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Source: China government network
Date: 2020-05-29
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Xinhua news agency, Beijing, May, 28 Title: Draw the great power from the people -- General Secretary Xi Jinping and the NPC deputies and CPPCC members attending the 2020 NPC and CPPCC National Committee are working together to compile the national record.
Xinhua News Agency reporter Huo Xiaoguang, Zhang Xiaosong, Zou Wei, Zhu Jichai
To block the outbreak of the new crown pneumonia and win the battle for a well-off society and a decisive battle. How hard it was in 2020, how extraordinary this year's national meetings are.
In recent days, general secretary Xi Jinping participated in the three session of the thirteen National People's Congress, the Inner Mongolia and Hubei delegations, attended the plenary meeting of the delegation of the PLA and the armed police force, visited the economic committee members who participated in the three session of the thirteen session of the CPPCC National Committee and participated in the joint meeting. We must win the battle to build a well-off society in an all-round way and fight for war.
The hearts of the people - "to protect people's lives and health, we can at all costs".
On May 24th, Roger, the deputy to the National People's Congress (NPC), took part in the deliberations and statements of the delegation of Hubei. Xinhua News Agency reporter Huang Jingwen photo
Roger, the Secretary of the Party committee and President of Shiyan Taihe Hospital in Hubei Province, did not realize that within three days, the two general secretary Xi Jinping mentioned him.
"This morning, I was impressed by what I said on behalf of a NPC deputy from Hubei on the channel." On the afternoon of May 22nd, Xi Jinping, general secretary of the hall of the Great Hall of the people, came to the Inner Mongolia delegation where he was attending the deliberation. Speaking of the new crown pneumonia epidemic prevention and control struggle, Xi Jinping described the details.
"The representative told reporters that there were more than 3000 new crowns with pneumonia over 80 years old in Hubei. Among them, a 87 year old man, who had 10 nurses and nurses carefully nurturing for decades, finally saved the life of the old man."
What is the supremacy of the people? So many people turn around a patient, which is truly at all costs. " General secretary Xi Jinping said with emotion.
The NPC deputy mentioned by general secretary is Roger. For several months, fighting against the epidemic front line and stepping into the victory of the fight against the epidemic and entering the hall of people's participation in politics and politics, Roger's heart was full of emotion.
Two days later, on the afternoon of May 24th, the delegation of Hubei considered the scene. Roger sat opposite to general secretary Xi Jinping, making the first speech. Just opening his mouth, he was recognized by the general secretary.
"Are you Roger?"
Since the outbreak of the epidemic, the situation of Hubei and Wuhan has always touched the heart of general secretary Xi Jinping.
In March 10th, at the critical moment of epidemic prevention and control, general secretary Xi Jinping flew to Wuhan to direct the war to encourage the people of Hubei.
After a lapse of two months, general secretary Xi Jinping once again came to the middle of the Hubei people.
"According to the needs of epidemic prevention and control, the leading comrades of the Central Committee have reduced the number of the next group. I particularly suggest that the delegation of Hubei must come. You are the representatives of the about 60000000 people of Hubei. I want to see you. " The general secretary said.
Special arrangements, special concerns, warmed the hearts of the delegates and warmed the hearts of the people of Hubei.
Looking back at the moment, I am filled with emotion.
In 2020, what a breathtaking chapter in the history of the Chinese nation?
In the face of the new crown pneumonia epidemic, since the founding of new China, the major public health emergencies such as the fastest speed of transmission, the widest range of infection and the most difficult prevention and control have been carried out. The Party Central Committee with Comrade Xi Jinping as its core is resolute, resolute and decisive, putting the safety and health of the people in the first place. Under the command and personal deployment of general secretary Xi Jinping, the 1 billion 400 million Chinese people started a people's war, a total war and a blocking war.
In order to fight the "epidemic" needs, this year's two sessions have been postponed for more than two months. This is a very unusual move.
In March 10th, the Wuhan Wuchang shelter hospital was formally suspended. Medical staff took pictures after taking the new crown pneumonia. Xinhua News Agency reporter Xiao Yijiu photo
Through arduous efforts, decisive achievements have been made in the battle of Wuhan and Hubei, and major strategic achievements in the prevention and control of the epidemic have been achieved.
In April 16th, people in and out of Hanzheng Street pedestrian street in Wuhan scanned health codes. Xinhua News Agency reporter Wang Yuguo photo
Now, the annual reunion of the country is a remarkable symbol of China's major strategic achievements in the fight against epidemic diseases.
Yangtze River bridge, Wuhan (UAV photo), taken at the morning rush hour in April 15th. Xinhua News Agency reporter Li He photo
In April 22nd, the "chefs" in Wuhan's grain road street, "chefs" every day, the chef in the red hot noodle shop was making hot and dry noodles. Xinhua News Agency reporter Shen Bohan photo
"The enterprises in Wuhan resumed their work and resumed their production. The children went to school one after another. The hospitals were in normal attendance, and the shopping malls and supermarkets were all open. The vigorous Wuhan returned." The 5 representatives spoke successively, and reported to the general secretary the situation of "restarting" and "reborn" in Hubei and Wuhan from the epidemic situation, and the words were full of excitement.
On May 24th, Yu Cheng, deputy to the National People's Congress, took part in the deliberations and speeches of the delegation of Hubei. Xinhua News Agency reporter Huang Jingwen photo
Yu Cheng, a representative of the second light industrial school in Wuhan, brought the hearts of the people of Wuhan. During the epidemic, she went to volunteer in the Wuhan AI family international community.
In March 13th, a batch of live fish transported to the Wahson city Hankou Plaza community in Wuhan, Hubei province were unloading. On the same day, the first batch of 100 tons of live fish deployed from Xianning arrived at 11 communities in Wuhan's Qikou district under the jurisdiction of the community. Xinhua News Agency reporter Cheng Min photo
"General secretary, when you visited Hubei, people in Wuhan liked to eat live fish and organize more supplies. Within a week, the live fish was sent to our community. A more than 80 year old uncle Liao said excitedly, "I didn't expect such a serious epidemic, and I could eat fresh fish. Thank you for the party and the government."
Speaking of the emotional situation, Yu Cheng choked. She rose from her seat and bowed deeply.
In April 30th, the constructor of the seven Bureau of China Railway Works on the construction site of Wuhan Wu nine North pipe gallery project. Xinhua News Agency reporter Xiao Yijiu photo
"Before coming, I must say" thank you ". I would like to thank the general secretary, the CPC Central Committee, and all the people and friends from all walks of life who support Hubei and Wuhan.
The applause lasted for a long time.
"I was very moved to hear your speech." General secretary Xi Jinping said with deep affection, "the people of Hubei and the people of Wuhan have made great contributions to the prevention and control of the epidemic and have made great sacrifices. Wuhan is worthy of being a hero city. The people of Hubei and Wuhan are worthy of the heroic people. I would like to extend my sincere greetings and heartfelt thanks to all of you here and to the cadres and masses of all ethnic groups in Hubei.
"The Chinese Communist Party is rooted in the people and blood in the people. The fundamental purpose of the party to unite and lead the people in revolution, construction and reform is to enable the people to live a good life. No matter how much challenges and pressures they face, no matter how much sacrifices and costs they have, this is unswerving and unwavering.
In the Inner Mongolia delegation, general secretary Xi Jinping, taking the "people" as the key word, starts from the party's ideals and beliefs, its nature and purpose, and its initial mission, and profoundly interprets the connotation of these two words.
Talking about upholding the supremacy of the people, general secretary Xi Jinping quoted the archaic language as saying:
"The ancients said," the world is the same as the rest of the world, and the people who are good at the world are seeking the world. The Party Constitution clearly stipulates that our party does not have its own special interests. The party always puts the interests of the masses first. "
In May 11th, farmers weeding in the yellow flower garden near TANGJIABAO village in Xiping Town, Datong, Shanxi province. Xinhua News Agency (Photo by Chai Ting)
General secretary Xi Jinping talked with everyone about the experience of going to Shanxi on the eve of the two sessions: "I am in Datong's organic yellow flower standardization planting base, and the villagers say that the CPC is the most important achievement in doing the work for the people and for the benefit of the people."
The supremacy of the people! This is precisely the beginning of the Chinese Communists' ambition to struggle, which is the ruling idea of general secretary Xi Jinping.
Only by standing at this "origin" can we truly understand the weight of this sentence: "to protect people's lives and health, we can at all costs."
For the benefit of the people, "common prosperity and all-round well-off society, let's take this road together".
Achieving the goal of tackling poverty in a timely manner is a major concern of general secretary Xi Jinping. Since 2013, he has been working with representatives of the NPC and CPPCC members every year to tackle poverty.
"At present, there are 52 poverty-stricken counties in the country who have not yet removed their cap, and 2707 poor villages have not yet been listed.
On the morning of May 23rd, Xi Jinping, general secretary of the economic circle, visited the Beijing railway building to attend the three session of the thirteen session of the National Committee of the Chinese people's Political Consultative Conference.
More than two months ago, in March 6th, when the NPC and CPPCC held the meeting in the past year, general secretary Xi Jinping attended the largest meeting of the party's eighteen major tackling difficulties since the end of the party. On the eve of the two sessions, he went to Shaanxi and Shanxi to investigate and understand the progress of tackling poverty.
Beat the drum and urge the army. Now, in 2020, nearly 5 months have passed.
"Although the total amount is not large compared with the past, they are all poor in poverty and trapped in difficulties, which is the hardest part to chew." General secretary Xi Jinping's words expressed a firm determination: "we must strive to overcome the adverse effects brought by the new crown pneumonia epidemic and resolutely win the battle against poverty. This is our party's solemn commitment to the people and history. "
"This is also a complex of mine." The general secretary spoke slightly, his eyes flashing:
"When I was a farmer for six or seven years, I asked the people around me when I was hungry. What kind of life do you think is the best and what is the specific target?"
The common people have given the goal of "three steps": "the first goal is to hope that they won't beg for food and have enough to eat. Don't worry about what to eat. Half a year's food is not a problem. Further, the local dialect is called "pure grain son", that is, it can eat pure sorghum rice and corn flour. The third goal is that they think it's unattainable. "They want to eat fine grain and eat fine grains, and they often eat meat," which is "the standard of heaven" and "the desire of next life".
"I said, you should do more to boldly think of what is more advanced." Recalls General Secretary Xi Jinping, "their answer is that they will pick up golden shoulder pole when they go to work in the mountains."
The audience burst into a knowing laugh.
"I think this goal is also being achieved. This "golden shoulder pole" is what I understand as agricultural modernization. General secretary Xi Jinping contains true feelings. "This generation of people has such a complex. We must help our people, especially our farmers, on the road of socialism, and we must join the road of common prosperity and all-round well-off society."
For whom? What kind of development will it achieve? Rooted in the long history of the loess land, the answer has long been placed in Xi Jinping's mind.
In this year's government work report, China, as the world's second largest economy, has not put forward specific targets for annual economic growth.
In the Inner Mongolia delegation, general secretary Xi Jinping expounded the considerations behind this major decision:
"If we set a goal, it will become a strong stimulus and a growth rate. This is not consistent with our economic and social development purposes. We have been talking about not simply talking about heroes with GDP growth rate. We are pursuing high quality development, carrying out the development of new development concept and pursuing the happiness and good life of the people.
It is precisely because of this kind of starting point and foothold for the people and for the benefit of the people that during the two sessions, general secretary Xi Jinping frequently asked the government for the people and asked the people.
I always care about the progress of poverty alleviation and consolidation of achievements.
On May 23rd, Liu Qi, a member of the CPPCC National Committee, spoke at the CPPCC joint meeting. Xinhua News Agency reporter Xie Huanchi photo
At the CPPCC joint meeting, Liu Qi members mentioned 16 years ago that Xi Jinping led the delegation of the Zhejiang party and government to Chongqing Wulong county.
"At that time, I served as the county magistrate in Wulong, and I reported to you the idea of developing tourism to help the poor. We will continue to work as a member of the government in order to develop the relevant industries through poverty alleviation through tourism. Wulong has removed the hat of poor counties, and the total disposable income of the poor population has reached 10027 yuan.
In March 11th, in Shangyu District People's Bureau of Shaoxing City, Zhejiang Province, the Shangyu area employing enterprises and Xiaojin County migrant workers in Sichuan province made special recruitment on the Internet. Xinhua News Agency reporter Weng Xinyang photo
General secretary Xi Jinping was very happy and asked, "what is the traffic situation there now?"
"Now that all the high-speed roads are connected, the high-speed rail is also being built."
Tourists enjoy the rural scenery on the viewing floor of Fuxing village, Huang Ying Township, Wulong District, Chongqing (photo taken in March 28, 2019). Xinhua News Agency reporter Liu Chan photo
"Chongqing's tourism resources are still very rich. In the past, many "remote areas" have now become "fairyland in the world", and transportation infrastructure is very important.
On May 22nd, Fei Dongbin, deputy to the National People's Congress, took part in the deliberations and speeches of the delegation of Inner Mongolia. Xinhua News Agency reporter Huang Jingwen photo
On behalf of the delegation of Inner Mongolia, Fei Dongbin, mayor of Wulanchabu, said that after the poverty alleviation, the proportion of poor people returning to poverty due to illness is still relatively high. General secretary Xi Jinping was concerned about what the high incidence of diseases was, and urged that we should continue to do our work well.
Focusing on the key points and solving the biggest livelihood problem of employment with innovative ideas.
In the early days of the epidemic, many migrant workers sat at home and sat on the mountain. After the epidemic slowed down, our Party branch immediately coordinated with the employing units to help everyone work safely and quickly.
On May 22nd, Xue Zhiguo, deputy to the National People's Congress, took part in the deliberations and speeches of the delegation of Inner Mongolia. Xinhua News Agency reporter Huang Jingwen photo
On behalf of the delegation of Inner Mongolia, Xue Zhiguo, Secretary of the mobile party branch secretary of Aohan banner stationed in Beijing, Chifeng, reported to the general secretary on the role of Party organizations in helping migrant workers transfer jobs.
"Are you back now?" General secretary Xi Jinping asked.
"Nine Chengdu returned to Beijing to resume work."
"What can we do if we are employed locally?"
"We Aohan millet and buckwheat noodles are very good, the villagers can participate in planting and breeding cooperatives."
After listening to Xue Zhiguo's reply, general secretary Xi Jinping nodded.
General secretary further stressed: "affected by the epidemic, this year's steady employment task is very heavy, we must do a good job of graduates, migrant workers, ex servicemen and other key groups employment."
Under the situation of normalization of epidemic prevention and control, we must solve the problem of employment and make good use of the situation.
"From January 20th to March 30th this year, the new registered cyclists in the US group reached 457 thousand and 800, with 257 thousand of the riders on the platform being the poor people who built the archives, and more than half of the drivers involved in the dripping nets were migrant workers."
On May 23rd, Wang Yiming, a member of the CPPCC National Committee, spoke at the joint session of the CPPCC. Xinhua News Agency reporter Xie Huanchi photo
Wang Yiming, member of the CPPCC Joint Committee, used such a set of data to support the "new employment form" to protect the "reservoir" and the "stabilizer" of the people's livelihood, and put forward relevant suggestions.
General secretary Xi Jinping responded immediately: "the epidemic is coming suddenly, and the" new form of employment "is also coming suddenly. In this regard, we must follow the trend and let it take its natural course and stand out. Of course, laws, regulations and policies and measures that are compatible with the "new form of employment" can not be tardy.
Consolidate the foundation and consolidate the long-term mechanism.
In the Hubei delegation, general secretary Xi Jinping was concerned about the "hematopoiesis" role of the rural industry.
On May 24th, Song Qingli, deputy to the National People's Congress, took part in the deliberations and speeches of the delegation of Hubei. Xinhua News Agency reporter Huang Jingwen photo
Song Qingli, a representative of Enshi minority farmers, referred to the development of the local selenium rich industry. General secretary Xi Jinping encouraged me: "I have just visited Shaanxi last month, and the area along Ankang is also a selenium rich area. You are also a selenium rich area. We must make good use of this valuable resource and transform it into a selenium rich industry."
In March 13th, at the Wu Jia Tai Fu Se tea company in Xuanen, Enshi, Tujia and Miao Autonomous Prefecture of Hubei Province, the tea maker made traditional handmade tea according to the traditional craft. Xinhua News Agency (Song Wen photo)
Food is the food of the people. At the CPPCC joint session, general secretary Xi Jinping emphasized the importance of "rice bag" and "vegetable basket" in combination with his work experience.
"When I was a city leader in Fuzhou, doing a good job of" vegetable basket "is the top priority. Fuzhou people called "three days do not see green two eyes and Venus", to eat fresh vegetables, vegetables. You can't fly without vegetables or vegetables. The mayor still needs to be aware of this. "There is grain in hand, and no fear in heart is the truth at all times."
On May 22nd, Huo Zhaoliang, deputy to the National People's Congress, took part in the deliberations and speeches of the delegation of Inner Mongolia. Xinhua News Agency reporter Xie Huanchi photo
On behalf of the delegation of Inner Mongolia, the representative of Xilinguole Meng, Huo Zhaoliang, introduced them to adhere to the ecological priority and green development, and put more than 60% of the league in the red line of ecological protection.
In March 13th, Tegus, a conservation worker, was feeding the red deer at the wild animal and plant rescue station at Su Nen in Xilinguole Meng, Inner Mongolia. Xinhua News Agency reporter Lian Zhen photo
In this regard, general secretary Xi Jinping affirmed and explicitly instructed: "stay there, future generations can use."
The general secretary pointed out: "the protection of the ecological environment is a hundred year plan for the benefit of the people. We must maintain the strategic strength of strengthening ecological civilization construction, firmly establish the guidance of ecological priority and green development, and continue to fight the war of blue sky, clear water and pure land, so as to build a more robust green the Great Wall in the north of Xinjiang.
Bit by bit, branches and leaves close.
From poverty alleviation to employment and livelihood, from ecological protection to food security, from "six stability" to "six guarantees", all tasks are aimed at realizing the "common prosperity and all-round well-off" dream.
"One by one, we must carry out the work one year after another, and strive to make the masses see changes and get tangible benefits." The words of general secretary Xi Jinping pointed out the Communists' initial mission and struggle character.
Relying on the people -- "cultivating new opportunities in crisis and opening up new situations in changing circumstances"
"I conducted research during the epidemic period, and received nearly 7000 questionnaires, which reflect some difficulties faced by the development of private enterprises. The most prominent is that the growth rate of private investment has declined, and the employment situation has also declined."
On May 23rd, Liu Yonghao, a member of the CPPCC National Committee, spoke at the joint session of the CPPCC. Xinhua News Agency reporter Xie Huanchi photo
Liu Yonghao, member of the CPPCC Joint Committee, reflected to the general secretary that the current private enterprises, especially small and medium-sized enterprises, are facing "three difficulties", which are difficult to produce and sell, difficult to invest and difficult to cash in.
Xi Jinping felt very much when he heard the three "difficult" words: "today's private enterprises are growing in the fields of hope in China, but at the beginning they are faced with barren land. It is difficult for them to survive in the crevice and go all the way. It is great to develop to today's scale and make today's contribution.
The general secretary said: "the further development of private enterprises will still be carried out in the 'difficult' situation. In the philosophical sense, "difficulty" is an eternal proposition on the road to advance in any field. At present, in the face of great changes in the world in the past hundred years, "difficulties" exist in all aspects in the course of achieving the goal of "two one hundred years". Only by continuous exploration and continuous struggle can we overcome the difficulties in the process and embark on a new road of development.
Facing the difficulties is a sober understanding of the current situation at home and abroad.
Looking at the country, our economy is at a critical stage of transforming development mode, optimizing economic structure and changing growth momentum. The prospects for economic development are good, but we are also faced with difficulties and challenges brought about by the intertwining of structural, institutional and cyclical issues. As the general secretary said, "in addition to the impact of the new crown pneumonia epidemic, China's economic operation is now facing greater pressure."
Looking around the world, we must face the deep recession of the world economy, the sharp shrinkage of international trade and investment, the turbulence of international financial market, the limited international contacts, the adverse economic globalization, the protectionism and unilateralism of some countries, and the rise of geopolitical risks. We must seek our own development in a more unstable and uncertain world.
The general secretary made an important judgement: "everything in the world has its two sides. The epidemic is a crisis, but it also breeds some new opportunities in a certain degree". General secretary Xi Jinping used a comprehensive, dialectical and long-term vision to make a deep analysis of the current economic situation and make strategic guidelines.
The general secretary cited a series of data to further clarify the point of view.
More than 100 million the main body of the market and more than 1.7 billion people who have received higher education or possess various professional skills;
The large scale domestic demand market formed by 1 billion 400 million people, including more than 400 million middle-income groups.
In March 10th, Liu Jincai, a junior student in the mangrove village of Longkou Town, Zhungeer Qi, Inner Mongolia, went online at home. Xinhua News Agency reporter Liu Lei photo
During the epidemic, new technologies and new formats emerged. In the first quarter, online retail sales of physical goods increased by 5.9%, accounting for 23.6% of the total retail sales of social consumer goods, and substantial growth in online conferences, online transactions and online education.
"We have many advantages." "Seize the opportunity to accelerate the development of strategic emerging industries and form more new growth points and growth poles." The analysis of breaking the ice calls for the clouds to see the sun.
Tackling difficulties, our greatest strength comes from the people.
"I have repeatedly stressed that the people are the greatest strength of our party in power."
In the Inner Mongolia delegation, general secretary Xi Jinping praised the power of the people: "the broad masses of the people understand the general situation, take the overall situation into consideration, consciously cooperate with the overall situation of the epidemic prevention and control struggle, and form the basic force of epidemic prevention and control". "Do a good job in coordinating epidemic prevention and control and economic and social development, and rely firmly on the people".
Depending on the people, this is the winning weapon for epidemic prevention and control of the people's war. To mobilize the people is a strong backing for all weathers.
On May 24th, Zhang Wenxi, deputy to the National People's Congress, took part in the deliberations and speeches of the delegation of Hubei. Xinhua News Agency reporter Huang Jingwen photo
Zhang Wenxi, Secretary of the Party branch of Chen Yu village, Xinzhou District, Wuhan, is a representative of the National People's Congress from the front line of anti epidemic. He made the general secretary's general experience to Xi Jinping for the importance of grass roots governance.
"Our village is 27 kilometers away from the main urban area of Xinzhou, with 1326 villagers, of whom 447 are returning to Wuhan. To tell you the truth, everyone was in a panic at the beginning.
"If the epidemic situation is not sealed, the key is to see the Party branch." Under the leadership of Zhang Wenxi, 5 party members and cadres summoned cadres and volunteers to form a team of 24 people, including Bao Bao, hanging banners, sowing trumpets, striking the gong and shouting, and mobilizing the masses to build up a "iron wall".
Zhang Wenxi also reflected to the general secretary of the "short board" of Grassroots Governance: Village and community cadres were less involved and more tasks, and their treatment did not catch up.
"This epidemic tests the level of governance capability of our grass-roots governance system, and further highlights the role of our street and community, township and village grassroots organizations." General secretary Xi Jinping pointed out that "from the perspective of peacetime and wartime integration, we must have a foundation and a mechanism system. It can be pulled out at the critical time.
At the end of last year, the the fourth Plenary Session of the 19th CPC Central Committee thematic study of the party promoted the important proposition of the modernization of the national governance system and governance capacity. The sudden outbreak of the new crown pneumonia further highlighted the urgency of promoting the modernization of the national governance system and governance capacity.
Focusing on this topic, delegates on behalf of the Committee have made suggestions.
On behalf of the delegation of Hubei, the 5 representatives put forward opinions and suggestions from different angles to weave the public health protection network.
After listening to your speech, general secretary Xi Jinping thoroughly analyzed the problems from 5 aspects, and put forward the countermeasures accurately: "reform the disease prevention and control system", "enhance the monitoring and early warning and emergency response capabilities of the epidemic", "improve the major epidemic treatment system", "improve the public health emergency laws and regulations", "carry out the Patriotic Health Campaign in depth". It is based on the current and long-term perspective.
In the Inner Mongolia delegation, general secretary Xi Jinping also put forward the proposition of "efficient governance".
"We should take the initiative and act as the foundation of the current and long-term vision. We should introduce some effective measures in pushing forward the construction of major projects, supporting the development of market entities, speeding up the adjustment of industrial structure, and improving the ability of grass-roots governance. In particular, we should study and plan long-term strategic tasks and strategic layout, with targeted deployment of high quality development and efficient governance. Great planning, major reforms and major policies should be taken to grasp the initiative and fight for development initiative in dealing with crises.
On the afternoon of 26, when attending the plenary meeting of the PLA and the Armed Police Corps, Xi Jinping stressed that the whole army should consciously integrate ideas and actions into the decision making and deployment of the Party Central Committee, strengthen confidence and face difficulties and push forward the work of the armed forces under the premise of normalization of epidemic prevention and control, and resolutely implement the objectives and tasks of national defense and army building in 2020, and resolutely accomplish the tasks entrusted by the party and the people. Various tasks.
In the course of tackling difficulties, we made progress and achieved historic progress in the historical examination. The speech of general secretary Xi Jinping was inspiring and inspiring.
"Party members and cadres should closely rely on the people and carry forward their mental outlook in the fight against epidemic diseases. They must really work hard, fight hard, be daring to be responsible and dare to fight. We must dare to overcome difficulties and win new and greater victories.
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Phone 0800 543 943 Waitematā DHB Child, Women & Family Services Services include Auckland Regional Dental Service, Child Development Services, Child Disability Services, Child & Family Services, Home Care for Kids and Maternity Services. Waitematā DHB Antenatal Classes - Pregnancy and Parenting Education Waitematā District Health Board (DHB) provides an enhanced programme for publicly funded pregnancy and parenting education The Cooper Clinic Offering a dedicated vasectomy clinic and treatment for erectile dysfunction in Auckland Closed today. Phone 0800 884 408 Waitematā DHB Older Adults & Home Health (OA&HH) OA&HH provides expertise and specialist care for older people both in an inpatient and community setting. Diabetes Foundation Aotearoa Diabetes Foundation Aotearoa emphasises partnership with the community to achieve better health for present and for future generations. Open today 8:30 AM to 4:00 PM. 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Malachi 2:5
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Malachi 2:5
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5 "My covenant was with him, a covenant(A) of life and peace,(B) and I gave them to him; this called for reverence(C) and he revered me and stood in awe of my name.Read full chapter
Malachi 4:2
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Malachi 4:2
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2 But for you who revere my name,(A) the sun of righteousness(B) will rise with healing(C) in its rays. And you will go out and frolic(D) like well-fed calves.Read full chapter
Hebrews 13:15
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Hebrews 13:15
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15 Through Jesus, therefore, let us continually offer to God a sacrifice(A) of praise—the fruit of lips(B) that openly profess his name.Read full chapter
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It was another big weekend of action over at the GGPoker World Series of Poker with four more online bracelet events…
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E044: NORMAN CHAD: This is the Worst WSOP of the Century!
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This article was originally published on PokerIndustryPRO.com


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The learners are presented to a variety of research methods and techniques needed for the preparation of their graduating and research projects in general. Starting off with critical analysis of scientific papers both from natural and social sciences, learners are preparing background information for their research, annotated bibliographies, literature review and selecting appropriate research methods. Quantitative and qualitative research methods will be presented along with examples for their application. Preparation and presentation of project proposal which also includes proposing sustainable solutions and recommendations are the main goal of the course
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Lisbon to Invest in Attracting Tourism to Offset Pandemic
19 June 2020 – The Lisbon Regional Tourism Board (ERT-RL) will invest up to 600,000 euros to attract Portuguese tourists to the region. Over the next three months, the public body will launch low-cost packages focusing on accommodations and experiences, all at lower prices than before the pandemic. In 2017, tourism accounted for 19.7% of Lisbon's GDP, according to a study by the management consultancy Roland Berger.
Packages of experiences (including, among others, boat trips, visits to wine cellars and urban art routes) will be offered for 20 euros. A meal and performance of fado will cost 25 euros, while a Lisboa Card, which provides access to the city's participating museums and monuments, is on sale for just 12.5 euros.
Accommodation and experience packages will consist of accommodation for two nights, a 24-hour Lisboa Card and two experiences, for two people. Prices, which vary depending on the accommodations, will cost between 160 and 240 euros.
In all, the programme will invest more than half a million euros, €300,000 of which will go to marketing campaigns. Another €150,000 will go to payments to the companies offering the experiences, with an additional €100,000-€150,000 going to fado houses.
Original Story: Jornal de Negócios – Rafaela Burd Relvas
Translation/Summary: Richard D. Turner


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15 DIY Projects To Make And Sell This Summer
Are you looking to make some extra money this spring or summer by making handicrafts? Here are 15 DIY projects you can make and sell to make extra money every month. These adorable craft ideas cost little in the way of materials and can be a great way to supplement your income. #sidehustles #craftstosell
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LOADSHEDDING IS BACK! (Interview with Ted Blom)
The President promised us that we will not have a dark Christmas, and kept his word. He also said that there will be no load shedding till the 13th of January, BUT, darkness greeted us over the weekend, in another round of rolling blackouts throughout the country. We engage energy expert Ted Blom on what's lighting the fires at Eskom


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A report on their Historical Background and Prospects for Conservation.
97 pages, numerous maps and other illustrations, original pictorial cream wrappers.
- Binding Condition: Very Good
- Overall Condition: Very Good
- Size: Square 8vo (200 x 200 mm)
- Sold By: Clarke's Africana & Rare Books
- Contact Person: Paul Mills
- Country: South Africa
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33 projects in result set. Displaying 20 per page. Projects sorted by activity ranking.
|0. rgl: 3D visualization support - rgl is a 3D visualization system based on OpenGL. It provides a medium to high level interface for use in R, currently modelled on classic R graphics, with extensions to allow for interaction.
|Activity Percentile: 69.23
Registered: 2008-08-16 13:50
|1. Fish Migration Monitoring (stacomiR) - stacomiR stands for migratory control stations. It enables to read informations in a database dedicated to the monitoring of fish migrations.It allows to build overviews of fish migrations and create charts with data.
|Activity Percentile: 61.54
Registered: 2011-02-23 20:04
|2. IPSUR - This project contains the IPSUR package, the textbook "Introduction to Probability and Statistics Using R", and RcmdrPlugin.IPSUR, an R Commander Plugin to accompany the textbook. IPSUR is distributed under the GNU-FDL.
|Activity Percentile: 0.00
Registered: 2007-08-16 17:14
|3. Causal Effects Sensitivity Analysis - Graphical Sensitivity Analysis for Assessing Surrogate Endpoints via Causal Inference
|Activity Percentile: 0.00
Registered: 2013-02-22 01:18
|4. Muste - the R implementation of Survo - Muste - Multiplatform Survo Type Editorial Environment for Data Analysis
|Activity Percentile: 0.00
Registered: 2011-06-27 14:20
|5. SciViews - SciViews: a R GUI API, and a R GUI extension for Komodo Edit.
|Activity Percentile: 0.00
Registered: 2008-05-26 16:41
|6. R2jags - A package for running jags from R
|Activity Percentile: 0.00
Registered: 2008-05-10 19:19
|7. radR - processing of radar data in R - radR retrieves data from your live radar or recorded archive and analyses it for features, which are saved in replayable files that are much smaller than raw data sets. You can extend radR via plugins.
|Activity Percentile: 0.00
Registered: 2007-11-20 20:30
|8. Modelling Peaks Over Threshold - The project consists in developing functions for peaks over threshold modeling. Most of results come from the extreme value theory (EVT). The EVT is currently extensively used, mostly in environmental sciences and finance.
|Activity Percentile: 0.00
Registered: 2007-09-21 14:29
|9. cvrcalc - A Cardiovascular Risk's Calculator by Scores
|Activity Percentile: 0.00
Registered: 2013-08-02 12:18
|10. RWinEdt - RWinEdt is a package that implements a quite ugly interface from WinEdt to R. Note that the License GPL is specific to the R package RWinEdt, not to the editor WinEdt itself (which is not shipped with the package).
Registered: 2007-09-14 14:22
|11. Toolbox for chemoinformatics - Basic tools for chemoinformatics, including manipulations with chemical structures, reading/writing files, computing descriptors, building SAR/QSAR/QSPR models, predicting properties of chemical compounds, etc.
|Activity Percentile: 0.00
Registered: 2012-10-01 20:42
|12. Isolation Forest - This package implements an anomaly detection method that detects data-anomalies using binary trees. Using the property that anomalies are more susceptible to isolation, anomalies can be detected as data points which have short expected path lengths.
|Activity Percentile: 0.00
Registered: 2009-07-14 06:02
|13. xPos-a - Development of a simulation-based optimization algorithm handling (hopefully) both uncertain and multicriteria evaluations.
|Activity Percentile: 0.00
Registered: 2009-07-02 00:56
|14. Request openblender.io API Services - Openblender.io is a platform that provides services such as blending open data, cleansing data and text vectorization, for mention some. This package looks forward to giving access to those services using R. Visit www.openblender.io for more information.
|Activity Percentile: 0.00
Registered: 2020-06-22 17:08
|15. Aquatic Modelling Environment (AquaEnv) - AuaEnv is an integrated development toolbox for aquatic chemical modelling focused on (ocean) acidification and CO2 air-water exchange.
It contains all necessary tools to model the pH, CO2 exchange, and aquatic acid-base chemistry in general.
|Activity Percentile: 0.00
Registered: 2008-10-24 09:56
|16. Multivariate Projection Methods - mpm is an R package by Paul Lewi and Luc Wouters for exploratory multivariate analysis of large data matrices, such as gene expression data from microarray experiments.
|Activity Percentile: 0.00
Registered: 2008-01-29 14:46
|17. Graphical Multiple Comparison Procedure - This package provides functions and a graphical user interface for graphical described multiple test procedures (sequentially rejective, Simes and parametric).
|Activity Percentile: 0.00
Registered: 2010-02-17 13:06
|18. Optimal Two-stage Phase II Design - This R package creates the optimal two-stage phase II designs with long-term endpoint.
A later expanded version of the package (OptInterim) can be downloaded on CRAN
|Activity Percentile: 0.00
Registered: 2008-12-14 01:13
|19. Mutoss - Mutoss (multiple hypotheses testing in an open software system) aims at providing a unified, extensible interface covering a large spectrum of multiple hypotheses testing procedures in R. Features a GUI and a simulation tool. Funded by PASCAL2.
|Activity Percentile: 0.00
Registered: 2010-02-11 12:32
33 projects in result set. Displaying 20 per page. Projects sorted by activity ranking.


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ATP7A delivers copper to the lysyl oxidase family of enzymes and promotes tumorigenesis and metastasis
Edited by Mary Lou Guerinot, Dartmouth College, Hanover, NH, and approved February 25, 2019 (received for review October 11, 2018)
The family of copper-dependent lysyl oxidase (LOX) enzymes contributes to tumor metastasis. In this study, we show that the ATP7A copper transporter is required to deliver copper to LOX family members. Deletion of ATP7A inhibited LOX activity in breast and lung cancer cell lines, resulting in a significant loss of tumor growth and metastatic potential of these cells in mice. Elevated expression of ATP7A was found to be associated with reduced survival of breast cancer patients. Our study suggests that blocking the function of ATP7A could be an approach to inhibiting LOX-dependent malignancies.
Lysyl oxidase (LOX) and LOX-like (LOXL) proteins are copper-dependent metalloenzymes with well-documented roles in tumor metastasis and fibrotic diseases. The mechanism by which copper is delivered to these enzymes is poorly understood. In this study, we demonstrate that the copper transporter ATP7A is necessary for the activity of LOX and LOXL enzymes. Silencing of ATP7A inhibited LOX activity in the 4T1 mammary carcinoma cell line, resulting in a loss of LOX-dependent mechanisms of metastasis, including the phosphorylation of focal adhesion kinase and myeloid cell recruitment to the lungs, in an orthotopic mouse model of breast cancer. ATP7A silencing was also found to attenuate LOX activity and metastasis of Lewis lung carcinoma cells in mice. Meta-analysis of breast cancer patients found that high ATP7A expression was significantly correlated with reduced survival. Taken together, these results identify ATP7A as a therapeutic target for blocking LOX- and LOXL-dependent malignancies.
Copper is an essential micronutrient required as a catalytic cofactor for several metalloenzymes, including those involved in cancer-promoting activities (1). Copper-dependent enzymes with roles in tumor growth or metastasis include mitogen-activated kinase 1 (2, 3), superoxide dismutase 1 (4), cytochrome c oxidase (5), and members of lysyl oxidase (LOX) family (6⇓–8). The discovery that copper concentrations in serum and tumors are elevated in cancer patients and are correlated with disease severity has prompted speculation that copper delivery to oncogenic enzymes may be rate-limiting for tumor growth and metastasis (9⇓–11). This idea has been supported by reports that copper chelation reduces tumor growth and malignancy in animal models of cancer (12, 13). Clinical studies indicate that copper chelation prolongs the survival of patients with late-stage breast cancer (14) and kidney cancer (15). These studies raise the possibility that blocking specific steps in the delivery of copper to oncogenic metalloenzymes may provide an approach to treating cancer.
LOX is a secreted copper-dependent amine oxidase that plays critical roles in the development of connective tissue and remodeling of the extracellular matrix by catalyzing the cross-linking of collagen and elastin (16). In addition to LOX, several LOX-like enzymes (LOXL1–4) have been identified that share a conserved catalytic copper-binding domain. To date, functional roles for LOX or LOXL enzymes have been documented in breast, colorectal, prostate, gastric, hepatic, pancreatic, and head and neck cancers, as well as in cancers of the skin, including melanoma (6, 17⇓–19). LOX has been shown to promote tumor cell migration and adhesion via the activation of focal adhesion kinase (FAK1) (20⇓–22). In addition, LOX and LOXL2 activity is required to facilitate the recruitment of myeloid cells to metastatic sites, creating a favorable environment for the subsequent invasion and growth of tumor cells (18, 19, 23).
The importance of LOX and LOXL proteins in cancer has led to intensive efforts to develop inhibitors of these enzymes as potential anticancer therapies. Since all LOX family members share a functional requirement for copper, we surmised that inhibiting copper incorporation into these enzymes may be an effective strategy to inhibit LOX-dependent metastasis. However, the mechanism of copper incorporation into LOX and LOXL proteins is poorly understood. The copper-binding catalytic domain of each LOX family member is lumenally oriented (extracytoplasmic), suggesting that copper insertion occurs via a conserved mechanism during the biosynthesis of these enzymes en route through the secretory pathway. Previous studies have shown that the Golgi-localized copper transporter ATP7A delivers copper to tyrosinase, a metalloenzyme with an extracytoplasmic copper-binding catalytic domain (24). In addition, LOX activity is reduced in Menkes disease fibroblasts, which contain mutations in the ATP7A gene (25). However, the extent to which ATP7A is required for the activity of different members of the LOX family of enzymes and, by extension, the importance of ATP7A in LOX-mediated pathologies remain unknown.
Here we demonstrate that ATP7A is required for the activity of multiple members of the LOX family, and that silencing ATP7A in two different cancer cell lines suppresses tumorigenesis and metastasis in mice. Importantly, elevated ATP7A expression was found to be significantly correlated with reduced survival in breast cancer patients, suggesting a role for ATP7A in human cancer. These studies identify ATP7A as a target for inhibiting LOX-dependent malignancies.
ATP7A Is Necessary and Sufficient for Copper-Dependent Activity of LOX Family Members.
The 4T1 cells were chosen as a model of metastatic human breast cancer because when orthotopically implanted into the mammary glands of mice, these cells readily metastasize to the lung in a LOX-dependent manner (7, 23, 26). We found that all known LOX family members were expressed in 4T1 cells under standard culture conditions (SI Appendix, Fig. S1A), and that 4T1 tumors grown orthotopically in the mammary fat pads of BALB/c mice expressed significantly higher levels of Lox, Loxl2, Loxl3, and Loxl4 mRNAs relative to the adjacent stromal tissue (SI Appendix, Fig. S1B).
To investigate the requirement for ATP7A in LOX activity, we silenced Atp7a gene expression in murine 4T1 breast carcinoma cells using a CRISPR-Cas9 approach. Immunoblot analysis confirmed the absence of ATP7A protein in two independent CRISPR clones, C3/ATP7A− and C8/ATP7A− (Fig. 1A). We used immunofluorescence microscopy to verify that ATP7A was localized to the Golgi apparatus in wild-type 4T1 cells (4T1-WT), a site where ATP7A is thought to metallate nascent metalloenzymes within the secretory pathway (24, 27). ATP7A was localized to the perinuclear region in 4T1-WT cells (Fig. 1B), which overlapped with antibodies against the Golgi marker protein, GM130 (SI Appendix, Fig. S2A). As expected, ATP7A was not detected in C3/ATP7A− and C8/ATP7A− cells by immunofluorescence microscopy (Fig. 1B). Consistent with the known role of ATP7A in copper export (28), copper concentrations were elevated in C3/ATP7A− and C8/ATP7A− cells compared with 4T1-WT cells (SI Appendix, Fig. S2B). However, the growth rates of C3/ATP7A− and C8/ATP7A− cells were not significantly different from the growth rate of 4T1-WT cells (SI Appendix, Fig. S2C), suggesting that the elevated copper concentrations were not cytotoxic. LOX activity was detected in the conditioned media of 4T1-WT cells and was diminished by pretreating these cells with β-aminopropionitrile (BAPN), a specific inhibitor of the LOX family of enzymes (29) (Fig. 1C). In contrast, LOX activity was virtually undetectable in the media of both C3/ATP7A− and C8/ATP7A− cells (Fig. 1C), suggesting that LOX activity is dependent on ATP7A expression.
These observations were not due to off-target effects of CRISPR-Cas9, because reduced LOX activity was also observed in 4T1 cells in which ATP7A was silenced using an RNAi approach (SI Appendix, Fig. S3 A and B). Furthermore, LOX activity was restored to wild-type levels in C3/ATP7A− cells transfected with a human ATP7A expression plasmid (Fig. 1D). LOX activity in C3/ATP7A− cells could be restored by the addition of supraphysiological copper concentrations to the culture medium (Fig. 1E). These observations suggest that the reduction in LOX activity in the absence of ATP7A is due to a disruption of copper delivery to LOX.
While these data support the hypothesis that ATP7A mediates copper delivery to LOX, a potential caveat to this conclusion is that commercial LOX assays, like the one used in our study, do not distinguish between the activities of different LOX family members. Thus, it was unclear from our results which of the LOX members' activities might be dependent on ATP7A. To investigate this further, we performed LOX activity assays on the conditioned media of 4T1-WT and C3/ATP7A− cells transfected with plasmids encoding three different LOX family members—LOX, LOXL1 and LOXL2—together with a GFP expression plasmid to control for transfection efficiency. After 24 h, the media were collected, and LOX activity was measured as a function of cellular GFP expression. For 4T1-WT cells transfected with each LOX plasmid, a significant increase in LOX activity was detected in the media relative to cells transfected with the GFP vector alone (Fig. 1F). In contrast, none of the LOX plasmids produced a significant increase in LOX activity when transfected into C3/ATP7A− cells (Fig. 1F). These observations suggest that ATP7A is necessary and sufficient to metallate multiple members of the LOX family.
ATP7A Deletion in 4T1 Breast Cancer Cells Attenuates Tumor Growth and Metastasis in Mice.
Because several members of the LOX family are known to promote tumor metastasis, we hypothesized that a loss of ATP7A might reduce metastasis of 4T1 cells. To test this hypothesis, 4T1-WT and the ATP7A-null cell lines were injected into the inguinal mammary fat pads of female BALB/c mice, and primary tumor growth and metastatic lung nodules were quantified after 4 wk. Both the C3/ATP7A− and C8/ATP7A− tumors were significantly smaller than wild-type tumors (Fig. 2A). Consistent with our findings in cultured cells, tumors derived from C3/ATP7A− and C8/ATP7A− cells accumulated significantly higher levels of copper compared with tumors derived from 4T1-WT cells (Fig. 2B). Importantly, there were markedly fewer metastatic lung nodules in mice bearing C3/ATP7A− and C8/ATP7A− tumors compared with mice bearing wild-type tumors (Fig. 2 C and D). A similar reduction in both primary tumor growth and metastasis was observed for shRNA-ATP7A cells in which ATP7A was silenced using RNAi (SI Appendix, Fig. S3 C–F). We considered the possibility that the reduced metastatic potential of tumors lacking ATP7A might be attributable to a reduction in primary tumor size. However, the number of metastatic lung nodules remained markedly lower even after the ATP7A-silenced tumors were permitted to grow until they reached the same size as wild-type tumors (SI Appendix, Fig. S4).
Previous studies have demonstrated that LOX and LOXL2 are necessary for the recruitment of CD11b+ and CD117+ myeloid cells to the lungs of tumor-bearing mice to promote formation of the premetastatic niche, a process that facilitates the invasion and growth of tumor cells (18, 23). Consistent with a loss of LOX activity, both CD117+ and CD11b+ myeloid cells were significantly reduced in the lungs of C3/ATP7A− tumor-bearing mice compared with 4T1-WT tumor-bearing mice (Fig. 2 E and F).
Previous studies also have shown that inhibition of LOX activity reduces the abundance of linear collagen fibrils associated with tumors (30). Using second harmonic generation imaging microscopy (30⇓–32), we investigated whether the loss of ATP7A reduced collagen fibril assembly in primary tumors and metastatic lung nodules. As expected, tumor-associated collagen was predominantly organized in long linear arrangements (>30 µm) in 4T1-WT tumors, but in C3/ATP7A− derived tumors, the collagen fibrils were significantly shorter and exhibited an overall reduced linear organization (SI Appendix, Fig. S5 A–C). This difference was also present in the metastatic nodules in the lungs of tumor-bearing mice in which there were significantly fewer collagen fibrils in C3/ATP7A− nodules compared with those derived from 4T1-WT cells (SI Appendix, Fig. S5 D–F). Taken together, these data suggest that ATP7A is required for copper delivery to LOX and for LOX-dependent prometastatic processes in 4T1 cells.
ATP7A Is Necessary for LOX-Dependent FAK1 Phosphorylation.
An important mechanism of LOX-mediated metastasis is the activation of FAK1, a key regulator of tumor cell adhesion and motility (21, 22, 33). Levels of phosphorylated FAK1 (p-FAK1) were found to be significantly elevated in 4T1-WT cells compared with C3/ATP7A− cells, C8/ATP7A− cells, and 4T1 cells expressing shRNA-ATP7A (Fig. 3A). Notably, FAK1 phosphorylation was restored to wild-type levels in C3/ATP7A− cells within 20 min after the addition of recombinant active LOX protein to the culture medium (Fig. 3 B and C). FAK1 is phosphorylated by the upstream tyrosine-protein kinase Src, which is activated by the epidermal growth factor receptor (EGFR) (34). Recent studies suggest that LOX activity can increase Src phosphorylation in response to EGFR signaling (35). Consistent with these findings, Src phosphorylation was reduced in untreated and EGF-stimulated C3/ATP7A− cells compared with 4T1-WT cells (SI Appendix, Fig. S6 A and B). FAK1 phosphorylation is known to regulate the assembly of proteins, including vinculin, at focal adhesion sites (36, 37). In agreement with a loss of FAK1 phosphorylation, there was a significant increase in vinculin-positive focal adhesions in C3/ATP7A− cells compared with 4T1-WT cells (Fig. 3 D and E). There were no differences in total vinculin between 4T1-WT and C3/ATP7A− cells (Fig. 3F).
We next investigated the effect of ATP7A deletion on cell motility using an in vitro scratch assay. The 4T1-WT cells exhibited increased motility compared with the C3/ATP7A− cells (Fig. 3 G and H and Movies S1 and S2), which were suppressed by the LOX inhibitor BAPN (Fig. 3H). However, supplementation of exogenous recombinant LOX to the media of C3/ATP7A− cells was not sufficient to restore motility in these cells (SI Appendix, Fig. S6C). Thus, despite the requirement for ATP7A for LOX-mediated FAK1 phosphorylation (Fig. 3B), LOX activity alone was not sufficient to restore motility in ATP7A-null cells.
Because ATP7A functions as a copper exporter (in addition to supplying copper to secreted cuproenzymes), we considered the possibility that the hyperaccumulation of copper in ATP7A-null cells may contribute to the tumor-suppressive effects of ATP7A silencing. At excess concentrations, copper can generate reactive oxygen species (ROS), potentially resulting in cytotoxicity. Consistent with this concept, ROS concentrations were found to be elevated in ATP7A-null cells compared with 4T1-WT cells (SI Appendix, Fig. S7A). Moreover, subtoxic concentrations of copper and H2O2 added in combination to the media were found to confer cytotoxicity in ATP7A-null cells, but not in 4T1-WT cells (SI Appendix, Fig. S7B) and to suppress the ability of ATP7A-null cells to form colonies in soft agar (SI Appendix, Fig. S7 C and D). These results suggest that in addition to a loss of LOX metallation, the accumulation of copper and ROS may also contribute to the inhibitory effects of ATP7A silencing on tumor growth and metastasis.
ATP7A Deletion in Lewis Lung Carcinoma Cells Inhibits LOX Activity, Tumor Growth, and Metastasis.
To determine whether our observations with 4T1 mammary carcinoma cells might be applicable to other cancer types, we ablated Atp7a in Lewis lung carcinoma (LLC) cells using CRISPR-Cas9. Immunoblot analysis verified the loss of ATP7A expression in two independent clones, LLC7A-1 and LLC7A-2 (Fig. 4A). Consistent with our observations in 4T1 cells, LOX activity was markedly reduced in LLC7A-1 and LLC7A-2 cells compared with wild-type LLC cells (Fig. 4B).
To determine the effect of ATP7A deletion on tumor growth, wild-type LLC, LLC7A-1, and LLC7A-2 cells were injected s.c. into C57BL/6 mice and permitted to grow for 2 wk. Primary tumors derived from the LLC7A-1 and LLC7A-2 cells were found to be significantly smaller than those from wild-type LLC cells (Fig. 4C).
To investigate the effect of ATP7A on metastasis, LLC, LLC7A-1, and LLC7A-2 cells were injected into the tail veins of C57BL/6 mice. After 3 wk, the metastatic burden of the ATP7A-null LLC cells in both lung and liver was significantly reduced compared with that in wild-type LLC cells (Fig. 4D). Taken together, these findings suggest that ATP7A is necessary for growth and metastasis in models of both mammary and lung carcinoma.
ATP7A Expression Is Correlated with Reduced Survival in Breast Cancer Patients.
Previous studies have shown that high LOX expression is correlated with reduced survival in patients with estrogen receptor (ER)-negative breast cancer (38). We used publicly available databases to investigate the degree to which patient mortality is correlated with ATP7A mRNA expression in breast tumors. As in the case of LOX, high ATP7A expression was found to be significantly correlated with lower metastasis-free survival in patients diagnosed with ER-negative breast cancer (Fig. 5A). Together with our experimental observations, these data suggest that ATP7A promotes breast cancer malignancy in humans.
Copper chelation therapy is an experimental approach that has been shown to significantly extend the life expectancy of patients diagnosed with stage III and stage IV breast cancer (14), raising the possibility that inhibition of copper delivery to specific oncogenic metalloenzymes might offer therapeutic benefits. Prometastatic metalloenzymes that are affected by copper chelation therapy include the LOX family of enzymes, which have well-documented roles in tumor malignancy (6). Previous studies using experimental mouse models have shown that silencing LOX reduces the metastatic spread of several breast carcinoma cell lines, including 4T1 cells (23).
The rationale for our study was two-pronged: first, to test the hypothesis that the resident Golgi copper transporter ATP7A is necessary to deliver copper to members of the LOX family, and second, to test whether ATP7A silencing would attenuate LOX-dependent metastasis. Suppression of ATP7A expression in 4T1 cells using two independent approaches—CRISPR and RNAi—resulted in the loss of LOX activity in the culture medium (Fig. 1C and SI Appendix, Fig. S3B). This was not the result of off-target events, because LOX activity was restored by transfection of a human ATP7A plasmid in ATP7A-null cells (Fig. 1D). The finding that excess copper added to the culture medium could restore LOX activity in ATP7A-null cells indicated that the lack of LOX activity was attributable to a failure of copper delivery to LOX (Fig. 1E). The finding that forced expression of different LOX family members increased LOX activity in 4T1-WT cells, but not in ATP7A-null cells (Fig. 1F), indicates that ATP7A is necessary and sufficient to metallate multiple LOX family members.
Consistent with a loss of ATP7A-dependent LOX activity, known pathways of LOX-dependent metastasis were attenuated in ATP7A-null 4T1 cells, including myeloid cell recruitment to the lungs, FAK1 phosphorylation, EGFR-mediated phosphorylation of Src, and tumor-associated collagen fibril assembly (Figs. 2F and 3C and SI Appendix, Figs. S5 and S6B). The finding that ATP7A deletion in LLC cells also suppressed tumorigenesis and metastasis suggests that, like members of the LOX family, ATP7A is a relevant prometastatic factor in multiple cancer types (Fig. 4). Future studies will evaluate the importance of ATP7A in other cancer models, including models of spontaneous cancer. Previous studies have demonstrated that elevated LOX expression is correlated with poor survival of ER-negative breast cancer patients (38).
Consistent with a requirement for ATP7A in LOX activity, we found that high ATP7A expression correlates with decreased survival in patients with ER-negative breast cancer (Fig. 5A). Taken together, these findings suggest a model in which ATP7A functions to metallate LOX proteins in the secretory pathway to promote LOX-mediated mechanisms of metastasis (Fig. 5B). Although FAK1 phosphorylation was restored by the addition of exogenous LOX to ATP7A-null cells (Fig. 3C), why this treatment did not restore migratory potential in these cells, as determined using the scratch assay, is unclear (SI Appendix, Fig. S6C). It is possible that the correction of cell migration in ATP7A-null cells requires the activity of multiple LOX family members. It is also possible that the loss of migratory potential in ATP7A-null cells is partly attributable to LOX-independent mechanisms. Indeed, the finding that ATP7A-null cells accumulated elevated levels of copper and ROS and were sensitive to a combination of subtoxic concentrations of copper and hydrogen peroxide is consistent with this hypothesis (SI Appendix, Fig. S7).
The importance of LOX and LOXL enzymes in different cancers has prompted intense efforts to develop small molecule inhibitors and immunotherapies against specific LOX family members (35, 39⇓–41). The results from our study raise the possibility of a new approach to treating LOX-dependent malignancies by blocking ATP7A-mediated copper delivery to this family of enzymes. Although we did not observe a correlation between ATP7A expression and patient survival for other cancer types, such correlations may be obscured by the expression of ATP7B, another copper transporting P-type ATPase in the Golgi complex that also metallates secreted cuproenzymes. As both ATP7A and ATP7B proteins share highly conserved functional motifs, drugs could be developed that block both ATPases to provide a therapeutic benefit against cancers caused by one or more LOX family members (42⇓–44). Furthermore, as previous studies have shown that ATP7A and ATP7B are involved in resistance to various chemotherapy drugs (45, 46), therapeutic targeting of ATP7A/B may offer additional benefits by reducing the development of chemoresistance.
Materials and Methods
Cell Lines, Plasmids, and Reagents.
The 4T1 and LLC cells were obtained from American Type Culture Collection. ATP7A knockout lines were generated by transfecting wild-type 4T1 or LLC cells with a CRISPR-Cas9/GFP construct targeting exon 16 (Sigma-Aldrich), using Lipofectamine 2000. ATP7A knockdown and control 4T1 cells were generated by stable transfection of a short hairpin RNA against ATP7A or GFP (Origene), respectively. Cells were grown in complete medium consisting of DMEM (Life Technologies) supplemented with 10% (vol/vol) FBS, 2 mM glutamine, and 100 U/mL penicillin-streptomycin (Life Technologies) in 5% CO2 at 37 °C. Purified LOX protein and expression plasmids for human LOX, LOXL1, and LOXL2 were purchased from Origene. BAPN was purchased from Sigma-Aldrich.
All animal procedures were performed with the approval of the University of Missouri's Animal Care and Use Committee. Female BALB/c mice and male C57BL/6 mice were purchased from The Jackson Laboratory. Mice were maintained on a 12-h light-dark cycle and fed with Picolab diet 5053 (13 ppm Cu; PMI International).
Cell Proliferation and Survival Assays.
Cells were seeded in 24-well plates (50,000 cells/well) and allowed to grow until ∼80% confluence. The Vybrant MTT Cell Proliferation Assay Kit (Thermo Fisher Scientific) was used to measure rates of proliferation in accordance with the manufacturer's instructions. Cell survival was determined using the crystal violet assay, as described previously (47).
In Vitro Scratch Assay.
In vitro scratch assays were performed as described previously (48). Live cell imaging and video recording were performed at 37 °C and 5% CO2 using a Nikon Eclipse Ti microscope. Photometrics software was used to calculate gap closure by measuring the change in gap distance over time.
Animal Tumor Models.
Orthotopic growth of 4T1 cells in of 6- to 7-wk-old female BALB/c mice was performed as described previously (26). Primary tumors and lungs were harvested at 4 wk unless indicated otherwise. Bouin's solution was used to identify surface metastatic nodules, which were then enumerated using dissecting microscopy. For the LLC model, 2 × 106 cells in PBS were injected s.c. into the right armpit. After 2 wk, the tumors were excised, weighed, and imaged. For investigating metastasis, LLC, LLC7A-1, and LLC7A-2 cells were stably transfected with a construct expressing mCherry and a puromycin resistance gene (Addgene plasmid 72264; Veit Hornung Lab) using Lipofectamine 2000 and selected with puromycin (1.25 µg/mL). Populations of LLC, LLC7A-1, and LLC7A-2 cells expressing equal levels of mCherry, as determined by fluorescence intensity and qRT-PCR, were isolated and injected i.v. into the tail vein. After 3 wk, the liver and right lung were excised and homogenized in TRIzol solution (Invitrogen). RNA was purified using the RNeasy Plus kit (Qiagen), and cDNA was synthesized using the RNA to cDNA EcoDry kit (Takara). qRT-PCR analysis was performed using 100 ng of template was used per sample. In the event of nondetectable genes, 40 PCR cycles were performed. Taqman probes for mCherry and GAPDH were obtained from Applied Biosystems.
Copper Measurements.
Tumors or cell pellets were dissolved in 70% (vol/vol) nitric acid and elemental analysis was performed by inductively coupled plasma optical emission spectrometry (ICP-OES). Values were acquired in triplicate for each sample and the results were normalized to weight.
Immunofluorescence Microscopy and Immunoblotting.
Immunofluorescence microscopy was performed as previously described (49), using the following antibodies: Alexa Fluor 488-conjugated anti-rabbit or anti-mouse (1:1,000; ThermoFisher), anti-vinculin (1:200, clone 7F9; EMD Millipore). Nuclei were stained with DAPI (ThermoFisher). Immunoblot analysis was performed as previously described (46). Primary antibodies used were rabbit anti-ATP7A (1:1,000), anti-tubulin (1:2,000, T8328; Sigma-Aldrich), anti-phospho-FAK1 (1:1,000, 3281S; Cell Signaling Technology), anti-FAK1 (1:1,000, ab40794; Abcam) and anti-vinculin (1:1,000, 7F9; EMD Millipore). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000, sc-2357; Santa Cruz Biotechnology) or goat anti-mouse IgG (1:1,000; Thermo Fisher Scientific31430).
LOX Activity Assay.
LOX activity in the conditioned media was determined using the LOX activity kit (Abcam), according to the manufacturer's instructions. Where indicated, plasmids encoding LOX, LOXL1, or LOXL2 (Origene) were transiently transfected into 4T1-WT or C3/ATP7A− cells together with a plasmid encoding GFP in pQCXIP to normalize for transfection efficiency. After 24 h, GFP fluorescence was measured in transfected cells and LOX activity was assayed in the media. Mean LOX activity was normalized to GFP fluorescence. All LOX assays were performed at least three times, with similar results.
Flow Cytometry.
Lung tissue (30–70 mg) isolated from mice bearing 4T1-WT or C3/ATP7A− tumors was minced and digested for 1 h at 37 °C in RPMI media containing 4 mg/mL Collagenase D (Roche) and 2.5 mM CaCl2. Cell suspensions were passed through a 40-µm nylon filter and red blood cells were lysed using RBC lysis buffer (Miltenyi Biotec). Cells were resuspended in PBS with 0.5% (wt/vol) BSA and 2 mM EDTA and then blocked for 10 min with Mouse BD Fc Block (553142; BD Biosciences). Cells were then incubated for 10 min at 4 °C with VioBlue-CD45 (130-110-664), Viobility 405/452 (130-110-206), PerCP-Vio700-CD11b (130-109-289) from Miltenyi Biotec and APC-CD117 (553356) from BD Bioscience. Spectral compensation was established with Ultracomp eBeads (01-222-42; Invitrogen). Fluorescence minus one samples for CD117 and CD11b were used as gating controls. Samples were run on a BD Fortessa ×20. Data analysis was performed using FlowJo v10.5.0 software.
Statistical Analysis.
Data are presented as a minimum of three biological replicates and expressed as mean ± SEM (SEM). Statistical analyses were performed with GraphPad Prism 7.0. Data were analyzed using standard Student's t test and were considered to be significant when P ≤ 0.05. Statistical significance representations: *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Clinical Association Analyses.
Distant metastasis-free survival of ER-negative breast cancer patients was stratified by ATP7A mRNA expression (Affymetrix ID: 205197_s_at) relative to the median based on publicly available databases (KMplot.com) (50).
We thank all members of the M.J.P. laboratory for their support and helpful comments. Confocal images were acquired at the University of Missouri Molecular Cytology Core facility. We thank Dr. Alexander Jurkevich for help with SHG microscopy. This work was supported by National Institutes of Health Grant CA190265 and a seed grant from the University of Missouri Life Sciences Center.
- ↵1To whom correspondence should be addressed. Email: petrism{at}missouri.edu.
Author contributions: V.S., K.J.-M., S.Z., G.A.W., and M.J.P. designed research; V.S., K.J.-M., S.Z., A.L.M., N.G., A.K., and E.L. performed research; V.S., K.J.-M., S.Z., A.L.M., N.G., A.K., E.L., K.S., G.A.W., and M.J.P. analyzed data; and V.S. and M.J.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1817473116/-/DCSupplemental.
Published under the PNAS license.
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