Combine the vegetables and salt in a large non-reactive container, cover loosely, and let stand overnight.
In the morning, drain the vegetables, pressing out the juice. Add the vinegar, brown sugar and the spices (tie up the spices in a cheesecloth bag). Bring to a boil and simmer until vegetables are clear and the syrup is thickened.
Discard the spice bag and seal the piccalilli in hot jars, allowing 1/4-inch head space in each jar.


text:
How to Create an FMEA and Use It in Six Sigma
by Bert Markgraf
FMEA helps reduce defects in manufacturing to Six Sigma levels.
Kim Steele/Photodisc/Getty Images
1 Organizational Influences & the Swiss Cheese Effect
2 Lean Manufacturing Standardizing Procedures
3 Assess the Strengths & Weaknesses of Internal Company Controls
4 Lean Six Sigma & Quality Assurance Methods
A Failure Modes and Effects Analysis is a powerful tool for analyzing production processes and determining where defects occur, their causes and how to reduce them. This analysis matches the goals of Six Sigma to reduce defects to insignificant levels. Using Six Sigma as a framework, you can analyze the stages of Six Sigma implementation with an FMEA to identify problems and determine what solutions will result in Six Sigma levels of quality. Such an analysis is especially relevant for small businesses because they can perform it in-house without special training or additional costs.
Use the definition phase of Six Sigma to identify the Critical-to-Quality characteristics for an FMEA by asking what the customer would expect to see in the product, and what characteristics the customer would regard as essential.
Define the tests or measurements that determine the level of each characteristic, such as a test for breakage or a measurement for strength or resistance. For small businesses aiming to keep costs low, use tests or measurements that your production already performs as part of its operations.
Define what level you will consider a defect when you carry out the tests or measurements. For these levels, refer back to customer expectations.
Use the measurement phase of Six Sigma to carry out the tests or measurements you identified for the FMEA.
Analyze the measurement and testing systems to verify that they are delivering the appropriate data.
Collect the verified data for further analysis.
Establish Six Sigma benchmarks relating the collected data to the desired levels of the essential characteristics. Tabulate the collected data to show to what degree you are meeting the critical quality characteristics.
Identify variations in the collected data and determine the causes of the variations.
Complete the failure modes analysis of the FMEA by asking which aspects of production cause the data variation can go wrong, and how the variations cause defects.
Analyze the production process to find ways to improve the measured data. Simulate changes in the failure modes you identified with the FMEA and verify the effects.
Once the measured data is acceptable, establish tolerances for later use in your Six Sigma program. Make small changes in the production process and measure the effects as part of the effects analysis of the FMEA to ensure that the tolerances you specify are realistic.
Implement the changes and complete a new set of measurements and tests. Compare to the original data and the data benchmarks to confirm that the changes generate improvements and match the desired results.
Implement controls to make sure the process continues to deliver the improvements in the product. Use the failure modes of the FMEA to determine points of verification that are key to keeping the parameter variations within the tolerances.
Identify the key measurements and the critical process variables from the FMEA to assign responsibility for maintaining quality levels within Six Sigma.
Put in place reporting and verification procedures that give ownership of the optimized process to the people responsible for the quality of the output. Assign Six Sigma black and green belt responsibilities to the appropriate operating personnel.
University of Chicago: Failure Modes and Effects Analysis (FMEA)
University of Utah: Six Sigma DMAIC 15-Step Process
Second World Conference on POM and 15 th Annual POM Conference, Cancun, Mexico, April 30 – May 3, 2004: A Review of the Causal Mapping Practice and Research Literature
BMGI: CASE STUDY - Baycare Health - Increasing On-time Starts for First Case Surgeries
Bert Markgraf is a freelance writer with a strong science and engineering background. He started writing technical papers while working as an engineer in the 1980s. More recently, after starting his own business in IT, he helped organize an online community for which he wrote and edited articles as managing editor, business and economics. He holds a Bachelor of Science degree from McGill University.
Markgraf, Bert. "How to Create an FMEA and Use It in Six Sigma." Small Business - Chron.com, http://smallbusiness.chron.com/create-fmea-use-six-sigma-44185.html. Accessed 18 July 2019.
Markgraf, Bert. (n.d.). How to Create an FMEA and Use It in Six Sigma. Small Business - Chron.com. Retrieved from http://smallbusiness.chron.com/create-fmea-use-six-sigma-44185.html
Markgraf, Bert. "How to Create an FMEA and Use It in Six Sigma" accessed July 18, 2019. http://smallbusiness.chron.com/create-fmea-use-six-sigma-44185.html
Robustness of a Quality Control Method
Audit Business Accounts
Organizational Influences & the Swiss Cheese Effect
Implement Universal SPC in a Manufacturing Firm
Leverage Six Sigma With Lean Manufacturing to Accelerate Business Performance
Analysis for Process Improvement
Definition of Performance Measurement
What Are the Standard Elements of a Quality Management Plan?
Design an HRD Audit
Process Audit Checklist
Plan a Process-Based Audit

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Chart.js-to-image in Laravel does someone use Chart.js-to-image in his laravel application?
i render a chart.js chart in my application and i will save it as a img in my public folder to use it later to render a pdf with dompdf. but im not able to implement the chartjs-to-image in my project. i have a error with
const fs =  require('fs')

i also try it with base64 image but there is the problem that my chart is not the same bevor and after base64img ( peak bevore goes up to 30 after only to 20).

redpajama_set_name: RedPajamaStackExchange


text:
Categories Tunisia
Tunisia Recent History
Due to its strategic position in the Mediterranean, Tunisia was disputed since ancient times by different peoples. In the III and II century BC it was already inhabited by the Phoenicians who had founded their largest colony: Carthage.
At that time important historical events took place such as the famous "Punic wars", which would then decide whether the center of the greatest empire in history would have been the Italian Peninsula or Tunisia, if the future civilization would have been Latin or African-Asian. According to Abbreviationfinder, an acronym site which also features history of Tunisia, the colossal duel ended with the radical destruction of Carthage and thus the Tunisian region was subjected to the sovereignty of Rome. The Carthaginians, descendants of those skilled merchants and navigators who were the Phoenicians, then knew who were the best colonizers who ever existed. The Romans, in fact, gave Tunisia a period of prosperity and peace, but all this collapsed with the fall of the Roman empire.
First the Vandals, Germanic barbarians who arrived in North Africa after having traveled and devastated the Italian peninsula, and then the Byzantines, put an end to the happiest period in Tunisian history. The Vandals remained in Tunisia until the middle of the sixth century after Christ; the Byzantines replaced them and dominated the region for another century, until they were driven out by the Arabs.
The Muslims brought together under their domination a territory comprising Algeria, Tunisia and Tripolitania. The indigenous population, consisting mainly of Berbers, was quickly converted to the Muslim religion, but the political dominance of the Arabs was not well accepted. The Berbers organized continuous riots against the Arabs; the most famous of these guerrillas was directed by a woman, Queen Khaina, who was eventually captured and killed. Despite these difficulties, the Arabs held the dominion of the region, which they entrusted to the care of governors.
A period of greater prosperity occurred instead during the Moroccan domination. Around 1160, in fact, the sovereigns of Morocco drove the Arabs from Tunisia and established their own viceroy. Tunis became the largest city in North Africa after Cairo during this period. There were, as still today, many lively streets covered with curtains, to protect them from the sun, the "Suks", in each of which certain craftsmen practiced their profession: perfumers, leather makers, booksellers, goldsmiths. Moroccan domination lasted over three centuries.
Towards the eleventh century life in the maritime cities of Tunisia was disturbed by the presence of fleets of Turkish corsairs; they occupied these ports to make them the bases from which to make incursions on the European coasts. Naturally, the European powers reacted to this state of affairs and thus the fleets of Emperor Charles V, led by the Genoese Andrea Doria, were seen to clash with the pirates in front of the Tunisian ports. The Tunisian caliph himself turned to the emperor for protection against the corsairs, who were supported by Turkish power. But it was a wrong move: indignant to see their caliph almost submissive to a Christian sovereign, the people rose in revolt by dismissing him. The Turks took advantage of all this to occupy Tunisia, bringing their dominion (1574).
With the arrival of the Turks, the government of Tunisia remained entrusted to an authority called "bey" or "pasha", which depended only formally on the sultan of Constantinople. The bey regime practically lasted to the present day.
During the 16th and 17th centuries, Tunisia's life was sometimes peaceful, sometimes turbulent, depending on whether there was a tyrant or benign ruler in government. In the 19th century, European powers began to take an increasingly active interest in Tunisia, where many European industries and banks had transferred large interests.
In the meantime, to remove all pretexts from intervention, Europeans did everything possible to adapt their country to modern civilization: slavery was abolished and a form of constitutional government was established; grandiose public works were also carried out, such as roads and aqueducts, but the excessive waste of state money brought Tunisia to the brink of economic ruin. Here is the pretext that the European powers awaited: Italy was also interested in the fate of Tunisia, but France was more ready to intervene and, in 1883, was able to establish its own protectorate on this country.
France nominally left the government to the bey but practically had all the powers in Tunisia. Under the French administration the country made great progress; about 100,000 Italian emigrants also collaborated in this work of civilization (also because the Sicilian channel separates Italy from this African country with only 140 kilometers).
Nationalistic unrest had arisen in the country since World War I, but the National Liberation Movement took shape organized in 1920 with the founding of the "Party of Destur" (the Constitution) which ran aground in sterile attempts to obtain reforms and concessions from France partial. A movement less inclined to compromise, the "Neo Destur" was founded in 1934 by Habib Burghiba, and immediately promoted unrest and strikes. The aspirations of the nationalists at the beginning were mainly aimed at obtaining a Franco-Tunisian government, with its own Constitution.
Then came the Second World War; North Africa was occupied by the Germans who, in order to definitively detach Tunisia from France, gave great freedom of action to the Neo Destur who, therefore, continued his liberation struggle. But when the territory was reconquered by the Allies, the New Desturian hopes were extinguished and the chief Burghiba had to repair in Cairo.
But in the meantime the party of Destur, despite having been prohibited, continued to gather proselytes, including many intellectuals, among the interest of the whole population.
Among the most serious problems that Tunisia had to face after the war, the priority was certainly the economic one since, although it could not count on great resources, during the war period it had been the most targeted region of North Africa. And with the economic problems there were the social ones: unemployment, low wages, exploitation of the indigenous workforce, shortage of electricity, population growth not followed by an increase in agricultural productivity, nor by industrial development.
These were the main reasons why Tunisia still had to remain within the French Union. The solution to Tunisian problems was entrusted to the United Nations in August 1946, but without success, as was also the hope placed in support from the United States. The situation of the Italians in Tunisia, both during and after the war, was also very precarious. At the fall of Tunis 1600 Italian heads of family were interned and forced to sell their land properties on unfavorable terms. Later they were expelled and France, having returned to good relations with Tunisia, asked Italy, in return, to renounce all the agreements already stipulated with the 1896 Convention.
The problem of Tunisian independence found its way into the summer of 1955 when Mendes France, having the courage to take note of the irresistible will of that people, began negotiations culminating in the declaration of 20 March 1956 with which the protectorate was ended. In July 1957 Tunisia officially decreed the state of the bey, proclaimed the Republic and with a popular plebiscite was appointed first president of Burghiba.
He immediately revealed himself to be an intelligent and balanced man and expressed a sincere desire for collaboration with France and with the whole western world. In June 1958 he obtained total eviction of French troops, minus the base of Bizerte, granted to NATO via France. But precisely because of Bizerte, in the long run, relations with France deteriorated so much that Burghiba, on July 17, 1961, in a speech to the National Assembly, declared that in a short time he would accelerate the process of decolonization of Tunisia with the definitive recovery of Bizerte and the definition of the border to the south of the country. The next day the Tunisian troops attacked the French troops, barricaded in the base, but were rejected after three days and the French occupied the whole city. On September 29th, thanks to mediation by the United States, an agreement was reached with which the French were ordered to leave Bizerte and return to the air base. And while Tunisia always maintained good relations with the other Arab countries of the west, Morocco and Libya, with Egypt instead had various disagreements as Burghiba was accused of defecting the Arab cause.
In June 1962 the base of Bizerte was returned to Tunisia and for some time things went quite well. But then disagreements returned when in May 1963, after a resolution to return part of the Tunisian lands still in the hands of the French, the National Assembly instead established the expropriation of all foreign possessions. The Assembly thus intended to introduce in the agricultural field that system which was called "Tunisian socialism", changed the name of the single party which became the "Desturian Socialist Party" and established a state socialism, however prudent, so as not to discourage the private enterprise..
Burghiba continued to apply a moderate policy and this stimulated foreign investments, particularly western ones, and contacts with the East were also possible, so much so that in 1964 diplomatic relations were established with popular China.
This attitude, however, created many disagreements with the other Arab countries, especially with Egypt, following the Arab League's decision to break relations with the Federal Republic of Germany; decision not shared. Relations with Egypt then eased at the outbreak of the 1967 Arab-Israeli war. The following year, however, he had a severe controversy with Syria and some friction with Algeria over mutual accusations of interference with internal affairs. But in principle Tunisia always expressed its solidarity with all Arab countries.
Inside, things did not go as desired and the agricultural collectivization experiments all failed, so in September 1969 the Minister of Finance and Planning, Ahmad Ben Salah, was ousted, but passed to the Ministry of Education. From here he was also ousted in November of the same year, tried and sentenced to forced labor (in February 1973 he managed to escape and take refuge in Europe).
In the meantime, another problem arose, that of the succession of Burghiba, seriously ill, and after several discussions ended up recognizing a valid project presented by Burghiba himself, namely that of being able to elect his successor himself. In 1974 the National Assembly, making an amendment to the Constitution, allowed the appointment as President for Life in Burghiba.
From 1976 to 1978 there were worsening relations with Libya and Algeria and there was a general strike in the country, called by the "Union Generale des Travailleurs Tunisiens", with great unrest, in January 1978, to stem which was proclaimed the state of siege and several trials were unpunished for the unionists who had decreed it. In June 1978, Burghiba's opponents founded a political party called the "Democratic Socialist Movement". In 1980 Muhamad Al-Mzali was elected prime minister who immediately decided on an opening policy allowing for multi-partyism. The Tunisian Communist Party was officially recognized and analogous what was promised for those parties which in the subsequent elections had exceeded 5% of the vote. In the November 1981 elections, the National Front, a coalition of the Socialist party from the Depression with the General Union of Tunisian Workers, had an overwhelming victory. Instead, the "Movement of Socialist Democrats" and the "Movement of Popular Unity", which were officially recognized only in 1983, did not reach 5%.
In 1984 popular uprisings started again due to a strong economic crisis. In order to stem the ever growing popular discontent, reshuffles were made in the government which they saw in April 1986 elected to Prime Minister Rasid Sfar and Minister of the Interior Zayn al-Abidin Ben Ali.
But Burghiba's main opponents were the proselytes of the "Islamic Tendency Movement". This caused a long period of difficult relations with Iran held responsible for all the excesses caused by that movement.
In 1987 there was a change of the guard in the upper government spheres as Burghiba deposed Prime Minister Sfar and replaced him with Ben Ali who, after an incurable friction with the old president, decreed his deposition.
In 1989, as soon as he was elected president, Ben Ali immediately began extensive reform work. First he abolished the life election of the President of the Republic and increased his office to 5 years; in the name of tolerance he promoted amnesties and openings towards opponents by freeing 5000 people, imprisoned by the past regime, to whom he returned all their rights. At the end of that same year he rejected the request to formalize the Islamic Tendency Movement and this provoked a violent protest especially in the universities, true hideouts of Islamic fundamentalists.
In foreign policy, Ben Ali maintained good relations both with the West and with the Arab states, with whom he began a close collaboration, especially with Libya.
On the occasion of the invasion of Kuwait in 1990 he condemned Iraq but also deplored the intervention of the multinational force led by the United States. Then it also approved the UN resolution to impose the economic embargo on Iraq. All this provoked unrest in the country by the Muslims and even more the following year at the time of the Gulf War.
Then he announced some changes to the electoral law in order to guarantee a wider representation to all the minor parties and, in March 1994, the political elections that took place together with the presidential ones, saw Ben Ali reconfirmed in the office of President of the Republic.
In order to give a highly personalized imprint to the regime, he constantly stained himself with violation of human rights. His police oppressed the defenseless population with his political controls, arresting and persecuting progressive intellectuals and activists. Many international humanitarian organizations, including Amnesty, denounced this state of affairs in Tunisia between 1995 and 1997.
After the Gulf War ended, Ben Ali, after obtaining various positive results in the economic and social field, devoted himself to diplomacy and wanted to increase his relations with the European Union. Then, when the process of détente began in the Middle East, and the headquarters of the Organization for the Liberation of Palestine moved from Tunis to Gaza, Ben Ali could also begin the normalization of relations with the state of Israel.
By the end of 1999 new presidential elections were called and Ben Ali was president for the third time and the legislatures were the prerogative of his party which obtained an amazing majority.
Previous Post » Sudan Recent History
« Next Post Benin Recent History


A northwest suburb of Phoenix, flourishing Peoria is located in Maricopa and Yavapai counties. With 154,065 residents, Peoria is a busy city in its own right, providing its citizens with world-class facilities, exciting entertainment and a captivating history through projects like the Peoria Performing Arts Center, the Peoria Sports Complex and maintaining one of the oldest continuously operating high school, Peoria High School.
Peoria has a knack for re-inventing itself. Much of the Peoria town site and business district burned in 1917, but the city rebuilt and began growing faster than ever. Nearly a century later, between 1990 and 2000, Peoria was one of the fastest growing cities in the nation. Today, its focus is on renewing historic old Peoria City and filling in vacant land responsibly, after having gained the Challenger Space Center of Arizona and building the Peoria Sports Complex that is the spring training site for the Seattle Mariners and San Diego Padres.
Peoria home values have increased 4.2 percent to $427,300 in average, and they're selling slightly faster than last year, too, staying on the market about 100 days after listing. The price per square foot has stayed fairly flat in the last year, hovering in the $120 per square foot range. The number of foreclosures has dropped by 11 percent during the same time period, although the city still has 460 properties that are in the foreclosure process. The median price of a foreclosed home is $165,000.
Peoria offers some terrific neighborhoods, and the area around North 107th Ave is a favorite. The Blackstone Country Club, Trilogy Gold Club and dozens of shops and restaurants are just a quick drive away. The area has mostly 3- and 4-bedroom homes and a wide variety of townhouses and apartments. The median home price is $306,558. Families love the safety, friendliness and convenient freeway access of Westgreen Estates so much that the population has jumped nearly 3 percent in recent months. A home in Westgreen Estates has a median price of $161,049. Affluent families also delight in the area around 99th Avenue, for its outstanding schools, low crime rate and median home price of $587,689.
There are plenty of differences between traditional home buying and rent-to-own arrangements, and many of them benefit buyers. Unlike conventional purchases, rent to own homes don't need an immediate down payment or mortgage. Instead, buyers pay monthly rent and a one-time option fee. This gives the buyer the chance to move into the home right away, yet gain some time to continue to save for a down payment and improve credit ratings. There's also a price advantage in markets where prices are rising because buyers pay today's home rate in tomorrow's higher market.
In spite of local housing expenses costing 19 percent above the national average, most necessities in Peoria are affordable and below the country's average. Groceries are just a tiny 2 percent more expensive, but healthcare is 1 percent cheaper, and utilities are 8 percent less expensive than in the rest of the nation. As a whole, Peoria residents will spend 7 percent more on living costs than the national average.
Gleaming with shiny new sports complexes and performing arts centers, Peoria is a shining example of how to create city-building developments that enrich citizens' lives. Noting all of Peoria's many benefits, Money magazine named Peoria to its "Top 100 Places to Live" in the U.S.. Those special town features also extend to the city's appealing homes, safe neighborhoods and abundant local activities. While home prices are rising, much of Peoria remains affordable, and that mix of costs is reflected in the city's cost of living that's 7 percent above the national average.


dubai travel guide plastic wooden deck suppliers in the uae. when fixing wood plastic garden decking with concealed wood decking company in dubai - landscape company in dubai wood decking company dubai : a wooden deck is a useful home perfection that adds beauty and value to your dwelling.
where can i buy decking in dubai where to buy wooden garden decking in dubaiwhere to buy wooden garden decking in dubai. eco deck.
patio deck out of 25 wooden pallets. patio decking out of 25 wooden pallets pallet flooring pallet terraces . diy wood pallet garden gazebo deck with furniture: here we have an idea for.


text:
Assessing Industrial Boiler Feedwater Pump Systems
Boiler feedwater pumping systems are critical to the efficient and reliable operation of power boilers. In many plants, these pumps were installed decades ago and need to be modernized and optimized.
Generally, plant maintenance staffs and aftermarket repair organizations have attributed boiler feedwater failures to internal component damage that needs to be addressed via repair, rebuild or replacement.
But in addition to evaluating the components, end users should consider boiler feedwater pump reliability issues in the context of system design. Pump component failures are often the result of external conditions that can be identified, addressed and modified.
Correcting or optimizing system issues can maximize the performance of the components and extend the pump's mean time between repair (MTBR). Reviewing a typical boiler feedwater system study can illustrate the importance of identifying and validating common performance issues.
In this example, an industrial plant requested an assessment of its boiler feedwater system to understand why one of two parallel pumps was delivering less flow than the other pump. The existing three-feedwater-pump system was originally designed to supply two identical power boilers that create steam for the various plant processes.
In this study, the No. 2 boiler feedwater pump was evaluated in relation to the overall pump system, which also included the No. 1 and No. 3 pump units. The No. 1 unit was off and served as a backup. The No. 2 pump was considered for efficiency loss—in this case, producing less flow than the No. 3 pump at the same load. At the time of the study, the No. 2 pump had been in operation, without a rebuild, for five years and the No. 2 pump for 10 years.
According to maintenance records, the automatic recirculation control (ARC) valve in the No. 2 pump discharge line had required three component replacements during the previous year. It was suspected that the bypass line remained open during periods when recirculation flow was not required, i.e., during high-demand periods. The ARC valves are designed to be fully open in high demand conditions and to protect the pump by increasing recirculation (bypassing) in low-flow conditions and preventing reverse flow.
The differential pressure orifice flow meters installed in the recirculation lines were not functioning properly. Maintenance planned to repair and calibrate both flow meters to determine the bypass flow condition of the operating pumps: Is pump No. 2 and/or No. 3 recirculating flow when the ARC valve for the recirculation (bypass) line should be closed, lowering efficiency and wasting energy?
Once the differential pressure orifice transmitters were recalibrated, accurate flow rates could be measured to support decisions about the operating status of pumps No. 2 and No. 3. Based on the steam production rate calculations and the flow readings from the pump suction lines, the initial indication was that flow from the No. 2 pump was being bypassed to the deaerator during high-demand periods. This scenario was reflected in the calculated efficiency loss for the No. 2 pump.
If both recirculation lines were verified to be closed and pumps No. 2 and No. 3 were operating synchronously under normal boiler load, and if the No. 3 pump was producing significantly more flow (at least 20 percent) than the No. 2 pump, then the No. 2 pump could be considered for repair or refurbishment. The vibration reading indicated that the No. 2 pump had 0.18 inches per second (ips) at peak, which was in the normal range.
In order to validate the flow rates, a portable clamp-on flow meter was used on the discharge line, downstream of the bypass, of the No. 2 and No. 3 pumps. Two readings, one midmorning and one midafternoon, were taken. The No. 2 pump flows were 518 gallons per minute (gpm) and 530 gpm, while the No. 3 pump flows were 606 gpm and 750 gpm. The amp draw, flow and head pressure from each pump was used to estimate pump efficiency. The calculations indicated that the No. 2 pump was about 36 percent efficient and the No. 3 pump was 49 percent efficient.
The flows for the No. 2 pump (518 and 530 gpm) are 73 percent of the best efficiency point (BEP = 720 gpm), and the pump is operating in the lower range for higher-speed centrifugal pumps. The flows for the No. 3 pump (606 and 750 gpm) are 84 percent and 104 percent of BEP, respectively.
An economic analysis revealed that the annual electrical cost for pump No. 2 was $746,748, while pump No. 3 cost $774,565. That equates to a net cost difference of $27,817, which represented a cost difference of 3.7 percent—a secondary indicator of efficiency loss.
At the conclusion of this study, maintenance personnel calibrated both orifice flow meters to the deaerator. This showed that the No. 2 pump was bypassing flow in high-demand periods with the No. 2 ARC valve open when it should have been closed. The No. 3 pump was operating normally; the ARC valve was closed during high-flow conditions and open in low-flow conditions. As a result, the No 2 pump was not sent for repair.
The loss in the No. 2 pump's efficiency resulted from bypassing flow in high-flow demand conditions. This outcome of the study demonstrated the importance of assessing the whole pump system in lieu of attributing pump performance issues strictly to internal component damage or failure. In this case, it saved the plant from repair costs and the potential for downtime costs if a second pump failed while one of the three pumps was out of service for repair.


Jugendstil .935 Silver and Amethyst Necklace, Theodor Fahrner, the shaped plaque set with an oval cabochon amethyst, suspending a drop, and suspended from paperclip chain, lg. 3, 20 in., Continental hallmarks, signed, (repairs).
Repair to join, noticed from reverse, bottom right. Acouple of other spots of possible repair, not obtrusive.


text:
Senior library tech assistant reminisces on 30 years at FAMU
April 10, 2019 7:58 pm by Lindsey Britton | Staff Reporter
Alumna Lillie Muse recalls her journey as a student then staff member on FAMU'S campus. Muse has worked at the university for over 30 years.
Photo Submitted by Lindsey Britton.
On, Feb. 9, senior library technical assistant Lillie Muse celebrated 30 years working at Florida A&M University's Coleman Library.
Muse has walked through FAMU's campus since her undergraduate days as a social work major eventually graduating in 1987. Prior to being a student, Muse was born on the university's campus inside the Florida A&M University Hospital, which is now the registrar's office known as Foote Hilyer.
Muse detailed her lifelong connection to the university has helped her cherish the memories she has made on campus.
"I love FAMU because I was born on this campus, and when I came to school on this campus, it was like family," Muse said. "At the beginning of those 30 years, it started like family. If you hurt, they hurt, if you were happy and did something great, they were great with you."
Currently, Muse works at the library located inside the School of Journalism & Graphic Communications. Each day, she greets students with a smile and reiterates the importance of staying positive. Muse explained she has a passion for helping others and seeing a smile on student's faces. She also discusses her purpose in life and what makes her happy.
"My purpose in life is to be the best person I can be. What makes me happy is seeing the joy in other people that come in my presence," Muse said.
Muse's office not only displays her love of being a helping hand but also her love of the university. Orange and green paraphernalia can be spotted the moment you step foot in her room. With a small towel that says, "I believe you FAMU," motivational quotes, and pictures of memorable moments on campus, anyone would know she is a proud alumna of FAMU.
Muse said her daily routine is what helped make her time working for the university enjoyable.
"Well I get up, I pray, and I try to get myself together so that I can meet people," Muse said. "So people will see what's inside of me on the outside of me."
Senior Library Technical Assistant, Carla Thomas, mentioned she and Muse respect one another and bare scars that help them understand each other. She described Muse as "jolly, helpful, and always willing to lend a hand."
"I commend her in her willingness to go above and beyond to help the students here, regardless of race, color, creed," Thomas said. "She goes beyond and above to assist the students. If it's something she doesn't know she challenges herself to seek the resources."
Over the years, Muse has run into lifelong friends who have created strong bonds with her.
Senior library technical associate supervisor, Alvin Lee, has worked with Muse for about nine years and explained he and Muse have a profound spiritual bond. When asked to compare his initial first impressions in relation to now, Lee expressed Muse has always been the same.
"Thinking back when I first met her, she hasn't changed," Lee said. "She has always been open and honest (and) the same applies today. She is more to me than a sister in Christ. Having no family here, Lille is like an extended family to me."


text:
A Brief History of the City of Salinas
Small tribes of Native Americans first made their home here in the Salinas Valley many centuries before the first European explorers came to California to build a president in Monterey and missions in Carmel, Soledad, San Juan Bautista and the San Antonio Valley.
During the time of Spanish rule settlements developed around the missions here on the Central Coast, but the area remained largely undeveloped until after Mexico overthrew their Spanish rulers and began granting lands in Alta California to its people.
"Salinas" is Spanish for 'salt marsh' because at that time there was a large slough that ran through the area. The Mexican government granted Sausal and Nacional among some of the first ranchos after Mexico seceded from Spain in 1822. It was on adjoining parts of these ranchos that "Salinas City" was born after California became a territory of the United States.
Early in the gold rush years James Bryant Hill bought Rancho Nacional and became a pioneer in the agricultural industry by farming wheat; the valley had primarily been used for cattle and other livestock from the time of some of it's earliest settlers until the 1920's. Sometime in 1854 Mr. Hill was named postmaster of Salinas, even though there was no town "officially" of that name yet. In 1852 Jacob Leese purchased Rancho Sausal for $600, later he sold 80 acres to Elias Howe. Howe built an inn called "Halfway House" at the intersection of the Monterey-San Juan Bautista and the Los Angeles-San Francisco stage lines in 1856.
In 1867 Alberto Trescony, Alan Riker and William Jackson formed a partnership with cattle rancher Eugene Sherwood and laid out a city plan of a half-square mile. Realizing the importance of the railroad to getting the agricultural products to market, Sherwood offered Southern Pacific Railroad free acreage for the right-of- way and a depot; which opened its doors for business in 1872, that was also the year that "Salinas City" became the seat of Monterey County. Two years later the name was changed to the "City of Salinas" and incorporated.
The agricultural industry began to change from hides and tallow to more of what we are know for to this date; products like grains, beans and of course sugar beets. After World War I the "green gold" of lettuce, broccoli and artichokes helped to make Salinas one of the wealthiest cities per capita in the U.S.
In our community today the Spanish heritage is reflected in the names of our streets, lakes, shopping centres, recreation areas and school districts. Salinas is a very multi-cultural city with contributions being made for many different groups such as: The Chinese, Mexican, Dutch, African Americans and Germans just to name a few.
DivCL 22/9/02
FREE CITY BUS RIDING PROGRAM Essay
Carthage was a city-state on the Greek model that
The history of PC RAM
AP History
The Man who makes History
History of Classroom Inclusion essays
A Brief History Of Buddhism
0 March 26, 2019
Previous: Previous post: Look Before You Leap
Next: Next post: Pratt 1


text:
Hair Art: The Morbid Trend That Dominated the Victorian Era
BY Molly MacGilbert
IN Living
Disembodied hair triggers an almost subconscious reflex of disgust in humans. While natural when attached to a scalp, an errant strand floating in a bowl of tomato soup or a damp wad clogging up a shower drain is enough to send a shiver down even the strongest of spines. So why is it that, two centuries ago, loose hair—and often, hair cut from a corpse—was the key ingredient in a popular household craft?
The creation of art from human hair, or "hairwork," reached its peak in the 19th century. "Hairwork" instructions and patterns were often featured in shops, ladies' magazines, and "fancywork" books, so the trend became a popular parlor craft among middle-class white women of the Victorian era. According to Artsy, strands of hair were braided into bracelets, molded into loops to create floral patterns, and ground up for pigments. Human hair can keep its color for centuries without decaying, making it a sustainable material for use in art, jewelry, wreaths, and beyond.
In 1854, Wilkie Collins wrote that bracelets made of human hair were "one of the commonest ornaments of woman's wear," per JSTOR Daily. In Charles Dickens' 1865 book Our Mutual Friend, the writer describes a man who wore a "decent silver watch in his pocket and its decent hair-guard round his neck [and] looked a thoroughly decent young man." The full sentence featured seven "decent"s, indicating that Dickens believed a "hair-guard"—a watch fob made of hair—helped make a man very decent indeed.
The trend grew in popularity in 1867, when a man named Mark Campbell published a book called Self-Instruction in the Art of Hair Work, Dressing Hair, Making Curls, Switches, Braids and Hair Jewelry of Every Description. The title reflects the length and detail of the manual itself, which was made up of 276 pages of instructions and illustrations. "Heretofore the Art of making these goods has been zealously guarded by a few dealers, who have accumulated fortunes, and would still retain it a profound secret but for the publication of this book," Campbell wrote. "Persons wishing to preserve and weave into lasting mementos, the hair of a deceased father, mother, sister, brother, or child, can also enjoy the inexpressible advantage and satisfaction of knowing that the material of their own handiwork is the actual hair of the 'loved and gone.'"
Infant mortality rates were high in the 1800s, so death was omnipresent. "Not only were people used to being around bodies that were dying and had recently died, but the kind of associations that we have today of the dead body being a gruesome thing or a frightening thing was a little different," Dr. Helen Sheumaker, author of Love Entwined: The Curious History of Hair Work, told National Geographic. Throughout 19th-century England, women would make wreaths from the hairs of a single deceased person as a symbol of mourning, or from the hair of multiple family members, dead and alive, to form an unorthodox family tree.
In Emily Brontë's 1847 novel Wuthering Heights, Heathcliff places a lock of his hair in the locket that hangs around the neck of his dead love, Catherine. "Rather than gathering a memento of Catherine for himself, Heathcliff sees to it that a material fragment of his body will go down into the grave with Catherine's corpse, to intermingle with her flesh," Deborah Lutz, Victorian literature scholar, wrote (via JSTOR Daily), adding that human-hair relics reflect a "desire to see death as not permanent, in that material remains might be proof that the loved one still exists somewhere, somehow."
Hair art was present throughout the 18th and 19th centuries, but according to Emily Snedden Yates (via Artsy), "it wasn't until the Victorian era that the 'cult of the dead' became almost a mania in Britain." In 1861, Queen Victoria's husband, Prince Consort Albert, died, leaving the queen shrouded in a dark period of mourning that would last until her own death. She had "at least eight pieces of jewelry made that incorporated Prince Albert's hair," per JSTOR Daily. The same year the prince died, the American Civil War broke out, resulting in a high death toll that further fueled "death culture" across the Atlantic.
Still, not all works of hair art had such morbid origins. In fact, most of the "hairwork" created throughout history has been rooted in a sweeter sentimentality. Mothers framed sweepings from their children's first haircuts, friends exchanged locks like friendship bracelets, and others braided and framed strands alongside poems. "[It wasn't] really about death, even when it's in memorial to someone who died," Sheumaker told National Geographic. "It's about sentiment and emotion."
A post shared by KY (@laetitiaky)
Emotions were directed elsewhere once World War I began in 1910, and the hair-raising craft craze soon died out. In the century since, "hairwork" has taken on a new life. In the 1980s and '90s, feminist artists used hair from their own heads or those of fellow women to make statements about gender roles, per National Geographic. While Victorian hair art was unambiguously white, hair has long been used in African-American and African-diasporic art to express the Black experience, as beautifully exemplified today by artist Laetitia KY. And the next time you bristle at the tangled clumps in your hairbrush, you can follow Rookie's direction and fashion them into a "CREEPY and BEAUTIFUL and semi-morbid" craft. Maybe the Victorians were on to something: human life is finite, but human hair is (almost) forever.
Top Image via Wikimedia Commons
These Dresses Could—And Did—Literally Kill
This U.K. Artist Turns Found Items Into Haute Art: BUST Interview
This Is How Victorian Men Were Advised To Treat Women
Molly MacGilbert is a British-born writer and editor based in New York City. She loves cats, carbs, and comedy. For more, visit mollymacgilbert.com.
Author photo by Allison Barr / @alliisonder
Tags: Queen Victoria , Wuthering Heights , Laetitia KY , Charles Dickens , Victorian era , hairwork , hair art


Whenever I hear the world "miracle" the first thought I have is of a huge transformation occurring that is often unexplainable such as someone with an incurable disease suddenly healing or someone in need receiving help at the right time to make a difference in their lives. Have you ever thought about how these miracles occur?
Belief and faith are powerful drivers of miracles as they are the fuel that empowers your soul light to shine. When you shine, you receive healing, and other souls receive this same light, the light which speaks only of love. Holding on to the strong energy of faith in your heart keeps this passageway of light open, connecting you to spirit, and giving you hope and strength. You receive the energy of the divine.
What if I told you that miracles happen every day in both big and small ways all around you? It is easy to overlook the everyday interactions which produce tiny sparks of healing light. These smaller miracles often happen so quickly that you may miss the connection. These tiny sparks unite, and when you allow your heart to open to the faith and belief of possibilities, then you create a chain of energy which creates the big miracles.
Recently I received messages from my angels and spirit guides who sent me information about miracles and how it is possible to bring more miraculous moments in our lives. In the spiritual realm, they refer to miracles as "everydays" because these energy shifts can truly happen every day of our lives, big or small.
Once you open your energy to believing that huge energy transformations are possible, then you give permission to the divine source, to the universe, and to your angels and guides to assist you in receiving the miracles destined for your soul. Your soul is a miracle, and right now, miracles surround you everywhere in your life.
Cherish the gift of your soul in this lifetime and the people and experiences which are part of your journey. There are many more miracles waiting for you as your heart expands and connects to the faith in infinite love. Live from your heart every day, and you stay in the miracle of you.


Is 67519 the best Kansas zip code for your business?
Now you can skip hours of internet research and jump straight to getting answers with our meaningful at-a-glance Demographics Spreadsheet Report. With the most current & most popular demographic data, it's the perfect starting point for your research about 67519 and the rest of Kansas.
All 67519 population data above are from the US Census Bureau.
The population data for 67519 are from the Census' American Community Survey 2017 5-year estimates. The demographic data for 66015, 66714, 66859, 66946, and 67673 are the most current, comparable demographics available from the US Census Bureau, are from the American Community Survey 2017 5-year estimates, and were downloaded on December 2017. Check out our FAQ section for more details.
With 232 people, 67519 is the 547th most populated zip code in the state of Kansas out of 694 zip codes. But watch out, 67519, because 66714 with 230 people and 66859 with 228 people are right behind you.
The largest 67519 racial/ethnic groups are White (59.1%) followed by Hispanic (39.7%) and Two or More (1.3%).
In 2017, the median household income of 67519 residents was $43,462. 67519 households made slightly more than 66015 households ($43,448), 66946 households ($43,438), and 67673 households ($43,438) . However, 5.6% of 67519 residents live in poverty.
The median age for 67519 residents is 41.3 years young.
Looking for 67519, KS Starter Report PDF? Click Here.


text:
Tweeted talk 10 caruso who female
|August 28, 2020
You understand and agree that you shall have no expectation of privacy or security in your use of this Service.
A team-by-team look at each of the 22 rosters.
To search for players who were born on a certain date, for example all players born on December 25, choose the month, day and year with the drop down boxes and then choose the 'Full Date Search' option.
Washington Football Team NFL $3B 16.
Specifically, Omen by HP gaming PCs and displays will be used exclusively for Overwatch League games, with PCs powered by Intel Core i7 processors.
Rodgers has remarked how he likes the 6-foot-4 Allison's experience, smarts and catching radius in that spot, and just mentioning the word slot brings a smile to Allison's face.
The platform will launch in Tokyo.
Los Angeles Dodgers MLB $3B 14.
Chicago Bears NFL $3B 14.
Jordan Crawford, LaMarcus custom team jersey Named NBA Players Of The Week Dec 9 2 PM Jordan Crawford and LaMarcus Aldridge have been named the Eastern Conference and Western Conference Players of the Week.
• The NBA and the National Basketball Players Association agreed on social justice messages that can be displayed on the back of jerseys.
Boston Red Sox MLB $3B 17.
There's always a bunch of guys who come and do these things; it's always, like, six people.
Guaranteed to add charisma to your event, their abilities, talents and congenial personalities will rock out your guests with wholesome entertainment, photographs and meet greets.
Alex Morgan 5.
Boston Celtics NBA $3B 20.
It doesn't matter if you have a 'C' on your chest or not.
Hulu LLC has been operated under the auspices of Disney, 21st Century Fox, NBC Sports owner Comcast and Time Warner.
Roger Federer $106 million $6 million salary winnings $100 million endorsements Tennis 2.
Westbrook dismissed Beverley's reputation for being a defensive stopper, saying the guard tricked everyone into thinking he was good on defense.
Forbes Top 25 World's Most Valuable Sports Franchises 2020, Dallas Cowboys NFL $5B 2.
ESPN will open its 17th consecutive year create your own jersey NBA coverage with a doubleheader.
They've played well recently.
4 • Super Bowl customize your own football gear TBD Feb.
So the fact that it's possible and we wouldn't get an opportunity at that, that's weak to me.
Derrick Henry RB Tennessee Titans Highest-Paid Athletes 1.
Christian McCaffrey RB Carolina Panthers 7.
Because of differences in how web browsers incorporate and activate this feature, it is not always clear whether users intend for these signals to be transmitted, or whether they even are aware of them.
Throughout that time, some players have built quite the body of work for themselves, going as far as reaching the Basketball Hall of Fame and having their jersey number retired in some instances.
LeBron James $88 million $28 million $60 million NBA 6.
He looks good.
Cristiano Ronaldo 4.


text:
Ancient Armenian books tell a story not written in their pages
Ancient Armenian books tell a story not written in their pagesOver a meter wide when opened and weighing 32 kilograms, the Homilies of Mush is the largest ancient Armenian book to be rescued from eastern Anatolia during anti-Armenian massacres in Ottoman Turkey almost a century ago.
Archivists say the story of how the manuscript and many others like it were saved could be more telling of the plight of the Armenian people then what the intricate Armenian lettering describes within the pages.
Armenia marks the 92nd anniversary on Sunday of mass killings by the Ottoman Turks, a slaughter that is among the most painful episodes of Armenia's history, the costs of which Armenians measure not only in lost lives but also a destroyed cultural heritage.
Some 9,000 rare manuscripts are estimated to have been destroyed as Armenians were driven from their homeland in World War I, but about 30 books currently on display in Armenia's Archive of Ancient Manuscripts are believed to have been rescued by fleeing peasants.
One of these texts are the Mush Homilies. In 1915 when Ottoman forces attacked Mush, an illiterate peasant woman is said to have found the massive book in the courtyard of a church.
Too heavy to carry whole, she cut the 800-year-old book in half and took one half, according to the director of the Archive, Sem Arevshatyan.
The unnamed woman initially brought the text to the seat of Armenia's Gregorian Apostolic church in Echmitzin where it was later to be joined by the other half, discovered by a retreating Russian colonel named Nikolai de Roberti.
"Many of these books were brought by illiterate, unread people, who nevertheless understood that these texts were immensely important," Areshatyan said.
"Instead of taking their personal belongings they carried these books."
Little today is left of the numerous Armenian settlements that once characterized Eastern Anatolia, known as Western Armenia to Armenians.
Many churches have since been converted to mosques or taken apart so their stones could be used to build homes, and the some 40,000 Armenians that remain in Turkey rarely speak the language outdoors.
According to Arevshatyan not all of the attacking Turks were willing to follow through completely on the alleged plan.
"Many books were destroyed but some were sold to collectors in Europe by Turkish officers who understood that they had value," Arevshatyan said.
A slow trickle of antique texts continues to fill the archive's shelves to this day as more Armenian works pillaged in Anatolia are discovered by collectors around the world and donated to the repository.
Earlier this week a Diaspora Armenian from Paris was able to convince the sister of a collector who recently passed away to donate a page from a lost tenth century bible to the archive.
"Hopefully when she sees that it is good hands she will be willing to donate more works from the collection," said Claude Mutafian, a 62-year-old historian on medieval Armenia who carried the sheepskin sheet to Yerevan from Paris.
Agence France-Presse / Press Trust Of India (adjusted)
First Published: 00:00 IST(3/1/2007)
http://www.hindustantimes.com/StoryPage/Print.aspx?Id=332d8e5c-52c7-41a4-a850-b131dff86c3b
Newer PostProfessor Fatma Müge Göçek's word during the 24 April 1915 commemoration
Older PostThe best flee market in the world
Anna Martin donates two icons to an Armenian town in Syria
《Շաբաթվա թեման》 հաղորդման հյուրերն են Գարեգին Զուռնաչյանն ու Գարսեվան Մալխասյանը։ Թեման՝ էսթետիկ հիմունքներով հասակի ավելացում:
Փոխադարձ պատկերացումներ (Հայաստան և Սփիւռք). Հրաչ Քալսահակեան


text:
Letters to the Editor: "The disingenuous argument that a no-deal is essential to force a good deal out of the EU has proven false"
Don't miss letters in the Swindon Advertiser each day
PLEASE keep your letters to 250 words maximum giving your name, address and daytime telephone number - even on emails. Email: letters@swindonadvertiser.co.uk. Write: Swindon Advertiser, Unit 1 and 2 Richmond House, Edison Park, Swindon, SN3 3RB. Phone: 01793 501806.
Anonymity is granted only at the discretion of the editor, who also reserves the right to edit letters.
'Eye-watering' cost of leaving with no deal
In the thirty five years I have been reading the Adver I have never read such an ill informed, no simply wrong, letter as D N Simpson wrote in our paper on April 4.
Where to start: We will not be leaving the EU on April 12.
If we did not pay the EU £38 billion we owe the EU we would lose our credit rating and borrowing would be eye-wateringly expensive.
We would not get all our territorial waters back as France and Spain would dispute large areas of them and continue fishing, protected by their navies.
There will still be restrictions and quotas as the fish stocks are at an abysmal level.
Banning European diesels would not bring down carbon emissions as diesels use slightly less fuel than petrol cars. It might improve the value of second hand cars, but this would increase carbon emissions as second hand cars are less efficient.
Food shortages would not cause a reduction in obesity as this is caused by cheap ready-made food and stodgy foods that this country produces, such as potatoes.
There is no wine lake or butter mountains as these problems were solved in 1984 - ie milk quotas.
Foreign criminals can be deported at present but this would not bring down the prison population as they would still have to serve their time.
Or we could send them home without them being punished.
Mr Simpson says 17,400,000 have been labelled stupid, ignorant or ill informed, well 17,399,999 of them haven't just shown they ARE ill informed.
Steve Thompson, Norman Road, Swindon
Why so reluctant?
Excellent to see MPs doing their job on Wednesday (April 3) in voting to force the government to delay Brexit and avoid a no-deal.
Their job as our elected representatives is to make decisions on our behalf. They have determined, based on all the evidence in front of them, that a no-deal would be hugely detrimental to the economic, political and logistical interests of their constituents and the country.
I feel embarrassed that a country with such a great international reputation as the UK would even consider walking away from its agreed international and financial commitments.
As Caroline Lucas pointed out in Parliament yesterday, such recklessness is hardly going to prove our trustworthiness to other countries around the world, who the Brexiteers optimistically assure us are desperate to sign trade deals with us.
The disingenuous argument that a no-deal is essential to force a good deal out of the EU has proven false.
Despite the billions we have spent preparing for a no-deal Brexit, the EU have been perfectly frank that they have finished negotiating.
Nearly three years on from the narrow result in the advisory referendum of 2016 (in which the winning side broke electoral law) it's increasingly obvious that a confirmatory vote is the best way to implement the final terms of Brexit.
It's a mystery to me why Brexiteers are so reluctant to hold another referendum.
If the country still wants Brexit then they will win and their mandate will be massively strengthened. Or perhaps the country has changed its mind, as democracies have the right to do.
Either way, it's the only way to enable us to move on from this issue which has created such chaos and disunity.
Neil Mercer, Maidstone Road, Swindon
Betrayal of Brexiteers
Much as it goes against the grain to burst fellow Brexiteers bubbles, I must take issue with some of their statements in Thursdays Adver.
Theresa May's deal was rubbish and I was surprised when Jacob Rees-Mogg and Boris folded and voted for it. The PM claimed her deal ensured the UK had control of its borders. This is untrue, the 310 mile border between South and Northern Ireland will be totally open to unchecked immigration.
The backstop means we are trapped in a customs union, so can't make our own trade deals with the rest of the world without EU OK. I can't understand how Ireland is being allowed to block Brexit.
The UK has to hand over £39 billion as a thank you for talking to us. That's three "red lines" gone for starters.
We actually had a strong negotiating hand but the Prime Minister failed to use it. Parliament's job was to accept the referendum result, as promised, and honour it, not constantly undermine it. What idiots took no deal off the table?
That tells the EU to offer us nothing and we still won't leave. The mind boggles!
True Brexiteers have been totally betrayed. Parliament is not fit for purpose.
Just out of interest, all readers should research the ITV programme of 7th June 2016 Cameron and Farage.
Cameron tells the live audience that the referendum vote is for "you the people" not the politicians, and the result will be honoured. This is true, watch it .
Fear not Brexiteers, if the 11.4 million leavers play their cards right and vote for the new Brexit Party at the next general election we will walk it, and smash the current two party system for ever.
Alan Spencer, Swindon
Letters to the Editor: What do you think?
Do you have an opinion on our reader letters? Get in touch.
Add your contribution now By uploading a contribution, for use online and in print, you accept our contributor terms. You will either own or have permission to use anything you provide.
SPECIAL REPORT: Swindon raids spotlight local drug gangs
It's the end of the road for Swindon Half Marathon (but this year's event will go ahead)
Pair spotted trying front doorhandles and stealing from vehicles in Haydon Wick
'Great Western Powerhouse' bid will turbo charge region's economy
Three car pile-up blocked Queens Drive
Lanes cleared after three crashes on M4 that caused nine miles of queues on A419
Three crashes caused miles of congestion on M4
FOOD REVIEW: Bandido Burrito, Elgin Drive, Swindon
AIR TATTOO: Your guide on how to get to the Fairford airshow by road, rail, coach and air


This chapter reviews current developments in the area of translational control in neurons. It focuses on the activity-dependent translational modulation by neuronal regulatory RNAs, including underlying interactions with eukaryotic initiation factors (eIFs), and on the role of such modulation in locally controlled protein synthesis in synapto-dendritic domains. It highlights the role of dendritic RNA targeting as a key prerequisite of local translation at the synapse and discusses the significance of these mechanisms in the expression of higher brain functions, including learning, memory, and cognition. The chapter concludes with discussion of anticipated future work to continue to elucidate these mechanisms and provide advances in the area of translational regulation in neurons and our understanding of how translational dysregulation contributes to neurological and cognitive disorders.
Control of gene expression is essential for the temporal and spatial modulation of cellular form and function (Mathews, Sonenberg, & Hershey, 2007). Gene expression is regulated at the levels of transcription, RNA processing, translation, and post-translational modification. Translation of mRNAs is an important mechanism in the control of gene expression as regulation of protein synthesis directly and rapidly impacts cellular function (Van Der Kelen, Beyaert, Inze, & De Veylder, 2009). Translation proceeds in the three phases of initiation, elongation, and termination, and various translation factors cooperate with mRNAs, tRNAs, and ribosomal subunits in these phases. Translational initiation, which typically is rate-limiting, is often a site of regulation in the translation pathway.
Translation initiation unfolds in three steps: (i) formation of the 43S preinitiation complex (PIC), (ii) formation of the 48S initiation complex, and (iii) assembly of the 80S ribosome. In eukaryotes, translation initiation requires participation of 12 or more eIFs in the pathway that concludes with the formation of 80S ribosomes (Bourgeois, Mortreux, & Auboeuf, 2016; Costa-Mattioli, Sossin, Klann, & Sonenberg, 2009; Hinnebusch, 2017).
A ternary complex, consisting of eIF2, Met-tRNAiMet, and GTP, initially associates with the small 40S ribosomal subunit, together with eIFs 1, 1A, and 3, resulting in the formation of the 43S PIC (Jackson, Hellen, & Pestova, 2010). Higher-order structures in the 5′ untranslated region (UTR) of an mRNA are unwound by eIF4A, a factor that is in part integrated in eIF4F and operates in conjunction with eIF4B. A heterotrimeric complex, eIF4F consists of eIF4E (a cap-binding protein), eIF4A (a DEAD-box RNA helicase), and eIF4G (a scaffold protein interacting with eIF4E, eIF4A, eIF3, and poly(A)-binding protein (PABP)). eIF4A is an ATP-dependent RNA helicase which, at times assisted by RNA helicases Ddx3 and Dhx29, unwinds mRNA higher-order structures between the 5′ end and the initiator codon (Hinnebusch, 2017). eIF4B plays a dual role: it (i) stimulates the helicase activity of eIF4A and (ii) mediates recruitment of the 43S PIC to the mRNA. The latter is achieved by interactions with eIF3 and with 18S rRNA, the latter a component of the 40S ribosomal subunit. eIF4B contains two RNA-binding domains, an RNA recognition motif (RRM) located in the N-terminal region and two arginine-rich motifs (ARMs) located in the C-terminal region (Méthot, Pause, Hershey, & Sonenberg, 1994; Méthot, Pickett, Keene, & Sonenberg, 1996). eIF4B unspecifically binds mRNAs through the ARMs and specifically binds stem-loop structures in 18S rRNA through the RRM (Méthot et al., 1996). Thus, eIF4B plays a role as a bridge between the 5′ end of the mRNA and the 43S PIC (Méthot et al., 1996). In addition, PABP stimulates translation initiation by binding to the mRNA 3' poly(A) tail and by simultaneously interacting with eIF4G, thus effectively circularizing the mRNA (Pestova, Lorsch, & Hellen, 2007).
Once the 43S PIC has attached to the mRNA, it translocates ("scans") through the 5′ UTR until it recognizes a competent start codon, resulting in 48S initiation complex formation. Subsequently, the 80S ribosome is formed following factor release and joining of the 60S large ribosomal subunit.
Various RNA helicases participate in splicing, nuclear export, translation, cytoplasmic mRNA decay, miRNA-induced gene silencing, and RNA transport (Bourgeois et al., 2016). Here we will focus on RNA helicases associated with translation initiation.
The paramount RNA helicase operating in translation initiation is eIF4A. The eIF4A family consists of eIFs 4A1, 4A2, and 4A3 of which eIFs 4A1 and 4A2 share 95% amino acid identity whereas eIF4A3 shares 65% identity with the other two isoforms (Bourgeois et al., 2016; Yoder-Hill, Pause, Sonenberg, & Merrick, 1993). Structured 5′ UTRs interfere with the ability of the 43S PIC to bind to and move along the mRNA, and eIFs 4A1 and 4A2 promote 5′ UTR unwinding during 43S PIC recruitment and scanning (Pestova et al., 2007). While eIF4A3 is a component of exon junction complex, it also participates in the unwinding of structured 5′ UTRs (Choe et al., 2014).
A CNS principal neuron typically entertains more than 1,000 synaptic connections with other neurons (Stiles & Jernigan, 2010). Synapses are regulated independently in an input-specific manner (Bartley, Huang, Huber, & Gibson, 2008). The term synaptic plasticity refers to the ability of synapses to strengthen or weaken in response to changes in the input they receive (Hughes, 1958). Long-lasting synaptic plasticity and long-term memory require new protein synthesis (Dahm, Kiebler, & Macchi, 2007; Kang & Schuman, 1996; Kindler, Wang, Richter, & Tiedge, 2005). Locally controlled translation allows select proteins to be synthesized at the synapse upon demand (Job & Eberwine, 2001). Regulators are therefore essential to control local translation at the synapse. The fragile X mental retardation protein (FMRP) (Bhakar, Dolen, & Bear, 2012), miRNAs (Fabian, Sonenberg, & Filipowicz, 2010) and brain cytoplasmic (BC) RNAs (Iacoangeli & Tiedge, 2013) have been identified as translational repressors in neurons in the basal default state. Mechanisms of translational control by FMRP and BC RNAs have been studied in depth and will be discussed in the following.
Fragile X syndrome (FXS), resulting from functional absence of FMRP, is a common cause of intellectual disability and autism (Bassell & Warren, 2008). Loss of FMRP is typically the consequence of transcriptional silencing induced by 200 or more CGG triplet repeats in the 5′ UTR of the FMR1 gene. Lack of FMRP results in impaired neuronal translational control, i.e. in inadequate translational repression of FMRP target mRNAs (Weiler et al., 1997). FMRP inhibits translation at the level of elongation by stalling translocation of ribosomes along the mRNA (Darnell et al., 2011). In addition, FMRP is associated with Dicer and Argonaute 2 (Ago2), components of the RNA-induced silencing complex (Chen & Joseph, 2015).
In Fmr1 KO animals, various proteins are expressed at elevated levels, including the α subunit of the Ca2+/calmodulin-dependent protein kinase II (CaMKIIα), striatal-enriched protein tyrosine phosphatase (STEP), potassium channels Kv3.1 and Kv4.2, microtubule-associated protein 1B, and PI3K enhancer (Goebel-Goody et al., 2012; Lee et al., 2011; Lu et al., 2004;Hou et al., 2006; Strumbos, Brown, Kronengold, Polley, & Kaczmarek, 2010). HITS-CLIP analysis (Darnell et al., 2011) identified 842 FMRP target mRNAs, including a subset encoding pre- and post-synaptic proteins. The data indicate that translation of selective mRNAs is regulated by FMRP to control expression of proteins associated with synaptic functions (Darnell & Klann, 2013). Lack of FMRP in Fmr1 KO mice results in increased susceptibility to audiogenic (sound-induced) seizures (Musumeci et al., 2000).
BC RNAs, constituting a subtype of non-protein-coding, small cytoplasmic RNAs (scRNAs), are neuronal regulatory RNAs that function as repressors of translation initiation in the basal default state (Eom, Berardi, Zhong, Risuleo, & Tiedge, 2011; Eom et al., 2014; Wang et al., 2002; Wang et al., 2005). BC RNAs are predominantly expressed in neurons (DeChiara & Brosius, 1987; Tiedge, Chen, & Brosius, 1993) as non-neuronal expression appears restricted to cells of the germ line (Muslimov et al., 2002) and subsets of cancer cells (Chen, Böcker, Brosius, & Tiedge, 1997; Iacoangeli et al., 2004).
BC1 and BC200 RNAs are of limited sequence similarity but feature identical 3' C-loop motifs that are critical for binding to eIF4B (Eom et al., 2011). BC RNAs repress protein synthesis by interacting with eIFs 4A and 4B and with PABP (Eom et al., 2011, 2014; Lin, Pestova, Hellen, & Tiedge, 2008; Wang et al., 2002; Wang et al., 2005). BC RNA translational repression is mediated mainly through interactions with eIFs 4A and 4B whereas interactions with PABP account for at most 20% of the repression mechanism (Eom et al., 2011; Lin et al., 2008). BC RNAs bind to eIF4A through their central A-rich regions and the 3' stem-loop interfaces, causing inhibition of eIF4A helicase activity (Eom et al., 2011; Lin et al., 2008). They bind to eIF4B through their 3′ C-loop motifs in a high-affinity interaction that competitively inhibits binding of 18S rRNA to the factor and, as a consequence, prevents recruitment of the 43S PIC to the mRNA (Eom et al., 2011).
Phosphorylation and dephosphorylation of proteins are known to be among the most frequent posttranslational modification events and play important roles as molecular switches to regulate protein function. In response to various extracellular stimuli and intracellular messengers, signaling cascades regulate translation of mRNAs by modulating phosphorylation of components of the translational machinery (Roux & Topisirovic, 2012).
Phosphorylation of FMRP occurs primarily at serine 499 (S499), and FMRP thus phosphorylated associates with stalled ribosomes (Ceman et al., 2003), that is, is linked to reduced translation elongation. In addition, phosphorylation of FMRP promotes formation of Ago2-miRNA inhibitory complexes on target mRNAs, causing translational repression (Muddashetty et al., 2011). S499-phosphorylated FMRP thus acts as a negative translational regulator in the basal state. Upon stimulation of group I metabotropic glutamate receptors (mGluRs), protein phosphatase 2A (PP2A) is rapidly activated to dephosphorylate FMRP at S499. This dephosphorylation enhances translation of FMRP target mRNAs as dephosphorylated FMRP does not stall polyribosomes and does not promote formation of miRNA inhibitory complexes (Muddashetty et al., 2011; Narayanan et al., 2007; 2008; Niere, Wilkerson, & Huber, 2012). Thus, phosphorylation and dephosphorylation of FMRP are key regulatory events in the activity-dependent control of neuronal protein synthesis (Figure 1).
Figure 1. Phosphorylation status in translational control.
BC RNAs and FMRP repress translation of their target mRNAs at the levels of initiation and elongation, respectively. During initiation, binding of BC RNAs to eIF4B competitively inhibits binding of 18S rRNA (and, consequently, the 43S PIC) to the factor, thus repressing translation initiation. FMRP stalls translocation of ribosomes along mRNAs in the elongation phase of translation. As a result, the two translational control systems (BC RNAs and FMRP) repress translation of a subset of mRNAs at the synapse in the basal state. Upon neuronal stimulation via group I mGluR signaling, PP2A is rapidly activated to dephosphorylate eIF4B at S406 and FMRP at S499. Dephosphorylation of eIF4B at S406 decreases its binding affinity for BC RNAs, releasing BC RNAs from the eIF4B-BC RNA complex (Eom et al., 2014). eIF4B engages with the 40S small ribosomal subunit in the 43S PIC to initiate translation. Dephosphorylation of FMRP ends polyribosome stalling, thus allowing elongation to resume.
Serine 406 (S406) and serine 422 (S422) are known phosphorylation sites on eIF4B (Raught et al., 2004; van Gorp et al., 2009). Phosphorylation of eIF4B at S422 enhances its affinity for the eIF3 complex and stimulates the helicase activity of eIF4A (Shahbazian et al., 2006). Several kinases phosphorylate eIF4B at S422, including S6 kinase (S6K), a downstream target of PI3K/mTOR signaling (Raught et al., 2004), p90 ribosomal protein S6 kinase (RSK), a downstream target of ERK1/2 MAPK signaling (Shahbazian et al., 2006), and protein kinase B (PKB), a component of the PI3K-mTOR pathway (van Gorp et al., 2009). In contrast, the significance of S406 eIF4B phosphorylation in translational control has remained poorly understood until recently.
Phosphorylation of eIF4B at S406 significantly increases the factor's affinity for BC RNAs (whereas phosphorylation at S422 does not), with the result that it is higher for BC RNAs than it is for 18S rRNA (Eom et al., 2014). S406 dephosphorylation reduces the factor's affinity for BC RNAs by almost two orders of magnitude. In neurons, such dephosphorylation occurs rapidly, within 1 min, following stimulation of group I mGluRs and activation of PP2A. As a result, the eIF4B-BC RNA complex dissociates, enabling recruitment of the 43S PIC and thus translation initiation. PP2A is subsequently deactivated (beginning after 2 min) and eIF4B is rephosphorylated at S406 (within 10 min of initial mGluR activation) (Eom et al., 2014; Narayanan et al., 2007). As a result, BC RNA translational control switches back from permissive to repressive (Figure 1). This mechanism establishes an activity-dependent window of opportunity for the synaptic synthesis of select proteins, effectively controlling the amount of protein that can be produced following one round of receptor activation. Tight control of local protein synthesis is essential, as excessive production may give rise to adverse phenotypic consequences (see the next section).
Which kinase is responsible for the rephosphorylation of eIF4B at S406? Maternal and embryonic leucine zipper kinase (MELK) phosphorylates eIF4B at S406 (but not at S422) in cancer cells (Wang et al., 2016). Synthesis of myeloid cell leukemia 1 protein, an anti-apoptotic protein important for cancer cell survival during cell division, is associated with phosphorylation of eIF4B by MELK (Wang et al., 2016). The eIF4B phosphorylation status may differentially impact translation in different cell types, owing to cell-type specific availability of additional interacting factors.
The dual phosphorylation status of eIF4B, at S406 and S422, is a determinant of the reversible, activity-dependent switch between repressive and permissive states of translation initiation in neurons (Eom et al., 2014). The available evidence is in support of the following scenario. In the repressive state, S406 is phosphorylated, allowing high-affinity binding of BC RNAs and thus disallowing interactions with the 43S PIC, as a consequence repressing translation initiation. At the same time, S422 is dephosphorylated, and binding to eIF3 is not enabled. Conversely, in the permissive state, S406 is PP2A-dephosphorylated, enabling eIF4B interactions with the 43S PIC, whereas S422 is phosphorylated, enabling interactions with eIF3. As a result, translation is initiated.
Absence of regulatory BC1 RNA in the BC1 KO animal model results in exaggerated protein synthesis that is dependent on group I mGluR activation and MEK-ERK signaling (Zhong et al., 2009). Thus, in the basal neuronal default state, BC1 RNA negatively regulates neuronal translation, acting as a break downstream of translational stimulation mediated by activation of group I mGluRs (Chuang et al., 2005; Huber, Kayser, & Bear, 2000; Zhong et al., 2009). Absence of this break gives rise to excessive cortical gamma oscillations, prolonged epileptiform discharges in hippocampal CA3 pyramidal cells, and convulsive seizures upon auditory stimulation in vivo (Zhong et al., 2009).
As cortical gamma oscillations have been implicated in prefrontal cortical mechanisms of perception and cognition (Cho, Konecky, & Carter, 2006; Cho et al., 2015), dysregulation of such oscillations in BC1 KO animals raised the question whether these animals are cognitively impaired. Recent work (Iacoangeli, Dosunmu, Eom, Stefanov, & Tiedge, 2017) indicates that this is indeed the case. BC1 KO animals excessively self-groom, an activity associated with autism-like behavior in rodents (Kalueff et al., 2016; McFarlane et al., 2008; Silverman, Tolu, Barkan, & Crawley, 2010). Furthermore, behavioral analysis using a modified Attentional Set Shift Task assay has shown that while learning and memory per se appear normal, BC1 KO animals are impaired in the cognitive control of acquired memories. When presented with a novel situation that conflicts with previously stored information, BC1 KO animals (but not corresponding WT animals) continue to operate in the framework of the older but now superseded memory. The cognitive errors are of the regressive type (Iacoangeli et al., 2017): a BC1 KO animal will, even after having made a serendipitously correct choice and having been rewarded for it, revert to making a series of consecutive incorrect decisions. The previously stored but now irrelevant information has remained dominant over subsequently acquired relevant but conflicting information (Figure 2). Thus, the animals display lack of behavioral flexibility as supervisory cognitive control is defective (Iacoangeli et al., 2017).
Figure 2. BC RNA control in animal cognition. (A) BC1 KO and WT animals perform equally well in discrimination learning. They learn and memorize that a scent of sage, but not of cinnamon, is predicting a reward (a cheerio). (B) BC1 KO animals are significantly impaired in cognitive flexibility. In conflict learning sessions, WT animals rapidly adjust their retrieval strategy as they learn and memorize that the reward-predicting odor has been switched from sage to cinnamon. BC1 KO animals, in contrast, operate on the basis of the earlier acquired information and continue to search for a reward in the sage-scented bowl even when confronted with repeated negative feedback. Thus, in the absence of BC RNA translational control, cognitive flexibility is impaired as animal behavior is dominated by previously established but now outdated outcome expectancies (Iacoangeli et al., 2017).
Adapted from Iacoangeli et al. (2017).
In addition, Briz et al. (2017) recently reported that BC1 KO animals exhibited impaired texture recognition and social interactions, similar to what has been observed with animal models of FXS and autism spectrum disorder (ASD) (Pasciuto et al., 2015; Santos, Kanellopoulos, & Bagni, 2014). The novel object recognition test (NORT), using visual NORT (vNORT) and texture NORT (tNORT), was applied to examine whether visual or sensory experience is associated with learning. BC1 KO animals preferred a novel object over a familiar one in the vNORT, but had no preference for a novel object in the tNORT (Briz et al., 2017). In a three-chamber test, BC1 KO animals showed deficits in sociability but not in social memory (Briz et al., 2017).
The question arises how BC1 RNA impacts animal behavior. BC1 RNA represses synthesis of a subset of synaptic proteins (including PSD-95, NR2B and mGluR5) in postsynaptic microdomains (Briz et al., 2017; Zhong et al., 2009). In BC1 KO mice, elevated levels of such proteins lead to increased spine density and decreased dendritic complexity, similar to what has been observed in Fmr1 KO mice, an FXS animal model (Restivo et al., 2005; De Rubeis, Fernandez, Buzzi, Di Marino, & Bagni, 2012)). Elevated PSD-95 levels cause increased postsynaptic clustering and stabilization of spines, and increase the number and size of spines along dendrites (De Roo, Klauser, Mendez, Poglia, & Muller, 2008; El-Husseini, Schnell, Chetkovich, Nicoll, & Bredt, 2000). In BC1 KO animals, significantly elevated levels of PSD-95 are associated with spine abnormalities, including enlarged spine heads and PSDs (Briz et al., 2017; Zhong et al., 2009). Such abnormalities may skew synaptic excitation-repression equilibria, causing neuronal hyperexcitability which, in BC1 KO animals, has been observed in the form of an increased propensity for prolonged epileptiform discharges and a susceptibility to audiogenic seizures (Zhong et al., 2009; Iacoangeli & Tiedge, 2013; Zhong et al., 2009).
In addition, excessive cortical gamma frequency oscillations are a hallmark of BC1 KO mice (Zhong et al., 2009). Gamma frequency band activity has been implicated in cortical mechanisms of cognitive processing (Fries, 2009), specifically as it relates to prefrontal cortical network functionality and cognitive flexibility (Cho et al., 2006; 2015). Impaired gamma synchrony may cause discoordination of network connectivity and reduced cognitive flexibility, thus contributing to autism spectrum disorder (ASD) phenotypes (D'Cruz et al., 2013; Uhlhaas & Singer, 2012. In summary, recent work (Briz et al., 2017; Iacoangeli et al., 2017) is in support of the notion that impaired neuronal translational control may give rise to cognitive phenotypes that resemble ASD manifestations (Aguilar-Valles et al., 2015; Gkogkas et al., 2013; Santini et al., 2013).
Strain type may constitute a confound in the phenotypic analysis of mice. Iacoangeli et al (2017) found that cognitive competence in the C57BL/6J strain is generally lower than that in a C57BL/6J and 129X1/SvJ mixed-background strain. Translational control may potentially be impacted in C57BL/6J mice as a result of a mutation in a CNS-specific tRNA gene which, in the presence of a second, interacting mutation, may cause ribosome stalling (Ishimura et al., 2014). Possible deleterious consequences of such an epistatic tRNA gene mutation may thus affect the readout of other mutations (Ishimura et al., 2014). This is directly relevant in cases where such other mutations impact neuronal translational control pathways, in particular considering the possibility that tRNA mutation-induced potential ribosome stalling may be exacerbated under conditions when rapid or local synthesis of proteins is required in the brain (Darnell, 2014). In conclusion, any mutation impacting neuronal translation in a mouse-model on the C57BL/6J background may potentially face phenotypic interactions with that strain's tRNA mutation. Caution is therefore advisable when performing translational control phenotypic work with mutant model mice on the C57BL/6J background (Darnell, 2014).
Once a neuron is fully differentiated, distal dendritic domains can be several hundred micrometers away from the nucleus. Dendritic arborizations of mature neurons may feature thousands of dendritic spines (Loew & Hell, 2013), micro-protrusions each of which typically forms a synapse with a presynaptic terminal.
The discovery of RNAs, ribosomes and translation factors in dendrites and dendritic spines suggested that synapse form and function can be modulated directly through regulation of local protein synthesis (Steward & Levy, 1982; Sutton & Schuman, 2006; Tiedge & Brosius, 1996). Today we know that RNA transport serves as an important cellular protein-sorting and distribution mechanism, and that mRNAs are locally translated in neurons as well as in other eukaryotic cell types (Grossman, Aldridge, Weiler, & Greenough, 2006; Martin & Zukin, 2006; Pfeiffer & Huber, 2006; Schuman, Dynes, & Steward, 2006; Wells, 2006). Through this mechanism, neurons acquire spatial and temporal control of mRNA translation in synapto-dendritic domains. Neurons are thus enabled to rapidly produce proteins at individual dendritic branches or even at single synapses in response to local stimulation (Besse & Ephrussi, 2008). Thus, RNA transport is requisite for rapid translational responses that are independent of transcription in the nucleus or translation in the cell body (Shav-Tal & Singer, 2005).
Local translation occurs during development in processes of immature neurons as well as in dendrites of fully differentiated neurons (Kennedy & Ehlers, 2006). The first step for such regulation is the selective transport of a specific set of RNAs (including mRNAs, tRNAs, and regulatory RNAs) to dendritic destination sites. Well-established dendritically localized mRNAs include those encoding microtubule-associated protein 2 (MAP2) (Garner, Tucker, & Matus, 1988), the α subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKIIα) (Burgin et al., 1990), inositol 1,4,5-triphosphate receptor type 1 (Furuichi et al., 1993), neurogranin (Landry, Watson, Kashima, & Campagnoni, 1994), activity-regulated cytoskeleton-associated protein (Arc, arg3.1) (Link et al., 1995; Lyford et al., 1995), dendrin (Herb et al., 1997), cAMP response element binding protein (CREB) (Crino et al., 1998), β-actin (Eom, Antar, Singer, & Bassell, 2003), and protein kinase M-zeta (PKMζ) (Muslimov et al., 2004). In addition, the synapto-dendritic presence of rRNAs, tRNAs and regulatory BC RNAs has been documented (Kleiman, Banker, & Steward, 1993; Tiedge et al., 1993; Tiedge & Brosius, 1996; Tiedge, Fremeau, Weinstock, Arancio, & Brosius, 1991). These discoveries raised questions concerning mechanisms of RNA transport and the nature of spatial determinants that specify RNA delivery to dendritic destination sites.
Neuronal RNAs located in synapto-dendritic domains contain cis-acting dendritic targeting elements (DTEs). Deletion of a DTE impairs dendritic localization of the RNA (Blichenberg et al., 1999; Mori, Imaizumi, Katayama, Yoneda, & Tohyama, 2000; Muslimov et al., 1997; Muslimov et al., 2004). In dendritic mRNAs, DTEs are found within UTRs as well as in protein-coding regions (Andreassi & Riccio, 2009; Moore, 2005; Muslimov et al., 2004). It is likely that localized neuronal RNAs use more than one type of DTE as combinations of different DTEs can mediate distinct steps in transport and localization (Blichenberg et al., 1999; Muslimov et al., 2004; Muslimov, Iacoangeli, Brosius, & Tiedge, 2006; Subramanian et al., 2011). Localization of BC RNAs and CaMKIIα mRNA appears to depend on multiple DTEs (Gao, Tatavarty, Korza, Levin, & Carson, 2008; Mori et al., 2000; Muslimov et al., 2004; 2006; Subramanian et al., 2011).
RNA transport is mediated by trans-acting RNA-binding proteins (RBPs) which recognize DTEs and specify RNA delivery (Gao et al., 2008; Huang, Jung, Sarkissian, & Richter, 2002; Kwon et al., 2002; Muslimov et al., 2006). RNAs with multiple DTEs can be recognized by different RBPs, further increasing the complexity of neuronal RNA targeting mechanisms (Bassell & Kelic, 2004; Bullock, Ringel, Ish-Horowicz, & Lukavsky, 2010). Dendritically localized RNAs and their cognate transport factors associate to form ribonucleoprotein (RNP) complexes. These complexes engage, directly or indirectly, with motor proteins that move them along the dendritic cytoskeleton (Doyle & Kiebler, 2011; Kanai, Dohmae, & Hirokawa, 2004).
Transport RBPs are mediators of subcellular RNA delivery. Such RBPs include zipcode binding protein (ZBP), Staufen, and heterogeneous nuclear ribonucleoprotein (hnRNP) A2 (Figure 3; Gao et al., 2008; Muslimov, Patel, Rose, & Tiedge, 2011; Shan, Munro, Barbarese, Carson, & Smith, 2003; Tang, Meulemans, Vazquez, Colaco, & Schuman, 2001; Zhang et al., 2001). Several neuronal mRNAs, including those encoding CaMKIIα, Arc, MAP2, and neurogranin, are delivered to dendrites via the hnRNP A2 pathway (which also mediates targeting of myelin basic protein mRNA in oligodendrocytes; Gao et al., 2008; Hoek, Kidd, Carson, & Smith, 1998; Shan et al., 2003; Tübing et al., 2010).
Figure 3. RNA transport in dendrites.
The hnRNPA2 and ZBP pathways are two major RNA targeting mechanisms in neurons. RNA DTEs are recognized by transport RBPs to form RNPs. RNP interactions with molecular motors may require participation of additional adapter proteins. ZBP interacts with the β-actin mRNA zipcode while hnRNP A2 interacts with various RNA DTEs, including those in BC RNAs and MAP 2 mRNA.
Inset: hnRNP A2 interacts with the GA-motif DTE in the 5′ stem-loop domain of BC1 RNA. A second transport factor, which remains currently unidentified (Protein X, represented in a hypothetical rendering), is likely participating in dendritic BC RNA transport via DTE interactions (Muslimov et al., 2011).
The double-stranded 5′ stem-loop domains of BC1 and BC200 RNAs contain similarly structured DTEs (Iacoangeli & Tiedge, 2013; Muslimov et al., 2006, 2011). BC RNA DTEs feature architectural motif components that underlie their dendritic targeting competence. In rodent BC1 RNA, the 5′ stem-loop domain harbors an apical GA motif (Muslimov et al., 2006). GA motifs are constructed around noncanonical (non-Watson-Crick) purine•purine nucleotide pairings, including in particular two G•A/A•G pairs arranged in tandem (Iacoangeli & Tiedge, 2013; Tiedge, 2006). These tandem pairs engage in the trans-Hoogsteen/sugar edge format of hydrogen bonding (Iacoangeli & Tiedge, 2013; Lescoute, Leontis, Massire, & Westhof, 2005). They, together with an additional non-Watson-Crick A•A pair, constitute the noncanonical core of the BC1 GA motif. This core is flanked on both sides by several G = C standard Watson-Crick base pairs, affording the motif with high structural stability (Muslimov et al., 2006).
The BC1 GA motif interacts with transport factor hnRNP A2 which is required for the delivery of BC1 RNA to synapto-dendritic domains (Muslimov et al., 2011). The motif is indispensable for dendritic targeting as conversion of its noncanonical purine•purine core to standard WC pairings abolishes hnRNP A2 binding and distal dendritic delivery (Muslimov et al., 2006, 2011). In addition, a GA motif-adjacent unpaired U residue at position 22 (U22) is requisite for dendritic targeting although it is not interacting with hnRNP A2 (Muslimov et al., 2006, 2011). Thus, other transport factors, in addition to hnRNP A2, may participate in BC RNA dendritic delivery.
Primate BC200 RNA, interacting with hnRNP A2 and being delivered to dendrites (Muslimov et al., 2011), copies the BC1 RNA DTE structural arrangements. A double-stranded 5′ stem-loop domain harbors a bipartite GA motif with noncanonical cores featuring A•A and tandem G•A/A•G pairings (Skryabin et al., 1998). As in BC1 RNA, these cores are flanked by standard Watson-Crick G = C pairs and a motif-adjacent unpaired U residue. The architectural attributes of BC1 and BC200 5′ motif structures are thus remarkably similar, and future work will provide further insight into the question of how interactions with RNA transport factors and dendritic delivery competence are encoded by BC RNA architectural motifs.
Recent work with transgenic mouse lines expressing mutant BC1 RNAs has confirmed a 5′ DTE requirement for dendritic localization (Robeck, Skryabin, Rozhdestvensky, Skryabin, & Brosius, 2016). However, apparent methodological insufficiencies are cause for concern (see accompanying technical comment, Sci. Rep. 6, 28300, 2016, published online). For example, the use of 35S-labeled probes with thick (30 μm) tissue sections is problematic as 50% of the emitted beta particles decompose within 25 μm (Bicknese, Shahrokh, Shohet, & Verkman, 1992), causing arbitrary variations in the spatial distribution of labeling intensities as a result of differential-absorption quenching artifacts (Smolen & Beaston-Wimmer, 1990).
Similar to BC RNA GA motifs, CGG-repeat RNA forms stable stem-loops that interact with hnRNP A2 (Sofola et al., 2007; Swanson & Orr, 2007). GA-motif BC RNA DTEs and CGG-repeat stem-loops both feature noncanonical purine•purine base pairing (G•G in the latter case) (Muslimov et al., 2006; Napierala, Michalowski, de Mezer, & Krzyzosiak, 2005; Sobczak et al., 2010; Tiedge, 2006; Zumwalt, Ludwig, Hagerman, & Dieckmann, 2007). GA targeting motifs and CGG-repeat stem-loops compete with each other for binding to hnRNP A2, causing reduced dendritic delivery of BC1 RNA in the presence of expanded CGG-repeat RNA (Muslimov et al., 2011). These observations are relevant with respect to the fragile X premutation disorder which is caused by CGG repeat expansion in the 5′ UTR of the FMR1 gene (Hagerman, 2013).
BC RNA genes originated through retroposition (Kim, Martignetti, Shen, Brosius, & Deininger, 1994; Martignetti & Brosius, 1993a). Gene duplication and retroposition are two major but distinct mechanisms of genomic diversification in eukaryotes. Whereas genes encoding mRNAs (and thus proteins) are frequently dispersed in the genome by gene duplication, genes encoding non-protein-coding (including regulatory) RNAs are often dispersed by retroposition (Brosius, 1991, 2005; Cordaux & Batzer, 2009; Herbert, 2004; Kazazian, 2004). In the retroposition mechanism, cellular transcripts are reverse transcribed and inserted into the genome as retroposons, often in high copy numbers (Cordaux & Batzer, 2009; Iacoangeli & Tiedge, 2013; Kazazian, 2004;. Non-protein-coding RNA genes are frequently transcribed by RNA polymerase III (Pol III). Because such genes—in contrast to Pol II transcribed protein-coding genes—carry promoters within their RNA-coding regions, a retroposon derived form a Pol III transcript may acquire the status of a transcriptionally competent new gene (Iacoangeli & Tiedge, 2013).
The retroposition mechanism has been an innovative force in the shaping and remodeling of eukaryotic genomes. About 45% of the human genome has been retroposition-generated (Herbert, 2004), and over two thirds of the genomic content has been generated by this and other RNA-to-DNA conversion mechanisms (Brosius, 1999; De Koning, Gu, Castoe, Batzer, & Pollock, 2011). In stark contrast, protein-coding genes make up little more than 1% of the human genome (Taft, Pheasant, & Mattick, 2007).
About three quarters of the human genome are transcribed into RNAs (Djebali et al., 2012), most of them likely with regulatory functions. The need for RNA regulators has apparently increased significantly during mammalian phylogenetic development as a result of growing organismal complexities (Mattick, 2003; Taft et al., 2007). The evolution of such RNAs is rapid and ongoing, and evolutionary constraints are often such that it is more likely higher-order than primary structure that is under selective pressure in phylogenetic RNA development (Noller, 2005; Leontis, Lescoute, & Westhof, 2006; Pang, Frith, & Mattick, 2006) Such constraints may include motif recognition by RBPs which is often mediated by RNA 3D structure rather than sequence (Grandin, 2010; Lescoute et al., 2005; Noller, 2005).
Also by retroposition, BC1 RNA has served as a master gene for ID element amplification and dissemination in rodent genomes (Kim et al., 1994). ID elements are identical or similar to the 5′ BC1 domain, including the cis-acting DTE (Kim et al., 1994; Muslimov et al., 1997). Thus, an ID element, retroposed into a host mRNA gene and transcribed in neurons, may confer dendritic targeting competence to that mRNA (Buckley et al., 2011). The retroposition mechanism has thus helped disseminate elements with DTE potential in rodent genomes during phylogenetic development (Iacoangeli & Tiedge, 2013; Muslimov et al., 2014).
ID elements have been identified in introns of cytoplasmic transcripts to which they confer dendritic targeting competence (Buckley et al., 2011). They are also contained in a number of neuronal mRNAs that are targeted to dendrites in an activity-dependent manner (Muslimov et al., 2014). This mechanism relies on high-affinity interactions of noncanonical RNA motif structures with hnRNP A2. The ID element 4 subtype (ID4) interacts with hnRNP A2 in a Ca2+ concentration range of 50 nM to 5 μM, and the binding affinity peaks with a KD = 200 pM at 500 nM Ca2+. mRNAs that carry ID-type noncanonical motif DTEs are delivered to dendrites upon β-adrenergic receptor activation which, in turn, causes influx of Ca2+ through voltage-dependent calcium channels (Muslimov et al., 2014). Intracellular Ca2+ waves can be triggered by transient rises in postsynaptic [Ca2+]i, reaching dendritic [Ca2+]i amplitudes of 1 μM or more (Berridge, Lipp, & Bootman, 2000; Grienberger & Konnerth, 2012; Ross, 2012). Once such a Ca2+ transient has reached a local threshold of 500 nM, it can cause a switch to the high-affinity conformation of the ID targeting element, enabling binding of hnRNP A2 and dendritic delivery (Muslimov et al., 2014). The activity-dependent ID dendritic delivery mechanism allows neurons to supply synapto-dendritic domains with select RNAs on demand.
In conclusion, dendritic RNA targeting is an essential prerequisite for the locally controlled, activity-dependent synthesis of proteins at the synapse. Impaired neuronal RNA transport mechanisms, increasing evidence indicates, may be contributing to physiological and cognitive dysfunction in neurological disorders.
In-depth understanding of molecular-cellular mechanisms that regulate dendritic RNA transport and local protein synthesis is critically important in dissecting how dysregulation of such mechanisms can cause disease. We anticipate that future work will elucidate mechanisms of (i) synapse-to-soma or synapse-to-nucleus signaling, (ii) proximal vs. distal dendritic RNA delivery, (iii) local transition from microtubule-based dendritic delivery to actin-based distribution and docking in postsynaptic microdomains, (iv) local regulation of protein synthesis in response to stimulation or injury, during development or in disease, and (v) local translational control in synaptic plasticity, learning, memory, and cognition.
Recent work has shown that translational dysregulation may underlie human cognitive and behavioral dysfunction (Darnell, 2011). A properly maintained excitation-inhibition balance is a hallmark of neuronal function as any skewing of such balance beyond physiological ranges may cause hypo- or hyperexcitability. Dysregulation of translation initiation causes ASD-like phenotypes in mouse models (Aguilar-Valles et al., 2015; Gkogkas et al., 2013; Santini et al., 2013). Recent genome-wide analyses reveal aberrant expression of non-protein-coding RNAs in ASD patients (Parikshak et al., 2016; Ziats & Rennert, 2013). BC1 KO mice exhibit ASD-like behavioral manifestations (Iacoangeli et al., 2017). Undoubtedly, future advances in the area of translational regulation in neurons will improve our understanding of how translational dysregulation contributes to neurological and cognitive disorders.
We thank the members of the Robert F. Furchgott Center for advice and discussion. Work in the authors' laboratory was in part supported by NIH grants NS046769 and DA026110 (to H.T.).