Isotope Coded Affinity Tag: Applications and Benefits

Isotope Coded Affinity Tag: Applications and Benefits Proteomics is a vital and necessary branch of science targeted at the in-depth study of proteins and their structure to understand their function; as an important pharmacological tool in drug discovery and drug development. The most widely used analytical approach to protein separation and quantification, usually involves integrating protein separation by 2D polyacrylamide gel electrophoresis with micro capillary reverse phase-liquid chromatography protein identification; and finally, detection by mass spectrometry. However, the presence of limitations such as the lack of automation and high costs associated within the combination technique led to the research and introduction of a better and more reliable technique involving the use of isotope coded affinity tags (ICAT). This report looks at the history of isotope coded affinity tags, its advantages over 2D electrophoretic techniques, the principles associated with the technique, its development over the years and finally its application and contribution to the growth and development of analytical science. It also aims to comment on future developmental routes for the technology. TABLE OF CONTENTS (Jump to) A. Background B. Introduction to protein quantification B.1. 2D Polyacrylamide Gel Electrophoresis B.2. Reverse Phase High Liquid Chromatography B.3. Mass Spectrometry B.4. Problems associated with 2DLC-MS combination technique C. Introduction to Isotope Coded Affinity Tags (ICATs)   C.1. Major advancements in isotope coded affinity tag approach D. Principles of Isotope Coded Affinity Tags (ICATs) D.1. Protein Sampling D.2. ICAT reagent Tagging D.3. Peptide Isolation D.4. Protein quantification D.5. Peptide identification E. Applications of Isotope Coded Affinity Tags (ICATs) E.1. Applications in the quantitative identification of cancer  biomarkers E.2. Applications in the quantification of antimalarial drugs  and their metabolites in biological fluids E.3. Quantification of protein expression in oxidative-stressed liver  cells as a therapeutic target for the treatment of liver disease E.4. Quantitative analysis of defaulted proteins present in the brain as  a therapeutic target for the treatment of brain diseases E.5. Applications in the proteomic analysis of recombinant proteins F. Future Development of Isotope Coded Affinity Tags (ICATs) BACKGROUND Proteins are very important components of biologically active systems and some of their functions include structural foundation (connective tissue), transportation (carrier proteins) or immunity (antibodies). Specific and selective protein-protein interactions within the body are the basis for key metabolic and kinetic pathways within living organisms. A disruption in a specific proteins interaction and function, leading to a small or large interference in the subsequent metabolic pathway within the body due to any number of reasons; is the major cause of disease which if not dealt with, can lead to fatality. For this reason, Proteomics is a vital and necessary branch of science targeted at the in-depth study of proteins and their structures; to understand their function as an important pharmacological tool in drug discovery and drug development. Developments in proteomics and genomics over the years through quantitative-structure activity relationship (QSAR) studies and computer aid ed drug design (CADD), has helped to identify novel drugs and their targets for action. INTRODUCTION TO PROTEIN QUANTIFICATION The use of Isotope coded affinity tags as a protein quantification method in proteomics was first developed in 1999 by Aebersold et al. to aid the detection and purification of recombinant proteins[1]. Before the research done in 1999, most widely used approaches to protein quantification were done by 2D Polyacrylamide Gel Electrophoresis (2D PAGE) combined with micro-capillary Liquid reversed phase liquid chromatography (2DLC) and novel electrospray ionization (ESI-MS) or tandem mass spectrometry (MS-MS) technique for detection [2]. B.1. 2D POLYACRYLAMIDE GEL ELECTROPHORESIS This is because 2D Polyacrylamide Gel Electrophoresis (2D PAGE) is very well known for its sensitivity and high resolving separation power. It is also a highly adaptable technique, and its resourcefulness makes it highly sort after for the separation of biological molecules including proteins, based on both physiochemical properties and other chemical-specific interactions. The limit of detection is well documented to a resolution of more than 7000 macromolecules in a singular separation. A large variety and combination of solvents and additives can be used with 2D-PAGE electrophoretic technique to ensure analytes solubility within complex protein mixtures. B.2. REVERSE PHASE LIQUID CHROMATOGRAPHY The inclusion of liquid chromatography as a second separation step also allows for the further separation of the protein mixtures based on difference in retention properties of the components. Recent breakthrough in the analytical approach to liquid chromatography involves the used of two HPLC pumps connected through a detailed 6-port valve system; which results in a more comprehensive separation by gradient elution of complex protein mixtures at high speed and quick run times. B.3. MASS SPECTROMETRY Finally, a mass spectrometric technique (Electrospray ionization (EIMS) or tandem mass spectrometry (MSMS)) which provides a UV detection of protein and measures the mass to charge ratios of the eluted peptides is employed. The detector produces a comprehensive chromatogram by plotting UV signals against their corresponding reverse phase retention times, and then the ESI-MS/MS-MS provides mass information for the eluted peptides. Figure 2: The construction of a 2DLC column and its interface with mass spectrometry. (A) A pressure bomb is used for column packing and sample loading. (B) The flow rate of in the 2-D column is controlled at 100-300 nL/min, and ESI is achieved by applying 2 kV to the gold wire.[4 5] B.4. PROBLEMS ASSOCIATED WITH THE 2DLC-MS COMBINATION TECHNIQUE However, in spite of the popularity of the combination technique, a number of limitations exist that makes the technique far from perfect. It has been documented that complex proteins and peptides with very high alkalinity or basicity and some trans-membrane proteins cannot be separated by this combination method. Also during total cell analysis, the combinatorial technique was found to readily accommodate highly abundant protein separation with the lower abundant proteins being scarcely detected. The over process also requires several sequential stages including difficult techniques such as in-gel digestion; making the combination technique highly labour intensive, difficult to automate and hence non-cost effective. This called for a further development in proteomic research to overcome these problems by possibly avoiding the separation step by electrophoresis and hence the introduction of the use of novel Isotope coded affinity tags (ICAT). INTRODUCTION TO ISOTOPE CODED AFFINITY TAGS The approach of isotope coded affinity tagging mainly combined with a form of high performance liquid chromatography and tandem mass spectrometry (LC-MS/MS) is a relatively new and improved method used in proteomics for the precise quantification and identification of protein sequences within simple or complex protein mixtures. It has been documented to be simpler as it is capable of directly quantifying the proteins from complex mixtures, eliminating the electrophoretic stage. This makes isotope coded affinity tagging more efficient, easily-automated and hence a lot less labour and cost intensive than the electrophoretic process. The use of ICAT is the new and preferred analytical method for protein quantification. Isotope coded affinity tagging is based on a class of chemical reagents called Isotope coded affinity tags (ICAT). The ICAT reagent occurs in two forms depending on the number of deuteriums; light containing none or heavy containing eight. ICAT reagents are made up of three major functional units: A distinct chemically reactive group responsible for the selective labelling of the SH groups of thiol (cysteine) residues, An isotope coded linker responsible for the soluble properties of the reagent and it also provides a site for the addition of the isotopic label, And a biotin affinity tag used to achieve protein isolation and identification. It depends on the principle of strong binding interaction of biotin and avidin. C 1. MAJOR ADVANCEMENTS IN ISOTOPE CODED AFFINITY TAG LABELLING Since the technique was initially introduced in 1999 for the labelling of protein mixtures at low levels, there have been valuable technological advancements in the approach using isotope coded affinity tags (ICATs) within the pharmaceutical industry. These include: The design and modification of affinity tags to improve on the chromatographic separation process. [25] The use of variable peptide specific affinity tags to maximise large-scale quantification on individual processes. [25] An introduction to the combination of different tags to achieve maximum proteome industry [21] The use of exopeptidases to efficiently remove the affinity tags from the peptides in the purification stage [22, 23] D. PRINCIPLES OF ISOTOPE CODED AFFINITY TAG (ICAT) APPROACH Isotope coded affinity tags are used for identifying and quantifying the protein content of two different cell states or population within a mixture. The technique is based largely on two concepts: The peptide sequence of the protein to be quantified (between 5-25 Amino acids long) contains sufficient information to identify that unique protein. And those peptides tagged with the light and heavy reagents respectively are chemically identical and hence serve as very ideal internal standards for quantification. Figure 4. A schematic diagram for the ICAT approach to protein quantification. The principles of Isotope coded affinity tags as documented by Aebersold et al. are divided into four stages: Sampling, Tagging, Isolation and Quantification. D.1. PROTEIN SAMPLING Firstly, two different protein samples containing reduced cysteine (thiol) side chains are individually derived; by breaking down the cell structure, and isolating and extracting the proteins required from the cell. D.2. ICAT REAGENT TAGGING For one of the protein samples, the light form of the ICAT reagent (containing zero deuterium) is introduced to covalently bind to the SH cysteine residues; whilst for the other, the heavy form of ICAT reagent (containing eight deuterium) is used. The individual labelled mixtures represent different cell states or populations. The two samples are then combined into one complex protein mixture and a protease enzyme is added to cut-up or cleave the larger protein molecules into tagged smaller peptides fragments. D.3. PEPTIDE ISOLATION Avidin is then introduced to the mixture to act as a magnet and due to the strong and highly specific binding interaction of biotin and avidin, the ICAT-tagged peptides are isolated from the mixture through affinity chromatography. The isolated peptides are then analysed and separated by micro-capillary high performance liquid chromatography- mass spectrometry (HPLC-MS/MS). D.4. PROTEIN QUANTIFICATION This is the most important step of the analytical process as the quantity and sequence identity of the proteins from which the tagged peptides originated, are automatically determined. Quantification is achieved by comparing the integrated peak intensities for simultaneously eluted pairs of identical, doubly charged peptide ions. The pair corresponds to the two different forms of the ICAT reagent with the mass spectrometer running successively in two modes. One mode measures the comparative fragmenting of peptides eluting from the micro-capillary column whilst the other records the sequence information of the tagged peptides in the same molar ratios as the corresponding proteins. This also means that the chemically identical ICAT-labelled peptide ions are readily identified because as they co-elute, they differ in mass-to-charge (m/z) ratio because of an 8 deuterium difference in the mass of the ICAT-reagents. D.5. PEPTIDE IDENTIFICATION The final stage of isotope coded affinity tagging involves an automated correlation with protein sequence data banks using algorithms and permutations, to identify the protein from which the sequenced peptide originated and hence identify the protein. A combination of all results generated on the chromatogram by the mass spectrometer; and analysis of the ICAT reagent-labelled peptides therefore determines the relative quantities as well as the sequence identities of the components of protein mixtures in a single automated operation. In mass spectrometry, the ratios between the intensities of the lower and upper mass components of these pairs of peaks provide an accurate measure of the relative abundance of the peptides (and hence the proteins) in the original cell pools because the MS intensity response to a given peptide is independent of the isotopic composition of the ICAT reagents. E. APPLICATIONS OF ISOTOPE CODED AFFINITY TAGS The use of ICAT reagent -labelled internal standards, has now become a common and fundamental practice in quantitative mass spectrometry. It has been researched to great advantage in a number of different fields of biochemistry. E.1. Quantitative identification of Cancer biomarkers [9,10] Analytical methods that employ isotope coded affinity tags are very useful and hence popular in the development of high throughput approach to early cancer detection in humans. [9]The significant quantification and identification of cancer biomarkers using ICAT reagents is a therapeutic target for cancer treatment. In this case, protein samples containing cancerous and non-cancerous cells are denatured and reduced to expose the cysteine -SH peptide residues contained. They can then subsequently labelled with the light or heavy forms of isotope coded affinity tags in vivo using stable isotopic labelling (SILAC; (e.g., 2H, 13C, 15N, and 18O)) or in vitro using isobaric tags (iTRAQ). This approach allows expressed proteins and peptides in malignant, cancer-derived cells to be compared with non-cancerous cells.[8] The use of labelled peptides as internal standards allows for relative and/or absolute estimation and quantification of the abundance of the differential proteins present. Emer ging technologies such as the use of protein microarrays are opportunities presently being researched and developed for future improvements in cancer biomarker identification. [10] E.2. Quantification of antimalarial drugs and their metabolites in biological fluids [7] Malaria is a deadly disease responsible for millions of deaths every year, in many tropical and developing countries. Antimalarial drugs such as chloroquine, mefloquine and pyrimethamine and their metabolites; interact with specific dihydrofolate enzymatic sites in plasmodium falciparum malaria. Since enzymes are largely made up of proteins, many enzymatic functions are made up of peptide peptide interactions. Isotope coded affinity tagging combined with high performance liquid chromatography has been documented by Kalpesh N. P. et al, 2010 [7] to be a reliable method for the selective determination and quantification of these potent antimalarial drugs in biological fluids. ICAT reagents are very useful in the extraction stage of the antimalarial drug from a biological matrix as they provide high peptide selectivity and specificity, to avoid interference from multiple antimalarial combination, or endogenous peptides that exist within the matrix. The use of the ICAT approach has grea tly aided research and development into the pharmacokinetics of different antimalarial drugs especially Chloroquine.[7,8] E.3. Quantification of protein expression in oxidative-stressed liver cells as a therapeutic target for the treatment of liver disease [12] A major pathogenic event recurrent in several variations of liver diseases in humans, involves oxidative stress of the liver caused by the formation of reactive oxygen species. Hepatocytes normally have mechanisms responsible for the regulation of oxidative and anti-oxidative molecules within the cell. However, the presence of reactive oxygen species in the liver affects major cellular components including cell proteins, and eventually, the cells regulatory ability. This leads to metabolic or proliferative liver disease and eventual cell fatality.[13] Reactive oxygen species (ROS) are largely represented by mitochondria and cytochrome P450 enzymes in liver cells. The expression of certain protein molecules termed as biomarkers within oxidative-stressed liver cells, and their subsequent quantification using ICAT reagents, can enable an early detection of liver disease. It can also allow for the progressive monitoring of liver damage as a therapeutic target to the treatment of liver disease.[15] E.4. Quantitative analysis of defaulted proteins present in the brain as a therapeutic target for the treatment of brain diseases. The brain is a very complex structure, vital to the existence of mankind. However, a lot of the underlying mechanisms responsible for the normal function and mis-function of the brain have not been fully researched. Research into quantitatively characterising the human brain proteome and using the analysis to understand important cell signalling mechanisms [16], is a very important area of neuropoteomics (i.e. proteomic research and development). The large scale use of stable isotope coded affinity tags in quantitative analysis of complex brain matrixes has helped to provide internal standards for relevant peptides that are chemically similar but isotopically different. These internal standards can be used to correctly identify important biomarkers present in the brain as in epilepsy[17]; or absent biomarkers as in the pathogenesis of Parkinsons disease[18]. E.5. Applications in the proteomic analysis of recombinant proteins High-throughput approaches to the quantification and identification of proteins, is widely applied in the industrial synthesis of therapeutic enzymes. [19] Proteomic analysis on most recombinant proteins, struggle with very low yields and poor solubility which greatly affects the ability to achieve high-throughput protein purification. Quantitative methods that employ isotope coded affinity tags have been documented to be the only way to achieve selective high-throughput protein purification with improved yields, solubility and folding of the recombinant protein, during the process [19]. This is because, purification processes by biotin affinity normal resulting in great yields of over 90%, making it very economically favourable. Combinations of two or more isotopic tags are typically needed to make the most of high-throughput screening.[1] THE FUTURE OF ISOTOPE CODED AFFINITY TAGS (ICATs) The main application area of isotope coded affinity approach is in the identification of biomarkers as a therapeutic target for disease treatment and prevention. The future of analytical techniques that use Isotope coded affinity tags for peptide-labelling includes:

Sylvia Plath :: Biography Biographies Essays

Sylvia Plath      Ã‚  Ã‚  Ã‚   Sylvia Plath was a remarkable twentieth century American poet. Her poetry focused on depression, aspects on suicide, death, savage imagery, self-destruction and painful feelings of women. Plath attempts to exorcise the oppressive male figures that haunted her life served as one of the fundamental themes in her poetry.    Her poetry is a good example on how "suffering and transformation could be within traditional poetic contexts" (Initiation p.142). She also believed that a poem "must give an expression to the poet's own anguish because suffering has become the central fact of historical and personal existence" (Initiation p.143). This is what she believed and how she dealt with her problems by expressing her feelings through poetry. Though what was expressed in her poems also portrayed her fate in suicide.    Sylvia Plath was born on October 27, 1932 in Boston, Massachusetts to Otto and Aurelia Plath. Her father, Otto Plath was a German biology professor at Boston University. Her mother, Aurelia, was a high school English teacher, until she married and became a homemaker. When Sylvia was only eight, her father died from complications of undiagnosed diabetes, which also scarred her for life. At this same age she started her career as writer she published her first couplet in the Boston Sunday Herald, and since then has persistently worked on poetry and her writings.    In high school, she was a remarkably intelligent, popular, student. She was the typical "Straight A's" girl. As a member of the National Honors Society, she received a scholarship to attend Smith College in 1950. While studying creative writing and graphic arts in her third year of college, she was a guest editor in Mademoiselle Magazine. Shortly after that, on August 24, 1953, because of extreme depression, she attempted to commit suicide for the first time by taking a large dose of sleeping pills. She was later treated with intense psychotherapy and electroshock therapy in a private hospital. After a long recovery, she returned to Smith College and graduated in 1954. This incident is well described in the Bell Jar, her second published novel.    By now her career as a poet and writer was not going well, after forty-five rejections from newspapers and magazines, Seventeen magazine agreed to have one of her stories to be published. Later, it was announced that she had received third place in Seventeen Magazine's writing contest. Sylvia Plath :: Biography Biographies Essays Sylvia Plath      Ã‚  Ã‚  Ã‚   Sylvia Plath was a remarkable twentieth century American poet. Her poetry focused on depression, aspects on suicide, death, savage imagery, self-destruction and painful feelings of women. Plath attempts to exorcise the oppressive male figures that haunted her life served as one of the fundamental themes in her poetry.    Her poetry is a good example on how "suffering and transformation could be within traditional poetic contexts" (Initiation p.142). She also believed that a poem "must give an expression to the poet's own anguish because suffering has become the central fact of historical and personal existence" (Initiation p.143). This is what she believed and how she dealt with her problems by expressing her feelings through poetry. Though what was expressed in her poems also portrayed her fate in suicide.    Sylvia Plath was born on October 27, 1932 in Boston, Massachusetts to Otto and Aurelia Plath. Her father, Otto Plath was a German biology professor at Boston University. Her mother, Aurelia, was a high school English teacher, until she married and became a homemaker. When Sylvia was only eight, her father died from complications of undiagnosed diabetes, which also scarred her for life. At this same age she started her career as writer she published her first couplet in the Boston Sunday Herald, and since then has persistently worked on poetry and her writings.    In high school, she was a remarkably intelligent, popular, student. She was the typical "Straight A's" girl. As a member of the National Honors Society, she received a scholarship to attend Smith College in 1950. While studying creative writing and graphic arts in her third year of college, she was a guest editor in Mademoiselle Magazine. Shortly after that, on August 24, 1953, because of extreme depression, she attempted to commit suicide for the first time by taking a large dose of sleeping pills. She was later treated with intense psychotherapy and electroshock therapy in a private hospital. After a long recovery, she returned to Smith College and graduated in 1954. This incident is well described in the Bell Jar, her second published novel.    By now her career as a poet and writer was not going well, after forty-five rejections from newspapers and magazines, Seventeen magazine agreed to have one of her stories to be published. Later, it was announced that she had received third place in Seventeen Magazine's writing contest.

Assignment 2 †Why Teaching Essay

It is important not only for you, but for your Field Supervisor and me, to know what led you to enter the teaching profession. For this assignment you are to write a 300-500 word essay on â€Å"Why I Elected to Travel the Road to Teaching. † ***The first part of your paper will explain your life experiences which led you to pursue a career in teaching. ***The second part of your paper will name/identify the 3 main keys to success as identified in â€Å"Keys to Success for New Teachers. † ***The third part of this paper will explain how the knowledge of these keys can help you be a successful teacher? (You will probably need to review these ‘keys’ found in the course material. ) Formal writing is required. Your paper must incorporate correct sentence structure, punctuation, capitalization and grammar. EDTC 5100 – Assignment 2 Name: Cameron Guidry Last 4 digits of SSN#: 2381 1. Why I Elected to Travel the Road to Teaching My road to becoming a teacher is perhaps less romantic than some would like. I didn’t enjoy school when I was younger and felt no inclination to be a teacher. I did, however, find a passion for learning and writing when I was earning my undergraduate degree at The University of Kansas. Meeting instructors who were excited about their subjects got me excited too, and eventually I was getting excited all on my own. The experience was entirely foreign to me. I was reading just to read and writing without being told. I had spent my four years of high school counting the hours until it was time to leave, and it was the realization that this didn’t have to be the high school experience that pushed me toward teaching as a career. I still have a passion for the subject, and I am currently earning a PhD in English, an endeavor that is entirely fueled by my own passion. It is my hope, and experience up to this point, that I can inject that enthusiasm into my classroom. I spent two years teaching at the university level, and found success. It was as I had imagined; I had students who entered my room uninterested and left my room well equipped readers. The issue I saw was that I would only have the opportunity to interact with those fortunate few that made it to my college classroom, and it was my desire to offer what I could to a more diverse group, perhaps illuminating a possibility that wouldn’t be clear without my presence. I don’t expect to create a graduating class of English majors, but I do believe that I can provide the role model that my students can benefit from. I feel an obligation, one that I created myself, to educate. It’s not an obligation I fulfill begrudgingly; it is one that I happily attempt to answer and is my road to teaching. 2. Name the Three Keys They are be reasonable, organize your life, and reflect.3. Tell how the keys can help you to be a successful teacher? These three keys are helpful for life in general, but as a teacher I am finding myself constantly on my back foot while answering questions and asking someone to take their seat. It can be overwhelming, but by finding my center outside of the classroom, and achieving some understanding before the bell rings, attempting to control the chaos becomes a managable thing.Additional Comments:

Sport Tourism in Manchester, United Kingdom The WritePass Journal

Sport Tourism in Manchester, United Kingdom Abstract Sport Tourism in Manchester, United Kingdom , pp. 1). Lacking the infrastructure support would hamper the overall implementation of this avenue, making regulatory partnership vital.   Manchester has found itself in positive area by building a partnership with both the social and legislative elements. Manchester has recognized that the area of sport is a very popular cultural draw with the potential to spread good will towards the entire city and region (Tallon 2010, pp. 239).   This facet of their plan not only built a solid foundation, but expanded the accompany revenue base.   Regulators instituted policies that have resulted in the construction of the City of Manchester Stadium, with the explicit goal of revitalizing the entire district.   This approach to the revitalisation effort has been heralded as a solid foundation upon which to build the regeneration of Manchester’s entire infrastructure (Tallon 2010, pp. 239). Underlying policies that are conducive to the sport arena are credited for energizing the tourist trade, transforming the image of the city itself as well as creating a sustainable form of industry that can serve to propel the city into the next era.   Each of these components adds value, yet, the potential for a substantial lack of cohesiveness do es exist (Tallon 2010, pp. 239) Employing governmental assistance that designated Greater Manchester an ‘Enterprise Zone’ in 1987, there has been a sustained drive to capitalize on the potential for urban tourism centred on the area of sport (Tallon 2010, pp. 52). This on-going assistance has been a tremendous benefit to the implementation of the urban renewal plan. As a result of the on-going campaign to build economic progress, regulators have actively worked to instil a tourism friendly element that is a continual boon to the urban recovery process in Manchester (Weed 2010, pp. 187). Alongside the recognition of the potential revenue to be found in the tourist trade, Manchester has worked unceasingly to put itself in a better strategic marketing position.   It is the combination of forward thinking policy in conjunction with an adaptable industry that demonstrates Manchester’s long term commitment to the area of urban tourism. Economic and Social Context In a very public effort to rebrand the city in the 1990’s, Manchester adopted the new motto â€Å"The life and Soul of Britain† (Spirou 2011, pp. 112). A demonstrative first step allows a positive perception to begin. This rededication was a precursor of the effort to improve the social and cultural perception of Manchester as a destination. Acting on the potential for an increase in   revenue, Manchester rightly pursued the industry of sport as a method to address this deficiency (Weed 2010, pp. 187). Succeeding in attaining their goal of the Common Wealth Games of 2002this economic activity served to lift the city above the dismal outlook with the further positive ramification of being judged relied upon to do the same for   many similar progressive endeavours   (Weed 2010, pp. 187).   This is a positive element in that each project further strengthens the infrastructure. Manchester’s demonstrated the widely felt social imperative to look beyond the current methods of creating revenue in order to make the most of the possibilities (Ashworth and Page 2010, pp. 1).   This approach is in line with   the working recognition for the need to employ an approach that connects the infrastructure of social sciences, with a focus on the element of urban studies to industry.   Manchester’s ability to embrace the social science aspect has led to a tourism industry that has the potential to continue to expand (Ashworth and Page 2010, pp. 1).   An associated increase in revenue and visitors is a positive credit to the underlying effort. The   implementing of the urban tourism plan has yielded substantial benefits to the social and cultural fabric of Manchester (Law 2010, p. 129).   Underlying infrastructure such as road and regional travel has drastically improved as a result of the urban tourism drive.   The establishment of stronger underlying elements enables the wide variety of non-sport related activities to benefit from the industry as well (Law 2010, pp. 129).   Through the increased capacity to travel easily, more visitors have been attributed with coming to the city, providing a wealth of revenue for many of the industries that rest well outside of the sport focus. Yet, this also brings in the potential for related issues that could detract from the sport centred focus of the city (Law 2010, pp. 130.)   The lack of proper application has the potential to result in the splitting of vital resources to the detriment of everyone. Manchester was able to learn from their bids for the Olympic Games, adapt and make a successful bid for the Commonwealth Games (Cook and Ward 2011, pp. 2519).   This is clear demonstration that the legislative and social perception was ripe for the development of an industry mechanism with the capacity to help them succeed. Combining the element of politics, social responsibility and ethical practice Manchester has managed to achieve a state of relative prosperity that will lend credence to the spirit of urban tourisms vital capacity to lend aid to struggling economies. Competing Interests Alongside the development of Manchester as ‘SportCity’, there are the separate industries that must compete in order to survive (Smith 2013, pp. 385). This recognition requires that any successful long term plan must include the capacity to bring in each of the disparate elements in such a manner that it promotes the whole. Modern studies on the benefits of creating a sport centred industry have been found to be beneficial, although the lack of adequate planning has the inherent capability of hobbling the industries that have little to do the with sport (Smith 2013, pp. 385).   In the drive to enable the full range of economic benefits, ill-considered actions can have a tremendous impact on the remaining components of any cities combined industry. An emerging market that has found turbulence in Manchester sport is the market for the gay community (Hughes 2003, pp. 152).   Many argue that the perception of the gay lifestyle is in direct contrast to the effort to establish a sport destination spot. Targeting a market with substantial revenue, yet possessing potentially negative aspects when interacting with sport centred marketing, there is a real need to develop an overall approach that provides an inclusive element for progress (Hughes 2003, pp. 152).   The ability to include the often contrasting positions of the competing groups in the city provides an ample illustration of the capability of the regional government to conduct large scale tourist attractions. In an effort to address the diverse industries vital to the city, Manchester created a council referred to as the Employment in Construction Charter with the focus of linking the public sector to the private companies in order to distribute the growing revenue (Spirou 2011, pp. 206). This is a direct reflection of the effort to balance the division of power in order to promote a policy of growth for each of the separate factions. During the bid for the Commonwealth Games, Manchester was credited with not only appealing to the larger international community, but providing substantial providence for the local retailers as well (Cook and Ward 2011, pp. 2525). This demonstrates that Manchester learned from the prior Olympic Games Bid and adjusted to meet the requirements of the diverse elements in a successful manner. Effectiveness of Policy The City of Manchester efforts to revitalize the region through the utilization of the urban tourist area of sport has yielded significant results.   The cities recognition that tourism is one of the highest revenue producing streams available has created a viable window of opportunity (Ritchie and Adair 2004, pp. 269).   This approach has begun to yield a substantial return in both the financial and social perception areas. Sports tourism has the unique ability to both unlock the heritage elements that benefit that region of industry and the underlying natural and cultural depth that lies alongside the city (Ritchie and Adair 2004, pp. 269).   These outcomes serve to illustrate the veracity of the path taken by Manchester in the effort to rejuvenate their city on every level. Working towards goals that include the International Olympics and the Commonwealth Games are a very effective policy for a wide variety of reasons (Ritchie and Adiar 2004, pp. 269).   Even the unsuccessful bids produce a litany of economic resources for the city.   With national entities such as Tomorrows Tourism, Britain’s National Tourism concern, actively participating in the drive to establish a positive outcome, the underlying infrastructure benefited on every level. Many of the internal improvements to the city are reflections of the efficacy of the urban renewal efforts.   With the increased amount of visitors, the number of museums in Manchester is on the rise, with the subsequent impact of improving the outlook for the Greater Manchester Museum of Science and Industry (Law 2010, pp. 76).   This is a direct result of the effectiveness of the policy in place. Manchester has successfully raised its profile to an international level that has the capacity to fuel the cities entire economic engine (Ritchie and Adair 2004, pp. 274).   Forging trade alliances in several fundamental industries outside of the sport centred world served to broaden the Manchester business base, which in turn will balance the long term sustainability outlook for the cities programs. Currently, Manchester boasts ultra-modern constructs such as the Lowry Centre, which alongside the rapidly developing art and culture industries have evolved into a very credible, and well balanced economic environment (Di-Toro 2010, pp. 1). This is a very apt demonstration of the ability of a savvy electorate to forge a business environment that is perceived to be beneficial by the majority of the populace. This concerted and progressive policy implementation has vaulted Manchester to third in the most visited cities in the UK, following London and Edinburgh (Di-Toro 2010, pp. 1). Together with the foundation of sport centred tourism, Manchester is rapidly becoming a well-rounded destination that has an appeal to many separate social levels, thereby increasing the cities overall value in terms of urban tourism potential. Areas of Improvement The ambitious drive by Manchester to lift their economic outlook via the instrument of sport centred tourism has not been without its significant detractions (Ritchie and Adair 2004, pp. 274).   With the most glaring error being the single minded approach to the marketing method, the availability of associated markets in Manchester has been deemed to be small as compared to other regions.   As a consequence, there is not a strong central or primary community that will allocate the overall distribution of income (Ritchie and Adair 2004, pp. 274).   As the sport industry continues to thrive without proper planning, there is the real potential for the other un-associated industries to flounder.   Davies (2010, pp. 1238) argues that there needs to be a far greater development of the role of sport within the underlying infrastructure before implementing any long term planning. The often volatile nature of the field itself can hamper the successful proliferation of progressive poli cy, instead hampering the growth due to poor performance. The focus on the building of ‘Sportcity’ in Manchester, while producing an economic viability option, has taken over much of the city itself, irrevocably altering the face of the city forever (Berg, Braun, and Otgaar 2002, pp. 56).   This fact has the potential to diminish the historical or creative aspects of the city’s tourism potential. The overall effort to instil a sense of long lasting progress will be reached through the development of a strategic plan that brings together the disparate elements of both the sport and tourism industries (Ritchie and Adair 2004, pp. 274).   The arena of tourism is dominated by the area of commercialism, bringing the very real potential for the base interests of the commercial industries to begin to take precedence over the needs of the local population. The addition of the sport element, with its series of oversight mechanisms enables the creation of a credible system of checks and balances with the depth to take the city forward into the next era. References Ashworth, G. and Page, S. 2010. Urban tourism research: Recent progress and current paradoxes. Tourism Management, 32 (1), pp. 1-15. Beioley, S. 2002. Metro land-The urban tourism market.  Tourism Insights, 1 (1), pp. 1-3. Berg, L., Braun, E. and Otgaar, A. 2002.  Sports and city marketing in European cities. Aldershot, Hampshire, England: Ashgate. pp. 1-125. Cook, I. and Ward, K. 2011. Trans-urban Networks of Learning, Mega Events and Policy Tourism.  Sage Journals, 48 (12), pp. 2519-2535. Davies, L. 2010. Sport and economic regeneration: a winning combination?.  Sport in Society, 13 (10), pp. 1438-1457. Di-Toro, M. 2010. Britain’s hip new tourist destinations? Manchester, Glasgow, Birmingham and Liverpool.  Visit Britain, 1 (1), pp. 1-3. Hughes, H. 2003. Marketing gay tourism in Manchester.  Vacation Marketing, 9 (2), pp. 152-163. Law, C. 2002.  Urban tourism. London: Continuum, pp. 1-200. Ritchie, B. and Adair, D. 2004.  Sport tourism. Clevedon, England: Channel View Publications. pp. 1-300. Smith, A. 2013. The Development of â€Å"Sports-City† Zones and Their Potential Value as Tourism Resources for Urban Areas.  European Planning Studies, 18 (3), pp. 385-410. Smith, A. 2013. REIMAGING THE CITY: The Value of Sport Initiatives.  Annals of Tourism Research, 32 (1), pp. 217-236. Spirou, C. 2011.  Urban tourism and urban change. New York: Routledge, p. 1-200. Tallon, A. 2010.  Urban regeneration in the UK. London: Routledge, pp. 1-200. Weed, M. 2010. Sport, Tourism and Image.  Journal of Sport Tourism, 15 (3), pp. 187-189.

My Last Duchess Essays

My Last Duchess Essays My Last Duchess Paper My Last Duchess Paper Robert Browning’s â€Å"My Last Duchess† Published 1842 Our world is changing and evolving at an astounding rate. Within the last 200 years, we have seen two World Wars and countless disputes over false borders created by colonialists, slavery, and every horrid form of human suffering imaginable!! Human lifestyles and cultures are changing every minute. While our grandparents and ancestors were growing up, do you ever think they imagined the world we live in today? What is to come is almost inconceivable to us now. In this world, the only thing we can be sure of is that things will change. With all of these transformations occurring, it is a wonder that a great poet like Robert Browning may write words so many years ago, that are still relevant to you and I in todays modern society. Browning’s first dramatic monologue â€Å"My last duchess† was written during the Italian Renaissance when egotism, marriage and aristocracy influenced the society. The monologue is loosely based on historical events involving Alfonso, the Duke of Ferrara, who lived in the 16th century. The Duke is the reciter of the monologue, and tells us he is entertaining an emissary who has come to negotiate the Duke’s marriage (he has recently been widowed) to the daughter of another powerful family. As he shows the visitor through his palace, he stops before a portrait of the late Duchess, apparently a young and lovely girl. The Duke begins reminiscing about the portrait sessions, then about the Duchess herself. His musings give way to a diatribe on her disgraceful behaviour: he claims she flirted with everyone and did not appreciate his â€Å"gift of a nine-hundred-years- old name. † As his monologue continues, the reader realizes with ever-more chilling certainty that the Duke in fact caused the Duchess’s early demise: when her behaviour escalated, â€Å"[he] gave commands; / Then all smiles stopped together. † But Browning has more in mind than simply creating a colourful character and placing him in a picturesque historical scene. Rather, the specific historical setting of the poem harbours much significance: the Italian Renaissance held a particular fascination for Browning and his contemporaries, for it represented the flowering of the aesthetic and the human alongside, or in some cases in the place of, the religious and the moral. Thus the temporal setting allows Browning to again explore sex, violence, and aesthetics as all entangled, complicating and confusing each other: the lushness of the language belies the fact that the Duchess was punished for her natural sexuality. The Duke’s ravings suggest that most of the supposed transgressions took place only in his mind. Like some of Browning’s fellow Victorians, the Duke sees sin lurking in every corner. The reason the speaker here gives for killing the Duchess ostensibly differs from that given by the speaker of â€Å"Porphyria’s Lover† for murder Porphyria; however, both women are nevertheless victims of a male desire to inscribe and fix female sexuality. The desperate need to do this mirrors the efforts of Victorian society to mould the behaviour- sexual and otherwise- of individuals. For people confronted with an increasingly complex and anonymous modern world, this impulse comes naturally: to control would seem to be to conserve and stabilize. The Renaissance was a time when morally dissolute men like the Duke exercised absolute power, and as such it is a fascinating study for the Victorians: works like this imply that, surely, a time that produced magnificent art like t he Duchess’s portrait couldn’t have been entirely evil in its allocation of societal control- even though it put men like the Duke in power.

Should healthcare workers be forced to get annual flu vaccines Research Paper

Should healthcare workers be forced to get annual flu vaccines - Research Paper Example However, for purposes of the article in question, the researchers attempt to answer how useful the flu shot is from a broad perspective of its ability to positively affect overall shareholder health. Moreover, the article makes a case for the manner in which the HCW (healthcare workers) should engage with the notion of the flu shot as a means to bolster their own health as well as provide for the safety and health of the patients they come in contact with. As such, the dual nature of the way in which healthcare workers must engage with this issue has to do not only with their responsibility to their own health but the responsibility they share with their profession and the individuals that rely on their careful attention to promoting healthcare best practices by ensuring they are as disease free as possible so as not to infect any of the patients they come into contact with. Clinical digest. Nurses more likely to take flu vaccination if they believe there will be a personal benefit. (2011). Nursing Standard, 26(12), 17. This article approaches the topic of healthcare workers obtaining flu vaccines from the perspective of gain that a potential flu shot might bring to the worker. In this way, the article discusses the construct of a dual paradigm that exists between the healthcare worker actively wanting to immunize themselves as opposed to the perceived gain that they might experience from this due to a reduced risk of infection from incoming patients. In this way, the article brings to the reader’s attention that although all nurses must swear the Hippocratic Oath, the fact of the matter is that selfish motivations still continue to define the manner in which healthcare professionals engage with decisions that should attempt to balance both the needs of the individual and the concern for patient health. In this way, the full breadth and scope of the issue is seen as something that must either be incumbent upon the individual healthcare professional or wo uld necessarily need to be mandated by the entity which employs them. As the authors note, both alternative measures have their distinct drawbacks. Firstly, with respect to the employer mandating such a course of action, a host of legal issues would dominate such a choice. Secondly, with respect to the individual themselves being responsible for self-administration, the degree to which this would actually occur may be insignificant. Evans, G., & Marill, M. (2012). A house divided: A muddled mandate on health care worker flu shots goes to HHS. Hospital Infection Control & Prevention, 39(3), 25-30. This particular article discusses the legal difficulties surrounding employer mandated flu shots. As previously discussed, when an employer mandates such a course of action, they are acting in the best interest of the patients they will be serving; however, many issues arise in forcing a given employee to take such a course of action. No the least of these issues is the fact that by demandi ng such an action from their shareholders, health care management is oftentimes venturing into the gray zone of what an employer can require of its employees. Due to the host of new healthcare legislation that has recently been passed in the form of HIPPA and FMLA, the extent to which employers can be involved in the medical practices of their employees is severely restricted. However, the case of mandatory flu shots, as these

Sharpe's Budget Problem Essay Example | Topics and Well Written Essays - 750 words

Sharpe's Budget Problem - Essay Example The cash budget should include the all the costs likely to be paid and cash to be received (Crosson, Power, and Needles, 2011). The direct cost are the expenses incurable which are directly associated with the revenue sources, and/or can be directly apportioned to that source of the revenue with a high degree of accuracy. The cash budget should be monitored closely so as to establish how closely the company has adhered to meeting its objectives with respects to its finances (Hilton, Thorne and Lang field-Smith, 2006). In order to achieve this, it is expedient to prepare a variance report. A variance statement will contrasts the expected cash outflow and expenditure. ... It will be imprudent for a company to fail to produce a cash budget. In this case, the sales of the company are increasing and collections are made at a rate of 10% of sales during the month, 60% of the sales during the month presiding the month of sales, and 30% in the second month following the sale. Rent and other expenditures are made each month and do not vary. Tax is paid only in the month of March and June, 2004 at the rate of $ 22,500 during the period under consideration. Workings NOTE 1 cash collections Jan Feb Mar Apr May Jun Jul Aug Sales 90,000 120,000 135,000 240,000 300,000 270,000 225,000 150,000 Cash sales 10% of sales 9,000 12,000 13,500 24,000 30,000 27,000 22,500 15,000 Notes – 2 collections Nov Dec Jan Feb Mar Apr May Jun Jul Aug sales 220,000 175,000 90,000 120,000 135,000 240,000 300,000 270,000 225,000 150,000 60% of sales 132,000 105,000 54,000 72,000 81,000 144,000 180,000 162,000 135,000 30% of sales 66,000 52,500 27,000 36,000 40,500 72,000 90,000 8 1,000 Total collections for the month 171,000 106,500 99,000 117,000 184,500 252,000 252,000 216,000 Notes – 3 purchases payments Nov Dec Jan Feb Mar Apr May Jun Jul Aug sales 220,000 175,000 90,000 120,000 135,000 240,000 300,000 270,000 225,000 150,000 Purchases 60% 54,000 72,000 81,000 144,000 180,000 162,000 135,000 90,000 - - Payments 54,000 72,000 81,000 144,000 180,000 162,000 135,000 90,000 - Payment of notes payable of $ 200,000 Sharpe requires cash of $ 200,000 to pay notes payable. There are two scenarios in this case, the first case is where the not payable are paid without interest. In this case, the company has ample cash to repay the notes. The following is the cash budget after factoring in the notes payable at the end of July. The assumption that the