Suspension Bridge Essay Example for Free

Suspension Bridge Essay What is meant by suspension bridge? A suspension bridge is a type of bridge in which the deck (the load-bearing portion) is hung below suspension cables on vertical suspenders. Outside Tibet and Bhutan, where the first examples of this type of bridge were built in the 15th century, this type of bridge dates from the early 19th century. Bridges without vertical suspenders have a long history in many mountainous parts of the world. Structure of suspension bridge – This type of bridge has cables suspended between towers, plus vertical suspender cables that carry the weight of the deck below, upon which traffic crosses. This arrangement allows the deck to be level or to arc upward for additional clearance. Like other suspension bridge types, this type often is constructed without false work. Can a suspension bridge be considered as a work on parabola? Yes, a suspension bridge can be called a work on parabola. As a suspension bridge is designed on parabola as the bridge is also in the â€Å"U† shaped figure as we can see it in our project. Parabola is a curved conical shaped open figure. Parabola can be defined in different ways. First, it is the graph of any quadratic function of the form y = ax2 + bx + c. Another way though, is to define it as the collection of all points that are equidistant from a line and a point not on the line. A parabola is a curve, much like a U shape. The most simple parabola is y = x^2. More info about parabola   and suspension bridge Some facts about parabola and suspension bridge:- The vertical line passing through the vertex is called the axis of symmetry. The eq. for the line is where D=0 Quadratic formula if ax2 + bx + c = 0 then x=-b ±b2-4ac2a Basic design of parabolic bridge:- Dutch scientist Christian Huygens believed that two cables running between two towers could support weight that was much greater than its own weight. The cables suspended between the two towers naturally took the shape of a parabola. The parabolic shape of the cable is created not only by gravity but also by the compression and tension forces acting on the bridge. Todays scientists believe that this is the most efficient design because it requires the least amount of materials to support a heavy load. The modern suspension bridge is composed of a deck, supports, foundations, long wire cables and hangers. Latest Research on Suspension Bridges:- Research on structural topology optimization on a suspension bridges parabolic cable has shown that it is possible to increase its capacity to withstand compressive forces. In order to help stabilize suspension bridges, truss systems are used. The truss system helps the deck resist bending, twisting and swaying. DO you KNOW: The longest suspension bridge in the world (as of 2010) is the Akashi Kaikyo Bridge in Kobe, Japan. It opened in 1998 and spans an incredible 1991 meters (6529 feet) Suspension Bridges Design technology Suspension bridges in their simplest form were originally made from rope and wood. Modern suspension bridges use a box section roadway supported by high tensile strength cables. In the early nineteenth century, suspension bridges used iron chains for cables. The high tensile cables used in most modern suspension bridges were introduced in the late nineteenth century. Today, the cables are made of thousands of individual steel wires bound tightly together. Steel, which is very strong under tension, is an ideal material for cables; a single steel wire, only 0.1 inch thick, can support over half a ton without breaking. Light, and strong, suspension bridges can span distances from 2,000 to 7,000 feet far longer than any other kind of bridge. They are ideal for covering busy waterways. With any bridge project the choice of materials and form usually comes down to cost. Suspension bridges tend to be the most expensive to build. A suspension bridge suspends the roadway from huge main cables, which extend from one end of the bridge to the other. These cables rest on top of high towers and have to be securely anchored into the bank at either end of the bridge. The towers enable the main cables to be draped over long distances. Most of the weight or load of the bridge is transferred by the cables to the anchorage systems. These are imbedded in either solid rock or huge concrete blocks. Inside the anchorages, the cables are spread over a large area to evenly distribute the load and to prevent the cables from breaking free. Suspension bridge: construction that allows automobiles to travel between two points separated by an obstacle. Side span: segment between two pylons at the ends of a bridge. Centre span: segment between two pylons at the centre of a bridge. Side pylon: tower-like vertical construction situated at the side, usually supporting the cables of a suspension bridge or a cable-stayed bridge. Foundation of a pylon: very durable lower part of a tower. Suspender: support cable. Suspension cable: set of braided wire that supports a bridge. Pylon: tower-like vertical support that usually supports the cables of a suspension bridge or a cable-stayed bridge. Stiffening girder: tightener beam

A Critical Analysis of Carol Ann Duffys Essay Example for Free

A Critical Analysis of Carol Ann Duffys Essay A Critical Analysis of Carol Ann Duffys In Mrs Tilschers Class In Mrs Tilschers Class by Carol Ann Duffy, many issues are addressed about her class that play a part in explaining the subjects of the poem. To begin with the content and subjects of the poem are based around a school year in Mrs Tilschers class. Her entire class is trapped and enthralled during the school day. They are taught information, into intricate detail. For example the poem begins with the line, you could travel up the blue Nile with your finger. This simply begins the world of imagination to which each and every child is subjected. Straight after that, the poem goes on to describe how the children are chanted the scenery of the world by Mrs Tilscher. She remarkably inserts images of geographical places, historical events and general knowledge firmly in the childrens brains. Tana, the great dam, Ethiopia, the last great King, Haile Selassie, Khartoum, where General Charles Gordon was assassinated on the step of the embassy and Lord Kitchener stepped in to relieve him at the siege. See more: how to write a critical analysis outline The great Aswi n dam was known about and also the great pyramids of Egypt. Children viewed books as enthralling, fascinating and enjoyable to read which was remarkable for children of such different backgrounds. Carol Ann Duffy often uses short, sharp sentences in this poem to get her message across quickly and clearly. The children viewed their life at school as better than home. In my view this must have been something remarkably different for the children to have rather spent more time at school time than at home. But why was this the case, there must have been something remarkably different for this to be true? In the second stanza we are probably told why, Mrs Tilscher loved you, we are told. She was kind, considerate, some mornings you found shed left a good gold star by your name. Although this is not much, it is the sentimental thought which counts, all the little things add up to the big factor that the children viewed her as another mother and she felt the same way. Surprisingly, within the second stanza Carol Ann Duffy inserts a reality with upsets the imaginary atmosphere. Brady and Hindley faded like a faint, uneasy smudge of a mistake. This poem was clearly written in the late sixties or early seventies as we are shown by the mention of Brady and Hindley. Ian Brady and Myra Hindley were convicted for abducting, sexually and mentally torturing and burying small children on the Lancashire moors. They were known as the moors murderers, at this stage in Britain all children were warned of such characters. Mrs Tilscher made it very clear to her children that there were such characters in the world and that they were safe within her classroom. She tried to warn them of the realities they may face in the future. The murderers are pictured in the poem as fading like a mistake. Perhaps Mrs Tilscher is trying to imply to the children that all people make mistakes and they are just fading in the distance. However it is clear that they will always be there and it is unlikely any of the children would forget that. Not only did Mrs Tilscher educate the children but she taught them vital lessons for example mistakes are the steppingstones to success and the theory and reasons behind forgiveness. Mentioning them in the middle of an idyllic situation reminded them of the unpleasantness which could not be kept away from the children. The use of alliteration is also used in this line with the words faded and faint. However in the third stanza the mood changes as the terms go on and the reality of future prospects draw nearer and nearer. The children are obviously growing up, both physically and mentally. The inky tadpoles changed from commas into exclamation marks, time is moving on. The children become more and more sexually inquisitive and the children become more and more fed up and tense. A dunce frees a few frogs simply to cause a bit of mischief, which amuses all the boys and a lot of girls, croaking around the playground. A rough boy told you how you were born this would have been a shock to many children as they were still young but the atmosphere in the class is constantly changing from one that is idyllic and seemingly unchangeable to one with an uncertain future and nervous inhabitants. Finally the final stanza is perhaps the key verse. A tangible alarm made you always untidy, hot, fractious under the heavy, sexy sky. This quote is once again emphasising the childrens natural sexual inquisitiveness as they gradually become aware of their hormones. You asked her how you were born and Mrs Tilscher smiled, then turned away. Mrs Tilscher and her children are in a state of innocence and what they ask is exactly what she tries to protect them from. She doesnt want them to become in contact with the outside world so therefore she is not going to tell them, as she does not want them to know. It is clear that Mrs Tilscher is looked on with affection and that she loves them and doesnt want them to grow up. The term comes to an end, all are impatient to grow up and gain more freedom, and prosper within the high hopes of their lives. You ran through the gates, impatient to be grown, as the sky split open into a thunderstorm, the sky splits, they are impatient to grow up and enjoy themselves. However it is clear that they do not know what quite to expect as a lot more comes with adulthood than meets the eye, for example life is complicated, decisions have to be made and many, many responsibilities are given to you. The language of the poem portrays few significant similes and metaphors. The only simile in the poem itself is in the second stanza, the classroom glowed like a sweetshop. This simile paints a fine, clear picture of what the atmosphere was like inside the classroom. We are informed that there is sugar paper and coloured shapes lining the walls, and glitter sparkling in the sunlight. The glowing sweetshop creates an atmosphere of happiness and security, almost as if it was possible to dip into. I find this simile very effective because this is a clear indication and description of the classroom. Enjoyable enough for all to delve into it and enter their imaginary world, this simile is the only one in the play, however it is extremely important and gives us a vivid impression of the room itself. Few metaphors are present throughout the poem; the laugh of a bell is significant by the fact that this was a privilege, to be happy, good and hardworking and to be awarded with the duty of bell ringing. From my view this was something most tried to achieve, not only was the bell laughing by the noise it made but the child swinging it, in a happy mood, swung the bell with enthusiasm and enjoyment. A xylophones nonsense which was heard coming from another class is a less significant metaphor, once again illustrating the picture of happiness that was achieved in Miss Tilschers class, as nonsense came from other classes. One other metaphor that appears in the fourth stanza is, the air tasted of electricity. This metaphor indicates a clear-cut change in the atmosphere. As storm clouds brew, a thunder and lightening storm is preparing to roar. The lassitude which penetrates the air is yet another indication that something different and new will soon happen. The final metaphor is that of, the sky split open, the sky splits, just like a crack in a bowl, and everyone is oblivious to what is going to come next. I find that this is the most effective of the metaphors as it is true. As one goes into adulthood, there is a sense of obliviousness and you often are ignorant to your surroundings, exactly what this metaphor implies. The first two stanzas of this poem are quite different to the last two. The poet divides the poem into four stanzas the first two have eight lines each, while the last two have seven lines each. The tone of the first two is pleasant and cosy, apart from the reality check of mentioning Brady and Hindley. The last two are very different. The tone changes and there is a sense of discomfort. A cosy, idyllic picture is painted in the first two stanzas with phrases such as, better than home, Mrs Tilscher loved you and gold stars. This is quite different from the last two as we are confronted be phrases such as feverish July, the air tasted of electricity, untidy, hot and heavy sexy sky. This poem is very descriptive by the use of many adjectives. Carol Ann Duffy constantly uses the word you. This suggests that she is implying it in a general sense towards each reader, as this is the sort of Primary School experience that most people went through. The poet has clearly made a division in the middle of the poem to emphasise the change, from good to bad. At the end of the poem, the metaphorical storm has been gathering since July. The sky splitting open suggests that knowledge and adulthood are nothing but a shock. YOU are subjected to the shocks of the thunderstorm of adulthood which all have to pass through. This is known as a rite of passage that everyone has to pass through. There is no obvious sense of rhythm nor is there a rhyming scheme in the poem. The sentences do not seem to flow clearly. This is probably because there are so many short sentences. Each line is about seven words long and very descriptive. This gives the impression that once again, great detail is used. The scent of a pencil slowly, carefully shaved, the use of alliteration here creates an atmosphere, which is so familiar to the readers. It is almost as if the reader finds it possible to enter Mrs Tilschers class just by the picture the poet paints in the poem. This poem is very true and realistic, as it happens to everyone, it is a rite of passage. The children leave Mrs Tilscher and remember her with great affection, as it is the end of an era, which shall never be forgotten by anyone who was taught by Mrs Tilscher. You grow up, from age to age, until you reach the thunderstorm of adulthood which all have to contend with.

Plant DCL Proteins

Plant DCL Proteins Abstract Dicer, a double-stranded RNA (dsRNA)-specific endoribonuclease, plays an essential role in triggering both transcriptional and post-transcriptional gene silencing in eukaryotes by cleaving dsRNAs or single-stranded RNAs bearing stem-loop structures such as microRNA precursor transcripts into 21- to 24-nt small RNAs. Unlike animals, plants have evolved to utilize at least four Dicer-like (DCL) proteins. Extensive genetic studies have revealed that each DCL protein participates in a specific gene silencing pathway, with some redundancy. However, a mechanistic understanding of how the specific action of each DCL protein is regulated in its respective pathway is still in its infancy due to the limited number of biochemical studies on plant DCL proteins. In this review, we summarize and discuss the biochemical properties of plant DCL proteins revealed by studies using highly purified recombinant proteins, crude extracts, and immunoprecipitates. With help from co-factor proteins and an ATP ase/DExH-box RNA-helicase domain, the microRNA-producing enzyme DCL1 recognizes bulges and terminal loop structures in its substrate transcripts to ensure accurate and efficient processing. DCL4 prefers long dsRNA substrates and requires the dsRNA-binding protein DRB4 for its activity. The short-dsRNA preference of DCL3 is well suited for short-RNA transcription and subsequent dsRNA formation by coupling between a plant-specific DNA-dependent RNA-polymerase IV and RNA-dependent RNA-polymerase 2 in the transcriptional gene silencing pathway. Inorganic phosphate also seems to play a role in differential regulation of DCL3 and DCL4 activities. Further development of biochemical approaches will be necessary for better understanding of how plant DCL proteins are fine-tuned in each small RNA biogenesis pathway under various physiological conditions. Introduction RNA silencing, also known as RNA interference (RNAi), is one of the fundamental molecular mechanisms conserved in most eukaryotes to regulate gene expression both transcriptionally and post-transcriptionally. In both situations, what triggers the RNA silencing pathway is a small RNA molecule, 21 to 24 nt in length, called small interfering RNA (siRNA) or microRNA (miRNA) depending on its origin and the downstream pathways involved. The class 3 endoribonuclease (RNase) III enzymes known as Dicer are responsible for producing siRNA from longer double-stranded RNAs (dsRNAs) and miRNA from single-stranded RNAs with internal stem-loop structures   by a dsRNA-specific endoribonuclease. Therefore, the activity and regulation of Dicer-family proteins in a cell are vital to many biological processes requiring flexible adjustments at the level of gene expression, such as development, organogenesis, the circadian rhythm, biotic and abiotic stress responses, and defense against viruses an d transposons. Biochemical characterization of Dicers in animals The Dicer family is a unique class of RNase III enzymes due to the presence of an ATPase/DExD/H-box helicase domain at the N-terminus, a Piwi/Argonaute/Zwille (PAZ) domain in the middle and dual RNase III domains followed by one or two dsRNA-binding domains in the C-terminal half (exception: Giardia intestinalis) (Figure 1) (Bernstein et al. 2001). In general, the helicase domain serves as a protein-protein interaction surface recruiting co-factor regulatory proteins (Lee et al. 2006; Ma et al. 2008; Ye et al. 2007). It also utilizes ATP hydrolysis to achieve processive cleavage of the long dsRNA substrate (Cenik et al. 2011; Welker et al. 2010). The PAZ domain contains a conserved pocket for recognizing the terminus of the dsRNA substrate, and the distance between PAZ and the RNase III catalytic center determines the product sizes (MacRae et al. 2007; MacRae et al. 2006). Each of the two RNase III domains cuts one of the dsRNA strands, leaving a characteristic 2-nt overhang at 3-end of the product (Elbashir et al. 2001; Takeshita et al. 2007; Zhang et al. 2004). The C-terminal dsRNA-binding domains (dsRBDs) serve as a protein-protein interaction interface and nuclear localization signals, in addition to having dsRNA-binding function (Doyle et al. 2013; Hiraguri et al. 2005; Wostenberg et al. 2012). The specific functionality of each domain differs depending on the Dicer protein. Since the first demonstration of in vitro small RNA-producing activity of Dicer in the fruit fly Drosophila melanogaster (Bernstein et al. 2001), its biochemical properties and regulatory machinery have been extensively studied in humans, D. melanogaster and Caenorhabditis elegans. In humans, there is only one Dicer-family protein (hDicer), which cleaves short-hairpin pre-miRNAs produced by Drosha and dsRNA substrates into 20- to 22-nt small RNAs in an ATP-independent manner (Myers et al. 2003; Provost et al. 2002; Zhang et al. 2002). The cleavage activity requires a divalent metal cation such as Mg2+, Co2+ or Mn2+, and recognizes mainly the 5-end of the substrate to dictate the product length (Park et al. 2011). This 5-counting rule is reliant on the conserved 3-pocket motif within the PAZ domain and the 5-pocket motif, which is less conserved in Dicers of other eukaryotes. The binding of Dicer to a dsRNA substrate and its cleavage are uncoupled, because Dicer can bind to dsRNA with out Mg2+ or under low temperature (Provost et al. 2002; Zhang et al. 2002). The helicase domain of hDicer has an autoinhibitory function (Ma et al. 2008). In line with this, the activity of recombinant full-length hDicer protein can be improved under limited proteolytic conditions (Zhang et al. 2002). hDicer is responsible for both siRNA and miRNA production, and co-factor dsRNA-binding proteins TRBP and PACT dictate hDicer function in the two distinct small RNA production pathways (Chendrimada et al. 2005; Haase et al. 2005; Kok et al. 2007; Lee et al. 2013; Lee et al. 2006). In particular, the hDicer complex containing PACT disfavors siRNA precursor dsRNA and shows different cleavage patterns on the same pre-miRNA substrate than the hDicer-TRBP complex (Lee et al. 2013). The interaction with TRBP occurs through the hDicer helicase domain, and stimulates the hDicers catalytic activity. (Ma et al. 2008). Similarly, it has been reported that the C. elegans Dcr-1 interacts with a dsRNA-binding protein RDE-4 which enhances the Dicer activity toward long dsRNA substrates in siRNA production, while RDE-4 is apparently dispensable in miRNA production pathway (Parker et al. 2006; Parker et al. 2008; Tabara et al. 2002). D. melanogaster has two Dicer proteins, Dcr-1 and Dcr-2, which produce miRNA and siRNA, respectively (Lee et al. 2004; Miyoshi et al. 2010). Dcr-1 alone can process dsRNA into siRNA in vitro, but its interaction with the dsRNA-binding protein Loquacious isoform PB (Loqs-PB) confers pre-miRNA substrate specificity to the Dcr-1-Loqs complex by suppressing cleavage of long perfect dsRNAs and enhancing pre-miRNA processing activity (Saito et al. 2005; Zhou et al. 2009). Dcr-2 interacts with Loqs isoform PD and another dsRNA-binding protein, R2D2, in the siRNA production pathway (Liu et al. 2003; Liu et al. 2006; Miyoshi et al. 2010; Zhou et al. 2009). Dcr-2 alone is also capable of cleaving a pre-miRNA precursor in an ATP-independent manner, but R2D2 significantly suppresses Dcr-2 activity toward pre-miRNA, whereas Loqs-PD enhances the cleavage activity of Dcr-2 toward long perfect dsRNA precursors by boosting its affinity to the substrate (Cenik et al. 2011; Miyoshi et al. 2010). The pr ocessive processing of long dsRNA substrates by Dcr-2 depends on ATP hydrolysis by its ATPase/helicase domain, implying that one of the functions of the helicase domain is to allow Dcr-2 to produce multiple siRNAs from a single long dsRNA molecule before it dissociates from the substrate (Cenik et al. 2011). Such differential regulation of Dicer activity through specific interaction with co-factor dsRNA-binding proteins in distinct pathways is commonly found in most of the systems studied, including plants. DCL proteins in plants Plant genomes contain at least four distinct classes of DCL family proteins (DCL1-4). Like their animal counterparts, each class of DCL has evolved to participate in its primary pathway (Figure 2), but the three siRNA-producing DCLs (DCL2-4) function redundantly as well, because defects in one class of DCL can be compensated for by other classes in some cases (Gasciolli et al. 2005; Mukherjee et al. 2013; Xie et al. 2004). Because DCL1 is the only Dicer protein that produces most 21-nt miRNAs (Kurihara and Watanabe 2004; Reinhart et al. 2002), knockout mutants of DCL1 are embryonic lethal (Schauer et al. 2002). DCL4 is the major producer of 21-nt antiviral siRNA and endogenous siRNAs such as trans-acting siRNA and phased siRNAs (phasiRNA) (Bouche et al. 2006; Gasciolli et al. 2005; Mukherjee et al. 2013; Qu et al. 2008; Xie et al. 2005; Yoshikawa et al. 2005). DCL2 can compensate for the loss of DCL4 (Bouche et al. 2006; Gasciolli et al. 2005; Parent et al. 2015), although its major function remains unclear. DCL3 mainly produces 24-nt repeat-associated siRNAs derived from transposons and DNA repetitive elements, and participates in transcriptional gene silencing (TGS) through RNA-dependent DNA methylation, suppressing proliferation of these elements (Henderson et al. 2006; Pontes et al. 2006; Xie et al. 2004). In addition to the four classes of DCLs, monocots have another distinct class of Dicer, DCL5 (also known as DCL3b) (Margis et al. 2006). DCL5 is specifically expressed in developing panicles and is responsible for 24-nt reproductive phasiRNAs, although the biological significance of a reproductive-organ-specific 24-nt phasiRNA pathway mediated by this specific Dicer remains to be elucidated (Borges and Martienssen 2015; Fei et al. 2013; Kapoor et al. 2008; Song et al. 2012). This pathway might be analogous to the Dicer-independent PIWI-interacting RNA (piRNA) pathway in vertebrates, which suppresses transposons and other genes specifically in germlines (H irakata and Siomi 2016). Both forward and reverse genetics and physiological studies have successfully dissected the major RNA silencing pathways and allowed identification of the function of DCL genes in each pathway in plants. However, investigations on the molecular and enzymatic characteristics underlying the functional diversification and specificity of the DCL proteins are still in their infancy. Detection of DCL activity in crude extracts of various plants Biochemical characterization of plant Dicer activity was first demonstrated in wheat germ extract (monocot) and cauliflower extract (dicot), which contain multiple DCL activities producing ~21 nt and ~24 nt small RNAs with 2-nt 3-overhangs in the double-stranded form (Tang et al. 2003). These activities are weaker in the absence of ATP, consistent with characteristics of Dicer family proteins from Drosophila and C. elegans. Long dsRNA competitors effectively suppress both activities in wheat germ extract. The 24-nt small RNA producing activity was inhibited by 25-nt synthetic siRNA duplexes, whereas 21-nt small RNA production was unaffected by 21-nt synthetic siRNA duplex competitors, suggesting that two different enzymes with active sites that have distinct size-dependent binding properties are in the wheat germ extract (Tang et al. 2003). A recent study on wheat germ extract characterized these activities in further detail, revealing (1) that the 21-nt activity could be found in a much larger (~950 kDa) complex than the 24-nt activity, which had maximum activity in an approximately 450 kDa complex; and (2) the biochemical properties associated with the activities, such as divalent cation and NTP requirements, optimum NaCl concentration, temperature, and pH, and substrate length dependence (Shivaprasad et al. 2015). The identities of the DCL enzymes responsible for these activities in the wheat germ extract remain to be identified. A better understanding of the biochemical characteristics of individual plant Dicer proteins has come from the model plant Arabidopsis thaliana, which has four DCL proteins: DCL1, DCL2, DCL3 and DCL4 (summarized in Table 1). The first in vitro DCL activity in A. thaliana was demonstrated using a suspension cell lysate, a crude extract of inflorescence tissue, and an immunoaffinity-purified protein complex (Qi et al. 2005). Similar to the previous study using wheat germ extract or cauliflower, extracts from both Arabidopsis cultured cells and inflorescence tissue contained DCL dsRNA-cleaving activity producing 21- and 24-nt small RNAs from 400-bp dsRNA (Qi et al. 2005). The 21-nt producing activity and 24-nt producing activity were found in >660 kDa and ~400 kDa fractions, respectively, suggesting that these Dicers reside in protein complexes composed of multiple co-factors (Qi et al. 2005). In agreement with previous genetic studies showing CARPEL FACTORY/DCL1 is responsible for 21-nt miRNA production in vivo (Kurihara and Watanabe 2004; Reinhart et al. 2002), the 21-nt small RNA producing activity was DCL1 immunoaffinity-purified from inflorescence-derived crude extract by an anti-DCL1 antibody (Qi et al. 2005). The 24-nt activity was associated with anti-DCL3 antibody immunoprecipitate, and the activity was abolished when purified from a dcl3-1 mutant, showing that DCL3 is responsible for the 24-nt activity in Arabidopsis inflorescence extract. The immunoaffinity-purified DCL1 activity required ATP, whereas the activity of the DCL3 immunoprecipitate was ATP-independent (Qi et al. 2005). Interestingly, the dcl1-7 mutation did not abolish the 21-nt small RNA producing activity in the extract or immunoprecipitates, implying that the substitution (P415S) in its N-terminal helicase domain did not alter the enzymes catalytic activity itself (Qi et al. 2005); this study also found that the activity of DCL4 responsible for formation of 21-nt siRNA was present in the inflorescence extract. The presence of DCL4 activity in an Arabidopsis crude extract was demonstrated in later studies using 2-week-old seedlings as the starting material (Fukudome et al. 2011; Nagano et al. 2014), and will be discussed later in this review. In-depth biochemical characterization of DCL1, a microRNA-producing enzyme in plants  DCL1 activity requires DRB1/HYL1 and SERRATE for accurate processing of the miRNA precursor Both in wheat germ and Arabidopsis extracts, DCL activities are associated with size fractions larger than DCL monomeric form, implying that these DCLs form functional protein complexes composed of multiple co-factors in vivo. As summarized in an earlier section, such interactions between a Dicer and a co-factor protein are commonly found in mammals, nematodes and insects. One of the most characterized classes of co-factor proteins is a dsRNA-binding protein (dsRBP) harboring multiple dsRNA-binding domains or motifs. The A. thaliana genome encodes five dsRNA-binding (DRB) family proteins: DRB1/HYL1, DRB2 DRB3, DRB4, and DRB5. Multiple genetic and biochemical studies have demonstrated two specific interactions between DCLs and DRBs in A. thaliana: DCL1-DRB1/HYL1 and DCL4-DRB4 (Han et al. 2004; Hiraguri et al. 2005; Kurihara et al. 2006; Nakazawa et al. 2007). Arabidopsis DCL1, DRB1/HYL1, and another co-factor, SERRATE (SE), constitute an essential microRNA production pathway in vivo (Han et al. 2004; Lobbes et al. 2006). Unlike animals, which utilize two distinct RNase III enzymes, Drosha and Dicer, for the first and second cleavage of microRNA precursors, plants do not employ Drosha. Therefore, the DCL1-complex is responsible for the processing of both primary and precursor miRNA substrates. The detailed molecular machinery of the dual miRNA processing mediated by DCL1 and the co-factor proteins have been extensively studied biochemically using highly purified recombinant proteins produced in heterologous systems (summarized in Figure 3). One of the systems utilizes baculovirus-mediated recombinant protein production in Sf21 insect cells, followed by two-step affinity purification (Dong et al. 2008). The highly purified recombinant DCL1 protein alone could process a 94-bp dsRNA substrate with a 2-nt 3-overhang into 21-nt small RNA in an ATP/Mg2+ dependent manner. The optimum NaCl concentration for the activity was 25-50 mM, and a NaCl concentration higher than 100 mM severely impaired the activity (Dong et al. 2008). While the recombinant DCL1 protein alone could produce 21-nt small RNA from both primary and precursor miRNA (pri-/pre-miR167b) substrates in vitro, DRB1/HYL1 and SE recombinant proteins co-incubated in the same reaction mixtures significantly increased both yield and accuracy of the processing (Dong et al. 2008). Without these co-factors, more than 80% of 21-nt small RNA products from the DCL1-alone reaction were due to incorrect processing from the end of the primary miRNA substrate, whereas the processing mediated by the DCL1-DRB1/HYL1-SE complex produced accurate 21-nt products with a sequence identical to miR167b/miR167b*, amounting for up to 81% of the products (Dong et al. 2008). This demonstrated that accurate processing of miRNA precursors by DCL1 requires the co-factors DRB1/HYL1 and SE. Cons istent with a previous study, the interaction between DCL1-DRB1/HYL1 through the second dsRNA-binding motif of DCL1 is important for the precise processing of pri-miRNA in A. thaliana (Dong et al. 2008; Kurihara et al. 2006). Also, using highly purified recombinant proteins and surface plasmon resonance analysis, it has been suggested that DCL1 changes its structural conformation when it binds RNA and exposes more binding sites for SE (Iwata et al. 2013). Binding to substrate dsRNA or miRNA precursors might be an important regulatory step for DCL1 dicing activity, as its dsRNA-binding domains exhibit the strongest binding to dsRNA among the four Arabidopsis DCLs (Hiraguri et al. 2005). ATPase/DExH-box RNA-helicase domain of DCL1 suppresses its dicing activity, confers ATP dependence, and influences processing accuracy In addition to its RNase III and dsRNA-binding domains, the helicase domain of DCL1 plays a significant role in regulating its dicing activity. Two independent forward genetic studies have identified two dcl1 mutant alleles, dcl1-13 (E395K) and dcl1-20 (R363K), as hyl1 suppressors, and the amino acid substitutions of both alleles occur within the ATPase/DExH-box RNA-helicase domain. These dcl1 mutations partially rescue the accumulation of some miRNAs in a hyl1-2 mutant (Liu et al. 2012; Tagami et al. 2009), and dcl1-13 was at least partially able to restore the phenotypic defects of hyl1-2 such as a reduced number of rosette leaves and a   leaf shape (Tagami et al. 2009). Highly purified recombinant DCL1-20 protein exhibited enhanced catalytic activity (Kcat/Km) toward pri-miRNA156a compared to wild-type DCL1 (Liu et al. 2012). Similarly, the helicase domain-deleted DCL1 recombinant protein (DCL1à ¢Ã‹â€ Ã¢â‚¬  Helicase) showed higher processing activity in vitro and was no l onger dependent on ATP for its activity toward pri-miRNA156a (Liu et al. 2012), suggesting that the helicase domain of DCL1 might have an autoinhibitory function like that of human Dicer (Ma et al. 2008; Provost et al. 2002). The in vivo miRNA processing imprecision in hyl1-2, however, was not restored by a dcl1-20 mutation, implying that the partial recovery of the hyl1-2 mutant, including miRNA accumulation, was due to the enhanced catalytic activity resulting from the substitution in the helicase domain (Liu et al. 2012). Interestingly, the effect and magnitude of DRB1/HYL1 and DCL1 helicase domain seem to vary among miRNA precursors. For example, the in vivo processing accuracy of miR156a is much more severely affected by hyl1-2 mutation than miR166b is (Liu et al. 2012). pri-miR156a is processed from the loop-proximal site to the loop-distal base in vitro (Liu et al. 2012), which is considered unusual for plant miRNAs (Addo-Quaye et al. 2009; Mateos et al. 2010). Accurate processing of pri-miRNA166b by native DCL1 is largely dependent on the presence of ATP, and processing by DCL1à ¢Ã‹â€ Ã¢â‚¬  Helicase is less accurate than that of native DCL1 (Liu et al. 2012). In contrast to miR156a, the processi ng precision of which is markedly affected by hyl1-2, that of miR166b was much more impaired by dcl1-20 mutation than hyl1-2 (Liu et al. 2012). Also, the effect of the other helicase mutant allele, dcl1-13, on miRNA production was shown to depend on the presence or absence of DRB1/HYL1 in vivo (Tagami et al. 2009). These observations indicate that efficient processing of different miRNA precursors by DCL1 have different reliance upon DRB1/HYL1 and DCL1helicase domain that potentially depends on structural determinants of the miRNA precursors. Structural determinants for efficient and accurate processing of miRNA precursors by DCL1 Primary transcripts of miRNA (pri-miRNA) have a characteristic secondary structure: a loop-distal stem (lower stem), a miRNA duplex, a loop-proximal stem (upper stem) and a terminal loop (Figure 3). Typical miRNA maturation from these precursors requires at least two cleavages occurring at the lower and upper stems. In animals, the single-stranded base region of the loop-distal stem is recognized by the dsRNA-binding protein DGCR8, which guides the processing center of Drosha to the correct position, which is 11 nt from the base of the stem (Han et al. 2006). However, this distance-from-base rule is not sufficient for plants because the length of the loop-distal stem of plant pri-miRNAs is highly variable (Song et al. 2010). Several structural features of pri-miRNAs that influence the activity, binding position and directionality of the processing by DCL1 have been elucidated genetically and biochemically (Figure 3a, b). One structural determinant lies within the loop-distal stem of pri-miRNA. For the first cleavage at the loop-distal stem, bulges and unpaired regions play a major role in the efficiency of miRNA processing. Mutant pri-miRNAs with closed bulges were processed at the correct position, but resulted in the accumulation of unprocessed pre-miRNAs in vivo, indicating that the rate of subsequent processing at the loop-proximal stem was impaired (Song et al. 2010). In pri-miR171a, which has a long loop-distal stem, the first cleavage position was determined by the distance from a relatively unstructured region instead of the base of the stem; the conserved distance from an unstructured region of the lower stem important for miRNA processing was found to be approximately 15 nt (Figure 3a) (Mateos et al. 2010; Song et al. 2010; Werner et al. 2010). The 15-nt rule was essentially reproduced in an in vitro miRNA processing system using highly purified DCL1-DRB1/HYL1-SE recombinant proteins and an artificial pri-miRNA substrate bearing another unstructured region in the elongated lower stem. In addition to the canonical processing, another type of processing occurred at 15 nt from the artificially introduced unstructured region, validating the functionality of the 15-nt rule (Song et al. 2010). The importance of bulges and unpaired regions in the lower stem for processing by DCL1 might explain why some miRNAs with a near-perfect matched stem seem to be DCL4-dependent, rather than DCL1-dependent (Rajagopalan et al. 2006; Song et al. 2010). On the loop-proximal and terminal loop side, a branched terminal loop (BTL) or a large terminal loop was found to be an essential structural factor that may alter directionality of processing by DCL1 and the resultant miRNA-accumulation (Figure 3b). BTL induces abortive processing of pri-miR166c both in vivo and in vitro (Zhu et al. 2013), meaning the first cleavage of the pri-miRNA occurs in the loop-proximal stem as opposed to the normal productive processing beginning in the loop-distal stem. The molecular basis of this bidirectional processing by DCL1 was further investigated using an in vitro system that reconstitutes the DCL1-processing machinery. For this purpose, DCL1, DRB1/HYL1 and SE harboring Agrobacterium tumefaciens were co-infiltrated to Nicotiana benthamiana leaves, and the transiently expressed DCL1-DRB1/HYL1-SE complex was immunoaffinity-purified two days after infiltration (Zhu et al. 2013). The reconstituted DCL1 complex cleaves the substrate pri-miRNA 16-17 nt fro m the unpaired region of the lower stem, supporting previous studies (Mateos et al. 2010; Song et al. 2010; Werner et al. 2010). By disrupting one of the two RNase III domains of DCL1 alternately and using 5- or 3-end labeled pri-miR166c substrates, the bidirectional nature of both productive and abortive processing was demonstrated (Zhu et al. 2013; Figure 3b). The helicase domain of DCL1 fine-tunes the position of both productive and abortive processing by DCL1 in an ATP-dependent manner (Zhu et al. 2013). DCL1à ¢Ã‹â€ Ã¢â‚¬  Helicase complex could not abortively process a substrate with BTL. Also, wild-type DCL1 required ATP for abortive processing, but not productive processing, indicating that the ATPase-driven helicase activity is necessary in abortive processing to unwind the structured BTL (Zhu et al. 2013; Figure 3b). In productive processing, the effect of helicase deletion and ATP depletion depend on the distance between the processing site and the bulge in the lower stem. Many potential byproducts of the abortive processing of pri-miRNA precursors with BTL can be found in publically available high-throughput small RNA sequencing data from both Arabidopsis and rice, implying that both substrate structure and the functionality of the ATPase/helicase domain of DCL1 are conserved mechanisms to regulate miRNA biogenesis in higher plan ts (Zhu et al. 2013). Dissecting distinct characteristics of DCL3 and DCL4 activities DCL4 activity requires the dsRNA-binding protein DRB4 In A. thaliana, DCL2, DCL3 and DCL4 are responsible for producing various siRNAs 21-24 nt in length. The dsRNA-cleaving activities of DCL3 and DCL4 can be detected in crude extracts prepared from 2-week-old seedlings (Fukudome et al. 2011). Extracts from wild-type seedlings cleave 500-bp dsRNA substrates into 21-nt and 24-nt small RNAs. In this system, the 21-nt and 24-nt small RNA-producing activities can be attributed to DCL4 and DCL3 respectively, because the dsRNA-cleaving activity of the corresponding size was abolished in each of the single mutants (Fukudome et al. 2011). Also, a mutation in the dsRNA-binding protein DRB4, which interacts with DCL4 (Hiraguri et al. 2005; Nakazawa et al. 2007), abolished DCL4 activity in seedling extracts. The DCL4 activity could be further purified by immunoprecipitation with anti-DCL4 or anti-DRB4 antibodies. The immunoaffinity-purified DCL4 requires Mg2+ and ATP for its activity, and is inhibited by >200 mM NaCl. This property is similar to t hat of recombinant DCL1 protein (Dong et al. 2008). The DCL4 complex immunoprecipitated from the drb4-1 mutant did not show dsRNA-cleaving activity, but the addition of recombinant DRB4 protein to the complex restored the 21-nt producing activity in vitro, showing that DRB4 functions as an essential co-factor for the dsRNA-cleaving activity of DCL4 (Figure 4b). In this system, mutant DRB4 proteins harboring substitutions in the conserved amino acid residues that form a hydrogen bond with the phosphodiester backbone of dsRNA at the dsRNA-binding site (H32A in the first dsRBD and K133A in the second dsRBD of DRB4) lost their ability to interact with dsRNA and DCL4, and did not restore DCL4 activity. The second substitution (K133A) alone impaired its interaction with the C-terminal half of DCL4 containing two RNase III domains and two dsRBDs in a GST pull-down assay using recombinant proteins, but was not sufficient to block restoration of DCL4 activity when added to DCL4 immunopurified from a drb4-1 mutant extract. There might be an add itional interaction surface between DCL4 and DRB4 involving dsRBD1 of DRB4 and the N-terminal half of DCL4, which contains an ATPase/DExH-box RNA-helicase domain and an RNA-binding domain (formerly known as domain of unknown function DUF283; Figure 1), as their specific interaction was reported in vitro (Qin et al. 2010). Short dsRNA preference of DCL3 activity orchestrates 24-nt siRNA biogenesis in TGS pathway Crude extracts from 2-week-old seedlings have also been used to characterize substrate specificity of DCL3 and DCL4. Consistent with the long dsRNA preference of Drosophila Dcr1 (Bernstein et al. 2001), DCL4 preferentially cleaves longer dsRNA substrates, and is less efficient in producing 21-nt siRNAs when the substrate is shorter than 50 nt (Nagano et al. 2014). On the other hand, DCL3 activity, producing 24-nt siRNAs, favors shorter substrates such as 30 nt and 37 nt dsRNA with a 1-nt or 2-nt 3-overhang (Nagano et al. 2014). It also favors substrate dsRNA with 5-adenosine or uridine. The 24-nt small RNA produced by DCL3 has a 2-nt 3-overhang, and the cleavage follows the 5-counting rule proposed for human Dicer (Park et al. 2011). DCL3 is not reliant on ATP hydrolysis for activity, as it can still process the short dsRNA substrate in the presence of a non-hydrolyzable ATP analog, adenosine 5-O-(3-thio)triphosphate (Nagano et al. 2014). Unlike DCL4, which targets long dsRNAs such a s RDR6-dependent TAS dsRNAs or exogenous viral dsRNAs in vivo (Bouche et al. 2006; Dunoyer et al. 2005; Qu et al. 2008; Yoshikawa et al. 2005), DCL3 may not need to perform a processive cleavage, which requires ATP hydrolysis, because the length of its targets allows only a single cut (Figure 4a). The DCL3 preference for short dsRNA substrate is consistent with the one precursor, one siRNA model for RNA polymerase IV (Pol IV)-dependent 24-nt siRNA biogenesis (Blevins et al. 2015; Zhai et al. 2015). In this model, a remarkably short (30- to 40-nt) transcript with 5-adenosine is produced by Pol IV and is simultaneously converted into double-stranded form by an RNA-dependent RNA polymerase, RDR2. The short dsRNA substrate is processed into 24-nt siRNA preferentially by DCL3 due to its length specificity, facilitating the subsequent RNA-directed DNA methylation process (Blevins et al. 2015; Zhai et al. 2015). The transcription of short RNAs by Pol IV, and the length and 5-adenosine substrate preference of DCL3 might be essential mechanisms to prevent other DCLs from processing specific dsRNA substrate needed for the TGS pathway. Such coupling of RDR-Dicer-RNAi is also known in fission yeast, where a Dicer physically interacts with an RNA-dependent RNA polymerase to form coupled ma chinery that drives siRNA-mediated TGS (Colmenares et al. 2007). In addition, DCL3 can participate in 24-nt siRNA production from longer transcripts with aid from another RNase III enzyme, RNase III-like 2 (RTL2). As a class II RNase III enzyme, RTL2 possesses one RNase III domain and two dsRBDs, and is involved in rRNA maturation [in vivo is implied]in A. thaliana (Comella et al. 2008). Recombinant RTL2 protein can cleave long dsRNA substrates into 25 bp or longer dsRNA in vitro (Kiyota et al. 2011). Recently, it has been shown that RTL2 processes a subset of Pol IV-dependent dsRNA into shorter intermediates, which are preferable for DCL3 activity in vivo (Elvira-Matelot et al. 2016). Although no direct interaction has been reported, RTL2 and DCL3 can be considered other examples of coordinated action of a dsRBD-containing protein and a Dicer in plants. DCL3 is also reported to physically interact with the dsRNA-binding protein DRB3 in the antiviral RNA-directed DNA methylation pathway (Raja et al. 2014). The function of DRB3 in DCL3 activity rem ains elusive. Inorganic phosphate, NaCl and KCl differentially regulate DCL3 and DCL4 activities In the same assay system using crude extracts, inorganic phosphate at a physiological concentration promotes DCL3 activity but suppresses DCL4 activity toward 50-nt dsRNA substrates (Nagano et al. 2014). The differential effe

Essay --

Human Trafficking Human trafficking which is commonly referred as â€Å"modern-day slavery† is a trade in humans in the form of prostitution, sexual exploitation, forced labor or even for the extraction of organs. Here in this country, people are being abducted, bought, and sold like slaves to be forced into all forms of servitude. The U.S Department of Justice stated that â€Å"human trafficking has become the second fastest growing criminal industry — just behind drug trafficking — with children accounting for roughly half of all victims† (Baldas, Human trafficking a growing crime in the U.S). It is not only happening in the States but it is also a worldwide phenomenon. To think that human trafficking may be happening even in our own neighborhood, for us being unaware as we go about our daily lives is unimaginable. Human traffickers generate billions of dollars per annum by victimizing countless people all around the world. Human trafficking is illegal in any way or anywh ere as it is a massive violation to human rights. People may still wonder that how can human trafficking still exist in the 21st century. There are various causes that make adults and children vulnerable to exploitation. However, human trafficking does not exist mainly because of the victims’ vulnerability to exploitation. Instead, just like drugs and arms trafficking, human trafficking is a market-driven industry which is based on the principles of supply and demand. Joyce Hart stated that â€Å"when there is a demand for services, human traffickers will eagerly fill it with slaves or very low-cost workers† (26). It is a highly sophisticated and organized crime industry with low risk and high profit, which are the main two factors that cause its demand to thrive. When the i... ...he 21st century, it is also expanding. Everyone should do their part in order to prevent more people being exploited and protect them. One of the main reasons that many of these traffickers are not being prosecuted is because a lot of victims are too traumatized or terrified to testify against their own abusers. Therefore, â€Å"police and criminal justice staff need standard working procedures to guarantee the physical safety of victims, protect their privacy and make it safe for them to testify against their abusers† (UNODC). Even for normal citizens, there are also ways to help fight against human trafficking such as contacting respective authorities if knowing of a potential human exploitation, changing purchasing options, or start a fundraiser and donate the funds to an anti-trafficking organization. These people need our help and together we can make a difference.

Long Distance Relationships Essay example -- essays research papers

Long Distance Relationships Relationships have been around since the beginning of mankind. There are many goals in life that people pursue and having a soul mate is one of them. One type of relationship that is practiced most among teenagers is long distance relationships. Due to the fact that so many teenagers fall in love so quickly and are immature, they will not let go of partners even though they may have to separate. Many people find it difficult to stay in one relationship for a very long time yet alone a long distance relationship. Long distance relationships in my view are the hardest to keep intact than any other types of relationships. Whether its interracial or interfaith relationships, it is a proven fact that the most difficult relationship to keep from falling apart are the...

The View from the Bottom Rail Essay -- essays papers

The View from the Bottom Rail The Lewinsky Scandal†¦ A perfect example as to why we cannot accept everything at face value before carefully examining it first. Everyone thought President Clinton was behaving himself in the White House, but, as it turns out, he was most definitely not. This can be the same for history. We must carefully consider different aspects of articles so that we do no make the mistake of believing everything we read. In order to fully understand an article, we must understand the author that wrote it. It is necessary to examine prejudices, sources, information left out, and missing background information before accepting an article. This method of critical analysis allows us to better understand the article and therefore history because we are more aware of the authors and their possible mishaps. â€Å"The View from the Bottom Rail†, an article in After the Fact, provides an opportunity to examine different aspects of analysis. If we look at it carefully, then we will be able to determine if the thesis was proven effectively. In â€Å"The View from the Bottom Rail†, the authors, James Davidson and Mark Lytle, proposed, â€Å"For several reasons, that debased position has made it unusually difficult for historians to recover the freedman’s point of view.† Within the article, Davidson and Lytle cycled through different aspects as to why it is hard for historians to determine the â€Å"view from the bottom rail†. They questioned the validity of many sources that, if accurate, would have contained the perspective of an ex-slave. These sources included both white and black testimony. In order to examine these sources, the authors traced the topics using microcosm. Because they were covering a topic and not an event, microcosm was the most appropriate method of examining the subject. Davidson and Lytle first introduced a source. Then, they pondered over the different ways that the source could be biased. They took small segments from the source and used those to demonstrate why the source could not be taken at face value. For example, when examining the proposed source of a slave master’s account, Davidson and Lytle examined one aspect of this to make a conclusion. They determined that, â€Å"With slaves so dependent on the master’s authority, they were hardly likely to reveal their true feelings; the dangerous consequences of such indiscr... ...s old. The authors do question whether her mental status was viable or not. However, further background information was needed. It was imperative for the authors to examine the validity of this source before using it. It is not know if the ex-slave was capable of recalling accurate details of her life. Since the authors’ argument was that the same slave told two different stories depending on the circumstances of the interview, how do we know if the stories varied because of the circumstances or because of a poor or maybe even imaginative memory? This background information would make the authors’ arguments even more convincing. However, if we assume that the ex-slave was capable, then the argument is flawless. Overall, the article was well written. Only minor aspects were left uncovered. In addition, not much background information was needed. Also, the authors’ only had sparse and subtle prejudices. A variety of sources was used effectively. In the end, the thesis was proven convincingly. Almost all audiences would be assured that, â€Å"For several reasons, that debased position has made it unusually difficult for historians to recover the freedman’s point of view.†

Modernism and the Visual Arts Essay

Originally titled as Le Bain, means the bath; Luncheon on the Grass was exhibited at the Salon de Refuses in 1863. It is painted with oil on a large canvas by Édouard Manet created in between 1862 and 1863. The painting represents the contrast between a female nude with fully clothed men in a rural setting. By that time, normally woman in paintings didn’t look at viewers straight, especially with nudes. Traditionally, nude models mostly looked away from the view. But however, Manet’s naked female model straightly stares at the viewer. This was shocking, but also challenging at the same time. The Luncheon on the Grass, received a great attention by causing public scandal with nude female and painting was success through setting, color, technique, composition and light. Luncheon on the Grass brought in much argument at its reveal. From this painting, the viewer is provoked by a naked female directly looking at the audience. By this time, this naked woman was obviously politically improper and especially, woman looking at the viewer as unashamed was totally unbelievable and offensive in a male-controlled, conquered society. This was the main reasons why it was forbidden and got teased from the public. Not only because picture of the naked female, but it was a commentary, and arguable as well. Painting it makes seem usual, and normal, besides something with sexualized. However, the two fully clothed men are not looking directly; it is only the naked female who grabs attention to be focus of the painting. Once again, the setting is very unusual, and it is unique. It seems like the models are just posing instead of having lunch together. The picnic basket lies on its side in the corner without any attention, and also the naked female has been completely left out along. The woman in far back in lake also seems left out from the lunching group too. None of these people seems gathered in the nature. However, some critics have inferred the painting as oddness with compositional sense, but it is delightful with the thought of the illusory. Manet’s use of color was influenced by the Impressionist movement, and it led the painting successful. Manet’s color use is combined with a very practical theme. He used mostly dark colors for the foreground such like dark greens, orange-browns and blacks for outlines. He also broke up of the movement of figure to add reality. Manet also inserted element of still life which is picnic basket in the bottom left corner of the painting. It subsidizes an added level of color. What is interesting in this painting is that he limited to use warm colors. And instead of having soft brush strokes and color mix, he uses vivid outlines around the figure. This concept lets the figure pop out of the landscape. Looking at the female figure, Manet painted in a flat tone with very little shading to capture the moment. The painting contains flat areas of color, and even in some places, Manet left it as a bare canvas. With limited of shading and contrast using of cool and warm colors prevent the tradition of depth and volume; also cause viewer’s attention to the surface of the painting. Manet’s light source through the painting was also interesting. This impression era is covered by the use of broad and detailed light source, which limits almost no shadows. However, the lightning in this scene of painting is unreliable and unusual. Compare to the two clothed men, the nude female takes the spotted light. She almost has no shadows besides strong outlines. Also because the two clothed men are wearing dark clothes, the nude female obviously pops out from the background and grabs viewers’ attention. I can see where the light hits in different areas, but the nude female takes the main light source, and therefore, the landscape background smoothed out the light. Tradition to this time era, Manet’s techniques were extraordinary and brush strokes become much less developed. Most of his brush strokes have been done in free handed. Focusing on the nude female, her skin is not smoothed out with value of color and brush techniques. Also the background is containing a rough brush strokes. During this time period, most of the artists are hiding brush strokes for their technique, but Manet did the opposites. It is obvious to see each brush stroke in nude female giving a rough complexion. In the background, also this style of rough brush strokes are made noticeable, and this even seems unfinished. Manet was free with technique. He did not try to hide or to follow the trends; indeed, some scenes in the painting seem unfinished. Through the composition, the figures are positioned in central. The interesting thing is that through this composition, I can draw myself a direct pyramid from. From the foreground nude female figure rises up to the lady in the water. With this compositional sense, attention falls between the two men. Men seem very odd and just stuck to be posing because compare to the women two men seems very dull and not interested in. They were not paying attention to the woman, and also not gathered at all. None of the figures seems relaxed. Rather than posing natural in the nature, these figures give tightness look. However, Manet successfully balanced the light against dark, use of cool color background and well blends the nude figure with warm tone, covers up the oddness and conveyed as successful painting. To sum up, Manet’s Luncheon on the grass is with mystery, oddness and wonder. He detailed his idea to the work, and all the elements he included in this painting lead great result follow in 1863. As an impressionist, Manet earned great media attention by causing public scandal and painting was success through setting, color, technique, composition and light. Artist: Édouard Manet Title: The Luncheon on the Grass Date: 1862 and 1863 Medium: Oil paint