Previous metaphor studies have paid much attention to nominal metaphors and predicative metaphors, but little attention has been given to adjective metaphors. Although some studies have focused on adjective metaphors, they only examined differences in the acceptability of various types of adjective metaphors. This paper explores the cognitive effects evoked by adjective metaphors. Three psychological experiments revealed that (1) adjective metaphors, especially those modified by color adjectives, tend to evoke negative effect; (2) although the meanings of metaphors are basically affected by the meanings of their vehicles, when a vehicle has a neutral meaning, negative meanings are evoked most frequently for adjective metaphors compared to nominal and predicative metaphors; (3) negative meanings evoked by adjective metaphors are related to poeticness, and poetic metaphors evoke negative meanings more easily than less poetic metaphors. Our research sheds new light on studies of the use of metaphor, which is one of the most basic human cognitive abilities. Immer klangen von dämmernden Türmen die blauen Glocken des Abends. (An einen Frühverstorbenen)‘The blue evening bells still ring from the twilit towers’Die weiße Stimme sprach zu mir: Töte dich! (Offenbarung und Untergang)‘The white voice talked to me: Kill yourself!’Schwemut und purpurnes Lachen (Abend in Lans)‘Gloom and deep red laugh’Trakl uses color adjective metaphors in a very striking way throughout the poem. (1) is an example of adjective metaphors created from blue ‘blau’ in German. There are other expressions using blue ‘blau’ in his poems; the blue cry ‘die blaue Klage,’ in the blue evening the figure of the dead ‘im blauen Abend der Toten Gestalt,’ and A blue animal is scared of death ‘Ein blaues Tier will sich vorm Tod verneigen.’ These expressions emphasize negative images working in negative contexts . The last example evokes a metaphorically negative image by describing an animal as blue. (2) is an example of an adjective metaphor created from white ‘weiß.’ There are other expressions such as white sorrow ‘weiße Traurigkeit’ and The white offspring’s dark future ‘Die weißen Enkel dunkle Zukunft.’ These expressions also emphasize negative images working in negative contexts . (3) is an example of an adjective metaphor created from deep red ‘purpurn.’ There are many other examples using deep to be found in Trakl’s poems; deep red plague ‘purpurne Seuche,’ The deep red curses ‘Die purpurnen Flüche,’ in deep red dreams pain and agony ‘in purpurne Träume Schmerz und Plage,’ and the deep red sufferings ‘die purpurnen Marten.’ These expressions also emphasize negative images.Sakamoto aoi kannen-teki-na keno-kan noho-ga tsuyo katta.‘I had a rather blue and ideological disgusted feeling.’kentai-no aoi uta‘weary blue song’nigotta, shiroi yokujou‘dark, white sexual desire’The expressions given above seem to evoke negative images, but in this paper we test this assumption through psychological experiments. Furthermore, although the above are examples of color adjective metaphors, we will examine not only color adjective metaphors but also other types of adjective metaphors more thoroughly to investigate whether evoking negative images is a unique feature of adjective metaphors through a comparison with nominal metaphors and predicative metaphors. We also address the issue of why these metaphors evoke negative images.Metaphors have traditionally been thought simply be devices of the poetic imagination that represent a rhetorical flourish―a matter of extraordinary rather than ordinary language. Metaphors have typically been viewed as characteristic of language alone, a matter of words rather than thought or action. Lakoff and Johnson My job is a jail” shot down all of my arguments” The following are expressions from German poetry by Georg Trakl open heart.The old woman had an cold heart.The rich man had a cold smell.The stone statue had a Adjective metaphors are sometimes called synesthetic metaphors. According to prominent theories of metaphor Many studies focusing on synesthetic metaphors have examined how the acceptability of synesthetic metaphors can be explained by the pairing of the modalities of adjective modifiers and head nouns. Ullmann Twenty years after Williams’ study was published, Day high spirits,” are grounded in correlations based on our real world experiences. The HAPPY IS UP metaphor is grounded in the experience that persons in a positive emotional state have an erect posture, that is, they carry themselves in a positive, tall manner. As for synesthetic metaphors, Taylor Other studies on adjective metaphors have investigated whether adjective metaphors are motivated by the perceived resemblance between the two perceptual domains or by correlations in experience. In cognitive linguistics research Very few studies, however, have attempted to explore the cognitive effects evoked by adjective metaphors. Therefore, this paper explores such cognitive effects, and in particular synesthetic metaphors as a subcategory of adjective metaphors.Metaphor comprehension has been recognized by interaction theorists as the process of finding relevant features that constitute the metaphorical meaning from the interaction between a source concept and a target concept The rest of this article is organized as follows. Section of Experiment 1 presents the first experiment (Experiment 1) conducted to examine the tendencies of cognitive effects associated with adjective metaphors, and whether adjective metaphors, especially those modified by color adjectives, tend to evoke negative images. Based on the results of Experiment 1, Section of Experiment 2 presents our main experiment (Experiment 2) conducted to examine whether the evoking of negative meanings is a unique feature of adjective metaphors through a comparison with nominal and predicative metaphors. Based on the results of Experiments 1 and 2, we explore the possible reasons why adjective metaphors were found to evoke negative meaning. The results of Experiment 3 suggest that negative meanings evoked by adjective metaphors are related to poeticness, and therefore that poetic metaphors evoke negative meanings more easily than less poetic metaphors. The last section discusses the significance and implications of this study and possible directions of future work.http://www.macromill.com/global/privacy/index.html). In total, 3,267 Japanese males and females, aged 20–75, agreed to participate in our experiment.All participants in Experiments 2 and 3 provided written informed consent prior to the experiments. Documents about the experimental procedures and written informed consents were presented to an internal organization of the University of Electro-Communications, Tokyo, Japan. Participants in Experiment 1 were recruited through Macromill, Inc., Tokyo, Japan, an organization that maintains a panel of more than 533,579 individuals who have provided informed consent and agreed to participate in web-based online survey research. Participants’ rights are protected based on the company’s privacy policy , touch (‘tezawari’), voice (‘koe’), taste (‘aji’), smell (‘nioi’), feeling (‘kimochi’), dream (‘yume’), uneasiness (‘fuan’), greed (‘yokubou’), affection (‘aijou’), and manner (‘taido’). Twenty-five expressions made by combining adjectives and nouns belonging to the same perceptual domains were excluded as non-metaphorical expressions. These 25 Japanese expressions denoting perceptions from the five sense modalities are shown in We conducted a pre-experiment to choose the materials to be used in the experiment. Thirty Japanese males and females, aged 21–25, participated in the pre-experiment. Materials used for the pre-experiment were 250 Japanese adjective metaphors. These metaphors were made by combining 25 Japanese adjectives denoting perceptions from the five sense modalities with 11 Japanese nouns; Participants were asked to evaluate how easily they understood each metaphor. The ratings were made on a seven-point scale ranging from −3 (very difficult) through 0 (not sure) to +3 (very easy). They were also asked to evaluate how conventional they felt each metaphor to be. The ratings were made on a seven-point scale from −3  through 0 (not sure) to +3 . We selected metaphors with a mean value within the range of −2.0 to +2.0 in both scales. This procedure reduced the possibility that differences in the cognitive effects evoked by the adjective metaphors would result from differences in the accessibility or conventionality among the materials used in the experiment. As a result, 158 Japanese adjective metaphors were chosen as the materials to be used in the experiment.The 3,267 participants were classified into 10 groups, and 18–20 linguistic expressions were assigned to each group. The linguistic expressions assigned to one group were randomly assigned to each participant in that group .dislike – like, uncomfortable – comfortable, not interesting – interesting, not appropriate – appropriate, dull – sharp, weak – powerful, heavy – light, coarse – delicate, ugly – beautiful, dark – light, unclear – clear, scary – safe, sad – glad, old – new, and not salient – salient. The 15 SD scales were selected from the high factor loading scales among the 34 SD scales used in Kusumi The participants of Groups 1 and 2 were each assigned 18 adjectives and nouns, and the remaining 8 groups were assigned 19 or 20 metaphorical expressions per participant. They were asked to rate the assigned expressions against the following 15 Semantic Differential  scales: We classified all of the mean values of the vehicles and topics rated on the 15 SD scales into T = V, T<V, and T>V, considering the prediction given in To compare the actual semantic changes resulting from our experiment with the predicted semantic changes, we classified the resulting actual semantic change as shown in 2  = 4.612, p<.05. Among the cases that showed a change different from the predicted change, the proportion of cases showing the change to the negative pole (-) was significantly higher than the proportion showing the change to the positive pole (+), χ2  = 214.158, p<.001.As for the cases which were predicted as no change (0), the proportion of cases showing the same actual change as the predicted change was significantly higher than that showing a change different from the predicted change, χ2  = 66.881, p<.001. This finding suggests that actual semantic changes do not follow the predicted changes toward the positive pole (+).As for cases that were predicted to change to the positive pole (+), the proportion of cases showing the same change as the predicted change was lower than that showing a different change, χ2  = 637.180, p<.001. This result suggests that the actual semantic changes do follow the predicted change toward the negative pole (−).As for cases that were predicted to change to the negative pole (−), the proportion of cases showing the same change as the predicted change was significantly higher than that showing a different change, χ2  = 15.094, p<.01 for touch; χ2  = 21.252, p<.001 for sound; χ2  = 8.574, p<.001 for smell. As for ‘taste’ adjective metaphors, there was no significant difference found, χ2  = 0.769, p = .380. On the other hand, for ‘color’ adjective metaphors that were predicted as having no change (0), the proportion of cases showing a change different from the predicted change was significantly higher than the proportion showing the predicted change, χ2  = 33.515, p<.001. Among the ‘color’ adjective metaphors that showed a change different from the predicted change, the proportion of cases showing a change toward the negative pole (−) was significantly higher than the proportion showing a change toward the positive pole (+), χ2  = 112.034, p<.001. This result suggests that only “color” adjective metaphors tend to change toward the negative pole against the prediction of no change (0).We also classified the full dataset of 2,370 cases into five types of adjective metaphors, as shown in 2  = 105.779, p<.001 for color; χ2  = 4.389, p<.05 for taste; χ2  = 28.451, p<.001 for smell. As for ‘touch’ and ‘sound’ adjective metaphors, the proportion of cases showing the predicted change as the predicted change was slightly higher than that showing a different change, but this difference was not significant. This finding indicates that all of the adjective metaphor types do not show the predicted change toward the positive pole (+).For the ‘color,’ ‘taste,’ and ‘smell’ adjective metaphors that were predicted to change toward the positive pole (+), the proportion of cases showing the predicted change was significantly lower than that showing a different change, χ2  = 103.313, p<.001 for color; χ2  = 53.765, p<.001 for touch; χ2  = 147.574, p<.001 for sound; χ2  = 114.722, p<.001 for taste; χ2  = 231.410, p<.001 for smell. This result confirms that actual semantic changes do in fact follow the predicted change toward the negative pole (−).Regarding all of the metaphor types that were predicted to change toward the negative pole (−), the proportion of cases showing the predicted change was significantly higher than that showing a different change, χ2  = 367.175, p<.001. This result suggests that semantic interactions between the vehicles and the topics of the adjective metaphors tend to evoke a negative effect. In addition, we analyzed the tendency of negative effects among the adjective metaphor types. To see more clearly whether there was a tendency for adjective metaphors to evoke positive or negative effects, we classified all the cases showing different changes from the predicted change into those having a positive effect and those having a negative effect. Cases showing no change against the predicted change to the negative pole (−) were regarded as evoking a weakly positive effect, and were classified into the positive effect category in the same way as those which showed a change toward the positive pole (+) against the predicted change to the negative pole (−). Cases showing no change against the predicted change toward the positive pole (+) were regarded as evoking a weakly negative effect, and were classified into the negative effect category in the same way as those which showed a change toward the negative pole (−) against the predicted change to the positive pole (+). As a result, the 848 cases that showed changes different from the predicted changes were classified into 145 positive effect cases and 703 negative effect cases. A Chi-square test showed that the cases showing a negative effect were significantly more frequent than those showing a positive effect, χ2  = 109.763, p<.001 for color vs. touch; χ2  = 117.848, p<.001 for color vs. sound; χ2  = 25.203, p<.001 for color vs. taste; χ2  = 71.947, p<.001 for color vs. smell. Differences among the color adjectives, yellow ‘kiiroi,’ blue ‘aoi,’ red ‘akai,’ black ‘kuroi,’ and white ‘shiroi,’ were not observed.We conducted a Chi-square test using Bonferroni correction  for the five types of adjective metaphors. The result showed that adjective metaphors created from adjectives denoting ‘color’ evoked the most negative effect. This result is consistent with the results of the analysis using the full dataset of 2370 cases. ‘Color’ adjective metaphors evoked a significantly more negative effect than the other four types of adjective metaphors, χ2  = 24.746, p<.001 for taste vs. touch; χ2  = 9.993, p<.005 for taste vs. smell; χ2  = 27.859, p<.001 for taste vs. sound. These negative effects were especially observed among adjective metaphors created from sour ‘suppai,’ bitter ‘shibui,’ and hot ‘karai.’The second most negative effect was observed for adjective metaphors created from the adjectives denoting ‘taste’. These metaphors evoked significantly more negative effects than ‘touch,’ ‘smell,’ or ‘sound’; χ2  = .250, p = .617 for touch vs. sound; χ2  = 3.160, p = .075 for touch vs. smell; χ2  = 4.775, p = .029 for sound vs. smell. As for ‘touch,’ adjective metaphors showing a negative effect were observed more frequently among those created from hard ‘katai’ and cold ‘tsumetai’ than among those created from light ‘karui,’ soft ‘yawarakai,’ and hot ‘atsui.’ As for ‘smell,’ negative effects were observed among those created from stinking (1) ‘kinakusai,’ stinking (2) ‘kusai,’ and smelly ‘namagusai.’Significant differences among adjective metaphors created from the adjectives denoting ‘touch,’ ‘sound,’ and ‘smell’ were not observed; χThe results of Experiment 1 showed that 848 cases did not show the expected shift toward the semantic value of the vehicle. One possible explanation of this unexpected change may be related to the imageability or frequency of the words. For example, metaphor ratings may shift towards the topic if the topic is more imageable than the vehicle. To examine whether such differences in imageability or frequency between topics and vehicle causes the unexpected semantic value shift, we conducted multiple regression analyses with semantic value shift as the dependent variable. One analysis was conducted for the cases showing the unexpected value shift. The imageability and frequency values are based on the NTT database: lexical properties of Japanese We also analyzed how the full dataset of 2,370 ratings are distributed amongst the 15 SD scales. The results are shown in We must acknowledge the possibility that the meaning of some of the scales may have been unclear to the participants, and thus we do not know with certainty if everyone interpreted the scales in the same way. The results of Experiment 1, however, showed that semantic interactions between vehicles denoting the different perceptual domains and adjective metaphor topics tended to evoke negative cognitive effects, and that there were differences among the types of adjective metaphors. This raises the question of why and through what processes such cognitive effects are evoked. One possible explanation for these observed differences among the types of adjective metaphors is different degrees of accessibility among the adjective metaphor types. According to Ullmann’s Our finding that the ‘color’ adjective metaphors evoked the most negative effect may be related to the fact that color is located in the highest position of the hierarchy. However, the second most negative effect was evoked by adjective metaphors created from ‘taste,’ which is located in a lower position in the hierarchy. Furthermore, as we described in Methods section, in our pre-experiment we asked participants to evaluate how easily they understood the metaphors proposed for the experiment materials as well as how conventional they felt the proposed metaphors were, and selected metaphors with a mean value ranging from −2.0 to +2.0 in both scales. Through this procedure, we reduced the possibility that differences in the cognitive effects evoked by the adjective metaphors would result from differences in the accessibility or conventionality among the materials used in the experiment. Therefore, the observed different effects evoked by the five types of adjective metaphors cannot be explained simply by differences in acceptability among the adjective metaphor types. This observation is supported by the result of the multiple regression analysis using imageability and frequency described above.yellow ‘kiiroi,’ blue ‘aoi,’ red ‘akai,’ and white ‘shiroi’ do not themselves have explicit negative meanings. Wierzbicka As stated above, the most negative effect was evoked by the adjective metaphors created from ‘color.’ This result is interesting because, according to Sakamoto The results of our experiment are not particularly unusual; in fact, they are consistent with those of Sakamoto Furthermore, as mentioned in the Introduction, Trakl has used color adjectives to emphasize negative concepts such as death and grief throughout his poems In Experiment 1 we analyzed how semantic interactions between the vehicles and topics of adjective metaphors function to shift the meanings of words to either the positive pole or the negative pole. The results showed that adjective metaphors, and especially those modified by color adjectives, tend to evoke negative images, thereby contradicting our prediction. In Experiment 2, therefore, we examined whether evoking negative meanings is a unique feature of adjective metaphors through a comparison with nominal and predicative metaphors.Because our aim was to examine whether the evoking of negative meanings is a unique feature of adjective metaphors, we selected the SD scales shown in smell (‘nioi’), (the current)moment (‘genzai’), footstep (‘ashioto’), and pose (‘shisei’). Details of the process in which nouns with neutral meanings were selected are given in We also selected topics and vehicles to construct Japanese metaphorical expressions. The topics were selected from nouns categorized as belonging to the highly abstract semantic level in a Japanese thesaurus Vehicles for the three types of metaphors were also selected from the Japanese thesaurus Next we constructed metaphors by combining the four topics and the 49 vehicles. We excluded 16 expressions that were not regarded as metaphors, such as “minor smell” and “moment of life.”Using the same procedure as Experiment 1, participants were asked to evaluate how easily they understood each metaphor. The ratings were made on a seven-point scale ranging from −3 (very difficult) through 0 (not sure) to +3 (very easy). They were also asked to evaluate how conventional they felt each metaphor to be. The ratings were made on a seven-point scale from −3  through 0 (not sure) to +3 . As a result, we confirmed that all the metaphors were rated from −2.0 to +2.0 in the accessibility and conventionality scales. This procedure reduced the possibility that differences in the cognitive effects evoked by metaphors would result from differences in the accessibility or conventionality among the materials used in the experiment.smell of dreams (‘yume no nioi’), predicative metaphors: rolling smell (‘korogaru nioi’), adjective metaphors: white smell (‘shiroi nioi’)). These metaphors were constructed by combining the 4 topics and the words associated with the 3 types of vehicles, namely, nouns, verbs, and adjectives. In the psychological experiment, participants were asked to evaluate the meaning of the metaphorical expressions. A total of 60 Japanese males and females, aged 20–28, were classified into two groups, and 90 metaphorical expressions were assigned to each group. Participants were asked to rate the assigned expressions against 7 SD scales given in We conducted a psychological experiment in which participants evaluated the meaning of all three types of metaphors , vehicles that had no significant difference between their mean value and value 0 were regarded as vehicles with a neutral meaning (0). In addition, vehicles that had a significant difference between their mean value and value 0 were classified into either vehicles with a positive meaning or those with a negative meaning.We assume that the meanings of metaphors result from the semantic interaction between their vehicles and topics. Because the meanings of the individual topics used in our study were neutral, we classified all of the metaphorical expressions into those using vehicles with neutral meanings, those using vehicles with positive meanings, and those using vehicles with negative meanings. 2 =  = 37.735, p<.01 (0 vs. +). However, there was no significant difference found between the number of neutral metaphorical expressions (0) and the number of negative (−) ones, χ2 =  = 2.513, p>.05 (0 vs. −).As for the metaphors in which the vehicles by themselves had neutral meanings, as shown in 2 =  = 15.211, p<.01 (0 vs. +); χ2 =  = 4.840, p<.05 (0 vs. −).As for nominal metaphors, the proportion of metaphors showing a neutral meaning was highest. The results of Chi-square tests showed that these metaphorical expressions (0) were significantly more common than the other expressions, χ2 =  = 0.154, p<.01 (0 vs. +); moreover, there was a slightly significant difference between the number of neutral (0) metaphorical expressions and the number of negative (−) ones, χ2 =  = 3.846, p = .05 (0 vs. −).As for predicative metaphors, the proportion of metaphors showing a neutral meaning was highest. The results of Chi-square tests showed that these metaphorical expressions (0) were significantly more common than the positive (+) metaphorical expressions, χ2 =  = 14.222, p<.01 (− vs. +). However, there was no significant difference found between the number of neutral expressions (0) and the number of negative (−) ones, χ2 =  = 1.815, p>.05 (0 vs. −).As for adjective metaphors, the proportion of metaphors showing a negative meaning was highest. The results of Chi-square tests showed that these metaphorical expressions (−) were significantly more common than the positive (+) metaphorical expressions, χThese results show that nominal metaphors and predicative metaphors are both affected by the meaning of their vehicles and tend to show neutral meanings, while adjective metaphors tend to show negative meanings.2 =  = 6.234, p<.05 for adjective metaphors vs. nominal metaphors; χ2 =  = 5.357, p<.05 for adjective metaphors vs. predicative metaphors).Chi-square tests were conducted among the three types of metaphors. The results showed that adjective metaphors showed significantly more frequent negative meanings than the other two types of metaphors  = 28.174, p<.01 (+ vs. −); χ2 =  = 8.067, p<.01 (+ vs. 0).As for metaphors with vehicles having positive meanings See , the pro2 =  = 22.154, p<.01 (+ vs. −); χ2 =  = 8.758, p<.01 (+ vs. 0).As for nominal metaphors, the proportion of metaphors showing a positive meaning was highest. The results of Chi-square tests showed that these positive metaphorical expressions (+) were significantly more common than the other expressions, χ2 =  = 5.286, p>.05.As for predicative metaphors, the proportion of metaphors showing a positive meaning was highest. However, there was no significant difference found among the other types of metaphors, χ2 =  = 2.235, p>.05.As for adjective metaphors, the proportion of metaphors showing a positive meaning was highest. However, there was no significant difference found among the other metaphors, χ2 =  = 1.239, p>.05 for nominal metaphors vs. predicative metaphors; χ2 =  = 3.477, p>.05 for nominal metaphors vs. adjective metaphors).Although the proportion of nominal metaphors showing positive meanings was found to be the highest, Chi-square tests showed that there was no significant difference among the nominal metaphors and other metaphors  = 23.148, p<.01 (− vs. +); χ2 =  = 10.939, p<.01 (− vs. 0).As for metaphors with vehicles having negative meanings See , the proAs for nominal metaphors, we were unable to find metaphors using vehicles that were rated negative by the participants of the experiment.2 =  = 11.267, p<.01 (− vs. +); χ2 =  = 5.556, p<.05 (− vs. 0).As for predicative metaphors, the proportion of metaphors showing a negative meaning was highest. Chi-square tests showed that negative metaphorical expressions (−) were significantly more common than the other expressions, χ2 =  = 5.400, p<.05 (− vs. 0).As for adjective metaphors, the proportion of metaphors showing a negative meaning was highest. Chi-square tests showed that negative metaphorical expressions (−) were significantly more common than the other expressions, χWe further classified all the metaphors into either metaphors showing negative meanings or metaphors having positive and neutral meanings, and then compared the predicative metaphors and adjective metaphors using a Chi-square test.2 =  = .186, p>.05.The results of the Chi-square test showed no significant difference between predicative metaphors and adjective metaphors, χThis result suggests that metaphors using vehicles with negative meanings tended to be affected by the meaning of their vehicles. This tendency is the same for both nominal and predicative metaphors using vehicles with neutral meanings as well as for all of the metaphors using vehicles with positive meanings.In Experiment 2 we analyzed whether the evoking of negative meanings is a unique feature of adjective metaphors through a comparison with nominal and predicative metaphors. The results showed that the meanings of metaphors are generally affected by the meanings of their vehicles, and that all types of metaphors using vehicles with positive meanings tend to evoke positive meaning while all types of metaphors using vehicles with negative meanings tend to evoke negative meanings. However, as for metaphors with vehicles of neutral meaning, adjective metaphors evoked negative meanings significantly more frequently than both nominal and predicative metaphors. The results of Experiment 2 were thus consistent with those of Experiment 1, thereby verifying our hypothesis that the evoking of negative meanings is a unique feature of adjective metaphors. However, it is still unclear why negative meanings are evoked by adjective metaphors. Because adjective metaphors evoking the most negative meanings are used in very striking ways, such as in Trakl’s poems as well as Japanese literary texts introduced in the Introduction, we hypothesized that negative meanings evoked by adjective metaphors are related to poeticness. We tested this hypothesis in Experiment 3.The results of Experiment 2 suggested that the meanings of metaphors are generally affected by the meanings of their vehicles, and previous studies such as Osgood We selected 15 vehicles that were evaluated as significantly positive in a pre-experiment . Similarred silence (“akai seijaku”), blue taste (“aoi aji”), white today (“shiroi genzai”), beautiful footstep (“utsukushii ashioto”), bright touch (“akarui shokkaku”), soft intention (“yawarakai honne”), warm thinking (“atatakai kangae”), sweet silence (“amai seijaku”), delicious intention (“oishii honne”), crispy footstep (“koobashii ashioto”), safe thinking (“anzenna kangae”), gentle today (“yasashii genzai”), quiet taste (“shizukana aji”), pleasant footstep (“tanoshii ashioto”), and new silence (“atarashii seijaku”).We combined the vehicles and topics selected through the pre-experiments. Among 105 expressions, five expressions regarded as non-metaphors were excluded and we selected 100 adjective metaphors, including the following: Using the same procedure as for Experiments 1 and 2, participants were asked to evaluate how easily they understood each metaphor. The ratings were made on a seven-point scale ranging from −3 (very difficult) through 0 (not sure) to +3 (very easy). They were also asked to evaluate how conventional they felt each metaphor to be. The ratings were made on a seven-point scale from −3  through 0 (not sure) to +3 . We confirmed all the metaphors were from −2.0 to +2.0 on the accessibility scale and the conventionality scale and we ensured that the metaphors used in the experiments were neither incomprehensible nor too conventional.In Experiment 3 we tested the hypothesis that negative meanings evoked by adjective metaphors are related to poeticness. First we conducted a psychological experiment to grasp the participants’ basic impressions of the adjective metaphors. In the experiment, 60 Japanese males and females, aged 18–25, were asked to rate the meanings of the 100 adjective metaphors on a seven-point scale ranging from −3 (extremely negative) through 0 to +3 (extremely positive).Next, we conducted another psychological experiment to assess the poeticness of the 100 adjective metaphors. Fifty Japanese males and females, aged 20–24, participated in the experiment. These participants were different from those who participated in the previous impression rating experiment described above. They were asked to rate how poetic they felt each metaphor was on a seven-point scale ranging from −3  through 0 (not sure) to +3 (highly poetic).r = −0.336 (p<.05), and that the more poetic metaphors were evaluated, the more negatively they were evaluated.We conducted a correlation analysis between the poeticness ratings and the meaning evaluations of the adjective metaphors. Our hypothesis that negative meanings evoked by adjective metaphors are related to poeticness predicted that adjective metaphors would be rated as more poetic, and thereby evaluated as more negative, that is, closer to the negative pole on the impression scale. Thus, our hypothesis predicted that there would be a negative correlation between the evaluations of poeticness and the meanings of the adjective metaphors. The results showed that the correlation coefficient for poeticness and a negative evaluation was The results of Experiment 3 suggested the possibility that negative meanings evoked by adjective metaphors are related to poeticness, and that poetic metaphors evoke negative meanings more easily than less poetic metaphors. However, we do not know with certainty whether participants who felt that a specific metaphor is poetic tended to rate that metaphor negatively, because the ratings were collected from different groups of participants.The results of this study seem to be related to the results of Utsumi et al. My job is a jail” shot down all of my arguments” While numerous other cognitive studies of metaphors have focused on nominal metaphors such as “In Experiment 3, we explored the reason why adjective metaphors evoked negative meanings. The results suggested the possibility that negative meanings evoked by adjective metaphors are related to poeticness, and that poetic metaphors evoke negative meanings more easily than less poetic metaphors. However, we do not know with certainty whether participants who felt that a specific metaphor is poetic tended to rate that metaphor negatively, because the ratings were collected from different groups of participants. This result may be related to the results found by Utsumi et al. red voice” created from the neutral vehicle “red,” for example, the adjective “red” first evokes an intermediate category “red things,” to which “blood,” “fire,” “passion,” “apple,” and “danger,” typically belong. Then, exemplars relevant to the noun “voice” are selected, and these then evoke a final abstract category of property, such as “scary,” “screaming,” and “dangerous.” In this way, adjective metaphors are understood as not being directly mapped onto their topics from ad hoc categories of vehicles, but rather by mediating to an intermediate category. Therefore, supposing that adjective metaphors are comprehended in this two-stage categorization process, it seems reasonable that the prototypical meanings of their vehicles do not directly affect the meanings of adjective metaphors, and that adjective metaphors are more likely to evoke different meanings compared with the meanings of their vehicles by themselves, namely, the exemplars with negative meanings are selected among the various exemplars belonging to the intermediate category evoked by the adjectives as vehicles.The mechanisms of the negative effects evoked by adjective metaphors can be discussed in relation to the comprehension process revealed by previous research. A considerable number of cognitive studies on metaphors have examined the cognitive mechanism of metaphor comprehension. Most of these studies have focused on the comprehension process of nominal and predicative metaphors. Glucksberg and his colleagues This paper explored the cognitive effects evoked by adjective metaphors. Through a series of psychological experiments, we showed that adjective metaphors evoke negative meanings and, with respect to the metaphors for which their vehicles themselves have a neutral meaning, adjective metaphors evoked negative meanings significantly more frequently than both nominal and predicative metaphors. Our research is one of only a few studies to examine adjective metaphors, while many cognitive studies on metaphors have focused on nominal and predicative metaphors. Our research is also one of only a few studies to focus on the cognitive effects evoked by adjective metaphors; most other studies examining adjective metaphors have focused on how the acceptability of adjective metaphors as synesthetic metaphors can be explained by the pairing of the modalities of the adjective modifiers and head nouns. The results of our study have implications for future studies of metaphors, which represent one of the most basic human cognitive abilities.File S1Details of the SD scales used in Experiment 2.(DOCX)Click here for additional data file.File S2Details of the nouns with neutral meanings used in Experiment 2.(DOCX)Click here for additional data file.File S3Vehicles used in Experiment 2.(DOCX)Click here for additional data file.File S4Vehicles with positive meanings used in Experiment 3.(DOCX)Click here for additional data file.File S5Topics with neutral meanings used in Experiment 3.(DOCX)Click here for additional data file.


The relationship between dietary glycemic index, glycemic load and risk of coronary heart disease (CHD), stroke, and stroke-related mortality is inconsistent.We systematically searched the MEDLINE, EMBASE, and Science Citation Index Expanded databases using glycemic index, glycemic load, and cardiovascular disease and reference lists of retrieved articles up to April 30, 2012. We included prospective studies with glycemic index and glycemic load as the exposure and incidence of fatal and nonfatal CHD, stroke, and stroke-related mortality as the outcome variable. Pooled relative risks (RR) and 95% confidence intervals (CI) were calculated using random-effects models.P for interaction = 0.003). Higher dietary glycemic load, but not glycemic index, was positively associated with stroke . There is a linear dose-response relationship between dietary glycemic load and increased risk of CHD, with pooled RR of 1.05 (95%CI 1.02−1.08) per 50-unit increment in glycemic load level.Fifteen prospective studies with a total of 438,073 participants and 9,424 CHD cases, 2,123 stroke cases, and 342 deaths from stroke were included in the meta-analysis. Gender significantly modified the effects of glycemic index and glycemic load on CHD risk, and high glycemic load level was associated with higher risk of CHD in women , but not in men . Stratified meta-analysis by body mass index indicated that among overweight and obese subjects, dietary glycemic load level were associated with increased risk of CHD (RR = 1.49, 95%CI 1.27−1.76; High dietary glycemic load is associated with a higher risk of CHD and stroke, and there is a linear dose-response relationship between glycemic load and CHD risk. Dietary glycemic index is slightly associated with risk of CHD, but not with stroke and stroke-related death. Further studies are needed to verify the effects of gender and body weight on cardiovascular diseases. High carbohydrate intake has adverse effects on lipid and glucose metabolism Dietary GI and GL have increased in recent years because of increases in carbohydrate intake and changes in food processing, especially in the lower- and middle-income countries of the Asia-Pacific region Accumulating epidemiological studies have suggested that high dietary GI and GL could be detrimental in regard to the risk of coronary heart disease We searched for all published prospective studies that described the associations between GL, GI and the risk of incident CHD, stroke, and stroke-related mortality. A systematic literature search was performed using the MEDLINE (Pubmed) and EMBASE databases and was supplemented through the manual review of reference list of obtained articles up to April 30, 2012. The following terms were used: ((“glycaemic index”[All Fields] OR “glycemic index”[MeSH Terms] OR (“glycemic”[All Fields] AND “index”[All Fields]) OR “glycemic index”[All Fields]) OR (“glycemic”[All Fields] AND “load”[All Fields])) AND ((“coronary disease”[MeSH Terms] OR (“coronary”[All Fields] AND “disease”[All Fields]) OR “coronary disease”[All Fields] OR (“coronary”[All Fields] AND “heart”[All Fields] AND “disease”[All Fields]) OR “coronary heart disease”[All Fields] OR “coronary artery disease”[MeSH Terms] OR (“coronary”[All Fields] AND “artery”[All Fields] AND “disease”[All Fields]) OR “coronary artery disease”[All Fields] OR (“coronary”[All Fields] AND “heart”[All Fields] AND “disease”[All Fields])) OR (“stroke”[MeSH Terms] OR “stroke”[All Fields]) OR (“cardiovascular diseases”[MeSH Terms] OR (“cardiovascular”[All Fields] AND “diseases”[All Fields]) OR “cardiovascular diseases”[All Fields] OR (“cardiovascular”[All Fields] AND “disease”[All Fields]) OR “cardiovascular disease”[All Fields])). No language restriction was applied for searching and study inclusion. Our systematic review was conducted according to the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines Studies were considered eligible for meta-analysis if they met the following criteria: the study had a prospective design; the exposure was dietary GL or GI; the outcome was incident CHD or stroke; and the study excluded participants with known pre-existing cardiovascular disease. Because the nested case-control study in a prospective cohort is just an efficient sampling of the same cohort study and thus retains the same prospective advantages of the cohort, and dietary information was collected among apparently healthy participants at baseline before the development of outcome of interest, the study by Pierucci et al Data extraction was conducted independently by 2 authors , using a standardized data extraction form. To resolve discrepancies, a third investigator (W.Z.) was consulted. We contacted authors of the original studies in the case of missing data. For each included article, study characteristics were recorded as follows: authors, publication year, country of origin, name of study, study design, features of study population , duration of the follow-up, mean  or median values for the GI or GL, reference food used for GI calculation, the criterion for ascertainment of outcomes, numbers of incident CHD or stroke cases, and confounding factors that were adjusted for in the multivariable analysis. Accepted standardized quality scores for observational studies are not available. Therefore, study’s quality was assessed by review of study design, including inclusion and exclusion criteria, assessment of exposure, assessment of outcome, control of confounding, and evidence of bias. Each of the 5 quality criteria was evaluated and scored on an integer scale  and summed. Quality scores from 0 to 3 were considered lower quality and 4 to 5 higher quality.In the original articles which used tertiles, quartiles, quintiles, deciles, or percentiles of GI and GL as categories for dietary GI and GL levels, we extracted median values, numbers of cases/noncases, relative risks (RRs), and 95% confidence intervals (CIs). For studies that reported several multivariable-adjusted RRs, we extracted the effect estimate that was most fully adjusted for potential confounders. If medians for categories of dietary GL and GI were not reported, approximate medians were estimated using the midpoint of the lower and upper bounds (or using the mean when the midpoint could not be estimated).We used the multivariate-adjusted odds ratio and hazard ratio reported in the original articles, and the odds ratios in the nested case-control study design were assumed to be accurate estimates of risk ratio. We therefore consider these estimates as relative risks.In CHD risk-related analysis, records from the studies by Sieri et al In stroke risk-related analysis, 3 studies I2 statistics were used to examine statistical heterogeneity across studies. I2 was calculated based on the formula I2 = 100%×(Q–df)/Q.Fixed- and random-effects models were used to calculate the pooled risk estimates and 95% CI by comparing the highest and lowest categories of exposure. In the fixed-effects model, the pooled RR was obtained by averaging the lnRRs weighted by the inverses of their variances. In the random-effects model, DerSimonian and Laird’s method was used to further incorporate between-study heterogeneity In the secondary analysis, we estimated the dose-response relationship based on available data for categories of dietary GL or GI on median dose, number of cases and participants, and effect estimates with corresponding standard errors using the generalized least-squares trend estimation (GLST) analysis P value <0.05 was considered statistically significant. All analyses were performed using STATA 10.1 software .Potential publication bias was assessed by using the Egger’s regression test The results of the literature search are shown in P = 0.005) compared with the lowest category of dietary GI levels . A pronounced association with CHD risk was observed for dietary GL. Compared with the lowest category of dietary GL levels, higher GL levels were associated with a significant 28% increased risk for CHD . Additional sensitivity analysis that excluded the study by Mursu et al P>0.05).A total of 12 separate estimates from 10 studies I levels . There iP for interaction = 0.006 for GI; P for interaction = 0.005 for GL). A significant 49% increased risk of CHD for higher GL diet was observed in women  (P<0.001) in women (P = 0.90) in men  . Similarin women  and 0.99) in men .2 in 4 studies 2 in the Mursu study 2 and 29 kg/m2 in the Liu study P for interaction = 0.003) but not for GI (P for interaction = 0.11).We next assessed the potential effect modification by BMI on the relations of dietary GI and GL to CHD risk. For this analysis, results by BMI were available from 5 studies for dietary GI P = 0.25) for the highest versus the lowest category of GI levels. High dietary GL level was associated with 19% increased risk for stroke . No evidence of heterogeneity across studies was observed.A total of 5 separate estimates from 3 studies P = 0.07; A total of 3 separate estimates from 2 studies P = 0.97 for nonlinear response test), and for dietary GI  for CHD risk and 1.03  for stroke risk in per 50-unit increment of dietary GL levels, respectively Click here for additional data file.Figure S2Relative risks for the association between dietary GI or GL and risk of CHD in men. All the risk estimates and 95% CI were calculated by comparing the highest category with the lowest.(TIF)Click here for additional data file.Figure S3Relative risks for the associateion between dietary GI and stroke-related mortality. All the risk estimates and 95% CI were calculated by comparing the highest category with the lowest.(TIF)Click here for additional data file.Figure S4Relative risks of CHD and stroke by continuous dietary GI levels. The 2-stage generalized least-squares trend estimation (GLST) method (TIF)Click here for additional data file.Table S1Multi-variable adjusted RRs and 95%CI for CHD in the original articles in this meta-analysis.(PDF)Click here for additional data file.Table S2Multi-variable adjusted RRs and 95%CI for stroke in the original articles in this meta-analysis.(PDF)Click here for additional data file.Table S3Multi-variable adjusted RRs and 95%CI for stroke-related mortality in the original articles in this meta-analysis.(PDF)Click here for additional data file.


Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera ( Ixodes scapularis, Rhipicephalus (Boophilus) microplus and Amblyomma variegatum  microplus  to silence protein expression in ticks or tick cell lines has been quite limited. Narasimhan et al.  achieveda et al.  silencedI. scapularis cell lines has previously been examined in studies conducted by Kurtti et al. (Renilla luciferase (RLuc) was used as a model system .Ten cell lines representing four ixodid tick genera of medical and veterinary importance Table  were groRenilla luciferase (RLuc) designated SFV4-stRLuc, was used  and 50 μl of this suspension was then added to the cells at a multiplicity of infection (MOI) of 3.A strain of SFV expressing 6 cells/ml in either sealed flat-sided culture tubes  or 24 well plates  and incubated in ambient air at 28 °C. The dsRNA (400 ng per tube or 200 ng per well of cells) or siRNA (50 nM concentration) was first incubated at room temperature in OptiMEM  for 5 min and then mixed with the transfection reagent (1 or 2 μl of transfection reagent (siRNA or dsRNA respectively) + 49 μl OptiMEM) and incubated for 20 min at room temperature. OptiMEM alone was used if no transfection reagent was required. Immediately before addition of test reagents, the volume of culture medium in each tube or well was reduced by half. Aliquots of 100 μl of incubated transfection mix were added to the medium in each tube or well and the cells were incubated at 28 °C for 24 h. Fresh complete culture medium was then added to restore the original volume if longer incubation periods were required.The transfection reagents Lipofectamine 2000 (Invitrogen), XtremeGENE siRNA transfection reagent (Roche), DharmaFECT 1 (Thermo Scientific), HiPerFect (Qiagen), Lipofectamine RNAiMAX (Invitrogen) and siPORT Amine transfection reagent (Ambion) were tested. Cells for transfection and RNAi experiments were seeded 24 h prior to use at 0.7–1.0 × 10RLuc gene from the SFV4-stRLuc genome, the endogenous gene subolesin  following the manufacturer’s instructions. dsRNA targeting enhanced green fluorescent protein (eGFP) was used as a negative control. The primers and PCR conditions used are listed in Table To make long dsRNA (approximately 600 bp) Ambion’s MEGAscript RNAi Kit was used. Gene-specific primers linked with T7 promoter regions were used to amplify regions of the Long dsRNAs were made as described above and then Shortcut RNase III (New England Biolabs) was used to cleave the long dsRNA into a heterogeneous pool of siRNAs. This cleavage was confirmed by gel electrophoresis. Block-it green fluorescent siRNA (Invitrogen) was used to analyse transfection efficiency. Scrambled, non-targeting siRNA (Thermo Scientific) was used as a negative control.RLuc virus 24 h later. The cultures were incubated for a further 48 h before RLuc production was measured by luciferase assay as follows. Equal numbers of cells from each replicate of each cell line to be assayed were isolated by centrifugation and lysed with a passive lysis buffer (Promega). RLuc expression levels were assessed using a Luciferase reporter assay system (Promega) and a GloMax Luminometer (Promega) following the manufacturer’s protocols.Cells were transfected with siRNA or dsRNA as described above and were then infected with SFV4-stCells were transfected with long dsRNA as described above and incubated for 24–96 h. RNA was then extracted from the cell cultures using an RNeasy kit (Qiagen). Equal amounts of RNA were reverse transcribed into cDNA and then real-time PCR using gene-specific primers was used to quantify levels of RNA transcripts. The housekeeping gene β-actin was used as a reference control gene. The primers and conditions used for the real-time PCR are listed in Table For fluorescence and light microscopy, a Zeiss Axiovert inverted microscope with observer D1 was used. The photomicrographs were taken and analysed using Zeiss Axiovision software. For each cell line a minimum of 400 cells were counted at 100× magnification with simultaneous normal transmitted light and UV reflected light 24 h post-transfection. Confocal microscopy was carried out using a Zeiss LSM710 confocal microscope with Zeiss Zen software.t test was used to determine significant differences between groups in experiments involving measurement of luciferase activity or real-time PCR. A p value of <0.05 was considered significant.The unpaired two-sample Generally, at least in mammalian cells, siRNAs are delivered with a transfection reagent such as liposomes. To investigate whether transfection reagents are required for tick cell lines and to determine transfection efficiency and toxicity, six different transfection reagents were tested on two tick cell lines, BDE/CTVM16 and IRE/CTVM19. Both cell lines grow as single cells rather than connected cell sheets and are therefore easy to count visually. The cells were incubated alone or with green fluorescent siRNA in the presence or absence of a transfection reagent and the number of green cells in each culture was quantified by fluorescence microscopy (minimum 400 cells counted); Fig. R. (B.) microplus line BME/CTVM23. There was, however, wide variation in transfection efficiency between cell lines with both reagents. For example, low transfection levels were achieved with IDE8 and ISE6 cells with both reagents while IRE/CTVM19 cells showed low levels of transfection with Lipofectamine 2000 but high levels with Xtreme. By day 4, the proportion of positive cells in all cultures had decreased by 10–30 %; by day 14 no green cells were present in HAE/CTVM9 and IDE8 for both reagents and RA257 for Lipofectamine 2000, while green cells remained until day 28, albeit in decreasing numbers, in AVL/CTVM13, BME/CTVM23 and RA257 transfected with Xtreme. It was also noticeable that Xtreme had little apparent toxic effect on the cells, while over time treatment with Lipofectamine 2000 adversely affected the cells, especially after the first week of treatment.Following the same protocol, Lipofectamine 2000 and Xtreme were tested on a further seven cell lines. Presence of fluorescence was quantified by microscopy Table . OverallRLuc has been engineered to express RLuc when it infects cells . RNA was extracted from the cells, total amounts of RNA were measured, equal amounts of RNA were reverse transcribed into cDNA and subolesin RNA transcript levels were then measured by real-time PCR. β-actin was used as a reference gene. Subolesin has been previously implicated in various functions including tick immunity and has been successfully silenced by RNAi in previous studies  microplus cell line BME26 which has also been used previously  microplus line BME/CTVM23, and the R. (B.) decoloratus line BDE/CTVM16. We also examined cell lines from four other economically important tick species: A. variegatum (AVL/CTVM13), H. anatolicum (HAE/CTVM9), R. appendiculatus (RA257 and RAE/CTVM1) and R. evertsi (REE/CTVM28).To address this we assembled a panel of cell lines derived from eight different tick species within four ixodid genera of medical and veterinary importance. We included three of the cell lines which have been used previously for gene silencing by other research groups, namely IDE8, ISE6 and IRE/CTVM19  or long dsRNA (with or without a transfection reagent) against RLuc and then infected with SFV-stRLuc. Both the long dsRNA and the siRNA treatments were effective. The levels of RLuc protein decreased significantly in many of the treated cultures. According to Garcia et al.  did not respond to dsRNA despite responding to siRNA. This suggests that these cells may be unable to process long dsRNA efficiently or correctly, possibly through a loss of Dicer functionality similar to that seen in the mosquito cell line C6/36  microplus cell line BME/CTVM23 was negligible with long dsRNA and poor with siRNA, even when a transfection reagent was used. In contrast, using dsRNA in the cell line BME26, derived from the same tick species, Zivkovic et al. (Because long dsRNA appeared to be more efficient than siRNA at knocking down virus protein expression in some cell lines, long dsRNAs were used to test other variables such as cell type and incubation time when trying to knock down an endogenous gene. The two genes selected, subolesin and 2I1F6 hematopoietic stem/progenitor cells protein-like, have both been successfully silenced previously in c et al.  reportedc et al.  achievedIn previous studies, the effect of long dsRNA on tick cells was measured after incubation for between 72 and 120 h (Blouin et al. Overall, our results indicate the importance of selecting the most suitable tick cell line for the intended research. While conditions for different cell lines varied, parameters have been established that can be followed and can consistently produce results. The methods described in this study provide a template that can be followed and adapted according to requirements as research with different tick cell lines increases.


Three lines of evidence support this possibility. 1) Shales ingest gas in amounts and selectivities consistent with gas-carbon equilibrium. There is a 50% increase in solid hydrocarbon mass when Fayetteville Shale is exposed to methane (450 psi) under moderate conditions (100°C): Rock-Eval S2 (mg g-1) 8.5 = > 12.5. All light hydrocarbons are ingested, but with high selectivity, consistent with competitive addition to receptor sites in a growing polymer. Mowry Shale ingests butane vigorously from argon, for example, but not from methane under the same conditions. 2) Production data for a well producing from Fayetteville Shale declines along the theoretical curve for withdrawing gas from higher hydrocarbons in equilibrium with carbon. 3) A new general gas-solid equilibrium model accounts for natural gas at thermodynamic equilibrium, and C6-C7 hydrocarbons constrained to invariant compositions. The results make a strong case for methane in equilibrium with carbon and higher hydrocarbons. If correct, the higher hydrocarbons in source rocks are gas reservoirs, raising the possibility of substantially more gas in shales than analytically apparent, and far more gas in shale deposits than currently recognized.Methane in source rocks may not exist exclusively as free gas. It could exist in equilibrium with carbon and higher hydrocarbons: CH Few questions in geoscience are as interesting and controversial as the origin of methane in natural gas. And fewer go as far back in time. Evans proposed thermal cracking in 1971 to explain oil trending to methane with depth in a Canadian basin . ThermalCatalysis by transition metals explains these properties -7, and tCatalysis accounts for the composition of natural gas, but other questions remain troubling. Shales ingest and expel gas in the laboratory in ways contradicting classical physics. Non-classical behavior is apparent in chaotic generation curves , and in 3/CaO, its pressure increases and falls with temperature as a thermodynamic function of the equilibrium constant. Methane could similarly exist in two states, gaseous methane and solid-state methane, at equilibrium. It accounts for the results presented here and the non-classical behavior in earlier experiments.The premise that methane should be independent of the solid hydrocarbons generating it could be flawed. If generation is catalytic and reversible, methane and its source would be more like carbon dioxide and calcite at equilibrium. Carbon dioxide is never independent of calcite. Ingested and expelled reversibly from CaCOTheory). The second part is the supporting experimental evidence (Ingestion), and the third contains a kinetic model of the theoretical equilibrium (Gas-Solid Equilibrium Kinetic Simulations). A new general equilibrium model for light hydrocarbon generation summarizes the results.An equilibrium hypothesis is presented in three parts. The general concept is laid out first  illustrates the concept in its simplest form.4’ should apply to the higher light hydrocarbons as well, although methane is the primary focus here.The equilibrium hypothesis and supporting evidence are discussed below. Here, we consider the origin of solid hydrocarbons, also referred to as ‘carbon pools’. ‘Methane’ is used throughout to illustrate relationships for simplicity. All references to ‘methane’ or ‘CHThe existence of solid hydrocarbons in source rocks is not new. They have been recognized by organic geochemists for decades. Generating gas from higher hydrocarbons necessarily generates a carbon deposit to balance hydrogen. It is the text-book explanation for the origin of pyrobitumen, for example, a ubiquitous organic mineral in sources rocks with H/C ratios ranging from about .5 to 1.6 . Pyrobit2 and graphite CH0. The activated carbon suggested by Alexander et al. . However, in contrast to adsorption and solution, hydrocarbons compete for [···]. Thus rates of ingestion are not independent of other hydrocarbons.Assume that hydrocarbons add to the solid hydrocarbons in source rocks differently. Hydrocarbon X adds to some receptor [···] generating the adduct [X], a process referred to here as ‘ingestion’. X disappears by second-order kinetics at rates proportional to X and [···]: independent of some other hydrocarbon Y. If, however, X reacts with [···], and Y competes with X for [···], X will go into the solid at rates inversely proportional to Y.We can distinguish between ingestion and either adsorption or solution through competitive addition. If solid hydrocarbons are mere solvents, hydrocarbon X will go into solid-solution at rates proportional to concentrations of X, Do hydrocarbons compete in their addition to solid hydrocarbons?iso-butane and n-butane (C2-C4). Both vials were heated at 75°C for 200 hours. One  was diluted with argon by 50% five times over the 200 hours and the other  was diluted with equal amounts of methane five times. The only difference in the two reactions was dilutions with argon in one  and dilutions with methane in the other . Thus, C2-C4 gas concentrations in each vial diminished equally with each argon and methane dilution. Concentrations over time should be about the same in A and B if Mowry Shale is passive and only removes C2-C4 hydrocarbons by adsorption or solution. If it removes them by ingestion, and C1-C4 hydrocarbons compete for [···], C2-C4 concentrations in Vial A should fall progressively below those in Vial B.To put this question to experimental test, two vials were charged with identical amounts of Mowry Shale and an equal molar mixture of ethane, propane, 2-C4 hydrocarbon mixture was added and the vial was heated and diluted with methane as was Vial B.Another vial (C) was charged with beach sand with a thin coating of n-octadecane (~ 1%) to assess adsorption and solution. The C2-C4)A / ∑ (C2-C4)B should remain constant over time. If, however, Mowry Shale ingests C2-C4, the ratio of slums ∑ (C2-C4)A / ∑ (C2-C4)B should progress to zero over time.If Mowry Shale consumes hydrocarbons classically through adsorption and solution exclusively, the ratio of sums ∑ (C2-C4) over time in the three vials are shown in Table A and B over time are dramatic. Mowry Shale consumed 163 μg C2-C4 g-1 in argon  compared to under 30 μg g-1 in methane . Figure A and B. Concentrations of n-butane in B fall exponentially consistent with sequential 50% dilutions, but% composition of C2-C4 remains essentially constant over time  fell sharply in A and remained essentially constant in B  consistent with acid-catalyzed isomerization promoted by the mild acidity of beach sand  where [···] denotes the concentration of open catalytic sites, the receptors noted above. [Cx] forms through reaction (3) from [Cn], a high molecular weight hydrocarbon bonded to an active site, for example (CH2)n-M where n > > x, an intermediate discussed elsewhere [lsewhere .n] and [Cn-x] in reaction 3 are indistinguishable when n is large (n ~ n-x), and that [Cn] is therefore a constant in the kinetics of [Cx] generation. Gas generation proceeds through reaction (4), a reversible reaction. [···] bonds selectively to various hydrocarbons, and only to hydrocarbons.We shall assume  + H2 = (Cx) + [···]) [x] generating (Cx) and from (Cx) to the pool generating [Cx].A hydrogen balance is ignored throughout this scheme. All hydrocarbons in brackets are unsaturated, C+ [···]) . We assuReactions (3) and (4) are within a closed system, a sealed source rock, for example. Gas escapes confinement through reaction (5).x] and [Cn].Reaction (6) summarizes the kinetic steps interconverting free gas and solid-state gas as symbolized by [Cx] generation is proportional to [···]*[Cn]. Because [Cn] is constant and [···] becomes constant over time (steady-state), the rate of gas generation from solid hydrocarbon is constant over time. It will continue generating [Cx] and free gas (Cx) until their concentrations become sufficiently high to promote reverse reactions at rates equal to forward reactions. At that point, reaction (6) is at steady-state (equilibrium). (Cx) and [Cx] are then at their maximum concentrations, and [···] is at its minimum concentration. This is the state of a sealed (closed) source rock in the subsurface. We are interested in the dynamics of that system when the rock is opened, and reaction (5) is significant. How will (Cx) decline over time when the system is at equilibrium and the rate of reaction (5) exceeds the rate of generation (reaction 3)?The rate of [Cx). [Cn], [Cx], and [···] are all zero. In the second, (Cx) and [Cx] interconvert at equilibrium, but [Cn] does not generate [Cx]; gas generation does not attend gas release, in other words. In the third, reaction (6) is fully operative, two reservoirs of gas, [Cx] and (Cx), at equilibrium, are depleted while [Cx] is generated at a constant rate from [Cn].Three possibilities are considered in the following kinetic simulations. The first is classical first-order expulsion without generation or equilibrium of any kind. The kinetic model thus contains only free, in-place gas  = (0.2*(Cx)), and all other concentrations altered accordingly as described in Figure x) over iterations simulates expulsion rates over time. ∆(Cx) is henceforth referred to as ‘rate’ denoted R and iteration as ‘time’ (t). Figure t is the expulsion rate at time t, (R)i is the initial expulsion rate for free gas (Cx), [R]i is the initial expulsion rate for solid hydrocarbon gas [Cx], and C is the constant for rate of generation from [Cn].Each possibility is treated assuming equal rate constants and equivalent intermediate concentrations. Figure Figure Gas production Miller Heirs well, Fayetteville Shale - Many gas wells decline classically, by simple exponential decay, but there are notable exceptions mainly in low porosity-permeability wells including unconventional wells  and [···]:It is in this context that methane-solid hydrocarbon equilibrium must be weighed. With methane in equilibrium with ethane and propane , it shoux] represents the intermediate shaping product structures and compositions. Metathesis of [Cx] brings C1 to C4 to equilibrium [x] bring C5 to C7 isomers to invariance [x] and thus to solid hydrocarbons, is the critical step in the proposed methane-solid hydrocarbon equilibrium. Mowry and Fayetteville Shales ingest light hydrocarbons in substantial amounts and with high selectivity. Mowry Shale in argon with open receptors ingests C2-C4 hydrocarbons vigorously, and the same shale in methane with pacified receptors totally rejects the same hydrocarbons under the same conditions . It therefore follows that Cuilibria -28, dispneration . UnconveProduction rates declining by DED1 is not in itself significant. But, the coincidence of theory and data in Figure 4. It does not emerge in RockEval analysis in the S1 peak at 300°C, but later as some higher hydrocarbon in the S2 peak at 443°C (Table 1 + [···] = > [C1]. The reactions between C1-C4 hydrocarbons and Mowry Shale at 70°C are equally striking. Adamantanes, like methane in thermal stability and reactivity, become highly reactive on carbon surfaces [Ingestion gives the hypothesis additional and independent support. First, it is unprecedented. Light hydrocarbons react with solid hydrocarbons in source rocks under conditions where they do not react with ordinary solid hydrocarbons. Methane, perhaps the least reactive hydrocarbon known, disappears in Fayetteville Shale at 100°C leaving no trace of CHOnly the question of reversibility and perhaps generation remain. Catalysis would seem a given since methane could not react with solid hydrocarbon at 70°C Table  without The author declares that there are no competing interests.Author thanks Petroleum Habitats and Worldwide Geochemistry for their generous support.


Differences in the squid “on” and “off” states are clearly distinguishable as bimodal and unimodal pair distance distribution functions respectively. These observations are consistent with a Ca2+-free squid heavy meromyosin that is compact, but which becomes extended when Ca2+ is bound. Further, the scattering profile derived from the current model of tarantula heavy meromyosin in the “off” state is in excellent agreement with the measured “off” state scattering profile for squid heavy meromyosin. The previous and current studies together provide significant evidence that regulated myosin's compact off-state conformation is an ancient trait, inherited from a common ancestor during divergent evolution.The overall conformations of regulated myosins or heavy meromyosins from chicken/turkey, scallop, tarantula, limulus, and scorpion sources have been studied by a number of techniques, including electron microscopy, sedimentation, and pulsed electron paramagnetic resonance. These studies have indicated that the binding of regulatory ions changes the conformation of the molecule from a compact shape found in the “off” state of the muscle to extended relationships between the tail and independently mobile heads that predominate in the “on” state. Here we strengthen the argument for the generality of this conformational change by using small angle X-ray scattering on heavy meromyosin from squid. Small angle X-ray scattering allows the protein to be visualized in solution under mild and relatively physiological conditions, and squid differs from the other species studied by at least 500 million years of evolution. Analysis of the data indicates that upon addition of Ca Vertebrate striated muscle contraction is regulated by the thin filament's troponin/tropomyosin complex. By contrast, myosin itself see  controls2+ yields a slower sedimentation velocity than that observed in the presence of EGTA Despite these different triggers for contractile activity in regulated myosins, both regulatory ions appear to produce a similar conformational change: from a generally compact off-state conformation, where the two heads are folded back towards the tail and contact each other asymmetrically, to an on state where the tail and independently mobile heads have a somewhat more extended relationship. Compact shapes of the off state of HMM, myosin, and/or the thick filament have been viewed directly, and in some cases to ∼20 Å resolution, by electron microscopy No atomic resolution structure of the myosin dimer or of HMM is available, and questions also remain concerning the current low-resolution descriptions. A technical concern, for example, is the conditions under which the compact conformation has been determined. The sedimentation, electron microscopy or EPR techniques that have been employed subject the protein to relatively unphysiological and/or extreme conditions. Sedimentation uses high g forces, and negative staining or freezing have been employed for the electron microscopy and EPR experiments. Another question about the compact off-state conformation involves its exact role in evolutionary biology. Although this conformation has been shown to be present in species separated by 600 million years of evolution We have now extended these studies to another species and have used another biophysical technique, with the aim of assessing both the current picture of how myosin changes its shape during regulation, as well as the evolutionary history of myosin-linked regulation. These studies involve examining the active and off-state conformations of HMM in solutions of physiological buffers using small angle X-ray scattering (SAXS). Moreover, these experiments were performed on HMM from squid, which is separated by at least 500 million years of evolution from any of the other species for which studies of the off-state myosin have been described previously. Our results and analyses have fortified our understanding of the overall conformational change that occurs for myosin upon activation.g) and intensity at zero scattering angle (I(0)) were computed using the Guinier method as implemented in RAW . Using the same analysis, the “on” state yields a Guinier-derived Rg value of 77±2 Å. The IFT analysis gives a somewhat higher value of 83.9±0.8 Å with maximum diameter of 290 Å. The “on” state is systematically larger than the “off” state as measured by Rg regardless of maximum diameter setting chosen.Radius of gyration , nor was any radiation damage observed upon comparing curves of different exposures . Kratky Molecular weight estimates based on a glucose isomerase standard (1.25 mg/ml) give 268±45 kDa for the “off” state and 241±12 kDa for the “on” state. Independent molecular weight estimates based on a corrected Porod volume give 317 kDa for the “off” state and 262 kDa for the “on” state 2-weighted distribution of all possible electron pair distances within the protein. The maximum diameter object observable in this experiment is set by the Shannon Limit π/qmin = 312 Å where qmin is the minimum observable q value. In the absence of calcium (EGTA+AMP.PNP), the protein assumes a unimodal P(r) distribution characteristic of a simple compact globular shape (solid line in 2++AMP.PNP) the protein gives a more extended bimodal P(r) distribution indicating the presence of two separated domains , for both states were calculated using the inverse Fourier method as implemented in the GNOM program 2+-free (EGTA) squid HMM shows excellent agreement  greement . The Ca22+ squid HMM is elongated and somewhat narrower, but otherwise similar to the non-Ca2+ case , squid is quite distinctive. Squid and scallop belong to different molluscan classes: scallop is a bivalve, whereas squid is a cephalopod — a uniquely “intelligent” class of mollusc. These two classes diverged from one another about 500 million years ago 2+  the descendants if the sample size is small, say consisting of fewer than about 6 cases . By contSuch is the result of our SAXS studies on the regulatory structure of squid myosin. Off-state regulated myosin  from two closely related vertebrate sources  were obtained from the Marine Biological Laboratory, Woods Hole, MA., where the funnel retractor muscle was harvested. Myosin was prepared as described Squid (Hydroxylapatite (HA) column purification of the crude HMM was performed as described 3, 2.5 mM MgCl2, 0.5 mM Tris(2-carboxyethyl)-Phosphine Hydrochloride (TCEP), 0.5 µg/ml leupeptin]. Pure fractions were combined, and 0.2 mM AMP.PNP was added.Previously frozen HA-purified HMM (100 mM K.Pi) was thawed for five minutes in a room temperature water bath, then run over a Pharmacia Superdex 200 column at room temperature in: [40 mM Na Malonate (pH 7), 20 mM 4-(N-Morpholino)butanesulfonic acid (MOBS) (pH 7), 3 mM NaNThis sample was divided in half, and 1 mM EGTA was added to the “off” sample. This was concentrated (Centricon 10), AMP.PNP was increased to 2 mM, and the sample was clarified . Protein concentration = 2.25 mg/ml, determined by the Bradford method (using BioRad Protein assay solution). The final AMP.PNP and EGTA molarities were added to a small portion of the column buffer for SAXS dilutions.2, this sample was concentrated (Centricon 10). AMP.PNP was increased to 2 mM final, and the sample was clarified . Protein concentration = 2.2 mg/ml. The final AMP.PNP and CaCl2 molarities were added to a small portion of the column buffer for SAXS dilutions.The other half of the 0.2 mM AMP.PNP pure fraction combination described above was used for the on state sample. After addition of 0.5 mM CaCl2 at 1.25 mg/ml. The concentration of the glucose isomerase standard was determined by the A280 method using the reported extinction coefficient of 45660 M−1cm−1Glucose isomerase used as the SAXS molecular weight standard  was prepared in 10 mM HEPES buffer at pH 4.0 with 1 mM MgCl10 photons/s. The X-ray beam was collimated to 250×250 µm2 diameter and centered on a 2 mm diameter vertical quartz capillary tube with 10 µm thick walls . The capillary tube and full X-ray flight path, including beamstop, were kept in vacuo to eliminate air scatter. Sample plugs of approximately 15–20 µl were delivered from a 96-well plate to the capillary using a Hudson SOLO single-channel pipetting robot . To reduce radiation damage, sample plugs were oscillated in the X-ray beam using a computer-controlled syringe pump . Images were collected on a Pilatus 100K-S detector  with sequential 180 s exposures being used to assess possible radiation damage. Sample and buffer solutions were normalized to equivalent exposure before subtraction using beamstop photodiode counts. The complete automated BioSAXS system is described elsewhereAll samples were centrifuged at 14,000 RPM for ten minutes prior to data collection. Each sample was prepared at three concentrations  to assess concentration dependence. SAXS data were collected on CHESS beamline F2 at 9.881 keV  at 1×10min = 0.01 Å−1 to qmax = 0.27 Å−1.Sample-to-detector distance was calibrated using silver behenate powder . Images were reduced to profiles and buffer subtracted using the BioXTAS RAW software g) was calculated using both the method of Guinier www.if.sc.usp.br/~saxs/) Radius of gyration (Rmax) with total estimate scores2 values being examined at each step. The maximum diameter of the protein is not a well-defined quantity, especially in the presence of mixtures of conformations, higher-order oligomers, or aggregates. Values of Dmax chosen for comparison of P(r) functions were the smallest values that yielded smooth decay of the curve to zero while staying below the Shannon limit set by the experimental apparatus: Dmax<π/qmin.The Inverse Fourier Transform (IFT) method implemented by GNOM was run multiple times over a range of possible values for the maximum diameter .(DAT)Click here for additional data file.Data S2Small angle x-ray solution scattering profile for squid heavy meromyosin+EGTA+AMP.PNP. The profile is plain text in three-column format .(DAT)Click here for additional data file.Figure S1Guinier plots of HMM samples in the presence and absence of Ca2+. Both samples show some systematic deviation from linearity at lowest q, which indicates the presence of larger components (aggregation or higher-order oligomers) in the preparation. All low q values are shown in the Guinier plots. In all cases except the most dilute profile, qRg≈1.3 for the highest q value used in the analysis. The three lowest q values in the HMM+EGTA+AMP.PNP profile were excluded from the fitting to minimize the influence of the apparent aggregation.(TIFF)Click here for additional data file.Figure S2Superposition of the Guinier curves for the highest concentrations of both “on” and “off” states. Significant linear regions exist in both profiles despite some apparent aggregation and the Rg values are significantly different. Note that the HMM+EGTA+AMP.PNP profile has been shifted down by a small arbitrary increment for clarity of comparison.(TIFF)Click here for additional data file.Figure S3Kratky plots for Squid HMM in both the absence (HMM+EGTA+AMP.PNP) and presence (HMM+Ca2+AMP.PNP) of Ca2+. The fall-off of the tails in the Porod region (high q) indicates that both states are folded.(TIFF)Click here for additional data file.Figure S4Comparison of experimental data to inverse Fourier solutions for the HMM+EGTA+AMP.PNP. Two different choices for the maximum diameter are shown here , but the {back transformed) inverse Fourier solutions are nearly the same within the range sampled by the experimental data. The slight deviation from linearity seen in the small-angle portion of the Guinier plot  appears (TIFF)Click here for additional data file.Figure S5Comparison of experimental data to inverse Fourier solution for HMM+Ca2+AMP.PNP.(TIFF)Click here for additional data file.Figure S6Alignment of homologous tarantula HMM structure with envelope for HMM+EGTA+AMP.PNP (blue). The HMM+Ca2++AMP.PNP state is similar, but somewhat more extended (pink).(TIFF)Click here for additional data file.Figure S7Experimental data for HMM+EGTA+AMP.PNP (blue) superimposed on the dummy atom (DAMMIF) model (red).(TIFF)Click here for additional data file.Figure S8Experimental data for HMM+Ca2+AMP.PNP (blue) superimposed on the dummy atom (DAMMIF) model (red).(TIFF)Click here for additional data file.Methods S1Detailed procedure, parameters and references for inverse Fourier transform calculations and molecular shape reconstructions of squid heavy meromyosin samples.(DOCX)Click here for additional data file.Table S1Guinier analysis parameters for all dilutions of HMM samples in the absence (HMM+EGTA+AMP.PNP) and presence (HMM+Ca2+AMP.PNP) of Ca2+ ions. Data are given for the first and second successive 180 s x-ray exposures (exposure 1 and exposure 2 respectively) when available. Parameter qStart gives the smallest q value in the fitting range. The product qRg is the value calculated at the maximum q value used in the fitting range. The parameter rsq is goodness of fit and I(0) is the intensity extrapolated to q = 0. Rg values computed for the inverse Fourier Transform (IFT) method are the real-space values reported by the program GNOM 1.(DOCX)Click here for additional data file.


The suspension module control system model has been established based on MIMO (multiple input and multiple output) state feedback linearization. We have completed decoupling between double suspension points, and the new decoupling method has been applied to CMS04 magnetic suspension vehicle in national mid-low-speed maglev experiment field of Tangshan city in China. Double suspension system model is very accurate for investigating stability property of maglev control system. When magnetic flux signal is taken back to the suspension control system, the suspension module's antijamming capacity for resisting suspension load variety has been proved. Also, the external force interference has been enhanced. As a result, the robustness and stability properties of double-electromagnet suspension control system have been enhanced. Maglev vehicle has minor noise, no pollution safety, and comfort many other advantages, so the maglev traffic has a bright prospect in the future. Maglev vehicle concludes several complex subsystems  with couEMS middle- and low-speed maglev vehicle applies modular suspension bogies, and one module concludes two suspension points. Double suspension points' suspension task is accomplished by one suspension controller. the leakage flux of magnetic winding is neglected;z(t) evenly;the magnetic resistances of the ferrite core and rail are neglected; namely, magnetic potential falls on air gap the inclination angle of magnetic rigid body is minuscule, and active point of magnetic force is considered invariable;the action line of load forces is superposed with direction of suspension gap measured;O.distribution of mass of the bracket is even, and the masses of two magnets are equal, so the action point of gravity is superposed on the geometry center Assume the following:Based on above assumptions, the force analysis and geometrical relationship of maglev system are showed in Signs of Fi = 0 and ∑Mi = 0. The kinematics equations of maglev system can be got as follows:When electromagnet module is in condition of balance, we can obtain the equation ∑Extract acceleration signals from :(2)z¨1=A = (−2L − 3l)/2ML, B = (−2L + 3l)/2ML, C = (L + 3l)/ML, and D = (L − 3l)/ML.Set Simplify :(3)z¨1=z1, z2 and real physical positions d1, d2 isWe can get geometrical relationship of parameters in Magnetic force equations are as follows:Voltage balance equations of electromagnet winding are denoted in :(6)u1=2Substituting  into 6)6), we geFrom , we obtaOn all accounts, the dynamic law of maglev system can be determined by the following equations:Choose Simplify :(11)x˙= MIMO affine nonlinear system is described asx0 ∈ X. The problem of MIMO nonlinear system feedback linearization will have a solution, if and only if a neighborhood U near x0 and m functions h1(x),…, hm(x) exists. The solution makes relative degree  of nonlinear system  and g(x):Define the equilibrium point of maglev system Namely, above equations satisfyH(x) = 4(A2 − B2) · x5x6/μ02N2 ≠ 0; then E(x) is not singular. Relative degree of maglev system is  = , andAt the moment, det  Therefore, f(x) and h(x) as follows:Compute the vector field generated by u = H−1(x)[−b(x) + v], where the terms u = [u1 u2]T, v = [v1 v2]T, andDesign feedback control value We can obtain the control value after linearization from :(20)u=HThe diffeomorphic mapping matrix isx):Choose the coordinates of transformation by matrix ΦMMc) = 6, maglev system after linearization is controlled completely. So we design double suspension controller for regulating control properties of suspension system:Because of rank  , which is showed in Standard maglev bogie's parameters are given in Decoupling property test comes true on one suspension point of maglev bogie. Set expected position 10 mm. When maglev system becomes stable, we add additional square signal with amplitude 0.5 mm and period 4 s. Observe suspension signals with two control algorithms S1 and S2. Experiment results are showed in Figures Experiment results illustrate that when double suspension control system applies algorithm S2, the dynamic decoupling problem has been worked out. S2 raises the robustness and stability properties of maglev control system, but method S1 has no decoupling effect. Double suspension control model has been founded with magnetic flux signal based on MIMO feedback linearization. The feedback linearization algorithm enables accurate linearizing model to keep all properties of original nonlinear system which overcome the disadvantages of Taylor's expansion linearization method. In order to work out dynamic coupling problem and external interference problem of EMS mid-low-speed maglev vehicle, we take magnetic flux signal back to maglev control system and design double suspension compensable controller. Some experiments about new algorithm have been done in maglev vehicle CMS04 designed by NUDT. Experiment results demonstrate that double suspension module is precise based on MIMO state feedback linearization theory. With magnetic flux feedback, the maglev control system has better robustness and adaptability than traditional algorithm.


Some modern agrochemicals with antioxidant potential were tested for their protective effect against ozone injury using clover and tobacco ozone-sensitive cultivars as model plants subjected to ambient ozone at two sites (Kyiv city in Ukraine and Szarów village in Poland). All used agrochemicals showed partial protective effects against ozone injury on clover and tobacco. Conducted studies confirmed the effectiveness of modern fungicides belonging to strobilurin group as protectants of sensitive crops against ozone damage. The effectiveness of new growth regulators “Emistym C” and “Agrostymulin” was showed for the first time. Out of the studied agrochemicals, fungicide “Strobi” and natural growth regulator “Emistym C” demonstrated the best protective effects. These agrochemicals present promise for further studies of their possible utilization for enhancement of ozone tolerance of sensitive crops.  Ozone is one of the major toxic to vegetation and human health gaseous air pollutants. It contributes to crop losses and forest decline. It was estimated that the present day global relative yield losses range between 7% and 12% for wheat, between 6% and 16% for soybean, between 3% and 4% for rice, and between 3% and 5% for maize (range resulting from different metrics used) [3-tolerant cultivars; (ii) application of chemical protectants. So far there has been only limited success in producing genetically transformed plants with increased tolerance to elevated ozone concentrations because plant resistance to ozone is a very complicated phenomenon involving multiple signaling pathways and defense responses [The ozone-induced crop losses could be mitigated by two ways: (i) selection of Oesponses .The phytotoxicity of ozone results primarily from the oxidative stress imposed by the pollutant on sensitive components of the plasmalemma , 5. In cPresently, modern agrochemicals containing antioxidant compounds, such as fungicides belonging to strobilurins and triazoles , are gaining increasing attention as possible ozone protectants . The aboAnother promising group of agrochemicals is growth regulators containing phytohormones or/and others antioxidants. Apart from their effects on plant development, some phytohormones, that is, cytokinins, gibberellins, salicylic acid and their synthetic analogs were shown to enhance tolerance of crops to abiotic stresses including ambient ozone , 7. The " 3-sensitive cv. Geraldton) and tobacco (Nicotiana tabacum L.) cvs. Bel-W3 (O3-sensitive) and Bel-B (O3-resistant) were used. In Szarów only subterranean clover was used. Prior to exposure to ozone, tobacco and clover were cultivated in ozone free air until they reach 4 leaves. In Kyiv plants were cultivated in the special chamber, made from organic glass, with the volume of 1.1 m3, at 25–28°C temperature, 65–70% RH, light illumination of 66 μmol m−2·s−1 PAR and 14 : 10 (L : D) h photoperiod. The air was pumped up through charcoal filter with the rate of 0.3 m3 per min. In Szarów plants were cultivated in the special ozone free greenhouse at temperature of 25 ± 2°C with a relative humidity of 75% under natural photoperiod. Afterwards, the plants were sprayed with distilled water (control) or solutions of one of the following agrochemicals: (1) EDU (ethylenediurea), in concentrations of 150 mg L−1; (2) “Emistym C”, a natural growth regulator containing exometabolites of micorhizal fungi , in concentration of 30 and 150 mg L−1; (3) “Agrostimulin,” containing synthetic growth regulator—ivin, in concentration of 30 and 150 mg L−1; modern fungicides belonging to the group of strobilurins such as (4) “Quadris” (containing 25% of azoxystrobin ) in concentration of 150 and 300 mg L−1; (5) “Flint” (containing 50% of trifloxystrobin) in concentration of 150 and 300 mg L−1; (6) “Strobi” (containing 50% of kresoxim-methyl) in concentration of 150 and 300 mg L−1. Ozone protective effect of “Emistym C” and “Agrostimulin” has been studied for the first time.The experiments were conducted simultaneously on two monitoring stations located in semiurban  and rural  sites. In Kyiv plants of subterranean clover  were studied. In Kyiv higher concentrations of the chemicals were applied only once (one day before exposure). In Sharów lower concentrations of agrochemicals were applied twice (one day before exposure and on 16th day of exposure). We used EDU as a standard ozone protectant, in concentration, whose effectiveness is well established on many plant species .The test plants were exposed into the field on the following day after application of agrochemicals. The duration of exposure of the test plants to ambient ozone was 2 and 4 weeks for tobacco and clover, respectively. On the last day of exposure ozone-induced foliar injury (%) and content of photosynthetic pigments  in leaves of test plants were evaluated. Photosynthetic pigments were extracted by 100% acetone and determined spectrophotometrically using “Spekol 11”  [During the period of the experiments ambient ozone concentrations were continuously monitored at the both locations. In Kyiv and Szarów ozone monitoring was conducted using the Thermo Environmental UV Photometric Ozone Analyzer Model 49-003 and Model 49 C, correspondingly. Average daily ozone and temperature concentrations as well as the doses of ozone, which test plants received during the period of experimentation were calculated using index of AOT 40 (accumulated exposure over a threshold of 40 ppb).Statistical analysis of the results obtained was conducted with the usage of descriptive statistics and analysis of variance. Treatment means were compared using ANOVA and LSD-test (Statistica 6.0 software) .3-sensitive tobacco plants demonstrated higher sensitivity to ozone than clover in terms of visible foliar injuries. All the applied agrochemicals  showed partial protective effect to O3-sensitive plants. The plants treated with the solutions of the mentioned agrochemicals had less foliar injuries, higher biomass and higher content of photosynthetic pigments (chlorophylls a and b) in leaves as compared to control (sprayed with distil water)  in lower concentration (in Szarów) than when they were applied once (one day before exposure) in higher concentration (in Kyiv). For biomass accumulation, this tendency was less defined. It could be supposed that repeated application of the tested agrochemicals even in lower concentrations is more effective as it allows protection of newly emerged leaves. Though, the differences in ozone doses, which plants received in both locations, could also contribute to the observed effects. The degree of protective effect for all tested agrochemicals was higher in clover plants as compared to tobacco. Evidently it was a consequence of higher degree of ozone injuries in tobacco as compared to clover.Thus, conducted studies confirmed the effectiveness of ozone protective effect of EDU and fungicides belonging to the group of strobilurins , 8, 10.