Aspergillus Fumigatus Identification and Molecular Character

Aspergillus Fumigatus Identification and Molecular Character IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF Aspergillus fumigatus FROM SOIL R. V. Shalini, and Dr. K. Amutha ABSTRACT: Soil was collected, serially diluted and pure culture obtained; slant was prepared in potato dextrose agar and maintained throughout the study. Morphological, microscopical and macroscopically identification were carried out on the isolated organism. DNA was isolated from the 24 hour culture, for ITS-PCR amplification. DNA was amplified by mixing the template DNA (50nm) with the polymerase reaction buffer, dNTP mix, primers and Taq. Polymerase chain reaction (PCR) was performed in a total volume of 50Â µL reaction mixture. The PCR product was mixed with loading buffer (8Â µL) containing 0.025% bromophenol blue, 40% w/v sucrose in water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and the amplified product was visualized under a UV trans illuminator for further examination. The PCR products were finally sequenced using the help of an automated DNA sequencer at progen Ltd (Salem, India) and analyzed with the BLAST program provided by the National Center f or Bio-technology information (NCBI) to confirm the fungal species. The current study demonstrates that DNA genome containing 18S rRNA has a high degree of analytical sensitivity and specificity (100%) for the detection of a wide range of fungi. OBJECTIVE: To isolate, identify and characterize Aspergillus fumigatus using molecular biological methods. MATERIALS AND METHODS: The soil was collected from different places, pooled together allowed to be dried at room temperature. The morphology based identification of Aspergillus was done which includes the size, shape, colour, ornamentation of spore and mode of attachment. Unfortunately a lot of difficulties arose for phenotypical identification of this fungus due to its unstable characteristics. Comparatively a DNA sequence-based identification format appeared to be the most promising in terms of its speed, ease, objectivity and reliability for species identification. RESULTS: The preliminary morphology based studies showed the isolated fungi as a species of aspergillus.However after the DNA isolation followed by sequencing it was concluded that the particular species identified as Aspergillus was Aspergillus fumigatus. KEY WORDS: Aspergillus, serial dilution, DNA, Sequenced. INTRODUCTION: The presence of organic matter in the soil affects the quantity and quality of microbes in the soil. The development of micro fungi in the soil is favoured by soils having acidic reaction and aerobic condition which is likely present in the soil. However the amount of degradation in the soil is brought about by the organisms present in the soil. 1The rate at which the organic matter is decomposed is inter related with soil microbes. (Arunachalam et al., 1997). Microorganisms come in various sizes and shapes and is determined by the soil ph., temperature, available moisture, degree of aeration, availability of nutrients in the soil etc. The genus of spore forming fungi is found worldwide out of which Aspergillus is the most dominant species and is ubiquitoes.Out of that 95% is occupied by Aspergillus fumigatus. The other pathogenic forms of Aspergillus species are Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus etc. This fungi exists only in mycelial f orm, and is thermo tolerant capable of growing at temperatures between 15-53Â °c.Being a spore producing fungi the spores gets dispersed by wind in the atmosphere. 2Aspergillus fumigatus is the most common among all the airborne saprophytic fungal pathogens in immune compromised patients mostly in developed countries (Latge, 1999). It is the main pathogenic agent of various diseases caused in humans including invasive pulmonary aspergillosis, aspergilloma and allergic bronchopulmonary aspergillosis (Tomee van der Werf, 2001) – the former is a frequent cause death in immune compromised patients. The possession of different virulent traits gives A. fumigatus the ability to cause these diseases. It is a known fact that other members of the genus Aspergillus are either less pathogenic or non- pathogenic. Identification of the most common and important species remains problematic due to the variability in the phenotypic characters. However a validated and a careful approach of phenotypic classification (taxonomy) together with phylogenetic treatment of DNA sequence data is a prerequisite for a reliable and a rapid identification. In our inve stigation we used the molecular techniques (sequencing) for the reliable identification rather the identification based on their microscopic and few physiological features. MATERIALS METHODS: Collection of soil samples: Soil samples were collected from different places (in and around Chengalpattu). The surface deposits were removed to a depth of about 10 cm and the exposed soil was collected to a depth of 2-3cm. The collected soil samples were stored in zip locked covers stored in refrigerator temperature for further analysis. The collected soil samples were passed through a sieve to remove the stones and other impurities. Isolation of fungi: The glass wares were sterilized in an autoclave to a temperature of 120Â °c for twenty minutes. The chemicals were of analytical grade (Himedia). The method used for the isolation of fungi from soil was serial dilution method. 1 gm of soil was weighed and mixed in 10ml of double distilled sterile water. This was used for preparing serial dilutions. 1 ml of the final dilution (10-6 ) was pipetted into the prepared potato dextrose agar media (PDA) amended with a suitable antibiotic Chloramphenicol (12mg/100ml). The plates were incubated at 30Â °c for about seven days. Fungi that appeared on petriplates were isolated. The isolates were picked up based on apparent dissimilarity of cultural characteristics and purified. The purified isolates were identified according to the genera on the basis of cultural characteristics such as nature of growth, spore colour, and pigment production, and on morphological characteristics of mycelia and fruiting bodies (Domsch etal., 1980; Raper and Fenne ll 1965) and maintained in agar slants for future use3. Isolation of DNA: Genomic DNA was extracted from 24 hour old culture. Measured 100 micro gram of mycelium into a sterile 1.5- micro centrifuge tube. Simultaneously ground 1 microgram of dried (vacuum filter mycelium first) in a mortar and pestle treated with liquid nitrogen 5-6 times. Poured the frozen powder into the Eppendorf tube. Added 660 750 Â µl of lysis buffer and 10 Â µl of B-mercaptor.Vortexed the mixture for a few seconds. And Incubated at 65Â °C for 1 hour. Used a water bath for incubation. Centrifuged at a speed of 3400 rpm for 5 minutes at room temperature and aspirated out the top layer.Transfered the top aqueous layer into a fresh Eppendorf tube discarded the bottom layer. Measured out 700 Â µl of chloroform, isoamyl alcohol (24:1) into Eppendorf tube and adjusted the volume to meet a 1:1 ratio of aqueous phase.Vortexed the mixture for a few seconds. Centrifuged at a speed of 12000 rpm for 10 minutes at room temperature and aspirated out 550 600 Â µl of the top layer. Transfered the top aqueous layer into Eppendorf tube and discarded the bottom layer. Added 0.1volume of 3m potassium acetate and 0.7 volume of isopropanol. Mixed well by inverting the tube not by vortexing.Centrifuged for about 10 minutes and discarded the supernatant. Added 0.5 mL of ice cold ethanol (70% and inverted the tube gently, again it was centrifuged for about 5 minutes in a spinner) finally the pellets were resuspended in 100Â µl of TE buffer (PH-8). After further purification DNA was quantified spectrophotpmetrically and the quality was analyzed in 0.9% agorose gel. Amplification of 18srRNA by PCR: For ITS-PCR amplification, DNA was amplified by mixing the template DNA (50nm) with the polymerase reaction buffer, dNTP mix, primers and Taq polymerase chain reaction (PCR) was performed in a total volume of 50Â µL reaction mixture containing Primer (2Â µM/Â µL) 8.0Â µL 10X Buffer 5.0 Â µL 2mM dNTP Mix 5.0Â µL Taq DNA polymerase (5U/Â µL) 0.5Â µL Template DNA (50ng) 2.0Â µL Sterile distilled water 29.5Â µL Total volume 50.0Â µL PCR amplification condition: Amplification was carried out in a primus advanced gradient thermocycler. The PCR was programmed with an initial denaturing at 94Â °C for 5 min, followed by 30 cycles of denaturation at 94Â °c for 30 seconds, annealing at 61Â °c for 30 seconds, and extension at 70Â °c for 2 minutes and a final extension at 72Â °c for 7 minutes. The PCR product was mixed with loading buffer (8Â µL) containing 0.025% bromophenol blue, 40% w/v sucrose in water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and the amplified product was visualized under a UV trans illuminator for further examination. (Sequencing) Sequencing of ITS region for identification of isolated fungi : Chosen Samples of the genomic DNA containing 18S rRNA were shortlisted for more specific species confirmation by using DNA sequencing. The sequenced PCR product was aligned with other isolate sequences from NCBI genbank for identification. The PCR products were finally sequenced using the help of an automated DNA sequencer at progen Ltd (Salem, India) and analyzed with the BLAST program provided by the National Center for Bio-technology information (NCBI) to confirm the fungal species. RESULTS: Macroscopic and Microscopic Analysis: Analysis of the isolated Aspergillus species showed variation in the colony colours, texture, and reverse side colours (table 1 and 2). The morphological microscopic and molecular characteristics showed that the isolate is Aspergillus fumigatus (details given in table 1and 2). Morphological characters of colony (table1) Characteristics Aspergillus fumigatus Surface colour Margins Reverse side Growth Green to dark green Entire Yellow Rapid Microscopic characteristics (table2) Characteristics Aspergillus fumigatus Hyphae Branched septate Conidiophore Present Vesicle Dome shaped Conidia Present Phialides Uniseriate Fruiting body Cleistothecia Fig A1 Fig A2 Morphological characterization of Aspergillus species on potato dextrose agar A1-Aspergillus fumigatus surface colour, A2-Reverse side of the colony. DNA sequencing of ITS region for identification of species: The species of fungi from the PCR sample was identified by DNA sequencing of the internally transcribed spacer (ITS) region of rRNA gene. Segments of the entire ITS regions, including partial 5.8S rRNA and internal transcribed spacer 2, complete sequence, 28S rRNA,partial sequence were amplified using the primer PGF04 5’-GGC ATC GGC C-3’. Amplification of the ITS region of strain Aspergillus fumigatus had a size of 1703bp. It was submitted to the NCBI and the accession number KC 119199 was received. M 1 2 Fig A3 represent the banding pattern of Aspergillus fumigatus from PCR reactions Lane M= Marker, Lane 1= Aspergillus fumigatus, lane 2=Aspergillus fumigatus. DISCUSSION: Detection of A. fumigatus is of great concern because it is a dangerous allergen associated with aspergillosis 5(Abraca et al., 1994; Schuster et al., 2002; Noonimabcet al.2009; Edwin et al., 2010; Gautam et al., 2011). This highlights the importance of correct identification and taxonomical differentiation between different species of Aspergillus. The taxonomy of Aspergillus has always been complex due to its great number of species (nearly 250), which have very few differences. The identification of different Aspergillus species, on the basis of their morphological characters (example, colony colours, and reverse side) is one of the oldest and most adopted methods. Some of the species of Aspergillus have the same morphological features which make it difficult to distinguish between them it is also a time consuming process and may not be accurate (Klich and Pitt, 1988; Samson et al., 2004)6. This shows that morphological and microscopical characters are not enough for fungal identification and it renders the need of molecular techniques for correct species identification. Molecular characterization on the other hand, is a rapid and a quick procedure which requires minimal handling of pathogens. It also helps in distinguishing morphologically, similar fungal species. Several similar studies on the application of PCR technology were used for the identification and detection of fungi, by using internal transcribed spacer (ITS) were already been studied and published7 by several scientist (Henson and French, 1993; Marek et al., 2003; Haughland et al., 2004; Druzhinina etal., 2005).. Many more such studies were also carried out very recently by God et and Munaut (2010) in the differentiation of Aspergillus flavus, A.parasiticus, A.tamarii and A.nomius by PCR-RAPD markers. Similarly, Leema et al. (2010) confirmed the species A. flavus by verifying; using the molecular methods that is, by amplification of the internally transcribed spacer regions. By using the help of RAPD-PCR, 8Khan et al. (2007) studied diversity in various Aspergillus niger isolates sourc ed from pigeon pea fields .Several molecular techniques have been tested to classify different Aspergillus species like random amplification of polymorphic DNA (RAPD) (Yuan et al., 1995), the internal transcribed spacer (ITS) region (Kumeda and Asao, 1996; Henry et al., 2000; Kumeda and Asao, 2001; Rigo et al., 2002) and the aflatoxin gene cluster (Chang et al., 1995; Watson et al., 1999; Tominaga et al., 2006). In this study care was taken to choose the genomic DNA containing 18sRNA specifics primers that were helpful in amplifying medically important fungi. The genomic DNA containing 18s rRNA was the right candidate for detection of fungus as it is a mutli-copy gene which evolves slowly and is conserved among fungi. The present study proves that the genomic DNA containing 18s rRNA based PCR is suitable for probing large range of medically significant fungi owing to its higher level of analytical sensitivity and specificity. CONCLUSION: In this present study we had shown that molecular techniques are rapid and best for identification of fungi than the traditional morphological methods for early diagnosis and treatment of fungal infections. The goal of our study was to identify a practical, quick, cheap, method for the identification of A. fumigatus, the most common of the Aspergillus pathogens. REFERENCES: Arunachalam K, Arunachalam A, Tripathi RS, Pandey HN. 1997 – Dynamics of microbial population during the gradation phase of a selectively logged subtropical humid forest in north east India. Tropical Ecology 38, 333–341. Sirida Youngchim,1,2 Rachael Morris-Jones,1 Roderick J. Hay3 and Andrew J. Hamilton1 Production of melanin by Aspergillus fumigatus Journal of Medical Microbiology 2004, 53, 175–181. Domsch, K.H.,Gams W. and Anderson T.H. 1980.Compendium of soil fungi, vol 1.IHW-Verlag,Eching.,Raper,K.B.andFennel,D.I.1965.The genus Aspergillus (Baltimore: Williams Wilkins). Ferrer C, Colom F, Frases S, Mulet E, Abad JL, Alio JL:Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA in eye infections. J ClinMicrobiol 2001, 39(8): 2873-2879. Abraca ML, Bragulat G, Cabanes FJ 1994. Ochratoxin A production by strains of Aspergillus flavus var. niger . Appl. Environ. Microbiol. 60:2650-2652. Klich MA, Pitt JI 1988. Differentiation of Aspergillus flavus from Aspergillus parasiticus and other closely related species.Trans. Br.Mycol. Soc.91:99-108. Henson J, French R 1993. The polymerase chain reaction and plant disease diagnosis. Ann. Rev. Phytopathol.31:81-109. Khan MR, Anwer MA, Mohiddin FA 2007. Molecular diversity in Aspergillus isolates collected from pigeonpea field in Aligarh region.Environ. Biol. Conserv. 12:59-64. Godet F, Munaut F 2010. Molecular strategy for identification in Aspergillus section flavi. FEMS Microbiol. Lett. 304:157-168. Leema G, Kaliamurthy J, Geraldine J, Thomas PA 2010. Keratitis due to Aspergillus flavus: Clinical profile, molecular identification of fungal strains and detection of Aflotoxin production.Mol. Vision 6:843-854. Yuan G, Liu C, Chen C 1995. Differentiation of Aspergillus parasiticus from Aspergillus sojae by Random Amplification of Polymorphic DNA. Appl. Environ. Microbiol. 61:2384-2387. Kumeda Y, Asao T 2001. Heteroduplex panel analysis a novel method for genetic identification of Aspergillus section flavi strains. Appl. Environ. Microbiol. 67:4084-4090. Chang PK, Ehrlich KC, Bhatnagar JD, Cleveland TE 1995. Increased expression of Aspergillus parasiticus aflR, encoding a sequence specific DNA-binding protein, relieves nitrate inhibition of aflatoxin biosynthesis. Appl. Environ. Microbiol. 61:2372-2377.

Was Anne Hutchinson a Threat to Puritan Elite?

â€Å"Was Anne Hutchinson a threat to the Puritan elite? † LaToya McSwain US History 1 November 20, 2012 Believing that the Church of England had strayed too far from its original doctrine, disgruntled group of churchgoers decided to withdraw from the church. In an effort to escape persecution they fled to America. This group became known as the Puritans. Though, in their opinion, the church had strayed from God’s word, they held firm to their position that the Bible was God’s blueprint for daily living.The Puritans believed the church of that day was too saturated with irrelevancy; therefore, in an effort to purify religion, they felt the need to peel off those irrelevant formalities. Hence, the name Puritans-purifying their religion, as well as, their lives. Their strong spiritual convictions served as both a blessing and a curse. It served as a blessing in that it unified them and provided hope during hardships. It served as a curse in that they developed a so mewhat judgmental view of others as worldly if their actions didn’t coincide with the Puritan faith.There is also the idea of â€Å"pre-destination†. Each individual’s lifestyle indicated your standing with God. If you were well off, it was assumed you were in good standing with God. If you were poor, you obviously were not in good standing with God. Placement in heaven and hell was already pre-determined by God and no one knew their destiny. This is uncertainty is what kept the Puritans on the straight and narrow path. If the way of Puritan life is not strictly adhered to, individuals face harsh criticism and discipline-both by the church and society.One of the first American feminist, Anne Hutchinson, believed each individual should have the right to freedom of worship, speech, and thought. She did not deviate from her beliefs, ethics, or ideas no matter what it would cost her. There was never any intention to make anyone angry or hurt anyone’s feelings with her beliefs. Anne Hutchinson was born in a town called Alford, Lincolnshire, England in July, day unknown, 1591. Her birth name was Anne Marbury. She was born to Francis Marbury, a deacon, and Bridget Dryden.Her father, a deacon at Christ Church, Cambridge, was arrested due to his open deplore of the clergy’s lack of competence. He was in jail for a year for his ideas and abrasive words. Because of Anne’s father’s beliefs, she took a strong liking to religion very young in her life. By age 21, she was married to Will Hutchinson. During her life, she bore 15 children with her husband. She and her family followed the word and teaching of John Cotton, Protestant minister. John Cotton had some of the same ideas her father had when she was growing up, but now these same ideas and teachings were accepted by Puritanism.

The Return: Shadow Souls Chapter 2

Elena burst out of the backseat of the Jaguar and ran a little way from the car before turning to see what had fallen on top of it. What had fallen was Matt. He was in the process of struggling to get up off his back. â€Å"Matt – oh, my God! Are you all right? Are you hurt?† Elena cried at the same time as Matt was shouting in tones of anguish: â€Å"Elena – oh, my God! Is the Jag all right? Is it hurt?† â€Å"Matt, are you crazy? Did you hit your head?† â€Å"Are there any scratches? Does the moonroof still work?† â€Å"No scratches. The moonroof is fine.† Elena had no idea if the moonroof worked, but she realized that Matt was raving, off his head. He was trying to get down without getting any mud on the Jag, but he was handicapped since his legs and feet were covered with mud. Getting off of the car without using his feet was proving difficult. Meanwhile, Elena was looking around. She herself had once fallen from the sky, yes, but she had been dead for six months first and had arrived naked, and Matt fulfilled neither requirement. She had a more prosaic explanation in mind. And there it was, lounging against a yellowwood tree and eyeing the scene with a very slight, wicked smile. Damon. He was compact; not as tall as Stefan, but with an indefinable aura of menace that more than made up for it. He was as immaculately dressed as always: black Armani jeans, black shirt, black leather jacket, and black boots, which all went with his carelessly windblown dark hair and his black eyes. Right now, he made Elena acutely aware that she was wearing a long white nightgown that she had brought with the idea that she could change her clothes underneath it if necessary while they were camping. The problem was that she usually did this just at dawn, and today writing in her diary had distracted her. And all at once the nightgown wasn't the correct attire for an early-morning fight with Damon. It wasn't sheer, being more akin to flannel than to nylon, but it was lacy, especially around the neck. Lace around a pretty neck to a vampire – as Damon had told her – was like a waving red cloak in front of a raging bull. Elena crossed her arms over her chest. She also tried to make sure that her aura was pulled in decorously. â€Å"You look like Wendy,† Damon said, and his smile was wicked, flashing, and definitely appreciative. He cocked his head to the side coaxingly. Elena refused to be coaxed. â€Å"Wendy who?† she said, and at just that moment remembered the last name of the young girl in Peter Pan, and winced inwardly. Elena had always been good at repartee of this kind. The problem was that Damon was better. â€Å"Why, Wendy†¦Darling,† Damon said, and his voice was a caress. Elena felt an inward shiver. Damon had promised not to Influence her – to use his telepathic powers to cloud or manipulate her mind. But sometimes it felt as if he got awfully close to the line. Yes, it was definitely Damon's fault, Elena thought. She didn't have any feelings for him that were – well, that were anything other than sisterly. But Damon never gave up, no matter how many times she rejected him. Behind Elena was a thump and squelch that undoubtedly meant Matt had finally gotten off the roof of the Jag. He jumped into the fray immediately. â€Å"Don't call Elena, Elena darling!† he shouted, continuing as he turned to Elena, â€Å"Wendy's probably the name of his latest little girlfriend. And – and – and do you know what he did? How he woke me up this morning?† Matt was quivering with indignation. â€Å"He picked you up and threw you on top of the car?† Elena hazarded. She talked over her shoulder to Matt because there was a faint morning breeze that tended to mold her nightgown to her body. She didn't want Damon behind her just now. â€Å"No! I mean, yes! No and yes! But – when he did, he didn't even bother to use his hands! He just went like this† – Matt waved an arm – â€Å"and first I got dropped into a mud hole and next thing I know I got dropped on the Jag. It could have broken the moonroof – or me! And now I'm all muddy,† Matt added, examining himself with disgust, as if it had only just occurred to him. Damon spoke up. â€Å"And why did I pick you up and put you down again? What were you actually doing at the time when I put some distance between us?† Matt flushed to the roots of his fair hair. His normally tranquil blue eyes were blazing. â€Å"I was holding a stick,† he said defiantly. â€Å"A stick. A stick like the kind you find along the roadside? That kind of stick?† â€Å"I did pick it up along the roadside, yes!† Still defiant. â€Å"But then something strange seems to have happened to it.† From nowhere that Elena could see, Damon suddenly produced a very long, and very sturdy-looking stake, with one end that had been whittled to an extremely sharp point. It had definitely been carved from hardwood: oak from the look of it. While Damon was examining his â€Å"stick† from all sides with a look of acute bafflement, Elena turned on a sputtering Matt. â€Å"Matt!† she said reproachfully. This was definitely a low point in the cold war between the two boys. â€Å"I just thought,† Matt went on stubbornly, â€Å"that it might be a good idea. Since I'm sleeping outdoors at night and a†¦another vampire might come along.† Elena had already turned again and was making appeasing noises at Damon when Matt burst out afresh. â€Å"Tell her how you actually woke me up!† he said explosively. Then, without giving Damon a chance to say anything, he continued, â€Å"I was just opening my eyes when he dropped this on me!† Matt squelched over to Elena, holding something up. Elena, truly at a loss, took it from him, turning it over. It seemed to be a pencil stub, but it was discolored dark reddish-brown. â€Å"He dropped that on me and said ‘scratch off two,'† Matt said. â€Å"He'd killed two people – and he was bragging about it!† Elena suddenly didn't want to be holding the pencil anymore. â€Å"Damon!† she said in a cry of real anguish, as she tried to make something out of his no-expression expression. â€Å"Damon – you didn't – not really – â€Å" â€Å"Don't beg him, Elena. The thing we've got to do – â€Å" â€Å"If anybody would let me get a word in,† Damon said, now sounding truly exasperated, â€Å"I might mention that before I could explain about the pencil someone attempted to stake me on the spot, even before getting out of his sleeping bag. And what I was going to say next was that they weren't people. They were vampires, thugs, hired muscle – but these were possessed by Shinichi's malach. And they were on our trail. They'd gotten as far as Warren, Kentucky, probably by asking questions about the car. We're definitely going to have to get rid of it.† â€Å"No!† Matt shouted defensively. â€Å"This car – this car means something to Stefan and Elena.† â€Å"This car means something to you,† Damon corrected. â€Å"And I might point out that I had to leave my Ferrari in a creek just so we could take you on this little expedition.† Elena held up her hand. She didn't want to hear any more. She did have feelings for the car. It was big and brilliantly red and flashy and buoyant – and it expressed how she and Stefan had been feeling on the day that he bought it for her, celebrating the start of their new life together. Just looking at it made her remember the day, and the weight of Stefan's arm around her shoulder and the way he'd looked down at her, when she'd looked up at him – his green eyes sparkling with mischief and the joy of getting her something she really wanted. To Elena's embarrassment and fury, she found that she was shaking slightly, and that her own eyes were full of tears. â€Å"You see,† Matt said, glaring at Damon. â€Å"Now you're making her cry.† â€Å"I am? I'm not the one who mentioned my dear departed younger brother,† Damon said urbanely. â€Å"Just stop it! Right now! Both of you,† Elena shouted, trying to find her composure. â€Å"And I don't want this pencil, if you don't mind,† she added, holding it at arm's length. When Damon took it, Elena wiped her hands on her nightgown, feeling vaguely light-headed. She shivered, thinking of the vampires on their trail. And then, suddenly, as she swayed, there was a warm, strong arm around her and Damon's voice beside her saying, â€Å"What she needs is some fresh air, and I'm going to give it to her.† Abruptly Elena was weightless and she was in Damon's arms and they were going higher. â€Å"Damon, could you please put me down?† â€Å"Right now, darling? It's quite a distance†¦Ã¢â‚¬  Elena continued to remonstrate with Damon, but she could tell that he had tuned her out. And the cool morning air was clearing her head a bit, although it also made her shake. She tried to stop the shivering, but couldn't help it. Damon glanced down at her and to her surprise, looking completely serious, began to make motions as if to take his jacket off. Elena hastily said, â€Å"No, no – you just drive – fly, I mean, and I'll hang on.† â€Å"And watch for low-going seagulls,† Damon said solemnly, but with a quirk at the side of his mouth. Elena had to turn her face away because she was in danger of laughing. â€Å"So, just when did you learn you could pick people up and drop them on cars?† she inquired. â€Å"Oh, just recently. It was like flying: a challenge. And you know I like challenges.† He was looking down at her with mischief in his eyes, those black on black eyes with such long lashes that they were wasted on a boy. Elena felt as light as if she were dandelion fluff, but also a little light-headed, almost tipsy. She was much warmer now, because – she realized – Damon had enfolded her in his aura, which was warm. Not just in temperature, either, but warm with a heady, almost drunken appreciation, as he took her in, her eyes and her face and her hair floating weightlessly in a cloud of gold around her shoulders. Elena couldn't help but blush, and she almost heard his thought, that blushing suited her very well, pale pink against her fair complexion. And just as blushing was an involuntary physical response to his warmth and appreciation, Elena felt an involuntary emotional response – of thankfulness for what he had done, of gratitude for his appreciation, and of unintentional appreciation of Damon himself. He had saved her life tonight, if she knew anything about vampires possessed by Shinichi's malach, vampires who were thugs to begin with. She couldn't even imagine what such creatures would do to her, and she didn't want to. She could only be glad that Damon had been clever enough and, yes, ruthless enough to take care of them before they got to her. And she would have to be blind and just plain stupid not to appreciate the fact that Damon was gorgeous. After having died twice, this fact did not affect her as it would most other girls, but it was still a fact, whether Damon was pensive or giving one of those rare genuine smiles that he seemed to have only for Elena. The problem with this was that Damon was a vampire and could therefore read her mind, especially with Elena being so close, their auras intermingling. And Damon appreciated Elena's appreciation, and it became a little cycle of feedback, all on its own. Before Elena could quite focus she was melting, her weightless body feeling heavier as it molded itself to Damon's arms. And the other problem was that Damon wasn't Influencing her; he was as caught up in the feedback as Elena was – more so, because he didn't have any barriers against it. Elena did, but they were blurring, dissolving. She couldn't think properly. Damon was gazing at her with wonder and a look she was all too used to seeing – but she couldn't remember where. Elena had lost the power to analyze. She was simply basking in the warm glow of being cherished, being held and loved and cared for with an intensity that shook her to the bone. And when Elena gave of herself, she gave completely. Almost without conscious effort, she arched her head back to expose her throat and closed her eyes. Damon gently positioned her head differently, supported it with one hand, and kissed her.

The Characters Of The Beloved Characters - 1881 Words

Everyone tries to be like the actors or characters they see on television or in movie theatres. Often times people can’t really see themselves as one character because they see too many negative aspects, and instead choose a character that they want to be more like because the traits of that character are more appealing. People often try to associate themselves with different aspects of a character. Some people may say something like â€Å"This character is so generous, I’m like this character because I am extremely generous†. Everyone wants to be associated with a character or more than one character, usually for better reasons rather than negative, but all in all, we try to find characters that help give us the appearance we look for as a†¦show more content†¦I have few friends, but the friends I have are true friends, they are always there for me and I am always there for them. I love them with all my heart and there isn’t anything we wouldnâ⠂¬â„¢t do for each other. I always have a very difficult time making new friends. Not because people don’t like me, but because starting conversations with people is hard for me. My friends found me when I was emotionally alone and they took me in. My life has been forever changed because of these few individuals. I care for them more than I care for myself. They are closer to me than some of my own family. Being socially awkward and shy is not a fun thing to be. I can never seem to be the kind of person that everyone wants to hang out with. At first, it did feel awful, but I’ve come to learn that I don’t have to get along with everyone, not everyone needs to be my friend. I have found my own friends and they are all I need. Being shy is just something that has always been with me. Going back to my first day in grade school, I was always shy to talk to people I’ve never seen before and if I did it was always an awkward moment. I naturally just became sort of an outcast and hung out with very few people. I’ve come to embrace who I am and the friends who I have are all the friends I would really care about or would want to have. Mac has a different personality from most characters in aShow MoreRelatedCharacter Analysis Of Beloved1342 Words   |  6 PagesDenver is the daughter of Sethe, the main character in novel Beloved by Toni Morrison. Denver is the most dynamic character in the novel. Denver is a young girl who spends hours alone. As a child, Denver’s dependence on others is an opposing force that she must realize and overcome and she must begin to rely on herself so that she can reach her fullest potential. As the relationship between Sethe and Beloved grows stronger, Denver has opportunities to break free and realize the danger of relyingRead MoreBeloved Character Analysis1122 Words   |  5 PagesIn the book Beloved, Toni Morrison gives the audience, many choices to think about what is really happening in each section to get the point of what’s going on. The audience has to ponder on each character. Let’s take Sethe for example, as a character to mainly focus on. In Beloved,Sethe is a mother to four children and a wife to Halle. Sethe has been through so much ever since the very beginning. She had to make a rough choice about whether she would kill her oldest daughter, Beloved. Sethe endedRead MoreBeloved Character Analysis867 Words   |  4 PagesIn the novel Beloved by Toni Morrison the character Sethe is faced with the traumatic experience of having to return to slavery at Sweet Home, in order to save her children she attempts to kill them. She succeeds in killing one by cutting the infant’s throat with a hacksaw. This â€Å"rough choice† revolves around the novel on whether or not, the choice was right or wrong. Sethe’s tough choice between the right or wrong in the murder of her child is right and was necessary for her to insure the safetyRead More Character of Beloved in Toni Morrisons Beloved Essays2510 Words   |  11 PagesThe Character of Beloved in Toni Morrisons Beloved Perhaps one of the most important issues in Toni Morrisons award-winning novel Beloved is Morrisons intentional diversity of possible interpretations. However the text is looked at and analyzed, it is the variety of these multiple meanings that confounds any simple interpretation and gives the novel the complexity. The debate rages on over many topics, but one issue of central and basic importance to the understanding of the novel is definingRead MoreBeloved As A Character Defies Classification.She Is Both1106 Words   |  5 Pages Beloved as a character defies classification. She is both the light and the dark, an all-encompassing duality that forces Sethe to recognize pain from her past, as well as deal with new pain in the present. Beloved’s shocking reappearance forces Sethe and others to confront old, painful memories whilst concurrently reconciling who or what Beloved is. For many, the character of Beloved embodies multiple generations of slavery and symbolizes the horrors of the past – seemingly back from the deadRead More The Character of Beloved from Beloved by Toni Morrison Essay2089 Words   |  9 PagesThe Character of Beloved from Beloved by Toni Morrison The character of Beloved, from Toni Morrison?s novel, Beloved, is an embodiment of the evils of slavery. Beloved, the daughter of a former slave, is a child who died before her time, therefore her existential search for identity parallels the search of self that slavery created in an innumerable amount of human beings. When reading the novel, Beloved, it is vital for the inexperienced reader to pay attention to the trials of Beloved, asRead MoreCharacter Analysis Of Toni Morrisons Beloved926 Words   |  4 Pagespassing day? Beloved is a story written by Toni Morrison about the hardships that lead the protagonist, Sethe, to kill her own daughter, who would later come back from the grave. Sethe is a middle-aged, former slave who has experienced the cruelest, most unjust torment in her life – slavery. She escaped this barbaric life, but when the chance of being taken away comes back, she has to murder her own daughter to save her. Through close examination of the book, movie, and many other character analysisRead MoreBeloved by Toni Morrison769 Words   |  3 PagesBeloved is the character who lends her name to the title of Morrison’s novel, isnt really a flesh-and-blood character at all, she’s is a ghost. It is this fact that makes the presence of the character a symbol in terms of her effect on the narrative and the other characters so profound. For a ghost, Beloved exerts a ton and control over most of the characters, affecting nearly every part of their lives, their ability to live in peace, their ability to love one another, and to move on with theirRead MoreAnalysis of Beloved, by Tony Morrison Essay1629 Words   |  7 Pages Beloved is a novel written by Tony Morrison and is based on the American Civil War. The plot of the novel is based on the effects, consequences and the results of the Civil War. The author uses characters that would effectively bring out the Civil War theme in terms of social circles and occupations in the society. The novel is based on the characters regarded as slaves or have undergone capture, slavery and escaped from their masters (Haskins Haskins 13). The main character in the novel, SetheRead MoreCritical Reviews On Beloved By Toni Morrison1131 Words   |  5 PagesBeloved Critical Reviews The past comes back to haunt accurately in Beloved. Written by Toni Morrison, a prominent African-American author and Noble Prize winner for literature, the novel Beloved focuses on Sethe, a former slave who killed her daughter, Beloved, before the story begins. Beloved returns symbolically in the psychological issues of each character and literally in human form. The novel is inspired by the true story of Margaret Garner, a slave in the 1850s, who committed infanticide