Migration Rate of Colo16 Cells Exposed to UV Light

Migration Rate of Colo16 Cells Exposed to UV Light Introduction: Cells exposed to ultraviolet (UV) rays of light often supress signalling pathways associated with inflammation as well as the immune system and as a result of activating the p38 mitogen activated protein kinase (MAPK) pathway. Radiation from UV light is from the electromagnetic spectrum and can be divided into two primary wavelengths; UVA 320 – 400 nm and UVB 290 – 320 nm. UVA rays are more dominant than UVB, however both UVA rays reaching the subcutaneous layer of skin and UVB rays reaching the dermis are known to cause the development of skin cancers via damaging the DNA of skin cells (1, 2, 3). The effect of UV light on cells can be measured by determining the time the cells take to migrate back to their normal positions after the creation of an artificial wound. An inexpensive and easy assay to perform to make such measurements is the scratch assay. The scratch assay uses a pipette tip to create an artificial gap between the cells under aseptic conditions and uses high resolution imaging devices connected to an inverted microscope to take pictures of the cells migrating at specific time intervals. The images can then be used to calculate the distances travelled under the different UV exposures and therefore a comment on how the cells were affected under the different conditions can be made (1, 2, 3, 4). In this experiment, Human squamous cell carcinoma (SSC) cells that are known to have a dysfunctional p53 proteins were taken from the epithelial Colo16 cell line and used to measure the migration distances of the cells under different UV exposures. Then the matrix metalloproteinase (MMP) inhibitor GM6001 was added to the Colo16 cells in order to determine if it had an effect on the cell migration distances under the same UV exposures (1, 3, 5, 6). GM6001 is a MMP inhibitor that inhibits the enzyme collagenase which destroys the peptide bonds of the protein collagen found in the extracellular matrix. Collagen is an important component of the connective tissue and helps the cells adhere to their surrounding surfaces. GM6001 has been shown to block the phosphorylation of the epidermal growth factor receptors (EGFR) and inhibit cell migration responses (3, 5). Aims: To determine the migration rate of Colo16 cells exposed to UV light at intervals of 12 hours over 48 hours after the creation of an artificial wound (gap). To determine the effect of the MMP inhibitor GM6001 on the migration rate of Colo16 cells exposed to UV light at intervals of 24 hours over 72 hours after the creation of an artificial wound (gap). Hypothesis: If cells from the Colo16 cell line are treated with the MMP inhibitor GM6001 then it is expected that the mean gap distance will increase when compared with untreated Colo16 cells under the same exposure to UV light due to the inhibition of collagenases that breakdown collagen and the blocking of EGFR phosphorylation. Materials and methods: As per the BIOL2299 2014 Prac 4 lab notes. Results: Raw class data for the untreated and treated Colo16 cell gap distance (mm) is shown in Table 1 and Table 2 respectively. From the class data our group was Group A shown in Table 3 and Table 4, and the mean class data along with its standard deviation values is shown in Table 5 to Table 8. Table 1. Class Untreated Colo16 Cell Gap Distance (mm). Table 2. Class Colo16 GM6001 Treated Cell Gap Distance (mm). Table 3. Group A Colo16 Untreated Cell Gap Distance (mm). Table 4. Group A GM6001 Treated Colo16 Cell Gap Distance (mm). Table 5. Mean Class Colo16 Untreated Cell Gap Distance (mm). Table 6. Class Standard Deviation (SD) of Colo16 Untreated Cell Gap Distance (mm). Table 7. Mean Class GM6001 Treated Colo16 Cell Gap Distance (mm). Table 8. Class Standard Deviation (SD) of GM6001 Treated Colo16 Cell Gap Distance (mm). Figure 1. Line graph showing the mean gap distance of Untreated Colo16 cells under different UV exposures along with the standard deviation values after the creation of a synthetic gap using a pipette tip at intervals of 12 hours over 48 hours post-irradiation. Figure 2. Line graph showing the mean gap distance of Colo16 cells treated with the MMP inhibitor GM6001 under different UV exposures along with the standard deviation values after the creation of a synthetic gap using a pipette tip at intervals of 24 hours over 72 hours post-irradiation. Discussion: From the line graph in Fig. 1, the Colo16 cells under UV radiation showed a slower decline in gap distance compared with the control. And the Colo16 cells exposed to UVA + UVB rays had the slowest decrease in gap distance over 48 hours after the creation of the artificial wound with a gap distance of only 0.200 mm suggesting that both UVA and UVB work with synergy together and the p38 MAPK pathway and therefore cause a delay in wound healing. From the line graph in Fig. 2, the Colo16 cells treated with the MMP inhibitor GM6001 exposed to different UV conditions had a delayed cell migration response when compared to the control GM6001 Colo16 cells that were not exposed to UV irradiation. This may be due to the inhibition of collagenases that help breakdown the collagen found in the extracellular matrix (ECM) of the Colo16 cells via the action of GM6001. GM6001 is a MMP inhibitor that inhibits the enzyme collagenase that normally is involved in the breakdown of collagen of the ECM. This inhibition may affect the cell migration of Colo16 cells during wound healing as a result of the collagen not being broken down by the collagenases in the ECM and therefore resulting in the cells being adhered to their surrounding for a longer time; increasing the time required to close the gap and complete the wound healing process. Discussion questions: What other ways are there to determine whether cells are migrating into the wound or proliferating into it? (Are there morphological characteristics of cell migration or proliferation?) Other ways of determining cell migration or proliferation include: 1 – Immunofluorescence staining: cells can be stained using fluorescence markers that use antibodies to bind to specific antigens associated with cell migration or proliferation such as the proliferating cell nuclear antigen (PCNA) (7). 2 – Laser scanning confocal microscopy: cells can be viewed under high resolution and the morphological features of migrating cells can be observed such as the formation of fibrin matrices, an increase of length, width and total spread of fibroblasts (7). 3 – Boyden chamber: which measures cell migration via the determination of a chemical gradient based on chemicals release via chemotaxis during cell migration (7). What is a Boyden chamber and why would you use it? Give 3 examples of where it can be used. A Boyden chamber is an in vitro chemotaxis assay that measures cell migration via the analysis of leukocyte chemotaxis, where cells are placed in pores at the top of a chamber and allowed to migrate through its pores containing chemotactic agents. After incubation the cells are stained and the number of cells that have migrated to the lower portion of the chamber (8). The Boyden chamber can be used to measure: 1 – Cell migration based on chemicals released to the cell’s surrounding, and determine if a specific chemical causes the cells to migrate towards or away from the stimulant (Chemotaxis) (8). 2 – Cell migration based on the gradient of specific extracellular matrix proteins, and determine if a particular protein causes the cells to migrate towards or away from the protein. This can be done via the coating of the chamber with the protein of choice (Haptotaxis) (8). 3 – Cell migration rate of tumour cells through the vascular endothelium toward specific chemokines (8). Describe 3 limitations of the scratch method. Some limitations of the scratch method include: 1 – Creation of an artificial wound of the same approximate width can be extremely difficult and any variance in the gap distance between the different cells at time 0 hr can greatly affect the significance of the results (4). 2 – The incubation of the cells after the creation of the wound (scratch) has to be long enough to help the cells migrate during the fastest time frame and any variance in this may lead to the cells not migrating equally, therefore affecting the results. Also, the temperature and environmental conditions of the incubator have to be at their ideal values for healthy cellular migration (4). 3 – Highly time consuming, in order for the readings to be statistically significant at least 100 readings of distance for each sample and each experiment must be repeated at least three times. As a result, this creates copious amounts of data that consumes a lot of time and energy to analyse (4). If you are investigating cell growth factors on cell migration, should serum be present in the media? Explain your answer. Yes, Serum containing the growth factors should be found in the media in order to study its effects and the growth factors should be soluble in the serum and added to the media before the addition of cells in order to be equally distributed in the solution (4). What are the main differences in investigating a scratch assay using non-transformed vs transformed cells? Transformed cells are transfected with a plasmid coding the gene of interest along with a plasmid marker before the conduction of a scratch assay, whereas non-transformed cells are investigated without being transfected with a specific plasmid (4). Apart from taking photos of the cells over time, propose another way you may be able to show cell migration. Cell migration can be shown via the conduction of an electric cell-substrate impedance sensing (ECIS) in vitro cell migration assay that measures cell migration via the creation of a wound by passing an electrical current through the cell and causing electroporation. Then cell migration is measured via the calculation of the cell’s impedance in ohms over a specific time as seen in Fig. 3 (9). Figure 3. Typical ECIS data involving the electric cell-substrate impedance sensing (ECIS) in vitro cell migration assay (http://www.biophysics.com/woundhealingpubs.php#ECIStheory). Which of the methods is the one that most labs would not use, explain why you think this would be so. Most labs would not use the ECIS in vitro migration assay due to its expensive cost and it’s relatively time consuming cell type dependant incubation times. Another in vitro migration assay that would not be used in labs is the microfluidics-based system due to its need for nanofabrication facilities and its reasonably very expensive cost (4). Conclusion: In conclusion, Colo16 cells under UV radiation showed a slower decline in gap distance compared with the control. And the Colo16 cells exposed to UVA + UVB rays had the slowest decrease in gap distance over 48 hours after the creation of the artificial wound with a gap distance of only 0.200 mm suggesting that both UVA and UVB work with synergy together and the p38 MAPK pathway and therefore cause a delay in wound healing. Whereas, the Colo16 cells treated with the MMP inhibitor GM6001 exposed to different UV conditions had a delayed cell migration response when compared to the control GM6001 Colo16 cells that were not exposed to UV irradiation. This may be due to the inhibition of collagenases that help breakdown the collagen found in the extracellular matrix (ECM) of the Colo16 cells via the action of GM6001. This inhibition caused by GM6001 may affect the cell migration of Colo16 cells during wound healing as a result of the collagen not being broken down by the collagenases in the ECM and therefore resulting in the cells being adhered to their surrounding for a longer time; increasing the time required to close the gap and complete the wound healing process. References: BIOL2299 Biology of Tissue Growth and Repair Manual, 2014. Reichrath J, Rass K. Ultraviolet damage, DNA repair and vitamin D in nonmelanoma skin cancer and in malignant melanoma: an update. Advances in experimental medicine and biology. 2014;810:208-33. Muthusamy V, Piva TJ. A comparative study of UV-induced cell signalling pathways in human keratinocyte-derived cell lines. Archives of dermatological research. 2013;305(9):817-33. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nature protocols. 2007;2(2):329-33. Grobelny D, Poncz L, Galardy RE. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry. 1992;31(31):7152-4. Moore GE, Merrick SB, Woods LK, Arabasz NM. A Human Squamous Cell Carcinoma Cell Line. Cancer Research. 1975;35(10):2684-8. Chen HC. Boyden chamber assay. Methods in molecular biology (Clifton, NJ). 2005;294:15-22. http://www.cellbiolabs.com/boyden-chamber-assays http://www.biophysics.com/woundhealingpubs.php#ECIStheory

The Twilight Saga 4: Breaking Dawn 19. Burning

The pain was bewildering. Exactly that – I was bewildered. I couldn't understand, couldn't make sense of what was happening. My body tried to reject the pain, and I was sucked again and again into a blackness that cut out whole seconds or maybe even minutes of the agony, making it that much harder to keep up with reality. I tried to separate them. Non-reality was black, and it didn't hurt so much. Reality was red, and it felt like I was being sawed in half, hit by a bus, punched by a prize fighter, trampled by bulls, and submerged in acid, all at the same time. Reality was feeling my body twist and flip when I couldn't possibly move because of the pain. Reality was knowing there was something so much more important than all this torture, and not being able to remember what it was. Reality had come on so fast. One moment, everything was as it should have been. Surrounded by people I loved. Smiles. Somehow, unlikely as it was, it seemed like I was about to get everything I'd been fighting for. And then one tiny, inconsequential thing had gone wrong. I'd watched as my cup tilted, dark blood spilling out and staining the perfect white, and I'd lurched toward the accident reflexively. I'd seen the other, faster hands, but my body had continued to reach, to stretch___ Inside me, something had yanked the opposite direction. Ripping. Breaking. Agony. The darkness had taken over, and then washed away to a wave of torture. I couldn't breathe – I had drowned once before, and this was different; it was too hot in my throat. Pieces of me shattering, snapping, slicing apart___ More blackness. Voices, this time, shouting, as the pain came back. ‘The placenta must have detached!† Something sharper than knives ripped through me – the words, making sense in spite of the other tortures. Detached placenta – I knew what that meant. It meant that my baby was dying inside me. â€Å"Get him out!† I screamed to Edward. Why hadn't he done it yet? â€Å"He can't breathe! Do it now!† â€Å"The morphine – â€Å" He wanted to wait, to give me painkillers, while our baby was dying?! â€Å"No! Now – ,† I choked, unable to finish. Black spots covered the light in the room as a cold point of new pain stabbed icily into my stomach. It felt wrong – I struggled automatically to protect my womb, my baby, my little Edward Jacob, but I was weak. My lungs ached, oxygen burned away. The pain faded away again, though I clung to it now. My baby, my baby, dying___ How long had passed? Seconds or minutes? The pain was gone. Numb. I couldn't feel. I still couldn't see, either, but I could hear. There was air in my lungs again, scraping in rough bubbles up and down my throat. â€Å"You stay with me now, Bella! Do you hear me? Stay! You're not leaving me. Keep your heart beating!† Jacob? Jacob, still here, still trying to save me. Of course,I wanted to tell him. Of course I would keep my heart beating. Hadn't I promised them both? I tried to feel my heart, to find it, but I was so lost inside my own body. I couldn't feel the things I should, and nothing felt in the right place. I blinked and I found my eyes. I could see the light. Not what I was looking for, but better than nothing. As my eyes struggled to adjust, Edward whispered, â€Å"Renesmee.† Renesmee? Not the pale and perfect son of my imagination? I felt a moment of shock. And then a flood of warmth. Renesmee. I willed my lips to move, willed the bubbles of air to turn into whispers on my tongue. I forced my numb hands to reach. â€Å"Let me†¦ Give her to me.† The light danced, shattering off Edward's crystal hands. The sparkles were tinged with red, with the blood that covered his skin. And more red in his hands. Something small and struggling, dripping with blood. He touched the warm body to my weak arms, almost like I was holding her. Her wet skin was hot – as hot as Jacob's. My eyes focused; suddenly everything was absolutely clear. Renesmee did not cry, but she breathed in quick, startled pants. Her eyes were open, her expression so shocked it was almost funny. The little, perfectly round head was covered in a thick layer of matted, bloody curls. Her irises were a familiar – but astonishing – chocolate brown. Under the blood, her skin looked pale, a creamy ivory. All besides her cheeks, which flamed with color. Her tiny face was so absolutely perfect that it stunned me. She was even more beautiful than her father. Unbelievable. Impossible. â€Å"Renesmee,† I whispered. â€Å"So†¦ beautiful.† The impossible face suddenly smiled – a wide, deliberate smile. Behind the shell-pink lips was a full complement of snowy milk teeth. She leaned her head down, against my chest, burrowing against the warmth. Her skin was warm and silky, but it didn't give the way mine did. Then there was pain again – just one warm slash of it. I gasped. And she was gone. My angel-faced baby was nowhere. I couldn't see or feel her. No!I wanted to shout. Give her back to me! But the weakness was too much. My arms felt like empty rubber hoses for a moment, and then they felt like nothing at all. I couldn't feel them. I couldn't feel me. The blackness rushed over my eyes more solidly than before. Like a thick blindfold, firm and fast. Covering not just my eyes but also my self with a crushing weight. It was exhausting to push against it. I knew it would be so much easier to give in. To let the blackness push me down, down, down to a place where there was no pain and no weariness and no worry and no fear. If it had only been for myself, I wouldn't have been able to struggle very long. I was only human, with no more than human strength. I'd been trying to keep up with the supernatural for too long, like Jacob had said. But this wasn't just about me. If I did the easy thing now, let the black nothingness erase me, I would hurt them. Edward. Edward. My life and his were twisted into a single strand. Cut one, and you cut both. If he were gone, I would not be able to live through that. If I were gone, he wouldn't live through it, either. And a world without Edward seemed completely pointless. Edward had to exist. Jacob – who'd said goodbye to me over and over but kept coming back when I needed him. Jacob, who I'd wounded so many times it was criminal. Would I hurt him again, the worst way yet? He'd stayed for me, despite everything. Now all he asked was that I stay for him. But it was so dark here that I couldn't see either of their faces. Nothing seemed real. That made it hard not to give up. I kept pushing against the black, though, almost a reflex. I wasn't trying to lift it. I was just resisting. Not allowing it to crush me completely. I wasn't Atlas, and the black felt as heavy as a planet; I couldn't shoulder it. All I could do was not be entirely obliterated. It was sort of the pattern to my life – I'd never been strong enough to deal with the things outside my control, to attack the enemies or outrun them. To avoid the pain. Always human and weak, the only thing I'd ever been able to do was keep going. Endure. Survive. It had been enough up to this point. It would have to be enough today. I would endure this until help came. I knew Edward would be doing everything he could. He would not give up. Neither would I. I held the blackness of nonexistence at bay by inches. It wasn't enough, though – that determination. As the time ground on and on and the darkness gained by tiny eighths and sixteenths of my inches, I needed something more to draw strength from. I couldn't pull even Edward's face into view. Not Jacob's, not Alice's or Rosalie's or Charlie's or Renee's or Carlisle's or Esme's†¦ Nothing. It terrified me, and I wondered if it was too late. I felt myself slipping – there was nothing to hold on to. No!I had to survive this. Edward was depending on me. Jacob. Charlie Alice Rosalie Carlisle Renee Esme†¦ Renesmee. And then, though I still couldn't see anything, suddenly I could feel something. Like phantom limbs, I imagined I could feel my arms again. And in them, something small and hard and very, very warm. My baby. My little nudger. I had done it. Against the odds, I had been strong enough to survive Renesmee, to hold on to her until she was strong enough to live without me. That spot of heat in my phantom arms felt so real. I clutched it closer. It was exactly where my heart should be. Holding tight the warm memory of my daughter, I knew that I would be able to fight the darkness as long as I needed to. The warmth beside my heart got more and more real, warmer and warmer. Hotter. The heat was so real it was hard to believe that I was imagining it. Hotter. Uncomfortable now. Too hot. Much, much too hot. Like grabbing the wrong end of a curling iron – my automatic response was to drop the scorching thing in my arms. But there was nothing in my arms. My arms were not curled to my chest. My arms were dead things lying somewhere at my side. The heat was inside me. The burning grew – rose and peaked and rose again until it surpassed anything I'd ever felt. I felt the pulse behind the fire raging now in my chest and realized that I'd found my heart again, just in time to wish I never had. To wish that I'd embraced the blackness while I'd still had the chance. I wanted to raise my arms and claw my chest open and rip the heart from it – anything to get rid of this torture. But I couldn't feel my arms, couldn't move one vanished finger. James, snapping my leg under his foot. That was nothing. That was a soft place to rest on a feather bed. I'd take that now, a hundred times. A hundred snaps. I'd take it and be grateful. The baby, kicking my ribs apart, breaking her way through me piece by piece. That was nothing. That was floating in a pool of cool water. I'd take it a thousand times. Take it and be grateful. The fire blazed hotter and I wanted to scream. To beg for someone to kill me now, before I lived one more second in this pain. But I couldn't move my lips. The weight was still there, pressing on me. I realized it wasn't the darkness holding me down; it was my body. So heavy. Burying me in the flames that were chewing their way out from my heart now, spreading with impossible pain through my shoulders and stomach, scalding their way up my throat, licking at my face. Why couldn't I move? Why couldn't I scream? This wasn't part of the stories. My mind was unbearably clear – sharpened by the fierce pain – and I saw the answer almost as soon as I could form the questions. The morphine. It seemed like a million deaths ago that we'd discussed it – Edward, Carlisle, and I. Edward and Carlisle had hoped that enough painkillers would help fight the pain of the venom. Carlisle had tried with Emmett, but the venom had burned ahead of the medicine, sealing his veins. There hadn't been time for it to spread. I'd kept my face smooth and nodded and thanked my rarely lucky stars that Edward could not read my mind. Because I'd had morphine and venom together in my system before, and I knew the truth. I knew the numbness of the medicine was completely irrelevant while the venom seared through my veins. But there'd been no way I was going to mention that fact. Nothing that would make him more unwilling to change me. I hadn't guessed that the morphine would have this effect – that it would pin me down and gag me. Hold me paralyzed while I burned. I knew all the stories. I knew that Carlisle had kept quiet enough to avoid discovery while he burned. I knew that, according to Rosalie, it did no good to scream. And I'd hoped that maybe I could be like Carlisle. That I would believe Rosalie's words and keep my mouth shut. Because I knew that every scream that escaped my lips would torment Edward. Now it seemed like a hideous joke that i was getting my wish fulfilled. If I couldn't scream, how could I tell them to kill me? All I wanted was to die. To never have been born. The whole of my existence did not outweigh this pain. Wasn't worth living through it for one more heartbeat. Let me die, let me die, let me die. And, for a never-ending space, that was all there was. Just the fiery torture, and my soundless shrieks, pleading for death to come. Nothing else, not even time. So that made it infinite, with no beginning and no end. One infinite moment of pain. The only change came when suddenly, impossibly, my pain was doubled. The lower half of my body, deadened since before the morphine, was suddenly on fire, too. Some broken connection had been healed – knitted together by the scorching fingers of the flame. The endless burn raqed on. It could have been seconds or days, weeks or years, but, eventually, time came to mean something again. Three things happened together, grew from each other so that I didn't know which came first: time restarted, the morphine's weight faded, and I got stronger. I could feel the control of my body come back to me in increments, and those increments were my first markers of the time passing. I knew it when I was able to twitch my toes and twist my fingers into fists. I knew it, but I did not act on it. Though the fire did not decrease one tiny degree – in fact, I began to develop a new capacity for experiencing it, a new sensitivity to appreciate, separately, each blistering tongue of flame that licked through my veins – I discovered that I could think around it. I could remember why I shouldn't scream. I could remember the reason why I'd committed to enduring this unendurable agony. I could remember that, though it felt impossible now, there was something that might be worth the torture. This happened just in time for me to hold on when the weights left my body. To anyone watching me, there would be no change. But for me, as I struggled to keep the screams and thrashing locked up inside my body, where they couldn't hurt anyone else, it felt like I'd gone from being tied to the stake as I burned, to gripping that stake to hold myself in the fire. I had just enough strength to lie there unmoving while I was charred alive. My hearing got clearer and clearer, and I could count the frantic, pounding beats of my heart to mark the time. I could count the shallow breaths that gasped through my teeth. I could count the low, even breaths that came from somewhere close beside me. These moved slowest, so I concentrated on them. They meant the most time passing. More even than a clock's pendulum, those breaths pulled me through the burning seconds toward the end. I continued to get stronger, my thoughts clearer. When new noises came, I could listen. There were light footsteps, the whisper of air stirred by an opening door. The footsteps gotcloser, and I felt pressure against the inside of my wrist. I couldn't feel the coolness of the fingers. The fire blistered away every memory of cool. â€Å"Still no change?† â€Å"None.† The lightest pressure, breath against my scorched skin. â€Å"There's no scent of the morphine left.† â€Å"I know.† â€Å"Bella? Can you hear me?† I knew, beyond all doubt, that if I unlocked my teeth I would lose it – I would shriek and screech and writhe and thrash. If I opened my eyes, if I so much as twitched a finger – any change at all would be the end of my control. â€Å"Bella? Bella, love? Can you open your eyes? Can you squeeze my hand?† Pressure on my fingers. It was harder not to answer this voice, but I stayed paralyzed. I knew that the pain in his voice now was nothing compared to what it could be. Right now he only feared that I was suffering. â€Å"Maybe†¦ Carlisle, maybe I was too late.† His voice was muffled; it broke on the word late. My resolve wavered for a second. â€Å"Listen to her heart, Edward. It's stronger than even Emmett's was. I've never heard anything so vital. Shell be perfect.† Yes, I was right to keep quiet. Carlisle would reassure him. He didn't need to suffer with me. â€Å"And her – her spine?† â€Å"Her injuries weren't so much worse than Esme's. The venom will heal her as it did Esme.† â€Å"But she's so still. I must have done something wrong.† â€Å"Or something right, Edward. Son, you did everything I could have and more. I'm not sure I would have had the persistence, the faith it took to save her. Stop berating yourself. Bella is going to be fine.† A broken whisper. â€Å"She must be in agony.† â€Å"We don't know that. She had so much morphine in her system. We don't know the effect that will have on her experience.† Faint pressure inside the crease of my elbow. Another whisper. â€Å"Bella, I love you. Bella, I'm sorry.† I wanted so much to answer him, but I wouldn't make his pain worse. Not while I had the strength to hold myself still. Through all this, the racking fire went right on burning me. But there was so much space in my head now. Room to ponder their conversation, room to remember what had happened, room to look ahead to the future, with still endless room left over to suffer in. Also room to worry. Where was my baby? Why wasn't she here? Why weren't they talking about her? â€Å"No, I'm staying right here,† Edward whispered, answering an unspoken thought. â€Å"They'll sort it out.† â€Å"An interesting situation,† Carlisle responded. â€Å"And I'd thought I'd seen just about everything.† â€Å"I'll deal with it later. We'll deal with it.† Something pressed softly to my blistering palm. â€Å"I'm sure, between the five of us, we can keep it from turning into bloodshed.† Edward sighed. â€Å"I don't know which side to take. I'd love to flog them both. Well, later.† â€Å"I wonder what Bella will think – whose side she'll take,† Carlisle mused. One low, strained chuckle. â€Å"I'm sure she'll surprise me. She always does.† Carlisle's footsteps faded away again, and I was frustrated that there was no further explanation. Were they talking so mysteriously just to annoy me? I went back to counting Edward's breaths to mark the time. Ten thousand, nine hundred forty-three breaths later, a different set of footsteps whispered into the room. Lighter. More†¦ rhythmic. Strange that I could distinguish the minute differences between footsteps that I'd never been able to hear at all before today. â€Å"How much longer?† Edward asked. â€Å"It won't be long now,† Alice told him. â€Å"See how clear she's becoming? I can see her so much better.† She sighed. â€Å"Still feeling a little bitter?† â€Å"Yes, thanks so much for bringing it up,† she grumbled. â€Å"You would be mortified, too, if you realized that you were handcuffed by your own nature. I see vampires best, because I am one; I see humans okay, because I was one. But I can't see these odd half-breeds at all because they're nothing I've experienced. Bah!† â€Å"Focus, Alice.† â€Å"Right. Bella's almost too easy to see now.† There was a long moment of silence, and then Edward sighed. It was a new sound, happier. â€Å"She's really going to be fine,† he breathed. â€Å"Of course she is.† â€Å"You weren't so sanguine two days ago.† â€Å"I couldn't see right two days ago. But now that she's free of all the blind spots, it's a piece of cake.† â€Å"Could you concentrate for me? On the clock – give me an estimate.† Alice sighed. â€Å"So impatient. Fine. Give me a sec – â€Å" Quiet breathing. â€Å"Thank you, Alice.† His voice was brighter. How long?Couldn't they at least say it aloud for me? Was that too much to ask? How many more seconds would I burn? Ten thousand? Twenty? Another day – eighty-six thousand, four hundred? More than that? â€Å"She's going to be dazzling.† Edward growled quietly. â€Å"She always has been.† Alice snorted. â€Å"You know what I mean. Look at her.† Edward didn't answer, but Alice's words gave me hope that maybe I didn't resemble the charcoal briquette I felt like. It seemed as if I must be just a pile of charred bones by now. Every cell in my body had been razed to ash. I heard Alice breeze out of the room. I heard the swish of the fabric she moved, rubbing against itself. I heard the quiet buzz of the light hanging from the ceiling. I heard the faint wind brushing against the outside of the house. I could hear everything. Downstairs, someone was watching a ball game. The Mariners were winning by two runs. â€Å"It's my turn† I heard Rosalie snap at someone, and there was a low snarl in response. â€Å"Hey, now,† Emmett cautioned. Someone hissed. I listened for more, but there was nothing but the game. Baseball was not interesting enough to distract me from the pain, so I listened to Edward's breathing again, counting the seconds. Twenty-one thousand, nine hundred seventeen and a half seconds later, the pain changed. On the good-news side of things, it started to fade from my fingertips and toes. Fading slowly, but at least it was doing something new. This had to be it. The pain was on its way out†¦ And then the bad news. The fire in my throat wasn't the same as before. I wasn't only on fire, but I was now parched, too. Dry as bone. So thirsty. Burning fire, and burning thirst†¦ Also bad news: The fire inside my heart got hotter. How was that possible? My heartbeat, already too fast, picked up – the fire drove its rhythm to a new frantic pace. â€Å"Carlisle,† Edward called. His voice was low but clear. I knew that Carlisle would hear it, if he were in or near the house. The fire retreated from my palms, leaving them blissfully pain-free and cool. But it retreated to my heart, which blazed hot as the sun and beat at a furious new speed. Carlisle entered the room, Alice at his side. Their footsteps were so distinct, I could even tell that Carlisle was on the right, and a foot ahead of Alice. â€Å"Listen,† Edward told them. The loudest sound in the room was my frenzied heart, pounding to the rhythm of the fire. â€Å"Ah,† Carlisle said. â€Å"It's almost over.† My relief at his words was overshadowed by the excruciating pain in my heart. My wrists were free, though, and my ankles. The fire was totally extinguished there. â€Å"Soon,† Alice agreed eagerly. â€Å"I'll get the others. Should I have Rosalie†¦ ?† â€Å"Yes – keep the baby away.† What? No. No! What did he mean, keep my baby away? What was he thinking? My fingers twitched – the irritation breaking through my perfect facade. The room went silent besides the jack-hammering of my heart as they all stopped breathing for a second in response. A hand squeezed my wayward fingers. â€Å"Bella? Bella, love?† Could I answer him without screaming? I considered that for a moment, and then the fire ripped hotter still through my chest, draining in from my elbows and knees. Better not to chance it. ‘Til bring them right up,† Alice said, an urgent edge to her tone, and I heard the swish of wind as she darted away. And then – oh! My heart took off, beating like helicopter blades, the sound almost a single sustained note; it felt like it would grind through my ribs. The fire flared up in the center of my chest, sucking the last remnants of the flames from the rest of my body to fuel the most scorching blaze yet. The pain was enough to stun me, to break through my iron grip on the stake. My back arched, bowed as if the fire was dragging me upward by my heart. I allowed no other piece of my body to break rank as my torso slumped back to the table. It became a battle inside me – my sprinting heart racing against the attacking fire. Both were losing. The fire was doomed, having consumed everything that was combustible; my heart galloped toward its last beat. The fire constricted, concentrating inside that one remaining human organ with a final, unbearable surge. The surge was answered by a deep, hollow-sounding thud. My heart stuttered twice, and then thudded quietly again just once more. There was no sound. No breathing. Not even mine. For a moment, the absence of pain was all I could comprehend. And then I opened my eyes and gazed above me in wonder.

Foreign Cultures Essay

The problem of the students who have to learn foreign cultures or not had concerned a number of people. Some individuals advocate that the students are just need to know their own country culture.However, their are a large group of opponents have an opposite perspective. As far as I concerned,The students in college are necessary to know the foreign cultures. Most of the student will go to other countries in the future for their jobs or visit their relatives or friends. So they have to know some foreign cultures for the convenience and live without troubles. If you are a tourist on that country, and you know the foreign cultures, you can enjoy your trip. If you are working on that country, you will put more focus on your work and getting more friends. But if you don’t know the cultures of the country which you live, you will have a lot of troubles. You can not pay any attention on you major work, you will not happy because some awkward things will happen. In addition, if you familiar with the cultures of foreign countries, wherever you are you will be the person who is most attractive. A lot of people might think you are really intelligent and know a lot of interesting things and knowledge they don’t know. So you might getting a lot of friends who interest you. If you are in the foreign country, you will have live easier and smoother. to get a good job. If you want to be a teacher in the school or a tour guide, you will need this culture a lot. In the conclusion, learning foreign cultures are really helpful, they are a sort of ability that can avoid some embarrassments, and can help you getting more opportunities to make new friends and good jobs in the future.

The Bretton Woods System Essay - 2073 Words

The period referred to as the Golden Age was between 1950 and 1973. There was rapid growth in the US economy during this time, the economies of Europe and Japan also had very high rates of growth. The world’s advanced countries spurred a commodities boom which benefited natural resource-rich countries such as the oil rich ones in the Middle East, as well as the industrial nations of East Asia i.e. Singapore, Thailand, China etc. The Bretton Woods Conference, which is officially known as the United Nations Monetary and Financial Conference took place from July 1 to July 22 in 1944, in Bretton Woods, New Hampshire in the United States. It was a gathering of delegates from 44 different countries that met in order to try and formulate a†¦show more content†¦The countries that joined the IMF between 1945 and 1971 had initially agreed to keep their exchange rates at rates that could be adjusted only in instances to correct a fundamental disequilibrium in the balance of payments, and only with the IMFs agreement. This system prevailed until 1971, when the U.S. government suspended the convertibility of the dollar, as well as the dollar reserves held by other governments, into gold. The International Bank for Reconstruction and Development (World Bank) is probably the world’s single most important source of financial aid for developing nations. It was one of the institutions founded in 1944 at the Bretton Woods Conference to help Europe recover from World War II. The International Bank for Reconstruction and Development (IBRD) is only one of five institutions that make up the organization known as the World Bank Group. IBRD is the part of the World Bank that works with creditworthy poorer as well as middle-income countries by promoting sustainable, equitable and job-creating growth, reduce poverty and address issues of regional and global importance. It ccommenced operations on June 25, 1946, and then approved its first loan on May 9, 1947. This third major institution was simply the set of rules for the monetaryShow MoreRelated The Bretton Woods System Essay1766 Words   |  8 Pages The theme of this essay outlines two things. One, the key elements of Bretton woods system and second, the characterisation of Bretton woods system by Ruggie as ‘embedded liberalism’, and how far he succeeds in it. The Bretton woods system is widely referred to the international monetary regime, which prevailed from the end of the World War 2 until the early 1970s. After the end of the World War 2, the need of international monetary framework to boost trade and economic; growth andRead MoreHistory Of Bretton Woods System And Jamaica1436 Words   |  6 PagesHISTORY OF BRETTON WOODS SYSTEM AND JAMAICA BRETTON WOODS SYSTEM The Bretton Woods System was formed as a result of the collapse of the Golden Standard and The Great Depression. These closely related events prompted the need to establish an international monetary system, whose main aim was to revive the economies of the Post World War as well as fostering international economic relations that would end inter-war conflicts. Furthermore this historic event took place in July 1944 in New Hampshire wherebyRead MoreWas the Bretton Woods System successful?2025 Words   |  9 PagesWhat was the Bretton Woods System? The end of the World War II marked the beginning of a new era for the world economy. The Bretton Woods System refers to an agreement made at an international conference between 44 nations in 1944 at Bretton Woods, New Hampshire, United States of America (hereby U.S.) on the 22nd of July 1944. It was aimed at maintaining stability in the monetary system in the post World War II period. â€Å"In an effort to free international trade and fund postwar reconstruction theRead MoreMechanism Behind The Foundation Of Bretton Woods System1693 Words   |  7 Pagesmechanism behind the foundation of Bretton Woods system and how this system worked from 1944 to 1973. Analysis includes the mechanism of the fixed exchange rate regime based on gold and the US dollar and also includes the factors and reasons that led the system to collapse. Background of Foundation of the Bretton Woods System As far back as the World War II, the United States has been attempting to replace the Great Britain, establishing a world currency system centred in US dollar. After the warRead MoreThe Bretton Woods System On The World s Natural Riches872 Words   |  4 Pagesmultinational corporations. The Bretton Woods system was aimed to boost the production and consumption of commodities. The World Bank and the IMF, led by the US, financed corporations and exploitation of resources. The Bretton Woods system was supposed to make use of the world’s natural riches. The fall of Communism in the world gave more power to the corporations and led to the creation of the free market system. The US government tried to help underdeveloped nations to export wood, livestock and other commoditiesRead MoreHow do you explain the rise and fall of the Bretton Woods system? and the creation of an optimal currency area?2535 Words   |  11 PagesHow do you explain the rise and fall of the Bretton Woods sys tem? How far the emergence of the Euro can be seen against the background of the need for exchange rate stability and the creation of an optimal currency area? 1) The rise and fall of the Bretton Woods system: The origins of the Bretton Woods system are to be found in the convergence of several key conditions: the shared experiences of the Great Depression, the concentration of power in a small number of states, and the presence of aRead MoreWhy did the Bretton Woods system came to an end?1504 Words   |  7 Pagesï » ¿Why did the Bretton Woods system come to an end? The Bretton Woods system was created in July 1944 by the United States and its allies in order to formulate a plan for European recovery and create a new postwar international monetary and financial system that was supposed to encourage grow and development (Balaam, Dillam 2011). The Bretton Woods financial and monetary structure was supposed to ensure exchange rate stability and encourage its member countries to eliminated exchange rate restrictionsRead MoreThe Stystem Developed in the The Bretton Woods Conference862 Words   |  3 PagesIntroduction: The Bretton Woods Conference in New Hampshire in 1944 was a unique event in which representatives of two nations influenced the planning of the world monetary system (Scammell, 1982). The Article of Agreement was a negotiation between Britain and The United States, which was accepted and signed by 44 other nations (Ikenberry, 1933). The conference saw the establishment of the two most significant financial institutions, The International Bank for Reconstruction and Development [WorldRead MoreThe Major Cornerstone For The U.s.s Economic Prosperity And International Hegemony1217 Words   |  5 PagesAs the major cornerstone for the U.S.’S economic prosperity and international hegemony, following the change of the Bretton Woods System to the Petrodollar system in 1971; I ve found the largest independent factor to influence American international interests has been the implementation and preserva The Petrodollar System From the Gold Standard To Black Gold By: Gibson J.T. Intro Behind the global power of the U.S. dollar, hides the most significant form of currency you ve probablyRead MoreRisk Is Everyone s Best Friend And Worst Nightmare1689 Words   |  7 Pagesgains. This causes market fluctuations, instability, and the like. We will examine these principles in the context of the pre- and post-1971 financial system with regards to the risk, innovation, and implications of the leaving the Bretton Woods system. The idea behind the Bretton Woods system was to standardize and stabilize the world financial system during and after WWII. This was done through a variety of means, the most important being regulating international exchange rates, tieing the US Dollar