text:
Rural adventures
Beaver safari on the Hortobágy
GPS adventure tour
The Spirit of the Vértes
Etyek gourmet day
Colorful medley in Orfű
Adventures at Vendvidék
Wine tasting in Mór
Farm visit and cheese making
Mountain adventure in the Bükk
CSR Team Building at Orfű
Tailor-made Trips
CSR Programmes
Eco-tours
Gourmet experiences at Etyek
Wildlife holiday at Lake Tisza
Hungary's hidden side
Village tour in Őrség
Daytrip to the Danube Bend
Mountain scooter tour in Bükk
Gastro & wine tour to Mór
Farm tour with cheese making
Paint the Trail – Charity
Travel Industry Partners
The spirit of the Vértes: sacral traditions, legends and ruins, gastronomy, herbs and mushrooms
Current, Events, Experiences, News / By Humtour Admin
We organize the program in English for individuals by appointment, contact us for an offer. Email us at info@humtour.com or:
(The photos are illustrations, the species and crop richness depends on the weather, can not be guaranteed!)
Wander among the hidden monasteries and castle ruins of the Vértes, while taking part in a nature and mushroom foraging tour. We will learn about the treasures of nature, collect mushrooms and fruits, learn about ecology, nature and environment with the help of our guide, and experience first-hand the natural phenomena caused by climate change. Depending on the weather, we will have the opportunity to collect edible mushrooms and herbs, and to fill our study baskets.
The hike is recommended in spring and autumn, but it is possible all year round.
The length is about 7 km, the difficulty level is medium, the ascent is 160 m.
The tour will take place in the "wilderness" of the Vértes, in the dense forests between Vértesszentkereszt and Pusztavám.
During the hike we will collect mushrooms and learn about wild flowers and herbs. Our guide is Attila Koszka, a conservation engineer, mushroom inspector and trophy evaluator, who will tell us about mushrooms, medicinal plants and forest ecology during the hike.
We will have lunch in Vértesszentkereszt, where Anita, the host of DoriNita Farm, will be waiting for us with a hot meal, cookies and syrup. At the place of lunch we can also get acquainted with the mushrooms and plants we have collected in the basket. Afterwards, we will visit the ruins of the former Benedictine Abbey of Vértesszentkereszt.
After lunch, a local guide will show the group around and talk about the monastery and its fascinating legends.
The ruins of the Benedictine abbey, built in the veneration of the Holy Cross, are hidden in the dense forest of the Vértes. At the top of a small rise, the former monastery appears like a vision from among the huge trees. The sacral site, hidden in the forest, has 8-10 metre-high wall remains, wide wall pillars and fragments of the doorway arch in their original position, yet it is one of the most beautiful and peaceful monuments in the country.
The first written record of the Benedictine abbey dates back to 1146. So in the first half of the 12th century, the monastery of the Csák clan in Vértes was already here. According to Anonymus, the estate itself was given by the chieftain Árpád to Előd and then by King Stephen I it was donated to the Csák family. The walls that have been discovered and the archaeological finds testify to the fact that the monastery was continuously expanded from the 12th century to the 16th century.
According to spiritual teachings, our planet is interwoven by so-called Saint George lines (Ley lines, dragon streams), along which healing energies are concentrated, especially at their intersections. Travellers visiting the ruins can absorb the healing energies of the Saint George lines.
After visiting the ruins, we head back to the cars and continue collecting mushrooms and forest treasures if possible.
Once back at the cars, Attila will inspect the mushrooms in everyone's baskets and only then can we depart.
Guided tour with renowned biodiversity researcher and mushroom expert
Home-made, hearty lunch with local flavours
Expert mushroom inspection
Entry to Vértesszentkereszt Abbey and guided tour
The tour requires some physical fitness due to its length, difficulty, elevation and duration.
The program can be adapted to the needs of the participants.
The price of the tour is calculated after having discussed the specific services and programs that are to be included.
Upon request, transfer can be provided.
To discuss your needs and request a quote, please contact us at info@humtour.com or click on the button at the bottom of the page.
We will send you an email within 24 hours to confirm your request for a quote, and once we have agreed on the exact details, you can pay for your tour by bank transfer.
If you have questions or need further information, please do not hesitate to contact us!
The Humtour Team
info@humtour.com
Check out our photos related to the tour! (Click on the gallery to enlarge!)
Farm visit, traditional pig feast-style lunch and cheese making at the foot of the Vértes – February 4, 2023
FULL! Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – March 18, 2023
Nature, Art & Gastronomy: Mushrooms in the forest, in the sketchbook and on the plate – March 25, 2023
FULL! Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – April 8, 2023
FULL! Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – April 22, 2023
Nature, Art & Gastronomy: Mushrooms in the forest, in the sketchbook and on the plate – Saturday, April 22, 2023
Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – May 6, 2023
FULL! Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – May 13, 2023
Nature, Art & Gastronomy: Mushrooms in the forest, in the sketchbook and on the plate – Saturday, May 13, 2023
Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – Saturday, June 3, 2023
Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – Saturday, May 6, 2023
FULL! Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – Saturday, May 27, 2023
Beaver safari and the magic of Hortobágy near the Nine-arched Bridge – Individual appointment and advance booking
Current (26)
Carpathian Tourism (1)


text:
Abkhaz Leaders Wary of Russian Integration Proposal
Russia's vassal in the South Caucasus is unwilling to give up its independence.
Nick Ottens November 19, 2014 November 23, 2021
A Russian proposal to deepen security cooperation with the breakaway Georgian territory of Abkhazia is meeting resistance not just from Tbilisi but the region itself. While the separatist enclave on the Black Sea coast has few allies besides Russia, it is reluctant to surrender sovereignty to it.
In an interview with Radio Free Europe/Radio Liberty, acting Abkhaz president Valeri Bganba said Russia's proposals were unacceptable. When lawmakers were presented with the details last week, the reaction was "close to" negative, he said, "because many points are about losing sovereignty."
Russia proposes to merge the Abkhaz and Russian forces stationed in the area and give itself the right to take command of the joint force in times of crisis.
Abkhazia has depended on Russian economic and military support since it seceded from Georgia in 1993. Georgia most recently attempted to retake control of the territory in 2008 when it also attacked South Ossetia, another breakaway province. Without Russian support, it is doubtful the two statelets would have been able to withstand the Georgian offensive.
Under a proposed treaty, Abkhazia would also harmonize its customs and tax regulations with those of the Eurasian Economic Union which is due to go into effect next year. Besides Russia, the trade bloc includes Armenia, Belarus and Kazakhstan. Kyrgyzstan, another former Soviet republic in Central Asia, has expressed an interest in joining.
In order to entice Abkhazia to accept the proposal, Russia has promised to pay for the increases in Abkhaz pensions and public-sector salaries needed to align them with those in Russia.
South Ossetia is more enthusiastic about integration. Following Russia's occupation and annexation of the Crimean Peninsula from Ukraine in March, its leaders openly called on Russia to annex them as well. So did lawmakers in the Moldovan breakaway region of Transnistria.
Russia is one of few countries that recognizes Abkhazia and South Ossetia as independent states. It does not recognize Transnistria as such.
Georgia described the proposed Russian treaty as yet another attempt to seize control of its territory. President Giorgi Margvelashvili called on parliament and the country's international partners to act.
Read more: Caucasus, Eastern Europe, Georgia, Russia, Small Regions and States

redpajama_set_name: RedPajamaCommonCrawl


text:
Socialist Evil
Government of the Stupid by the Stupid over the Stupid.
Elementary School Massacre - Why didn't "God" intervene?
Keep reading to the end for some "Mind-Blowing" information.
The senseless massacre of children hits all of us. Loss like this crushes. For the Parents, the pain seems beyond endurance. Life stops.
Living becomes just taking the next tight breath and crying... crying... Why?
Are we really here? What did we do to deserve this? What point is there anymore?
All our assumptions are open for review. We are confused.
And then we start imagining what we could have done to stop this. What can we do now to prevent this happening again.
Who's at fault? Where was the protection? Nothing is safe anymore.
The ultimate fault lies with us not following the basic moral standards of civilization.
We do not have a built in instinct to differentiate between what is good and what is bad other than basic pain avoidance.
We need an external standard.
The standard we teach and live by determines the consequences, good or bad.
The reason all these murderers commit these atrocities is because they were never taught a standard of morality that holds life to be sacred.
When I was a child, raised with the standard of the 10 realities of civilization passed down to us by Moses, I could never even imagine why anyone would ever murder someone.
But after years of exposure to the moral relativism and narcissism of current society, it has entered my consciousness.
My training allows me to choose not to follow this possibility but the shooter had no such constraint.
If God can do anything why didn't God stop the massacre of children?
Really? Why didn't YOU teach your children how to behave? Why did YOU allow society to become corrupted by alien ideologies that worship false moral standards?
Why did YOU give up personal responsibility to the State? Why did you allow Politicians to convince so many that they were selfishly entitled to take other's wealth?
If they can take wealth without responsibility, why not Life?
Why did YOU denigrate the Religious? Why did YOU pervert equality under the law to mean equalizing all results and all people? Why did YOU let Politicians foster Jealousy and covetousness for Political gain? Why did YOU allow movies and TV depicting the joy of theft, murder, jealousy, revenge and adultery? Why did YOU allow conniving rascals to be portrayed as Heroes?
God didn't stop it because YOU willed it to happen! You willed the consequences when YOU did not follow the moral standards given to you through Noah and Moses.
It really is a cycle.
People see tragedy and conclude there is no God or that God is evil.
Therefore they question the moral principles they were taught and even question the point of existence.
Either they form a new opinion based on their own confused mind or they follow a strong leader's confused ideas that promise safety and order.
Ideas which come from minds with no clear differentiation between good and bad, result in a confused morality without certainty of a good outcome.
When people act from a confused morality there is a probability of massacre of children and suicide.
When a society adopts a confused morality , like the current United States, the probability becomes a certainty. Then...
People see tragedy and conclude there is no God or that God is evil... and the cycle begins anew.
Based on history and human nature, only through internal self control guided by the 10 moral principles, in a society informed by the same guiding moral principles and free choice, can people act for the good and avoid atrocities of bad.
External control does not work as well.
We can ban weapons and the murderers will adapt.
We can post armed guards on our schools and the murderers will adapt.
They will kill the guard or use explosives or find some other way around anything we do externally.
We have armed guards to protect banks, not from the robbers who adapt and rob the bank anyway, but to maintain order among the regular people and deter the amateurs.
Outside policing and laws cannot stop bad actions without becoming a complete Police state.
A Police State is slavery for it's people and the powerful rulers commit even worse atrocities anyway.
The massacre is a wake up call to address the cause of the moral confusion that motivated this person's actions and our society's moral confusion.
If we do not, then we will be like Israel, which faces a daily threat from it's own citizens, raised with the alien suicidal and homicidal moral standards of Islam, and have to post an armed guard at every school, restaurant, mall and community gate.
If the question still remains...
Who says God is responsible to do that?
A human being of his own free will did the shooting.
Should God take away free will?
What would that do to all of us and the very nature of the Universe.
If God stopped every act of evil, there would be no heroic acts. In fact there would be no will to act at all.
Asking a powerful outside agency to fix everything is motivated by the pain of suffering.
If we felt nothing or just a small amount we wouldn't ask the question.
Centuries ago, when children routinely died from disease there wasn't this clamor.
We expected death and protected our emotions until the children reached a certain age.
Older Civilizations expected men to prove themselves on the battlefield and die in glory.
We didn't ask why God allows wars. We prayed that God would create more wars.
Calling on God or even government seems not to be based on a particular act but rather on how much we suffer.
We want something to make us feel better if the suffering is too great.
I wouldn't ask "why didn't God stop me from forgetting my 30% off coupon for Barnes and Noble".
We accept that question as silly. That's just too trivial.
We may be sad or beat ourselves up and lament the loss.
But the suffering is a faint echo of how we feel for those elementary school children.
For the loss of a coupon, it is far easier to accept that free choice means we make mistakes.
If we are mature, we can accept responsibility for that lapse without blaming others.
We can see it as a lesson and devise some sort of way of remembering next time.
It's far harder to accept the consequences of free choice, in the case of murdered children.
Logically though...
If God can do anything and does not interfere with my 30% off coupon, why should God interfere with the children's murder?
If God can do anything and we expect interference with the children's murder, why not with my 30% off coupon?
Saving 30%? What's the big deal in the scheme of things?
But a child's death and the loss we feel, that affects us greatly.
But in the scheme of things... is there a difference?
What if there is reincarnation? What if one of the children was spared the pain of a wasting disease or tragedy later in life, to begin a new life right way.
What if another Stalin or Hitler or worse would have descended from someone in that kindergarten class?
We really don't know the bigger scheme, do we?
What is the standard to judge the "best" action for God to take?
Hitler's mother might feel the same pain as we would in losing a child.
But would the best action be not to allow him to commit suicide and spare the mother's feelings?
Have we ever considered that if the rules of Nature can be broken then maybe it would de-stabilize nature?
If nature is broken to prevent murder, there may be consequences that are worse.
Suffering does not go on forever. We do eventually feel better and regain purpose and zest for living.
Healing. Is that how God interferes?
Maybe God interferes with evil through giving us principles to follow by our own will and an active guidance to tune into.
Still think it's God's fault because...
If God can do anything why didn't God stop the massacre of children or the Holocaust or the WTC bombing or all the bad?
OK Kiddo. Here it goes.
The premise that "God can do anything" is false.
God can't do anything in the same way that we can't just keep making exceptions and altering a system we set up or the system falls apart.
In the same way that we are bound by our word and our morality not to do just anything.
In the same way that a grown person can't get into the jungle gym pipes in a play space made for children.
The images we have formed of what we think is God, are inaccurate.
These images and premises come from the faulty translation of The Hebrew Torah (five books of Moses).
OK get ready for your world to turn upside down.
God does not exist in our universe.
Our universe exists in God.
That is what the Hebrew of the Torah (Five books of Moses) tells us.
What is God? God is what is.
There is no image we can create that is God. All images are false.
The powers of nature and the Universe are not Gods although they are the manifestation of what is.
There is no bearded man sitting on a throne waiting to smote and punish.
No Hell of infinite torture.
None of the images we created in our mind.
What was translated as Lord or God, YHVH in Hebrew, means "existence/being".
Existence/being is intelligent in a way that is different from our intelligence.
("My thought are not your thoughts")
The intelligence of existence (for lack of a better phrase) is the seed of the intelligence of nature/the powers of the Universe (Elohim) and us.
As science is realizing, the Universe is more like a brain that processes information. Guess what? The first word in Genesis, "Be-Raishit", that was translated as "In the Beginning" also means With Intelligence. "Raishit" contains the root meaning "Head". Be-Raishit also means inside the Head, which is the brain.
The first Letter "Bet" means a shelter/house/container.
We could not live in "infinite existence" (again, for lack of a better phrase).
The being/existence – makes a sheltered place bounded by time and space where we can live.
A shelter protects but also conceals what is outside of it. We only see a small amount through the windows, so to speak. (Adam, Moses, the Prophets and the still small voice of guidance we all have)
Our sheltered Universe was created with a flexibility for how energy is transmitted and where it will interact (the science of Quantum Mechanics), which allows enough uncertainty at the sub-atomic level for there to be Free Choice. Our Universe is partially determined by the will of infinite existence, so that it holds together, but can be influenced by our will.
We are co-creators as the cliché goes.
According to the Torah, our first Human ancestors were given a choice of an implanted, clear differentiation with what would be good and what would be bad - or mixing the good and bad in our knowledge so that we would have to use our own free will to differentiate. We would act according to the good with the goal of eventually returning to this original Eden level by our own will. Our ancestors chose to consume the fruit of mixed good and bad and exert their will rather than be programmed with no choice. The consequences of confusion of good and bad are, that there will be death and suffering but also the possibility of magnificent achievement motivated by our will to good.
So it is WE who have to fix what bad leads to, not YHVH or the powers of the universe (Elohim).
Part II - History
In general, we have sucked at avoiding the bad.
Luckily a fail safe mechanism was put in that will put back clarity, after a certain time period is exceeded, if we do not achieve it through our will.
That fall back is referred to as Messiah or Mashiach which means "to pull" our consciousness to clarity.
It also refers to the anointing of kings, another lesson to help us pull back to clarity.
The Israelites asked for the false protection of Kings and were told of the consequences – death and taxes. Children forced into battle, economic hardship etc. Our will - our responsibility for the consequences.
"We have met the enemy and it is us" – Pogo
If WE finally take responsibility and stop looking to "God" or the State, we can change the consequences.
Biblical History is there for us to learn from. Have you? When did you last hear this, if ever?
I'm skipping a lot (also a pun for Lot and the story of Sodom and Gomorrah :)
Cain kills his brother. The rest of the society that formed still persisted in being unable to differentiate good and bad and allowed the bad influences to dominate.
They robbed and murdered putting their interest above other's suffering.
We were implanted with a language that described reality perfectly and in which we all understood each other perfectly.
Noam Chomsky, a famous atheist linguist, proved that language is innate. It follows that the first language was also innate.
We see this first language reflected in Biblical Hebrew and the word roots in Akkadian and other ancient languages.
Corrupt Leaders took advantage of the capacity of our original implanted language to influence people to be one with the leader's goal of mastery over the human race.
They even found a place where all could gather together and not be dispersed physically as they were not dispersed with language.
Together they built a tower to spread their name and serve as a beacon.
The tower became the people's obsession, more precious than their lives or wives and they lost all discernment of good and bad.
At that point, the one language was broken into 70 to prevent gathering together to implement that which is inspired by the bad inclination.
But... that still didn't work. So, existence (YHVH) interferes, through the intelligent universe (Elohim). Elohim speaks to Noah to build an ark.
Elohim, through Nature on Earth, flooded away that society and started over again with Noah's family.
The Ark was the incubator of learning to care for others. Noah and his family had thousands of species to care for. They had not one moment's rest.
Out of the lessons learned, Noah's children founded academies to teach 7 principles that would guide society away from the inclination to bad outcome.
They are: Appoint unbiased judges, don't murder, don't steal, don't be cruel and eat an animal alive, don't worship and gather under the false power of an idol/image/Leader, don't hump everything in sight and don't pooh pooh any of the above.
The rainbow was the symbol that there would be no further interference by Elohim using nature. So don't count on big disasters to give us a moral leg up anymore.
Despite the babel and the principles of Noah, we still made statues and continued to appease all the powers of Nature like the Sun and the Moon and the weather. Abraham was able to perceive an inkling of the true nature of our universe and realized that all the forces of nature that people worshipped out of fear were really one and that some sort of unifying power must be responsible. He taught this monotheism, how to achieve this insight (meditation, contemplation and awe) and moral principles for 75 years. His influence appears in many of the Eastern schools of thought and religious practices.
Abraham's descendants through his son Isaac and grandson Jacob, kept their focus on the 7 principles and did not lose their language, moral teachings and culture despite abject humiliation and slavery in Egypt. Or vice versa despite glory as rulers or princes in Egypt (Joseph and Moses). These Israelites were entrusted with 10 principles of civilization, through Moses, to guide away from the bad. (Expanding on the 7 principles of Noah and adding: explicit identification of YHVH, telling the truth, managing jealousy and covetousness and accepting the burden of parental/ancestral teaching and history)
The Israelites were appointed as guardians of the words transmitted to Moses and have preserved them exactly as written.
They were given a religious practice, to reinforce living with the 10 principles, and serve as a light to the rest of the nations. The rest of the Israelite screw ups are documented in the Prophets for people to learn from mistakes as well as moral achievement.
Eventually most of the nations either adopted the 7 principles or in the case of the Christians the 10 principles.
Islam adopted the 10 principles, as mangled by Muhammad, among themselves but added a Political system that reinforces everything bad towards all others.
We were instructed to teach these principles to our children and remind ourselves of them daily. If we chose our actions, government and mores of society according to the guiding principles we would not have holocausts or tyranny or massacres of children. When we fail to imbue these principles in our children, government and society, we suffer bad consequences.
When we organized society, for the first time since Moses, according to those 10 principles in the United States, we had an outpouring of technology to cure disease and improve the material wealth of the impoverished. The less of those guiding principles that we teach and follow, the more technology is turned to bad, as the shooter did with his rifle, Hitler with trains, death camps, computers and gas chambers, Stalin with his blockade of Ukraine and Muslims with planes and bombs.
History is repeating itself and we are responsible to get it right this time around.
www.TheSocietyProject.org
Posted by BrerRabbi at 2:33 PM
The whole world just turned upside down
Elementary School Massacre - Why didn't "God" inte...
Conspiracy theory revealed
BrerRabbi
A small, weak, but ingenious force can overcome a larger, stronger, but dull-witted power. Brer Rabbit continually outsmarts his bigger animal associates, Brer Fox, Brer Wolf, and Brer Bear. Yes, we who are oppressed by the bigger, larger, stronger but dull witted ideas that have taken over the world can use our ingenuity and humor to outsmart the ideology of the extreme Left and extreme Right, Multi-Culturalism, Political Correctness, Socialism, Communism, Despotism, Collectivism, Big Government, Government Social Programs, Taxation without representation, Islamism and Terrorism, Jew Hatred, Racism, Reverse-Racism, Bureaucracy, Climate Change, Nanny State, Moral Relativism, Enviro-mentalism, Fanaticism, Secularism and the overuse of "ism". All writings copyright 2009 - 2014 L.S.Rosner.


I'm going to be honest here. I hate SEO. It changes so much and always has. Some people totally geek out at that, I am not one of them. I work hard at just doing the basics. Thank goodness there are a lot of people that love it. They are great resources and if you want killer SEO, you can hire them.
It's crucial to have top-notch SEO practices in place for your business blog. The difference between a well optimized site and a poorly optimized one could be tens, if not hundreds of thousands of dollars. I shudder to think of all the money I let slip away by not listening to my many friends in the early years and put better habits in place from the beginning. That was back at the beginning of time when SEO was actually much more simple.
So when I write an article about SEO here on the site. It's about me getting better at it and creating better habits as well. "Do as I say, not as I do" drives me batty. I know I need to be better again and this is how and why.
Why is it so important for a business blog to have phenomenal SEO?
Way back when, it used to be okay to have poor SEO. It wouldn't hurt you much because other sites in the industry had similarly poor SEO. In fact, if anyone was doing SEO it rocketed them to the top. That's not the case for any industry now. Even if it WAS the case, why wouldn't you want to have a major jump on the competition?
No matter what your specific topic is, chances are you're going to be facing people who really know what they're doing around search engines. There will be other blogs run by experts in that field that have been around longer. Upping your SEO knowledge will put you up there as the expert as well.
If you don't have top-notch SEO practices, your chances of ranking quickly are slim. Sometimes, it's very possible to accidentally rank. If you do accidentally get a good rank, how are you going to keep that rank and maybe copy it for another keyword?
Pay attention to SEO, if for no other reason than the fact that your competition is paying attention.
How can you talk about building a great business if your own website isn't properly optimized?
It's not just about rankings. A business blog needs to be well optimized to set an example and to demonstrate credibility.
One of the biggest advantages of working with business people is that they have money to spend. The downside, however, is that you really have to demonstrate that you know what you're talking about before they'll part with their money.
Showing that you know what you're doing with SEO can help establish that credibility.
The biggest benefit to running a blog is often not the actual sales or advertising revenue.
Instead, it comes in the form of contacts. Your future mentor might meet you through your blog. Or someone might like what you're up to and decide to invest $100,000 into your business.
They may also decide they like your product enough to buy it in bulk. Or they might be just one degree of separation away from someone who you really want to meet.
The bottom line is, you never know who might be reading your blog. That's why it's so crucial that you do everything in your power to get as many visitors as possible.
Yes, it takes time, energy and effort to optimize your blog. If that's what it takes to get your blog in front of the right people, then is it worth it? If the answer is "yes," then you owe it to yourself to put in that time.


text:
Home About Current page
Institutional Excellence
Washtenaw Community College was established in 1965, when Washtenaw County voters passed a millage to provide financial support for a countywide community college. WCC opened with a 6-member Board of Trustees, a newly-appointed president, and a parcel of land in Ann Arbor and Superior Townships. Classes started in September 1966 on a temporary campus at Willow Run, with 1200 students enrolled in 30 occupational programs and a transfer curriculum.
From this modest beginning, WCC has grown into one of the nation's premier community colleges with an annual enrollment of more than 18,000 students in credit classes each year, and thousands more in Economic and College Development classes. Our diverse student body includes more than 1,000 students from 100+ foreign countries. Our 285-acre campus and strategically located extension centers, operating resources, technology-enabled facilities, and instructional quality are the envy of post-secondary education institutions near and far.
LEARN ABOUT OUR MISSION, VALUES, AND VISION
President Bellanca
Since 2011, WCC has been led by Dr. Rose B. Bellanca. She is the fourth president of the College. Learn more about Dr. Bellanca.
Meet Dr. Bellanca
WCC Board of Trustees
WCC is governed by a 7-member board of trustees, who are each publicly elected to a 6-year term. Learn more about the current trustees, their roles, and their meetings.
WCC nursing student
We have built a reputation for being a top-quality organization by participating in regular voluntary review processes and transparent budgeting.
Policies express the Board's values and intentions for the College. Our policies are established by the Board of Trustees and carried out by the president and staff.
Faculty and staff at WCC
WCC seeks to recruit and retain a diverse workforce. We are committed to creating an inclusive, welcoming environment.
student reviewing WCC publications
We strive to maintain a college brand and identity that is clear and consistent. Questions about which colors, fonts, or logo to use? It's all in our visual identity standards guide.
Get a glimpse of WCC's history and see how the faces, tuition, and landscape have changed from yesterday to today.
WCC's History
See what it's like to be a student in one of our programs.
Jobs @ WCC
Public Bids and Request for Proposals RFPs


text:
Penn is a borough in Westmoreland County, Pennsylvania, United States. The population was 475 at the time of the 2010 census.

The borough was named for Pennsylvania founder William Penn.

Geography
Penn is located at  (40.329018, -79.640141).

According to the United States Census Bureau, the borough has a total area of , all of it land.

Demographics

At the time of the 2000 census there were 460 people, 182 households, and 133 families living in the borough.

The population density was 2,974.7 people per square mile (1,184.0/km²). There were 187 housing units at an average density of 1,209.3 per square mile (481.3/km²).

The racial makeup of the borough was 96.52% White, 1.52% African American, 1.09% from other races, and 0.87% from two or more races.

Of the 182 households 37.4% had children under the age of 18 living with them, 51.1% were married couples living together, 18.7% had a female householder with no husband present, and 26.4% were non-families. 25.8% of households were one person and 12.6% were one person aged 65 or older.

The average household size was 2.52 and the average family size was 2.99.

The age distribution was 27.6% under the age of 18, 6.1% from 18 to 24, 32.2% from 25 to 44, 18.0% from 45 to 64, and 16.1% 65 or older. The median age was 35 years.

For every 100 females, there were 81.8 males. For every 100 females age 18 and over, there were 77.1 males.

The median household income was $35,962 and the median family income  was $40,481. Males had a median income of $32,031 compared with that of $22,875 for females.

The per capita income for the borough was $14,312.

Roughly 4.4% of families and 7.4% of the population were below the poverty line, including 9.1% of those under age 18 and 7.0% of those age 65 or over.

References

External Links
Borough of Penn, Pennsylvania (official Facebook page)

Boroughs in Westmoreland County, Pennsylvania
Populated places established in 1859
Pittsburgh metropolitan area
1859 establishments in Pennsylvania

redpajama_set_name: RedPajamaWikipedia


Attorney and member of the Police Service Commission (PSC) Martin George has expressed support for the call that acting Police Commissioner Stephen Williams make public the full report which was submitted after the investigation into what was nationally dubbed, a total day of policing which occurred on March 23.
Speaking during a telephone interview yesterday, George said there were still a number of "ominous" questions that remained unanswered. George spoke in the capacity of an attorney. On Friday, the T&T Chamber of Commerce issued a release in which they criticised Williams for opening up, both himself and the contents of the report to public scrutiny. This, after Williams provided a statement on the issue during the weekly police media briefing last Wednesday.
Responding to questions, Williams reportedly said, "The investigator has reported prima facie there is the issue of disciplinary breach and he has identified the divisional commanders in the various areas for which road exercises were conducted and some of the station commanders who may be subject to issues around discipline." He also said despite the disciplinary breaches by the various heads of divisions for certain stations, the report would not be made public.
Claiming this was the essential question which needed to be answered, George added: "Can the executive really crucify the rest of the Police Service and wash their hands of it, saying we had no part in it, we knew nothing about it, we did not sanction it and we are going to take action after the fact?" George said the public would have a hard time accepting this logic.
"Is it that they knew and turned a blind eye to it? Is it that they were afraid to confront the situation? Is it that they were waiting for a scenario such as when the Minister of National Security gave his directive, for them to then say the instructions came from higher up so therefore we will now put a stop to it?" George asked.
Crediting the public for being more intelligent and discerning in this matter, George said, "This entire operation showed tremendous coordinated activity by the Police Service, and I think the members of the public would love to see the police employ this approach in fighting crime and solving some of the high profile outstanding criminals matters such as the Dana Seetahal murder. If the police were to mobilise like that, immediately upon the execution of Ms. Seetahal, I am confident they would have caught the criminals by now.


A second story addition to this 1960's flat-roofed house used the existing rafters for the new floor. The original broad eaves, horizontal lines and brick wainscot were incorporated into a new design featuring galvalume low-sloped roofs, cedar and natural gray cement stucco and enough windows to naturally daylight every space. Ground floor rooms were maintained in-situ but updated and the former garage was converted into a family room (a new garage was added fronting the alley). The original house had single pane windows and NO insulation...we remedied that!


I NOW proceed to perform the promise I made, of presenting the public with farther particulars relative to the poetry of Spain and Portugal.
Towards the close of the fifteenth century, was born Mosen Juan Boscan Almogavar,  the reformer of Spanish poetry; and, in the year 1503, his more celebrated assistant and friend Garcilaso de la Vega.  Boscan was tutor to the great duke of Alva:  "the heroic virtues that adorned the mind of the pupil prove with what diligence and success the tutor performed his duty;" so says one of his biographers.  Let not the reader detest the poet Boscan because he had the misfortune to educate the detestable duke of Alva! Alexander had listened to the lessons of Aristotle,  and the son of Antoninus must have heard the precepts of his father;  but no culture can render the night-shade innocent.
This innovation, like all other improvements, was not introduced without opposition. Inigo Lopez de Mendoza,  the celebrated marquis of Santillana, had made use of the Italian metres many years before. Don Diego de Mendoza,  of the same noble house, had the honour of co-operating with Boscan and Garcilaso in a more successful attempt; though such is the caprice of Fame, that he is better known in England as the author of Lazarillo de Tormes,  than as the historian, the poet, and one of the reformers of his country's literature: to the disgrace of mankind, whatever work is lively and loose, will certainly be popular. The name of Garcilaso has eclipsed that of his assistants, and he is to this day esteemed the best of the Spanish poets, yet the little volume of Garcilaso's productions is more distinguished by melody of versification than sublimity of thought. The volume consists of 184 pages, of which 110 are taken up by three eclogues!  In the present æra of taste, no poet possessed of common sense would ever commit a pastoral; and none but a Spanish or Portuguese poet would ever have extended one to upwards of seventeen hundred lines!
"Nature has hidden in rough shells, at the bottom of the sea, those pearls to which man has affixed such value; she has hidden that gold with which our souls are fettered, in the bowels of the earth, amid barbarous nations, and in distant countries: she has guarded the sea with rocks, and sown it with dangers, to place boundaries to our desires, and lengthen the period of our lives: but Evil, to deprive us of our tranquillity, laid open these secrets, and hid from us the true knowledge where real tranquillity is to be found. Then did this malignant spirit disfigure the shepherds with coarse vile garments, and represent their life of contentment as a life of mean and despicable labour; and by these magical delusions were we taught to despise the only treasure which the earth affords to render the mind happy: but when this fascination is removed, and we see things clearly, how much more beautiful appear the various colours with which the fields are apparelled, and the trees, and the sun, and the horizon beautiful when he sets, than all the deceitful trappings of Vanity! How much more delightful to our ears is the song of innocent birds, than the sound of flattering tongues, that endeavour to entrap our reason! Is not the rock that hangs over the stream, in whose caverns the birds dwell, and under whose shade the fishes sport, more to be admired than the sumptuous and superb edifice, that cannot so well resist the force of the tempest, or the secret sap of time? Where can life pass more delightfully or more tranquilly than among the flocks and herds? How much more secure is the enjoyment of these than the hopes of the court, and the deceits of the city! And if we have so often sighed for that happy age of gold, it is for this advantage, exceeding all others, that men lived then like shepherds, and followed their flocks, and cultivated the earth: and this truth is clearly proved; for the first man whom God created held this office, and the title which God gave him, was that of lord of the animal world; and Abel, the first martyr, in whom the church began, and the other children of Adam, tended their flocks: so likewise did Abraham and Isaac, and Jacob with his beloved Rachel, and Esau; Joseph and his brethren were shepherds, as they confessed to Pharaoh. Moses and Zipporah, Saul and David, kings of Israel, and Mesa, king of Moab, had executed this honourable office; and king Cyrus had exercised it among the ancient Persians. Romulus and Remus, the founders of Rome, with Faustulus, who educated them, kept sheep; and among those valiant Romans, the fame of whose exploits has echoed over the world, we read of many whose names discover their origin, such as the Vituli, the Vitellii, the Porcii, the Capri, the Tauri and the Bubulci. Many persons have risen to the highest dignities, from the pastoral state: Giges, king of Lydia; Sophy, king of the Turks; Primislaus, king of Bohemia; Tamerlane, emperor of the Scythians; Justin, emperor of the Romans; Viriatus, captain of the Portuguese; and Sixtus the first, the Roman pontiff: and, in truth, what is the life of a shepherd, but the similitude of empire? but a system of government, with moderation and mildness? For what can be more similar to the government of a kingdom than the management of a flock? To defend them from wild beasts, to secure them from robbers, to guide them to good pastures, cool shades, and clear waters; to threaten them with his voice, to chastise with the crook those who stray; to amuse them with the pipe and with the song, to cure them with herbs when they are sick; to be clothed with their wool, to feed upon their milk, and thus to pass peaceably through life? Among the vain deities whom the blinded Gentiles worshipped, Apollo, Mercury, Daphne, and Pan, and Proteus, and Paris, and Polyphemus, were shepherds; and the true God whom we serve, is frequently styled a shepherd, in the holy Scriptures; so ancient and so honourable is the pastoral life, which the avarice of men has now made despicable!
"Much knowledge is certainly necessary for a shepherd; an acquaintance with the nature of soils and pastures, the virtues of herbs, the changes of weather, the movements of the heavenly bodies, the effects of the sun, and the qualities of animals; and this life, though the most quiet, produces in its employments all things necessary for our subsistence: wool, milk, skins, the flesh of animals, herbs, grain, fruit. What life, then, can be more delightful than the pastoral life? or what prejudice can be greater than that which denies this truth?
So curiously has this ingenious Portuguese defended pastoral poetry! But though we may agree with him that the life he describes is the most natural and most honourable state of man, we shall be very far from acknowledging, that either his eclogues, or those of any other poet, fairly represent it.
Garcilaso de la Vega, in the most enormous of his eclogues, has introduced almost action enough for a drama.  Albanio opens it, with a soliloquy of lamentations, and then he falls asleep. Salicio then enters, singing a translation of Horace's favourite ode, "Beatus ille qui procul negotiis,"  of which there are above twenty versions in the Spanish language. In the middle of it, he stops short, on seeing a man sleeping, dilates upon the excellence of sleep; and then, recognizing Albanio, informs the reader, that he knows him, that he was once very happy, and is now very miserable, but that he had not yet learned the cause. Albanio now talks in his sleep and Salicio interrupts and wakes him. He now requests him to relate his history, and Albanio tells a very long story of his being the intimate friend of a young female relation, with whom he used to hunt; how he one day told his love; she left him, and he is dying with despair. After they are gone out, Camille enters, and lies down by a fountain to take her siesta — her evening's nap. Albanio finds her, and seizes her, but releases her on her solemn promise to remain and hear him, which she, as soon as released, breaks, and runs away, and Albanio runs mad. Salicio now enters, with Nemoroso: Nemoroso tells a long story about a magician, which is a panegyric upon the family of Alva; and the eclogue concludes with their resolution to get Albanio cured by this magician.
And trim our cot with neatness.
The joys of kings excelling.
In this the objection to the Moorish metre is removed, by the alternation of a regular rhyme.
The charms that make me love.
Sadly she sits, and weeps what she has done.
Boscan paraphrased the Hero and Leander of Musæus, of course he injured it; for to paraphrase is to dilate, and to dilate, to weaken.  He survived his friend, Garcilaso (who was killed in battle) but a few years: they both died young; but their celebrity will always last; for though Spain may hereafter produce better poets, the glory of reforming the national poetry must still remain.


text:
Home Breaking James DeGale: I will show Eubank Jr boxing skills
James DeGale: I will show Eubank Jr boxing skills
Degale is confident he will show Eubank Jr levels.
DeGale v Eubank Confirmed For February 26. Photo Credit: SkySports
Two domestic rivals with a lot to say about each other will finally meet on the 23rd February, as former IBF Super Middleweight Champion, James DeGale (25-2-1, KO15) and Chris Eubank Junior (27-2, KO21) face off at the O2 Arena in London.
It's been a badly kept secret for a while now, with the pair rumoured to meet in December 2018, but the match-up now has a date and a venue confirmed.
The fight is a box office attraction, with ITV Box Office picking up the rights as part of their recent exclusive agreement with Premier Boxing Champions. Also, as now seems tradition, a spurious belt in the form of the vacant IBO Super Middleweight Title will be on the line.
DeGale: The loser of this fight retires
DeGale has described the contest as a "retirement fight" with the loser seemingly having nowhere to go going forward. St Albans based DeGale relinquished his IBF World Title last year, after avenging a shock 2017 loss to Caleb Truax in April last year. Rather than face a dangerous mandatory in Jose Uzcategui for little money or reward, "Chunky" decided to return the belt in pursuit of money spinning fights. He has only fought once since the Truax rematch, a three round knockout win way down the card on a small bill in California in September.
DeGale-Eubank Jnr face off. Photo Credit: Evening Standard
What makes the match even and interesting, is that DeGales last few performances have been below par, and he has shown signs of decline, and Eubank will see this as an ideal opportunity for a significant win to be added to his ledger.
After an up and down draw against then WBC Champion, Badou Jack in a unification fight in January 2017, DeGale disappointed in dropping his IBF belt in a shock loss to Truax in December the same year at the Copper Box Arena in London. DeGale was flat throughout, and allowed Truax to push him back and dictate the pace of the fight, and the Minnesota man took the IBF belt with a deserved majority decision. The rematch saw DeGale cut in the third, and was troubled while he adjusted. He managed to do enough to regain his Title by one point on two cards, and by seven on the third.
Eubank Jr believes he will be too much for DeGale
Eubank Junior will see weaknesses in DeGale that he hopes he can exploit, but will also know that this is a must win fight for him too. The 29 year old Brighton resident suffered the second defeat of his career in February 2018, when eliminated at the semi-final stage of the World Boxing Super Series. Eubank worked hard throughout the contest with George Groves, but "The Saint" was always in control and took a deserved comfortable points win in defence of his WBA (Super) Super Middleweight Title. He returned in Jeddah, Saudi Arabia, on the undercard of the final of the tournament, retiring the limited JJ McDonagh in three rounds.
The weight may also prove key, as Eubank is a small Super Middle, and more naturally a Middleweight, with DeGale a bona fide 168 pounder. DeGale hopes a win will secure a trilogy fight with Groves, but must first take care of Eubank, which will be no easy task. At this point, I believe if the best DeGale turns up, it should be enough to see off the relentless pace of Junior.
chris eubank jr
itv box office
james degale
premier boxing champions
Previous articleWarrington v Frampton – Big Fight Preview & Prediction
Next articleRICHARDS – JAKE BALL IS A TIME-WASTER


We are a group of healthcare professionals teaching mindfulness in Hong Kong. Coming from different professional backgrounds, we are drawn together by our interest and practice in mindfulness training and our aspiration to share its benefits with the people we serve. All of us have teacher training in Mindfulness-based Stress Reduction (MBSR) or Mindfulness-based Cognitive Therapy (MBCT), in addition to other professional and meditative training.
We offer this web site as an avenue for people to find out information on mindfulness training programs in Hong Kong. As you will find out in the Upcoming Events page, we work in different settings and serve people through various channels that our lives open for us. While we have not formed ourselves into any formal organization, we feel very much connected with each other as we support each other in nurturing our wisdom and compassion and living a fuller life through mindfulness practice, and in our work in bringing this wonderful practice to you.
Please also see: Hong Kong Mindfulness Teachers Network.


NEW ON MARKET! Own a piece of paradise on 1.75 Acres in Olentangy Schools with approx 3400 Sq.Ft of livable space, just N of Hyatts, off Liberty! Incredible 2 Story Great Rm w/Gas Log Fireplace opens to a spacious Kitchen w/Stainless Steel Appliances, Corian Countertops, Eating Space w/Bay Window & French Doors to your Paver Patio. 1st Floor Master Suite w/Vaulted Ceiling & Ledge, Walk-in Closet, Double Sinks, Kidney Shaped Soaking Tub & Shower Stall. Upstairs is a huge Loft, 2 Spacious Bedrooms & a Full Bath w/Double Sinks. Beautifully Finished Lower Level w/Half Bath, Dry Bar W/Mini Refrig. 1st Floor Laundry & Half Bath. Immaculate!!


New Julia Hunter Product now available at Medifine Aesthetics UK.
Exfoliating Pads containing natural organic acids, Lactic AND all the shielding and EMPOWERING aged grapeskin exfoliating elements are ESSENTIAL to help resolve as much as SCIENTIFICALLY possible, in COMBINATION with all the other SKIN THERAPY MAXIMAL THERAPY POSSIBLE external therapies that penetrate as DEEPLY and COMPLETELY as possible due to CHIRALITY, CONCENTRATION and PUREST SOURCING, resulting in as much as can be formulated, Healthier, More Youthful Functioning and APPEARING Skin.
WHOLE BODY RENEW should be used DAILY OR EVERY OTHER DAY – on ENTIRE face, ears, neck, chest, back, arms, legs, and ENTIRE body for MAXIMUM Results and Skin Renewal. SAFE and Perfect for all Skin Types, Colors and Genders.


text:
Home Game Reviews Click Clack Lumberjack Review
Click Clack Lumberjack Review
Tyler Nichols
Review of: Click Clack Lumberjack
Board Game Review By:
Reviewed by: Tyler Nichols
Last modified:Jul 10, 2014
We review the dexterity game Click Clack Lumberjack. Based on the game Toc Toc Woodman from Korea, Click Clack Lumberjack will have players trying to debark a plastic tree with careful swings of an axe.
Jenga was a game that my sister and I played with frequently when we were kids. We had fun building the tower of blocks and waiting until one of us messed up to watch the whole thing come tumbling down. Click Clack Lumberjack from Mayday games gave me a nostalgic feeling when I first laid eyes on it. In this 2-7 player dexterity game, players take turns hitting a stack of trunk cores attempting to knock the bark off without knocking the whole thing down. If you have heard of the game Toc Toc Woodman from Korea, it is exactly the same game with just a new name. Will this filler game stand up with other dexterity games or will it fall quickly to the ground. Read on to find out!
Click Clack Lumberjack is a dexterity game for 2-7 players that plays in about 15 minutes. Click Clack Lumberjack plays best with 5 players.
Once setup, the tree actually looks really nice. Mayday Games did a great job with the components.
The object of this game is to get as many points as possible. In the center of the table is a mighty tree that players are attempting to debark without cutting down the tree. Because the trunk is made up of different core pieces, they are able to shift around as players hit them with a plastic axe included in the game. If players are able to move the trunk cores far enough off center, the bark pieces will fall to the ground gaining them a point. If they knock the core of the tree off they get negative five points. The player who manages to knock of the most bark and avoids taking down the core will win the game.
The pieces for Click Clack Lumberjack are top notch. They are all made from very thick plastic and will stand up to many games and even the hardest of hitters. I like how the core trunk pieces has cuts to make it almost look like rings in the wood. Even the way that the bark is designed makes the finished stacked pieces look like an actual tree, minus the leaves of course. The quality of the components makes the game look very inviting to players and as realistic as you can with plastic pieces. Plus the game comes with a plastic axe, which I have caught my kids playing with on numerous occasions. Overall the components in Click Clack Lumberjack are well made and work perfectly for the game.
First thing for players to do is construct the tree. There are a total of 9 core pieces that allow for 4 piece of bark to slide in on the sides of each core piece. These combination core/bark pieces are all stacked on top of each other on the base trunk piece. On a players turn they take the axe and get two hits on the tree. When a player manages to get a bark piece or a trunk core to fall off they take that piece and put it in front of them. On four pieces of the bark there are small grubs that give the player an extra turn hitting the tree. Play will continue until all pieces of bark are off the trunk or the entire thing is on the table. At that point, players will total up their gained pieces with bark, worth a point for each, and subtract five for each core piece and the player with the most points is the best lumberjack ever. That is it, the entire game in one short paragraph. That has to be some kind of a record. (Publisher's Note: It is).
I like that Click Clack Lumberjack takes a deft hand. You can't just smack the tree as hard as you want without knocking it all over.
I had a lot of fun playing this game. The way that this game is designed makes this type of dexterity game quite different than anything else I have played. Instead of just trying to knock things down or trying to not make the structure fall, you are forced to skirt the line in the middle. By forcing players to use more finesse with their hits, it adds a lot of tension to the game. Every miss hit could cause most of the stack to fall on the ground. There are a lot of entertaining moments when players best laid plans go to waste. Because of that, people don't turn away from the game. You don't want to miss those. I do like how the game doesn't end the first time that some of the core pieces fall. People laugh and continue on with the game. This helps to keep the game flow going.
As the game progresses, the tree will get more and more unstable.
I'm used to using my fingers in a game like this, but using the axe is a different experience. It takes some time to get used to the amount of force needed to move the cores around, however, once you get a few games in, it feels very natural. The axe, plus the design on the trunk core and bark pieces, also helps to connect the players to the theme. The game is a little intimidating out of the box, but within five minutes you can have your tree ready to go. I was amazed with the components that come in the game. They are extremely high quality with very thick pieces and when you complete the tree it just looks awesome. If you were playing this at a party or with a large group, the game play and components will draw people to the game just wondering what you are playing.
This game also works quite well with kids. My oldest is a little young for this game, but she gets the idea and finds it fun. I think kids over 5 will have a blast with this game and so will their parents.
Click Clack Lumberjack embodies the essence of what a filler game should be. It's simple to learn, a blast to play and doesn't take the whole night to play. I honestly think that Click Clack Lumberjack needs to be available for the masses in big chain stores and would easily take the place of Jenga in my collection if I owned it. Click Clack Lumberjack is a great vertical dexterity game that you can play with people of all ages and one I think most should own.
If you are interested in getting a copy for yourself, it's about $20
Final Score: 3.5 Stars – A uniquely designed dexterity that will be a joy to play with all age groups.
• Easy to learn
• Makes a good party game
• Tons of fun to play
• Can be challenging at first
• Players might dislike the lack of strategy
Dexterity Game
Filler Game
Non-Gamer Friendly
For Tyler, games that present interesting decisions and memorable moments are ones that he will play again and again. He also has the responsibility to teach his two younglings the ways of board games.


text:
CRISPR-Cas9 Gene Editing in Lizards Through Microinjection of Unfertilized Oocytes
Ashley M. Rasys, Sungdae Park, Rebecca E. Ball, Aaron J. Alcala, View ORCID ProfileJames D. Lauderdale, View ORCID ProfileDouglas B. Menke
Ashley M. Rasys
Department of Genetics, University of Georgia, Athens, GA 30602, USADepartment of Cellular Biology, University of Georgia, Athens, GA 30602, USA
Sungdae Park
Department of Genetics, University of Georgia, Athens, GA 30602, USA
Rebecca E. Ball
Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA
Aaron J. Alcala
James D. Lauderdale
Department of Cellular Biology, University of Georgia, Athens, GA 30602, USANeuroscience Division of the Biomedical Health Sciences Institute, University of Georgia, Athens, GA 30602, USA
ORCID record for James D. Lauderdale
Douglas B. Menke
ORCID record for Douglas B. Menke
For correspondence: dmenke@uga.edu
CRISPR-Cas9 mediated gene editing has enabled the direct manipulation of gene function in many species. However, the reproductive biology of reptiles presents unique barriers for the use of this technology, and there are currently no reptiles with effective methods for targeted mutagenesis. Here we present a new approach that enables the efficient production of CRISPR-Cas9 induced mutations in Anolis lizards, an important model for studies of reptile evolution and development.
Squamates (lizards and snakes) comprise a diverse group of reptiles represented by over 10,000 recognized species1. However, mechanistic studies of gene function in squamates and other reptiles lag behind other major vertebrate groups. While the adoption of CRISPR-Cas9 mediated gene editing has enabled direct manipulation of gene function in many fish2,3, amphibian4,5, avian6, and mammalian species7,8, there remain no reptilian model systems with established methods for the production of targeted sequence alterations. To date, attempts to manipulate gene function in reptiles have been limited to a small number of studies employing whole embryo culture coupled to viral-or electroporation-based methods to alter gene expression9,10. These methods produce transient, localized, and highly mosaic patterns of transgenesis. Moreover, these techniques have not been used to engineer targeted gene modifications in any reptile species.
Among squamates, Anolis lizards are compelling candidates for the establishment of gene editing methods. Over the past 50 years, anoles have become one of the central model systems for studies of reptile evolution, physiology, and development11. This group has experienced an extensive adaptive radiation in the Caribbean with hundreds of described species that display a wide range of morphological, behavioral, and physiological differences. Studies of the convergent evolution of similar sets of Anolis "ecomorphs", or habitat specialists, on different Caribbean Islands has produced a rich-literature on the biology of Anolis lizards. Although many Anolis species can be successfully raised in the lab, we chose to develop genome editing in the brown anole lizard, Anolis sagrei. This invasive lizard is now found far beyond its native Caribbean range and is ideal for genetic studies due to its small size, ease of husbandry, long breeding season, and relatively short generation time.
CRISPR-Cas9 mediated genome editing is an effective method for producing genetically modified vertebrates12. Typically, CRISPR-Cas9 components are microinjected into vertebrate embryos at the one-cell stage to generate individuals potentially harboring alterations at the locus of interest. However, there are significant challenges associated with microinjection of Anolis zygotes. These challenges include internal fertilization and the long-term storage of sperm within the oviducts of adult females, which makes timing the microinjection of single cell embryos extremely difficult. At the time of ovulation Anolis eggs are also quite large (∼8 mm in length) and are filled with substantial amounts of yolk; these oocytes are fragile and are difficult to manipulate without rupturing. Furthermore, after fertilization the egg shell must be deposited around the egg and embryonic development is initiated before the egg is laid. Finally, unlike the hard shells of birds, the egg shells that enclose Anolis embryos are pliable and no air space is present within the egg, presenting obstacles for embryo manipulation within the egg shell. Most of these reproductive challenges for microinjection are not unique to anoles, but are features that are typical of many reptiles. Here we demonstrate the use of CRISPR-Cas9 to mutate the tyrosinase gene in Anolis sagrei using an approach that is generalizable to other loci. The procedure we have developed involves microinjection of CRISPR-Cas9 components into immature, unfertilized Anolis oocytes within the ovaries of female lizards.
Reproductively active A. sagrei females lay approximately 1 egg every week, similar to Anolis carolinensis13. Each ovary contains a series of approximately 10 maturing ovarian follicles arranged by size, with the smallest follicle closest to the germinal bed and the largest vitellogenic follicle positioned distally (Fig. S1). With the exception of the largest follicle, these follicles are previtellogenic or in the early stages of vitellogenesis and are largely similar in size between the left and right ovaries. In our approach, female lizards are anesthetized and are placed on a surgical platform underneath a standard dissecting scope. Left and right ovaries are separately accessed via vertical incisions positioned along the left or right flank, respectively (Fig. 1). During surgery, the ovary can be gently moved to allow easy observation and injection of the oocytes under a dissecting microscope. Oocytes are microinjected with Cas9 ribonucleoprotein complex (Cas9 RNP) while remaining associated with the ovary (Fig. 1, Fig. S1, and Video S1). In our hands, oocytes that are 0.75 to 5 mm in diameter yield the highest frequency of mutant animals (Fig. S2). A typical anole ovary has 4 to 6 oocytes in this range. Therefore, approximately 10 oocytes in this size range can be injected per animal. Oocytes equal to or larger than 6 mm in diameter are not injected due to the increased risk of rupturing these large, yolk-filled oocytes. After microinjection of oocytes is completed on one side, the incision is sealed with veterinary glue. The procedure is then repeated on the opposite side. We routinely inject 5 females per day for a total of about 50 oocytes per injection session.
Gene editing in lizards through microinjection of ovarian follicles. Flow diagram detailing CRISPR design, surgical procedure, collection periods, and screening strategy. CRISPR design shows the placement and sequence of CRISPR guides A (blue), B (pink), and C (cyan) within exon 2 of the tyr gene; PAM sites are underlined. The surgical procedure panel depicts lizard anesthesia and surgical steps to access and microinject ovary follicles. The collection periods panel highlights the time between gathering eggs and raising hatchlings. The screening strategy panel illustrates the steps used to detect tyr-crispants including: 1) PCR primer design, 2) PAGE analysis, which can reliably detect 2-3 bp changes, and 3) Sanger sequencing.
Following recovery, the microinjected oocytes continue to mature within the female and are eventually ovulated and fertilized through natural mating with an introduced male or via stored sperm from previous matings. The ovaries have an ovulatory cycle that ranges from two to four weeks, but the left and right ovaries are out of phase relative to each other, which results in eggs laid in alternating order from follicles on the left and right ovaries14. Because of the alternating follicle contributions from the left and right ovaries, a putative "follicle train" can be established by ranking follicles based on size and by designating ovarian turns. By ordering follicles in this manner, one can infer the range of follicle sizes which yielded crispant lizards. For instance, follicles in the 3 to 5 mm range develop into eggs that are laid within three to four weeks post injection (Fig. S2).
To assess the effectiveness of our approach, we targeted the second exon of the tyrosinase (tyr) gene. Tyrosinase was chosen for this study because loss-of-function mutations are viable in a wide-range of vertebrates, the resulting pigmentation phenotypes are readily detected, and this allowed us to develop a new Anolis model to investigate human albinism. Cas9 protein coupled to a mixture of three different synthetic tyr guide RNAs was injected into immature oocytes. The decision to simultaneously inject three guide RNAs was motivated by the presence of a number of single-nucleotide polymorphisms (SNPs) in tyr exon 2 within the population of lizards used for these experiments. For these experiments, a total of 146 oocytes from 21 reproductively active females were microinjected over the course of eight surgical sessions.
We obtained nine F0 animals harboring mutations in tyr exon 2. Four of these animals were phenotypically albino and harbored loss-of-function mutations at both tyr alleles. Five animals carried heterozygous loss-of-function mutations and exhibited normal pigmentation. Mutant alleles could be visualized by PAGE after PCR amplification across tyr exon 2 using genomic DNA prepared from F0 embryos or hatchlings as a template. The changes associated with each of the CRISPR-Cas9 induced mutations was determined by sequencing the amplicons from each animal and comparing the sequence to that of wild-type lizards in the colony. The overall mutation frequency in terms of mutant lizards per follicle injected was 6.2%. A mutation frequency of 9.7% was obtained from microinjected follicles that were 1.5 to 2.5 mm in diameter, while follicles 0.75 to 1.0 mm and 3 to 5 mm in diameter yielding frequencies of 9.3% and 5.6%, respectively. No mutations were obtained from microinjection into follicles smaller than 0.5 mm in diameter. Consistent with results in other vertebrates, CRISPR-Cas9 genome editing in lizards resulted in indels that typically ranged in size from 3 to 17 base pairs.
Our discovery that microinjection of genome editing reagents into oocytes generates F0 lizards carrying bi-allelic mutations in the targeted gene, demonstrates that our approach offers an efficient path for directly testing gene requirement in anoles. For genes where loss of function mutations are homozygous lethal, or studies requiring heterozygous animals, we expect that naturally occurring SNPs can be used to facilitate targeting of one allele only.
The establishment of CRISPR-Cas9 editing in this inexpensive reptilian system will finally permit mechanistic studies of gene function to be performed in reptiles. We anticipate the gene-editing strategy we have developed in anoles can also be successfully applied to many other squamate species.
A.M.R., J.D.L., and D.B.M conceived and designed the experiments. A.M.R., performed the majority of the experiments with support from S.P., A.J.A. and R.E.B. Data interpretation was carried by A.M.R., S.P., R.E.B., J.D.L., and D.B.M. The manuscript was written by A.M.R., D.B.M., and J.D.L. with help from all other authors.
The authors declare no competing interests.
Figure S1.
Anole lizard ovary. (A) Dissected ovary showing previtellogenic (PF) and vitellogenic (VF) follicles. (B) Same ovary prior to dissection showing microinjection of a 1.5 mm diameter follicle (asterisk in panels A and B).
Inferred relationship between follicle size and crispants. (Top) the distribution of follicles sizes within ovaries at time of injection depicted as a "follicle train." (Below) a distribution graph showing inferred follicle sizes over weeks post-surgery. Non-injected eggs and large yolky follicles are shown in grey, while injected follicles are in black and follicles that likely produced crispant lizards are in cyan.
Animals used in this study were wild-caught Anolis sagrei from Orlando, FL. Lizards were housed at University of Georgia following published guidelines15. Breeding cages housed up to 4 females and 1 male together. Twenty-one adult females from cages that consistently produced eggs were selected for this study. All experiments followed the National Research Council's Guide for the Care and Use of Laboratory Animals and were performed with the approval and oversight of the University of Georgia Institutional Animal Care and Use Committee (A2016 09-008-Y2-A3).
Selection of crRNA guide sequences and preparation of Cas9 RNP
The CRISPOR target selection tool (version 4.4) was used to select target regions with efficiency scores of 50% or greater within the second exon of the A. sagrei tyr gene16. The tyr gene reference sequence was obtained from a draft genome assembly of Anolis sagrei. Alt-R CRISPR-Cas9 crRNAs, tracrRNA, and Cas9 V3 were purchased from Integrated DNA Technologies, Inc. The crRNA target sequences were as follows:
AsagTyrEx2A: 5′-TTGCCGGGGTTTCGAAGAAT-3′
AsagTyrEx2B: 5′-ATGATAAAGGGAGGACACCT-3′
AsagTyrEx2C: 5′-GAAGTTAGCCATTTTGTCCA-3′.
Cas9 RNP was prepared by following manufacturer recommendations. A 5 µM injection solution was made using standard microinjection solution (10 mM Tris-HCl, pH 7.4) containing phenol red to help verify that injected solutions entered the oocytes.
Anesthesia and analgesia
Lizards were anesthetized by administering 30 mg/kg of Alfaxalone (Alfaxan, 10 mg/mL, Jurox) in combination with 0.1 mg/kg of dexmedetomidine (Dexdomitor 5 mg/10 mL, Zoetis/Orion). To ensure accurate dosing, these drugs were administered by subcutaneous injection in the cervical area as an Alfaxalone/Dexmedetomidine (A/D) mixture. Preoperative analgesia was obtained by subcutaneous injection of meloxicam (0.3 mg/kg, Loxiject, 5 mg/mL, Henry Schein) in the dorsal epaxial area just above the shoulder and topical application of lidocaine (2.0 mg/kg, Lidocaine HCL, 2%, Hospira) over the surgical site. See video for injection sites. The anesthetic combination A/D provided approximately 30 min of surgical anesthesia time. Lizards typically recovered about 40-45 min following A/D administration. If a longer anesthesia time was required, a second dose of 30 mg/kg of alfaxalone alone was administered 25-30 min post induction dose, providing an additional 30 min of surgical anesthesia.
The method and location where injections were made were specifically chosen to avoid some of the challenges with administering drugs to reptiles. One such issue to be aware of is the hepatic-first pass effect which is a phenomenon found in many reptiles where, drugs, if administered in hindlimb or caudal regions, are rapidly cleared by the ventral abdominal and hepatic portal veins and metabolized by the liver, inhibiting wide systemic circulation. We have found that administering A/D subcutaneously in the dorsal epaxial area just above the shoulder region resulted in only moderate to light levels of anesthesia. Contrary to this, lizards administered subcutaneously in cervical area are rapidly induced and reach a surgical plane of anesthesia within 1 min. Because injection volumes can be large, a subcutaneous method was also preferred over an intramuscular approach. For these reasons, the cervical area and a subcutaneous route were used in this study.
Another important factor that can influence drug metabolism in reptiles is body temperature. Lizards are ectothermic and are dependent upon the environmental temps to regulate their body warmth which in turn impacts their metabolic rate. A decrease in body temperature will lead to a decrease in drug metabolism potentially resulting in a persistence of circulating drugs. In such an instance, this can result in an animal responding poorly, prolongment of anesthesia recovery time, or in some cases lead to death. The converse is also true. Animals maintained at too high a temperature may metabolize and clear drugs quickly resulting in insufficient anesthesia time. To avoid this, anesthetized anoles should be maintained on a heating source at around 32°C until fully recovered.
Surgery and microinjection
After successful anesthesia induction, the lizard becomes non-responsive to any noxious stimuli (i.e., an absence of response to a cloacal/tail clamp that normally induces severe discomfort). The anesthetized lizard was placed into right lateral recumbency and the left flank was aseptically prepared by alternating disinfection with 70% ethanol and 7.5% povidone-iodine (Surgical Scrub Solution, 16 fl. oz. 473 mL, Dynarex) wipes for 5 minutes.
Following standard surgical practices, sterile iris scissors (FST, item 15023-10) were used to make an 8-10 mm vertical cutaneous incision on the left side, in the mid-coelom region. A second incision between the ribs was made through the musculature (i.e., internal/external intercostal and pigmented coelom muscle layers) to enter the coelom. The ovary can be found dorsally in the mid-coelom region and was easily accessible by shifting intestines gently aside using blunt forceps (FST: 45° angled forceps, item 00649-11; FST: strait forceps, item 00632-11). Once located, the ovary was carefully rotated and repositioned to expose immature follicles ranging anywhere from 0.25 mm to 5 mm in size.
Using the blunt forceps to clasp and hold the ovary in place, a microinjection needle was visually guided into the follicle center at an angle between 35-45° degrees relative to horizontal. 5 µM Cas9 RNP solution was then injected into follicles at differing volumes ranging from as little as 15 nL to as much as 575 nL which was dependent upon needle and follicle size. Retrospectively, ideal injection volumes were determined (3≤5 mm, 300-500 nL; 2≤2.5 mm, 200-250 nL; 1≤1.5 mm, 100-150 nL; and 0.75 mm, 25 nL) based on surgical sessions that produced mutants. Large yolky follicles greater than 5 mm in diameter, and eggs already present in the oviduct, were not injected. Sterile drops of P-Lytes solution (Veterinary Plasma-Lyte A Injection pH 7.4) were applied directly on the ovary or in coeliotomy opening throughout the procedure to prevent tissue dehydration.
After injection, the ovary was gently returned into the coelom and overlying musculature and skin was lightly pulled together to close the cavity. Tissue adhesive (3M Vetbond, #1469SB) was carefully applied only to only the external surface of the skin, avoiding the underlying musculature. Once the tissue adhesive was dry, the lizard was re-positioned into left lateral recumbency and the procedure was repeated for a right coeliotomy.
During recovery, triple antibiotic ointment (Bacitracin Zinc, Neomycin Sulfate, Polymyxin B Sulfate) was applied topically to the surgical wounds. Lizards were monitored daily for 1 week for any signs of infection, pain, or inflammation. After recovery from anesthesia, females were housed together with their previous female mates and allowed to recover for 7 days prior to reintroducing the male.
All surgeries were performed using sterilized equipment and tools (i.e. forceps and iris scissors) under a dissecting scope (Zeiss Stemi SV11) with a top light (AmScope 80-LED illuminator). Body temperature was maintained throughout the procedure by placing lizards on a heating platform (Fisher Scientific: model 77, serial # 802N0041CAT 12-594) with surgical towels draped between the heat source and the lizards to provide a barrier. The contact surface temperature was held at 32°C and readings continuously taken using thermometer strips. Each laparotomy was performed within 10-12 minutes. Follicle injections were carried out using a standard zebrafish/xenopus microinjection rig (Harvard Apparatus PLI-100 Pico-Injector) set at 20 PSI with an injection time of 50-60 msecs. Initially, a manual micromanipulator (Märzhäuser Wetzlar; MMJ-rechts: 00-42-107-0000) was used to perform steady needle injections. However, use of this limited the degrees of freedom to inject the ovary from multiple directions and angles, therefore, a simple hand-guided technique using no micromanipulator was ultimately preferred and proved to be more efficient. Injection needles with a gradual taper typically used for zebrafish microinjection were made following the Sutter Instrument Company Pipette Cookbook guidelines using a Flaming/Brown Micropipette Puller (Model P-97) and cut to have a 20 to 40 µm diameter opening.
Cages were monitored for a specified number of weeks following surgery which was based on the highest number of follicles injected in an ovary (e.g., if 8 and 5 follicles were injected in the right and left ovaries of one lizard, respectively, a cage housing this lizard would be monitored for n=16 weeks). Because these females often had 1 or 2 eggs in the oviduct as well as 2 large (>5mm) un-injected large follicles, cages were monitored for an additional 3-4 weeks following surgery.
Embryos and hatchlings from surgery cages were screened via PCR PAGE analysis under conditions that reliably detect a 2-3bp change17. DNA was extracted from tail clips from hatchlings or from tissue collected from embryos following standard protocols. PCR was performed using the following primers: P1, 5-CAAGAACTTTGCAATGGAACAAATG-3'; P2, 5'-GAATTCAACGTCTGCTGAAGATG-3'; P3, 5'-TGTTTAAGTCTGACTCAGTACGAAG-3'; P4, 5'-GGATTACCTTCCAAAGTATTCCTG-3'. See Figure 2 for primer location relative to targeting sites. Sanger sequencing was performed on PCR products to fully characterize mutations.
Detection and sequencing of tyr-crispants. (A) albino tyr-crispant (left) and wildtype (right) aged-matched hatchlings. (B) PCR primer placement (P1-P4) relative to CRISPR target sites A (blue), B (pink), and C (cyan). Representative PAGE results are shown for seven of the mutant lizards. Colored arrows denote bands with altered mobility relative to wild-type (WT). (C) Sequences of CRISPR-Cas9 induced indels from representative tyr-crispants. (Top) Mut1 and Mut4-7 sequences with deletions. (Below) Mut2 and 3 sequences with insertions. Targeted guide sites A (blue), B (pink), and C (cyan) are highlighted in wildtype reference sequences. tyr-crispants deletions/insertions are indicated in black and sequences matching wildtype in grey.
Follicle train assignment
Follicles were ranked by size and by alternating left and right ovarian contributions to infer a probable timeline of egg lay for a given follicle size at time of injection. This method of follicle train assignment assumes 1) eggs present in the oviduct will be laid within one week, 2) follicles greater than 8-10 mm in 2 weeks, and 3) follicles less than or equal to 5mm in diameter will be laid no sooner than 3 weeks. Our reason for including these 3 underlying assumptions derives from the observation that females who had 1-2 eggs present in their oviduct, also had a follicle greater than 8-10 mm in diameter followed by a follicle between 3-5 mm in each ovary, suggesting at least a week interval between these sizes. As each lizard possesses a "leading" ovary and "lagging" ovary in follicle sizes, the leading ovary is given preferential ordering in train position. It is important to note that this method of ordering does not account for any potential loss of follicles accidently destroyed in the microinjection process and assumes that if such an event occurred, the follicle developmental timeline of that ovary is unaffected.
The data that support the findings of this study are available from the corresponding author upon request.
We thank S. Divers for his guidance on reptile anesthesia and surgical techniques and J. Eggenschwiler and C. Sabin for comments on this manuscript. This work was funded by National Science Foundation awards #1149453 to D.B.M. and #1827647 to D.B.M. and J.D.L., and a Society for Developmental Biology Emerging models grant to A.M.R. A.M.R. was supported by NIH training grant T32GM007103 and by an ARCS Foundation Scholarship.
Uetz, P. & Stylianou, A. The original descriptions of reptiles and their subspecies. Zootaxa 4375, 257–258 (2018).
Hwang, W. Y. et al. Brief communications. 31, 227–229 (2013).
Ansai, S. & Kinoshita, M. Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open. 3, 362–371. (2014).
Flowers, G. P., Timberlake, A. T., Mclean, K. C., Monaghan, J. R. & Crews, C. M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development 141, 2165–2171 (2014).
Blitz, I. L., Biesinger, J., Xie, X. & Cho, K. W. Y. Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system. Genesis 51, 827–834 (2013).
Oishi, I., Yoshii, K., Miyahara, D., Kagami, H. & Tagami, T. Targeted mutagenesis in chicken using CRISPR/Cas9 system. Sci. Rep. 6, 23980 (2016).
Wang, H. et al. One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Cell 153, 910–918 (2013).
Honda, A. et al. Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9. Exp. Anim. 64, 31–37 (2015).
Nomura, T., Yamashita, W., Gotoh, H. & Ono, K. Genetic manipulation of reptilian embryos: toward an understanding of cortical development and evolution. Front Neurosci 9, 45 (2015).
Tschopp, P. et al. A relative shift in cloacal location repositions external genitalia in amniote evolution. 516, 391–394 (2014)
Sanger, T. J. & Kircher, B. K. Model Clades Versus Model Species: Anolis Lizards as an Integrative Model of Anatomical Evolution. Methods Mol Biol 1650, 285–297 (2017).
Komor, A. C., Badran, A.H. & Liu, D.R. CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes. Cell 168, 20–36 (2017).
Andrews, R.M. Oviposition Frequency of Anolis carolinensis. Copeia 1985, 259 (1985).
Smith, H. M., Sinelnik, G., Fawcett, J. D. & Jones, R. E. A Survey of the Chronology of Ovulation in Anoline Lizard Genera. Trans Kansas Acad Sci (1903-) 75, 107–120 (1972).
Sanger, T. J., Hime, P. M., Johnson, M. A., Diani, J. & Losos, J. B. Laboratory protocols for husbandry and embryo collection of Anolis lizards. Herp Review 39, 58–63 (2008).
Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol 17, 148 (2016).
VanLeuven, A. J., Park, S., Menke, D. B. & Lauderdale, J. D. A PAGE screening approach for identifying CRISPR-Cas9-induced mutations in zebrafish. BioTechniques 64, 275–278 (2018).
You are going to email the following CRISPR-Cas9 Gene Editing in Lizards Through Microinjection of Unfertilized Oocytes
Ashley M. Rasys, Sungdae Park, Rebecca E. Ball, Aaron J. Alcala, James D. Lauderdale, Douglas B. Menke