Harvard Medical School, Boston, USA) Presented at Symposium on collagen and skin repair Reims, France Sept. 12-13 1991
Human - Diabetic skin ulcers Massey P, Patt L, D'Aoust JC, The effects of glycyl-l-histidyl-l-lysine copper chelate on the healing of diabetic ulcers, Wounds 4:21-28, 1992
Human - Diabetic skin ulcers Tested GHK-Cu cream on healing of skin ulcers GHK-Cu increased skin ulcer re-epithelialization The combined effects of glycyl-l-histidyl-l-lysine copper (II) and Cell-Tak on the healing of linear incision wounds. Schmidt SP, Resser JR, Sims RL, Mullins DL and Smith DJ (University of Akron, Ohio, USA) Wounds 6, 62-67, 1994
Wound healing and fibroblast production of collagen in guinea pigs The effects of GHK-Cu and three synthetic analogues on wound healing of the guinea pig dorsal skin, as well as on cultured fibroblasts, were examined. Hydroxyproline, proteins, DNA and semicarbazide- sensitive amine oxidase, with a high affinity for benzylamine, were measured, and the histology of the wounds was observed after staining with hematoxylin/eosin.GHK-Cu and the analogues caused a decrease of the activity of semicarbazide sensitive amine oxidase, with a high affinity for benzylamine, 4-8 days after surgery, followed by an increase on day 11 that was higher than in the control group. A slower reorganization of the skin and a delayed activation of fibroblasts are observed with these peptides-Cu complexes. The peptides had a direct effect on fibroblasts. The products at a concentration of 10exp (-7) M, decreased cell reproduction and increased collagen expression. Effect of tripeptide-copper complexes on the process of skin wound healing and on cultured fibroblasts. Buffoni, Pino and Dal Pozzo (Department of Pharmacology, University of Florence, Firenze, Italy) Arch Int Pharmacodyn Ther 1995 330(3):345-60
Mice The use of breakdown resistant copper peptides to stimulate skin repair and wound healing US Patent 5,382,431 Tissue protective and regenerative compositions Pickart
Human skin repair In vivo nickel contact dermatitis: human model for topical therapeutics. Zhai, Chang, Singh, and Maibach (University of California, San Francisco, USA) Contact Dermatitis Vol. 40, pp. 205-208, 1999
Human skin repair Stripped skin model to predict irritation potential of topical agents in vivo in man. Zhai, Poblete, and Maibach (University of California, San Francisco, USA) International Journal of Dermatology, Volume 37, pages 386-389, 1998
Human skin repair Sodium lauryl sulfate damaged skin in vivo in man: a water barrier repair model. Zhai, Leow, and Maibach (University of California, San Francisco, USA) Skin Research and Technology, Volume 4, pages 24-27, 1998
Human skin repair Human barrier recovery after acute acetone perturbation: an irritant dermatitis model. Zhai, Leow, and Maibach (University of California, San Francisco, USA) Clinical and Experimental Dermatology, Volume 23, pages 11-13, 1998
Effect of GHK-Cu on healing of ischemic open wounds in rats. GHK-Cu caused a significant decrease in wound area (64.5% GHK-Cu vs 28.2% control) by day 13. GHK-Cu also significantly lowered concentrations of TNF-alpha and MMP-2 and MMP-9.

Canapp SO Jr, Farese JP, Schultz GS, Gowda S, Ishak A.M, Swaim SF, Vangilder J, Lee-Ambrose L, Martin FG, The effect of topical tripeptide-copper complex on healing of ischemic open wounds, Vet Surg. 2003 Nov-Dec32(6):515-23
Rat dermal skin repair Biotin was attached to GHK then bound to collagen films. This gave increased wound contraction, increased cell proliferation, and produced a high expression of the antioxidant superoxide dismutase. Tissue copper levels were increased 9-fold. Arul V, Gopinath D, Gomathi K, Jayakumar R. Biotinylated GHK peptide incorporated collagenous matrix: A novel biomaterial for dermal wound healing in rats. Biomed Mater Res B Appl Biomater. 2005 May73(2):383-91
Compare GHK-Cu and zinc oxide creams for wound healing The unhealed wound area was smaller and wound contraction was higher in the GHK-Cu than in the zinc oxide group and the control. Median time for the coverage of the wound bed with granulation tissue was significantly shorter in the GHK-Cu group than in the other groups. Filling of the open wound with granulation tissue to skin level was significantly slower in the control group than in the other two groups. Neovascularization was best observed in the GHK-Cu group. The authors suggest that GHK-Cu is a better choice in the treatment protocols of open wounds than zinc oxide.
Biotinylated GHK peptide (BioGHK) incorporated collagen biomaterial (PIC) was tested for wound healing in diabetic rats. In diabetic rats treated with BioGHK collagen, healing was hastened with an increased rate of wound contraction. Glutathione (GSH) and ascorbic acid levels in the skin of streptozotocin-induced diabetic rats were higher in the PIC group as compared to control (Untreated) and collagen (Collagen Film--CF) treated groups. Superoxide dismutase (SOD) and catalase (CAT) activity was altered in all the groups. Fibroblast cell culture studies suggest that PIC promotes fibroblast growth, revealed epithelialization, increased synthesis of collagen and activation of fibroblasts and mast cells in the PIC group. BioGHK incorporated collagen may be an approach to enhance diabetic wound healing.

Photograph:
Left - Control Graft about 20% of skin graft (at center) established -this is a typical graft "take".
Right - Graft soaked in GHK-Cu in liposomes. Original graft overgrew transplant area

.

Study Result Reference
Improvement of skin grafts in pigs Pickart US Patent 4,760,051 Compositions containing glycyl-1-histidyl-1-lysine copper(II) enhance the wound healing process without evoking an antigenic response.
Tested GHK-Cu on skin transplants in mice Transplanted 1.5 cm diameter full thickness skin grafts in mice. This is considered an "impossible" transplant experiment 40% of full thickness transplants became permanent grafts Pickart Iamin: A Human Growth Factor with Multiple Wound Healing Properties. in Biology of Copper Complexes , Clifton, NJ, 1987, pp. 273-285.
Human hair transplants .Studied the effect of GHK-Cu analog on hair transplants. Treated patients saw new hair growth in six weeks, versus the normal 10 to 14 weeks. In most cases, skin crusting after transplantation is reduced from 10 to 14 days to five days. Increased the degree of hair outgrowth from human hair transplants Perez-Meza et al, (International Journal of Cosmetic Surgery (Vol. 6, 1998, pp 80-84)
Human hair transplants 30 hair transplant patients, found GHK-Cu analog reduced the shedding of transplanted hair from 30 percent with saline to 10 percent. The healing time of the transplanted grafts was cut in half. Regrowth of new hair from the transplants occurred in six to eight weeks with saline and four to six weeks with GHK-CU analog. Patient satisfaction after transplantation rose from 80 percent to 95 percent. Hitzig,G. Enhanced healing and growth in hair transplantation using copper peptides, Cosmetic Dermatol 2000 (June) 13, 18-21

GHK was originally isolated by its actions on increasing the survival of rat liver organ cultures. GHK also increases repair of liver damage in rats.

GHK-Cu was tested for healing of bone tissue

Growth of bone chondrocytes

The effect of GHK-Cu on the stimulation of new bone production in guinea pigs was studied.The authors prepared 7.5% and 12.5% collagen gels, supplemented with the tripeptide GHK-Cu, perfloxacine and hypersulphated glycosaminoglycan (HSGAG).

GHK-Cu stimulates bone healing in animals and the functions of bone repair cells in culture. The development of GHK-Cu for clinical use is being conducted under the direction of Prof. Milan Adam (University of Prague, Photograph - on the left) . Adam developed a collagen-graft-glycosaminoglycan copolymer supplemented with GHK-Cu for bone healing.


Anti-Inflammatory Actions: Damaged Tissue

The anti-oxidant actions of GHK and GHK-Cu that help to protect injured tissue appear to have multiple actions. These are (1) a direct anti-inflammatory of the copper-peptide complex, (2) an activity that blocks the release of free iron from ferritin molecules, (3) an ability to block tissue damage caused by interleukin-1 at a GHK-Cu concentration of about 10exp(-10) M, and (4) an ability to block the oxidation of low density lipoproteins (LDL) by free copper.

Anti-Oxidant Actions

The reactions between nickel ions and GHK and similar oligopeptides were characterized by spin trapping experiments.

GHK-Cu possessed superoxide dismutase and catalase-like activities.

Beretta G, Artali R, Regazzoni L, Panigati M, Facino RM, Glycyl-histidyl-lysine (GHK) is a quencher of alpha,beta-4-hydroxy-trans-2-nonenal: a comparison with carnosine. insights into the mechanism of reaction by electrospray ionization mass spectrometry, 1H NMR, and computational techniques.
Chem Res Toxicol. 2007 Sep20(9):1309-14.

Beretta G, Arlandini E, Artali R, Anton JM, Maffei Facino R.

Acrolein sequestering ability of the endogenous tripeptide glycyl-histidyl-lysine (GHK): Characterization of conjugation products by ESI-MS(n) and theoretical calculations.
J Pharm Biomed Anal. 2008 Jul 1547(3):596-602.

Anti-Inflammatory Actions: General

Anti-Oxidant and Anti-Inflammatory Actions of GHK and GHK-Cu

GHK and GHK-Cu may function as the circulating human non-steroidal anti-inflammatories (NSAIDs). In human plasma there is about 200 nanograms per milliliters of GHK and GHK-Cu at age 20. This declines to about 80 nanograms per milliliter at age 60 but these levels are highly variable. Given the respective binding constants for copper(+2) between GHK and albumin in human plasma, it is likely that only about 10% of circulating GHK is chelated with copper(+2). In areas of tissue damage, this ratio could be higher because of lowered albumin concentrations. There are very close similarities between the three dimensional chemical structures of GHK-Cu and H2-Receptor antagonists used as anti-ulcer medicines such as cimetidine, ranitidine, famotidine and nizatidine. Since GHK-Cu is a normal component of saliva present at about 40 nanograms/milliliter, it may function a natural protector of gastrointestinal linings. Also, most common anti-ulcer drugs are potent binders of ionic copper (II).There are also similarities, though less obvious, between most Non-Steroidal Anti-Inflammatory Drugs (NAISDs) and GHK. Virtually all NSAIDs avidly bind copper(+2).

Study Result Reference
Development of tissue protective analogs of GHK-Cu GHK-Cu and analogs were tested for anti-oxidant and tissue protective properties GHK-Cu and analogs were found to enhance or restore resistance to oxidative or inflammatory damage. Certain analogs were 100-fold more effective than GHK-Cu. US Patent 5,118,665 New anti oxidative and anti-inflammatory metal peptide complexes - containing glycine, histidine and lysine residues used to enhance or restore resistance to oxidative or inflammatory damage. Pickart
Blocking of iron oxidation A study of whether some of the wound healing properties of GHK-Cu are due to an affect on iron metabolism. The presence of iron complexes in damaged tissues is detrimental to wound healing, due to local inflammation, as well as microbial infection mediated by iron. The effects of GHK:Cu(II) on iron catalyzed lipid peroxidation. GHK:Cu(II) inhibited lipid peroxidation if the iron source was ferritin. Whereas GHK:Cu(II) inhibited ferritin iron release it did not exhibit significant superoxide dismutase-like or ceruloplasmin activity. It appears that GHK-Cu binds to the channels of ferritin involved in iron release and physically prevents the release of fee). Thus, a biological effect of GHK:Cu(II), related to wound healing, may be the inhibition of ferritin iron release in damaged tissues, preventing inflammation and microbial infections. Effects of glycyl-histidyl-lysyl chelated Cu(II) on ferritin dependent lipid peroxidation. Miller, DeSilva, Pickart, Aust. Pickart and Aust (Biotechnology Center, Utah State University, Logan, UT, USA) Adv. Exp Med Biol 1990264:79-84
Finding of superoxide dismutase and catalase-like activities in GHK Nickel complexes Redox chemistry of complexes of nickel) with some biologically important peptides in the presence of reduced oxygen species. Coterie N Tremolieres E Berliner JCL Cattier JP Henichart JP (INSERM, Ill, France) J Internat BioPharm 1992 Apr46(1):7-15
Cytoprotective actions against oxygen free radicals GHK-Cu markedly inhibited intestinal mucosal tissue from lipid peroxidation by oxygen-derived free radicals. Alberghina M, Lupo G, La Spina G, Mangiameli A, Gulisano M, Sciotto D, Rizzarelli E, Cytoprotective effect of copper(II) complexes against ethanol-induced damage to rat gastric mucosa, J Inorg Biochem. 1992 Mar45(4):245-59.
Anti-oxidant protection of insulin secreting cells after injury. Interleukin beta (IL-1 beta) is released during injuries and after tissue damage. IL-1 inhibits insulin release by pancreatic cells. The study tested whether GHK-Cu would block the IL-1 damage to insulin secreting pancreatic cells. Rat pancreatic islet cells were incubated with or without 50 U/ml IL-1 beta, in the presence or absence of various concentrations of Cu(II)-GHK or CuSO4 (1-1000 ng/ml). After incubation, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets, basal insulin secretion was 92 +/- 11 ( pg/islet) and glucose-induced release was 2824 +/- 249. In islets pre-exposed to 50 U/ml IL-1 beta, basal insulin release was not significantly affected but glucose- induced insulin release was greatly reduced (841 +/- 76 ). In islets incubated with IL-1 beta and Cu-GHK (0.4 mumol/l, maximal effect) basal secretion was 119.0 +/- 13 and glucose-induced release was 2797 +/- 242. CuSO4 was without protective actions. Copper addition prevents the inhibitory effects of interleukin 1-beta on rat pancreatic islets, Vinci, Caltabiano, Santoro, Rabuazzo, Buscema, Purrello, Rizzarelli, Vigneri and Purrello (University of Catania Medical Endocrinology, University of Catania Medical School, Italy) Diabetologia 1995 38(1):39-45
Effect of GHK on blocking oxidative damage that produces Alzheimer's disease Loosely bound copper(II) can produce oxidation of amyloid protein of Alzheimer's disease and cause neurodegeneration Loosely bound copper(II) can produce oxidation of amyloid protein of Alzheimer's disease and cause neurodegeneration The amyloid precursor protein of Alzheimer's disease in the reduction of copper(II) to copper(I). Multhaup Schlicksupp Hesse Beher Ruppert Masters Beyreuther ( ZMBH-Center for Molecular Biology University of Heidelberg, Germany) Science 1996 Mar 8271(5254):1406-9
Increase superoxide dismutase in wounds Biotin was attached to GHK then bound to collagen films. This gave increased wound contraction, increased cell proliferation, and produced a high expression of the antioxidant superoxide dismutase. Tissue copper levels were increased 9-fold. Arul V, Gopinath D, Gomathi K, Jayakumar R. Biotinylated GHK peptide incorporated collagenous matrix: A novel biomaterial for dermal wound healing in rats. Biomed Mater Res B Appl Biomater. 2005 May73(2):383-91
GHK detoxifies 4-hydroxy-2-nonenal, a toxic mlecule GHK blocks the toxic actions of 4-hydroxy-2-nonenal (HNE), a fatty acid decomposition product felt to be causative in the development of diabetes, nephropathy, retinopathy, and neurodegenaerative diseases.
GHK blocks block acrolein production Acrolein is a toxin created by carbonyl radicals from polyunsaturated fatty acids. GHK detoxifies acrolein. The authors suggest that GHK may be of value in prevention of atherosclerosis, diabetes, neuropathy, and Alzheimer disease.
Similarities between GHK and Anti- Ulcers Pharmaceutics
Note the 3 main components -
(1) an N-terminal side change,
(2) and central imidazole ring,
(3) a C-terminal - lysine-like - very basic group

Intestinal and Stomach Healing

GHK-Cu has potent effects on the healing of stomach ulcers and intestinal inflammations. One small human study found a very positive effect of GHK-Cu on the healing of intestinal lesions in persons with refractory inflammatory bowel disease.

Effects of GHK-Cu on gastric acidity, mucous production and the development of ulcers
(Shay gastric ulcer model (95% ethanol) in rats)
Dosage of GHK-Cu Stomach pH Rats with visible gastric ulcers Gastric mucous production
none 2.3 72% Unobservable
1 milligram 3.8 33% ++
3 milligrams 4.7 none ++++
10 milligrams 6.7 none ++++

Likewise, GHK-Cu produced a similar blockage of rat duodenal ulcer formation (cystamine induced).

Healing of Intestinal and Stomach Ulcers

GHK-Cu was tested for healing of experimental stomach ulcers and intestinal damage in rats.

GHK-Cu healed experimental stomach ulcers and intestinal inflammation and damage

Alberghina M, Lupo G, La Spina G, Mangiameli A, Gulisano M, Sciotto D, Rizzarelli E, Cytoprotective effect of copper(II) complexes against ethanol-induced damage to rat gastric mucosa, J Inorg Biochem. 1992 Mar45(4):245-59.

16 patients with refractory inflammatory bowel disease were treated with rectally administered solutions of GHK-Cu.

After the 12 weeks treatment, there was a 60% reduction in severity bas measured by endoscopy, histopathology, and symptoms

Study Result Reference
Healing of stomach and intestinal ulcers Pickart US Patent 4,767,753 Copper complexes of histidyl-lysine polypeptide(s) for reducing stomach secretions, increasing stomach mucous and preventing ulcers. US Patent 5,023,237 Use of polypeptide or its copper complex for cytoprotection in treatment of intestinal and stomach ulcers, and to facilitate wound healing. US Patent 5,145,838 Methods and compositions for healing ulcers and peptide derivatives.
Cytoprotective actions against oxygen free radicals GHK-Cu markedly inhibited intestinal mucosal tissue from lipid peroxidation by oxygen-derived free radicals An open study of PC1020 (GHK-Cu) rectal solution in treatment of distal inflammatory bowel disease. Levine, Patt, Koren (University of Washington) World Congress of Dermatology, October 1994, Further details presented at 25th Annual Meeting of DDW, Levien, Patt, Koren,Joslin (University of Washington) May 1995

Anti-Pain, Anti-Anxiety, Self Confidence

GHK possesses anti-pain, anti-anxiety, and probably increased self-confidence actions. Such actions could have arisen as an evolutionary mechanism to increase the survival of an animal after physical injuries.

The anti-pain effect in rats are started at 0.5 milligrams/Kg while the anti-anxiety effects start at 0.5 micrograms/Kg, which is the level that the Russian and Ukranian groups observed dermal wound and bone healing.

Bobyntsev II, Chernysheva OI, Dolgintsev ME, Smakhtin MI, Belykh AE. [Effect of Gly-His-Lys peptide and its analogs on pain sensitivity in mice]. [Article in Russian]. Eksp Klin Farmakol. 201578(1):13-5.

Application of peptide gly-his-lys for analgesic effect in pain caused by thermal irritation.

See also RU patent 2421235.

Bobyntsev II, Chernysheva OI, Dolgintsev ME, Smakhtin, Belykh AE. Anxiolytic effects of gly-his-lys peptide and its analogs. Bull Exp Biol Med 2015 Apr 158:72608.

Chernysheva O.I. 1, Bobyntsev I.I. 1, Dolgintsev M.E. THE TRIPEPTIDE GLY-HIS-LYS INFLUENCE ON BEHAVIOR OF RATS IN THE TEST «OPEN FIELD»

Kursk State Medical University

A trainer called Skin Biology and said that the fighters were injecting themselves with 1 milligram of GHK before major fights to increase their self confidence and heal faster after the match.

It is possible that the GHK does increase self confidence. In some of the rat experiments, the effective dosage was 0.5 micrograms/Kg. If this is recalculated for a human weight of 70 KG, then 0.035 milligrams would be effective. Then the 1 milligram used by the fighters may be sufficient.

SRCP Biochemistry Related to Tissue Regeneration

When wound healing is inadequate, the healed area is often devoid of sensory abilities.

Studied the effect of GHK on nerve growth.

Gly-His-Lys supported chick neuron differentiation and viability in cell culture of various neurons - chick embryo PNS (ganglion trigeminale) and from CNS of embryonal rats (hippocampus) and dissociated cells from chick embryo cerebral tissue. The optimum concentrations of GHK for neuron outgrowth was 10 ng/ml. GHK increased the ratio of neurons to glial cell in culture.

GHK and cellular energy production

GHK was tested for effects on phosphoylase A activation.

GHK stimulated in dose-dependent fashion the activity of phosphorylase a in isolated rat hepatocytes. This effect was associated with increases in both IP3 production and [Ca++]. These effects of Gly-His-Lys were antagonized by losartan, a nonpeptide angiotensin II receptor antagonist (AT1 selective), which suggested that these receptors were involved in its effect. Binding competition experiments clearly indicated that Gly-His-Lys interacts with AT1 receptors.

Study Result Reference
Databank search for source of human GHK - Effect of GHK-Cu on collagen synthesis Determined possible sources of GHK and effect of GHK-Cu on collagen synthesis in two strains of human fibroblasts and embryonic lung fibroblasts. GHK is a very rare sequence appearing in only 8 human proteins sequences as of 1990. It appears three times in collagen and also exist in several inflammatory proteins - All fibroblast lines stimulated collagen synthesis at 10exp (-12) M and maximally at 10exp (-9) M Glycyl-l-histidyl-l-lysine, a triplet from the a2 (I) chain of human type I collagen, stimulates collagen synthesis by fibroblast cultures Maquart, Gillery, Monboisse, Pickart, Laurent and Borel Ann. N.Y. Acad. Sci. 580:573-575, 1990
Search for source of GHK Studies on a skin structural protein called SPARC. GHK and HGHK, two peptides with very high copper-binding affinity are angiogenic. They are generated by the breakdown of SPARC, a structural protein in skin which is rich in cysteine. SPARC may function at several levels to control the progression of neovessels. Proteolysis of SPARC by plasmin results in the release of peptides containing the sequence Gly-His-Lys, which are angiogenic in vitro and in vivo. At later stages of angiogenesis when endothelial cell proliferation ceases, SPARC may exert inhibitory effects on angiogenesis. Regulation of angiogenesis by extracellular matrix: the growth and the glue. Sage and Vernon (University of Washington School of Medicine, Seattle, WA, USA) J Hypertens Suppl 1994 12(10):S145-52
Effect of GHK on chemoattraction of healing associated immune cells Compared GHK to known potent chemoattractants and GHK analogs. GHK was most potent chemoattractant tested for mast cells, but Gly-His-Gly, His-Lys, and His-Gly-Gly were inactive. Stimulation of rat peritoneal mast cell migration by tumor derived peptides. Poole and Zetter (Harvard Medical School) Cancer. Res. 43, 5857-5861, 1983
Effect of GHK on chemoattraction of healing associated immune cells Compared GHK to known potent chemoattractants and GHK analogs. An implantable device was used for the study of leukocyte chemoattraction in rats for up to 18 days. GHK attracted wound healing immune cells (mast cells, macrophages, polymorphonuclear leukocytes) at about 10exp (-10) M. An in vivo assay for chemoattractant activity. Zetter, Rasmussen and Brown (Harvard University Medical School, Boston, MA, USA) Lab Invest 1985 53(3):362-8
Heparin is felt to mediate some healing events GHK-Cu and heparin which is a natural anti-coagulant and a mediator of wound healing. GHK found to bind to heparin in nuclear magnetic resonance spectroscopy studies. Binding of the growth factor glycyl-L-histidyl-L-lysine by heparin. Rabenstein, Robert and Hari (University of California at Riverside, USA) FEBS Lett 1995, 376, pp. 216-20
Angiogenesis in rabbit cornea model and capillary cell migration GHK-Cu tested for angiogenic activity. Copper complexes of glycyl-L-histidyl-L-lysine and heparin induced angiogenesis in rabbits. Copper deficient rabbits cannot induce angiogenesis. Ceruloplasmin, copper ions, and angiogenesis. Raju, Alessandri, Ziche and Gullino (National Cancer Institute, Bethesda, MD, USA) J Natl Cancer Inst 1982 69(5):1183-8
Angiogenesis in rabbit cornea model and capillary cell migration GHK-Cu tested for angiogenic activity and effect on capillary cell migration. Increased angiogenesis in rabbit cornea model - Increased in vitro migration of capillary cells by 8-fold Characterization of a chemoattractant for endothelium induced by angiogenesis effectors. Raju, Alessandri, Gullino (National Cancer Institute, Bethesda, MD, USA) Cancer Res. 44:1579-1584, 1984
Nerve Growth - comment
Nerve Growth Studied the effect of GHK on nerve growth. Gly-His-Lys supported chick neuron differentiation and viability in cell culture and increased nerve outgrowth . The optimum concentrations of GHK for neuron function were 100-400 ng/ml. Effects of synthetic tripeptide on the differentiation of dissociated cerebral hemisphere nerve cells in culture. Sensenbrenner Jaros Moonen, Mandel (University of Strausbourg, France) Neurobiology 1975 5(4):207-13
Nerve Growth Uber die Wirkung eines synthetischen Tripeptids auf in vitro kultiviertes Nervengewebe (The effect of a synthetic tripeptide nervous tissue cultured in vitro), Lindner, Grosse, Halle and Henklein (Karl Marx University, Berlin, Germany) Z Mikrosk Anat Forsch 197993(5):820-8
Nerve Growth Severed nerves are placed in a collagen tube impregnated with GHK. This caused an increased the production of Nerve Growth Factor and the neurotrophins NT-3 and NT-4 Ahmed MR, Basha SH, Gopinath D, Muthusamy J, Jayakumar RJ, Initial upregulation of growth factors and inflammatory mediators during nerve regeneration in the presence of cell adhesive peptide-incorporated collagen tubes, J Peripher Nerv Syst. 2005, 10:17-30
GHK was tested for maintaining fibroblast viability in serum free medium Chick fibroblast were maintained in serum free medium after the addition of GHK. The GHK containing culture medium allowed studies of factors affecting collagen metabolism without the complications of the proteins from serum. An in vitro model of fibroblasia - Simultaneous quantification of fibroblast proliferation, migration, and collagen synthesis. Graham, Diegelmann and Cohen (Medical College of Virginia, Richmond, VA, USA) Proc Soc Exp Bio Med 176, 302-308, 1984
Collagen synthesis in cultured fibroblasts The effect of GHK-Cu was determined. GHK-Cu stimulated collagen synthesis in cultured fibroblasts. The stimulation began between 10exp (-12) and 10exp (-11) M, maximized at 10exp (-9) M, and was independent of any change in cell number. The presence of a GHK triplet in the alpha 2(I) chain of type I collagen suggests that the tripeptide might be liberated by proteases at the site of a wound and exert in situ healing effects. Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+. Maquart, Pickart, Laurent, Gillery, Monboisse and Borel (Laboratoire de Biochimie, CNRS URA 84, Faculte de Medecine, Reims, France. FEBS Lett 1988, 238(2):343-6
Collagen synthesis in cultured fibroblasts The effect of GHK-Cu on collagen production was determined. GHK-Cu increased collagen synthesis at 10exp (-9) M Requirement of Copper and Tripeptide Glycyl-L-Histidyl-L-Lysine Complex Formation for Collagen Synthesis Activity in Normal Human Dermal Fibroblasts. Oddos, T, Jumeau-Lafond, A Johnson & Johnson, Val de Reuil, France, Ries, G, Johnson & Johnson, Dusseldorf, Germany Abstract P72, American Academy of Dermatology Meeting, February 2002
Sulfated glycosaminoglycan (water-holding proteins) synthesis in cultured fibroblasts. The effect of GHK-Cu on the synthesis of sulfated glycosaminoglycan (water-holding proteins) in cultured fibroblasts was determined. GHK-Cu induced a dose-dependent increase of the synthesis of total GAGs secreted into the culture medium and those associated with the cell layer. The effect of GHK-Cu increased with dosage and was optimal at 10exp (-9) to 10exp (-8) M. Higher concentrations had less effect the rate of synthesis. GHK-Cu preferentially stimulated the synthesis of extracellular dermatan sulfate and cell layer associated heparin sulfate. Stimulation of sulfated glycosaminoglycans by the tripeptide copper complex glycyl-l-histidyl-l-lysine copper(II). Wegrowski, Maquart, Borel (University de Reims, France) Life Sci. 51, 1049-1056, 1992
The effects of GHK-Cu on the synthesis of glycosaminoglycans and small major proteoglycans was determined. A study of the effects of GHK- Cu in vivo, using the wound chamber model. Stainless steel wire mesh cylinders were implanted subcutaneously on the back of rats.GHK-Cu treated cultured fibroblasts and rat wound chambers had an increase in messenger RNA for decorin by not for biglycan. In both systems, GHK-Cu increased the synthesis of of decorin, dermatin sulfate and chondroitin sulfate. Expression of glycosaminoglycans and small proteoglycans in wounds: Modulation by the tripeptide copper complex glycyl-histidyl-lysine Cu(II). Simeon, Wegrowski, Bontemps and Maquart J Invest Dermatol 2000 Dec115(6):962-968
Wound healing events in "wound chambers" were studied in rats A study of the effects of GHK- Cu in vivo, using the rat wound chamber model. GHK-Cu treated rat wounds had a concentration dependent increase of dry weight, DNA, total protein, collagen, and glycosaminoglycan. The stimulation of collagen synthesis was twice that of noncollagen proteins. Type I and type III collagen mRNAs were increased. An increase of the relative amount of dermatan sulfate was also found. In vivo stimulation of connective tissue accumulation by the tripeptide- copper complex glycyl-L-histidyl-L-lysine-Cu2+ in rat experimental wounds. Maquart, Bellon, Chaqour, Wegrowski, Monboisse, Chastang, Birembaut and Gillery (Universite de Reims, France) J Clin Invest 92: 2368-76, 1993
Metalloproteinases that remove damaged proteins and scar tissue Metalloproteinases are a family of proteins that remove damaged proteins and scar tissue. The expression and activation of matrix metalloproteinases where investigated in a model of experimental wounds in rats, and their modulation by GHK-Cu. Wound chambers were inserted under the skin of rats and were injected daily with either injections of either 2 mg GHK-Cu or the same volume of saline. The wound fluid and the neosynthetized connective tissue deposited in the chambers were collected and analyzed for matrix metalloproteinase expression and/or activity. Interstitial collagenase increased in the wound fluid throughout the experiment and GHK-Cu did not alter its activity. Matrix metalloproteinase-9 (gelatinase B) and matrix metalloproteinase-2 (gelatinase A) were the two main gelatinolytic activities expressed during the healing process. Pro-matrix metalloproteinase-9 was strongly expressed during the early stages of wound healing (day 3) but decreased rapidly whereas in GHK-Cu treated chambers it persisted until day 22. Pro-matrix metalloproteinase-2 increased progressively until day 7, then decreased until day 18. Activated matrix metalloproteinase-2 increased until day 12, then decreased progressively whereas GHK-Cu increased pro-matrix metalloproteinase-2 and activated matrix metalloproteinase-2 during the later stages of healing. GHK-Cu increased metalloproteinases activity up to 4-fold which may increase the activity of wound remodeling processes which remove damaged protein and scar tissue. Expression and activation of matrix metalloproteinases in wounds: modulation by the tripeptide-copper complex glycyl-L-histidyl-L-lysine- Cu2+, Simeon Monier Emonard Gillery Birembaut, Hornebeck and Maquart (Faculte de Medecine, Reims, France) J Invest Dermatol 1999 112(6):957-64
Metalloproteinases and antiproteinases - commen t GHK-Cu increas es MMP-2 in cultured fibroblasts but in rat wounds it decreases MMP-2 and MMP-9. GHK-Cu also increases inhibitors of metalloproteinases TIMP-1 and TIMP-2 in cultured fibroblasts. The overall actions would appear to reduce proteolysis but perhaps maintain it at a lower level than in early stage wounds.
Metalloproteinases and antiproteinases that remodel tissue The effect of GHK-Cu on the induction of metalloproteinases in cultured wound fibroblasts. GHK-Cu at 10exp(-10) M increased MMP-2 mRNA and also inhibitors of metalloproteinases TIMP-1 and TIMP-2. The authors argue this indicate GHK-Cu modulates tissue remodeling. The tripeptide-copper complex GHK-Cu stimulates matrix metalloproteinases 2 expression by fibroblast cultures. Simeon, Emonard, Hornebeck & Maquart Laboratoire de Biochimie-UPRESA CNRS 6021, Faculte de Medecine, Reims, France. Life Sci 2000 Sep 2267(18):2257-65
Metalloproteinases and antiproteinases that remodel tissue GHK-Cu caused a significant decrease in wound area (64.5% GHK-Cu vs 28.2% control) by day 13. GHK-Cu also significantly lowered concentrations of TNF-alpha and MMP-2 and MMP-9. Canapp SO Jr, Farese JP, Schultz GS, Gowda S, Ishak A.M, Swaim SF, Vangilder J, Lee-Ambrose L, Martin FG, The effect of topical tripeptide-copper complex on healing of ischemic open wounds, Vet Surg. 2003 Nov-Dec32(6):515-23
Glycyl-histidyl-lysine interacts with the angiotensin II AT1 receptor. Garcia-Sainz JA, Olivares-Reyes JA. Departamento de Bioenergetica, Universidad Nacional Autonoma de Mexico, Mexico D. F. Peptides 199516(7):1203-7
Review of Copper Peptide Tissue Regeneration - Year 2000 Overview of biological and clinical results. Link to abstract of talk Prague, Czech Republic, May 19, 2000
GHK is a matrikine that stimulates tissue remodeling The term of "matrikine" has been proposed to designate such ECM-derived peptides able to regulate cell activity. GHK is a matrikine and a potent activator of ECM synthesis and remodeling Regulation de l'activite cellulaire par la matrice extracelulaire: le concept de matrikines. Maquart FX Simeon A Pasco S Monboisse JC. J Soc Biol 1999193(4-5):423-8
GHK-Cu and reduction of keloid scar formation GHK-Cu was tested for effects on the production of the keloid-producing hormone TGF-beta1 by normal and keloid-producing fibroblasts. GHK-Cu, at 10exp (-9) M reduced the production of TGF-beta1 in both normal and keloid-producing fibroblasts. In contrast, retinoic acid (Retin-A, Tretinoin) increases the production of scar producing TGF-beta1 in these lines. The effect of copper tripeptide and tretinoin on growth factor production in a serum-free fibroblast model. McCormack, M., Nowak KC, Koch, J. Arch Facial Plast Surg 2001 3: 28-32
Review of Copper Peptide Tissue Regeneration - Year 2002 Overview of biological and clinical results Pickart, L. Copper Peptides for Tissue Regeneration. Specialty Chemicals Oct. 9, 2002, 29-31.

Stimulation of Fingernail Growth

During prolonged rainy periods in Washington State, horses often develop severe irritations in their lower extremities &ndash especially where the hair covered skin joins to the hooves. This skin area can develop irritations, infections and bleeding. During experiments to heal such irritated skin with creams containing copper peptides, the application of the copper-peptide cream was often imprecise due to movements of the horses and the cream were generously applied to the lower skin of the leg and part of the upper hooves. While the creams were observed to rapidly heal the skin areas, we also unexpectedly observed that damaged hooves appeared to improve markedly in health. Later, we experimented with more controlled application of the copper peptide creams into cracks in badly damaged hooves. We found that the copper peptide cream usually produced a remarkable healing of the hooves and closure of the cracks.

Since the hooves of horses and the nails of humans are similar in terms of their biochemistry and cellular biology, we tested the application of such copper peptide creams to damaged human fingernails and toenails. We observed that such treatment produced in humans, as in the horses, a remarkable improvement in nail health and growth. Such types of copper-peptides, when applied to the nail matrix and nail bed area, enhance the process of nail growth resulting in stronger, thicker and smoother nails. Such types of copper-peptides have previously been found to strongly enhance the production of the protein collagen and also accelerate the repair of damaged skin. However, since nails are primarily composed of the hard protein keratin, it was not expected that copper-peptides would increase the production of keratin and nail growth.

We then studied the actions of a copper-peptide cream in an informal pilot study on nail growth in humans. The Placebo Control nails were treated with a similar cream that contained no copper-peptide. For testing experiments, the fingernail growth rates of the index finger were used as a measurement. In the first set of experiments, the copper peptide was applied to the index fingernail and cuticle on the right hand while the left hand nail was untreated and used as a control. In the second set of experiments with different volunteers, the cream was applied to the index fingernail and cuticle on the left hand while the right hand nail was treated with the placebo cream and used as a control. Nails were treated for four weeks. Nail length was measured from the end of the nail bed to the tip of the nail at its center by pushing a small plastic ruler under the nail and firmly against the nail bed. The nails were also visually inspected to determine that the treated nails appeared to be longer on the treated hand and this was readily apparent in all cases. The effect of nail growth stimulation was approximately similar if either the right hand treated group or the left hand treated group was used.As seen in Table 1, Nails treated with the copper-peptide complex had better nail growth. However, it must be emphasized this was not a blinded study.

Table 1 - Effect of Copper Peptide Product on Nail Growth
First Experiment (Sex: M or F) Finger Nail Growth with Copper Peptide Cream
(Millimeters in four weeks)
Finger Nail Growth with Placebo cream
(Millimeters in four weeks)
Right Hand Left Hand
Person 1M 4.2 2.7
Person 2M 3.2 2.1
Person 3F 4.3 3.2
Person 4F 3.8 2.9
Person 5F 3.9 2.6
Second Experiment Left Hand Right Hand
Person 6M 2.8 1.5
Person 7M 3.7 2.6
Person 8F 4.1 3.0
Person 9F 4.1 2.6
Person 10F 3.5 2.2

Improving Suntanning and Reducing Skin Peeling

This patent contains methods for increasing the efficiency of melanin formation and reducing post-tanning peeling damage. Decreasing the time in sunlight and post-tanning peeling would reduce overall ultraviolet exposure.

The entire approach to suntanning and sun protection is in a state of change. Many chemical sunscreens are being banned because of estrogenic actions and other toxicities. The combination of skin repair creams plus ultraviolet reflectors such as zinc oxide or titanium dioxide may be a better approach to sun protection.

Table 9. Improving Suntanning and Reducing UV Damage to Skin Cells

Improved suntanning methods Compositions and methods for skin tanning and protection. US Patent 5,698,184 Pickart
Blocks lethal ultraviolet radiation damage and inhibits the damaging glycation GHK blocks lethal ultraviolet radiation damage to cultured skin keratinocytes by binding and inactivating reactive carbonyl species such as 4-hydroxynoneal, acrolein, malondialdehye, and glyoxal. See microphotographs below which are courtesy of Lipotec Corporation (www.lipotec.com) . This protection was found at a relatively high level of GHK, 20 mg/ml or 0.2%, but this concentration can be easily added to protective sunscreens. Current sunscreens are built around molecules, such as oxybenzone, avobenzene, menthyl anthranilate, octyl methoxycinnamate, that strongly absorb ultraviolet radiation they later shed the energy by releasing it as free radicals. Such free radical absorbing chemicals prevent sunburn but many of these chemicals have strong estrogenic activities and there is concern over their accumulation in human tissues. GHK also inhibits the damaging glycation of copper, zinc-superoxide dismutase caused by fructose. GHK is more active than carnosine in this respect. J. Cebrian, A. Messeguer, R.M. Facino and J.M. Garcia-Anton, Inter. J. Cosm. Sci. 27, 271-278 (2005)

UVB radiation + HNE (4-Hydroxynonenal)

GHK + UVB + HNE (4-Hydroxynonenal)

GHK-Cu emerged during my attempts to reverse certain changes that occur during human aging. The goal was to suppress the synthesis of the blood fibrinogen, a protein that rises with age and rises even more after myocardial infraction. Its blood concentration is an excellent predictor of mortality. Elevated fibrinogen levels increase blood coagulation and decrease tissue perfusion, by increasing the thixotropic properties of blood in the microcirculation.

I found that the albumin fraction of human blood plasma has a suppressive action on fibrinogen synthesis and also improved the survival of the cultured liver cells that produce fibrinogen. Further isolations found these activities concentrated in a low molecular weight fraction that contained GHK-Cu. Subsequent work defined the three dimensional solution structure of GHK-Cu and the binding affinities between GHK and copper (II). My colleagues at the University of Washington (Seattle) and I used the structure of GHK-Cu to create analogs that were very potent cell growth inhibitors, inhibiting fibroblast replication at concentrations equivalent to chemotherapeutic drugs such as cisplatin and bleomycin. During surgical procedures to test these inhibitors on the suppression of tumor growth in mice, GHK-Cu was used as a control substance. It became apparent that GHK-Cu was rapidly healing the surgical incisions.

Later research found that Iamin is generated during tissue damage and suggests that, after tissue damage, GHK-Cu serves as a human feedback signal that has potent tissue protective properties and stimulates tissue remodeling after the initial phase of wound healing. It is postulated that a localized generation of GHK-Cu after tissue damage causes an influx of skin repair cells called macrophages which initiate skin repair mechanisms. The decrease in the blood concentrations of GHK-Cu during human aging may be a factor in the decreased tissue repair and subsequent increased organ failure that occurs during aging.

GHK-Cu - Function in Human Body

A number of names have been used in the scientific literature for GHK.

Names and identifications used for GHK
Systematic name N(2)-(N-Glycyl-L-histidyl)-L-lysine
CAS Registration number 49557-75-7
Molecular Formula C14-H24-N6-O4
General name Glycyl-histidyl-lysine
Synonyms Copper glycyl-histidyl-lysine
Copper(II)ghk
Glycylhistidyllysine
Gly-his-lys
Iamin
NSC 379527
Prezatide (or Prezatide Copper as a complex)

What is the function of GHK-Cu in the human body? The molecule is present blood plasma, saliva, and urine in physiologically active quantities. Evidence suggests that it is a biochemical feedback signal that is generated after tissue injury. It appears to have two main functions: (1) first as a potent tissue protective, anti-inflammatory agent that limits oxidative damage after tissue injury, and (2) as a signal that activates tissue remodeling, that is, the processes for removal of damaged protein and scar tissue and their replacement by normal tissue. Thus, GHK-Cu links the processes of removal of damaged scar tissue and deposition of new tissue.

GHK, which is generated in damaged tissues, directly accelerates many healing associated properties at concentration of around 10exp(-10)M. Some of the GHK stimulated effects seem to be directly mediated by GHK or GHK-Cu after it obtains copper (II) from albumin. Other actions of GHK and GHK-Cu are likely to arise from GHK-Cu's attraction of wound macrophages and other healing-associated immune cells which, in turn, release families of growth factor proteins appropriate to the repair of the damaged tissue.

Francois Maquart and colleagues at Reims have argued that GHK-Cu acts on the second phase of healing as an inducer of tissue remodeling processes. Further support for this concept is that the molecular weight of collagen fragments induced by GHK-Cu are much smaller than those produced in the early phase of wound repair. This suggests that, with the copper complex, collagen synthesis and degradation are simultaneously occurring. Also, in cell culture, GHK-Cu reduces the secretion of the scar producing protein, TGF-beta-1 by normal fibroblasts and keloid-producing fibroblasts. This, combined with GHK-Cu's healing activities, suggests that scar-free healing needs both, an activation of metalloproteinases, and a reduction in TGF-beta-1 production.

Proposed Mechanism Copper-Peptide GHK-Cu Actions After Tissue Injury

Information from a variety of sources allows us to propose a mechanism for GHK-Cu effects.The sequence of events of GHK-Cu induced effects appear to be as follows:

1. Initially after tissue damage, the first stage of wound healing processes is activated. These include localized blood coagulation, an early neutrophil invasion that secretes sterilizing oxygen radicals, and later an induction by growth factors, such as TGF-beta-1, of copious amounts of scar-forming collagen to form a protective covering over the injury.

2. A second stage of healing begins to be activated as disrupted cells release proteases that generate a population of peptides that include Gly-His-Lys and His-Gly-His-Lys, both of which have a very high affinity for copper (+2) ion.

3. The Gly-His-Lys and His-Gly-His-Lys begin to accumulate copper (+2) ion from albumin and form GHK-Cu and HGHK-Cu.

4. The accumulation of peptide-bound copper ion produces multiple anti-inflammatory effects that help to stop the actions of sterilizing oxygen radicals and permit the initiation of healing events. GHK-Cu blocks ferritin channels and the release of free (oxidative) iron, thus blocking iron catalyzed lipid peroxidation that occurs after injury. GHK-Cu blocks also interleukin-1 damage to tissue cells.

5. GHK-Cu released into the blood stream raises the body's production of, and circulating blood concentration of, wound macrophages that enhance repair.

6. GHK-Cu suppresses the synthesis of scar development by repressing fibroblast production of TGF-beta-1.

7. GHK-Cu also chemoattracts wound macrophages to the wound area. These macrophages act directly to stimulate healing by removing cellular debris and secreting a family of approximately 20 growth factor proteins.

8. GHK-Cu acts directly on fibroblasts to stimulate m-RNAs for collagen, elastin, proteoglycans, metalloproteinases, and TIMP-1 and TIMP-2. This in turn raises the levels of these proteins. This results in a condition whereby protein synthesis and deposition is occurring concomitant with protein breakdown that removes scar tissue and cellular debris remaining from the tissue disruption. Thus, GHK-Cu links scar reduction and the rebuilding of tissues.

9. GHK-Cu induces angiogenesis by serving as a chemoattractant to direct new blood capillaries to the wound area and by inducing the production of several protein essential for angiogenesis.

10. GHK-Cu induces neuronal outgrowth and re-innervation of the damaged tissues.

11. This mechanism of copper-peptide induced tissue repair appears to function for skin, hair follicles, the stomach lining, the intestinal lining, bone tissue, and hooves and fingernails.

12. This copper-peptide regeneration mechanism is different from most known biochemical hormonal response patterns such as the insulin-glucose system or the erythropoetin-red blood cell system. With these systems, a small change in the concentrations of glucose or red blood cells results in a precisely controlled release of the hormones to re-establish normal glucose or red blood cell levels.

13. The copper-peptide tissue remodeling is a much looser stimulus-response system - somewhat like a "fuzzy logic" response. Traumatic tissue damage is an inherently messy business - damage many be slight or massive. The bodily repair systems do not always receive rapid and clear information as to the extent of tissue damage - damages may be sudden and acute - or the result of a slow degenerative disease. This may explain why dermal scars and lesions last so long in adults the body just does not recognize the need to remove the imperfections.

The Need for Improved Skin Regenerative Copper Peptides

The first generation products designed around GHK-Copper performed well in many controlled tests, however, the products failed in FDA clinical trials on the healing of very difficult-to-heal human wounds (as have many other approaches). The fragility and rapid breakdown of GHK and similar peptides is the major problem in developing products for clinical and cosmetic use. In the human body, the GHK-Cu complex can be constantly generated. However, when used as a single dose therapy, its fragility leads to rapid breakdown, clearance from the dermis, and a loss of effectiveness.

In 1975, during attempts to isolate GHK from human blood, we found that the molecule was especially vulnerable to carboxypeptidases and was rapidly degraded by blood enzymes. Intradermal injections of GHK are cleared from the skin in approximately 30 seconds. If added to blood, GHK is rapidly degraded into constituent amino acids by blood enzymes. Endo, Miyagi, and Ujie also reported that GHK was rapidly degraded by blood plasma and rapidly eliminated from rats. (Reference, Endo, Miyagi, and Ujie, Kissei Pharmaceutical Co., Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization.J Chromatogr B Biomed Sci Appl 1997 Apr 25692(1):37-42)

This fragility and rapid breakdown of GHK and other simple copper peptide complexes is the major problem in developing products for clinical and cosmetic use. In the human body, the GHK-Cu complex can be generated constantly. However, when used as a single dose therapy, its fragility leads to rapid breakdown, clearance from the dermis, and a loss of effectiveness. A variety of chemical modifications to GHK have produced bioactive copper complexes with enhanced breakdown resistance. The problem with this classical organic chemistry approach is that each new chemical becomes, in FDA regulatory terms, a new chemical entity. This increases the possibility of undesirable side effects and much slower regulatory approvals.

Current Development of Improved Copper Peptides

Currently more effective copper peptides with tissue regenerative actions have been developed. Several hundred copper-peptide complexes were evaluated but none were significantly better than GHK-Cu. Some complexes, such as f-Met-Leu-Phe-Cu actually produced more scar formation. The principal defects of single peptides were a lack of stability and poor adhesion to the skin's surface.

We then tested fractions of peptides remaining after enzymatic digestion of various proteins. These peptides, being the end products of enzymatic digestion, proved to be very resistant to further enzymatic digestion. Further testing found the most promising peptides were a fraction of peptide fragments that remained after partial proteolysis of soy proteins. Such soy peptides have a very low antigencity and long history of safe use in cosmetic products and in solutions used clinically for intravenous alimentation. To increase adherence to skin, a peptide fraction with a significant percentage of sugar residues was used. Peptide digests with attached sugars have mucus-like properties and peptide fragments from collagen have been used as glues such as the "Le Pages" glues once used by school children. This helps adhere the active components of the cream to the skin or to wet wounds.

Similar mixed peptide digests of proteins were used to attract and concentrate wound macrophages in the 1930s. The digest was injected intraperitoneally into rats and 24 hours later the macrophages concentrated in the intraperitoneal cavity and were harvested.

When copper (II) is chelated to this peptide fraction, this produces copper peptides with very strong skin repair properties. In studies persons with nickel allergy, these copper peptides were also found to have strong anti-inflammatory actions, approximately equal to that observed for cortisone. Because of their breakdown resistance, such copper peptides can be used with skin exfoliating hydroxy acids for more rapid skin renewal and for scar reduction. These peptides have enhanced potency, breakdown resistance, a longer duration of action and very high adherence to skin.

In veterinary studies, creams made from these new copper complexes produced rapid and scar-free healing in dogs after spaying operations, and in young horses after leg-straightening operations. This allowed the dogs to be returned to their owners in four days instead of the usual five, while the foals were returned in five days instead of seven. In humans, four small, placebo-controlled studies found faster skin healing after skin injuries induced by tape stripping, acetone burns (removal of skin lipids), 24-hour detergent irritation, and nickel allergy inflammation.

Chemical Synthesis vs Biological Methods for Creation of Long-Acting Copper Peptides

Replaced histidine residue with either a synthetic amino acid, L-spinacine, or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid. Investigated with potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies.All the ligands formed copper complexes having different stoichiometries and stabilities.

After 3 hours in serum, GHK was degraded but the synthetic compounds showed no significant degradation.

Method to Develop Long Acting Copper Peptides Result Potential Problems Reference
Chemical Synthesis. A partially retro-inverso analogue of GHK was synthesized, in which the -CONH- bond between histidine and lysine was modified as -NHCO-. The new peptide analogue showed approximately a ten-fold increase in stability versus the parent peptide. This is a new chemical entity - Would require extensive safety testing. May lose anti-inflammatory activity H-Gly-His psi (NHCO)Lys-OH, partially modified retro-inverso analogue of the growth factor glycyl-L-histidyl-L-lysine with enhanced enzymatic stability. Dalpozzo A, Kanai K, Kereszturi G, Calabrese G. Int J Pept Protein Res 1993 Jun41(6):561-6
Chemical Synthesis. Synthetically modified GHK with breakdown resistant groups - These are new chemical entities - Would require extensive safety testing. May lose anti-inflammatory activity Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogs: chemical behavior and biological activity, Conato et al, Biochim Biophys Acta 1526, 199-210, 2001
Biological Synthesis. Use fraction of soybean protein enzymatic digest - these end result peptides are resistant to further enzymatic breakdown After 3 hours in serum, copper complexes of the soybean digest retained wound healing and anti-inflammatory activities when compared to native serum. None found - Passed all safety tests. These types of peptides have been extensively used for intravenous feeding, in food supplements, and in shampoos and conditioners Pickart US Patent 5,382,431 Tissue protective and regenerative compositions US Patent 5,554,375 Tissue protective and regenerative compositions US Patent 5,698,184 Compositions and methods for skin tanning and protection US Patent 5,888,522 Tissue protective and regenerative compositions .

Howard Maibach directed four studies on human skin repair using these breakdown resistant copper peptides formulated into a non-ionic cream base. These placebo-controlled, double-blinded studies gave statistically significant positive results that the copper-peptide creams markedly accelerated the rate of skin recovery and reduced irritation after severe skin damage. The studies included (1) Acetone damaged skin has had the fats removed from the skin and produces damage similar to a very dry and cracking skin, (2) Detergent damaged skin has many fats removed plus extensive damage to the outer layer of skin barrier proteins. This is the standard test for contact dermatitis, (3) Nickel allergy damaged skin is produced by applying nickel salts to the skin of sensitized patients. This is similar to the skin damage caused by other allergic responses such as poison ivy, poison oak and other allergens, and (4) Tape stripped skin which is similar to damaging scraps, abrasions, and small cuts on the skin's surface. A strip of tape is put on the skin, then quickly ripped off. This process is repeated about 50 times in the same place, ultimately producing a small wound on the skin.


The mixed copper-peptides uses a different approach than the single molecule GHK-Cu method. The digest contains millions of variant peptide copper ion complexes, more like the situation that arises after tissue damage and subsequent proteolysis. The physiological mechanism whereby the skin or other organs signal for repair is quite complex. However, for therapeutic use, a much simpler approach often suffices to induce tissue regeneration. In vivo, the active peptides must have a very high affinity for copper (+2) to obtain copper ion and form copper peptide complexes. But exogenously, we can easily complex mixtures of peptides, even with much lower affinity for copper ion, to copper (+2) simply by adding copper salts to a solution of the peptides.The scientific rational for the bioactivity of such copper peptides is that numerous peptide fragments are generated by proteolytic enzymes. A fraction of these peptides will have a high affinity to copper(+2) and a portion may have properties similar to GHK-Cu.

Comparison of attributes of GHK and HGHK vs Enzymatic Protein Digests
Biological Parameter GHK and HGHK Newer Enzymatic Protein Digests
Wound Macrophage Chemoattraction- Very High High
Copper Binding Affinity - Very High Moderate, Must add copper ion to mixture
Antigenicity - None None in type currently used / Passed human antigenicity testing and animal tests
Healing activity in animals models - Moderate High
Adherence to skin and wounds- Low Very high, forms a biological "glue"
Clearance from applied area - Rapid Slow

Importance of Careful Formulation of Copper Peptide Complexes

With copper-peptide creams, great care must be taken to produce a cream that has minimal interactions with the ionic copper in the cream. Formulators who prepare skin creams usually have only a limited knowledge of chemistry and know virtually nothing about cell biology, hormonal interactions, and general biochemistry. It is easily possible to produce copper-containing creams, lotions, and solutions that neutralize copper-peptide effects by the interaction of various components with ionic copper. It is also possible to create copper-complexes that inhibit cell replication. It should be emphasized that the GHK-Cu work arose from National Cancer Institute funded projects for the development of growth inhibitory analogs of GHK-Cu. During this project, GHK-Cu analogs were synthesized that inhibited fibroblast replication at 10exp (-16) M. Some companies have recently sold cosmetic skin products using EDTA-copper, but this complex inhibits fibroblast function and skin repair. Any product should be carefully tested for its effect on skin repair.

For the copper-peptide studies, we formulated a non-ionic, but stable cream. For cream preservation, Germaben II was used which our testing found to have no influence on the wound repair process. This product contains diazolinydinyl urea, methylparaben, and propylparaben and produces self-sterilizing creams with excellent preservation for at least four years at room temperature. A drawback of the preservative system is that some persons are sensitized to parabens but there is no perfect preservative for wound healing products. Benzoyl alcohol should never be used as a preservative for wound healing products, since it inhibits the replication of wound fibroblasts.

GHK Suppresion of Cancer Metastasis Genes, Decorin, and Growth Inhibitory Analogs of the GHK copper binding region

GHK supresses human cancer metastasis genes but because of the cell culture system used, it is likely that the GHK was converted to GHK-copper. GHK-copper also increases the protein Decorin (Named because it "decorates" the collagen strands). Decorin has a perplexing mixture of effects: Regeneration of muscles and nerves, reduction of scarring, and inhibition of tumor growth and cancer metastasis in animals.

GHL Suppression of Cancer Metastasis

The authors searched for substances that can reverse expression of those genes involved in human colon cancer metastasis.

To determine gene expression of human normal and cancerous cells, they measured RNA produced by cells. To find substances that can reverse expression of those genes involved in metastasis, they used very reliable database developed at the Broad Institute (Massachusetts Institute of Technology and Harvard) of 7000 genome wide expression profiles after treatment of 4 human cell lines with 1309 bioactive substances. Only two substances, GHK and securenine (an alkaloid) from the choice of 1309 were able to quiet down the genes involved in tumor spreading. The authors also mention low toxicity of GHK and the low concentration (1 micromolar) that produces desired effect.

GHK suppresses human cancer metastasis genes

Clin Exp Metastasis. 2010 Feb27(2):83-90. Epub 2010 Feb 9.

A 'metastasis-prone' signature for early-stage mismatch-repair proficient sporadic colorectal cancer patients and its implications for possible therapeutics.

Hong Y, Downey T, Eu KW, Koh PK, Cheah PY.,Department of Colorectal Surgery, Singapore General Hospital, Singapore, 169608, Singapore.

Metastasis is the major cause of cancer mortality. We aimed to find a metastasis-prone signature for early stage mismatch-repair proficient sporadic colorectal cancer (CRC) patients for better prognosis and informed use of adjuvant chemotherapy. The genome-wide expression profiles of 82 age-, ethnicity- and tissue-matched patients and healthy controls were analyzed using the Affymetrix U133 Plus 2 array. Metastasis-negative patients have 5 years or more of follow-up. A 10 x 10 two-level nested cross-validation design was used with several families of classification models to identify the optimal predictor for metastasis. The best classification model yielded a 54 gene-set (74 probe sets) with an estimated prediction accuracy of 71%. The specificity, sensitivity, negative and positive predictive values of the signature are 0.88, 0.58, 0.84 and 0.65, respectively, indicating that the gene-set can improve prognosis for early stage sporadic CRC patients. These 54 genes, including node molecules YWHAB, MAP3K5, LMNA, APP, GNAQ, F3, NFATC2, and TGM2, integrate multiple bio-functions in various compartments into an intricate molecular network, suggesting that cell-wide perturbations are involved in metastasis transformation. Further, querying the 'Connectivity Map' with a subset (70%) of these genes shows that Gly-His-Lys and securinine could reverse the differential expressions of these genes significantly, suggesting that they have combinatorial therapeutic effect on the metastasis-prone patients. These two perturbagens promote wound-healing, extracellular matrix remodeling and macrophage activation thus highlighting the importance of these pathways in metastasis suppression for early-stage CRC.

GHK-copper increases decorin which suppresses cancer growth and metastasis

Metastases in breast cancer are a vital concern in treatment, with epidermal growth factor receptor and ErbB2 strongly implicated in mediating tumor invasion and spreading. In this study, we investigated the role of decorin in suppressing both primary breast carcinomas and pulmonary metastases. We show that decorin causes marked growth suppression both in vitro and in vivo using a metastatic breast cancer cell line and an orthotopic mammary carcinoma model. Treatment with decorin protein core reduced primary tumor growth by 70% and eliminated observed metastases. An adenoviral vector containing the decorin transgene caused primary tumor retardation of 70%, in addition to greatly reducing observed metastases. Moreover, we demonstrate that ErbB2 phosphorylation and total receptor protein levels are reduced in this model system upon de novo expression of decorin under the control of a doxycycline-inducible promoter. Primary tumor growth in vivo was reduced by up to 67% upon decorin induction, and pulmonary metastases were markedly hampered as well. These effects are likely occurring through decorin's long-term downregulation of the ErbB2 tyrosine kinase cascade. These results demonstrate a novel role for decorin in reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapeutics for metastatic breast cancer.

Decorin, a member of the small leucine-rich proteoglycan gene family, down-regulates members of the ErbB receptor tyrosine kinase family and attenuates their signaling, leading to growth inhibition. We investigated the effects of decorin on the growth of ErbB2-overexpressing mammary carcinoma cells in comparison with AG879, an established ErbB2 kinase inhibitor. Cell proliferation and anchorage-independent growth assays showed that decorin was a potent inhibitor of breast cancer cell growth and a pro-apoptotic agent. When decorin and AG879 were used in combination, the inhibitory effect was synergistic in proliferation assays but only additive in both colony formation and apoptosis assays. Active recombinant human decorin protein core, AG879, or a combination of both was administered systemically to mice bearing orthotopic mammary carcinoma xenografts. Primary tumor growth and metabolism were reduced by approximately 50% by both decorin and AG879. However, no synergism was observed in vivo. Decorin specifically targeted the tumor cells and caused a significant reduction of ErbB2 levels in the tumor xenografts. Most importantly, systemic delivery of decorin prevented metastatic spreading to the lungs, as detected by novel species-specific DNA detection and quantitative assays. In contrast, AG879 failed to have any effect. Our data support a role for decorin as a powerful and effective therapeutic agent against breast cancer due to its inhibition of both primary tumor growth and metastatic spreading.

Lung metastasis is the most crucial event affecting the therapeutic outcome of osteosarcoma. The prevention of lung metastasis is therefore important in improving the prognosis of patients with osteosarcoma. Decorin is a major extracellular matrix protein which has become the focus of various cancer studies. The biological role of decorin in osteosarcoma has yet to be clarified. The aim of this study was to examine the potential of decorin as a novel biological target for the treatment of osteosarcoma. In this study, the LM8 murine osteosarcoma cell line (LM8) with high metastatic potential to the lung was used. The two cell lines established were LM8-DCN which stably expressed human decorin (hDCN) and LM8-mock, established as a control. The LM8-DCN cell line was subcutaneously injected into the backs of mice. Significantly fewer pulmonary metastases were observed in mice with LM8-DCN compared to mice inoculated with LM8 and LM8-mock (P<0.001). In addition, the mice in the LM8-DCN inoculated group survived significantly longer than those in the LM8 and LM8-mock inoculated group, based on the Kaplan-Meier survival analysis and log-rank tests (P<0.005). The effect of decorin on the growth rates, motility and invasion ability of LM8 was investigated in vitro. There was no difference in the morphology and growth rates, but the motility and invasion of LM8 were inhibited by decorin. These results suggest that decorin has the therapeutic potential to prevent lung metastasis in osteosarcoma.

Oncology. 200977(2):92-9. Epub 2009 Jul 7.
Decorin suppresses bone metastasis in a breast cancer cell line.

Araki K, Wakabayashi H, Shintani K, Morikawa J, Matsumine A, Kusuzaki K, Sudo A, Uchida A. Department of Orthopaedic Surgery, Mie University Graduate School of Medicine, Mie, Japan.

Decorin, the prototype of an expanding family of small leucine-rich proteoglycans, is involved in a number of cellular processes including matrix assembly, fibrillogenesis and the control of cell proliferation. In this study, we investigated the role of decorin in suppressing tumor aggressiveness and bone metastases. We used a metastatic breast cancer cell line, MDA-MB-231, to show that decorin causes marked growth suppression bothin vitro and in vivo. A cytomegaloviral vector containing the decorin transgene caused greatly reduced cell growth, motility and observed metastases. Bone metastases were decreased by >90% upon decorin transfection. These results demonstrate a novel role for decorin in the reduction or prevention of tumor metastases in this breast cancer model and could eventually lead to improved therapies for metastatic breast cancer.

Epidermal growth factor receptor (EGFR) and androgen receptor (AR) pathways play pivotal roles in prostate cancer progression. Therefore, agents with dual-targeting ability may have important therapeutic potential. Decorin, a proteoglycan present in the tumor microenvironment, is known to regulate matrix assembly, growth factor binding, and receptor tyrosine kinase activity. Here, we show that in prostate-specific Pten(P-/-) mice, a genetically defined, immune-competent mouse model of prostate cancer, systemic delivery of decorin inhibits tumor progression by targeting cell proliferation and survival pathways. Moreover, in human prostate cancer cells, we show that decorin specifically inhibits EGFR and AR phosphorylation and cross talk between these pathways. This prevents AR nuclear translocation and inhibits the production of prostate specific antigen. Further, the phosphatidylinositol-3 kinase (PI3K)/Akt cell survival pathway is suppressed leading to tumor cell apoptosis. Those findings highlight the effectiveness of decorin in the presence of a powerful genetic cancer risk and implicate decorin as a potential new agent for prostate cancer therapy by targeting EGFR/AR-PI3K-Akt pathways.

Decorin, the prototype member of the small leucine-rich proteoglycans, resides in the tumor microenvironment and affects the biology of various types of cancer by downregulating the activity of several receptors involved in cell growth and survival. Decorin binds to and modulates the signaling of the epidermal growth factor receptor and other members of the ErbB family of receptor tyrosine kinases. It exerts its antitumor activity by a dual mechanism: via inhibition of these key receptors through their physical downregulation coupled with attenuation of their signaling, and by binding to and sequestering TGFbeta. Decorin also modulates the insulin-like growth factor receptor and the low-density lipoprotein receptor-related protein 1, which indirectly affects the TGFbeta receptor pathway. When expressed in tumor xenograft-bearing mice or injected systemically, decorin inhibits both primary tumor growth and metastatic spreading. In this review, we summarize the latest reports on decorin and related molecules that are relevant to cancer and bring forward the idea of decorin as an anticancer therapeutic and possible prognostic marker for patients affected by various types of tumors. We also discuss the role of lumican and LRIG1, a novel cell growth inhibitor homologous to decorin.

P63 as a Cancer Inhibitor

There is some evidence that protein P63 also inhibits cancer growth.

Arch Dermatol Res. 2009 Apr301(4):301-6. Copper-GHK increases integrin expression and p63 positivity by keratinocytes. Kang YA, Choi HR, Na JI, Huh CH, Kim MJ, Youn SW, Kim KH, Park KC. Department of Dermatology, Seoul National University College of Medicine, Yeongeon-dong, Jongno-gu, Seoul, Republic of Korea.

Glycyl-L-histidyl-L-lysyl (GHK) possesses a high affinity for copper(II) ions, with which it spontaneously forms a complex (copper-GHK). It is well known that copper-GHK plays a physiological role in the process of wound healing and tissue repair by stimulating collagen synthesis in fibroblasts. This study was conducted to investigate the effects of copper-GHK on keratinocytes. Proliferative effects were analyzed and hematoxylin and eosin staining and immunohistochemistry were conducted to evaluate the effects of copper-GHK in skin equivalent (SE) models. In addition, western blotting was performed. In monolayer cultured keratinocytes, copper-GHK increased the proliferation of keratinocytes. When the SE models were evaluated, basal cells became cuboidal when copper-GHK was added. Immunohistochemical analysis revealed that copper-GHK increased proliferating cell nuclear antigen (PCNA) and p63 positivity. Furthermore, the expression of integrin alpha6 and beta1 increased in SE models, and these results were confirmed by Western blotting. The results of this study indicate that treatment with copper-GHK may increase the proliferative potential of basal keratinocytes by modulating the expression of integrins, p63 and PCNA. In addition, increased levels of p63, a putative stem cell marker of the skin, suggests that copper-GHK promotes the survival of basal stem cells in the skin.

Cell Death Differ. 2010 Apr 9. [Epub ahead of print]

p53-family proteins and their regulators: hubs and spokes in tumor suppression.
Collavin L, Lunardi A, Del Sal G. Laboratorio Nazionale CIB (LNCIB), AREA Science Park, Trieste, Italy, Dipartimento di Scienze della Vita, Università degli Studi di Trieste, Trieste, Italy.

Abstract
The tumor suppressor p53 is a central hub in a molecular network controlling cell proliferation and death in response to potentially oncogenic conditions, and a wide array of covalent modifications and protein interactions modulate the nuclear and cytoplasmic activities of p53. The p53 relatives, p73 and p63, are entangled in the same regulatory network, being subject at least in part to the same modifications and interactions that convey signals on p53, and actively contributing to the resulting cellular output. The emerging picture is that of an interconnected pathway, in which all p53-family proteins are involved in the response to oncogenic stress and physiological inputs. Therefore, common and specific interactors of p53-family proteins can have a wide effect on function and dysfunction of this pathway. Many years of research have uncovered an impressive number of p53-interacting proteins, but much less is known about protein interactions of p63 and p73. Yet, many interactors may be shared by multiple p53-family proteins, with similar or different effects. In this study we review shared interactors of p53-family proteins with the aim to encourage research into this field this knowledge promises to unveil regulatory elements that could be targeted by a new generation of molecules, and allow more efficient use of currently available drugs for cancer treatment.

Inhibition of the growth of cultured cells and an implanted fibrosarcoma by aroylhydrazone analogs of the Gly-His-Lys-Cu(II) complex. Biochem Pharmacol 1983 Dec 1532(24):3868-71.

Pickart L, Goodwin WH, Burgua W, Murphy TB, Johnson DK, Biochem. Pharmacol., 32, 1983, 3868-71.

Aroylhydrazones of pyridoxal and of salicylaldehyde, a series of tridentate chelating agents, are potent inhibitors of DNA synthesis and cell growth in a number of human and rodent cell lines grown in culture. A copper(II) complex of the most potent of the chelators, salicylaldehyde benzoyl hydrazone (SBH), exhibits significantly greater inhibitory activity than does SBH itself. Although the bioactive forms and mechanism of action of these agents are uncertain, their cytotoxic activity can equal or exceed that of many chelators and chelates previously known to possess such properties, including compounds used clinically. SBH and its copper complex are relatively non-toxic to mice and show some measure of selectivity in their effects on different cell types. It is possible that aroylhydrozones of this type and/or their metal complexes could prove to be useful therapeutic agents.

Hydrazone analogs of the GHK copper binding region are very potent inhibitors of DNA synthesis and cell growth. Furthermore, the wound repairing cell, the fibroblast, seems to be exceptionally sensitive to certain types of copper-complex inhibitors. This makes the preparation of tissue regenerative copper complexes difficult since even some types of peptide copper complexes inhibit fibroblasts as do other small complexes such as diethylamine-copper and triethylamine-copper.


GHK-Cu and hydrazone analogs of GHK copper binding region that that are very potent inhibitors of cell replication

Top: Copper binding region of GHK

Middle: PCPH-Cu (2-Pyridinecarboxaldehyde 2''-pyridylhydrazone hydrazone copper(2+))

Bottom: SBH-Cu (Salicylaldehyde benzoyl hydrazone copper(2+))

Concentrations of PCPH-Cu, SBH-Cu, Cisplatin and Bleomycinsynthesis 50%

Cell Types in Culture PCPH-Cu
Nanograms per ml for 50% cell reduction SBH-Cu
Nanograms per ml for 50% cell reduction Cisplatin
Nanograms per ml for 50% cell reduction Bleomycin
Nanograms per ml for 50% cell reduction Normal human fibroblasts 0.00003 0.1 Not done - Normal human kidney 45 - - Mouse fibrosarcoma 0.511 2.4 32 Human bladder cancer 12 34 95 7 Human lung epithelial cancer 580 410 870 44 Human melanoma 0.05 270 6,000 80,000

References on chemistry of GHK-Cu

Studies on GHK and structure and activity. A variety of GHK-Cu analogs were synthesized and tested for their stimulation of DNA synthesis.

While many analogs similar to GHK-Cu had bioactivity, none equaled the bioactivity of GHK-Cu

Growth modulating human plasma tripeptide: Relationship between molecular structure and DNA synthesis in hepatoma cells. Pickart and Thaler FEBS Lett 1979, 104(1):119-22 Cell culture studies of the effects of GHK complexed with copper and iron on cell growth patterns. Experimental observations that GHK is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate.At physiological pH in vitro, GHK associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHK acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures. These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHK in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHK. GHK and transition metals, which appear to form complexes prior to interaction with cells. Growth modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes. Pickart and Thaler (University of California, San Francisco, USA) J Cell Physiol 1980, 102(2):129-39 GHK and copper(II) transport into cells. The association of GHK with copper and a homology similarity between the tripeptide and the copper transport sites on albumin and alpha- fetoprotein, where the cupric atom is bound to a histidyl residue adjacent to a basic residue, suggested that GHK may act as a copper transport factor.It was found that GHK readily forms complexes with copper(II) and enhances the uptake of the metal into cultured cells. Growth modulating plasma tripeptide may function by facilitating copper uptake into cells. Pickart, Freedman, Loker, Peisach, Perkins, Stenkamp and Weinstein Nature 1980, 288, 715-7 A review of information on the biological actions and structure function relationships as understood in 1981. At this time GHK's chemoattractant actions on white cells were unknown. Glycylhistidyllysine (GHK), a tripeptide from human plasma, has been shown to alter the growth rate of many cell types and organisms in culture systems. GHK is optimally active at concentrations between 10 and 200 nanograms/milliliter. Present information suggests that GHK functions as a transporter of transition metals, in particular copper, to the cell surface for uptake into the cell. The use of glycylhistidyllysine in culture systems. Pickart In Vitro 1981,17(6):459-66 Determination of the effectiveness of GHK chelation for copper under physiological conditions. The interaction between Cu(II) and the growth modulating tripeptide GHK in the presence and absence of L-histidine was investigated by potentiometric titration and visible-absorption spectrophotometry at 25 degrees C in 0.15 M-NaCl. Analyses in the pH range 3.5-10.6 indicated the presence of multiple species in solution in the binary system and extensive amounts of the ternary complexes in the ternary system. The species distribution and the stability constants were evaluated. The results obtained from the equilibrium dialysis experiments showed that GHK was able to compete with albumin for Cu(II) at pH 7.5. At equimolar concentrations of albumin and the peptide, about 42% of the Cu(II) was bound to the peptide. At the physiologically relevant concentrations of Cu(II), albumin, L-histidine and this peptide, about 6% of the Cu(II) was associated with the low- molecular-weight components. The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth- modulating tripeptide from plasma. Lau and Sarkar Biochem J 1981 199(3):649-56 Review of GHK-Metal Interactions Available evidence suggests that GHK acts as a complex with transition metals Peptide and protein complexes of transition metals as modulators of cell growth. Pickart In: Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins (Marcel Dekker Pub.) 1982, 75-104. Interaction of Cu(II) and Gly-His-Lys was determined by 13C- and 1H-NMR and EPR spectroscopy. Interaction of Cu(II) and GHK, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-NMR and EPR spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The NMR line-broadening was interpreted in terms of major and minor species formed as a function of pH. The EPR parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration. N.m.r. and e.p.r. investigation of the interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma. Laussac JP Haran R Sarkar B Biochem J 1983 Feb 1209(2):533-9 Magnetic resonance studies of GHK and copper Determine the stability of GHK-Cu in solution. GHK and GHK-Cu slowly exchange copper(II) in solution. PMR studies of Cu(II) and Zn(II) interaction with glycyl-l-histidyl-l-lysine and related peptides. Kwa E, Bor-Sheng L, Rose N, Weinstein B, Pickart L. Peptides 1983, 8, 805-808 Studies on the structure of GHK-Cu in solution. Optical, electron paramagnetic resonance, and electron spin-echo envelope spectroscopies were used to examine the structure of the Cu(II) complex of glycyl-L-histidyl-L-lysine (GHK) in solution.At neutral pH, GHK forms a mononuclear 1:1 Cu(II) compound having an EPR spectrum resembling that of Cu(II) equatorially coordinated by two or three nitrogen atoms. Electron spin-echo studies demonstrate that one of these is located in the histidyl imidazole ring. A pH titration of Cu(II)-GHK shows three optical transitions with apparent pKs of 3.6, 9.2 and 11.4 and molecularities, with respect to protons, of 2, 2, and 1, respectively. At the lowest pK, GHK binds Cu(II), forming the species present at physiological pH. These solution studies are consistent with nitrogen coordination of Cu(II) in Cu(II)-GHK. Structure of the glycyl-L-histidyl-L-lysine copper(II) complex in solution. Freedman, Pickart, Weinstein, Mims and Peisach (Albert Einstein Medical School, New York, USA) Biochemistry 1982 21(19):4540-4

Determination of the X-ray structure of aqueous crystals of GHK-Cu.

The structure of a copper complex of the growth factor glycyl-l-histidyl-l-lysine at 1.1 angstrom resolution. Perkins CM, Rose NJ, Weinstein B, Stenkamp RE, Jensen, LH, Pickart L. Inorg. Chem Acta 1984, 82, 93-99. Studies of cell inhibitory copper complexes A series of hydrophobic analogs of the GHL-Cu structure were synthesized and tested. Hydrophobic analogs of the GHL-Cu structure were inhibitory to cell DNA synthesis and growth Cytotoxic chelators and chelates inhibition of DNA synthesis in cultured rodent and human cells by aroylhydrazone analogs of the gly-l-his-l-lys copper(II) complex. Johnson, Pickart and Rose Inorg Chem Acta 67, pp. 159-165, 1982 Synthesis of growth inhibitory analogs of GHK-Cu. A variety of copper binding growth inhibitory analogs of GHK-Cu very synthesized and tested. A variety of copper binding analogs of GHK-Cu had potent activity in the inhibition of fibroblasts and fibrosarcoma cells at concentrations of 10exp (-16) M. Inhibition of the growth of cultured cells and an implanted fibrosarcoma by aroylhydrazone analogs of the Gly-His-Lys-Cu(II) complex. Pickart, Goodwin, Burgua, Murphy and Johnson Biochem Pharmacol 1983, 32(24):3868-71 A review of information on the biological actions and structure function relationships as understood in 1983. At this time GHK's chemoattractant actions on white cells were unknown The biological effects and mechanism of action of the plasma tripeptide glycyl-l-histidyl-l-lysine. Pickart Lymphokines 8, pp. 425-446, 1983 Electron spin resonance studies of the molecular action of growth inhibitory copper analogs of GHK-Cu with cells. The interaction of two cytotoxic hydrophobic GHK-Cu analogs SBH-Cu (salicylaldehydebenzol hydrazone-Cu) and PCPH-Cu (pyridine 2-carboxyl aldehyde 2'-pyridyl hydrozonate-Cu) with cells was determined. SBH-Cu(II) and PCPH-Cu(II) first interact with sufhydyl groups in the cells. The Cu(II) is first reduced to Cu(I), then re-oxidized to Cu(II) Formation of adducts between cupric complexes of known antitumor agents and ehrlich ascites cells. Antholine, Lyman, Petering and Pickart (University of Wisconsin Milwaukee, USA) Biological and Inorganic Copper Chemistry, Adenine Press, pp. 125-137, 1985

GHK forms stable complexes with palladium (II) The binding of palladium by GHK was determined.

Conclusion: GHK may possibly participate in the transport of Pd(II) in the tissues and its elimination through the kidneys

NMR studies on binary and ternary Pd(II) complexes formed by the growth modulating tripeptide glycylhistidyllysine and nucleotides. Laussac, Pasdeloup and Hadjiliadis J Inorg Biochem 30:227-38, 1987 The authors characterized the mechanism of copper accumulation by the brain, using rat hypothalamic tissue slices incubated with 67Cu as a model system.Two ligand-dependent processes were discerned: a high affinity, low capacity process and a low affinity, high capacity process. The two processes were similar in that each exhibited: (a) a requirement for complexing of copper for optimal 67Cu accumulation and (b) a broad ligand specificity with respect to amino acids (histidine, cysteine, threonine, glycine) and peptides (Gly-His-Lys, glutathione) and ineffectiveness of albumin in serving as a facilitatory ligand. Brain tissue accumulates 67copper by two ligand dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process. Hartter and Barnea (Department of Obstetrics and Gynecology, University of Texas Health Science Center, Dallas, USA) J Biol Chem 1988 Jan 15263(2):799-805 Study of GHK degradation in rats A selective and sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of GHK. and its metabolite, L-histidyl-L-lysine in rat plasma. The limit of detection for GHK and HK were 50 and 15 ng/ml, respectively, and the calibration curves were linear in the range 0.1-5.0 microg/ml. The developed method was applied to the pharmacokinetic study of GHK after a single dose was administered intravenously to rats. GHK was rapidly degraded to HK, which was eliminated rapidly. Simultaneous determination of glycyl-L-histidyl-L-lysine and its metabolite, L-histidyl-L-lysine, in rat plasma by high-performance liquid chromatography with post-column derivatization. Endo, Miyagi and Ujiie (Pharmacological Laboratories, Kissei Pharmaceutical Co., Ltd., Minamiazumi, Nagano, Japan) J Chromatogr B Biomed Sci Appl 1997, 692(1) :37-42

The Aging Reversal Experiments and GHK

The discovery of GHK-Cu arose from studies on human aging and a search for methods to reverse certain deleterious biochemical changes that occur with aging and are intensified in heart attack survivors. The initial target was to find methods of reducing the concentration of the blood protein fibrinogen. Elevated blood fibrinogen reduces blood flow through the tissues and raises the death rate.

Studies on fibrinogen metabolism concluded that there were factors in the blood protein albumin fraction that reduced fibrinogen synthesis and strongly stimulated liver metabolism. Some of these effects were due to albumin-bound free fatty acids but analysis of this albumin fraction found a tripeptide-copper complex (GHK-Cu) that improved liver cell metabolism and survival in culture. The plasma concentration of GHK-Cu was higher in men of age 20 (

200 ng/ml) than in men of age 70 (

80 ng/ml). Ultimately, this tripeptide copper complex (GHK-Cu) was found to have multiple actions that activated tissue regeneration and remodeling. The improvement of skin quality and turnover in older persons by application of GHK-Cu could be considered an aging reversal effect.

Observation and Question Asked Type of studies Assumed Cause at Start of Studies. Result/Actual Cause References Human blood fibrinogen levels increase with age and disease - This increases blood coagulation and is associated with a sharply increased death rate.

Why was the fibrinogen increased?

Expected cause - Fibrinogen was not being used and accumulated in the blood

Actual cause - Human fibrinogen turnover studies found that the blood increase was due to an increased liver synthesis of the protein in older persons and heart attack survivors.

Pilgeram LO, Pickart, L Bandi, Z, Fatty acid control of fibrinogen turnover in aging and atherosclerosis. 7th International Congress of Gerontology, June 1964, pp. 451-460. Pickart L, Pilgeram LO. The role of thrombin in fibrinogen biosynthesis. Thromb Diath Haemorrh. 1967 May 3117(3-4):358-64. Pilgeram LO, Pickart L. Control of fibrinogen biosynthesis: the role of free fatty acid. J Atheroscler Res. 1968, 8:155-66. Fibrinogen is synthesized in the liver.

Was the increased fibrinogen synthesis observed in older persons and heart patients due to changes in the liver or was it due to factors in the blood that perfused the liver?

Measure fibrinogen synthesis in isolated human liver tissue from persons aged 20 to 30 and in persons aged from 60 to 80. Incubated the young (20-30) liver tissue with blood from either the young (20-30) or the old group (60-80). p (60-80)

Assume cause - The increase in fibrinogen synthesis with age was due to changes in the liver tissue.

Actual cause - The increase in fibrinogen synthesis in the older persons was not due to changes in the liver tissue but almost totally due to factors in the older blood.

Pickart L, Thaler MM. Suppression of tumor-associated hyperfibrinogenemia and free fatty acidemia with p-phenoxybenzalbutyrate (clofibrate). Cancer Res. 1979 Oct39(10):3845-8. Pickart, L. Fat metabolism, the fibrinogen/fibrinolytic system and blood flow: new potentials for the pharmacological treatment of coronary heart disease. Pharmacology 198123(5):271-80 Pickart L Suppression of acute-phase synthesis of fibrinogen by a hypolipidemic drug (clofibrate). Int J Tissue React. 1981 3(2):65-72.

What is the factor(s) in the blood that stimulated or inhibited fibrinogen synthesis? Studied the effect of various plasma protein fractions that affected fibrinogen synthesis in mice. No. The albumin fraction of plasma had a suppressive action on fibrinogen synthesis that returned synthetic patterns to that of a younger person. Some effects appeared to be due to free fatty acids but this albumin fraction itself also stimulated liver cell metabolism. Pickart L, Thaler MM. Fatty acids, fibrinogen and blood flow: a general mechanism for hyperfibrinogenemia and its pathologic consequences. Med Hypotheses. 1980 May6(5):545-57. Pickart L, Thaler MM. Free fatty acids and albumin as mediators of thrombin-stimulated fibrinogen synthesis. Am J Physiol. 1976 Apr230(4):996-1002. What was the factor in the albumin fraction that stimulated liver metabolism? Studies the protein albumin and various small molecules associated with the protein.

Expected cause - The effects were due to the albumin molecule.

Actual cause - The effects were not due to albumin but rather due to a small peptide-metal complex associated with the protein.

A tripeptide in human plasma that increases the survival of hepatocytes and the growth of hepatoma cells. Pickart Ph.D. Thesis in Biochemistry, University of California, San Francisco, 1973 Thaler MM, Pickart L Metabolic and growth promoting properties of serum tripeptide and its synthetic analog. In: Gene Expression and Carcinogenesis in Cultured Liver. (Academic Press, 1975) pp. 292-310. What was the structure of the peptide-metal complex? Isolation and chemical analysis. The complex was composed of a tripeptide, glycyl-l-histidyl-l-lysine plus approximately equimolar copper 2+ or GHK-Cu Schlesinger DH, Pickart L, Thaler MM. Related Articles, Protein Growth-modulating serum tripeptide is glycyl-histidyl-lysine. Experientia. 1977 Mar 1533(3):324-5. What was the function of GHK-Cu? Synthesized variants of GHK-Cu that inhibited cell growth in cell culture, wound repair, and some tumors in animals. During studies of growth inhibitory copper analogs of GHK-Cu on wound repair, it was observed that GHK-Cu, which was used as control substance, was healing wounds while the inhibitors stopped wound healing. It was concluded that GHK-Cu had a stimulatory action on wound healing. Pickart L, Lovejoy S. Biological activity of human plasma copper-binding growth factor glycyl-L-histidyl-L-lysine. Methods Enzymol. 1987147:314-28.

GHK-Cu remains the best molecule for internal medical treatments. The physiological function of GHK-Cu in the human body appears to be as a protectant and repair signal for damaged tissues. It is possible that GHK-Cu could be used clinically to protect and speed repair of damaged organs. H. Paul Ehrlich found that intra-muscular injection of GHK-Cu into the thigh muscle of rabbits raised circulating wound macrophages in the blood and accelerated the healing of distant wounds in the rabbit ear. Patients might be pre-treated with GHK- Cu before surgery to enhance post-surgical repair. Based on rabbit models, a dosage of 30 milligrams of GHK-Cu should suffice. The molecule is also very beneficial on kidney organ culture. Thus, GHK-Cu might be infused into patients with kidney failure to exert its tissue protective and repair actions. Potential clinical targets would be injuries to, or system failure of, tissues such as skin, bone, kidneys, and the gastrointestinal intestinal tract. GHK-Cu might be administered after traumatic tissue injury such as automobile accidents.The newer breakdown resistant, highly adhesive copper peptides under development should prove better for cosmetic and superficial uses such as post-procedure dermatological healing, and development of scarless surgical procedures. The copper peptides appear to be very useful for post-procedure recovery after skin peels, dermabrasion and laser resurfacing. The combination of hydroxy acids and these peptides slowly, over a period of several months, reduces old scars and skin lesions. This method is economical and avoids the complications that often occur after chemical peels or laser treatments. In experimental studies, the use of such types of copper peptides after surgical procedures often results in scarless or nearly scarless healing.

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Discussion

The norovirus capsid protein VP1 contains determinants of genome encapsulation and particle assembly as well as membrane penetration and entry, thereby representing a portion of the molecular machinery required for infection. Here we have structurally defined the interaction between the MNoV P domain and the newly discovered entry receptor CD300lf, an interaction that appears to require interfacial coordination of metal ions. Further, we herein identify bile acids as cofactors for MNoV infection and establish the P domain structural determinants used in the engagement of the bile acids GCDCA and LCA. These results are supported by a series of biophysical assays that have incorporated analysis of structure-based mutations. These structural and functional studies have significant implications for our understanding of norovirus biology.

Interactions Between the P Domain and Its Cellular Receptor CD300lf.

Because of its position on the outer face of the viral capsid and its targeting by neutralizing antibodies (18 ⇓ ⇓ ⇓ ⇓ –23, 29, 34, 35), the P2 subdomain was a prime candidate for CD300lf receptor engagement. Consistent with this, we show that CD300lf contacts a cleft in P2 formed by the AB and DE loops, with two receptors splayed away from the P domain dimer interface (Fig. 1). Feline calicivirus (FCV) and Hom-1 calicivirus employ junctional adhesion molecule A (JAM-A) and related proteins as receptors (36, 37). The cryo-EM structure of the FCV capsid in complex with the two Ig-like extracellular domains of fJAM-A indicates this receptor also binds to the P2 subdomain, although the receptors appear to engage the top of the P domain near the dimer interface (38). This same dimer interface proximal region of P2 is used by some human noroviruses to bind HBGA glycans (25, 28, 39, 40). Our structural data are robustly supported by quantitative binding analysis (SPR, BLI, and ITC), where mutation of critical determinants in the CD300lf CRD3 and CC′ loops along with mutations in the P domain AB loop, DE loop, and E strand all result in disruption of the interactions.

CD300lf binding to the P domain of MNoV was potently blocked using the neutralizing antibody A6.2.1. Cryo-EM models suggest that A6.2.1 binding site spans both the AB and EF loops of P2, a conclusion supported by both escape mutants and site-directed mutagenesis studies (21, 26, 34, 41). This predicts that the A6.2.1 contact site overlaps with the CD300lf contact site on the P domain seen in our cocomplex structure (SI Appendix, Fig. S6), strongly supporting the concept that A6.2.1 inhibits MNoV infection by blocking cell attachment. The monoclonal antibody 2D3.7 broadly neutralizes MNoV strains and by cryo-EM binds very close to the A6.2.1 site although at an angle that allows it to make more contact with the AB and EF loops (21). Based on our structural studies it seems likely that 2D3.7 also neutralizes MNoV by blocking the CD300lf binding site.

The Role of Multivalency in Cellular Attachment.

The MNoV P domain buries ∼630 Å 2 of solvent accessible surface at the CD300lf contact interface, an area notably smaller than that buried in many other receptor–virus interfaces. For instance, the contact area buried by HIV gp120 and its receptor CD4 is ∼742 Å 2 (42), between rhinovirus HRV14 and its receptor ICAM‐1 is ∼990 Å 2 (43), between coxsackievirus A21 and its receptor ICAM-1 is ∼970 Å 2 (44), and between poliovirus and the poliovirus receptor CD155 is ∼1,300 Å 2 (45). Consistent with this small contact area, the affinity between the P domain and CD300lf is relatively weak with a KD of ∼25 μM for monotypic binding as measured in solution by ITC. The low-affinity binding observed between the P domain and CD300lf suggests that cell attachment might be avidity-driven, requiring that P domains on a virion simultaneously engage multiple CD300lf proteins on the cell surface. We therefore docked the X-ray structure of the complex onto a cryo-EM–derived model of the MNoV particle. We found that CD300lf domains cluster in threes on the virion surface (Fig. 7A). The distribution of CD300lf contacts can be seen by removing the CD300lf molecules from the model (Fig. 7B). This distribution makes clear that each CD300lf cluster is composed of receptors from three different P domain dimers (Fig. 7C). This docking model results in some minor overlap of CD300 domains (Fig. 7C), which may be resolved by mobility of P domains, which are flexibly connected to the shell domain of VP1. These results support a model in which multiple CD300lf engagements can be made by each virion to drive MNoV binding and entry. We undertook several binding studies using an Fc–CD300lf fusion protein, revealing that the bivalent reagent can bind recombinant P domain and MNoV from infected cell lysates with an avidity at least 50-fold higher than the monotypic binding affinities we observed. Because our data are most consistent with a need for multivalent interactions between the P domain and several cell surface CD300lf receptors, the strength of these bivalent interactions may be physiologically relevant.

Docking of CD300lf–P domain complex onto a cryo-EM–derived model of the MNoV virion. (A) The solvent accessible surfaces of CD300lf are shown in yellow, with the P domain subunits in blue and green. The CD300lf domains group in threes across the surface of the virion. Each CD300lf trimer is made up of receptors from three different P domain dimers. (B) The CD300lf contacts mapped in yellow on the model surface after removing CD300lf. Fivefold, threefold, and twofold symmetry is marked with a white pentagram, triangle, or ellipse, respectively. (C) (Right) A top-down view (same orientation as in A) for three P dimers in a trimer cluster with the shell region below in blue. (Left) A side view of the three P dimers. Note how the CD300lf C termini (marked by red cylinders) cluster at the center of each trimer and overlap slightly. The magenta ovals with a white letter B mark visible bile acid binding pockets.

Molecular Mimicry by MNoV in Receptor Engagement.

Our structure of CD300lf in complex with phosphocholine has defined the host ligand binding site used by the receptor. The ligand head group is orientated so that the fatty acid tail would extend out of the pocket away from the CD300lf-bearing cell surface, consistent with the function of CD300lf as a lipid receptor. The ligand orientation and metal coordination are very similar to that used by TIM-4, a related immune regulatory molecule, to bind phosphatidylserine (SI Appendix, Fig. S8) (46). By comparing the structures CD300lf bound to either phosphocholine or the MNoV P domain, we have revealed in precise atomic detail how the viral capsid mimics binding of a CD300lf ligand and the role of divalent cations in both of these processes. This form of molecular mimicry predicts that the interaction between the P domain and CD300lf during infection would be incompatible with simultaneous binding of ligands to the lipid-binding pocket of CD300lf, suggesting that infection might be regulated by high-affinity receptor ligands. Bacterial ligands may also bind to CD300 family members (10), raising the possibility that interactions between bacteria and the virus may involve binding of ligands to CD300lf that in turn compete for interactions with the P domain.

The Role of Bile in MNoV Infection.

We previously showed that efficient MNoV binding to cells and infection requires a small molecular weight heat stable serum cofactor (5). Here we show that the bile acid GCDCA can serve as a cofactor by increasing cell binding and infection. We do not know if GCDCA is responsible for the activity originally observed in serum. The role for this bile acid is likely to involve direct binding to the viral capsid protein because our crystal structures of the CD300lf–P domain complex bound with both GCDCA and LCA revealed two binding pockets per P domain dimer that can accommodate these bile acids. The location of the bile acid pocket was surprising for several reasons. First, the bile acid pocket is remote from the CD300lf–P domain interface, suggesting that if bile acids function by altering the CD300lf–P domain interaction, this must be via a long-distance allosteric effect. Second, the bile acid pocket is distinct from the glycan-binding sites observed on human and murine norovirus P domains (25, 40), suggesting that norovirus P domains are promiscuous in their ability to bind diverse biologic small molecules and further arguing that the interaction between noroviruses and target cells is regulated at multiple levels. It seems likely that the efficiency of norovirus infection of cells in culture requires that each of these different levels of control be perfectly matched to avoid abortive infection. Third, binding of GCDCA and LCA to MNoV P domain did not induce significant structural rearrangements in the P domain or in the CD300lf interface. We did find that addition of GCDCA had a small (approximately twofold) but reproducible effect on binding of soluble CD300lf to the P domain in the presence of divalent cations. It is possible that this small effect, summed over the many copies of the P domain on the virion, has biological significance. However, this is speculative, and the molecular mechanism of cofactor function remains an open question. It is intriguing to consider that bile acids, rather than directly modulating receptor:P domain interactions, instead alter the structural conversions that occur as the virus enters the cell to release its RNA into the cytoplasm or the conformation of the virion itself in ways not detected by biding studies of the P domain and CD300lf in solution.

We also found that addition of calcium and magnesium also augments infection. Our structural data indicate that there are at least three divalent cation binding sites associated with CD300lf binding to each P domain monomer. Two of these are in the binding interface between the P domain and CD300lf and likely facilitate the interaction. A third metal ion is located midway between the receptor and bile acid binding sites of the P domain, directly participating in neither interaction. Crystal structures and ITC binding of the MNoV P domain with CD300lf showed that either magnesium or calcium can support complex formation. Of particular interest, Ca 2+ is present in high concentrations in bile (47), making it reasonable to propose that calcium might be present at sites of MNoV infection (48). It is intriguing to speculate that the availability of divalent cations in the body, which may not be available in the environment, might foster infectivity of the virus once it reaches its target.

Implications of These Studies.

These studies support the concept that noroviruses have evolved to be dependent on small molecules either by molecular mimicry of host lipids or by depending on cofactors such as bile acids and divalent cations for critical steps in infection. Physiologically, it is attractive to consider that the presence of bile acids and Ca 2+ in the intestine downstream of the common bile duct, in combination with tropism of the virus for tuft cells as the sole epithelial cell expressing CD300lf (7), explains the tropism of the virus. This adds to the prior finding that tuft cell tropism is conferred by the combinatorial effects of the host cytokine IFNλ and the MNoV nonstructural protein NS1 (49). Further, although we could not reproducibly observe an effect of addition of LCA on binding or infectivity due to solubility issues, this secondary bile acid can also bind to the MNoV P domain in the pocket used by GCDCA. This indicates that secondary bile acids might serve a physiologically important role in vivo. However, enhancement appears to be rather specific because multiple other bile acids did not augment infection. Because we have not exhaustively sampled primary and secondary bile acids in the intestine, it is quite possible that additional cofactors or even inhibitors of norovirus infection will be discovered in the future.

In addition to identifying that the norovirus P domain interacts with a soluble cofactor, the structural studies presented here raise the possibility of designing effective orally bioavailable bile acid analogs for the treatment of human norovirus infection, which has also been shown to be enhanced by bile (30). Further, we previously showed that intestinal bacteria play a critical role in the establishment of persistent enteric MNoV infection (50). Because intestinal bacteria are involved in metabolizing primary bile acids into secondary bile acids, this raises the possibility that transkingdom interactions between norovirus and enteric bacteria are via alterations in bile acids in the intestine. Indeed, broad-spectrum antibiotics might alter norovirus infection via alterations in bile acid metabolism (50, 51).


Methods

Ethics statement

All participants provided written informed consent. This protocol was approved by the Fred Hutchinson Cancer Research Center Institutional Review Board, the Makerere University School of Medicine Research and Ethics Committee (SOMREC), and the Uganda National Council on Science and Technology (UNCST).

Study cohort and specimen collection

Specimens were obtained from participants enrolled in the “HIPPOS” Study, an ongoing prospective cohort study, begun in 2012, of KS patients initiating treatment at the Uganda Cancer Institute (UCI) in Kampala, Uganda. Participants were eligible for the HIPPOS study if they were >18 years of age with biopsy-proven KS, and naïve to antiretroviral therapy (ART) and chemotherapy at enrollment. Participants attended 12 study visits over a one-year period and received treatment for KS consisting of ART and chemotherapy (a combination of bleomycin and vincristine or paclitaxel) per standard protocols by UCI physicians. At each visit, participants received a detailed physical exam to assess clinical response using the ACTG KS response criteria [57].

Participants provided plasma samples at each visit for KSHV, CD4 and HIV viral load testing, and in addition, provided up to 12 biopsies of KS lesions before, during, and after KS treatment. KS tumor biopsies were obtained using 4mm punch biopsy tools after cleaning the skin with alcohol, and either snap-frozen at the clinic site and stored in liquid nitrogen (LN2) or placed in RNAlater (Sigma-Aldrich, Cat. # R0901) and stored at -80°C. Study clinicians collected swabs of the oral mucosa at each study visit and participants self-collected oral swabs at home for 1 week after the visit after education on the sample collection technique, as previously validated by our group in Uganda [58]. Briefly, a Dacron swab is inserted into the mouth and vigorously rubbed along the buccal mucosa, gums, and hard palate. The swab is then placed in 1 mL of filter-sterilized digestion buffer [59] and stored at ambient temperature [60] before being placed at -20°C for storage.

DNA preparation

DNA was extracted from 300μL homogenized tumor lysates using the AllPrep DNA/RNA Mini Kit (QIAGEN, Cat. # 80204) and eluted into 100μL EB Buffer. For oral swab specimens, DNA was extracted from the swab tip eluate using the QIAamp Mini Kit (Qiagen, Cat. # 51304) following the manufacturer’s protocol. Purification of DNA from saliva stabilized in RNAprotect Saliva Reagent (Qiagen) was performed following the manufacturer’s protocol with the following modifications: There was no initial pelleting or PBS wash, 20 μL proteinase K was used per 200 μL specimen, and DNA was eluted in 50 μL water. DNA was quantified using a NanoDrop 1000 Spectrophotometer (ThermoScientific).

All PCR preparations were done in a PCR-clean room, except for the addition of control templates. PCR was conducted using the PrimeSTAR GXL kit (Takara, Cat. # R050B) with ThermaStop (Thermagenix) added. Cycling conditions were: 98°C for 2 mins 35 cycles of 98°C for 10 secs, 60–65°C (depending on primer) for 15 secs, 68°C for 1min/kb 68°C for 3 mins and then hold at 4°C. Primer sequences are listed in S1 Table.

Copy number quantification

KSHV genome copy numbers were quantified by droplet digital PCR (ddPCR) using the QX200 Droplet Generator and Reader (Bio-Rad), with ddPCR SuperMix for Probes (No dUTP) (Bio-Rad, Cat. # 186–3024). Primers and probes (S1 Table) were designed to detect 4 KSHV-unique genes K2/vIL-6, ORF16/vBCL-2, ORF50/RTA and ORF73/LANA (Fig 1A), with KSHV genome copy number reported as the average of the 4 measures. 420 ng BCBL-1 cell line DNA diluted 1:10,000 (

475 genome copies) was used as positive control, 1 ng human genomic DNA (Bioline, Cat. # BIO-35025) as negative control, and water as no template control. Cycling conditions were: 95°C for 10 mins 40 cycles of: 94°C for 30 secs, 56°C for 30 secs, 60°C for 1 min one cycle at 98°C 10 for mins, and then hold at 12°C. The KSHV on-target percent was calculated using the copy number quantification by ddPCR normalized to the total nucleic acid concentration.

(A) Schematic representation of a linear KSHV genome, with genes colored in green and the major repeat regions in orange. The locations of the K1, vIL-6, vBCL-2, RTA, LANA and K15 genes used for genome quantitation are indicated in green type. (B) Raw (light blue), sUMI-consensus (blue) and dUMI-consensus (dark blue) read coverage along the de novo assembled, BCBL-1 KSHV genome. (C) Bubble plot of minor sequence variants. Each bubble represents a position within the genome at which a variant base or indel was detected, colored by whether they were predicted to be silent or protein-altering mutations. Mutations likely to be silent included synonymous and intergenic point mutations, while protein-altering mutations included non-synonymous, nonsense and frameshift mutations. Bubble height represents variant base frequency among dUMI-consensus reads at that position. Vertical grey columns represent masked repeat regions in which no reliable alignments were possible.

UMI-addition and library preparation (Fig 2)

500-bp DNA fragments, 10–20 ng/μL of DNA extract in 100 μL chilled TLE buffer (10mM Tris, pH8.0, 0.1mM EDTA) was sheared using a Bioruptor (Diagenode) on high power for up to 15 min. Fragment sizes were assessed on 1.5% agarose gels. Sheared DNA was bead-purified using 1.2X volume of Agencourt AMPure XP Beads (Beckman Coulter Cat. # A63880) and eluted in 50 μL water. Library preparation (end repair, A-tailing and adapter-ligation) was performed using the KAPA HyperPrep Library Preparation Kit (Cat. # KR0961/KK8503). Double-stranded DNA adapters contained a random 12-bp dUMI sequence and a defined 5-bp spacer sequence added to Illumina TruSeq adaptor sequences [61] (S1 Fig). Subsequently, DNA was bead-purified with 1X volume of beads and eluted in 50 μL water.

Each study participant contributed multiple KS tumors and oral swabs, but only those samples with the highest viral loads were reported here. Sequencing libraries were prepared from DNA extracts from each sample with adaptors containing duplex Unique Molecular Identifiers (dUMIs, see S1 Fig). Adaptor-labelled DNA libraries were enriched using biotinylated RNA baits homologous to KSHV sequences. Captured DNA was PCR-amplified to levels sufficient for Illumina HiSeq sequencing. For most samples, libraries were subjected to a second round of enrichment and PCR amplification. Upon sequencing, whole KSHV genomes were first assembled de novo from each sample without the use of dUMIs. The sample-specific genomes generated (sample-consensus) were then used as reference to map the consensus of reads with identical dUMI-tags (i.e., dUMI-consensus reads).

DNA libraries were subjected to pre-enrichment amplification with primers mws13 & mws20 (S1 Table and S1 Fig). PCR conditions were: 95°C 4 mins 5–8 cycles of 98°C 20 sec, 60°C 45 sec, 72°C 45 sec 72°C 3 mins, 4°C hold. If the bead-purified elution from the end repair and adapter step had more than 240 ng total, it was divided into 50 μL PCR reactions of ≤240 ng and pooled after amplification. PCR products were then bead-purified as above with 1.2X volume beads and elution in 100 μL water, quantified with Nanodrop, and their sizes assessed using a Bio-analyzer (Agilent DNA 7500) or Qiaxcel (QIAGEN AM420).

Library enrichment & sequencing

Biotinylated RNA baits for enriching KSHV sequences in the library were those designed in [62] and were obtained from Agilent, Inc. (Santa Clara, CA). The design was a 120-bp, 12X tiling of the genome of KSHV isolate GK18 (Genbank ID: AF148805.2). The diversity of the bait library was further increased by including K1, ORF75, K15, ORF26 and TR sequences of strains JSC-1 (Genbank ID: GQ994935.1), DG1 (Genbank ID: JQ619843.1), BC-1 (Genbank ID: U75698.1), BCBL-1 (Genbank ID: HQ404500.1), Sau3A (Genbank ID: U93872.2), and of all Western and African isolates in [29,33] (Genbank ID: AF130259, AF130266, AF130267, AF130281, AF130305, AF133039, AF133040, AF133043, AF133044, AF151687, AF171057, AF178780, AF178810, AF220292, AF220293, AY329032, KT271453-KT271468).

Target enrichment was performed using SureSelect Target Enrichment Kit v1.7 (Agilent) with all suggested volumes reduced by half. DNA hybridized to biotinylated-RNA baits was captured with streptavidin beads (Dynabeads MyOne Streptavidin T1, Invitrogen) and resuspended in 20μL water. The DNA-streptavidin bead mixture was used directly in post-enrichment PCR amplification with primers mws13 and mws21, the latter of which includes a sample index sequence (S1 Table and S1 Fig). The PCR cycle number ranged from 10–16, with products monitored every 2 to 3 cycles on a TapeStation (Agilent) to ensure correct fragment sizes (

500bp). When over-amplification resulted in library fragment sizes much larger than expected, a single “reconditioning”PCR cycle with fresh reagents was done [63]. PCR products were cleaned using 1.2X volume AMPure XP beads and the eluted DNA library was sequenced using Illumina HiSeq 2500 with 100-bp paired end reads. For some tumor samples with low KSHV copy numbers and all oral swab samples, a second library enrichment was performed.

De novo assembly of sample-consensus genomes

Initially, a sample-consensus KSHV genome (Fig 2) was generated de novo from paired-end reads of each sample using custom scripts (S2 Fig, https://github.com/MullinsLab/HHV8-assembly-SPAdes). At this stage, the first 17-bp from read ends were trimmed to remove dUMI sequences. Next, reads were subjected to windowed quality filtering using sickle pe [64] with a quality threshold of 30 and a window size 10% of read length. Filtered reads were aligned to a human genome (GRCh38 p12, GenBank GCA_000001405.27) using bwa mem [65]. Unmapped reads were used as input into the de novo assembler SPAdes v3.11.1 [66], with the setting -k 21,35,55,71,81. This often yielded 3 to 4 scaffolds that together encompassed the entire 131-kb unique sequence regions of KSHV, bounded by the major repeat regions: Internal Repeat 1 (IR1), Internal Repeat 2 (IR2), LANA central repeat and Terminal Repeats (TR) (Fig 1A). Next, all scaffolds over 500 bp were aligned using bwa mem to the genome of reference KSHV isolate GK18. From the aligned scaffolds a draft genome was generated in Geneious (Biomatters, Ltd) with manual correction as needed. To finish the assembly, GapFiller v1.1 [67] was used, setting bwa as the aligner and filtered paired-end reads as the input library. The genomes were annotated in Geneious based on the GK18 reference, also adding the annotation for long non-coding RNA T1.4 based on [68]. The major repeat regions were masked with Ns since they were poorly resolved by assembly of short reads that can map to multiple locations within the repeat regions.

Variant identification from dUMI-consensus reads

Paired-end reads, including their dUMI sequence tags, were mapped by bwa to sample-consensus genomes (Fig 2) using a Makefile adapted from [61] (https://github.com/MullinsLab/Duplex-Sequencing). Briefly, all reads mapping to the same genomic position were collapsed by single strand UMIs (sUMI) to make sUMI-consensus reads (S2 Fig). Complementary UMI tags from opposing strands were matched to create dUMI-consensus reads, thus removing nearly all PCR polymerase misincorporation and chimera artifacts. Nine bases from both read ends were then trimmed to minimize read end artifacts. Discrepancies between mapped dUMI-consensus reads and the sample-consensus genomes were manually inspected in Geneious and misalignments around homopolymer runs were corrected. Only the remaining discrepancies were considered to be sequence variants that existed prior to PCR amplification.

All genome and subgenome sequence alignments were generated using MAFFT [69] [algorithm FFT-NS-i x1000, scoring matrix 1PAM/k = 2], and all phylogenetic trees were inferred using RAxML [70] (-f d, GTR gamma, N = 100 starting trees), using a representative KSHV genome from each individual. The NeighborNet phylogenetic network was generated using SplitsTree5, excluding gap sites [71]. Consensus genome sequences were deposited in GenBank (Accession numbers MT510648—MT510670, MT936340, see Table 1) annotated with the genomic rearrangements, when present.

Integration analysis

Systematic searches for KSHV integration into the human genome were done in two ways. First, each library was searched using local BLASTN against both human and KSHV sequences and then using the Perl script SummonChimera [72] to extract coordinates of potential integration sites. Second, a sample-consensus KSHV genome was appended as an extra chromosome to the human genome reference GRCh38 p12. The appended human genome reference was used to map sUMI consensus reads via Speedseq [73] to generate alignment files with only discordant or split reads. These were input into LUMPY for structural variant analysis [74]. Human chromosome sequences linked to KSHV sequences were taken to be putative integration sites.


Structure of DNA

In the 1950s, Rosalind Franklin, Maurice Wilkins, Francis Crick and James Watson were actively investigating the molecular structure of DNA. Franklin was using X-ray crystallography in Wilkins’ lab to observe the patterns formed by X-rays shot through crystalized DNA fibers. Watson and Crick were able to piece together the puzzle using Franklin’s data and key pieces of information about the pairing of nucleotides in a DNA molecule. In 1953, both Wilkins and Franklin published papers on their X-ray data in the same Nature issue with Watson and Crick’s paper on the structure of DNA. Franklin died from cancer in 1958, and although Franklin’s research on viruses was appreciated in her lifetime, her contributions to the discovery of the structure of DNA were largely recognized posthumously. In 1962, the Nobel Prize in Physiology or Medicine was awarded to Watson, Crick, and Wilkins for solving the structure of DNA.

DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. Nearly every cell in a person’s body has the same DNA. The majority of DNA is located in the cell nucleus (where it is called nuclear DNA ), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA). A cell’s complete complement of DNA is called its genome. Normally, DNA molecules are double-stranded, linear, thread-like molecules bound with proteins in the nucleus of each cell. Stretched end-to-end, the DNA molecules in a single human cell are estimated to be about 6 feet in length.

The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and more than 99% of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences.

DNA bases pair up with each other, adenine with thymine (A-T) and cytosine with guanine (C-G), to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral ladder called a double helix. The double helix’s ladder structure is formed with the base pairs as the rungs and the sugar-phosphate molecules as the vertical sidepieces (Figure 2.12).

An important property of DNA is that it serves two functions: to replicate, or make copies of itself, and to produce proteins. During replication (Figure 2.13), each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell. DNA replication begins with the separation of the bonds holding the base pairs together in the parent strand, creating two template strands.

Figure 2.12: Differing chemical structures of DNA and RNA

Figure 2.13: Process of DNA replication resulting in two complete DNA molecules

Unpaired bases on the template strands attract and bond with complementary free nucleotides in opposite directions. When all of the new base pairs are formed on each strand, replication is complete, and there are now two complete DNA molecules.

There is a second nucleic acid in all cells called ribonucleic acid, or RNA (Figure 2.12). Like DNA, RNA is made up of nucleotides that include a base, sugar and phosphate. RNA nucleotides contain the chemical bases adenine, cytosine, and guanine, but they do not contain thymine, which is instead replaced by uracil (U). In addition, RNA exists as a single-stranded molecule rather than a double-stranded helix. This molecule plays a key role in transporting amino acids for protein chain construction during protein synthesis.

During the production of proteins, RNA bases are arranged in sequences of three, which are called codons. These sequences determine the assembly of different amino acids. For example, the combination and arrangement of thymine, adenine, and guanine encode the amino acid isoleucine, which helps rebuild muscle tissue. Amino acids join together in chains to produce different proteins, which are chemical compounds that are necessary for the structure and operational processes of the body’s cells. There are 20 different amino acids that can come together in differing combinations and amounts to produce millions of different proteins basic to life.


Background

Maize (Zea mays) is one of the most widely planted crops in the world, used for human consumption, animal feed, and as raw material for biofuels and other products (reviewed in [1]). In addition to its agricultural importance, maize is a model plant for monocot crop genetics, with complete genome sequences of B73 [2] and 34 other maize genomes deposited in Maize Genetics and Genomics Database (maizeGDB.org). However, few genomic tools for phenotyping impacts of individual genes are available. Maize transformation is technically difficult, expensive, and time consuming (reviewed in [3]). Thus, alternatives to transformation of stable transgenics are valuable. Virus-based tools developed for high-throughput virus-induced gene silencing (VIGS) could help fill the gap for maize gene function studies.

Many plant viruses have been developed as beneficial tools for expression of protein or VIGS (reviewed in [4,5,6,7,8,9,10]). VIGS has been used for gene functional analyses in host plants [11,12,13,14], utilizing target gene fragments in viral vectors in sense or antisense orientations or hairpin structures (see examples in [15]), with insertion sequence fragments as small as 100–300 nt being effective to silence target genes [13]. Plant viruses have also been widely used for protein expression in many different plant species (reviewed in [5, 16,17,18]). In one case, a plant virus vector derived from bean pod mottle virus (BPMV) was even developed for simultaneous single gene expression and single gene silencing [19]. For maize, several viruses have been developed as tools for gene expression or silencing, including brome mosaic virus (BMV) [20, 21], cucumber mosaic virus (CMV) [13], and maize rayado fino virus (MRFV) [22] for gene silencing [23], wheat streak mosaic virus (WSMV) [24,25,26] and sugarcane mosaic virus [27] for protein expression, foxtail mosaic virus (FoMV) for gene silencing [28] or protein expression [29] and guide RNA delivery for CRISPR/Cas9 gene editing [30], barley stripe mosaic virus for gene silencing [23] or protein expression [31] or for guide RNA delivery [32] and barley yellow striate mosaic virus (BYSMV) for simultaneous guide RNA and high cargo-capacity protein expression [33].

Viruses in the family Potyviridae, or potyviruses, have been developed as heterologous gene expression vectors in various plant hosts, enabling tracking of virus movement and other experiments. Viruses in the family Potyviridae have flexuous, rod-shaped virus particles with a single-stranded positive-sense RNA genome encapsidated by viral coat protein, and are the largest group of plant viruses (reviewed in [34]). The potyvirus genome encodes a polyprotein which is cleaved by its own viral proteases to produce 10 mature functional viral proteins for virus replication, pathogenicity, aphid transmission and other functions through interaction with many plant host proteins (reviewed in [34]). Potyviruses developed for protein expression include tobacco etch virus (TEV) [35, 36], zucchini yellow mosaic virus (ZYMV) [37], plum pox virus (PPV) [38], potato virus A (PVA) [39,40,41], turnip mosaic virus (TuMV) [42, 43], tobacco vein banding mosaic virus (TVBMV) [44], WSMV [24], and SCMV [27]. Because of their strong silencing suppressor, potyviruses were not favored for development of VIGS vectors, however, PVA was developed for transient gene silencing [45]. The most common insertion sites in these potyvirus-based vectors are the P1/HCPro and NIb/CP coding sequence junctions, with four other junction sites in the polyprotein open reading frame (ORF) or at the 5′ end of the polyprotein ORF utilized in some vectors.

Since more than one sequence insertion site can be utilized for some potyviruses, and their expandable rod-shaped virions are generally more tolerant of insertion sequences than viruses with icosahedral virions and inherent packaging constraints, expression of two to five proteins from a single vector has been achieved in some of these systems [36, 41,42,43].

Given the features of potyviruses and interest in further tools for maize, maize dwarf mosaic virus (MDMV) was an attractive option for development. MDMV was first reported in Ohio in 1963 [46]. MDMV is naturally transmitted by aphids in a nonpersistent manner [47], and is readily transmissible by rub inoculation in the laboratory [48]. MDMV infected plants have mosaic symptoms on leaves of susceptible cultivars of maize that can be visible as early as 5–7 days after rub inoculation. MDMV is common in the United States and is capable of reducing yields, but successful resistance breeding and disease management have limited major yield loss (reviewed in [49]). In addition to maize, the host range of MDMV includes some sorghum cultivars and Johnsongrass (Sorghum halepense L.), which is a major overwintering virus reservoir [50, 51]. Near-complete consensus sequence of a lab-maintained isolate MDMV OH2 (GenBank accession no. JQ403609) and complete sequence of a derived infectious clone (MDMV OH1 GenBank accession no. JQ403608) from Ohio were previously reported [52]. Here we report the cloning and sequencing of a new Ohio MDMV isolate from Johnsongrass, MDMV OH5, development of MDMV OH5 infectious clones, and engineered constructs for simultaneous gene expression and multi-gene silencing in maize. We report the first development of a virus-based vector for simultaneous gene expression and multi-target VIGS in maize.


RESULTS

Coupling of transcription with RNA detection

Here, we present S HERLOCK-based pr ofiling of in vitro t ranscription (SPRINT) and show how this methodology can measure concentrations of various effector molecules and overcome limitations of existing methods. In an isothermal, one-step and one-batch reaction, Cas13a is targeted to an RNA site that is transcribed from a DNA template. As Cas13a does not cleave DNA, it will only detect its target sequence when it is transcribed as RNA. Cas13a subsequently cleaves fluorescently labeled pentauridine oligonucleotides, yielding a fluorescent signal. This assay was used to quantify the activation or repression of transcription under various conditions. Because transcription can be regulated by aTFs or riboswitches in a ligand-dependent manner, SPRINT can be used to quantify those ligands in samples with a fluorometric output (Figure 1D).

Optimization and benchmarking of SPRINT

As proof of principle, the well-described synthetic guanine-responsive riboswitch xpt/pbuE*6U ( 30) was used to establish reaction conditions for detecting transcription with Cas13a. The xpt/pbuE*6U contains the aptamer domain from the B. subtilis xpt-pbuX riboswitch which responds to the ligand guanine and a few related compounds such as hypoxanthine ( 61, 62). The xpt aptamer is coupled to the pbuE*6U expression platform, which is derived from the B. subtilis pbuE adenine-responsive riboswitch ( 63). The nomenclature of aptamer/expression platform for chimeric riboswitches will be used throughout the manuscript. In the absence of guanine, the intrinsic terminator within the xpt/pbuE*6U riboswitch causes RNA polymerase to abort synthesis in presence of guanine, full-length readthrough transcripts are synthesized that can be detected by Cas13a.

To couple in vitro transcription and Cas13a-mediated RNA detection, assay conditions had to be established for a reaction that is isothermal, one-step and one-batch. However, combining the reaction components at 37°C in SHERLOCK buffer ( 44) yielded a high background signal in the absence of ligand (Figure 2A). To overcome this issue, we tested a buffer that was used previously to assay synthetic riboswitches ( 30), referred to as SPRINT buffer. Unmodified SPRINT buffer reduced the background signal and enabled detection of the ligand with a 2.8-fold signal induction (Figure 2A). To further reduce the background signal, the contribution of Cas13a to the background signal was assessed. SHERLOCK reactions were run in SPRINT and SHERLOCK buffer (Figure 2B) and RNA could be reliably quantified in both buffers with negligible background signal ( Supplementary Figure S1a ). This indicates that the SHERLOCK components function efficiently in SPRINT buffer and instead in vitro transcription should be the focus for optimizing the dynamic range of the SPRINT assay.

Optimization of conditions and benchmarking of signal. All fluorescence measurements were background corrected, bars indicate mean value and error bars indicate s.d. from the mean n = 3. All measurements were normalized to 125 nM fluorescein. (A) SPRINT reactions were carried out with the guanine riboswitch xpt/pbuE*6U in SPRINT buffer or SHERLOCK buffer. Transcription of target RNA was regulated by the riboswitch in response to ±100 μM hypoxanthine. The measurements displayed were taken at 20 minutes reaction time. (B) SHERLOCK reactions were carried out in SHERLOCK buffer or SPRINT buffer at varying concentrations of added target RNA. The measurements displayed were taken at 20 min reaction time. (C, D) SPRINT reactions were carried out with the guanine riboswitch xpt/pbuE*6U with varying concentrations of rNTP and transcription template. Fluorescence was measured in response to ±10 μM guanine at 25 minutes reaction time. (E) SPRINT reactions were regulated by the guanine riboswitch xpt/pbuE*6U in response to ±10 μM guanine. Single turnover assays were conducted with 66 μg/ml heparin, multiple turnover assays were conducted without heparin.

Optimization of conditions and benchmarking of signal. All fluorescence measurements were background corrected, bars indicate mean value and error bars indicate s.d. from the mean n = 3. All measurements were normalized to 125 nM fluorescein. (A) SPRINT reactions were carried out with the guanine riboswitch xpt/pbuE*6U in SPRINT buffer or SHERLOCK buffer. Transcription of target RNA was regulated by the riboswitch in response to ±100 μM hypoxanthine. The measurements displayed were taken at 20 minutes reaction time. (B) SHERLOCK reactions were carried out in SHERLOCK buffer or SPRINT buffer at varying concentrations of added target RNA. The measurements displayed were taken at 20 min reaction time. (C, D) SPRINT reactions were carried out with the guanine riboswitch xpt/pbuE*6U with varying concentrations of rNTP and transcription template. Fluorescence was measured in response to ±10 μM guanine at 25 minutes reaction time. (E) SPRINT reactions were regulated by the guanine riboswitch xpt/pbuE*6U in response to ±10 μM guanine. Single turnover assays were conducted with 66 μg/ml heparin, multiple turnover assays were conducted without heparin.

The SPRINT buffer was systematically altered to identify improved conditions ( Supplementary Figure S1B ). SPRINT buffer is Tris-based, whereas SHERLOCK buffer is HEPES-based and when using HEPES instead of Tris buffer for SPRINT reactions, signal suppression in absence of ligand was abolished. This indicates that absence of HEPES in the SPRINT buffer is essential to enable transcription termination by the xpt/pbuE*6U riboswitch. Because bovine serum albumin (BSA) did not affect the assay, it was removed from the buffer ( Supplementary Figure S1C ) to decrease the risk of RNase contamination from BSA preparations. Since many hydrophobic small molecules of potential interest cannot be dissolved in water but can be dissolved in DMSO, the response of SPRINT reactions was also measured in increasing amounts of DMSO ( Supplementary Figure S1D ). Although DMSO slightly increases both background and on-signal, final concentrations of 10% DMSO did not inhibit the assay. However, most changes to the SPRINT buffer composition resulted either in no change or a decrease in detection of ligand-induced readthrough transcription ( Supplementary Figure S1B ).

To further optimize both signal intensity and reaction time, the concentration of NTPs and DNA template in the SPRINT reaction was systematically altered. The lowest concentrations of DNA and NTPs resulted in the largest fold change when transcription readthrough was induced with guanine (Figure 2C). However, because these conditions led to relatively weak signals (Figure 2D), we decided to compromise a higher fold induction for stronger signals and chose 2.5 nM DNA template and 20 μM NTP to enable reliable signal detection at 20 min reaction time. This modified SPRINT buffer (Figure 2A) is used in all experiments in this study, unless indicated otherwise. As the concentrations of NTPs and DNA template have a strong effect on inducibility and speed of the signal, they can be considered prime parameters for optimizing and adapting SPRINT assays.

The effect of heparin on SPRINT was assessed because in vitro transcription assays usually contain heparin to restrict RNA polymerase to a single turnover of transcription. SPRINT assays with the xpt/pbuE*6U guanine riboswitch were tested with and without heparin (Figure 2E). Repression of background signal is more efficient in the single turnover assays with heparin, but the induced signal in the multiple turnover assays was ∼35-fold larger and reached its maximum approximately twice as fast. This shows that multiple turnover transcription is mainly responsible for the strength of the fluorescent signal as opposed to the multiple turnover reactions of the Cas13a enzyme. This observation is consistent with previous results that showed amplification of the RNA input is necessary for a strong SHERLOCK signal ( 44, 45). For this reason, most SPRINT assays were conducted without heparin but note that SPRINT can be run as a single turnover assay whenever a minimal background signal is preferred over a fast response.

To assess its sensitivity, the SPRINT reaction was compared to the gold standard approach of quantifying transcription products by separating 32 P-labeled RNA transcripts using denaturing polyacrylamide gel electrophoresis ( 58). The transcriptional response of the guanine riboswitch xpt/pbuE*6U and the adenine riboswitch pbuE/pbuE ‡ ( 64) to their respective ligand was measured by SPRINT and the radiolabeling method (Figure 3A, B). The T50 value, the ligand concentration at half-maximal activation of transcription, was obtained from a fit to the data and used to compare the methods. For both riboswitches, the T50 was similar between multiple turnover SPRINT, single turnover radiolabeling, and values from the literature that were also obtained with single turnover radiolabeling. This indicates that results obtained with SPRINT are comparable to those obtained with radiolabeling while increasing speed, ease, and throughput of transcription assays. Together, these data establish that we have effectively combined small-molecule dependent transcriptional regulation and Cas13a-mediated RNA detection into a single reaction.

Dose-response of various riboswitches measured with SPRINT. All fluorescence measurements were background corrected, bars indicate mean value and error bars indicate s.d. from the mean n = 3. (A, B) The responses of the guanine riboswitch xpt/pbuE*6U and the adenine riboswitch pbuE/pbuE ‡ to their cognate ligands were measured. Results from SPRINT measurements are marked in blue, results from radiolabeling measurements are marked in red. The measurements displayed were taken at 20 min reaction time. Responses of various riboswitches to their cognate ligands and chemically similar compounds were measured: (C) a SAM sensing riboswitch, (D) a flavin mononucleotide sensing riboswitch, (E) a fluoride sensing riboswitch (signal measured at 60 minutes), and (F) a serotonin sensing riboswitch. (G) The native metE SAM sensing riboswitch is an OFF switch and attenuates transcription with increasing concentrations of ligand. Heparin was added to 66 μg/ml. Signal of the metE reaction was measured at 100 minutes reaction time. *(30), # (68), ‡ (23), § (73), † (31).

Dose-response of various riboswitches measured with SPRINT. All fluorescence measurements were background corrected, bars indicate mean value and error bars indicate s.d. from the mean n = 3. (A, B) The responses of the guanine riboswitch xpt/pbuE*6U and the adenine riboswitch pbuE/pbuE ‡ to their cognate ligands were measured. Results from SPRINT measurements are marked in blue, results from radiolabeling measurements are marked in red. The measurements displayed were taken at 20 min reaction time. Responses of various riboswitches to their cognate ligands and chemically similar compounds were measured: (C) a SAM sensing riboswitch, (D) a flavin mononucleotide sensing riboswitch, (E) a fluoride sensing riboswitch (signal measured at 60 minutes), and (F) a serotonin sensing riboswitch. (G) The native metE SAM sensing riboswitch is an OFF switch and attenuates transcription with increasing concentrations of ligand. Heparin was added to 66 μg/ml. Signal of the metE reaction was measured at 100 minutes reaction time. *(30), # (68), ‡ (23), § (73), † (31).

SPRINT can be implemented with various riboswitches

To test the versatility and specificity of SPRINT, detection of other small molecules and ions via riboswitches was assessed. The adenine riboswitch pbuE/pbuE ‡ responded selectively to adenine but not guanine (Figure 3B). The S-adenosylmethionine (SAM)-responsive riboswitch yitJ/pbuE*6U detected SAM but not the related compound S-adenosylhomocysteine (SAH), as expected ( 30). The T50 value for SAM was comparable to prior measurements using the 32 P-labeling assay (Figure 3C). The flavin mononucleotide (FMN) riboswitch ribD/pbuE*7U riboswitch was observed to have a high-affinity response to FMN with a T50 value around 1.01 ± 0.03 μM, a low-affinity response to the closely related flavin adenine dinucleotide (FAD) ( 30, 65), and no response to ribocil, which is a selective agonist of the related ribB riboswitch ( 66, 67) (Figure 3D). This is consistent with published results ( 30, 65–67). All four riboswitches (xpt/pbuE*6U, pbuE/pbuE ‡ , yitJ/pbuE*6U and ribD/pbuE*7U) are synthetic with variations of the pbuE expression platform ( 30), highlighting the utility of artificial riboswitches as robust biosensors.

Occasionally, reaction conditions needed to be optimized to accommodate new riboswitches. The ligand-dependent induction of the FMN riboswitch was initially very weak. To improve this, the magnesium concentration in the SPRINT reaction was increased. Of the four riboswitches that were tested at higher MgCl2 concentrations, only the ribD riboswitch showed a significant increase in fold induction ( Supplementary Figure S2 ). Therefore, SPRINT reactions with the ribD/pbuE*7U riboswitch were conducted at 10 mM MgCl2 instead of 2.5.

Some ligands inhibited the SPRINT assay when added to high concentrations. The fluoride riboswitch crcB turns on transcription readthrough by selectively responding to fluoride. Using the SPRINT assay, we measured a T50 value of 11 ± 1 μM (Figure 3E), which is, surprisingly, lower than previously observed KD values of ∼60 μM resulting from in-line probing ( 68). This may be explained in part by an observed inhibition of the assay at fluoride concentrations above 100 μM. To understand whether this inhibitory effect stems from inhibition of the E. coli RNAP or inhibition of Cas13a, SHERLOCK reactions were conducted at varying concentrations of sodium fluoride. Cas13a was gradually inhibited with increasing fluoride concentrations and the signal sharply dropped ∼600 μM ( Supplementary Figure S3A ). To measure the response of E. coli RNAP to fluoride, a two-batch protocol was developed ( Supplementary Figure S3B ) to first transcribe RNA in presence of fluoride, then wash out the fluoride and add the washed transcripts to a SHERLOCK reaction. Fluoride induced transcription from the fluoride riboswitch in a linear manner up to concentrations of 100 μM, but above concentrations of 125 μM fluoride drastically reduced overall transcriptional activity ( Supplementary Figure S3C ). This suggests that E. coli RNAP itself is the most fluoride-sensitive component in the SPRINT reactions. Studies with crcB knockout strains of E. coli support these findings by showing significant growth inhibition when the fluoride concentrations in the media exceed 100 μM ( 68). These results show how the two-step assay can be used to separate in vitro transcription reactions from Cas13a reactions or potentially to wash out Cas13a-inhibiting compounds after transcription that would otherwise interfere with the assay.

Novel RNA aptamers are routinely generated via s ystematic e volution of l igands by ex ponential enrichment (SELEX) against compounds of interest ( 23, 69–71) and the incorporation of such synthetic aptamers into SPRINT can greatly expand its biosensing repertoire. A synthetic aptamer that binds 5-hydroxytryptophan (5HTP) ( 23) was previously incorporated into the expression platform pbuE’ to generate an ON-riboswitch that responds to both 5HTP and serotonin ( 72). This riboswitch, called P1/pbuE’7U, was used with SPRINT to successfully detect serotonin. The T50 value was measured as 5 ± 3 μM for serotonin and the riboswitch discriminated against the related compound tryptamine (Figure 3F). Surprisingly, serotonin inhibited both the transcription and Cas13a reaction at concentrations >100 μM, whereas similar compounds such as 5HTP did not cause inhibitory effects ( Supplementary Figure S3D–F ). For this reason, the displayed dose-response curve should be interpreted with caution as inhibitory effects distort the signal at concentrations ∼100 μM. Despite the idiosyncratic inhibition by serotonin, these results demonstrate how expression platforms can be combined with synthetic aptamers in a plug-and-play manner to create novel biosensors for the SPRINT platform.

SPRINT can rapidly assess native riboswitch function

All riboswitches described in this study thus far are ‘ON’ switches that induce transcription in presence of ligands ( 30, 31, 68, 72) and are thus predisposed to work well in the context of the SPRINT assay, whereas most native riboswitches are ‘OFF’ switches. To assess whether SPRINT can robustly monitor OFF switches, we examined the metE riboswitch from B. subtilis that aborts transcription in response to SAM ( 73). The native riboswitch sequence was amplified from the B. subtilis genome via PCR and in a second PCR step the tac promoter was appended to the 5′-end and the Cas13a target transcript to the 3′-end so that the resulting PCR product could be used directly as DNA template for SPRINT ( Supplementary Figure S4A ). Initially, very high concentrations of SAM were required to repress the signal ( Supplementary Figure S4B ). To circumvent this issue, heparin was added to the SPRINT assay to restrict the RNA polymerase to a single turnover of transcription. Although the reaction was slowed down, the sensitivity of the assay was greatly improved. Titrating SAM yielded a T50 value of around 1 μM (Figure 3G), which is similar to previously established values of 0.5 and 2 μM ( 31, 73). This demonstrates that both, ON and OFF riboswitches can be integrated into a DNA template for SPRINT.

SPRINT can sense small molecules via transcriptional repressor proteins

Previously developed methods use allosteric transcription factors (aTFs) to detect compounds. Jung et al. ( 54) developed a system called ROSALIND that senses compounds with aTFs to regulate T7 RNAP-driven transcription of a fluorophore-binding aptamer ( 74) and thereby yielding a fluorometric output (Figure 1A). In this manner, 16 diverse compounds, including tetracyclines, macrolides, small molecules and metal ions were detected. To incorporate the broad repertoire of aTF-biosensors into SPRINT, we explored conditions that enable an isothermal, one-batch and one-step reaction that detects compounds with aTFs and generates a signal via Cas13a.

The ROSALIND buffer was adopted for aTF-based SPRINT reactions, although some modifications were made to the buffer in order to optimize sensitivity and speed of the reaction. The inorganic pyrophosphatase (IPPase) enzyme was deemed unnecessary and removed from the ROSALIND buffer. Further adaptions include reducing the concentrations of rNTP from 2.85 mM to 40 μM, DNA template from 25 to 15 nM, and T7 RNAP from 10 to 6.7 ng/μl.

Using the adapted buffer and conditions, the responses of the repressors TetR and SmtB were measured with SPRINT. The repressor SmtB enabled transcription in a zinc-dependent manner with a T50 of 4.8 ± 0.3 μM and showed a high selectivity against copper (Figure 4A). The repressor TetR responded to increasing concentrations of anhydrotetracycline with a T50 of 1.9 ± 0.2 μM which is within the range of 1–2.5 μM previously measured ( 54) (Figure 4B). This shows that SPRINT can not only measure regulation of E. coli RNAP by riboswitches, but also regulation of T7 RNAP by transcription factors. Combining these two mechanisms of detecting ligands greatly expands the scope of molecules that can be detected with SPRINT.

Transcription regulation by transcription factors. Fluorescent signal of the SPRINT reactions was measured at 20 minutes reaction time. Background was subtracted and signal normalized to 125 nM fluorescein. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. (A) SmtB de-represses transcription in response to zinc, but not copper. (B) TetR de-represses transcription in response to anhydrotetracycline. *(54).

Transcription regulation by transcription factors. Fluorescent signal of the SPRINT reactions was measured at 20 minutes reaction time. Background was subtracted and signal normalized to 125 nM fluorescein. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. (A) SmtB de-represses transcription in response to zinc, but not copper. (B) TetR de-represses transcription in response to anhydrotetracycline. *(54).

SPRINT can screen compounds in a high-throughput format

There is a growing interest in drugging RNA structures ( 75–79) including targets such as the HIV TAR element ( 80), human expansion repeats ( 75, 81), or riboswitches ( 82–85) but developing platforms for high-throughput screening of RNA-drug interactions remains challenging. As a proof of concept, the response of the guanine riboswitch xpt/pbuE*6U to 30 different small organic molecules was measured in a high-throughput screening format (Figure 5A, Supplementary Figure S5A ). For all compounds, the SPRINT signal at high concentration was plotted against the signal at low concentration to visualize the dose-response relation for each compound. Exact concentrations and full names of the compounds can be found in Supplementary Table S3 . Compounds such as guanine that elicited an equally strong transcriptional activation at low and high concentrations are found in the upper right quadrant of the plot and are expected to be strong agonists of the target. Compounds such as N2-methylguanine that are found in the upper left quadrant are expected to have less agonistic activity towards the RNA target. Non-binding compounds such as N6-methyladenine are in the lower left quadrant. Compounds such as 7-deazaguanine that showed some activation of transcription at low concentrations but reduced or no activation at high concentrations are suspected to inhibit the assay at higher concentrations. Notably, the results of this screen are consistent with prior reports on binding of individual compounds to the xpt aptamer domain using ITC or footprinting approaches ( 61, 86–89). Therefore, the compound screen with SPRINT enabled us to quickly assess the effect of various compounds on the RNA target to find compounds of interest.

SPRINT can be used for compound screens and enzyme-coupled assays. Fluorescent signal of the SPRINT reactions was background subtracted and normalized to 125 nM fluorescein. (A) The guanine riboswitch xpt/pbuE*6U regulated transcription in response to 30 different compounds. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent mean value, n = 3. (B) The effect of the compounds on transcription from a constitutive promoter was measured. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent one biological replicate. (C) SPRINT measured constitutive transcription of Cas13a target as transcription was inhibited at increasing concentrations of rifampicin. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. (D) Diagram of an enzyme-coupled assay. The enzyme hPNP converts inosine to hypoxanthine, which is detected by the guanine riboswitch xpt/pbuE*6U and triggers the SPRINT signal. (E) The enzyme-coupled assay was used to measure enzymatic activity of hPNP. Concentration of inosine was 1 mM, hPNP was added to an activity of 10 mU/μl, concentration of Immucillin-H was 10 μM. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. p-value was calculated using a two-tailed t-test. (F) The enzyme-coupled assay was used to measure concentration-dependent substrate conversion. hPNP was added to an activity of 1 mU/μl. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3.

SPRINT can be used for compound screens and enzyme-coupled assays. Fluorescent signal of the SPRINT reactions was background subtracted and normalized to 125 nM fluorescein. (A) The guanine riboswitch xpt/pbuE*6U regulated transcription in response to 30 different compounds. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent mean value, n = 3. (B) The effect of the compounds on transcription from a constitutive promoter was measured. Signal with solvent only was subtracted from signal with ligand to correct for differences in solvents of ligands. Dots represent one biological replicate. (C) SPRINT measured constitutive transcription of Cas13a target as transcription was inhibited at increasing concentrations of rifampicin. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. (D) Diagram of an enzyme-coupled assay. The enzyme hPNP converts inosine to hypoxanthine, which is detected by the guanine riboswitch xpt/pbuE*6U and triggers the SPRINT signal. (E) The enzyme-coupled assay was used to measure enzymatic activity of hPNP. Concentration of inosine was 1 mM, hPNP was added to an activity of 10 mU/μl, concentration of Immucillin-H was 10 μM. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3. p-value was calculated using a two-tailed t-test. (F) The enzyme-coupled assay was used to measure concentration-dependent substrate conversion. hPNP was added to an activity of 1 mU/μl. Bars indicate mean value, error bars indicate s.d. from the mean. n = 3.

When screening for target inhibitor drugs, it is essential to reliably identify pan-assay interference compounds (PAINS) ( 90) that might show up as false positives. For this purpose, the drug panel was tested in SPRINT reactions that constitutively transcribe a Cas13a target without regulatory components such as riboswitches or aTFs. This setup only measures effects of compounds on the core assay itself. In this way, compounds such as 7-deazaguanine, N2-methylguanine, 2,5,6-triaminpyrimidin-4-one (2,5,6-TAP) and to a lesser extent 2-fluoroadenine could successfully be identified as assay-interfering compounds (Figure 5B, Supplementary S5B, Supplementary Table S3 ).

Previous studies were aimed at identifying novel antibiotics by targeting the transcriptional machinery of bacteria ( 91, 92). This can include targeting riboswitches ( 66, 82–84, 93), transcription factors ( 94, 95) or the RNA polymerase itself ( 96, 97). Rifampicin inhibits the bacterial RNAP ( 98) by blocking elongation ( 99) and is the leading drug to treat mycobacterial infections such as tuberculosis ( 100). We measured the inhibitory effect of rifampicin on the RNAP with SPRINT. A constitutive transcription reaction was titrated with different concentrations of rifampicin (Figure 5C). A T50 value of 5.9 ± 0.6 nM was obtained, which is very close to the established KD of 3 nM ( 101). This demonstrates how SPRINT can be used to screen for drugs that target transcription and rapidly quantify drug efficacy.

Enzyme-coupled assay

SPRINT can generate fast fluorescent signals in response to various compounds and therefore could be used to detect products of enzymatic reactions. Most enzyme coupled assays are limited to the detection of one particular metabolite such as ADP generated in a kinase reaction or NADH generated in a redox reaction ( 102). Detecting enzymatic products with a riboswitch or transcription factor is an attractive alternative because of the large diversity of compounds that can be detected with these systems. We examined human purine nucleoside phosphorylase (hPNP) ( 103, 104), part of the purine salvage pathway, which catalyzes the phosphorylation of the ribose moiety of various purine nucleosides and concurrent removal of the sugar from the nucleobase and assessed conditions for an isothermal, one-batch and one-step enzyme-coupled SPRINT assay.

The conversion of inosine to hypoxanthine by hPNP was coupled to the synthetic guanine riboswitch xpt/pbuE*6U (Figure 5D). The guanine riboswitch responds to hypoxanthine ( 30) but does not bind nucleosides such as inosine. This enzyme-coupled assay enabled the observation of enzymatic activity by the hPNP enzyme (Figure 5E) without changing any components of the SPRINT buffer, except adding the substrates inosine and phosphate ( Supplementary Figure S6 ). Titration of the coupled reaction with inosine showed a concentration-dependent increase of hypoxanthine production by the hPNP enzyme (Figure 5F). The conversion of deoxyguanosine to guanine by hPNP can be inhibited with nucleoside analogs such as Immucillin-H (forodesine) which leads to accumulation of deoxyguanosine and consequent apoptosis in activated T-cells. Therefore, hPNP is an important drug target for the treatment of leukemia, arthritis, multiple sclerosis and transplant rejection ( 105–111). Adding the competitive inhibitor Immucillin-H at 100-fold lower concentration than the substrate inosine caused a significant reduction in the enzymatic activity as measured in the SPRINT assay (Figure 5E). Together, these results demonstrate how SPRINT can be used to assess the activity of enzymes, measure the inhibition of such enzymes with drugs and indirectly detect compounds such as inosine that are not bound by aTFs or riboswitches.

Portable assay formats

To address the need for point-of-care diagnostic devices, SPRINT needs to be adaptable to portable devices such as lateral flow assays (LFA) ( 45, 112) or portable fluorometric devices ( 37, 54, 113, 114). An inexpensive, hand-held device that can be made with a 3D-printer was previously developed for detecting water contaminants via fluorescence ( 54). This portable device illuminates reaction tubes with blue light ∼470 nm and fluorescent probes can be seen through a yellow film used as a long-pass optical filter. Fluorescence from SPRINT reactions was easily visible to the human eye using the device and no adaptations were required except increasing the concentration of FAM-labeled RNA in SPRINT reactions to 1.25 μM ( Supplementary Figure S7 ). Using the fluoride riboswitch, different fluoride concentrations could be differentiated by eye after 20 minutes of reaction time (Figure 6A) at 30°C which can be easily achieved by tightly holding the tubes in the hand. Further, the zinc-responsive transcription factor SmtB was used to detect zinc in municipal water samples that were collected, filtered and shipped from Paradise, California where zinc levels were affected by 2018 Camp Fire ( 54) (Figure 6B). By comparing the fluorescence of the samples with a calibration curve, the zinc concentration of the samples could be approximated by eye to be around 0–1 μM (I), 2.5–5 μM (II) and ∼5 μM (III), respectively. The zinc concentration in the municipal water samples was previously determined by flame atomic absorption spectroscopy (FAAS) ( 54) for comparison. These results demonstrate that SPRINT can be adapted to inexpensive portable formats and shows how the assay could be used for field testing.

Detection of analytes with a handheld device. SPRINT assays were assembled and added to water samples containing varying concentrations of the analyte. The concentrations indicated refer to the final concentration of analyte after mixing sample with the assay components. After sample and assay components were mixed, the tubes were placed in the LED device and photographs were taken right afterwards (0 min). The different appearances of the fluorescent samples in A and B are due to variations of the ambient light when the pictures were taken. (A) SPRINT reactions were run with fluoride riboswitch crcB. (B) SPRINT reactions were run with the transcription factor SmtB that responds to zinc. The five reactions on the left contained ddH2O with added ZnCl2. The three reactions to the right labeled I, II, and III contained municipal water samples from Paradise, California.

Detection of analytes with a handheld device. SPRINT assays were assembled and added to water samples containing varying concentrations of the analyte. The concentrations indicated refer to the final concentration of analyte after mixing sample with the assay components. After sample and assay components were mixed, the tubes were placed in the LED device and photographs were taken right afterwards (0 min). The different appearances of the fluorescent samples in A and B are due to variations of the ambient light when the pictures were taken. (A) SPRINT reactions were run with fluoride riboswitch crcB. (B) SPRINT reactions were run with the transcription factor SmtB that responds to zinc. The five reactions on the left contained ddH2O with added ZnCl2. The three reactions to the right labeled I, II, and III contained municipal water samples from Paradise, California.


Supporting information

S1 Fig. Location and climatic conditions of the accessions collecting sites.

(A) Distribution of the 44 natural accessions used in this study. Color of the points represents the altitudinal gradient. The map is shaded following the temperature gradient. (B) Mean annual precipitation and mean annual temperature for the sites where accessions were collected, in relation to major biome types of the world following Whittaker’s classification. 1–9: Tundra, Boreal forest, Temperate Grassland Desert, Woodland Shrubland, Temperate Forest, Temperate Rain Forest, Tropical Forest Savana, Tropical Rain Forest, and Desert. (C) Relationships altitude and climatic indicators at the collections sites.

S2 Fig. Rosette expansion rate of 39 A. thaliana accessions in control conditions at 8-h day length.

Bars are mean ± 95% CI of rosette expansion rate (mm 2 d -1 ) at 30 days post inoculation (n = 3) of plants grown in mock-inoculated:well-watered conditions.

S3 Fig. Least-square means for aboveground dry mass following ANOVA of 39 A. thaliana accessions grown under contrasted viral infection and watering levels.

Aboveground dry mass has been log-transformed prior analysis. Bars are lsmeans ± 95% CI. for plants grown under four treatments: mock-inoculated:WW, CaMV-infected:WW, mock-inoculated:WD and CaMV-infected:WD conditions. WW: well-watered WD: water deficit. Data are from Experiment 1.

S4 Fig. Relationship between relative change of aboveground dry mass (ADM) of 39 A. thaliana accessions in response to CaMV-infection and water deficit.

Each point represents the mean relative change of each accession. Point colors refer to the different response groups as determined in Fig 1. Solid lines represent significant linear regressions: (A) r = 0.39, P = 0.015 (B) r = 0.41 P = 0.003. Dashed lines represent identity(1:1) line, i.e. the line of equal response. Data are from Experiment 1. WW: well-watered conditions WD: water deficit.

S5 Fig. Relationship between R-score and relative change of aboveground dry mass (%) in response to CaMV-infection and water deficit for five response groups.

Each point represents a response group as determined in Fig 1. Plants were CaMV-infected or mock-inoculated under well-watered (WW) and water deficit (WD) conditions Colors are similar to response groups I-IV in Fig 1. Lines represent significant linear regressions.

S6 Fig. Effects of CaMV infection and watering on leaf mass per area (LMA) of 39 A. thaliana accessions.

(A) Bars are means ± 95% CI of leaf mass per area (LMA mg mm -2 ) at 30 dpi (n = 3) of plants grown in mock-inoculated:WW (white bars), CaMV-infected:WW (dark grey bars), mock-inoculated:WD (light grey bars) and CaMV-infected:WD (black bars) conditions. (B) Relative change (± se) of LMA (%) in mock-inoculated:WW (white circle), CaMV-infected:WW (dark grey triangle), mock-inoculated:WD (light grey diamond) and CaMV-infected:WD (black square) conditions. Data are from Experiment 1.

S7 Fig. Effects of CaMV infection and watering on leaf dry matter content (LDMC) of 39 A. thaliana accessions.

(A) Bars are means ± 95% CI of leaf dry matter content (LDMC mg g -1 ) at 30 dpi (n = 3) of plants grown in mock-inoculated:WW (white bars), CaMV-infected:WW (dark grey bars), mock-inoculated:WD (light grey bars) and CaMV-infected:WD (black bars) conditions. (B) Relative change (± se) of LDMC (%) in mock-inoculated:WW (white circle), CaMV-infected:WW (dark grey triangle), mock-inoculated:WD (light grey diamond) and CaMV-infected:WD (black square) conditions. Data are from Experiment 1.

S8 Fig. Comparison of the relationships between LMA and LDMC and inherent growth rates at 8-h day length and 12-h day length.

(A) Relationship between leaf mass per area (LMA mg mm -2 ) and leaf dry matter content (LDMC mg g -1 ) in experiment 1. (B) Relationship between LMA and LDMC in experiment 2. Each point represents the mean relative change of each genotype. Lines represent significant linear regressions (P < 0.001). Mock-inoculated:WW (white circle), CaMV-infected:WW (dark grey triangle), mock-inoculated:WD (light grey diamond) and CaMV-infected:WD (black square). Data are from Experiment 1 and 2 (n = 39, 8-h day length and n = 20, 12-h day length, respectively). (C) Relationship between expansion rate (mm 2 d -1 ) at 8-h and 12-h day length on 15 A. thaliana accessions (r = 0.71, P = 0.003).

S9 Fig. Relationship between vegetative growth and leaf morphological traits of 39 A. thaliana accessions.

Each point represents an accession under well-watered (WW dark blue circle) or water deficit (WD light blue triangle) conditions. (A) Relationship between relative change of aboveground dry mass production (ADM %) in CaMV-infected plants and leaf dry matter content (LDMC mg g -1 Pearson’s r = –0.43, P < 0.007 for WW and r = –0.20, P = 0.23 for WD). (B) Relationship between relative change of ADM (%) in CaMV infected plants under WW and under WD and leaf mass per area (LMA mg mm -2 r = –0.52, P < 0.001 for WW and r = –0.35, P = 0.031 for WD). Lines represent significant linear regressions at P < 0.05. Data are from Experiment 1.

S10 Fig. Effects of CaMV infection and watering treatment on phenological traits of 20 A. thaliana accessions.

(A) Days to bolting. (B) Days to flowering. Bars and error bars are means ± bootstrapped 95% confidence intervals of plants grown under mock-inoculated:WW (white bars), mock-inoculated:WD (dark grey bars), CaMV-infected:WW (light grey bars) and CaMV-infected:WD (black bars) conditions. WW: well-watered conditions WD: water deficit Accessions are ordered according to their final projected area of the rosette in condition of viral infection. Data are from Experiment 2.

S11 Fig. Relationships between leaf morphological traits, ruderality and phenology of 20 accessions of A. thaliana.

Relationships of days to bolting vs. (A) leaf dry matter content (LDMC mg g-1), (B) leaf mass per area (LMA mg mm-2), and (C) ruderal score (R %), and days to flowering vs. (D) LDMC, (E) LMA, and (F) R. Each point represents an accession grown under the control condition (well-watered x mock-inoculation). Lines are significant linear regressions at P < 0.05. Data are from Experiment 2.

S12 Fig. Dynamics of viral symptoms in 20 accessions of A. thaliana infected with CaMV under well-watered and water deficit conditions.

(A) Lag time of symptoms appearance. (B) Rate of symptoms appearance. Bars and error bars are means ± 95% confidence intervals extracted from sigmoidal curve fitting of symptom dynamics under well-watered (grey bars) and water deficit (black bars) conditions. Marks above the bars indicate 5%-level significant decrease (–) or increase (+) in response to the water deficit. Accessions are ordered according to increasing final projected area of the rosette. Data are from Experiment 2.