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In 2008, a curious find was discovered down a coal mine in the Ukrainian city of Donetsk. As it could not be safely or successfully cut out due to the nature of the sandstone in which it was embedded, the mysterious artifact looking much like an ancient wheel remains in situ down the mine. The following article is extracted from The Myth Of Man by J.P. Robinson.SMXL

Whilst drilling the coal coking stratum named J3 ‘Sukhodolsky’ at a depth of 900 meters (2952.76 feet) from the surface, workers were surprised to find what appears to be the imprint of a wheel above them in the sandstone roof of the tunnel that they had just excavated.

Thankfully, photographs of the unusual imprint were taken by the Deputy Chief V.V. Kruzhilin and shared with the mine foreman S. Kasatkin, who brought news of the find to light. Without being able to further explore the site and inspect the imprint at close hand, we are left with only the photographs as evidence of their existence (there was more than one imprint) and the word of a group of Ukrainian miners.

Without being able to definitively date the strata in which the fossilized wheel print was found, it has been noted that the Rostov region surrounding Donetsk is situated upon Carboniferous rock aged between 360-300 million years ago, and the widely distributed coking coals have derived from the middle to late Carboniferous; suggesting a possible age of the imprint at around 300 million years old. This would mean that an actual wheel became stuck millions of years ago and dissolved over time due to a process called diagenesis, where sediments are lithified into sedimentary rocks, as is common with fossil remains.

The following is an extract from a letter written by S. Kasatkin (translated from Ukrainian) in reference to his testimony of having been witness to the anomalous wheel imprint discovered by his team of miners in 2008:

‘This finding is not a PR action. In due time (2008), we as a team of engineers and workers asked the mine director to invite scientists for detailed examination of the object, but the director, following the instructions of the then owner of the mine, prohibited such talks and instead only ordered to accelerate work on passing through this section of lava and on fast ‘charging’ of the section with mining equipment.

Owing to that, this artifact and the smaller one found during further work came to be in a tunnel blockage and could not be taken out and studied. It is good that there were people, who in spite of the director’s prohibition, photographed this artifact.

I have connections with the people who first discovered these imprints and also with those who photographed them. We have more than a dozen witnesses. As you understand, the admission in the mine is strictly limited (it is dangerous on sudden emissions) and to obtain such permit is rather difficult.

The ‘wheel’ was printed on sandstone of the roof. Guys (drifters) tried to ‘cut away’ the find with pick hammers and to take it out to the surface, but sandstone was so strong (firm) that, having been afraid to damage a print, they have left it in place. At present the mine is closed (officially since 2009) and access to the ‘object’ is impossible - the equipment is dismantled and the given layers are already flooded.’

Ancient Tracks

Evidence for the existence of wheeled vehicles in antiquity has surfaced in other parts of the world, as petrified ancient tracks found in France, Spain, Italy, Malta, Kazakhstan, Ukraine, and even North America reveal. A prehistoric site known formally as Misrah Ghar il-Kbir meaning the Great Cave in Maltese (and commonly referred to as Clapham Junction), is located at Siggiewi, near the Dingli Cliffs in Malta.

It is at this now famous site that what have been termed ‘cart ruts’ cut into the limestone have mystified all that have visited the area. Likewise, a number of unusual tracks in stone are also visible on the island of Sicily at the Greek amphitheater called the Great Theater of Syracuse. Interestingly, most archaeologists have suggested that the Maltese tracks were probably created by Sicilian settlers who traveled to Malta around 2000 BC at the start of the Bronze Age.SML

Yet more tracks are to be found in Turkey. Some at Sofca cover an area roughly 45 by 10 miles (72.42 by 16.09 km), and also in Cappadocia, where several pockets of tracks can be seen. The many ruts discovered around the world have caused a great deal of controversy as to their purpose, age, and origin. These mysterious factors remain up for debate, but due to the association and close proximity with megalithic structures, in Malta particularly, and due to the fact that many tracks are now submerged below the sea in that region, many researchers have concluded that the fossilized lines show signs of great antiquity.

Bizarrely, considering the anomalous wheel print discovered in Ukraine that we have just discussed, a medieval city-fortress in the Crimean Mountains of Ukraine called Chufut-Kale lies in ruins, but also plays host to a number of cart ruts in stone like those at the nearby site of Eski-Kermen.

Dr. Alexander Koltypin is a geologist and director of the Natural Science Research Center at Moscow’s International Independent University of Ecology and Politology. He has spent a great deal of time visiting these sites and comparing them to one another in search of similarities.

“I first saw tracks in stone - fossilized car or terrain vehicle traces (usually called cart ruts) on Neogen plantation surface (peneplene in Phrygian) plain in May 2014 (Central Anatolia Turkey). They were situated in the field of development of Middle and Late Miocene tuffs and tuffites and according to age analysis of nearby volcanic rocks, had middle Miocene age of 12-14 million years,” wrote Koltypin.

This particular region which Koltypin has researched further is relatively unknown and the guide books offer nothing in the way of information. Whilst orthodox researchers claim that the tracks are simply the remnants of old petrified cart ruts from the kind of wheeled vehicles which donkeys or camels would have pulled, Koltypin has other ideas. “I will never accept it,” he explained when confronted with the standard explanations. “I myself will always remember . . . many other inhabitants of our planet wiped from our history.”

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https://www.ancient-origins.net/unexplained-phenomena/ancient-wheel-0010997

The details of the story depicted in the cave art surprised the researchers. Previously, the oldest known cave art first appeared in Europe 40,000 years ago, showcasing abstract symbols. By 35,000 years ago, the art became more sophisticated, showing horses and other animals.

But detailed scenes that share a story and therianthropes didn’t appear until 20,000 years ago – until this discovery. It “suggests that there was no gradual evolution of Paleolithic art from simple to complex around 35,000 years ago – at least not in Southeast Asia,” Aubert said.

“The hunters represented in the ancient rock art panel are simple figures with human-like bodies, but they have been depicted with heads or other body parts like those from birds, reptiles and other faunal species endemic to Sulawesi,” said Adhi Agus Oktaviana, study co-author and a PhD student at Griffith University in Australia, who has also studied rock art in Borneo, Sumatra, Raja Ampat and Misool.

https://www.cnn.com/2019/12/11/world/oldest-rock-art-humans-scn/index.html

A 100,000-year-old workshop used to mix and store the reddish pigment ochre has been discovered in Blombos Cave on the rugged southern coast near Cape Town. At the same site, scientists have found some of the earliest sharp stone tools, as well as evidence of fishing.

The latest find is reported in Friday's edition of the journal Science. It includes pieces of ochre, grinding bowls, shells for storage and bone and charcoal to mix with the pigment.

Lead researcher Christopher Henshilwood of the University of Bergen, Norway, said the find represents an important benchmark in the evolution of complex human mental processes.

The ochre could have been used for painting, decoration and skin protection, according to the researchers.

The discovery shows that even at that time "humans had the conceptual ability to source, combine and store substances that were then possibly used to enhance their social practices."

Two separate tool kits for working ochre were found at the site, the researchers said.

Henshilwood, who is also affiliated with the South Africa's University of Witwatersrand, said in a statement that researchers believe that pieces of ochre were rubbed on rock to make a fine red powder, and that was mixed with crushed bone, charcoal, stone chips and a liquid. The mixture was put into abalone shells and stirred with a bone.

https://www.foxnews.com/science/100000-year-old-art-studio-found-in-south-african-cave

Rock art dated to a minimum age of almost 40,000 years has been discovered in the Maros region of southern Sulawesi, Indonesia. This is an incredible result, published in Nature today, because one of the biggest challenges in rock art research is dating.

Consequently, every time we get dates for rock art, wherever from and no matter how old or young, it is important. But when we get really old dates outside Europe it is both highly significant and very exciting.

Specifically, the earliest minimum age for a hand stencil was found to be at least 39,900 years at the site of Leang Timpuseng and the oldest animal painting, of a babirusa “pig-deer” at the same site, dates back to at least 35,400 years.

A second animal painting (probably a pig) at another site has a minimum age of 35,700 years.

Obtaining 36,000 to 40,000 year minimum ages for paintings of animals and hand stencils of Sulawesi is an especially important rock art dating result because it has long been argued that the origin of art began in the deep caves of Europe more than 30,000 years ago.

Rock art is found all over the world. It is an archive of Indigenous arts and history stretching back tens of thousands of years and in this sense is a major component of world art history.

Rock art typically consists of paintings, drawings, engravings, stencils, prints, bas-relief carvings and figures made of beeswax in rock shelters and caves, on boulders and platforms.

Rock art sites are special, often spectacular places that reflect ancient experience and sometimes spirituality. They are locations where aspects of ceremony, belief and history are recorded in visual form. They are a testament to thousands of years of Indigenous culture and cultural interaction with other peoples, other creatures and the environment.

Where does the first rock art come from?

The Sulawesi dates show that the making of rock art did not originate in Europe, that it is more likely a much older behaviour brought by the first humans to both Europe and Southeast Asia. Or that rock art practices of making hand stencils and skilfully executed depictions of wild animals were independently invented in far flung parts of the world many tens of thousands of years ago.

Both possibilities are equally exciting as they force us to rethink many things about our most ancient modern human ancestors. They significantly change debates about the origin of art, the behavioural practices modern humans brought with them when they left Africa more than 60,000 years ago and what it is to be human.

Certainly, it appears that when modern humans reached new lands in vastly different parts of the world they literally put a human stamp on the new landscapes.

From southwest China to Malaysia, from Indonesia to the north of Australia, research by all three of us indicates the oldest surviving rock art to invariably consist of naturalistic paintings of animals.

In many places we also find hand stencils among the oldest surviving art forms. All attempts to date this early art have indicated considerable antiquity with various minimum ages but the new results from Sulawesi show this early widespread practice may have begun almost 40,000 years ago right across the region.

Australian rock art

In Australia there are at least 100,000 rock art sites, most across the north of the continent. But unlike Sulawesi, the oldest paintings are mostly in sandstone shelters rather than limestone, making them much more difficult to date.

Many researchers have suggested the oldest paintings include depictions of long extinct animals but we can never be absolutely sure of this. Used pieces of ochre, “crayons”, are found in the lowest levels and throughout excavated rock shelter floor deposits in Australia.

At more than one location they have been dated to up to 50,000 years ago. Sulawesi is not far from northern Australia and the first people to reach Australia’s shores more than 50,000 years ago would have passed through that region of Southeast Asia.

The ochre crayons from sites in northern Australia combined with the new dates of similar-looking imagery from Sulawesi give us strong circumstantial evidence that the oldest naturalistic animal paintings and hand stencils from Australia may also rival those of Europe in terms of age.

According to the Guardian, the destroyed site was a rock shelter located in Juukan Gorge in Western Australia that had been continuously occupied by the early inhabitants of the territory dating back over 46,000 years.

The cave was one of the oldest in the western Pilbara region and the only inland site with evidence of continual habitation which lasted through the last Ice Age.

“It’s one of the most sacred sites in the Pilbara region…we wanted to have that area protected,” said Burchell Hayes, the director of the Puutu Kunti Kurrama and Pinikura (PKKP) Aboriginal Corporation which oversees the land. .....

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Hematite Mining in the Ancient Americas: Mina Primavera, A 2,000-Year-Old Peruvian Mine

Kevin J. Vaughn, Moises Linares Grados, Jelmer W. Eerkens and Matthew J. Edwards

Mina Primavera, a hematite (Fe2O3) mine located in southern Peru, was exploited beginning approximately 2,000 years ago by two Andean civilizations, the Nasca and Wari. Despite the importance of hematite in the material culture of ancient Americas, few hematite mines have been reported in the New World literature and none have been reported for the Central Andes. An estimated 3,170 tonnes of hematite were extracted from the mine for over 1,400 years at an average rate of 2.65 tonnes per year, suggesting regular and extensive mining prior to Spanish conquest. The hematite was likely used as pigment for painting pottery, and the mine demonstrates that iron ores were extracted extensively at an early date in the Americas.

"Iron mining in the Old World, specifically in Africa, goes back 40,000 years.

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https://www.purdue.edu/uns/x/2008a/080129VaughnMine.html

Archaeologists have discovered a 12,000-year-old iron oxide mine in Chile that marks the oldest evidence of organized mining ever found in the Americas, according to a report in the June issue of Current Anthropology.

A team of researchers led by Diego Salazar of the Universidad de Chile found the 40-meter trench near the coastal town of Taltal in northern Chile. It was dug by the Huentelauquen people -- the first settlers in the region -- who used iron oxide as pigment for painted stone and bone instruments, and probably also for clothing and body paint, the researchers say.

The remarkable duration and extent of the operation illustrate the surprising cultural complexity of these ancient people. "It shows that [mining] was a labor-intensive activity demanding specific technical skills and some level of social cooperation transmitted through generations," Salazar and his team write.

An estimated 700 cubic meters and 2,000 tons of rock were extracted from the mine. Carbon dates for charcoal and shells found in the mine suggest it was used continuously from around 12,000 years ago to 10,500 years ago, and then used again around 4,300 years ago. The researchers also found more than 500 hammerstones dating back to the earliest use of the mine.

"The regular exploitation of [the site] for more than a millennium … indicates that knowledge about the location of the mine, the properties of its iron oxides, and the techniques required to exploit and process these minerals were transmitted over generations within the Huentelauquen Cultural Complex, thereby consolidating the first mining tradition yet known in America," the researchers write. The find extends "by several millennia the mining sites yet recorded in the Americas."

Before this find, a North American copper mine dated to between 4,500 and 2,600 years ago was the oldest known in the Americas.

Indexed and Archived from University of Chicago Press Journals. "Archaeologists uncover oldest mine in the Americas." ScienceDaily. ScienceDaily, 20 May 2011. by Dragonfly Kingdom Library 

https://www.sciencedaily.com/releases/2011/05/110519101231.htm

The veins of copper that ripple through its bedrock drew the attention of early Native Americans, who used the metal to make tools. However, many details of their activities—such as when they mined—remain hidden behind the thick haze of time.

Now, new research suggests that Isle Royale’s mining boom peaked about 6000 years ago and left a legacy of aquatic pollution. The high levels of copper, lead, and potassium in sediments from a cove on the island point to a long and intense period of indigenous mining. Researchers presented these results, published recently in the journal The Holocene, in a poster session on 16 December at the 2014 American Geophysical Union Fall Meeting.

Evidence of Ancient Mining

European explorers first noticed evidence for indigenous copper mines back in the 1800s. In some places, miners had dug down more than 20 meters into bedrock—an impressive feat considering their limited tools. However, without a way to date the pits directly, the timing of these mining activities could only be loosely constrained by the ages of copper artifacts found across the Great Lakes region. Archaeologists have dated many objects associated with the so-called Old Copper Complex, but the objects span thousands of years.

The mines caught the interest of David Pompeani, a Ph.D. student at the University of Pittsburgh, who went looking for clues beneath the chilly waters of Lake Superior. He hypothesized that chemicals released during the process of mining and annealing copper would leak into the lake and settle in its sediments. These sediments could then be dated using carbon-14 and other radioactive isotopes.

One such indicator of mining activity is lead, which would have leached from mine tailings and vaporized when miners heated copper to shape the metal, only to collect again in nearby waters. Pompeani and his colleagues previously found lead pollution in 8000- to 5000-year-old sediments along the south shore of Lake Superior. They interpreted this pollution as evidence of an extended era of widespread copper mining on Michigan’s Keweenaw Peninsula.

A Focus on Isle Royale

Several questions followed. How far did these ancient miners range? Did they migrate from one mine to another? To learn more, Pompeani’s team jumped across the lake to Isle Royale, a streak of rock that lies just off the Canadian shore. There, indigenous miners excavated the largest known pre-Columbian copper mine on top of Minong Ridge.

In nearby McCargoe Cove, a deep inlet that cuts diagonally across the island’s glacial striations, the researchers found elevated levels of lead and copper, along with potassium—a by-product of the fires used for mining and annealing. At their peak, lead and copper concentrations reached values an order of magnitude greater than background levels and about half as high as those associated with modern contamination.

The spike in pollution began 6500 years ago and lasted for about a millennium. Then, abruptly, it ended, suggesting mining ended too. Pollution did not rise again until the mid-1800s, when mining resumed on Isle Royale, smelting began on the Keweenaw Peninsula, and leaded gasoline emissions grew.

The scientists do not know why mining screeched to a halt. They speculate that miners may have exhausted all the easily accessible veins and moved on. Climate changes may have also played a role—evidence from lake sediments around the Midwest suggests climate began to get dryer. In time, geologic clues may continue to provide more information.

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https://eos.org/articles/miners-left-pollution-trail-great-lakes-6000-years-ago

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Researchers analyzing one of the radial Archaeopteris tree root systems at the Cairo, New York, site CHARLES VER STRAETEN

Scientists have discovered the world’s oldest forest—and its radical impact on life

By Colin BarrasDec. 19, 2019 , 11:00 AM

Scientists have discovered the world’s oldest forest in an abandoned quarry near Cairo, New York. The 385-million-year-old rocks contain the fossilized woody roots of dozens of ancient trees. The find marks a turning point in Earth’s history. When trees evolved these roots, they helped pull carbon dioxide (CO2) from the air and lock it away, radically shifting the planet’s climate and leading to the atmosphere we know today.

“The Cairo site is very special,” says team member Christopher Berry, a paleobotanist at Cardiff University in the United Kingdom. The quarry floor, about half the size of a U.S. football field, represents a horizontal slice through the soil just below the surface of the ancient forest. “You are walking through the roots of ancient trees,” Berry says. “Standing on the quarry surface we can reconstruct the living forest around us in our imagination.”

Berry and colleagues first discovered the site in 2009 and are still analyzing the fossils it contains. Some of the fossilized roots there are 15 centimeters in diameter and form 11-meter-wide horizontal radial patterns spreading out from where the vertical tree trunks once stood. They seem to belong to Archaeopteris, a type of tree with large woody roots and woody branches with leaves that is related in some way to modern trees, the team reports today in Current Biology. Previously, the oldest Archaeopteris fossils were no more than 365 million years old, Berry says, and exactly when the tree evolved its modern-looking features has been unclear. ....... 

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https://www.sciencemag.org/news/2019/12/scientists-have-discovered-world-s-oldest-forest-and-its-radical-impact-life

Abstract

The term “diagenesis” refers to essentially everything that happens to sediments and rocks after their deposition but prior to metamorphism. There are a variety of diagenetic processes, biological, chemical and physical, that ultimately convert sediments into sedimentary rocks. The earliest of those events are covered in this chapter on near-surface diagenesis; subsequent chapters cover processes and products that occur primarily during later stages of diagenesis (mainly mesogenesis). Those include mechanical and chemical compaction, cementation, dissolution, replacement and structural deformation. All these processes can profoundly affect the porosity, permeability and hydrocarbon reservoir potential of clastic terrigenous deposits, and most of them are a function of initial sediment composition and the changes in pressure, temperature and water chemistry that accompany progressive burial. Less explicitly covered, but potentially no less important, is diagenesis that can occur during one or more episodes of local or regional uplift and consequent exposure (telogenesis). These events also introduce changes in the pressure/ temperature/water chemistry regime of rocks, and thus can cause major diagenetic changes, especially grain dissolution and cementation. In an attempt to address the impacts of the various diagenetic events that rocks may experience, this book includes both a section on the recognition of porosity types as well as one on paragenesis (i.e., the placement of diagenetic events into a temporal sequence related to the burial/uplift history of rocks). Burial diagenesis is critically important in controlling the porosity of clastic terrigenous rocks and is, in the main, porosity destructive—that is, almost all rocks lose porosity with increased burial depth. Nonetheless, several factors can retard or inhibit porosity loss, including early grain-coating cements, that block later overgrowth cementation, regional overpressuring of basins that reduce effective overburden stresses and, under some circumstances, hydrocarbon entry that can reduce rock-water interactions. In addition, the processes of dissolution and fracturing may, under the right circumstance, lead to actual increases in subsurface porosity. So the discussion of porosity destruction, preservation and creation pervades all chapters in the diagenesis section, and emphasis is placed on recognition of key features associated with anomalous porosity retention or creation.

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https://pubs.geoscienceworld.org/books/book/1526/chapter/107223237/Diagenesis

A RUSSIAN academic has claimed an ancient non-human civilisation drove GIANT CARS on the Earth's surface 12 to 14 MILLION years ago - with the tracks still visible TODAY.

Dr Alexander Koltypin, a leading geologist in his home country, claims that mysterious groove-like markings in the Phrygian Valley of central Turkey were artificially made by all-terrain vehicles and NOT created by any natural process.

The scientist, a director of the Natural Science Scientific Research Centre at Moscow's International Independent University of Ecology and Politology, has just returned from a field trip to the site in Anatolia with three colleagues.

He described the markings as "petrified tracking ruts in rocky tuffaceous deposits" made from compacted volcanic ash.

He said: "We can suppose that ancient vehicles on wheels were drove on soft soil, maybe a wet surface.

"Because of their weight the ruts were so deep.

"And later these ruts - and all the surface around - just petrified and secured all the evidence.

Describing what could have made such tracks, he added: "All these rocky fields were covered with the ruts left some millions of years ago... we are not talking about human beings."

"We are dealing with some kind of cars or all-terrain vehicles.

"The pairs of ruts are crossing each other from time to time and some ruts are more deep than the others."

According to Dr Koltypin's observations, "the view of the ruts does not leave any doubt that they are ancient, in some places the surface suffered from weathering, cracks are seen here".

The age of the ruts is between 12 and 14 million years old, the academic believes.

"The methodology of specifying the age of volcanic rocks is very well studied and worked out," he said.

"As a geologist, I can certainly tell you that unknown antediluvian all-terrain vehicles drove around Central Turkey some 12-to-14 million years ago."

Dr Koltypin claimed archeologists "avoid touching this matter" because it will "ruin all their classic theories".

He added: "I think we are seeing the signs of the civilisation which existed before the classic creation of this world.

"Maybe the creatures of that pre-civilisation were not like modern human beings. "

He said the ancient "car tracks" are one of a number of clues "which prove the existence of ancient civilisations" but are often ignored by mainstream scientists.

There was no comprehensible system for the tracks but the distance between each pair of tracks "is always the same", he added.

He suggested "this distance very much fits that between the wheels of modern cars" but that the tracks are too deep for today's vehicles.

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https://www.express.co.uk/news/weird/598948/Russia-geologist-Dr-Alexander-Koltypin-non-humans-cars-Earth-tracks-Turkey

Controversial Claim by Geologist: Mysterious tracks in Turkey caused by unknown civilization millions of years ago

Dr. Alexander Koltypin , geologist and a director of the Natural Science Scientific Research Centre at Moscow's International Independent University of Ecology and Politology has recently completed investigations at the site in Anatolia which is marked with strange ruts, described as “petrified tracking ruts in rocky tuffaceous deposits’ made from compacted volcanic ash,” according to MailOnline.

The tracks cut across the landscape of the Phrygia Valley, dating back to various historical periods, according to conventional academia. The earliest roads are thought to have been made during the Hittite Empire (circa 1600 BC – 1178 BC). As time went on, paths were cut deeply into the soft rock by the Phrygians, then by the Greeks, and Alexander the Great with his armies. They eventually became part of the Roman road network, writes Culture Routes in Turkey

Koltypin and colleagues have examined the rocky fields interlaced with deep grooves, and have suggested that it was indeed vehicles which caused the tracks, but not lightweight carts or chariots. Instead he suggests the “unknown antediluvian all-terrain vehicles” were huge and heavy. In addition, he dates them back to approximately 14 million years ago, and claims they were driven by an unknown civilization.

He told MailOnline, “All these rocky fields were covered with the ruts left some millions of years ago....we are not talking about human beings.”

The geologist says with certainty that the ruts are prehistoric without a doubt, due to the weathering and cracks observed.

“The methodology of specifying the age of volcanic rocks is very well studied and worked out,” Koltypin said.

The scientist notes that the distance between each pair of tracks remains consistent, and that the measurement fit that between the wheels of a modern vehicles. However, the tracks are much too deep for today’s cars, raising more questions about what sort of transport device was being used.

The deepest ruts are three feet (one meter), and on the walls of these ruts are horizontal scratches, very much appearing to have been left by the ends of axels poking out of ancient wheels.

News site Express reports that Koltypin believes the deep channels were cut into the soft, wet soil and rock due to the sheer weight of the large prehistoric vehicles. He says, “And later these ruts - and all the surface around - just petrified and secured all the evidence. Such cases are well known to geologists, for example, the footprints of dinosaurs were ‘naturally preserved’ in a similar way.”

Koltypin is aware that his claims are controversial, but says mainstream academia will not address the subject matter as it will “ruin all their classic theories.”

“I think we are seeing the signs of the civilization which existed before the classic creation of this world. Maybe the creatures of that pre-civilization were not like modern human beings,” he proposes.

Very similar interesting and mysterious tracks exist in other locations of the world, notably in the Maltese archipelago. These ancient grooves continue to puzzle researchers. Some of the strange tracks of Misrah Ghar il-Kbir deliberately plunge off cliffs or continue off land and into the ocean. It is not yet known who made the tracks, or why.

Like the channels at Malta, questions remain surrounding the deep tracks cut into the stone in the Phrygian Valley.

Koltypin’s research work continues as he investigates anomalous sites, but it will likely be some time before established academia embraces his unconventional theories.

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https://www.ancient-origins.net/news-mysterious-phenomena/controversial-claim-geologist-mysterious-tracks-turkey-caused-unknown-020489

How a giant tree's death sparked the conservation movement 160 years ago

160 years ago a giant sequoia in California was cut down, becoming the inspiration for the national park system

On Monday, 27 June, 1853, a giant sequoia – one of the natural world's most awe-inspiring sights - was brought to the ground by a band of gold-rush speculators in Calaveras county, California. It had taken the men three weeks to cut through the base of the 300ft-tall, 1,244-year-old tree, but finally it fell to the forest floor.

A section of the bark from the "Mammoth Tree", as newspapers soon described it, had already been removed and was sent to San Francisco to be put on display. The species had only been "discovered" (local Native American tribes such as the Miwok had known of the trees for centuries) that spring by a hunter who stumbled upon the pristine grove in the foothills of the Sierra Nevada whilst chasing an injured bear. Word of the discovery quickly spread.

In the age of PT Barnum's freak shows, the speculators, mostly gold miners, had sensed a commercial opportunity. The section of bark – re-erected using scaffold, with a piano inside to entertain paying visitors - would later be sent to Broadway in New York, as would the bark from a second tree felled a year later. The bark of the "Mother of the Forest" – as the second tree was named – would even go on to be displayed at London's Crystal Palace causing great excitement and wonder in Victorian England before it was destroyed by fire on 30 December 1866. (The bark of the original mammoth tree was also lost to fire as it lay in storage in New York in 1855. A fitting end, perhaps, as fire plays such a crucial role in the life cycle of giant sequoias.)

The fame of the trees was such that a hotel was quickly built at the site to host the influx of tourists. To entertain the guests, tea dances were regularly held on the stump of the mammoth tree and a bowling alley was built on the now prone trunk. (This page has a wonderful range of images of the Mammoth Tree and the Mother of the Forest.)

The remarkable, engaging story of these two doomed trees is too detailed to be told here, but what is worth recalling on this anniversary is the reaction their destruction caused in the media at the time – and its subsequent effect on some progressive politicians a decade later when they cited their felling and exploitation as an inspiration to establish what later came to be known as the US national park system.

Was the outrage expressed by some in the popular media of the day evidence of the first stirrings of an environmental consciousness in the US? It would be wrong to assess such statements without noting the historical context of that age – a time of the "manifest destiny" when nature was viewed as a God-given resource for Mankind to exploit – but it is also hard to ignore the clear outrage and bemusement among some commentators that such magnificent natural specimens had been brutalised in this way.

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According to Gary D Lowe, a local historian, author and "Big Tree" aficionado, the first-known negative commentary came a month before the tree was felled. An article in the Sonora Herald, a local newspaper, reported that Captain Hanford, the man leading the enterprise, "is about stripping off the bark". The report went on: "This will of course kill the tree, which is much to be deprecated."

On 27 June, 1853 – the same day the tree finally fell - a report in San Francisco's Placer Times and Transcript also noted an article, again in the Sonora Herald, expressing regret that Captain Hanford was preparing for a "portion of the mammoth tree" to be sent to New York.

"Amator" [Latin for "friend"] is dreadfully shocked at the vandalism and barbarity of flaying that giant of the woods, and depriving California of its greatest "growing" exponent.

However, the same report also goes on to say that the stripping of the tree's bark is "characteristic of California enterprise" and that Hanford's efforts to exhibit the bark in New York will allow "millions of the inhabitants of the earth to see it, has rendered his adopted state a lasting benefit, given to science a page, and the world a natural curiosity". So any sadness at the tree's demise was counteracted by the boost to local pride.

But these were reports in local newspapers with little influence outside the communities they served. A far more significant report came that autumn when Maturin M Ballou, the Boston-based editor of Gleason's Pictorial Drawing Room Companion, one of the most widely read magazines of the day, printed an illustration of the "largest tree yet discovered in the world" on 1 October, 1853. The accompanying text said:

To our mind it seems a cruel idea, a perfect desecration, to cut down such a splendid tree…In Europe, such a natural production would have been cherished and protected, if necessary, by law; but in this money-making, go-ahead community, thirty or forty thousand dollars are paid for it, and the purchaser chops it down, and ships it off for a shilling show! We hope that no one will conceive the idea of purchasing the Niagara Falls with the same purpose!...But, seriously, what in the world could have possessed any mortal to embark in such speculation with this mountain of wood? In its natural condition, rearing its majestic head towards heaven, and waving in all its native vigour, strength and verdure, it was a sight worth a pilgrimage to see; but now, alas, It is only a monument of the cupidity of those who have destroyed all there was of interest connected with it.

Five months later, on 11 March, 1854, Ballou printed a further remark in his magazine:

A tree of such gigantic proportions as well might excite the wonder and curiosity of the world. Although the destruction of such a magnificent object was an act of vandalism not to be forgiven, yet the desecration has been committed, and it is useless now to reiterate our vain regrets.

However, the ripples of outrage took a further year – and the stripping of the Mother of the Forest – to really gain traction. Then came this editorial in the New York Herald, dated 17 December, 1855:

The finest, the most beautiful and symmetrical of these trees, (though not the largest) has been cut down…From this beginning, unless the Goths and Vandals are arrested in their work, the destruction of the incomparable forest will probably go on till the last vestige of it is destroyed. In this view, the point that we make is, that the State of California and the Congress of the Union should interpose to preserve these trees, as the living proofs that the boasted monarchs of the wood of the Old World are but stunted shrubbery compared with the forest giants of our own country. We say that Congress should interpose, upon the presumption that these trees are public property, are on the public lands of California, and because Congress has already interposed to protect the public live oak forests of Florida from the rapacity of unscrupulous speculators…We repeat, that it is the duty of the State of California, of Congress, and of all good citizens, to protect and to preserve these California monuments of the capabilities of our American soil. Let it be the law that this…Mammoth Grove shall stand.

The next notable article was printed in the March 1859 issue (pdf) of Hutchings' California Magazine. It was also later reprinted the following year in the popular tourist guide, Scenes of Wonder and Curiosity in California:

In our estimation, it was a sacrilegious act; although it is possible, that the exhibition of the bark, among the unbelievers of the eastern part of our continent, and of Europe, may have convinced all the "Thomases" living, that we have great facts in California, that must be believed, sooner or later. This is the only palliating consideration with us for this act of desecration.

And then, in 1864, came the culminating moment when John Conness, the senator from California, rose in Congress to make a speech urging his colleagues to pass a bill that would see the now nationally famous Yosemite Valley and its neighbouring grove of sequoias in the mountains above Mariposa secured and protected "inalienable forever". In making his case, he directly referenced the fate of the felled trees at Calaveras just over a decade earlier:

From the Calaveras grove some sections of a fallen tree were cut during and pending the great World's Fair that was held in London some years since…The English who saw it declared it to be a Yankee invention, made from beginning to end; that it was an utter untruth that such trees grew in the country; that it could not be; and, although the section of the tree was transported there at an expense of several thousand dollars, we were not able to convince them that it was a specimen of American growth. They would not believe us. The purpose of this bill is to preserve one of these groves from devastation and injury. The necessity of taking early possession and care of these great wonders can easily be seen and understood.

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The bill passed and the "Yosemite grant" paved the way for the first official national park being established at Yellowstone in 1872. Celebrated conservationists such as John Muir would all later visit the stump of the original "mammoth tree" to reflect on both its fate and influence. However, the grove of sequoias at Calaveras – where the story of the US conservation movement arguably began – did not become a state park until 1931 following a decades-long fight to see off the desires of lumber companies.

Today, the trees are now safe from the "Goths and Vandals", but not, alas, some of the side-effects of modern civilization: urban ozone, climate change, uncontrolled frequent fires, to name but a few.

Indexed and archived from the Guardian by Dragonfly Kingdom Library

https://www.theguardian.com/environment/blog/2013/jun/27/giant-tree-death-conservation-movement

Behavioural pharmacology

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Oxytocin for the treatment of drug and alcohol use disorders

Mary R. Lee and Elise M. Weerts

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Abstract

There is growing interest in the use of oxytocin (OT) as a potential treatment for alcohol and other substance use disorders. OT is a neuropeptide that modulates adaptive processes associated with addiction including reward, tolerance, associative learning, memory, and stress responses. OT exerts its effects via interactions with the hypothalamic–pituitary–adrenal (HPA) axis, and multiple neurotransmitter systems including the dopamine mesolimbic reward and corticotrophin-releasing factor stress systems. Oxytocin effects on stress systems are of high interest given the strong link between stress, drug use and relapse, and known dysregulation of HPA-axis activity associated with substance use disorders. At the same time, the oxytocin system is itself altered by acute or chronic drug exposure. This review summarizes the preclinical and clinical literature on the oxytocin system, and its relevance to drug and alcohol addiction. In addition, findings from recent clinical trials conducted in participants with cocaine, cannabis or alcohol use disorder are included and evidence that oxytocin may help to normalize blunted stress responses, and attenuate withdrawal associated hypercortisolism, negative mood and withdrawal symptoms are summarized.

Keywords: Oxytocin, addiction, dependence, substance use disorder, alcoholism, treatment

Introduction

Oxytocin (OT) is a 9 amino acid polypeptide hormone that acts via a specific receptor and is widely distributed in the central nervous system (CNS) and peripheral tissues (Gimpl and Fahrenholz 2001). OT is involved in the regulation and release of adenohypophyseal hormones including prolactin, adrenocorticotropin (ACTH), gonadotropins, and corticotrophin-releasing factor (CRF). Initially, OT was thought to be primarily involved in sexual behaviors, female parturition and lactation. Subsequent research has determined that OT is also involved in emotional regulation, pain and stress, and modulates response to rewarding behaviors promoted by food, sex and drugs (Meyer-Lindenberg et al. 2011; Onaka et al. 2012). The co-modulation of both stress and motivational processes is believed to be due to the important role of OT to shift salience to social, affiliative processes, both by increasing the salience itself of rewarding stimuli and/or by reducing stress, allowing for attention to social bonding (Baskerville and Douglas 2010). This is obviously relevant to addiction, where salience of drug stimuli overshadows motivation for social affiliation, and where stress may trigger drug seeking and relapse (Sinha 2008). In the current review, we will focus on the role of the oxytocin system in drug and alcohol addiction and highlight key findings to date on the use of intranasal OT to treat substance use disorders.

Oxytocin and stress

The influence of OT to dampen stress responses is important. Neuroendocrine pathways that modulate the response to stress include three interconnecting circuits, the HPA axis, the adrenomedullary system, and the extra-hypothalamic CRF system. The HPA axis releases CRF from paraventricular neurons within the hypothalamus, stimulating the synthesis and release of adrenocorticotropin (ACTH) by the anterior pituitary, which in turn stimulates the synthesis and release of corticosteroids (CORT) (cortisol in human and nonhuman primates and corticosterone in rodents) via the adrenal cortex. The sympathetic adrenomedullary system, which releases norepinephrine and epinephrine, and CRF expression in the extra-hypothalamic brain regions including limbic regions, are key substrates involved in anxiety and other stress-related behaviors. Stress, defined as any stimulus that disrupts physiological homeostasis, triggers a cascade of adaptive responses involving any or all of these pathways to return the organism to homeostasis.

There is strong evidence from the preclinical literature that stress exposure is an important contributor to relapse. In rats and monkeys, acute stress enhances alcohol preference and reward (Funk et al. 2004), and increased alcohol intake is correlated with stress-induced increases in CORT levels (Fahlke et al. 2000; Fish et al. 2008). In addition, following repeated social stress exposure (e.g., defeat, low social rank, and maternal separation), rats and monkeys subsequently show greater alcohol intake when compared to non-stressed cohorts (Fahlke et al. 2000; Cruz et al. 2008; Fish et al. 2008). Current theories suggest that CORT release induced by stress augments drug reinforcement. Indeed, in rodents, CORT increases drug reward by increasing mesolimbic dopamine transmission (Piazza and Le Moal 1996), rats self-administer CORT itself at levels similar to those elicited by stress, and intracerebroventricular infusions of CORT enhance the reinforcing effects of alcohol (Fahlke et al. 1996).

Studies in laboratory animals have demonstrated that OT has marked anti-stress effects. When administered centrally, OT decreases stress-induced increases in CORT levels (Lang et al. 1983; Windle et al. 1997; Neumann et al. 2000) and reduces stress-induced behaviors in rodent models of anxiety and depression (Arletti and Bertolini 1987; Insel and Winslow 1991; Windle, et al. 1997; Neumann et al. 2000). At the same time, the endogenous OT system appears to be sensitive to stressors. In rats, exposure to acute stress increased OT levels in blood and in hypothalamic and extra-hypothalamic brain regions (Lang et al. 1983; Neumann et al. 1998; Ebner et al. 2000; Ondrejcakova et al. 2010) and increased OT mRNA levels (Jezova et al. 1995). Thus, OT appears to play a protective role in homeostatic regulation of stress responses, and OT administration may attenuate the effects of stress on drinking/drug use and relapse (Uhart and Wand 2009; Koob et al. 2014).

Investigations in human subjects are in line with the preclinical literature. When administered via the intranasal route, OT produces changes in measures of autonomic arousal and mood (MacDonald et al. 2011), increased positive communication during couples’ conflict discussions (Ditzen et al. 2009) and improved recognition and processing of positive facial expressions (Di Simplicio et al. 2009; Marsh et al. 2010; Lischke et al. 2012). The anti-stress effects of OT have been also been investigated using the Trier Social Stress Test, a well-validated laboratory procedure for induction of stress responses in human subjects (Foley and Kirschbaum 2010). This test, which includes components of public speaking component and oral mental arithmetic, produces a robust increase in CORT and self-reported psychological stress and these effects are attenuated by OT (Heinrichs et al. 2003; Quirin et al. 2011; Simeon et al. 2011; de Oliveira et al. 2012; Kubzansky et al. 2012). Consistent with an OT anti-stress hypothesis, a recent study that measured both OT and CORT after the TSST found that salivary OT levels increased immediately following social stress exposure, prior to increases in salivary CORT (Jong et al. 2015). Taken together, these data suggest that OT treatment may be useful to normalize the HPA-axis and reduce stress-related physiological and subjective responses (e.g., anxiety, craving) that increase drug and alcohol use and trigger relapse. ......

Indexed for NIH by Dragonfly Kingdom Library

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5089904/

Natural triterpenes modulate immune-inflammatory markers of experimental autoimmune encephalomyelitis: therapeutic implications for multiple sclerosis

R Martín,* M Hernández, C Córdova, and ML Nieto

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Abstract

BACKGROUND AND PURPOSE

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases that develop as a result of deregulated immune responses causing glial activation and destruction of CNS tissues. Oleanolic acid and erythrodiol are natural triterpenes that display strong anti-inflammatory and immunomodulatory activities. Oleanolic acid beneficially influences the course of established EAE. We now extend our previous observations to erythrodiol and address the efficacy of both compounds to protect against EAE, given under different regimens.

EXPERIMENTAL APPROACH

The utility of both triterpenes in disease prevention was evaluated at a clinical and molecular level: in vivo through their prophylactic administration to myelin oligodendrocyte protein-immunized C57BL/6 mice, and in vitro through their addition to stimulated-BV2 microglial cells.

KEY RESULTS

These triterpenes protected against EAE by restricting infiltration of inflammatory cells into the CNS and by preventing blood–brain barrier disruption. Triterpene-pretreated EAE-mice exhibited less leptin secretion, and switched cytokine production towards a Th2/regulatory profile, with lower levels of Th1 and Th17 cytokines and higher expression of Th2 cytokines in both serum and spinal cord. Triterpenes also affected the humoral response causing auto-antibody production inhibition. In vitro, triterpenes inhibited ERK and rS6 phosphorylation and reduced the proliferative response, phagocytic properties and synthesis of proinflammatory mediators induced by the addition of inflammatory stimuli to microglia.

CONCLUSIONS AND IMPLICATIONS

Both triterpenes restricted the development of the characteristic features of EAE. We envision these natural products as novel helpful tools for intervention in autoimmune and neurodegenerative diseases including MS.

Keywords: encephalomyelitis, neuroimmunology, inflammation, microglia, pharmacology, triterpenes

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Introduction

Multiple sclerosis (MS) is an autoimmune demyelinating disease directed against myelin proteins of the brain and spinal cord, and is considered as one of the major neurological diseases in young adults (Noseworthy et al., 2000). The precise cause of MS is unknown, but one theory is that it might be triggered by exposure to a viral infection or environmental influences. The disease takes dissimilar courses in different people and can go into four main pathological subtypes, even leading to death in the very progressive form (Lassmann et al., 2001).

Experimental autoimmune encephalomyelitis (EAE) induced in susceptible strains of animals provides the best available model for understanding events in MS and to test new drugs that could lead to novel therapies (Steinman, 1999). MS/EAE pathogenesis is driven mostly by a Th1-mediated autoimmune response. The development of the disease includes breakdown of the blood–brain barrier (BBB), infiltration of the CNS – brain and spinal cord – by myelin-reactive T cells and macrophages, activation of resident CNS cells (microglia and astrocytes), demyelination and axonal loss (Merrill and Benveniste, 1996; Benveniste, 1997; Engelhardt, 2006).

Microglial cells are active participants throughout the MS disease process. ‘Activated’ microglia produces inflammatory cytokines, free radicals and attracts immune cells into the CNS. A diffuse activation of microglia throughout the brain serves as a source of inflammation inside the CNS in chronic MS/EAE, while at latter stages of the disease a chronically activated microglia is associated with impaired neural function (Rasmussen et al., 2007).

Other components of the immune system that play crucial roles in MS/EAE pathogenesis include dendritic and B cells, antibodies, as well as inflammation-related enzymes, cytokines and chemokines. Thus, COX-2 and inducible nitric oxide synthase (iNOS) enzymes and pro-inflammatory cytokines such as IFN-γ, TNF-α or IL-17 are considered to be pathogenic, while the Th2 cell-related cytokines IL-4 and IL-10 have been shown to down-regulate the immune response in acute EAE (Hafler, 2004; Imitola et al., 2005; Sospedra and Martin, 2005). Much progress has been made over the past decade in elucidating the causes and molecular basis of MS, but in spite of the extensive research performed to develop new pharmacotherapeutic approaches to slow down the disease progression, there are still no optimal therapies available, due to both unwanted side effects of the drugs and the clinical and immunopathological heterogeneity of this disease (Hemmer et al., 2006).

Oleanolic acid and erythrodiol are two natural triterpenes of the oleanane group present in many vegetables, including the leaves and fruits of Olea europea (the olive tree). They have been recognized to have hepatoprotective, anti-inflammatory and antihyperlipidemic properties. Indeed, oleanolic acid has been promoted in China as an oral drug for human liver disorders. Data correlated well with the traditional use of O. europea in African and European Mediterranean countries, where this plant has been utilized widely in folk medicine as a diuretic, hypotensive, hypoglycaemic, emollient, febrifuge and tonic, for urinary and bladder infections, for headaches, as well as a therapy for inflammatory pain (Dold and Cocks, 1999). Recently, a number of synthetic oleanane triterpenoid derivatives have been synthesized based on oleanolic acid with more potent activities, some of which are currently being developed for the treatment of chronic kidney diseases (Pergola et al., 2011) or as an attractive new therapeutic option for cancer patients by enhancing the effect of immunotherapy (Nagaraj et al., 2010). In the last years, a variety of novel pharmacological properties of triterpenoids have been reported: (i) beneficial effects on cardiovascular system due to antioxidant and vasorelaxant activities (Rodriguez-Rodriguez et al., 2006); (ii) interaction with cytochrome P450s; (iii) anti-proliferative activities on tumoural cells by activating apoptotic programmes (Martín et al., 2007; 2009); (iv) effects on intracellular redox balance and protective effects against lipid peroxidation; as well as (v) immunomodulatory effects (Marquez-Martin et al., 2006). Besides, we have shown that oleanolic acid has a therapeutic effect on an experimental model of MS (Martín et al., 2010), demonstrating that i.p. administration of oleanolic acid, in mice with established EAE, is capable of reducing important biomarkers related to EAE disease. However, the potential of these biologically active molecules on maintenance of health has not been addressed in depth, although disease prevention is a major goal on public health, particularly because of the shifting of the concept from ‘disease care’ to ‘health care’. Therefore, it has been of interest in the present study to assess the influence of early administration of oleanolic acid and erythrodiol, an intermediate from which oleanolic acid is formed and on which no previous data exist, on health promotion in our EAE model. Our findings confirmed that both erythrodiol and oleanolic acid markedly slowed the clinical manifestations of the disease and we were able to correlate the magnitude of improvement for EAE with the decrease of the immuno-inflammatory responses.

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Methods

Disease induction and treatment

All animal care and experimental protocols were reviewed and approved by the Animal Ethics Committee of the University of Valladolid and complied with the European Communities directive 86/609/ECC and Spanish legislation (BOE 252/34367-91, 2005) regulating animal research. C57BL/J6 mice (from Charles River Laboratories, Barcelona, Spain) were housed in the animal care facility at the Medical School of the University of Valladolid and provided food and water ad libitum.

Immunization

EAE was induced in 8 to 10-week-old female C57BL/J6 mice by subcutaneous immunization with 100 µg of myelin oligodendrocyte glycoprotein (MOG)35–55 peptide (MEVGWYRSPFSRVVHLYRNGK; from Dr F. Barahona, CBM, Madrid) emulsified in complete Freund's adjuvant containing 0.4 mg Mycobacterium tuberculosis (H37Ra; Difco, Detroit, MI, USA) on day 0. Additionally, mice received 300 ng of Pertussis toxin i.p. on days 0 and 2. Clinical signs of EAE were assessed daily in a double-blind manner on a scale of 0 to 5, with 0.5 points for intermediate clinical findings: grade 0, no abnormality; grade 0.5, partial loss/reduced tail tone, assessed by inability to curl the distal end of the tail; grade 1, tail atony; grade 1,5, slightly/moderately clumsy gait, impaired righting ability or combination; grade 2, hind limb weakness; grade 2,5, partial hind limb paralysis; grade 3, complete hind limb paralysis; grade 3,5, complete hind limb paralysis and fore limb weakness; grade 4, tetraplegic; grade 5, moribund state or death. Scores from two investigators, both unaware of the treatments, were averaged. Data were plotted as daily mean clinical score for all animals in a particular treatment group. Scores of asymptomatic mice (score = 0) were included in the calculation of the daily mean clinical score for each group. Mice scoring at level 4 for 2 days were automatically given a disease severity grade of 5 and killed.

Triterpene treatment procedure

MOG-Immunized mice were treated daily with 50 mg kg–1 day–1 of oleanolic acid or erythrodiol by i.p. injection beginning at different times.

Groups OA0 and ERY0: triterpene treatment started at the immunization day.

Groups OA-7 and ERY-7: triterpene treatment started on day -7, before EAE induction.

Groups OA12 and ERY12: triterpene treatment started on day 12 after EAE induction.

Control groups (without EAE induction):

Group control, C: treated daily with 0.2% w/v DMSO.

Groups OA and ERY: healthy mice treated with the triterpenes for the same time as the corresponding EAE mice.

Animals were studied at two different times:

30 days after immunization, when EAE mice showed hind limb paralysis, or

at the day when severe symptoms (score 5) in each animal group were apparent. This was at day 40 in untreated EAE mice and at day 110 for triterpene-treated EAE mice, after immunization.

Control mice (without EAE induction) were also injected daily with oleanolic acid or erythrodiol for an equivalent period of time.

Oleanolic acid and erythrodiol (Extrasynthese, Genay Cedex, France) were first dissolved in 2% w/v DMSO and then diluted with PBS for each experiment (the final concentration of DMSO was 0.2%, w/v).

Histological studies

Spinal cord tissue was obtained from five representative animals of the different experimental groups on day 30 after immunization. Tissues were fixed and embedded in paraffin, cut on a microtome (5 µm thicknesses), stained with eosin-haematoxylin. Histological examination was performed with a Nikon Eclipse 90i (Nikon Instruments, Inc., Amstelveen, the Netherlands) connected to a DXM1200C digital camera (Nikon Instruments Inc). Sections from 4–10 segments per mouse were examined by one investigator, without knowledge of the treatments.

Intravital microscopy in mouse brain

Intravital microscopy of the mouse cerebromicrovasculature was performed as previously described (Martín et al., 2010). Briefly, mice were anaesthetized at day 30 post-immunization by i.p. injection of a mixture of 100 mg·kg−1 ketamine and 10 mg·kg−1 xylazine, and the tail vein was cannulated for administration of fluorescent dyes. A craniotomy was performed using a high-speed drill (Dremel, Madrid, Spain) and the dura matter was removed to expose the underlying pial vasculature. The mouse was maintained at 37°C throughout the experiment and the exposed brain was continuously superfused with artificial CSF buffer at 37°C.

Leukocytes were fluorescently labelled by i.v. administration of rhodamine 6G (5 mg·kg−1 body weight) and visualized by a Zeiss Axioplan 2 imaging microscope (Hertfordshire, UK) connected to an AxioCam MR digital camera using the AxioVision AC imaging software and an Acroplan 20x/0.50W Ph2 lens. Eight different post-capillary venules of diameter between 30 and 70 µm were chosen for observation. Rolling leukocytes were defined as white cells moving at a velocity less than that of erythrocytes. Leukocytes remaining stationary for 30 s or longer were considered adherent to the venular endothelium. Leukocyte adhesion was expressed as cells/mm2 of venular surface area, as shown previously (Martín et al., 2010).

Evaluation of cytokines and MOG-specific antibodies by elisa

Anti-MOG-specific IgM and IgG isotypes were detected in serum samples collected from animals on day 30 after immunization, using elisa. In brief, 96-well polystyrene microtitre plates were coated with 0.5 mg per well of MOG35–55 peptide diluted in PBS overnight in a humidified chamber followed by PBS washing and blocking for 1 h with 5% BSA in PBS. Wells were incubated in duplicate with serum samples diluted 1:60 in PBS for 2 h at room temperature. After washing, HRP-labelled rat anti-mouse IgM, anti-mouse IgG, anti-mouse IgG1 and anti-mouse IgG2a (1:2000) from Serotec (Sigma-Aldrich, St Louis, MO, USA) were subsequently added for 90 min. After another washing, adding the substrate, and arresting the reaction with 0.1N HCl, absorbance was read at 450 nm. Data are expressed as mean optical density at 450 nm.

Leptin levels in serum samples and spinal cord tissue were determined by elisa (RayBiotech, Norcross, GA, USA). For cytokine quantification (IL-4, IL-6, IL-10, IL-17, TNF-α, and IFN-γ), cell culture medium, serum and spinal cord tissue were analysed by elisa according to the manufacturer's protocols (eBioscience, San Diego, CA, USA). Spinal cords were removed on day 30 after immunization or at the severe stage of the disease (score 5), weighed and then frozen at −80°C. SC tissue was homogenized by using a tissue homogenizer (Cole-Parmer Instrument, Vernon Hills, IL, USA) in an ice bath in 0.5 mL ice-cold PBS supplemented with 0.4 M NaCl, 0.05% Tween 20, 0.5% BSA and a protease inhibitor cocktail: 20 µg·mL−1 of leupeptin, 20 KI units of aprotinin, 0.1 mM phenylmethylsulphonyl fluoride (Sigma-Aldrich), and centrifuged at 3000× g for 10 min at 4°C. Supernatant were stored at −80°C until cytokine assays were performed. Total protein was assayed using the Bradford method. A 50 to 100 µL sample of each supernatant was used for tests.

Data were processed and expressed as pg of cytokine per mg of spinal cord wet weight, or pg of cytokine per mL for serum samples.

BBB permeability measurement

To evaluate BBB disruption, we measured the extravasation of Evans blue (EB) dye as a marker of albumin extravasation. At 30–31 days following EAE induction, mice were injected i.p. with 1 mL of 4% w/v EB. After 4 h, mice were killed, perfused, and brain and spinal cords were removed. Dye was extracted for 2–3 days in formamide (4 mL·g−1 of wet tissue) at room temperature. Extracted dye concentration was determined by measuring the absorbance at 650 nm. CNS tissue was dried 24 h at 60°C and weighed. Calculations were based on external standard readings and extravasated dye was expressed as mg of EB per mg dried weight of tissue.

Cell culture

Murine BV-2 cells, an immortalized murine microglia cell line, exhibit phenotypic and functional properties comparable with those of primary microglia and hippocampal neurons (Bocchini et al., 1992). BV-2 cells (a gift from Prof J. Bethea, Miller School of Medicine, Miami, FL, USA) were cultured in Dulbecco's modified Eagle's medium high sucrose, supplemented with 10% fetal bovine serum (FBS), 100 U·mL−1 penicillin and 100 µg·mL−1 streptomycin, and kept at 37°C in 5% CO2. Cells were seeded in 96-well plates (5 × 104 cells per well) or 60 mm culture dishes (3 × 106 cells per well.).

Proliferation assay

Cell proliferation was quantified by using the Promega kit (Madison, WI, USA), Cell Titer 96® Aqueous One Solution Cell Proliferation Assay, according to the manufacturer's recommendations. Briefly, cells were seeded in 96-well plates and serum starved for 24 h. Then, cells were treated in triplicate with IFN-γ, leptin or LPS, in the presence or absence of the triterpenes. After 24 h of incubation, formazan product formation was assayed by recording the absorbance at 490 nm in a 96-well plate reader (OD value). Formazan is measured as an assessment of the number of metabolically active cells and expressed in percentages relative to FBS-stimulated cells. Cell viability was assessed by Trypan blue exclusion.

Western blot analysis

Cells were washed with PBS and harvested in Laemmli SDS sample buffer. Protein extracts were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA-TBST at room temperature and then incubated for 18 h at 4°C with the indicated antibodies including ERK 1/2 (Zymed Laboratories, South San Francisco, CA, USA), rabbit p-ERK1/2, p-rS6 (Cell Signaling Technology, Danvers, MA, USA), COX-2 (sc-1745, Santa Cruz Biotech, Santa Cruz, CA, USA), actin (sc-8432, Santa Cruz Biotech) and iNOS (BD Biosciences, Lexington, KY, USA). After washing with TBST buffer, a 1:2.000 (v/v) dilution of horseradish peroxidase-labelled IgG was added at room temperature for 1 h. The blots were developed using enhanced chemiluminescence.

Phagocytosis assays

Cells were stimulated in serum-free media with or without 100 UI·mL−1 of IFN-γ, 1 µg·mL−1 of LPS or 0.5 µM of leptin for 24 h, in the presence or absence of different doses of oleanolic acid or erythrodiol and then exposed to 0.1 mg·mL−1 of FITC-labelled dextran (MW 40 000) for 2 h. Non-internalized particles were removed by vigorous washing with cold PBS (pH 7.4) prior to measuring fluorescence at 480 nm excitation and 520 nm emission on either a Flow Cytometer (Gallios™; Beckman Coulter, Fullerton, CA, USA) or a Fluoroskan multiwell plate reader (TECAN Genios Pro; Tecan Group Ltd, Zurich, Switzerland). Cultures without fluospheres were used (blank wells) as background. Each culture condition was done in triplicate, and three independent experiments were performed. To confirm that the fluospheres were accumulated intracellularly, a Leica TCS SP5X confocal microscope was used with the Leica LAS AF acquisition software (Wetzlar, Germany) and a ×60 oil objective.

Statistical analyses

Statistical analysis was performed with the GraphPad Prism Version 4 software (San Diego, CA, USA) by anova. Analyses were performed using repeated measures anova (or two-way anova) for comparison of clinical parameters, and one-way anova for comparison of parameters such as cytokines, extravasation, leukocytes and MOG antibodies. A post hoc analysis was made by the Bonferroni's multiple comparison test. P < 0.05 was considered statistically significant.

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Results

Effects of preventive treatment with oleanolic acid or erythrodiol on clinical EAE

Female C57BL/6 mice exhibit active EAE after immunization with the MOG35–55 peptide. In this experimental model we compared the effects of two pentacyclic triterpenes, oleanolic acid and erythrodiol given at a dose (50 mg·kg−1) previously proven to be both safe and therapeutically relevant in rodents (Jeong, 1999; Senthil et al., 2007; Martín et al., 2010) in two regimens: 7 days before immunization (day -7; OA-7, ERY-7) or at the day of induction (day 0; OA0, ERY0). The clinical analysis of the different groups of animals is shown in Figure 1. The placebo-treated animals developed neurological symptoms of active EAE after 12 to 31 days, consisting of tail limpness and a mild-to-moderate paraparesis, as well as progressive weight loss. Interestingly, when oleanolic acid or erythrodiol were administered from the day of induction, clinical disease was markedly less severe and mice had a later onset of the clinical signs compared with untreated animals with EAE (Figure 1A). First neurological symptoms (score 1) were observed at day 11 with mean day of onset 13.5 ± 2 in untreated EAE mice, while OA0 or ERY0 animals showed no clinical signs at that time and a similar score (tail atony) was first reached on day 27 (mean values 33 ± 2 and 34 ± 2 days respectively). When the triterpenes were given as a pre-treatment, starting 1 week before EAE induction, clinical disease remained mostly suppressed for the duration of the experiment (until day 30 post-induction)........... https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3419913/

A human skull that is prominently displayed at the National Museum here has been attracting crowds and controversy in equal measure since it was first unveiled early this month. After two decades in storage, the fossilized cranium has now been identified by Brazilian scientists as the oldest human remains ever recovered in the Western Hemisphere.

The skull is that of a young woman, nicknamed Luzia, who is believed to have roamed the savannah of south-central Brazil some 11,500 years ago. Even more startling, a reconstruction of her cranium undertaken in Britain this year indicates that her features appear to be Negroid rather than Mongoloid, suggesting that the Western Hemisphere may have initially been settled not only earlier than thought, but by a people distinct from the ancestors of today's North and South American Indians.

''We can no longer say that the first colonizers of the Americas came from the north of Asia, as previous models have proposed,'' said Dr. Walter Neves, an anthropologist at the University of Sao Paulo, who made the initial discovery along with an Argentine colleague, Hector Pucciarelli. ''This skeleton is nearly 2,000 years older than any skeleton ever found in the Americas, and it does not look like those of Amerindians or North Asians.''

If the date is confirmed, the find could transform thinking about the peopling of the Americas. It may be some time before that work is completed, but meanwhile, archeologists here and abroad say the find is potentially very important.

The finds, along with recent discoveries in North America like those of the so-called Kennewick Man and Spirit Cave Man, are forcing a reassessment of long-established theories as to the settling of the Americas. Based on such evidence, Dr. Neves suggests that Luzia belonged to a nomadic people who began arriving in the New World as early as 15,000 years ago.

Luzia's Negroid features notwithstanding, Dr. Neves is not arguing that her ancestors came to Brazil from Africa in an early trans-Atlantic migration. Instead, he believes they originated in Southeast Asia, ''migrating from there in two directions, south to Australia, where today's aboriginal peoples may be their descendants, and navigating northward along the coast and across the Bering Straits until they reached the Americas.''

About one-third of Luzia's skeleton has been recovered, enough to indicate that she appears to have perished in an accident or perhaps even from an animal attack. She was in her 20's when she died, stood just under five feet tall, and was part of a group of hunter-gatherers who appear to have subsisted largely on whatever fruits, nuts and berries they came across in their meanderings, plus the occasional piece of meat.

AMER'ICAN, adjective Pertaining to America.

AMER'ICAN, noun A native of America; originally applied to the aboriginals, or copper-colored races, found here by the Europeans; but now applied to the descendants of Europeans born in America.

The name American must always exalt the pride of patriotism. - Washington 

COVID-19 and therapy with essential oils having antiviral, anti-inflammatory, and immunomodulatory properties

Muhammad Asif, Mohammad Saleem, Malik Saadullah, Hafiza Sidra Yaseen & Raghdaa Al Zarzour

Inflammopharmacology volume 28, pages1153–1161(2020)Cite this article

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Abstract

Coronavirus disease of 2019 (COVID-19) has emerged as a global health threat. Unfortunately, there are very limited approved drugs available with established efficacy against the SARs-CoV-2 virus and its inflammatory complications. Vaccine development is actively being researched, but it may take over a year to become available to general public. Certain medications, for example, dexamethasone, antimalarials (chloroquine/hydroxychloroquine), antiviral (remdesivir), and IL-6 receptor blocking monoclonal antibodies (tocilizumab), are used in various combinations as off-label medications to treat COVID-19. Essential oils (EOs) have long been known to have anti-inflammatory, immunomodulatory, bronchodilatory, and antiviral properties and are being proposed to have activity against SARC-CoV-2 virus. Owing to their lipophilic nature, EOs are advocated to penetrate viral membranes easily leading to membrane disruption. Moreover, EOs contain multiple active phytochemicals that can act synergistically on multiple stages of viral replication and also induce positive effects on host respiratory system including bronchodilation and mucus lysis. At present, only computer-aided docking and few in vitro studies are available which show anti-SARC-CoV-2 activities of EOs. In this review, role of EOs in the prevention and treatment of COVID-19 is discussed. A discussion on possible side effects associated with EOs as well as anti-corona virus claims made by EOs manufacturers are also highlighted. Based on the current knowledge a chemo-herbal (EOs) combination of the drugs could be a more feasible and effective approach to combat this viral pandemic. .........

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https://link.springer.com/article/10.1007/s10787-020-00744-0

Electrohypersensitivity as a Newly Identified and Characterized Neurologic Pathological Disorder: How to Diagnose, Treat, and Prevent It

Dominique Belpomme and Philippe Irigaray

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Abstract

Since 2009, we built up a database which presently includes more than 2000 electrohypersensitivity (EHS) and/or multiple chemical sensitivity (MCS) self-reported cases. This database shows that EHS is associated in 30% of the cases with MCS, and that MCS precedes the occurrence of EHS in 37% of these EHS/MCS-associated cases. EHS and MCS can be characterized clinically by a similar symptomatic picture, and biologically by low-grade inflammation and an autoimmune response involving autoantibodies against O-myelin. Moreover, 80% of the patients with EHS present with one, two, or three detectable oxidative stress biomarkers in their peripheral blood, meaning that overall these patients present with a true objective somatic disorder. Moreover, by using ultrasonic cerebral tomosphygmography and transcranial Doppler ultrasonography, we showed that cases have a defect in the middle cerebral artery hemodynamics, and we localized a tissue pulsometric index deficiency in the capsulo-thalamic area of the temporal lobes, suggesting the involvement of the limbic system and the thalamus. Altogether, these data strongly suggest that EHS is a neurologic pathological disorder which can be diagnosed, treated, and prevented. Because EHS is becoming a new insidious worldwide plague involving millions of people, we ask the World Health Organization (WHO) to include EHS as a neurologic disorder in the international classification of diseases. ...... https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7139347/

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