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Under SPTO, the cost of the space allocated for the Pacific Village is offered at PATA member's rate and in addition, there is a 5% loyalty discount currently offered, which is valid only until end of March. Therefore, SPTO would like to request member countries for their interests and confirmation to participate at PTM 2006 before Friday 24th February 2006. This deadline of 24th February will also enable SPTO to negotiate a larger space should there be more interests from members to participate at this year's PTM 2005. As clearly stated by PATA, the current allocated space of 54 square meters only caters for 7 sellers (6 companies/NTOs and SPTO). There will be six sets of appointments with buyers. Therefore, SPTO would appreciate early confirmation of interests so that all the necessary arrangements and negotiations can be done on time to capitalize of the special rates and discounts.


The Liberal Party has for years been trying to raise the pension age from 67, but Mr Morrison announced yesterday he would no longer pursue the change.
"Next week, cabinet will be ratifying a decision to reverse taking the pension – the retirement age – to 70. It will remain at 67," he told the Nine Network.


(NAPSI)—The next time you visit someone in a hospital or other health care setting, you may be surprised at just how much of what happens there is done by nurses.
Recently released results from a University of Phoenix College of Health Professions survey found that the role of nurses has grown dramatically in recent years. More than eight in 10 registered nurses (RNs) say health care professionals besides physicians (nurse practitioners, registered nurses and so on) are playing or will play a larger role in the overall management of patient care.
University of Phoenix, which offers leading-edge graduate, undergraduate, certificate and nondegree programs aimed at preparing students to improve the quality of health care in their communities and the industry, sought to understand the evolving role of nurses and what this changing environment means for the future of health care.
In addition to playing a larger role in managing patient care, it found, about a third of RNs say they've seen an increased role in doing tasks traditionally done by a physician. This may be due in part to specialty tracks available to nurses, including nurse practitioner programs.
• Increasingly greater leadership role (36 percent).
About a third of RNs say they'll be focusing more on the emotional well-being of patients, while nearly three in five strongly agree that good people skills are just as important as technical skills when giving quality care.
As opportunities continue to increase in the profession—the Bureau of Labor Statistics notes that employment of registered nurses is projected to grow 15 percent by 2026—RNs say their facilities should focus on preparing health care professionals with greater leadership skills.
For further information about the College of Health Professions, visit www.phoenix.edu. For full survey information and details about College of Health Professions programs, please visit phoenix.edu.


You are eligible to access the money in your MNDCP account at any age once you end employment (whether by retirement, resignation, permanent disability, or termination) .
Withdrawals may begin 30 days after your termination date.
If you return to work with a Minnesota public employer and commence contributing to your MNDCP account, you are not eligible to take a withdrawal from your MNDCP account until you end employment .


The focus of the third edition of the 'Museums in Arabia' conference encourages participants to engage in a broad discussion around architecture, art and design within the context of museums and galleries in the Gulf. The conference will be hosted by the College of Architecture, Art and Design at the American University of Sharjah in Sharjah, United Arab Emirates, April 18-20, 2017.
Authors are invited to submit paper proposals relating to architecture, art and design within the context of museums, galleries and heritage institutions in the Gulf region. This is an interdisciplinary conference and, as such, we welcome paper proposals from scholars and practitioners from a broad range of disciplines.
This conference will be of interest to academics and students working in the field of museums and cultural heritage in the region and globally, museum and gallery practitioners, art historians, artists, designers, architects, and, more broadly, those with an interest in the sociocultural, economic and political landscape of the region.
Please note that selected conference papers will be developed into a book, co-edited by conference organizers Dr. Sarina Wakefield and Seth Thompson. If interested in participating, please indicate so on your paper proposal submission. Details regarding the potential publication will be communicated after accepted participants have been notified.
For more information, please visit: http://www.aus.edu/mia2017 or download the attached document.


MONTREAL, Canada -- Mercedes driver Lewis Hamilton took a determined Sunday drive and dominated from start to finish to take his 56th career victory at Sunday's Canadian Grand Prix.
It's had already been a magical weekend for the "Jedi Knight" after taking his 65th career pole and equaling his hero Ayrton Senna. On top of this achievement, Hamilton received the love and respect of the Senna family in form of the F1 legend's 1985 race helmet, which signifies another huge milestone in Hamilton's prestigious career.
"It's been such an incredible weekend. I just couldn't be happier with how it's gone and I'm so grateful for this result. We came away from Monaco and we were scratching our heads, but we pulled together and look what we achieved. We came here with a much better understanding of the car and we delivered a real blow to the Ferraris. Valtteri did a fantastic job too and this is our first one-two finish together. We've scored a big load of solid points and it's well deserved. It's crazy to think I had my first pole and win here 10 years ago. The race actually felt very reminiscent of 2007, in terms of how it unfolded," said Hamilton.
Hamilton's teammate Valtteri Bottas took second and his 13th career podium giving Mercedes their first one-two finish of the season.
I'm so happy for us as a team to bounce back the way we did this weekend. It's so impressive to see how the team has reacted in the last two weeks – how it's worked and improved," said Bottas.
Red Bull's Daniel Ricciardo took third with a solid drive to make his third podium in a row this season. In response to the crowd, Ricciardo performed a "shoey" alongside iconic actor Sir Patrick Steward who happily joined in.
"Not the easiest third place but when I saw the chequered flag I was like: yes I can finally breathe. I had zero intention of doing a shoey again, but the crowd was chanting and then Sir Patrick (Stewart) got involved so I thought: alright, we might be on here. I think it turned out pretty good," said Ricciardo.
Ricciardo's teammate Max Verstappen was forced to retire after an amazing start from fifth to second when his car suffered a technical issue. Ferrari's Sebastian Vettel made a solid comeback to finish fourth as he was able to take advantage of the battle between Force India drivers Sergio Perez and Esteban Ocon.
Force India, who came out the box with a very strong chance at a podium, may have fallen apart when the team did not enforce orders on Sergio Perez. Perez was ordered to release teammate Esteban Ocon so Ocon could take a crack at Daniel Ricciardo. But, Perez did not follow these orders, which opened the door for Vettel to pass both drivers leaving them in fourth and fifth.
Vettel's teammate Kimi Raikkonen finished seventh after running into trouble with Renault's Nico Hulkenberg, who finished the day in eighth.
Williams' Lance Stroll took his first career points at his home race with an amazing drive to finish ninth. After what has been an incredible week for the 18yr filled with love and support from his fellow countrymen, he said "It means the world to me being able to finish a race in the points and be able to see those Canadian flags up in the air at my race."
Haas' Romain Grosjean took the final points-paying spot in tenth while Jolyon Palmer finished 11th in the second Renault. Twelfth went to Grosjean's Haas teammate Kevin Magnussen ahead of Sauber's Marcus Ericsson in 13th, who said after the race "I got the maximum out of the car in the race. We made a step forward with our tire management throughout the race."
McLaren-Honda's Stoffle Vandoorne finished in 14th, and Pascal Wehrlein in15th made up the final two finishing positions, with five other drivers forced to retire. The most notable retiree was Fernando Alonso who fought strongly for 10th before running into what he said was an "engine failure" and was forced to retire. Shortly after pulling off the track, Alonso made his way to the grandstands throwing his gloves into the crowd before heading back to the garage.
Hamilton's dominant win this weekend, along with Vettel's recovery leaves the championship down to 12 points with Mercedes back at the top of the constructors' championship by eight points.
This weekend's Canadian Grand Prix finished on a high note with an increase in attendance by 20% from last year as the sport continues to evolve under the new ownership of Liberty Media who continue to find new ways to get fans more involved.
Spectators in Montreal could follow all the action and analysis in real time as they were the first to experience free Wi-Fi throughout the weekend, a feature that was greeted with surprise and delight across social media.
This year's Canadian Grand Prix also saw the return of the raft race, which hasn't been around since the late 1990s. These additions, along with the exciting championship battle this season, brought many fans to the GP and kept them engaged. Many can hardly wait for the Azerbaijan Grand Prix in two weeks.


Full evaluation report of Yorkshire Wildlife Trust's programme to improve the water quality and biodiversity of natural heritage - Clifton Beck in West Yorkshire specifically. The project was funded by Heritage Lottery Fund (now the National Lottery Heritage Fund). The report is broken down in to exploration of volunteer, landowner and environmental outputs, outcomes and impact. Shared with permission from Yorkshire Wildlife Trust.
THE REPORT INCLUDES THE MAJORITY OF INFORMATION, THERE WILL BE MORE LEGACIES AND ACTIVITIES NOT DETAILED IN THESE PAGES.
A SEPARATE SUMMARY REPORT IS ALSO AVAILABLE.
close to the beck; liaise with partners; and oversee delivery of the work on natural habitats.
• Strand 1, Inform the community so that they understand the importance of the area.
of the water quality issues in the local area.
children understand where things they put down the drain could end up, and open the door to discussing wider issues.
increase access and raise the profile of the beck, changing the way it is viewed and cared for and giving them a greater sense of place.
• Strand 2, Engage local people to be more active in and informed about the beck.
and reporting on invasive species of plants along the beck.
o Recruit a local project trainee to develop conservation skills and work towards improved employability after the project.
• Strand 3, Collaborating with Land Managers to change the way land is looked after.
o Work with land managers of the three sites to create a plan for the managing the habitat on a longer-term basis.
• Strand 4, Act to Make a Difference improving habitat along the river.
supporting improved conditions for biodiversity.
o Install bat and bird boxes to support increasing populations.
to the intended outcomes of the project. School holidays and delays to the booking of school workshops prevented the in-depth teacher interviews.
period and he requested not to be contacted during that time.
(i.e. 24%) gave feedback which therefore provides an indication of the project but is not representative of all participants.
root and leaving to rot in a dark place or stomping on larger piles of pulled plants to ensure they can't develop.
Beech Whips: Infant beech trees which are removed so native bio-diversity nurturing plants can grow.
Brush Cutter: A large electrical strimming type tool for removing larger plants and high-volume foliage.
Coppicing: Cutting trees down to just above ground level, to help healthy new shoots grow.
weatherproof box. Finders log the find online and add feedback for others.
heritage for the benefit of the British public.
threaten the health of other species.
letting small amounts through – for instance through gaps deliberately built into piles of branches and leaves.
excessively fast flowing flood water.
documented. From this, a score is calculated identifying the health levels of the water.
This table shows the planned outputs of the project strands, with the actual totals on the right2.
75 children, 5 schools 146 children and young people engaged to date in 5 schools + 2 youth groups.
10 water monitoring points 9 locations identified. Monitoring occurred and will continue after this project (with Calder Rivers Trust).
additional voluntary hours. Now in full time employment as an Ecology Assistant.
22 trained, and now registered on a long term basis for future monitoring with Calder Rivers Trust.
15 landowners / managers 34 land owners and managers attended one of two land management workshops.
24 hectares improved and 1.3 km buffer zone created.
Woodland: 33 hectares improved 33 hectares improved.
Parkland: 6 hectares improved 9 hectares improved.
however all partners are keen to emphasise the real results will take much longer to see.
Final as of May 31st 2018, however the final total could be larger since some activity continued for weeks afterwards.
Other plans were also described out throughout the original funding application and project plan. These are reported below.
days / week) for 18 months trust to draw down additional funding and make the post full-time in latter stages.
partnership and Friends of Judy Woods.
Native trees planted 2356 trees planted across three sites over 14 tree planting days.
created stages of the project.
Beech tree whips removed 6 beech whip clearing days held.
days for Himalayan balsam 11 balsam pulling days held.
Fencing installed 790m fencing installed at Bobby Green Farm.
celebration events engaged through casual conversation during volunteer task days.
139 volunteers. 58 attended more than once and are now on YWT's ongoing volunteer register.
190 volunteer days invested 308 volunteer days recorded (approx. 1200 hours).
to it for a further three years.
100 postcards about the project printed and distributed.
Blackshaw Beck Lane as well as further work in Judy Woods and Bobby Green Farm.
Additional work at Whinney Hill. Being upstream from Wellholme Park, the benefits will filter down to the important habitats in the park.
Additional upstream highway improvements with installation of silt traps and gulleys to alleviate sedimentation, upstream from the 3 main sites.
Additional landowner engaged, and land improved on one of the farms to address high levels of sediment input at a cattle crossing point.
Additional events: bat walk, led by project officer; archaeology walk through Judy Woods led by Friends of Judy Woods.
Leaky dams installed at Judy Woods and Friends of Judy Woods trained in installation of Leaky Dams, to help control flooding.
Wildflower planting added to improvement measures at Judy Woods, for visual and pollinating benefits.
4 days of step installation at Wellholme Park to steer visitors (and dogs) towards more manageable, less damaging parts of the beck.
Willow spiling at Bobby Green Farm and Wellholme Park to reinforce riverbanks and provide habitat for native wildlife.
Litter picking across the sites on 8 occasions.
180 hours additional volunteering by project officer to Calder Rivers Trust for Clifton Beck and surrounding areas due to this project.
New 10-point plan resource for improved land management methods created specifically for urban users, land managers and horse riders.
One volunteer has already now led a further 5 task days with other volunteers in her local community.
or impairment that affects daily life?
and economic value to the partner areas, through business growth, employment and wellbeing – themes explored further in the outcomes section.
of volunteering on this project which will be likely to limit the numbers of those who think they can take part.
than the other. A tactical change to reach a new audience in the second workshop was very successful.
travelled from outside of West Yorkshire to take part.
Much of the evidence about community responses comes anecdotally from staff and volunteers (who are also part of the community of course).
the local walking (especially dog walking) community has become more informed.
An upsetting incident deflated the team part way through, but also demonstrated the strength of feeling about the project from the local community.
3. Local People Have Helped and Will Continue to Help Interpret the Work. Other Locals Are Already Understanding More and Changing Perceptions.
engagement with new and hyper-local audiences, and regular maintenance already being reported and acted on.
scores out of 10 for how much heritage learning they had gained, the results were as follows8.
• 44% scored 7 or above for new knowledge about social or industrial heritage (e.g. buildings, people and manufacturing from the past).
• 39% scored 7 or above for agricultural heritage (such as farming, crops or landscape design of the past).
for partners and funders to decide.
though more time and evidence are needed.
ensure outcomes could still be met.
3. Anecdotally the school workshops appear to have been effective though feedback from pupils directly would help verify this.
4. The biggest surprise is the success of innovative interpretation brought about in response to vandalism, in the form of geo-caching.
the YWT team who have not worked with geocaching before.
5. Community generated and shared interpretation has been especially effective.
ensure resources and opportunities are accessible for a variety of audiences.
• 139 volunteers gave over 1200 hours to the project.
• 19% had never volunteered before9.
• 58% volunteered more than once.
• Almost 10% volunteered more than 15 times.
above), including some hours outside of his formal role.
180 hours to their capacity.
• Many volunteers attended in a one-off or 'micro-volunteering' capacity.
their second visit. 70% of survey responses are from repeat volunteers. 30% are from micro-volunteers.
1. Skills were successfully gained across all the areas monitored.
leading volunteer balsam bashing task days.
• The water quality monitoring training was the best attended, attracting 20% of volunteers.
• Around 7% attended sessions learning how to identify invasive species, and how to manage invasive species.
• A small number (c3%) also attended the land management workshops (detailed later in the report).
of Judy Woods, this perhaps isn't surprising).
the Environmental Outcomes section on page 30.
1. Everyone achieved what they wanted to get out of the volunteering experience from a personal perspective.
towards a Duke of Edinburgh award.
health and wellbeing showed the strongest increases of all.
these outcomes occurred more strongly than any other area of development such as learning about heritage and learning new skills.
This shows the importance of getting to understand volunteer's personal aims and the strength of benefit that a tailored set of opportunities can enable.
This way of working was a factor volunteers drew attention to in their responses12.
commitment for the area is now being spread via his grandson who proudly tells his friends in school that Grandad is planting the trees in the park.
ongoing water quality monitoring in the area, and now volunteers on other YWT projects, and with the RSPB.
trainee with one of the trust's nature reserves.
having clarified aspects of the offer (such as curriculum links and free workshop), bookings for the following term started to take shape.
was completed within two months and has helped the team work more effectively across their many different projects thanks to a more efficient system.
use of pesticides; and the support of his line manager in helping him become more work-ready when the apprenticeship ended.
work, so the knock-on environmental and economic impact spreads further still.
1. The project attracted 139 volunteers who gave over 1200 hours.
3. Commitment levels were good, with over half repeat volunteering, and around 10% volunteering more than 15 times.
planting were the most prolifically attended task days with around 60% of volunteers having developed these skills.
5. Around 20% of volunteers attended training workshops.
6. Skills development was very well enabled informally on task days, and this was highlighted as one of the things YWT do best.
activity, more social activity, and a greater sense of connectivity to people, community and place.
recognised and contribute greatly to this success.
8. Through the traineeship, Joe developed many new skills, from improved communication, to project management and administration experience.
He now has a full-time job thanks to the project.
secured thanks to this project. In time Joe would like to focus on conservation work, continuing the impact of this traineeship.
workshop for land managers from farms, local authorities and environment agencies; one for horse riders and owners.
training in the future, there were no responses.
position in terms of relevant knowledge and made much larger gains in understanding by the end.
with workshop one, confidence in being able to do the right thing did show some progress, but at a lower level than other outcomes.
this project, the team hope to build more ambitious funding application plans with local councils to extend and expand the work.
upper reaches of the beck around Blackshaw Lane. YWT was able to lever match funding from an external source to double the council's own investment.
activity more compelling than explanations on paper and in meetings can achieve.
Calderdale Council gained new knowledge about techniques such as the installation of fascines to help structure the flow and crossing of the beck.
herself, and a trustee on their board of directors; adding approximately 180 hours extra capacity to the organisation during this period.
replaced with native species, and willow spiling has been put in place along the banks of the beck.
native species like birch and hawthorn.
results. More traditional land managers have been harder to get on board.
managers. Because commitment and confidence were lower than other outcomes for land managers, the potential to achieve change is far less certain.
have to unlearn other ways to be able to absorb new thinking.
2. Land will be better managed thanks to new and deeper relationships, immediate results, additional capacity, and structured future development.
partners to explore more ambitious fundraising and implementation for the future, hoping to expand similar work across other areas of the region.
legacies listed below help move the beck closer to the 'good' classification YWT and their partners hope to reach in due course.
• Improved water quality in the beck, particularly near Wellholme Park where the volume and number of species have doubled since 2016.
location have also been identified. As a non-native invasive threat to other species, these also need further work.
on an annual basis to reduce its presence, spread, and negative impact on other wildlife.
chemical levels in the water, in turn improving its quality and the biodiversity benefitting from it.
adjoining Bobby Green Farm also got involved, leading to additional hectarage benefitting.
pollinating flowers will enable more native species to thrive, improving the biodiversity of the area.
benefits further down the beck. So, the work will have wider and longer-term impact.
enable the officer to continue working in the area.
along other sections of Clifton Beck and as part of other work across the region.
keep going, if resources can be found.
of the natural heritage already being in better condition already exist.
3. Water quality at one monitoring point has significantly improved.
part of plans to tackle this issue.
through the water and shielded from threats from surrounding farmlands.
species with sympathetic native alternatives.
stabilize banks and reduce sedimentation entering the water.
upstream where the benefits will flow down through all the main sites.
and delivering what was agreed, on time, without needing reminders or supervision.
discussing and sharing ideas together.
their work, showing the power of their resilience.
ever increasing and improving is a rare achievement.
its own timetables. The project must fit with natural cycles not just administrative periods.
more successful than the first in all ways.
commitment, capacity, impact and added value – growing resources rather than draining them.
contingency / succession plans in mind as work develops.
appeal to those who can only commit one-off or very occasional contributions would help create a much more diverse and inclusive body of support.
volunteers would yield much greater resources for the trust and its partners.
activities. Additional work on extra sites upstream from the planned sites was developed so that knock-on impact was possible all the way downstream.
work. Of course, this only applies to skills which are appropriate to be carried out informally and unsupervised.
of the project. The specialist conservation expertise could be learned from colleagues.
combined recommendations below will help project officers feel more able to adapt when needed.
agreements. If funders know what is happening, they usually understand the need for things to change.
deciding what additional work to commit to. In this project extra volunteer hours and additional funding provided the extra resources needed.
successful than the originals and brought new practice, ideas, enthusiasm and audiences to YWT.
of their parts. For others, it may not be the right thing at the right time and trying to keep them engaged can drain rather than multiply resources.
8. Plan activity at strategic locations to have the highest possible impact: Activity is mostly very carefully planned to have a strong effect.
involved in advising what activity takes place where. Local knowledge can often spot issues that don't appear on paper.
advocacy for the project high; and can help them achieve life changing results such as getting back into work or securing new employment.
attracting and monitoring the micro-volunteering market of one-off helpers is equally valuable. It is also a more accessible and inclusive model.
YWT volunteers, so YWT will benefit from organisations who have supported micro-volunteering habits elsewhere.
work given its capacity to bring new audiences to specific sites, convey information, and nurture community response and conversations online.
remove many potential barriers to access.
work brings health, wellbeing and social rewards. This could and should be on the radar of more people who could benefit.
connected to and caring about the area.
The project is summarised using a logic model format of inputs – activities – outputs – outcomes – impact below.
* Items marked with an asterisk are Heritage Lottery Fund commissioned outcomes and outputs.
• £110k financial *66+ hectares improved.
Environment Agency; Collaboration *16km beck benefitting. *Heritage is better managed.
Trust; Bradford 11 Himalayan balsam bashing days and explained.
• Volunteer time Fencing, revetments, willow spiling, steps and More stable riverbanks.
• Natural environment leaky dams made and installed; litter picked; Reduced invasive species threat.
(6 locations) native flowers and trees planted. Improved habitats for wildlife.
• Machinery / New patches of invasive non-native species Better managed public access.
equipment/ identified. New natural heritage volunteers.
*6 land manager partners engaged. New and stronger partnerships.
*3 land management plans updated (in progress). *New interpretation skills.
3 ten-point plans created for horse riders; urban *New natural flood management skills.
*1 project officer + 1 project trainee recruited. conserving natural heritage.
Additional 180 volunteer hours to Calder Rivers Increased motivation / momentum.
monitoring. evidence for fundraising and advocacy.
schools + 2 youth groups. behaviour (new volunteers).
100 information postcards printed / distributed. management / conservation skills).
*139 volunteers over 60+ task days (average c5 Increased time outdoors.
days each). Increased physical activity.
Which volunteering activities did you take part in ?
personal reasons for getting involved?
gain any of these skills? (0 = not at all. 10 = Extremely).
TABLE 10 WHAT WERE YOUR OWN PERSONAL REASONS FOR VOLUNTEERING - WHAT DID YOU HOPE TO GET FROM IT?
Woods on a Wednesday which is our normal work morning.
help wildlife. Help keep fit. Meet people.
To benefit the community. Outdoor exercise in a team. To learn new and develop existing skills. To gain a sense of achievement.
I wanted to gain experience in different land management/NFM practices, for a hopefully future career in conservation.
getting a job in the Environmental Sector again.
Work experience and improved knowledge of Wildlife.
TABLE 11 IF YOU HAVE / WILL APPLY THIS LEARNING TO OTHER SITES OUTSIDE OF THIS PROJECT, PLEASE SAY MORE ABOUT THE SITE(S) AND WHAT YOU WILL DO?
How do I know what is going to come round the bend?
I am a member of Judy Woods (Friends of) any skills acquired will potentially utilised there.
I shall listen attentively where I can and attempt to emulate further skills I have learnt in coming task days.
TABLE 12 WHAT WAS THE BEST THING ABOUT VOLUNTEERING FROM YOUR PERSPECTIVE?
To learn the latest ideas on woodland up keep.
Satisfaction in doing valuable conservation work, and the confidence to do more in the future.
Being outdoors and giving back to the environment.
It improved my self-esteem, I'm proud that I have volunteered.
TABLE 13 WHAT WERE THE BIGGEST CHALLENGES ABOUT VOLUNTEERING, FROM YOUR PERSPECTIVE?
None as a FoJW we already do a lot of similar work.
If I can't attend on the days that task days are run on a regular basis.
Having spare time to go out and volunteer.
Finding out about volunteering opportunities in the area I live.
I'm not yet in a position to say.
TABLE 14 WHAT DID YORKSHIRE WILDLIFE TRUST AND PARTNERS DO ESPECIALLY WELL REGARDING THE VOLUNTEERING EXPERIENCE?
Seonaidh and Joe were excellent team leaders and well organised so the task days I attended were well run.
Dunno. Was quite well organised so I felt to be part of a competent organisation.
Extremely friendly staff. No matter your physical capabilities, they made tasks doable and adapted them to each individual.
Make you feel welcome, explain the tasks both the reason and how to carry them out.
attitude towards the project and about the work done and thanking for the help.
Explaining the benefits of doing the tasks I underwent.
TABLE 15 WHAT COULD YORKSHIRE WILDLIFE TRUST AND PARTNERS DONE TO HAVE IMPROVED THE VOLUNTEERING EXPERIENCE?
For myself I cannot think of anything.
Well, can't think of anything.
They did a great job, if I had more time available I would have been out a lot more.
They could have provided the upcoming tasks by email a bit more in advanced.
They could have involved volunteers in the preparation work and educated more about the area and theory behind the project and tasks.
I cannot comment as of yet.
I have already gained knowledge about Judy Woods.
a lot of useful and interesting knowledge.
TABLE 17 WHAT ELSE IS IMPORTANT FOR US TO KNOW ABOUT YOUR EXPERIENCE ON THIS PROJECT?
Seonaidh has kept the FOJW up to date through her presence and organised presentations. Many thanks.
do this type of work. I feel it is important for it to continue.
I believe the group has made a positive difference to the catchment and river system already during a short period of 1=1.5 years.
But it is important that the project carries on in some form to ensure it doesn't start deteriorating again.
I have thoroughly enjoyed. Life enhancing.
I'm not sure as of now.


Book 4? Any info or release date?
Hi, I was just looking through the demo version of book 3 in iBooks and saw that it referenced topics that are to be covered later in the series.
Do you have any estimate on release date etc for this (these?) book(s)?
Re: Book 4? Any info or release date?
We have a watchKit book in the works, but when Apple completely revamped the architecture for watchOS 2, it derailed us for a while. We're back at it again, but we currently don't have a release date.


text:
Eastern Arkansas deals with broken power lines, ice-covered bridges, and traffic jams for miles from ice storm
How eastern Arkansas is dealing with issues from the ice storm and winter weather
Electoral ballots 'rescued' as Trump supporters riot at Capitol
The Electoral College ballots were inside the Capitol as Congress was in a joint session to affirm President-elect Joe Biden's victory.
Published: 5:09 PM CST January 6, 2021
Updated: 5:28 PM CST January 6, 2021
As supporters of President Donald Trump violently clashed with law enforcement and stormed the U.S. Capitol Wednesday, Senate staffers acted quickly to grab the Electoral College vote certificates on the day Congress convened to affirm President-elect Joe Biden's victory.
"Electoral college ballots rescued from the Senate floor," Sen. Jeff Merkley, D-Ore tweeted along with a photo. "If our capable floor staff hadn't grabbed them, they would have been burned by the mob."
In what is a Constitutionally-mandated measure, a joint session of Congress convened Wednesday to count the electoral votes. As promised, dozens of House Republicans -- but still a minority of the party's Congressional members -- objected when the state of Arizona was read.
RELATED: US Capitol secure hours after Trump supporters riot, storm building
RELATED: President Trump tells supporters to 'go home' after violent clashes at Capitol
RELATED: Biden calls on Trump supporters to 'pull back,' urges decency
After members retreated to their respective chambers for the mandated two-hours of debate, rioters -- incited by President Donald Trump's baseless claims that the election was stolen due to widespread fraud -- made their way to the Capitol and forced their way inside.
The pro-Trump supporters were seen fighting with officers both inside the building and outside.
A woman who was shot inside the U.S. Capitol has died. That's according to two officials familiar with the matter who spoke to The Associated Press on Wednesday on condition of anonymity because they weren't authorized to speak publicly.
Vice President Mike Pence, who was presiding over the proceedings, was evacuated. House and Senate members were either evacuated or told to shelter in place before they could be taken from the building.
Credit: Sen. Jeff Merkley, D-Ore.
Electoral College ballots on the floor of the U.S. Senate that were secured after Trump supporters stormed the Capitol building, Jan. 6, 2021.
As darkness began to set in, law enforcement officials were working their way toward the last holdouts, using percussion grenades to try to clear the area around the Capitol. Big clouds of tear gas were visible.
Elections officials across the country, the former head of the federal Cybersecurity and Infrastructure Security Agency, and former Attorney General William Barr have said there is no indication of widespread fraud on a scale that would change the outcome of the November election. Biden won by 7 million votes over Trump and in the Electoral College by a 306-232 margin.

redpajama_set_name: RedPajamaCommonCrawl


If you like HDR images then you need to check out this set! I found them at pixelandpoly. There are some many nice pictures but these two are my favorite.
Its the beginning of December and I am already sick of winter. So today I successfully transported my mind away from the dark days of winter with the wonderful colors in the forest.
Winter to some people isn't very fun. I turn 30 this winter and every time it snows at my house I feel like a little kid again. Winter can also make for some amazing photos as you will see in this post. The very first one is my favorite!


In many countries, governments spend substantial amounts on measures of labour market policy (LMP) for unemployed workers with the aim of increasing their chances and speed of finding a job. Spending on LMP may go above 1% of GDP in some OECD countries (OECD 2015), reaching maximum burdens during recessions. During the Great Recession in the US, the OECD strongly recommended spending additional resources on supportive LMP, notably training and job search assistance (OECD 2012).
Surprisingly, empirical evidence on the effectiveness of LMP as a system is broadly missing, despite its widespread use in many OECD countries. In the last two decades a considerable empirical literature on the effects of single LMP measures – like training, job search assistance, or sanctioning – has emerged (see Card et al. 2015 for an overview). But in the real-world context of current unemployment insurance (UI) systems, several LMP programs run at once, following the policy strategies defined by public employment service (PES) authorities and implemented by caseworkers with discretionary powers. Depending on the goals of (local) policymakers, the weights on different types of programmes and 'philosophies' may differ – do they provide support or put pressure on job seekers?
We show how regime effects can be identified and estimated using detailed large-scale register data on job seekers in Switzerland. We discuss how to measure policy regime intensities and develop a framework to jointly estimate different effects – of regimes by PES agencies and caseworkers on the one hand, and of specific treatments on programme participants on the other hand. Moreover, we distinguish the effects of supportive policies ('carrots') – notably, training and job search assistance programs – from the effects of punitive policies ('sticks') – sanctions and workfare programmes. Assessing all these effects in one model offers the crucial advantage that we can evaluate the relative importance of different elements in the system as well as their interplay. This offers a substantially more comprehensive picture of the impacts that labour market policy really exerts on concerned individuals.
We exploit detailed register data for 130,000 individual job search spells for men of all ages in Switzerland between 2000 and 2005. Switzerland is particularly suitable for such an exercise since the different LMP intensities vary substantially across the country. We combine registers that report LMP events in daily precision, detailed socio-demographic and UI benefit-related eligibility information as well as individual social security earnings data for several years before and after unemployment. Importantly, we observe policy implementers – PES agencies and caseworkers – in the data as well. The study considers two individual labour market outcomes, which cover the mid run and the short run: average monthly earnings within the 3.5 years after unemployment exit on the one hand, and unemployment duration on the other.
Figure 1 visualises some key results on the effects of different types of regimes and treatments on mid-run earnings. As to be expected from existing evidence, being treated by at least one 'stick' significantly reduces individual earnings after unemployment; on the other hand, participating in a supportive programme affects earnings positively. However, on top of these effects directly arising from programme participation, we find economically relevant effects of supportive and punitive policy regimes. Both PES agencies and caseworkers contribute to these additional LMP effects. Increasing the intended frequency of use of restrictive policies by 10 percentage points (i.e., about half a standard deviation) reduces realised earnings by about 4%. Increasing the use of supportive policies, on the other side, improves earnings by about 2.5%. Interestingly, more intense use of both carrot and stick regimes causes significant reductions in unemployment duration. It is important to note that these regime effects apply to everyone at any time, not only to programme participants after treatment as in the case of the 'classical' treatment effects. This underlines the economic relevance of these newly identified LMP effects.
Notes: Effects are expressed in percent of average monthly earnings within 3.5 years after unemployment (3547 CHF = 3290 EUR = 3575 USD in sample). Treatment effects: effects of being exposed to at least one carrot (job search assistance, training) or stick (sanction, workfare program). Regime effects: marginal effect of changing policy intensity by 0.1.
We also address the question how labour market policy mixes could be optimised. Our study provides a set of 'experiments' which simulate how marginal policy regime changes would affect earnings and unemployment durations of the job seekers as well as the cost of UI. Figure 2 summarises the results for two 'experiments'. The first assumes that PES agencies and caseworkers would decide to increase the use of both types of regimes – carrots and sticks – by half a standard deviation (about 10 percentage points) above median policy intensity. The second assumes an increase in the supportive policy regime and a decrease in the punitive regime of the same size. The results are striking in documenting the relevance of policy regimes and their interplay. Intensifying both carrots and sticks generates a relevant reduction in individual unemployment duration (-20 days), at the expense of achieving lower earnings (-3%) after unemployment. UI saves about 6.2% of cost, mostly due to the reduced unemployment durations. The second experiment, which puts more weight on supportive regimes, is favourable for the individual's pay, at the expense of some additional cost for UI.
Notes: Marginal effects of changing the regimes by 0.5 standard deviation (i.e., about 0.1 in policy intensity). Earnings changes: in percent of average monthly earnings of non-treated individuals. Cost changes: in percent of total benefit cost per person.
This novel empirical approach of jointly assessing the effects of labour market policies demonstrates that we miss out on an important part of LMP impacts when only focusing on the consequences of single treatments. Policy regimes matter – their effect, which comes on top of classical programme effects, is significant. Considering regime effects can change the assessment of the total effect of certain types of policies on individual labour market outcomes. For instance, supportive policies are positive when taking into account regime effects. Only considering short-run treatment effect results in a negative evaluation, since unemployment duration appears to be prolonged (due to the so called 'lock-in' effect). However, when including regime effects the picture changes – supportive regimes as a whole can reduce unemployment durations and show positive impacts on individual earnings.
Applying a comprehensive framework to UI or welfare registers in different countries can offer instructive new empirical tools for policy optimisation.2 The framework can easily be extended to other different types of assessed policies. Applying it on a large scale, covering different policy intensities, can produce useful sets of estimates of marginal effects of LMP. These can be used as inputs for policy optimisation exercises. Policymakers need, however, to first fix their objectives. What is most important – reducing unemployment durations, improving individual job stability and welfare, or saving on the UI budget?
Arni, P, Lalive, R, and G J van den Berg (2015) "Treatment versus regime effects of Carrots and Sticks", IZA Discussion Paper 9457.
Blundell, R, M Costa Dias, C Meghir and J van Reenen (2004) "Evaluating the employment impact of a mandatory job search program", Journal of the European Economic Association, 2(4): 569–606.
Card, D, J Kluve and A Weber (2015) "What works? A meta-analysis of recent active labor market program evaluations", IZA Discussion Paper 9236.
OECD (2015) "OECD Employment Outlook", OECD, Paris.
OECD (2012) "OECD Economic Surveys: United States", OECD, Paris.
Pavoni, N, O Setty and G L Violante (2013) "Search and work in optimal welfare programs", NBER, Working Papers 18666.
van den Berg, G J, A H Bergemann and M Caliendo (2009) "The effect of active labor market programs on not-yet treated unemployed individuals", Journal of the European Economic Association, 7: 606–616.
1 There is a small empirical literature on anticipatory effects of possible future treatments on not-yet-treated individuals. See, e.g., Blundell et al (2004) for job search assistance programmes and van den Berg et al (2009) for training programs. Note that these effects only cover one particular part of regime effects and only the sub-group of not-yet-treated individuals for the specific programme.
2 There are some theoretical contributions to the issue of optimal policy program mixes. A current example is Pavoni et al (2013) who discuss the optimal combination of work-first and job-search-first programs.


text:
10 Black Ballerinas Other Than Misty Copeland Who Are Also Changing the Face of Ballet
Ayesha K. Faines
Kimberly Braylock, San Francisco Ballet
A New Jersey native, Kimberly received a full scholarship to the San Francisco Ballet school before earning a spot in the company in 2010. Since then, she's toured in London and China, and danced featured roles in The Nutcracker and in Christopher Wheeldon's Cinderella. In her spare time, Kimberly models for Discount Dance Supply, a popular dance wear catalog. She recently married San Francisco Ballet Orchestra musician Rufus Olivier.
Dara Holmes, The Joffrey Ballet
Dara Holmes is a mesmerizing dancer with a tragic past. When she was three, she pointed to a picture of a ballerina and said, "Mama, teach me this." Dara was eager to dance, but formal lessons were out of the question for her cash-strapped family. That is, until five years later when Dara attended an outreach program at the Wilmington School of Ballet. The school's owner, Elizabeth Hester, took notice of Dara's innate ability and awarded her a scholarship for as long as she needed. But several years later, a life-changing event threatened to derail all that she'd accomplished. On a November night in 2004, she and her mother were driving to a dance rehearsal when a speeding car struck theirs. Dara broke her collarbone. Her mother was killed. Plagued with feelings sorrow and guilt, Dara poured herself into her craft, treating dance like therapy. She won a scholarship to train with the prestigious Harid Conservatory where she spent three years polishing her skills. After graduation, she earned a spot with The Joffrey Ballet where she has since performed lead roles in Cinderella, Swan Lake, Paquita and Le Corsaire.
Black women in ballet
List of Black Ballet Dancers
Pictures of Black Ballet Dancers
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text:
Q: PHP equivalent to jsp:include PHP has include and require_once which are equivalent to JSP's include directive(<%@ include ..%>)
JSP also has a jsp:include which only includes the output from the included file, keeping the included file in a servlet of its own.
I am looking for something similar in PHP, so that the main page's variables and other content don't mess with those of the included files.
Does one exist?

A: An easy solution is to include the file inside a function to prevent the scope of the file from littering the global namespace.
function jsp_include($file) {
    include($file);
}

A: Doing it in OOP manner should also do the trick, in a more neatly manner.
servlet.php
class Servlet{

  private $servletVar1 = "Some string";
  private $servletVar2 = 2150;

  public function html(){
     echo "
Hello World!
";
  }

}

main.php
include("servlet.php");

class MainPage{

   private $title = "Page Title";

   public function html(){
       echo "<!DOCTYPE html>";
       echo "";
       echo "";
       echo "<title>".$this->title."</title>";
       echo "";
       echo "";
       $servlet = new Servlet();
       $servlet->html();
       echo "";
       echo "";
   }

}

$page = new MainPage();
$page->html();

A: You could always do a file_get_contents() and call the URL of that PHP script on your server, and then echo the results.  I would caution you though that this is very bad from a security standpoint.  If your DNS records get changed and what not, someone could really mess with things.  It is best to avoid this problem altogether by using OOP as "thephpdeveloper" suggested.  You can also use namespaces.

A: There's Runkit_Sandbox
Instantiating the Runkit_Sandbox  class creates a new thread with its own scope and program stack. Using a set of options passed to the constructor, this environment may be restricted to a subset of what the primary interpreter can do and provide a safer environment for executing user supplied code.
But I've never used it and therefore cannot say how reliable it is.

redpajama_set_name: RedPajamaStackExchange


Continuing the string of Ubisoft's live action trailers, with Assassin's Creed and The Division previously getting their own, Far Cry 5 has its own live action trailer, which you can watch above. It tells the story of how Joseph Seed (aka The Father, the antagonist) and Jerome Jeffries, a pastor in Hope County, came to be opposed to one another.
We've given Far Cry 5's co-op a shot, and it looks to be the key thing that will make Far Cry 5 stand out over its somewhat tired Far Cry clichés.
It appears that Jeffries is more than justified in not being Seed's biggest fan, as the trailer shows how Seed went into Jeffries' church, brought his congragation out and into Eden's Gate, and, eventually, killed a young woman in a baptism. The implication is that the young woman is Jeffries' daughter, and his congregation have watched on and followed Seed after he killed her.
It's a little bit grim, but hey, it's Far Cry, it's delighted in having some rather sick antagonists ever since Vaas graced our screens with his definition of insanity. Links to Ophelia, the famous painting, from the end of the trailer are also welcome.
Far Cry 5 comes out next month, March 27, for PC and consoles. Baptismal murders may or may not be involved.


Looking for a way to improve your coaching? How about attending a USATF Level 1 Coaching Course!
The USATF Level 1 Course is 21.5 hours on October 13th & 14th, 2018 at the University of Southern Maine. Instructors are Dave Kerin, Joel Pearson, and Katie Adams. More information, including registration is found here.


One of my favorite things to do in Pattaya during the day is to explore the city on foot. To wander up and down the sois and alleys looking for anything new or interesting. There is always some side street I haven't seen or new store's and restaurants opening up. Pattaya is never boring, and there is always something that engages me on my journey's about town. I always have my camera with me and enjoy documenting my time in Pattaya over the years.
This particular day my walk started mid-morning after fueling up on food before having a coffee over at Starbucks at the Pattaya Avenue Mall.
I noticed a new Craft Brewery had opened, at least it was new to me, called Beer Fest. It wasn't open yet, otherwise, I would have checked it out. I'll need to make plans to head back over there for a beer.
After having a look around I headed down Second Road, trying to stay in the shade of the stores as it was barely 11 AM and already starting to get hot. I headed up South Pattaya road stopping at a few places along the way. First up was the Honda shop.
While I'd love to have a scooter to zip around town, I just don't think it is safe driving one in Thailand. For now, I'll stick to walking and public transportation. This shop had a lot of scooters on display.
I found a place where I couldn't possibly get my laundry done. Though it may be appropriate for some.
If you are in the mood for some Italian food while in Pattaya, Volare on Soi Buakhao is a good place to try.
Since I brought up the topic of food. While I was part way into my walk, I got a call from a friend asking if I wanted to meet for lunch. I broke off my walk and headed down to Hooter's on Beach Road to meet him for some food and a cold beer.
I went all out and had their sample platter which had two types of hot wings and buffalo shrimp. Washed it down with a cold San Miguel Lite and I was a happy man. Though that platter set me back a few bucks, it is definitely not the cheapest thing on the menu. I'm sure I paid the same price I would have back home. But it was yummy.
Now that I was well fed and had a couple beers in me it was time to resume my photo walk. The Pattaya sun was really starting to beat down and I knew that I wasn't going to be out for much longer. I took a walk down Beach Road taking in the sights and sounds.
I kept walking down Beach Road when the craving for ice cream hit me. I ducked into Mike Shopping mall to cool off with a little air conditioning and see if they had a Dairy Queen. While they didn't seem to have a DQ on site, I did find a brand of Vodka that wouldn't be so well received back home.
Plus they had hats for dogs. Who hasn't thought to themselves, "what my dog really needs is a cowboy hat!" I'm going to file this under 'Only in Thailand' – at least I hope so.
After cooling off a little and having a good look around the shopping mall, I decided to head out and finish my walk. It's the only exercise I get so I don't like to slack on it. I decided to hike to the North end of Beach Road. The breeze off the ocean really helps keep me feeling cool.
I passed by the Hard Rock Hotel, which reminded me it's been a while since I've visited. They have good food and a live band that plays. At least they used to. I need to go back and check it out again.
I really like the renovation they did to the beach promenade a few years ago. There is plenty of room and the trees lining the side provides a bit of shade from the afternoon sun. They also put up a few interesting pieces of art.
Unfortunately, they have become another place for the poor and almost homeless expats to sit drinking their bottle of Leo in the afternoon. But this day they were all clear.
These heads on a stick really creep me out. At least this one wasn't staring at me.
I still can't figure out how they can fill all these high-rise condos that have been going up. With the crash in the Russian economy, my guess is they aren't.
I finally make it to the small shrine on Beach Road. I'm not sure who is being worshiped here, but I always see a few locals praying in making donations everytime I pass by.
My walk is stopped by some workers using a truck-mounted crane to lift bundles of heavy things over a makeshift wall blocking off the beach front. Their crane is going right over the sidewalk. I stop. Getting something heavy dropped on my head is not my idea of a good time. The worker operating the crane sees me and waves me through when it is all clear.
Of course, the wall telling me to keep out only piqued my curiosity. A little bit further down I found a place where I could have a peek inside. All I saw was bags of some unknown substance piled high and some pipes. I have no idea what they were doing.
I finally make it to the North end of Beach Road and I'm hot sweating and tired. It's time to head back home.
After I quickly shower and clean up, I decided to head back to Beach Road and walk on down to the Pattaya Beer Garden for a beer and some Penang Curry as the sun goes down. A perfect end to the day.


text:
Q: how to remove double white space at the end of string in R? I'm trying to fix some cells in a tibble. The column of my interest contains strings with white spaces and double white spaces at begin or end.
I saw these posts before asking

*

*Merge Multiple spaces to single space; remove trailing/leading spaces

*How to remove extra white space between words inside a character vector using?

*How to remove all whitespace from a string?

*How to trim leading and trailing whitespace in R?
This is a reproducible example of what I'm doing
library(dplyr)

mtcars2 = tbl_df(mtcars) %>%
   mutate(name = rownames(mtcars)) %>%
   mutate(name = gsub("^ *|(?<= ) | *$", "", name, perl = TRUE)) %>%
   mutate(name = gsub("^\\s+|\\s+$", "", name)) %>%
   mutate(name = iconv(name, from = "", to = "ASCII//TRANSLIT", sub = ""))

head(mtcars2, 3)

And the result is
# A tibble: 3 × 12
    mpg   cyl  disp    hp  drat    wt  qsec    vs    am  gear  carb          name
  <dbl> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl> <dbl>         <chr>
1  21.0     6   160   110  3.90 2.620 16.46     0     1     4     4     Mazda RX4
2  21.0     6   160   110  3.90 2.875 17.02     0     1     4     4 Mazda RX4 Wag
3  22.8     4   108    93  3.85 2.320 18.61     1     1     4     1    Datsun 710

But in my dataset after doing that some double white spaces persist !!
Is there a more general command to remove white spaces at the end of string? Many thanks in advance !

A: Have you tried the trimws() function?
> trimws('   hello   ')
[1] "hello"


text:
NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS| Volume 273, ISSUE 13, P7382-7389, March 27, 1998
Disruption of the DT Diaphorase (NQO1) Gene in Mice Leads to Increased Menadione Toxicity*
Venugopal Radjendirane
Pius Joseph
Ying-Hue Lee
Shioko Kimura
Andres J.P. Klein-Szanto
Frank J. Gonzalez
Anil K. Jaiswal
To whom correspondence should be addressed: Dept. of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498. Tel.: 713-798-7691; Fax: 713-798-3145
* This work was supported by National Institutes of Health Grant RO1 ES07943.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ The first two authors contributed equally to this work.
Open AccessPublished:March 27, 1998DOI:https://doi.org/10.1074/jbc.273.13.7382
Previous ArticleTn5 in Vitro Transposition *
Next ArticleThe Role of the α3(VI) Chain in Collagen VI Assembly
NAD(P)H:quinone oxidoreductase 1 (NQO1) is a flavoenzyme that catalyzes two-electron reductive metabolism and detoxification of quinones and their derivatives leading to protection of cells against redox cycling and oxidative stress. To examine the in vivo role of NQO1, a NQO1-null mouse was produced using targeted gene disruption. Mice lacking NQO1 gene expression showed no detectable phenotype and were indistinguishable from wild-type mice. However, NQO1-null mice exhibited increased toxicity when administered menadione compared with wild-type mice. These results establish a role for NQO1 in protection against quinone toxicity. The NQO1-null mice are a model for NQO1 deficiency in humans and can be used to determine the role of this enzyme in sensitivity to toxicity and carcinogenesis.
NQO1,
The abbreviations used are: NQO1, NAD(P)H:quinone oxidoreductase 1; bp, base pair(s); kb, kilobase pair(s); HSV, herpes simplex virus; TK, thymidine kinase; ES, embryonic stem.
1The abbreviations used are: NQO1, NAD(P)H:quinone oxidoreductase 1; bp, base pair(s); kb, kilobase pair(s); HSV, herpes simplex virus; TK, thymidine kinase; ES, embryonic stem.
previously known as DT diaphorase, is a flavoprotein that catalyzes two-electron reduction of quinones and their derivatives including azo dyes and nitroaromatic compounds (
Ernster L.
Chem. Scripta. 1987; 27A: 1-13
Talalay P.
Fahey J.W.
Holtzclaw W.D.
Prestera T.
Toxicol. Lett. 1995; 82–83: 173-179
Radjendirane V.
Jaiswal A.K.
Cadenas E. Forman H.J. Oxidative Stress and Signal Transduction. Chapman and Hall, New York1997: 441-469
). NQO1 is a cytosolic protein that is found ubiquitously in eukaryotes (
Li R.
Bianchet M.A.
Amzel L.M.
Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8846-8850
). In mammals, it is present in many organs but is most abundant in liver (
). However, in man, NQO1 activity is significantly higher in many extrahepatic tissues (
Martin L.F.
Patrick S.D.
Wallin R.
Cancer Lett. 1987; 36: 341-347
J. Biol. Chem. 1994; 269: 14502-14508
). The expression of the NQO1 gene is induced in response to a variety of agents including xenobiotics, oxidants, antioxidants, UV light, and ionizing radiation (
Xie T.
Xu Y.
Oncol. Res. 1994; 6: 525-532
). The obligatory two-electron reduction of quinones catalyzed by NQO1 competes with the one-electron reduction of quinones by enzymes such as NADPH-cytochrome P450 oxidoreductase and protects cells against redox cycling and oxidative stress (
Kappus H.
Sies H.
Experientia (Basell). 1981; 37: 1233-1241
Tampo Y.
Yonaha M.
Arch. Biochem. Biophys. 1996; 334: 163-174
). This protection is the result of conversion of quinones to hydroquinones compared with semiquinones and reactive oxygen species. The role of NQO1 in cellular protection is well documented for menadione and benzo(a)pyrene quinones (
). However, some hydroquinones can autoxidize to generate reactive oxygen species or alkylate DNA directly (
Cadenas E.
Biochem. Pharmacol. 1995; 49: 127-140
). In these instances, NQO1 catalyzes activation of such compounds to their ultimate toxic forms. This property of NQO1 along with the observation that NQO1 is expressed at higher levels in certain tumor types compared with normal tissues has been used to develop bioreductive chemotherapeutic agents (
Workman P.
Recent studies have characterized a C → T (nucleotide position 609, NQO1P203S) mutation in the NQO1 gene which results in loss of NQO1 activity (
Traver R.D.
Horikoshi T.
Danenberg K.D.
Stadlbaur T.H.W.
Danenberg P.V.
Ross D.
Gibson N.W.
Cancer Res. 1992; 52: 797-802
). This mutation in the NQO1 gene has been found in a human bladder carcinoma cell line (
Eickelmann P.
Schulz W.A.
Rohde D.
Schmitz-Drager B.
Biol. Chem. Hoppe-Seyler. 1994; 375: 439-445
), colon cancer (
Kolesar J.M.
Kuhn J.G.
Burris III, H.A.
J. Natl. Cancer Inst. 1995; 87: 1022-1024
), and fibroblasts taken from a cancer-prone family (
Kuehl B.L.
Paterson J.W.E.
Peacock J.W.
Paterson M.C.
Rauth A.M.
Br. J. Cancer. 1995; 72: 555-561
). In addition, Rosvold et al. (
Rosvold E.A.
McGlynn K.A.
Lustbader E.D.
Beutow K.H.
Pharmacogenetics. 1995; 5: 199-206
) demonstrated that the same C → T mutation was overrepresented significantly in lung cancer. More recently, Rothmanet al. (
Rothman N.
Smith M.T.
Hayes R.B.
Hoener B.
Campleman S.
Li G.L.
Dosemeci M.
Linet M.
Zhang L.
Xi L.
Wacholder S.
Lu W.
Meyer K.B.
Titenko-Holland N.
Stewart J.T.
Yin S.
Cancer Res. 1997; 57: 2839-2842
) reported an increased frequency of C → T mutations in the NQO1 gene associated with benzene poisoning. The presence of a mutation, together with the fact that the physiological function of NQO1 is to detoxify potentially mutagenic compounds, poses the question of whether or not a deficiency in NQO1 activity predisposes individuals to certain types of cancer.
In the present report, we used homologous recombination in embryonic cells to disrupt the NQO1 gene and generated knockout (NQO1−/−) mice that lack expression of the NQO1 gene. NQO1−/− mice showed no detectable phenotype and were indistinguishable from wild-type mice or heterozygous (NQO1+/−) and normal littermates. However, NQO1−/− mice exhibited increased toxicity to menadione compared with wild-type mice.
Isolation and Characterization of Mouse NQO1 Gene
A full-length rat NQO1 cDNA was used to screen a 129SVJ mouse λ genomic library from Stratagene (La Jolla, CA) by procedures described previously (
Biochemistry. 1991; 30: 10647-10653
). Several λ clones were characterized, and one, designated λEMBL3-mNQO1g-10, was used for further studies and analysis. To determine the nucleotide sequences encompassing the exon-intron junctions of mouse NQO1 gene, oligonucleotides 15–17 bp in length were selected at random from the corresponding mouse NQO1 cDNA (
Vasiliou V.
Theurer M.
Puga A.
Reuter S.F.
Nebert D.W.
) and used for sequencing NQO1 gene fragments subcloned into pUC18 by procedures described previously (
Sal G.D.
Manfioletti G.
Schneider C.
BioTechniques. 1989; 7: 514-519
). The process of sequencing continued until the coding region from mouse NQO1 gene was completely covered.
Construction of the Targeting Vector
The targeting vector was constructed using the pPNT vector having positive selection neomycin (G418) resistance and negative selection thymidine kinase markers (
Tybulewicz V.L.J.
Crawford C.E.
Jackson P.K.
Bronson R.J.
Mulligan R.C.
Cell. 1991; 65: 1153-1163
Scopus (1159)
). To disrupt the NQO1 gene, a targeting plasmid containing a deletion of exon 6 of the NQO1 gene was constructed. The 2.0-kbBamHI fragment containing a portion of intron 5, exon 6, and a small portion of the 3′-flanking region of the NQO1 gene was replaced by the 2.0-kb BamHI fragment from the pPNT vector containing the bacterial phosphoribosyltransferase II gene conferring G418 resistance (Fig. 1). The targeting vector contained 3.4 kb of homologous 5′-sequence and 1.4 kb of homologous 3′-sequence of the neo-cassette. A herpes simplex virus thymidine kinase (HSV-TK) gene inserted at the 3′-end of the construct allowed the use of a positive-negative selection scheme. To construct the targeting vector, a 7.0-kb XbaI-EcoRI fragment was isolated and subcloned at the XbaI-EcoRI site of pUC19 to convert an XbaI-SalI site. In the plasmid pUC19, the polylinker region contains an SalI site 5′ to the XbaI site. Therefore, the SalI site was simply added to the XbaI site during subcloning of the 7.0-kb XbaI-EcoRI fragment in pUC19. The resultant plasmid pUC19-mNQO1 gene (SalI-EcoRI) was opened at the EcoRI site, made blunt ended, and SalI adapters were added. These manipulations resulted in conversion of the XbaI and EcoRI ends toSalI ends and loss of the BamHI site in the polylinker region of pUC19. The loss of the BamHI site from pUC19 was achieved during digestion of pUC19 withXbaI-EcoRI to generate a vector plasmid to subclone the 7.0-kb mNQO1 gene. The BamHI restriction site is located between the XbaI and EcoRI sites of the polylinker region in pUC19. The digestion of pUC19 withXbaI and EcoRI removed the portion of the polylinker region between XbaI and EcoRI containing the BamHI site, resulting in loss of the BamHI site. The plasmid pUC19-mNQO1 gene (SalI-SalI) was digested with BamHI to remove 2.0 kb of the mouse NQO1 gene containing a portion of intron 5, exon 6, and a small portion of the 3′-flanking region. The 2.0 kb of the mNQO1 gene in the plasmid pUC19-mNQO1 gene was replaced with 2.0 kb of neo-cassette (derived from pPNT vector by digestion withXhoI, addition of BamHI linkers, and redigestion with BamHI to obtain a 2.0-kb neo-cassette). The 7.0 kb of the SalI-SalI fragment from the NQO1 gene containing 2.0 kb of the neo-cassette instead of exon 6 of the mNQO1 gene was isolated and subcloned at the SalI site of the pMC1tk-pA plasmid to generate the targeting vector pPNT-mouse NQO1 gene (Fig. 1). Four plasmids were produced with various orientations of neo- and HSV-TK cassettes. The HSV-TK cassette was under the control of the HSV-TK promoter/enhancer.
Figure 1Genomic sequence and targeting construct of the mouse NQO1 gene. Black boxes represent exons 1–6 of the mouse NQO1 gene. N, NcoI; X,XbaI; B, BamHI, E,EcoRI. The EcoRI-NcoI probe used to screen ES cells for homologous recombination is indicated.
Electroporation, Selection of ES Cells, and Generation of Chimeric Mice
Genome Systems (St. Louis, MO) mouse embryonic stem (ES) cells were used for homologous recombination and deletion of exon 6 from the NQO1 gene. The ES cells were thawed and diluted with ES cell medium (HEPES-buffered Dulbecco's modified Eagle's medium, 15% fetal bovine serum (Hyclone), 55 μm β-mercaptoethanol, 0.1 mm nonessential amino acids, penicillin-streptomycin, 1,000 units of leukemia inhibitory factor/ml), pelleted by centrifugation, and resuspended in ES cell medium. The ES cells were plated onto a 60-mm Petri dish previously seeded with mitotically inactive, γ-irradiated mouse embryonic fibroblasts. After 2 days, fresh medium was added to the ES cells, incubated for 4 h, treated with 0.25% trypsin and EDTA buffered with HEPES, resuspended at 2.5 × 105/60-mm-diameter plates with electroporation buffer (Hanks' balanced salt solution, 20 mm HEPES buffer, 0.11 mm β-mercaptoethanol, pH 7.2) and used for electroporation.
Supercoiled targeting plasmid DNA was prepared by banding twice with cesium chloride and linearized with HindIII. The DNA was cleaned, precipitated, and resuspended at a concentration of 2 μg/μl sterile distilled water. 50 μg of linearized plasmid was ethanol precipitated and resuspended in 50 μl of electroporation buffer and electroporated in ES cells with a Bio-Rad gene pulser. The electroporation was carried out at 250 V, 250-microfarad capacitance. The electroporated ES cells were plated onto 60-mm Petri dishes containing mitotically inactive mouse embryonic fibroblasts with ES cell medium. One day after electroporation, cells were doubly selected with the drugs G418 (300 μg/ml) and gancyclovir (2 μm). After 1 week of double selection, the ES cell clones were picked up under a dissecting microscope, transferred to individual wells of a 96-well plate containing 25 μl of 0.025% diluted HEPES-buffered trypsin-EDTA, and dissociated by pipetting up and down six times. Dissociated clones were transferred to wells of a 24-well plate containing mitotically inactive mouse embryonic fibroblasts with continued double-selection pressure. The clones were expanded and treated with trypsin. One-fourth of each clone was used to make DNA for analysis, and three-fourths were frozen in 10% dimethyl sulfoxide and 20% fetal bovine serum in ES medium in liquid nitrogen.
The ES cells with homologous recombination were detected by genomic Southern analysis using an EcoRI-NcoI fragment (280 bp) of the mouse gene from the 3′-flanking region as probe (the position of the probe is shown in Fig. 1). Positive ES cell clones with homologous recombination and deletion of exon 6 were detected in the ratio of 1:140 to ES cell clones. The ES cells from the positive clone mNQO1g-46 were injected into blastocysts, and chimeric animals were then bred to produce F1 mice. Southern blots were used to identify germ line transmission, and the heterozygous animals were interbred to produce homozygous knockout mice that do not express the NQO1 gene.
Southern and Northern Blot Analysis
DNA was isolated from ES cells and mouse tails by the procedure described by Laird et al., (
Laird P.W.
Zijderveld A.
Linders K.
Rudnicki M.A.
Jaenisch R.
Berns A.
Nucleic Acids Res. 1991; 19: 4293
). DNAs were digested overnight with NcoI, electrophoresed on 1.0% agarose gel, blotted, and hybridized with a 280-bp EcoRI-NcoI fragment from the 3′-flanking region of the mouse NQO1 gene by standard procedures (
Sambrook J.
Fritsch E.F.
Maniatis T.
Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989
). Southern blots were washed, exposed to x-ray films, and subjected to autoradiography. In a related experiment, the DNAs from wild-type (NQO1+/+), heterozygous (NQO1+/−), and NQO1−/− mice were digested with BamHI, run on agarose gel, blotted, and hybridized with 1.1 kb of human NQO1 cDNA (complete coding region) and the 2.0-kb neo-cassette probes. The NQO1 cDNAs are highly conserved among humans, rats, and mice and are known to hybridize each other in Southern analysis (
McBride O.W.
Adesnik M.
). The blots were washed and autoradiographed.
Total RNA was isolated from various tissues including liver, lung, kidney, colon, and skeletal muscle by the procedures described (
Chirgwin J.M.
Przybyla A.E.
MacDonald R.J.
Rotter W.J.
Biochemistry. 1979; 18: 5294-5304
Scopus (16653)
). 30 μg of the RNA was subjected to electrophoresis on a 1.0% agarose gel containing formaldehyde, blotted, and hybridized with 310 bp of exon 6 probe from human NQO1 cDNA (
Church G.M.
Gilbert W.
NQO1 Activity and Western Blot Analysis
The various tissues (liver, lung, kidney, colon, and skeletal muscle) from wild-type, NQO1+/−, and NQO1−/− mice were homogenized in 50 mmTris, pH 7.4, containing 0.25 m sucrose and centrifuged at 105,000 × g for 1 h to obtain cytosolic fractions. Dicoumarol-sensitive NQO1 activity was measured in all cytosolic fractions by a method reported earlier (
Shaw P.M.
Reiss A.
Schembri J.
Eur. J. Biochem. 1991; 195: 171-176
). The various cytosolic fractions were also analyzed for the presence or absence of NQO1 protein by Western blot analysis as described previously using antibodies against purified rat liver NQO1 protein (
). The rat NQO1 antibody is known to cross-react with mouse and human proteins (
Burnett P.
). Western blots were developed with ECL (Amersham) reagents by the procedure suggested by the manufacturer.
Menadione Toxicity
6–8-week-old wild-type, NQO1+/−, and NQO1−/− mice were used. Menadione (vitamin K3) was dissolved in dimethyl sulfoxide and was administered intraperitoneally at doses of 0, 2.5, 5, 10, and 20 mg/kg of body weight. Animals were given a single dose every day for 3 consecutive days. Animals were observed daily for symptoms of toxicity and mortality. 24 h after the last dose of menadione, blood was drawn from the surviving animals. The activities of alanine aminotransferase and aspartate aminotransferase were analyzed in the serum of each animal using the diagnostic kit purchased from Sigma.
Genomic Structure of Mouse NQO1 Gene
The structure of the mouse NQO1 gene was found to be similar to that reported for the human NQO1 gene. Like the human NQO1 gene, the mouse NQO1 gene contained six exons interrupted by five introns. The splice junctions and nucleotide sequences in the various exons were highly conserved between the human and mouse genes (Table I).
Table IComparison of human and mouse NQO1 genes
Exon no. human/mouse
Exon size
Sequence at the beginning of exon
Sequence at the end of exon
Human 118 CCCACA TGGTCCgt
Mouse 115 CTGAGC TGGCGGgt
Human 165 agGCAGAA TCACAGgt
Mouse 165 agCGAGAA TCACAGgt
Human 131 agGTAAAC TTCCAGgt
Mouse 131 agGTGAGC TTTCAGgt
Human 114 agTTCCCC TTCCGGgt
Mouse 114 agTTCCCA TTCCAGgt
Human 102 agAGTAAG ATTCAGgt
Mouse 102 agAATAAG ATTCAGgt
Human 1833 agAGTGGC CTGCAG(A)n
Mouse 901 agAGTGGC CACATC(A)n
Production of NQO1−/− Mice
The structure of the targeting vector pPNT-mouse NQO1 gene is shown in Fig. 1. This construct was used successfully to generate NQO1−/− mice. The 5′- and 3′-homologous genomic sequences were 3.4 kb and 1.4 kb long, respectively. In the targeting vector, a 2.0-kbBamHI fragment containing exon 6 of the NQO1 gene was replaced with 2.0 kb of neo-cassette. This replacement was engineered to delete the carboxyl 101 amino acids of the NQO1 enzyme. This design would effectively disrupt NQO1 gene function. The decision to delete exon 6 was based on two important observations. First, deletion of 73 amino acids from the COOH terminus of the NQO1 cDNA resulted in a shorter protein and complete loss of NQO1 activity in transfected COS1 cells.
P. Joseph and A. K. Jaiswal, unpublished observations
Second, the C → T mutation resulting in the loss of NQO1 activity was reported to be in exon 6 of the NQO1 gene (
DNA from selected ES cells was analyzed by Southern blotting to screen for homologous recombinants (Fig. 2 A). The digestion of DNA from ES cells (NQO1+/+) and wild-type mouse with NcoI and hybridization with 280 bp of EcoRI-NcoI fragment from the mouse NQO1 gene revealed the presence of a 9.4-kb band (Fig. 2 A). Of the 140 ES clones analyzed, one NQO1+/− heterologous ES cell clone (mNQO1g-46) was identified. The presence of a 2.4-kb NcoI fragment in a genomic Southern (Fig. 2 A) indicated that exon 6 is replaced with the neo-cassette as depicted in Fig. 1. The homologous recombination-positive ES cells were used to generate chimeric mice, and germ line transmission was detected. Heterozygous mice from the F1 generation were normal and were interbred to generate homozygous NQO1−/− mice. The heterozygous and homozygous mice DNAs were analyzed for the presence of mutant allele(s) of NQO1 gene carrying deletion of exon 6 (Fig. 2 A). The absence of a 9.4-kb NcoI band and the presence of a 2.4-kbNcoI band clearly indicated that homozygous NQO1−/− mice were born (Fig. 2 A mice). In addition, the absence of exon 6 and the presence of the neo-cassette were confirmed by digestion of genomic DNA with BamHI followed by Southern analysis and hybridization with NQO1 cDNA and the neo-cassette probes (Fig. 2 B). Southern analysis of DNA from the wild-type mice digested with BamHI and hybridization with NQO1 cDNA showed two bands of 8.0 and 2.0 kb (Fig. 2 B). The 8.0-kb band contains exons 1–5 of the mouse NQO1 gene, and the 2.0-kb band contains exon 6 of the mouse NQO1 gene as shown in Fig. 1. The wild-type mice DNA did not hybridize to the neo-cassette probe (Fig. 2 B). In a similar Southern analysis experiment, the NQO1−/− mice DNA showed only an 8.0-kb BamHI band hybridizing to the NQO1 cDNA (Fig. 2 B). The 2.0-kbBamHI band containing exon 6 of the mouse NQO1 gene was absent in NQO1−/− mice DNA upon hybridization with NQO1 cDNA (Fig. 2 B). However, the NQO1−/− mice DNA hybridized with the neo-cassette probe and, as expected, showed a band of 2.0 kb upon digestion with BamHI. This clearly indicated that the 2.0-kb neo-cassette has replaced the 2.0-kb BamHI fragment containing exon 6 from the mouse NQO1 gene in NQO1−/− mice. In the same experiment, as expected, NQO1+/− DNA hybridized with both exon 6 and neo-cassette probes.
Figure 2Southern blot analysis of DNA. Panel A, 10 μg of DNA from the homologous recombination-positive ES cell clone mNQO1g-46 and mouse tails were digested withNcoI, electrophoresed on 1.0% agarose gel, blotted to nylon membranes, probed with 32P-labeledEcoRI-NcoI DNA fragment from the 3′-flanking region of the NQO1 gene, and subjected to autoradiography. Two different sets of mice data are shown. +/+, wild-type; +/−, heterozygous; −/−, NQO1-null allele. Panel B, 10 μg of DNA from wild-type, heterozygous NQO1 +/−, and NQO1−/− mice were digested with BamHI, run on agarose gel, Southern blotted, and probed with human NQO1 cDNA and the neo-cassette probes.Numbers on the left indicate estimated sizes of DNA fragments (in kb).
Analysis of NQO1−/− Mice
Viability and Fertility
The NQO1−/− mice were found to be normal in appearance and showed no discernible difference in their weight, development, or in their behavior compared with their wild-type NQO1+/+ littermates. This was true for both male and female mice. At 6 weeks of age, wild-type NQO1+/+, heterozygous NQO1+/−, and homozygous NQO1−/− mice were killed for gross and histological examination. The organs and tissues examined histologically included liver, lung, kidney, colon, stomach, duodenum, spleen, thymus, lymph nodes, heart, brain, and skeletal muscle. No obvious anatomical differences in these organs were seen. In addition, the knockout animals appeared to have normal reproductive capacity compared with wild-type mice.
Northern Analysis
Analysis of RNA from five different tissues (liver, lung, kidney, colon, and skeletal muscle) by hybridization with the exon 6 probe indicated that NQO1 mRNA was present in wild-type and heterozygous NQO1+/− animals and absent in NQO1−/− mice (Fig. 3 A). The NQO1 mRNA was lower in heterozygous mice compared with wild-type mice.
Figure 3Northern and Western analysis. Panel A, Northern analysis. 30 μg of RNA isolated from liver, lung, kidney, colon, and skeletal muscle of wild-type, heterozygous NQO1+/− and NQO1−/− mice were electrophoresed on formaldehyde gel, blotted to nylon membranes, hybridized with 310 bp of exon 6 probe from human NQO1 cDNA, and subjected to autoradiography. The blot after stripping off the NQO1 probe was rehybridized with a β-actin probe. Results are shown only for liver RNA. RNA from other tissues produced results similar to those of liver RNA and are not shown. The exposure time for NQO1 and β-actin mRNA was 24 h. Panel B, Western analysis. Cytosolic proteins from liver, lung, kidney, colon, and skeletal muscle were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. Western blots were probed with antibodies against rat NQO1 and developed with ECL (Amersham) reagents. +/+, wild-type; +/−, heterozygous; −/−, NQO1-null allele.
Analysis of NQO1 Protein
Analysis of cytosolic proteins from the various tissues (liver, lung, kidney, colon, and skeletal muscle) by SDS-polyacrylamide gel electrophoresis and Western blotting indicated the absence of the 32-kDa NQO1 protein in all of the tissues of NQO1−/− mice (Fig. 3 B). In similar experiments, NQO1 protein was detected in all of the tissues of wild-type mice. The NQO1 protein was also detected in all of the tissues of heterozygous mice. However, the NQO1+/− mice demonstrated the presence of intermediate amounts of NQO1 protein between NQO1−/− and wild-type mice. Western analysis of the various tissues with NQO1 antibody also showed the presence of a <30-kDa cross-reacting band in all tissues of wild-type, heterozygous NQO1+/−, and NQO1−/− mice (Fig. 3 B).
NQO1 Activity
The levels of dicoumarol-sensitive cytosolic NQO1 activity in the various tissues of wild-type, heterozygous NQO1+/−, and NQO1−/− mice are shown in Fig. 4. Among the five tissues tested, the highest levels of cytosolic NQO1 activity were observed in kidney followed by colon of wild-type mice. Livers from wild-type mice showed only one-fifth of the NQO1 activity observed in kidney. The level of NQO1 activity in lungs and skeletal muscles of wild-type mice was reduced further to 10% of NQO1 activity in kidney. The kidney and colon showed the complete absence of NQO1 activity in NQO1−/− mice. However, liver, lung, and skeletal muscle showed some NQO1 activity, which was no more than 15% of NQO1 activity in the respective tissues of wild-type mice. Heterozygous NQO1+/− mice tissues showed levels of NQO1 which were intermediate between NQO1−/− and wild-type animals.
Figure 4NQO1 activity. The cytosolic fractions from the liver, lung, kidney, colon, and skeletal muscle of wild-type NQO1+/+, heterozygous NQO1+/−, and NQO1−/− mice were analyzed for dicoumarol-sensitive NQO1 activity by procedures as described (
). The NQO1 activity in various tissues is expressed as nmol of 2,6-dichlorophenolindophenol reduced/min/mg of cytosolic proteins.
Survival of Animals
Exposure of wild-type, heterozygous (NQO1+/−), and knockout (NQO1−/−) mice to different concentrations of menadione revealed a dose-dependent response for animal survival. A significant difference in the survival of wild-type and NQO1−/− mice was observed with a doses of 5, 10, and 20 mg of menadione/kg of body weight (Fig. 5 A). The survival rate was determined to be 100% wild-type, 70% heterozygous NQO1+/−, and 30% knockout NQO1−/− mice with 10 mg of menadione/kg of body weight.
Figure 5Menadione toxicity of wild-type (NQO1+/+), heterozygous NQO1+/−, and NQO1−/− mice. The wild-type, heterozygous, and knockout mice were injected intraperitoneally with different doses of menadione dissolved in dimethyl sulfoxide. It included 0 (dimethyl sulfoxide control), 2.5, 5.0, 10, 20, and 30 mg of menadione/kg of body weight (b.w.). 10 animals were injected in each group. The various doses were injected once a day for 3 consecutive days. Survival of the animals was followed every day of the experiment. 24 h after the last dose, blood was drawn from the various surviving animals and analyzed for serum levels of aspartate aminotransferase and alanine aminotransferase activities. Panel A, all of the animals in the various groups receiving 2.5 mg and lower doses of menadione/kg of body weight survived. However, significant differences in the survival of animals between NQO1−/− and wild-type were observed with 5, 10, and 20 mg of menadione/kg of body weight. The differences in the percentage of surviving animals with 10 mg of menadione/kg of body weight are shown. Panels B and C, levels of aspartate aminotransferase and alanine aminotransferase activities in the serum of wild-type NQO1+/+, heterozygous NQO1+/−, and NQO1−/− mice as a function of dose of menadione. The values are the mean ± S.E. of 10 animals. No measurement of enzymes could be done in the case of NQO1−/− mice administered 20 mg of menadione/kg of body weight because none of the animals survived the 3 days of treatment.
Levels of Aspartate Aminotransferase and Alanine Aminotransferase in Serum of Wild-type, Heterozygous, and Knockout Mice as an Indication of Liver Damage
The levels of aspartate aminotransferase and alanine aminotransferase in the serum of wild-type, heterozygous, and NQO1−/− mice are shown in Fig. 5 B. Both of these enzymes were found elevated in NQO1−/− mice with doses of 2.5 and 5 mg of menadione/kg of body weight. The corresponding doses of menadione had no effect on these levels in the serum of wild-type mice. Heterozygous mice showed intermediate sensitivities to menadione. 10 mg of menadione/kg of body weight produced elevated levels of aspartate aminotransferase and alanine aminotransferase in the serum of both wild-type and NQO1−/− mice. However, the magnitude of elevation was significantly more in the NQO1−/− mice compared with wild-type mice.
Several lines of evidence indicate that modification of the NQO1 gene by replacing exon 6 with the neo-cassette resulted in a null mutation. NQO1 mRNA and protein were not detected in NQO1−/− mice. The NQO1 activity dropped from very high levels in kidney and colon of wild-type mice to zero in NQO1−/− mice. The NQO1 activity also dropped significantly (>85%) in other tissues including liver, lung, and skeletal muscle. We believe that residual amounts of NQO1 activity observed in liver, lung, and kidney tissues of NQO1-null mice are caused by NQO1-related protein(s) rather than NQO1. This is clearly evident from the fact that the NQO1 protein was absent in these tissues of NQO1-null animals as determined by Western analysis. These NQO1-related protein(s) must be tissue-specific because NQO1 activity was not detectable in kidney and colon tissues of the NQO1-null animals. The small amount of NQO1 activity detected in liver, lung, and skeletal muscle of these mice is probably not caused by a <30-kDa band detected just below the 32-kDa NQO1 bands in Western blot analysis because this band was also present in kidney and colon tissues of NQO1-null mice when no measurable NQO1 activity was found. The <30-kDa band may be NQO1-related protein with no NQO1 activity or an artifact of cross-reaction of the NQO1 antibody with an unrelated protein. It remains a possibility that the <30-kDa band is cytosolic NQO2 (
). Human NQO2 is a 27-kDa protein that cross-reacts with antibody against NQO1 (
). It is also known that NQO2 requires reduced dihydronicotinamide riboside instead of NAD(P)H as cofactor (
Zhao Q.
Yang X.L.
). Its activity with NAD(P)H as cofactor is also very low as measured in the present studies. The identification of the <30-kDa band as NQO2 awaits additional experimentation.
The loss of NQO1 in knockout mice did not affect the development and viability of mice. This was expected because a small percentage of the adult human population is known to be homozygous for mutant alleles of NQO1 (
). The C → T mutation in exon 6 of these individuals results in a proline → serine change and the loss of NQO1 activity. Humans with the NQO1-null genotype exhibited no developmental or physiological abnormalities. However, it is expected that NQO1−/− mice and humans with the NQO1−/−-null genotype would be more susceptible to free radical damage and development of toxicity from exposure to quinones, their derivatives, and redox cycling compounds.
Interestingly, the NQO1 was shown to play an important role in the maintenance of the reduced form of coenzyme Q in membranes which provides protection to the membranes against free radical damage (
Beyer R.E.
Segura-Aguilar J.
DiBernardo S.
Cavazzoni M.
Fato R.
Fiorentini D.
Galli C.
Setti M.
Landi L.
Lenaz G.
Dallner G.
Biochim. Biophys. Acta. 1995; 1271: 195-204
). Thus, NQO1−/− mice lacking the expression of NQO1 would be expected to have a significantly lower capacity to reduce coenzyme Q, leading to free radical-induced damage to membranes and DNA and premature aging of the animals. However, the extent of damage would also depend on the presence of other NQO1-related protein(s) that catalyze the reduction of coenzyme Q. This is clearly an area of great future interest.
NQO1 activity is also a part of defensive network within the cells which protects against redox cycling, oxidative stress, and other toxic effects caused by exposure to quinones and their derivatives (
O'Brien P.J.
Chem.-Biol. Interact. 1991; 80: 1-41
Riley R.J.
Biochem. Pharmacol. 1992; 43: 1657-1669
). NQO1 activity was shown to prevent the formation of highly reactive quinone metabolites (
Chesis P.L.
Levin D.E.
Ames B.N.
), detoxify benzo(a)pyrene quinones (
Lind C.
), and reduce Cr(VI) toxicity (
De Flora A.
). Recently, benzene poisoning, a risk factor for hematological malignancy, has been shown to be associated with the NQO1 C → T mutation (
). It is a well known fact that induction of NQO1 and other detoxifying enzymes is one mechanism of critical importance in chemoprevention (
Rushmore T.H.
Pickett C.B.
Biochem. Pharmacol. 1994; 47: 25-37
). Therefore, many compounds that block toxic, mutagenic, and neoplastic effects of carcinogens share in common the ability to elevate levels of detoxifying enzymes including NQO1, glutathioneS-transferase, and UDP-glucuronosyltransferases (
Wattenberg L.W.
Cancer Res. 1985; 45: 1-8
Cancer Res. 1992; 52: 2085S-2091S
). Studies with NQO1-null mice lacking the expression of NQO1 gene clearly support a protective role of NQO1 against menadione toxicity. NQO1-null mice were more sensitive to menadione than wild-type mice. 70% of the null mice died when exposed to 10 mg of menadione/kg of body weight. On the other hand, no deaths were observed with a similar dose of menadione administered to wild-type mice expressing the NQO1 gene. In addition, serum levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase as markers for hepatotoxicity were found to be significantly different in NQO1-null animals compared with wild-type animals. Elevation of these liver enzymes, which are considered to be a measure of liver cell death, were detected at doses of 2.5 and 5 mg/kg of body weight in NQO1-null animals but were more or less unchanged at these doses in wild-type mice. These data indicated that liver damage is involved in mediating the toxicity of menadione in knockout mice and at higher doses in wild-type mice. The toxicity of menadione in other organs of the NQO1-null mice will be expected but remains to be determined.
In conclusion, a NQO1-null mutant mouse was produced which develops normally and is completely viable and fertile. However, NQO1−/− mice exhibit significantly increased sensitivities to menadione toxicity compared with wild-type mice. The generation and establishment of NQO1−/− mice provide very important tools in determining the in vivo role of NQO1 in protection against redox cycling-activated compounds and whether these mice are sensitive to cancer when exposed to environmental carcinogens including benzo(a)pyrene (benzo(a)pyrene quinones) and benzene (benzoquinone). The data obtained with NQO1-null mice may be used to design further studies in humans to determine if individuals carrying null alleles of the NQO1 gene are at risk for developing cancer related to exposure to chemicals via diet and occupation. In addition, the NQO1−/− mice will be an invaluable tool to study the role of NQO1 in activation of antitumor drugs such as mitomycin C and indoloquinone. This is especially important knowing that NQO1 gene is overexpressed in several kinds of tumors (
). Finally, it will be of interest to determine if mice lacking NQO1 have life spans that differ from wild-type mice since accumulation of oxidative damage has been considered as a factor in aging.
We are thankful to Drs. Harris Busch, Kurt Randerath, and Hagop Youssoufian, all from Baylor College of Medicine, Houston, for reading the manuscript critically. We also thank Dr. Hagop Youssoufian for the use of equipment and reagents and for help in preliminary Western immunoblot experiments.
Received in revised form: December 5, 1997
DOI: https://doi.org/10.1074/jbc.273.13.7382
© 1998 ASBMB. Currently published by Elsevier Inc; originally published by American Society for Biochemistry and Molecular Biology.
Creative Commons Attribution (CC BY 4.0) |
Creative Commons Attribution (CC BY 4.0)


text:
Sports Ray Lewis joining Showtime's 'Inside The NFL'
Ray Lewis joining Showtime's 'Inside The NFL'
Published on Thursday, 07 September 2017 16:08
Written by BARRY WILNER
By BARRY WILNER
AP Pro Football Writer
Football has been Ray Lewis' passion since he was a child. One of his lasting memories is watching "Inside The NFL" when it was hosted by Nick Buoniconti and Len Dawson.
On Tuesday night, Lewis steps into the Showtime studios to sort of follow those Hall of Famers' footsteps.
Lewis joins James Brown, Boomer Esiason and Phil Simms as a regular on the weekly program where frank talk and strong opinions are as prominent as game highlights.
"The first thing that attracted me to the show is I have been watching it for years," Lewis said. "I always appreciated that they would actually sit down and talk football with guys who know football.
"Then, the moment they reached out and said we want to have you on the show, it is the perfect platform for me."
A platform which the former Ravens linebacker who very well could be entering the Hall of Fame himself next year promises he won't let go to waste.
"I think I bring an aspect to the show they have not had, a true defensive perspective," Lewis said. "They have had some over the years from the guys who made a living reading defenses and trying to beat them. But from everything I did on the football field, I became the quarterback from the defensive side.
"And one thing I know better than defense is offense and quarterbacks and what they think and how they think and what they are trying to do and how to stop it."
Lewis retired after winning his second Super Bowl on what he dubbed his "last ride" in February 2013. For 17 seasons, including two Defensive Player of the Year awards and seven All-Pro selections, Lewis was the measuring stick for his position. Esiason, who was approaching the end of his career when Lewis broke into the NFL in 1996, told Lewis "guys like him" were the reason Esiason was leaving the game.
"If they are making them this big and this fast," Esiason said, "it is time for me to go."
Now, Lewis gets to trade barbs with Esiason, who certainly is no slouch in that department, Brown and Simms.
Lewis admits he has a lot to learn about television, but because "Inside The NFL" is taped, the educational curve shouldn't be too steep. His familiarity with the two quarterbacks doesn't hurt, either.
Mostly, though, the folks at Showtime are counting on Lewis' passion for the game to come through and connect him with viewers.
"On paper, he has everything you look for in an NFL analyst," producer Pete Radovich said. "Besides being one of the greatest players of our generation and a huge personality and never being at a shortage of words, there are a million reasons for hiring Ray.
"It was a safe bet to go with Ray. He checks off on everything you could want. There is an emphasis on not being sensitive on this show and being able to dish it out and take it. That may have been the first thing I brought up in our first meeting was that we like to have fun and bust each other's chops. He lit up and said: 'That is all we do.'"
Lewis also brings a voice from another generation. Simms' and Esiason's heydays were in the 1980s and early '90s.
It won't hurt Showtime that Lewis will be on the Hall of Fame ballot for the first time. Radovich calls Lewis being voted into the Canton shrine "a slam dunk." You can bet his candidacy will be discussed on "Inside The NFL."
"When you are playing, it's hard to even think about those things," Lewis said. "This game is hard. Now, can you imagine if your name is called in February, you sit among the people you sat and admired as a youngster. I admired LT (Lawrence Taylor) and Willie Lanier and Ronnie Lott and Mel Blount, and now to get to sit back and say I get to join this fraternity? That is highest reward you can ever attain."


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