Glycosilation of Recombinant Protein in Hamster Ovary Cells

Glycosilation of Recombinant Protein in Hamster Ovary Cells GLYCOSILATION OF RECOMBINANT PROTEIN IN CHINESE HAMSTER OVARY CELLS MANISHA YADAV INTRODUCTION: GLYCOSYLATION OF PROTEINS The phenomenon of addition subsequent processing of carbohydrates in most of the plasma- membrane and secretory proteins is called glycosylation. This is the principle chemical modification or post- translational modification to most of such proteins. Glycosylation reactions occur in the lumen of Endoplasmic Reticulum (ER) and can also occur in the Golgi cisternae [1]. There are two different forms of oligosaccharide chains: O-linked oligosaccharides and N-linked oligosaccharides. The O-linked oligosaccharides are short structures and contain 1-4 sugar residues whereas; the N-linked oligosaccharides always contain mannose along with N-acetyl glucosamine. The structures of N-linked oligosaccharides have various branches terminating with sialic acid residues which are negatively charged groups [1]. Approximately 50% of the proteins manufactured in the eukaryotic cells are glycosylated. Many glycoproteins are produced from different glycoforms with different glycan structure which are attached to a single peptide backbone [2]. These glycan structures affect the biological properties of various glycoproteins which include pharmacokinetics, secretion, solubility, bioactivity, in vivo clearance, antigenicity and receptor recognition [2]. CHINESE HAMSTER OVARY CELLS (CHO CELLS) For the production of biopharmaceuticals, animal cell cultures are used due to their abilities of post-translational modification of proteins involving glycosylation [2]. Chinese Hamster Ovary (CHO) cells were first used in the year 1919 as a laboratory specimen for typing pneumococci [3]. CHO cells are the most commonly used cell lines for studying gene expression (particularly to express recombinant proteins), toxicity screening, genetics and nutrition. They are frequently used mammalian hosts for production of recombinant protein therapeutics at an industrial level [3]. Due to their adaptability to several culture conditions, relatively fast generation time, easy to maintain and also due to their plasticity in the surroundings of genetic alterations, CHO cells are technologically more prominent and highly useful [2]. DIFFERENT EXPERIMENTS AND STUDIES ON GLYCOSYLATION OF RECOMBINANT PROTEIN IN CHO CELLS Alterations in the glycosylation of recombinant proteins depend on a range of parameters which include: metabolic flux, cellular metabolism and the efficiency of the glycosylation process [4]. CHO cell lines are used in identifying the biological roles of mammalian glycans and the pathways to synthesise them [5]. An analysis of N- glycans and major O- glycans in CHO mutants was performed using glycomic analysis by MALDI-TOF (Matrix Assisted Laser Desorption Ionization- Time of Flight) and time of flight- Mass Spectroscopy (TOF-MS). To enhance the applications of CHO mutants and to obtain its insights, alterations in the glycosyltransferase activity was done. It was found that lectin resistant CHO cells make very large and various complex N-glycans. Also it was observed that terminal addition of sialic acid enhances polylactosamine extension whereas fucose addition reduces polyLacNAc extension [5]. To determine the structural characteristics of recombinant envelope glycoprotein (rgp120) present in human immunodeficiency virus type-1 (HIV- 1), the virus was produced by expression in CHO cells. To confirm the primary structure of rgp120 protein, enzymatic cleavage was done to assign intrachain disulphide bonds and the potential sites for N-glycosylation were determined. To carry out such technique reverse-phase high performance liquid chromatography method was employed [6]. For tryptic peptide mapping studies, N-glycosylation of 24 potential sites were characterized by determining the susceptible carbohydrate structures attached to the peptides which include: endo-ÃŽ ²-N-acetyl glucosaminidase H and N- glycosidase F [6]. It was found that in all the 24 sites of gp120 protein, 13 consisted of complex-type oligosaccharide structures and the remaining 11 consisted of mannose-type oligosaccharide structures. It also contained certain hybrid-type oligosaccharide structures [6]. Analys is of the carboxyl terminal using carboxypeptidase digestions demonstrated that the glutamic acid residue 479 is the carboxylterminus molecule secreted by CHO cells [6]. The gp120 protein has a polypeptide core of approximately 60,000 Daltons, an extensive modification in this polypeptide by N-linked glycosylation results in an increase in its molecular weight to 120,000 Daltons [6]. Glycosylation is a cellular process in which achieving a precise glycan distribution is challenging, because it is a non-template driven process. Hence, it results in significantly uncontrolled variability in the glycan distributions [7]. Different cell types differ in their ability to carry out specific post-translational modifications; for example: glycosylation, which affects the different processes such as receptor binding, bioactivity, susceptibility to proteolysis, clearance rate of therapeutic recombinant protein in vivo and immunogenicity [8]. As a result of this difference, various forms of glycans are formed and their attachment to proteins increases variability and non-uniformity [7]. Hence, an experiment was conducted to study the heterogeneity of a recombinant protein in different animal systems including Chinese hamster ovary cells [8]. CHO cells have proven produce proteins with glycoforms which are both bioactive and compatible in humans [3]. CHO cells produce recombi nant IFN-ÃŽ ³ which has 3 sources of variation: site-specific N-glycan microheterogeneity, variable N-glycosylation site occupancy and C-terminal polypeptide truncations [8]. It has examined that for efficient secretion and dimerization of IFN-ÃŽ ³, glycosylation of Asn25 is necessary [8]. For optimal enzymatic activity, glycosylation is required. Neutrophils release granules such as Myeloperoxidase (MPO) into the phagosome during the process of phagocytosis of exogenous structures. Respiratory burst is initiated which reduces oxygen and releases superoxide radical anions. These superoxides upon dismutation produce Hà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡Oà ¢Ã¢â‚¬Å¡Ã¢â‚¬Å¡ which mediates MPO-driven oxidation of chloride to antimicrobial HOCl [9]. To increase the production of MPO protein, recombinant human MPO (r-MPO) are produced by expression in CHO cell lines. However, the r-MPO in CHO cell lines consists of some missing proteolytic steps as compared to the dimeric leukocyte enzyme (h-MPO). As a result, the r-MPO protein differs in the glycan structure and is a monomeric protein [9]. Tryptic digestion of reduced carboxamido- methylated proteins was carried out to determine the glycan structures. A method like con A-immobilized affinity chromatography was used to enrich the glycopeptide s. These glycopeptides were fractionated and analyzed by HPLC. The N-deglycosylated forms along with their glycopeptides were then identified by MALDI-MS. The result indicated that both r-MPO and h-MPO consists of high-mannose structures. The r-MPO has high amount of complex-type glycans like di- trisialylated triantennary structures [9]. A glycoprotein hormone called human erythropoietin (EPO) regulates the level of circulating erythrocytes in humans. They play an important role in maturation of erythrocytes. In normal human, EPO is produced in the kidney. Various recombinant human erythropoietins are being produced in non-mammalian mammalian cells. There are two forms of EPO (erythropoietin), EPO-bi and EPO- tetra. Each one of them have different biological activities. These were isolated from culture medium of CHO cell line (B8-300) encoding human EPO [10]. The culture was purified by reverse-phase chromatography, anion-exchange chromatography and gel filtration. The highly purified recombinant human erythropoietin from mast cells was used as a control and it is the standard EPO [10]. The results obtained showed that EPO-bi has only 1/7 in vivo activity and the invitro activity was 3 times higher than in vivo activity. Whereas EPO-tetra have similar in vivo and in vitro activities as the standard EPO. EPO-tetra, t he standard EPO and EPO-bi had same immunoreactivity and amino acid composition. The structural analysis of N-linked sugar chains showed that EPO-tetra and the standard EPO contain tetraantennary complex-type as their major sugar chain, whereas EPO-bi contains biantennary complex-type as their major sugar chain [10]. Heparin is most widely used in modern medicine to control blood coagulation and is in great demands. Therefore, CHO cells are used for producing heparin. CHO cells are capable of producing HS (Heparan Sulfate) naturally, which is a related polysaccharide as heparin [11]. Human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotranferase 1 were sequentially transfected into CHO host cells. Screening of these transfectant cells was carried out using quantitative RT-PCR and western blotting technique. The heparan sulphate produced by CHO cells is a less sulfated glycosaminoglycan (GAG). This is due to the glycosylation process resulting in heterogeneity in glycan structures. This HS has similar disaccharide units as heparin, but HS has lower anticoagulant activity than heparin [11]. TREATMENT OF DISEASES: Glycosylation process has been proved efficient in treating various diseases. Few examples are as follows: Fabry disease: it is an X-linked genetic disease. It arises due to a deficiency in the lysosomal enzyme called ÃŽ ±-galactosidase A (GLA). This enzyme hydrolyzes the terminal ÃŽ ±-galactosyl moieties from glycoproteins and glycolipids. An enzyme replacement therapy is carried out to treat the disease. CHO cell lines are used to produce recombinant GLA with very high productivity. When the recombinant GLA was compared to an approved GLA (agalsidase beta), the charge and size of the recombinant GLA was found to be more neutral and smaller respectively. This difference was due the absence of terminal sialic acid. To increase the sialic acid content, combined reaction of sialyltransferase, galactosyltransferase and their sugar substrates was developed. This product generated had same isoelectric points, size and sialic acid content as in approved GLA (agalsidase beta) [12]. Gaucher disease: it is an inherited metabolic disorder and is caused due to impaired activity of glucocerebrosidase (GCR) enzyme. This is a lysosomal enzyme which is responsible for hydrolysis of glucocerebroside to ceramide and glucose. The treatment of this disease consists of enzyme replacement therapy in which exogenous GCR is administered in the patients. This protein was expressed in CHO-DXB11 (dhfr ) cells. The glycosylated GCR produced due to transfection and gene amplification with methotrexate is then detected by immunoblotting assay. CHO cells are the highest producer of glucocerebrosidase enzyme [13]. CULTURE PARAMETERS THAT CAN AFFECT GLYCOSYLATION: Culture conditions such as pH, nutrient content, temperature, ammonia and oxygen have an important effect on distribution of glycan structures in recombinant protein [2,4,10]. pH: any adverse change in the external pH conditions can change the internal pH of Golgi apparatus, resulting in reduction in the activity of glycosylating enzymes [2]. Oxygen: to maintain optimum growth and metabolism of producer cells in bioprocesses, the dissolved oxygen level should be under control. At high oxygen level there is an increase in sialyltransferase activity. By controlling dissolve oxygen (DO) set points, the galactosylation of IgG immunoglobulin was changed efficiently and a decrease in digalactosylated glycans was observed [2,4]. Effect of producer cell line on protein glycosylation: in the Golgi of the cell, the protein glycosylation pattern depends on the expression of several glycosyltransferase enzymes. Differences in the activities of these enzymes result in significant changes in the protein structure [2]. Fucosylation: the Fucosylation pathway synthesizes the fucose donor called GDP-fucose. ÃŽ ±-1,6-fucosyltransferase enzyme is a glycosyltransferase enzyme which catalyzes the transfer of fucose to Asn-linked GlcNAc residue from GDP-fucose [2,6]. This enzyme is found in the N-glycans of several glycoproteins. Few studies suggest that fucose residues play a vital role in defining the oligossacharide conformations which are required for specific protein- carbohydrate interactions [2]. CONCLUSION: The properties of a mature protein depend on the carbohydrate moiety of glycoproteins. These properties include protein stability, solubility, molecular recognition or immunogenicity [9,4]. CHO cells are being widely used in biopharmaceutical industry. By optimizing glycosylation, the therapeutic efficacy can be improved. The bioprocesses to produce monoclonal antibodies and fusion proteins have shown complex relationship between these variables and its protein quality [14]. Glycosylation can be controlled by understanding the cellular and metabolic alterations which results in the modification of protein structures [4]. It has been observed that by decreasing the concentration of glutamine to 0mM, there is reduction in fucosylation, sialylation and antennarity, but an increase in neutral N-linked glycans has been observed [4, 7]. Stable CHO cells wih functional recombinant proteins which are properly glycosylated and secreted can be easily generated. The screening strategies can be used to produce biopharmaceuticals and other biological products which are highly cost-effective and is used for public health value [12, 13]. REFERENCES: Lodish, H., Berk, A. and Zipursky, S. L. 2000. Molecular cell biology, New York: Freeman, 4th edition. Butler, M. 2006. Optimisation of the cellular metabolism of glycosylation for recombinant proteins produced by mammalian cell systems. Cytotechnology, 50 (1-3), pp. 57-76. Jayapal, K. P., Wlaschin, K. F., Hu, W., Yap, M. and GS, A. 2007. Recombinant protein therapeutics from CHO cells-20 years and counting. Chemical engineering Progress, 103(10), p. 40. Burleigh, S., Van De Laar, T., Stroop, C., Van Grunsven, W., Odonoghue, N., Rudd, P. and Davey, G. 2011. Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells. BMC biotechnology, 11 (1), p. 95. North, S. J., Huang, H., Sundaram, S., Jang-Lee, J., Etienne, A. T., Trollope, A., Chalabi, S., Dell, A., Stanley, P. and Haslam, S. M. 2010. Glycomics profiling of Chinese hamster ovary cell glycosylation mutants reveals N-glycans of a novel size and complexity. Journal of Biological Chemistry, 285(8), pp. 5759-5775. Leonard, C. K., Spellman, M. W., Riddle, L., Harris, R. J., Thomas, J. N. and Gregory, T. 1990. Assignment of intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1 recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in Chinese hamster ovary cells.Journal of Biological Chemistry, 265 (18), pp. 1037310382. Amand, M. M., Tran, K., Radhakrishnan, D., Robinson, A. S., Ogunnaike, B. A. 2014. Controllability Analysis of Protein Glycosylation in Cho Cells. Plos One, 9(2), p. 87973. James, D. C., Goldman, M. H., Hoare, M., Jenkins, N., Oliver, R. W. A., Green, B. N., Freedman, R. B. 1995. Posttranslational processing of recombinant human interferon-ÃŽ ³ in animal expression systems. Protein Science, pp. 331-340. Van Antwerpen, P., Slomianny, M., Boudjeltia, K. Z., Delporte, C., Faid, V., Calay, D., Rousseau, A., Moguilevsky, N., Raes, M., Vanhamme, L. and Others. 2010. Glycosylation Pattern of Mature Dimeric Leukocyte and Recombinant Monomeric Myeloperoxidase GLYCOSYLATION IS REQUIRED FOR OPTIMAL ENZYMATIC ACTIVITY.Journal of Biological Chemistry, 285 (21), pp. 1635116359. Takeuchi, M., Inoue, N., Strickl, Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S. and Kobata, A. 1989. Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells.Proceedings of the National Academy of Sciences, 86 (20), pp. 78197822. Baik, J. Y., Gasimli, L., Yang, B., Datta, P., Zhang, F., Glass, C. A., Esko, J. D., Linhardt, R. J. and Sharfstein, S. T. 2012. Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin. Metabolic engineering, 14 (2), pp. 8190. Sohn, Y., Lee, J. M., Park, H., Jung, S., Park, T. H., Oh, D. and Others. 2013. Enhanced sialylation and in vivo efficacy of recombinant human ÃŽ ±-galactosidase through in vitroglycosylation.BMB reports, 46 (3), pp. 157162. Novo, J. B., Morganti, L., Moro, A. M., Paes Leme, A. F., Serrano, S. M. D. T., Raw, I. and Ho, P. L. 2012. Generation of a Chinese hamster ovary cell lineproducing recombinant human glucocerebrosidase.BioMed Research International, 2012. Hossler, P., Khattak, S. F. and Li, Z. J. 2009. Optimal and consistent protein glycosylation in mammalian cell culture.Glycobiology, 19 (9), pp. 936949.

The Wisdom of Franz Kafka’s On Parables :: Kafka On Parables Essays

The Wisdom of Franz Kafka’s On Parables Is it even possible to gain a better life through knowledge and wisdom? Should we listen to the words of the wise? Franz Kafka tries to answer these questions in his short essay ``On Parables,'' with a resounding ``No!'' In this Kafkan world, one filled with the daily struggles and cares of life, the only thing we can know is the incomprehensibility of it all. He states that all wisdom is expressed in parables then destroys any hope we may have by trouncing the authenticity of parables. But then he does something strange, vividly illustrating his point by using the very method he hoped to discredit. The first paragraph of ``On Parables'' provides Kafka's main point: ``the words of the wise are always merely parables and of no use in daily life.'' How does he arrive to this conclusion? He first first asserts that the words of the wise are always parables then explains why all parables are useless. Finally, he concludes that the words of the wise, since they are all parables, are all useless for daily life. Kafka first assumes that ``the words of the wise are always merely parables,'' and expects the reader to follow this assumption rigidly throughout the work. Notice that there is no wiggle room for the wise; their words are always parables. So don't complain, don't object. It is so, at least within the scope of his essay. Kafka does however state why the sages use parables. Since sages themselves are incapable of communicating wisdom, they speak in imperfect parables in a futile attempt to communicate that wisdom. Kafka further hints that this may be because even the sage doesn't understand such wisdom. The words of the sage, ``Go over,'' indicate that the sage is not currently where he wants the people to go, but may himself yet be stuck in this world of daily cares, struggles, and hardships. If he were where he wants us to be, he would have used the words ``come over'' instead. ``Over where?'' you may ask. Kafka answers this question by posing what I will refer to as three lands of life related to parables. The first, the most real one, refers to the state of a person's life before encountering a parable. The second, a potential land, is the parable itself and the resulting actions and consequences a person derives from the parable.

Early Christian, Jewish, and Byzantine Art

Early Christian art spans from the first to fifth centuries followed by the vast era of Byzantine art from the fifth century to the 16th century in Eastern Europe. Much of the art during this period had a religious context or enacted a religious purpose. The paintings and mosaics were meant to remind worshippers of their God, and the architecture was meant to serve both functional and aesthetic purposes. When Constantine I issued the Edict of Milan in 313 and moved the center of the Roman’s empire from Rome to Constantinople, the Byzantine Empire and Constantinople became the center of power and culture. The architecture of the Christian era came in two forms: the basilica and central plans. The basilica plan typically contains a large nave, an apse and an atrium on either end, clerestory windows, and two side aisles along the nave. This plan is found in The Church of Santa Sabina. The central plan, or tholos, served as tombs, martyrs’ churches, or baptisteries. These plans typically contain either a sarcophagus or altar at the center and most often have a large dome on top. This plan is found in the Church of Santa Costanza and the Mausoleum of Galla Placidia, which employs the cruciform, or cross-shaped, style. Mosaics during the Christian era sometimes used syncretic images such as in Harvesting of Grapes in the ambulatory of the Church of Santa Costanza. The architecture of Byzantine art is characterized much by the Church of Hagia Sophia, or â€Å"Holy Wisdom. † This church combines the two floor plans of the Christian period, basilica and central. It has pendentives to hold its massive dome, flowing layers of half domes along its exterior wall, and many windows to let the gold on the mosaics shimmer. The Church of San Vitale is designed in a central plan but is interesting in that it has a very modest, unassuming exterior, but a complex, mosaic-covered interior. Two mosaics depicting Emperor Justinian and Empress Theodora represent how artists employed reverse perspective. The Transifguration of Christ as St. Catherine’s Monastery in Sinai depicts Christ in a blue mandorla which represents royalty and glory. The mosaics in later centuries depict Christ in a Pantokrator fashion: a bust level portrait of Christ with book in one hand and â€Å"peace† in other. This Late Byzantine art also depicts Christ in new ways, such as the Crucifixion and together with Mary in Virgin of Vladmir.

Executive Order 9835 Demanded Loyalty

Executive Order 9835 Demanded Loyalty In 1947, World War II had just ended, the Cold War had just begun, and Americans were seeing communists everywhere. It was in that politically-charged atmosphere of fear that President Harry S. Truman on March 21, 1947, issued an executive order establishing an official â€Å"Loyalty Program† intended to identify and eliminate communists in the U.S. government. Key Takeaways: Executive Order 9835 Executive Order 9835 was a presidential executive order issued by President Harry S. Truman on March 21, 1947. The so-called â€Å"Loyalty Order† created a controversial â€Å"Federal Employee Loyalty Program† charged with eliminating communists from all areas of the U.S. government. The order empowered the FBI to investigate federal employees and created presidentially-appointed Loyalty Review Boards to act on reports from the FBI.Between 1947 and 1953, more than 3 million federal employees were investigated, with 308 fired after being declared security risks by the Loyalty Review Boards.   Truman’s Executive Order 9835, often called the â€Å"Loyalty Order,† created the Federal Employee Loyalty Program, which authorized the Federal Bureau of Investigation (FBI) to conduct initial background checks on federal employees and carry out more in-depth investigations when warranted. The order also created Presidentially-appointed Loyalty Review Boards to investigate and act on the findings of the FBI. â€Å"There shall be a loyalty investigation of every person entering the civilian employment of any department or agency of the executive branch of the Federal Government,† the Loyalty Order decreed, also providing that, â€Å"equal protection from unfounded accusations of disloyalty must be afforded the loyal employees.† According to the paper The Second Red Scare, Digital History, Post-War America 1945-1960 from the University of Houston, the Loyalty Program investigated over 3 million federal employees, 308 of whom were fired after being declared security risks. Background: Rise of the Communist Threat Shortly after the end of World War II, not only had the entire world learned the horrors of nuclear weapons, America’s relationship with the Soviet Union had deteriorated from wartime allies to staunch enemies. Based on reports that the USSR had succeeded in developing its own nuclear weapons, Americans, including government leaders, were gripped by a fear of the Soviets and communists in general, whoever and wherever they might be.  Ã‚   Growing economic tension between the two nations, along with fears of uncontrolled Soviet spy activity in America began to influence ​U.S. foreign policy and, of course, politics. Conservative groups and the Republican Party sought to use the so-called â€Å"Red Scare† threat of Communism to their advantage in the 1946 midterm Congressional elections by claiming that President Truman and his Democratic Party were â€Å"soft on Communism.† Eventually, the fear that communists were beginning to infiltrate the U.S. government itself became a key campaign issue. In November 1946, Republican candidates won sweeping victories nationwide resulting in Republican control of both the House of Representatives and the Senate.   Truman Responds to the Red Scare Two weeks after the election, on November 25, 1946, President Truman responded to his Republican critics by creating the Presidents Temporary Commission on Employee Loyalty or TCEL. Made up of representatives from six Cabinet-level government departments under the chairmanship of a Special Assistant to the U.S. Attorney General, TCEL was intended to create federal loyalty standards and procedures for the removal of disloyal or subversive individuals from federal government positions. The New York Times printed the TCEL announcement on its front page under the headline, â€Å"President orders purge of disloyal from U.S. posts.† Truman demanded that the TCEL report its findings to the White House by February 1, 1947, less than two months before he issued his Executive Order 9835 creating the Loyalty Program. Did Politics Force Truman’s Hand? Historians contend that the timing of Truman’s actions, taken so soon after the Republican Congressional victories, show that both the TCEL and the subsequent Loyalty Order had been politically motivated.   Truman, it seems, was not as worried about Communist infiltration as the terms of his Loyalty Order indicated. In February 1947, he wrote to Pennsylvania’s Democratic Governor George Earle, â€Å"People are very much wrought up about the communist bugaboo but I am of the opinion that the country is perfectly safe so far as Communism is concerned- we have too many sane people.† How the Loyalty Program Worked Truman’s Loyalty Order directed the FBI to investigate the backgrounds, associations, and beliefs of any of the approximately 2 million executive branch federal employees. The FBI reported the results of their investigations to one or more of the 150 Loyalty Review Boards in various government agencies. The Loyalty Review Boards were authorized to conduct their own investigations and to collect and consider testimony from witnesses whose names were not disclosed. Notably, the employees being targeted by the loyalty investigations were not allowed to confront the witnesses testifying against them. Employees could be fired if the loyalty board found â€Å"reasonable doubt† regarding their loyalty to the U.S. government or ties to communist organizations. The Loyalty Order defined five specific categories of disloyalty for which employees or applicants could be fired or rejected for employment. These were: Sabotage, espionage, spying or the advocacy thereofTreason, sedition or the advocacy thereof;Intentional, unauthorized disclosure of confidential informationAdvocacy of the violent overthrow of the U.S. governmentMembership in, affiliation with or sympathetic association with any organization labeled as totalitarian, fascist, Communist or subversive The Subversive Organization List and McCarthyism Truman’s Loyalty Order resulted in the controversial â€Å"Attorney Generals List of Subversive Organizations† (AGLOSO), which contributed the second American Red Scare from 1948 to 1958 and the phenomenon known as â€Å"McCarthyism.† Between 1949 and 1950, the Soviet Union demonstrated that it had indeed developed nuclear weapons, China fell to Communism, and Republican Senator Joseph McCarthy famously declared that the U.S. Department of State employed more than 200 â€Å"known communists.† Despite having issued his Loyalty Order, President Truman again faced charges that his administration was â€Å"coddling† communists. Results and Demise  of Truman’s Loyalty Order According to historian Robert H. Ferrell’s book Harry S. Truman: A Life, by mid-1952, the Loyalty Review Boards created by Truman’s Loyalty Order had investigated more than 4 million actual or prospective federal employees, of which 378 were fired or denied employment. â€Å"None of the discharged cases led to discovery of espionage,† noted Ferrell. Truman’s Loyalty program has been widely criticized as an unwarranted attack on innocent Americans,  driven by the Red Scare. As the Cold War’s threat of nuclear attack grew more serious during the 1950s, Loyalty Order investigations became more common. According to the book Civil Liberties and the Legacy of Harry S. Truman, edited by Richard S. Kirkendall, â€Å"the program exerted its chilling effect on a far larger number of employees than those who were dismissed.† In April 1953, Republican President Dwight D. Eisenhower issued Executive Order 10450 revoking Truman’s Loyalty Order and dismantling the Loyalty Review Boards. Instead, Eisenhower’s order directed the heads of federal agencies and the U.S. Office of Personnel Management, supported by the FBI, to investigate federal employees to determine whether they posed security risks.

How to Write an SEO Article †Part 2

How to Write an SEO Article – Part 2 How to Write an SEO Article Part 2 How to Write an SEO Article Part 2 By Colin In part one of How To Write An SEO Article, we looked at how important it is to get the basics of an SEO article correct from the start. We looked at the importance of writing relevant content to the user and not the search engine, and we also looked at the research that goes into keywords and keyphrases that are to be integrated into the article. Nothing that was described in part one should be considered difficult by anyone. If it is, please email me individually, making sure to swallow a pinch of salt before you do. In part two of How To Write An SEO Article, I’m going to take everything we have learned in part one and apply it to the article that needs to be written. I’ll also cover that slightly boring, but all-important, ‘techy bit’. Using Keywords and Keyphrases Okay, so you have your keywords either by your own intuition or from a client. Then what? Well, the first step is to go back to step one, and remind yourself what the actual article that is going to be so helpful and informative to the reader, is all about. Then, based on this remit, write the article, and along the way, include the keywords and keyphrases among the text in as natural and logical a manner as possible. It may be you can build the article around the keywords themselves, but the most important thing is not to over use them. It makes the article appear like an attempt at SEO article writing gone mad. Also, it won’t read well and a reader will give up before he gets half way through. If you are struggling to find suitable places for the keywords, never force it into the text just so you can include it. If it doesn’t flow with the natural rhythm of the piece, leave it out or re-write the text. If you still find it impossible to use it elsewhere in the piece, wipe the slate clean and re-write the article from an entirely new angle. Obviously, if you are writing to the demands of a client who has given you a difficult remit with awkward keywords, this rule may have to be bent a little, but being a creative freelance writer means being able to adapt to situations like this, so trust yourself and go with the flow. Point of Note #1: they wouldn’t need someone to do it for them, if they could do it themselves. Point of Note #2: remember you’re the expert, so write the article for the reader, not the algorithm. Where possible (although this may not be part of your remit), try to include one of the keyphrases in the headline, and if possible, the sub-headline. This is a neat way of taking the pressure off the article content, while keeping the keywords at the front of the search engine algorithm results when it comes around. Hypertext Links Creating hypertext links that are embedded within the content of an article, is a great way of increasing general interest, as well as allowing a higher chance of search engine spiders being able to reach it. Too many hypertext links within your text will only annoy readers, though, so creating links to every keyword is not recommended. Linking to other interesting articles or websites from other words or phrases, that may not be specific to the remit given to you, may help to build a higher percentage of user interest, as well as boost the SEO ranking of the page the article is published on. For example, here is a section from an SEO article for the music industry I recently wrote: â€Å"Bidding for concert tickets from an unknown Internet source, is not a good way of buying your all important seats to see Britney Spears.† The keyphrase in this article is ‘concert tickets’ and as you can see, has been used within the article’s context. However, it doesn’t take a genius to work out that more searches are run for the phrase ‘Britney Spears’ than for ‘concert tickets,’ therefore, not only is it mentioned in the same paragraph, but there is also a hypertext link included which links to her official site. Having both phrases, ‘concert tickets’ and ‘Britney Spears,’ alongside each other, is quite clearly a potent combination for anyone concerned who might want to run an Internet search like it; Britney fans, music fans, memorabilia collectors, ticket touts, etc. Therefore, this sentence when published, will look like this: â€Å"Bidding for concert tickets from an unknown Internet source, is not a good way of buying your all important seats to see Britney Spears.† Keyword Density Keyword density is a term often used in the context of SEO, and is often stressed as being a vital component of every SEO article writer’s armoury. The phrase came into being because keywords, which provide the foundation on which SEO copywriting is based, led many people to conclude that the more keywords included in a SEO article, the higher chance there is of that web page being ranked during a search. Scientific formulae were developed to try and prove this, and thus an industry was born. Unfortunately, in this writer’s experience, this is almost always complete rubbish. Generally, one will find that no matter how many times the phrase ‘Britney Spears,’ is used in an article, when published, it simply will not shoot into the top ten in Google’s search ranking. Good keyword usage is supported not by the amount of times it is used in a web article, but by the combinations of other relevant keywords it sits beside, as in the example given earlier of ‘Britney Spears’ and ‘concert tickets.’ Each individual, but related keyword, helps narrow the search down even further. However, even with the most advanced SEO keyword placement strategies in place, there is still one remaining factor that is vital to help support the use of keywords and keyphrases: web page construction. Which takes me neatly onto the ‘techy bit’. Web Page Optimisation (The ‘Techy Bit’) As a freelance writer this section isn’t really required, but I though it wouldn’t hurt to come to a better understanding of the different ways that keywords and SEO can be applied, to increase the page rank of a website. Within the construction of a simple HTML web page, is a section at the top called the Header. Within this section there are various meta tags, the most important of which is the Title tag, and the Meta description tag. Search engine spiders read the meta data within a webpage, and are attracted to those sites which have keywords present early on. Search engine spiders and web browsers read HTML code from top to bottom (meta data then content), so what comes first gets read first and is therefore output first. It is therefore very important for good web page optimisation to occur, that an emphasis be placed on the usage of keywords in the Title tags and Meta tags, and to have the most important ones placed near the beginning. For example, here is an extract of HTML data taken from a website that might have published the article on Britney Spears concert tickets: html head titleBritney Spears Concert Tickets/title meta name="Keywords" content="Britney Spears, concert tickets, Britney on tour, gigs, stadium tour, Toxic, Oops I Did It Again† /head body Bidding for bconcert tickets/b from an unknown Internet source, is not a good way of buying your all important seats to see a href=†britneyspears.com†Britney Spears/a †¦..etc. /body /html As you can see, the most important keyphrases were used in the web page title, as well as at the front of the meta keywords line. And in the content we had the combination of keywords and keyphrases, including a logical hypertext link to what happens to be a very popular website on the Internet, for whatever reason. And that, in a nutshell, is about all you need to know to get started writing SEO articles. Want to improve your English in five minutes a day? 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