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11 Amazing Day Trips to Take From Tokyo
Isabel Putinja August 14, 2017
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Once you've covered Tokyo's major sights and had your fill of its big city bustle, it's time to hop on a train and plan a day-long getaway. This is easy to do, thanks to Japan Railway's efficient network: There's a surprising variety of destinations within easy reach of the capital. In no time you can be in a completely different scene exploring temple towns, pottery villages, Zen-like gardens, tiny islands, and blissful beaches. Here are 11 enticing day trips to take from Tokyo.
1. Chichibu-Tama National Park
Stairs to Mitsumine shrine. Courtesy of WorldContributor/Wikimedia.
Nature lovers and outdoor enthusiasts are often drawn to Chichibu-Tama National Park (just 75 miles west of Tokyo) for the exceptional hiking possibilities that take trekkers through river valleys and mountain landscapes. A popular climb is to the top of Mount Kumotori — at 6,620 feet, this is the highest mountain in the Tokyo Metropolitan area, and Mount Fuji can be seen from here on clear days. A less demanding but equally rewarding hike is up the 3,609-foot-high Mount Mitsumine. The scenic forest pathway to the top takes hikers up a flight of stairs to an ancient Shinto shrine at summit of the mountain.
Another impressive sight (that doesn't require a vigorous hike!) is Hitsujiyama Flower Park with looming Mount Buko as a backdrop. This is where a sea of "shibazakura" (moss phlox) is in bloom every spring, creating a stunning carpet of pink and white blossoms. This sight can only be enjoyed in late April and early May, which is also when the park's 1,000 cherry trees bloom, creating a magnificent natural spectacle.
2. Nikko
Toshogu Shrine. Courtesy of Fg2/Wikimedia.
About 70 miles north of Tokyo lies Nikko, an 17th-century UNESCO Heritage Site of 103 shrines, temples, and other historical buildings. The complex is set in a national park of dense forests, mountain landscapes, waterfalls, lakes, and hot springs. This is a popular day trip with Tokyoites who come to escape the hustle and bustle of the city and take in the special spiritual vibe here. The lavishly decorated Toshogu Shrine is embellished with wood carvings and gold leaf and is the most popular attraction here. But there are other fascinating sights, like the the Shin-kyo Bridge, painted a bright red and spanning a rushing stream, and the Kanman-ga-Fuchi Abyss, a forest path lined with a neat row of jizo stone statues which represent the figure of Buddha as a monk.
3. Kamakura
Buddhist statues at Kamakura. Courtesy of Chris 73/Wikimedia.
This small seaside town is a former Japanese capital and is famous for its giant 15th-century bronze statue of the Great Buddha of Kamakura (Kamakura Daibutsu) — this is Japan's second tallest Buddhist statue at 44 feet. Another important attraction is the massive Tsurugaoka Hachiman-gu, Kamakura's largest Shinto shrine, while the more peaceful Jomyo-ji temple is worth visiting for its beautiful Zen-style garden and traditional teahouse. The town's many other Buddhist temples, shrines, and monuments can be easily explored thanks to Kamakura's own mini railway system, which shuttles visitors around from one sight to the next. Kamakura is located only 37 miles southwest of central Tokyo, making this an easy day trip.
4. Mount Fuji
View of Mount Fuji from Shojiko Lake. Courtesy of Halfd/Wikimedia.
Mount Fuji is Japan's most iconic image and its highest mountain at 12,388 feet. Interestingly, this cone-shaped volcano was recognized by UNESCO in 2013 as a cultural heritage site instead of a natural one because the mountain is considered to have "inspired artists and poets and been the object of pilgrimage for centuries."
Located 62 miles southwest of Tokyo, a day trip to Mount Fuji is certainly doable, but those wishing to scale the mountain will have to set aside more time and plan their ascent carefully. The climb is actually a challenging one, and the climbing season is only limited to the months of July and August, with most climbers making the ascent at night to arrive at the summit in time for sunrise.
But outside the climbing season, the scenic lakes situated at the base of Mount Fuji provide the perfect vantage point to admire the majestic mountain. Many visitors also make a visit to the 400-year-old Fujiyoshida Sengen Shrine set in a dense forest on the north side of the mountain. This is where hikers traditionally offer their prayers before passing under the wooden "torii" gate and beginning their ascent.
5. Enoshima Island
Sunset at Enoshima Island. Courtesy of Abendstrom/Wikimedia.
This tiny 1.5-square-mile island is located 36 miles southwest of Tokyo. It can be reached on foot or by car via a causeway that links it to the mainland. Once on the island, visitors will find beaches, hot springs, a botanical garden, and the Iwaya Caves, where statues of the Buddha and a mythical dragon are concealed underground. A climb up the Sea Candle lighthouse rewards visitors with panoramic views over the island. Another must-visit is the Enoshima Shrine dedicated to Benzaiten, the Buddhist goddess of music. A popular local delicacy the island is known for is "shirasudon": rice topped with a tiny worm-like white fish called "shirasu."
6. Narita
A street in Narita. Courtesy of CGP/Wikimedia.
Most travelers to Japan know Narita as the home of one of Tokyo's major international airports, situated about 38 miles east of central Tokyo. But Narita itself is worth a stopover for its many atmospheric temples and picturesque streets lined with traditional buildings. The airport even has organized tours for passengers on a layover, which include a guided two- or three-hour tour of the area.
A must-visit is the 1,000-year-old Naritasan Shinshoji Temple set in vast and quiet grounds full of multi-tiered pagodas. A "goma" fire ritual is performed here every day, during which prayers and chanting are offered to the gods. A highlight is the open-air museum at nearby Boso no Mura, which is a replica village from the Edo period (17th to 19th centuries), made up of traditional merchant houses and workshops showcasing local handicrafts.
7. Izu Peninsula
One of Izu's beaches. Courtesy of Saigen Jiro/Wikimedia.
The Izu Peninsula is a popular resort destination thanks to this coastal region's mild climate and its many beaches and hot springs. The towns of Atami, Shuzenji, and Ito are known for their "onsen" resorts where locals love to go to soak in the natural hot springs while enjoying the sea air and mountain scenery. The eastern coast of the peninsula attracts beach lovers and surfers, while the more rugged west coast is popular with hikers looking to take in the impressive views of Mount Fuji from here. With only a distance of 100 miles to cover from Tokyo, the Izu Peninsula attracts plenty of weekend leisure travelers.
8. Mashiko
Potters' kilns in Mashiko. Courtesy of Patrick.charpiat/Wikimedia.
Charming, rural Mashiko is synonymous with pottery. Here, a community of potters work their wheels creating the town's famous ceramic pottery, called mashiko-yaki. It's fired in giant kilns that can be spotted outside the town's many workshops. The town's main street is lined with shops where the potters' wares can be purchased, as well as galleries and museums showcasing the local artisans' crafts. At the Mashiko Museum of Ceramic Art, visitors can even try their hand at the wheel. The town is at its busiest during its biannual pottery fair held here every May and November. With only 86 miles to cover from Tokyo, Mashiko is an easy day trip.
9. Kawagoe
Candy Alley in Kawagoe. Courtesy of At by At/Wikimedia.
Just 32 miles northwest of Tokyo is Kawagoe, a former city of castles dating back to the Edo period. The warehouse district is worth exploring for its many fine examples of historic warehouses built of clay and traditional merchant houses with sloped roofs covered in glazed ceramic tiles. Always crowded with shoppers is the narrow "candy alley" lined with stores selling traditional Japanese sweets like sugared coated and deep fried "karinto" biscuits, small cakes made of red bean paste and sweet potato, and flavored rice crackers.
Kawagoe's most visited attraction is the Kitain Buddhist temple, built on the remains of an Edo-period castle — the only remaining vestiges of the city's many erstwhile castles. In a small courtyard on the temple grounds are 540 stone statues depicting "gohyaku rakan," the Buddha's disciples. Each figure has a different facial expression: Some are laughing or scowling, while others are pouting or weeping.
10. Kawasaki
Traditional houses at Nihon Minka-en Open Air Museum. Courtesy of Fg2/Wikimedia.
Twelve miles southwest of Tokyo is Kawasaki, a largely industrial city that also offers visitors a taste of traditional Japan and its rustic architecture of times past. Located here is Nihon Minka-en, an open-air museum comprising a collection of 25 traditional Japanese buildings and houses from the Edo period. These were brought from all over the country and reassembled here, with the oldest building dating back to 1688. Visitors can see intricately restored examples of traditional farmhouses, merchant houses, samurai dwellings, a water mill, a Shinto shrine, and even a kabuki stage.
Kawasaki is also known for one of Japan's most outlandish festivals. Each April, the "Kanamara Matsuri" is held here, also known as the "Festival of the Iron Phallus." This is a day-long festival celebrating fertility that involves parading giant penises through the city streets all the way to the local Kanayama shrine, where worshipers come to ask for protection from sexually transmitted diseases, increased fertility, long marriages, and healthy births.
11. Mito
Kairakuen Gardens. Courtesy of Lukas/Wikimedia.
A trip to Japan is incomplete without the experience of visiting one of its atmospheric Zen-like gardens. One of the country's three most famous gardens is located in Mito, just 74 miles from central Tokyo. The Kairakuen Gardens (meaning "a garden to be enjoyed together") were first planted in 1842 and cover an area of 18 acres with bamboo groves, woods of fragrant cedars, and 3,000 "ume" plum trees. These bloom in late February and early March, creating brilliant canopies of white, pink, and red blossoms and attracting crowds of visitors.
Need a place to stay in Tokyo? Check out our reviews and photos of 376 hotels.
You'll Also Like:
6 Must-Visit Hidden Gems in Japan
The Best Itinerary for Japan
7 Unexpected Things You'll Find in Your Hotel Room in Japan
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Beautiful fish and a beautiful shot.
Great!! Such clearity of the focus shot and colorful. Thanks.
Well captured Percula Clownfish, I have a pair in my tank but have ye to be able to get a good shot of them.


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Buford, Gwinnett and state work together to widen S.R. 20/Buford Drive from South Lee Street to S.R. 13/Buford Highway. Google Maps
Buford, Gwinnett and state work together to widen S.R. 20/Buford Drive
Karen Huppertz, For the AJC
Gwinnett County Commissioners recently approved a construction contract to widen State Route 20/Buford Drive that is jointly funded by Buford, the Georgia Department of Transportation and Gwinnett County.
The commissioners approved a $5.5 million contract with C.W. Matthews Contracting Co. Inc. to widen State Route 20 from South Lee Street to State Route 13/Buford Highway. S.R. 20 will be widened from two to three lanes in both directions with the installation of a raised concrete median and sidewalks. The S.R. 20 widening project will also include intersection traffic signal upgrades along with drainage improvements, including curb and gutter.
C.W. Matthews will also install sidewalks along the west side of Old Suwanee Road from S.R. 13 to Chatham Road as part of this contract. The Old Suwanee Road improvements include drainage improvements, along with curb and gutter.
Part of the funding for the S.R. 20 project comes from a $1 million Local Maintenance and Improvement Grant from the Georgia DOT that is in addition to the $450,000 LMIG funding previously approved by Georgia DOT. Commissioners also voted to apply for and accept the additional LMIG funds.
Additional funding for the project is provided through SPLOST contributions by Gwinnett and Buford. Gwinnett commissioners voted to approve an intergovernmental agreement with the city, which provided funding for the S.R. 20 widening contract, with additional funds identified to widen Thompson Mill Road from S.R. 13/Buford Highway to Brickton North Drive. This project is currently in the concept phase.
These projects, which are located in commission districts 1 and 4, will be managed by the county. Funding for Gwinnett's share comes from the 2009, 2014 and 2017 SPLOST programs.

redpajama_set_name: RedPajamaCommonCrawl


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home MLB Free Picks Mets vs Rockies Preview & Prediction September 16, 2019
Mets vs Rockies Preview & Prediction September 16, 2019
Play on: Mets ML (-142)
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The New York Mets have won five of their last seven contests but might have to go perfect over the last weeks of the season in order to make up ground in the National Leauge Wild Card race. They've struggled in recent meetings with the Rockies, but I think at motivated Mets side will get the job done here Monday night in the first of a three-game set.
Mets' southpaw Steven Matz (10-8, 3.84 ERA) has ugly road splits this year, but he has shown signs of improvement allowing just three earned runs over 11 innings of work in his last two starts away from home. Matz is 2-0 with just four runs allowed over his last three starts overall, all team wins. Additionally, we can note that the Rockies are 7-20 in their last 27 games vs. a left-handed starter.
As for the Mets, they're 12-3 in their last 15 road games vs. a right-handed starter and should have no trouble to manufacture runs against Colorado's Antonio Senzatela (9-10, 6.87 ERA). The 24-year-old right-hander has been tagged with five runs or more in six of his last seven starts overall and he owns an ugly 6.59 ERA in 10 starts home at Coors Field on the season.
Free pick on New York Mets.
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Mike Lundin is arguably the hottest NFL handicapper in the world following an ABSOLUTELY ASTONISHING 9-1 (90%) start to the season. Additionally, Mike has really picked up the pace at the ballpark in recent weeks, particularly with his runline plays as he has picked a STUNNING 29-8 MLB RLs since July 15.
Mike already has a couple of NFL premium picks for Thursday and Sunday up for grabs, and subscribers can count on multiple MLB picks every day. Invest in a subscription to get ALL plays.


text:
MMS Staff News
Newsletter | Issue 11 | April 2019
Developing skills to navigate for success
Upon graduation, medical students should have accumulated the necessary foundational knowledge and skills required to deliver patient care.
There is no clear roadmap, however, on how to navigate the rapidly changing healthcare system as a medical professional to optimise career choices.
Surgical training, for example, is highly competitive and there are many extracurricular components which can strengthen an applicant's chance of success which are not widely known.
The course will prepare students for a successful medical career
To address this gap and support emerging healthcare professionals with a plan for career success, two passionate University of Melbourne based surgeons have developed a course to clarify the Pathway to Career Progression.
Course Directors, Associate Professor Justin Yeung and Mr Krinal Mori, chose a mobile-based learning platform to deliver their course to assist time-poor emerging medical professionals to acquire a competitive advantage.
"I've always been passionate about mentorship and assisting junior doctors to develop as successful medical professionals," Associate Professor Yeung, a colorectal surgeon at Western Health, said.
"A significant number of non-accredited registrars do not have a secure job after several years of training, following qualification from medical school, as there is limited exposure to developing professional career skills as part of early learning," he said.
"This course will empower junior doctors because they will be safe in the knowledge they have done as much as they can to optimise their career choices."
Pathway to Career Progression covers how to make the first steps towards a successful medical career including insights on the recruitment process.
"We have assembled a team of recent trainees and experienced practitioners to describe how trainees can maximise their learning and development strategies to fast track career progression in the field of their interest," Mr Mori, a laparoscopic and bariatric surgeon at Northern and Austin Hospitals, said.
"A lot of junior doctors know what they want to do, what their interests are, but they don't know how to reach them," he said.
"They often gather information from various sources, and in some cases, it can be conflicting advice, which can generate even more confusion."
Pathway for Career Progression evolved from a one-day session, where medical students and junior doctors heard presentations on how to prepare a CV, develop a successful research project and prepare for the interview process.
Students will gain mentorship in planning their medical career
"We had such incredible feedback from the one-day session, but participants wanted more knowledge, and we knew we would be able to develop a more comprehensive resource, and explore topics in more depth, if we developed a structured online course," Mr Mori said.
"By bringing the detailed learning package together, where this resource is accessible on a mobile phone, students will be able to focus more on their interests rather than worrying about the process," he said.
The course addresses a wide range of topics including how to develop sound research methodology skills and identify high impact projects to improve publication and presentation opportunities.
"A glowing deficit in applications we see is a lack of experience in completing research at a standard which can be published and presented," Associate Professor Yeung said.
"For surgical selection, the metrics change slightly each year, however, this is one element which could set a candidate apart from the rest," he said.
Additional course highlights include how to maximise in-field experience; understand which conferences are worth targeting; and develop a strategy to meet the challenge of difficult questions associated with professional interviews and public presentations.
"This course is about how to work smarter and develop a positive aura around yourself to ensure you are best positioned to achieve targeted career goals," Mr Mori said.
Pathway to Career Progression is suitable for second year medical students through to junior doctors as it covers a wide range of topics, delivered by peers, who are similarly motivated to share their insights.
"It's the A, B, C to a medical career; trainees are often expected to run before they can walk and this course provides 12 hours of learning, which goes back to basics, and is essential for a successful career in the healthcare system," Associate Professor Yeung said.
Find more information on Pathway to Career Progression here or contact the Mobile Learning Unit at mobile-learning@unimelb.edu.au.


To install aspell-pt just follow these instructions.
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Which will install aspell-pt and any other packages on which it depends.


Unitil, a provider of natural gas and electricity to customers in New England, and the American Red Cross of N.H., Maine and north central Massachusetts renewed their partnership for a third year. Unitil and the Red Cross will work together to enhance their respective commitments to emergency response and community safety.
"We are incredibly proud to support the American Red Cross and its work to provide essential emergency and safety services," said Carol Valianti, Unitil's vice president of communications and public affairs. "We look forward to expanding our efforts to increase safety awareness and provide meaningful assistance to those in our community who need our support."
Unitil is committed to supporting the Red Cross in its efforts to provide life-saving blood products to meet the needs of accident victims, cancer patients, children with blood disorders and others. In response to the constant need for blood donations, Unitil held three Red Cross blood drives in 2012 at which Unitil employees in Massachusetts and New Hampshire donated a total of 65 pints of blood.


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Transparent and Open Social Science...
"False-positive Psychology" by Simmons, Nelson, and Simonsohn
False-positives in social science research can have particularly negative impacts when research is used to inform policy or other outcomes. These outcomes can range, from the endorsement of the idea that voters' attitudes about same-sex marriage can be changed by just a short conversation with a gay canvasser, to the suggestion that increasing austerity measures leads to economic growth.
In this article, authors Joseph Simmons, Leif Nelson, and Uri Simonsohn, address the costly issues and frequency of false positives in social science research and suggest six requirements for authors that they believe are simple, low-cost, and straight-forward solutions to the problem.
They first define a false positive as the "incorrect rejection of a null hypothesis". The problem with false positives is that once they are published, they are persistent and there is little incentive to replicate findings to test their significance. The authors write: "..false positives waste resources: they inspire investment in fruitless research programs and can lead to ineffective policy changes. Finally, a field known for publishing false positives risks losing its credibility."
Simmons, Nelson, and Simonsohn argue that, with the current standards of disclosure, false positives are, in fact, "vastly more likely", due to the ease at which researchers can publish "statistically significant" evidence for any hypothesis.
Many of these issues can be attributed to researcher degrees of freedom, or the multitude of decisions researchers can make regarding the design and details of their experiments. Such freedom increases the likelihood that analyses will produce false positives, and can be attributed to two factors: "ambiguity in how to best make decisions, and the researcher's desire to find a statistically significant result", the latter factor referring to the temptation and likelihood of researchers coming to conclusions that are consistent with their own desires or beliefs.
The authors performed computer simulations to estimate the influence of researcher degrees of freedom on the probability of a false-positive result. They focused on four common degrees of freedom that increase the likelihood of a researcher falsely detecting a significant effect:
Flexibility in choosing among dependent variables
choosing sample size
using covariates, and
reporting subsets of experimental conditions.
They showed that if an effect is significant with a small sample size, then it would be significant with a larger one, too.
They also suggest six requirements as a solution to the high incidence false-positives, encouraging appropriate conduct of research, transparency in methods, and holding readers accountable to make informed decisions about the credibility of findings.
Authors must:
decide the rule for terminating data collection before data collection begins and reports this rule in the article – This prevents authors from adding additional observations and further testing for significance if initial results are insignificant.
collect at least 20 observations per cell or else provide a compelling cost-of-data-collection justification – Small samples are "simply not powerful enough to detect most effects" and are "more likely to reflect interim data analysis and a flexible termination rule".
list all variables collected in a study – This prevents researchers from only reporting convenient subsets of measurements and allows readers to identify degrees of freedom.
report all experimental conditions, including failed manipulations – This "prevents authors from selectively choosing only to report conditions that yield results consistent with their hypothesis."
report the statistical results of eliminated observations if those observations had been included – This requires authors to explain why they eliminated the data and encourages readers to consider the validity of the data exclusion.
report the statistical results of the analysis without the covariate if the analysis includes a covariate – This requires authors to "justify use of the covariate," reveals "the extent to which a finding is reliant on the presence of a covariate," and, again, encourages readers to practice discernment in whether the covariate is warranted.
Finally, Simmons, Nelson, and Simonsohn present four guidelines for reviewers to abide by.
Reviewers should…
ensure that authors follow the requirements;
be more tolerant of imperfections in results (false-positive findings could be due to an "unreasonable expectation" imposed by reviewers for data to turn out as predicted);
require authors to demonstrate that their results do not hinge on arbitrary analytic decisions; and
require the authors to conduct an exact replication if justifications of data collection or analysis are not compelling.
While there has been some criticism of the authors' proposed solution, they still conclude that the requirements advocated in this article impose minimal costs to all involved in research and review and are a step towards the goal of discovering and disseminating valid research.
Think about the various researcher degrees of freedom for an experiment. Can you think of a time when degrees of freedom might've affected your own research?
You can read the entire article here: http://journals.sagepub.com/doi/pdf/10.1177/0956797611417632.
Simmons, Joseph P., Leif D. Nelson, and Uri Simonsohn. 2011. "False-Positive Psychology Undisclosed Flexibility in Data Collection and Analysis Allows Presenting Anything as Significant." Psychological Science 22 (11): 1359–66. doi:10.1177/0956797611417632.
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text:
Publicity for Hu Na contrasts with quiet action on other cases
By Frederic A. Moritz Staff correspondent of The Christian Science Monitor
With a smile that has made the front page of many newspapers, photogenic tennis star Hu Na is by now nearly as famous in the United States as in her native China.
The reason: Last summer she defected at a Santa Clara, Calif., match. This week the US granted her ''political asylum'' despite high-level Chinese warnings that Peking may retaliate by reducing cultural exchange programs.
This public rebuff to China follows growing publicity on the Hu Na case. The issue has reopened debate over just what should be the priorities in US policy toward China.
On one side are the promoters of closer ties between the US and China who do not want Peking provoked to the point of lessening athletic, educational, and other cultural relations. On the other side are those who are concerned that the US State Department is tempted to close the door to Chinese asylum seekers, while welcoming as useful propaganda tools applications from Soviet bloc citizens.
The publicity in the Hu Na case increased pressure on the State Department and the White House from conservative backers who have supported President Reagan's policy of arms sales to Taiwan. A rejection of Miss Hu's bid would have left the Reagan administration vulnerable to charges of abandoning its firm policy toward China. On the other hand, tensions with Peking over a host of issues, including arms sales to Taiwan and trade, have left concern that grants of asylum could add one more strain to the relationship.
In recognizing Hu Na's argument that she had ''a well-founded fear of persecution'' in China, the Reagan administration in effect rejected Chinese assurances that Miss Hu would not be punished if she returned to China. The decision also appears to give credence to some of Miss Hu's bleak description of sports life in China.
Miss Hu has claimed that she defected because, as a notable athlete, she was being pressured to join the Communist Party and thus get caught in dangerous factional fighting. In the application she reportedly argued that she was under the pressure of political study sessions aimed at improving her tennis in harmony with Marxist-Leninist principles. To some China specialists, this revived memories of the 1960s and '70s, when prominent Chinese athletes were promoted by one political faction, only to be purged and sometimes sent to forced labor when the sponsoring faction fell from power.
The Hu Na case stands out because it contrasts with the quiet, diplomatic method generally followed by China and the US in cases where Chinese in the US choose to stay on.
There are now some 10,000 Chinese students and scholars in the US (split about evenly between privately sponsored and government-sponsored students), according to the State Department. Since 1979, hundreds of such Chinese students and scholars have stayed on in the US after their periods of study or work were over. For many of these relatively young and privately sponsored students, the private F-1 visa can be smoothly adjusted without applying for ''political asylum.''
Since 1979, some 10 mainland Chinese have been granted ''political asylum'' in the US out of 300 or 400 applications, according to one well-placed source. Of more than 1,000 pending applications from Chinese asylum seekers, the number of applicants from mainland China has not been separately classified according to the State Department. The State Department says, however, that a ''large proportion'' are from Taiwan and Hong Kong.
Asylum applications are made privately, and there is no public discussion. This is to ensure that if asylum is denied, the applicant is less likely to be punished after returning to China. Also, if the US denies asylum, the applicant can arrange to go to a third country, instead of returning to China. Decision on asylum bids can be put off indefinitely, and until there is a final decision, the applicant may remain in the US.
A Chinese Embassy official in Washington says China had long expected that a number of privately sponsored students would stay on in the US for a variety of reasons. But China takes a different position on the often older, officially sponsored Chinese with the J-1 visa. ''These people owe it to their country to come home. We spend $5,000 to $10,000 a year on them, so it is not fair for them just to run off,'' he says.
Moreover officially sponsored Chinese are considered less likely to stay because they are usually older, with more professional and family ties at home. Also, unlike the F-1 visa, the J-1 cannot be adjusted for a longer stay without the approval of the Chinese government. Thus, for those on the J-1, the only way to stay may be to apply for ''political asylum.''
This requires the Immigration and Naturalization Service to rule, with State Department advice, that the applicant has ''a well-founded fear of persecution based on race, religion, nationality, group membership, or political opinion.''
China police chief sought asylum in US, says Chinese media
How defiant churches are challenging Australia's strict asylum policy
Opinion Chinese dissident – bad timing for Washington, Beijing


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            var podSpec = m[2].replace(/^\'|\'$/g, '');
            obj[m[1]] = podSpec; // i.e pod 'Foo', '1.2' ==> { 'Foo' : '1.2'}
        }

        return obj;
    }, {});
};

/*
Podfile.prototype.getTemplate = function() {
    return util.format(
            '# DO NOT MODIFY -- auto-generated by Apache Cordova\n' +
            'platform :ios, \'8.0\'\n' +
            'target \'%s\' do\n' +
            '\tproject \'%s.xcodeproj\'\n' +
            '%s\n' +
            'end\n',
             this.projectName, this.projectName, this.podToken);
};
*/
Podfile.prototype.getPodfileTemplate = function() {
    return util.format(
      // 'source \'git@github.com/CocoaPods/Specs.git\'\n' +
      'platform :ios, \'7.0\'\n' +
      '# inhibit_all_warnings!\n' +
      'def common\n' +
      '\t' +
      '%s\n' +
      'end\n' +
      'target \'%s\' do\n' +
      '\tcommon\n' +
      'end\n',
      this.podToken, this.projectName);
};

Podfile.prototype.getPodfileTemplateForAli = function() {
    return util.format(
        // 'source \'git@gitlab.alibaba-inc.com:alipods/specs.git\'\n' +
        // 'source \'git@gitlab.alibaba-inc.com:alipods/specs-mirror.git\'\n' +
        // 'source \'https://github.com/CocoaPods/Specs.git\'\n' +
        'platform :ios, \'7.0\'\n' +
        '# inhibit_all_warnings!\n' +
        'def common\n' +
        '\t' +
        '%s\n' +
        'end\n' +
        'target \'%s\' do\n' +
        '\tcommon\n' +
        'end\n',
        this.podToken, this.projectName);
};

Podfile.prototype.addSpec = function(name, spec) {
    name = name || '';
    spec = spec; // optional

    if (!name.length) { // blank names are not allowed
        throw new CordovaError('Podfile addSpec: name is not specified.');
    }

    this.pods[name] = spec;
    this.__dirty = true;

    events.emit('verbose', util.format('Added pod line for `%s`', name));
};

Podfile.prototype.removeSpec = function(name) {
    if (this.existsSpec(name)) {
        delete this.pods[name];
        this.__dirty = true;
    }

    events.emit('verbose', util.format('Removed pod line for `%s`', name));
};

Podfile.prototype.getSpec = function(name) {
    return this.pods[name];
};

Podfile.prototype.existsSpec = function(name) {
    return (name in this.pods);
};

Podfile.prototype.clear = function() {
    this.pods = {};
    this.__dirty = true;
};

Podfile.prototype.destroy = function() {
    fs.unlinkSync(this.path);
    events.emit('verbose', util.format('Deleted `%s`', this.path));
};

Podfile.prototype.writeUseAliSource = function() {
    var text = this.getPodfileTemplate();
    var self = this;

    var sourceString = "";

    this.source = [
        'git@gitlab.alibaba-inc.com:alipods/specs.git',
        'git@gitlab.alibaba-inc.com:alipods/specs-mirror.git'
    ]

    if(this.source){
        for(var i=0; i<this.source.length; i++){
            sourceString += 'source \'' + this.source[i] + '\'\n'
        }
    }

    if(this.source){
        text = sourceString + text;
    }

    var podsString =
        Object.keys(this.pods).map(function(key) {
            var name = key;
            var spec = self.pods[key];

            if (spec.length) {
                if (spec.indexOf(':') === 0) {
                    // don't quote it, it's a specification (starts with ':')
                    return util.format('\tpod \'%s\', %s', name, spec);
                } else {
                    // quote it, it's a version
                    return util.format('\tpod \'%s\', \'%s\'', name, spec);
                }
            } else {
                return util.format('\tpod \'%s\'', name);
            }
        })
            .join('\n');

    text = text.replace(this.podToken, podsString);
    fs.writeFileSync(this.path, text, 'utf8');
    this.__dirty = false;

    events.emit('verbose', 'Wrote to Podfile.');
}

Podfile.prototype.write = function() {
    var text = this.getPodfileTemplate();
    var self = this;

    var sourceString = "";

    if(this.source){
        for(var i=0;i<this.source.length;i++){
            sourceString += 'source \'' + this.source[i] + '\'\n'
        }
    }

    if(this.source){
        text = sourceString + text;
    }

    var podsString =
    Object.keys(this.pods).map(function(key) {
        var name = key;
        var spec = self.pods[key];

        if (spec.length) {
            if (spec.indexOf(':') === 0) {
                // don't quote it, it's a specification (starts with ':')
                return util.format('\tpod \'%s\', %s', name, spec);
            } else {
                // quote it, it's a version
                return util.format('\tpod \'%s\', \'%s\'', name, spec);
            }
        } else {
            return util.format('\tpod \'%s\'', name);
        }
    })
    .join('\n');

    text = text.replace(this.podToken, podsString);
    fs.writeFileSync(this.path, text, 'utf8');
    this.__dirty = false;

    events.emit('verbose', 'Wrote to Podfile.');
};

Podfile.prototype.isDirty = function() {
    return this.__dirty;
};

Podfile.prototype.before_install = function() {
    // Template tokens in order: project name, project name, debug | release
    var template =
    '// DO NOT MODIFY -- auto-generated by Apache Cordova\n' +
    '#include "Pods/Target Support Files/Pods-%s/Pods-%s.%s.xcconfig"';

    var debugContents = util.format(template, this.projectName, this.projectName, 'debug');
    var releaseContents = util.format(template, this.projectName, this.projectName, 'release');

    var debugConfigPath = path.join(this.path, '..', 'pods-debug.xcconfig');
    var releaseConfigPath = path.join(this.path, '..', 'pods-release.xcconfig');

    fs.writeFileSync(debugConfigPath, debugContents, 'utf8');
    fs.writeFileSync(releaseConfigPath, releaseContents, 'utf8');

    return Q.resolve();
};

Podfile.prototype.install = function(requirementsCheckerFunction) {
    var opts = {};
    opts.cwd = path.join(this.path, '..'); // parent path of this Podfile
    opts.stdio = 'pipe';
    var first = true;
    var self = this;

    if (!requirementsCheckerFunction) {
        requirementsCheckerFunction = Q();
    }

    return requirementsCheckerFunction()
    .then(function() {
        return self.before_install();
    })
    .then(function() {
        return superspawn.spawn('pod', ['install', '--verbose'], opts)
        .progress(function (stdio){
            if (stdio.stderr) { console.error(stdio.stderr); }
            if (stdio.stdout) {
                if (first) {
                    events.emit('verbose', '==== pod install start ====\n');
                    first = false;
                }
                events.emit('verbose', stdio.stdout);
            }
        });
    })
    .then(function() { // done
        events.emit('verbose', '==== pod install end ====\n');
    })
    .fail(function(error){
        throw error;
    });
};

module.exports.Podfile = Podfile;

redpajama_set_name: RedPajamaGithub


South City Kitchen Vinings is bringing the Bayou to you! Now through Fat Tuesday on Tuesday, March 4, South City Kitchen will celebrate Mardi Gras with its exclusive "Taste of New Orleans" menu. Small plate selections will showcase "Nawlins" delicacies including Louisiana gumbo with catfish, crawfish, Andouille, okra and brown roux broth, gulf crab and apple beignets with bacon mayo and oysterman's stew with fennel, fingerling potatoes, bacon, basil butter and oyster crackers. Crispy buttermilk catfish served alongside Gulf blue crab maque choux and a celery salad and red fish napoleon with crawfish mousse, green rice and crawfish etouffee will be featured entrees. A New Orleans classic, the Ambrosia "Pain Perdu" with coconut bread pudding, pineapple-banana rum caramel and High Road vanilla ice cream will end the meal on a decadent note. Les bons temps rouler!


Adara Nunn is determined to prevent her cousin from having an abortion, even if it means putting her own dreams on hold … for years. When Christopher Dean loses a patient, he decides he wasn't meant to be a surgeon after all. So what IS he meant to do with his life?
A change of scenery gives Chris a new outlook, especially after he meets Adara Nunn. It looks like Adara's troubles are coming to an end too, until flashing red and blue lights bring their plans for the future to a screeching halt.
After the tragic drowning of his cousin, James Cooper Knight spends his days trying to make up for his past mistakes. He not only dedicates his life to addiction counseling, but guilt drives him to the water, searching for others who've been caught unaware of the quickly rising tides of St. Simons.
Miss Beryl Angsley, relishing a final few months of freedom before marriage, accompanies her father across the globe on a mission of diplomacy only to find herself kidnapped by Chinese pirates in the South China Sea. British privateer, Philip Carruthers, on a secret undertaking for Queen Victoria, has at last found the treasure he's sought for two years. Unfortunately, he also finds a captive Englishwoman.
Sometimes, what we hunt can capture us… He wants her dead. She knows she should be afraid. After all, he's a contract hit-man. But someone has to stop his killing spree. An old locket with a painful history, connects their minds. She is aware of his plans far more than he is aware of her presence inside his head.
Lady Lora Ransdale… Keeping her scheming cousin away from her and her inheritance requires drastic measures. Pretending to be mad–in a harmless sort of way–works well. Until she meets Hawk Martin. When her cousin demands that she marry Hawk, she assumes the handsome architect is an accomplice in the plot–not a dashing and charming protector. Despite Lora's resolve to keep her new husband at a distance, his comfort and tender touch make her yearn for a true marriage and lasting love.
Hawk Martin… A self-made man, he never dreamed he would marry the daughter of an earl, even one who might or might not be slightly unhinged. But he is determined to keep her safe. If they outwit her nefarious cousin, will this new life – and his heart – be shattered when she learns of his past? Or can an orphan from the slums of London find enduring love with a lady of quality?
Paxton Terrence, a retired Navy SEAL is apprehensive after losing the woman he thought he'd spend forever with. At first glance, Annie, the woman he agreed to watch and protect is the woman who could heal his broken heart. The only problem, his rich cousin, Kai, jumped in front of him to get the girl.
Annie has a deadline to keep. Jilted in love–again–she decides to train Kai, an unsuspecting and adorable beau, to be her perfect mate utilizing standard doggy training techniques. What Annie doesn't expect is the undeniable attraction she feels for Paxton, a retired Navy SEAL who pops up everywhere she goes.
Crooked Moon, first and foremost, is a human drama, an emotional journey into the lives of two women and those closest to them. As children, Frankie Cavalese and Callie Mason were the very best of friends, growing up together behind opposite walls of a row home in a blue-collar neighborhood of Philadelphia called Rainytown.


As you've no doubt heard by now, we no longer plan to use Modern as a Pro Tour format as of 2017. Modern will continue to be a big part of our Organized Play offerings, both at the premier level and otherwise, but it is no longer a good fit for the Pro Tour.
So why isn't it right for the Pro Tour? It comes down to our goals for the events. The first is that we want to reward good drafting, innovative deck building, and tight gameplay in unestablished environments. Second, we want to highlight the newest card set. To those ends, we positioned the Pro Tour events just a couple weeks after each new set comes out, which both provides the fresh new proving ground for our players and showcases each new set in a premier-level setting right at the beginning of its life cycle.
As time has passed since Modern's inception, some cracks started to appear that made us question its relevance to the Pro Tour. Our top players pointed out to us that Modern wasn't often about innovating or solving the puzzles presented by a new card set, but rather it rewarded huge numbers of repetitions with established decks, and while that kind of play can be interesting and is relevant to a lot of the Magic audience, it wasn't what the Pro Tour was supposed to be about.
In order to try to present the players with a new environment to explore, we'd implement the changes to the banned list that we had identified throughout the previous year right before the Pro Tour, which often cast a shadow of dread over the impending Pro Tour for many of the format's fans, as the spotlight of a Pro Tour accelerated the rate at which we'd ban problematic cards in the format. On top of that, the skill of the pro players combined with the high incentives of the event really accelerated the tuning and development of the best decks (such as this year's Eldrazi menace) to a large degree, which isn't great for a format that is designed to change very slowly over time. We'd rather let those deck evolutions play out over months on Magic Online or at store-level events, as that accelerated metagame pace often just means speeding up more changes to the banned list as well.
For all those reasons, we made a move to eliminate the Modern Pro Tour once before, for the 2015 season. That decision was met with a chorus of boos, and was quickly rescinded. So why are we doing so again?
The main reason is that we've made Standard a lot more dynamic than it was in 2015, primarily by moving to two blocks per year (with corresponding format rotations). Now four Standard Pro Tours should deliver a large variety of decks across the year, and there is significantly less need to break up potential stagnation with a variant format.
In the wake of the recent Splinter Twin banning / Eldrazi dominance / Eye of Ugin banning / Ancestral Visions and Sword of the Meek unbanning series of events, I am frequently asked what we want Modern to be as a format. My answers to these questions should be seen as guidelines that we use to help our thinking internally, but they are not infallible policies. Should players' attitudes toward the format change over time, we're likely to adjust our guidelines as well.
There's room for interpretation in many of those statements—intentionally—but this paints a clearer picture of how we see Modern.
There you have it—Standard and Modern, our two most-played Constructed formats. We want to use each one in the places that are best for that format and for Magic as a whole—hopefully you'll agree that shifting the Pro Tours all to Standard does just that.


text:
Bieber's Baby Mama Got Busted -- Here's Your Morning Gossip
Dana Eisenberg
TMZ has proof that Justin Bieber's alleged baby mama was just looking for some cash. A friend of Mariah Yeater's revealed texts from her in which she asked him to delete texts from her mom where she mentioned someone named Tristan as the father.
And Bieber's girlfriend Selena Gomez should be careful. The stalker who's been showing up at her workplace and threatening to kill her has been released.
Dave Hester from "Storage Wars" used the word "yuup!" on his show, which is problematic since evidently T rey S ongz begins all of his songs with that word. The two are now in a legal battle over the word, which is totally worth the time, money and effort. Here are some more absurd celebrity lawsuits.
Hugh Jackman is getting rave reviews and big bucks for his new one man show. Too bad it's only a limited run.
Joe Jonas is dating a mystery woman. He was seen leaving a restaurant with the brunette in NYC the other night.
More: Justin Bieber Selena Gomez


Electrical Wire & Cable Specialists If you're looking for immediate shipment of the wire and cable that you need, you've come to the right place with Electric Wire and Cable Specialists. EWCSwire.com is your one stop shop for all kinds of electronic wire & cable in Phoenix, Arizona.
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text:
Ньютаунгор (; , ранее известен как Ducarrick) — деревня в Ирландии, находится в графстве Литрим (провинция Коннахт) на трассе .

Примечания

Населённые пункты графства Литрим

redpajama_set_name: RedPajamaWikipedia


text:
Special Feature Articles - Most Recent
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Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron–electron resonance
Maxie M. Roessler, Martin S. King, Alan J. Robinson, Fraser A. Armstrong, Jeffrey Harmer, and Judy Hirst
aCenter for Advanced Electron Spin Resonance, and
bInorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3QR, United Kingdom; and
cMedical Research Council Mitochondrial Biology Unit, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge, CB2 0XY, United Kingdom
See allHide authors and affiliations
PNAS February 2, 2010 107 (5) 1930-1935; https://doi.org/10.1073/pnas.0908050107
Maxie M. Roessler
Martin S. King
Alan J. Robinson
Fraser A. Armstrong
Jeffrey Harmer
For correspondence: [email protected] [email protected]
Judy Hirst
Edited by David Britt, University of California, Davis, Davis, CA, and accepted by the Editorial Board November 25, 2009 (received for review July 20, 2009)
Figures & SI
In oxidative phosphorylation, complex I (NADH:quinone oxidoreductase) couples electron transfer to proton translocation across an energy-transducing membrane. Complex I contains a flavin mononucleotide to oxidize NADH, and an unusually long series of iron-sulfur (FeS) clusters, in several subunits, to transfer the electrons to quinone. Understanding coupled electron transfer in complex I requires a detailed knowledge of the properties of individual clusters and of the cluster ensemble, and so it requires the correlation of spectroscopic and structural data: This has proved a challenging task. EPR studies on complex I from Bos taurus have established that EPR signals N1b, N2 and N3 arise, respectively, from the 2Fe cluster in the 75 kDa subunit, and from 4Fe clusters in the PSST and 51 kDa subunits (positions 2, 7, and 1 along the seven-cluster chain extending from the flavin). The other clusters have either evaded detection or definitive signal assignments have not been established. Here, we combine double electron-electron resonance (DEER) spectroscopy on B. taurus complex I with the structure of the hydrophilic domain of Thermus thermophilus complex I. By considering the magnetic moments of the clusters and the orientation selectivity of the DEER experiment explicitly, signal N4 is assigned to the first 4Fe cluster in the TYKY subunit (position 5), and N5 to the all-cysteine ligated 4Fe cluster in the 75 kDa subunit (position 3). The implications of our assignment for the mechanisms of electron transfer and energy transduction by complex I are discussed.
NADH:quinone oxidoreductase
pulsed EPR
Complex I (NADH:quinone oxidoreductase) is an essential respiratory enzyme. It is an entry point to the electron-transport chains of many aerobic organisms, and its dysfunction is linked to numerous human diseases by mitochondrial DNA mutations, decreased respiratory capacity and increased oxidative stress (1). Complex I is a complicated, membrane-bound enzyme that couples NADH oxidation and quinone reduction to proton translocation across an energy-transducing membrane. It comprises a membrane extrinsic (hydrophilic) domain, and a membrane intrinsic (hydrophobic) domain. A chain of iron-sulfur (FeS) clusters in the hydrophilic domain is essential in transferring electrons from the flavin mononucleotide at the site of NADH oxidation to the hydrophobic site of quinone reduction. The mechanism by which electron transfer drives proton translocation is not known. The 3.3 Å structure of the hydrophilic domain of Thermus thermophilus complex I, the only high resolution structure available, reveals how the FeS clusters and the flavin are arranged (2). Seven clusters form an extensive chain from the flavin to the quinone binding site; the eighth cluster, of unknown function, is on the opposite side of the flavin (see Fig. 1). A ninth cluster, more than 20 Å from the cluster chain, occurs in only a few species, including T. thermophilus and Escherichia coli. Current knowledge of the properties of the clusters, most obviously their reduction potentials, has been derived almost exclusively from X-band continuous wave (CW) EPR spectroscopy on complex I from B. taurus (bovine) heart mitochondria (3–6), which is closely related to human complex I.
Arrangement of the redox cofactors in B. taurus complex I, based on the structure from T. thermophilus, and possible assignments of EPR signals to clusters. Left: FeS clusters in the structure of the hydrophilic arm of T. thermophilus complex I [2FUG.PDB (2)] conserved in B. taurus, named according to their type (2Fe or 4Fe), subunit [according to the standard nomenclature for B. taurus, 75 = 75 kDa subunit, 51 = 51 kDa subunit, 24 = 24 kDa subunit, PS = PSST subunit, TY = TYKY subunit (7)], and differentiated when necessary by C (all cys ligated) or H (one his ligand), or as cluster 1 or 2. Distances are center-to-center for the FeS clusters or the central ring of the isoalloxazine system. (Right): Extant assignments of the five EPR signals observed in B. taurus complex I reduced by NADH (3–6). N5 is not addressed here.
To understand the mechanism of energy transduction by complex I, it is essential to assign the set of spectroscopically determined cluster properties to the set of structurally defined clusters. The correct assignment will define the properties of individual clusters (reduction potentials) and of the cluster ensemble (a basis for the free-energy profile for electron transfer along the chain) as required to interpret intramolecular electron transfer rates and identify possible energy coupling sites (8). However, not all of the FeS clusters have been detected by EPR, and the assignments of the observed EPR signals to the structurally defined clusters remain controversial (3–6). Five reduced FeS clusters are typically observed in complex I from B. taurus reduced by NADH, and their CW EPR spectra are denoted N1b, N2, N3, N4 and N5. Signal N1a, from 2Fe[24] (see Fig. 1 for our nomenclature), is exhibited by the 24 kDa subunit and the 24 + 51 kDa subcomplex, but not by complex I (6), so it is not relevant here. Fig. 1 presents the three contending assignments of signals to clusters. Signal N1b is from a 2Fe cluster, but not from 2Fe[24], so it is from 2Fe[75] (position 2 in the seven-cluster chain from 4Fe[51] to 4Fe[PS], see Fig. 1). Signals N3 and N2 are from 4Fe[51] (position 1) and 4Fe[PS] (position 7), respectively, because interactions with the flavosemiquinone (9) and ubisemiquinones (10) have been observed. Ohnishi's model (A) assigns N4 and N5 to 4Fe[75]C and 4Fe[75]H (positions 3 and 4), respectively (3, 4), whereas Hirst's models (B and C) assign N4 to one (or both) of 4Fe[TY]1 and 4Fe[TY]2 (positions 5 and 6), and N5 to 4Fe[75]C (position 3) (5, 6). Importantly, the assignments of N4 and N5 rely on data from overexpressed subunits, enzyme fragments, and point mutations, and thus on circumstantial evidence rather than on direct measurements on intact B. taurus complex I.
Here, we assign N4 directly by DEER spectroscopy (11). DEER uses the electron-electron dipolar interaction between two paramagnetic centers to ascertain their separation and relative orientation; it is well established for determining the distances between organic radicals (such as NO•) with S = 1/2 ground states and precisely known magnetic moments (12, 13). In contrast, applying DEER to metal sites in multicentered enzymes is a relatively new application that builds on the seminal work of Bertrand (14) and Noodleman et al. (15) on FeS clusters, and on the application of DEER to a [NiFe]-hydrogenase by Bittl and coworkers (16, 17). First, the magnetic moment of a cluster results from ferromagnetic and antiferromagnetic coupling between the Fe subsites and varies with the cluster type and environment (14). Second, FeS EPR spectra are much broader than the microwave (mw) pulse excitation profiles of the DEER sequence, so each pulse selects only a limited range of molecular orientations. Therefore, a series of experiments, in which both the magnetic field and pulse frequencies are varied, must be combined. Here, we use the cluster positions defined by the structure of the hydrophilic domain of T. thermophilus complex I to simulate DEER traces for each of the models A, B and C, and compare them to our experimental data. Once N4 is assigned, the origin of N5 is deduced. Our assignment of N4 has direct implications for thermodynamic and kinetic models of electron transfer in complex I, and for future investigations of the mechanism of energy transduction.
Determination of Principal g values and Linewidths for Signals N1b, N2, N3, and N4.
EPR spectra of complex I from B. taurus recorded at X- and W-band are shown in Fig. 2. The spectra comprise four overlapping signals (N1b, N2, N3, and N4) from four reduced FeS clusters. Each signal is described by three principal g values and three g-strain parameters to describe the linewidths (see Table 1). As expected from their reduction potentials (3), N2, N3, and N4 contribute significantly at both -0.4 and -0.3 V (see Table 1). Signal N1b is substoichiometric (it is small at -0.4 V and essentially absent at -0.3 V) hence the cluster responsible has a more negative potential (6). Signal N5 is not observed because it is too fast-relaxing (it can only be observed clearly at lower temperatures, below 7 K (3, 6)). Importantly, the X- and W-band spectra essentially overlay when plotted on a g-value axis to remove the mw frequency/magnetic field dependence (see Fig. 2), indicating that the spectral peak positions correspond to the principal g values. Furthermore, any dipolar or exchange interactions between the EPR visible clusters must be less than ∼50 MHz, the X-band linewidth, indicating an intercluster distance greater than ∼10 Å (depending on the magnetic moments, see below).
Principal g values, g-strain parameters, and relative intensities of spectra from the four reduced FeS clusters observed in B. taurus complex I
EPR spectra of complex I from B. taurus, and simulations for N1b, N2, N3, and N4. X-band CW EPR spectra of complex I reduced to -0.4 V and -0.3 V were recorded at 12 K, at 9.408 and 9.393 GHz, respectively. The W-band (93.875 GHz) echo-detected EPR spectrum (numerical 1st derivative representation) of complex I reduced to -0.4 V was recorded at 8.5 K. The three spectra (Black) are compared to one another and to their simulated spectra (18) (Red, comprising N1b, N2, N3, and N4, see Table 1), by using the g-value scale. N1b, N2, N3, and N4 are shown for the -0.4 V X-band spectrum. At -0.3 V, N1b is essentially absent. See Experimental Methods section for the measurement parameters.
DEER Traces from B. taurus Complex I.
DEER spectroscopy employs two mw frequencies to isolate the dipolar interactions (see Fig. 3). The pulse sequence at frequency ωA produces a refocused echo from the detection (A) spins; the pulse at frequency ωB inverts the pump (B) spins, and changes the local field at the detection spins. The resulting DEER traces (see Figs. 4 and 5) show how the refocused echo intensity (modulated by the dipolar coupling frequencies) depends upon the timing of the pump pulse, t, within the detection pulse sequence. Different frequency combinations for ωA and ωB select different sets of orientations of a cluster pair with respect to the spectrometer's magnetic field, B0 (see Fig. 6 and Fig. S1), and each DEER trace comprises a set of dipolar frequencies, ωdd, from the contributing cluster pair orientations:
[1]The superscript m denotes a single orientation of the unit magnetic field vector
with respect to the molecular frame (see Fig. 6 for the polar angles θ and ϕ); g values gA and gB are from cluster A (detection) and B (pump), respectively;
defines the angle between
and the FeAi-FeBj unit vector nij; rij is the distance between i and j; and, kAi and kBj are the spin projection factors (SPFs) of each Fe (i of cluster A, j of cluster B). SPFs describe how the spins of the Fe subsites in a cluster couple magnetically to produce the observed S = 1/2 spin state. Then, the simulated DEER oscillation from a pair of clusters is:
[2]The term
describes the extent to which the A (detection) spin is excited by the detection pulse sequence with the field orientation
is the corresponding modulation or oscillation depth, describing the extent to which the pump pulse is resonant with the B (pump) spin for
. To account for the random distribution of molecules in the frozen-solution samples, a summation is performed over all orientations of
with respect to the molecular frame.
The four-pulse DEER sequence. The mw detection subsequence at ωA is applied to the detection (A) spins, and the pump pulse at frequency ωB is applied to the pump (B) spins. The refocused echo from the detection spins at time 2(τ1 + τ2) is observed as a function of time (t), where t0 = 2τ1 is outside the spectrometer deadtime of ≈100 ns.
X-band echo-detected EPR spectrum of complex I reduced to -0.4 V recorded at 10 K and simulated with N1b, N2, N3, and N4. Data in black, simulation (see Table 1) in red. The markers correspond to the pump (Red) and detection (Black) pulse positions for the DEER traces in Fig. 5. Positions 3* and 4* refer to the -0.3 V sample; all others refer to the -0.4 V sample. See Experimental Methods section for the measurement parameters.
DEER spectra from B. taurus complex I reduced to -0.4 V and -0.3 V measured at 10 K. DEER traces (Black) and best-fit simulations (Red) for models A, B, and C. Traces 3* and 4* refer to the -0.3 V sample, all others are from the -0.4 V sample, using the following field (mT), detection (ωA, GHz), and pump (ωB, GHz) pulse positions (see also Fig. 4): (1) B0 = 337.7, ωA = 9.1104, ωB = 9.7269; (2) B0 = 340.5, ωA = 9.1888, ωB = 9.7267; (3) B0 = 340.4, ωA = 9.1810, ωB = 9.4500; (3*) B0 = 345.9, ωA = 9.3382, ωB = 9.5998; (4*) B0 = 345.9, ωA = 9.3382, ωB = 9.5998; (5) B0 = 347.3, ωA = 9.3682, ωB = 9.1888. All traces are normalized to the intensity at t = 0, and intensity changes are denoted by the scale bar. The fits shown are for k1,2 = +1.17 and k3,4 = -0.67; the detection-pump spin pairs used in each model are given below the figures, along with the mean intercluster distances.
Orientation selective DEER simulation parameters for a [2Fe-2S]-[4Fe-4S] pair. The unit magnetic field vector,
, is defined by the angles θ and ϕ. nij is the unit vector between Fe ions i and j of the two clusters, rij is the distance between them, and
is the angle between
and nij. The g3 axis from the [2Fe-2S] spin is along the Fe-Fe vector. The g3 axis from the [4Fe-4S] spin is normal to the faces assigned to the Fe2+ and the Fe2.5+ pairs, and, in this example, the g1 and g2 axes are normal to the Fe2+-Fe2.5+ containing faces.
First, we evaluate qualitatively the experimental DEER traces in Fig. 5. The intercluster distances can be estimated from the dominant periods of oscillation, ranging from 0.5 to 0.7 μs (ωdd/2π = 2.0–1.4 MHz). Assuming that the dominant oscillation corresponds to the intense (perpendicular) feature in the powder-averaged dipolar spectrum, and taking reasonable values for the SPFs (k1,2 = 1.35, k3,4 = -0.85, see below), then 10 Å equates to 70–150 MHz, 20 Å to 2–5 MHz, 30 Å to 1–3 MHz, and 40 Å to 0.7–1.3 MHz (the exact values depend on the SPF assignments): the traces in Fig. 5 suggest an intercluster distance of ≈20–35 Å. The cluster pairs that contribute most intensity to the observed traces can also be identified. In Fig. 5, traces 3* and 3 (matching traces from samples at -0.3 and -0.4 V, respectively) are essentially identical. Signal N1b is only observed at -0.4 V (see Table 1) so the close match suggests that N1b does not contribute significantly. The DEER electron-spin echo from N5 is not observed at 10 K because it relaxes too rapidly: It contributes only to signal decay, not to the DEER oscillations. The remaining signals, N2, N3, and N4, yield three pairs: N2-N3, N2-N4, and N3-N4. The 4Fe[PS] (N2) and 4Fe[51] (N3) clusters are too far apart (61 Å, see Fig. 1) to contribute more than a weak, indiscernible monotonic decay. In model A the N2-N4 and N3-N4 distances are 49 and 17 Å, in model B 26 and 41 Å, and in model C 14 and 49 Å, respectively. In models A and C neither distance is around 20 to 35 Å, already indicating that model B is most likely.
A quantitative evaluation of each model requires the numerical simulation and analysis of a comprehensive set of DEER traces, as described below. Importantly, because the dipolar frequencies depend on the orientation of a cluster pair with respect to B0 (as well as on the SPFs and on 1/r3), a unique description of the data is only obtained when a sufficient set of orientations are interrogated, by varying the pump and detection pulse positions (see Fig. S1). Fig. 4 depicts the pulse positions used to measure the DEER traces in Fig. 5; additional data, which were included in the analysis, are described in Figs. S2 and S3. Specifically, the simulations (SI Text and ref. (19) for details) require the spin Hamiltonian parameters in Table 1, the coordinates and SPFs for each Fe of each paramagnetic cluster, and the g-axis orientation for each paramagnetic cluster, relative to the molecular frame. The latter quantities are not known precisely, and our procedures to account for them are described next.
The Fe Coordinates: The Structure of the Hydrophilic Domain from T. thermophilus Complex I as a Template for B. taurus Complex I.
Here, we use the structure of the hydrophilic domain of T. thermophilus complex I (2) (the only available structure) as our basis for interpreting EPR data from the B. taurus enzyme (the best characterized enzyme spectroscopically). To evaluate our structural model the sequences of the T. thermophilus and B. taurus core hydrophilic proteins were compared (see SI Text for details). The sequence identities and similarities (see Table S1) are high in all cases. The subunits are generally conserved in length, and there are few large alignment gaps (except for in the large domain of the 75 kDa subunit). Multiple sequence alignments and comparative protein models, generated for the B. taurus structure, support the close structural identity of the two proteins. They show also that essentially all the insertions/deletions comprise surface loops or structures, and the few that do not are readily accommodated without significant alterations in structure (see Fig. S4).
There are three reasons to challenge T. thermophilus complex I as a viable structural model for the B. taurus enzyme: i) the Nqo15 subunit of T. thermophilus (2) is not found in B. taurus, and the hydrophilic domain of B. taurus complex I contains seven subunits that are not found in T. thermophilus (7); ii) the 75 kDa subunit of T. thermophilus complex I ligates a 4Fe cluster (4Fe[75]*) that is not found in B. taurus; iii) T. thermophilus complex I has not been well characterized by EPR, but available spectra (20) do not closely resemble those from B. taurus (notably, the gz component of N4, usually evident at g ∼ 2.10 (5), has not been observed). Interestingly, the EPR spectra of E. coli complex I resemble those from B. taurus more closely (21), although the sequence identities between all three species are comparable (see Table S1). E. coli complex I does not contain any extra subunits, and it does contain 4Fe[75]*, so neither feature is a significant determinant of the EPR spectrum. Although the immediate cluster environments are very similar in T. thermophilus and B. taurus complex I, they are not identical (see SI Text and Fig. S5) and residue differences close to one or more clusters will affect their spectra and/or reduction potentials (different clusters are reduced in different species). However, it is highly unlikely that these changes affect the structure. Consequently, we consider the cofactor arrangement in T. thermophilus complex I an excellent model for the interpretation of spectral data from B. taurus complex I.
Spin Projection Factors (SPFs) and g-Axis Orientation Combinations.
The reduced 2Fe[75] cluster (N1b) has an axial g matrix. For completeness we considered two g3-axis orientations, one along the Fe-Fe vector and one normal to the [2Fe-2S] plane; the g1 and g2 axes can take any direction perpendicular to the g3 axis without changing the simulations (see Fig. 6). In 2Fe clusters like 2Fe[75], it is accepted that strong antiferromagnetic coupling between the Fe2+ (S = 2) and Fe3+ (S = 5/2) subsites produces a well separated Stotal = 1/2 ground state, and the SPFs are k1(Fe2+) = -4/3 and k2(Fe3+) = +7/3 (22). However, there are two possible assignments for k1 and k2 because the Fe2+ and Fe3+ could be in either subsite.
In reduced 4Fe clusters there are many possible spin coupling schemes for the (formally) three Fe2+ (S = 2) and one Fe3+ (S = 5/2) (23). Mössbauer spectroscopy (24) and theoretical studies (15) have indicated that 4Fe clusters contain delocalized valence electrons distributed over an Fe2+-Fe2+ pair (k3 = k4) and a mixed valence Fe2.5+-Fe2.5+ pair (k1 = k2) (see Fig. 6). The six ways of assigning the two pairs produce six arrangements of SPFs, and in each arrangement the g3-axis is considered perpendicular to both the Fe2+-Fe2+ and Fe2.5+-Fe2.5+ vectors (25, 26). Signal N2 is close to axial, so there are only six possible combinations of g axes and SPFs. Signals N3 and N4 have rhombic g matrices so the orientations of the g1 and g2 axes are important. We tested two generally accepted possibilities: the g1 and g2 axes point along the Fe2+-Fe2+ and the Fe2.5+-Fe2.5+ vectors, or they are rotated by 45° (to be normal to the faces that contain an Fe2+ and an Fe2.5+). Therefore, N3 and N4 both have 6 × 2 × 2 = 24 possible SPF/g-axis combinations.
The SPFs of the Fe2+ and Fe2.5+ pairs in 4Fe clusters are variable also (27). In reduced ferredoxin-type clusters, they may result from the |5/2|2|1/2〉 state with k1,2 = +1.17, k3,4 = -0.67, the |7/2|3|1/2〉 state with k1,2 = +1.50, k3,4 = -1.00, or a mixed state with average values of k1,2 = +1.35, k3,4 = -0.85 (notation: spin state of Fe2.5+ pair | spin state of Fe2+ pair | spin ground state of cluster). All three possibilities were tested. As a sensitivity test we also investigated the |9/2|4|1/2〉 state (k1,2 = +1.83, k3,4 = -1.33) that is thought to contribute to the ground state of aconitase-type clusters. Finally, to ensure a manageable computation time, the SPFs of each 4Fe cluster in a given simulation were the same.
In summary, the number of SPF/g-axis orientation combinations are N1b (4), N2 (6), N3 (24), and N4 (24), and there are four sets of 4Fe SPFs. For example, to simulate the N2-N4 pair, there are 6 × 24 × 4 = 576 possible combinations.
Simulation of Observed DEER Traces and Assignment of N4.
The observed DEER traces are weighted sums of the refocused echo intensity from each paramagnetic cluster,
[3]The terms ANx and
are the refocused echo intensity and the simulated DEER trace (from Eq. 2) for the detection spin, Nx, respectively. Note that ANx depends on B0, but B0 does not vary during an experiment so ANx is a constant in Eq. 3. The N2-N3, N2-N4, and N2-N1b pairs all contribute to
, but the N2-N3 and N2-N1b distances (61 and 51 Å, respectively) are too long to yield oscillations (in any model, see Fig. 1), so only N2-N4 is included. The N3–N4 pair is expected to contribute to
, but the N3-N2 distance is again too long. The N3-N1b distance is too short (14 Å) and, N1b is present only substoichiometrically, so the oscillation depth, cmod, of the N3-N1b contribution is low. Three pairs, N4-N2, N4-N3 and N4-N1b, may contribute to
, but the N4-N1b contribution (like N3-N1b) is only of low intensity. Finally, the contribution from
is small because AN1b is small and only the unknown N1b-N4 distance merits consideration (model A, 13.5 Å; model B, 35.0 Å, and model C, 41.2 Å). Finally, because our simulations do not consider the additional complexity of a single detection spin being influenced simultaneously by two pump spins (the large g-matrix anisotropies make this case unlikely), each simulation comprises only four cluster pairs (one for each detection spin). The pair combinations which were tested are listed in Table S2, and the pairs which produced the best-fit simulations for each model are shown in Fig. 5, along with the intercluster distances.
Finally, each simulated DEER trace from Eq. 3 is dampened by an exponential decay to account for signal relaxation, and a baseline offset is added, to account for unmodulated intensity that is not correctly described (resulting primarily from inaccuracies in ANx and the g-axis orientations). The residual, R, is calculated by Eq. 4, and the best fits to the data traces, yexp(t), were identified by inspecting the fits with the lowest squared residual values (the sum of R2 over all the DEER traces):
The DEER simulation algorithm has been described previously (19), and its particular application to a set of FeS clusters is summarized in SI Text. In accord with previous work on, for example, NO radicals (12), we chose to fit our data directly in the time domain, to allow simultaneous fitting of both the baseline and the DEER oscillation, and to avoid difficulties associated with the Fourier transformation of DEER traces with large unmodulated contributions. First, an initial screen was used to test all possible combinations of the SPFs, g-axis orientations and cluster pairs, for each model. The constant c2 was included to maximize the sensitivity to the oscillation period (to the SPFs and the intercluster distances). The initial screen excluded ≈95% of the combinations. Then, the remaining combinations were reduced by examining fits with c2 = 0, to maximize the sensitivity to the oscillation depth (to the g-axis orientations). The best fits to the data, for each of models A, B, and C (see Fig. 1), are displayed in Fig. 5A, B, and C, respectively.
The fits shown in Fig. 5 (additional data are shown in Fig. S3) include c2, so they are most sensitive to the oscillation period. Clearly, model B describes the experimental data well, but models A and C produce only simulations with completely inappropriate oscillation periods: the intercluster distances in models A and C are entirely inconsistent with the data. In contrast, ≈1% of the total number of possible combinations produced satisfactory fits for model B. All these fits contained the same g-axis orientations for N4 and N2 (see Fig. S6). Changing the g-axis orientation of N3 had little effect because the N3–N4 distance is too long; changing the SPFs for N1b had essentially no effect. The best-quality fits (see Fig. 5B) used the SPFs from the |5/2|2|1/2〉 state, but those from the mixed |7/2|3|1/2〉 and |5/2|2|1/2〉 state produced similar quality fits. The |7/2|3|1/2〉 state produced only lower quality fits, and the |9/2|4|1/2〉 state did not produce any satisfactory fits. Imperfections in the best fits from model B may arise from small inaccuracies in the structural model (for example, a cluster may be tilted slightly in the B. taurus enzyme, relative to in T. thermophilus), or from our imprecise knowledge of the g-axis orientations and SPFs (see Fig. S7). These uncertainties limit the accuracy of our data fits, but they do not detract from the unambiguous assignment of signal N4 to the 4Fe[TY]1 cluster (position 5). DEER clearly discriminates between the models because of its 1/r3 sensitivity to distance.
We conclude that N4 originates from the reduced 4Fe[TY]1 cluster in complex I (position 5) and thus model B is correct.
The application of DEER to measure the distances between complex paramagnetic metal centers in enzymes is a natural extension from established studies of the distances between organic S = 1/2 radicals (11–13). In reduced complex I there are five paramagnetic FeS clusters, and it would be unrealistic to attempt to define their spatial relationships ab initio using DEER. Not only do five interacting paramagnets lead to a complicated network of relationships, but the complex magnetic interactions within the clusters and the orientation selectivity of individual DEER measurements complicate the analysis significantly—for even a single pair of clusters. Here, we combine DEER spectroscopy with a structural model for the FeS cluster ensemble. Our success has relied on using structural information to restrict both the spatial arrangements of the paramagnetic centers and the g-axis orientations. Our results present a definitive assignment of EPR signal N4 to a structurally defined cluster in complex I, because they are based on direct measurements, rather than on data from overexpressed subunits and subcomplexes—which may not retain their structural or spectral integrity.
Our analysis concludes unambiguously that N4 derives from 4Fe[TY]1 (position 5), as suggested by Hirst and coworkers (5), and supports the consensus assignment of N1b to 2Fe[75] (position 2), N2 to 4Fe[PS] (position 7), and N3 to 4Fe[51] (position 1). It is entirely inconsistent with the assignment of N4 to 4Fe[75]C (position 3), as contended by Ohnishi et al. (3, 4), or with the assignment of N4 to 4Fe[TY]2 (position 6), an alternative suggestion by Hirst and coworkers (5). Although our analysis does not address directly the assignment of N5, mutation of the histidine ligand of 4Fe[75]H to alanine did not affect N5 in Yarrowia lipolytica complex I (although it did prevent catalysis) (28). Therefore, the assignment of N5 to 4Fe[75]H (position 4) by Ohnishi et al. (3, 4) is not correct. Hirst and coworkers concurred that N5 derives from the 75 kDa subunit but assigned it instead to 4Fe[75]C (position 3) (5), an assignment that is consistent with all extant data and with the analysis described here. Signal N5 is unusual: it is observed only in a subset of eukaryotic enzymes, and it relaxes more rapidly at -0.4 V than at lower potential (6).
The assignment of EPR signals to structurally defined clusters (model B, see Fig. 1) provides an alternating or 'roller-coaster' potential energy profile for electron transfer along the cluster chain between the active sites. Alternating the low and high potential clusters optimizes the rate at which a single electron traverses the empty chain, whereas two or more adjacent low potential clusters would impede electron transfer significantly (8). Furthermore, in the mitochondrial matrix the NAD+ pool is typically 90% oxidized, and, because NADH/NAD+ interconversion and intramolecular electron transfer in complex I are fast, the FeS clusters are probably, on average, reduced to the NAD+ potential (≈-0.3 V) during turnover. Consequently, four electrons occupy the seven FeS cluster chain [for N3 (4Fe[51]), N5 (4Fe[75]C), and N4 (4Fe[TY]1) EpH7 = -0.25 V; for N2 (4Fe[PS]) EpH7 = -0.1 V (3)]; the other three clusters are oxidized. An alternating occupancy along the chain (positions 1, 3, 5, and 7) further optimizes the rate at which an electron is transferred out to quinone, following input of an electron from the flavin (in analogy to the Grotthuss mechanism for proton transfer). More importantly, the alternating occupancy may be key to efficient energy conversion by complex I; the energy of the incoming electron is conserved in the outgoing electron (in analogy to the conservation of energy in Newton's cradle), allowing close to the full redox potential drop from NADH to quinone to be trapped in proton translocation.
Experimental Methods
Preparation of EPR Samples of Complex I from B. taurus.
Complex I was isolated from B. taurus heart tissue as described previously (29), except ethylene glycol, not glycerol, was used in the membrane preparation, phenylmethanesulfonyl fluoride was omitted, and asolectin was omitted from the ion-exchange chromatography. Where possible ultrapure chemicals (Fluka) were used, to minimize contamination by manganese and eliminate the Mn2+ signal from W-band spectra. The ultrapure chemicals were dithiothreitol (Mn ≤ 5 ppm), ethylenediaminetetraacetic acid (Mn ≤ 5 ppm), ethylene glycol (Mn ≤ 0.05 ppm), KCl (Mn ≤ 0.01 ppm), NaCl (Mn ≤ 0.01 ppm), and Trizma base (Mn ≤ 5 ppm). Purified complex I was concentrated to ∼70 mg mL-1 (quantified by the Pierce bicinchoninic acid assay) using Vivaspin centrifugal concentrators with a 100 kDa cutoff, and imported into an anaerobic glovebox (O2 ∼ 2 ppm). Typically, it was reduced by 10 mM NADH (to ≈-0.4 V), or by 140 mM NAD+ and 2 mM NADH (to ≈-0.3 V), to a final concentration of ∼55 mg mL-1 (∼55 μM), then the samples were frozen anaerobically in liquid nitrogen.
EPR Spectroscopy.
X-band CW EPR experiments were performed on a Bruker BioSpin GmbH EMX spectrometer equipped with a high sensitivity Bruker probehead and a low-temperature Oxford Instruments CF935 helium-flow cryostat. The magnetic field was calibrated at room temperature with an external 2,2-diphenyl-1-picrylhydrazyl standard (g = 2.0036). Experiments were conducted at 12 K, with 0.5 mW power, and 0.5 mT amplitude at 100 kHz. Pulsed EPR measurements were performed on a W-/X-band Bruker Elexsys 680 spectrometer using a Bruker 3 mm split ring resonator (ER-4118X_MS3) at X-band and a EN600-1021H TeraFlex resonator at W-band, both equipped with an Oxford Instruments cryostat and a variable temperature unit. DEER measurements employed a four pulse sequence, π/2–τ1–π–τ1–τ2–π–τ2–echo, for the detection pulses at frequency ωA, and a single pump pulse at frequency ωB moved within the refocused echo sequence (see Fig. 3). The detection pulses were phase cycled according to [+x,+y,+y,+,-x,+y,+y,-], and employed lengths of tπ/tπ/2 = 40 ns/40 ns. The pump pulse (tπ) had a duration of either 16, 20, or 24 ns, depending on the achievable excitation magnetic (B1) field. A variable τ1 time (from 140–164 ns, or from 180–204 ns) was applied to dampen nuclear modulations, and time τ2 was 1.1–1.6 μs. Echo-detected EPR spectra were recorded by integrating the echo intensity either from a refocused echo (using the same parameters as in the DEER experiments), or from the pulse sequence π/2–τ–π–τ–echo, with tπ/2 = 48 ns, tπ = 96 ns, and τ = 200 ns at X-band, and with tπ/2 = 24 ns, tπ = 48 ns, and τ = 140 ns at W-band. All pulsed X-band experiments were carried out at 10 K with a repetition time of 2 ms, and W-band measurements were performed at 8.5 K with a repetition time of 1.5 ms.
This work was supported by the Medical Research Council and the Engineering and Physical Sciences Research Council (Grant EP/D044855D/1 supporting the Oxford Center for Advanced Electron Spin Resonance).
1To whom correspondence may be addressed. E-mail:jeffrey.harmer{at}chem.ox.ac.uk or jh{at}mrc-mbu.cam.ac.uk.
Author contributions: F.A.A., J. Harmer, and J. Hirst designed research; M.M.R., M.S.K., and A.J.R. performed research; M.M.R., J. Harmer, and J. Hirst analyzed data; M.M.R., M.S.K., A.J.R., F.A.A., J. Harmer, and J. Hirst wrote the paper.
This article is a PNAS Direct Submission. D.B. is a guest editor invited by the Editorial Board.
This article contains supporting information online at www.pnas.org/cgi/content/full/0908050107/DCSupplemental.
Freely available online through the PNAS open access option.
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Maxie M. Roessler, Martin S. King, Alan J. Robinson, Fraser A. Armstrong, Jeffrey Harmer, Judy Hirst
Proceedings of the National Academy of Sciences Feb 2010, 107 (5) 1930-1935; DOI: 10.1073/pnas.0908050107
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The Opposition People's United Party (PUP) today issued a call, via the PUP's United Women's Group, for the resignation of the United Democratic Party's (UDP) Mesopotamia area representative – Minister of Housing Michael Finnegan, for "an unprecedented barrage of viciously crude [attacks] and [an] explicitly sexual innuendo" he made in Parliament, directed at the only woman in the House – the PUP's Belize Rural Central area representative Dolores Balderamos-Garcia.
The controversial comments—which promptly went viral on social media—were made last Wednesday, August 7, at a Special Sitting of Parliament which, paradoxically, had as its highlight a motion to "deprecate" the actions of former PUP leader Said Musa and current PUP leader, Opposition Leader Francis Fonseca, in giving a secret tax concession to British billionaire Michael Ashcroft.
However, the public discourse has since been focused on comments Finnegan made to Balderamos-Garcia, when she rose to protest the public conduct of the Sergeant of Arms: Brian Audinett, popularly known as Yellow Man.
She said that the mace borne by the Sergeant of Arms is the symbol of dignity and authority entrusted by the Speaker in the House, and even the police on duty at the House are under the direction of the mace bearer. However, she said, she has been observing Yellow Man's conduct since last year.
"So she is going around having a camera on Yellow Man to see where he urinates?" Finnegan questioned.
It appears someone did have a camera following Yellow Man: the PUP posted online this afternoon a photo which they say is Yellow Man urinating in public, on a Government vehicle parked in front of the Heritage Bank on Princess Margaret Drive in Belize City.
Yellow Man is "out of the hood," and "You know how you behave out of the hood…," Finnegan remarked.
At this afternoon's press conference called by the PUP women's group, Balderamos-Garcia also indicated that the Sergeant of Arms has been recently accused of assaulting a police officer. The case in question was reported by Phillip "Fawda" Henry – who has also alleged a recent assault.
While she protested in Parliament, Port Loyola area representative Anthony "Boots" Martinez rose on a point of order, saying that Balderamos-Garcia was getting personal when she raised issue with the Sergeant of Arms' drinking habits.
"They can take a point of order, but they won't shut me up," Balderamos-Garcia replied, restating her concern about Yellow Man's decorum and conduct.
When we spoke with Finnegan today, we asked him if he could at least concede to the general concern Balderamos-Garcia had raised. He still resisted doing so.
"I will say to you, if she had a problem with the Sergeant of Arms, or if I have a problem with a Senior Clerk or Clerk of the National Assembly, I don't take that to the Parliament; I go to the Speaker… then I have a discussion with the Speaker with what I don't like," he said.
Finnegan accused Balderamos-Garcia of having had "a personal beef" with Yellow Man, stemming from disputes they had during the last village council election.
"According to some experts, this disrespectful behavior is attributed to certain events that happened earlier in the person's life. It is caused by an abusive or negligent mother, sister, school teacher or girlfriend that planted a seed deep down in his subconscious mind," they added.
"What I did Wednesday had nothing to do with women. It had to do with Dolores' conduct on Yellow Man," Finnegan replied.
The public upset, though, has been over the language Finnegan chose to use in defending Brian "Yellow Man" Audinett.
"We strongly believe that this is a trend by the said area representative. He has disrespected the following women: Madam Speaker Hon. Elizabeth Zabaneh; Miss Rhenae Nunez and now Hon. Dolores Balderamos-Garcia. Who is the next woman in line who will be disrespected?" they question.
"We also called on the area representative to once again apologize to Hon. Dolores Balderamos-Garcia and all the women he has disrespected and to conduct himself honorably in the House," the PUP mayors added.
They also call on all the organizations in Belize who advocate for women's rights to stand up and condemn the area representative's actions, and point to the fact that Belize is a signatory to the Convention on the Elimination on the Discrimination of Women (CEDAW).
When we spoke with him today, Finnegan did not apologize, though; although he joked about resigning to contest a by-election against the PUP – confident of another win.
However, he did signal that he may have been hasty in responding and could "maybe" have used different language.


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Doliops imitator is a species of beetle in the family Cerambycidae. It was described by Schultze in 1918.

References

Doliops
Beetles described in 1918