We're calling for campus and city-wide bans on single use plastic products, including bags, straws and take-out foam cups and containers.
At Rutgers University, students spend an average of $1,500 a year on textbooks and course materials alone. Publishers are constantly implementing new methods to jack up prices, from using expensive online passcodes to creating costly special editions that make purchasing of used books a hassle. These tactics leave students trapped, with textbook prices rising four times as fast as the rate of inflation.
A new approach is needed to create a long-lasting difference. Luckily, NJPIRG has won real change with our Open Textbooks campaign, which focuses on the cost-saving power of open source textbooks which are published under an open license, making them available at low or no cost! In February 2016, the President of Rutgers University agreed to fund a $12,000 grant program to encourage faculty to adopt open textbooks in their classes.
Since its start, the program has already saved Rutgers students $2.1 million. Our mission is to continue and expand this program so that passing a class isn't decided by your bank account.
We know we can have healthier communities and a livable future for generations to come. But to get there, we must transform the way we produce and consume energy. That's why we're calling for a commitment to 100 percent renewable power and expanding access to clean energy for all. It's a big, bold goal – and it's a goal that's 100 percent possible.
We're working to get Rutgers to commit to 100% renewable energy by 2050, with 100% of electricity coming from renewable sources by 2030. In Fall 2018, we successfully had a charge passed through the University Senate Executive Committee to do just that– as well as create a Rutgers Office of Sustainability.
We're continuing to build support from faculty and students to send a clear message that the Scarlet Knight community is 100% committed to 100% renewables.
As we prepare for 2020, we're working with our campus communities to make it easier and more accessible for students to vote and we're working with campus administrators to make voting and civic engagement more institutionalized in residence halls, class registration, and other aspects of student life.
It's our future. Let's vote on it.
We rely on bees to pollinate 71 of the 100 crops that provide 90% of most of the world's food.
We're working to get campuses and cities to ban the sale of bee-killing pesticides.
Learn how to make your campus bee-friendly!
No one should have to worry about whether they will have food on their plate or a roof over their head. But the reality is that hunger and homelessness are widespread problems that affect far too many people.
Poverty isn't exclusive to any one community. Even college campuses are not immune. Our latest research suggests that one in five students currently experience hunger and 55% of food insecure students choose between buying food and textbooks.
We have the resources and knowledge to eliminate hunger and homelessness in our time, but it remains to be seen whether we have the political will to do so. Fortunately, on campuses across the country, there are students like us who are dedicated to making their communities a better place, and improving the lives and well-being of those around them. Our Hunger Campaign takes this passion and dedication and harnesses it through a combination of direct service, fundraising events, and political advocacy.


Davis Polk brings unique experience to high-profile matters involving transactional and international trade issues faced by companies engaged or investing in international business.
As U.S. national and economic security concerns increase, they affect a wider variety of our clients' commercial, corporate and investment transactions, including exports, funds transfers, securities issuance and underwriting, international trade, insurance, and inbound and outbound direct-investment transactions. We are leaders in helping clients with: reviews by the Committee on Foreign Investment in the United States ("CFIUS") of deals affecting U.S. national security or the protection of critical infrastructure; and compliance of U.S. economic sanctions and anti-money laundering laws and regulations, including the Bank Secrecy Act and USA PATRIOT Act, the Foreign Corrupt Practices Act ("FCPA") and the Exon-Florio statute (as amended by the Foreign Investment and National Security Act of 2007).
Our lawyers have served in senior positions in the White House, the Departments of Justice and Treasury, including the Office of Foreign Assets Control ("OFAC") and the Financial Crimes Enforcement Network ("FinCEN"), the FBI and CIA, and the U.S. Securities and Exchange Commission, as well as in trade-related positions at the State Department.
We are well-known for representing global financial institutions and corporations in high-stakes OFAC sanctions investigations and compliance advisory matters, as well as in parallel civil and criminal enforcement proceedings.
We counsel clients on the intricacies of Russian, Ukrainian and Syrian sanctions, on the rapidly developing openness to Cuba and on emerging frictions between the U.S. and EU over implementation of the Iran nuclear energy agreement.
We have extensive experience advising clients – including sovereign wealth funds, state-owned enterprises, hedge funds, private equity funds and multigovernment-owned entities – on national security issues in connection with proposed U.S. acquisitions or investments.
A month-by-month recap of the U.S. Department of Justice's criminal enforcement and policy developments and other significant issues.
FinReg is a Davis Polk blog that offers updates and insights on financial regulation topics.


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Maryland Department of Health creates GoVAX 'Real Kids' campaign to encourage parents to vaccinate children 5 to 11 years old
Children describe in their own words why it's important to be vaccinated
Baltimore, MD – The Maryland Department of Health (MDH) has created a new series of television, radio and social media ads featuring "Real Kids" — children aged 5 to 11 explaining why they got vaccinated against COVID 19 — as part of its statewide GoVAX campaign. Interviews with the children were conducted over the past few weeks at vaccination clinics in Baltimore City and Howard County and at a private pediatrics practice in Columbia.
"We know some parents are taking a 'wait and see' approach, even though they intend to get their children vaccinated eventually. We urge these parents not to wait and to get their children vaccinated as soon as possible. The vaccines are safe and effective for children and continue to be our best defense against serious illness, hospitalization and death," said MDH Secretary Dennis R. Schrader.
Children aged 5 to 11 were authorized by the U.S. Food and Drug Administration (FDA) to receive the Pfizer COVID vaccine on October 29, 2021. To date, 40 percent of Maryland children in this age group have received at least one dose of the two-dose regimen.
"It's important to stress to all parents and guardians that vaccines help protect their children against serious illness," said MDH Deputy Secretary of Public Health Dr. Jinlene Chan. "We've seen an uptick in outbreaks in schools recently, and we know how important it is to keep children in school for in-person learning. The more children who get vaccinated, the better our chances of keeping kids in the classroom and allowing them to participate in the activities they enjoy."
Children appearing in the "Real Kids" videos give various reasons for wanting to get vaccinated, ranging from returning to soccer practice and having sleepovers with friends to wanting to visit grandparents or taking a cruise with their parents. Nearly all understood the importance of being protected against the disease and described being vaccinated as "getting a little pinch."
Seven-year-old Roslyn from Baltimore said she wanted to get vaccinated to help protect her little sister, who is too young to get the vaccine herself. "It was easier than I thought it was going to be," Roslyn said. "I was a little more nervous than scared."
"Since I have the vaccine, I believe my parents will worry less," said Chandler, 10, of Columbia, who also reported being happy and relieved to be vaccinated.
The state's multipronged GoVAX campaign, first launched a year ago, encourages all Marylanders to protect themselves, their families and their communities by getting vaccinated as soon as they become eligible.
Since its debut, the campaign has featured TV, radio, outdoor and digital advertising, social media, grassroots outreach through door-to-door canvassing, virtual town halls, the use of sound trucks in targeted neighborhoods and community-based messaging through doctors' offices, barbershops and salons.
The campaign has incorporated testimonials from dozens of "trusted leaders" throughout the state as GoVAX Ambassadors, including physicians, first responders, faith and community leaders, sports figures, research scientists, health officers, elected officials and others.
"As a pediatrician and a parent, I think about all the routine vaccines we give our kids. We know shots are effective and they keep kids safe," said Dr. Letitia Dzirasa, Health Commissioner for Baltimore City and GoVAX Ambassador. "Protecting this population is critical to keeping them in school. They've missed so much having to be virtual, so we want to make sure they are able to stay in school and learn safely."
"We've been doing pretty well vaccinating 5- to 11-year-olds so far, but we have to keep pushing. We also need to continue pushing vaccination with 12- to 17-year-olds," said Dr. Monique Soileau-Burke, a pediatrician in Columbia, Vice President of the Maryland Chapter of the American Academy of Pediatrics and GoVAX Ambassador. "And for the college kids, well, I'm going to talk directly to them — Get your shot and do your part."
To date, Maryland has administered almost 11 million doses of the COVID-19 vaccine, with more than 4.9 million Marylanders receiving at least one dose.
For more information about COVID-19 vaccines and the GoVAX campaign, visit covidLINK.maryland.gov.
For the most recent Maryland COVID-19 vaccine data, visit coronavirus.maryland.gov.
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redpajama_set_name: RedPajamaCommonCrawl


AN AUSTRALIAN energy company says it is financially worse-off because of its decision to lock in a long-term deal to sell gas to Santos to ship internationally from Curtis Island.
AGL said in a blog this week it would profit more from selling gas to Australian customers, instead of Santos' $17.5 billion GLNG venture.
It's because AGL, like most in the industry, did not anticipate the extent of the gas crisis and partly blamed its poor deal-making decision on the closure of Hazelwood Power Station.
The comments were in response to a Daily Telegraph news article that AGL said implied the company was making an ongoing decision to hand the reserves to GLNG instead of the domestic market.
According to its first quarter report for 2017, 58% of Santos' international exports are sourced from third party gas, like AGL's.
"The fact is, AGL would profit more from not selling to GLNG but making the gas available to the Australian market," AGL's Richard Clifton-Smith said.
The three Curtis Island-based projects, known as the "big three", have come under fire for filling six LNG trains with international exports while Australian customers seemingly cry dry.
AGL's comments come off the back of the kingpins of each Curtis Island site making good with Prime Minister Malcolm Turnbull about their commitment to the domestic gas market.
The gas exporting trio signed a deal with the Federal Government confirming their promise to supply 54 petajoules of gas, and more if needed, to shake off the predicted shortage.
On top of the tight domestic market, Mr Clifton-Smith said the big change was caused by the closure of Hazelwood Power Station in Victoria.
"...Which significantly increased the demand for gas power generation," Mr Clifton-Smith said.
He said if AGL had known of the 1700 MW power station's closure the GLNG deal may not have been made in 2015.
"Clearly if AGL had not sold the gas to GLNG, we would have been in a better position to service our customers," Mr Clifton-Smith said.
"At the beginning of 2017, AGL was in a position where it no longer had adequate gas available to supply to its existing customers coming off contract."
Mr Clifton-Smith said since Mr Turnbull's warning of a gas export cap or ban, new short term contracts have been made available.
Meanwhile AGL, which is copping pressure to keep its coal-fired power station Liddell open, plans to build a $250 million LNG import project to help supply the Victorian market.
Shell chairman Zoe Yujnovich shed light on the gas export industry yesterday, telling a crowd at an energy summit, Australia can not have a thriving export industry without a well-functioning east coast domestic gas market.
"Blaming the LNG industry alone for recent issues with the supply and price of gas is in itself a selective intentionally misleading reading of the gas market," she said.


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Expunging or sealing records Article
Setting Aside Convictions and Restoring Civil Rights
After you have been convicted of a crime and completed your sentence (jail, prison, probation, fine and fees), you may be eligible for a Set Aside. The article provides information about setting aside both felony and misdemeanor convictions, restoring civil rights and restoring gun rights, based on Arizona law.
Each case is unique and we cannot guarantee that a Set Aside will be granted. Success in receiving a Set Aside depends on the laws at the time of conviction and the assigned judge's discretion. Since the outcome of a case depends on many different things, this is intended for educational purposes and is not legal advice. An attorney can provide additional information and legal advice. Therefore, questions about an individual situation should be discussed with an attorney.
What is "set aside"?
A Set Aside does not change the fact that you have a criminal record but it does show that you competed your sentence and probation, paid your fines and fees and have no pending charges.
A Set Aside is not the same as an expungement: Arizona expungement laws only apply to marijuana related offenses. You interactions with the court system can be found on-line. Anyone searching your history will still see the conviction but also the designation of Set Aside.
If I committed a crime in California, can I get it Set Aside in Arizona?
No. Each individual state decides what expungement and/or set aside means in its state. You must address the court in the state you committed the crime in. You can contact the court or an attorney in that state for more information. This article only addresses Arizona law.
How will employers and landlords view Set Asides?
Policies may vary, but some employers view Set Asides very positively and request that an applicant receive a Set Aside. Others have less flexibility and will only see the conviction. The same is true for landlords.
Expungement does not wipe out the conviction. The record of conviction is not destroyed. A conviction may be used to deny certain kinds of employment, licenses, permits, certificates as well as used against a person in future criminal cases, even though the conviction was set aside.
In Arizona, after a conviction is set aside, does a person have to disclose the conviction on a job application or in a job interview?
Yes, while it is your choice with internet background checks, the record of the conviction remains. There are state and local government agencies and some private employers who "Ban the Box" and only ask about criminal convictions at the in-person interview. This allows the applicant to explain any interaction with the criminal court system.
Where are Arizona's laws about setting aside a conviction?
The laws about setting aside a conviction are presently found in. A.R.S. § 13-907 which permits a person convicted of a felony or misdemeanor to request a "set aside" of the conviction under certain circumstances.
Who might be able to set aside a conviction in Arizona?
A set aside may be available if a person was convicted y in an Arizona Court and that person has successfully prison or jail time, probation and paid fines and fees.
A set aside is not available to a person convicted of a criminal offense:
Involving the infliction of serious physical injury,
Involving the use or exhibition of a deadly weapon or dangerous instrument,
For which the person is required or ordered by the court to register as a sex offender, A.R.S. § 13-3821,
For which there has been a finding of sexual motivation under A.R.S. § 13-118
In which the victim is a minor under 15 years of age, or
In violation of A.R.S. § 28-3473, any local ordinance relating to stopping, standing or operation of a vehicle or title 28, chapter 3, except a violation of §28-693 or any local ordinance relating to the same subject matter as A.R.S. § 28-693.
What happens if the Court grants a person's request to set aside a conviction?
If the Court grants the application to set aside a conviction, the Court will set aside the judgment of guilt. Then the Court will order the person's release from all penalties and disabilities resulting from the conviction, except:
The penalties and disabilities imposed by the department of transportation under A.R.S. §§ 28-3304, 28-3306, 28-3307, 28-3308 or 28-3319, except that the conviction may be used if the conviction would be admissible had it not been set aside;
The set aside conviction may be pleaded and proved in any subsequent prosecution of that person for any offense or
It may be used by the department of transportation to enforce A.R.S. §§ 28-3304, 28-3306, 28-3307, 28-3308 or 28-3319 as if it had not been set aside.
When might a person be eligible to get their civil rights restored after a felony conviction?
A felony conviction results in the suspension of some civil rights which may include the right to vote, hold public office, serve on a jury, and possess firearms. Usually, these civil rights are not suspended for misdemeanor convictions so this process may not apply. Generally, if you've completed probation or finished your prison term, your civil rights will be restored automatically. If you have more than one felony offense, then you should apply for your civil rights back. You can apply for your civil rights for cases that you cannot get Set Aside.
The restoration of civil rights may be available to felony offenders convicted in a County Superior Court in Arizona or in federal court, Arizona Courts do not have the authority to set aside convictions or restore guns rights to persons convicted in federal court.
Will a person's gun rights be restored as part of restoring civil rights?
Whether a person's gun rights are restored may depend upon the date of conviction, any subsequent convictions and the judge's discretion.
Requests to restore civil rights and restore gun rights are separate orders, but the requests can be submitted to the Court at same time as the request to set aside the conviction.
How does a person apply to have a conviction set aside, civil rights restored and gun rights restored?
To restore these rights, a person would file a request with the Superior Court in the county where the conviction occurred. For a federal conviction, a person would submit an application to the Superior Court in the county where that person lives. Instructions for setting aside a conviction, restoring civil rights, and restoring gun rights can be found at the following websites:
Coconino County: update
Maricopa County: update links
If the county in which the person was convicted does not have these forms online, contact the Clerk's Office in the county where the conviction occurred to see which forms could be used. Contact information for County Superior Court Clerks can be found at update
What happens after a person submits the application to set aside a conviction, to restore civil rights and to restore gun rights?
The Court will review the application and may grant the request, deny the request, set a hearing in some counties or set orders the Court believes is appropriate in the person's case. For questions about the hearing, call the Court that set the hearing or contact an attorney.
What can a person do if the Court denies the request?
If the Court denies the request, a person can file a request for reconsideration, depending upon the reason for the denial. Instructions and forms for requesting a reconsideration can be found on the Courts' websites. A person planning to request a reconsideration may want to discuss the request with an attorney before filing it.
On 8/16/10
AZLAwHelp.org said
Coconino County Superior Court serves individuals in Flagstaff.
Bob said
Where is the form for Flagstaff Justice court? Or, do I simply use the Maricopa Superior court form?
Law Librarian said
The Coconino County web address has changed to http://www.coconino.az.gov/lawlibrary.aspx?id=19434
NM has a bill in progress now concerning this very topic. I hope it makes it way through the senate and house. I am 47 now, but still am considered a danger, I guess, even though I have no post record since 1990.
On 12/27/08
Sarah said
Why isn't there any information about how many years court records are available to the public.
If I was arrested for domestic violence, but my case was dismissed. Can the case be expunging?
If when signing the plea the judge states "I will allow the felony to be set aside." After completing probation I was denied. The felony is Agg. Assault and I know that violent crimes are not eligible to be set aside. But I feel there should be accountability. What are the options?
I was convicted of Theft, A class 6 undesignated offense back in the mid 90's. I completed 3 years on probation. My probation officer put in a order of discharge from probation in the late 90's . He signed it and the Judge signed it. It was never checked on if it was a misdemeanor or a felony. I never recieved and paperwork that stated anything about it, just that I was released from probation. So what do I need to do to get this removed? If the paperwork was not completed correctly my the courts can I get it off my record? Thanks
convictions were in the 1970's how do I go about getting my civil rights back?
How would I have a set aside convication sealed from public access? Currently, the set aside convication is listed on the Pima County Justice Court website? Thank you.
i had two class 6 undesignated possession of narcotic/paraphanelia charges in 2000 I completed my probation and just got my charges designated to a class one misdemeanor can I work in the medical field? I used to be a cna before that, my licence lapsed but was never taken away because of this that's why I am asking.
can i get my covictions set aside in arizona if i now live in WI and do i have to be in AZ to apear in court for them to be set aside
My brother was charged and served 10 years in prison for recieving and selling stolen property in MI. He moved to AZ to find a job 2 months ago and he is looking forward to going back to school to become a paralegal. Will he be able to become a paralegal and he can he become a lawyer? He has no record in AZ nor any other state. Any advice is helpful. Thanks!
Can I use an AZ lawyer to expunge my record on a felony case from 2005 in Missouri?
Is it helpful to get set asides on misdemeanor convictions besides paraphinalia or drug related. Misdemeanor convictions.bcuz on an Arizona website it says it would be less help with other types of misdemeanor s besides drug related. Either way what are my career options with several misdemeanors that are5 to 2 years old? ? :( I don't think I'll be Abe to maintain a level one finger print clearance card a level one card to be exact so I need to know all of my career options for Arizona. Or would I be better off moving out of state. Or is there enough forgiveness in this state for wide options
Gila County Housing Dept.
Scottsdale Granite Reef Senior Center
Arizona Coalition to End Sexual & Domestic Violence
Arizona Secretary Of State
Arizona South Asians For Safe Families (ASAFSF)
Governor Ducey's Second Chance Box
Arizona Courts Locator
Maricopa County Judicial Branch Self-Service Center Online
Pima County Courts Self Service Center
Victim's Rights
Crime Victim Compensation
Expunging or sealing records


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redpajama_set_name: RedPajamaGithub


SHENZHEN SUN GLOBAL GLASS CO., LTD manufacture high quality vinyl backed safety mirror with PE glossy film CAT I and PVC woven fabric film CAT II. This product is widely used as gym mirror, dance room mirror, wall mirror, bathroom mirror, etc. Edge working includes flat edge, round edge, and beveled edge with width 5mm to 60 mm. Other process such as cutting custom shapes, silk screen printing, drilling holes, sandblasting patterns, tempering mirror, could all be well processed by our factory. Custom OEM service in shape (round, oval, square, rectangular, and irregular shapes) and extra large size is also available.


text:
Posts by philipmatthews
Hard News: Friday Music: Sweet Inspiration, 11:45 Aug 19, 2016
PA readers of a certain age might enjoy this story by Charlie Gates in the Press about the time The Fall ("the mighty Fall") played Christchurch in 1982, and somehow found themselves on the front page of the paper. That photo of "happy Fall guitarist" Marc Riley was on the cover of Flying Nun's Fall in a Hole and so on. It's all legendary now. I think there's an entire chapter in the Roger Shepherd book.
The bit I love is the detail from Bruce Russell about some friends spraypainting "bang fucking bang, the mighty Fall are coming" on a billboard in Chch. I wonder if anyone took a pic?
Hard News: About Chris Brown, in reply to Russell Brown, 11:23 Oct 4, 2015
Yeah, it is. And you come up against absolutes very quickly.
I was thinking yesterday that one of the problems with Twitter is that it forces you to take sides when you really want to be in the grey area in the middle.
Hard News: About Chris Brown, in reply to Danielle, 11:15 Oct 3, 2015
I think my larger point, which I ranted about on Twitter earlier, is: we all have to accept that we are soaking in all manner of horribleness like misogyny in ALL pop culture. Because it reflects AND inspires us, it's hard to draw causal relationships.
Thanks Danielle. That is what I wanted to say yesterday when I got into an ill-advised Twitter discussion on this. Twitter is probably too rushed and chaotic a medium for it.
There was a line in Andrea Vance's column about both Chris Brown and the Lorde revenge video, which is that "pop culture defines acceptable social norms". That seems utterly wrong to me. There is a place for violence in entertainment; pop culture is not a set of instructions for everyday behaviour. Sometimes that violence might be fantasy or catharsis.
Hard News: About Campbell Live, 22:54 Apr 9, 2015
I've also been surprised at the number of entirely reasonable people who have said today that would stop watching 3 News if Campbell Live was axed. From the days of Ralston and Nightline on, 3 News always felt like the underdog and I think a lot of loyal people are feeling betrayed.
Serious question: who here regularly watches Campbell Live? And by that, I mean the full episode, live to air. Not occasionally watching a video someone has shared on Facebook.
I do, most nights. Have been a steady TV3 news watcher since the 90s and Campbell has just naturally followed on.
Hard News: Media Take: The Easter Show, in reply to Alan Perrott, 20:06 Apr 8, 2015
there is not even the teeniest tiniest jot of evidence (in a time not exactly short of written records) aside from the self-serving, chosen for purpose, books of the bible, that jesus ever existed.
No, the majority of historians would say that he did.
Hard News: Media Take: The Easter Show, 12:06 Apr 8, 2015
A very interesting show, thanks. As someone who sometimes covers religion as a journalist, I can see the value in what Francis Ritchie is doing with NewsLeads. It's a bewildering area to write about if, like most reporters (I guess), you are generally secular or actively atheist and you're either trying to prop up a routine, seasonal "Christianity is in decline" story or you're writing about the Tamakis or Catholic child abuse. More ordinary religious stories don't get a look in and the churches haven't been very good at communicating beyond their own communities. There is often a defensiveness and wariness, and I don't entirely blame them.
Interesting too to see the Tamakis in a Maori context. I think they have mellowed and what they do is more nuanced and positive than has been depicted by an often negative media. But Brian continues to be a pretty terrible spokesman for the church; Hannah is much clearer and more articulate. It's like watching the Clintons in action.
Hard News: Friday Music: It's good to…, in reply to Hilary Stace, 22:35 Mar 13, 2015
Chris Knox's hair!
Hard News: Friday Music: It's good to…, 14:22 Mar 13, 2015
Songs that make people cry. Well, this one gets me anyway.
Hard News: Friday Music: Love Unknown Orchestra, in reply to Patrick Xavier, 12:09 Feb 13, 2015
Also the thing about Shayne not being especially clear in his enunciation. Until this week, I thought it was "And I quietly count with a gun ..."
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In the quiet of the morning the group we have travelled to Ghana with using a grant from the US Department of State to study extractive industries find ourselves swapping stories – wistfully thinking of American life back home. We find ourselves constantly comparing and contrasting the political environment in which public policy around extractive industries are crafted in both nations. Scratch beneath the surface and you will find that Ghana and the US are not that different after all.
The pernicious influence of special interest money permeates throughout American political culture just as it does in Ghana; compulsory Integration models in the states have allowed for mineral rights to be taken from American citizens, and confusion over the leasing of mineral rights for natural gas extraction has led to uprooted communities. Environmental degradation and costs to local communities have been paramount in both the US and Ghana. These two nations separated by the Atlantic are struggling to balance new extractive industries as an engine for economic growth and protecting communities from the pitfalls associated with the "resource curse." As both nations forge ahead in developing their oil and gas sectors, how they manage the risk associated with natural gas development will ultimately define how the citizenry thinks about the role of government and government institutions.
Below is a short comparison of some key aspects affecting constituencies in the US and Ghana.
During a recent discussion with a local royal chief in a small village I am reminded that corporate influence is not localized to any specific nation. Here, a gold mining company publicly presents the tribal leaders of a village with keys to two Land Rovers. In America, special interest money floods campaign coffers, exceedingly so in the wake of Citizen United. According to Open Secrets individuals and political action committees affiliated with oil and gas companies have donated $238.7 million to candidates since 1990. From 2010-2011 Exxon Mobil and The Koch Brothers, one of the largest oil and gas conglomerates in the US, spent $384,030 and $318,800 respectively on campaign donations on both sides of the partisan divide to influence environmental legislation aimed at regulating the oil and gas sector. In Pennsylvania, Common Cause's www.marcellusmoney.org has tracked the significant campaign cash contributions that have flooded campaign coffers, and in New York our recent report "Deep Drilling, Deep Pockets," highlighted the large amounts industry has spent to lobby our elected officials.
In Ghana only 5% of royalties paid by the extractive industry sector is paid to the State. Out of that 80% of the money goes into the government's general fund, with only 9% trickling down to effected communities. In the US, Congress has historically rewarded energy companies and those involved in the extractive industry sector with tax breaks, without tangible realization of positive benefits to communities. Nearly two-thirds of US corporations don't pay any income taxes. According to a study from the non-partisan Government Accountability Office, 83 of the top 100 publicly traded corporations that operate in the US exploit corporate tax havens. Since 2009, America's most profitable companies, such as ExxonMobil, General Electric, Bank of America and Citigroup, all paid a grand total of $0 in federal income taxes. Even as we write this, Congress is considering offering major subsidies to promote natural gas extraction methods and providing major tax incentives to the industry, speeding up the timeline for extraction and feeding the natural gas boom (and possibly bust) cycle.
At the heart of the debate over natural gas extraction in the US is the right of landowners to either retain their land or sign leases with companies with the hope of negotiating lucrative contracts for their mineral rights. In Ghana, Article 257 of the Constitution states that public lands and public property are "vested in the President on the behalf of, and in trust for, the people of Ghana." In essence, the state has claims to mineral rights, not the individual. On the surface the situation in Ghana appears anathema to American values. Forced resettlement programs of thousands of fisherman, farmers and landowners offends our notion of private property and ownership as inviolate. Yet areas in New York and Pennsylvania have allowed for Compulsory Integration where companies were granted the right to drill on lands for which they did not hold leases. Some residents that may own the surface rights but not the mineral rights experience the effects of a "split estate." Additionally, population displacement often occurs near areas of heavy drilling either because of fear of health effects, noise or pollution, or due to harassment by companies. The important benefits and drawbacks that result from personal ownership of mineral rights must be considered seriously. Further, neither the United States or Ghana require companies to disclose the exact composition of the chemical mixtures used in the process, shrouding it in a cloud of secrecy from the public.
In Ghana, as farmland is turned over to industry to pave the way for rapid development, food productivity has begun decreasing – causing food and commodity prices to rise. Housing prices have been steadily increasing as foreigners flock to the areas surrounding the Jubilee oil field, causing a surge in demand for those residences. Prostitution and crime has been on the rise, as well. Even smoking has increased as foreigners bring with them new social norms. In the US we have seen similar externalities imposed on host communities by the extractive industry sector. In Pennsylvania we have already seen the rise in housing shortages due to workers being brought in from out of state, traffic incidences, and roadway degradation. Air quality concerns, drinking water contamination, and stress-related health effects are being documented. Both nations lack clear and updated standards for hazardous waste removal of drilling fluid or drill cuttings. Each country will have to address new pressures placed on transportation infrastructure, including increasing maintenance costs as new roads are created and old roads need constant repair to handle the increase in heavy truck traffic.
Residential and operational waste – regardless of its country of origin – is a common postcard to receive from the presence of extractive industries. Improperly handled waste contributes to a multitude of public health issues, such as tainted drinking water, disease transmission, air pollution, and threats to the food supply. One of the differences between Ghana and the U.S. lies in the awareness of where our waste goes. Americans are physically separated from the sources and end products of our distracted commercial lives. Trash is collected by a contractor and taken to dump sites, incinerators, or overseas. Ghanaians face their (and others') waste on the country's busy sidewalks, in open sewers, and floating in their magnificent waterways. They witness the neocolonial exploitation of their local resources for the imbalanced consumption and financial gain of other countries. While our processes for extraction and waste disposal differ somewhat, we share a common problem – how to reduce our demand on the entire cycle. Many of the earth's resources are finite and severely threatened, and so sustainability must be the prescription for healthy development.
This list is not exhaustive, nor it is it meant to be. To move toward a best-practices model for developing extractive industry sectors and managing the high risks associated with doing so means paying close attention to the pace and scope of development, as well as attempting to ameliorate negative externalities imposed on communities. This must include mechanisms for proper oversight and regulation, sustainable planning and development, enhanced civic societal input at the decision- making table, and realistic expectations about the financial promises of oil and gas. In nations such as Ghana, managing these revenues will require more transparency and better management to ensure that revenues do not create large wealth distribution imbalances. In the US, ensuring that industry and government do not form cozy relationships that undermine independent oversight regimes is a major concern.
Deanna Bitetti is the Associate Director of Common Cause/NY. Samantha Malone is the Communications Specialist at the Center for Environmental Healthy Environments and Communities and a doctorate student in the University of Pittsburgh Graduate School of Public Health. They are currently in Ghana as part of a State Department funded research trip on resource extraction hosted by Duquesne University (Pittsburgh, Pennsylvania) and the University of Ghana (Legon, Ghana).
Marcellus Shale Advisory Commission Report Released Does Thickness of Shale Predict Production?


The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene-specific, unbiased transcriptome wide amplification accurately maintains proportionality among all RNA species within a given specimen. This allows the utilization of clinical material obtained with minimally invasive methods such as fine needle aspirates (FNA) or cytological washings for high throughput functional genomics studies. This review provides a comprehensive and updated discussion of the literature in the subject and critically discusses the main approaches, the pitfalls and provides practical suggestions for successful unbiased amplification of the whole transcriptome in clinical samples.
Quantification of gene expression is a powerful tool for the global understanding of the biology underlying complex pathophysiological conditions. Advances in gene profiling analysis using cDNA or oligo-based microarray systems uncovered genes critically important in disease development, progression, and response to treatment [1–12]. While the expression of a single or a limited number of genes can be readily estimated using minimum amount of total or messenger RNA (mRNA) from experimental or clinic samples, gene profiling requires large amount of RNA which can only be generated from global RNA amplification when using often limited amount clinical material. Conventionally at least 50 – 100 μg of total RNA (T-RNA) or 2 – 5 μg poly(A)+ RNA are generally necessary for global transcript analysis studies though efforts to enhance signal intensity and fluorochrome incorporation have reduced the amount of total RNA needed for array analysis to 1–5 ug . Large amounts of RNA are not usually obtainable from clinical specimens. Thus, they pertain to experimental endeavors where cultured cell lines or tissues from pooled experimental models are used while only occasionally they are obtainable from large excisional biopsies . However, most biological specimens directly obtained ex vivo for diagnostic or prognostic purposes or for clinical monitoring of treatment are too scarce to yield enough RNA for high throughput gene expression analysis. Needle or punch biopsies provide the opportunity to serially sample lesions during treatment or to sample lesion to identify predictors of treatment outcome by observing the fate of the lesion left in place. In addition, the simplicity of the storage procedure associated with the collection of small samples which can be performed at the bed side provides superior quality of RNA with minimum degradation . Finally, the hypoxia which follows ligation of tumor-feeding vessels before excision is avoided with these minimally invasive methods, therefore, obtaining a true snapshot of the in vivo transcriptional program. These minimally invasive sampling techniques yield generally few micrograms of total RNA and most often even less [15, 16]. Similarly, breast and nasal lavages and cervical brush biopsies, routinely used for pathological diagnosis, generate insufficient material far below the detection limit of most assays. Acquisition of cell subsets by fluorescent or magnetic sorting or laser capture micro-dissection (LCM) for a more accurate portraying of individual cell interactions in a pathological process generate even less material, in most cases, nanograms of total RNA [17–20].
Efforts have been made to broaden the utilization of cDNA microarrays using two main strategies: intensifying fluorescence signal [13, 21–24] or amplifying RNA. Signal intensification approaches have reduced the requirement of RNA few folds but cannot extend the utilization of microarray to sub-microgram levels. RNA amplification in turn has gained extreme popularity based on amplification efficiency, linearity and reproducibility lowering the amount of total RNA needed for microarray analysis to nanograms without introducing significant biases. Methods aimed at the amplification of poly(A)-RNA  via in vitro transcription (IVT)  or cDNA amplification via polymerase chain reaction (PCR)  have reduced the material needed for cDNA microarray application and extended the spectrum of clinical samples that can be studied. Nanograms of total RNA have been successfully amplified into micrograms of pure mRNA for the screening of the entire transcriptome without losing the proportionality of gene expression displayed by the source material. Curiously, the most important advances were made by Eberwine whose main goal was not to use clinical material for high-throughput studies but rather to amplify enough material from single cells for individual or few gene analysis [28, 29]. His revolutionary contribution has, however, provided a striking opportunity to explore the function of the human genome ex vivo and has exponentially opened the frontiers of clinical investigation. Modifications, optimizations and validations of RNA amplification technology based on Eberwine's pioneering work are still actively explored.
In this chapter, we will summarize efforts to optimize RNA amplification and describe in detail current amplification procedures that have been validated and applied to cDNA microarray analysis.
Samples used for RNA isolation and amplification should always be collected fresh and immediately processed. Excisional biopsies should be handled within 20 min and stored at -80°C (for instance with RNAlater™, Ambion, Austin, TX) if RNA isolation cannot be performed right away. Material from FNA should be collected in 5 ml of ice cold 1 × PBS or other collection medium without serum at the patient's bedside to minimize RNA metabolism or degradation. After spinning at 1,500 rpm for 5 minutes at 4°C, 2.5 ml of ACK lysing buffer should be added with 2.5 ml of 1 × PBS and incubated for 5 minutes on ice to lyse red blood cells (RBC) in case of excessive contamination. Cell pellets should be washed in 10 ml 1 × PBS and then re-suspend in small volumes of RNAlater followed by snap freezing or prior lysis of the pellet in 350 μl of RLT buffer with fresh addition of 2-mercaptoethanol (2-ME) (RNeasy mini kit, QIAGEN Inc, Valencia, CA USA) before snap freezing at -80°C. For LCM, good results can be obtained by lysing cells directly in 50 μl RLT buffer with 2-ME. Total RNA (T-RNA) and poly A RNA can both be used as starting material for RNA amplification.
The RNA isolation method strongly affects the quality and quantity of RNA. T-RNA can be isolated using commercially available RNA isolation kits. The T-RNA content per mammalian cell ranges between 20 to 40 pg of which only 0.5 – 1.0 pg are constituted by messenger RNA (mRNA) [30, 31]. Sample condition, viability, functional status and phenotype of the cells are the major reasons for differential yield of T-RNA. Sample handling with precaution for RNase contamination always improves the quality and quantity of the RNA obtained. Measurement of T-RNA concentration can be performed with a spectrophotometer at OD260. An OD260/280 ratio above 1.8 is to be expected. When a very limited number of cells is available such as from LCM or FNA, very low or even negative OD readings may be observed. In this case, OD reading can be omitted. When RNA is isolated from archived samples or from samples whose collection and storage conditions were not controlled and optimized, it is preferable to estimate RNA quality and quantity using Agilent Bioanalyzer (Agilent Technologies Inc. Palo Alto, CA) or RNA gels. Clear 28S and 18S ribosomal RNA bands indicate good quality of RNA. Since 28S rRNA degradation occurs earlier than 18S rRNA and mRNA degradation in most cases correlates with 28S ribosomal RNA, the ratio of 28S versus 18S rRNA is a good indicator of mRNA quality . 28S/18S rRNA ratios equal or close to 2 suggest good RNA quality.
A critical step in RNA or cDNA amplification is the generation of double stranded cDNA (ds-cDNA) templates. First strand cDNAs are reverse transcribed from mRNA using oligo dT or random primers. In order to generate full length first strand cDNA, oligo dT(15–24 nt) with an attachment of a bacterial phage T7 promoter sequence is commonly used to initiate the cDNA synthesis [25, 29, 33–36]. In case of degraded RNA , random primers with attachment of T3 RNA polymerase promoter (T3N9) have been used for first and second strand cDNA synthesis . To prevent RNA degradation while denaturing and during the reverse transcription (RT) reaction, it is useful to denature the RNA (65°C for 5 minutes or 70°C for 3 minutes) in the presence of RNasin® Plus RNase Inhibitor (Promega, Madison, WI) which forms a stable complex with RNases and inactivates RNase at temperatures up to 70°C for at least 15 minutes.
To enhance the efficiency of the RT reaction and reduce incorporation errors, the temperature of the RT reaction can be maintained at 50°C [39, 40] instead of 42°C to avoid the formation of secondary mRNA structures. This can be done by using thermo-stable reverse transcriptase (ThermoScript™ RNase H- Reverse Transcriptase, Invitrogen, Carlsbad, CA) or regular RTase  in the presence of disaccharide trehalose [42–44]. Disaccharide trehalose not only can enhance the thermo-stability of RTase but also posses thermo-activation functions. This modification greatly enhances the accuracy and the efficiency of RT with minimum impact on the DNA polymerase activity . The utilization of DNA binding protein T4gp32 (USB, Cleveland) in RT reactions also improves cDNA synthesis [40, 41, 45, 46]. T4gp32 protein may essentially contribute to the qualitative and quantitative efficiency of the RT reaction by reducing higher order structures of RNA molecules and hence reduce the pause sites during cDNA synthesis.
In Van Gelder and Eberwine's T7 based RNA amplification , the amount of oligo dT-T7 primer used in the first strand cDNA synthesis can affect the amplified RNA in quantity and quality. Excessive oligo dT-T7 in the RT reaction could lead to template independent amplification . This phenomenon is not observed when the template switch approach is combined to in vitro transcription (Wang, E. unpublished data).
RNA amplification methods differ according to the strategies used for the generation of ds-cDNA as templates for in vitro transcription or PCR amplification. There are two basic strategies that have been extensively validated and applied for high throughput transcriptional analysis. The first is based on Gubler-Hoffman's  ds-cDNA synthesis subsequently optimized by Van Gelder and Eberwine [28, 29]. This technology utilizes RNase H digestion to create short fragments of RNA as primers to initiate the second strand cDNA elongation under DNA polymerase I. Fragments of second strand cDNA are then ligated to each other sequentially under E. Coli DNA ligase followed by polishment using T4 DNA polymerase to eliminate loops and to form blunt ends. Amplifications based on this methods have been widely used in samples obtained in physiological or pathological conditions and extensively validated for its fidelity, reproducibility and linearity compared to un-amplified RNA from the same source materials [29, 33, 47, 49–52].
The alternative ds-cDNA synthesis approach utilizes retroviral RNA recombination as a mechanism for template switch to generate full length ds-cDNA. The method was initially invented for full length cDNA cloning and, therefore, the main targets of this method are undegredated transcripts. Gubler-Hoffman's ds-cDNA synthesis has the potential of introducing amplification biases because of a possible 5' under-representation. In addition, the low stringency of the temperature in which ds-cDNA synthesis occurs may introduce additional biases . Although 5' under-representation could, in theory, be overcome by hairpin loop second-strand synthesis , the multiple enzymes (4) used in the reaction could also in turn cause errors.
To ensure generation of full-length ds-cDNA,  synthesis is performed taking advantage of the intrinsic terminal transferase activity and template switch ability of Moloney Murine Leukemia Virus RTase . This enzyme adds non-template nucleotides at the 3' end of the first strand cDNA, preferentially dCTP oligo nucleotides. A template-switch oligonucleotide (TS primer) containing a short string of dG residues at the 3' end is added to the reaction to anneal to the dC string of the newly synthesized cDNA. This produces an overhang that allows the RTase to switch template and extend the cDNA beyond the dC to create a short segment of ds-cDNA duplex. After treatment with RNase H to remove the original mRNA, the TS primer initiates the second stranded cDNA synthesis by PCR. Since the terminal transferase activity of the RTase is triggered only when the cDNA synthesis is complete, only full-length single stranded cDNA will be tailed with the TS primer and converted into ds-cDNA. Using the TS primer, second strand cDNA synthesis is carried at 75°C after a 95°C denaturing and a 65°C annealing step in the presence of single DNA polymerase . This technique, in theory, overcomes the bias generated by amplification methods depending only on 3' nucleotide synthesis and hence it is, in theory, superior to the Gubler-Hoffman's ds-cDNA synthesis. However, no significant differences in correlation coefficients of amplified versus non amplified RNA were observed when the Gubler-Hoffman's ds-cDNA method was compared with the TS ds-cDNA amplification using high throughput analysis [40, 56] The fidelity of template switch-based amplification methods has been assessed by numerous gene profiling analyses on different type of microarray platforms, real time PCR and sophisticated statistical analyses and it has been well accepted for high throughput transcriptome studies.
Amplification of mRNA without skewing relative transcript abundance remains a focus of research. Linear amplification methods have been developed that in theory should maintain the proportionality of each RNA species present in the original sample. IVT using ds-cDNA equipped with a bacteriophage T7 promoter  provides an efficient way to amplify mRNA sequences and thereby generate templates for synthesis of fluorescently-labeled single-stranded cDNA [25, 26, 28, 29, 33, 53]. Depending upon the T7 or other (T3 or SP6) promoter sequence position on the ds-cDNA, amplified RNA can be either in sense or antisense orientation. Oligo dT attachments to the promoter sequence, for example oligo dT-T7, prime first strand cDNA positioned the promoter at the 3' end of genes (5' end of cDNA) and, therefore, lead to the amplification of antisense RNA (aRNA) or complement RNA (cRNA). Promoters positioned at the 5' end of genes by random  or TS primers (Wang E, unpublished observation) generate sense RNA (sRNA). Amplified sRNA can be also produced by tailing of oligo dT to the 3' of the cDNA followed by oligo dA-T7 priming for double stranded T7 promoter generation at the 5' end of genes . The singularity of this approach resides in the utilization of a DNA polymerase blocker at the 3' of the oligo dA-T7 primer which prevents the elongation of second strand cDNA synthesis while priming for the elongation of the double stranded promoter. In this fashion, only sense amplification can be achieved by the presence of the 5' ds-T7 promoter followed by single strand cDNA templates.
Two rounds of IVT are commonly required when sub micrograms of input total RNA are used. It has been estimated that after two rounds of amplification the frequency of only 10% of the genes in a specimen is reduced  and more than two rounds of amplification may still retain at least in part the proportionality of gene expression among different RNA populations . However, we, generally, do not recommend going over two rounds of amplification unless necessary for extremely scant specimens such as when processing single or few cell specimens, to avoid unnecessary biases related to amplification. The fidelity of IVT has been extensively assessed by gene profiling analysis, quantitative real-time PCR and statistical testing by comparing estimates of gene expression in amplified versus non-amplified RNA .
Pitfalls have been also associated with IVT. The fidelity of the first round amplification decreases when the input starting material is less than 100 ng because of the intrinsic low abundance of transcripts (particularly those under represented in the biological specimen). This can be rescued by two rounds of IVT if sufficient RNA species are present in the input material . In addition, two rounds of amplification tend to introduce a 3' bias due to the use of random primers in the cDNA synthesis for ds-cDNA template creation. This should not affect the usefulness of the technique for high throughput gene profiling analysis since cloned cDNA arrays are 3' biased and even oligo arrays are designed to target the 3' end of each gene. Sequence-specific biases introduced during amplification are generally reproducible and, although negligible, could mislead data interpretation only when amplified RNA is directly compared with non amplified RNA on the same array platform. This type of error can be easily circumvented by using samples processed in identical conditions. Degradation of amplified RNA during prolonged (more than 5 hours) IVT may result in lower average size of aRNA and decreased yields . This results from residual RNase in the enzyme mixture used for IVT reaction and can be prevented by the addition of RNase inhibitor in the reaction if a prolonged amplification is needed.
IVT is burdensome time consuming and may, theoretically, produce a 3' bias especially when two rounds of amplification are employed. Exponential amplification (PCR-based) may avoid these drawback and it has shown promise since, contrary to the IVT, is simple and efficient. However, PCR-based amplification has its own drawbacks.
The limitations of PCR-based amplification stem from the characteristics of the DNA-dependent DNA polymerase enzymatic function. The function of this enzyme is biased towards a lower efficiency in the amplification of GC rich sequences compared with AT rich sequences. In addition, as previously discussed, not only creates errors more frequently than RNA polymerase but also amplifies these mistakes because the reaction utilizes the amplicons as templates for subsequent amplification . In addition, due to the exponential amplification, the reaction could reach saturation in conditions in which excess input template quantities are used or because of the exhaustion of substrate. This would favor the amplification of high-abundance transcripts which would compete more efficiently for substrate in the earlier cycles of the amplification process resulting in loss of proportionality of the amplification process. Optimization of PCR cycle number to avoid reaching the saturation cycle and adjustments in the amount of template input could overcome the problems . The utilization of DNA polymerase with proofreading function could eradicate errors created in the cDNA amplification . This approach preserves the relative abundance of transcript  and it may outperform IVT when less than 50 ng of input RNA are available as starting material [66, 67].
PCR-based cDNA amplification can be categorized as template switching (TS)-PCR [52, 68, 69], random PCR  and 3' tailing with 5' adaptor ligation PCR  based on the generation of a 5' anchor sequence which provides a platform for 5' primer annealing. TS-PCR employs the same template switch mechanism in ds-cDNA generation and in the amplification of ds-cDNA using 5' TS primer II (truncated TS primer) and 3' oligo dT or dT-T7 primers (depending upon the primer used in the first strand cDNA synthesis). Random PCR utilizes modified oligo dT primers (dT-T7 or dT-TAS (T arget A mplification S equence) or random primers with an adaptor sequence for the first strand cDNA initiation and random primers with an attachment of the same adaptor, for example dN10-TAS , for second strand cDNA synthesis. The attached sequence, such as TAS, generates a 5' anchor on the cDNA for subsequent PCR amplification with a single TAS-PCR primer. This approach is more suitable for RNA with partial degradation and with the risk of under representation of the 5' end. The third exponential amplification utilizes terminal deoxynucleotidyl transferase function to add a polymonomer, for example poly dA, tail to the 5' end of the gene. The tailed poly dA provides an annealing position for the oligo dT primer which lead the second strand cDNA synthesis. Ds-cDNA can then be amplified under one oligo dT primer or dT-adaptor primer if an adaptor sequence is attached . Direct adaptor ligation is another alternative way to generate ds-cDNA with a known anchor sequence at the 5' end . In this way, single strand cDNA is generated using oligo dT primers immobilized onto magnetic beads and second strand cDNA is completed by Van Gelder and Eberwine's ds-cDNA generation method. A ds-T7 promoter-linker is then unidirectionally ligated to the blunted ds-cDNA at the 5' end. PCR amplification can then be performed using the 5' promoter primer and the 3' oligo dT or dT-adapter primer, if an adapter is attached. PCR amplified ds-cDNA is suitable for either sense or antisense probe arrays.
The combination of PCR amplification to generate sufficient ds-cDNA template followed by IVT [70, 71]. is an attractive strategy to amplify minimal starting material since it takes advantage of the efficiency of the PCR reaction and the linear kinetics of IVT while minimizing the disadvantage discussed above. Validations of PCR-based RNA amplification methods are fewer than those for IVT but have been so far persuasive in spite of the prevalent expectations. Skepticism concerning the reproducibility and linearity are still one of the key factors preventing the extensive application of this approach.
The generation of high quality cDNA microarray data depends not only on sufficient amount and highly representative amplified target, but also on the target labeling efficacy and reproducibility. Steps involved in the targets preparation such as RNA amplification, target labeling, pre-hybridization, hybridization and slides washing are imperative in enhancing foreground signal to background noise ratios. Linear spectrum of signal intensity that correlates with gene copy numbers without having to compensate detection sensitivity is one of the key factors for high quality cDNA analysis. Therefore, target labeling is a critical step to achieve consistently high signal images.
Typically, fluorescently-labeled cDNA is generated by incorporation of conjugated nucleotide analogs during the reverse transcription process. Depending upon the detection system, labeled markers can be either radioactive, color matrix or florescent. Florescence labeling outperforms the other labeling methods because of the versatile excitation and emission wave length. In addition, it has the advantage of not being hazardous. Among the fluorochrome, Cy3 (N, N8-(dipropyl)-tetramethylindocarbocyanine) and Cy5 (N, N8-(dipropyl)-tetramethylindodicarbocyanine) are most commonly used in cDNA microarray applications due to their distinct emission (510 and 664 respectively). Cy5 labeled dUTP and dCTP are less efficient in incorporation during the labeling reaction compared to Cy3 labeled dUTP or dCTP and they are more sensitive to photo bleach because of their chemical structure. Therefore, labeling bias needs to be accurately analyzed and results should be normalized according to standard normalization procedures.
Target labeling can be divided into two major categories: direct fluorescence incorporation and indirect fluorescence incorporation. The first category utilizes fluorescence-labeled dUTP or dCTP to partially substitute unlabeled dTTP or dCTP in the RT reaction to generate Cydye-labeled cDNA. This label incorporation method is suitable for cDNA clone microarray using amplified aRNA as templates or oligo array using amplified sRNA as template.
A limitation of direct labeling consists in the fact that fluorescent nucleotides are not the normal substrates for polymerases and some may be particularly sensitive to the structural diversity of these artificial oligonucleotides. The fluorescent moieties associated with these nucleotides are often quite bulky and, therefore, the efficiency of incorporation of such nucleotides by polymerase tends to be much lower than that of natural substrates. An alternative is to incorporate, either by synthesis or by enzymatic activity, a nucleotide analog similar to the natural nucleotide in structure featuring a chemically reactive group, such as 5-(3-Aminoallyl)-2'-deoxyuridine 5'-triphosphate (aa-dUTP), to which a fluorescent dye, such as Cydye, may then be attached . The reactive amine of the aa-dUTP can be incorporated by a variety of RNA-dependent and DNA-dependent DNA polymerases. After removing free nucleotides, the aminoallyl labeled samples can coupled to dye, purified again, and then applied to a microarray . The optimized ratio of aa-dUTP versus dTTP in the labeling reaction should be 2 to 3 respectively.
In theory, indirect outperforms direct labeling by reducing of the cost and maximizing signal intensity through increases in incorporation of fluorochrome or through signal amplification using fluorescence-labeled antibody or biotin-streptavdin complexes. However, more steps are involved in the purification of the labeled target prior to hybridization which make this strategies less frequently used.
The protocols presented here are routinely used in our laboratory in response to several inquires by interested investigators, The protocol is based on a combination of strategies discussed in the previous section that have been used for RNA amplification that we have applied to optimize TS-IVT following the original Eberwine's RNA amplification protocols.
Dilute stock solution to the appropriate working concentration.
Oligo dT-T7 primer (5' AAA CGA CGG CCA GTG AAT TGT AAT ACG ACT CAC TAT AGG CGC T(15) 3') (0.125–0.25 μg/μl for the first round amplification depending on the amount of input total RNA and 0.5 μg/μl for the second round amplification) in RNase free water. Synthesized primer should be SDS-PAGE purified to insure the full length. The concentration of primer is varied according to the starting material used. This promoter sequence is much longer than the consensus sequence defined by Dunn and Studier (1983) and can be purchased from New England Biolabs and Stratagene Inc. In the extended sequence shown here, the consensus sequence is embedded in the between a 5'flanking region that provides space for the T7 RNA polymerase to bind and a 3'-flanking trinucleotide that stimulates transcription catalyzed by the enzyme.
TS primer (5' AAG CAG TGG TAA CAA CGC AGA GTA CGC GGG 3') (0.25 μg/μl) SDS-PAGE purified. According to the Chenchik's  data, ribouncleotide GGG at the 3' end should give the best TS effect instead of deoxinucleotide GGG. We have used TS primer with dGGG at the 3' end in multiple experiments and achieved satisfying results. The amount of TS primer used in the second strand synthesis can be varied according to the amount of starting material. We generally use 0.25 μg/μl when 3–6 ug of total RNA used and 0.125 μg/μl when less total RNA used.
Random hexamer (dN6) (8 μg/μl).
Microcon YM-30 column (Millipore; Cat# 42410).
50°C for 90 min in thermal cycler.
Cycle at 37°C for 5 min to digest mRNA, 94°C for 2 min to denature, 65°C for 1 min. for specific priming and 75°C for 30 min for extension.
Note: Since the TS primer which initiates the second strand cDNA synthesis is already present in the first strand cDNA synthesis reaction and has been primed to the extended part of the cDNA, no additional primer is required in this step.
Stop reaction with 7.5 μl 1 M NaOH solution containing 2 mM EDTA and incubate at 65°C for 10 min. to inactivate enzymes.
Add 1 μl Linear Acrylamide (0.1 μg/μl) as DNA carrier to the sample to enhance double stranded-cDNA precipitation. Add 150 μl Phenol: Chloroform: Isoamyl alcohol (25:24:1) to the double stranded cDNA tube and mix well by pipetting (be careful not to spill or contaminate). Transfer the slurry solution to Phase lock gel tube and spin at 14,000 rpm for 5 min at room temperature. Transfer the aqueous phase to RNase/DNase-free 1.7 ml tube and add 70 μl of 7.5 M ammonium acetate first and then 1 ml 100% ethanol (EtOH). Mix well. Centrifuge right away at 14,000 rpm for 20 min at room temperature to prevent co-precipitation of oligos. (A visible small white pellet should be seen at the bottom of the tube even if nano grams of starting material have been used. This pellet suggests successful precipitation.) Wash pellet with 800 μl 100% EtOH and spin down at maximum speed for 8 min. Repeat this washing step one more time. Air dry or speedvac and re-suspend double stranded cDNA in 8 ul DEPC H2O.
According to Ambion, the incubation can be interrupted by storing reaction tube at -20°C and resuming the incubation later without losing efficiency.
Monophasic reagent such as TRIzol reagent from GibcoBRL, (Cat#15596) are used here based on the efficient recovery of aRNA (RNeasy mini kit could be used for aRNA purification instead of TRIzol but, in our experience, RNA recovering is about 50% of that recovered with the TRIzol method.).
a. Add 0.5 ml of TRIzol solution to the transcription reaction. Mix the reagents well by pipetting or gentle vortexing.
b. Add 100 μl chloroform. Mix the reagents by inverting the tube for 15 seconds. Allow the tube to stand at room temperature for 2 – 3 minutes.
c. Centrifuge the tube at 10,000 g for 15 min at 4°C.
d. Transfer the aqueous phase to a fresh tube and add 250 μl of isopropanol.
e. Store the sample on ice for 5 minutes and then centrifuge at 10,000 g for 15 minutes.
h. Measure the quantity of RNA concentration spectrophotometrically.
From here, follow the previously described procedure for second strand cDNA synthesis, double stranded cDNA cleanup. In the second IVT, 40 ul of IVT reaction mixture are suggested to use instead of 20 ul. RNA isolation is followed.
Mix well and heat to 65°C for 5 min then cool down to 42°C.
Add 1.5 μl SSII. Incubate for 90 min at 42°C. Add 2.5 μl 0.5 M EDTA and heat to 65°C for 1 min. Add 5 μl 1 M NaOH and incubate at 65°C for 15 min to hydrolyze RNA. Add 12.5 μl 1 M Tris immediately to neutralize the pH. Bring volume to 70 μl by adding 35 μl of 1 × TE.
Note: The amounts of aRNA used for labeling depends on the size of the array. If the array with 2000–8000 genes, 3 ug aRNA will be sufficient while a larger chip such as 16–20 k will need 6 ug of aRNA. The labeling reaction components do not need to be changed.
Prepare Bio-6 column and run target solution through it. Collect flow through and add 250 μl 1 × TE to it. Concentrate target to ~20 μl using Microcon YM-30 column.
Combine Cy3 labeled reference sample and Cy5 labeled target sample (adjust the color to purple) and then complete dry the sample using speedvac. Resuspend sample in 37 μl volume (for 22 mm × 40 mm printing surface) containing 1 μl 50× Denhardt's blocking solution, 1 μl poly dA (8 μg/μl), 1 μl yeast tRNA (4 mg/ml), 10 μl Human Cot I DNA, 3 μl 20× SSC, 1 μl of 10% SDS and 20 μl of DEPC treated water. Heat sample for 2 min at 99°C and apply target mixture to array slide, add coverslip, place in humidified hyb chamber, and hybridize at 65°C over night.
1. Wash with 2 × SSC + 0.1% SDS to get rid of the cover slide.
2. Wash with 1 × SSC for 1 min.
3. Wash with 0.2 × SSC for 1 min.


text:
Inter-Korean summit 2018
Pope Francis hopes for success of inter-Korean summit
Pope Francis has prayed for peace on the Korean Peninsula, expressing his support to the upcoming inter-Korean summit, South Korea's presidential office said Thursday.
The pope held a special session of prayer Wednesday (local time) at St. Peter's Square of the Vatican with thousands of Catholics as President Moon Jae-in and North Korean leader Kim Jong-un will hold a summit Friday at the border truce village of Panmunjom.
"The meeting will be an auspicious opportunity that leads to a journey for reconciliation and the restoration of fraternity," Pope Francis was quoted as saying by Cheong Wa Dae. "It will help guarantee peace on the Korean Peninsula and in the world."
The pope expressed hope that Moon and Kim can serve as "craftsmen of peace" as they've embarked on the path toward peacemaking.
Pope Francis sent a similar message on April 1 to mark Easter, expressing hope for peace on the peninsula.
"We implore fruits of dialogue for the Korean Peninsula, that the discussions under way may advance harmony and peace within the region," the pope was quoted as saying.
"May those who are directly responsible act with wisdom and discernment to promote the good of the Korean people and to build relationships of trust within the international community." (Yonhap)
Nation hoping for successful summit
The government is seeking to promote the flying of the "unification flag," in the hopes of creating the mood for a successful inter-Korean summit. President Moon Jae-in and North K...


text:
Q: Выбор in-memory базы данных Необходимо сделать эффективный поиск в коллекции кастомных объектов по нескольким полям. Коллекция содержит около 1 млн. объектов.
Сейчас сделено через словари/хеш-таблицы в приложении.
Коллеги, предлагают посмотреть в сторону in-memory базы данных.
Какие порекомендуете?
Смущают дополнительные накладные расходны на IPC из приложения в БД.

A: Hypersonic SQL DB , он же HSQLDB вне конкуренции.
Года 3-4 назад писал аппу, где как раз нужен был in-memory DB. После различных мучений остановился на HSQLDB.
Есть еще Derby, который входит в состав Java, но мне показался тяжеловатым.
В последнее время стал модным SQLite в телефонах Android/iOS он понужается вовсю.

redpajama_set_name: RedPajamaStackExchange


We asked journalist Aaron Wiener to go out and understand how this effort took on a life of its own in each community. He came back with a vivid portrait of an effort that is both changing community and changed by community, an effort now as disparate as the places where it lives.
Photo courtesy of the Akron Community Foundation.
Lilly Weinberg is director for community foundations at Knight Foundation. Below, she highlights a recent report detailing the impact of the Knight-supported On the Table initiative, which brings together community residents over mealtime conversations to discuss pressing community issues.
Akron is growing. That is a phrase we haven't been able to say since the 1960's when population peaked at 292,000. Since then, the city has lost roughly one-third of its population. Yet Akron grew by 135 people in 2015, according to the most recent Census estimate. It's not a lot – you could line them up, count them, pose for a group picture. But we believe it's a key sign of what will come, a turning of the tide for a city that has struggled for decades to reverse the forces of decline.
Ana Paula González, who leads 500 Startups' Miami operation, writes about Miami's entrepreneurial evolution and why the best is yet to come.
Strong democracies depend on freedom of expression and access to accurate information about community and public affairs. This is as true today as when freedom of the press was enshrined by the framers of the U.S. Constitution in the first correction they made to the governing principles of our country — what we call the First Amendment.
Photo by J.C. Burns on Flickr.
Cities across the country – big and small – are investing in linear parks and urban trails. Communities are prioritizing these important and substantial investments for a variety of important reasons: they effectively connect public assets - like parks and libraries - with diverse neighborhoods; activate underused spaces (think New York City's High Line crafted from a former rail line); and spur economic development in nearby areas.
But designing, promoting and funding linear parks can be challenging, often spanning miles of multiple municipalities, costing hundreds of millions of dollars, with support from complicated funding models.
So, what happens when three unique cities get together to talk about their signature linear parks and trails? A whole lot of learning. Knight Foundation funded an information exchange between two Knight cities, Lexington, Ky. and Miami, and Atlanta to do just that . Last month, a team from The Friends of The Underline (a 10-mile linear path in Miami-Dade) and Townbranch Commons (a 3-mile linear trail and park in downtown Lexington) met in Atlanta to have a deep-dive exchange about their future projects with the city's Beltline team. As many know, Atlanta's Beltline is a multi-billion dollar, 22-mile light rail and bike/pedestrian trail that has transformed the communities it passes through. While most community members love their Beltline, not everyone is thrilled. We wanted to hear it all: the good, bad and indifferent. And while we came from very different communities with unique projects, we had four shared takeaways.
As one travels across San Jose's sprawling 180-square-mile landscape, it's hard to believe this is America's tenth most populous city. The low-rise suburban city hosts seemingly endless single-family homes, strip malls, freeways and suburban office parks, but too few vibrant and well-used public spaces that welcome and celebrate our one million residents.
In San Jose, Knight seeks to change that by creating one of the nation's most engaged cities driven by a focus on public life — drawing people out of their cars and homes and into the community. In doing so, we aim to place people at the center of the city's present and future. By helping to build a San Jose for people of all ages, backgrounds, and abilities, we aim to create a vibrant and welcoming city that makes being out and in public irresistible and celebrates the collision of diverse people and ideas.
Building stronger democracies starts with local communities. It is at the local level where we make the decisions that have the most immediate impact on our lives. Knight Foundation's Community and National Initiatives program focuses on supporting more informed and engaged communities through investments that attract and nurture talent, enhance opportunity and foster civic engagement.
Photo: A young resident gives their input on shaping civic life in Charlotte at a pop-up event at Eastland Mall. Courtesy of the Center for Active Design.
A groundbreaking playbook from the Center for Active Design is sparking conversations among residents on how to shape civic life and create fantastic public spaces in their cities.
Akron, Ohio, residents explore downtown using Pokémon GO. Photo by Tim Fitzwater.
In summer 2016, Pokémon GO took the world by storm. Large crowds teemed in public spaces around the globe at all hours. We saw a prime opportunity to learn more about how our society, and its ubiquitous digital technology, might further spark public life in the communities where we work.
Veggielution First Friday. Photo by Danny Harris on Twitter.
In San José, Knight seeks to create one of the nation's most engaged cities. We seek to place people at the center of the city's present and future. From walkable and bikeable neighborhoods to more user-friendly design of city services to building vibrant public spaces for all, Knight's work aims to help San Joseans fall in love with and work in support of their city every day.
To advance these efforts, Knight is launching Speak Up San José, a yearlong initiative that invites all residents for conversation and action to advance our city's future. Specifically, the foundation is committing $150,000 to sixteen community groups to host 28 events over the coming twelve months. From community dinners to street parties to salons, each event is conceived by local organizations, community groups and neighborhood know-it-alls to advance locally relevant issues. Most importantly, every event is free, open to the public and/or engages a diverse group of individuals, and built around a specific action point.
A panorama view of Miami. Used under CC0 Creative Commons from Pixabay.
We're proud to be a part of what the Miami community has accomplished in such a small window of time. Miami's innovation economy is growing. Yet much remains to be achieved. In a spirit of continuous learning, we want to hear from you – those building the future Miami – about what it will take to succeed.


Well, what´s to say about our new DIVINE SL? It is simply the lightest production dropper post in the world with a total length of 400mm and 80mm infinite drop.
Tailored to the needs of XC and marathon racers, the DIVINE SL is the youngest offspring of our dropper family. Based on the award-winning and patented REVIVE technology, we´ve modified the internals to work perfectly with the shorter travel.
Of course, you can mount and use your DIVINE SL also on bikes outside the XC department. However, the DIVINE SL was specifically designed in respect of XC riders' requirements.
One of the features, is the "Tech-Climb" feature. While our DIVINE SL is completely locked in place in the top-out and bottom-out position for consistent riding position, the post can slightly spring in the middle positions. We´ve found out, that extremely steep and technical (e.g. rooty or rocky) uphill sections can be conquered with much more ease, when the saddle is just slightly dropped (lets say about 1-2cm). Having the saddle dropped just a bit (and not completely), the "Tech-Climb" feature" also adds some suspension to the post, flitering the impacts of roots and rocks. Back fully extended or fully dropped, the post is rigid again.
Titanium saddle bolts, carbon and oval rail compatible forged clamping plates, tapered tubes. All of this comes stock on the DIVINE SL to achieve a sensational low weight of 385g (30.9/400mm).
You can easily cut it to a short as 283mm, if you can spare the extra length with your frame and saddle height. The cut weight can go to a minimum of 345g!
The shorter travel allows the DIVINE SL to come with a design, that doesn´t require manual resets like the REVIVE.
Of course, our DIVINE SL requires internal routing of the cable through your frame.
The Divine SL seatpost comes with a standalone Triggy cable actuated remote lever. Alternative remote clamp options are listed in related products below.


text:
The new papa paradigm
Posted on June 13, 2007 June 20, 2014 by Hal L. Millard
For expectant dads, the act of conception was the fun part (let's be real here, fellas).
Rad dad: Jason Pradway's parenting group, Radical Dads, seeks to challenge and discuss traditional perceptions of fatherhood. photo by Jonathan Welch
But, oh, to hold your newborn and dream of his or her future and your new life as a parent … well, for a lot of guys, the conclusion of the journey into fatherhood can be not only exhilarating and joyous but downright scary, at least the first time.
A slew of books are available on that try to ease men into parenting, maybe what dads really need is a way to connect with other like-minded dads to provide help and moral support. If so, then Jason Pradway, and his fledgling group, "Radical Dads," might be the ticket to realizing a 21st century ideal of fatherhood where "nurturing" and other squishy feminine concepts can be freely embraced by the XY set.
"I had just seen so many fathers who, after their child was born, they would become complacent," says Pradway, who has a two-year-old son, Nico. "I wanted to be the opposite of complacent."
Pradway's group, which meets each Tuesday from 7 to 9 p.m. at the N.C. School of Natural Healing (1054 Riverside Drive in Asheville), is a left-leaning group that includes everything from punk-rock dads to hippie dads, he says. (And while more-conservative dads might feel uncomfortable, they certainly are welcome and encouraged to attend, he adds.) The group has a political and spiritual bent that is tied to its liberal inclinations, he says, but adds that many of the intimate talk sessions tend to focus on such ordinary topics as sleep tips, nutrition and how fathers can not only become a bigger part of their children's lives—but also how they can guide children to reach their maximum potential as human beings.
And lest you think these dads advocate a mushy, soft approach to raising children, more traditional concepts such as discipline also are addressed, Pradway says, adding that he's aghast at how undisciplined today's children are.
And though Pradway says he still struggles with getting members to open up, for those who do open up, the group acts as a sounding board for their problems and offers a way to share proven parental tips and techniques.
Pradway says his ultimate goal is to spread the Radical Dad concept nationwide.
For more details, check out Pradway's videos at www.youtube.com/skinnyboxer or call him at at 450-2333.
like849 viewsCommunity NewsNews
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3 thoughts on "The new papa paradigm"
jason Treadway
Why would any Dad or expectant Dad refuse to come to radical dads after this great story!! Kudos to Hal.
edtomfish
if you take the prad way and i take the tread way, who gets to Scotland 1st?
nice article jas
A media alias never hurt no one.


Living history. Special Savings. Family fun. All this and more, make Plimoth Plantation Homeschool Day, a perfect opportunity to travel back in time and experience life in 1620.
A week ago, the kids and I finally took a field trip to a destination we have always wanted to visit. What homeschooler hasn't read, and read, and read some more, about the Pilgrims sailing across the Atlantic Ocean on the Mayflower - the hardships they endured, their quest for freedom, their joy at sighting land, and then finally arriving at Plymouth Rock, in 1620?!
When we first started homeschooling, my two oldest girls and I got "stuck" in the Early American time-period; we literally "beat" the subject to death. Have you ever done that? Studied every little aspect of a subject because you're scared that you're going to miss something?
Anyway, it was perfect timing for us to attend Plimoth Plantation Homeschool Day. We have been (once again) studying Early American history with our two youngest, and just finished a unit on the Plymouth and Massachusetts Bay colonies.
One thing I love about living in New England, is the fact that we are so close to so many historical sites. Since moving to NH, our family has taken time to visit some of the many places in the Northeast that we have read and learned about. Not only is New England a beautiful place to live, it is filled with so much history just a short drive away.
Anyway, our day started around 6:30am, with our rain gear and umbrellas in case it rained, and a picnic lunch, to minimize our eating out costs.
A nice money-saving perk for Plimoth Plantation Homeschool Day, is the special discount you will receive on admission. A Heritage Pass which allows you to tour the Mayflower, Grist Mill, and Plimoth Plantation, is discounted.
We enjoyed touring the Mayflower and wondering what it must have been like for 102 people, with their belongings, food, and animals to journey across the ocean in such a small, contained space. When you see the size of the ship, it's really hard to fathom how they fit everything on, including an additional boat, called a shallop, that was stored ON the Mayflower, as well!
This boat (shallop) was also stored on the Mayflower!
Aboard the ship, there were a couple re-enactors that told stories and answered any questions our kids had. All played the part well.
The size of the Mayflower - it was very small.
The size of the captain's quarters, compared to the Pilgrims'.
Seeing where the animals were kept, in relation to the people.
Next, one of the guides directed us to the place where the Pilgrims went to get their fresh water. The river (Town Brook) runs through a pretty memorial garden, called Brewster Gardens. On our way to the garden, we passed Plimoth Rock, statues of William Bradford, Massasoit, and a large memorial for the Pilgrims who died during the first year.
After a 5-10 minute walk along the river, you will arrive at Plimoth Grist Mill - a replica of the corn-grinding mill the Pilgrims built in 1636. We found the tour guides helpful and knowledgeable; they engaged my children, asking them different questions to help them learn how the mill worked. We just finished a unit on Simple Machines, which was perfect, as the mill had lots of examples of the machines the kids had studied.
A replica of the corn-grinding mill the Pilgrims built in 1636.
Once we were done, we walked back to our car and headed to Plimoth Plantation, just a few miles away.
A note on parking: There's metered parking all along the ocean (Water Street) where the Mayflower ll is located, as well as on many side streets. We parked downtown where you will find free two-hour parking on Court Street (3A). It took us just over two hours to tour the Mayflower, and Plimoth Grist Mill, although we cut our time short at the mill, (to my kids disappointment) to make it back to the car in time.
Upon entering the parking area of Plimoth Plantation, I noticed quite a few very large charter buses, as well as school buses in the parking lot. Although I thought it was odd for a homeschool day event, I didn't think much of it until later. I looked around for the typical homeschooler-type vehicles = mini-vans, full-size vans, and large SUVs. To my surprise, there were very few in the parking lot. I thought, "Maybe homeschoolers on the east coast don't have as many kids as homeschoolers in the midwest, and drive smaller cars."
As we walked up to the Visitor's Center, we saw oodles and oodles of school kids lining up outside the Center - 163 to be exact (I overheard the group leader tell the women at the ticket counter that he had 163 kids with him.) Needless to say, we spent the remaining time at Plimoth Plantation dodging this very large group of middle schoolers. There were other school groups as well, but this one, in particular, went through as a "mob".
During our time at Plimoth Plantation, we saw but a few families - maybe five or so. I can't tell you how surprised I was that more homeschooling families weren't at Plimoth Plantation Homeschool Day. Perhaps it wasn't well advertised? Or, perhaps others who have gone to Plimoth Plantation Homeschool day in previous years, had the same (and a little disheartening) experience as us... I'm not sure.
At any rate, we made the most of the afternoon. We tried to avoid this large group, with no luck, and finally waited for them to leave. We spent quite some time in the community meeting house/tower, looking over the plantation and out at the ocean. What a gorgeous view of the ocean!
Oceanview from the Community Meeting House/Tower.
Once the large school groups left the village area, we finished our tour.
It was fun to listen to the re-enactors speak the Old English. We got a kick out of the words, brother, which they said, "brover" and feathers, which they said, "feavers".
Each villager was very engaging, informative, and answered a myriad of questions from us, as well as other visitors. Some allowed my kids to help them at the different tasks they were doing.
Old English sayings and words.
My kids asked about and learned some new Billington Boys stories.
I couldn't get over that they slept on the dirt floor of their homes.
Their bed was, by far, the most expensive item in their house.
As we were leaving, we met a guy who was working in the field. They had planted corn the day before, and yes, they still use fish in the planting process. He was preparing to plant tobacco and would have asked us to help him, but it had started raining.
Part of the living museum, also includes a Wampanoag Village, where your kids will experience the life of an Wampanoag Indian. You can walk through their summer and winter homes, watch them cook meals, and craft different items. We watched a woman weave a basket and also saw (from a distance) the burning of a huge tree into a canoe.
We finished our tour by visiting the Nye Barn, where you will see some rare breed animals.
Despite the over-abundance of school groups and lack of homeschoolers at Plimoth Plantation Homeschool Day, we enjoyed ourselves and learned a lot. The experience enriched our Early American studies, and gave us a greater appreciation for those who first settled in America. We were very appreciative of the Plimoth Plantation Homeschool Day discount as well. If you would like to become a member, homeschoolers receive 20% off a yearly membership.


text:
Q: Property value assignment I am not understanding why the first block of the code is valid but not the latter:
var e = new Exception()
{
    Data =
    {
        {"a","a"}
    }
};

Compile-time error here:
e.Data =
{
    {"a","a"}
};

A: Well Data property has been declared as readonly property:
public virtual System.Collections.IDictionary Data { get; }

That's why you can initialize it (i.e. set in constructor)
  // Create an instance, initialize `Data` with { "a", "a" }
  Exception e = new Exception() {
    Data = { { "a", "a" } }
  };

But you can't set it:
 // Trying to set Data as { { "a", "a" } };
 e.Data = { { "a", "a" } }; // <- Compile time error here

Error CS0200  Property or indexer 'Exception.Data' cannot be assigned to -- it is read only (bold is mine, Dmitry Bychenko)

Please note, that you can just add required items into Data:
  Exception e = new Exception();

  // Add {"a", "a"} to existing Data
  e.Data.Add("a", "a");

A: The other answer isn't 100% accurate. Yes, Data is a readonly property. However, a readonly (i.e., getter-only) property can only be given an initial/default value (in which case, the value is assigned to the compiler-generated backing field), but it cannot be assigned to; period. Not in the object initializer, not in the constructor, and not anywhere else.
Note that readonly fields, on the other hand, can be assigned to in the constructor.
In order to assign to a property, it must have a setter (e.g., set;, private set;, etc.) and if you're using C# 9.0 or above, you could use an init-only setter (i.e., init;) which will allow you to assign to the property in the object initializer but not after that.
As long as the property doesn't have a setter, you can never directly change its value. To prove this, try setting Data to null in the object initializer:
var e = new Exception()
{
    Data = null  // Nope, compiler won't allow it because Data is readonly
};

So, why does it work in the first block?
That's because you're initializing Data using a Collection Initializer. When you do that, the compiler doesn't actually assign a value to the property. Instead, it calls the Add() method. You can see this for yourself here. The first (i.e., working) block is translated by the compiler to:
Exception e = new Exception();
e.Data.Add("a", "a"); // <-- Ah! `Data` was never assigned to.

..which, by the way, would normally throw a NullReferenceException (because we're calling the Add() method on a property that should be null). But if you look at the source code of Exception.Data, you see that its backing field is always initialized in the getter so that it's never null:

public virtual IDictionary Data {
    [System.Security.SecuritySafeCritical]  // auto-generated
    get {
        if (_data == null)
            if (IsImmutableAgileException(this))
                _data = new EmptyReadOnlyDictionaryInternal();
            else
                _data = new ListDictionaryInternal();

        return _data;
    }
}

A: you can not assignt data because Data is a dictionary. You could assignt the new dictionary object if it had a setter, but Data has only a getter and it is created as an empty collection on init
public class ABC
{
 public virtual System.Collections.IDictionary Data { get; } =  new Dictionary<object,object> ();
}

you can only init it by some data the same way as any another collection using a Collection Initializer
    var abc = new ABC()
    {
        Data = {
        {"a","a"}
       }
    };

or add new item by two common ways, the same as you are using any another dictionary
    abc.Data["c"] = "c";
    //or
    abc.Data.Add("d", "d");
}