Ederly Parents Should Live in Nursing Homes or Not? Essay

Vietnam’s population is older and older and it is important for young people, especially politicans, to seriously think about the issue of nursing homes. Should elderly parents live in nursing homes or not? Sending elderly parent into nursing homes is the best choice for them in the rest of their lives for three main reasons. First, elderly parents live in nursing homes will be good for their children. Nowadays, young people are too busy. They have o go to work and have no time to take care of their parents. They leave their parents at home lonely for whole day and feel nervous about their parents. Consequently, they can not concerntrate on their work and nursing homes are a good choice for them to send their parents into. Second, when elderly line in nursing homes, they can make friends with the people at the same age. They will fell very happy because they can chat whole day with their friends, do some helpful excercises such as jogging, tai chi, instead of being lonely at h ome when their children go to work. Third, the most important reason, is that nursing homes can provide elderly parents with modern health care facilities. In nursing homes, professional doctors, nurses and high quality equipments are always available to take care of elderly parents. Doctors here also give them a suitable diet which is good for their health. Opponents of sending elderly parents into nursing homes say that elderly parents will fell lonely when they live far from their children and it is also against Vietnamese moral value. However, as stated previously, nursing homes are the place which provide old people with the best health care. Moreover, their children can come and visit them whenever they want. In conclusion, there are undoubtly many benefits to send elderly parents into nursing homes. Most of old people who live in nursing homes show that they live better and happier with their friend here. Sending elderly parents into nursing homes becomes an important issue for both young people and elderly pare nts themselves to think about.

Pirates of the Caribbean Critical Essay

A key character that ensures we enjoy the film throughout is captin jack sparrow. The first time we meet jack he is portrayed as a powerful figure standing tall on top of his ship. As the scene progresses we see that he is drunk and unorganised sailing on his own on a sinking boat. The director shows us this by using a wide array of camera shots and other techniques. When we first see jack he is standing on top of his ship. Although he is standing tall and proud his clothes are weather beaten and he has clearly been at sea for a long time.The low angle camera shots that the director uses help the impression that he is powerful and strong. The director also uses an over the shoulder shot to help connect the audience to jack. The director then thatters this illusion by panning down the mast to reveal that he is on a small boat, that is quickly taking on water ,all on his own. The director does this to make jack an instantly likeable character and to make us want to fing out more about him. The change from jack being a drunk to a skilled swordfighter makes the character of jackmore interesting.The second time we meet jack he is in the blacksmiths where William turner is an apprentice. The camera angle that the director uses a wide shot so that you see that the workshop is small and to emphasize that he is agile and quick. The director does this so we find out slightly more about jack and become more interested. Through out the film jack is portrayed as an anti- hero and this is emphisized as jack does not want to shoot will as he says ‘’this bullet is not ment for you ‘’ meaning he went to port royal with a mission. The director does this to reveal a major plot point. he director of â€Å"the pirates of the caribbean† portrays miss elizabeth swann as a naive and dependent girl but this impression is ruined the second time we meet her. in the first encounter we see elizabeth trying on a dress her father has bought her from london. s he seems very feminine, well educated and wealthy but she has something to hide. a medallion she had taken off of will whe she and her majestys navy first found him. this adds to the enjoyment of the film by adding a twist that changes the whole story as this medallion lands elizabeth and the man she loves in alot of trouble. Read Critical Essay about Skurzynski’s NethergraveIn this scene she appears quite rebellious in the way she wants will to speak to her using her first name instead of miss swann but will has to remind her that this cannot happen because there is a difference between their two classes. the director does significant and clever he has elizabeth standing at the top of a stair case and will at the bottom during this conversation subtly  signifying that elizabeth is of a higher class but as the conversation progresses elizabeth walks down the staircase as she thinks that her and will are of the same class. he reason elizabeth treats will like this is because ever since  she laid her eyes on him six years ago on the crossing from england to port royal she hhas loved him madly. howthis makesthe film more enjoyable is by putting in a romantic element to the story meaning the two cant live with out the other so they are both trying to save the other by putting their own lives on the line creating excitement and suspense.

Roanoke: the Lost Colony

Roanoke: The Lost Colony Roanoke was the first attempt at creating an English settlement in the New World. It began in 1587 when Sir Walter Raleigh gathered over 100 people to voyage to Roanoke Island on North Carolina’s coast. Three years later, all of Roanoke’s inhabitants were gone with barely any evidence that they even lived there. The only clues found were the letter â€Å"CRO† carved into the trunk of a tree. On the nearby palisades, John white found the word â€Å"CROATOAN† carved into the surface.I believe the colonists of Roanoke voyaged to the nearby island of Croatoan and were absorbed by the native tribes. White, finding the clues, then began trying to identify the fate of the villagers. Before leaving, he told the villagers to leave clues as to their location should they have to leave the colony. White knew of an island called Croatoan south of Roanoke. A storm forced White and his men back to England before he could search for his family.Ano ther clue as to where the colonists went is the fact that several of the natives in Croatoan describe their ancestors as white people. Later, there were natives found with gray eyes which were only found among these natives and no others. The natives also claim their ancestors came from â€Å"Roanoke in Virginia. † The Pembrokes, the natives of Croatoan, spoke pure Anglo-Saxon English and bore the last names of many of the lost colonists. The natives even had fair eyes, light hair, and Anglo bone structure.These clues all point to the colonists voyaging into Croatoan and making a settlement there. Apart from telling the colonists to leave Clues, White also told them to carve a Maltese cross above their destination. Because no cross was found at Roanoke, it leads me to believe the colonists had to leave in a hurry. There could have been a threat of invasion which gave the colonists little time to escape and leave clues to their whereabouts. The lost colony of Roanoke was aband oned and its inhabitants voyaged into the wilderness and joined local native tribes.

Health Management Systems Case Study Example | Topics and Well Written Essays - 1500 words

Health Management Systems - Case Study Example Taking HMS as a perfect sample, this paper seeks to answer critical questions regarding the company, among them concerning the management structure, management strategies, use of technology and the company’s viability. The Organizational Structure of Health Management System (HMS) Like most successful companies, HMS has a solid management structure. The management is arranged in a hierarchy. There are different departments that are interrelated although they provide different services. Currently, the company is headed by Steve Starkey as the president. Down the hierarch are vice presidents of key departments. Some of these departments include; Sales, accounts, product development, human resource and finance. Each department has its own cluster of employees who see the smooth running of each department. For example, in the accounts department, there are other sub-departments like document management system, contract management systems and patient statements and e-payment soluti ons. The company has subordinate members of staff who are in charge of various auxiliary services. The staff mainly works for the well being of other employees. They carry out services like preparing meals, cleaning and driving. Ownership of the company HMS is arguably a big company, judged by the number of employees, space and the scope of the market it serves the company has embraced partnership with different organization in efforts to realize its mission. The major partners of the company include IBM premium business partner, Vision solutions, 3M, Capsule Technologies, Passport Health Communications, and Wolters Kluwer. All these partners are software producing companies. The partners hold a considerable amount of shares in the company. However, the board of executives consists of members with shares in the company. Management The organization’s administration is a core pride of the company. For managers to carry out their stipulated duties, they understand their stipulat ed roles properly. A role is defined as an expected set of activities resulting from a job (DuBrin J. A. 2008). The company is headed by high caliber members of the board of executives. This leadership stretches to various departments each headed by a manager. In most departments, there is a president and a vice president. Marketing Strategy The company tactfully implements its marketing strategy via a well established partnership between marketing and sales departments. These are some of the strategies that the company employs: 1. Use of research analysts These analysts are specially trained to carry out market analysis and determine the viability of a market. The analysts carry out interviews in different community hospitals and determine the kind of technology used in carrying out their activities, information systems and patient documents systems. 2. Seminars and Trade Shows The same department promotes its products through seminars and trade shows to potential community hospita l owners and health practitioners. 3. Education Education is one of the best ways to mobilize, inform and market a company’s products. HMS has effectively used this tool as a marketing strategy. The company has an Education Administrative Assistant working in partnership with the marketing department to carry out the duty of advertising the courses offered by the company. This platform opens an opportunity for the company to sell not only its computer related products

Global Warming Essay Example | Topics and Well Written Essays - 750 words - 3

Global Warming - Essay Example If the inactions of the past several decades is an indication, then the answer is no. Essentially, the greenhouse effect functions in the following manner. When sunlight pierces the atmosphere and hits the earth’s surface, not all of the sun’s solar energy is absorbed. Approximately a third of it is reflected back into space. Specific atmospheric gases serve in much the same way as does the glass of a greenhouse, thus the terminology. These gases allow sunlight to penetrate then trap some of the solar energy which heats the earth (Breuer, 1980). It is a delicate balance and because these greenhouse gases have been artificially augmented by man-made sources, more build up in the atmosphere has occurred thus trapping more of the sun’s energy and reflecting less back in to space. This occurrence is causing the earth to warm. Carbon Dioxide (CO2) is the most prevalent of the greenhouse gases. Trees absorb CO2 and when they die, CO2 is restored to the atmosphere. The clearing of forests by mass burning, which is happening at a phenomenal rate in the tropical rain forests, is decreasing the amount of CO2 that is absorbed and increasing the amount that is added to the atmosphere. CO2 supplies about half of the total gases that create the greenhouse effect (Breuer, 1980). Although deforestation is contributing heavily to the excess of CO2 in the atmosphere, a larger portion is caused by the burning of fossil fuels such as oil and coal. Fossil fuels are burned by factories, vehicles and electricity-producing power plants to name a few sources. Other greenhouse gases include methane, which is released when vegetation is burned during land clearing, during oil exploration activities and the coal-mining process; chlorofluorocarbons (CFCs), which is the substance that cools refrigerators and provides the propulsi on in aerosol cans and nitrous oxide (N2O) which is the lesser cause of CO2 (Breuer, 1980). It is generated from both

Benefits of Assessment Centers for Organizations Research Paper

Benefits of Assessment Centers for Organizations - Research Paper Example This research will begin with the statement that the commonest application of assessment is in measuring management employees’ performance, especially for interpersonal competencies. The assessment centers help determine the specific competencies in an individual employee through a series of techniques and methods that include both individual and group activities. The assessors determine an individual’s performance against other employees. Many analysts affirm that assessment centers are very effective in measurement interpersonal skills. An assessment center puts employees through work-like conditions involving individual and team activities and tests that offer the best simulation of real work situations for monitoring and measurement of key competencies. Assessment centers are critical in determining the competency of employees. Competency is a broad term referring to a set of skills, knowledge, individual behaviors and how they auger with the job specification of th e employee under consideration. The interplay of factors in every employee is critical for the overall success of the organization. In addition, assessment centers help organizations to determine the most important skillsets for the organization with regard to the management. For instance, an employee’s willingness to delegate, works in a team, take risks, and take personal initiative. Assessment centers are also great in selecting employees who can provide the best performance for particular jobs. The organization can therefore choose employees for jobs that are most fitting and improve the organizational performance. An assessment center also aids in developing and identifying fresh potential for the organization’s top supervisory jobs (Boehm, 1982), particularly in cases where other methods fail, given its superior ability to reveal an employee’s interpersonal skills. Finally, an assessment center helps identify competencies needing further development and co nsequently help in appraising of employees, which can help in career development. This report analyzes assessment center as an assessment tool and examines its implementation considerations for an organization. A description of the Assessment Center Approach A number of factors, which articulate its goal, underlie the assessment center approach to measuring organizational performance. For an organization to implement an effective assessment center, it needs to identify the competencies necessary for its future employees (Rupp & Reynolds, 2009). In addition, the management has to come up with means to determine the competencies that the current employees possess. Finally, one of the most vital phases in the overall design of the assessment center is the identification of competencies gap, and the eventual process of developing means of bridging this gap. By identifying gaps in an individual’s set of competencies, the organization can help nurture these skills in employees to i mprove their performance, for instance with regard to knowledge and skills. Implementing an Assessment Center for the Organization A number of factors come into play before an organization can effectively implement an assessment center. First, the organization has to find appropriate and experienced raters to conduct the assessment. According to Coffee (2005), in the industrial settings, 3 to 6 assessors assess 6 to 12 candidates over a period of 3 days. Secondly, the organization has to identify an all-encompassing set of assessment techniques, which ensure that the assessment goes on successfully and comprehensively covers all areas of relevance in the study. Normally, the assessment methods are either written or oral. The written exercises include in-basket

Chose an interesting Topic - Make it strong or Use Bridging the Essay

Chose an interesting Topic - Make it strong or Use Bridging the Digital Divide - Essay Example Such is the case with the urbanized communities, who are in a better position to access such technologies, as opposed to the rural communities. The access to computer and other forms of technology comes with its educational advantage, where the societies accessing the computers have higher chances of becoming educated than those who do not have the access (Bridging the Digital Divide, 49). Owing to digital divide, Nicholas Negroponte started the One Laptop per Child Program (OLPC), whose main objective was to bridge the digital divide, by making computers available and accessible to all children all over the world. Despite facing various challenges, the program has registered considerable success, by making computers accessible to many children all over the world, with some countries such as Uruguay completely meeting the objective of providing every child with a computer (Bridging the Digital Divide, 50). Due to the power and electricity accessibility problems in different parts of the world, as well as the internet infrastructural issues, the program focuses on availing computers that are not entirely dependent on electricity as a source of power and with inbuilt internet accessibility. This has seen the development of Computer models such as the OX-I, OX-1.75 and OX-3(Bridging the Digital Divide, 50). This initiative has seen other technology companies such as Intel launch similar programs, with its Classmate-PC initiative that has since made computers even more accessible to different parts of the world. The importance of such programs in bridging the digital divide cannot be overemphasized. Question 1 There are various reasons why digital divide matters to children and their families. First, digital divide places some sections of the community at an advantage over the others, through providing them with opportunities for education and information accessibility, which the other sections lack (Servon, 48). Digital divide also matters to children and their pa rents, since it serves to increase the social and economical inequality, through making computer technology affordable to the families that are at a higher economic level, enabling their children to enjoy the benefits that come with computer and internet skills, while the poor families are left behind. This translates to the rich families dominating the poor families in social aspects, such as economical and political (James, 15). Digital divide also determines the infrastructural development of the society. This means that the societies that have access to computer technology will also enjoy other infrastructural development such as electricity, compared to societies with no computer technology access. This translates to families and children who have no computer technology access remaining disadvantaged by underdevelopment (Yelton, 33). Question 2 The OLPC project will be successful. This is because; the program has translated into reality in countries such as Uruguay and parts of the US, with the same also happening in Australia and Rwanda, and in other countries in the near future (James, 23). Additionally, the initiative to provide every child with a laptop is attractive to many philanthropists, NGOs, charitable organizations and even governments, which can easily translate such a program to

Management-managing change in organisation Essay

Management-managing change in organisation - Essay Example The expectations and changes that are within the organization when expanding into the global market are affecting the way that the organization operates as well as the developments that are currently a part of the system. Defining how Gap has been able to associate with the organizational change management is then able to provide a stronger basis for the needs that are within the store. Gap Inc began in 1969 by Doris and Don Fischer in San Francisco, California. The main concept was to provide new trends in clothes with the first line of clothing being based on fashion jeans. The Gap store was built not only to provide specialty clothing from their own line of production, but also provided a consolidated area for other jeans and clothing items, such as Levis brands. The store was able to develop into providing a space that offered other lines of clothes as well as the Gap brand as well as other pop culture concepts that were popular during the time. Music, records, tapes and other smaller items were initially placed in the store to ensure that there was the ability to have different popular concepts and ideas as a part of the store (Gap, 2010). The main concept that is associated with Gap Inc comes from the vision slogan, â€Å"wearing your passion.† They have developed several outlets that provide insight into what customers need for popular wear and different cultural concepts. More importantly, there is a connection to providing quality clothing with creative results that are a part of the store. Gap also focuses on specific ethical considerations that are able to enhance the lines of clothing that are offered through the store. The priorities that are built from Gap include the ability to deliver results while keeping a sense of responsibility toward the consumers that are interested in the line and brand of clothing. To follow

Contradicting and Acquisition Assignment Example | Topics and Well Written Essays - 500 words

Contradicting and Acquisition - Assignment Example Appropriation does not necessarily represent cash. Instead, it represents the amount for a given purpose as stated in the appropriation act which the companies are obligated to during a given period as noted in the appropriation act. It is most important for it helps the companies spend federal funds (Warde, 2010). Difference between authorization and appropriation: The constitution usually grants its Congress the power of appropriation for agencies and federal spending. The federal programs are authorized by some Senate rules and activities of appropriation follows. Congress usually breaks the rules because the rules carry no constitutional weight or no statutory weight. The authorization-appropriation process serves as a highway in enacting federal spending. So the Congress is authorized before appropriation takes place whereby budget authority is provided to federal agencies that incur obligations and make payment out of the treasury (Gibbler, 2006). Obligations: Legal actions or responsibilities made by federal agencies at a given period. The actions may include different transactions made by them and they may include the number of orders received, services received, different contracts awarded among others. Obligation results to the amount of payment made including advances, amount of checks issued, reimbursement and net of refunds during the same period or in the future (Smith, 1976). Expenditure: It is a charge on current cash that results when agencies settle an obligation as evidenced by receipts, voucher, and invoice. Federal agencies spend authorized funds in some of the federal programs. Gross outlays are disbursement that is, cash, and fewer funds received. Net outlays involve disbursement minus reimbursement collected.  

Nazi Ideology Essay Example for Free

Nazi Ideology Essay Nazism also officially known as the National Socialism is defined as an ideology and practices that are influence by the National Socialist German Worker’s Party that is under the leadership of Adolf Hitler. In relation to this, Nazism is also regarded as political policies that were adopted by the dictatorial of Nazi Germany that took place from 1933 to 1945. Nazism strongly advocates the superiority of an Aryan race that makes the Germanic people stand above others. During the leadership of Hitler, Nazis supported the centralized government that is led by the Fuhrer that claimed to have the responsibility of defending and protecting Germany and the German people in their country and abroad against the forces of Communism and Jewish subversion (Thomas). As such, the recurring themes of Nazism include extreme nationalism, xenophobia, and the glorification of the Aryan race (Levy 497). These themes of Nazism have put many negative effects for people that do not belong to the Aryan race especially for Jews as well as to the German themselves. The themes of this ideology created prejudices and stereotypes against other races especially for the Jewish people that often become the cause of conflict and violent practices. Due to this, Jewish people and other races that Hitler deems as dirty or is a threat to the Aryan race are hunt down. The holocaust is a clear example of the violent practices that he implemented in line of the Nazis ideology. Nevertheless, the German people also experience the negative effect of this ideology as some of them experience xenophobia. They think that they should not interact with other races especially those that they believe is a threat to their racial superiority. Lastly, Germans also have experience difficulties in interacting and relating with other races especially when they have the mentality that they are better as compared to them (Levy 497-498). Works Cited Levy, Richard S. Antisemitism. California: ABC-CLIO, 2005. Thomas, Robert. â€Å"The Nature of Nazi Ideology. † 11 June 2009 http://www. libertarian. co. uk/lapubs/histn/histn015. pdf.

Current Diagnostic Methods for Human Immunodeficiency Virus

Current Diagnostic Methods for Human Immunodeficiency Virus Abstract: Detection of human immunodeficiency virus (HIV) infection is essential for diagnosis and monitoring of the infection. There are several different types of diagnostic tools available that are based on detection of HIV-specific antibodies, virus antigen, or nucleic acid. Sensitivities and specificities of assays utilized for HIV detection have improved. Newer HIV testing technologies such as third-generation enzyme immunoassay (EIA) which detect HIV-specific IgG and IgM antibodies, fourth-generation EIA which detect both anti-HIV antibodies and HIV p24 antigen, and nucleic acid-based tests (NATs) for HIV RNA, have significantly decreased the window period. This review study provides an overview of current technologies for the detection and monitoring of HIV infection and recent advances in the field of HIV diagnosis. Keywords: HIV diagnosis; HIV antibody test; human immunodeficiency virus; Immunoassay; Polymerase chain reaction (PCR) Introduction: Diagnosis of HIV infection contributes to evaluating the progression of disease, monitoring the effectiveness of antiretroviral therapy (ART), and prevention and control of HIV/AIDS. The diagnosis of HIV is associated with decrease in risky behaviors, reduced HIV transmission, and improved survival linked to increased case detection, earlier care and treatment. HIV-negative persons can also protect themselves from HIV when making sexual decisions by engaging in safer sex behaviors and sometimes, taking pre-exposure prophylaxis (PrEP). Early diagnosis of HIV infection provides an opportunity for risk reduction counseling and preventing further transmission of the disease, while late diagnosis of HIV infection is detrimental to infected patients and to the public health, and is associated with an increased rate of morbidity, mortality, and healthcare costs. Since the start of the epidemic, it is estimated that 78 million people have become infected with HIV and 35 million people have died from AIDS-related illnesses. In 2015, 2.1 million people became newly infected, 36.7 million people were living with HIV and 1.1 million people died from AIDS-related illnesses. New HIV infections have fallen by 6% since 2010. Sub-Saharan Africa, which bears the heaviest burden of HIV/AIDS worldwide, accounts for 65% of all new HIV infections. Other regions significantly affected by HIV/AIDS include Asia and the Pacific, Latin America and the Caribbean, and Eastern Europe and Central Asia (Table 1) [9]. The present study aims to conduct a narrative review to summarize and discuss the current diagnostic methods for HIV and recent developments. We start with a brief overview of HIV infection, follow by a description on the development of virological and immunological markers following HIV infection. Thereafter, we introduce current algorithms for laboratory HIV testing with different kind of current diagnostics techniques including various generations of enzyme immunoassays, rapid or point-of-care tests, and qualitative/quantitative PCR assays. Overview of HIV Infection: HIV-1 causes chronic infection which is usually characterized by progressive immune deficiency, a long period of clinical latency, and appearance of opportunistic infections [1, 2]. Characteristics of HIV include infection and viral replication in T lymphocyte expressing CD4 antigen. Qualitative defects in CD4 cell response and a gradual decline in their numbers increase the risk of opportunistic infections like Pneumocystis carinii pneumonia, and neoplasms such as Kaposis sarcoma and lymphoma [3-5]. HIV infection can disrupt functions of blood monocytes, tissue macrophages, and B lymphocytes, and also increase the potential of encapsulated bacteria for developing infections [6, 7]. Direct invasion of CD4 cells in the peripheral and central nervous systems can cause meningitis, peripheral neuropathy, and dementia [8]. The prognosis is variable between people infected with HIV-1. In adults, the average time between HIV exposure to AIDS stage is 10-11 years, but a remarkable proportion of individuals (~20%) progresses rapidly to AIDS within 5 years after HIV exposure. On the other hand, it is estimated that 12% of infected individuals will remain free of AIDS for 20 years [10]. Prophylaxis and in particular antiretroviral therapy (ART) significantly enhanced the overall prognosis of HIV disease against opportunistic infections [11]. The most common route of HIV infection is sexual transmission at the genital mucosa via direct contact with infected body fluids, such as blood, semen, and vaginal secretions. Infection may also occur via inoculation of infected blood, transfusion of infected blood products, transplantation of infected tissues, from an infected mother to her infant during pregnancy, or by reuse of contaminated needles [12]. The probability of transmission after a single exposure with an uncontrolled HIV source has been estimated to be 1/150 with needle sharing, 1/300 with occupational percutaneous exposure, 1/300-1/1000 with receptive anal intercourse, 1/500-1/1250 with receptive vaginal intercourse, 1/1000-1/3000 with insertive vaginal intercourse, and 1/3000 with insertive anal intercourse. The average risk is 12-50% for congenital (mother-to-child) transmission, 12% for breast-feeding, 90% for a contaminated blood transfusion, and 0.1-1.0% for nosocomial transmission [13]. The risk of HIV transmission during early or acute HIV infection appears to be greater than during chronic infection (251). Available data suggest that the viral load is an important predictor of the risk of heterosexual transmission, and patients with levels less than 1500 copies of HIV-1 RNA per milliliter are at lower risk of HIV transmission, whereas the probability of transmission is increased dramatically with increasing vira l loads (250). Laboratory markers for HIV-1 infection: Several immunological and virological blood markers can be monitored during the course of HIV infection. These markers appear highly consistent between different individuals in a chronological order and allows classification of HIV infection into distinct laboratory stages including eclipse period, seroconversion window period, acute HIV infection, and established HIV infection (Figure 1) [14, 15]. Shortly after exposure to HIV-1, no viral markers are consistently detectable in plasma, but low levels of HIV-1 RNA can be found intermittently [16]. This period is called the eclipse phase. About 10 days after infection, HIV-1 RNA becomes detectable by NAT in plasma and quantities rise to very high levels [17], which subsequently decline rapidly until reaching a set point, a stable level that may persist for years. This stable level of HIV RNA represents an equilibrium between HIV and host immune responses and is an important indicator of subsequent disease progression, and potential transmission of HIV. It has been shown that the higher HIV-1 RNA plasma level is associated with faster progression to AIDS [18]. The set point plasma HIV-1 RNA level can be a helpful clinical tool for determining the timing of initiation of antiretroviral therapy for HIV-infected patients. For instance, patients with high set point levels can be started on aggressive antiretroviral therapy and patient s with low set point levels can be monitored without initiating therapy [19]. HIV-1 p24 antigen is expressed and quantities rise to levels that can be measured by fourth-generation immunoassays within 17 days after infection (typical range 13-28 days) [15, 20]. Due to high titers of p24 antigen present in the sera of acutely infected patients during the interval prior to seroconversion, p24 Ag assay can be utilized to diagnose the primary HIV-1 infection [21]. Nevertheless, detection of p24 antigen is transient because, as antibodies begin to develop, they bind to the p24 antigen and form immune complexes that interfere with p24 Ag assay [22, 23]. The time interval between infection with HIV and the first detection of antibodies is known as the serological window period. The detection of HIV-specific antibodies indicates the end of the window period and the individual is known as seropositive [24]. The length of the window period depends on the design and the sensitivity of the immunoassay. Expression of IgM antibodies can be detected by immunoassays within 10 to 13 days after the appearance of viral RNA, 3 to 5 days after detection of p24 antigen, and peak between the 4th and the 5th week [15, 20, 25, 26]. Thereafter, the emergence of IgG antibodies occurs at about 3-4 weeks after infection and persist throughout the course of HIV infection [21]. Nevertheless, the immune responses are highly dependent on the ability of the individuals immune system to produce the antibodies. Approximately, 50% of patients within 3-4 weeks and about 100% of them within 6 months have detectable antibodies, although there are reports indicating that a small percentage of patients may require up to 6 months for the appearance of antibodies [27]. Laboratory HIV testing algorithms: Since 1989, the diagnostic algorithm for HIV testing recommended by CDC and the Association of Public Health Laboratories (APHL) relied on the confirmation of a repeatedly reactive HIV immunoassay with the more specific HIV-1 antibody test, either the HIV-1 Western blot or HIV-1 indirect immunofluorescence assay (IFA). The Western blot was previously considered to be the gold standard for the diagnosis of HIV infection by Clinicians [29, 30]. It should be noted that both the Western blot and IFA are first-generation assays that detect only IgG antibodies against HIV proteins. Retrospective testing of specimens from high-risk individuals pointed that antibody testing alone may miss a significant percentage of HIV infections detectable by virologic tests such as HIV antigen and nucleic acid assays. In 2013, the CDC and the APHL released new guidelines on HIV testing that have led to the earlier diagnosis of HIV infection when compared with the previous diagnostic algorithm. The new recommended algorithm starts with a fourth-generation HIV-1/2 Ag/Ab immunoassay to screen for HIV infection that detects both HIV-1/2 antibodies and the HIV-1 antigen. When the result of initial immunoassay is nonreactive, further testing is not required for samples. Instead, testing with an HIV-1/HIV-2 antibody differentiation test is needed when the sample is reactive on the screening fourth-generation assay. Reactive results with the initial fourth-generation assay and the HIV-1/HIV-2 antibody differentiation immunoassay should be considered as reactive for HIV-1 antibodies, HIV-2 antibodies, or HIV antibodies, undifferentiated. Reactive results with the initial fourth-generation assay and nonreactive or indeterminate on the HIV-1/HIV-2 antibody differentiation immunoassay should be tested with an FDA-approved HIV-1 NAT to differentiate early HIV infection from a false-positive screening result [28] (Figure 2). HIV diagnostic tests: Serological diagnostic assays: Enzyme Immunoassays (EIA):Significant advances in the development of HIV immunoassays have been created since the discovery of HIV in 1983. Based on different design principles, HIV immunoassays are generally classified into generations. The earliest immunoassays (first-generation) are indirect EIAs that used coated viral lysate antigens derived from cell culture on a solid phase for antibody capture and an indirect format that detected antibody utilizing an enzyme-conjugated antihuman IgG [36]. Antibody can be detected within 8-10 weeks postinfection by first generation immunoassay. These assays have 99% sensitivity and 95-98% specificity for HIV infection. Second-generation immunoassays use synthetic peptide or recombinant protein antigens alone or in combination with viral lysates to bind HIV antibodies, and they use an indirect immunoassay format that employs conjugated antihuman IgG, which binds to IgG with high affinity, to detect IgG antibodies [37]. Utilizing recombinant anti gens in the second-generation assays improves sensitivity for HIV-1, HIV-1 group O, and HIV-2, allowing earlier detection of IgG antibodies. The sensitivity and specificity of second-generation assays have been reported to be ˃99.5% and ˃99%, respectively. First and second generation immunoassays can only detect IgG antibody to HIV. The window period was decreased to 4 to 6 weeks postinfection by second-generation assays. Third generation immunoassays also utilize synthetic peptide or recombinant antigens to bind HIV antibodies, but in an immunometric antigen sandwich format; HIV antibodies in the specimen bind to HIV antigens on the assay substrate and to antigens conjugated to indicator molecules. This allows detection of both IgM and IgG antibodies which leads to increase in sensitivity and specificity of the test. Lower sample dilutions and the ability to detect IgM antibodies (which are expressed before IgG antibodies) further decrease the window period to 2-3 weeks postinfection [38]. The reported sensitivity and specificity of third-generation assays is ˃99.5%. Combination or fourth-generation tests use synthetic peptide or recombinant protein antigens in the same antigen sandwich format as third-generation assays for the detection of IgM and IgG antibodies, and also monoclonal antibodies for the detection of p24 antigen [39]. Inclusion of p24 antigen capture allows the detection of HIV-1 infection before seroconversion and further decreases the window period. Most fourth-generation antigen/antibody immunoassays (termed combo assays) do not distinguish antibody reactivity from antigen reactivity [39]. Recent published data has shown that the fourth-generation assay was able to establish HIV infection in more than 80% of patients who tested NAAT positive but either nonreactive or indeterminate by other tests like Western blot, first to third generation immunoassays, and rapid tests [40-42]. Delaney et al. found that the fourth-generation immunoassay are able to detect HIV infection 1-3 weeks earlier than the first, second, and third generation immunoassay which cannot detect p24 antigen. The results of their study revealed that the median duration of the eclipse period was 11.5 days and 99% of specimens from HIV-infected patients could be reactive with Ag/Ab combination tests within 45 days of exposure. Moreover, for detection of antibodies by the IgG/IgM-sensitive and other plasma screening assays, 50 days or longer were required and after 3 month of exposure, infection could be detected by all tests. Several studies have reported sensitivities of 100% for fourth-generation immunoassay, whereas other surveys reported transient sensitivities range from 62-89% when assessed against HIV RNA assays. This decreased sensitivity can be attributed to the presence of a second diagnostic window. This situation is rare but it can happen. Second diagnostic window period lies between the p24 antigen detection and the anti-HIV antibody detection, and is associated with reduction in the p24 antigen and antigen/antibody complexes levels, as well as a delay in HIV-specific antibody development which totally may affect the sensitivity of fourth-generation immunoassays. So, it is possible that many acute HIV infections have been missed using fourth-generation assays. Despite negative results from a fourth-generation immunoassay in high-risk populations with suspected acute HIV infection, it is needed to repeat the test on new blood samples obtained several days later, as well as testing for HIV anti body alone, p24 antigen or use of an HIV RNA assay. In 2015, an improved version of immunoassay, BioPlex 2200 HIV Ag-Ab screening test system, received FDA approval in HIV screening which detects both HIV antibody and the HIV-1 p24 antigen by providing separate results for each analyte. This test also provides separate results for HIV-1 and HIV-2 antibodies, so there is no need for a HIV-1/2 differentiation assay for antibody reactive samples. It was reported that the sensitivity and specificity of BioPlex 2200 HIV Ag-Ab assay were 100 and 99.5%, respectively [43]. HIV Confirmatory Tests:Screening tests must be highly sensitive to produce few false-negative results, whereas confirmatory assays are characterized with high specificity to produce few false-positive results [44]. If the result of a screening test is repeatedly reactive, this has to be confirmed by (at least) one confirmatory assay. Western blot or indirect IFA traditionally have been employed as confirmatory assay due to their higher specificity. The probability that both ELISA and Western blot would give false-positive results is extremely low (

Role of Elevated Levels of Homocysteine in Blood Plasma

Role of Elevated Levels of Homocysteine in Blood Plasma Homocysteine, a non-protein amino acid, is an intermediate in the metabolism of methionine and biosynthesis of cysteine. It has gained prominence in the past half-century because its accumulation in the body has been linked to increased risk and occurrence of atherosclerosis and cardiovascular disease. Homocystinuria is due to errors in metabolism and homocysteinemia is attributed to polymorphisms in the genes involved in methionine metabolism and cysteine biosynthesis, and deficiencies in the nutrients folic acid, and vitamins B12 and B6. Proof of these can be found in the results of genetic and dietary studies. The clinical quantification of homocysteine has evolved through the years and routine tests are currently available. Baseline homocysteine concentration for normal adults has been identified to be between 12-15 Â µmol/L. Elevated levels of homocysteine can be reduced with folate and B vitamins supplementation, but this intervention does not work in patients who have suffere d stroke or heart attacks. More research studies have supported the hypothesis that elevated homocysteine causes CVD and is not just a marker for the disease. However, despite the amount homocysteine research performed, many issues remain to be resolved, among which are the elucidation of the molecular mechanism of the direct action of homocysteine and the standardization of techniques for the quantification of homocysteine levels. Brief introduction. 200 w The role of elevated levels of homocysteine in blood plasma has been the subject of intense study and literature reviews for more than 50 years since an association between defects in homocysteine metabolism and thromboembolism was observed. This role was further verified in patients with homocystinuria and abnormalities in vitamin B12 metabolism with general vascular damage and widespread thrombosis. Subsequently, the homocysteine theory of arteriosclerosis was formulated by McCully and Wilson in 1975. Since then, the positive association between the risk of cardiovascular disease and homocysteine levels in the general population was validated in many epidemiological studies (Boushey, et al., 1995; Verhoef, et al., 1996; Eikelboom et al., 1999; Humphrey et al., 2008). Significantly, these studies found that small increases of homocysteine levels in blood increase the risk of coronary heart disease. Another important finding was the role of diet, vitamins and folic acid in lowering h omocysteine levels. Some authors have questioned the direct homocysteine-cardiovascular disease link, basing their conclusions on a review of longitudinal, prospective studies (Kaul, Zadeh and Shah, 2006). Accordingly, the evidence is not strong enough to warrant a causal effect, the mechanisms for how homocysteine causes cardiovascular disease has not been elucidated, and that there has been no proof showing that interventions of decreasing homocysteine levels have modified the risk for atherosclerosis (Kaul, Zadeh and Shah, 2006). Moreover, homocysteine has been proposed to be a marker, and not a cause of CVD (Wierzbicki, 2007). Recent studies have proposed a mechanism on increased risk of cardiovascular disease (CVD) due to elevated homocysteine. Inhibition of the growth of endothelial cells and promotion of vascular smooth muscle cell proliferation results in damage of vascular cells (Zou, 2007). The abnormality in the production of endothelial cells was caused by homocysteines inhibition of DNA methylation in the promoter region in the gene of an activator of the cell cycle (Jamaluddin, et al., 2007). Amino acid and focus on cysteine. 300 w All living cells contain the biomolecules proteins, carbohydrates, nucleic acids and lipids (Mathews and Van Holde, 1996; McKee and McKee, 2004). These are made up of the repeating sub-units of amino acids, sugars, nucleotides and fatty acids respectively. Cells also contain small organic molecules that are involved in complex biosynthetic and regulatory pathways that are tightly controlled at the molecular level. Amino acids are naturally occurring compounds containing an amino group, a carboxyl group, and a unique side chain or R group (Figure 1). The chemical and functional properties of an amino acid are largely determined by its R group (McKee and McKee, 2004; Mathews and Van Holde, 1996). Although there are hundreds of amino acids, only twenty have been identified to be building blocks of proteins. Among these is cysteine, which contains a sulfhydryl, or thiol (SH) in its R group (-CH2-SH) (Berg, Tymoczko and Stryer, 2002). The sulfhydryl group is very reactive and could react with another thiol group, forming disulfide bonds or sulfide bridges. These bridges are important in increasing the stability of some proteins. Two sulfhydryl moieties of two cysteine bonds can oxidize forming cystine. This commonly occurs in extracellular fluid like blood and urine. However, the solubility of cystine is very low, and in large amounts, cystine can cause kidney stones (McKee and McKee, 2004).The sulfhydryl moiety also forms weak bonds with nitrogen and oxygen. Another amino acid which contains a sulfhydryl group is methionine. Its side chain is -CH2CH2SCH3. The sulfur in methionine can form bonds with electrophiles (Berg, Tymoczko and Stryer, 2002). The methyl group (-CH3) can be activated and is involved in many reactions where just one carbon atom is being added to another compound. Cysteine is considered a non-essential amino acid because it can be synthesized de novo in both plants and animals (Figure 2). In animals, cysteine is formed from the cleavage of cystathionine through the action of ÃŽ ³-cystathionase. Cystathionine is produced when serine condenses with homocysteine that is derived from methionine (McKee and McKee, 2004). Methionine, an essential amino acid, cannot be synthesized in humans, and thus, have to be provided in the diet. Its metabolism involves homocysteine. Homocysteine structure and metabolism. 200 w Homocysteine is a non-protein amino acid and thus, it is not utilized in protein synthesis. Its structure is similar to cysteine but it has an additional carbon before the sulfhydryl moiety (R group: -CH2 -CH2 -SH). The tendency of homocysteine to form cyclic compounds may have limited its potential as a protein building block. Homocysteine is not provided in the diet, but is biosynthesized from methionine in a process involving several steps. Methionine is first adenosylated to form S-adenosyl methionine (SAM), and then the methyl group is transferred to an acceptor molecule to form S-adenosyl homocysteine (SAH) in a process called transmethylation. Adenosine is then removed thereby forming homocysteine (Figure 3) (Durand et al., 2001; Selhub J. , 1999; Marinou, et al., 2005). Transmethylation is the only pathway for producing homocysteine in the body. Homocysteine then undergoes two pathways: remethylation to methionine, and transsulfuration to produce cystathionine, cysteine, pyruvate and taurine. Remethylation requires folate and cobalamin (vitamin B12), and transsulfuration requires pyridoxine (vitamin B6). Homocystinuria results from genetic errors in the metabolic pathways (Kluijtmans, et al., 2003; Klerk, et al., 2002), but the current focus is on the more common hyperhomocysteinemia that results fr om genetic variations or nutritional inadequacy (Finkelstein and Martin, 2000). Polymorphisms in the gene methylenetetrahydrofolate reductase (MTHFR) which catalyzes the transfer of a methyl group to homocysteine to re-form methionine have been found in many studies to increase homocysteine levels (Klerk, et al., 2002). Among the proposed effects of increased homocysteine levels resulting in CVD are oxidation of low density lipoprotein, decrease in the thrombomodulin expression inhibiting the anticoagulant pathway leading to thrombosis, platelet activation and aggregation, and smooth muscle cell proliferation (Eikelboom, et al., 1999; Jamaluddin, et al., 2007). Normal Homocysteine levels. 200 w The concentration of circulating total homocysteine (tHcy) is an accurate marker of low folate and vitamin B12. In 1999, 3563 male participants and 4523 female participants were surveyed to determine the normal blood homocysteine levels (Selhub, et al., 1999; Selhub, 1999). The survey found that homocysteine increased with age and was higher in adult males than and females. Screening of babies confirmed that homocysteine levels are higher in baby boys (Refsum, et al., 2004). It was recommended that blood homocysteine levels equal to or greater than 11.4 Â µmol/L (males) and 10.4 Â µmol/L (females) are to be considered above normal and was associated with low vitamin concentrations in two-thirds of the subjects surveyed (Selhub, et al., 1999). This verified that an assessment of homocysteinemia requires the knowledge of the health status of the patient. The degree of homocysteinemia is classified based on determination fasting levels of serum homocysteine. Currently, a plasma tHcy level of 15 Â µmol/L is considered as normal (Refsum, et al., 2004). In healthy adults with good folate and B vitamins status, the upper reference limit is 12 Â µmol/L. Accordingly, homocysteinemia is classified as moderate (15-30 Â µmol/L), intermediate (30-100 Â µmol/L) and severe (> 100 Â µmol/L). This categorization is necessary to come up with a decision regarding the treatment of the condition. Homocysteine and nutrition. 300 w Studies have shown that the enzymes regulating the metabolism of homocysteine are activated by the B vitamins and folate which can be sourced from the diet. The balance of the remethylation and transmethylation pathways are coordinated by nutritional inputs, specifically methionine (Selhub, 1999). Decreasing dietary methionine increased homocysteine remethylation, while increased methionine increased the production of cystathionine through upregulation of the transsulfuration pathway. The molecular mechanisms for the effects of dietary methionine are based on the capacity of S-adenosyl methionine SAM to inhibit methylenetetrahydrofolate reductase (MTFHR) and activate cystathionine synthase (Selhub and Miller, 1992). Thus, when dietary methionine is high, there is a rapid conversion of methionine to SAM. The high levels of SAM promote the inhibition of MTHFR and subsequently, remethylation of homocysteine is also depressed. The pathway is channeled to the transsulfuration pathway, to produce cysteine. In opposite conditions of low dietary methionine, low SAM levels are not enough to inhibit MTFHR activity, resulting in the remethylation of homocysteine. The role of folate in homocysteine metabolism has been the subject of many research studies (Antoniades, et al., 2009; Boushey, et al., 1995; Lonn, et al., 2006). A diet that is poor in folate was also found to impair remethylation and synthesis of SAM (Miller, et al., 1994). Folate deficiency increased the concentration of tHcy and hepatic SAM concentration. Introducing dietary folate and methionine also decreased tHcy. Since vitamins and trace minerals are lost due to food processing, fortification is proposed in order to replenish the lost nutrients. Folic fortification of breakfast cereals when consumed daily was recommended to be the most effective means of decreasing serum folate (Riddell, et al., 2000). High-dose folic acid supplementation also decreased tHcy levels in cases where there is vitamin B12 deficiency (Min, 2009). However, a high methionine diet is not recommended since it can decrease body weights and HDL-cholesterol production in mice (Velez-Carrasco, Merkel and S mith, 2008). Aside from the observed effects of folic acid on reducing tHcy concentrations, folic acid is also thought to be involved in ameliorating endothelial dysfunction through its action of maintaining endothelial nitric oxide synthase in its coupled state which favors the formation of nitric oxide, and not oxygen radicals (Moens, et al., 2008). Role of Homocysteine and vitamins. 200 w The enzymes involved in metabolism of homocysteine require the participation of vitamin cofactors B6 and B12, and folic acid. The direct association between low folate, vitamin B6 and B12 status and hyperhomocysteinemia have been validated many times Hao, et al; 2007; (Hao, et al., 2007; Boushey, et al., 1995; Kluijtmans, et al., 2003; Koehler, et al., 2001). Investigations have geared towards the probability of using these vitamins in the prevention and treatment of homocysteinemia in the general population, and in those who already have cardiovascular disease. The use of multivitamins supplementation for several months improved the concentration of the B vitamins and folate acid in plasma, and decreased homocysteine and LDL cholesterol levels in 182 study participants (Earnest, Wood and Church, 2003). Similar homocysteine-lowering effects were also observed in patients with celiac disease, who have malabsorption problems, who take vitamin supplements (Hadithi, et al., 2009). However, the case for the vitamins is different in patients who already have CVD. Although the treatment of different combinations of vitamins B6, B12 and folic acid of patients with coronary artery disease resulted in a 30% reduction of tHcy one year after receiving vitamin B12 and folic acid, follow-up measurements of homocysteine levels showed that the vitamin supplementation did not significantly affect the total cardiovascular events (Ebbing, et al., 2008). Thus, the findings do not support the use these vitamins for secondary prevention of coronary artery disease. The Norwegian Vitamin Trial (NORVIT) also found that vitamin supplementation did not reduce the risk of cardiovascular disease recurrence after a heart attack (BÃ ¸naa, et al., 2006). Moreover, preliminary results of the NORVIT suggested that the B vitamin treatment increased risk of cancer, and that such treatment should not be given. Role of Homocysteine in atherosclerosis. 500w Many studies since the 1960s have validated the relationship between hyperhomocysteinemia and risk of atherosclerosis (Eikelboom, et al., 1999). Severe hyperhomocysteinemia (homocysteine levels greater than 100 Â µmol/L) can be caused by several inherited genetic disorders. Foremost among the disorders are mutations in the gene encoding for the major transsulfuration enzyme cystathionine ÃŽ ²-synthase, remethylation enzymes methylenetetrahydrofolate reductase, methionine synthase in the methionine cycle, or defects in vitamin B12 metabolism (Finkelstein, 1998; Kraus, 1998). These genetic conditions lead to extreme elevations of plasma homocysteine and early atherothrombotic disease, where the typical pathologic features of endothelial injury, vascular smooth muscle cell proliferation, and progressive arterial stenosis are observed. While these genetic errors in metabolism are rare, they gave researchers a model for studying cardiovascular injury that was associated with high homocys teine levels. The clinical and pathologic features observed under the conditions of homocystinuria resulted in the homocysteine theory of atherosclerosis that declared high plasma homocysteine concentrations to be responsible for vascular damage. Results of individual studies and meta-analysis have come up with support for the homocysteine theory of atherosclerosis. However, there are still controversy over whether homocysteinemia causes CVD or vice versa, or is it that homocysteinemia is just a marker or indicator of CVD. Mounting evidence for the causal effect of homocysteine was backed up by basic and cellular studies which utilized advanced molecular and genetic techniques. Early studies infer that homocysteine damages the endothelial cells lining the blood vessels and increase the production of vascular smooth muscle (Berg, Tymoczko and Stryer, 2002). From a pathophysiologic point of view, homocysteinemia is associated with increased oxidative stress in the cells, and development of thrombosis (Tyagi, et al., 2005), impaired endothelial function (Stuhlinger, et al., 2001) and the activation of inflammatory pathways that are sensitive to changes in cellular redox states (Weiss, et al., 2003). It was originally proposed that homocysteinemia promotes the accumulation of S-adenosyl homocysteine, a potent inhibitor of cellular DNA methylation (Zou, 2007). The hypomethylation of DNA could affect promoter activity resulting in remodelling of chromatin and changes in the transcription of certain genes. However, it was found that In the case of homocysteinemia, increased tHcy levels cause the hypomethylation of DNA promoter region of the cyclin A gene (Jamaluddin, et al., 2007; Zou, 2007). Cyclin A protein is a sub-unit of cyclin-dependent kinases (CDK) that activates CDK during the cell cycle. In the presence of high tHcy concentration, methylation was inhibited at two CpG sites in the cyclin A promoter, resulting in a 6-fold increase in promoter activity, and therefore increased proliferation of endothelial cells. Homocysteine directly inhibited the activity of DNA methyltransferase I (DNMT1) by 30% (Jamaluddin, et al., 2007). Furthermore, homocysteine reduced binding of methyl CpG binding protein 2, while it also increased the binding of cyclin A promoter to histones H3 and H4 which led to chromatin remodelling. Review methods of analysing homocysteine and differences between measurements. 2800 w Total homocysteine, tHcy, is the sum of all chemical species of homocysteine that is present in the blood (Figure 4) (Ueland, et al., 1993; Refsum, et al., 2004). As mentioned earlier, only a small portion of the tHcy comes from free homocysteine, a large portion comes from mixed disulfides and protein-bound homocysteine. All the assays used to quantify tHcy cleave and convert all these forms into a single species by adding a reducing agent, therefore what is measured is the reduced form. Blood for homocysteine determination is collected in tubes. If plasma homocysteine is to be measured, the tube contains anticoagulants like EDTA, heparin or sodium citrate. If serum homocysteine levels are to be determined, then the samples are allowed to coagulate, which takes thirty minutes and not more, to reduce the risk of increased homocysteine levels coming from the cellular fraction of blood (comprised of red blood cells and platelets). Increased release of tHcy from cells also makes it necessary to centrifuge and separate blood components within 30 minutes of collection. Centrifugation time and speed are relatively low (5 min and 300g respectively, for example) and is performed at 4Â ° C. The supernatant (serum/ plasma fraction) that is collected may be analyzed immediately, or stored for 2-3 weeks in the refrigerator or frozen for 2-3 months (Homocysteine, 2008). It is possible to store the samples once processed because the homocysteine levels have been shown to be stable after separation of plasma/serum from the cellular fraction (Refsum, et al., 2004). There are reports showing that tHcy in serum can remain stable for several years if stored at -70 Â °C. Scheme for the steps followed in the determination of total homocysteine from blood serum or plasma. (Hcy-SR, Hcy-mixed disulfide; EC, electrochemical detection; LC, liquid chromatography; CE-LIF, capillary electrophoresis with laser-induced fluorescence detection; Ab, antibody; EIA, enzyme immunoassay; FPIA, fluorescence polarization immunoassay; MS-MS, tandem mass spectrometry). Figure from Refsum et al., 2004. The first step is to generate free homocysteine by chemical reduction of disulfide bonds using dithioethritol, sodium borohydride, n-tributyl phosphine, 2-mercaptoethanol and phosphine tris (2-carboxyethyl) phosphine, which is water-soluble (Jacobsen, 1998). After the reduction, homocysteine is separated from the cysteine, glutathione and cysteinylglycine, low molecular weight thiols, by HPLC. Homocysteine can also be derivatized for gas chromatography determination/ HPLC or subjected to immunoassay (Jacobsen, 1998; Refsum, et al., 2004). The development of the methodologies for determining total homocysteine started in the 1980s. There are many variants of the methodologies, but they can be categorized into three general methodologies: (1) chromatographic, (2) enzyme immunoassays and, (3) enzyme cycling. Chromatographic methods The earliest methods used were the chromatographic methods, of which high pressure liquid chromatography (HPLC) is more used compared to gas chromatography (GC). Variations in the methodology include the combination of liquid chromatography (LC) with mass spectrometry (MS) and GC with MS. The equipment and the skill requirements for these techniques have limited their use mainly in research laboratories. In the HPLC method, the reduced samples are deproteinated and then directly separated on an ion exchange column. The separated homocysteine molecules undergo derivatization with ninhydrin (a chromophore for colorimetric quantification), or fluorescent compounds (e.g. fluorescamine, SBD-F). Alternatively, the reduced homocysteine molecules are first derivatized before separation through reversed-phase HPLC. 6-Aminoquinolyl-n-hydroxylsuccinimidyl carbamate (AQC) is a fluorophore that gives higher sensitivity when used to derivatize cystathionine and homocysteine (Seo, 2005).The derivatized samples are then passed through an ion exchange column. The length of time that it takes for the sample to go through the column is called the retention time. The samples leaving the column pass through a detector, which could be a UV absorbance detector (spectrophotometer), a fluorescence detector or a mass spectrometer which is a powerful detector. The detectors quantify the samples, and send signa ls to a recorder which produces the chromatogram, where quantities can be seen as peaks. An internal standard of known quantity is used in the determination of the actual quantity of the sample. Normally, the internal standard should have a retention time that is the same as homocysteine. Internal standards are added to the sample tubes and derivatized together with the reduced homocysteine. To quantify homocysteine, the tHcy peak area is compared with the peak area of the standard eluted at the same retention time. Although there are many advancements in the HPLC technology, tHcy quantification using this method is relatively slow, time-consuming, requires very careful sample preparation and handling, and skilled technicians. Sample pre-treatment requires 30-60 minutes, while an HPLC run takes 10-30 minutes. Samples are run sequentially, which makes HPLC not suitable for high throughput determinations. Some laboratories use gas chromatography or gas chromatography with mass spectrometry to get more sensitive results. The principles behind GC and HPLC determination are similar, except that in GC, the samples are vaporized and passed through a gas before elution from a column. Similar to HPLC, GC is also more time-consuming compared to other methods. Homocysteine of dried blood samples have been determined using reversed phase HPLC (Bowron, et al., 2005). In this technique, blood samples were collected in tubes that contain potassium EDTA. After mixing gently, the blood was spotted on filter paper cards that are used in standard neonatal screening. The cards were air-dried and stored at room temperature until analysis. Six-mm discs of the cards were punched out, and incubated in tubes with an internal standard and a deproteinization agent. The produced thiols were then derivatized for fluorescent detection, and separated by reversed phase HPLC. The resulting measurements showed that the method was linear up to a homocysteine concentration of 140 Â µmol/L, in comparison with HPLC of plasma homocysteine which showed a linearity up to 200 Â µmol/L. The coefficient of variation within a batch of measurement was 5% and within batches was 8%. HPLC of plasma samples was only 1% within batch and 4% between batches. Storage of the dried blood sample spots for more than 24 hours in room temperature decreased the homocysteine concentration, probably due to the hemolysis of whole blood during drying. The hemolysis could have been accompanied by the denaturation of enzymes that release homocysteine from blood cells. Overall, although the use of dried blood samples offers flexibility in sampling and storage, the assay had lower precision compared to HPLC. It may not be able to quantify dried blood homocysteine in newborns because its lower limit of detection is above the levels that some healthy neonates have. The method can be used in the detection of homocysteinuria. A more rapid protocol for tHcy measurement is liquid chromatography electrospray ionization-tandem mass spectrometry (LC/MS/MS) (Gempel, et al., 2000). It removes the derivatization step and can perform 400 analyses daily. Blood samples are applied to filter cards that are used for neonatal homocysteine screening. The cards are incubated in the presence of a reducing agent and a deuterated internal standard. The tHcy is then extracted in an acetonitrile solution and loaded on a cyano column. Eluates are ionized with an ion spray device before passing through a mass spectrometer. The procedure makes use of an autosampler, which hastens the analysis. The advantage of the method is the removal of the derivatization step, because the mass spectrum is determined directly. Impurities of the samples are removed by passing them through the cyano column. Since the procedure takes only 3 minutes to finish 1 sample, more samples can be processed in less time compared to HPLC (Gempel, et al., 20 00). An innovation in a high throughput liquid chromatography-tandem mass spectrometry method is the use of a 96-well plate format that skips precipitation, centrifugation and derivatization. Plasma samples, calibrator and control were mixed well in 96-well plates. From each well, an aliquot was transferred to another plate with sodium hydroxide solution (diluent) and dithioethritol (or another suitable reductant). After thorough shaking and a short incubation period, the plate was placed in the autosampler for LC-MS/MS analysis. Comparison of the results with those obtained by HPLC showed that the LC-MS/MS method gave consistently higher homocysteine readings, which was attributed to the use of different standards by the two methods. The biggest advantage of the new method over HPLC was the expense, because LC-MS/MS reduced the costs for material and manpower by more than 50% (Arndt, et al., 2004). Enzyme Immunoassay The immunoassay-based tests for homocysteine were developed in the 1990s. Most assays employ a mouse monoclonal antibody against S-adenosylhomocysteine (SAH), which is formed when adenosine and S-adenosylhomocysteine hydrolase are added to reduced total homocysteine of the plasma/serum sample. The earliest report on the procedure involved the use of flouresceinated SAH as tracer. This method is fluorescent polarization immunoassay or FPIA. After SAH is generated, it is mixed with a fluorescein-tagged SAH. The mixture is coated onto a microtiter plate, then made to bind with mouse anti-SAH (Figure 6). When antibody-SAH bind, the changes in fluorescent polarization of the SAH-tracer are detected and serve as basis for the quantification after a standard curve of known homocysteine calibrators is constructed (Shipchandler and Moore, 1995). The newer versions of enzyme immunoassays were performed on microtiter plates and used the formation of colored complexes to measure absorbance which was used as the basis of the quantification (Figure 7) (Frantzen, et al., 1998). After the conversion of reduced tHcy to SAH, another enzyme is added to the mixture to hydrolyze the excess adenosine. After reaction with a monoclonal anti-SAH antibody, a specific anti-mouse-antibody is added, which forms a colored product that can be detected spectrophotometrically for quantification. The immunoassays are simpler to perform compared to chromatography because they do away with the derivatization step and the expensive equipment needed in chromatographic separation. The method has been proven to be rapid and precise. The coefficient of variation between and within assay is 8% and less than 6% respectively, and the results also correlated well with those obtained using HPLC. The advantage of the immunoassay over HPLC is speed and ease of determination (Frantzen, et al., 1998). Many samples can be run simultaneously on several microtiter plates, which make the immunoassay ideal for routine screening. Furthermore, the technique can be and has been fully automated using robotics technology, and is highly flexible and can be used on different analytical platforms. Other similar imunoassays have been developed. In a variant of the assay, recombinant homocysteine lyase, converts homocysteine into ÃŽ ±-ketoglutarate, with the evolution of ammonia and H2S (Tan, et al., 2000). A second reaction reacts H2S with N,N-Dibutylphenylene diamine (DBPDA) resulting in the formation of form 3,7-bis(dibutyl amino)phenothiazine-5-ium chloride, a highly fluorescent compound. Both highly purified recombinant homocysteine lyase and DBPDA were synthesized in the authors laboratory, which somehow limits the utility of the procedure because their production requires skill and specialized equipment. Another assay utilizes the recombinant enzyme, methionine ÃŽ ³-lyase, and the commercially available fluorophore N,N-diethyl-p-phenylenediamine (Chan, et al., 2005). The rest of the procedure is similar to that used in typical immunoassays. The method can be used in an automated analyzer or manually, which makes it ideal for laboratories that cannot afford the expensive automated analyzers. Although the recombinant enzyme was purified only from a crude extract of E. coli, the results compared well with those that were obtained using HPLC. These results showed that the need for tedious and careful purification procedures can be removed. However, a main drawback was the non-specificity of the enzyme for homocysteine; it also reacts, although with less specificity, with cysteine which is present in larger amounts than homocysteine in the plasma. Thus, the use of pure homocysteine as calibrator is not suitable for the assay. Instead, the recommendation is to use pooled plasma as the cali brator, but this requires the determination of the tHcy by HPLC, which complicates the procedures for small laboratories who do not have access to HPLC equipment. Moreover, the precision of the procedure is also lowered if samples are diluted, because this also reduces the amount of cysteine in the sample relative to its concentration the calibrators. The presence of cysteine is also the cause for the lower sensitivity of this assay, because at lower tHcy levels the background fluorescence due to cysteine is amplified (Chan, et al., 2005). Enzyme cycling The most recent method for tHcy determination is the enzyme cycling method which amplifies the detection signal to improve the sensitivity of the detection (Figure 8) (Dou, et al., 2005). Similar to the previous methodologies, all protein-bound and oxidized forms of homocysteine are first reduced to free homocysteine. Next, methionine and S-adenosyl homocysteine (SAH) are formed from the methylation of free tHcy by the action of the enzyme homocysteine methyltransferase with S-adenosyl methionine as methyl donor or co-substrate. The SAH so formed comes from SAM and the transmethylation reaction, and not from the free homocysteine molecules. Upon formation, SAH is hydrolyzed immediately to homocysteine and adenosine by SAH hydrolase. The homocysteine originating from SAM enters the homocysteine transmethylation reaction catalyzed by homocysteine methyltransferase to form homocysteine and methionine again, resulting in an enzymatic cycle and an increase in detection signals. The reacti ons caus

Western Influence on Japan Essay -- Geography

Western Influence on Japan Japan, as a nation, is a continually changing society. Ever since western nations became involved with Japan, its changes over recent times have increased at a substantial rate. Japan now faces cultural, economical and social differences as a result of the western involvement. The involvement was initiated by the Japanese themselves, beginning during the Meiji Period1 through current times. As time increases, Japan is slowly becoming more 'westernized' because of western involvement. Western involvement is greatly affecting Japan. Western involvement began in Japan during the late 1800's. The Meiji dynasty helped to carry it through, seeing the importance of western ideas. "Learn all you can from the Europeans and Americans"2 was what Emperor Meiji was saying to the Japanese in 1867. During this period, Japan agreed to change it's hereditary authority and class barriers between its people.3 Japan also opened their ports during this period4 and sold fuel to other countries such as the Us.5 The Japanese language took a major turn, too, with the addition of borrowed words from all over the world.6 Japan borrowed the American education system of elementary, middle school and universities during this time.7 A new western style army and universal military conscription program were soon set up by General Yamagata Autamo as well.8 The Meiji period was an important part of Japan's changing western ways. After World War II, Japan changed forever. With the Atomic bomb physically destroying their cities, and their defeat destroying their imperialistic mentalities, Japan was deeply wounded. Japan lost the respect of the world during the war and few people felt sorry f... ...R. Japan. p.113. 22 Davidson, Judith. Japan - Where East Meets West, p.107. 23 Pitts, Forrest R. Japan. p.46. 24 Davidson, Judith. Japan - Where East Meets West, p.101. 25 p.94. 26 Miller, Richard J. and Katoh, Lynn. Japan, p.57-58. 27 Greene, Carol. Enchantment of the World, Japan, p.97. 28 Pitts, Forrest R. Japan. p.78. 29 Davidson, Judith. Japan - Where East Meets West, p.48. Works Cited: Davidson, Judith. Japan - Where East Meets West. Minnesota: Dillon Press Inc., 1983. Greene, Carol. Enchantment of the World, Japan. Chicago: Regensteiner Publishing Enterprises Inc., 1983. Miller, Richard J. and Katoh, Lynn. Japan. New York: Franklyn Watts Inc., 1969. Pitts, Forrest R. Japan. Canada: Fideler Company., 1974. Reischauer, Edwin O. The United States and Japan. Massachusettes: Harvard University Press, 1965.

The Atomic Bomb Helpful or Harmful Essay -- essays papers

The Atomic Bomb Helpful or Harmful There used to be a time in America when the name â€Å"Atomic Bomb† seemed fictional to some, non existent to others,and seemed only a dream to those in the science world. That time is long gone.The day that changed all ideas and opinions about what war was and what is has evolved to be was August 6,1945. President Truman had decided to drop the Atomic bomb in order to end the war and save as many lives as possible. The United States had dropped the bomb on Hiroshima in order to end the war almost instantly and avoid bloody invasion, thereby saving both American and Japanese lives. Whether or not to drop the atomic bomb was president Truman’s decision, faced with this decision Truman researched and asked advisors to share their ideas about the bomb and then made the best decision for the American and Japanese people. â€Å" In an invasion of Japan,the fighting would have been more savage, and the number of lives lost on both sides would of been tragic.†(O’Neal 35) The Atomic bomb was essential in helping to put and en d to the war and saving lives. The decision to drop the atomic bomb was made by President Truman . In his decision he states, â€Å" The only thing that was the right thing to do for the American and Japanese people was to end the war through the use of the atomic bomb.† ( O’Neal 36) Why all the controversy over the bomb? Historians and people today continue to ask this question along with others. Were the Japanese seriously considering surrendering before the bomb was let off, and was the only way the war could of been ended ended was throughout the bomb? The atomic bomb saved lives on both sides and served it’s purpose of helping to end the war. The facts remain the Atomic Bomb that was dropped on Hiroshima killed many civilians, but if the war continued to go on it would of killed many more. When President Truman walked in to the White House he had problems and decisions lying ahead of him that he knew little about.In his first few days of briefing from advisors and committee he had said,†I have to decide Japanese strategy-shall we invade Japan proper or shall we bomb and blockade? That is my hardest decision to date .but I’ll make it when I have all the facts. â€Å"(Takaki 26) As you can see Truman had no intention of making uneducated decisions and he was by no means in a rush to bo... ...tomic bomb was being assembled at the time and it was to be let off on Tokyo, that bomb was never used. The 500,000 lives that were saved in bombing Hiroshima compared to the 100,000 that died in the bombing shows that Truman did in fact make the best decision when lives were at stake. Bibliography: Alperovitz, Gar. The Use of the Atomic Bomb. Chicago : D.C. Heath and Company, 1974. Blow, Michael. The History of the Atomic Bomb. New York ,New York:American Heritage Publishing and Company Inc., 1968. Feis, Herbert. The Atomic Bomb and the end of World War II. Chicago: Princeston University Press, 1866. Kurzman, Dan. Day of the Bomb. New York: McGraw- Hill Book Company, 1986. Nardo, Don. Modern Japan. California: Lucent Books, 1995. O’Neal, Michael. President Truman and the Atomic Bomb. San Diego,California Greenhaven Press, Inc., 1990. Takaki, Ronald. Hiroshima. Canada: Little Brown and Company Limited, 1995. Wheeler, Keith. World WarII Time Life Books. Virginia: Time Life Books Inc., 1983. Wyden, Peter. Day One Before Hiroshima and After. New York: Simon and Schustler, 1984.

Analyzing Dickinson’s Poetry Essay

To analyze Dickinson’s poetry, this paper will involve the analysis of three of her works, `Safe in their Alabaster Chambers†, â€Å"I Heard a Fly Buzz-when I died†, and â€Å"The Brain-is wider than the Sky†. 1. The poems were written in the first person. Since most of her poems tackled the depressing situation of death, the speaker of the poem can in fact be a dead person. However, it seemed that ED may also be assuming an all-observing, all-seeing speaker like God. In the Brain-is wider than the sky, it even seemed that God was in fact the speaker since â€Å"the weight of God† was compared to the â€Å"brain†.   As for the poem’s audiences, it may be that the literary works were directed towards the ‘living’ – people who are not safe within alabaster chambers and who have not heard the buzzing fly as they lay on their deathbeds. 2. In the â€Å"The Brain – is wider than the sky†, there is really no definite setting, it can be likened to any moment of rationalization. In â€Å"I heard a fly buzz when I died†, the setting was in a deathbed while it was perhaps in the cemetery for the poem â€Å"Safe in the alabaster chambers†. The situation was related to dying. It may be that the speaker is already dead, or nearing his death. Nonetheless, the action in the poems remains the same – surrendering to the abyss. 3. Most of the poems had their verbs in the present tense, and in the indicative mood. The style may be to emphasize that the speaker is actually experiencing whatever situation is being imparted in the poems. Such style makes the poems more contemporary and typical, and thus engaging to read and easier to relate to despite the fact that they were written centuries ago. The syntax may also indicate that the poems will be eternal since the action involved is always presented as a current situation. 4. In her poems, Dickinson uses two formal patterns alternatively- tetrameter and trimeter. In every stanza, the first and third lines always have four stresses while there are only three stresses in the second and fourth lines. The rhyme schemes come in the ABCB form. 5. Dickinson uses the slant rhyme in the second and fourth lines of the first two or three stanzas to provide a sense of association and form. In the last stanza however, she then uses a true rhyme also in the last words of the second and fourth stanzas to emphasize conclusions to the proposed action. 6. In â€Å"The Brain is deeper than the sky†, the phrases â€Å"The brain is† and â€Å"The one the other will† were repeated thrice and twice, respectively, to give both indicative and comparative effects. The repetition emphasizes the subject of the poem – â€Å"the brain† – and stresses its association with other elements – the sea, the sky, and the weight of God. 7. To extensively describe the subjects of her poems, Dickinson The poem contained metaphors and personifications to describe her chosen subjects. In one poem, she likened a fly to death perhaps to stress out the repugnance of not being able to experience the simple joys of living. It is also important to note that she always compared the poem’s settings to universally recognizable elements of nature. For example, she likened the stillness of being dead to â€Å"heaves of storm†. 8. The effectiveness of Dickinson’s poems in relaying thematic obsessions may rely on the fact that she uses a mixture of images to convey the setting of her works. In `Safe in their Alabaster Chambers†, Dickinson describes the situation of the dead through their inability to be â€Å"touched by morning†, feel the sunshine, and hear the birds and the bees. She also totally equates death to â€Å"soundlessness†, darkness, and numbness. The same image associations can also be observed in â€Å"I Heard a Fly Buzz-when I died†. However, in contrast to the first poem, the latter’s scenario of soundlessness exempted the buzzing of the fly.   In â€Å"The Brain-is wider than the Sky†, visual comparisons were made with the brain and major elements of nature. 9. In most of the poems, the speaker just describes poem subjects in relation to what she sees, feels, or hears. In the process, she narrates her observations and seemingly creates an underlying story for her works. In these stories, the climactic moment is death and the resolution is one’s total submission to the darkness and numbness of losing her life. 10. Dickinson’s poems are mostly playfully dreadful as they deal with death in relation to bees, sunshine, and castles. Death was portrayed as a very awful situation of being deprived of the small things which make living simply a pleasant experience. Although not portrayed as something gruesome, the description of a death as a natural and inevitable experience adds dread to poem’s tone.

Humor in Midsummer Night’s Dream Essay

Historical records from the late sixteenth century are marked by the severe outcomes of the plague, which spread throughout most of Europe. The increased death rate and famine suppressed cultural development and thus very few works of art or literature were produced. During these devastating years, the young poet William Shakespeare refused to travel to the provinces, away from London, where the plague had reached enormous extents. On the contrary, in 1593 and 1594 he composed the poems Venus and Adonis and Lucrece which were both met by great interest and excitement. These positive responses encouraged Shakespeare and with even stronger efforts he wrote the play A Midsummer Night’s Dream in the beginning of 1595, when the plague was brought under control. This work not only marks the end of famine and suffering in London during the late sixteenth century, but also signifies that the Master is now â€Å"confident in his art, at ease with it, as a man in his dressing-gown†. Categorized as a festive comedy, A Midsummer Night’s Dream is a considerable advance in Shakespeare’s work. The most remarkable evidence for this advance is the skilful usage of different variations of humor in one play. Another distinctive aspect of the work is the incorporation of another play within the borders of A Midsummer Night’s Dream. Analysis of different characteristics of the work should inevitably focus on the story of the craftsmen performing â€Å"Pyramus and Thisbe†, since this part is a representative of Shakespearean comedy and all of its patterns. Therefore, it also portrays the impressionable usage of humor, or more specifically, of literary devices and social characteristics, not only in A Midsummer Night’s Dream but also throughout all Shakespearean comedies. The poet’s works can be roughly categorized in four categories: tragedies, comedies, histories, and romance. All of them are having separate characteristics, tone and style, involving different literary devices and mechanisms. Comedies are usually stories with happy ending, lighthearted patterns and often involving a marriage. They all have a distinct usage of humor, which can be defined as innovative and typically Shakespearean. The plays usually present the struggle of young lovers who have to get over many obstacles in pursue of their feelings and often involve mistaken identities,  separation and unification, heightened tensions within a family, and multiple plots. One very significant trait of the poet’s comedies is that they do not involve any satire, which makes them more lighthearted and easy to process. The common setting of the plots tends to be nature, more specifically the â€Å"green world†. This is one of Shakespeare’s preferred cites since it creates a more casual atmosphere, where jokes and puns easily find their way to the public, unbound by the social norms present the city. Another significance of the setting is the current time period, which constitutes the pattern of the society, its expectations and rules, and its literacy. The poet carefully takes advantage of the prejudices and the ideologies of the public and hence makes his comedies very contemporary, discussing topics that are relevant to the time period. Shakespearean comedies imply the participation of the audience and therefore they are dependent on people’s way of viewing life and way of expression. A play performed during the period of its setting would be far more funny and entertaining than the same play performed nowadays because of the evolved human way of thinking. Nonetheless, Shakespearean comedies contain certain literal devices creating humor, which function in the same way now, as four centuries ago. The story of the amateur and awkward actors in A Midsummer Night’s Dream contains a great deal of those devices, which make it independent upon the public, still involving it in its plot. One of those devices is the use of prose throughout the play within Shakespeare’s work. The poet removes the whole rhyme pattern in order to emphasize the puns and jokes in the course of the plot. When the actors are talking in a plain manner the humor is more easily transferred to the public, since they do not have to perceive the rhyming. Furthermore, the prose contributes to the image of the craftsmen. Their illiteracy and simple-mindedness can be identified easily through their way of expression. The artisans speak in a simple manner, corresponding to their social class standing. Although they are striving to use a more sophisticated language, they mix up grammar and spelling in a very comic way: â€Å"Have you the lion’s part written? Pray you, if it be, give it me; for I am slow of study†. The usage of prose in the play also creates plain humor, which does not need to be interpreted in any way. This is simple poetic device that creates comedy, which is equally funny now and four centuries ago. Sentences as â€Å"I will undertake it† bring about the same reaction of the public regardless of the time period. This mechanism may be the most frequently used one in the part with the artisans, but probably the most efficient device is the word play, created through the misspellings and wrong pronunciations of the craftsmen. Shakespeare takes advantage of the sound similarities between words with completely different meanings and adds the alternatives to the mechanicals’ speech. In their efforts to sound more sophisticated, the artisans mix up words as â€Å"obscenely†(Shakespeare, 68), â€Å"parlous† (Shakespeare 87), â€Å"disfigure† (Shakespeare 88), â€Å"savours, odours† (Shakespeare 89), â€Å"translated† (Shakespeare 91), â€Å"presently† and so forth. Those confusions create different connotations and meanings, thus entertaining the audience. For example, Bottom misuses obscenely, meaning seemly, or more precisely, â€Å"fitly†; instead of â€Å"parlous† Snout should have said â€Å"perilous†, and Bottom completely mixes â€Å"odours, savours, odorous†. All these mistakes build a very efficient humor mechanism that Shakespeare uses in most of his comedies. Another effective literary device that Shakespeare had mastered is the mix of short and long sentences, presenting the interrupted lines of thought of the artisans, their inability to express themselves and their lack of education. It also contributes to the authentic tone of their natural conversation: â€Å"A calendar, a calendar! Look in the almanac – find out moonshine, find out moonshine† (Shakespeare 69). Bottom’s excitement disables him to form complete and meaningful sentences; he wants to express himself as fast as he can, regardless of the way of expression. The frequent usage of â€Å"and†, instead of â€Å"if†, in the beginning of the sentences, shows that the artisans’ thoughts and ideas are flowing during the course of their speech: â€Å"Thus hath he lost sixpence a day during his life: he could not have ‘scaped sixpence a day. And the Duke had not given him sixpence a day for playing Pyramus, I’ll be hanged† (Shakespeare 122). The obvious repetition here also signifies the awkwardness and narrow-mindedness of the mechanicals. Their personalities are also involved in the creation of the remarkable comedy. Except for their illiteracy, simple and narrow mindedness and their awkwardness, the artisans have more traits that build their image of comic characters. Nick Bottom, for example, is a overconfident weaver, who is the central figure in the plot of the story. He has an incredible belief in his abilities and skills and pretends to  dominate over the other mechanicals. Bottom does not accept the fact that he is illiterate and therefore his confused words make him sound ridiculous. He loves to exaggerate and over-dramatize, praising his own self-esteem. The weaver does not realize that the others are not taking him seriously and is not aware of his foolishness. His image may seem absurd and worthy of pity, but it also gains the public’s attention and sympathy. In this way the audience is in the same time laughing at his words and perceiving them as something honest and simple. Bottom is always ready to undertake anything that would make him important. From the play it can be derived that he developed a complex of inferiority, which, nonetheless, does not seem dispiriting but comic. When Peter Quince is giving out the roles for â€Å"Pyramus and Thisbe† Bottom is repeatedly showing his will to act for everyone. After every single role is announced, he is awkwardly trying to portray himself as the most appropriate actor for it: â€Å"That will ask some tears in the true performing of it. If I do it, let the audience look to their eyes: I will move storms, I will condole, in some measure. †( Shakespeare 65). There is an interesting comic reference here to one of the main themes of the play, the use of one’s eyes in love. This is evidence that the story of the amateur actors is representative of the whole play and the humor used is relevant to the main course of A Midsummer Night’s Dream. Finally, Bottom is assigned the role of Pyramus, who is â€Å"a lover that kills himself, most gallant, for love†. There is an obvious contradiction between the images of the mechanical and Pyramus, which adds funny absurdity to the comedy. â€Å"Gallant† is probably the least appropriate description of the tall and clumsy craftsman. Bottom’s confidence often diminishes the role of Peter Quince, another character that entertains the public through his speech and attitude toward the future performance. He is an ordinary carpenter who is not experienced in anything else than his craft. He tries to be the leader of the group that is attempting to put together the play â€Å"Pyramus and Thisbe†. Even though his directions are respected, they usually meet the disapproval of the other artisans. Peter Quince takes advantage of the short-mindedness of his fellows and comes up with irrational justifications of his decisions, which, nonetheless, seem true to the craftsmen: â€Å"That’s all one: you shall play it in a mask, and you may speak as small as you will. † (66 Shakespeare); â€Å"You can play no part but Pyramus; for Pyramus is a sweet-faced man, a proper man as one shall se in a summer’s day, a most lovely, gentlemanlike man† (68 Shakespeare). This is how Peter Quince succeeds in convincing Bottom that he is perfect for the role of Pyramus – using his high self- esteem and need to be granted recognition. Peter Quince further contributes to the comedy by selecting Francis Flute, a bellows-mender, for the role of Thisbe. Even before the actual performance, the audience imagines how the ordinary craftsman is speaking in a â€Å"small† voice, wearing a mask which is supposed to hide his beard. Flute’s determination to overcome his sense of masculinity, which is ridiculed in the play, is a remarkable sacrifice for the success of the performance. His extreme readiness fascinates the public and presents the seriousness of the artisans. Also from this scene it can be derived that the craftsmen are putting all their efforts into the performance. Robin Starveling, first chosen to play Thisbe’s mother, readily accepts his role, which would make him more than an ordinary tailor. Later on in the play, he is assigned the role of the moonshine, which does not discourage him. The same comic enthusiasm is also shared by Tom Snout, the thinker who is chosen to play Pyramus’s father but later on receives the role of the wall separating Pyramus and Thisbe. Together with Snug, the joiner chosen to play a lion, they both feel they have significant parts in the performance, and every one of them is concerned about his role. For instance, Snug is worried that his roaring may frighten the ladies in the audience. He is determined that his acting will convince the public that he is a real lion and the other craftsmen agree with him. Their funny concerns are followed by even more comic solutions – there will be a Prologue who will reveal the true personalities of the craftsmen before the performance, and Snug will show a part of his face to comfort the ladies in the audience. In this way the suspense of the whole performance will be ruined, but the artisans are afraid that their acting skills may be too sophisticated. The craftsmen’s low level of education, short and narrow mindedness and awkward speech play a great role in the development of the comedy. They are all ordinary characters, from the low working class who have one and the same goal – to achieve something remarkable and worthy of respect. Despite their comic lack of sophistication, they succeed in entertaining the audience in another, more original and funny way. The play of â€Å"Pyramus and Thisbe† is a tragedy but their performance can be more precisely defined as a tragic comedy. The actual performance is played during the celebrations of Theseus’s marriage and even though it is inappropriate for such occasion, it turns out to be successful. In the beginning the audience is confused by the strange and amateur acting, but finally the craftsmen appear to be the culmination of the whole festival. The performance is welcomed by a â€Å"flourish of trumpets†, which are very inappropriate for the following fiasco. There is a distinct comic contrast between the flourish of the trumpets, usually meant to signify a glamorous work, and the impressions following the play. Peter Quince, taken the role of the Prologue, confidently steps onto the scene and starts reading from the scroll, unintentionally changing its meaning by mistaking its punctuation: â€Å"That you should think, we come not to offend, but with good will. To show our simple skill, that is the true beginning of our end. † Although this seems funny to the reader, the audience in the play is first confused by the illiteracy of the Prologue: This fellow doth not stand upon points† (Shakespeare 129), meaning that he is not â€Å"punctilious†. At first, Theseus is embarrassed by the awkward performance of Quince and wonders if there is a reason for his mistakes. Without being worried about his weak performance, the determined craftsman continues his important speech, full of comic sentences, as â€Å"he bravely breached his boiling bloody breast†. The true meaning of â€Å"breached† is actually stabbed but Shakespeare uses this word to create a comic alliteration. After the Prologue has reassured the audience, the reader encounters another comic effect of the play – the characteristics of the Wall, played by Snout. It is intentionally described as alive and moving: â€Å"Wall parts its fingers† (Shakespeare 130). The Wall is personified which makes the whole performance even more ridiculous and funny. As the other craftsmen, Snug also wants to be noticed in the performance and thus overacts, making his role more than absurd. Robin Starveling and Snug, acting as the moonshine and the lion, also take their parts more seriously than they should have and contribute to the comic tragedy that the play â€Å"Pyramus and Thisbe† eventually becomes. The audience, although confused by the whole performance, is entertained and the efforts of the craftsmen are justified. They reached their goal of producing something for which they will be acknowledge and respected, even though their intentions were not fulfilled. The story of the artisans performing the play â€Å"Pyramus and Thisbe† contains plenty of humor devices and comic scenes, which make it representative of Shakespearean comedies in many ways. First of all the small play represents the struggle of young lovers to overcome the difficulties set by the circumstances in pursue of their feelings, which is a typical theme for a tragedy, but after the performance it is also associated with humor and comedy. Furthermore, the amateur actors represent the illiteracy of the low class. Shakespeare recognizes the importance of this widespread for the particular time period problem, but in the same time he portrays the comic part of it and praises the artisans’ strive to achieve something meaningful. By his usage of humor he is not criticizing the artisans; he rather justifies their difficulties with grammar and expression in general. The play â€Å"Pyramus and Thisbe† is not a lyrical digression from the main topic of A Midsummer Night’s Dream. It presents another outcome of the situation with the lovers, more undesirable and lacking a happy end. Shakespeare produces a play within the play to further entertain the audience and show the comedic character of his work. Another interpretation of the story of â€Å"Pyramus and Thisbe† may be the initial intention of Shakespeare to write a tragedy, not a comedy. By the time he writes A Midsummer Nights Dream, he has mastered to an extent the tragic genre, and feels more comfortable producing such pieces of literature. The occasion though, a marriage celebration, requires from him to create a rather entertaining work. Maybe through the incorporation of the small play, Shakespeare implies that his original idea was to create another tragedy. He does not allow the audience to feel the tragic nature of â€Å"Pyramus and Thisbe† by using a great deal of humor in it. Even though Shakespeare produces A Midsummer Night’s Dream in a period of time when people are not concerned about art and literature, but about their survival, the play has great success ever since then. The conventional humor used to portray the lovers’ struggles seems untouched by time and the evolving human way of thinking. Four centuries after it was written, the work still fascinates with its characteristics and mechanisms. Comedies are generally a very hard genre because what is funny today may not be funny at all tomorrow. Despite that fact, Shakespeare’s genius manages to create a universal humor, using comical devices that are still interesting and entertaining. By those devices he creates a parallel world where time is a relative term and where humor and laughter are the driving forces – world where people are entertained in spite of the social norms and rules. Bibliography Biscay, Matt. â€Å"A Midsummer Night’s Dream: A Comedy. † Skyminds. net. 2006. Skyminds. net. 08 Nov 2006 . Mahony, Simon. â€Å"Simon Mahony Academic Stuff. † Pyramus and Thisbe in Shakespeare and Ovid. 2002. King’s College London. 5 Nov 2006 . McFarland, Thomas. Shakespeare’s Pastoral Comedy. North Carolina, USA: The University of North Carolina Press, 1972. Parrot, Thomas. Shakespearean Comedy. New York, USA: Russel & Russel, 1962. Shakespeare, William. A Midsummer Night’s Dream. 2003. Editor: Brian Gibbons. United Kingdom: Cambridge University Press, 2006. Teague, Francis. Acting Funny: Comic Thoery and Practice in Shakespeare’s Plays. London: Associated University Presses, 1994.