Big Changes at Playboy Magazine

Big Changes at Playboy Magazine For decades Playboy magazine has been known for its titillating nude photo spreads and centerfolds. However, a new era is upon us. The magazine will no longer include nude photos as of March 2016 issue.   The U.S. print edition of Playboy will be modernized to look more like men’s magazines, such as Esquire or GQ, which currently carry more PG-13-type pictures. However, Playboy’s international editions will still publish nude photos. A New Era In a letter to readers on Playboy.com, the magazine addressed the momentous change: â€Å"The question everyone will likely be asking is, â€Å"Why?†Ã‚  Playboy  has been a friend to nudity, and nudity has been a friend to  Playboy, for decades. The short answer is: times change. When Hef created  Playboy, he set out to champion personal freedom and sexual liberty at a time when America was painfully conservative. See: any popular movie, TV show or song from that era. Nudity played a role in the conversation about our sexual liberties, and over 62 years the country made great strides politically and culturally. We like to think we had something to do with that.† Playboy, like other forms of print media, has also seen a marked decrease in readership. In its heyday, Playboy had a circulation of 5.6 million in 1975.     According to the Alliance for Audited Media, its circular is a mere  800,000 now. Last year Playboy launched a safe-for-work website that can be viewed any where without fear of pornographic images popping up, which has resulted in younger viewers and more readership overall- quadrupling from 4 million to 16 million visitors. The ubiquity of nudity in today’s world- versus when Playboy launched in 1953- has forced the magazine to get with the times. Pay-per-view soft core porn images have a very limited audience in a world where one can view full-length hardcore films for free in a matter of a few keystrokes. What does this mean for women? For one, the magazine will feature a new sex columnist, one that Playboy’s chief content officer Corey Jones has said will be a â€Å"sex-positive† woman who will write enthusiastically about sex. This particular change is not insignificant and suggests that discussions of sex in the magazine have the potential to be transgressive. Playboy, which calls itself a cultural arbiter of beauty, taste, opinion, humor and style, will also continue its tradition of investigative journalism, in-depth interviews, and fiction. They are hoping that the de-emphasis on nudity will court big name stars and writers that were previously put off by the magazine’s racy content. Since the magazine is no longer relying on nude photos to draw in readers, their choices for future cover girls are reflecting the shift in focus. According to the Hollywood Reporter, openly feminist pop songstress Taylor Swift is Playboy’s first choice for the inaugural non-nude edition in April 2016. It remains to be seen if Swift will agree to the cover. Nevertheless, opponents of pornography, whether hard or soft core  Ã¢â‚¬â€¹and those who believe that media outlets like Playboy exploit women are unlikely to be swayed by Playboy’s move away from nude pictures.   And, indeed, considering that the magazine’s target demographic is young men, one can imagine that the magazine’s impact will be not unlike other men’s magazines such as Maxim, GQ, or Esquire- none of which are known for woman-friendly content and entertainment.

Words Ending in -ance and -ence

Words Ending in -ance and -ence Words Ending in -ance and -ence Words Ending in -ance and -ence By Maeve Maddox A reader asked if there were some easy-to-remember spelling tip for dealing with words ending in -ence and -ance. Both endings derive from Latin nouns. Words from Latin nouns ending in -entia affluence from affluentia, â€Å"abundance† audience from audientia, â€Å"a hearing† benevolence from benevolentia, â€Å"good will† continence from continentia, â€Å"self-control† diligence from diligentia, â€Å"accuracy† Words from Latin nouns ending in -antia elegance from elegantia, â€Å"neatness† petulance from petulantia, â€Å"forward conduct† significance from significantia, â€Å"an indication, a sign† vigilance from vigilantia, â€Å"watchfulness† tolerance from tolerantia, â€Å"enduring† Here are some of the most common English words with these endings. The only way to spell them correctlyapart from using a spell checkeris to memorize them. Words ending in -ence: absence affluence audience coherence conference confidence conscience consequence consistence correspondence dependence diligence evidence existence influence obedience occurrence patience persistence preference reference Words ending in -ance: acquaintance allowance ambulance annoyance appearance appliance arrogance disturbance dominance extravagance grievance guidance ignorance instance nuisance relevance remittance resistance significance substance tolerance Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Spelling category, check our popular posts, or choose a related post below:The Yiddish Handbook: 40 Words You Should Know20 Pairs of One-Word and Two-Word FormsDrama vs. Melodrama

Education of Professionals in the Light of the Changing Nature of Essay - 1

Education of Professionals in the Light of the Changing Nature of Professional Practice - Essay Example The concept of a profession is a developing one and is not stationary. So, the definition should not be static defining just a few people as professionals. The expansion in the definition should indeed be made considering the criteria like core values and knowledge which will be discussed later on in this essay. This expansion is important because people involved in newly emerging professions like packing and transporting are also considered as professionals (Downie, 1990) There is so much importance in the practice of professionals that the quality of a professional is dependent on the practice of how one exercises his or her duties following the ethical codes and must have sufficient concern for the society. For example, often the new technological developments by professionals are double-edged(Mike W..Martin, Roland Schinzinger, 2005). The advent of nuclear power has increased our power capability, but at the same time, it has posed atomic bomb threat. – briefly explain what you mean and try to give an example. This, in turn, can be managed by means of good training and education on ethical behavior and also attaining proper education should be viewed in all aspects and not just in attending courses or a mere qualification. Governments, politicians and social activists take an active part in improving the quality of professionals’ practice (Becher, 1993). On the other hand, there are also objections from some communities over their value to their society, the way in which their projects increase the value of the society. Some projects may degrade the society very much, for example, the nuclear tests. – such as?. So, the process of building such quality in educating professionals requires a careful analysis and the work should proceed from the grass root level so that a solid foundation is laid in professional ethics helping all through their career.

Research the roles of African Americans in the military history of Essay

Research the roles of African Americans in the military history of World War I. How were African Americans recruited How were t - Essay Example This paper seeks to discuss African American soldiers’ role in the military history of the First World War, starting with the process of recruitment, moving along to their experiences in the armed forces, as well as in combat, and finally exploring the psychological impact it had on African Americans, in addition to their view of racial discrimination. 2.0 African Americans and Recruitment In the spring of 1917, The United States of America had to face a war of unsurpassed magnitude, requiring it to harness all its resources -- material, intellectual, and human. Hence, this was the mobilization of the colored people as a part of the country’s line of defense in the First World War (Williams 15). However, the path towards the fulfillment of their patriotic duty was not paved clear. Opposition in the person of members of the senate and southern democrats existed (Orr 90). The aforementioned officials resisted the idea of including African American draftees in the armed fo rces of the United States. Senator James K. Vardaman was adamant in his idea that millions of armed colored men served only as an unparalleled peril to the South (Ellis 11). However, because of black leaders’ efforts, 367,710 blacks were drafted (Orr 90). These African American draftees consisted of a variety of professions from common workers and farmers to physicians and attorneys. They were issued drafts on the months of June and September, and were ordered to join the 1,200 enlistees in Fort Dodge, Des Moines, Iowa in a Colored Officers Training Camp, regardless if they were willing to do so or not (Lentz-Smith 41). The aforementioned training camp was made possible through the resoluteness of the National Association for the Advancement of Colored People (NAACP) in pushing for the setting up of a training school for African American officers (Orr 91). 2.1 African Americans in the Armed Forces and in Combat African Americans’ participation in military defense was a n unheard of concept even if it was in service to America (Lentz-Smith 21). Majority of white people greatly opposed this on the premise that colored people could not be capable soldiers (Williams 2). Some even went so far as to consider the colored man as more like a farm animal such as a horse instead of a man; an example was Ely Green who decided to enlist in the war upon hearing that very discriminating statement from white farmers (Lentz- Smith 38). Even with their entry to the armed forces, African Americans experienced the said discrimination. 200, 000 of those black soldiers were relegated to the American Expeditionary Force and were assigned labor functions -- something as menial as digging up trenches (Roberts and Tucker 2318). Hence, the reality of shouldering shovels in place of guns (Williams 2). The Navy further highlighted this by only including black men as mess boys or attendants. However, no such emphasis compared to the Marines’ complete rejection of them ( Roberts and Tucker 2318). Racial discrimination was underlined in what is now known as the Battle of Anniston. The soldiers of the Third Alabama Colored Infantry experienced such blatant discrimination as they were driven back to camp by white military men and civilians when they went out on their first night there. They endured this

Alpha Lipoic Acid what is it, and how does it improve (help) Essay

Alpha Lipoic Acid what is it, and how does it improve (help) peripheral neuropathy and insulin resistance - Essay Example The metabolic dysregulation associated with DM causes secondary pathophysiologic changes in multiple organ systems that impose a tremendous burden on the individual with diabetes and on the health care system. The two broad categories of DM are designated type 1 and type 2. ALA has been shown to be useful in type 2 diabetes mellitus, and in order to understand the mechanism of action of ALA in control of DM and DM-associated complicating conditions, such as, neuropathy, it is important to understand the pathophysiologic and biochemical mechanism of these conditions (Boulton, 2005). A prominent biochemical feature of type 2 DM is insulin resistance. This group of disorders is characterised by a pathogenic process that leads to hyperglycemia through variable degrees of insulin resistance, impaired insulin secretion, and increased glucose production. Type 2 DM is characterized by three pathophysiologic abnormalities: impaired insulin secretion, increasing peripheral insulin resistance, and excessive hepatic glucose production. Obesity, particularly visceral or central as evidenced by the hip-waist ratio, is very common in type 2 DM. Adipocytes secrete a number of biologic products, namely, leptin, TFN-alpha, free fatty acids, resistin, and adiponectin that modulate insulin secretion, insulin action, and body weight and may contribute to the insulin resistance. As expected, in the early stage of the disease, glucose tolerance remains normal despite insulin resistance since the pancreatic beta cells compensate by increasing the output of insulin. As insulin resista nce and compensatory hyperinsulinemia progress, the pancreatic islets in certain individuals are unable to sustain the hyperinsulinemic state (Huebschmann et al., 2006). Diabetic Neuropathy Diabetic neuropathy occurs in approximately 50% of individuals with long-standing type 1 and type 2 DM. It may manifest as polyneuropathy, mononeuropathy, and/or autonomic neuropathy. As with other complications of DM, the development of neuropathy correlates with the duration of diabetes and glycemic control; both myelinated and unmyelinated nerve fibers are lost. Because the clinical features of diabetic neuropathy are similar to those of other neuropathies, the diagnosis of diabetic neuropathy should be made only after other possible etiologies are excluded (Boulton et al., 2004). The most common form of diabetic neuropathy is distal symmetric polyneuropathy. It most frequently presents with distal sensory loss. Hyperesthesia, paresthesia, and dysesthesia also occur. Any combination of these symptoms may develop as neuropathy progresses. Symptoms include a sensation of numbness, tingling, sharpness, or burning that begins in the feet and spreads proximally. Neuropathic pain develops in some of these individuals, occasionally preceded by improvement in their glycemic control. Pain typically involves the lower extremities, is usually present at rest, and worsens at night. Both an acute and a chronic form of painful diabetic neuropathy have been described. As diabetic neuropathy progresses, the pain subsides and eventually disappears, but a sensory deficit in the lower extremities persists. Physical examination reveals sensory loss,

Strains of ESBL Producing E. Coli | Investigation

Strains of ESBL Producing E. Coli | Investigation Introduction Background of Study Extended Spectrum Beta- Lactamases (ESBL) are beta lactamases which are mainly produced by family members of Enterobacteriaceae derived from mutations of the previous broad-spectrum beta-lactamase (Sharma et al., 2010). This enzyme works by hydrolysing and destroying the ÃŽ ²- Lactam ring of all cephalosporins, penicillins and monobactams (Sharma et al., 2010). In recent years, the emergence of ESBL producing Escherichia coli has posed a very serious problem to the management of diseases caused by this organism as only limited choice antibiotics can be given to patients. Carbapenems are the drugs of choice for the treatment of ESBL producing E.coli, however, carbapenamase resistance has recently been reported (Paterson and Bonomo, 2005). Prolonged use of antibiotics was suggested as the main cause of the emergence of ESBL E.coli and the fact that the genes coding for ESBLs are easily transferred from one organism to another organism via conjugation, transduction and transformation ma ke the spread even quicker (Vaidya et al., 2011). ESBL producing organisms were first reported from a patient in Germany in 1983 and since then , several outbreaks have been reported worldwide usually one particular â€Å"super† strain has been involved presumably combining not only the capability to produce ESBLs but also possessing various other virulence factors that contribute to their pathogenic success. (Harada et al., 2013). These pathogenic ESBL producing Escherichia coli in recent years have become a major concern and their emergence is now become alarming in clinical fields and subjected to comprehensive studies worldwide. The most common infections caused by pathogenic ESBL producing E.coli are urinary tract infections (UTI), bloodstream infections, gastrointestinal infections (Fatima et al., 2012; Bekat et al., 2002). According to Petty et al., (2013), globally, E.coli sequence type ST131 is the multidrug resistant clone strain which is responsible for ESBL CTX-M15 bearing genes, and it is the most alarming pathogenic ESBL producing E.coli associated in causing UTIs and septicaemia in hospital community acquired infections. ? in UK or worldwide? As genes coding for ESBL in Escherichia coli are known to be transferable this raises further fear of the spread of these genes to other strains, continuous monitoring of the predominant strains of E.coli which carry the ESBL genes is therefore important. Problem statement Studies of ESBL producing Escherichia coli in the South Manchester population have been carried out previously. This study will investigate strains of ESBL producing E. coli currently circulating in the Stockport Population of South Manchester and compare them to those delineated in the previous studies using a molecular typing and pulse-field gel electrophoresis. Objectives The objectives of the project are: Screen for ESBL Escherichia coli clinical isolates Identify strain using PFGE Assess the relatedness of the strains by PFGE analysis Determine Escherichia coli plasmid profile Identify Escherichia coli phylotyping group 1.0.4. Significance of study Finding from this study will contribute to the existing data and the body of knowledge on the molecular relationship of predominating of E.coli isolates from South Manchester populations. 1.0.5. Scope and Limitations There are no data on the antibiotics consumed by the patients in which the clinical isolates originates from. The availability of this data might help in understanding relationship between an exposures of certain antibiotics to the emergence of ESBL producing E.coli strain. PFGE also has several limitations in which the method assess visual relatedness of an isolates and not using a phylogeny relationship which provide more accurate molecular relationship between an isolates. Escherichia coli Escherichia coli is a motile gram negative rod, facultative anaerobe, non- spore forming bacteria taxonomically belong to the family of Enterobacteriaceae. It is considered as a normal inhabitants of gut and intestine in almost all warm blooded mammals and found as a faecal contaminant in the environment (Brennan et al., 2010; Darnton et al., 2007; Diniz et al., 2005). Most varieties of E.coli are harmless and do in the most part contribute to the normal and healthy intestine condition, while a few cause limiting abdominal cramp associated with diarrhoea. However, there are some serotypes that becoming a major threat to the human health, because they have acquired certain genetic material and virulence factors which enabling them transformed into pathogenic E.coli causing broad spectrum of disease (Clarke et al., 2003; Kaper et al., 2004). Pathotypes of E.coli are classified by specific mechanism in which they causing a disease, presence of certain virulence genes and their clinical manifestations (Chang et al., 2004). Growth requirements E.coli are non- fastidious bacteria, thus it can be cultured in artificial media with various altered physical and nutritional growth factors. It can be isolated easily from clinical samples by culturing into culture media and incubated at optimum temperature of 37 ºC anaerobically or aerobically as it is a facultative organisms (Yunlin et al., 2004) Uropathogenic Escherichia coli According to Pitout et al., (2005) E. coli is a frequent cause of the urinary tract infections (UTIs) of a hospitalised and non- hospitalised patients. UTIs are usually self- limiting but untreated lower urinary tract infections such as simple cystitis (bladder infection) can lead to much more severe illness known as pyelonephritis (renal infections) mainly among adult women (James et al., 2011). Infections occur by ascending movement of E. coli up the periurethral area colonising the bladder or infections by movement down from the intestinal tract. Due to anatomical complexities in women, they are more prone to be diagnosed with UTIs for at least once in their lifetime (James et al., 2001) 1.3  Escherichia coli typing 1.3.1  Plasmid profiling Multidrug resistant bacteria including ESBL producing Escherichia coli acquire their resistance by various gene transfer mechanisms which include transformation, horizontal transfer either by transduction, and conjugation, transposon and most often, are plasmid mediated (Carattoli et al., 2005) Plasmids are an extra chromosomal fragments of self- replicating DNA present in most of the bacterial species. Plasmids contain genes that are an essential for the replication of genes that promotes resistance to agents such as antibiotics, ultraviolet radiation, metals and bacteriophages. 1.3.2  Pulse-field gel electrophoresis PFGE was developed and described first by Schwartz and Cantor (1984). It is a molecular technique of typing a bacteria especially pathogenic Escherichia coli 0157:H7, non 0157: H7, Salmonella serotypes, Shigella sonnei and Shigella flexneri. PFGE uses a gel electrophoresis- based technique that allows separation of large molecular weight DNA up to 2Mb- 10Mb using a standard PFGE method (CDC, 2013; Hansen et al., 2002; Vimonet et al., 2008) PFGE is different to conventional gel electrophoresis as the large genomic DNA is digested with restriction enzyme that recognise and cleave specific sequences of DNA known as restriction site in an organism to produce a multiple DNA fragments which differ in size of their molecular weight (Van der Ploeg et al., 1984). The fragments are then run through constant changing electric field of PFGE resulting in a formation of DNA at various discrete size bands. This typing method has also been shown to have more discriminating power and reproducibility between laboratories than the newer molecular typing method such as ribotyping and multi- locus sequence typing (MLST) which confer more on the global epidemiology and revolutionary relationship between bacterial species (Vimonet et al., 2008) 1.3.3.  Escherichia coli phylogenetic group 2.0  Materials and Methods 2.0.1  Bacterial Isolates Bacterial isolates used in this study were Escherichia coli clinical isolates which was collected from Stepping Hill Hospital. Isolates undergo an anonymisation numbering of 1 to 20. 2.0.2.  Bacteriological Media The media used in the study were a selective differential medium for UTI Escherichia coli which is Chromogenic agar and nutrient agar which was used as a medium for growth and maintenance of isolates. 2.0.3  Antibiotic disks Table 1: Antibiotic disks used in this study was obtained from Oxoid.Ltd. Antibiotics Antibiotic Group Gentamicin (10 µg) Aminoglycosides Ciprofloxacin (5 µg) Quinolone Amoxicillin (25 µg) Penicillin Cefpodozime (10 µg) Cephalosporin Mecillinam (10 µg) Beta lactam Trimetophrim (2.5 µg) Bacteriostatic ESBL Disk kit (Mast Diagnostics) 2.0.4  Buffers and solutions Tris Borate EDTA (TBE X1 and X0.5) (Sigma) pH 8.2 was used as a running buffer in agarose gel electrophoresis. 2.0.5  Commercial kits The commercial kit used in this study was QIAprep Spin Miniprep Kit (Qiagen) and DNeasy Blood and Tissue Kit (Qiagen) 2.1.  Screening for multidrug resistance and potential ESBL producers in Escherichia coli clinical isolates Antibiotic susceptibility of Escherichia coli to six antibiotics (Table 1) were tested using the Kirby Bauer disk diffusion method. A 24 hour cultures from Nutrient agar was used. Then, a single colony was taken and transferred into 5ml Mueller Hinton Broth. It was then incubated at 37 °C to develop a heavy suspension of overnight cultures. A sterile cotton swabs were used to streak onto the Mueller Hinton agar and the rotation were repeated for three times. A final sweep was made around the rim of the agar. The plates were allowed to dry for several minutes. Using antibiotic dispenser, the disk that has been impregnated with a fixed antibiotic concentration was placed on the surface of the agar surface. After 24hr of an incubation period, the plates were checked for the presence of inhibition zone. Each recorded inhibition zone was compared with antimicrobial susceptibility testing disc chart provided by The British Society for Antimicrobial Chemotherapy (BSAC). The inhibition zon e of each antibiotic was reported as ‘sensitive’, ‘intermediate’ or ‘resistance’. Isolates showing resistance to three or more classes of antibiotics were considered as multidrug resistance (Falagas, 2007). ESBL producers were detected by testing sensitivity of isolates against a pair discs (cefpodoxime 10 µg and cefepime 10 µg) with and without clavulanic acid placed oppositely on an agar. According to manufacturer (Mast diagnostics), isolates were considered as an ESBL if there is a presence of 5mm larger inhibition zone in disks with clavulanic acid rather than the disks without the clavulanic acid. 2.2. Determination of plasmid profiles in MDR and ESBL Escherichia coli 2.2.1  Plasmid Extraction Prior to Plasmid DNA extraction, a fresh overnight cultures of E.coli after an incubation at 37 ºC in a Mueller Hinton broth were harvested. Plasmid DNA extraction was carried out using QIAprep Spin Miniprep Kit (Qiagen) following the manufacturer’s instructions. Extracted plasmid DNA was stored at -20 ºC until use. 2.2.2  Detection of plasmid by agarose gel electrophoresis The profiles of the plasmid DNA was determined on a 0.7% agarose gel electrophoresis which has been carried out at 70 Vcm-1 for 120 minutes. The size of DNA bands was estimated using Hyper ladder 1 (Bioline) as a reference molecular weights marker. The bands were visualized under UV transilluminator and photographed with digital camera connected to visualisation unit (Alpha Innotech) and the size of the plasmid were measured by visual comparison to the reference marker. 2.3  Escherichia coli pathotypes determination 2.3.1.  Genomic DNA extraction Primary cultures on the nutrient agar was inoculated into 3ml Mueller Hinton broth for 24 hours at 37 ºC. The cells was then harvested by centrifugation at 12, 000 for 3 minutes. Genomic DNA extraction was carried out using DNeasy Blood and Tissue (Qiagen) kit following the manufacturer’s instructions. Final volume of 150 µl genomic DNA were collected and kept at -20 ºC until needed. 2.3.2  Multiplex PCR for Escherichia coli phylotyping PCR reaction mix preparation must be carried out on ice. PCR was performed in 0.2ml PCR tubes on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems ®) with a total 25 µl of reaction volume as described in Table 2 and PCR condition according to Table 3. The negative control reaction lacking the DNA was included. Table 2:  PCR reaction mix Components Required concentrations Volume ( µl) per reaction Biomix Red 2X 12.5 Primer (forward) chuA yjaA tspE4.c2 20pmol 20pmol 20pmol 1 1 1 Primer (reverse) chuA yjaA tspE4.c2 20pmol 20pmol 20pmol 1 1 1 DNA 2 Ultrapure sterile water 4.5 Total volume per reaction 25 Table 3: Conditions for PCR gene amplification Genes Primer sequence PCR condition chuA Forward 5’-GACGAACCAACGGTCAGGAT-3’ Reverse 5’-TGCCGCCAGTACCAAAGACA-3’ Initial denaturation: 94 °C for 4 mins Denaturation: 94 °C for 25 secs Annealing: 52 °C for 40 secs 30 cycles Extension: 72 °C for 50sec Final extension: 72 °C for 6 mins yjaA Forward 5’-TGAAGTGTCAGGAGACGCTG-3’ Reverse 5’-ATGGAGAATCGGTTCCTCAAC-3’ tspE4.c2 Forward 5’-GAGTAATGTCGGGGCATTCA-3’ Reverse 5’-CGCGCCAACAAAGTATTACG-3’ 2.3.3  Detection of by agarose gel electrophoresis After completion of the multiplex PCR, the amplification product were separated by dry electrophoresis system. 15 µl of amplified product was mixed with 5 µ 5X DNA loading buffer (Bioline) and loaded onto 2% agarose gel incorporated with SYBR green dye. After electrophoresis, the gel was visualised by exposing the gel under UV light and was photographed with a digital UV camera connected together with the visualisation unit (AlphaInnotech). The size of the amplicon were measured by visual comparison to the 1kb DNA marker (Bioline). Phylogenetic typing analysis were carried on the basis of the presence or absence of an amplicon sized 279bp, 211bp and 152bp which belong to chuaA, yjaA and tspE4.c2 genes respectively. 2.4.  Pulse- field gel electrophoresis (PFGE) 2.4.1.  DNA extraction Each isolates was inoculated into 5ml Mueller Hinton Broth and incubated overnight at 37 ºC with gentle agitation. Cells were then harvested by placing 1ml of culture into 1.5ml microcentrifuge tube and was centrifuged at 13, 000 rpm for one minutes. The supernatant was discarded and the process was repeated until all the 5ml of culture finished. The supernatant was again discarded and pellet of cells was resuspended in 500 µl of 0.5M EDTA buffer (see appendix) and was centrifuged at 13, 000rpm for one minutes to removes broth debris that might be interfering with the extraction processes. The washing step was repeated twice to ensure complete removal of debris. The supernatant was discarded once again and pellet was resuspended in 500 µl of suspension buffer. 2.4.2.  Preparation of low melting point (LMP) agarose To prepare the LMP agarose, 3g of SeaKem PFGE agarose (BioRad) were dispensed into 100ml of TE buffer (see appendix) in a universal bottle. It were then heated to dissolve. Agarose was transferred to a 56 ºC waterbath until needed. 2.4.3.  Preparation of the bacterial plugs The wells of PFGE plug molds were numbered. 3 plugs was prepared for each isolates. Then, 750 µl of LMP agarose was added immediately into each cell- buffer suspension and carefully mixed by pipetting up and down several times and be careful not to induce any formation of bubbles. The mixture of cells and agarose was quickly pipetted into the well of a plastic PFGE plug molds (BioRad). The wells was filled to the rim and plugs were allowed to solidify at room temperature or chilled for 5 minutes in the refrigerator. 2.4.4.  Lysis of the cells The cells were lysed by adding a mixture of 1ml of proteolysis buffer with 10 µl of Proteinase K stock solution (50mg/ml) (see appendix) into a 1.5ml new labelled microcentrifuge tube. The plugs were removed from the plug molds by peeling the sealant tape below the wells until all tape was removed. The PFGE plastic arm was used to push the plugs out of the molds into the microcentrifuge containing the mix of proteolysis buffer-proteinase K solution. All plugs for one isolates were transferred into the same tubes. Care was taken while pushing the plugs out of the molds as not to tear the fragile plugs. Tubes was then incubated in a heating block at 50 ºC for 24 hours for digestion to take place. 2.4.5.  Washing of the plugs After completion of an overnight incubation, the proteolysis buffer and Proteinase K activity were eliminated by carefully pipetting out the volume, care taken not to tear the plugs. The plugs were then washed with TE buffer. The washing steps was repeated three times, for every half an hour and were held at room temperature to equilibrate the plugs. 2.4.6.  Restriction enzyme digestion After completion of the washing steps, wash buffer was removed in the final wash leaving only agarose gel in the tubes. Then, 300 µl of 1X restriction enzyme buffer specific to the enzyme used was pipetted in each tubes containing the agarose plugs and was let to equilibrate at room temperature for 10 minutes. The restriction buffer was then discarded, taking care not to tear the plugs. Next, 300 µl of restriction buffer containing 50U of Xbal enzyme was added into the tubes and was incubated in an incubator for 24 hours at 37 ºC specific to the optimal temperature for Xbal enzyme. 2.4.7.  Pulse- field gel electrophoresis 2.4.7.1.  Electrophoresis gel preparation. After incubation, restriction enzyme reaction was stopped by addition of 200 µl of 50mM EDTA. Plugs were cooled at 4 ºC until needed. Then, a (1%) agarose gel was prepared by heated to dissolved 3g of PFGE grade agarose (BioRad) into 300ml of 0.5X TBE buffer over magnetic hot plate with constant stirring or in the microwave and swirl to dissolved. The agarose was then poured into a casting tray that has been placed with PFGE comb and let to solidify at room temperature. The enzyme- buffer was aspirated and one plug of each isolates was loaded into the gel. Care was taken not to tear the plugs. Then, a thin slice high range and mid- range lambda molecular weight marker (New England Biolabs) was loaded into the wells next to each other. After all samples was loaded into wells, the wells were sealed with melted LMP agarose. 2.4.7.2.  Electrophoresis Run The electrophoresis was performed by using a CHEF mapper (BioRad) which subsequently was filled with approximately 3 liters of 0.5ml TBE buffer. The running buffer was let to cool approximately at 14 ºC before turning on the pump. The run time was set for 24 hours at 6 Vcm-1 with 120 º angle using switch time of 2.16 sec to 54.17 sec. 2.4.7.3.  Gel staining Once the run was complete, the gel was stained with 3X Gel red nucleic acid stain (Biotium) with approximately 200ml distilled water and was gently agitated on rotary shaker for 20 minutes. The gel was then visualised under UV transilluminator and a picture was taken once optimal image obtained.

Atticus As A Model Parent :: essays research papers

In To Kill a Mockingbird, Harper Lee suggests that Atticus is a model parent. Atticus gives guidance to Jem and Scout, and he treats them with fairness and honesty. He tries to bring them up as best he can as a single parent. Atticus is always guiding Jem and Scout with advice so that they will become more compassionate people. Atticus sets a good example for the children when Mr Ewell confronts him. Even though he is provoked and insulted, Atticus simply has a â€Å"peaceful reaction†. This shows the children never to get into fights with people when they are upset about something. Atticus shows children about courage and all the forms it appears in. When Jem is told to read for Ms Dubose and she dies, Atticus explains to Jem about her morphine addiction, and how she died â€Å"free†. This shows Jem that courage isn’t always where you expect to find it, and that if you have some compassion, you see people for who they really are. The most important piece of advice he gives his children is that â€Å"you never really understand a person until you consider things from his point of view†¦ until you climb into his skin and walk around in it.† This is important for the childre n to know, because it helps them to be more caring people, and they use this advice throughout the novel. Atticus treats everyone with fairness. He always hears both sides of the story. He does this after Scout has gotten into a fight with Francis Hancock. After a time Atticus hears Scout’s side of the story and realises that it wasn’t totally Scout’s fault. Scout also tells Uncle Jack that when she and Jem get into fights Atticus stops to hear both sides of the argument before placing the blame, if any. When he is confronted by awkward issues Atticus never tries to hide or cover up the truth. He tells Uncle Jack at Finches landing that when a child asks you something, â€Å"answer him, for goodness sake.† After Atticus is confronted by the mob outside the county jail, he doesn’t try to pretend that they weren’t there to hurt him. He admits that Mr Cunningham might have â€Å"hurt me a little.† When Scout asks Atticus if they are poor, the usual response would be to say no, so as not to scare Scout.