Genetic Influences on Salmonella Formation

Genetic Influences on Salmonella Formation IHF Gene Influences Salmonella Enteritidis Biofilm Formation Integration Host Factor (IHF) is important for biofilm formation by Salmonella enterica Enteritidis Bruna Leite, Catierine Hirsch Werle, Camila Pinheiro do Carmo, Diego Borin Nbrega, Guilherme Paier Milanez, Cristina E. Alvarez-Martinez, Marcelo Brocchi Abstract Salmonella enterica Enteritidis forms biofilms and survives in agricultural environments where it infects poultry and eggs. Once established, biofilms are difficult to eradicate, due to their high resistance compared to planktonic cells, causing serious problems in industry and public health. In this study, we evaluated biofilm formation in wild-type strains of S. enterica Enteritidis and in ihf mutants employing different microbiology techniques. Our data indicate that ihf mutants display impaired biofilm formation, with a reduced of matrix formation and a decrease in CFU and metabolic activity. Phenotypic analysis indicated a deficiency in curli fimbriae expression and in cellulose production and pellicle formation. These results show that IHF has a regulatory role in biofilm formation in S. enterica Enteritidis. Keywords: Biofilm, Salmonella enterica Enteritidis, Polysaccharide matrix, Curli fimbriae, Cellulose, Integration Host Factor. Introduction A biofilm is defined as a bacterial colony adherent to a solid surface, which secretes a protective exopolysaccharide matrix. Every natural wet surface is a potential substrate for microbial biofilms. These sessile multicellular microbial consortia are embedded within self-produced extracellular polymeric substances (EPS). In food handling facilities, biofilms can be particularly problematic The ability to form biofilms is also an important factor in the virulence of S. Enterica. S. enterica subspecies I serovar Enteritidis is a leading cause of salmonellosis worldwide, and has emerged as one of the most important foodborne pathogens for humans. It is mainly associated with consumption of contaminated meat and eggs of poultry. A number of studies have demonstrated that S. enterica is capable of forming biofilms on a wide variety of contact surfaces, and the formation of biofilms may improve the ability of these organisms to resist stresses such as desiccation, extreme temperatures, antibiotics, and antiseptics. Biofilm formation allows S. enterica to survive for long periods in a poultry farm environment and to contaminate poultry meat and eggs, which remain the leading vehicles of salmonellosis outbreaks Many factors are involved in biofilm development. Curli fimbriae and cellulose are the major components of biofilm formed by S. enterica, whereas capsular polysaccharide, other polysaccharide-rich compounds such as lipopolyssaccharide (LPS), and a large secreted protein, BapA, also contribute to biofilm formation. Several regulatory genes involved in biofilm formation have been identified The expression of curli fimbriae and cellulose can be assayed phenotypically by growing enteric bacteria on Congo red indicator plates Bacteria may live in planktonic form in liquid media or as biofilms on biotic or abiotic surfaces. They need to adjust their genetic programs in order to switch from one lifestyle to another. The production of bacterial products and behaviours associated with environmental adaptation must be tightly coordinated to optimize the energy consumption. In bacteria, gene expression regulation is exerted primarily at the level of transcription initiation using a large array of transcription factors whose concentrations and activities change depending on specific environmental or metabolic signals. Topological changes in DNA also influence promoter recognition, open complex formation, and gene expression Nucleoid-associated proteins (NAPs) are global regulators of gene expression in bacteria. They alter the topology of DNA by bending, bridging, or wrapping it, leading to DNA transactions and multiple cellular effects that culminate in the modulation of gene expression. Integration-host factor (IHF) is a dimeric NAP that binds DNA in a sequence-specific manner and introduces curvatures of up to 180 °, which influence many aspects of bacterial physiology, including global gene expression, DNA topology, site-specific recombination, and DNA replication. In E. coli and S. enterica Typhimurium, the two IHF subunits-IHFÃŽ ± and IHFÃŽ ²-can assemble as hetero- or homo-dimers. There is also evidence indicating that the different dimeric forms of IHF regulate different but overlapping sets of genes Based on the global regulatory role of IHF, we hypothesized that this NAP can influence or directly regulate genes involved in biofilm formation in S. enterica Enteritidis. This hypothesis is supported by previous observations demonstrating that IHF activates curli production in S. enterica Typhimurium. Therefore, in this study, we evaluated the role of IHF genes in the initial stages of biofilm formation in S. Enteritidis. To this end, we performed phenotypic studies using isogenic deletion mutants of individual ihf genes (ihfA or ihfB) and a double mutant strain with deletions in both IHF subunits (ihfAB double mutant). Materials and methods Bacterial strains In this study, the S. enterica Enteritidis wild-type strain PT4SEn (IOC4647) provided a by the Fundaà §Ãƒ £o Oswaldo Cruz (FIOCRUZ, Rio de Janeiro, Brazil) was used. The draft genome of this strain was recently published (Milanez et al. 2016). It was found to be pathogenic in a mouse model assay (Carmo et al., unpublished results). The mutants of S. Enteritidis PT4SEn were previously constructed (Carmo et al., unpublished results) by deletion of ihf genes using the lambda Red system by transduction with P22HT phages. Mutant strains were designated as S. enterica Enteritidis PT4SEn ΔihfA, PT4SEn ΔihfB, and PT4SEn ΔihfAB. Bacterial growth conditions and storage Bacteria were cultivated in Luria-Bertani broth (LB) and on Luria-Bertani agar (LBA) plates prepared according to the method of Sambrook and Russell. All strains were stored at -80 °C in 30% glycerol All strains were inoculated from fresh LBA plates into 15 mL LB and grown for 18  ± 2 h at 37 °C in an orbital shaker at 140 rpm. Cells were harvested by centrifugation (for 5 min at 9,500 g and 4 °C) and resuspended in NaCl (0.9%) adjusted to 0.5 McFarland scale equivalent to 1.5 108 cells/mL prior to use in subsequent assays. Complementation of S. enterica Enteritidis ΔihfA and ΔihfB mutants Sequences corresponding to the ihfA and ihfB genes and their regulatory regions were obtained by PCR from the PT4SEn genome using the primers listed in Table 1. The DNA fragments were cloned in the pACYC184 vector (New England Biolabs, USA) between the NcoI and EcoRI restriction sites (restriction enzyme sites in the DNA fragments were introduced via the primers) and the vector was subsequently electroporated into the respective S. enterica Enteritidis mutant strains. Cloning, PCR amplification, electroporation, plasmid extraction, and agarose gel electrophoresis were performed as suggested by Sambrook and Russell (2001). After DNA purification using the Wizard ® Genomic DNA Purification Kit (Promega Corporation, Madison, USA), Sanger sequencing was performed using 3730XL Applied Biosystems (Foster City, California, USA) by the High Performance Technologies Central Laboratory in Life Sciences (LACTAD, University of Campinas UNICAMP, Campinas, Brazil). Biofilm formation on polystyrene plates Biofilms were formed in 96-well plates (Cell Culture Plate, Nest, Biotechnology Co, China) containing 200 ÃŽ ¼L of cell suspension (1 106 cells/mL) of S. enterica Enteritidis PT4SEn wild-type or mutant strains in LB supplemented with 0.25% of glucose. Plates were incubated at 37 °C with orbital shaking at 140 rpm for 48, 72, and 120 h. At the end of the incubation period, planktonic cells were carefully removed, and biofilms were washed twice with 200 ÃŽ ¼L of saline solution (0.9% NaCl). The crystal violet staining method was used to assess total biofilm biomass. Each well of the biofilm plates was incubated with 200 ÃŽ ¼L of methanol for 15 minutes. Subsequently, methanol was removed and 1% (v/v) crystal violet solution was added, followed by a 5-min incubation period. Wells were washed with distilled water and finally 33% (v/v) acetic acid was added. The absorbance was measured at 570 nm. The colorimetric method based on the reduction of XTT (2,3- bis(2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino)carbonyl-2H tetrazolium hydroxide; Sigma-Aldrich, USA) was used to determine cell activity (XTT is converted to a coloured formazan salt in the presence of metabolic activity). To each well of the biofilm plate, 200 ÃŽ ¼L of a solution containing 200 mg/L of XTT and 20 mg/L of phenazinemethosulphate (PMS; Sigma-Aldrich, Ukraine) was added. Microtiter plates were incubated for 3 h at 37 °C in the dark. The absorbance was measured at 490 nm. To assess the number of viable cells in biofilms, 200 ÃŽ ¼L of saline solution was added to each well before removal of the biofilm by scraping. For each sample, an aliquot of 1 mL (5 wells) was sonicated (20 s with 22% of amplitude; Ultrasonic Processor, Cole-Parmer, Illinois, USA) to promote biofilm disruption. The number of colony forming units (CFU) in biofilms was determined by performing 10-fold serial dilutions in saline solution, plating on LBA plates in triplicate, and incubating for 24 h. Scanning electron microscopy (SEM) of biofilm cells Biofilms of S. enterica Enteritidis PT4SEn wild-type and mutant strains formed in 24-well plates (Well Cell Culture Cluster, Costar) were dehydrated by a 15-min immersion in increasing ethanol concentrations (70, 95, and 100% ethanol [v/v]) and placed in sealed desiccators. The samples were mounted on aluminium stubs with carbon tape, sputter-coated with gold, and analysed with a JEOL JSM-5800LV scanning microscope. All experiments were carried out in duplicate. Biofilm formation at the air-liquid interface Biofilm formation at the air-liquid interface was assessed in S. enterica Enteritidis PT4SEn strains by inoculation of LB cultures without NaCl, followed by incubation at 28 °C without shaking. Every day for 10 days, each isolate was visually examined for pellicle formation. Experiments were performed in triplicate. Expression of curli fimbriae Bacterial colony morphology of S. enterica Enteritidis PT4SEn wild-type and mutant strains was analysed on LB agar without NaCl, supplemented with Congo red (1.01340.0025, Sigma-Aldrich, Germany; 40 ÃŽ ¼g/mL) and Coomassie brilliant blue G (B0770-5G, Sigma-Aldrich, China; 20 ÃŽ ¼g/mL). Bacterial cultures were spread on agar plates and the colour and degree of colony rugosity were determined after 96 h of growth at 28 °C. Images were captured with a camera (Nikon P500) and under an HBO 100 Carl Zeiss Illuminating microscope system. Cellulose production The fluorescence exhibited by bacteria after growth of S. enterica Enteritidis PT4SEn wild-type and mutant strains in LB plates with Calcofluor (Fluorescent Brightener 28; F3543-1G, Sigma-Aldrich, China; 200 ÃŽ ¼g/mL) served as an indicator of cellulose production. Fluorescence was analysed visually using an UV light (366 nm) after 48 h of growth at 37 °C. Statistical analysis Data were analysed using STATA software, version 13.0 (Stata Corp, College Station, TX, USA). Data from all assays were compared using one-way analysis of variance (ANOVA). Sidaks adjustment for multiple comparisons was performed after a significant fitting. The significance level was set at 5%. Results ihf mutants display reduced viability, biomass, and metabolic activity A decrease of about 1-2 log10 in number of viable cells was observed for the ihf mutants in comparison with the wild-type S. enterica Enteritidis PT4SEn strain by CFU counting (Figure 1-A). The differences observed were statistically significant (P < 0.05) for all periods of time evaluated. The introduction of the pACYC184 plasmid carrying ihfA or ihfB was generally associated with an increase in CFUs, but complementation did not completely restore the values to those obtained with the wild-type strain. No statistical differences were observed at 48 and 72 h of incubation between ΔihfAc and the wild-type strain. The same observation is valid for ΔihfB after 120 h of incubation (Figure 1-A). These results show that the restoration of ihfA or ihfB gene copies in mutant strains is generally associated with an increase in CFUs in biofilms. The total biofilm biomass, assessed by CV staining of S. enterica Enteritidis PT4SEn and mutant strains is presented in Figure 1-B. An increase in biomass is observed for the wild-type strain over time. However, this effect was not observed for the correspondent PT4SEn ihfAB double mutant. None of the mutants presented an increase in biofilm matrix density at 48 and 72 h of incubation (P < 0.05). The complemented PT4SEn ihfA and ihfB mutants (ihfAc and ihfBc) showed an increase in total biofilm biomass in comparison to the non-complemented mutants (Figure 1-B). All mutant strains exhibited a significant reduction in metabolic activity measured by the XTT assay for cells in biofilm (P < 0.05). In addition, the double mutant (ihfAB) showed the greatest reduction in metabolic activity at 72 and 120 h (Figure 1-C). ihf genes are essential for biofilm structure To further characterize biofilm formation and structure in strains lacking ihf genes, we performed scanning electron microscopy (SEM) analysis of cells in biofilms. As shown in Figure 2, the absence of ihfA or ihfB drastically affects biofilm formation, as null mutants of S. enterica Enteritidis PT4SEn (Figure 2-D, E and F) exhibited a low amount of matrix and small number of cells compared to the wild-type (Figure 2-A). Complementation of ihf gene deletions by a wild-type copy of the corresponding gene promoted a significant restoration of biofilm formation (Figure 2-B and C). Pellicle formation at the air-liquid interface To further characterize the mutant strains with respect to their ability to form biofilms we analysed the biofilm formation at the air-liquid interface of cultures of the different strains. Cultures of the wild-type strain formed a thick and rigid pellicle after 10 days of growth (Figure 3-A). On the other hand, PT4SEn ihfA or PT4SEn ihfB mutant strains formed a less compact and fragile pellicle (not shown). Interestingly, the double mutant strain PT4SEn ihfAB did not form a visible pellicle at all at the air-liquid interface. Instead, cell deposition was observed at the bottom of the tube (Figure 3-B). Complementation with the wild-type copy of ihfA and ihfB restored the phenotype of the single mutants (PT4SEn ΔihfAc and PT4SEn ΔihfBc strains), which now formed a thick and rigid pellicle (not shown). Curli and cellulose Since curli and cellulose are important components in biofilm formation, we evaluated the role of IHF on their production. To this end, colony morphology was analysed on LBA plates supplemented with Congo red and Coomassie brilliant blue, as previously described.. enterica Enteritidis PT4SEn wild-type and PT4SEn ΔihfA and ΔihfB complemented strains exhibited a phenotype consistent with curli fimbriae and cellulose production, with red, dry, and rough (rdar) colony morphology (Figure 4-A to D). However, the PT4SEn ΔihfA, PT4SEn ΔihfB, and PT4SEn ΔihfAB mutants of S. enterica Enteritidis did not display the same colour and roughness, but instead exhibited a similar, but not identical, smooth and white (saw) morphotype, indicating a deficiency in the expression of curli fimbriae and probably also of cellulose (Figure 4-E to H). The expression of cellulose was also tested by screening the colonies for Calcofluor binding Cellulose production was observed for all strain s evaluated by this method, except for the double mutant ihfAB that was not fluorescent under an UV light source and was considered a poor producer of cellulose (Figure 5). Discussion The presence of microorganisms on food contact surfaces is one of the most common causes of food spoilage and transmission of foodborne diseases. Inadequate cleaning and disinfection of food-processing environments is the cause of major economic losses and represents a serious danger to public health. The ability of microorganisms to adhere and form biofilms makes disinfection even more difficult and challenging Infections with Salmonella enterica Enteritidis represent a major health problem and a significant burden on the food industry. About 80% of the infections are caused by biofilm formation In the matrix of a biofilm, bacteria grow on either biotic or abiotic surfaces, attaching to the surface and to each other, conferring resistance to immunity responses as well as antimicrobial agents As a consequence, antimicrobial treatments typically fail to eradicate biofilms. The need to create effective therapies to counteract biofilm infections is a pressing challenge in the food indus try The growing interest in understanding the regulatory network of gene activities during the transition from a planktonic to a sessile cellular lifestyle, prompted us to investigate the role of IHF in S. enterica Enteritidis biofilm formation. IHF has an important role in the regulation of gene expression and environment adaptability of S. Enterica Therefore, S. Enteritidis deletion mutants for ihfA, ihfB, or both genes (ihfAB) were employed in different assays to analyse biofilm formation. The logic behind this approach is based on the fact that IHF can act as a homodimer (IHFÃŽ ±ÃŽ ± or IHFÃŽ ²ÃŽ ²) or as a heterodimer (IHFÃŽ ±ÃŽ ²) The results presented here indicate an important role of this NAP in the formation of biofilms in S. enterica Enteritidis. All typical biofilm characteristics analysed in this study (CFU, biomass, and cellular metabolic activity) were significantly decreased in S. enterica Enteritidis mutant strains for ihfA, ihfB, or ihfAihfB. The biofilms formed by mutant strains exhibited a decreased matrix density compared with the wild-type strain. Therefore, these results indicate that IHF can influence the initial stage of biofilm formation by S. enterica Enteritidis, as the matrix is necessary in this phase. This is also supported by CV staining and SEM. The colony morphotypes observed in Congo red among wild-type and complemented strains exhibited the rdar morphotype, an indication of curli and cellulose production, while the mutant strains exhibited a similar but not identical saw morphotype, suggesting an altered expression of curli and probably also of cellulose. In fact, bacterial growth in calcofluor-containing medium indicated that the single ihf-mutants were able to produce cellulose, but the ihf-double mutant exhibited some deficiency in the production of this polysaccharide. Previously, Gerstel, Park, and Rà ¶mling demonstrated that the ΔihfAB double mutant of two S. enterica Typhimurium strains caused a reduction in CsgD expression and an altered rdar morphotype suggesting a role for IHF in curli expression in S. enterica Typhimurium. Curli is expressed by two divergent operons, csgBAC and csgDEFG. CsgD is a major regulator of curli expression and biofilm formation. This gene activates transcription of csgA and csgB that encodes the major (CsgA) and the minor (CsgB) curli subunits In addition, csgD also regulates cellulose production Therefore, IHF plays an important role in biofilm formation in S. enterica Typhimurium. Our results demonstrate a similar role for IHF in the biofilm formation of S. enterica Enteritidis. Despite high genetic similarity, the Enteritidis and Typhimurium serovars differ in various ecological and host-relationship parameters However, the regulation of biofilm formation by IHF in both serovars suggests that IHF plays a cen tral role in S. enterica biofilm biogenesis. However, additional studies of IHF function on biofilm biogenesis in other S. enterica serovars are needed to further clarify this question. In addition, the single ihf mutants also exhibited a phenotypic alteration in biofilm formation, indicating that both subunits are necessary for appropriate biofilm production. In our results, all the ihf mutants showed a deficiency for curli fimbriae production by phenotypic tests. To some extent, a deficiency in cellulose production was also observed, particularly in the double ihf-mutant. The complementation of the ihfA and ihfB mutants by the introduction of a pACYC184 plasmid carrying the wild-type genes reverted the deficiency in biofilm biomass, cell metabolism, and CFUs, but in the majority of the tests the values did not reach those observed for the wild-type strain. This is probably due to a dose effect of IHFÃŽ ± or IHFÃŽ ², despite the low copy number (about 15 copies per cell) of the plasmid used. In fact, the expression of ihf genes is finely regulated and depends on the growth phase The two operons bcsABZC and bcsEFG are responsible for cellulose biosynthesis in both S. enterica Enteritidis and S. enterica Typhimurium. This was demonstrated by the construction of non-polar mutants of bcsC and bcsE genes that formed a fragile pellicle at the air-liquid interface of LB medium The same authors also showed that cellulose-deficient mutants were more sensitive to chlorine treatments, indicating that the deficiency in the production of extracellular matrix can leave the cells more susceptible to the action of some chemical agents. In our study, IHF mutant strains formed a less compact pellicle in LB compared to wild-type strains. In addition, the ihf double mutant did not form an air pellicle at all, suggesting a role for IHF in the expression of cellulose. These findings corroborate a previous study in which S. enterica Typhimurium ihfAB mutants exhibited reduced bcsC transcription when evaluated by microarray analysis, but further studies are needed to better charact erize the underlying molecular mechanisms. Karaca, N Akcelik, and M Akcelik (2013) also evaluated pellicle formation at the air-liquid interface of 31 S. enterica isolates. They showed that the growth rate of isolates with a rigid pellicle was greater than that of the ones forming a fragile pellicle. Biofilm production at the air-liquid interface can facilitate and contribute to gas exchange, while enabling the acquisition of nutrients and water from the liquid phase. Biofilms at air-liquid and solid-air interfaces can cause serious problems in industrial water systems. In conclusion, our results indicate that IHF has an important regulatory role in biofilm formation of S. enterica serovar Enteritidis. Moreover, both IHF subunits appear to have a role in this process. Our data pave the way for further studies investigating the mechanisms involved in the regulation of biofilm formation by IHF. Acknowledgements This work was supported by grants from Fundaà §Ãƒ £o de Amparo à   Pesquisa do Estado de Sà £o Paulo (FAPESP 2014/13412-8) and Conselho Nacional de Desenvolvimento Cientà ­fico e Tecnolà ³gico (CNPq), Brazil. BL, DBN, and GPM were supported by a FAPESP fellowship (FAPESP 2012/25426-8, 2012/10608-3, and 2012/05382-6, respectively). CHW and CPC were supported by fellowships from CNPq (141629/2012-6 and 140786/2012-0, respectively). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest or conflict.

Dreams: Winnie-the-pooh and Vision Center Essay

We have prepared this handout of actual essays written by current Harvard students who attended secondary schools in the UK (with names changed for anonymity) in order to provide some guidance to schools and applicants. Because the university admissions processes in the US and the UK are markedly different, we have received requests for some sample essays and tips for writing them and hope they will be helpful. Here is the official description of the personal essay requirement: Please write an essay (250 words minimum) on a topic of your choice or on one of the options listed below. This personal essay helps us become acquainted with you as a person and student, apart from courses, grades, test scores, and other objective data. It will also demonstrate your ability to organize your thoughts and express yourself. 1. Evaluate a significant experience, achievement, risk you have taken, or ethical dilemma you have faced and its impact on you. 2. Discuss some issue of personal, local, national, or international concern and its importance to you. 3. Indicate a person who has had a significant influence on you, and describe that influence. 4. Describe a character in fiction, a historical figure, or a creative work (as in art, music, science, etc.) that has had an influence on you, and explain that influence. 5. A range of academic interests, personal perspectives, and life experiences adds much to the educational mix. Given your personal background, describe an experience that illustrates what you would bring to the diversity in a college community, or an encounter that demonstrated the importance of diversity to you. 6. Topic of your choice. Your essay for a US university might be the same one you would write for the UCAS system, but perhaps not. We are interested in your academic successes and future plans, but also want to understand what makes you tick as a person. What are your hopes, dreams and fears? Our advice is to think of two or three possible topics, write a quick first draft of each essay and then show them to your best friend, mother, teacher or anyone who knows you well. Ask that person if your voice and personality come through in the essays and which one sounds the most like you. Then take that essay and polish it off! As you will see from the following sample essays, these students have written about learning to ride a bike, culture shock at coming to the UK, music, public service, and a favourite book. What will you write about? Sample College Essay #1 I never imagined that by swimming, a Vision Center in India would be built. And I certainly never thought so many people could be cured of blindness there. For the past twelve years of my life, my passion has been competitive swimming. Mile after mile I train almost every single day in the hope of becoming that much faster, that much more powerful in the water, that much closer to my goals. (My classmates tell me I am better adapted to live in the water than on land!) I have reached more athletic goals than I ever imagined when I first splashed into the water as a timid six-year old. I have won several Texas state titles, been ranked nationally in both the US and the UK, set numerous International Schools Tournament records, and captained both my school and club swim teams. This past year, I decided to combine my love of swimming with a fundraising target. My older brother worked as an intern on the Flying Eye Hospital run by the international sight-saving ch arity ORBIS. I was horrified by his description of the magnitude of curable, but untreated eye diseases. I knew I had to take action. To help those who have or will lose their sight for no fault of their own, my triplet siblings and I organized, planned, publicized, and successfully led a community-wide Swim-a-thon that raised funds for ORBIS. The goal of our event was not only to raise funds for this very worthwhile cause, but more importantly, to raise awareness about avoidable blindness. Our theme â€Å"Every minute a child goes blind†, caught the attention of the community. The word spread. People were surprised to know that we have the medical capability to cure millions of people with a simple surgery or eye droplets, yet hundreds of people lose their sight every day. As a two-year class vice president and student member of the Athletic Advisory Board, I was able to gain permission from the Head of School to plan the event. I convinced the Athletic Director to set aside pool time and recruited life guards. In order to garner support, I placed ads in the school newspaper, hung posters throughout my school, and persuaded my coach to replace an afternoon workout with the Swim-a-thon. After weeks of preparation, swim mates, school faculty, and parents logged thousands of laps. It was an immensely successful day. Enough money was collected to build a Vision Center in India, with surgical equipment, medicines, and training materials. The new Vision Center will not only treat thousands of patients, but will create a permanent site to train doctors and other medical personnel. As Treasurer of my school’s chapter of the National Honor Society, I plan to allocate charity funds this year to ORBIS for the continuing operational costs of the Vision Center. Every minute a child goes blind. Thirty-seven million people in the world are blind. Remarkably, an overwhelming 28 million of them do not need to be. When I think about the Vision Center we funded, I am overwhelmed with a sense of accomplishment and pride. Even though I will never meet the many people who will receive medical treatment there, the satisfaction of knowing that I have helped change the lives of thousands of people is astonishing. More meaningful than any swim race or trophy, we have brought hope where there was darkness. Sample College Essay #2 If we speak the same language, then why don’t I understand you? Why are the clothes you wear so different and the expressions you say so unclear to me? It was my first day in England and a â€Å"Bank Holiday† at that. With only one sport on television, I was determined to watch and study a game I had no idea how to play. I didn’t know what an â€Å"over† was, or even the job of the bowler. I didn’t know what a â€Å"wicket† was, or how many a team needed to win. But I didn’t care. I was living across the pond now, and if I was going to fit into my new surroundings, understanding the rules of cricket seemed like a fine starting point. I persevered, and eventually I was explaining the now familiar game of cricket to my family; baseball analogies helped a great deal. I was proud of myself. I had conquered the English culture. Maybe England wasn’t so bad after all. I soon realized how naive these thoughts were. Cricket was just the beginning. A whole world of different traditions and customs was thrown in front of me. July Fourth was exchanged for Guy Fawkes Day and the â€Å"the celebration with the turkey† was erased from the calendar. Where would I fit in? Rugby and Premier League Football dominated the sports channels. Where was my beloved ESPN? Why is the television show, Little Britain, so hysterical? The movie theatres were smaller than a British mini cooper, and the Super Bowl kicked off at four a.m. The warmth of the Texan sun was replaced by the rainy days of Wimbledon. I was surprised to see that some parts of life abroad were better. Friends became mates. The frenetic pace of Piccadilly Circus and the splendour of St. Paul’s Cathedral are unsurpassed. Roundabouts make the traffic run smoothly. I like the sound of â€Å"Cheers†. Over sixty different nationalities and over thirty languages are represented at the International School I attend. The culture shock was overwhelming. But I refused to yield. I was going to start by mixing into the English culture. I reported on local and national events as an Editor of the school newspaper. In addition I met swimmers from all over the U.K. through my British swim team, all with different backgrounds and lifestyles from mine. The cultures that engulfed me when I first came to England are part of me now. London is at my disposal. The people, the pubs, the expressions, and the entertainment are all a part of what makes living in England a once-in-a-lifetime experience. Yes, I have missed several Thanksgiving feasts and numerous Astros games, but I have come to understand and enjoy a completely new place. I wouldn’t change any of my experiences. Living in Europe has broadened my perspective on life and opened my eyes to so may wonderful people and ideas. People have similar goals wherever they come from. I’m glad I know that there is no single right way to achieve them. Sample College Essay #3 I wake up and there is a rhythm in my head: it’s hazy. I climb into the shower and the water tapping on my scalp reminds me. As I sit on the bus to go to school, I get strange looks from passengers as I tap the rhythm onto my knees, but it’s not yet fully formed. Throughout the school day, I feel it evolve and develop until I inevitably sit down at my drums and play. From my brain via my heart it enters my muscles; they transfer it to the sticks which relay it to the drum. Eventually, the air gets my gift and the rhythm returns through my ears. Even after the sounds are gone, the rhythm is not. Until I go to bed, a day’s repetition keeps it rebounding inside my cranium, in my own private concert hall. This is the journey of my daily rhythm. I wake up and there is a rhythm in my head: it’s not straight for this rhythm swings. At 6:00 am in Germany I get on a coach and as the wheels rotate beneath me I get closer. My coach has thirty five other people in it, each one is carried forward in their own sense of time, but in less than an hour they must all merge; seven hundred people will not accept a big band not keeping the pulse. Butterflies are roused in my gut and nerves take over. I’ve never played a solo in front of so many people yet somehow my fear must be quelled. My imagination, my sticks and my drum-set have to communicate my inner rhythm; the audience must be able to feel it or else I have failed. Rhythm is the barrier to embarrassment. As the opening to â€Å"Sing, Sing, Sing† begins to take shape, all my trust is placed in the pattern I have within me transporting me safely to the end. If this vessel sank, I too would go with it. A standing ovation confirmed that this time, the barrier held strong. I wake up and there is a rhythm in my head: but it is quiet. In fact, no-one hears it; it makes no noise and never will. Between the hours of 0845 and 1545 I have 4 beats: each one signaling another unit of learning. This phrase is repeated 5 days a week for 40 weeks a year and the chorus goes on 6 more times. My song is my school, and in it I am caught up in its inner rhythms that I cannot control – I must give in to them. The melodies that are assigned to these rhythms are made up of Virgil, esters and numbers that don’t exist. From these, cadences form that give me a chordal progression through education. Each part of my song has been given a name; there are no verses, no choruses but consecutive Key Stages. The rhythm indicates when I should make the transition: there is a series of fills, but they are not called fills. They call them exams and as the stages progress, the fills get more intense. In fact, they get more frequent and at the end of my school career, I look forward to a year where exams punctuate my calendar. In January, I will have moved to the dominant, only to complete the progression in June when I descend and finish on the tonic: a perfect cadence. I wake up and there is rhythm: the rhythm is life. The cycle of night and day and the constant pulsing in my chest are rhythms, and as the Earth revolves around our local star it is in time with the universe. I think in meter: a man crosses the street and his steps divide the distance between one curb and the next – they provide a beat in the asphalt bar, or at least that is how I picture it. If animals could not use the rhythm of the seasons, then they would surely die. Life is a rhythm and all that it contains is in time. When the rhythm ceases to exist, so will I. Sample College Essay #4 Magicians are not truly magical, though they like others to think they are. So what inspires this â€Å"deception†? Some think it is the money and others, the glamour of performing on stage and mystifying the audience. But for me it has always been a question of identity. Magic has helped me develop my confidence and communication skills so when the time comes to stand up and address a crowd, such as the school debate or a Model United Nations conference, it is no effort at all. However, I can say that one unusual circumstance in my life has given me a new sense of direction for my magic. At first, I thought magic was mere entertainment, but Horace, a man from the local Spastics Center diagnosed with autism in his late twenties, changed my perspective on my art. So often when we think of the disabled, we imagine children, and we sympathize with them. With Horace, I was faced with a situation largely unknown to the general public’s experience: an unemotional adult who ra rely spoke to or acknowledged others around him. When I tried to engage him, he mumbled to me uninterested and somewhat detached. But then when I produced my deck of cards, when through several routines and then showed him how he, too, could create â€Å"miracles†, he smiled and laughed. This reaction highlighted the most rewarding aspect of magic because he accepted me into his world and responded to me. The Center’s staff even commented, â€Å"We have never seen Horace behaving in such an emotional way!† For the first time, Horace had been given hope that he too could, perhaps, achieve and live a meaningful life. The magic had broken a myth of futility and dispelled it forever. I saw that magic could provide a driving force for pursuing change, and this realization overwhelmed me. I had witnessed something so unusual that the force of it took my breath away. The essence of magic is establishing a connection between the audience and the performer. With Horace, the ordinary had become the extraordinary, and for a moment, we connected in a state called â€Å"Astonishment†. This experience brings about a revisit to our most basic form, unaltered by culture or society. Indeed, that instant is so special because as adults, we are all too rarely astonished, and this moment returns us to our days as children when we were clueless and laughed at everything. This unusual encounter showed me that in this moment of astonishment, magic has the power to inspire. Since the encounter with Horace I have founded a society that brings all the magicians at my school together to perform magic for the elderly and the less fortunate in the nearby community. If I can show them, for instance, how to produce onepound coins from thin air, then contrary to what they have been told, perhaps they can challenge their â€Å"limits†. Then, dare I say that my passion for magic would be enhanced by a touch of true magic, generated perhaps from a truly unusual moment of astonishment. Sample College Essay #5 The ball ricocheted off the wall and disappeared into the black hole under my bed. It had been some years since the Hoover had been granted visiting rights and a heavy cloud of fluff covered every inch of the 4’ x 6’3† rectangle. Slightly nervous of what I might find, I ventured in slowly with an outstretched hand. The ball was nowhere to be found but I felt a small box-shaped object. I dragged it out, dusted it off and there looking somewhat the worse for wear, was my old leather book trunk. As I eased open the lid, the familiar smell (slightly musty with time) transported me immediately back ten years. Inside, in pride of place on top, staring boldly back at me, was my old friend Winnie the Pooh. The familiar, faded yellow face, the shrunken red tshirt with tummy protruding proudly from beneath, an empty honey pot and by his side, as ever, was Piglet. It is to Winnie the Pooh that I owe my greatest debt. It was this funny bear of little brain and large appetite who first sparked my interest in the literary world. He taught me about friendships and Woozles and how to make the best Heffalump traps. Many a happy hour was spent with Pooh and his friends facing adversity with his ever optimistic demeanour. Although I haven’t seen this treasured copy for many a year, I must admit to a weekly dose of life in a Hundred Acre Wood. Each week I share my passion for reading with Class 2A at the local village primary school. We start off with one of Pooh’s adventures – richly embellished with different voices that perhaps A.A. Milne may not have intended but, nevertheless, seem to get the seal of approval from my six-year-old audiences. After this we get down to the nitty gritty – the business of learning to read – or as I like to call it â€Å"Discovering How to Lose Yourself†. I go round the class taking turns to listen to them read. Although the range of their abilities is surprisingly large they all make a huge effort and really enjoy themselves. Progress is made and more and more pupils get lost each week. All too soon it is time to go. I say my goodbyes and rush back to school for my next lesson. I hum a little happiness tune and as I round the corner into the school a large thundercloud looms above. In my head a very Pooh-like voice says â€Å"Tut, tut, looks like rain!†. Sample College Essay #6 Nothing of much significance ever happens on the Isle of Wight. And to those of you for whom island travels only involved the palm trees and pink sand variety, let me enlighten you. The Isle of Wight is a small, chalky lump that broke off the south coast of England and came to a halt one mile out. It is caught in a rather charming time warp – circa. 1955. No palm tree could ever survive the freezing easterly winds that blast through from Siberia every winter and the only pink sand would be the result of a small child falling over a sandcastle on the way back from the Mr. Whippy ice cream van. It has sand and it has trees but of the altogether more hardy type. The sand is yellow and coarse, the trees are sturdy and solid with waxy leaves to withstand the salty air. The people are sturdy and solid too with an accent inherited from their pirate ancestors that sets them apart from those on the mainland. Life ambles along and nobody rushes. Pity anyone caught in a queue at the butchers behind Mrs. Singleton as she recounts, far too vividly, details of her latest health scare. Don’t get me wrong, it has its virtues – the Victorian pier pointing like a lace gloved finger out to sea and the promenade with shops selling pink and white sticks of rock with â€Å"Isle of Wight† running through it (how do they do that?), â€Å"Kiss me Quick† sunhats and inflatable boats. And on the corner of the High Street, the Cod Father fish and chip shop with a sign in the window saying â€Å"We batter anything!† It was on this sleepy little island that I, as a small boy, spent most of my summers. Summers that, due to the temperature, would be called autumn anywhere else – but nothing a long wetsuit and hat couldn’t disguise. One day in early July, arriving back from another bracing trip to the beach, I saw my mother and sister sitting side by side on the grassy bank overlooking the lawn. Nex t to them was a large bag of sweets and a bicycle. My mother announced that today was the day she was going to teach me how to ride a bike. There had been many attempts before but today I could tell she meant business. A succession of sweets was laid out at regular intervals along the lawn marking the route I was to take. Each time I made it to that point without falling off I got the sweet. Knowing that I really didn’t have any choice, I climbed awkwardly onto the enormous bike. Mother steadied me with one hand and then with a shove I was hurtling down the bank and onto the lawn. A blur of screaming and clapping preceded the first of many spectacular falls and my shins took on the blue hue of an uncooked lobster with blobs of grease from the chain splattered amongst the bumps. My sister, younger than me and already a whiz on her pink Barbie bike, gave patronizing advice trying to sound encouraging but, I suspect, rather enjoying the moment. An hour and a half later, battered and bruised with tears streaking through the mud on my face, I climbed onto the beast one more time. I steadied myself, focused on the hedge at the other end of the lawn (where the biggest chocolate bar lay) and with an almighty push I launched myself forward peddling with fury. Before I knew what had happened, there was a rush of green as I landed headfirst into the hedge. The bike, free of its incompetent rider, did an elegant ark to the left before coming to a halt. As I cycle confidently (but still can’t do the no hands thing) to the boathouse every day, I think of the words of Claude Pepper who said, â€Å"Life is like riding a bicycle. You don’t fall off unless you plan to stop peddling.† I won’t stop peddling. – and am proud to announce that on the 3rd July 1998 something of great significance did, at last, happen on the Isle of Wight.

Term Paper about What is Platonic Love

Introduction The fact that the society has been involved in advancing feminism and gender equality, there is a fact that gender cannot ignore; no man is an island. The love and want of a soul mate keeps every man and woman searching for the ‘other half’. However, if this is necessary for the human race, why is it that the purpose and the meaning of it has eluded us? From the Plato’s symposium, in the epoch when the seeking for knowledge was as a pathway to enlightenment, the term love was a concept that was not well understood. This symposium serves as a pamphlet that shows the guidelines as seen by the philosophers at the time of Plato. The intervention of the Greek gods in their speeches is interpreted to mean different aspects of love and even their effects on the people. The symposium is a gathering of academically diverse and actually wise men who share same mind about love. The love between women and men is disparaged as lewd and procreant and the love between boys and men are praised as beneficial to wisdom. Main Discussion What is Real Love – Plato on Love Plato postulates that, humans are made up of three planes: Body (Soma), Soul (Psiche) and the divine consciousness/ spirit (Nous). In all these three dimensions, humans search for eternity and completeness. This is because as divine creatures, humans would have an innate wish to go back to their original state i.e. the state of union with God. From that, it can be deduced that, love is force that drives man towards the original condition- the creator. From the physical plane, beauty is a key attribute of the divine. According to Plato, people have a natural tendency to consider beauty and even to search for beautiful bodies. Gorgeous forms instigate the humans to spread the seeds of eternity so as to give life to another being who will continue apiece of those who brought him to life. In a higher realm, the psychological plane, Man has a natural tendency to seek for the divine and should feel encouraged towards upright people so as to give birth to moral virtues and even to spread the seeds of eternity through building projects and also reaching objectives that will end up leaving traces of their existence. Eventually, in the highest realm, love seems to be the sentiment that rolls back everything back to unity. So, as Plato defended, real love is when one   finds another one whom he/she can   manifest love in the three levels of existence and in addition feel fulfilled in all the dimensions i.e. Physiological, physical and the spiritual planes. Ideals of Love in Platos Symposium Plato considers love as a necessary in the life of man that enables him/ her to acquire joy and courage while living or dead. What is love? How does it feel like? How does it come about? These questions are difficult to explain, yet they are somehow understood. The Plato’s symposium, which dates back in the start of the middle period, Plato introduced his Eros theory (Eros usually was translated to mean love). This dinner party had the discussion of love as the main topic. The notion of love was understood at the end of the party came about gradually. This transformed from all speakers who spoke at the party and this was in comparison of the whole process of understanding the love that Socrates was trying to explain in his own speech. It is in the same symposium that Plato introduces his Eros theory. Fro these dialogues the western culture was also discussed- for instance the image of two lovers as being the others half, which Plato assigns to Aristophanes in the Symposium. Besides, the â€Å"ladder of love,† through which a lover can ascend to direct cognitive contact with beauty itself. The Phaedrus reveals love as being the great â€Å"divine madness† through which the wings of the lover’s soul may sprout, allowing the lover to take flight to all of the highest aspirations and achievements possible for humankind. In both these two cases, Plato regards sexual or physical contact between the lovers as not only wasteful but also degraded forms of erotic expression. Since the goal of love/ Eros is real beauty and that real beauty is the form of beauty (what Plato calls beauty itself) so eros only finds its fulfillment in Platonic philosophy. He further postulates that, unless it channels its lo ve power into what he calls higher pursuits, which result in the knowledge of the form of beauty, eros is subject to frustration. It is form this reason there for that Plato thinks that, many people squander the power of love by restricting themselves to the pleasures of the physical beauty. Why do People Suffer for Love? From this point of view, it is effortless to identify the reasons behind unhappy relationships. Humans focus on the physical dimension of love that is seeking for only the physical fulfillment and in turn neglecting the other aspects of psychological and the spiritual. Where a couple can give rise to off springs in the physical realm, but they cannot give rise to virtues together, or even to be united in a higher plane, then according to Plato this is not true love. This is simply satisfaction of the instincts, which is not only temporal but also fades away in the long run. Unluckily, many become aware of the spiritual and psychological dimensions of love when it’s too late and also when the two people have taken different bearings. In such at times the couple is united by merely the obligations of their physical existence but spiritually they are not together. Is Platonic Love Impossible? Soul mates are people (couple) who walk together towards the superlative world, they are spiritually, physically and psychologically together- in that, they complement each other in each of the three realms of existence. Some people think that platonic love is sexless, but rather, it is love that is not commanded by instincts, not ruled by the satisfactions of the body, but a kind of love that fulfills the spiritual needs and that brings forth not only off springs but also virtues to the world. In the contemporary society, people are used to love that is just based on the physical realm, that this form of love that transcends the frontiers of the earthily or material life is related to the impossible love. May be the contemporary society would have a lot to borrow from the ancient world. References Michael, J. Platonic love: Poems. New York: Orchises Press, 1991. Plato, C. Plato on love. New York: Hackett Publishing, 2006. Plato, Christopher, G. The symposium. New York: Penguine Classics, 2003. Price, A. Love and Friendship in Plato and Aristotle. London: Oxford University press, 1990. Campos, Thais. What is Platonic Love – Platos Philosophy and the Art of Loving. 10 Aug 2010. 9 October 2010 http://www.suite101.com/content/what-is-platonic-loveplatos-philosophy-and-the-art-of-loving-a260700#ixzz12535H6gi.

The Leadership Styles Of The Executive Staff - 1383 Words

â€Å"Was Enron the Work of a Few Bad Men or Dark Shadow of the American Dream?† In August 2000, Enron, an American energy corporation, stock had reached a high of $90.75 per share. However, by November 2001, the price had plummeted to less than a dollar amidst the collapse of one of analysts’ most highly recommended investments. On December 2, 2001, Enron became the largest American corporate bankruptcy to date. The company was deceptive, even fooling Fortune Magazine into naming it â€Å"America’s Most Innovative Company† for six consecutive years. The leadership styles of the executive staff fostered a cutthroat and unethical business subculture for the climate which inevitably led to Enron’s demise. Two of Enron’s fearless leaders†¦show more content†¦They were skilled communicators who developed a comprehensive and captivating vision for their company. They had referent power; their employees, stock analysts, and shareholders t rusted them. Analysts were blinded by the company’s fraudulent practices because they took the financial statements from the management on faith. In â€Å"Enron: The Smartest Guys in the Room,† Amanda Martin, a top executive at the company, described Skilling saying, â€Å"Jeff was like the prophet. He came in and said there s a whole new world out there...The excitement was palpable. You cannot imagine how proud we all were to be there, and then, of course, we had a leader who imbued us with a sense of confidence.† Skilling controlled his employees using rational persuasion and inspirational appeals. He used logic but also appealed to their values, further blurring the line between the achievements of employees and the subsequent success of Enron. Skilling and Lay were clearly charismatic leaders but it was their lack of other leadership skills, including altruism and integrity, that exposed the dark potential of charismatic leaders. Lay and Skilling also were transactional leaders, â€Å"leaders who guide or motivate their followers in the direction of established goals by clarifying role and task requirements† (TB). They implemented strict work schedules to ensure that each employee was maximizing