GREEN FLUORESCENT PROTEIN (GFP) Essays

GREEN FLUORESCENT PROTEIN (GFP) Essays GREEN FLUORESCENT PROTEIN (GFP) Essay GREEN FLUORESCENT PROTEIN (GFP) Essay GREEN FLUORESCENT PROTEIN ( GFP ) MUTANTS WITH ALTERED FLUORESCENCE INTENSITY AND EMMISSION SPECTRA Introduction: Now-a-days GFP is making revolution in the field of scientific discipline by its applications and properties.GFP is a stable protein extracted from the exposure variety meats of the jellyfish Aequoria Victoria by Shimomura et Al in 1962. In 1992 the cloning of GFP has done. It is found in a assortment of cnidarians ( both Hydrozoa and Anthozoa ) and it emits light by using energy from the Ca2+ activated photoprotein aequorin [ 1 ] . Energy transportation and the emanation spectra of GFP can be affected by dimerization. Structure of GFP is cylindrical ?-can construction and has a chromophore located centrally. The chromophore is responsible for the fluorescence and the formation is independent of species but chiefly depends on O. GFP is a little protein and has been made up of 238 aminic acids. Deletion of any seven amino acids either from C-terminus or N-terminus may ensue in the loss of fluorescence. Amino acerb replacing is responsible for the alteration in colors of GFP. It has a molecular weight of 27 KDa and has an soaking up scope at 488 nanometer and an emanation scope at 509 nanometer. It can carry through high temperatures ( 65 ?c ) and basic PH scope of 6-12 [ 2 ] . Increase in PH consequences in the lessening of fluorescence. Increase in the fluorescence and exposure stableness can be achieved by individual point mutant at S65T. Fluorophore of the GFP is generated by utilizing auto-catalytic procedure of uninterrupted mechanisms. Visible excitement is one of the optical belongingss of GFP. Its derived functions are produced from the mutagenesis experiments like random and directed mutagenesis [ 3 ] . GFP is majorly used as a newsman in showing cistrons. Protein and chromophore folding besides constitutes as a major advantage of GFP. It can besides be used in protein merger by using recombinant DNA engineering. : Aim of this research is to analyse belongingss of GFP by cloning, mutants, look of proteins and purification. Aims of this research are to sub-clone GFP into a vector and mutants are carried out by assorted mutagenesis experiments followed by look of proteins and purification. Finally after purification belongingss are analyzed. Materials and methods: Initially DNA is isolated and GFPuv is sub-cloned into the pET28c vector from pET23 plasmid by speectrophotometric analysis. 5 µg of pET23GFPuv DNA is digested by utilizing NdeI and HindIII limitation enzymes. And the digests are analysed by utilizing Agarose gel cataphoresis. GFP fragment is extracted and purified utilizing QIA speedy gel extraction kit from QIAGEN and the cured DNA is estimated. Recombinant protein is expressed in E.coli by ligation and transmutation. To corroborate the presence of GFP in the pET28c plasmid, settlement PCR is used. Further mutagenesis experiments are carried out by planing oligonucleotide primers which will change the spectral belongingss of the protein. Complementary primers incorporating same mutants are generated. Mutagenic primers are prepared with a liquescent temperature of ? 78?C, length between 25 and 45 bases and primers longer than 45 bases are by and large used. Introduction and designation of mutants within GFPuv cistron: Mutants are created in the GFPuv insert by site-directed mutagenesis Site-directed mutagenesis: 5 µl 10 ten PCR buffer 5 µl 20 millimeter dNTP mixes 15 ng GFPuv-pET28c templet Deoxyribonucleic acid 125ng oligonucleotide primer F+ 125ng oligonucleotide primer R+ 2 µl 25mM MgSo4 32 µl unfertile H2O 1 µl KOD hot start polymerase ( 1U/ µl ) * All the above are added to 0.2ml PCR tubings and incubated in a PCR machine for 24 rhythms: 94?C 30s 94?C 30s 55?C 1min 68?C 4min 20s 68?C 10 min * Reaction is so kept on ice for 2 min and 1 µl ( 1U ) of Dpn1 is added and incubated for 60 min at 37?C Alliance of amino acid sequences is carried out utilizing: hypertext transfer protocol: //www.ebi.ac.uk/Tools/clustalw2/index.html Merchandise of site-directed mutagenesis ( pET28c DNA ) is transformed into XL-1 supercompetent cells. Transformed settlements are extracted utilizing QIAprep Mini prep kit Qiagen [ 5 ] . Concentration and pureness can be checked by utilizing Agarose gel cataphoresis. For this 5 µl of plasmid readying and 10U HindIII are digested at 37?C for 1h. Sequencing is so carried out by utilizing 10 µl of Deoxyribonucleic acid at a concentration of 50ng/ µl. E.coli BL21 ( DE3 ) cells are prepared and are transformed into the pET28cGFPuv plasmid for look Auto-induction method: Wild type protein ( GFPuv ) and the mutant protein are expressed in the look vector [ BL21 ( DE3 ) ] utilizing auto-induction method. For this transformed settlements are inoculated into 3ml of LB-1D + antibiotic media and incubated at 37?C at 300 RPM for 6 hour and O.D is taken. Inoculum is taken into the flask incorporating SB-5052 auto-induction medium along with antibiotic and incubated at 28?C at 300 RPM for 20 hour. Cultures are so cooled for 1 hour. Entire induced sample is prepared by taking 100 µl of chilling civilization and 900 µl of SB-5052 media. Cells are so pelletized by centrifugating it with both entire induced and non-induced samples and are resuspended in 100 µl of SDS-PAGE ( Na dodecyl sulfate ( SDS ) polyacrylamide gel cataphoresis ( PAGE ) ) sample buffer. 12 % of polyacrylamide gel is prepared and the Soluble and indissoluble samples are prepared by cell fractional process utilizing BUGBUSTER. For this 1 µl of DNAase1 is used along with reagents. Cell suspension is so centrifuged at 13000rpm for 20mins. Supernatant is so used as soluble sample and indissoluble is prepared by resuspending the pellet in 2ml binding buffer. SDS-PAGE buffer and binding buffer are added to the soluble and indissoluble fractions. At 95?C all samples are heated for 5 min. Gel is so loaded as: Molecular weight standard-5 µl Uninduced sample 5 µl Induced entire sample 5 µl Soluble sample 5 µl Gel has to run for 1 hour. And is transfered to a box of Coomassie blue discoloration. Western blotting: GFP protein presence can be verified utilizing western blotting technique. Protein samples are foremost seperated by SDS-PAGE and are transferred to the nitrocellulose membrane. GFP edge to nitrocellulose membrane is so visualised by incubating the smudge with His-probe which is linked to a HRP ( Equus caballus radish peroxidase ) enzyme ( HisprobeTM-HRP solution is diluted to 1:5000 ( 1 µl in 5ml ) ) . His-tag of GFP protein is bound to examine. Smudges are kept in TBST and investigations and therefore investigations are visualised by chemiluminescence and these are photographed by chemiluminescent reader. Ni-NTA chromatography: His labeled GFP can be purified by Ni-NTA ( nickel nitrilo triacetic acid ) chromatography method. In this, sample of soluble protein is loaded on column packed agarose rosin and the non-specific protein binding is removed by rinsing rosin with buffer and is eluted by high concentrated iminazole of elution buffer. After elution the purification of protein is done by SDS-PAGE and Coomassie staining. The concentration of the protein is measured by Bradford check. Fluorimetry and mass spectroscopy: Properties of GFPuv protein are analysed by Fluorimetry and mass spectrometry. Fluorimetry: In this wavelength and strength of a molecule at specific wavelength are measured utilizing fluorimeters. Perkin Elmer LS50B is the fluorimeter used to mensurate GFP. Quartz cuvettes are placed in a chamber to mensurate the concentration and strength. The parametric quantities set to mensurate GFP are: Excitement 440nm Emission 460-550nm Slit widths 4 and 4 Accretion 5 20 µg/ml of protein concentration is used. The emanation and excitor wavelengths are set at 509nm and 395nm. Mass spectroscopy: GFPuv belongingss and molecular mass can be analysed by mass spectrometry. The type of mass spectrometry used here is electron spray ionisation ( ESI ) . ESI is a type of atmospheric force per unit area ionization technique ( API ) which is used for biochemical analysis. JEOL HX110/HX110A equipped with electron ion beginning tandem mass spectrometers are used to analyze structural belongingss [ 7 ] . 1-10 pmol/ µl of protein concentration is used. Solvents used are: MeOH MeCN TFA During ionization sample is dissolved in a dissolver and is pumped through a steel capillary at a rate of 1 µl/min and electromotive force of 3 or 4KV is applied [ 8 ] . Ion current is amplified by the sensor and the information system will enter signals in the signifier of mass spectrum. Consequence: Site-directed mutagenesis: Primers used for site directed mutagenesis ( Mutant ) Forward primer: 5-CACTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCC-3 Rearward primer: 5-GGAAAAGCATTGAACACCCCAAGAGAAAGTAGTGACAAGTG-3 Alliance of the amino acerb sequence of the mutation with the GFPuv amino acid sequence GFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 mGFPuv MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTL 60 ************************************************************ GFPuv VTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120 mGFPuv VTTFSWGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFEGDTLV 120 ***** : ****************************************************** Y66W GFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180 mGFPuv NRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGSVQLAD 180 ************************************************************ GFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238 mGFPuv HYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK- 238 ********************************************************** Amino acerb permutation: Y66W Belongs to Class 5, indole in chromophore ( bluish green fluorescent proteins ) [ 6 ] eCFP CATATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT 60 GFP -ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGAT 57 ********************************************************* eCFP GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC 120 GFP GGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATAC 117 ************************************************************ eCFP GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACA 180 GFP GGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACA 177 ************************************************************ eCFP CTTGTCACTACTTTCTCTTGGGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAA 240 GFP CTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCATATGAAA 237 ******************* ******************************** ****** Mutant eCFP CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCT 300 GFP CGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCT 297 ************************************************************ eCFP TTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTT 360 GFP TTCAAAGATGACGGGAACTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTT 357 ************************************************************ eCFP GTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACAC 420 GFP GTTAATCGTATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTCGGACAC 417 ************************************************************ eCFP AAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT 480 GFP AAACTCGAGTACAACTATAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAAT 477 ************************************************************ eCFP GGAATCAAAGCT 492 GFP GGAATCAAAGCTAACTTCAAAATTCGCCACAACATTGAAGATGGATCCGTTCAACTAGCA 537 ************ eCFP GFP GACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCAT 597 eCFP GFP TACCTGTCGACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTC 657 eCFP GFP CTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAATAA 717 SDS-PAGE: Coomassie staining gel of ( Sample 6 ) : Marker GFP protein ( soluble sample ) Western blotting ( Sample 11 ) : Induced entire sample GFP protein Ni-NTA chromatography: Fluorimetry: Mass spectroscopy: Wild-type: Mutant: Discussion: Site-directed mutagenesis: In the site-directed mutagenesis mutant is carried out at the right topographic point i.e. , at 197 and 198 topographic points. Tyrosine ( TAT ) is mutated to tryptophan ( TGG ) , Y W. During this mutant protein undergoes many alterations particularly in the fluorescence. GFP turns into CFP ( Cyan fluorescent protein ) hence the visible radiation emitted will non be precisely green. CFP will hold many curious characteristics like instead than individual excitement and emanation extremums it possess double hunching. Tag CFP possess some belongingss like: Structure monomer Molecular weight 27KDa Polypeptide length 239aa Fluorescence coloring material Cyan Maximal excitement 458nm Maximal emanation 480nm Excitation coefficient 37000M-1 cm-1 Pka 4.7 Quantum yield 0.57 Brightness 21.1 Brightness is produced by the quantum output and extinction coefficient. Double color visual image of the protein expressed is enabled by the CFP. This has led to the Fluorescence Resonance Energy Development ( FRET ) . SDS-PAGE: SDS-PAGE is carried out to divide proteins harmonizing to their cataphoretic mobility and experimental repetitions will ensue in the pureness appraisal of the protein. Four Wellss are loaded with samples and 2 and 4 Wellss show protein consequence and as 1 and 3 Wellss do nt incorporate protein they will be normal without any sets. Consequences shows that small sum of GFP has been observed in the indissoluble and big sum of protein has been observed in the soluble sample. Uninduced sample can non happen GFP. Western-blotting: Western-blot is performed to do certain the presence of protein. Histidine tagged investigation is added to corroborate the protein nowadays was GFP or non. pET28c plasmid contains T7 RNA polymerase booster sequence. But this booster is blocked by the represser. Hence lactose incorporating medium is required for E.coli growing. Because milk sugar is used as C beginning, glucose is converted into allolactose. This allolactose will adhere to repressor by unblocking booster, and expresses GFP. Hence presence of glucose will ensue in Lac-I and is binds to the operator. Band observed in the smudge is likely GFP and it has high degree of strength after initiation. And it is necessary to corroborate this by executing blotting technique utilizing His investigation to observe His labeled GFP. Sets are observed in the induced and soluble samples after executing western blotting corroborating the presence of GFP. Ni-NTA chromatography: Purification of GFP can be done by Ni-NTA chromatography. For a recombinant protein the amino acid adhering site with 6 or more His residues in a row acts as metal adhering site. So hexa-his sequence is called as His-tag. His-tag sequence is present in the N-terminal of the mark protein and is located in the booster part adjacently to the GFP cistron. During this procedure enzyme HRP is besides bound to the investigation. This HRP-probe will respond with luminal 4 peroxidase buffer which is further used for sublimating GFP by Ni-NTA chromatography. Purification by His-tagged GFP can be done by utilizing several methods like Ni2+-poly ( 2 acetomidoacrylic acid ) hydrogel. Supplanting of GFP can be done by adhering Ni to imidazole. This is chiefly because of high affinity of Ni towards imidazole compared to GFP.Distinctive sets are supposed to detect in the elute1, elute 2 and besides in the entire soluble fraction. Bands formed states the presence of the GFP mutation. Absence of the s ets states mutant absence. In the consequences sets are observed at the sum induced and the soluble samples which province the protein presence. Even little sums of sets are besides observed in the indissoluble sample. GFP protein produced in the induced entire sample is about at 27KDa. Little sets are observed in the indissoluble sample as it may be because of some drosss. Finally the GFP protein has been detected. Mentions: 1. Davenport D, Nichol JAC: Luminescence in Hydromedusae. Proceedings of the Royal Society, Series B 1955, 144:399-411 2. Ward. W. , Prentice, H. , Roth, A. Cody. C. and Reeeves.S.1982.Spectral disturbances of the Aequoria green fluorescent protein. Photochem. Photobiol. 35:803-808 3. Cormack, B. P. , Valdivia, R. H. , Falkow, S. ( 1996 ) . FACS-optimized mutations of the green fluorescent protein ( GFP ) . Gene, In imperativeness 4. Darelle Thomson, Greg Smith. ( 2001 ) .PCR-based plasmid vector building for coevals of recombinant viruses. Journal of Virological Methods 94, 7-14 5. Vogelstein, B. , and Gillespie, D. ( 1979 ) Preparative and analytical purification of Deoxyribonucleic acid from agarose. Proc. Natl. Acad. Sci. USA 76, 615-619. 6. HEIM, R. , PRASHER, D. C. A ; TSIEN, R. Y. 1994. Wavelength Mutations and Posttranslational Autoxidation of Green Fluorescent Protein. Proceedings of the National Academy of Sciences of the United States of America, 91, 12501-12504. 7. HARUKI NIWA, SATOSHI INOUYE et, al. , Chemical nature of the light emitter of the Aequorea green fluorescent protein. Vol. 93, pp. 13617-13622, November 1996. Proc. Natl. Acad. Sci. USA. 8. â€Å"Mass Spectroscopy: A Foundation Course† , K. Downard, Royal Society of Chemistry, UK, 2004.

My Happiest Day Essay Example | Topics and Well Written Essays - 500 words

My Happiest Day - Essay Example The opening ceremony was emphatic and exciting. One side of the stadium was full of Atletico Madrid fans while the other side Real Madrid fans were chanting and singing victory songs. Believe me; it was magnificent and gigantic especially because I was one of the fans present in that stadium. Once the referee blew the whistle, the high profile match commenced with hype. Real Madrid’s players were showing a lot of enthusiasm and gave the fantastic performance. Motivational slogans and songs from fans of the two teams were conspicuous. By the end of the first match, Atletico Madrid scored. For me, this was a sad moment but I still had nope that my team will emerge the winners. This was the only goal in the first halftime and the second half of the match. Real Madrid players had many chances to score, but ninety minutes erupted without a single goal. The referee extended the match by five more minutes and Real Madrid got a corner kick. I closed my eyes in hope that my team will s core and all I heard was the rumbling of the ground as thousands of fans celebrated the equalizing goal. Despite the fact that my eyes were shut, I shouted and celebrated with my fellow fans. My happiness was evident and felt as one of the happiest person in the world. It was evident that the match was going to have extra time. It was not only surprising but overwhelming because Real Madrid scored three more goals in twenty minutes. My favorite team was finally crowned as the Champion after twelve years.

Human Resource Managment Assignment Example | Topics and Well Written Essays - 500 words

Human Resource Managment - Assignment Example The other issue to look into is the ease in the flow of information from the management to the employees on matters pertaining to their welfare. When the management does the above well then the employees will have the drive to work better and harder. This is, in other words, a motivation which is all about the inner drive to act in a certain way. In this case, this inner drive is externally created by the management through good relations. When these two aspects are combined, the employees find themselves working harder than before toward the realization of organizational goals and objectives. This is the end result of a workforce that has good relations within itself and with the management leading to their high motivation, which is productivity as stated by (Martin 2005). A good example to show how the three relate will be illustrated using a Sales and Marketing Company. Sales and marketing firms are known to be requiring employees who are highly motivated as well as productive for the firms to gain market share and maintain profitability (Khan & Soverall 2007). However, the basic hindering factor is the poor employee relations that exist. For the firms that have been successful, it is the relationship between the management and the employees that act as a catalyst to direct employees’ motivation toward higher productivity (Dyer 2007). Sales and marketing is not an easy job for the employees as targets are the order of the day and failure to achieve one's targets almost always leads to imminent dismissal or low income. The firm, therefore, needs to come up with ways that will ensure that employees achieve their target with relatively manageable difficulty.

Innate Immune System Components

Innate Immune System Components There are individual systems of the immune system, innate immunity which we are born with and it is non-specific. It is genetically based and passed on to our offspring and adaptive immunity in which we acquire through humoral and cell mediated immunity. Innate and adaptive immune systems are distinct systems but act together at numerous levels to develop a complete defense against invading pathogens. Both systems have mechanisms for distinguishing self from non-self, therefore, under normal situations they are not directed against the hosts tissues and cells. Innate Immunity Elements of the innate immune system (figure 1.8) have been known for many years. However, in the past few years there has been a greater focus on innate immunity and its role in protection against infection and tissue injury and its role in tolerance to self-antigens. Innate immunity defines a collection of protective mechanisms the host uses to prevent or minimize infection. The innate immune system operates in the absence of the specific adaptive immune system but is tied to adaptive immunity in many ways. The innate immune system is characterized by a rapid response to an invading pathogen or foreign or effete cells. In addition to the rapid response, it is also non-specific and usually of a short duration. Innate immunity lacks immunological memory and there is no clonal expansion of lymphocytes as seen in the adaptive immune response. The innate immune response is also important in directing the specific, long-lived adaptive immune response. The host defense mechanisms associated with innate immunity consist of a number of physical barriers (intact skin) and secretions accompanied by a number of serum factors such as complement, certain cytokines, and natural immunoglobulins. The cellular components of innate immunity include a number of cell types, many of which are found at potential points of entry of pathogens. Examples of these cells include natural killer (NK) cells, (figure 1.2), polymorphonuclear neutrophils (PMNs), macrophages (figure 1.3), and dendritic cells (DCs), (figure 1.2). The intact skin and mucosal tissues provide considerable protection against invading infectious agents. However, once the agents pass through the skin a number of important events take place. This includes activation of the complement cascade that triggers the development of a number of substances to attract phagocytes to the area. A number of antimicrobial peptides are produced at epithelial cell surfaces. These antimicrobial peptides play an important role in local defense mechanisms, disrupt bacterial cell membranes, and probably play a role in preventing skin infections. Antimicrobial Peptides (figure 1.4) Human ÃŽÂ ²-defensins are produced by epithelial cells in the mucous membranes of the airways and intestinal tract. Defensins are small cationic peptides that have broad antimicrobial activities against a number of microbial agents including Gram-positive and Gram-negative bacteria, fungi, and enveloped viruses. Defensins are non-glycosylated peptides containing approximately 35 amino acid residues, and ÃŽÂ ²-defensins have six cysteine residues that provide a distinct structure. Stimulation of the epithelium by certain cytokines can induce defensin production. The exact mode of action of defensins antimicrobial activity is unknown. There are three defensin subfamilies: ÃŽÂ ±-defensins, ÃŽÂ ²-defensins, and ÃŽÂ ¸-defensins The Complement System The complement system (figure 1.5) is another important component of innate immunity. The system consists of 30 proteins found in serum or on the surface of certain cells. Activation of the complement system results in a cascade of biochemical reactions that ultimately ends in lysis and disruption of foreign or effete cells. Without activation, the components of the complement system exist as pro-enzymes in body fluids. As a by-product of the activation of the cascade, a number of biologically reactive complement fragments are generated. The complement fragments can modulate other parts of the immune system by binding directly to T lymphocytes and bone marrow-derived lymphocytes (B lymphocytes) of the adaptive immune system and also stimulate the synthesis and release of cytokines. Natural Antibodies Natural antibodies have been recognized for some time but recently they were described as a component of the innate immune system. Natural antibody is defined as an antibody that is found in normal, healthy individuals who have no evidence of exogenous antigenic stimulation. Natural antibodies are believed to develop in a highly regulated manner; they are usually found in low titer in serum and are low-affinity antibodies. A high percentage of the natural antibodies found in serum are of the IgM class. These antibodies are produced by a primitive B lymphocyte, called the B-1 lymphocytes. B-1 cells are usually CD5+ and considered to be long-lived and self-replicating. Natural antibodies play an important role as a first line of defense against pathogens and other types of cells, including precancerous, cancerous, cell debris, and some self-antigens. Toll-Like Receptors (TLR) TLRs (figure 1.6) are found on phagocytic cells, including mononuclear phagocytes, circulating monocytes, tissue macrophages, and endothelial cells, and are important components of the innate immune system. TLRs make up a family of cell surface protein receptors present on several cell types that function to recognize certain conserved molecular components of microorganisms and signal that microbes have breached the bodys barrier defences. TLRs serve as first responders in a mammalian host to recognize the presence of an invading pathogen. They also generate an inflammatory response to attempt to remove the invading agent. Phagocytosis (figure 1.7) Polymorphonuclear neutrophilic leukocytes have been well-known components of the innate immune system for many years. Detailed studies of PMN phagocytosis and intracellular killing of microorganisms have led to a better understanding of important defense mechanisms against invasion by pathogenic bacteria, fungi, and enveloped viruses. PMNs are attracted to the site of microbial invasion, recognize the microbe, become activated, kill the microorganisms, resolve the infection, undergo apoptosis, and are then ingested and removed by either macrophages or neighbouring endothelial cells to resolve the inflammatory response. PMNs arise as myeloid progenitors in the bone marrow. Specific growth factors and cytokines mediate the differentiation of myeloid precursors into mature PMNs. After entering the circulation, the PMNs have a half-life of about 8-12 h before undergoing a programmed cell death (apoptosis) and are reabsorbed through endothelial walls. The PMN turnover is about 1011 cells per day. Cytokines and Chemokines Cytokines and chemokines are small, secreted polypeptides that regulate essentially all functions of the immune system. Cytokines participate in determining the nature of the immune response by regulating or controlling cell growth, differentiation, activation, immune cell trafficking, and the location of immune cells within the lymphoid organs. Cytokines are a group of intercellular messengers that contribute to inflammatory responses through activation of the hosts immune cells. Cytokines are host-derived products that enhance the recruitment of circulating leukocytes as a response to the presence of pathogens. Cytokines also play important roles in leukocyte attraction by inducing the production of chemokines, which are known to be potent mediators of chemo-attractant activity for inflammatory cells. Chemokines and cytokines provide a complex network of signals that can either activate or suppress inflammatory responses Natural Killer Cells Initially, NK cells were referred to as non-specific lymphocytes because NK cells could kill certain virally infected and malignant cells without known prior sensitization. NK cells were known to resemble large lymphocytes morphologically and were referred to as large granular lymphocytes. Approximately, 10-15% of the lymphocytes circulating in peripheral blood are NK cells. NK cells are distinct from T- and B lymphocytes because they express neither immunoglobulin receptors nor T-cell antigen receptors. There are other distinctions including phenotype and function. NK cells have receptors that recognize major histocompatibility complex (MHC) class I antigens. Because NK cells have cytotoxic properties, their function is highly regulated in their interactions in both the innate and adaptive immune systems. NK cells play important roles in innate immune responses and immune regulation. They communicate with other cells through a complex of both activation and inhibitory signals through cell surface receptors. Dendritic Cells The DCs develop in the bone marrow from hematopoietic pluripotential stem cells. Precursor DCs are constantly generated in the bone marrow and are released into the peripheral blood. After leaving the bone marrow, the precursor DCs home to a number of different tissues where they reside as sentinels waiting to interact with antigen. The precursor DCs express low-density MHC class II antigens and after encountering a proper stimulus differentiate into highly endocytic and phagocytic iDCs. Precursor DCs circulate in the environment and on contacting a pathogen produce cytokines, that is, ÃŽÂ ³-interferon, and undergo maturation to iDCs. The iDCs increased phagocytic and endocytic capabilities that lead to binding antigen by the iDCs and then maturation to mature DCs. Adaptive Immunity In contrast to innate immunity, adaptive immunity (figure 1.8) is flexible, specific, and has immunological memory, that is, it can respond more rapidly and vigorously on a second exposure to an antigen. Immunologic memory provides a more powerful response to a repeated exposure to the same foreign substance or antigen. Adaptive immunity is more complex because it provides the ability to respond very specifically. Innate and adaptive immunity responses interact effectively to enhance the bodys defense mechanisms against foreign or damaged host cells. Inherent in both innate and adaptive immune responses are the mechanisms to distinguish self from non-self. The primary blood cell elements of the adaptive immune system are T lymphocytes and B lymphocytes. These T- and B-cells provide the unique specificity for their target antigens by virtue of the antigen-specific receptors expressed on their surfaces. The B- and T-lymphocyte antigen-specific receptors develop by somatic rearrangement of germline gene elements to form the TCR genes and the immunoglobulin receptor genes. This recombination mechanism provides unique antigen receptors capable of recognizing almost any antigen encountered, and provides the specific immunological memory for a rapid, vigorous, and specific response to a later exposure to the same antigen. It is estimated that millions of different antigen receptors may be formed from a collection of a few hundred germline-encoded gene elements. For many years, innate and adaptive immune responses were studied as separate systems because of their different mechanisms of action. However, it is now understood that synergy between the two systems is required to provide adequate immune reactivity against invading pathogens. Innate immune responses, through their barrier and relatively broad types of actions, represent the first line of defense against pathogens. At the time the innate system is getting activated, the adaptive system becomes activated also. The adaptive response becomes evident a few days later because it requires time for sufficient antigen-specific receptors to be generated through clonal expansion/proliferation. There are multiple interactions occurring between the two systems, which results in the co-amplification of each respective response and leads to the ultimate destruction and elimination of the invading pathogen. B lymphocytes The primary function of B lymphocytes is the production of antibodies that are specific for a given antigenic component of an invading pathogen. Antibodies are encoded by the heavy (H)- and light (L)-chain immunoglobulin genes. Antibodies may be secreted or cell surface-bound on B lymphocytes. There are five classes of immunoglobulins: IgM, IgG, IgA, IgD, and IgE; and the classification is based on the isotypes of the H chain. B lymphocytes represent roughly 10-15% of the peripheral blood lymphocyte population and free immunoglobulins make up a considerable proportion of serum proteins. After an encounter with a specific pathogen and an antibody response is generated, the level of specific antibodies to that antigen decreases in serum over a relatively short period of time. However, immunological memory persists in the B-cell population, which is capable of rapid clonal expansion upon re-exposure to that same antigen. T lymphocytes Whereas B lymphocyte products recognize extracellular pathogens, T lymphocytes are adept at identifying and destroying cells that have been infected by intracellular pathogens. For T cells to recognize antigenic peptides, the peptide must be presented in the context of cell surface MHC class I or class II proteins. In other words, T cells can only recognize molecular complexes consisting of the antigenic peptide and a self-structure, that is, the MHC. Depending on whether the antigenic peptide has been synthesized within the host cell or ingested by the cell and modified by proteolytic digestion, either MHC class I or class II proteins are required. Proteins of the MHC are intimately tied to T-lymphocyte responses and recognition of antigenic peptides. The MHC class I proteins consist of three HLA classes: HLA-A, HLA-B, and HLA-C with hundreds of allelic variants of each. Structural studies have shown that class I molecules exist as cell surface heterodimers with a polytransmembrane ÃŽÂ ±-chain associated (noncovalently) with a nonpolymorphic ÃŽÂ ²2 microglobulin protein. The protein chains are folded in such a way as to form a physical groove capable of binding up to an 11 amino acid long peptide. Antigenic proteins are degraded by proteolytic enzymes to about this size for binding to th e MHC class I proteins for antigenic presentation. Antigenic peptides are bound in the groove of the HLA molecule and expressed to the cell surface for presentation to initiate a T-cell response. Humoral Immunity (figure 1.9) The human immunoglobulins are a family of proteins that confer humoral immunity and perform vital roles in promoting cellular immunity. Five distinct classes or isotypes of immunoglobulins (IgG, IgA, IgM, IgD, and IgE) have been identified in human serum on the basis of their structural, biological, and antigenic differences.1-4 IgG and IgA have been further subdivided into subclasses IgG1, IgG2, IgG3, and IgG4 or subclasses IgA1 and IgA2 on the basis of unique antigenic determinants. Multiple allotypic determinants in the constant region domains of human IgG and IgA molecules as well as kappa (ÃŽÂ º) light chains indicate inherited genetic markers. Finally, there are several immunoglobulin-associated polypeptides such as secretory component (SC) and J chain that have no structural homology with the immunoglobulins, but serve important functions in immunoglobulin polymerization and transport across membranes into a variety of secretions (e.g., saliva, sweat, nasal secretions, breast milk, and colostrum). This diversity of the immunoglobulin components of the humoral immune system provides a complex network of protective and surveillance functions. Human IgA Polymeric secretory IgA (figure 1.10) is composed of two four-chain basic units and one molecule each of SC and J chain (approximately 400,000 MW). It is the predominant immunoglobulin in colostrum, saliva, tears, bronchial secretions, nasal mucosa, prostatic fluid, vaginal secretions, and mucous secretions of the small intestine. In contrast, 10% of the circulating serum IgA is polymeric, whereas 90% is monomeric (160,000MW). Together, they constitute approximately 15% of the total serum immunoglobulins. Trimers and higher polymeric forms can exist, but in small amounts. Two subclasses of IgA have been identified (IgA1 and IgA2), which differ by 22 of the 365 amino acids. In terms of complement activation, IgA poorly activates the classical pathway. This process has been hypothesized as a host mechanism for attenuating inflammatory responses induced by IgG antibodies at the mucosal surface. In contrast, IgA reportedly activates the alternative pathway of complement to provide some direct protective functions. IgA, once bound to a bacterial or parasitic surface antigen, may bind CD89 (IgA receptor) on infl ammatory cells (monocytes, macrophages, neutrophils, and eosinophils), leading to their destruction by means of antibody dependent cell-mediated cytotoxicity (ADCC). Moreover, its binding to viral or microbial surface antigens may restrict the mobility of microorganisms and prevent their binding to mucosal epithelium. Finally, secretory IgA can play an important first line of defense in antigen clearance by binding to antigens that leak across an epithelium and transporting them back across to prevent their entry. To summarize,  IgAs unique structure resists proteolysis and it functions to block uptake of antigen, bacterial o r viral attachment, limit inflammation induced by classical pathway complement activation, and promote microbial destruction through ADCC by binding to leukocyte receptors. Human IgD IgD (figure 1.11) is a four-chain monomer of approximately 180,000 MW with a long hinge region that increases its susceptibility for proteolytic cleavage. Although IgD is normally present in serum in trace amounts (0.2% of total serum immunoglobulin), it predominantly serves as a membrane-bound antigen receptor on the surface of human B lymphocytes. Despite suggestions that IgD may be involved in B-cell differentiation, its principal function is as yet unknown. As such, IgD is rarely quantified in a general workup of an individual suspected of a humoral immune deficiency or a B-cell dyscrasia. Hyperimmunoglobulinemia D with serum IgD levels >100 U/mL, however, has been noted in conjunction with periodic fever syndrome. This condition is a rare, autosomal recessive disorder that is characterized by recurrent episodes of fever accompanied by abdominal distress, lymphadenopathy, joint involvement, and skin lesions. It appears to be particularly responsive to anti-tumor necrosis factor ( TNF) treatment. Mutations that lead to this disease occur in the mevalonate kinase gene, which encodes an enzyme involved in cholesterol and nonsterol-isoprenoid biosynthesis. Human IgE IgE (190,000 MW) was identified in 1967 as a unique immunoglobulin that circulates in serum as a four-chain monomer. Although IgE constitutes only 0.004% of the total serum immunoglobulins, it possesses a clinically significant biological function by binding through its Fc region to the alpha chain on high-affinity receptors (FcÃŽÂ µR1) on mast cells and basophils. On subsequent exposure to relevant protein allergens from trees, grasses, weeds, pet dander, molds, foods, or insect venoms, IgE antibodies on mast cells become cross-linked. This process triggers the production and release of vasoactive mediators (e.g., histamine, prostaglandins, and leukotrienes) that can induce mild to severe immediate type I hypersensitivity reactions in sensitized  atopic individuals. Human IgG In healthy adults, the four polypeptide chain IgG monomer (150,000 MW) constitutes approximately 75% of the total serum immunoglobulins. IgG is approximately equally distributed between intra- and extravascular serum pools. Moreover, IgG possesses the unique ability to cross the placenta, which provides protection for the fetus and newborn. Human IgG has been subdivided into four subclasses on the basis of unique antigenic determinants. IgG1, IgG2, and IgG4 possess an MW of approximately 150,000, whereas IgG3 is heavier (160,000 MW) as a result of an extended 62-amino acid hinge region that contains 11 interchain disulfide bonds. IgG3s highly rigid hinge region promotes accessibility of proteolytic enzymes to sensitive Fc cleavage sites, which results in an increased fractional catabolic rate and a shorter biological half-life (7-8 days) than has been observed for IgG1, IgG2, and IgG4 (21-24 days). In terms of complement activation, IgG1 and IgG3 are the most effective, whereas IgG4 due to its compact structure does not readily activate the classical pathway of complement. IgG4 antibodies are also unique in that they appear to be functionally monovalent due to in vivo exchange of IgG4 half-molecules. As such, thi s is believed to lead to the formation of small IgG4 immune complexes that have a low potential for inducing immune inflammation. Moreover, IgG4 antibodies have the ability to interfere with immune inflammation caused by the interaction of complement-fixing IgG subclasses with antigen. Researchers in the field of allergy have speculated that IgG4 antibodies also scavenge antigen that prevents mast cell-bound IgE antibody from being cross-linked by antigen, and thus blocking IgE-mediated hypersensitivity reactions in atopic individuals who have undergone immunotherapy. Other important structural and biological differences among the human IgG subclasses relate to their Fc receptor binding, and the different binding sites on the constant region domains for rheumatoid factors, complement components, and bacterial proteins (protein A and protein G). Human IgM IgM (figure 1.12) is a pentameric immunoglobulin of approximately 900,000 MW that is composed of a J chain and five IgM monomers. Pentameric IgM constitutes approximately 10% of serum immunoglobulins in healthy individuals. Along with IgD, monomeric IgM is also a major immunoglobulin that is expressed on the surface of B cells where it serves as an antigen receptor. The C-terminal portion of pentameric secreted IgM differs from that of its monomeric cell-bound form. Secreted IgM has a mu chain with a 20-amino acid hydrophilic tail and a penultimate cysteine that facilitates polymerization. Cell membrane-bound IgM has a 41-amino acid membrane tail that contains a hydrophobic 26-amino acid segment that anchors the IgM molecule in the B-cell membrane lipid bilayer. IgM antibodies are clinically important because they predominate as an antigen receptor in early immune responses to most antigens. With a functional valency of 10, IgM antibodies are highly efficient in activating the classi cal complement pathway. IgMs actual functional valency, however, is only 5 due to steric hindrance among its many antigen-binding sites. Cell Mediated Immunity Cell Mediated Immune response (CMIR) (figure 1.9) is the functional effectors pf the immune response for phagocytosis, cell killing by cytotoxic T cells, NK and K cells Macrophage Activation While the production of antibody through the humoral immune response can effectively lead to the elimination of a variety of pathogens, bacteria that have evolved to invade and multiply within phagocytic cells of the immune response pose a different threat. Cell Mediated Cytotoxicity Cell Mediated Cytotoxic immune response is implicated in refusal of foreign grafts and the exclusion of tumors and virus-infected cells. The cells involved in these methods are cytotoxic T-lymphocytes, NK-cells and K-cells. NK cells Also known as the large granular lymphocytes are normally non-specific, MHC-unrestricted cells involved mainly in the elimination of neoplastic or tumor cells.   Once the target cell is recognized, killing occurs. K cells K-cells contain immunoglobulin Fc receptors. They are involved in Antibody-dependent Cell-mediated Cytotoxicity (ADCC). ADCC occurs as a result of an antibody being bound to a target cell surface via specific antigenic determinants expressed by the target cell. Once bound, the Fc portion of the immunoglobulin can be recognized by the K-cell. This type of CMIR can also result in  Type II hypersensitivities.

Engage in personal development in health Essay

1.1 My Duties and responsibilities as a senior support worker include: following policies and procedures, attending regular meetings in order to progress and in the interest of the service users within the Gofal services, personal care, recording temperatures, administering medication, promoting independence for service users and ensuring a healthy lifestyle, ensuring all domestic duties are complete, delegating tasks, key working tasks, mentoring new members of staff and competing all paperwork daily. 1.2 As a senior support worker I always expect and ensure my work is completed to a very high standard, ensuring all current legislation is complied with via C.S.S.I.W and care standards. I should always be accountable by making sure I can answer for my actions. Promote and uphold the privacy, dignity, rights, health and wellbeing of people who use health and care services and their carers at all times. Work in collaboration with my colleagues to ensure the delivery of high quality, safe and compassionate healthcare, care and support. Communicate in an open, and effective way to promote the health, safety and wellbeing of people who use health and care services and their carers. Respect a person’s right to confidentiality. Strive to improve the quality of healthcare, care and support through continuing professional development. Uphold and promote equality, diversity and inclusion. As a Support Worker, I make a valuable and important contribution to the delivery of high quality healthcare, care and support. Following the guidance set out in this Code of Conduct will give me the reassurance that you are providing safe and compassionate care of a high standard, and the confidence to challenge others who are not. This Code will also tell the public and people who use health and care services exactly what they should expect from me as a support worker. 2.1. The importance of reflective practice in continuously improving the quality of service provided is vital as it identifies weaknesses that can then be turned into strengths that will enable staff members to put action plans and guidelines into place to avoid any errors that may occur. This  also enables us to make sure that high standards are met without any errors. Even our service users or us as staff may change and things such as personal care, support or medication may also need to be re-evaluated. It is especially of importance to staff that may have been in the industry for some time as new staff members may be able to visualise things that you may have become too rigid or set about. 2.3. Own values, belief systems and experiences may affect working practice due to relationship breakdowns or personal problems at home this may have a significant affect on peoples general day to day mood and personality. This without a doubt will have a detrimental affect on the service users we are supporting. This is why you need to keep your personal life and working life separate. Working for a Muslim orientated company we have to respect the Wishes of our services so therefore Halal meat plays a major role within our services. Our meat must be Halal and no pork is to be consumed on the premises. Our service users are able to eat what they want outside of Gofal and whilst out on day services. Working a 24/7 rota can sometimes have an impact on those who have religious beliefs for example. Muslims attend mosque on a Friday where as Christians attend church on a Sunday and it is not always possible to accommodate everyone’s needs as the service users come first and the staff ratio always needs to correct in order to support to a high standard. 4.1. At Gofal we have regular supervision and team meetings where we can discuss anything of importance to us as support workers. If we are having any problems with staff members or service users then this gets discussed. As a result further training may be offered or different technique methods may be offered as well as reading the files again to go over any details that may have been missed or to refresh if you haven’t worked with a certain service user for a long time. In regards to progression I always aim to complete relevant courses and complete qualifications in order to further my career. If I complete my work to a high standard then at Gofal you will get recognised for your achievement and possibly work towards a higher grade. 5.1. Learning activities have affected practice as it builds confidence and experience in knowing what you are dealing with it may be in the form of an observation. Initially when I started Gofal I completed two shadowing shifts and this allowed me to observe different staff members approaches and how it may differentiate between maybe a male member of staff and a female member of staff. Depending on the service user we are supporting it may be a case of someone with a religious belief that may work well and build an excellent rapport with the service user. For example we have a female Muslim member of staff who works excellent with a Muslim service user where as he may not work as well with myself. Appraisals and supervisions play a major role as they reflect on areas of improvement as you may have concerns regarding your support or the support that you are giving the service users. For example the ratio of females/males, age, religious beliefs, drivers and medication trained.

Massive Software: A Film Innovation

It has been noted that crowds are an important feature in everyday living. Collectively, people assemble together in order to observe, protest, or celebrate various happening.Since the 19th century, crowds have become one of the most important objects of scientific inquiry, as realistically speaking, they share a collective behavior within the environment where a certain event is taking place (Magnenat-Thalmann, 2001).Nonetheless, crowd formation has been an imperative factor in the film industry. It plays a significant role in providing realistic approach for building large scenes that require numerous people in order to carry out the objective of stimulating the emotions of the audiences to make them feel as if such events are actually taking place.Back then, in order to carry out large-scale production scenes such as battle sequences and stadium spectators, myriads of extras are commissioned. One of the many disadvantages of such commissioning is apparent in the amount of time and finances it consumes.Realistically, two or three days of the scheduled production are wasted just to move the extras around the locations. At times, given directions are not followed by the extras most especially in terms of behaviors and reactions required in the set.These are just some of the key issues faced by most of production teams. However, with the advent of a technology called â€Å"Multiple Agent Simulation System in Virtual Environment† or â€Å"Massive Software,† as it is popularly referred to, enhanced creativity as well as faster and efficient production can now be achieved.Massive software is the fruition of the five years work of software developer and crowd specialist Stephen Regelous. The said technology is identified as a high-end computer simulation and visualization system that serves as the solution for generating massive yet realistic crowds, capable of performing reactions and behaviors. Instead of developing animated characters that are neede d to be manipulated all throughout, Massive creates autonomous agents, which can be people, animals, or even non-human characters.The use of fuzzy logic by Massive enables the agents to respond to their environment, and their reactions can simulate emotive qualities ranging from bravery to fear. As each agent is designed in accordance to arranged set up, massive agents are more unique and less robotic compared to other animated characters. In addition to this, Massive’s dynamic features also include smart stunts and cloth simulation which add up to the realistic environment created by Massive itself (Massive Software, 2009).First used in the feature film â€Å"Lord of the Rings† trilogy, Massive is considered as an invaluable technology that helped in shaping the epic battle in the said movie. Compared to traditional movie casting, the Lord of the Rings trilogy managed to deviate away from the customary commissioning of hundreds of actors in order to give life to some of the most intense sequences in the story through the use of Massive (Bares, 2005).Additionally, Massive is not only a crowd tool but also a tool for choreography, allowing directors to have a qualitative edge on their films. On an actual shoot, directors continuously give instructions to actors together with the extras. With the vast number of people, the scene would not eventually work as is.However, such is not the case with Massive. Through the said software, directors can move the camera as needed and at the same time put the agents with sophisticated behavior without necessarily repeating the scenes all over again (Bares, 2005).Currently, Massive is being used in many productions, may it be feature films or commercials, due to its capability to produce photo-real crowd and intense cinematic quality scenes that were never possible before (Bares, 2005).Massive is also being introduced to non-entertainment markets, specifically in fields that require strong visual effects such a s engineering and architecture, as it is perceived as a transformative technology that could impact buildings and public space design, pedestrian planning, disaster prevention and recovery, consumer behavior research, environmental impacts, and other life sciences (Thomson Reuters, 2009).In general, the introduction of Massive Software in the film industry is a premiere solution to the issue of crowd-related visual effects in both film and television. The said innovative technology marks a great change in the entertainment history as a whole because it deviates from the traditional commissioning of extras which are usually unmanageable and costly. Similarly, not only is Massive a solution to crowd effects, but it is also perceived as a useful tool in various fields, as it helps to produce a much more efficient and flexible outputs.   

Children’s Author Declines Amazon-Backed Award

British author of children’s books, Allan Ahlberg, has declined the inaugural Booktrust Best Books Lifetime Achievement Award on a pretext that it is sponsored by Amazon. He states that his dislike of Amazon is based on ‘ethical grounds’: because of numerous reports of Amazon’s tax evasion in the UK Ahlberg says that he cannot allow himself to be associated with this company. In recent years Amazon has been subject to heavy criticism due to the fact that it uses a loophole in the UK tax legislation, paying low corporation tax instead of high sales tax and stating that it sells goods from its headquarters located in Luxembourg. Allan Ahlberg is an author of more than 140 children’s books, most of which he wrote in collaboration with his wife, Janet Ahlberg, until she died of cancer in 1994 (he wrote stories and she illustrated them). He also wrote numerous books working with other illustrators. In his letter explaining this decision Ahlberg states that ‘tax, fairly applied to us all, is a good thing’, because it pays for libraries, hospitals and suchlike. Using legal loopholes to avoid paying it is, according to him, a disgrace, and he believes that Booktrust made a mistake by associating themselves with a company like Amazon. And Ahlberg himself feels that for him to receive a â€Å"lifetime achievement† award sponsored by Amazon would have been completely unacceptable. The reward is not merely symbolical – together with it Ahlberg turned down  £5,000 prize money. Booktrust’s CEO, Viv Bird, replied that she was sorry to learn about Ahlberg’s refusal, but it is the author’s personal decision and she doesn’t want to criticize his judgement. On the subject of Amazon she can only say that their organization works with a wide range of partners, and Amazon is and will be an important sponsor for them – the help from this company allows them to promote the best children’s books, encourage children to read and, in general, celebrate good fiction and its authors. She also added that thousands of children participated in voting for their favorite books for the Best Book Awards, and this year three hundred of them took part in the event itself, getting an opportunity to meet people who created all these wondrous fictional worlds for them. It is not the first time the Booktrust prize sponsor becomes the subject of controversy. For example, in 2003 a number of authors, including Gillian Cross and Melvin Burgess, published a letter stating that they don’t want to be associated with a prize sponsored by Nestle, thus protesting against the company’s selling their infants’ formula in third world countries. Ironically, in spite of his stand against Amazon â€Å"on moral grounds† and refusing to take a prize from the company’s metaphorical hands, Allan Ahlberg doesn’t seem to mind selling his books through its online store and making use of its enormous client base. So perhaps rejecting prize money won’t be too much of a loss for him.