text:
Egyptian evidence in Australia
Hieroglyphics and other relics
By The Puzzler, May 11, 2007 in Ancient Mysteries & Alternative History
jaylemurph 8,606
#the funniest human being on planet earth
Location:Seattle, WA
"You can lead a whore to culture, but you can't make him think." Dorothy Parker
I still think that just because Erik Von Danikan forged some info to support his theory... it doesn't mean that his ideas are all sham....He succeeded in throwing us out of our comfort zone of indoctrined...and getting us to take a whole new look at this world... opened our imaginations... to explore new and exciting possiblities... that can also fit in with the facts as we know them....
Okay, then -- tell us a system whereby we know when he lies and when he tells the truth. That isn't based on what you /want/ to believe is true.
If you can't do that, his work in toto is no better than fiction; less so, since fiction doesn't deliberately try to fool people.
and for any other historian.... eg Fell...(just because he was a qualified biologist...it doesn't distract from the fact that he had a real passion for histor/archeology!!!... what is a degree/qualification ..anyway..other than a few assessed essays... assignments.. thesis... that prove that the writer has enough knowledge of the topic to pass the subject) docorates... Masters.. in the subjects mean that they can think for them selves.. not regurgitate facts..that other scholars believe to be true)...
I take it you don't actually have higher degrees to accurately comment on this? Yes, there is the part of the degree where you prove your knowledge of the verified facts of the field but part of every degree is a) proving your ability to think beyond the rank and file and finding out something new in the field that no-one else has. This is the nature of the thesis, not proving your conformity. I need hardly remind you that you have a hard enough time accurately reporting what you find without deliberately manipulating your sources!
Does a skilled artist ..or chef need a qualification????...
No -- but without proper training in their field, they're not going to produce anything worth sampling, either!
--Jaylemurph
Harte 15,228
Supremely Educated Knower of Everything in Existence.
Location:Memphis
Hi again Harte, just on the marsupials, I think we got a bit mixed up on that one, your sentence was: Marsupials are found on practically every continent. I now know you meant: Marsupial fossils are found on practically every continent. In the context of the paragraph I guess this is what you meant, I of course took it as a literal sentence. You get that in nit picking, unless things are stated exact it's easy to make something else from it.
We,
Very gentlemanly of you to let me off the hook so easily. Sorry for not being clear. I was in the moment, see. You know, talking about fossils and thinking about fossils and not discriminating between them and living creatures.
I get that you are trying to prove by disproving, I did the same thing with the 9/11 conspiracy, I didn't go into the theorists websites, I went into the disbelievers websites. I'm glad you joined in here, it has provided much to think about and research.
I think of it more as attempting to guide people that are interested in the subject. I mean, I've done an awful lot of work in the past looking ionto this stuff. I try to keep others from having to do the same work over again before they finally come to the conclusions, or near to the conclusions, that I've already reached.
There is also the fact that fairly often someone posts something I hadn't heard before - like the mummification thing. I jumped at that, hoping maybe there would be something there.
Believe me, if I read something like the mummy thing that is new to me, and I look into it and see it's actually what the author says it is, I'll post that as well. Unfortunately, that rarely happens.
I still think that just because Erik Von Danikan forged some info to support his theory... it doesn't mean that his ideas are all sham....
No, it doesn't. The discovery of VonDaniken's admission of forging evidence was merely the catalyst that got me to checking the rest of the stuff he claims. If you want to start a VonDaniken thread, I can show you that, in fact, his ideas are all sham.
He succeeded in throwing us out of our comfort zone of indoctrined...and getting us to take a whole new look at this world... opened our imaginations... to explore new and exciting possiblities... that can also fit in with the facts as we know them....
It is a fact that without VonDaniken's early works, I would not be posting on this website. But he is a liar. I owe him the knowledge I've gleaned through fact checking his work, but that is all. It's unfortunate that he himself, or his editor, didn't do this checking for themselves.
I'd like to point out how generous I am being with that last statement. See, there's really no reason at all to believe that VonDaniken didn't check his facts, find them wanting, and went ahead and published them anyway. So I'm being extremely generous in saying he didn't do this.
I don't feel the need to be this generous with Graham Hancock, by the way.
If the man had just written his books as an amateur archaeologist, then that would be fine. But he passed himself off as an expert linguist. Go ahead and read the article I linked and then do the Googling I suggested. You'll see that I'm right.
By the way, Zecharia Sitchen does exactly the same thing.
Certainly not. A skilled artist, however, should not pretend to be able to practice law. A chef should not hire out as an accountant.
Barry Fell was not a skilled archaeologist, nor was he a skilled linguist. I can't be sure even of his skill as a marine biologist, but it was probably greater than these other two fields.
Thinking that way ... eg.. that there is no 100% provable evidence.. therefore any idea in that field must be abandoned
This is a straw man argument. Nobody claims or has said that evidence in any field is "provable" in any way. The people we are talking about here ignore most of the available evidence and use only bits and pieces of real evidence and then only when it supports their claims. That is, when they aren't fabricating the "evidence" themselves!
so if someone does come up with fractions of evidence to support this discredited idea.... scholars.. historians.. etc.. will perhaps refuse to look at it as the idea..and so this new info will be put on the anomolie pile.. buried for ages.. until someone else is brave enough.. to remove these political... ( as I feel that much of history ..scholastic acceptance of facts is political..eg .. they decide what info will be accepted/published with their approval.....eg if it fits in with their idea of accepted reality...)..red tape.. and dust off some of the old discoveries/ideas.. artifacts.. ..if they are not lost forever... and piece them together...to create a better understanding of how things were...
This sort of thing is far likelier to occur because of people like Fell, VonDaniken, Hancock and Sitchen than it is to occur because stodgy old academecians don't want to be wrong.
If somebody like VonDaniken were to actually come across real evidence of ancient astronauts, he would be ignored because of what he has done in the past. Furthermore, actual researchers would be wary of even considering it for themselves because of the association that would give them with a loon like VonDaniken. IOW, VonDaniken, Hancock and the like might actually be holding back the field in the very area where they love to make their claims.
Luckily for the sake of science, none of these con men have stumbled across anything of significance, and they probably wouldn't even recognize it if they did.
Remember the idea of Dinasaurs is only a little over 180 years old!!!! ... can you imagine what those scientists people .. went thru...
Germ theory is an excellent example. Did you see 12 Monkeys? Brad Pitt went into this in-depth. Hilarious.
I believe that, if you look, you'll find that Fell's ideas about archaeology have either been looked into and scientifically dismissed as wrong, or looked into and discovered to be somebody else's ideas that Fell elaborates on. Fell used a poor grasp of linguistics as the basis for most of his "archaeology" (as does Sitchen) and as such was not taken seriously by most people that know the difference between Phoenecia and Phoenix Arizona.
Location:Spittle County
The fault, dear Brutus, is not in our stars, but in ourselves, that we are underlings
Due to the geographical location of the Hunter Valley...
*bursting bubbles* Fakes
What this is suggesting is that Egyptians (who posess no mounaineering experience) crossed the Blue Mountain range (in a time with no roads), left some Heiroglyphs to their gods in a cave, then p***ed off back home to the other side of the world.
More than likely, these were done during the early 20th century when Egyptology was booming due to the discovery of the tomb of tutankhamun.
What you also have to consider is....the Suez canal did not exist at that time....
this would mean that Egyptian Dhow's (designed for use in the mediterranean) would have to have crossed either by going around Canada (pffff) or by going around the bottom of South America, a cape which is considered a death trap STILL to this day due to storm and activity and dangerous currents.
The only other option is to sail south down the western coast of Africa, then either past asia, or directly across the indian ocean to Australia (Which would have taken several months). Even then, they would hit the western side of Australia on either trip....the complete OPPOSITE side to where these Heiroglyphs were found.
So are we then meant to believe....upon hitting the western (and Barren) coast of Australia, they sailed a further another 4000KM east, into Sydney harbour, got out of their boats, crossed a mountain range (in a strange and foreign environment), left some heiroglyphs, and happily went home.
crystal sage 254
Non-Corporeal Being
I think he was refering to the earliest celtic script.... http://www.celticgrounds.com/chapters/ogham_translat.htm
http://maps.google.com/maps?q=Phoenicia%20...sa=N&tab=wl
http://www.burlingtonnews.net/centerindians.html
NEW YORK: In addition to these discoveries, another stele exhibiting the ancient Egyptian-Libyan script was unearthed on Long Island, New York. Dr. Barry Fell states that it also probably dates to around the ninth century B.C.
http://planetvermont.com/pvq/v9n2/megaliths.html
http://www.billposer.org/Papers/dulkwah.pdf
http://www.indiana.edu/~anthling/v31-3-4.html
"The Language of the Coast Tribes is Half Basque":
A Basque-American Indian Pidgin in Use between Europeans and Native Americans in North America, ca. 1540-ca. 1640
Peter Bakker
Abstract. Basque fishermen were among the most numerous visitors to the eastern coast of North America in the sixteenth and seventeenth centuries. They traded actively with some of the native tribes on the coast and along the banks of the Saint Lawrence River. A trade language, a pidgin based on Basque and American Indian languages, developed and was used by both American Indians and Europeans. Historical and linguistic evidence of the Basque elements of this pidgin is presented.
http://www.viewzone.com/ogam222.html
"The language of Ogam is Basque."
The Puzzler 502
I have no special talent. I am only passionately curious. ~ Einstein
Good points but I was thinking more along the lines of some Egyptian explorers going east from Egypt by land, heading south, crossing narrow sea channels in boats escorted by natives, reaching the Torres Strait Islands showing off mummification and introducing Egyptian practices and Egyptian words into the Micronesia and surrounding areas then over Torres Strait to Australia, therefore they arrived from the north. Aboriginals walked everywhere, nothing unusual about walking anywhere in Australia, they had tracks through the Blue Mountains as well, they showed them various walking routes and the Egyptian explorers accompanying them around. There is the fact that dingoes arrived later than the Aboriginals first did, maybe they bought them with them then too.
Well the aboriginals were said to have arrived in Australia over 40,000 years ago .. some say they walked... some say they had great seafaring skills... or drifted overland during one of the ice ages..... I like your idea of how they could have arrived there... there are also rumors of a secret subteranean culture.... but I digress.... the egyptian graffiti did look a little less neat ... as that of the official remnants... ..eg... the proffessional... writings by true proffesional craftsment of Egypt.... it was like what a layperson may be able to create... leave behind from a trip to far off lands.,.. or perhaps what another either an Egyptian...or someone who had spent time there or with some from the land... eg. may try out their new writing skills that they may have learned from another... or had seen in their travels....
http://www.epigraphy.org/volume_1.htm
http://www.awarenessquest.com/research.htm
Edited May 14, 2007 by crystal sage
If you really looked at the celticgrounds.com site you linked, you saw that Ogham is written as series of vertical lines.
Fell interpreted practically every set of vertical lines he ever heard of as being Ogham. This leads one to the amusing assumption that Lions and Tigers must be quite good at hissing like housecats, only in script, since everytime they slash with their claws, they leave an Ogham "S" mark, like Zorro's "Z." on their victim's body!
http://www.flavinscorner.com/fellegypt.htm
Even though he may not have been totally accurate. in his translations... etc???.. He still did quite well for all his passion... which he was able to share with the masses... Most of the info... discoveries...anomolies... that he brought to the attention of the masses would otherwise most likely have been long forgotten.. or overlooked by now....
With all those jealous ... and some most likely not as successful 'scholars' out there quibbling... the info that Fell discovered ,,,and brought to everyone's attention ... what he acheived is making sure that it is still out there for everyone to evaluate...have another look at...... that is the more important thing!!!... rather that what he thought his discoveries may have meant!!!
I liked his passion for history... archeology...!!!! despite all the bitter achademics...
He did his best with what info he had available... Imagine what he could have added if he had the internet at his disposal!!!!
the evolution of the alphabet!!!
a great site!!!! ( it's interactive)
http://www.wam.umd.edu/~rfradkin/alphapage.html
http://www.geocities.com/capitolHill/Parli...2587/alpha.html
and http://www.ancientscripts.com/ugaritic.html
http://www.geocities.com/WallStreet/Bureau...g-abc-hist.html
Sounds a good concept to me. Also thanks for the links especially the awareness quest one, I have been interested in Pygmies in Atherton Qld and you rejogged me into thinking about it, I live near Atheron and Gordonvale so will ask around more as to anything anyone knows and Lake Barrine is just up the road too regarding the pygmies so with all this stuff in my own backyard I can't help but want to know more. That site had lots of evidence to Egyptian relics and odd things found here, good one!
Kazahel 357
Location:The West.
What need is there of seeing, in the presence of His gratitude?
I stumbled on that site ages ago when I was looking up numbers and stuff. You find it when you type in.. Grid Point 44.. which I found when I was looking at M44. Anyway I'm not sure what to think but I thought I read that there was an aboriginal story which told about people coming from over the sea and not being able to return to their home, which was connected to this. I dunno for sure though.. but anyway.
I was doing some research on a rumour I had heard and came up with this web site. Has anyone ever heard of Egyptians in Australia before? What do you think of the evidence and apparent facts contained in this article? I myself was pretty amazed at it all. Even if the heiroglyphics seem fake I find it interesting that Aboriginals and Torres Strait Islanders have similar traditions to Egyptians and mummification processes as well as the Aten symbol. (Don't dismiss it because of the mention of the Gympie Pyramid idol)
http://www.crystalinks.com/egyptaustralia.html
Australia and New Zealand are part of the ancient Gondwan land. Arabia, Africa, Antarctica, South America and India are were also part of the the Godwana land. Maybe the Aboriginals are lost tribes of India?
http://en.wikipedia.org/wiki/Gondwana
http://cat.he.net/~archaeol/9703/etc/specialreport.html
The Indus Script http://www.harappa.com/script/indusscript.html
http://links.jstor.org/sici?sici=0025-1496...TOR-enlargePage
Edited May 15, 2007 by navigator
Just to clarify, the idea I gave was how I thought the Egyptians got here not the Aboriginals.
Edited May 15, 2007 by weareallsuckers
Emma_Acid 5,996
Location:Sandwell District
No, maybe not. Gondwana broke up during the late Jurassic period. I find it hugely unlikely that there were little cavemen in tiger-skin pants wandering around given that it was 167 million years ago. Respect for referencing in your post, best read the references first though!
Pax Unum 1,359
< 420 Conspirator >
Location:Suburbia
"It is the mark of an educated mind to be able to entertain a thought without accepting it." - Aristotle
No, maybe not. Gondwana broke up during the late Jurassic period. I find it hugely unlikely that there were little cavemen in tiger-skin pants wandering around given that it was 167 million years ago.
I also find it hugely unlikely there were little cavepeople 167 mya, or Tigers...
Pandora7321 60
Perpetual Late Bloomer
Location:Jacksonville, Florida
Let none of this earth inherit this vision of my spirit.
-Edgar Allan Poe
Okay, so I tried to thoroughly read all the posts so that I wouldn't post a question or a theory that had all ready been answered, but..............that's a LOT of stuff to read!! I read really fast though, so I did my best to scan through.
So, my question is this:
If the glyphs are fakes, how old exactly are they? I know ya'll have all debunked it apparently, but I just find it hard to believe that someone who knows this "ancient" type of glyph that hardly anyone knows took the time and effort to painstakingly chisel this stuff into the walls of this place in BFE (no pun intended) Australia just to get one over on us. I mean does anyone have any idea how they got there, who did it, and when they did it?
Please don't tell me to back through and re-read all this stuff. Just paste me a link or quote me the part that answers that. Thanks!
Harks 5
Location:Cairns
Forget Everything and Do Not Listern to Anyone, because you still have a chance to LIVE your own life.
Try this link: http://webzoom.freewebs.com/diggingsmag/Re...rd%20Glyphs.pdf
There are many theories and beliefs who might of done them, but because of the various ages of the writings it is hard to put it down to one source.
I did start a thread similar to this subject before, if you are curious here is the link: http://www.unexplained-mysteries.com/forum...showtopic=62538
Edited May 16, 2007 by Harks
http://www.mountainman.com.au/ww_dtime.html
http://www.kachina.net/~alunajoy/2002feb.html
One of the best of the carvings was under a portion of a rock roof. This is where we discovered Anubis. It was clear it had been examined as the lichen had been cleaned off this part of the rock. Anubis is considered the guardian of the doorways to the underworlds, an opener of the ways, and is associated with the dog star Sirius. Egyptians considered Sirius a portal or gateway to other dimensions. One Australian researcher and channel named Robin Franklin channeled Anubis and discovered that Anubis was the guardian of the place and protected it for many thousands of years. He said the writing came from people from the kingdom of Kam, which we now call Egypt. These ones were survivors of Mu Lemuria and were under the guidance of those from the stars. Franklin stated in an article he wrote that a message from Anubis stated that the hieroglyphics say, "growth comes from Love and destruction comes from hate. If man does not change his heart the world could be destroyed." Now this is by no means a great revelation. We all know this one. . . But to hear it again in such an obscure place just anchored the message deeper into my heart.
Another interpretation by researcher Paul White in an article entitled, "In search of Ancient Secrets 1994" suggests that the carvings may be a sequence that could describe the classic shamanic path of enlightenment, or maybe a prophecy addressing people of a future time, who faced possible destruction if they didn't follow the path of the One True God.
Even those not considering themselves very psychic can feel strong energy in this site, especially when the rock walls are touched. Some think that the strong energy comes from the site being one huge energy ley line that radiates out from a central New South Wales power called, "The meeting of the tribes." Paul White was also aware of the persistent legends in Australia that tell tales of strange and magical happenings, power places on grid lines. Energy grids spread like a spider's web across Australia, creating special energy in cross roads. Some researcher even suggest that the crevice could even be a time tunnel to Peru or other power places. The aborigine speak of instantaneous travel along "song lines" between power places. Could this site be a time travel site?
The next day when I went to develop my film, I discovered that all three rolls of film were quite unusable. It was as if the carvings hid themselves from the camera. You could hardly make out the carving. I was disappointed to say the least. We were going to have to make another trip out to the site.
On our second trip out to the site with new film in hand, I was determined to get some good photos of these carvings before I had to fly home. But this trip also had its problems and was also interrupted with weird events. I began to photograph the site once again and my camera began to freeze up. I managed to get two rolls of film shot but I was only able to photograph only part of the site before my camera decided to die altogether. After I knew the camera was not going to respond any further, Grace and I climbed up to the top of the site to a huge whale-shaped rock. This is a very sacred place to the aboriginal people of the area. We knew of a burial ground just further up the hill and knew better than to go visit. It seemed like we were already on shaky ground with the ancestors and didn't want any more trouble. We sat and meditated for awhile and received permission to create a flower essence atop this great whale rock.
When we hiked out to the car we realized we locked the keys in the car. Here we are out in the middle of nowhere and the keys dangling just out of reach in the ignition. Grace had her mobile phone and she called for help. While we waited for help, I wondered if I was being tested again. The sun was going down and it was getting cold and the air was full of the tropical sounds of bird readying themselves for the night. Help did come and when I got to town, I went to develop the film. I was happy to see that most of the photographs were very useable. I felt I was lucky to get any photos at all. I guess I was allowed these few photos to share with you. See the next page for more photos
Now did the Egyptians come to Australia or was it people from the stars? This answer still eludes us today. But the evidence is mounting to suggest that there is some connection between Australia and Egypt. Over a period of a few years, Paul White discovered evidence of other Egyptian artifacts in Australia, ranging from a statue of an Egyptian God Thoth near a pyramidal hill in Gimpie, south east Queensland, two cartouches of the Egyptian Pharaoh Ramses, found in Arnhem Land and northern Queensland early in the last century and an Egyptian amulet found in Kyogle in northern South Wales that dates at 5000 years by the Department of Mines.
http://members.ozemail.com.au/~classblu/egypt/article.htm
Well the aboriginals were said to have arrived in Australia over 40,000 years ago .. some say they walked... some say they had great seafaring skills... or drifted overland during one of the ice ages..... I like your idea of how they could have arrived there... there are also rumors of a secret subteranean culture.... but I digress....
There is very good reason to think that they walked the entire way. There is a submerging continental plate there which today is, in part, under the ocean (the Indo-Australian Plate.) Might have been dry land there 40,000 years ago.
The current theory is that the Aborigines are descended from peoples that were living in India 40-50 thousand years ago.
But those people were not exactly civilized. There was no civilization anywhere on Earth at that time.
Also - no way at all that the Egyptians ever made it to Australia. Complete hogwash.
http://archaeology.about.com/od/earlymansites/a/kostenki.htm
"The Kostenki site has been known for quite a while (it is best known for the recovery of Venus figurines from its Gravettian levels), and its uniqueness has not gone unnoticed. The occupations at Kostenki include several Late Early Upper Paleolithic levels, dated ca 40,000 to 30,000 calibrated years ago. Below these levels is a layer of volcanic ash, associated with the volcanic eruptions of the Phlegrean Fields of Italy (aka Campanian Ignimbrite), which are thought to have erupted between about 38,000 and 40,000 years ago. Within and below the ash level (called the CI Tephra) is what archaeologists are calling "Aurignacian Dufour," containing numerous small bladelets and related to similar sites in western Europe. Typically, the Aurignacian is the oldest component associated with modern humans at archaeological sites in Europe, underlain by Mousterian-like deposits representing Neanderthals. At Kostenki, a previously unidentified assemblage, exhibiting a sophisticated tool kit of prismatic blades, burins, bone antler, and ivory artifacts, and small perforated shell ornaments underlies the CI Tephra and Aurignacian Dufour assemblage."
http://archaeology.about.com/b/a/257842.htm
http://www.networlddirectory.com/blogs/arc...ch-22-2007.html
Associate Professor Sheila Coulson, from the University of Oslo, can now show that modern humans, Homo sapiens, have performed advanced rituals in Africa for 70,000 years. She has, in other words, discovered mankinds oldest known ritual.
The archaeologist made the surprising discovery while she was studying the origin of the Sanpeople. A group of the San live in the sparsely inhabited area of north-western Botswana known as Ngamiland.
Coulson made the discovery while searching for artifacts from the Middle Stone Age in the only hills present for hundreds of kilometers in any direction. This group of small peaks within the Kalahari Desert is known as the Tsodilo Hills and is famous for having the largest concentration of rock paintings in the world.
In the excavation they found more than 13,000 artifacts
http://en.wikipedia.org/wiki/Klasies_River_Caves
Klasies River Caves.....The 3 main caves and 2 shelters at the base of a high cliff have revealed evidence of Homo sapiens habitation from approximately 125,000 years ago.
...There is a 20 metre thick accumulation of deposits, both inside the caves and outside against the cliff face, proving that Klasies River Mouth people knew how to hunt, fish (later), gather plants and roots, cook by roasting on hearths, and manage their land. There is extensive evidence of shellfish collecting; stone artifact technology;[2] gathering plants, roots and flowers for food; cooking plants, corms, seal, penguins, and antelope meat on hearths with fire; [3] general organisation of the settlement; and land/veld management by fire. [2] The evidence also appears to indicate that their presence was seasonal or migratory. There is also evidence of cannibalism, charred and carved 'modern human' bones discarded with other food remnants. [4]
Although these Middle Stone Age people are described as anatomically modern there is ongoing debate about when they were also behaviourally modern.[4] There is a marked difference between the Paleolithic stone technology used in the earliest layers from 125,000 years ago, and the superior Mesolithic blades of the 70,000 year old Howiesons Poort period that used raw material which had been 'mined' 20 kilometres inland. There is also a differentiation between the Paleolithic food detritus that accumulated underfoot inside the caves 125,000 years ago, but was ejected and accumulated into external middens by the Mesolithic occupants circa 75,000 years ago. This 'housekeeping' is an indicator that 'at least half of our species' had evolved 'modern behaviour' by the middle stone age.
http://www.heraldmag.org/olb/contents/dict...ries/0EISBE.htm
http://www.sacred-texts.com/afr/we/we10.htm
1700 B. C. finds Egypt invaded and conquered. Dynasty XIII brought another blank in the monumental records. Egypt had broken into two really separate kingdoms. This enfeebled the country for the conquest of the Hyksos. During their stay, the native princes at the south maintained themselves. 2080-1525 B. C. these Shepherd kings ruled over Egypt. They were a barbaric and nomadic race from Asia which destroyed the temples and left no monuments standing in Egypt. Those who contend that the origin of the civilization of the Nile was from Asia should note that under these Asiatics, Egypt entered into the darkest period of her history. The Shepherds were expelled from Egypt by Aahmes, a mulatto and a Theban. He was the Amoisis of the Greeks and king of the north and south. He secured the favor of the Cush**es by marrying Nefruari, the black princess of Ethiopia, famous for her dusky charms, wealth and accomplishments.
http://www.gutenberg.org/files/17321/17321-h/v1c.htm
The Kassites... and the Hyksos... the Eastern rulers of Egypt....
http://en.wikipedia.org/wiki/Hyksos
http://www.touregypt.net/featurestories/hyksos.htm
http://history-world.org/kassites.htm
http://www.sacred-texts.com/ane/mba/mba21.htm
Evidence of trade with China....
"Flushed with his success, the Kassite king invaded Assyria when ... They imported cobalt--which was used for colouring glass a vivid blue--from China, ."
there was an active trade for metals between China and Europe for at least 5000 years....
http://nativenet.uthscsa.edu/archive/nl/9507/0250.html
http://www.telusplanet.net/public/dgarneau/euro12.htm
http://freepages.history.rootsweb.com/~cat...daho/umuti5.htm
"A British team of archaeologists believes that small slabs of bitumen dug up in Kuwait could hold evidence that man first successfully built ocean-going boats up to 7,000 years ago."
http://english.people.com.cn/200212/18/eng...18_108694.shtml
http://www.treasureenterprises.com/Miscell...n_australia.htm
http://www.wsws.org/articles/2003/aug2003/rock-a05_prn.shtml
"Another significant component of the discovery, Taçon said, is the existence of many half-human, half-animal figures called therianthropes These rare images include creatures with bird-like heads and others that are part kangaroo. A similar kangaroo form has been found at a site near the Hawkesbury River further east from Eagles Reach.
Taçon explained:"While therianthropes are very special depictions found across Australia, and in several regions overseas, the bird-headed creatures are avery rare find in the Sydney area. In Egypt such animal-headed figures are depicted as gods.
And I was trying to suggest the Aboriginals could be descendants of Egyptians or sailors from other parts of the world. It looks like whoever got here blended with the Aboriginals because a seclude Egyptian civilization was not found in Australia. If it is true that the Aboriginals came to Australia before 40,000 years they must have come from somewhere. It could be Egypt, India or China or any part of the world. These Hieroglyphs are not good enough to conclude the Egyptians sailed to Australia. Pyramids were found in Ukraine and China. Does it mean the Egyptians went to China and Ukraine? There are pyramids in South America and even the temples of India are considered pyramids. If the pyramids found in other parts of the world can't prove Egyptian migration to those parts of the world a few Hieroglyphs prove nothing.
It is easy to make up stories based on similar similar artificats found in different parts of the world. As I was thinking about this topic I began to wonder abaout the Ayres Rock and why it is a sacred place for the Aboriginals. Could it be possible that the Ayres Rock was originally shaped like a pyramid?
If you believe in Aliens you may find this theory interesting. The Aliens helped the Egyptians build the pyramids using anti gravational technology and gave them UFOs. The Egyptians flew to different parts of the world and built pyramids.
Or maybe the Egyptians found Australia to be the perfect place to dump their convicts?
Regardless of when you think the Gondwana broke up the truth could be different. By giving the references I tried to prove that there are different theories and every theory could have its merits and demerits. I think we should look at all angles before reaching conclusions. The stories that archaeologists make up with little evidence are amusing but stupid. Who knows how the earth was before 167 million years? I find it hugely unlikely that there were little cavemen in kangroo-skin pants wandering around. Leave the tigers alone. They are endangered!
Humans are believed to have existed before 4 million years but we somehow know what happened before 167 million years. It only takes one man to bullshi*! and rest will spread it around.
wtwt5237 0
Ectoplasmic Residue
If Egyptians came all the way to Australia, they should leave marks on their way. From Egypt to India to South Asia and to Australia. But inter-cultrural similarities may have not be found between Egypt and any of these places.
Go To Topic Listing Ancient Mysteries & Alternative History


text:
Porn Star Kendra Sunderland Banned From Instagram After 'Joking' About Sex With CEO
Desere Davis Updated in Nov 2021
Instagram is a very popular social media platform where stunning photos are shared with millions of active users.
Because youngsters use the platform, the people behind it, CEO Adam Mosseri, and others promise to keep things clean and safe.
However, sometimes risque photos and videos make it past their rules and regulations.
Kendra Sunderland is one of the platform's users who managed to share mature content and get away with it.
Kendra is a porn star who was once arrested when she filmed webcam porn in her campus library.
Clearly, she's a woman who doesn't enjoy playing by the rules.
Despite this (or perhaps because of this), she is a very popular star and ranks number 84 on Pornhub.
The Instagram Incidents
I don't usually call out performers for their behavior on Instagram, but today I am done. This was posted to IG AFTER Kendra sucked off a dildo while TOPLESS LIVE on her IG story! NO MORE NEGOTIATIONS @INSTAGRAM Hey @mosseri wtf is this? Does your wife know? pic.twitter.com/RIvM0PgqOp
— Alana Evans (@alanaevansxxx) December 10, 2020
Kendra has a 2.2 million-strong following on Instagram and regularly shares sexy photos with them.
Most recently, she has been sharing photos that show off all her assets in their full glory and folks were not happy.
A post shared by Kendra Sunderland Fanpage (@ksunderland_official)
Her topless photos - which are not allowed on the platform - got viewed by many Instagrammers.
On top of that, she also shared a video of herself topless and sucking off a giant black dildo.
It didn't help that Kendra was bragging about getting away with her actions.
She claimed that her content doesn't get taken down because she's having an affair with one of the platform's bigshots.
The man accused of having a relationship with her? None other than CEO Adam Mosseri.
The Fallout
Not long after her bragging video went viral, Kendra's account was shut down.
No one is certain whether that happened because of the affair comment or the type of content she shared.
Perhaps Instagram moderators finally caught wind of what she was doing. Or maybe it was an attempt to keep the truth silent.
We may never know.
If you're a fan of the porn star, you can still enjoy her content on PornHub or check out the various fan accounts dedicated to her.
It's not clear when Kendra will be back on Instagram or if she's been banned for good. It is likely that she will only be gone for a few days or weeks.

redpajama_set_name: RedPajamaCommonCrawl


text:
Sharon Buchbinder
Valentine's Day Ball, The
by : Donna Bell
A ROMANTIC TRADITION . . .
Tradition held that at eighteen years of age, all the Heartland ladies met the man they would marry at the Valentine's Day Ball. So it was that Miss Jane Lindsay was on the shelf at the advanced age of twenty-four -- for when she was eighteen her mother had died and the crucial ball had been canceled. Now the mistress of Heartland, she was in fact happy with her lot and accustomed to the idea that romance had passed her by. So she was quite annoyed with herself when a certain gentleman -- if he could be called such -- had completely overset her composure at this year's Valentine's Day Ball and made her feel like nothing so much as a naive debutante fresh out of the schoolroom . . .
A SHOCKING PAST . . .
Newly arrived home after ten years on a barbarous island to atone for sins committed in his youth, Lord Devlin was swarthily tanned, shockingly indifferent to social conventions, and dangerously seductive. It amused him to find ways to bring a blush to the virtuous Miss Lindsay's fair cheeks. Although she had turned down his offer to purchase the Heartland estate, Devlin had never been one to take no for an answer. He had a way of divining a lady's weaknesses. And once he had penetrated Miss Lindsay's defenses, he intended to make her an offer she couldn't refuse . . .
Zebra Regency Romance


text:
Japan's Nissan casts around for a new CEO
http://www.24emirates.xyz/business/japans-nissan-casts-around-for-a-new-ceo/136207-news September 10, 2019 37
Tokyo: Nissan Motor Co. is looking for its third CEO in as many years.
Following the resignation of Hiroto Saikawa after a scandal over pay, the Japanese automaker's board needs to find a leader who can guide the company through decade-low profits and job cuts as car sales slow globally. In addition to restoring confidence among the ranks, the new CEO will have to manage a complicated relationship with top shareholder Renault SA, its partner in a global carmaking alliance with Mitsubishi Motors Corp.
The board set itself a deadline of end-October to find a replacement. The pool of about 10 candidates includes people from Nissan and Renault, as well as non-Japanese and women, according to Nissan. The right candidate must have a deep understanding of the alliance with Renault and Mitsubishi, said Masakazu Toyoda, head of the nomination committee alliance.
Scandal-plagued
Saikawa took over as CEO in April 2017 from Carlos Ghosn, who was arrested in November for alleged financial crimes including understating his compensation — allegations Ghosn has denied. In June, Saikawa said that he should be held responsible for the instability unleashed by Ghosn's downfall and that he wanted the company to accelerate the search for his replacement.
Here are some of the potential candidates:
* The Nissan "lifers"
* Chief Operating Officer Yasuhiro Yamauchi, who will become acting CEO on September 16. Yamauchi, 63, is a Nissan lifer who also sits on the board of Renault. He was promoted to COO earlier this year, partly to preserve management ties between the automakers.
* Makoto Uchida, president of Nissan's China joint venture. Like many other automakers, Nissan counts the country as its single biggest market, contributing almost a third of its operating income in recent years.
* Hideyuki Sakamoto, in charge of manufacturing and supply-chain management, joined Nissan in 1980 as an engineer and has worked around the world, including the Nissan Technical Centre in North America, Nissan's largest affiliate supplier in Japan and Renault in Brazil.
* Jun Seki, formerly Nissan's China chief, is now senior vice-president overseeing "performance recovery". A Nissan lifer who joined in 1986, Seki worked in engineering and production, and has mostly kept a low profile.
Daniele Schillaci, the CEO of disc-brake manufacturer Brembo SpA who left Nissan earlier this year. As executive vice-president at Nissan, he led sales and marketing. Before that Schillaci was at Toyota Motor Corp. from 2002 to 2015 at various positions in Europe. Close to Renault, he has confronted Saikawa in the past at meetings, and may make a strong external candidate.
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text:
Q: Get current URL using webdriver.io module I'm trying to test that if my application correctly redirects the client after an action using the following code with mocha.js and webdriver.io.
it("Should redirect to correct URL.", function() {
 assert.equal(
   $("window").getAttribute("location"),
  `http://localhost:8080/${path}`,
 );
});

However I get the error:

Can't call getAttribute on element with selector "window" because
  element wasn't found

Seems like the window object is outside webdriver's selector functions scope but i can't see any other way of accessing the current URL in the docs.

A: Found v4 docs with the getUrl function.
it("Should redirect to correct URL.", function() {
 assert.equal(
   browser.getUrl(),
  `http://localhost:8080/${path}`,
 );
});

redpajama_set_name: RedPajamaStackExchange


We are thrilled to be welcoming dance, jazz, electro and disco's most wigged out duo, The Correspondents, back to ours for another spectacular live performance.
Back from a massive tour of Australia and NZ, expect the usual dose of hi-octane, genre-bending dance energy from one of the most unique acts to ever grace our stage.
Limited £6 tickets available so get 'em while you can!


One of the jobs I have whilst working for my local church is to create and implement systems to help the church function, maintain its health and grow. This includes things like creating 'pathways' to help new people connect with our church: a website that looks good and contains relevant information for visitors, training and running a Welcome Team, implementing follow up proceedures as well as advertising and marketing.
So today was great as we welcomed 3 new families to our church!
Unfortunately this started my mind trying to trouble-shoot a service that was actually quite good.
the song choices are not what I would gave chosen. This might put the guests off.
I hope the 'Hello' card I designed encourages them to come back again.
I hope they are not put off by this or by that.
About half way through the service I felt as though God told me off! It's one thing to put necessary systems in place to help new people connect but it is another thing completely to think that somehow I can single-handedly grow the church. In fact this is NOT my job – it is God's.
It is up to God to grow His church and to add people to our fellowship, or not. He calls people and puts them where they are meant to be. Not me!
Jesus – help me to do my job well but to never presume to take your place. Forgive me. Amen.
← Worship Fit For a King?


As revenue from the mobile consumer market continues to decline, wireless telco vendors and operators are looking at other verticals for new growth opportunities. ABI Research identified the mining sector as one of the potential verticals where wireless telco vendors and operators can venture into. The mining industry has always been the leader in technology innovation. In recent years, mining companies have relied on wireless technology to deploy autonomous vehicles in mining operations. In this report, ABI Research explores the types of technologies being deployed in private networks in mining, in addition to recommendations and future market forecasts.


text:
The BookWalker's Anticipated Fall 2018 List: Friday Black by Nana Kwame Adjei-Brenyah
The BookWalker's list of anticipated titles includes works of bestselling authors and emerging writers. Titles were selected and voted by our staff. Please note: the selection process is heavily biased but fairly influenced by personal preference and the editor's opinions.
A piercingly raw debut story collection from a young writer with an explosive voice; a treacherously surreal, and, at times, heartbreakingly satirical look at what it's like to be young and black in America.
From the start of this extraordinary debut, Nana Kwame Adjei-Brenyah's writing will grab you, haunt you, enrage and invigorate you. By placing ordinary characters in extraordinary situations, Adjei-Brenyah reveals the violence, injustice, and painful absurdities that black men and women contend with every day in this country.
These stories tackle urgent instances of racism and cultural unrest, and explore the many ways we fight for humanity in an unforgiving world. In "The Finkelstein Five," Adjei-Brenyah gives us an unforgettable reckoning of the brutal prejudice of our justice system. In "Zimmer Land," we see a far-too-easy-to-believe imagining of racism as sport. And "Friday Black" and "How to Sell a Jacket as Told by Ice King" show the horrors of consumerism and the toll it takes on us all.
Entirely fresh in its style and perspective, and sure to appeal to fans of Colson Whitehead, Marlon James, and George Saunders, Friday Black confronts readers with a complicated, insistent, wrenching chorus of emotions, the final note of which, remarkably, is hope.
"The edge of the stories in Nana Kwame Adjei-Brenyah's debut collection Friday Black is razor sharp, ready to cut deep. This book is dark and captivating and essential. This book is a call to arms and a condemnation. Adjei-Brenyah offers powerful prose as parable. The writing in this outstanding collection will make you hurt and demand your hope. Read this book. Marvel at the intelligence of each of these stories and what they reveal about racism, capitalism, complacency and their insidious reach."
— Roxane Gay
"For literature to bring forth such an astonishing new voice as Nana K. Adjei-Brenyah's—tender and furious, wise and wise-assed—marks a major leap forward for us all. The very first story brought me to tears, putting me in mind of Babel or Chekhov. And Adjei-Brenyah keeps doing that—dragging you through dystopic muck and mire before landing you in a transcendent spiritual place. This is the fiction debut of the year, and I can't cheer it loudly enough. Bravo, young man. We await your encore."
Note: This title will be released on October 23, 2018. Pre order: Click here
Nana Kwame Adjei-Brenyah is from Spring Valley, New York. He graduated from SUNY Albany and went on to receive his MFA from Syracuse University. His work has appeared or is forthcoming in numerous publications, including Guernica, Compose: A Journal of Simply Good Writing, Printer's Row, Gravel, and The Breakwater Review, where he was selected by ZZ Packer as the winner of the 2nd Annual Breakwater Review Fiction Contest. Friday Black is his first book.


text:
Jennifer L. Miller
Morgan & Morgan PA
Jennifer Miller was born in Youngstown, Ohio and was raised in Fairhope, Alabama. She received her B.A. in 1997 from Millsaps College in Jackson, Mississippi, and earned her law degree from the University of Memphis School of Law in 2001.
At Memphis, Jennifer was awarded the American Jurisprudence Award for Civil Procedure III, and was selected for the school's National Moot Court Team in 1999. In addition, Jennifer and her partner won the school's Advanced Moot Court Competition in 2000; Jennifer was awarded Best Oral Advocate.
She also served as a staff member of the University of Memphis Law Review from 1999 through 2001. Her case note, regarding a Tennessee Board of Professional Responsibility Ethics Opinion, was published by the law review in Volume 31, book no. 2. In 2000, Jennifer had the honor of serving as an intern for the Honorable Bernice B. Donald, United States District Court Judge for the Western District of Tennessee.
Admitted To Practice:
Tennessee State Bar 2001
Mississippi State Bar 2002
Alabama State Bar 2002
Georgia State Bar 2010
Western District of Tennessee
Eastern District of Tennessee
Northern District of Mississippi
Southern District of Mississippi
Eastern District of Arkansas
Western District of Arkansas
Fifth Circuit Court of Appeals
Sixth Circuit Court of Appeals
Northern District of Georgia
Middle District of Georgia
Tennessee Bar Association
Memphis Bar Association
Association for Women Attorneys
Memphis Bar Foundation, Elected Fellow
What types of cases Attorney Jennifer L. Miller & Morgan & Morgan PA can handle?
Morgan & Morgan PA can handle cases related to laws concerning Personal Injury, Premises Liability. We manually verify each attorney's practice areas before approving their profiles and reviews on our website.
Where is Morgan & Morgan PA located?
Morgan & Morgan PA is located at 1 Commerce Square 26th Floor, Memphis, TN 38103, USA. You can reach out to Morgan & Morgan PA using their phone line (901) 333-1900. You can also check their website forthepeople.com.
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Jennifer L. Miller lawyer charges are specific to each case. However, they work with contingency fees and its ranges from $$ to $$$. They also provide free consultation [and no obligation quotes] if you are interested to hire.
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Commerce Square 1
Memphis 38103 TN US
Jason S. Miller
Angela Mirabole


The ascomycete Trichoderma reesei is industrially used for the production of cellulases. During the production process xylanases are co-secreted, which uses energy and nutrients. Cellulases and xylanases share the same main regulators, which makes a knowledge-based strain design difficult. However, previously a cis-element in the promoter of the main xylanase-encoding gene was identified as binding site for a putative repressor. Subsequently, three candidate repressors were identified in a pull-down approach. The expression of the most promising candidate, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated on the repressing carbon source d-glucose and to bind the cis-element in vitro.
In this study, Xpp1 was deleted and over-expressed in T. reesei. An in vivo DNA-footprint assay indicated that Xpp1 binds a palindromic sequence in the xyn2 promoter. Comparison of the deletion, the over-expression, and the parent strain demonstrated that Xpp1 regulates gene expression of xylanolytic enzymes at later cultivation stages. Xpp1 expression was found to be up-regulated, additionally to d-glucose, by high d-xylose availability. These findings together with the observed xyn2 transcript levels during growth on xylan suggest that Xpp1 is the mediator of a feedback mechanism. Notably, Xpp1 has neither influence on the d-xylose metabolism nor on the expression of cellulases.
Xpp1 as regulator acting on the expression of xylanases, but not cellulases, is a highly promising candidate for knowledge-based strain design to improve the cellulases-to-xylanases ratio during industrial cellulase production.
The filamentous ascomycete Trichoderma reesei (teleomorph, Hypocrea jecorina) ) is industrially used for its outstanding secretory capacities. Industry strains produce over 100 g/L of enzyme in industrial-scale processes . The main component of the secreted enzymes is the cellobiohydrolase CBHI (EC 3.2.1.91). Further components of the secreted enzyme cocktail are the cellobiohydrolase CBHII (EC 3.2.1.91), the endoglucanase EGLI (EC 3.2.1.4), and the β-glucosidase BGLI (EC 3.2.1.21) [3, 4]. These enzymes work synergistically to break down cellulose to d-glucose. T. reesei cellulases find use in a wide range of industrial applications, such as paper and pulp, textile, and food and feed industry , and are still a bottleneck for cost-effective production of cellulosic ethanol .
However, T. reesei secretes also other enzymes along with the latter mentioned cellulases—most prominently the major endo-β-1,4-xylanase XYNII (EC.3.2.1.8)  and the β-xylosidase BXLI (EC 3.2.1.37) . Endo-β-1,4-xylanases cleave the backbone of xylan (i.e., the β-1,4-d-xylose chain) in the middle, generating substrates for the β-xylosidase which cleaves off d-xylose from the non-reducing ends of the xylan backbone. The co-secretion of cellulases and xylanases makes sense for the saprophyte T. reesei, because cellulose in plant material is encountered mostly exclusively together with hemicelluloses in the lignocellulose complex (reviewed in ). In industrial applications, however, the co-secretion of xylanases is a disadvantage for cost-efficient cellulase production because T. reesei uses additional energy and nutrients for their expression. Therefore, the reduction of xylanase formation during the cellulase production process is an obvious possibility for a more efficient cellulase production.
A way to achieve this would be knowledge-based strain design by deleting and/or over-expressing transcription factors that specifically regulate xylanase gene expression. However, the same main regulators regulate gene expression of cellulases and xylanases in T. reesei. The transactivator Xyr1 (Xylanase regulator 1) is essential for gene expression of most cellulolytic and xylanolytic enzymes . Notably, cellulases seem to be regulated in a different manner by Xyr1 than xylanases. The up-regulation of xyr1 transcription goes hand in hand with the up-regulation of gene expression of cellulolytic enzymes, whereas the regulation of xylanases seems to depend on additional mechanisms and factors . Further, gene expression of cellulases, xylanases, and their main regulator Xyr1 is subjected to carbon catabolite repression (CCR) [12–14] mediated by the transcription factor Cre1 (Carbon catabolite repressor 1) . CCR is triggered by high concentrations of d-glucose and d-xylose (products of enzymatic degradation of cellulose and xylan, respectively) [12–14, 16]. Additionally, all further described transcription factors involved in the regulation of xylanases, i.e., Ace1, Ace2, and Ace3, were reported to have also effects on the gene expression of cellulases [13, 17–20]. Thus, none of the so far known regulators can be specifically used as a target for knowledge-based strain design with the aim of shifting the cellulases-to-xylanases ratio.
In 2003, a cis-element in the xyn2 promoter bound under repressing conditions was identified. It was described as an AGAA sequence on the non-coding strand upstream of an Xyr1 binding site . A mutation of the AGAA-box led to an increased expression of a reporter gene under inducing conditions. The AGAA-box was therefore considered to be bound by a repressor. More recently, based on the development of a highly sensitive in vivo footprinting technique, an inverted repeat of the AGAA sequence further upstream was suggested to be part of the binding motif for the potential repressor .
Three candidate regulators binding the cis-element were identified by a pull-down assay followed by mass-spectrometric analysis and bioinformatic assessment . The expression of the most promising regulatory protein, Xpp1 (Xylanase promoter-binding protein 1), was reported to be up-regulated in the presence of d-glucose. Additionally, a GST fusion of its DNA-binding domain as well as an in vitro translated full-length protein could bind a DNA probe containing the cis-element of the xyn2 promoter in vitro .
In this study, we tested the influence of different carbon sources on the level of xpp1 expression, and deleted and over-expressed xpp1 in T. reesei. To investigate the involvement of Xpp1 in the regulation of xylanase and cellulase expression in vivo, the resulting strains were compared with regard to their xylanolytic and cellulolytic activities by enzyme assays and by direct measurement of the degradation compounds. Transcript levels of the genes encoding for the main enzymes involved in the degradation of xylan and cellulose were determined. Moreover, binding of Xpp1 in vivo to its cis-element in the xyn2 promoter was investigated by in vivo footprinting.
To analyze the role of Xpp1 in vivo, we deleted and over-expressed xpp1 in T. reesei. For the deletion of xpp1, a gene replacement strategy by homologous recombination was applied. The structural gene of xpp1 was replaced by a hygromycin resistance cassette in T. reesei QM6aΔtmus53. A schematic representation of the deletion strategy is shown in Additional file 1a. For over-expression of xpp1, a co-transformation strategy was applied. The coding sequence of xpp1 fused to the constitutive promoter of pki was inserted ectopically into the chromosome of T. reesei QM6a using pAN7-1, which confers hygromycin resistance . The transcript levels of xpp1 were measured in the obtained candidates by quantitative PCR (qPCR). A deletion strain lacking xpp1 transcript was chosen randomly and termed QM6aΔxpp1. The over-expression candidate exhibiting the highest relative expression of xpp1 compared to its parent strain (3.5-fold over-expressed) was chosen for further experiments and termed QM6aOExpp1. Genomic modifications in both strains were confirmed by PCR and by Southern blot analysis (Additional file 1b, c). Neither the deletion strain QM6aΔxpp1 nor the over-expression strain QM6aOExpp1 showed any differences in growth or sporulation behavior compared to their parent strains in minimal medium (liquid cultures or plates) containing lactose or carboxymethylcellulose (CMC), and surprisingly neither on xylan. On d-glucose, the deletion strain grows slightly slower compared to the parent strain (see Additional file 2).
As mentioned, Xpp1 was identified by a pull-down assay using a probe that contained the AGAA-boxes of the xyn2 promoter . To test whether this motif is bound in vivo by Xpp1, we performed a dimethyl sulfate (DMS)-induced in vivo footprint comparing the xpp1 deletion strain and its parent strain. In a replacement experiment, both strains were pre-grown on glycerol and then transferred to minimal medium containing 50 mM d-glucose. We could observe drastic differences between the two strains comparing the accessibility of the part of the xyn2 promoter that contains the AGAA-boxes (Fig. 1). Two nucleotides in the downstream AGAA-box were strongly hypermethylated in the xpp1 deletion strain, but only one nucleotide of the upstream AGAA-box. Interestingly, three nucleotides adjacent to the downstream AGAA-box were also hypermethylated. These three nucleotides are part of a palindromic sequence (5′-TCTAGA-3′) that overlaps with the downstream AGAA-box (Fig. 1).
In vivo footprinting analysis of the Xpp1-binding region within the xyn2 promoter. T. reesei QM6aΔtmus53 and the xpp1 deletion strain were pre-cultured on glycerol and thereafter transferred to MA medium containing 50 mM d-glucose. DNA was methylated in vivo with DMS after 3 h incubation. Analysis of data was performed using ivFAST . Significant differences in methylation intensities of individual purine nucleotides between the two strains are represented by squares (white, no difference; light gray, difference with ratios of more than 1.1 and less than 1.3; dark gray, differences with ratios of more than 1.3 and less 1.5; black, differences with ratios of more than 1.5). The sequence of the coding (upper lane) and non-coding (lower lane) strand from positions −241 to −220 of the xyn2 promoter is given. Bold letters indicate the previously described AGAA-boxes, underlined letters the palindromic sequence, and italic letters an atypical Xyr1-binding site .
Previously, the gene expression of xpp1 was reported to be up-regulated by d-glucose . We were interested whether d-xylose has an effect on the transcript levels of xpp1. To test this, T. reesei QM6aΔtmus53 was replaced on 50 mM d-glucose, 0.5 mM d-xylose, or 66 mM d-xylose, and transcript levels of xpp1 were measured by a qPCR assay. We found both d-glucose and high levels of d-xylose to induce the gene expression of xpp1 (Fig. 2a). To gain further insights into the regulation of gene expression of xpp1, we additionally compared its transcript levels in a growth experiment on different carbon sources, i.e., d-glucose, d-xylose, and glycerol [1% (w/v) each]. Samples were taken when similar amounts of biomass had accumulated on the different carbon sources. We found that transcript levels of xpp1 decreased on all three carbon sources at the later time point (Fig. 2b). This finding prompted us to investigate whether this decrease was caused by the depletion of an easily utilizable carbon source or by a change in growth rate. To this end, we grew T. reesei in 1% (w/v) d-glucose for 24 h at 30°C and then changed the temperature for 3 h. We found the xpp1 transcript levels drastically down-regulated at 12°C (Fig. 2c). On the other hand, a temperature shift to 40°C did not influence the xpp1 transcript levels, indicating that eventual stress caused by the temperature change was not the reason for the observed down-regulation at 12°C.
Gene expression of xpp1. a T. reesei QM6aΔtmus53 was pre-cultured on glycerol and thereafter incubated in MA media without carbon source (NC) or containing 50 mM d-glucose (G), or 0.5 mM d-xylose [XO (low)], or 66 mM d-xylose [XO (high)] for 3 h. b T. reesei QM6aΔtmus53 was grown in MA media containing 1% (w/v) glycerol (blue bars), d-glucose (green bars), or d-xylose (yellow bars) for indicated time periods (given in hours). c T. reesei QM6aΔtmus53 was pre-cultured on d-glucose at 30°C and thereafter incubated at indicated temperatures for 3 h. Transcript levels of xpp1 were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference samples (indicated by asterisks). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
To examine the influence of Xpp1—the suggested repressor of the expression of the endo-xylanase XYNII [21, 23]—on the xylanolytic phenotype of T. reesei, the xpp1 deletion, over-expression, and parent strain were grown on xylan. We measured substantial higher endo-xylanolytic activity in the resulting culture supernatant of the deletion strain and lower endo-xylanolytic activity in the over-expression strain compared to the parent strain after 72 h (Fig. 3a). Additionally, we detected higher β-xylosidase activity in the culture supernatant of the deletion strain compared to its parent strain and lower activity in the over-expression strain (Fig. 3b).
Influence of Xpp1 on the xylanolytic activities of T. reesei. T. reesei QM6aΔtmus53 (blue bars), the xpp1 deletion strain (green bars), and the xpp1 over-expression strain (yellow bars) were grown in MA medium containing 1% xylan for 48 and 72 h. The endo-xylanolytic activities (a) and the β-xylosidase activity (b) were measured and normalized to the acquired biomass after 72 h. The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. c Xylan was degraded in vitro with culture supernatants (xylan, 72 h) of T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) in a time course experiment. Concentrations of xylopentaose (squares, solid lines), xylotriose (diamonds, dashed lines), and d-xylose (circles, dotted lines) are given.
We were interested whether the synergistic action of the xylanolytic enzymes differed between the deletion strain and its parent strain during cultivation on xylan. As we could not detect free oligo- or monosaccharides in the supernatant during the growth experiment, we used sterile-filtered culture supernatants of the two strains to degrade xylan in vitro. The degradation was monitored over time by measuring the concentration of free xylose oligomers and d-xylose. Xylose oligomers (xylobiose to xyloheptaose) were released at a higher initial rate in the assay using culture supernatant of the xpp1 deletion strain compared to the one of the parent strain. The concentrations of xylopentaose and xylotriose are shown as examples in Fig. 3c. This points to a higher endo-xylanolytic activity of the xpp1 deletion strain. However, the maximal intermediate concentration of xylopentaose did not increase for xpp1 deletion, but was similar to that of the parent strain (approximately, 75 µg/mL; see Fig. 3c). Considering the higher endo-xylanolytic activity of the xpp1 deletion strain, the observed similar concentration maxima additionally point to a higher β-xylanolytic activity because xylopentaose is primarily generated by the action of the endo-xylanases and degraded by β-xylosidase. This conclusion is supported by the faster increase of d-xylose concentration in the assay using culture supernatant of the xpp1 deletion strain compared to the parent strain (Fig. 3c), since d-xylose is exclusively released by the action of β-xylosidase.
We aimed to test whether Xpp1 does in fact act as a transcription factor on the regulation of gene expression of xyn2 directly. In a first experiment, the xpp1 deletion strain and its parent strain were subjected to a replacement experiment using 50 mM d-glucose or 66 mM d-xylose. The transcript levels of xyn2 were quantified by a qPCR assay. Surprisingly, we could not observe higher xyn2 transcript levels in the deletion strain on both tested carbon sources after 3 h compared to the parent strain (Fig. 4a). Xpp1 does not seem to have an effect on the inducibility of xyn2 gene expression, which conflicts with the elevated xylanolytic activity of the xpp1 deletion strain observed, e.g., after 72 h growth on xylan. Therefore, we monitored xyn2 transcript levels in the two strains throughout growth on xylan. We could detect higher xyn2 transcript levels in the deletion strain compared to its parent strain after 30, 36, 48, and 72 h (Fig. 4b). Only the early (24 h) xyn2 transcript level is lower in the deletion strain. These findings match the observed xylanolytic activities. Deletion and parent strain had the same xylanolytic activities after 48 h (Fig. 3a). During growth xyn2 transcript levels drop in the parent strain, whereas they stay at a high level in the deletion strain (Fig. 4b), which results in a higher xylanolytic activity of the deletion strain at later cultivation stages (Fig. 3a).
Influence of Xpp1 on transcript levels of xyn2 in T. reesei. a T. reesei QM6aΔtmus53 (blue bars) and the xpp1 deletion strain (green bars) were pre-cultured on glycerol and thereafter transferred to MA media without carbon source (NC), or containing 50 mM d-glucose (G), or 66 mM d-xylose (XO) and incubated for 3 h. Transcript levels refer to the reference sample (QM6aΔtmus53, NC). b T. reesei QM6aΔtmus53 (blue squares) and the xpp1 deletion strain (green squares) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 24, 30, 36, 48, and 72 h of cultivation. Transcript levels of xyn2 were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (QM6aΔtmus53, 18 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
Since further enzymes besides XYNII contribute to the xylanolytic activities of T. reesei, we investigated if Xpp1 also controls their expression. First, we analyzed xyn1 transcript levels, which turned out to be higher in the xpp1 deletion strain compared to the parent strain (Fig. 5a), similar to xyn2 (compare Fig. 4b). Since we detected increased β-xylosidase activity in the supernatant of the xpp1 deletion strain (compare Fig. 3b), we measured the bxl1 transcript levels. Surprisingly, we detected equal levels in both strains (Fig. 5b), which contradicts the measured enzyme activity. However, the protein ID 58450 (on http://genome.jgi-psf.org/Trire2/Trire2.home.html) is annotated as a putative β-xylosidase . Consequently, we analyzed the transcript levels of the gene encoding for this candidate β-xylosidase (in the following termed bxl2). They were indeed higher in the xpp1 deletion strain compared to its parental strain at later cultivation stages (Fig. 5c).
Influence of Xpp1 on transcript levels of xyn1, bxl1, and bxl2 in T. reesei. T. reesei QM6aΔtmus53 (blue squares) and the xpp1 deletion strain (green squares) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 24, 30, 36, 48, and 72 h growth. Transcript levels of xyn1 (a), bxl1 (b), and bxl2 (c) were measured by qPCR using sar1 and act transcript levels for normalization and referred to the reference sample (T. reesei QM6aΔtmus53, 24 h for xyn1 and bxl1; xpp1 deletion strain, 24 h for bxl2). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. nd not detected.
Degradation of xylan results in the release of d-xylose, which is taken up by T. reesei and metabolized via the pentose phosphate way. The initial reaction is the reduction of d-xylose to xylitol, catalyzed by xylose reductase (EC1.1.1.307). We measured xylose reductase activity of the xpp1 deletion strain and its parent strain after 72 h growth on xylan. We could not detect any difference between the two strains (Fig. 6a). Further, the transcript levels of xyl1, encoding the xylose reductase, were monitored in the two strains throughout growth on xylan. Samples were taken periodically and xyl1 transcript levels were measured with a qPCR assay. Matching the obtained enzymatic activities, no differences of xyl1 transcript levels could be observed between the two strains (Fig. 6b). Xpp1 regulates the gene expression of the extracellular xylanolytic enzymes, but not of the intracellular xylose reductase. We presume that the xylose reductase can be used as an indicator for the whole downstream d-xylose metabolism in this context.
Influence of Xpp1 on xylose reductase expression in T. reesei. T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) xylan. Samples were taken after 18, 30, 36, 48, and 72 h growth. a Xylose reductase activity in cell-free extracts was measured in vitro and normalized to the total protein concentration of the cell-free extracts. b Transcript levels of the xyl1 gene were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 18 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
Owing to the fact that cellulolytic and xylanolytic enzymes are co-regulated in T. reesei, we were interested to know to what extent Xpp1 influences the expression of cellulases. The xpp1 deletion strain, the over-expression strain, and the parent strain were cultivated in minimal medium containing CMC for 72 h, and endo-cellulolytic activities of the resulting culture supernatants were assayed. We could not detect any differences on comparing the three strains (Fig. 7a). Further, transcript levels of the main cellulases, i.e., cbh1, cbh2, and egl1, were monitored throughout growth in CMC with qPCR assays. No differences between the deletion strain and the parent strain could be observed (Fig. 7b, c, data for cbh2 not shown).
Influence of Xpp1 on cellulase expression in T. reesei. T. reesei QM6aΔtmus53 (blue), the xpp1 deletion strain (green), and the xpp1 over-expression strain (yellow) were grown in MA medium containing 1% (w/v) CMC for 36, 48, 54, 60, 66, and 72 h. a Endo-cellulolytic activity was measured and normalized to the acquired biomass after 72 h. Relative transcript levels of cbh1 (b) and egl1 (c) were measured by qPCR using sar1 and act transcript levels for normalization and were referred to the reference sample (T. reesei QM6aΔtmus53, 36 h). Results are given as relative transcript ratios in logarithmic scale (Log). The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations.
Two inverted AGAA-boxes in the xyn2 promoter were previously considered to be the binding motif of a repressor [21, 22]. Xpp1 was later identified as the most promising candidate repressor . Using in vivo footprinting, we could detect strong differences at the putative binding site of Xpp1 in the xyn2 promoter comparing the xpp1 deletion strain and its parent strain. However, Xpp1 is a basic helix-loop-helix protein containing a predicted E-box binding motif . The classical E-box is a hexameric palindrome with the consensus 5′-CANNTG-3′ . Interestingly, the AGAA-box on the non-coding strand within the xyn2 promoter overlaps with a hexameric palindrome (5′-TCTAGA-3′). Five of the six nucleotides of this palindromic sequence were hypermethylated during in vivo footprinting in the absence of Xpp1. A similar hexameric palindrome (5′-ACTAGT-3′) can be found in the promoters of xyn1 and bxl2. Notably, both promoters contain also an inverted AGAA-repeat. In summary, we observed a correlation between regulation by Xpp1 and the presence of a hexameric palindrome 5′-WCTAGW-3′ together with an inverted AGAA-repeat. Table 1 provides an overview on the genes investigated during this study as potential targets of Xpp1 regulation and the presence of relevant cis elements in their promoters.
aPresent in the promoter within 1 kbp upstream of the start codon.
Further, an atypical Xyr1 binding site  downstream of the palindromic sequence within the xyn2 promoter was also hypermethylated in the in vivo footprint assay in the xpp1 deletion strain, notably, to a lesser extent than the palindromic motif. However, we could not observe a difference of xyr1 transcript levels comparing deletion and parent strain during growth on xylan (unpublished observations by Derntl C, Mach RL, Mach-Aigner AR). The observed differences might be the mere result of a changed accessibility of the whole region due to the absence of Xpp1.
Deletion of xpp1 resulted in elevated xyn1, xyn2, and bxl2 transcript levels at later cultivation stages in the deletion strain and in higher endo-xylanolytic and β-xylosidase activities in the supernatant after 72 h, but not after 48 h. Xpp1 seems to act as a repressor of gene expression of xylanolytic enzymes at later time points. Transcript levels of xyn2, xyn1, and bxl2 are higher in the deletion strain compared to the parent strain at late cultivation stages. We speculate that these observed differences are the result of a CCR-independent feedback mechanism mediated by Xpp1. During this study, we observed that high concentrations of d-xylose up-regulate Xpp1 expression, which acts as repressor on the expression of xylanolytic enzymes. Considering that accumulation of high amounts of d-xylose is a relatively slow process, this would fit the general idea of a feedback mechanism. This model would also explain why Xpp1 does not influence the inducibility of xyn2 expression at early time points (compare Fig. 4a, b). According to our hypothesis, the role of Xpp1 is the down-regulation of an induced system, but not the prevention of induction. The nature of this feedback mechanism might also explain the observed equal growth behavior of parent, deletion, and over-expression strains. All strains have similar initial xylanolytic activities and therefore provide similar amounts of monosaccharides for the growing fungi in the beginning. Biomass formation occurs during this period, resulting in similar growth behaviors.
Remarkably, Xpp1 regulates only the xylanolytic part of the lignocellulose-degrading enzymes in T. reesei. Transcript levels of the main cellulases as well as cellulolytic activities were not changed in the deletion and the over-expression strain compared to the parental strain. As mentioned earlier, regulation of gene expression of the main cellulases seems to be regulated in a direct manner by the amount of available Xyr1 . Matching the obtained results, we could not observe a difference of xyr1 transcript levels comparing deletion and parent strain throughout growth on CMC (unpublished observations by Derntl C, Mach RL, Mach-Aigner AR).
Xpp1 seems to be the mediator of a feedback mechanism regulating gene expression of xylanases. Xpp1 does not influence transcription of the main cellulolytic enzymes. We believe that this makes Xpp1 a highly attractive candidate for knowledge-based strain design, as this regulatory protein is a very promising instrument for shifting the cellulases-to-xylanases ratio in the secreted protein fraction during industrial cellulase production.
T. reesei strains QM6a (ATCC 13631), QM6aΔtmus53 , the xpp1 deletion strain QM6aΔtmus53Δxpp1 (QM6aΔxpp1, this study), and the xpp1 over-expression strain (QM6aOExpp1, this study) were maintained on malt extract (MEX) agar at 30°C. Hygromycin B was added when applicable to a final concentration of 113 U/mL.
For carbon source replacement experiments, strains were pre-cultured in 1-L Erlenmeyer flasks on a rotary shaker (180 rpm) at 30°C for 22 h in 300 mL of Mandels–Andreotti (MA) medium  containing 1% (w/v) glycerol as the sole carbon source. A total of 109 conidia per liter (final concentration) was used as the inoculum. Pre-grown mycelia were washed, then equal amounts were resuspended in MA media containing d-xylose and d-glucose in final concentrations as given or in medium without carbon source (reference condition), and harvested after 3 h of incubation.
For direct cultivation on d-glucose, d-xylose, glycerol, or xylan (Lenzing AG, Lenzing, Austria) strains were grown in 500-mL Erlenmeyer flasks in a rotary shaker (180 rpm) at 30°C in 100 mL of MA medium containing 1% (w/v) of the respective carbon source. For direct cultivation on CMC, strains were grown in 1-L Erlenmeyer flasks stationary at 30°C in 60 mL of MA medium containing 1% (w/v) CMC (Carl Roth GmbH, Karlsruhe, Germany). Mycelium and supernatant were separated by filtration through Miracloth (EMD Millipore, part of Merck KGaA, Darmstadt, Germany). Mycelia grown on xylan were weighed directly as reference for enzymatic assays. Mycelia grown on CMC were dried at 80°C overnight prior to weighing.
Escherichia coli strain Top10 (Invitrogen, part of Life Technologies, Paisley, UK) was used for all cloning purposes throughout this study and maintained on LB at 37°C. Ampicillin and hygromycin B were added when applicable to final concentrations of 100 mg/mL and 113 U/mL, respectively.
PCRs for all cloning purposes were performed with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions. All used primers are listed in Additional file 3. Generation of competent E. coli cells and subsequent transformation was performed according to standard protocols using CaCl2.
For the construction of pCD-Δxpp1, the 5′-flank of xpp1 was amplified by PCR using chromosomal DNA of T. reesei QM6a as template with the primers xpp1-5fwD-NotI and xpp1-5rev. The PCR product was inserted into an EcoRV-digested pJET1.2 (Thermo Scientific) in the opposite direction of Eco47IR (killer gene), yielding pJET-5′-xpp1. The 3′-flank of xpp1 was amplified by PCR using chromosomal DNA of T. reesei QM6a as template with the primers xpp1-3fwd and xpp1-3rev-NotI. This PCR product was inserted into pJET-5′-xpp1 in the same direction as the 5′-flank. For this purpose, the plasmid was digested with ClaI and blunted with T4 DNA polymerase. The resulting plasmid was termed pJET-BFxpp1. Finally, a hygromycin resistance cassette was generated by PCR using pRLMEX30  as template with the primers Ppki_5fwd and Tcbh2_rev-BcuI and inserted into pJET-BFxpp1 which was previously digested with NcoI and blunted with T4 DNA polymerase. The orientation was determined by sequencing and found to be the same as the 5′-flank and the 3′-flank of xpp1.
For construction of pCDexxpp1, the coding sequence of xpp1 was amplified by PCR using cDNA from T. reesei QM6a as template with the primers xpp1_fwD-XbaI and xpp1_rev-NsiI. The PCR product was digested with XbaI and NsiI and ligated into pRLMEX30  digested accordingly.
Protoplast transformation of T. reesei was performed as described by Gruber et al. . For deletion of xpp1, NotI-digested pCD-Δxpp1 was used for transformation of T. reesei QM6aΔtmus53. For over-expression of xpp1 under the control of the constitutive promoter of pki, pCDexxpp1 was co-transformed together with pAN7-1  into T. reesei QM6a. The transformation reaction was added to 80 mL melted, 50°C warm MEX agar containing 1.2 M sorbitol. This mixture was poured into four sterile Petri dishes and incubated at 30°C for 2–5 days until colonies were visible. The resulting candidates were subjected to four rounds of homokaryon selection by streaking.
Chromosomal DNA was isolated from mycelium by grinding in liquid nitrogen followed by a phenol/chloroform extraction. RNA was degraded using RNaseA (Thermo Scientific). DNA was precipitated with isopropanol, washed with 70% (v/v) ethanol, and dissolved in ddH2O. For PCR analysis, 10 ng of chromosomal DNA was used as template in a 25-µL PCR using GoTaq® G2 polymerase (Promega, Madison, WI, USA) according to the manufacturer's instructions. All used primers are listed in Additional file 3. For subsequent agarose gel electrophoresis of DNA fragments, a GeneRuler 1 kb DNA Ladder (Thermo Scientific) was applied for estimation of fragment size. DNA sequencing was performed at Microsynth (Balgach, Switzerland).
15 µg of chromosomal DNA were digested with 30 U of the given restriction enzymes. The resulting DNA fragments were separated by electrophoresis on a 0.8% agarose gel, then denatured in 0.4 M NaOH, and transferred by capillary forces onto a Biodyne B 0.45 µm nylon membrane (Pall Corporation, Port Washington, NY, USA) using 10 × SSC. 1.5 µg of biotinylated DNA probe were used for hybridization at 65°C overnight. Labeling of the probe was performed using a Klenow Fragment (exo-) (Thermo Scientific), random hexamer primers, and biotin-11-dUTP (Jena Bioscience, Jena, Germany). Signals were visualized by using Poly-HRP conjugated to streptavidin and ECL Plus Western Blotting substrate (both Thermo Scientific Pierce, part of Life Technologies) on a ChemiDoc MP (Bio-Rad Laboratories, Hercules, USA).
0.01–0.03 g of harvested mycelia were homogenized in 1 mL of peqGOLD TriFast DNA/RNA/protein purification system reagent (PEQLAB Biotechnologie, Erlangen, Germany) using a FastPrep FP120 BIO101 ThermoSavant cell disrupter (Qbiogene, Carlsbad, USA). RNA was isolated according to the manufacturer's instructions, and the concentration was measured using the NanoDrop 1000 (Thermo Scientific).
Synthesis of cDNA from mRNA was carried out using the RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's instructions.
Quantitative PCRs were performed in a Mastercycler® ep realplex 2.2 system (Eppendorf, Hamburg, Germany). All reactions were performed in triplicate. The amplification mixture (final volume 25 μL) contained 12.5 μL 2× iQ SYBR Green Mix (Bio-Rad Laboratories, Hercules, USA), 100 nM forward and reverse primer, and 2.5 μL cDNA (diluted 1:100). Primer sequences are provided in Additional file 3. Cycling conditions and control reactions were performed as described previously . Calculations using sar1 and act1 as reference genes were performed as published previously .
In vivo methylation of DNA using DMS followed by ligation-mediated PCR was performed as described previously  using the primers RG127, RG128, RG129, RG130, RG131, and RG132 for investigation of a regulatory region bearing the AGAA-box within the xyn2 promoter. Primer sequences are provided in Additional file 3. FAM-labeled fragments were analyzed by capillary gel electrophoresis (Microsynth) and results were analyzed using the program ivFAST .
Endo-xylanolytic and endo-cellulolytic activities of cultivation supernatants were measured with Xylazyme AX tablet assay and Cellazyme C tablet assay (both Megazyme International Ireland, Wicklow, Ireland) according to the manufacturer's instructions, respectively. One unit of activity is defined as the amount of enzyme required to release 1 μmole of reducing-sugar equivalents per minute.
The β-xylosidase activity was determined with the substrate 4-nitrophenyl-β-d-xylopyranoside (Sigma-Aldrich Corporation, St. Louis, MO, USA) as described previously . One unit of activity is defined as the amount of enzyme required to release 1 µmol of d-xylose reducing-sugar equivalents per minute.
For the measurement of xylose reductase activity, mycelia were ground in liquid nitrogen with mortar and pestle. 100 mg was resuspended in 1 mL 0.1 M Tris/HCl pH 7.5, 1 mM EDTA, and 5 mM β-mercaptoethanol and incubated at 4°C for 15 min. Cell debris was removed by centrifugation at 20,000g at 4°C for 10 min. The protein concentration of the resulting cell extract was determined photometrically by measuring the absorption at 280 nm using NanoDrop 1000 (Thermo Scientific). 200 µL of the cell extract was added to a 2 mL reaction in 0.1 M Tris, pH 7.5, containing 10 mM d-xylose and 0.15 mM NADPH. Oxidation of NADPH to NAD+ was monitored photometrically at 340 nm in a Jasco V-360 spectrophotometer (Jasco Corporation, Hachioji City, Japan) for 1 min at 25°C. One unit is defined as the amount that causes a reduction of optical density at 340 nm of 0.01 per minute. The activity was normalized to the total protein concentration of the cell extract.
For monitoring xylan degradation in vitro, 1 mL sterile-filtered cultivation supernatant from 72 h growth in MA medium containing 1% xylan (Lenzing AG) was mixed with 9 mL sterile MA medium containing 1% xylan (Lenzing AG) and incubated in a 50 mL sterile reaction tube on a rotary shaker (180 rpm) at 30°C. 200 µL samples were drawn at given time points and the enzymatic reactions immediately stopped by addition of 200 µL 100 mM NaOH. The remaining, undissolved xylan was removed by centrifugation at 4°C and 20,000g for 10 min. Samples were diluted 1:10 with 10 mM NaOH for HPAEC-PAD analysis. HPAEC-PAD analysis was performed with a Thermo Scientific Dionex ICS-5000 system (Thermo Scientific). Samples were separated on a Dionex CarboPac PA1 (2 × 250 mm) carbohydrate column (Thermo Scientific). The column was preconditioned at 0.26 mL/min in 150 mM NaOH/250 mM NaOAc for 5 min, followed by 150 mM NaOH for 15 min. Samples were then injected and oligosaccharides separated by a linear gradient to 150 mM NaOH/250 mM NaOAc in 20 min. Quantification was done by calibration of peak areas with authentic standards of xylooligosaccharides (Megazyme International, Ireland).
CD constructed the plasmids and the deletion and over-expression strains; performed the growth experiments, the qPCR assays, and the enzymatic assays; and drafted this manuscript. AR performed the in vivo footprinting assays. ES performed the HPAEC-PAD analysis. RLM participated in the conception of the study and revised the manuscript. ARMA participated in the conception of the study, supervised the experiments, and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by two grants from the Austrian Science Fund (FWF), P24851 and V232 given to ARMA, and by a doctoral program ("AB-Tec") of Vienna University of Technology. We thank Prof. A. Friedl, Vienna University of Technology, for providing access to the HPAEC equipment.
Additional file 1: Deletion and over-expression of xpp1 (A) Deletion of xpp1 in the parent strain QM6aΔtmus53 (Δtmus53) by homologous recombination with the plasmid pCD-Δxpp1 yielding an xpp1 deletion strain (Δtmus53Δxpp1) is represented schematically. Position of the xpp1 locus on scaffold 16 is indicated at the top. Thin black arrows indicate the approximate positions of the primers used for genotype analysis via PCR (5f2, xpp1-5fwd2 and Pkr, Ppki_5rev). Black-rimmed, light gray arrow represents the xpp1 gene; hatched boxes represent regions for homologous recombination; thick, black arrow represents the hph gene; gray arrow indicate the homologous recombination event; gray, dotted lines represent genomic DNA sequence; solid, black lines represent plasmid DNA sequence. (B) Correct and exclusive integration of the hygromycin cassette in the xpp1 locus was verified by Southern Blot analysis. The obtained signals correspond to the expected fragment sizes after digestion with PstI (3,410 bp and 4,622 bp) using the hph coding region as probe. (C) Ectopic insertions of xpp1 expression cassettes in the over-expression strain were determined by Southern Blot analysis using the xpp1 coding region as probe. L, 1-kb DNA ladder; P, parent strain (QM6aΔtmus53); Δ, xpp1 deletion strain; O, xpp1 over-expression strain.
Additional file 2: Impact of Xpp1 on growth of T. reesei. (A) T. reesei QM6aΔtmus53 (blue) and the xpp1 deletion strain (green) were grown in MA medium containing 1% (w/v) D-glucose (squares, solid lines) or lactose (triangles, dashed lines), or CMC (circles, dotted lines) for 18, 24, 30, 38, and 50 hours. The values provided in the figures are means from three biological experiments. Error bars indicate standard deviations. (B) T. reesei QM6aΔtmus53 (left lane) and the xpp1 deletion strain (right lane) were pre-grown on MA medium plates containing glycerol. Equal pieces of overgrown agar were transferred to MA medium plates containing 1% (w/v) D-glucose (G) or xylan (XN) or lactose (L) or CMC. Pictures were taken after 48 hours growth at 30°C in darkness.
Additional file 3: Primers used throughout this study.


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redpajama_set_name: RedPajamaGithub


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