Golden Age of Capitalism Essay Example | Topics and Well Written Essays - 750 words

Brilliant Age of Capitalism - Essay Example This was when trade was being upset with the development of new innovations in transport and correspondence which encouraged the global markets. Railways and steamships were utilized for land transportation of merchandise. This brought forth the universal market with products being shipped between nations, for example, America and England. The improvement of worldwide trade in fabricates on such a scale was just conceivable on account of an expansion in global progressions of capital and the universal arrangement of money related administrations (Milward 22). This was apparent in post world war II economy of Europe. By 1950 increasingly an incredible level of the salary of remote countries such Denmark was from outside exchange with different countries. The conversion scale between national monetary forms was steady regardless of the force among warring countries that was developing. Thus countries had the option to thrive and develop financially and advance reliance. The way of life for some, residents was at the time much better and kept on improving with the expansion in the national pay per capita because of monetary achievement. Over the period 1950-70, the pace of development of yield in the development division was somewhere in the range of 4 and 7 percent a year over Western Europe as entire, and in this way to some degree higher than the pace of development of all out yield (Milward 52). This additional to the achievement and monetary reliance of countries after World War II. Along these lines various divisions of various countries that had practical experience in an exchange to encourage development, rose extensively. The exchange shortage with the dollar zone expanded triple somewhere in the range of 1951 and 1957 (Milward 182). This expanded yield and western Europe had an excess that was remarkable. Help reached out to the residents of a country post war was vital in setting up the state as a methods for the individuals to better themselves. The general change of the German benefits frameworks in 1955-7 , an offer by the Christian Democratic Union for a drawn out working class appointive help, shared little for all intents and purpose, for instance with the augmentation of open government assistance in Sweden or Norway aside from an adherence to the view that the soundness of the state required a positive reaction to the interest for government assistance (Milward 47). This was significant in passing on to the open that as per the government assistanc e arrangements set up, they also were to add to the development of the economy in the nations referenced. In my point of view, this was a period of extraordinary hardships, those getting more extravagant were remote financial specialists, investors and vendors. Governments managed a hand, and drenched their economies in the developing scene advertise. This period in this way offered ascend to a whirlwind of new position openings in transportation and correspondence. The post-war reproduction of countries helped by mechanical progressions was intended to modify the economy, advance industrialization and modernization. Because of the achievement accomplished in this undertaking, it was important for the legislatures to keep up it for reasons for accomplishing financial reliance. Political solidness of this age put taking an interest nations in the cutting edge of global exchange, ventures, travel and movement which expanded more than some other period. It is inconceivable how countries had the option to encounter a period that was high in financial development because of transportation and correspondence progressions. This period was interesting and can't right now be

The Snowman Tradition free essay sample

He was constantly unbalanced. That was inescapable. Be that as it may, we were constantly pleased, in any case. We made him from our own hands, and he never looked half-terrible, on the off chance that I don’t state so myself. Of course, his eyes swell from his head, and truly, his face was unnaturally round, his nose irrationally pointed, and his arms ridiculously slim. He was a personification of life, however we wondered about him, our creation. It used to be that each winter, my kin and I would assemble a snowman in our patio. Mother would make reference to in passing that the patio was looking forceful unfilled, after the primary large snowfall, and we would all really try to understand, toss on our snow apparatus, and race out into the glaring white of another winter. Developing a snowman is an artistic expression. It takes care and persistence. One beginnings by pressing a snowball, and afterward moving it in the new day off. We will compose a custom article test on The Snowman Tradition or on the other hand any comparative subject explicitly for you Don't WasteYour Time Recruit WRITER Just 13.90/page Like exploding an inflatable, the ball mysteriously becomes bigger. My kin and I would race to see who could make theirs the biggest, albeit an infrequent snowball battle would breakout to occupy us from our underlying objective. In the long run, a victor was pronounced, and their mammoth snowball was utilized as our base. After this unique rivalry stage, it was all collaboration. My twin sibling Evan and I would take the next in line snowball, it being the second biggest, and equalization it on the base. The ideal middle. My more seasoned sibling Stephen would pack snow between the spaces, while my sister got water to solidify the recently pressed day off. The procedure was continued utilizing another snowball for the head. Two branches were then roughly pushed into the sides for arms. Rocks from beneath the patio shaped a slanted grin, a line of catches, and a couple of evil eyes. A carrot for a nose did what needs to be done. At the zenith of these underlying endeavors, we hurled a pail of water over our snowman for permanency, giving him a frosty covering that shone in the light. A scarf and gloves acquainted a human touch with our chilly companion. We would remain back, hands on our hips, and wonder about our aggregate endeavors. Hot cocoa back at the house was our last prize. This snowman making process was a custom all through my adolescence. One winter, notwithstanding, we halted; we were excessively old, it was a lot of work, it had lost its allure. Next, sledding was relinquished, and afterward, we quit going out into the snow by any means. We didn’t increase a loathing of winter. We were growing up, and all the more essentially, becoming separated. Furthermore, I loathed it. So a year ago, I rejuvenated the snowman convention. I attempted to enroll individual constructors, however they were occupied with different interests. So I meandered out into the snow alone, and I manufactured the base. I hunched down and moved my ever-developing snowball over the terrace, leaving a path afterward. I made a middle, and a head. I stuck in the branches, and the stones, and the carrot. I tossed a pail of water over my creation, and wore a baseball top on his head. Completed, I removed my gloves, happily offered them to the snowman, and ventured back to respect my workmanship. It took much longer alone, than it did as a group. My snowman was particularly disproportionate and despicable, obviously, I was glad for him. Mother snapped an image, an enduring token of my endeavors. I love my family, and I have felt that we have consistently been close. My kin and I are comparative in age, and we have grown close bonds with one another. My endeavors to revive the snowman convention reflect who I am: mindful, dedicated, and perhaps only a smidgen wistful. I have voiced my interests to my kin about the developing separation among us, and I constantly attempt to unite us back, be it through an outing to the films, or an infrequent game. I just need to recollect our winters together and our endeavors during the time to recall why I need to revive our association. The winter is quick drawing nearer, particularly up here in Connecticut. Maybe you’ll discover a snowman in our terrace this year, after the main huge snowfall. I’m sure it will have been made by a bigger number of hands than one.

[Guest Post] Olivia S. 20 en Monterrey

[Guest Post] Olivia S. ‘20 en Monterrey While some of us have spent the first week of freedom from classes doing essentially nothing (Im @ing myself), other MIT students have hit the ground running with meaningful projects such as the one that the brilliant Olivia S. 20 describes below. Enjoy the read! _,.-~-.,__,.-~-.,__,.-~-.,__,.-~-.,__,.-~-.,__,.-~-.,__,.-~-.,__,.-~-.,__,.-~-.,_ Monterrey, Nueva León, México- After finals, I decided to immediately flee the country, which is what I did through the Big Dreams program. I stumbled into this opportunity by accident when scrambling for a summer UROP by the direct funding deadline (weve all been there). When my soon-to-be supervisor, Dr. Abel Sanchez, saw on my resume que yo hablo español, he asked if I wanted to join a teaching innovative that he founded. Big Dreams started last year with 50 girls, 2 teachers, and 1 city. This year it is 600 girls, 30 teachers, 60 local TAs, and 6 cities. Next year, we plan to grow 10-fold again to more countries, but then the growth pattern will (likely) pause. Speaking of patterns, Patrones Hermosas or Beautiful Patterns is the name of the curriculum that is being taught in all of these locations. The curriculum is designed around algorithmic thinking; the first day we dove right into the stable matching problem. First, the girls worked with decks of cards to match 4 Reyes (Kings) and 4 Reinas (Queens) while trying to maximize the happiness of the matches based on the preferences of the royalty. Each group came up with their own method, some strategic and some less so, about how to solve this problem. Déspues, I, my co-teacher, Nidhi, and Aracely, our wonderful TA, taught the algorithm that can always be used to maximize happiness, called Gale-Shapley. Here is a picture of the girls working on this problem: Livin The Dream with Gale-Shapley The rest of the week was dedicated to more offline exercises in the morning, and coding exercises in the afternoon. There definitely were some bumps in the road, the language barrier being one of the biggest ones. Luckily, most of my Spanish came back from high-school and I was able to communicate with the girls and get around in the city in a mix of Spanglish. Translation only went awry once, when I tried to order fried plantains at a restaurant banana on a plate Computer science education, especially to young girls, is a cause that everyone involved with Big Dreams cares about and wants to see improve. Tech companies like Softtek, which is where I taught, who are opening their doors y cafeterías to this type of initiative are role-models for the rest of the industry. Thank you to Rosario and Hilda at Softtek for taking care of everything this week. Aracely, the best TA ever, in the funky chairs at Softtek Graciás a la University of Monterrey for hosting us on your beautiful campus. Thank you to Abel and the rest of the organizers for making this happen. Oh, and to Monterrey para las montañas Priya in the Sierra Madre Pankhuri and me in Barrio Antiguo Post Tagged #summer

Argumentative Essay On Nuclear Waste - 1712 Words

A world filled with uncertainty is already a scary place to live. Murphy’s law only makes that uncertainty destined for disaster where anything can go wrong, will undoubtedly go wrong. The United States has placed itself under the mercy of Murphy’s law, not because of Trump’s threats of war with North Korea, but because it has no safe way of disposing of its nuclear waste. Radiation is a silent killer; if exposed into the environment it could lead to genetic mutations, cancer, and if long enough, premature death. Furthermore, radioactive material can work its way up from the environment to animals, to humans up the food chain nowhere near the site of exposure. We have been complacent in finding a permanent solution to nuclear waste†¦show more content†¦That one percent of waste can be devastating to any ecosystem it is exposed to. Radiation is invisible, it has no scent, it cannot be cleaned by soap, and it should be of concern as much as any disease or virus. The scientific community unanimously agrees that exposure to high levels of radiation is a health hazard. Many people feel unconcerned because they feel it’s not a relevant problem in their lives. However, there are over 71,000 tons of nuclear waste simple in temporary storage at the U.S.’s 104 nuclear reactors (Oliver). Nearly one out of three Americans live within a 50-mile radius of a reactor filled with waste (Oliver). Still many are skeptical remaining ignorant of the potential dangers of radiation exposure. The severity of the damage to a person’s heath due to radiation is based on how much radiation and how long they were exposed to the amounts of radiation. The amount of radiation a person receives is measured in â€Å"rem† and depending on that the health effects could range from no observable health effects, to cancer, to even death (Effects of Radiation). Even a fraction of the amount of radiation to kill a person could cause nausea, fatigue, v omiting, loss of appetite, loss of hair and abnormalities in blood cells (Effects of Radiation). Nuclear waste from a typical fuel rod will still have 2,000 times more rem than the required dosage to kill a person even after 10 years (USNRC). Clear examples of why people should be worried about exposure toShow MoreRelatedI Am The Product Of Clark County Educational System Essay1503 Words   |  7 Pageshow to write essays correctly. I am the product of Clark County Educational system. I have always struggled through my last classes and hoped to get out of my struggles this semester. One the greatest challenges that I faced was the distinction that existed among different types of writings that are performed within the English language. 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Judge (2011) Organizational Behaviour 15th Edition New Jersey: Prentice Hall393164 Words   |  1573 Pagesthe job of managing people seem much simpler than it is. Most are based on the author’s opinions rather than substantive research, and it is doubtful that one person’s experience translates into effective management practice for everyone. Why do we waste our time on â€Å"fluff† when, with a little effort, we can access knowledge produced from thousands of scientific studies on human behavior in organizations? Organizational behavior is a complex subject. Few, if any, simple statements about human behavior

Kassatly Chtaura - 1648 Words

Kassatly Chtaura ABSTRACT In April 2013, Kassatly Chtaura, a family-owned company of both alcoholic and non-alcoholic beverages headquartered in Chtaura, Lebanon, faces a dilemma. It is doing well with regard to sales and market share and has succeeded in building a solid income. Meanwhile, the prior year’s numbers show dim growth performance, and the family is unsatisfied about sales of its syrups, juices, ready-to-drink beverages and wines have reached a plateau. Should the firm extend its supply network or manufacture a new factory and move some operations to Angola or Saudi Arabia? They are in distant locations with distinct cultures, although these are assuring markets. Or should it stay put and expand its operations by introducing†¦show more content†¦Undoubtedly, one of the advantages that Kassatly Chtaura has, is how they shape their strategy, implement new drinks according to market trends and its innovative position in alcoholic and non-alcoholic beverages. They have been receiving much recognition for the ingredients that are used in its production. The same laws have specified restrictions on alcohol and energy drinks, and thats where Kassatly Chtaura could find the right time to launch its various products in Lebanon and in turn be related to Saudi Arabia, which provides an advantage to position itself in the group GCC in the Middle East. However, the tension is handled in the country of Lebanon and in the closest countrie s. Is a rapid change that may arise from the overnight, so achieving manufacturing in another country where it is not fully understood the culture, the rules and laws governing, is a risk that can be significant in the formation of an expansion in Saudi Arabia or in Africa as an emerging market that is growing nowadays. Kassatly Chtaura is a company that has taken advantage of the situation, resources and the regions where they can make some investment to prop up the finances and not fall into a low development of innovation in the products they handle. Implementation of vodka, energy drinks and even some alignment with the wines have kept the name Kassatly Chtaura outside the Lebanese community, and internationally, they have achieved some recognition.

Sop Biomedical Engineering Free Essays

The wonders that science and technology have unfolded before us are not limited to time and space . My aim in life is to accumulate this diverse knowledge from all over the world. With this cherished goal in my mind, I wish to state in the following paragraphs, my viewpoints and the rational behind my application to the graduate studies program in Bio-Medical Instrumentation at **** university. We will write a custom essay sample on Sop Biomedical Engineering or any similar topic only for you Order Now Throughout my ten years of school life and two years of high school study, I have maintained a consistent and good academic record and was always amongst the top five of my class. Coming to extra-curricular activities, I was the head sports captain for girls and also participated in the state level U. N. Celebrations. Aspiring for the best undergraduate course that all good science students do and also influenced bythe fact that I come from a family of engineers and scientists-I appeared for the EAMCET ( Engineering and Medical Common Entrance Test)and opted for Instrumentation and Control Engineering at ***** College, affiliated to JNTU , Hyderabad in Andhra Pradesh. During my undergraduate years I could learn the fundamentals in Transducers and Instrumentation Components, Electrical and Electronic measurements, Electronic Equipment design, Analytical Instrumentation, Process Control Instrumentation, Power plant Instrumentation , Digital and optimal Control Systems. I observed on various occasions , especially in transducers and instrumentation components which is a vast subject, that there existed a wide enough rift between learning and practical implementation. While the undergraduate courses enable a student to grasp the fundamentals involved in a particular discipline of engineering, they do not present the opportunity to learn the ropes with new technologies that are current within the industry. Specialization becomes a necessity to understand new technologies and to improve upon the existing ones. It is to achieve this sophistication that I set out to pursue my post-graduate (M. S. ) studies in Bio-Medical Instrumentation. During my third year holidays I have done a project on  Computerized  respiration analysis  through embedded systems, a part of Bio-Medical instrumentation, one of the electives in the final year. This short stint has given me invaluable practical experience. It has given me the confidence to pursue a master’s degree and also kindled a desire to do research. The undergraduate course in Instrumentation and Control Engineering and with the advice of widely respected teachers at the college has given me the motivation to pursue a career in Bio-medical Instrumentation . The sharing of ideas and new findings has always been a part of my undergraduate life. Presentations and seminars were a perfect opportunity for me to explore beyond the syllabus and were instrumental in giving me a competitive edge over my peers. I relish a chance to indulge my creative side and gaining a deeper understanding of my work in process of presentations. I enjoy diving into a flood of data, picking out relevant information and delivering it all to an appreciative audience! In second year of my under-graduation I got a chance to present a paper at national-level on the topic  Robotics. The dynamic nature of scientific research was revealed to me as I worked on my presentation. Often new theories were replaced by old ones so fast that I was updating my work right up till the morning I had to present. By this experience I could learn that before beginning the first robotics project, prospective robotic hobbyist and robotic sports enthusiasts must have a basic understanding of the field of robotics and the issues surrounding robotic systems, including mechanical design, sensory systems, electronic control and software. A basic understanding of micro-controller systems including serial and memory-mapped interfacing, as well as some available open source software options should also be high on the list. Quest for knowledge needs considerable persistence and an unquenchable desire to learn. Whatever I have achieved till now can be attributed to my diligence and perseverance, which I have learned from my close knit family and culture. With the same indefatigable spirit, I am ready to utilize and direct all my physical and mental abilities to achieve the same in my future studies and research work. I believe the field of instrumentation offered so immense a potential that given me an opportunity to opt for Bio-Medical Instrumentation, I would be interested to work in the area of bio-medical instrumentation which deals with its development. I bring along a strong grasp of fundamentals, an aptitude for teaching and team work, a zest for challenges and an enthusiastic desire to learn all I can. In addition I would like to take with me a network of strong and lasting relationships that I maintain with my teachers and fellow students. I hope that my qualifications and background are found suitable for MS in Bio-Medical Instrumentation at ******* university. Having been enlightened by many professors and several graduating seniors about the rigorous course work besides possessing state-of-the-art research facilities, I had no dilemma in choosing **** university as my dream university for pursuing my graduate studies. I am sure that *** university with dynamic faculty, well equipped laboratories and world renowned graduate programs would definitely help me in achieving my goals and contribute something of my own in my field of interest. I believe that with my capacity for hardwork, commendable logic and dedication to achieve my goals, I will be able to do very well in my Graduate course. I aver that I take it as a challenge and spare no effort of mine in utilizing the resources available at your university. I am sure that you will share my confidence and give me an opportunity to continue with my further studies at your esteemed university. How to cite Sop Biomedical Engineering, Papers

Strategic Management-Chapter 9 Essay Example

Strategic Management-Chapter 9 Paper Most strategists believe that an organizations well-being depends on evaluation of the strategic-management process. True Adequate and timely feedback is important to effective strategy evaluation. true Too much emphasis on evaluating strategies may be expensive and counterproductive. True Strategy evaluation should have a long-run focus and avoid a short-run focus. False According to Richard Rumelt, consonance and consistency are mostly based on a firms external assessment. False According to Rumelt, consistency and feasibility are largely based on a firms internal assessment. True Consistency, distinctiveness, advantage, and feasibility are Richard Rumelts four criteria for evaluating a strategy. False Strategy evaluation is becoming increasingly easier with the passage of time, given technological advances. False The decreasing time span for which planning can be done with any degree of certainty is a reason strategy evaluation is more difficult today. True Strategies may be inconsistent if policy problems and issues continue to be brought to the top for resolution. True Competitive advantages normally are the result of superiority in one of three areas: feasibility, consistency, or consonance. False Regardless of the size of the organization, a certain amount of management by wandering around at all levels is essential to effective strategy evaluation. True Evaluating strategies on a continuous rather than on a periodic basis allows benchmarks of progress to be established and more effectively monitored. true It is most effective to conduct strategy evaluation annually, at the end of the fiscal year. False Changes in the organizations management, marketing, finance and accounting, production and operations, research and development (RD), and management information systems (MIS) strengths and weaknesses should all be the focus of a revised EFE matrix in strategy evaluation. False In strategy evaluation, a revised IFE matrix should indicate how effective a firms strategies have been in response to key opportunities and threats. False Strengths, weaknesses, opportunities, cost and threats should continually be monitored for change because it is not really a question of whether these factors will change, but rather when they will change and in what ways. True When measuring organizational performance, you need to compare expected results to actual results. True Criteria for evaluating strategies should be measurable and easily verifiable. True Financial ratios are rarely used as criteria to evaluate strategies. False Measuring organizational performance includes comparing expected results to actual results, investigating deviations from plans, evaluating individual performance, and examining progress being made toward meeting stated objectives. True Intuitive judgments are almost always involved in deriving quantitative criteria. True Most quantitative evaluation criteria are geared to long-term objectives rather than annual objectives. False Measuring organizational performance requires making changes to reposition a firm competitively for the future. False Taking corrective actions does not necessarily mean that existing strategies will be abandoned, or even that new strategies must be formulated. True Corrective action in strategy evaluation is necessary to keep an organization on track toward achieving stated objectives. True Alvin Toffler argues that environments are becoming so dynamic and complex that they threaten people and organizations with future shock, in his thought-provoking books entitled Future Shock and The Third Wave. True Future shock occurs when the nature, type, and speed of changes overpower an individuals or organizations ability and capacity to adapt. True According to research, participation in strategy-evaluation activities is one of the best ways to overcome individuals resistance to change. True The form of a Balanced Scorecard does not vary for different organizations or industries. False The Balanced Scorecard approach deals with the question, How satisfied are the firms customers? True Each year, Fortune publishes strategy-evaluation research on both the United States and other countries. True Yahoo was one of the firms most admired in its industry according to Fortunes 2012 evaluation False Strategy-evaluation activities must be meaningful, that is, they should specifically relate to a firms objectives. True Timely approximate information is generally more desirable as a basis for strategy evaluation than accurate information that does not depict the present. true The test of an effective evaluation system is its complexity. false Small organizations require a more elaborate and detailed strategy-evaluation system because they are still evolving. false There is no one ideal strategy-evaluation system for all organizations. true Contingency plans are alternative plans that can be put into effect if certain key events do not occur as expected. true Organizations should prepare contingency plans just for unfavorable events. False Strategists should try to cover all bases by planning for all possible contingencies. false Contingency plans should be as simple as possible. true Alternative strategies not selected for implementation should be discarded, as they have a tendency to contaminate the contingency plans. false Identifying both good and bad events that could jeopardize strategies is the first step of effective contingency planning. true A frequently used tool in strategy evaluation is the audit. true The U.S. Chamber of Commerce is against the accounting switch from GAAP to IFRS, saying it will cause cross-border commerce to decline. false Public accounting firms usually avoid strategy-evaluation services. false The accounting switch from GAAP to IFRS in the U.S. is going to cost businesses millions of dollars in fees and upgraded software systems and training. true Believing it will make it easier for investors to compare firms across countries and make it easier to raise capital globally, most large accounting firms and multinational firms favor the switch to IFRS. true The U.S. Chamber of Commerce supports the change from GAAP to IFRS, saying it will help the U.S. compete in the world economy. true IFRS standards comprise 25,000 pages, whereas GAAP standards comprise 5,000 pages. false Most executives believe that some strategic information should remain confidential to top managers. true Increased education and diversity of the workforce at all levels are reasons why the top-down approach should be favored in organizations. false Which of these is a basic activity of strategy evaluation? A) Reviewing the underlying bases of current strategies B) Comparing expected results with actual results C) Taking corrective actions D) Choices B and C only E) All of the above E) All of the above Too much emphasis on evaluating strategies A) may be expensive and counterproductive. B) is not possible the more emphasis the better. C) is good for morale, as employees like being closely evaluated. D) is always advised if the firm can afford it. E) is worse than too little or no evaluation. A) may be expensive and counterproductive. What is the cornerstone of effective strategy evaluation? A) Adequate and timely feedback B) Quality and quantity of managers C) Smaller ratio of top- to lower-level management D) Evaluation preceding implementation stage E) Punitive corrective actions A) Adequate and timely feedback All of these are Richard Rumelts criteria to evaluate a strategy EXCEPT A) advantage. B) consistency. C) feasibility. D) distinctiveness. E) consonance. D) distinctiveness. With the passage of time strategy evaluation is becoming A) increasingly difficult. B) much simpler. C) very convenient. D) an unnecessary activity. E) less important. A) increasingly difficult. All of the following are reasons strategy evaluation is more difficult today EXCEPT A) a dramatic increase in the environments complexity. B) the increasing number of variables. C) the increase in the number of both domestic and world events affecting organizations. D) the increasing time span for which planning can be done with any degree of certainty. E) the rapid rate of obsolescence of even the best plans. D) the increasing time span for which planning can be done with any degree of certainty. Which of the following is NOT a reason for the increasing difficulty of evaluating strategies? A) Product life cycles are longer. B) Domestic and world economies are less stable. C) Product development cycles are shorter. D) Technological advancement is more rapid. E) Change is occurring more frequently. A) Product life cycles are longer. Success today A) guarantees success tomorrow. B) is no guarantee of success tomorrow. C) should lull a firm into complacency. D) is all that really matters. E) none of the above. B) is no guarantee of success tomorrow. According to Rumelt, the final broad test of strategy is its A) advantage. B) feasibility. C) consonance. D) consistency. E) distinctiveness. B) feasibility. Competitive advantage normally is the result of superiority in resources, skills, or A) employees. B) position. C) consistency. D) feasibility. E) governance. B) position. What term refers to the need for strategists to examine sets of trends, as well as individual trends, in evaluating strategies? A) Consistency B) Consonance C) Synergy D) Feasibility E) Advantage B) Consonance Rumelts criteria of consonance refers to the need for strategists to examine A) inconsistent goals. B) sets of trends. C) impractical objectives. D) competitive advantages. E) the costs associated with particular strategies. B) sets of trends. If success for one organizational department means failure for another department, then strategies may be A) synergistic. B) advantageous. C) trendy. D) feasible. E) inconsistent. E) inconsistent. Modern organizational realities demand that employees demonstrate greater A) flexibility. B) innovation. C) creativity. D) initiative. E) all of the above E) all of the above Strategy-evaluation activities should ideally be performed A) just on a periodic basis. B) only at the onset of a problem. C) on a continuous basis. D) solely upon completion of major projects. E) annually only. C) on a continuous basis. Corrective actions are almost always ________ except when external and internal factors have not significantly changed and the firm is progressing satisfactorily toward achieving stated objectives. A) unnecessary B) needed C) undesirable D) prohibitively expensive E) futile B) needed If you discover during the course of strategy evaluation that major changes have occurred in the firms internal strategic position, you should A) continue on the present strategic course. B) wait until the next quarter to see if things revert. C) take corrective actions. D) follow the original strategic plan. E) none of the above C) take corrective actions. A revised ________ should focus on changes in the organizations management, marketing, finance and accounting, production and operations, research and development (RD), and management information systems (MIS) strengths and weaknesses. A) mission B) IFE matrix C) vision D) EFE matrix E) EPM matrix B) IFE matrix A revised ________ should indicate how effective a firms strategies have been in response to key opportunities and threats. A) IFE matrix B) mission C) EFE matrix D) vision E) CPM matrix C) EFE matrix Which of the following is NOT included in measuring organizational performance? A) Comparing results to competitors expectations B) Examining progress being made toward meeting stated objectives C) Investigating deviations from plans D) Evaluating individual performance E) Comparing expected results to actual results A) Comparing results to competitors expectations Which of the following is a corrective action a company might take to correct unfavorable variances? A) Divesting a division B) Revising objectives C) Raising capital with stock or debt D) Allocating resources differently E) All of the above E) All of the above Quantitative criteria commonly used to evaluate strategies are A) cash budgets. B) Balanced Scorecards. C) Capital Asset Pricing Models. D) financial ratios. E) present value analyses. D) financial ratios. Which of these is a potential problem associated with using only quantitative criteria for selecting strategies? A) Most quantitative criteria are geared to long-term objectives rather than annual objectives. B) Different accounting methods can provide different results on many quantitative criteria. C) Intuitive judgments are never used in deriving quantitative criteria. D) Quantitative criteria include human factors that may be underlying causes of declining performance. E) Quantitative criteria are not able to compare the firms performance over different period of time. B) Different accounting methods can provide different results on many quantitative criteria. Also important in evaluating strategies are ________ criteria, like high absenteeism and turnover rates, or low employee satisfaction. A) numerical B) qualitative C) quantitative D) accounting E) financial B) qualitative Financial ratios are used to compare a firms performance over different time periods, to compare the firms performance to industry averages, and to compare a firms performance with A) overall business standards. B) projected goals. C) the performance of suppliers. D) non-financial ratios. E) the performance of competitors. E) the performance of competitors. Most quantitative criteria are geared to ________ objectives rather than ________ objectives. A) top-management; employee B) short-term; annual C) annual; long-term D) environmental; community E) long-term; short-term C) annual; long-term What corrective actions might a firm take during strategy evaluation? A) Revise the business mission B) Issue stock C) Revise objectives D) Sell a division E) All of the above E) All of the above According to author Alvin Toffler, what occurs when the nature, types, and speed of changes overpower an individuals or organizations ability and capacity to adapt? A) Corporate insecurity B) Corrective actions C) Future shock D) Corporate agility E) Projected performance C) Future shock Corrective actions should always A) strengthen an organizations competitive position in its industry. B) streamline asset holdings. C) have no risk. D) involve abandoning existing strategies. E) all of the above A) strengthen an organizations competitive position in its industry. Research suggests that which of the following is one of the best ways to overcome individuals resistance to change in strategy evaluation? A) Participation B) Command-and-control C) Laissez-faire system D) Rational argument E) Emotional reactions A) Participation According to researchers, all of the following encourage individuals to accept change EXCEPT A) having a cognitive understanding of the changes. B) having a sense of control over the situation. C) having an awareness that necessary actions are going to be taken to implement change. D) participating in strategy-evaluation activities. E) being overpowered by the nature, types, and speed of changes. E) being overpowered by the nature, types, and speed of changes. Corrective action should do all of the following EXCEPT A) capitalize upon internal strengths. B) avoid external opportunities. C) avoid external threats. D) improve internal weaknesses. E) strengthen an organizations competitive position. B) avoid external opportunities. Which of the following is NOT one of the four perspectives from which the Balanced Scorecard allows firms to evaluate strategies? A) Social responsibility B) Financial performance C) Customer knowledge D) Internal business processes E) Learning and growth A) Social responsibility What aims to balance long-term with short-term concerns, financial with nonfinancial concerns, and internal with external concerns? A) Contingency planning B) The Balanced Scorecard approach C) Taking corrective action D) Benchmarking E) Consonance B) The Balanced Scorecard approach The Fortune 50 includes all of the following EXCEPT A) the largest retailers. B) the largest transportation companies. C) the largest utilities. D) the largest banks. E) the largest hospitals. E) the largest hospitals. Which of the following is NOT a key attribute that serves as one of the evaluative criteria for Fortunes Americas Most Admired Companies? A) People management B) Innovativeness C) Financial soundness D) Amount of physical resources E) Social responsibility D) Amount of physical resources The strategy-evaluation process should A) dominate decisions. B) be complex. C) be cumbersome. D) foster mutual understanding and trust. E) be restrictive. D) foster mutual understanding and trust. Controls need to be ________ rather than ________. A) action-oriented; information-oriented B) cultural; political C) qualitative; quantitative D) measurable; timely E) universal; diverse A) action-oriented; information-oriented The strategy-evaluation process should foster A) mutual understanding. B) doubt. C) corporate culture. D) complexity. E) division. A) mutual understanding. Which of the following is true regarding the design of a firms strategy-evaluation system? A) There is a one-size-fits-all system that works for all companies. B) It does not need to take into account the organizations size. C) The management style of a firm has no bearing on the design. D) It should be determined based on the unique characteristics of a company. E) There is one ideal system. D) It should be determined based on the unique characteristics of a company. ________ organizations require more elaborate and detailed strategy-evaluation systems than ________ ones because it is more difficult to coordinate efforts among different divisions and functional areas. A) Non-profit; for-profit B) For-profit; non-profit C) Large; small D) Small; large E) Foreign; domestic C) Large; small ________ plans can be defined as alternative plans that can be put into effect if certain key events do not occur as expected. A) Agile B) Scenario C) Evaluation D) Contingency E) Forecast D) Contingency Which of the following statements about contingency plans is NOT true? A) Contingency plans should be as simple as possible. B) Only high-priority areas require the insurance of contingency plans. C) Contingency plans should be developed for favorable and unfavorable events. D) Strategists should plan for all possible contingencies. E) Contingency plans minimize the impact of potential threats. D) Strategists should plan for all possible contingencies. What has been shown to permit quick response to change, prevent panic in crisis situations, and make managers more adaptable? A) Auditing B) Implementing a Balanced Scorecard C) Contingency planning D) Taking corrective actions E) Measuring performance C) Contingency planning What term is defined as a systematic process of objectively obtaining and evaluating evidence regarding assertions about economic actions and events to ascertain the degree of correspondence between these assertions and established criteria, and communicating the results to interested users? A) Auditing B) Innovation C) RD D) Strategic Management E) Financial ratios A) Auditing International financial reporting standards (IFRS) comprise approximately ________ pages. A) 1,000 B) 5,000 C) 10,000 D) 25,000 E) 100,000 B) 5,000 Already the European Union and 113 nations including Australia, Mexico, and Canada have ________ IFRS rules. A) adopted and then abandoned the use of B) been prohibited from adopting C) spoken out publicly against the adoption of D) revised the standard version of the E) adopted or soon plan to use E) adopted or soon plan to use With regard to the visible or hidden issue, most executives agree that A) the SEC should regulate whether or not companies make their strategic information visible. B) some strategic information should remain confidential to top managers. C) a company has gone too far when it takes steps to ensure that strategic information is not disseminated beyond the inner circle. D) the potential benefit of improved employee and stakeholder motivation and input is not worth the risk of rival firms easily knowing and exploiting a firms strategies. E) keeping strategies secret from employees and stakeholder will likely improve communication, understanding, and commitment. B) some strategic information should remain confidential to top managers. A particularly important twenty-first-century challenge facing all strategists today is A) deciding whether the process should be more an art or a science. B) deciding whether strategies should be visible or hidden from stakeholders. C) deciding whether the process should be more top-down or bottom-up in their firm. D) all of the above E) none of the above D) all of the above Increased education and diversity of the workforce at all levels are reasons why A) the top-down approach is preferred. B) the bottom-up approach is untenable. C) only top executives have the experience and acumen to make strategy decisions. D) middle- and lower-level managers, and even nonmanagers, should be involved in the strategic planning process. E) the horizontal approach is the most pragmatic choice. D) middle- and lower-level managers, and even nonmanagers, should be involved in the strategic planning process. Most strategy literature advocates that strategic management is A) more of a science than an art. B) more of an art than a science. C) based on analysis rather than research. D) based on intuition rather than analysis. E) based on creativity rather than intuition. A) more of a science than an art. All of the following are reasons to be completely open as opposed to secretive with the strategy process EXCEPT A) Managers, employees and other stakeholders can readily contribute to the process. B) Stakeholders have greater basis for understanding and committing to a firm that is open. C) All levels of todays workforce exhibit increased education and diversity, making such contributions valuable. D) Participation and openness enhance understanding, commitment and communication within the firm. E) Openness limits rival firms from imitating or duplicating the firms strategies. E) Openness limits rival firms from imitating or duplicating the firms strategies. Mintzbergs notion of crafting strategies A) is consistent with the view that strategic management is more a science than an art. B) contends that firms need to assess their environments, do research, evaluate alternatives, analyze, and then choose a course of action. C) suggests that strategic decision making be based primarily on holistic thinking, intuition, creativity, and imagination. D) reject strategies that result from subjective imagination in favor of objective analysis. E) insists on formality. C) suggests that strategic decision making be based primarily on holistic thinking, intuition, creativity, and imagination.

Plot, Setting, Comparing Themes, Character, Style, Tone, and Mood, Irony, Narrator and Voice Symbolism and Allegory Essay Example

Plot, Setting, Comparing Themes, Character, Style, Tone, and Mood, Irony, Narrator and Voice Symbolism and Allegory Essay Example Plot, Setting, Comparing Themes, Character, Style, Tone, and Mood, Irony, Narrator and Voice Symbolism and Allegory Paper Plot, Setting, Comparing Themes, Character, Style, Tone, and Mood, Irony, Narrator and Voice Symbolism and Allegory Paper Essay Topic: Literature Plot A series of related events in a story, each connected to the next. Sequence The order in which a storys events take place. Exposition The opening of a story, when the characters and their conflicts are introduced. Conflict A struggle between two forces. External Conflict Two characters, character and a group, character and an animal, or character and a force of nature. Internal Conflict A struggle that takes place in the characters mind or heart. Climax The most exciting part of a story; the moment when the outcome of the conflict is determined. (usually at the end of a short story.) A major decision. Resolution (denouement) The very end of the story, when loose ends are tied up. Flashback An action that interrupts the story to introduce an event that took place in the past. Flash-Forward An action that jumps ahead of the story to narrate an event that happens at a later time. Foreshadowing Hints in the story that certain events are going to happen later. Setting The time and place in which the story happens. Atmosphere Mood or feelings brought on by a storys setting. Subject The topic of a work of literature. The subject can usually be started in a single word or phrase. Theme The general idea or insight about human life that a work of literature reveals. The theme can be stated in one or more sentences. Generalization A broad statement that applies to many individuals, experiences, situations, or observations. A kind of conclusion that is drawn after considering as many facts as possible. Themes are expressed as generalizations. Genres The different forms of literature. (Short stories, novels, plays, poems) Universal themes Can be found in literature from different times, countries, and cultures. Universal themes cross genres as well as national boundaries, languages, customs, and historic periods. Characterization The way writers create characters in a story. Direct Characterization The writer tells us directly what a character is like (good or evil or lazy) Indirect Characterization The writer tells us clues in the story to decide what kind of person the character is. Protagonist The main character in a story. Antagonist The character that the main character struggles against. Subordinate Characters Minor characters on the story. Motivations The reasons behind a characters actions and feelings. Flat Characters A character who is not fully developed in the story. A flat character is almost never the main character. Round Character A character who is fully developed, just as a person in actual life. Dynamic Character A character who changes during the story. The change might involve recognition of some truth about life. Static Character A character who does not change during the story. Dialogue The conversations characters have with other characters. First-Person Narration A story told be an I(character) narrator. Style The particular way a writer uses language. (Word choice, sentence structure, and tone.) Diction The writers choice of words- an essential element of a writers style. Connotations Meanings and emotions associated with a word that go beyond its dictionary definition. Figures of Speech Imaginative comparisons in which one thing is described in terms of another. They are not meant to be understood on a literal level. Imagery Language that appeals to one or more of the 5 senses. Tone The writers attitude toward a subject or character, or toward the audience. Mood The overall feeling or atmosphere of a work of literature. Irony The difference between what we expect or what seems suitable and what actually happens. Verbal Irony When someone says something but means the opposite. Situational Irony An event that is contrary to, or the opposite of, what we expected. Dramatic Irony When we know what is going to happen to a character but the character doesnt know. Ambiguity A quality that allows something to be interpreted in several different or conflicting ways. Narrator The teller of a story. Omniscient Point Of View The narrator can tell us everything about the characters, including how they think and feel. First-Person Narrator A character in the story who refers to himself or herself as I or me. It only tells what the narrator know and chooses to reveal. Unreliable May not always tell the truth about characters or events in the story. Diction The writers choice of words. Tone The writers attitude toward the subject of a story, toward a character, or towards the audience. Voice The writers use of language and overall style, created by ton and choice of words. Symbol An object (a person, animal, or event) that stands for something more than itself. Public Symbol A symbol that has become widely recognized, such as the bald eagle (a symbol of the United States) or the olive branch (a symbol of peace). Invented Symbol A symbol invented by the writer, which usually stands for something abstract, such as evil, innocence, or love. Allegory A story which characters and settings stand for something beyond themselves, usually virtues and vices. Sometimes the characters in an allegory are given names that indicate what they stand for.

Edible Candle - Fire Science Demonstration

Edible Candle - Fire Science Demonstration For this science magic trick, you light a candle, blow out the flame and then eat the candle. Its safe, fun and even nutritious. Edible Candle Materials This project requires a few simple ingredients from the home: bananapecan nutcandle stick or candle holder with wax drippingsmatch or lighter Set Up The Trick Cut a banana so that it resembles a small candle. You want a candle that you can eat in one or two bites.Set your banana-candle on a candle stick or holder. If the candle stick has white wax drippings, this will add realism to the trick.Cut the pecan into a wick shape and set it into the top of the banana.When you are ready to do the trick, light the pecan, just like an ordinary candle wick. Blow out the flame and eat the candle. How It Works Pecans, as well as other nuts, are high in oils. The nut can be lit like a candle wick and will burn for several minutes. You could even use nuts on a cake as birthday candles!

Australia study Annotated Bibliography Example | Topics and Well Written Essays - 250 words

Australia study - Annotated Bibliography Example The book is notable in that it was written to be an aid for those who teach Aboriginal culture and languages. This Victorian-era work is a broad stroke examination of the cultural history and practices of the Aboriginal peoples of Australia. It provides a quite detailed look at native languages and details specific phonological and grammatical aspects that have challenged English speakers. This venerable book presents an interesting overview of factors that aggravated the racist impulses of the ruling Anglo-Australian society. This work utilizes the work of linguists and anthropologists from around the world who have studied the demise of native languages in Australia. It pays special attention to the particular social contexts in which Aboriginal languages have been used and are used today. Languages are examined from a largely functional standpoint and from the perspective that government policies have damaged the vast majority past the point of reclamation. This seminal report is one of the most comprehensive compilations of the latest data concerning the state of Aboriginal languages. It presents a chronological account of the erosion of Aboriginal lifestyles and cultural traditions. It paints a bleak picture of the state of native languages in Australia, though it doesn’t waver from a strictly fact-based recitation. Co-written by an anthropologist and linguist, this book focuses on the close relation between the environment, culture and language. This highly academic work makes a convincing case for the preservation of native languages, explaining what is at stake in a world where more languages are at risk than ever before. The authors make the interesting point that losing native languages is wasteful in that rich scientific knowledge is lost forever. Schmidt’s disciplined academic approach to the subject includes an analysis of

Federalist No. 69 and the Executive Branch Essay

Federalist No. 69 and the Executive Branch - Essay Example A government consists of three branches; the Executive, the Legislative, and the Judicial. The President of America has the right to revisit a bill, which undergoes two of the branches and forms into a law if approved by two-thirds of the people from both houses. On the contrary, the King of Britain has â€Å"an absolute negative† regarding approval from the houses. This naturally means agitation both from the Parliament and the public before a law is implemented rather by play than force. This authority is only invested in the King and not the President. Then Hamilton states four concrete points of difference between the King and the President: 1. The President has the authority to command the militia occasionally unlike the King of Great Britain and the governor of New York, who can command the whole militia at all times remaining in their numerous jurisdictions. Hence the power of the President is second-rate as compared to the other two. 2.   It is the duty of the Presid ent to be commander-in-chief of the United States’ army and navy. This article brings the President on equal grounds with the King and the governor, however lesser in substance. The President cannot declare war or regulate fleets and armies. Only the legislature has this authority. 3. The President can pardon call cases except for the impeachment case. This is not the case with the governor of New York who can even deal with the impeachment cases except for assassination and treachery. This is aggressively questioned by Hamilton for its undeserving distribution of power vested in the President. 4. The King of Great Britain and the governor of New York respectively have the influence to disband the Parliament or the legislature of the State which may be a purposeful control employed by the governor in certain delicate matters. The President, however, remains at the level of just adjourning only the national legislature under a single case of disagreement circumstance (Hamilton , 1992). These differences have been pointed out to display the real character of the executive, the President of the United States, who may not be compared to the King of Great Britain, not even the governor of New York. In reality, the U.S. Constitutions only recently gained power as the American Legislators made a conscious effort to regain the executive branch through the trust of the people. The President is the magistrate elected by the people of the United States. He is not completely independent because he is chosen for four years through fair elections (Tocqueville, 1945). The power as it is seen in the constitution did not exist prior to the introduction to the Constitutions of the United States. The people lived under the Articles of Confederation, which did not have an executive branch and it lasted for forty years (Prince, 1867). It was considered weak without the executive branch.  Ã‚  

The Central Dogma of Molecular Biology

The Central Dogma of Molecular Biology The molecule we know today as deoxyribonucleic acid was first observed in 1869 by Swiss biologist Friedrich Miescher, who stumbled upon a substance which was resistant to protein digestion. At the time he referred to the molecule as nuclein (Pray, 2008). Though Miescher remained in obscurity, Russian biochemist Phoebus Levene continued work with this substance and in 1919 discovered the three major components of a nucleotide: phosphate, sugar, and base. He noted that the sugar component was ribose for RNA and deoxyribose for DNA, and he proposed that nucleotides were made up of a chain of nucleic acids (Levene, 1919). He was largely correct, and in 1950 Erwin Chargaff, after reading a paper by Oswald Avery in which Avery identified the gene as the unit of hereditary material (Avery, 1944), set out to discover whether the deoxyribonucleic acid molecule differed among species. He found that although, in contrast to Levenes proposal that nucleotides are always repeated in the same order , nucleotides appear in different orders in different organisms, these molecules maintained certain characteristics. This led him to develop a set of rules (known as Chargaffs Rules) in which he states that the total number of purines (Adenine and Guanine) and the total number of pyrimidines (Cytosine and Thymine) are almost always equal in an organisms genetic material. In 1952 Rosalind Franklin and Maurice Wilkins used X-ray crystallography to capture the first image of the molecules shape, and in 1953 James Watson and Francis Crick finally proposed the three dimensional model for DNA (Watson, 1953). The four main tenants of their discovery still hold true today: 1) DNA is a double-stranded helix, 2) the majority of these helices are right-handed, 3) the helices are anti-parallel, and 4) the DNA base pairs within the helix are joined by hydrogen bonding, and the bases can hydrogen bond with other molecules such as proteins. The Central Dogma of Molecular Biology, first proposed by Francis Crick (Crick, 1958), describes the directional processes of conversion from DNA to RNA and from RNA to protein. This gene expression process starts with DNA, a double-stranded molecule consisting of base-paired nucleic acids adenine (A), cytosine (C), guanine (G), and thymine (T) on a sugar-phosphate backbone. This genetic material serves as the information storage  for life, a dictionary of sorts that provides all of the necessary tools for an organism to create the components of itself. During the process of transcription, the DNA molecule is used to make messenger RNA (mRNA), which carries a specific instance  of the DNA instructions to the machinery that will make protein. Proteins are synthesized during translation  using the mRNA molecule as a guide. Gene expression is a deterministic process during which each molecule is manufactured using the product of the previous step. The end result is a conversion fr om the genetic code into a functional unit which can be used to perform the work of the cell. As you can imagine, this process must be controlled by an organism in order to make efficient use of resources, respond to environmental changes, and differentiate cells within the body. Gene regulation, as it is sometimes called, occurs at all stages along the way from DNA to protein. Regulation falls into four categories: 1) epigenetic (methylation of DNA or protein, acetylation), 2) transcriptional (involves proteins called transcription factors), 3) post-transcriptional (sequestration of RNA, alternative splicing of mRNA, microRNA (miRNA) and small interfering RNA (siRNA)), and 4) post-translational modification (phosphorylation, acetylation, methylation, ubiquitination, etc. of protein products). Epigenetic regulation of DNA involves a reversible, heritable change that does not alter the sequence itself. DNA methylation occurs on the nucleic acid cytosine. Arginine and lysine are the most commonly methylated amino acids. When proteins called histones) contain certain methylated residues, these proteins can repress or activate gene expression. Often this occurs on the transcriptional level, and thus prevents the cell from manufacturing messenger RNA (mRNA), the precursor to proteins. Proteins are often referred to as the workhorse of the cell and are responsibl e for everything from catalyzing chemical reactions to providing the building blocks for skeletal muscles. Some proteins, called transcription factors), help to up- or down-regulate gene expression levels. These proteins can act alone or in conjunction with other transcription factors and bind to DNA bases near gene coding regions. This is a general schema for gene expression. DNA is a double-stranded molecule consisting of base-paired nucleic acids A, C, G, and T on a sugar-phosphate backbone and is used as information storage. mRNA is made during transcription and carries a specific instance of the DNA instructions to the machinery that will make the protein. Proteins are synthesized during translation using the information in mRNA as a template. This is a deterministic process during which each molecule is manufactured using the product of the previous step. mRNA requires a 5 cap and a 3 poly(A) tail in order to be exported out of the nucleus. The cap is critical for recognition by the ribosome and protection from enzymes called RNases that will break down the molecule. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by other enzymes called exonucleases. What can be gained by studying gene regulation? In general, it allows us to understand how an organism evolves and develops, both on a local scale (Choe, 2006,Wilson, 2008), and on a more global network level. There are, however, more specific reasons to investigate this process more closely. Failure in gene regulation has been shown to be a key factor in disease (Stranger, 2007). Additionally, learning how to interrupt gene regulation may lead to the development of drugs to fight bacteria and viruses (McCauley, 2008). A clearer understanding of this process in microorganisms may lead to possible solutions to the problem of antimicrobial resistance (Courvalin, 2005). There are two major factors that motivate the studies herein. Firstly, the size and quality of biological data sets has increased dramatically in the last several years. This is due to high-throughput experimental techniques and technology, both of which have provided large amounts of interaction data, along with X-ray crystallography and nuclear magnetic resonance (NMR) experiments which have given us the solved three-dimensional structure of proteins. Secondly, machine learning has become an increasingly popular tool in bioinformatics research because it allows for more sound gene and protein annotation without relying solely on sequence similarity. If a collection of attributes which distinguish between two classes of proteins can be assembled, function can be predicted. In this work we focus mainly on regulation at the transcriptional level and the components which play a commanding role in this operation. So-called nucleic acid-binding (NA-binding) proteins, which includes transcription factors, are involved in this and many other cellular processes. Disruption or malfunction of transcriptional regulation may result in disease. We identify these proteins from representative data sets which include many categories of proteins. Additionally, in order to understand the underlying mechanisms, we predict the specific residues involved in nucleic acid binding using machine learning algorithms. Identification of these residues can provide practical assistance in the functional annotation of NA-binding proteins. These predictions can also be used to expedite mutagenesis experiments, guiding researchers to the correct binding residues in these proteins. Toward the ultimate goal of attaining a deeper understanding of how nucleic acid-binding proteins facilitate the regulation of gene expression within the cell, the research described here focuses on three particular aspects of this problem. We begin by examining the nucleic acid-binding proteins themselves, both on the protein and residue levels. Next, we turn our attention toward protein binding sites on DNA molecules and a particular type of modification of DNA that can affect protein binding. We then take a global perspective and study human molecular networks in the context of disease, focusing on regulatory and protein-protein interaction networks. We examine the number of partnership interactions between transcription factors and how it scales with the number of target genes regulated. In several model organisms, we find that the distribution of the number of partners vs. the number of target genes appears to follow an exponential saturation curve. We also find that our generat ive transcriptional network model follows a similar distribution in this comparison. We show that cancer- and other disease-related genes preferentially occupy particular positions in conserved motifs and find that more ubiquitously expressed disease genes have more disease associations. We also predict disease genes in the protein-protein interaction network with 79% area under the ROC curve (AUC) using ADTree, which identifies important attributes for prediction such as degree and disease neighbor ratio. Finally, we create a co-occurrence matrix for 1854 diseases based on shared gene uniqueness and find both previously known and potentially undiscovered disease relationships. The goal for this project is to predict nucleic acid-binding on both the protein and residue levels using machine learning. Both sequence- and structure-based features are used to distinguish nucleic acid-binding proteins from non-binding proteins, and nucleic acid-binding residues from non-binding residues. A novel application of a costing algorithm is used for residue-level binding prediction in order to achieve high, balanced accuracy when working with imbalanced data sets. During the past few decades, the amount of biological data available for analysis has grown exponentially. Along with this vast amount of information comes the challenge to make sense of it all. One subject of immediate concern to us as humans is health and disease. Why do we get sick, and how? Where do our bodies fail on a molecular level in order for this to happen? How are diseases related to each other, and do they have similar modes of action? These questions will require many researchers from multiple disciplines to answer, but where do we start? We take a bioinformatics approach and examine disease genes in a network context. In this chapter we analyze human disease and its relationship to two molecular networks. First, we find conserved motifs in the human transcription factor network and identify the location of disease- and cancer-related genes within these structures. We find that both cancer and disease genes occupy certain positions more frequently. Next, we examine the human protein-protein interaction (PPI) network as it relates to disease. We find that we are able to predict disease genes with 79% AUC using ADTree with 10 topological features. Additionally, we find that a combination of several network characteristics including degree centrality and disease neighbor ratio help distinguish between these two classes. Furthermore, an alternating decision tree (ADTree) classifier allows us to see which combinations of strongly predictive attributes contribute most to protein-disease classification. Finally, we build a matrix of diseases based on shared genes. Instead of using the raw count of genes, we use a uniqueness) score for each disease gene that relates to the number of diseases with which a gene is involved. We show several interesting examples of disease relationships for which there is some clinical evidence and some for which the information is lacking. We believe this matrix will be useful in finding relationships between diseases with v ery different phenotypes, or for those disease connections which may not be obvious. It could also be helpful in identifying new potential drug targets through drug repositioning.

Inoculation of an Egg

1. EGG INOCULATION The fertile hen’s egg can be used to cultivate and propagate various types of viruses. Because of the ability to alter their tropism and to adapt to a new host species, many viruses become capable of growing in chick embryo tissues wherein they frequently attain a much higher concentration than in the tissues of the natural host. STRUCTURE OF AN EGG The extra-embryonic membranes of the chick embryo arise from three germinal layers: the endoderm, mesoderm and ectoderm (Fig. 1).The dorsal somatopleure consists of ectoderm on one side and mesoderm on the other side while the splanchnopleure consists of mesoderm and endoderm. By a process of folding, the somatopleure gives rise to the chorion and amnion while the allantois and yolk sac membranes develop from the splanchnopleure. The amnion arises from the head and caudal regions of the embryo, the membrane being reflected back to form the chorion. the amniotic membranes grow rapidly and fuse to form the amniotic sac by the 5th day. The allantois grows out as a bud from the hind gut of the embryo and enlarges rapidly.By the 10th day the allantois becomes attached to the outer layer of the amniotic sac and the inner layer of the chorion to form the chorioallantoic sac (CAS) which separates the chorion from the amnion. The fused chorionic and allantoic membranes are referred to as the chorioallantoic membrane (CAM). Because the CAS represents a diverticulum of the gut, it serves as the excretory receptacle for the embryo. It contains from 5 to 10 ml of fluid with dissolved solids, the solution being clear in early stages but becoming turbid after the 12th day due to the presence of urates.The CAM is the respiratory organ of the embryo and thus is richly supplied with blood vessels. The embryo is surrounded by the amniotic sac and lies bathed in about 1 ml of amniotic fluid. The amniotic fluid, which contains much of the albumin in the egg, serves as a source of protein which is ingested durin g swallowing movements the embryo is seen to make from the 9th day onward. The air-sac is present in the blunt end of the egg. Underlining the shell is the fibrous egg shell membrane. In the beginning stages of development, the chick embryo can be recognized with difficulty as a small dark area attached to the yolk sac.After 4-5 days the embryo can be readily detected by candling. After the 10th day, the embryo development, rapidly increase in size and feathers appear. The respiratory tract develops between the 12th and 15th days. If the egg remains uninoculated and is maintained in a humid 38oC environment, it will hatch on the 21st day of life. Inoculation Procedures The methods described below for the inoculation of the chick embryo do not comprise a complete list but represent those that are practiced most commonly. Likewise, while there are a number of techniques for inoculation by each of the routes listed, only the one most widely used is described.A. Yolk Sac Chlamydia and r ickettsia grow readily in the yolk sac (YS) membranes. Although some of the smaller viruses are inoculated by the YS route, they invade and replicate in the tissues of the embryo itself rather than in the YS tissues. a. Candling and drilling. Fertile eggs that have been incubated for 5 to 7 days are suitable since the YS is relatively large at this time. The eggs are candled and the boundary of the air sac penciled in. The shell over the air space, which is referred to as the shell cap, is disinfected by an application of iodine to one small area.When the iodine is dried, a hole is made through the shell over the center of the natural air space by means of a drill or egg punch. b. Inoculation and incubation. By means of a syringe fitted with a one and one-half to two inch 23 gauge needle, the inoculum is deposited in the YS by passing the needle through the hole in the shell cap and directing it downward to its full length parallel to the long axis of the egg. From 0. 2cc to 0. 5cc is usually inoculated. the hole in the shell is then sealed with tape and the eggs are incubated at 37oC. c. Harvesting Procedure.The egg is placed in a container which maintains it in the upright position during the harvesting procedure. The shell is cracked with sterile forceps and the cap lifted off. The exposed membranes are torn away. If the YS membranes are to be harvested. The contents of the egg are quickly emptied into a sterile petri dish. The YS is usually ruptured in the process. The YS membranes, which are easily recognized by their deep yellow color, are detached from the embryo and separated from the chorioallantois with sterile forceps and quickly transferred to a sterile petri dish.When the embryo is to be harvested, it is withdrawn by hooking the curved end of a dental probe around the neck. It is then separated from the adherent membranes with sterile scissors and transferred to a sterile petri dish. B. Chorioallantoic Sac (CAS) The influenza and the newcastle dis ease viruses and most other viral agents which cause respiratory infections grow readily in the endodermal cells of the allantoic sac wall and are liberated into the allantoic fluid. The encephalomyelitis viruses and the mumps virus also multiply readily when inoculated by this route. . Candling and drillings. Embryonating eggs which have received a preliminary incubation from 9 to 11 days are candled and the boundary of the air space penciled in. The eggs are held in the upright position with the air sac uppermost. A point is selected a few millimeters above the floor of the air space on the side of the egg where the chorioallantois is well-developed but free of large vessels. Iodine is applied to the area around the site. A hole is then drilled or punched through the shell. b. Inoculation and incubation.A one-half inch 26 gauge needle, fitted to a small syringe containing the inoculum, is inserted into the allantoic cavity by passing it through the hole in the shell parallel to th e long axis of the egg or at an angle directed towards the apical extremity. From 0. 1cc to 0. 2cc of inoculum is injected into each egg. The hole in the shell is then sealed with tape and the eggs are incubated. c. Harvesting of allantoic fluid (AF). In order to avoid hemorrhage into the AF while harvesting, the eggs are chilled in the refrigerator from 4 to 6 hours prior to the harvesting procedure.While harvesting, eggs are held in an upright position and the shell over the air sac is removed with sterile forceps. The floor of the air space is exposed. With a pair of small sterile curved forceps these membranes are torn away. In order to facilitate the harvesting of the AF the embryo is displaced to one side by placing the forceps against the embryo with the tips toward the shell wall. The AF can then be readily aspirated with a 5 ml or 10 ml sterile pipette. C. The Chorioallantoic Membrane (CAM) Nine to 12 days old embryonating eggs are candled and an area over the most vascular portion on the side of the egg marked with a pencil.The shell is disinfected with iodine over this point and also the air sac end. A hole is carefully punched over both these locations. The hole on the side of the egg must penetrate both the shell and the inner shell membrane. A small amount of fluid may exude from the hole if the inner shell membrane is penetrated. While candling the egg with its long axis in the horizontal position, a piece of rubber tubing is placed firmly over the hole in the end of the egg. Suction is applied until the air sac collapses in the end but reappears on the side of the egg.When this false air sac is confirmed by candling, the CAM is ready for inoculation. The CAM and inner shell membrane are usually tightly adherent by 9 days of incubation, and the inner shell membrane may consequently by dropped as well as the CAM. This is unacceptable since the inoculum will fall on the inner shell membrane and not the CAM. To avoid this, a drop of sterile PBS is placed over the newly punched hole in side of egg to soften the membranes. An alternate method is to drop the CAM at 7-8 days of incubation, then wait until 11-12 days before inoculation. a. Inoculation and incubation.By carefully passing the needle through the inner shell membrane from 0. 1cc to 0. 2cc of inoculum is dropped on the chorioallantois with a 1cc syringe fitted with a 22 or 23 gauge one-half inch needle. In very critical studies the egg should be candled during this procedure to insure that the inoculum is deposited on, rather than through, the membrane. After inoculation the egg is gently rocked in order to spread the inoculum uniformly over the surface of the CAM. The opening in the shell is covered with a small square of scotch tape and the inoculated eggs are incubated in a horizontal position with the hole uppermost. . Harvesting of the membrane tissues. The egg is placed in the horizontal position with the hole uppermost. Iodine is applied to the area around the w indow with a cotton swab and the tape then peeled off. The surrounding shell is broken away with sterile forceps and the chorioallantois exposed. The membrane is grasped with forceps, detached with scissors and quickly transferred to a sterile Petri dish. D. Amniotic Sac This method is used principally for the isolation of the influenza virus from throat washings. The embryo during the course of its development wallows the amniotic fluid, thereby bringing the inoculated virus which it contains into contact with the tissues of the respiratory and intestinal tracts where multiplication presumably occurs. After incubation amniotic fluid is then â€Å"subpassaged† by the CAS route (Fig. 2). The amniotic route of inoculation is used also for the isolation of the encephalomyelitis virus. a. Candling and drilling. Embryos from 13 to 15 days of age are used. The position of the embryo is determined by candling and a point on the shell over the air space on the side of the egg on whic h the embryo is situated is marked.The site is prepared in the usual manner and a hole is drilled or punched as for yolk sac inoculation. b. Inoculation and incubation. A 1cc syringe fitted with 1 3/4 inch 24 gauge needle is used for the inoculation. The egg is placed horizontally on the candler, the needle is introduced and gently stabbed in the direction of the embryo. Penetration of the amniotic sac is indicated by a sudden movement of the embryo. The needle is then withdrawn slightly and from 0. 1cc to 0. 2cc of the inoculum injected. the hole in the shell is sealed with tape and the eggs are incubated in the vertical position. . Collection of amniotic fluid. The shell is removed as for the allantoic and yolk sac routes of inoculation. A few drops of saline are placed on the floor of the air space to render the membrane transparent. Using the eyes of the embryo as a reference point, the amniotic fluid is aspirated by means of a Syringe fitted with a short 23 gauge needle. E. Mis cellaneous Routes ofInoculation a. Intravenous. This method is not used commonly, although it is the method of choice for the isolation of bluetongue virus. A large vein is located and marked in 12-14 day embryos.A rectangular piece of shell directly over the vein is removed and a droplet of sterile mineral oil is placed on the inner shell membrane so as to render it transparent. A 27-30 gauge five-eight inch needle fitted to a small syringe is introduced through the membrane into the vein in the direction of blood flow. From 0. 1 to 0. 5cc of inoculum is then injected. Incubation and harvesting of the embryo is carried out as already described. b. Intracerebral. This route may be used in the studies of pathologic alterations of the brain following infection. Eight to 14 day embryos are usually used.The viruses of herpes simplex and rabies may be cultivated by this method. Egg Inoculations. Materials needed: Embryonated eggs, 11-12 days A paramyxovirus, PI3 or Sendia virus Vaccinia virus Crystal violet Appropriate syringes and needles Egg candlers, egg punches Iodine disinfectant and swabs, cellophane tape Instruments, petri dishes Procedure: 1. Inoculation of dye into CAS a. Candle 11-12 day embryonated egg, mark boundaries of air sac with pencil. Just above air sac, choose a point devoid of vessels and mark with a pencil. bDisinfect egg shell at this point with iodine.Let dry before next procedure. c. Drill a small hole with an egg punch at the appropriate marked point (be careful-don’t break the shell). d. Inject 0. 1 – 0. 2ml dye into the CAS as described and illustrated. e. Place a small piece of cellophane tape over the hole. The egg would be ready to incubate if the inoculum had been virus. f. Candle the dye-inoculated egg to establish that the inoculum is in the correct place. Watch the inoculum spread through-out the confines of the CAS. g. Break the egg and pour the contents into a petri dish. Observe where the dye is.Identify the CAM, YS, amnion and embryo. If inoculated properly only the CAS should contain dye. 2. CAS inoculation of virus a. Follow procedures for CAS inoculation of a dye, except the inoculation should be 0. 1 ml of a live paramyxovirus. Place tape over the inoculation hole and incubate. b. Candle egg daily to determine embryo viability. If the embryo dies within 2 days of inoculation, it usually indicates bacterial contamina-tion or trauma. c. If the embryo dies after 2 days, refrigerate as soon as death is noted until the next laboratory period. 3. CAM inoculation of vaccine virusWarning;If you have not had a successful smallpox vaccination, have eczema or evidence of immune deficiency, contact the instructor before handling this virus. Be careful! Do not get this virus in your eyes! Vaccinia virus is the live virus vaccine for smallpox. While less pathogenic than smallpox or variola virus it can still cause serious or uncomfortable lesions if mishandled. a. Two 11-12 days embryonated eggs will be supplied to each group of students. Drop the membrane on both of the eggs according to the instructions and illustrations. b. Inoculate 0. ml vaccinia virus onto the dropped CAM. be sure to go through the inner shell membrane but not through the CAM. Rock the egg to distribute the inoculum over the entire floor of the false air sac. Cover the hole with tape and incubate in a horizontal position with the hole uppermost. 4. Harvesting of embryonated eggs (next laboratory). a. Follow instructions for removal of CAS fluid. Try to keep blood vessels from rupturing. Remove CAS fluid aseptically in a sterile pipette. Expel fluid into a sterile vial. This fluid will be used for the hemaggulatination exercise later.It can be frozen if necessary. Use the last drop of CAS fluid to inoculate bacteriological media to check for contamination. b. Harvest CAM as per instructions. Place the membrane in a petri dish and lightly pour PBS over the membrane until it flattens out and the pocks are cl early visible. c. Important. All fluids, instruments, and other things that have come into contact with virus-infected tissues must be properly sterilized. Follow carefully the instructor’s remarks for proper disposal of all materials. Be sure to disinfect your workspace with disinfectant when cleaning up. 1. INFECTIVITY ASSAYS The concentration of a suspension of virus is usually determined by measuring its infectivity. There are two types of infectivity titrations: the quantal assay, which depends upon an all-or-none does response, and the quantitative assay, which utilizes a plaque, pock or lesion count in which the effect of a single infectious virus particle is seen as a visible localized change in a background of normal cells. A. Quantitative Assay2 This method determines the actual number of infectious units (virus particles) in a given suspension.This type of enumerative response is assessed from focal lesions such as plaques in cell cultures, pocks on the CAM of chic k embryos or local necrotic lesions on a plant leaf. The number of infectious units per unit volume can be calculated, and this is referred to as the titer. With plaque assays, the titer of the original virus suspension is stated in terms of the number of plaque forming units (PFU) per ml. Ex. Fifty plaques on a 10-5 dilution of original suspension were counted. A 0. 1ml inoculum was used. #PFU/ml. of original volume No. of plaques = ——————————— dilution) x (Vol. of inoculum) =5. 0 x 107 PFU/ml=50 x 106 = or 105 x 0. 1 B. Quantal Assay This assay estimates the concentration of infectious particles by allowing them to replicate in a suitable host so that one infectious unit can be detected by the amplification effect of the infection. The actual number of infectious particles introduced into the test unit is unknown and may vary even between duplicates of the same dilution. To determine quantal infectivity t iters, mutiple replicate tests are used for each dilution of original suspension until the infectivity is diluted out.The result gives the dose necessary to produce a defined response. This response is usually based on a 50% end point, which is the dilution at which 50% of the test animals, eggs, or cell cultures react to the virus. Computation of the 50% end point is based on the presence or absence of a predetermined criterion, i. e. death (Median Lethal Dose or LD50), infectivity (Median Tissue Culture Infective Dose or TCID50, Median Egg Infective Dose or EID50), etc. The criterion must be either present of absent: either the animal is dead or alive, or the cell culture is infected or not infected.There are no plus/minus or graded reactions. This method does not measure the exact number of virus particle but only whether or not virus is present at a particular dilution. There are two formulas that can be used to determine 50% endpoints; the Reed-Muench and the Spearman-Karber me thods. Both are demonstrated here using the same data. 1. Reed-Muench Method Accumulated Values |Virus Dilution |Morality Ratio | | | | | | | |(a) |(b) |Died |Survived Died |Survived |Ratio |Percent | | | |(c) |(d) |(e) |(f) |(g) |(h) | |10-1 |6/6 |6 |0 |17 |0 |17/17 |100 | |10-2 |6/6 |6 |0 |11 |0 |11/11 |100 | |10-3 |4/6 |4 |2 |5 |2 |5/7 |71 | |10-4 |1/6 |1 |5 |1 |7 |1/8 |13 | |10-5 |0/6 |0 |6 |0 |13 |1/13 |1 | | | | | | | | | | At the 10-3 dilution, 5/7 or 71% of the accumulated test animals died, and at the 10-4 dilution, 1/8 or 13% died (columns g and h). The 50% endpoint, therefore, lies somewhere between the 10-3 and 10-4 dilutions. The final calculation requires interpolation between these two values. The formula for doing this is: (% mortality at dilution next above 50%) – (50%) ————————————————————— = proportionate distanc e (% mortality at dilution next above 50% – Mortality at at dilution next below or 71-50 21 —- = — = 0. 36 proportionate distance 71-13 58The dilution factor must also be considered, i. e. , 2-fold, 4-fold, 10-fold, etc. and the proportionate distance corrected (multiplied) by the log10 of the dilution factor (2-fold = 0. 3, 5-fold = 0. 7, 10-fold = 1, etc. ) The final estimate is determined by this formula: Negative log10 of LD50 end point = negative log of dilution above 50% mortality plus the proportionate distance factor (corrected for dilution series used) Negative log of dilution above 50% mortality –3. 00 Proportionate distance (0. 36) x dilution factor (log10-1= -1)= – 0. 36 Negative log LD50= -3. 36 LD50=10-3. 36 antilog of 10. 36=2. 29 LD50 titer -3. 36 =2. 29 x 103 / volume inoculated LD50 Calculation Inoculum: |DILUTION |DEAD |ALIVE |CUMULATIVE DEAD |CUUMULATIVE ALIVE |LD50= | |EXAMPLE |10-1 |6 |0 |17 |0 |(a-b)(c+d) | | | | | | | |2[(ax d)-(bxc)] | |Test System: |10-2 |6 |0 |11 |0 |=(3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | |Date Inoculated |10-3 |4 |2 |5 |2 |=24 =0. 36 | | | | | | | |66 | | |10-4 |1 |5 |1 |7 |LD50 = 3. 36 | | |10-5 |0 |6 |0 |13 | | |Inoculum |Dilution |Dead |Alive Cumulative Dead ( |Cumulative Alive ( | | | | | | | | |LD50= | |Test Systems: | | | | | |(a-b) (c+d) | | | | | | | |2((a x d) – (b x c)( | | | | | | | | | |Date Inoculated: | | | | | |= (3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | | | | | | | |=___________ | | | | | | | | | Inoculum |Dilution |Dead |Alive |Cumulative Dead ( |Cumulative Alive ( | | | | | | | | |LD50= | |Test Systems: | | | | | |(a-b) (c+d) | | | | | | | |2((a x d) – (b x c)( | | | | | | | | | |Date Inoculated: | | | | | |= (3) (8) | | | | | | | |2[(5Ãâ€"7)-(2Ãâ€"1)] | | | | | | | |=___________ | | | | | | | | | 2. Spreaman-Karber Method Estimation of the 50% endpoint by the Spearman-Karber method is much simpler. The formula is: Negative log10 of LD50 = X – d (P-0. 5) here X = log10 of the highest concentration used (lowest dilution); d = log10 of the dilution factor, and p = sum of % mortality at each dilution100 Using the same data chart above the following number are obtained: d(10-1)(do-2)(10-3)(10-4) Neg log : LD50 = 1. 0 -1(100 + 100 + 66 + 17)-0. 5 100 = -1 [1(2. 84-0. 5)] = -1 2. 34 = -3. 34 LD50 antilog of 10. 34 = 2. 19 LD50 titer= 2. 19 x 103 volume inoculated Note that the two methods produce slightly different results using the same data. The Spearman-Karber method is considered to be the more accurate. The Spearman-Karber method can be simplified even more if signs are neglected and common sense used.This formula is: Neg log LD50 = X + d (P + 0. 5) Where X = log10 of highest dilution showing 100% mortality; d = log10 of dilution factor; p = proportion of positives above dilution X Xdp Neg. log LD50 = 2 + 1 (4/6) = 1/6 + 0. 5) = 2 + 1 (. 67 + . 17 + 0. 5) = 2 + 1. 34 = 3. 34 The appropriate sign can then be inserted: LD50 = 10-3. 34 These formulas can also be used to estimate 50% endpoints in neutralization tests. Here, absence of the predetermined criterion is counted and used for the calculations. III. SEROLOGIC TECHNIQUES A. Hemagglutination Many viruses or viral antigens are capable of specifically and non-covalently binding to receptors on the surface of red blood cells (RBCs).When the right volumes of these viruses and RBC’s are mixed, the viruses bridge the RBCs to form a lattice which settles out of suspension in a uniformly thin shield on the bottom of a test tube or conical well. This phenomenon was first described by Hirst in 1941 and is known as hemagglutination (HA). the HA titer of a virus can be determined by mixing serial dilutions of a virus with a constant amount of RBCs which are usually prepared as a 0. 25%-1. 0% suspension in physiological saline. the highest dilution which agglutinates the RBCs is the endpoint. The HA titer is the reciprocal of the endpo int dilution, and that dilution is said to contain one HA Unit (HAU) of virus in the original volume.Unagglutinated RBCs sediment to a packed disc (â€Å"button†) on the bottom of the test tube or well. The viruses known to cause hemagglutination are heterogeneous but can be grouped according to the nature of their hemagglutinating protein (hemagglutinin). The hemagglutinin on the virion of influenza and the paramyxoviruses is a glycoprotein. These viruses, but not any others, also carry an enzyme, neuraminidase, which destroys the glycolipid receptors on the RBC surface and allows the virus to elute (unless the HA is carried out at a temperature too low for the enzyme to act). Certain toga and Coxsackie viruses possess a hemagglutinin but do not possess a neuraminidase-like enzyme. astidious conditions are necessary for these viruses to hemagglutinate and usually cells from only a very few species can be can be used. Vaccinia virus has a lipoprotein hemagglutinin associated with a soluble fraction separable from the viral particle itself. Some viruses agglutinate RBCs from a limited number of course and some HA reactions require careful control of pH, temperature and ionic conditions (see Table 5-1, p. 100-101, Rovozzo & Burke). We will perform the HA test with a paramyxovirus which will agglutinate human type O, bovine, guinea pig or chicken RBCs over fairly broad ranges of pH (6. 0-8. 0) and temperature (4-25oC). Material needed: 1 cc syringes 0. 025 ml microtiter tips 0. 25 ml microdiluters Microtiter plates with 96-V-bottom wells Phosphate buffered saline (PBS) Paramyxovirus, in form of allantoic fluid harvested 48-96 hr after infection of 10-day embryonating eggs Washed RBCs, 0. 5% in PBS Go-no-go test papers Dilution tubes and 1 ml pipets *Use only the top half the microtiter plate. Procedure: 1. With the micropipet (a microtiter tip attached to a 1cc syringe) held vertically, dispense 0. 025 ml (1 drop) PBS each into columns 2 through 12 of rows A. B, C, and D of the microtiter plate. Also put 0. 025 ml PBS into wells 1 through 4 of row H for controls. 2. Make a 1:10 dilution of virus in PBS in a dilution tube.With the same pipet used to dispense PBS, put 0. 025 ml of the 1:10 virus dilution into each well of columns 1 and 2, rows A-D. 3. Test the delivery volume of the microdiluter by immersing the tip in PBS then touching to the center of a circle on the go-no-go paper. Now begin dilutions by immersing the dry diluter in well two, rotating to mix and pick up 0. 025 ml of fluid, and transferring to well three. Continue rotating and transferring through row 12. Two lines may be diluted simultaneously if desired. After removal of 0. 025 ml from row 12, dip the diluter in disinfectant, then distilled water, then flame. Do not flame with protein or salt in the diluter.Do not add virus to the controls. 4. With a new syringe and tip add 0. 025 ml (1 drop) 0. 5% RBCs (mix suspension well before pipetting) to every well, includin g controls. Mix well by running a hard object down the underside of the plate. 5. Allow to stand at room temperature until the controls and higher virus dilutions have RBCs settled into a â€Å"button† in the point of the V, and positive wells have RBCs uniformly spread over the entire bottom of the well. This will take 1-2 hr. Then refrigerate the plate. 6. Read the HA titer as the reciprocal of the dilution of the last well showing positive HA. Calculate the dilution which contains 4HAU/0. 25ml for use in the HI test.Be sure to check controls for spontaneous agglutination. B. Hemagglutination Inhibition Viral hemagglutination may be inhibited in several ways. By combining with viral antigens which normally interact with RBC receptors, specific anti-viral antibodies can prevent the virus cell interaction which normally brings about hemagglutination. Since infection with a virus will elicit production by the host animal of antibodies directed against each virus-induced protei n, including the hemagglutinin, inhibition of hemagglutination by an animal’s serum indicates that the animal has been infected by the virus. A high HI titer may indicate that the infection was recent.A four-fold rise in titer between two serum samples taken a few weeks apart (as during acute and convalescent phases of a disease) indicates that infection occurred during the period between the sampling times. If the viral hemagglutinin is also the protein by which the virus attaches to cells susceptible to infection, a high HI titer shows an animal to be immune to reinfection. HI is carried out in much the same way has HA. The serum is diluted in microtiter plates and each dilution is allowed to react with a constant dose of virus (usually four HAU) for an interval of 15 min to one hr before RBCs are added. The reciprocal of the highest serum dilution which inhibits HA is the HI titer.Several controls are necessary (1) The lowest dilution of serum used in the test must be incu bated alone with RBCs to determine if it contains heterophile antibodies which cause RVC agglutination. (2) The virus must be back titrated to see that the proper dose was added to the test wells. (3) A known non-immune serum from the same animal species must be titrated (usually before the HI test is performed) to see if it contains non-specific inhibitors. Heterophile antigens are a group of shared antigens with over-lapping specificities. They are found in some plants (corn, spinach) some microorganisms (Pneumococcus, E. coli), and some fish and animal tissues (carp, toad, guinea pig, horse, man etc. Heterophile antibodies against these antigens will cross react with cells and fluids from the above-listed species. In the case of RBCs as the heterophile antigen, if heterophile antibodies against them are present, hemagglutination will occur, possibly masking the presence of the hemagglutination-inhibition reaction caused by anti viral antibodies. Nonspecific inhibitors of hemagglu -tination may also be found in the serum of man and animals. Their nature differs for different viruses and even for different strains of viruses, such as influenza virus. Serum inhibitors also differ in different species. Inhibitors may be of low titer, or in some cases higher than the actual antibody titers, thus masking its diagnostic importance.Methods which have been used to remove inhibitors include: (1) Heating at 56-1/4 for 30 min, (2) treatment with receptor-destroying enzyme (neuraminidase), trypsin and/or periodate, (3) absorption with kaolin, (4) extraction with acetone, (5) precipitation of beta-lipoproteins with heparin and manganous chloride or with dextran sulphate and calcium chloride. No single method is universally applicable. Sometimes more than one method must be used. Antibody titers can be depressed by some of these procedures. The final control used is the RVC saline control to check for self-agglutinating RBC. Table 1. Example of HA and HI |Virus |Antigen So urce |RBC |Temperature |Non. Sp. Inhib.Removal | |Influenza A and B |CAS fluid or cell culture|Chicken, Human O |Room |Neuraminidase | | |fluid | | | | |Mumps |CAS fluid |Chicken |Room |Neuraminidase | |Coxsackie |Cells culture fluid |Fowl |Room |Kaolin | |Rubella |Cell culture fluid |one-day old chicken, goose |4oC |Heparin and Manganous chloride| |Adenoviruses |Cell culture fluid |Rat, rhesus monkey |37oC/room |Not required | Procedure 1.Add one drop (0. 025 ml) PBS diluent to all wells of the microtiter plate. 2. You will be given three serums. One will be untreated, one treated for removal of non-specific inhibitors and/or heterophile antibodies, and one known negative serum. Your results will be compiled with the class results to clarify the total experiment. using the microdiluters, add 0. 025 ml test serum to well A of row 1 and 2. Dilute out to well H. The first well is a 1/2 dilution of serum and row H is 1/256. Add 0. 025 ml of the same test serum to row 7, the serum contr ol well. Dilute to well H as before. Carefully rinse the diluters and repeat with test serum 2 and test serum 3, sing rows 3, 4, 8 and 5, 6, 9 respectively. 3. Add 0. 025 ml of the challenge virus with the microdiluters to well A of rows 10 and 11. Dilute to well H. This is the antigen (virus) back titration and control. The highest dilution with complete hemagglutination is 1 HA unit. 4. Using the same â€Å"micro-pipet† as in #1, add another drop of PBS to all wells of rows 7-12. Empty the pipet and refill with the hemagglutinin (virus). Add one drop hemagglutinin to all wells of rows 1-6. Mix well. 5. Incubate at room temperature 30-60 minutes. 6. Using a new pipet, add one drop 0. 5% bovine RBC to all wells. Mix well. Store at 4oC and read the next day.Antibody titers are the highest dilution that inhibits hemagglutination (forms a distinct button). C. Hemadsorption Certain enveloped hemagglutinating viruses cause the insertion of viral hemagglutinins into the plasma memb rane of cells in which they are replicating. These modified areas of the cell surface are the sites at which progeny virus particles will mature. If agglutinable RBCs are brought into contact with hemagglutinin-containing surfaces of cultured cells, the RBCs will specifically bind to the infected cells. This phenomenon, known as hemadsorption, is particularly useful in detecting infection by viruses which cause little morphological change in infected cells. Procedure 1.Pour off medium from a tube of cultured cells infected with an orthomyxovirus or a paramyxovirus and from a tube of uninfected cells. 2. Wash monolayer thoroughly but gently with two rinses of 3 ml of physiological saline. 3. Add 0. 2 to 0. 5 ml 0. 5% bovine RBCs in saline. Allow RBC suspension to cover cell layer. Incubate at room temperature for 10-15 minutes. 4. Pour off RVC suspension and wash 2x with 2-3 ml saline. 5. Examine under microscope. Infected cells should have entire surface covered with RBCs. Non speci fic binding will cover only a few sites per cultured cell. SERUM NEUTRALIZATION The neutralization test estimates the capacity of a specific serum antibody to neutralize a virus biological activity.Major uses for this test include the identification of unknown virus or antibody, the determination of antibody levels, the comparison of antigenically related viruses and the study of the kinetics of antigen-antibody reactions. Viruses and the study of the kinetics of antigen-antibody reactions. Neutralization can occur by several mechanisms. Virus adsorption to cells may be inhibited by alteration of the configuration of cell receptor sites or by prevention of viral attachment. Virus degradation may be enhanced by interference with post-engulfment stages of virus replication, by prevention of release of functional virus cores into the cytoplasm or by the degradation of virus-Ab complexes within phagosomes.Also, complement-mediated reactions may enhance neutralization by production of le sions in the viral envelope. Several factors must be considered when performing a neutralization assay. Sensitivity of the test is related to the degree of susceptibility of the indicator host system to infection with the virus. The neutralization reaction is readily reversible by dilution with saline, by ultrasonic treatment or by lowering pH. Finally, the time required to reach equilibrium may vary with different systems. When performing the neutralization test two systems are used. The reaction system is incubation of virus and specific antisera until equilibrium is reached. The indicator system is the inoculation of the virus-Ab mixture into a susceptible host.If neutralizing anti-bodies are not present lesions such as pocks or plaques will be seen in the host. If neutralizing antibodies are present there will be no lesions. There are two techniques commonly used for the neutralization test. In the alpha procedure a constant serum concentration is added to serial log dilutions o f virus. The mixture is incubated and inoculated into an appropriate host system. In the beta procedure a constant virus concentration is incubated in serial two-fold dilutions of serum before inoculation into the host. The beta procedure is most commonly used because of its sensitivity, ability to measure antibody titer and its economical use of serum.The alpha procedure is not as sensitive and may be more subject to non-specific inhibition. It is more frequently used for comparative studies. Alpha Neutralization test Materials Needed: Flat-bottomed MT plate with bovine cell monolayer MT transfer plate with lid and holder MT tips 1 cc syringes serum samples stock virus MEM diluent dilution tubes sterile distilled water in beaker Procedure: Use aseptic technique. a. Make serial 10-fold dilutions of stock virus to 10-8 using MEM and dilution tubes (0. 2 plus 1. 8 ml). b. Using sterile 1 cc syringe and microtip add 1 drop (0. 025 ml) diluent (MEM) to rows 7 and 8 wells A-H, and rows 9 , 10 and 11 wells A and B of the transfer plate. c. Using the same syringe and microtip add 0. 25 ml of the virus dilutions to rows 1-8 as follows: 10-8 into wells H, 10-7 into wells G, and so on finishing with 10-1 in wells A. d. Using a new syringe and microtip add 0. 025 ml test serum A to rows 1 and 2 wells A-H, and row 9 wells A and B. Rinse syringe and microtip with sterile water and add 0. 025 ml serum B to rows 3 and 4 wells A-H, and row 10 wells A and B. Again rinse out syringe and microtip with sterile water and add 0. 025 ml serum C to rows 5 and 6 wells A-H, and row 11 wells A and B. (Row 9, 10, and 11 are serum controls). e. Tissue culture controls are the unused portion of the plate. f. Incubate virus and serum at room temperature for 30 minutes, then transfer reagents to cell cultures. SerumVirusSerum Controls A B C 123456789101112 ABC A |10-1 |No | |B |10-2 |Virus | |C |10-3 | | |D |10-4 Virus | | |E |10-5 | | |F |10-6 | | |G |10-7 | | |H |10-8 | | Beta Neutral ization Test Materials Needed: Flat-bottomed MT plate with lid 1 cc syringes MT tips Mt diluters sterile distilled water in beaker MEM diluent serum samples virus, 25-50 TCID50 bovine cell suspension Procedure: Use aseptic technique. a. Add 1 drop (0. 025 ml) diluent (MEM) to rows 1-8 wells A-H. b.Make 2-fold dilutions of serum through row H (final dilution 1:256), cleaning microdiluters in sterile distilled water between serums. c. Using the same syringe and microtip as in step a, fill with pretitrated (25-50 TCID50) IBR virus and add 0. 025 ml to rows 1-4 wells A-H, and to rows 7 and 8 wells A. d. Using rinsed microdiluters make 2-fold dilutions of the virus in rows 7 and 8 wells A-H. e. Incubate at room temperature for 30 minutes. f. Add 2 drops (0. 05 ml) of bovine cell suspension using a new 1 cc syringe and microtip to all wells of the test plus a few extra for tissue culture controls. Controls Serum ASerum BABVirus 123456789101112 A |1:2 | | | |B |1:4 |No | | |C |1:8 |Virus | | |D |1:16 | | | |E |1:32 | | | |F |1:64 | | | |G |1:128 | | | |H |1:256 | | | IV. CELL CULTURE Because viruses are obligate intracellular parasites, they cannot replicate in any cell-free medium, and thus require living cells from a suitable host within which to multiply.Animals such as mice and embryonating avian eggs may be used for the propagation of viruses, but for various reasons (time, cost, ease of handling, etc. ) the propagation of most viruses in a cultural medium of living cells is the method of choice today. More than half a century has elapsed since animal cells were first grown in vitro. In 1912 Carrel began growing bits of chick heart in drops of horse plasma. The cells at the edge of the explant divided and grew out of the plasma clot. The explants died within a few days, and Careel reasoned that their death was due to exhaustion of nutrients. He found that cells from a given explant could be maintained indefinitely if they were periodically subdivided and fed with a sterile aqueous extract of whole chick embryos.In the early 1950’s, Earle developed a technique for dissociating cells from a whole chick embryo from each other with trypsin. When this suspension of single cells was mixed with plasma and embryo extract and placed in a sterile glass container, the cells adhered to the glass and divided to form a primary culture. The primary culture contained a variety of cell types including macrophages, muscle fibers, etc. The cells grew to a monolayer, a thin sheet of cells (one layer in thickness) which covered the entire bottom surface of their culture vessel, and then stopped dividing. The cells could then be redispersed with trypsin and planted in new culture vessels containing fresh media.These secondary cultures contained fewer cell types than did the primary cell cultures, as many of the differentiated primary cells were out-competed and did not survive the transfer. Often, secondary cultures are composed entirely of spind le-shaped cells called fibroblasts because of their similarity to cultured connective tissue. Cells derived from kidneys and from certain carcinomas have a polygonal appearance in culture. Because of their tissue of origin, they and other cells with similar morphology are call epithelial. Cells may be grown in vitro in several ways. Organ cultures, if carefully handled, maintain their original architecture and functions for several days or sometimes weeks.Slices of organs (which are actually tissue cultures) consisting of respiratory epithelium have been used to study the histopathogenesis of infection by respiratory viruses that can only be grown outside of their natural host by using organ cultures. The term tissue culture was original applied to explants of tissue embedded in plasma. the term subsequently became associated with the culture of cells in general and is now obsolete in its original sense. Cell culture is the term most widely used today. It refers to tissue dissociate d into a suspension of single cells, which after being washed and counted, are diluted in growth medium and allowed to settle on to the flat bottom surface of a specially treated plastic or glass container.Most types of cells adhere quickly, and under optimum conditions they will undergo mitosis about once a day until the surface is covered with a confluent cell monolayer. There are three main types of cultured cells. The difference in these types lies in the number of times the cells can divide. 1. Primary cell cultures When cells are taken freshly from animals and placed in culture, the cultures consist of a wide variety of cell types, most of which are capable of very limited growth in vitro, usually fewer than ten divisions. These cells retain their diploid karyotype, the chromosome number and morphology of their in vivo tissues of origin. They also retain some of the differentiated characteristics which they possessed in vivo. Because of this, these cells support the replicatio n of a wide range viruses.Primary cultures derived from monkey kidney and mouse and chick embryos are commonly used for diagnostic purposes and laboratory experiments. 2. Diploid cell strains. Some primary cells can be passed through secondary and several subsequent subcultures while retaining their original characteristics. After 20-50 passages in vitro, these diploid cell strains usually undergo a crisis in which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production. 3. Continuous cell lines. Certain cultured cells, notably mouse embryo fibroblasts and human carcinoma cells, are able to survive the growth crises and undergo indefinite propagation in vitro.After an initial slowing down, these continuous cell lines grow more rapidly than before, their karyotype becomes abnormal (aneuploid) and other poorly understood changes take place which make the cells immortal. The cells are now â€Å"dedifferentiated†, having lost the specialized morphology, and biochemical abilities they possessed as differentiated cells in vivo. Continuous cell lines such as KB and Hela, both derived from human others derived from mice (L929) and hamsters *BHK), are widely used in diagnostic and experimental virology. The development during World War II of antibiotics simplified long-term animal cell culture by minimizing the problems of bacterial and fungal contamination.Another important discovery was made by Eagle in the 1950’s when he determined the minimal nutritional requirements of cultured cells. He began by showing that Hela and Mouse L-cells would grow in a mixture of salts, amino acids, vitamins and cofactors, carbohydrates and horse serum. By eliminating one component at a time, he then determined which nutrients were essential for cell growth. His minimal essential medium (MEM) contains 13 amino acids (human tissue in vivo requires only 8), 8 vitamins and cofactors, glucose as any energy source and a physiological salt solution which is isotonic to the cell. The pH is maintained at 7. 2-7. 4 by NAHCO3 is equilibrium with CO2.The pH indicator phenol red is usually incorporated into the medium, which turns red-purple if the medium is alkaline, yellow if the medium is acidic, and remains red if the pH is suitable. Serum in concentrations of 1-10% must beaded to the medium to provide the cells with additional undefined factors, without which most cells will not grow. Most animal cells must be kept incubated at 37oC. If cells are grown in vessels open to the atmosphere, their incubator must be humidified and contain an increased CO2 concentration. Some nonvolatile phosphate or substituted sulfonic acid buffers (HEPES, TES) eliminate the requirement for incubators to be gassed with CO2. With the advent of cell culture, many animal viruses have been propagated in vitro, and hundreds of previously unknown viruses have been isol ated and identified.The discovery of the adenoviruses, echoviruses, and rhinoviruses, for example, is directly attributable to the use of cultured cells, as is the revolution in the diagnosis of viral diseases and the development of poliomyelitis, measles, and rubella vaccines. A. Culture of Primary Chick Embryo Fibroblasts (CEF) Materials 10-12 days old embryonated eggs Forceps and scissors Sterile petri dishes Sterile 250ml flask with magnetic bar Sterile 30 oz prescription bottles containing MEM & 5% lamb serum Sterile PBS Sterile 0. 5% trypsin (STV) Sterile 15ml centrifuge tubes containing 0. 5 ml serum Hemocytometers 1ml and 10ml pipets Sterile Dulbecco’s saline Procedure 1. Disinfect the surface of the egg over the air sac.With scissors or blunt end of forceps, break shell over air sac. Sterilize forceps by dipping in alcohol and flaming. Peel away shell over air sac, resterilize forceps and pull back shell membrane and chorioallantoic membrane to expose embryo. 2. Rest erilize forceps, grasp embryo loosely around neck, and remove from egg to sterile petri dish. 3. Using two forceps, or scissors plus forceps, decapitate and eviscerate embryo. Mince remainder of embryo to very small fragments. 4. Add about 10ml sterile Dulbecco’s saline to tissue fragments in petri dish, swirl to suspend fragments, and carefully pour into 250ml flask. With flask covered, continue swirling for 2-3 min. to wash tissue fragments.Tilt flask, allow fragments to settle, and gently decant saline. 5. Add 12ml sterile trypsin to fragments in flask, cover, and stir with magnetic bar for 15 min. Tilt flask, allow fragments to settle, and pour trypsin cell suspension into 15ml centrifuge tube containing 1ml serum. The serum contains a trypsin inhibitor which will prevent further damage to cell membranes but he enzyme (note: it is preferable to treat the tissue with multiple short applications of trypsin rather than a few long ones, in order to minimize enzymatic damage t o cell membranes. However, limitations of time require us to use the shorter method. ) 6. Add 12ml sterile trypsin to fragments and repeat step 5.At the end of this second treatment, size of tissue fragments would be greatly reduced and a large number of single cells should be suspended in trypsin. 7. Balance centrifuge tubes against one another and centrifuge at 1500 rpm for 10 min. Carefully decant off supernatant and resuspend pooled cell pellets in 1ml MEM. Make a 1:10 dilution of the cell suspension in MEM for counting in a hemocytometer. 8. In most hemocytometers each heavily etched square in 1mm on each side. The depth of the chamber is 0. 1mm. Count the cells in 0. 13 mm and calculate the number of cells in your original suspension. Dilute to give 8ml with 2-8 x 105 cells/ml in MEM, place in prescription bottle, replace cap tightly, and incubate on flat side at 37oC. 9. Be sure to examine cells periodically.Actively growing cells produce acidic metabolic by-products, and thu s the pH of the medium may need to be adjusted by the addition of a few drops of 7. 5% NAHCO3. If floating (dead) cells are present the medium may need to be changed. B. TRANSFER OF CELL CULTURES After cultured cells have formed a confluent monolayer on the surface of their culture vessel, they may be removed from the surface, diluted, and seeded into new vessels. If the initial culture was primary, the new cultures are called secondary, and are likely to consist of fewer cell types. Removal of cells from glass surfaces may be by either physical methods – scraping with a sterile rubber policeman – or chemical methods – proteolytic enzymes or chelating agents – or a combination of the two.After removal, cells are pipetted up and down and diluted appropriately in fresh secondary culturing, and after one becomes familiar with the growth characteristics of a certain cell types, counting can usually be dispensed with. We will transfer a cell line of bovine cel ls by use of a mixture of trypsin and EDTA (versene) in physiological saline (STV = saline, trypsin, versene): 1. Pour off the medium from a 3 oz. prescription bottle containing a confluent cell monolayer. 2. Wash the monolayer with 5-10 ml of physiological saline (Saline A) rinse well without shaking (shaking produces bubbles) and pour off. 3. Add 0. 5 ml STV to the bottle and incubate, with STV covering cells, at 37oC for 2-15 min.Observe periodically to determine when cells are loosened from glass (note: STV will contain a pH indicator and should have a pH of 7. 0-8. 0. Below pH 7. 0, trypsin is inactive. A pH above 8. 0 is damaging to cells. ) 4. When cells are seen to detach from glass upon shaking, add 6 ml fresh medium and suspend cells by pipetting up and down a few times. 5. Add 10ml more medium and mix to get even cell suspension. 6. Seed 1 ml cell suspension in to each of 8 culture tubes, stopper tightly, and incubate in rack which holds tubes at slight angle from horizon tal. Seed remaining 8 ml cell suspension into a new 3 oz. prescription bottle or a 25 cm2 plastic flask. C.PRESERVATION OFCULTURED CELLS BYFREEZING Viability of viruses and bacteria is preserved during freezing, but originally attempts to preserve animal cells by freezing resulted in cell death. This was first thought to be due to laceration of cell plasma membranes by ice crystals, but more recent evidence suggests the cause may be osmotic changes during freezing which give rise to irreversible changes in lipoprotein complexes in intracellular membranes. In any event, the answer to animal cell preservation has proved to be addition of glycerol, ethylene glycol, or dimethyl sulfoxide (DMSO) to the medium and slow freezing, ideally at a cooling rate of one centigrade degree per minute.Cells must be stored at 70oC or lower (ideally in liquid N2 at 196oC), and when they are recovered, thawing must be rapid. With careful technique, 50-80% of the cells of a healthy culture will survive f reezing. Procedure 1. Remove confluent cell monolayer from culture vessel by method described in cell transfer procedure. After centrifugation, resuspend cells in 1 ml medium containing 15% serum and 7. 5% DMSO and placed in small snap-top tube. 2. Immediately place tubes in an ice bath. They will then be transferred to a styrofoam container and refrigerated. After 20-30 min, when cells have dropped to 4o, they will be transferred to a 20o freezer for 20-30 min, then to the 70o freezer for storage.Alternatively, the tubes can be placed in cotton-or polystyrene-insulated containers and placed directly in the 70o freezer for slow cooling. If cells are to be stored in liquid N2, they must be placed in sealed ampoules. 3. To recover, cells, remove tubes from 70o and place directly in 37o water bath. When thawing is barely complete, add contents of tube to a 25 cm2 flask containing 15 ml MEM + 10% fetal calf serum. Culture medium will be changed for your approximately 4 hrs. later (after cells have attached) to reduce the toxicity of DMSO for cells at 37oC. D. Effect of Viral Infection on the Host Cell During the time that synthesis of viral components is occurring in the infected cell, the cell undergoes characteristic changes.These changes are usually observed in tissue culture where infection of cells is more easily synchronized and where the cells can be observed frequently during the course of infection. Morphological changes in cells caused by viral infection are called cytopathic effects (CPE): the responsible virus is said to be cytopathogenic. The degree of visible damage to cells caused by viral infection varies greatly. Some viruses cause very little or no CPE. Their presence can be detected only by hemadsorption (already discussed) or interference, in which infected cell cultures showing no CPE inhibit the replication of another virus subsequently introduced into the cultures.On the other hand, some viruses cause a complete and rapid destruction of the cell monolayer after infection. The histological appearance of the CPE caused by some of these cytocidal viruses may be sufficiently characteristic to allow provisional identification of the virus. Some CPE can be readily observed in unfixed, unstained cells, under low power of the light microscope, with the condenser down and the iris diaphragm partly closed to obtain the contrast needed for viewing translucent cells. Several types of CPE are distinguishable in living cultures, but fixation and staining of the cells is necessary to see such manifestations of viral infection as inclusion bodies and syncytia.Recognizing CPE and using it as a diagnostic tool requires much experience in examining both stained and unstained cultures of many cell types. Listed below are several general types of CPE. Keep in mind that a given virus may not conform to the norm for its family, or it may produce different CPE in different host cell types. The best knowledge of viral CPE comes from experience . 1. Total destruction of the cell monolayer is the most severe form of CPE. All cells in the monolayer rapidly shrink and become dense (Pyknosis) and detach from the glass within 72 hours. This CPE is typical of most enteroviruses. 2. Sub-total destruction consists of detachment (death) of some but not all of the cells in the monolayer.The alpha-togaviruses, some picorna viruses, and some of the paramyxoviruses may cause this type of CPE. 3. Focal degeneration is characteristic of the herpesviruses and poxviruses. Instead of causing a generalized destruction of the cell monolayer, these viruses produce localized areas (foci) of infection. The focal nature of these lesions is due to direct cell-to-cell transfer of virus rather than diffusion through the extra-cellular medium. Cells initially become enlarged, rounded, refractile (more easily seen), and eventually detach from the glass, leaving cleared areas surrounded by rounded up cells as the infection spreads concentrically. Stran ding of the cytoplasm is usually pronounced and cell fusion may be evident. 4.Swelling and clumping of cells before detachment is typical of adenoviruses. Infected cells greatly enlarge and clump together in â€Å"grape-like† clusters. 5. Foamy degeneration (vocuolization) is due to the production of large and/or numerous cytoplasmic vacuole. Several virus families including certain retroviruses, paramyxoviruses, and togaviruses may cause vocuolization. 6. Cell fusion (syncytium or polykaryon formation) involves the fusion of the plasma membranes of 4 or more cells to produce one enlarged cell with 4 or more nuclei. Polykaryon formation may be the only detectable CPE of some paramyxoviruses; herpesviruses may also produce syncytia. 7. Inclusion bodies are areas of altered staining in cells.Depending on the causative virus, these inclusions may be single or multiple, large or small, round or irregularly shaped, intranuclear or intracytoplasmic, eosinophilic (pink staining) or basophilic (blue-purple staining). In most cases they represent areas of the cell where viral protein or nucleic acid is being synthesized or where virions are being assembled, but in some cases no virus is present and the inclusion bodies represent areas of viral scarring. V. BIOCHEMICAL AND BIOPHYSICAL CHARACTERIZATION OF VIRUSES There are many biochemical and biophysical tests which can be used for classification of viruses. We will perform four of these test using â€Å"unknown† viruses: viral sensitivity to lipid solvents, determination of virus size, determination of virus nucleic acid type, and viral sensitivity pH and heat.The chart on p. 127 of your lab book may help in the identification of your virus. A. Viral Sensitivity to Lipid Solvents. The lipid sensitivity test is one of the most basic tests for characterization of viruses. There is a correlation between the presence of an envelope and the susceptibility of viruses to lipid solvents such as ether, chloroform, and detergents. Enveloped viruses require their lipid membrane for infectivity; because the test measures destruction of viral infectivity vs. untreated viral controls, it is an indirect test. All lipid coated viruses are sensitive to chloroform, whereas all but a few poxviruses are sensitive to ether.This is because the lipid components of the poxviruses are much diffe