The First World War Ended the European Domination of the World Essay Example Essay Example

The First World War Ended the European Domination of the World Essay Example Paper The First World War Ended the European Domination of the World Essay Introduction The First World War in the beginning of the twentieth century is remembered as horrific and the savagery of mankind. It was the clash between the powers and the show off stage for their national pride. It was truly a struggle for dominance and delivered nothing but destruction and their downfall. The First World War surely ended the European domination of the world. It was a fair cycle of history that took away the baton of prolonged European imperialism to other rising regions such as Asia and America. It was surely the war that caused European downfall in economy, political unstableness, and change in their tension toward internal and external neighboring borders. The war was a controversial warfare in an immense scale that had no differentiation in both military combatants and civilians. This type warfare also known as the â€Å"total war† had brought European economies in demolition due to the over concentration on military spending. Politically, rise of republican governm ents along with new socialist ideas and the decline of monarchist system which was a strong centralized power contributed in the part of the shift in power. The First World War Ended the European Domination of the World Essay Body Paragraphs The war provided the foothold for newly industrialized states: the United States and Japan to join the race along with the European powers. Aftermath of the war gave opportunity to the worldwide independent movements especially in India and Arabian nations once firmly controlled by mighty British regime. Also, Chinese May 4th movement which was cultural and socio political reform proved the once European dominated regions were no longer in the hands of their influence. The war that crudely consumed colonies’ rights and possessions not only brought the resistance but heightened the nationalistic ideas within the colonized states. The process of European downfall in their dominance of the world originated within their inevitable competence. Vigorously outstretched European imperialism between the late 19th century and the First World War brought overwhelming power to the European states. Rivalry was impossible to be avoided which then divided Europe into major alliance systems i n attempts to balance each other’s power. This system in Europe created tension that changed their worldly interest into their close borders. Thus, the First World War was the event that ceased the European egocentric behavior followed by the shift of powers and their gradual falls in their dominance of the world. The First World War tremendously changed the balance of world powers. The war brought nothing but destruction to the entire European nations and inevitably brought economical downfall in Europe. This demolition gave rise to the other regional powers: the United States of America and Imperial Japan. During the war, each European power used the slogan of ‘total war† which was the mobilization of all their available resources, in order to render beyond use their rival’s capacity for resistance. The total war mostly occurred in the land of Europe and it was truly a severe damage to the European 19th century’s prosperity. The total war expenditu re by the European participators was listed as 190 billion dollars including the other indirect spending which was approximately 160 billion dollars. This was close to thirty percent of the European countries’ national wealth. Within the war period, accumulated European wealth that was built on through a long imperialism which maintained European dominance in the world was wastefully evaporated. Also, the post war wound was prevalent throughout the European continent. Industrialized German and French cities fell into ruins and Britain who was less damaged in their homeland also lost about 7,759,000 tons of merchant values throughout the war. Thoroughly, the European economies wrecked each other while passing their capital to an emerging America. By contrast, The United States whose exports were not even close to the half of British market in the late 19th century ascended greater than any other countries in the world. The First World War constructed America into a frontier of exporting weapons and provisions that promptly grasped the country into the position as the major power. Also, debenture bonds were enormously issued to European states by American government to heal the post war wounds. This event led American industries and agriculture into the most dominating structure in the world. From this economical gain, American government could construct powerful military organization that eventually influenced the Pacific, Asia and European regions. Similarly, Imperial Japanese exports were prevalent in the war where it blasted Japanese economy. The war founded the foothold for Japan to implement their imperialistic dreams in Asia. Backboned by these economical achievements, Japan was promptly able to develop its military systems. Whilst European powers turned its eyes to the continent of Europe, Japanese expansion in Asia was flourished. For example, Japanese military seized German settlements in Tsingtao China and German pacific colonies such as Marian a and Caroline in the year of 1914. Thus, another European power that dominated the region fell on the grip of a new emerging power in Asia. Thus, the First World War donated non-European powers such as America and Japan opportunities to emerge as the next influence of power. Moreover, political effect by the First World War was also as great as economical downfall in Europe. Imperialistic governments once dominated the world were fallen apart by the end of the war. Revolution in Russia caused Russian Empire to be torn down and replaced with a socialist styled government that killed millions of people in the process. Austro-Hungarian Empire, the representation of old Holy Roman Empire was no longer exists. Also, the maps of Eastern Europe and the Middle East were redrawn causing minorities to limit power of the pre-existed empires. When European Imperialism began to fall behind, the most popular type of government to gain influence was the republic. Before the war, Europe contained 19 monarchies and 3 republics, yet only a few years afterward, had 13 monarchies, 14 republics and 2 regencies. Evidently, revolution was in the air and people began to more ardently express their desires for a better way of life. Also, Socialism erupted as a major political force mainly through Russia. The introduction of Socialistic ideas which emphasized peace: â€Å"a strong desire to avoid conflicts and loss,† and â€Å"all workers united† began to implant the humanistic terms into the minds of Europeans which limit the idea of Imperialism throughout the world. Thus, the societies of European nations changed by their class systems altered the lower class consciousness to be evolved as well as forging a non-imperial identity. This political turmoil during the post war, Europe was mostly engaged in their domestic issues that paid lesser attention to rest of the world. While The First World War caused Europe to be engaged in their economical and political reorganizat ion, the European colonized states sought the opportunity to regain their oppressed identities. Additionally, The Fourteen Points that was delivered by the United States President Woodrow Wilson’s speech at a joint session of congress in 1918 greatly influenced the independence movement throughout the world. Basically, the speech emphasized n the â€Å"national self-reliance† which constructed a foundation that annihilated the 19th century styled European world order. Fall of Ottoman Empire once vastly controlled the Middle Eastern region introduced new nationalistic movements. New Turkish and Iranian nationalists ceased the prolonged Islamic activities and sought their national identity and pride that caused resistance to European influences. Mustafa Kemal Ataturk, the founder of Republic of Turkey resisted the European influence by beating back the British and French supported Greek invasion. In the case of India, British government anxiously persuaded the Indian sta tes to be part of the war in return for their independency. However, the promise was eventually declined by Britain that provoked Hindu nationalists and their provincial governments to be engaged in anti-British movement. Many Indian resistances were significantly prevalent throughout the region. â€Å"Salt March† organized by Gandhi was the important part of the Indian independence movements. It was a non-violent protest that triggered the wider â€Å"Civil Disobedience Movement. The movement encouraged Indian people for taking disobedience action toward the colonial rule. This event inspired many other nations and minorities who were under the oppressors. Also, sphere of influence by the European powers on China was no longer stayed as a presiding effect when the war ended. â€Å"May Fourth Movement,† often referred to as â€Å"New Cultural Movement† in China was anti-imperialist, cultural, and political movement that triggered Chinese nationalism. Similar to Indian independence movement, the movement was originated when the European powers did not vow to acknowledge the Chinese effort in the war. From this incident, Chinese were led to create a new strong nationalist party that resisted all of imperialistic terms that were forced by the European powers and Japan. Weakened European influence throughout the world due to the First World War caused political organization of once oppressed states. The war surely ended the sole of European domination of the world. The First World War brought changes in the order of the world. Prolonged European dominated world was surely a different place when the war ended. Over confidence from European economical, political, and military astonishments plunged Europe into rivalry states to each other that led them into a disaster. Also, total war wastefully used up almost half of the European national wealth that was accumulated during 19th century when European powers were at its climax of prosperity. Thes e expenses seriously damaged the European economy which meant declination of their power in the world and the rise to non-European states: the United States of America and Imperial Japan. These two countries profited enormously by exporting weapons and agricultural products during the war. On the other hand, harsh regulations implemented on colonies during the war, by the nature of total war, gave permanent reasons for nationalistic movements to occur throughout the world. It was independence movements that were against the idea of Imperial power in search for their identity. As well as the economic downfall, political instability along with the fall of European empires was another leading source of Europe’s losing its power. Emergence of Socialism and the creation of Republican states brought Europe into turmoil. All of these events took place by the end of the war that forced Europe to donate the baton of power to non-European emerging states: the United States of America a nd Imperial Japan. Thoroughly, the First World War affected European economy and its politics that surely closed the curtain on European domination of the world. We will write a custom essay sample on The First World War Ended the European Domination of the World Essay Example specifically for you for only $16.38 $13.9/page Order now We will write a custom essay sample on The First World War Ended the European Domination of the World Essay Example specifically for you FOR ONLY $16.38 $13.9/page Hire Writer We will write a custom essay sample on The First World War Ended the European Domination of the World Essay Example specifically for you FOR ONLY $16.38 $13.9/page Hire Writer

The Ben Franklin Essay Sample

The Ben Franklin Essay SampleThe Ben Franklin essay sample is a great one, but there are some things you should know about it before you begin to work with it. There are many ways to get your essay published, but there are a few rules that you will want to follow when you are creating your own.Your essay will go through many different phases throughout the entire process of publishing it. The first phase is editorial editing. This stage will involve editors reviewing and rewriting the essay for grammar and punctuation errors. These mistakes can take away from the essay's overall quality, so be sure to hire a professional editor to keep your essay in top shape.After the editors get through with the main parts of the writing, they will then begin revising and editing your thesis statement. Your thesis statement is the section of the essay that outlines the main points you want to make in your essay. It is important that you clearly define and demonstrate what the essay is trying to say and then make sure to cite supporting evidence and references in this part of the essay.The next phase of the writing process is the copy-editing phase. Here you will use the copy editing process to remove and replace factual errors or grammatical mistakes you have found throughout the entire writing process. You may also need to delete and replace several sentences to fit the tone of the essay. Once the copy editing process is completed, you will go back through the essay to proofread it for any further mistakes.Finally, you will need to submit your finished essay to an editor. Once the proofreading and revision processes are complete, the editor will review the final draft of the essay.While this process may seem tedious and time consuming, it is necessary for the essay to be edited and submitted to an editor. The reason that editors will edit your essay is to make sure that it is properly written and consistent. By following the above steps, you will ensure that your essay is co rrectly formatted and will be a great success.Before submitting your essay to an editor, there are a few things you should do first. The first step is to have a copy of your essay professionally proofread by a professional. Proofreading will not only ensure that the essay is correct, but it will also give you a better idea of how well the essay flows smoothly throughout the entire process.The Ben Franklin essay sample will tell you that the best way to create an effective essay is to follow a pattern of writing every day. The writer must be able to create a pattern for his or her piece. The structure of a good essay is crucial, and this is why you need to have a schedule that you follow every day so that you can get your work done.

Project Definition essays

Project Definition essays Located at the heart of Beulah Bog County and established in 1990, Exodus Twp. currently has a population of three hundred. The township is faced with the problem of enticing businesses and residents to their locale. Previous residents and businesses vacated their homes and store fronts due to high taxes and crime rates. Since the current Administration has been in office the crime rates have dropped to acceptable levels and the operating budget has been streamlined in addition to being balanced enabling the Trustees to reduce taxes. The Board of Trustees, Perkins, Hardy and Markham, want to reach prospective businesses and residents through the Internet, but do not have a web page for the township. With the absence of any Intra/Internet in place, the new Trustees cannot efficiently develop or support a web page. Once this web page is implemented, prospective residents and businesses can make contact with the Trustees and voice their concerns and opinions of current policies and prac tices. They will also have twenty-four hours access to their representatives and services. The objective of the township is to have this web page operational by the beginning of the next physical year, November 2002. The Township Trustees are setting aside $10,000 dollars for the development of the web page. Once the web page has been designed and implemented the townships Data-Processing Unit will be responsible for the maintenance. The township is faced with the risks of this project not being completed within set budget limits and time frame. They are also faced with the possibility that the web page will not draw the potential businesses and residents to their location. The purpose of the Exodus Township web-page is to attract people to the fine living available in their community. It should also attract businesses as well. ...

Swordfish Facts

Swordfish Facts Swordfish (Xiphias gladius) was made famous in the late 1990s by Sebastian Jungers book The Perfect Storm, which was about a swordfishing boat lost at sea. The book was later made into a movie. Swordfishing captain and author Linda Greenlaw also popularized swordfishing in her book The Hungry Ocean. Swordfish is a popular seafood that may be served as steaks and sashimi. Swordfish populations in U.S. waters are said to be rebounding after heavy management on a fishery that once overfished swordfish and also resulted in a large bycatch of  sea turtles. Swordfish Identification These large fish, which are also known as the broadbill or broadbill swordfish, have a distinctive pointed, sword-like upper jaw that is over 2 feet long. This sword, which has a flattened oval shape, is used to stab prey. Their genus  Xiphias comes from the Greek word xiphos, which means sword. Swordfish have a brownish-black back and light underside. They have a tall first dorsal fin and distinctly forked tail. They can grow to a maximum length of over 14 feet and weight of 1,400 pounds. Females are larger than males. While young swordfish have spines and small teeth, adults do not have scales nor teeth. They are among the fastest fish in the ocean and are capable of speeds of 60 mph when leaping. Classification Kingdom: AnimaliaPhylum: ChordataSubphylum: VertebrataSuperclass: GnathostomaSuperclass: PiscesClass: ActinopterygiiOrder: PerciformesFamily: XiphiidaeGenus: XiphiasSpecies: gladius Habitat and Distribution Swordfish are found in tropical and temperate waters in the Atlantic, Pacific and Indian Oceans between the latitudes of 60 °N to 45 °S. These animals migrate to cooler waters in the summer, and to warmer waters in the winter. Swordfish may be seen at the surface and in deeper waters. They can swim in deep, cold parts of the ocean due to specialized tissue in their head that warms their brain. Feeding Swordfish feed primarily on small bony fish and cephalopods. They opportunistically feed throughout the water column, taking prey at the surface, in the middle of the water column and at the ocean bottom. They may use their sails to herd fish. Swordfish appear to swallow smaller prey whole, while larger prey is slashed with the sword. Reproduction Reproduction occurs by spawning, with males and females releasing sperm and eggs into the water near the ocean surface. A female may release millions of eggs, which are then fertilized in the water by a males sperm. The timing of spawning in swordfish depends upon where they live - it may either be year-round (in warmer waters) or during the summer (in cooler waters). The young are about .16 inch long when they hatch, and their upper jaw becomes more noticeably longer when the larvae are about .5 inch long. The young dont begin to develop the sailfishs characteristic elongated jaw until they are about 1/4 inch long. The dorsal fin in young swordfish stretches the length of the fishs body and eventually develops into a large first dorsal fin and a second smaller dorsal fin. Swordfish are estimated to reach maturity at 5 years and have a lifespan of about 15 years. Conservation Swordfish are caught by both commercial and recreational fishermen, and fisheries exist in the Atlantic, Pacific, and Indian Oceans. They are a popular game fish and seafood, although mothers, pregnant women, and young children may want to limit consumption due to the potential for a high methylmercury content. Swordfish are listed as of least concern on the IUCN Red List, as many swordfish stocks (except for those in the Mediterranean Sea) are stable, rebuilding, and/or being adequately managed. Sources Arkive. Swordfish. Accessed July 31, 2012.Bailly, N. (2012). Xiphias gladius. In: Nicolas Bailly (2012). FishBase. Accessed through: World Register of Marine Species on 2012-07-31 on July 31, 2012.Collette, B., Acero, A., Amorim, A.F., Bizsel, K., Boustany, A., Canales Ramirez, C., Cardenas, G., Carpenter, K.E., de Oliveira Leite Jr., N., Di Natale, A., Die, D., Fox, W., Fredou, F.L., Graves, J., Guzman-Mora, A., Viera Hazin, F.H., Hinton, M., Juan Jorda, M., Minte Vera, C., Miyabe, N., Montano Cruz, R., Masuti, E., Nelson, R., Oxenford, H., Restrepo, V., Salas, E., Schaefer, K., Schratwieser, J., Serra, R., Sun, C., Teixeira Lessa, R.P., Pires Ferreira Travassos, P.E., Uozumi, Y. Yanez, E. 2011. Xiphias gladius. In: IUCN 2012. IUCN Red List of Threatened Species. Version 2012.1. . Accessed July 31, 2012.FishBase. Xiphia gladius. Accessed July 31, 2012.Gardieff, Susie. Swordfish. FLMNH Icthyology Department. Accessed November 9, 2015.Gloucester Times. The Perfect Storm: The History of the Andrea Gail. Accessed July 31, 2012.

HealthWise Requirement Analysis Case Study Example | Topics and Well Written Essays - 2000 words

HealthWise Requirement Analysis - Case Study Example Ltd. to conducts a requirement analysis to establish the needs and requirements of the proposed Information System. Requirement analysis is basic software development activity. The purpose of this phase is to outline business needs based on user interviews, study of existing system of information conducted in the client environment and through other information gathering techniques. Requirement analysis provides the foundation for a solid application development. It is a technical document prepared and finalized by a requirement analyst, which provides user specific details of the proposed system to help software engineers in the development of actual application. It contains several functional and non-functional requirement, user analysis and details of required forms and reports that are deemed essential to fulfill operational needs of the organization and the implementation of the system in client’s environment. Grady (2006, p. 4) states the detail of this process in these words, â€Å"System requirement analysis is a structured or organized methodology for identifying an appropriate set of resources to satisfy a system need and the requirement for those resources that provide a sound basis for the design or selection of those resources.† 2. ... Secondly, the HealthWise is rapidly gaining customers’ confidence and the management can foresee a major expansion of the business network around the globe in the near future. This expansion will not only require a streamlined information system in place but it should also be able to accommodate a fair amount of information supposed to be generated with the business growth. Therefore, HealthWise new Information System (HWIS) is required to be design with this future expansion in mind and should be reasonably sizeable to support the functions of a medium sized enterprise. Keeping in view the problems with the current paper-based system a web-based management information system is proposed by the Technologies & Solutions Pvt. Ltd. The system will facilitate decision-making process through its predefined and custom queries and reports. The system will store all information related to distributor, customers, sales and payments in a database. The system will accept input in structu red web forms and will provide on the fly reports pertaining information about distributors, sales volume in dollars, distributor groups and relative performances etc. HWIS will store and present information on all six product lines in an efficient manner. Moreover, HWIS will also accept direct purchase orders from the customer and will provide support to HealthWise staff and distributors to log into the system remotely through Virtual Private Network (VPN) connections to access web-based system. HWIS will provide web-based access to its database on verification of authorized login credential in an efficient and timely manner. 3. Design Specifications The system will be designed to support all business processes

Civil and Criminal Punishments Assignment Example | Topics and Well Written Essays - 500 words

Civil and Criminal Punishments - Assignment Example The paper will also explain the differences between civil penalties and criminal punishments, explaining whether punishments between a case tried at criminal level and civil level should correlate. A civil court is one that deals with cases related to disputes between organizations or individuals. Civil courts award victims with compensation. Civil courts deal with civil or common wrongs that are not criminal in nature such as disputes relating to credit card payments, allegations of breach of contract, compensations disagreements between tenants and landlords (Scaros, 2004). According to Currier & Eimermann (2009), lawsuits are usually filed in civil courts by organizations or individuals who believe they have been physically and financially hurt by another organization or individual. The types of remedies available in civil courts depend on several factors such as the type of claims made, the authority of the court, the defendant’s conduct and the harm that has been suffered or may be suffered. The remedies in civil courts usually depend on the strengths of one’s case and the opponent’s case, one’s resourcefulness and ability to present facts and the ability of the attorneys of both sides to represent their clients as required (Scaros, 2004). The types of remedies available in civil courts include: This is normally the usual and standard remedy for damages in civil courts. They are the payments awarded by a civil court, paid as compensation for injury or loss (Currier & Eimermann, 2009). Money damages usually consist of nominal damages, attorney’s fees, interest, punitive damages and compensatory damages. They are usually intended to settle the grief of the aggrieved party in civil cases (Powell, 1993). These are usually given so as to stop certain conducts of the accused. The civil court orders or prohibits against conditions or acts that have been requested. The orders given are called injunctions and they require one to either do

Glutamate Transporter Excitatory Amino Acid Experiment

Glutamate Transporter Excitatory Amino Acid Experiment Abstract N-(2-18F-Fluoropropionyl)-L-glutamate(18F-FPGLU) is a potential amino acid tracer for tumor imaging with positron emission tomography (PET). In this study, the  relationship between glutamate transporter excitatory amino acid carrier 1 (EAAC1) expression and 18F-FPGLU uptake in rat C6 glioma cells line and human SPC-A-1 lung adenocarcinoma cells line was investigated. The uptake of 18F-FPGLU in C6 cells increased significantly after induced by ATRA for 24, 48, and 72 h, which was closely related to expression of EAAC1 in C6 cells (R=0.939). Compared with the SPC-A-1(NT) control cells, the uptake of 18F-FPGLU on EAAC1 knock-down SPC-A-1(shRNA) cells significantly decreased to 64.0%. In the PET imaging of 18F-FPGLU of SPC-A-1 and EAAC1 knock-down SPC-A-1(shRNA)-bearing mice models, the uptake of 18F-FPGLU in SPC-A-1(shRNA) xenografts was significantly lower than that in SPC-A-1 xenografts, with Tumor/Muscle ratio of 1.67  ± 0.1 vs. 3.01  ± 0.3 at 60 min post-injection. The result s suggest that transport mechanism of 18F-FPGLU in glioma C6 and lung adenocarcinoma SPC-A-1 cells lines mainly involves in glutamate transporter EAAC1, which is an important transporter of 18F-FPGLU in tumor cells and may be a novel hallmark of tumor glutamate metabolism PET imaging. Keywords: N-(2-18F-fluoropropionyl)-L-glutamate; tumor imaging; glutamate transporter; excitatory amino acid carrier 1 Introduction As the most commonly used positron emission tomography (PET) tracer for tumor diagnosis, 18F-fluoro-2-deoxy-D-glucose (18F-FDG) also has certain false negative and false positive results(Shreve et al. 1999; Fletcher et al. 2008). It has been reported that 18F-FDG negative tumors may use a different metabolic pathway called glutaminolysis(DeBerardinis et al. 2007; Ward et al. 2012). Glutamine and glutamate play key roles in the adapted intermediary metabolism of tumors(Gao et al. 2009; Rajagopalan et al. 2011; Shanware et al. 2011). Several 18F-labeled glutamic acid and 18F-labeled glutamine have been used for metabolic PET imaging of tumor in humans (Baek et al. 2013; Venneti et al. 2015). High uptake of these amino acid tracers in tumor cells is likely related to the increased expression of amino acid transporters. For example, the upregulated system ASC, especially ASCT2 might contributed to the uptake of 18F-labeled (2S,4R)-4-fluoro-L-glutamine(Ploessl et al. 2012), and 18F-fluoro glutamic acid (BAY 85-8050) transport involved in Na+-dependent XAG- and Na+-independent XC- systems with XC- possibly playing a more dominant role, but both of them showed defluorination in vivo(Krasikova et al. 2011). 18F-labeled (4S)-4-(3-[18F]fluoropropyl)-L-glutamate (BAY 94-9392), another derivative of glutamic acid, whose transport was due mostly to upregulation of system XC-, a potential biomarker for tumor oxidative stressà ¯Ã‚ ¼Ã…’can be useful for detecting system XC- activity in vivo and is considered to be a potential tracer for tumor imaging(Koglin et al. 2011). Our recently developed novel N-18F-labeled glutamic acid, N-(2-[18F] fluoropropionyl)-L-glutamate (18F-FPGLU), seemed to be a potential amino acid PET tracer for tumor metabolic imaging, with high tumor-to-background contrast in several tumor-bearing mice models. Preliminary studies showed that 18F-FPGLU was primarily transported through Na+-dependent high-affinity glutamate transporter system XAG-(Hu et al. 2014), but the accurate transport mechanism is unknown. Glutamate transport system includes Na+-dependent excitatory glutamate transporter XAG- system and Na+-independent glutamate transporter XC- system(Avila-Chà ¡vez et al. 1997). System XC- (xCT) is overexpressed on tumor c ells and is a potential biomarker for tumor oxidative stress. As an important member of XAG- system, excitatory amino acid carrier 1 (EAAC1), also called excitatory amino acid transporter 3 (EAAT3), localizes to the post-synaptic structure of neurons and surrounding glial cells as regulator of excitatory neurotransmission, and also exists in peripheral tissues, perhaps as metabolic regulators(Bailey et al. 2011). The expression of EAAC1 was known to be regulated by several mechanisms that modify carrier abundance on the plasma membranes and was markedly induced by all tans-retinoic acid (ATRA) in rat C6 glioma cells, which led to strikingly stimulate amino acid influx(Bianchi et al. 2008). However, EAAC1 transporter may be a potential biomarker for tumor molecular imaging. It has not been reported so far. This study investigated the relationship between EAAC1 expression and 18F-FPGLU uptake in C6 rat glioma cells line and SPC-A-1 human lung adenocarcinoma. The uptake of 18F-FPGLU was assessed in ATRA-treated and untreated C6 cells lines, and also in shRNA-mediated EAAC1 knock-down SPC-A-1 cells and the non-targeted (NT) control cells in vitro. Further prospective researches of PET imaging of tumor-bearing mice models with C6, SPC-A-1 and EAAC1 knock-down SPC-A-1(shRNA) xenografts were performed to reveal the correlation between the uptake of 18F-FPGLU and the expression of EAAC1. Materials and methods Materials All reagents, unless otherwise specified, were of analytical grade and commercially available. All chemicals obtained commercially were used without further purification. Inveon small-animal PET/computed tomography (CT) scanner was purchased from Siemens (Germany). Synthesis of 18F-FPGLU The synthesis of 18F-FPGLU from 4-nitrophenyl-2-18F-fluoropropionate (18F-NFP) via a two-step reaction sequence has been described in detail by the earlier paper(Hu et al. 2014). Cell Culture and Animal Models The C6 rat glioma cells, SPC-A-1 human lung adenocarcinoma cells were obtained from Shanghai Institute of Cellular Biology of Chinese Academy of Sciences (Shanghai, China). The cells were cultured in culture flasks containing DMEM medium(for C6 cells) or RPMI 1640 medium (for SPC-A-1) supplemented with 10% FBS and 1% penicillin/streptomycin at 37oC in a humidified atmosphere of 5% CO2 and 95% air. 24 hours before the experiments in vitro, C6 cells lines or SPC-A-1 cell lines were trypsinized and 2105 cells per well were seeded into 24-well plates. All animal experimental studies were approved by the Institutional Animal Care and Utilization Committee (IACUU) of the First Affiliated Hospital, Sun Yat-Sen University (approval No.[2013]A-173). All efforts were made to minimize animal suffering, to reduce the number of animals used, and to use alternatives to in vivo techniques, if available. The nude mice were obtained from Laboratory Animal Center of the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). The C6, SPC-A-1 and EAAC1 knock-down SPC-A-1(shRNA) tumor models were made using previously described methods(Deng et al. 2011). Tumor cells (2-5-106) were injected subcutaneously and allowed to grow for 1 to 3 weeks. When the tumor reached 6-10 mm (diameter) micro P ET/CT scans were done. C6 induced by ATRA The rat glioma C6 cells were treated by all trans-retinoic acid (ATRA) 12 h after the passage. Culture medium was substituted with fresh medium (containing DMEM medium supplemented with 10% FBS) in the absence or in the present of ATRA at a concentration of 10 ÃŽ ¼M from a 10 mM stock solution in DMSO according to the literature16. After the treatment of ATRA for 24, 48 and 72 h, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to monitored the mRNA and protein expression levels of EAAC1 in ATRA treated C6 and non-treated C6 cells. Generation of shRNA-mediated EAAC1 knock-down cells. The method of generation of shRNA-mediated EAAC1 knock-down cells was similar to the literature(Youland et al. 2013). SPC-A-1 human lung adenocarcinoma cells was used for shRNA-mediated EAAC1 knock-down experiment. SPC-A-1 cells were transduced with lentivirus ecoding EAAC1-targeted short hairpin RNAs (shRNA). shRNA sequences were selected from human EAAC1 mRNA NM_004170 and the shRNA fragments were cloned in a lentivirus vector pGLV3 plasmid with the sequence 5-GCATTACCACAGGAGTCTTGG-3. A non-specific targeting (NT) shRNA for control was cloned in the same lenvirus plasmid backbone. Lentiviral packaging was performed with trans-lentiviral packaging mix in 293T cells according to the manufacturers instructions. SPC-A-1 cells were plated on 6-well plates at 2-105 cells per well. After 24 hours, medium was aspirated and replaced with 100 ÃŽ ¼L of virus-containing solution was added to each well and incubated at 37oC for 24 h. Cells were selected with puromycin and monitored for green fl uorescence protein (GFP) expression. The EAAC1 mRNA expression level was monitored by quantitative real-time polymerase chain reaction (qRT-PCR). The EAAC1 protein expression level was quantized by western blotting. qRT-PCR for the expression of EAAC1 Relative expression levels of EAAC1 mRNA in C6 and SPC-A-1 cells were calculated using the fluorescence quantitative real-time polymerase chain reaction (qRT-PCR) (Stratagene Mx3000P Real time PCR, Agilent). Total cellular RNA was isolated with the Rneasy mini Kit (TAKARA). 1 ÃŽ ¼g of RNA was synthesized to cDNA in a 20 ÃŽ ¼L reaction system with reverse transcriptase buffer, RT Enzyme Mix and primer MIX (Bestar qPCR RT kit, DBI). Conditions for reverse transcription were 5 min at 65oC, 5 min on ice, then 60 min at 37oC and 10 min at 98oC. Oligodeoxynucleotide primers of EAAC1 gene for PCR amplification was 5-AGTTCAGCAACACTGCCTGT-3 (forward) and (5-GTTGCACCAACGGGTA ACAC-3(reverse). PCR was programmed as follows: 2 min at 94oC, 20 s at 94oC, 20 s at 58oC à ¯Ã‚ ¼Ã…’ then 20 s at 72oC à ¯Ã‚ ¼Ã…’ for 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a initial control and each sample was amplified in triplicate. The relative expression of EAAC1 mRNA compared with GAPDH was calculated by comparative threshold method (2 -ΔΔCt ). Western blotting for EAAC1 Cells were lysed in a detergent-containing buffer with protease inhibitors for 20 min at 4oC. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) was used as a reference protein. After solubilization, cell lysates were collected and centrifuged at 14000 rpm for 10 min. The supernatants were transferred into new tubes, quantification of proteins was performed with Pierce BCA Protein Assay Kit (Thermo) and aliquots of 25 ÃŽ ¼g were loaded on an 10% gel for SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluorideà ¯Ã‚ ¼Ã‹â€ PVDFà ¯Ã‚ ¼Ã¢â‚¬ °membranes (Millipore) . The membranes with EAAC1 or GAPDH were departed at the middle position, and were blocked and incubated with deferent antibody, respectively. Non-specific binding sites were blocked with an incubation in Tris-buffer saline containing 5% of bovine serum albumin (BSA) for 1h at room temperature. Then the blots were exposed to EAAT3 antibody (rabbit monoclonal antiserum, 1:1000, Abcam) or anti-GAPDH rabbit monoclonal antibody(1:3000, Abcam) diluted in blocking solution for at 4oC overnight. After washing, the blots were exposed for1h at room temperature to goat anti-rabbit IgG HRP diluted 1:5000 in blocking solution. Cellular uptake of 18F-FPGLU Cells were plated in 24-well plates (2x105cells/well) and uptake studies were performed at 24 h after the passage. The cellular uptake of 18F-FPGLU studies was similar to the methods described previously(Krasikova et al. 2013). The medium was aspirated and the cells were washed 3 times with 1 mL warm PBS. 18F-FPGLU was dissolved in PBS solution and was added to each well (74-111 KBq/0.2 mL/well). After incubated with 18F-FPGLU at 37oC for 30 min, the radioactive medium was removed and the cells were washed 3 times with ice-cold PBS. Then, the cells were dissolved in 0.5 mL of 1 N NaOH and the activity was measured by ÃŽ ³ counter (GC-1200, USTC Chuangxin Co. Ltd. Zonkia Branch, China). The cell lysate (25ÃŽ ¼L) was used for determination of protein concentration by BCA protein assay. The uptake data are based on the amount of activity added to each well and the total amount of protein in each well. Each experiment was done in triplicate, averaged and was repeated 5 times on different days. The uptake of 18F-FPGLU was assessed on the ATRA-treated or untreated C6 cells, and on EAAC1 knock-down SPC-A-1(shRNA) cells or SPC-A-1(NT) control cells. The relative uptake ratios were calculated compared to the control cells. Small-animal PET-CT imaging Small-animal PET-CT imaging studies with tumor-bearing mice were carried out using the Inveon small-animal PET/CT scanner (Siemens). 3.7-7.4 MBq of 18F-FPGLU were injected intravenously in conscious animals via the tail vein. The mice were anesthetized with 5% chloral hydrate solution (6 mL/kg) and were kept warm throughout the procedure. Imaging started with a low-dose CT scan, immediately followed by a PET scan. PET images were acquired at 30, 60, 90, 120 min post-injection. For a comparative study, mice were kept fasting for 4 h and were anesthetized with 5% chloral hydrate solution (6 mL/kg) and imaged with 18F-FDG (3.7 MBq) at 60 min after intravenous injection. The images were reconstructed by two-dimensional ordered-subsets expectation maximum (OSEM). For each small-animal PET scan, ROIs were drawn over the tumor and muscle of the thigh on decay-corrected whole-body coronal images using Inevon Research Workplace 4.1 software. The quantification was performed according the meth ods described previously(Hu et al. 2014). Radioactivity concentration within a tumor or other tissue was converted to MBq/g and then divided by the administered activity to obtain an imaging ROI-derived percentage of injected dose per gram of tissue (% ID/g). Then, the ttumor/muscle (T/M) and tumor/brain (T/B) uptake ratios were calculated, respectively. Immunohistochemistry Expression of EAAC1 was assessed by immunohistochemistry on formalin-fixed paraffin embedded rat brain tissues and C6 xenograft samples. Immunohistochemistry experiments were carried out according to the literature(Wang et al. 2013). Normal rat brain tissues and C6 glioma tissues were fixed in 10% neutral buffered formalin overnight at room temperature. Tissues were then dehydrated, embedded in paraffin, and cut into 3-ÃŽ ¼m sections. After antigen retrieval, tissue sections were subject to immunohistochemical incubated with antibodies against EAAC1(Abcam), DAB was stained before mounted onto microscope slides. Tissues were analyzed with a Nikon E800M microscope. Statistical analyses Data were expressed as mean+/-SD. Statistical analysis was performed with SPSS software, version 16.0 (SPSS Inc.), for Windows (Microsoft). Student t test was used to assess differences in the magnitudes of samples from two measurements. A P values of less than 0.05 was considered to indicate statistical significant. A scatter plot was drawn with the relative mRNA expression and the relative uptake of 18F-FPGLU in C6 cells treated with ATRA for 24h, 48h, 72h. Spearman correlation analysis and a linear regression analysis was performed between them. Results EAAC1 expression and 18F-FPGLU uptake in C6 cells induced by ATRA The EAAC1 mRNA relative expression levels in ATRA-treated C6 cells assessed by quantitative real-time polymerase chain reaction (qRT-PCR) are shown by Figure 1A. Compared with the untreated C6 cells, the EAAC1 mRNA relative expression level in ATRA-treated C6 cells treated with ATRA at 10 ÃŽ ¼M for 24, 48 and 72 h was increased to 1.72  ± 0.11à ¯Ã‚ ¼Ã…’3.22  ± 0.22à ¯Ã‚ ¼Ã…’4.0  ± 0.21 times, respectively( Fig. 1A). Meanwhile, the western blotting results also showed that EAAC1 protein expression in ATRA-treated C6 cells was increased gradually(Fig. 1B). Corresponding with the high EAAC1 expression in ATRA-treated C6 cells, 18F-FPGLU uptake was significantly increased to 1.47  ± 0.11à ¯Ã‚ ¼Ã…’2.14  ± 0.29à ¯Ã‚ ¼Ã…’2.12  ± 0.16 times in C6 cells treated by ATRA for 24, 48 and 72 h, respectively(Fig. 1C). There was a high correlation between the relative EAAC1 mRNA expresion and the relative 18F-FPGLU uptake in ATRA treated C6 cells (R = 0.939, Fig. 1D). To summ arize, EAAC1 expression was markedly induced by ATRA in C6 cell lines. As a result, there was more 18F-FPGLU uptake in ATRA-treated C6 cells line which has more EAAC1 expression at both mRNA and protein levels. Figure 1 PET imaging on C6 glioma-bearing mice Small-animal PET-CT scan was performed on C6 glioma-bearing nude mice models 1h post-injection of 18F-FPGLU. PET-CT fusion imaging of the mice models demonstrated that 18F-FPGLU could intensely accumulate in C6 glioma (Fig. 2A). The tumor/brain uptake ratio of 18F-FPGLU on C6 glioma-bearing mice was higher than that of 18F-FDG at 1h post-injection of radiotracers(n = 3, P < 0.05, Fig. 2B). However, the tumor/muscle uptake ratio of 18F-FPGLU in C6 glioma-bearing mice was lower than that of 18F-FDG (n = 3, P < 0.05). Immunohistochemistry showed that widely diffuse EAAC1 transporter staining was shown in C6 glioma, however there was minimal EAAC1 staining in normal rat brain write matter tissue (Fig. 2C). Figure 2 EAAC1 expression and 18F-FPGLU uptake in EAAC1 knock-down SPC-A-1 human lung adenocarcinoma cells The influence of EAAC1 expression on 18F-FPGLU uptake was specifically investigated using RNA interference-mediated EAAC1 knock-down SPC-A-1 human lung adenocarcinoma cells. Lentivirally delivered shRNA significantly reduced EAAC1 mRNA expression in SPC-A-1(shRNA) cells, as compared to the non-targeted (NT) shRNA control cells (SPC-A-1(NT) cells), EAAC1 shRNA reduced EAAC1 mRNA expression by 72% in SPC-A-1(shRNA) cells (P < 0.01) (Fig. 3A). At the protein expression level, EAAC1 shRNA significantly decreased EAAC1 expression in SPC-A-1(shRNA) cells by 59.6% (P < 0.01) (Fig. 3B). Knock-down of EAAC1 expression was associated with a significantly lower 18F-FPGLU uptake by 36% in SPC-A-1(shRNA) cells (P