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https://openalex.org/W2097538272
https://www.nature.com/articles/srep05498.pdf
English
null
The image-scratch paradigm: a new paradigm for evaluating infants' motivated gaze control
Scientific reports
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cc-by
6,875
* These authors contributed equally to this work. OPEN SUBJECT AREAS: COGNITIVE NEUROSCIENCE HUMAN BEHAVIOUR Michiko Miyazaki1,2*, Hideyuki Takahashi1,3*, Matthias Rolf3, Hiroyuki Okada1 & Takashi Omori1 Received 28 October 2013 Accepted 12 June 2014 Published 30 June 2014 1Brain Science Institute, Tamagawa University, 2School of Social Information Studies, Otsuma Women’s University, 3Graduate School of Engineering, Osaka University. Human infants show spontaneous behaviours such as general movement, goal-directed behaviour, and self-motivated behaviour from a very early age. However, it is unclear how these behaviours are organised throughout development. A major hindrance to empirical investigation is that there is no common paradigm for all ages that can circumvent infants’ underdeveloped verbal and motor abilities. Here, we propose a new paradigm, named the image-scratch task, using a gaze-contingent technique that is adaptable to various extents of motor ability. In this task, participants scratch off a black layer on a display to uncover pictures beneath it by using their gaze. We established quantitative criteria for spontaneous eye-movement based on adults’ gaze-data and demonstrated that our task is useful for evaluating eye-movements motivated by outcome attractiveness in 8-month-olds. Finally, we discuss the potential of this paradigm for revealing the mechanisms and developmental transitions underlying infants’ spontaneous and intentional behaviours. I I nfants show various spontaneous behaviours from early in life, such as general movement1, self-exploration2, goal-directed behaviour3,4, motivated behaviour5,6, and intentional behaviour7,8. In this article, we refer to some behaviour as ‘spontaneous’ when it is independent of external inputs but is driven by lower- or higher-level internal (e.g. non-reflective, habitual, motivated, or intentional) states. This is a broad term including various behaviours from non-meaningful bodily movement to intentional behaviour. In the early stages of life, infants move their hands and arms arbitrarily and independently of external stimuli, which is referred to as general movement1. Several months later, infants gradually begin to generate actions that are driven by their own motivation or intention8 (i.e. intentional control). Clarifying when and how these spontaneous behaviours pass from lower to higher levels is one of the great issues of developmental science. In particular, it is important to illustrate using quantitative measurements how these behaviours develop. In the case of experiments with adults, verbal reports or complex hand manipulation are usually used to evaluate the inner state of the subject9,10. However, infants’ large variance of motor abilities throughout various developmental stages prevents us from using such measures for cross-age comparisons11. Correspondence and requests for materials should be addressed to H.T. (hideyuki@ams. eng.osaka-u.ac.jp) OPEN The number of those who detected the gaze-contingency was 27. Twenty-four of them were controlling the red circle intentionally. Questionnaire report of spontaneous eye movement in adults. We categorized those individuals who satisfied the following conditions as the spontaneous group: the individual who felt they were control- ling their gaze intentionally, plus they detected the gaze-contingency. The number of those who detected the gaze-contingency was 27. Twenty-four of them were controlling the red circle intentionally. The ratio of adults showing spontaneous scratching in the with- gaze-point condition (16 out of 19) was significantly higher than that observed in the no-gaze-point condition (8 out of 17; Fisher’s exact test; x2 5 5.57, df 5 1, p 5 0.033, Q 5 0.23; Table 1). y g y The ratio of adults showing spontaneous scratching in the with- gaze-point condition (16 out of 19) was significantly higher than that observed in the no-gaze-point condition (8 out of 17; Fisher’s exact test; x2 5 5.57, df 5 1, p 5 0.033, Q 5 0.23; Table 1). screen, by comparing the infant data to adults’ experimental results on the same task. We established quantitative measures for spontan- eous gaze control based on the adults’ experimental results. We then evaluated the eye movements of the 8-month-old infants by these measures (Experiment 1). Eye movements reflecting spontaneity in adults. The eye movements of adults showing spontaneous scratching contrasted with the eye movements of adults showing passive viewing (typical eye move- ments of both groups are shown in Supplemental Videos S1 and S2). Figure 2 shows typical eye movement trajectories of adults show- ing spontaneous and passive viewing. The left and middle panels of Figure 2 show that the exposed areas in the second phase (Phase II) are larger for the spontaneous adult than for the passive adult. The right side of Figure 2 depicts samples of the gaze trajectories of both an adult showing spontaneous scratching and an adult showing passive viewing during the interruption phase. The spontaneously scratching adult looked at the unscratched black layer and moved her/his gaze rapidly, whereas the adult showing passive viewing only gazed at the already exposed area. We interpret rapid, black-directed eye movement during the interruption phase as exploratory beha- viour caused by a prediction error that indicates acquisition of the gaze-scratch contingency. OPEN A non-verbal experimental method and criterion that is adaptable to various ages, producing comparable measurements across ages, is required for a precise understand- ing of the process of emergence of spontaneous behaviour. Here, we propose a new experimental task using the gaze-contingent technique (i.e. online interactive eye tracking) to quantify spontaneous behaviour, including higher-order processing (e.g. motivation or intention), in infants. The task was named the image-scratch task (Fig. 1A). In this task, colourful, attractive pictures covered by a black layer are presented on a display equipped with an eye tracker, and participants are able to scratch off the black layer by gaze control and expose the picture beneath the layer. We hypothesised that, if the participants become aware of the novel contingency between their gaze coordinate and the change of stimulus, they will try to scratch off the black layer by controlling their gaze, being motivated by exploring the hidden, colourful picture. The gaze-contingency paradigm has the advantage of being able to investigate the development of spontaneous behaviours in infants regardless of the variance of motor abilities in infancy because voluntary gaze control develops earlier than limb control12–15. This task is expected to adapt to various ages in the same manner and become a promising method for quantitative cross-age comparison of spontaneous behaviour in infants. To demonstrate the usefulness of the image-scratch task for evaluating the extent of spontaneous, motivated behaviour, we examined whether 8-month-olds can control their gaze spontaneously to scratch the layer from the SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 1 www.nature.com/scientificreports www.nature.com/scientificreports Figure 1 | The image-scratch task. (A) Participants are able to scratch off the black layer using their gaze. This photo courtesy of Michiko Miyazaki with permission of the participants. (B) Two experimental conditions. In the with-gaze-point (WGP) condition, a small red circle is presented simultaneously at the point where the participants’ gaze is. In the no-gaze- point (NGP) condition, the red circle is not presented. (C) The task includes five trials; each displaying one picture for 30 s. Pictures in trials 1 and 4 are solid simple geometric patterns (Phases I and II). To reveal subjective prediction errors indicating acquisition of the gaze-scratch contingency, a 10-s interruption phase, in which eye movements cannot scratch off the black layer, is inserted in the middle of the fifth trial. OPEN We define the exploration rate during this phase as the proportion of gazes aimed at the black layer in relation to all gazes directed towards the display. Several previous studies have used the gaze-contingent tech- nique16,17. However, none of these studies has managed to distinguish infants spontaneous gaze control from reflex-based eye movements induced by the mere visual saliency of the stimuli. To evaluate whether the eye movements of infants are truly driven by outcome attractiveness regardless of the visual saliency factor, we conducted two additional control experiments. Firstly, we reduced the outcome attractiveness in Experiment 2 by using monochromatic (greyscale) images as hidden pictures instead of colourful pictures (See Supplemental Information) in order to investigate whether the infants were truly motivated to scratch off the black layer and to expose the hidden pictures. Secondly, to examine whether infants were truly influenced by the gaze-scratch contingency instead of mere visual saliency, we performed a yoked control experiment using the presentation of a movie that showed exactly what had been on the screen of other infants classified as showing spontaneous scratching (See Supplemental Information, Experiment 3). Figure 3 shows individual plots of the exploration rate during the scratch interruption phase for each participant group (adults/ infants) and each condition (no-gaze-point/with-gaze-point). In OPEN To collect sufficient samples of adults in both spontaneous scratching and passive viewing, we prepared two conditions that differed in difficulty (easy/difficult-to-detect contingency). In the easy condition, a small red circle was presented simultaneously at the point where the participants’ gaze was, making it easier for the participants to find the gaze–scratch contingency (the with-gaze- point [WGP] condition). In the difficult condition, the red circle was not presented (the no-gaze-point [NGP] condition) (Fig. 1B). Forty adults participated in this study. The adults were instructed to sit in front of the display and look at it, with no further instructions regarding the task. In the image-scratch task, five pictures were pre- sented one-by-one on the display for 30 s each (Fig. 1C). In the last trial, a 10-s interruption phase was inserted within 20 s of normal operation, the onset of the interruption being chosen randomly for each participant between 7 s and 10 s after beginning of the trial. In this phase, the eye movements of participants did not scratch off the black layer, even if a fixation was detected. Instead, the image remained unchanged. Before and after the interruption phase, gazes scratched off the image as in all other trials. After the task, the adults were asked to complete a questionnaire asking whether they noticed the gaze–scratch contingency and scratched off the black layer intentionally. Figure 1 | The image-scratch task. (A) Participants are able to scratch off the black layer using their gaze. This photo courtesy of Michiko Miyazaki with permission of the participants. (B) Two experimental conditions. In the with-gaze-point (WGP) condition, a small red circle is presented simultaneously at the point where the participants’ gaze is. In the no-gaze- point (NGP) condition, the red circle is not presented. (C) The task includes five trials; each displaying one picture for 30 s. Pictures in trials 1 and 4 are solid simple geometric patterns (Phases I and II). To reveal subjective prediction errors indicating acquisition of the gaze-scratch contingency, a 10-s interruption phase, in which eye movements cannot scratch off the black layer, is inserted in the middle of the fifth trial. Questionnaire report of spontaneous eye movement in adults. We categorized those individuals who satisfied the following conditions as the spontaneous group: the individual who felt they were control- ling their gaze intentionally, plus they detected the gaze-contingency. Results Therefore we tested a range of different thresholds and evaluated the rate of correct classification for each threshold value. In order to estimate the optimal threshold we interpolated the resulting curve by means of a third order poly- nomial and searched for its peak value within admissible thresholds. The optimal performance was reached at 88.9% correct classifica- tions for a threshold value 0.187. Hence, defining a threshold of 0.187 on the exploration rate allows to distinguish adults showing spon- taneous scratching from adults showing passive viewing with 88.9% accuracy. We verified our method ex post by means of a leave-one- out cross validation, which showed the same classification rate (88.9%) as the direct data-driven estimation, which shows the robustness of the estimation. the two plots on the left side of Fig. 3, the dark-orange plots refer to the exploration rates of the spontaneously scratching adults. The dark-green plots refer to those of the passively viewing adults. We combined the data from the no-gaze-point and with-gaze-point con- ditions based on these latter two categories. Results show that the exploration rates of the adults showing spontaneous scratching are significantly higher than those of the adults showing passive viewing (t[33] 5 27.54, p 5 0.00000001, r 5 0.80). To find a criterion that provides the best discrimination of adults showing spontaneous scratching from adults showing passive viewing, we tested how well the exploration rate could predict the questionnaire outcomes by means of a simple threshold decision. Therefore we tested a range of different thresholds and evaluated the rate of correct classification for each threshold value. In order to estimate the optimal threshold we interpolated the resulting curve by means of a third order poly- nomial and searched for its peak value within admissible thresholds. The optimal performance was reached at 88.9% correct classifica- tions for a threshold value 0.187. Hence, defining a threshold of 0.187 on the exploration rate allows to distinguish adults showing spon- taneous scratching from adults showing passive viewing with 88.9% accuracy. We verified our method ex post by means of a leave-one- out cross validation, which showed the same classification rate (88.9%) as the direct data-driven estimation, which shows the robustness of the estimation. showing spontaneous scratching and passive viewing, the index does not exhibit an optimal threshold that would allow a prediction of the questionnaire reports with high accuracy. Results Taken together, the results obtained in the adult experiments show the existence of two indices for spontaneous eye movement: size of scratched area and exploration rate. Among them, the exploration rate in the interruption phase has the best threshold for a high- accuracy prediction of the questionnaire reports (88.9%). On the other hand, the size of the scratched area has no threshold allowing a better than 80%-accurate prediction of the questionnaire reports. Thus, we conclude that the exploration rate is the best predictive indicator of spontaneous eye movement. Estimation of eye movement in infants. Twenty-two 8-month-old infants participated in either the with-gaze-point or the no-gaze- point condition. We adopted the threshold that was established in the adult experiments to evaluate the eye movements of the infants (see Fig. 3; above threshold, infants classified as showing spon- taneous scratching; below threshold, infants classified as showing passive viewing). Typical eye movements of spontaneous- and pass- ive-classified infants can be seen in the Supplemental Informa- tion (Supplemental Videos S3 and S4). The ratio of infants showing spontaneous scratching in the with-gaze-point condition (8 out of 12) is significantly higher than that in the no-gaze-point condition (1 out of 9; see Table 1; Fisher’s exact test; x2 5 7.25, df 5 1, p 5 0.011, Q 5 0.58). This pattern is consistent with that observed in the exam- ination of adults. This result suggests that most infants in the with- gaze-point condition show eye movement similar to the adults who executed the image-scratch task spontaneously. To validate our find- ings further, we analysed whether the size of the scratched area sup- ports our categorisation of infants. As mentioned above, the typical adult showing spontaneous scratching scratched off a larger amount of the black layer by shifting her/his fixation quickly compared to the typical adult showing passive viewing (see Fig. 2). We found similar tendencies in the size of the scratched area between the spontaneous and passive participants in both the adult and infant groups. Cases of high exploration rates could also be explained by the scratched area being rather small at the onset of the interruption. To examine this issue, we tested whether the exploration rate was correlated to the size of the scratched area at the onset of interrup- tion. We found no correlation between the scratched area and the exploration rate both in adults (r 5 2.11) and infants (r 5 2.02). Results The exposed areas are represented by the white circles. The exposed areas in Phase II are larger for the spontaneous adult than for the passive adult. The fixation duration is represented by the size of circles, with larger circles indicating longer fixation time. The fixation circles obtained in Phase II are smaller for the spontaneous adult than for the passive adult. In the interruption phase, the spontaneous adult gazes at the unscratched black layer and moves her/his gaze rapidly, whereas the passive adult only gazes at the exposed area. Figure 2 | Typical eye-movement trajectories (A) adult showing spontaneous scratching and (B) adult showing passive viewing. The exposed areas are represented by the white circles. The exposed areas in Phase II are larger for the spontaneous adult than for the passive adult. The fixation duration is represented by the size of circles, with larger circles indicating longer fixation time. The fixation circles obtained in Phase II are smaller for the spontaneous adult than for the passive adult. In the interruption phase, the spontaneous adult gazes at the unscratched black layer and moves her/his gaze rapidly, whereas the passive adult only gazes at the exposed area. Figure 3 | Exploration rate during scratch interruption. Individual plots of gazing at the black area during the scratch interruption phase for each participant group (adults/infants) and condition (no-gaze-point/with- gaze-point). The dark-green, dark-orange, light-green, and light-orange plots indicate adults in passive viewing, adults in spontaneous scratching, infants in passive viewing, and infants in spontaneous scratching, respectively. The dotted line refers to the threshold established for the discrimination of intentionality in adults, which is used for the estimation of spontaneity in infants. the two plots on the left side of Fig. 3, the dark-orange plots refer to the exploration rates of the spontaneously scratching adults. The dark-green plots refer to those of the passively viewing adults. We combined the data from the no-gaze-point and with-gaze-point con- ditions based on these latter two categories. Results show that the exploration rates of the adults showing spontaneous scratching are significantly higher than those of the adults showing passive viewing (t[33] 5 27.54, p 5 0.00000001, r 5 0.80). To find a criterion that provides the best discrimination of adults showing spontaneous scratching from adults showing passive viewing, we tested how well the exploration rate could predict the questionnaire outcomes by means of a simple threshold decision. Results Experiment 1: efficient measurement of spontaneous eye move- ment for scratching in adults and evaluation of eye movement in 8-month-old infants. To establish a measure of spontaneous gaze control in adults, we compared the eye movements of those adults who subsequently reported that they had discovered the action– outcome contingency and scratched the layer with intention (adults showing spontaneous scratching) with the behaviour of those who reported that they did not notice the rule and simply looked at the display without any intention (adults showing passive viewing). Subsequently, we evaluated the eye movement of infants using this measure. Table 1 | The numbers of adult and infant participants assigned to spontaneous and passive groups Condition Spontaneous scratching Passive viewing Adults with-gaze-point (n 5 19) 16 3 no-gaze-point (n 5 17) 8 9 Infants with-gaze-point (n 5 12) 8 4 no-gaze-point (n 5 10) 1 9 Table 1 | The numbers of adult and infant participants assigned to spontaneous and passive groups Table 1 | The numbers of adult and infant participants assigned to spontaneous and passive groups SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 2 Figure 3 | Exploration rate during scratch interruption. Individual plots of gazing at the black area during the scratch interruption phase for each participant group (adults/infants) and condition (no-gaze-point/with- gaze-point). The dark-green, dark-orange, light-green, and light-orange plots indicate adults in passive viewing, adults in spontaneous scratching, infants in passive viewing, and infants in spontaneous scratching, respectively. The dotted line refers to the threshold established for the discrimination of intentionality in adults, which is used for the estimation of spontaneity in infants. www.nature.com/scientificreports Figure 2 | Typical eye-movement trajectories (A) adult showing spontaneous scratching and (B) adult showing passive viewing. The exposed areas are represented by the white circles. The exposed areas in Phase II are larger for the spontaneous adult than for the passive adult. The fixation duration is represented by the size of circles, with larger circles indicating longer fixation time. The fixation circles obtained in Phase II are smaller for the spontaneous adult than for the passive adult. In the interruption phase, the spontaneous adult gazes at the unscratched black layer and moves her/his gaze rapidly, whereas the passive adult only gazes at the exposed area. Figure 2 | Typical eye-movement trajectories (A) adult showing spontaneous scratching and (B) adult showing passive viewing. SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 Discussion The present study proposes a new paradigm named the image- scratch task. It uses a gaze-contingent technique for quantifying spontaneous behaviours by young infants to adults by means of common measurements. Quantified behaviours include those driven by higher-order processes (i.e. motivation or intention). Based on results from adults, we have established a quantitative measure of spontaneous gaze control in infants and have demonstrated that at 8 months, an infant’s gaze is driven, at least partially, by their motiva- tion (see details in Supplemental Information, Experiment 2). We found that the exploration rate during the interruption phase is an efficient measure for detecting spontaneous gaze in adults. We optimised a threshold on this rate for predicting participants’ ques- tionnaire reports, with good results. Interestingly the ratio of spontaneous adults was significantly higher in the with-gaze-point condition than in the no-gaze-point condition. A seminal study by Wang et al. using the gaze-contingent para- digm has addressed the quantification of infants’ gaze control using a similar approach16. In their task, infants could press the button on an eye tracker’s display by their gaze to play an attractive movie. The authors demonstrated that 6- to 8-month-old infants would choose a controllable button over an uncontrollable one, and that their eye movements are similar to the eye movements of adults who realise the contingency between their gaze and movie playing. Wang et al.’s approach is innovative; however, the similarity of eye movements between the infants and the adults who detect the contingency may be no more than a superficial approximation and may not reflect a common mechanism. Our findings suggest that the 8-month-olds can control their gaze, which is driven by their motivation. However, our current findings have not decided the issue of whether infants actually have an explicit expectation of the outcome of their own behaviour17–19. Several pre- vious studies have shown that younger infants can execute motivated behaviours such as goal-directed behaviours20,21. However, such behaviours are not necessarily to be interpreted as being accompan- ied by explicit expectations22. Instead, they can be explained as habit- ual responses23. For example, after learning that pulling a supporting blanket is needed for reaching an out-of-reach toy, 16- and 24- month-old infants soon stop pulling the blanket when the toy is gone; in contrast, 8-month-old infants keep pulling the blanket5. Results These results suggest that high exploration rates were not caused by the larger black area at the onset of interruption. The analysis of the adult eye movements yielded another signifi- cant index of spontaneous eye movement in addition to the explora- tion rate. This index is the difference in the size of the scratched area from the first trial (Phase I) to the fourth trial (Phase II), which is here termed the ‘‘intra-individual change of gaze control’’ from former to latter trials (see details regarding the estimation of eye movement in infants). As can be seen in the left and middle panels of Fig. 2, the exposed areas in Phase II are larger for adults showing spontaneous scratching than they are for adults showing passive viewing. Although the scratched area clearly differentiates between the adults A two-by-two, (Spontaneity: spontaneous vs. passive) 3 (Phase: Phase I vs. Phase II) ANOVA analysis was conducted on the size of the scratched area, revealing a significant interaction between Spontaneity and Phase in both adults and infants (adults, F [1, 34] 5 4.77, p 5 0.036, g2 5 0.14; infants, F [1, 20] 5 6.29, p 5 0.02, g25 SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 3 www.nature.com/scientificreports Figure 4 | Mean proportion of the scratched area in Phases I and II. (A) In adults and (B) infants. The error bars indicate standard error. Figure 4 | Mean proportion of the scratched area in Phases I and II. (A) In adults and (B) infants. The error bars indicate standard error. 0.32; see Fig. 4). From the simple main effect test, the effect of Spontaneity in Phase I is not significantly different between the two groups (adults, F [1, 34] 5 2.12, p 5 0.21, g2 5 0.06, non- significant [n.s.]; infants, F [1, 20] 5 1.94, p 5 0.24, g2 5 0.09, n.s.), whereas in adults, this effect is significantly different between the groups in Phase II. In infants, this effect is marginally significant (adults, F [1, 34] 5 16.34, p 5 0.001, g2 5 0.32; infants, F [1, 20] 5 3.21, p 5 0.18, g2 5 0.14). Results The effect of Phase in Passive group is not significantly different between the two groups (adults, F [1, 34] 5 0.07, p 5 0.80, g2 5 0.002, n.s.; infants, F [1, 20] 5 0.07, p 5 0.79, g2 5 0.003, n.s.), whereas this effect is significantly different between the groups in the Spontaneous group (adults, F [1, 34] 5 8.02, p 5 0.016, g2 5 0.19; infants, F [1, 20] 5 14.52, p 5 0.004, g2 5 0.42). in Supplemental Information, Experiments 2 and 3). In Experiment 2, we evaluated whether infants’ eye movements were truly derived from the motivation for exploring attractive pictures. If infants’ eye movements were not passive, but spontaneous, the size of the scratched area should be reduced when the attractive colourful pic- tures were replaced by monotone grayscale images (non-attractive condition). The results suggest that the size of the scratched area is indeed reduced in the non-attractive condition, whereas the explora- tion rate remains high. In Experiment 3, we examined the possibility that infants’ spontaneous eye movements are driven by reflex-based gaze evoked by the saliency of visual images. Most infants categorised as spontaneous were continuously exposed to a red circle indicating their gaze point during the image-scratch task. The movement and visual saliency of the red circle possibly evoked reflex-based gaze and led to their different gaze pattern. To exclude this possibility, we performed a yoked control using playback movies of the screen contents seen by the spontaneous infants, but in a non-contingent condition. The result supports the contention that the spontaneous infants controlled their gaze independently of the saliency of the visual stimulus. Taken together, these findings suggest that 8- month-old infants spontaneously control their gaze, which is driven by motivation in our task. Furthermore, the proportion of short fixations shows similar tend- encies in both adults and infants (see Supplemental Information, Figure S1). In the second phase, the proportion of short fixations is higher in the spontaneous group than in the passive group. SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 Discussion Finally, in the last trial, another colourful picture was pre- sented, and a 10-s interruption phase was inserted into 20 s of normal operation, the interruption onset chosen randomly for each participant between 7 s and 10 s after the beginning of the trial. In this phase, the eye movements of participants did not scratch off the black layer, even if a fixation was detected (Fig. 1C). Instead, the image remained unchanged. Before and after the interruption phase, gazes scratched off the black layer as in all other trials. Several studies have shown that infants older than 1.5 years can control their actions depending on goal evaluation7,8,23; these infants increase/decrease the pulling action depending on the attractiveness of the reward toy. However, such a task cannot be applied to younger infants because of their immature motor ability. Our task may be a useful tool for examining goal evaluation in young infants. Although in the present study we simply demonstrate that the difference in outcome attractiveness leads to different behaviour, we can in the future conduct goal evaluation experiments with infants younger than 1.5 years using our paradigm. To get the participants’ attention, a movie with a voice saying ‘Look! Look!’ was presented for 3 s before the presentation of each picture. Pleasant background music was played while the eye tracker collected the eye-movement data. The music faded out when the participant was not looking at the display. Both adults and infants executed the same task. Another interesting topic is that the explicit visual presentation of the gaze point as a red circle enhances spontaneous gaze control in both adults and infants. It will be important to reveal the reason underlying this finding, considering that both conditions are fun- damentally identical with regard to the presence of an action– outcome association. We consider that the visual feedback of the gaze point may not only be a controllable object, but may also be an aid to attributing the effect to the self-generated action (i.e. an attribution of agency). Future studies should aim to clarify the rela- tionship between the visual feedback of eye movement, the detection of an action–outcome contingency, and the sense of agency in both adults and infants. Procedure. In all experiments, participants were seated approximately 60 cm away from the display and eye tracker. Discussion In the latter case, the mere presence of the blanket is sufficient to evoke the pulling behaviour that has been reinforced previously. If the action of infants is based on explicit expectations, their behaviour should instead be controlled according to behavioural goals. Their behaviour would be exhibited/inhibited depending on the expected We adopted this threshold to estimate whether an infant scratched the black layer off by motivation. The exploration rate of half of the infants was over the threshold in the with-gaze-point condition (but not in the no-gaze-point condition) and the validity of our method was further supported by the mean size of the scratched area in spontaneous infants, which was significantly higher than the size of the area scratched by passive infants (see Experiment 1). From these results, we can conclude that 8-month-old infants can control their gaze for exploring the picture behind the black layer. Our method is based on a direct comparison of eye movements between infants and adults. However, a possible limitation of this direct comparison is that infants’ eye movements could be only superficially similar to those of adults. That is, similar eye move- ments could be driven by mechanisms (e.g. reflective eye movement) different from those of adults, mechanisms that are not directly accessible since infants cannot report directly as adults can. To check for this issue, we conducted two additional experiments (see details SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 4 4 www.nature.com/scientificreports with a black layer. When an eye gaze was detected as a point on the display, a circular area with a radius of 50 pixels was scratched off, and the corresponding area of the picture became visible (Fig. 1A). There was no requirement of gaze fixation for the scratching. the value of the outcome7,23. In the near future, we intend to test with our image-scratch task whether infants’ spontaneous behaviour is accompanied by explicit expectations. Verification would be inter- esting because it strongly relates to the big issue of developmental science: how self-consciousness and self-agency emerge through the developmental process. Figure 1C shows an actual visual stimulus used in Experiment 1. To compare eye movements between the first and fourth trials, the same pictures of solid simple geometric patterns were presented in the first (Phase I) and fourth (Phase II) trials. To increase the participants’ rate of exploration, colourful pictures were presented in the second and third trials. Discussion The adults were only instructed to look at the display, with no further instructions regarding the rules of the task. The infants, who were fastened in a baby carrier to prevent them from standing up, sat on their mothers’ lap, facing the display, and completed the same task. Stimuli were presented immediately after the 9-point calibration was executed. After task execution, adult subjects were asked to complete a questionnaire asking whether they had noticed the gaze–scratch contingency and scratched off the black layer intentionally. Data rejection. We rejected data from those participants who gazed outside the display for more than 80% of the mean time from the first to the fourth picture and during the interruption phase. In Experiment 1, 5 adults and 16 infants were excluded from further analyses. In addition, 13 infants were excluded because they did not complete the task. We believe that the image-scratch task has great potential in devel- opmental science because it permits quantification of infants’ spon- taneous behaviour accompanied by motivation regardless of differences in motor ability. We believe that our task design can be refined further and can become a powerful tool for quantifying vari- ous infants’ spontaneous behaviours, including those that are orga- nised by higher-order cognitive processes. Furthermore, our results seem consistent with results and concepts developed within con- structive disciplines such as machine learning and cognitive devel- opmental robotics24. Several studies have highlighted the importance and functional significance of intrinsically motivated behaviour25,26, such as behaviour driven by acquisition of novel information27 and goal-directed behaviour driven toward task fulfilment despite initial failure28,29. Integrating such findings into a cross-disciplinary con- ceptualisation seems to be a promising goal for future work, and could lead to improved hypothesis formulations and refined experimental designs. For such progress, we must evaluate the image-scratch task from an inter-disciplinary viewpoint, in light of behavioural psychology, neuroscience, philosophy, and constructive robotics. 1. Prechtl, H., Fargel, J., Weinmann, H. & Bakker, H. Postures, motility and respiration of low-risk pre-term infants. Dev. Med. & Child Neurology 21, 3–27; DOI:10.1111/j.1469-8749.1979.tb01577.x (1979). 2. Rochat, P. & Striano, T. Emerging self-exploration by 2-month-old infants. Dev. Sci. 2, 206–218; DOI:10.1111/1467-7687.00069 (1999). 3. Butterworth, G. & Hopkins, B. Hand-mouth coordination in the new-born baby. Br. J. Dev. Psychol. 6, 303–314; DOI:10.1111/j.2044-835X.1988.tb01103.x (1988). 4. van der Meer, A. L., van der Weel, F. R. & Lee, D. N. The functional significance of arm movements in neonates. Discussion Science 267, 693–695; DOI:10.1126/science.7839147 (1995). 5. Frye, D. in: Children’s Theories Of Mind: Mental States And Social Understanding. (eds Frye, D. & Moore, C.) 15–38. (Lawrence Erlbaum Associates, 1991). 6. Willatts, P. Development of means-end behavior in young infants: pulling a support to retrieve a distant object. Dev. Psychol. 35, 651–667; DOI: 10.1037/ 0012-1649.35.3.651 (1999). 7. Klossek, U. M. H., Russell, J. & Dickinson, A. The control of instrumental action following outcome devaluation in young children aged between 1 and 4 years. J. Exp. Psychol. Gen. 137, 39–51; DOI:10.1037/0096-3445.137.1.39 (2008). 8. Klossek, U. M. H. & Dickinson, A. Rational action selection in 1K- to 3-year-olds following an extended training experience. J. Exp. Child Psychol. 111, 197–211; DOI:http://dx.doi.org/10.1016/j.jecp.2011.08.008 (2012). 9. Fourneret, P. & Jeannerod, M. Limited conscious monitoring of motor performance in normal subjects. Neuropsychologia 36, 1133–1140; DOI:http://dx. doi.org/10.1016/S0028-3932(98)00006-2 (1998). Author contributions M.M. and H.T. developed the study concept and design. Testing, data collection, and analysis were performed by M.M. and H.T. M.M., H.T., M.R., H.O. and T.O. wrote the paper and approved the final version of the paper for submission. M.M. and H.T. developed the study concept and design. Testing, data collection, and analysis were performed by M.M. and H.T. M.M., H.T., M.R., H.O. and T.O. wrote the paper and approved the final version of the paper for submission. p 22. Bargh, J. A. Goal and intent: goal-directed thought and behavior are often unintentional. Psychol. 1, 248–251; DOI:10.1207/s15327965pli0103_14 (1990). 23. Kenward, B., Folke, S., Holmberg, J., Johansson, A. & Gredeback, G. Goal directedness and decision making in infants. Dev. Psychol. 45, 809–819; DOI:10.1037/a0014076 (2009). Acknowledgments 17. Deligianni, F., Senju, A., Gergely, G. & Csibra, G. Automated gaze-contingent objects elicit orientation following in 8-month-old infants. Dev. Psychol. 47, 1499–1503; DOI:10.1037/a0025659 (2011). This study was supported by the H. Nakayama Foundation for Science &Technology grant to M.M., MEXT KAKENHI ‘‘Origins of the Social Mind’’, JSPS KAKENHI (23700322) to M.M., and (23700321) to H.T. This study was also supported in part by MEXT KAKENHI ‘‘Constructive Developmental Science’’ (25119510), and JSPS KAKENHI ‘‘Constructive Developmental Science Based on Understanding the Process from Neuro-Dynamics to Social Interaction’’ (24000012). We are deeply thankful to K. Shibata, K. Matsumoto, K. Samejima, M. Sakagami, S. Shimojo and K. Izuma for their helpful comments; to C. Murai, J. Kanero, A. Isawa, and K. Takata for research assistance; to I. Iguchi and M. Saito for data collection; and to all the parents and infants who participated in this study. 18. Kenward, B. 10-Month-olds visually anticipate an outcome contingent on their own action. Infancy 15, 337–361; DOI:10.1111/j.1532-7078.2009.00018.x (2010). f y j 19. Verschoor, S. A., Weidema, M., Biro, S. & Hommel, B. Where do action goals come from? Evidence for spontaneous action–effect binding in infants. Front. Psychol. 1, 201; DOI:10.3389/fpsyg.2010.00201 (2010). y p yg 20. Lewis, M., Alessandri, S. M. & Sullivan, M. W. Violation of expectancy, loss of control, and anger expressions in young infants. Dev. Psychol. 26, 745–751; DOI:10.1037/0012-1649.26.5.745 (1990). 21. Kalnins, I. V. & Bruner, J. S. The coordination of visual observation and instrumental behavior in early infancy. Perception 2, 307–314; DOI:10.1068/ p020307 (1973). Methods Participants. Forty adults and 22 infants participated in Experiment 1: 21 adults were included in the with-gaze-point condition (mean age 6 SD, 21.7 6 3.7 years; 12 women and 9 men) and 19 adults were included in the no-gaze-point condition (mean age, 20.7 6 1.2 years; 13 women and 6 men); 12 infants participated in the with-gaze-point condition (mean age, 8.6 6 0.4 months; 5 females and 7 males) and 10 participated in the no-gaze-point condition (mean age, 8.9 6 0.5 months; 4 females and 6 males). 10. David, N., Newen, A. & Vogeley, K. The ‘‘sense of agency’’ and its underlying cognitive and neural mechanisms. Conscious Cogn. 17, 523–534; DOI:10.1016/ j.concog.2008.03.004 (2008). 11. Rovee-Collier, C. The development of infant memory. Curr. Dir. in Psychol. Sci. 8, 80–85; DOI: 10.1111/1467-8721.00019 (1999). 12. Aslin, R. N. in: Eye Movements: Cognition And Visual Perception (eds Fisher, D. F., Monty, R. A. & Senders, J. W.) 31–51. (Erlbaum, 1981). All participants were recruited from the participant pool of Tamagawa University (Tokyo, Japan) via telephone calls or email. All adults and parents of infants gave written informed consent before participating in the study. This study was carried out in accordance with the guidelines approved by the Ethics Committee of Tamagawa University. 13. Johnson, M. H. in: The Cognitive Neurosciences (ed Gazzaniga, M. S.) 735–747. (MIT Press, 1995). 14. Morrone, M. C. & Burr, D. C. Evidence for the existence and development of visual inhibition in humans. Nature 321, 235–237; DOI:10.1038/321235a0 (1986). 15. van de Weijer-Bergsma, E., Wijnroks, L. & Jongmans, M. J. Attention development in infants and preschool children born preterm: a review. Infant Behav. Dev. 31, 333–351; DOI:http://dx.doi.org/10.1016/j.infbeh.2007.12.003 (2008). Apparatus and stimuli. Eye gaze was measured using a Tobii near-infrared eye tracker (T120; Tobii Technology AB) which was integrated with a 17-inch LCD monitor. The display resolution was 1024 3 768 pixels and the sampling rate was 60 Hz. A standard 9-point calibration was used. The task programming was completed in Visual Basic 6.0 and Tobii Eye Tracker SDK. 16. Wang, Q. et al. Infants in control: rapid anticipation of action outcomes in a gaze- contingent paradigm. PLoS One 7, e30884; DOI:10.1371/journal.pone.0030884 (2012). In the image-scratch task, five pictures were presented sequentially on the display for 30 s each. Just after the presentation of a picture, the whole display was covered SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 5 Additional information 24. Asada, M. et al. Cognitive developmental robotics: a survey. Auton. Ment. Dev. IEEE Trans. 1, 12–34; DOI: 10.1109/TAMD.2009.2021702 (2009). Supplementary information accompanies this paper at http://www.nature.com/ scientificreports 25. Schmidhuber, J. Formal theory of creativity, fun, and intrinsic motivation (1990– 2010). Auton. Ment Dev. IEEE Trans. 2, 230–247; DOI: 10.1109/ TAMD.2010.2056368 (2010). Competing financial interests: The authors declare no competing financial interests. Competing financial interests: The authors declare no competing financial interests. How to cite this article: Miyazaki, M., Takahashi, H., Rolf, M., Okada, H. & Omori, T. The image-scratch paradigm: a new paradigm for evaluating infants’ motivated gaze control. Sci. Rep. 4, 5498; DOI:10.1038/srep05498 (2014). How to cite this article: Miyazaki, M., Takahashi, H., Rolf, M., Okada, H. & Omori, T. The image-scratch paradigm: a new paradigm for evaluating infants’ motivated gaze control. Sci. Rep. 4, 5498; DOI:10.1038/srep05498 (2014). 26. Oudeyer, P. Y., Kaplan, F. & Hafner, V. V. Intrinsic motivation systems for autonomous mental development. Evol. Comput. IEEE Trans. 11, DOI: 265-286; 10.1109/TEVC.2006.890271 (2007). 27. Gottlieb, J. Attention, learning, and the value of information. Neuron 76, 281–295; DOI: http://dx.doi.org/10.1016/j.neuron.2012.09.034 (2012). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permissionfrom the licenseholderin order toreproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ Von Hofsten, C. An action perspective on motor development. Tre 28. Von Hofsten, C. An action perspective on motor development. Trends. Cogn. Sci. 8, 266–272; DOI: http://dx.doi.org/10.1016/j.tics.2004.04.002 (2004). 29. Rolf, M. Goal Babbling For An Efficient Bootstrapping Of Inverse Models In High Dimensions. PhD thesis, Bielefeld University, (2012). SCIENTIFIC REPORTS | 4 : 5498 | DOI: 10.1038/srep05498 6
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Research trends, challenges, and emerging topics of digital forensics: A review of reviews
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Aalborg Universitet Research Trends, Challenges, and Emerging Topics in Digital Forensics A Review of Reviews Casino, Fran; Dasaklis, Thomas K.; Spathoulas, Georgios P.; Anagnostopoulos, Marios; Ghosal, Amrita; Borocz, Istvan; Solanas, Agusti; Conti, Mauro; Patsakis, Constantinos Published in: IEEE Access Citation for published version (APA): Casino, F., Dasaklis, T. K., Spathoulas, G. P., Anagnostopoulos, M., Ghosal, A., Borocz, I., Solanas, A., Conti, M., & Patsakis, C. (2022). Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews. IEEE Access, 10, 25464-25493. https://doi.org/10.1109/ACCESS.2022.3154059 Citation for published version (APA): Casino, F., Dasaklis, T. K., Spathoulas, G. P., Anagnostopoulos, M., Ghosal, A., Borocz, I., Solanas, A., Conti, M., & Patsakis, C. (2022). Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews. IEEE Access, 10, 25464-25493. https://doi.org/10.1109/ACCESS.2022.3154059 Aalborg Universitet Aalborg Universitet General rights C i h d General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. - Users may download and print one copy of any publication from the public portal for the purpose of private st Y t f th di t ib t th t i l it f fit ki ti it i l i - Users may download and print one copy of any publication from the public portal for the purpose of private study or research. - You may not further distribute the material or use it for any profit-making activity or commercial gain y p py y p p p p p p - You may not further distribute the material or use it for any profit-making activity or commercial gain - You may freely distribute the URL identifying the publication in the public portal - y p py y p p p - You may not further distribute the material or use it for any profit-making activity or co Y f l di t ib t th URL id tif i th bli ti i th bli t l Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. Take down policy If you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access to the work immediately and investigate your claim. Citation for published version (APA): Casino, F., Dasaklis, T. K., Spathoulas, G. P., Anagnostopoulos, M., Ghosal, A., Borocz, I., Solanas, A., Conti, M., & Patsakis, C. (2022). Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews. IEEE Access, 10, 25464-25493. https://doi.org/10.1109/ACCESS.2022.3154059 FRAN CASINO 1,2, (Member, IEEE), THOMAS K. DASAKLIS3, GEORGIOS P. SPATHOULAS 4, MARIOS ANAGNOSTOPOULOS 5, AMRITA GHOSAL 6, ISTVÁN BO ¨RO ¨CZ7, AGUSTI SOLANAS 1, (Senior Member, IEEE), MAURO CONTI 8,9, (Fellow, IEEE), AND CONSTANTINOS PATSAKIS 2,10 FRAN CASINO 1,2, (Member, IEEE), THOMAS K. DASAKLIS3, GEORGIOS P. SPATHOULAS 4, MARIOS ANAGNOSTOPOULOS 5, AMRITA GHOSAL 6, ISTVÁN BO ¨RO ¨CZ7, AGUSTI SOLANAS 1, (Senior Member, IEEE), MAURO CONTI 8,9, (Fellow, IEEE), AND CONSTANTINOS PATSAKIS 2,10 1Department of Computer Engineering and Mathematics, Universitat Rovira i Virgili, 43007 Tarragona, Spain 2Information Management Systems Institute, Athena Research Center, 151 25 Marousi, Greece 3Hellenic Open University, 570 01 Patras, Greece 4Norwegian University of Science and Technology (NTNU), 2802 Gjøvik, Norway 5Aalborg University, 9220 Copenhagen, Denmark 6CONFIRM Centre, University of Limerick, Limerick, V94 T9PX Ireland 7Vrije Universiteit Brussel, 1050 Brussels, Belgium 8Department of Mathematics, University of Padua, 35122 Padua, Italy 9Faculty of Electrical Engineering, Mathematics and Computer Science, Delft University of Technology, 2628 CD Delft, The Netherland 10Department of Informatics, University of Piraeus, 185 34 Piraeus, Greece Corresponding author: Constantinos Patsakis (kpatsak@unipi.gr) 1Department of Computer Engineering and Mathematics, Universitat Rovira i Virgili, 43007 Tarragona, Spain 2Information Management Systems Institute, Athena Research Center, 151 25 Marousi, Greece 3Hellenic Open University, 570 01 Patras, Greece 4Norwegian University of Science and Technology (NTNU), 2802 Gjøvik, Norway 5Aalborg University, 9220 Copenhagen, Denmark 6CONFIRM Centre, University of Limerick, Limerick, V94 T9PX Ireland 7Vrije Universiteit Brussel, 1050 Brussels, Belgium 8Department of Mathematics, University of Padua, 35122 Padua, Italy 9Faculty of Electrical Engineering, Mathematics and Computer Science, Delft University of Technology, 2628 CD Delft, Th 10Department of Informatics, University of Piraeus, 185 34 Piraeus, Greece Corresponding author: Constantinos Patsakis (kpatsak@unipi.gr) This work was supported in part by the European Commission under the Horizon 2020 Programme (H2020), as part of the projects LOCARD under Grant 832735, HEROES under Grant 101021801, and the CyberSec4Europe under Grant 830929; and in part by the European Commission (call ISFP-2020-AG-TERFIN) as part of the CTC Project under Grant 830929. The work of Fran Casino was supported by the Beatriu de Pinós programme of the Government of Catalonia under Grant 2020 BP 00035. ABSTRACT Due to its critical role in cybersecurity, digital forensics has received significant attention from researchers and practitioners alike. The ever increasing sophistication of modern cyberattacks is directly related to the complexity of evidence acquisition, which often requires the use of several technologies. To date, researchers have presented many surveys and reviews on the field. However, such articles focused on the advances of each particular domain of digital forensics individually. Received January 22, 2022, accepted February 16, 2022, date of publication February 24, 2022, date of current version March 10, 2022. eceived January 22, 2022, accepted February 16, 2022, date of Digital Object Identifier 10.1109/ACCESS.2022.3154059 FRAN CASINO 1,2, (Member, IEEE), THOMAS K. DASAKLIS3, GEORGIOS P. SPATHOULAS 4, MARIOS ANAGNOSTOPOULOS 5, AMRITA GHOSAL 6, ISTVÁN BO ¨RO ¨CZ7, AGUSTI SOLANAS 1, (Senior Member, IEEE), MAURO CONTI 8,9, (Fellow, IEEE), AND CONSTANTINOS PATSAKIS 2,10 Therefore, while each of these surveys facilitates researchers and practitioners to keep up with the latest advances in a particular domain of digital forensics, the global perspective is missing. Aiming to fill this gap, we performed a qualitative review of all the relevant reviews in the field of digital forensics, determined the main topics on digital forensics topics and identified their main challenges. Despite the diversity of topics and methods, there are several common problems that are faced by almost all of them, with most of them residing in evidence acquisition and pre-processing due to counter analysis methods and difficulties of collecting data from devices, the cloud etc. Beyond pure technical issues, our study highlights procedural issues in terms of readiness, reporting and presentation, as well as ethics, highlighting the European perspective which is traditionally stricter in terms of privacy. Our extensive analysis paves the way for closer collaboration among researcher and practitioners among different topics of digital forensics. I. INTRODUCTION under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 1 Downloaded from vbn.aau.dk on: October 24, 2024 Downloaded from vbn.aau.dk on: October 24, 2024 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/b I. INTRODUCTION principle is relatively straightforward, it is difficult in many cases to apply. This is why Locard introduced forensics labs in Law Enforcement Agencies (LEAs) over the first decade of the 20th century [3]. According to Edmond Locard’s exchange principle, in every crime, the perpetrator will alter the crime scene by bringing something and leaving something else [1], [2]. Therefore, these changes can be used as forensic evidence. While this While procedures that resemble digital forensics are men- tioned in computer science literature quite early, the domain was not fully defined until 1980s when it started to gain attention. The introduction of the IBM PC generalised the The associate editor coordinating the review of this manuscript and approving it for publication was Ilsun You . 25464 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews use of computing machines; thus, more interest was focused on digital evidence and many people came together and cre- ated a digital forensics community, which eventually became more formal in 1993 when the FBI hosted the First Interna- tional Conference on Computer Evidence [4]. Initially, the main activity was examining standalone computers to recover deleted or destroyed files from the disks. However, since the early 2000s, the digital forensics domain has expanded steadily, maturing along with regulations [5], [6]. Nowadays, users tend to utilise multiple digital devices and access tenths of digital services per day [7], [8]. The digital footprint of our everyday life has become enormous, and accordingly the probability that illegal activities leave digital evidence behind is very high. The need for forensic investigators has increased, and this have led to multiple academic education and certification programs related to digital forensics [9]. Additionally, the complexity of the tasks to be carried out and the required compliance with law and courts’ regulations has led to the establishment of strict protocols and proce- dures to be followed [10]–[12]. The continuous appearance of new forms of cybercrime also requires adaptive investigation process models, new technology, and advanced techniques to deal with such incidents [13]–[15]. Moreover, research directions and approaches, that could be applied in several domains, remain explored in a topic- wise manner, lacking interoperability, and denoting a lack of collaboration between researchers in different forensics domains. 1https://ec.europa.eu/commission/presscorner/detail/en/MEMO_18_ 3345 B. CONTRIBUTION According to a thorough methodological research, we collect all relevant surveys and reviews in the field of digital foren- sics, analyse them, and answer a set of research questions, listed in Table 1, by performing the following actions: • Analysing the current state of the art and practice, and identifying the challenges of each domain individually. • Assessing whether the current state of the art is aligned with the technological evolution in digital forensics. • Using the previously collected information to identify common issues, gaps, best strategies and key focus areas in digital forensics, trying to span across different domains. • Assessing technological advances to highlight emerging challenges in digital forensics. • Assessing technological advances to highlight emerging challenges in digital forensics. Beyond the rise of cybercrime, where the evidence is expected to be digital, digital evidence is underpinning almost all modern crime scenes. For instance, mobile devices have become a primary source of digital evidence as almost all our communications are performed through them [6]. In fact, according to EU,1 the bulk of criminal investigations (85%) involve electronic evidence. Thus, emails, cloud service providers, online payments, and wearable devices are often used to extract digital evidence in various circumstances. In addition to suggesting promising research lines in the field based on the above analysis, we cover other dimensions of digital forensics, including frameworks and process mod- els, standardisation, readability and reporting, as well as legal and ethical aspects. To the best of our knowledge, this is the first review of reviews covering the state of the art in digital forensics and showcasing the actual state of practice from a global perspective. The remainder of the article is organized as follows: Section II details our research methodology, providing a descriptive analysis of the retrieved literature, which is then complemented with a taxonomy of digital forensics in Section III. Section IV analyses the current state of practice regarding forensic methodologies and their phases, standards, and ethics. Relevant open issues, trends, and further research lines are discussed in Section V. The article concludes in Section VI with some final remarks. I. INTRODUCTION We sustain that the above is a serious gap in current literature, and we aim to fill it in this article. To this end, we present a review of reviews in the field of digital forensics. A. MOTIVATION Digital evidence has become a norm and underpins most modern crime investigations. However, there are digital evi- dence to which different methods and methodologies apply. Some principles may remain the same; however, they cannot be applied to all types of evidence. For instance, collect- ing evidence from the Cloud bears no resemblance to IoT forensics or image forensics. This has led to a huge amount of research, which addresses the challenges raised in each domain individually, with the bulk of the work devoted to the development of novel tools and algorithms to extract digital evidence and intelligence from heterogeneous sources. Currently, investigators devote many efforts to provide a systematic overview of the literature and the advances in each domain, with focused surveys and reviews. Despite the importance of these surveys, an analysis considering the chal- lenges and issues of the different digital forensics domains as a whole is still missing. In other words, each of these surveys is focused on a specific domain and, as a result, common issues, challenges and methods are not identified. used to bring together, appraise, and synthesise the results of related systematic reviews when multiple systematic reviews on similar or related topics already exist [17], [18]. Therefore, a review of reviews or an umbrella review compiles evidence from multiple reviews or survey papers into a single docu- ment. Syntheses of previous systematic reviews are known by a variety of names, one of which is an umbrella review. Other descriptions include the terms (‘‘review of reviews,’’ ‘‘sys- tematic review of reviews,’’ ‘‘review of systematic reviews,’’ ‘‘overviews of reviews,’’ ‘‘summary of systematic reviews,’’ ‘‘summary of reviews,’’ and ‘‘synthesis of reviews’’) [19]. FIGURE 1. Detail of the research methodology steps. Despite their growing popularity, no standardized report- ing guidelines currently exist for umbrella reviews. How- ever, various multidisciplinary teams around the globe work together to develop relevant standardized reporting guidelines that will soon be available [20]. In our case, we rely upon an entirely systematic way to conduct our umbrella review. In particular, we have used various features of the approach presented in [21] to conduct our review of reviews and pro- vide a transparent, reproducible and sound overview of the scientific literature on digital forensics from a global perspec- tive. Our review protocol consists of five steps, as shown in Figure 1: 1) Planning the review 2) Defining research ques- tions 3) Searching literature databases 4) Applying inclusion and exclusion criteria and 5) Synthesising and reporting the results of the literature analysis. FIGURE 1. Detail of the research methodology steps. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. TABLE 1. Summary of research questions and the corresponding sections devoted to answer them. s and the corresponding sections devoted to answer them. II. RESEARCH METHODOLOGY In recent years, academic publishing has significantly increased both in terms of volume and speed. At the same time, new channels for publication, such as conference pro- ceedings, open archives and numerous scientific journals, are rapidly expanding, thus allowing today’s researchers to publish their work in a multitude of venues [16]. According to recent studies, approximately 22 new systematic reviews are published daily [17]. New methodological approaches for synthesising this evidence have been developed to keep up with the proliferation of systematic reviews across dis- ciplines. Besides, conducting reviews of existing systematic reviews has become a logical next step in providing evidence in domains where a growing number of systematic reviews is available. Overviews or umbrella reviews are most commonly 25465 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews B. SELECTION OF STUDIES We used various pre-defined exclusion and inclusion crite- ria as described in Table 2 to assess the eligibility of the retrieved literature; both academic and grey. Some exclu- sion criteria were used before introducing the literature into the bibliographic manager (language, subject area and doc- ument type restrictions). It is also worth noting that we have only examined review papers and reports written in English. The distribution of publications over time is depicted in Figure 2. In particular, Figure 2 shows a year-by-year analysis of the selected papers. It is worth noting that the number of publications has increased significantly after 2017. Until the end of 2017, there were only about 38 review papers address- ing issues of digital forensics. However, from 2017 onwards, the number of reviews published in the scientific literature has risen to nearly 70. As a result, over the last four years, research in the area of digital forensics has slowly but steadily increased. This upward trend reflects the key public and policy impact of digital forensics nowadays. Our overall selection process steps are the following: (i) We initially evaluated the relevance of the titles of all scientific articles and reports. Articles/reports fulfilling one of the exclusion criteria were removed from the analysis and sorted according to the reason for their removal, (ii) In the sequence, we evaluated the relevance of all paper abstracts and report introduction sections (grey literature). Articles and/or reports that met one of the defined exclusion criteria were excluded from the analysis, and we documented the reason for exclusion, (iii) We also did a full-text reading, and some additional articles/reports were excluded and sorted by reason of exclusion during this step. We resolved any poten- tial disagreements among authors about the relevance of the retrieved articles/reports through discussion until reaching a unanimous consensus. We omitted several studies because they were not reviews or surveys (for example, papers rel- evant to financial forensics investigation, business forensics). We also discarded from the analysis articles that did not meet the inclusion criteria. Figure 2 also shows the domain-specific distribution of the 109 review papers included in our analysis. It is worth noting that we have identified seven (7) prevalent areas of research interest in digital forensics: Blockchain, Cloud, Filesystem and databases, Multimedia, IoT, Mobile, Networks. D. BIBLIOGRAPHIC ANALYSIS In this section, we present a descriptive analysis of the scientific papers included in the challenges-based and domain-specific classification (see Figure 2). The descrip- tive analysis includes 109 research papers published from 2006 until the end of November 2021. The purpose of the descriptive analysis presented is three-fold: We discovered additional studies using the so-called snowball effect (backward and forward), which involved searching the references of key articles and reports for addi- tional citations [23]. For instance, additional grey litera- ture was discovered by manually searching the reference lists in several reports, particularly research and committee reports or policy briefs from private and public sector institu- tions/organizations. For this study, we take into consideration 109 research papers and 51 reports. The 109 papers are used for identifying relevant challenges/trends across different dig- ital forensics domains (see Section III ). The 51 reports were used to derive further insights about the state of practice regarding digital forensics methodologies, practices and stan- dards, as well as discussing future trends and open challenges from a policy perspective (see sections IV and V). 1) It enhances the statistical description, aggregation, and presentation of the constructs of interest or their asso- ciations of the relevant literature (publications per year and domain etc.). 2) It contains insights to current research trends in the area of digital forensics and a critical discussion of the challenges identified. It, therefore, supports the classi- fication structure presented in Section III 3) It allows us to visually demonstrate the diverse research approaches used up to this point in the scientific lit- erature regarding the proliferation of digital forensics review papers. C. ANALYSIS AND REPORTING Electronic searches using Google also turned up relevant grey literature, such as unpublished research commissioned by governments or private/public institutions. In particular, we looked at the first 200 Google results for the queries digital forensics and reviews and digital forensics and surveys to find the published grey literature. It is worth noting that we used Google searches as a supplement to our primary search strategy (especially for streamlining the assessment), and Scopus and WoS were our primary source for finding scientific-related literature. Furthermore, compared to the bibliography retrieved from Scopus and WoS, the total num- ber of documents retrieved from Google was relatively low. All articles and/or reports that met the inclusion criteria were analyzed (in emerging themes) using a qualitative analysis software (MAXQDA11). The authors carried out the the- matic content analysis independently. We applied various qualitative analysis methods (such as narrative synthesis and thematic analysis) to classify and synthesise the extracted data in a sound and comprehensive manner. The results of our analysis are presented in sections III and IV. A. SEARCH STRATEGY As previously stated, our overall survey process is based on several predefined research questions relevant to the dig- ital forensics literature. We conducted extensive research addressing the various technical/functional/security chal- lenges of the digital forensics literature guided by these research questions. To this end, we performed a systematic literature search without time constraints in May 2021 which was subsequently updated in November 2021. The main search engines used were Web of Science (WoS), Scopus and Google. Scopus and WoS were used to locate all scientific- related literature due to their multidisciplinary coverage and scope [22], while Google was used to locate relevant stan- dards and best practices (grey literature). We queried Scopus and WoS using the terms ‘‘digital forensics and review or survey’’ in the title, keywords, and abstract of all articles. It is worth noting that first bulk search query yielded 536 unique results (combining both sources). 25466 25466 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews A. CLOUD represent research conducted in areas such as social media, smart grid, unmanned aerial vehicles and etc. Researchers, as well as government agencies, have thor- oughly explored many of the challenges in cloud forensics, though some challenges still remain to be addressed. For example, the diversity of embedded OSs with shorter product life cycles, as well as the numerous smartphone manufactur- ers around the world present, are challenges in this research area. In the literature, we can find research works that have addressed challenges in cloud forensics and their solutions from different perspectives. Purnaye et al. [7] explored the different dimensions of cloud fornesics and categorised the main challenges of this topic. Alex et al. [24] discussed chal- lenges in cloud forensics related to data acquisition, logging, dependence on cloud service providers, chain of custody, crime scene reconstruction, cross border law and law presen- tation. Khanafseh et al. [25] pointed out several challenges in cloud forensics, such as the unification of logs format, miss- ing terms and conditions in Service Level Agreement (SLA) regarding investigations where service level agreement is the main point and condition between the user and the cloud B. SELECTION OF STUDIES Multimedia forensics attracts most of the cur- rent digital forensics research (38 out of the 109 review papers), followed by Filesystem and database forensics papers (18 out of 109). Both streams justify that the widespread use of mobile devices with lower-cost storage and increased bandwidth has resulted in a massive generation of multimedia-related content. Furthermore, various miscella- neous review papers (applications that do not fit into any of the above categories) demonstrate the digital forensics mul- tidisciplinary nature. These multidisciplinary review papers 25467 25467 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 2. Selection criteria of the retrieved literature. TABLE 2. Selection criteria of the retrieved literature. FIGURE 2. Year-wise analysis of the selected literature per domain. FIGURE 2. Year-wise analysis of the selected literature per domain. FIGURE 2. Year-wise analysis of the selected literature per domain. III. TAXONOMY OF CHALLENGES-BASED DIGITAL FORENSICS RESEARCH In this section, we summarise the surveys/literature reviews collected following a rigorous statistical methodology based on the literature, as described in Section II. The topics of this classification have been systematically selected according to the contents of reviewed literature, and thus reflect the digital forensics research landscape and illustrates with high fidelity the heterogeneity of digital forensic solutions. The classifi- cation of digital forensics topics is graphically represented in Figure 3. In each case, we discuss the main limitations and challenges proposed in the literature. More precisely, we extract the challenges at a research field domain level (i.e., we group in a higher hierarchical level, when possible, the limitations of the methods presented in the surveys) to give a more comprehensive perspective and to enable further cross-topic comparisons in Section III-I. 25468 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews FIGURE 3. Challenges-based and domain-specific mindmap abstraction of digital forensics topics identified in the literature. FIGURE 3. Challenges-based and domain-specific mindmap abstraction of digital forensics topics identified in the literature. forensic challenges based on the cloud forensic process stages. Amminezhad et al. [31] described the different chal- lenges in cloud forensics that were addressed by other authors by performing an exploratory analysis. Rahman et al. [32] broadly classified the existing challenges in cloud forensics, classifying the literature into three categories, namely, multi- tenancy, multi-location and scope of user control. Finally, the authors in [33] identified and discussed the major challenges that occur at each stage of the cloud forensic investigation, according to well-known forensic flows. service provider, lack of forensics expertise, decreased access to forensic data and control over forensics data at all level from the customer side, lack of international collaboration and legislative mechanism in cross-nation data access and exchange, and lack of international collaboration and legisla- tive mechanism in cross-nation data access and exchange. Pichan et al. [26] considered the Digital Investigative Pro- cess (DIP) model [27] for describing the challenges emerging at each phase of the digital investigation process and pro- vided solutions for the respective identified challenges. The challenges identified by the authors in cloud forensics are unknown physical location, decentralized data, data dupli- cation, jurisdiction, encryption, preservation, dependence on CSP, chain of custody, evidence segregation, distributed stor- age, data volatility and integrity. Similar to the works of Khanafseh et al. B. NETWORKS Data monitoring and acquisition from network traffic are mandatory to prevent most of nowadays cyber-attacks [36]–[38], including, but not limited to, Distributed Denial of Service (DDoS), phishing, DNS tunnelling, Man-in-the- middle (MitM) attacks, SQL injection and others [39], [40]. Regardless of the orchestration mechanism behind them (i.e., single attackers or orchestrated botnets), the analysis and mitigation mechanisms rely on the proper monitoring and analysis of computer network traffic to collect information, evidence and proof of any intrusion detection or vulnerabil- ity. For this purpose, several well-known tools exist, such as network forensic analysis tools which provide function- alities such as traffic sniffing, Intrusion Detection Systems (IDS), protocol analysis, and Security Event Management (SEM) [40]–[43]. Nevertheless, one of the challenges of network forensics is to achieve accurate and efficient packet analysis in encrypted network traffic since it is far more chal- lenging than the analysis of unencrypted traffic. As authors stated in [40], [44], utilizing machine learning in packet analysis is evolving into a complex research field that aims to address the analysis of unknown features and encrypted network data streams. q y The diversity of embedded OSs with shorter product life cycles, as well as the numerous smartphone manufacturers around the world, stand out as significant challenges in the MF domain [47]. In general, MF presents various challenges due to a multitude of reasons. For example, in [48] the authors identify the following limitations for successfully carrying out MF investigations: 1) data-related issues (anonymity- enforced browsing and other anonymity services, and the considerable volume of data acquired during an investiga- tion) 2) forensic tools-related issues (MF research approaches have long focused on acquisition techniques, while minor importance was given to the other phases of MF investigative process) 3) device and operating systems diversity 4) security aspects (development of new and more sophisticated anti- forensic methods from the manufacturers) 5) cloud-related issues (current MF tools do not consider cloud aspects, cloud investigation barriers such as access to forensics data due to multi-jurisdictional legal frameworks, forensics data security) and 6) process automation. It is worth noting that MF faces significant challenges concerning the focus of the overall MF processes. For example, it is not clear whether investigation procedures should be model-specific for each device or should be generic enough to form a standardized set of guidelines applicable to forensics procedures [49]. C. MOBILE Smartphones and mobile devices may contain valuable infor- mation for a plethora of investigation purposes. Mobile foren- sics (MF) is a sub-branch within the digital forensics domain relevant to the extraction of digital evidence from portable and/or mobile devices. Mobile forensics processes could be broken down into the following three categories: seizure, acquisition, and examination/analysis. III. TAXONOMY OF CHALLENGES-BASED DIGITAL FORENSICS RESEARCH and Pichan et al., the authors in [28] also identified the challenges in cloud forensics and ana- lyzed them on the basis of their significance. Park et al. [29] discussed the different challenges within cloud forensic investigations highlighting the relevance of proactive mod- els, and discussing the integration of smart environments to enhance the robustness of forensic investigations. The authors in [30] provided a categorization of the cloud g As evident from the large number of publications in lit- erature reviews/surveys, cloud forensics is quite an explored research topic. Despite the considerable amount of research in cloud forensics, there still exist a number of chal- lenges/limitations that need much attention, as discussed by NIST [34]. In Table 3, we present a summary of the extracted challenges in the cloud forensic review/survey articles. From this summary, we observe that there is a dearth of research work focusing on cloud forensic stan- dard tools and technologies in the cloud environment. Also, very limited works have concentrated on pointing out the feasible solutions related to the challenges present in cloud forensics. 25469 25469 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 4. High level extraction of challenges in network forensics. TABLE 4. High level extraction of challenges in network forensics. TABLE 3. High level extraction of limitations in cloud forensics. TABLE 3. High level extraction of limitations in cloud forensics. D. IOT Although significant in terms of improved data availability and operational excellence, the broad adoption of IoT devices and IoT-related applications have brought forward new secu- rity and forensics challenges. IoT forensics is a branch of digital forensics dealing with IoT-related cybercrimes and includes the investigation of connected devices, sensors and the data stored on all possible platforms. According to the literature, several of the current limita- tions of IoT forensics include the management of different streams of data sources, the complicated three-tier architec- ture of IoT, the lack of standardized systems for capturing real-time logs and storing them in a valid uniform form, the preparation of highly detailed reports of all information gathered its corresponding representation, the preservation and acquisition of evidence considering its volatility and value of data, and the adoption of routine forensic tasks in the IoT ecosystem [52]–[56]. Data encryption trends also present additional challenges for IoT forensic investigators, and arguably cryptographically protected storage systems is one of the most significant barriers hindering efficient dig- ital forensic analysis [54], [57], [58]. Other studies high- light additional limitations of IoT forensics processes such as interoperability and availability issues related to the vast amount of connected IoT devices [54]–[56], [59], the Big Data nature of the IoT forensics evidence (Variety, Velocity, Volume, Value, Veracity) [55], [58], [60] and the various security storage challenges of IoT forensics evidence, espe- cially when related to biometric data [61]. Finally, various regulatory-related challenges also exist in the IoT forensics domain, particularly issues relevant to the ownership of data in the cloud as defined by region-specific laws [54]–[56], [58], [59]. For instance, service-level agreements stipulating the ‘‘terms of use’’ of the cloud resources between the cloud customer and the cloud service provider do not incorporate B. NETWORKS Another challenge is the need to perform live forensics (mobile device should be powered on) [50]. In addition, an important barrier for actually conducting MF investiga- tions relates to the various networking capabilities of smart- phones, which render the overall MF processes difficult to manage, particularly due to the complex structure of the cloud computing environment [51]. Finally, due to the security measures inherent to modern mobile devices, an investigator must actually break into the device using an exploit that will most likely alter the device data. Clearly, the latter violates the Association of Chief Police Officers (ACPO) principle Regarding the research and forensics-related surveys tack- ling such issues, several reviews recall the primary method- ologies and tools for network forensic analysis, such as the works seen in [36], [45], yet they were conducted almost a decade ago. Therefore, taxonomies classifying forensic frameworks suitable for Network Forensics are crucial [40]. An interesting review focusing on the attackers perspective, in terms of attack behaviour and plan identification, as well as prevention mechanisms, can be found in [46]. Finally, some protocol-oriented reviews, analyzing IEEE 802.11 protocol [43], and more recently, 5G networks [42], dis- cuss specific vulnerabilities in their corresponding contexts. In general, the main challenges of network forensics, as iden- tified by the authors in the aforementioned works, are classi- fied in Table 4. 25470 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews and introduces numerous procedural issues for a forensic investigation. In Table 5, we provide a classification of MF approaches’ current challenges. forensic investigations’ provisions. Legislative frameworks adopted in specific regions, such as the GDPR in Europe, also pose significant challenges for IoT forensic investigations, particularly data privacy provisions [53]–[56]. Finally, the use of blockchain and its capability to enhance IoT forensic investigations has been also discussed in [54]. In Table 6 we provide a classification of the current challenges of IoT forensics approaches. TABLE 5. High level extraction of limitations in mobile forensics. TABLE 5. High level extraction of limitations in mobile forensics. TABLE 6. High level extraction of limitations in IoT forensics. TABLE 6. High level extraction of limitations in IoT forensics. E. FILESYSTEMS, MEMORY AND DATA STORAGE FORENSICS A system’s memory can contain evi- dence related to the usage of the system, including the list of running processes, network connections, or the keys for the driver’s encryption. Usually, such data are not stored in the permanent storage of the system and are completely lost when the system is turned off or unplugged from the power. In the literature, we can find surveys devoted to the analysis of the memory acquisition techniques [66], [67] (i.e., both hardware and software-based), the subsequent memory analysis [68], and the available tools [67]. The main challenges of memory forensics derive from the fact that memory is volatile, so it has to be acquired when the system is running and thus probably modified by the running applications. This can lead to the page smearing issue [68], i.e., inconsistencies between the state of the memory as described by the page tables compared with the actual contents of the memory. Another issue that can occur during the memory acquisition is the incorpora- tion of pages, which are not present in the memory due to page swapping or demand paging [68]. Finally, although the memory acquisition techniques should be OS and hardware agnostic [66], each OS architecture handles the memory dif- ferently and is equipped with distinctive tampering protection mechanisms that hinder access to memory. aggregation and analysis, as well as their structural architec- ture to benefit forensic procedures [69], [73]. Digital triage is of special relevance here since reviewing many poten- tial sources of digital evidence for specific information by using either manual or automated analysis is mandatory to enhance investigations [73]. Nevertheless, the authors high- light that the legitimacy of several acquisition procedures is constrained by the applicable legislation and that the current state of practice requires more efficient solutions, especially when dealing with huge amounts of data. In [74], the authors presented a framework for database forensic investigations enhanced by forensic experts’ opinions with the aim to over- come the main issues that investigator’s face, such as the lack of standardized tools and different data structures and log structures. y A database (DB) is the most traditional way to organise and store data. The majority of applications and online ser- vices deploy some type of DB to store records about their customers, financial records, inventory, etc. E. FILESYSTEMS, MEMORY AND DATA STORAGE FORENSICS Forensic analysis of large filesystems requires efficient meth- ods to manage the potentially large amount of files and data contained in them. System logs are one of the most used information sources to leverage forensic investigations. In [62] the authors provide a review of the publicly available datasets used in operating system log forensics research and taxonomy of the different techniques used in the forensic analysis of operating system logs. The taxonomy is cre- ated based on a common investigation format that includes event logs recovery, event correlation, event reconstruction and visualization. Distributed filesystem forensics is even a more challenging task, such as in the case of identifying the malicious behaviour of the attackers by analysing cloud logs [63]. Nevertheless, the accessibility attributes associated with cloud logs impede the goals of investigating such infor- mation, as well as other challenges, similar to those extracted in Section III-A. Another challenging area is the analysis of proprietary systems such as SCADA systems. In [64] the authors present a survey on digital forensics that are applied to SCADA systems. The survey describes the challenges that involve 25471 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 7. High level extraction of challenges in file system, memory and data storage forensics. TABLE 7. High level extraction of challenges in file system, memory and data storage forensics. applying digital forensics to SCADA systems as well as the range of proposed frameworks and methodologies. The work also focuses on the research that has been carried out to develop forensic solutions and tools that can be tailor- made for the SCADA systems. Recent research has revealed that malware developers have been using a broad range of anti-forensic techniques and escape routes in-memory attacks and system subversion, including BIOS and hypervisors. In addition, code-reuse attacks such as returned oriented programming pose a serious remote code execution threat. To neutralise the effects of malicious code, specific tech- niques and tools such as transparent malware tracers, system- wide debuggers were proposed. In [65], authors present a survey on the state-of-the-art techniques that demonstrate the capability of thwarting the anti-forensic strategies previously mentioned. Memory forensics refers to the forensic analysis of a sys- tem’s memory dump. E. FILESYSTEMS, MEMORY AND DATA STORAGE FORENSICS Besides the vast amount of data that could be contained in a DB, a database management system (DBMS) which allows the end-users to administer the DB and store and access the data in a specific format, can also provide evidence of actions in user- level granularity. For instance, it can reveal who and when stored/accessed specific records. Therefore, digital forensics for DB has attracted the attention of the research commu- nity [69]. From this perspective, several surveys focused on database digital forensics based on the log files, metadata, and similar types of artefacts for the case of relational and NoSQL DB [70]–[72]. Furthermore, other authors addressed the digital forensic opportunities on the procedure of data Considering the increasing amount of IoT technologies and small devices that require live data analysis due to the volatility of the data stored in them, it is crucial to develop new strategies to enhance data acquisition procedures [75]. In the context of database forensics and data acquisition, the challenges of big data analysis and data mining techniques for digital forensics [76], [77], and text clustering [78] were investigated. Moreover, a survey of techniques to perform similarity digest search is provided in [79]. Table 7 summarises the main limitations and challenges extracted from the literature analysed in this section. 2https://wearesocial.com/blog/2020/01/digital-2020-3-8-billion-people- use-social-media, https://www.cisco.com/c/en/us/solutions/collateral/ executive-perspectives/annual-internet-report/white-paper-c11-741490. html F. BLOCKCHAIN Besides the financial domain to which it was initially applied, through bitcoin, blockchain technology is currently used in various other use cases such as supply chain management, cybersecurity enhancement, document/certificates validation, crowdfunding campaigns, and more [80]. Additionally, because financial system set on blockchain provide more privacy than traditional pay- ment systems, it is common for cryptocurrencies to be used for criminal activities [81]. This sets blockchain forensics methodologies as a necessity [82] due to the large volume of data that are stored in blockchain systems and the number of processes that are managed by such systems. The main prop- erty of blockchain-based systems is the guaranteed protection of data integrity, which is directly related to forensic analysis. On the one side, this property makes forensic analysis more manageable. However, on the other side, this may complicate the process as users may be more cautious when interacting with such systems. It has to be noted that a large portion of blockchain systems are public, allowing access to everybody and thus making forensic analysis a surplus process. A forensics investigator can set up a node in a public blockchain network, sync it with the rest of the nodes and obtain a local copy of the ledger. Even in such cases, the structure of the information stored in the ledger of blockchain systems is not optimal with respect to retrieving all required data (e.g., for a specific account or a specific smart contract), so efficient mechanisms are required [83] to extract valuable information out of the large volume of data stored in public ledgers [84]. In the case of private blockchain systems, the ledger data are not publicly available and traditional forensics approaches have to be applied to blockchain nodes to extract data. TABLE 8. High level extraction of challenges in blockchain forensics. TABLE 8. High level extraction of challenges in blockchain forensics. F. BLOCKCHAIN Blockchain technology has been constantly integrated into existing systems or used as the basis to rebuild systems from 25472 VOLUME 10, 2022 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews While the data stored in the ledger are of great impor- tance, there are more data to be considered when analyzing a blockchain node. The ledger holds all committed transac- tions, but a blockchain node stores more information with respect to its interactions with other nodes or clients. For example, the IP of the client that has connected to a node to submit a transaction or the activity of a specific node in the network (e.g., sync requests) are not included in the ledger’s data. On top of those, multiple security blockchain attacks are mainly targeted against the infrastructure or the network’s backbone and not against its content. Mining attacks, network and long-range attacks [89], [90] target at taking control of the blocks formation process, to maliciously alter past committed transactions and achieve double-spending attacks. In such cases, digital evidence from deployed nodes is the only way to prove malicious activity. At the same time, the size of the network in public blockchain systems makes it even harder to retrieve the required evidence. Table 8 summarises the main challenges extracted from the blockchain forensics literature. scratch in various domains. Besides the financial domain to which it was initially applied, through bitcoin, blockchain technology is currently used in various other use cases such as supply chain management, cybersecurity enhancement, document/certificates validation, crowdfunding campaigns, and more [80]. Additionally, because financial system set on blockchain provide more privacy than traditional pay- ment systems, it is common for cryptocurrencies to be used for criminal activities [81]. This sets blockchain forensics methodologies as a necessity [82] due to the large volume of data that are stored in blockchain systems and the number of processes that are managed by such systems. The main prop- erty of blockchain-based systems is the guaranteed protection of data integrity, which is directly related to forensic analysis. On the one side, this property makes forensic analysis more manageable. However, on the other side, this may complicate the process as users may be more cautious when interacting with such systems. scratch in various domains. G. MULTIMEDIA Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 9. High level extraction of challenges in multimedia digital forensics. TABLE 9. High level extraction of challenges in multimedia digital forensics. several examples of anti-forensic methodologies [129], such as encryption, data obfuscation (e.g., trail obfuscation), arti- fact wiping, steganography and image tampering [130], protected/hidden communications (e.g., tunnelling, onion routing), malware anti-sandbox/debug, VM and in general anti-analysis methods [131]–[134], and spoofing. As stated in [135], anti-forensic methods exploit the dependence of human elements on forensic tools, as well as the limita- tions of the underlying hardware in terms of architecture and computational power. Therefore, enhancing the train- ing and knowledge level of investigators and more robust forensic procedures (e.g., anti-anti forensic techniques [130]) are critical to minimise the impact of anti-forensics. In this line, some authors argue that the use of proactive foren- sics models could help enhancing the robustness of forensic investigations [136]. TABLE 9. High level extraction of challenges in multimedia digital forensics. g [ ] Another emerging topic in digital forensics is related to unmanned aerial vehicles (UAVs), or more commonly known as drones [137]. The applications and versatility of these devices are becoming more popular in a myriad of contexts, from industrial to military applications. One of the main challenges of drone forensics is the set of different hardware components that are part of a drone [138], and the partic- ular treatment that they require (i.e., with special regard to advanced anti-forensic techniques taking place [139], as well as the necessity of live forensics [137], [140] in this context). For instance, drones consist of sensors, flight controllers, electronic and hardware components, on-board computers, and radiofrequency receivers, each one linked to one or many evidence sources in terms of, e.g., data storage (the differ- ent memory sources present in the drone, such as memory cards storing media, or other software), data communications and other logs and data stored in sources related to the drone, such as the drone controller and external cloud-based sources [141], [142]. At the moment of writing, there are no baseline principles, standards, nor legislation covering all the particularities of forensic drone investigations [137], [142]. Thus, efforts towards the establishment of sound protocols, specific forensic frameworks, as well as drone-based forensic tools are critical [137]. G. MULTIMEDIA passive techniques to detect forgery [105], or carving on specific file formats such as JPEG [106]. Other image foren- sics surveys analysed topics such as hyperspectral image [92], [107], image authentication [108], the affectation of noise in images [109] and image steganalysis [110]–[114]. Another set of surveys focus on the specific context of child abuse material and its detection through image and video analysis [115]–[118]. More recently, the advent of deep learning techniques has enhanced the capabilities of image integrity detection and verification, outperforming tra- ditional methods in several image-related tasks, especially in these where anti-forensic tools were used [113], [114], [119]. In the context of video files, we can find surveys on video steganalysis [113], [114], [120], video forgery detection [95], [96], [98], [114], [121], [122], video forensic tools [95], [113], [123], [124], video surveillance analysis [125], [126], and video content authentication [127]. Finally, digital audio forensics has also been studied in [128]. Table 9 summarises the main limitations and challenges extracted from the multi- media forensic literature. In [143], authors surveyed the different dimensions and concerns which digital forensics should cover in the context of social networks. The authors discussed several aspects of social networks, such as privacy and security issues, the criminal and illegal acts that can occur, and the attacks on the underlying platform and the users. In addition, they describe several strategies to detect such abnormal behaviours along with the necessity to develop both pro-active and reactive mechanisms. In terms of community detection, graph analytic methods and tools are crucial to detect criminal networks in different contexts, such as finance, terrorism, and other het- erogeneous sources [144]. In [8], authors surveyed the efforts done so far on the analysis of social network shared data according to source identification, integrity verification and platform provenance. Moreover, authors discussed the cur- rent methodologies, and highlighted the current challenges G. MULTIMEDIA Due to the increasing number of ubiquitous technologies (e.g., IoT devices, smartphones, wearables) leveraged by the 4th industrial revolution, as well as a substantial improvement in the connectivity capabilities in smart scenarios due to 5G, the amount of multimedia prosumers (i.e., both producers and consumers of data) is increasing dramatically year after year.2 Nevertheless, such multimedia content growth is a double-edged sword. On the one hand, it is a synonym of opportunities for the industry, companies and users. On the other hand, it augments the possible vulnerabilities and attack vectors of such systems, which malicious users can exploit. Even if data are by default publicly available, it is still challenging to identify malicious activity on such platforms. It is common for deployed smart contracts to suffer from var- ious vulnerabilities either due to poor implementation or not properly configured blockchain networks [85]. In such cases, users can take advantage of such vulnerabilities, mainly aim- ing at financial profit. It is challenging to detect such activity and identify the actors that have initiated it. Smart contracts execution is not a straightforward process, and past execution cannot be easily repeated in a forensic sound way [86]. Apart from that, smart contracts may also get self-destructed by a special OPCODE that makes following past transactions even harder [87]. Digital forensics in the context of multimedia has received substantial attention from the research community. There exist numerous image forgery detection surveys exploring the topic from a global perspective [91]–[99]. In this con- text, pixel-based image forgery detection is one of the main topics [100], including image splicing forgery [101], and copy-move forgery [102]–[104], which is a well-known tech- nique in which parts of the current images are used to cover/hide specific characteristics. Some authors focused on Furthermore, privacy concerns have been raised concern- ing early open public blockchain systems, and thus, there have been multiple alternative systems that make use of var- ious privacy-enhancing techniques such as zero-knowledge proofs, onion routing or ring confidential transactions to pro- tect users privacy [88]. In such cases, forensics analysis of either network nodes or users’ wallets is required to retrieve either logs or cryptographic keys that can be used along with data existing on public ledgers and provide more information about the transactions that have taken place. 25473 25473 VOLUME 10, 2022 VOLUME 10, 2022 F. H. MISCELLANEOUS This section is devoted to the digital forensics reviews that fall beyond the domain categorisation of the previous paragraphs. As observed in most topics, anti-forensics can be under- stood as a standalone concern in digital forensics, which requires investigation in each context. The term anti-forensics refers to methods and strategies that prevent forensic inves- tigators and their tools from achieving their goals. There are 25474 25474 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews along with the need for multidisciplinary approaches to over- come them. reproducibility and pushes the advancement in the state of the art, which is needed to keep up with the pace of tech- nology development [148], [149]. The next most challenging issue is related to anti-forensics methods, which has been discussed in several sections of the taxonomy as well as in Section III-H. Anti-forensic strategies leveraged by malicious actors include adversarial methods such as obfuscation or encryption applied to, e.g., data and storage systems, as well as hardware-related technological challenges, such as mobile phones due to their inherent security measures, or in the case of drones due to their specific particularities, and software, as well as in the case of malware. In the case of tools and eval- uation benchmarks, it is evident that the community needs to devote more efforts towards fighting novel cybercrime, especially in topics where, e.g., different data sources and technologies are present. For instance, in the case of IoT and UAVs, different data sources may necessitate different digital forensics strategies, including tools related to device level forensics, network forensics, and cloud forensics. Another challenge that affects digital forensics is the lack of juris- dictional and legal requirements for different investigation scenarios such as ethics and data management of confidential and personal data. This is particularly relevant nowadays due to the widespread use of distributed systems such as blockchain and the cloud. The latter means that software and data may reside in different countries, and thus, specific cross-border collaborations are required, adding another layer of complexity to digital investigations. Moreover, this sce- nario impedes the adoption of proactive measures due to the difficulty of applying measures that conform to different legal frameworks. I. CHALLENGE ANALYSIS AND AGGREGATED RESULTS The classification of challenges and limitations according to each topic of the taxonomy has been conducted to keep a balance between accurate descriptions of challenges and hier- archical classification. On the one hand, we want to facilitate identifying the gaps and limitations of each topic and pro- vide a clear path for both new and experienced investigators towards the corresponding literature. On the other hand, and as stated in Section I, we provide the reader with a clear overview of the research lines that should be strengthened in the digital forensics ecosystem, as well as their interre- lations according to each topic of our taxonomy. Therefore, we used the extracted challenges of each topic and merged the ones appearing more than once (i.e., the ones appearing only in their corresponding topic were ignored due to their specificity) to create a comprehensive overview of the digital forensics challenges in Table 10. As it can be observed, we identified several limitations of digital forensics that can be applied in several topics or contexts and thus, indicate the need to devote more research efforts towards them. Note that, for instance, the last topic of the Table 10 appears to be only affecting IoT, yet we identified this challenge in the miscella- neous topic, and thus, we decided to include it. Nevertheless, since several topics are analysed in such a category, we did not represent them in Table 10. A proper understanding between all the actors involved in the digital forensics context, including stakeholders, LEAs, and court members, is mandatory to ensure the success- ful prosecution of perpetrators. In this regard, one of the highlighted challenges is to ensure that all partners have a sufficient level of training (including technical knowledge and standardised guidelines) and a proper understanding, including readable reports to enable a fruitful collaboration. Moreover, while it seems procedural, the chain of custody is still a challenge. This can be attributed to multiple reasons, such as obvious negligence of the corresponding personnel to properly report evidence acquisition and/or handling, cor- rupted officers, or even gaps in the process. Nevertheless, all of them cause severe issues in a court as a case can be missed or misjudged. Secure and auditable means of storing and processing the chain of custody, as proposed by LOCARD3 with the use of blockchain technology seems like a logical and stable solution. 3https://locard.eu/ H. MISCELLANEOUS A sector that is receiving increasing attention due to its crit- ical relevance to the proper functioning of our society is the energy sector, and more concretely, the smart grid. In [145], authors explore practical cybersecurity models and propose some guidelines to enhance the protection of the smart grid against cyber threats. Moreover, they explore software- defined networks and their main benefits and challenges. Finally, the authors propose a conceptual forensic-driven security monitoring framework and highlight the relevance of forensics by design in development phases. Context-aware scenarios such as smart cities have been also receiving increased attention due to their complex structures, requiring the continuous data collection, processing and interaction between a myriad of devices [146], [147]. Digital forensics in this particular scenario is a recent paradigm which requires further efforts from the research community to enhance cyber resilience and to provide efficient incident response mechanisms [147]. overcome them. The investigation and forensic analysis cat- egory contains the highest number of challenges. Therefore, the topics identified in the taxonomy share similar technical concerns in their corresponding contexts, and more multidis- ciplinary collaboration is needed towards such direction. The reporting and presentation category highlights one yet crit- ical challenge since the proper reporting of an investigation affects the outcome of the whole investigation. We further dis- cuss about forensic readability and reporting in Section IV-C. challenges, since logging and data acquisition in specific scenarios may pose technical challenges. This issue is exacerbated when coupled with cross-border investigation requirements due to data fragmentation. Moreover, once data corresponds to multiple forensic contexts, the complexity of performing digital investigation grows exponentially, leaving aside the need to perform live forensics according to the par- ticularities of the hardware. Additionally, the availability of some devices due to their resource-constraint nature is a fur- ther challenge. For instance, IoT botnets have high volatility, and UAVs may implement self-defence mechanisms, even at the physical level. In the case of the Miscellaneous category, we included the challenges and limitations of anti-forensics, drone forensics, smart grid, smart cities and social networks. I. CHALLENGE ANALYSIS AND AGGREGATED RESULTS A more thorough description of forensic read- ability and its challenges is discussed later in Section IV-C. The most reported challenge is the sound data acquisition from heterogeneous sources and its interpretation, includ- ing different hardware and monitoring processes collecting data and logs dynamically. Note that data acquisition and management is a challenge affecting activities related to dig- ital forensics. Moreover, data fragmentation, a common sce- nario nowadays, hinders investigations further. It is important to note that data acquisition is critical to creating bench- marks, which help researchers and practitioners to evalu- ate their models. The latter enables characteristics such as Data acquisition, as previously stated, is not only a chal- lenge in terms of the existing heterogeneous data sources and context but also in terms of size. The big data era comes with a myriad of opportunities but also with their corresponding 25475 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. TABLE 10. Cross-domain abstraction of the challenges and limitations of digital forensics, ordered by relevance according to the amount of times they were found in the topics of the taxonomy. For the sake of fairness, the general column Miscellaneous has been omitted. IV. DIGITAL FORENSICS METHODOLOGIES, PRACTICES AND STANDARDS In addition to the topic-based taxonomy presented in Section III, we collected a set of literature reviews, included in our research methodology, that analysed forensic frame- works and process models, and the adaptability and forensic readiness of the actual practices. In the following sections, we analyse the content of such reviews by extracting the chal- lenges and identifying the main qualitative features required to achieve forensically sound investigations. According to the outcomes depicted in Table 10, we can observe that topics such as IoT, cloud, and mobile are affected by the highest amount of challenges. Therefore, we believe that researchers and practitioners should devote more efforts to solving such topics’ challenges by leveraging cross-domain collaborations to enhance the quality and appli- cability of their outcomes. Similarly, other challenges which appear in several topics could be tackled more quickly if they were targeted with a multidisciplinary approach, with experts from the different digital forensics topics. A. FORENSIC FRAMEWORKS AND PROCESS MODELS A digital forensics framework, also known as a digital foren- sics process model, is a sequence of steps that, along with the corresponding inputs, outputs and requirements, aim to sup- port a successful forensics investigation [150], [151]. A digi- tal forensics framework is used by forensics investigators and other related users to ease investigations and the identification and prosecution of perpetrators. In addition to a set of specific steps identifying each investigation phase, the use of digital forensic frameworks enables timely investigations, as well as a proper reconstruction of the timeline of events and their associated data. In this regard, one of the most critical aspects of a digital investigation is the proper preservation of the evidence chain of custody, since it could lead to unsolvable inconsistencies, risking the admissibility of evidence in court. To create a visual representation of these challenges, we believe that mapping each challenge into different cate- gories will highlight which need to be reinforced. Therefore, Figure 4 presents the outcomes of our taxonomy in terms of topic challenges mapped into different categories repre- senting different phases, from the creation of the legal basis and framework of an investigation to the final reporting of the outcomes. As it can be observed, the challenges most cited in the literature are present in the evidence acquisition and data pre-processing category. They are mainly related to data acquisition issues and anti-forensics. Notably, these challenges affect the forensic procedures from the beginning (i.e., if we do not consider the standards, legislation and procedural category), and thus, it is crucial to devote efforts to According to their phases and their granularity, there are different investigation models suitable for different types 25476 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews FIGURE 4. Main digital forensic challenges mapped into different categories according to their application context, from the initial steps of an investigation (left) to the final ones (right). The size of each circle denotes the times it appeared considering the topics of the taxonomy. FIGURE 4. Main digital forensic challenges mapped into different categories according to their application context, from the initial steps of an investigation (left) to the final ones (right). The size of each circle denotes the times it appeared considering the topics of the taxonomy. of investigations. In this regard, Kohn et al. A. FORENSIC FRAMEWORKS AND PROCESS MODELS provide [152] an integrated suitability framework that maps a set of require- ments derived from an ongoing investigation to the most suitable forensic procedure. Moreover, the authors also use a graph-based approach to associate the most well-known forensic frameworks and their interrelationships regarding the number of phases and their content. Other well-known frameworks include the Analytical Crime Scene Procedure Model (ACSPM) [153], the Systematic digital forensic inves- tigation model (SRDFIM) [154], and the advanced data acquisition model (ADAM) [155]. In general, law enforce- ment agencies follow variants of the ACPO (Association of Chief Police Officers) guidelines [156]. Finally, other foren- sic guidelines and models proposed by NIST and INTERPOL can be found in [5], [157]. The most well-known digital forensic frameworks are summarised in Table 11. digital investigation models and proposed some measures to palliate them. In [177] the authors presented a chronological review of the most well-known forensic frameworks and their characteristics. The work presented in [178] evaluates the cur- rent frameworks among European law enforcement agencies, identifying and defining elements of robustness and resilience in the context of sustainable digital investigation capacity so that organisations can adapt and overcome deviations and novel trends. In [175], the authors identified the need to define specific models according to the forensic context, such as in the case of Mobile Forensics [175]. Moreover, the authors proposed a specific forensic framework to improve Mobile Forensics investigations. Further reviews of the most used forensic frameworks and their features can be found in [179], [180]. Table 14 reports the main challenges in forensic frameworks identified by each literature review. In parallel to forensic guidelines and frameworks, stan- dards are crucial to ensure conformance and mutual compli- ance across geographical and jurisdictional borders. There are currently numerous standards and established practices pro- vided by organisations worldwide using accepted methods. The technical details on how to forensically approach a given investigation differ depending on the device. The analysis of electronic evidence is typically categorised into the phases stated in Table 12. However, the exact phases naming may vary due to different forensic models’ usage according to each organisation’s needs. In general, the procedures summarised in Table 11 have a common hierarchical structure [165], [166], which can be divided in the steps described in Table 12. 4https://caseontology.org/ A. FORENSIC FRAMEWORKS AND PROCESS MODELS Note that some of the models may include more granular approaches to some of the steps, which are necessary due to the investigation context (e.g., specific devices and seizure/acquisition constraints). In the case of the chain of custody and trail of events preservation, a forensically sound procedure needs to ensure features such as integrity, traceability, authentication, veri- fiability and security [167], [168]. In this regard, Table 13 provides a description of each feature. While not an official standard, the Cyber-investigation Analysis Standard Expression (CASE)4 is a community- driven standard that aims to develop an ontology that can effi- ciently represent all exchanged information and roles within the context of investigations regarding digital evidence. The International Organization for Standardization (ISO) has released a series of standards to assist in this effort by providing the family of ISO 27000, focusing on informa- tion security standardisation procedures. In what follows, In the past, several authors identified several challenges in digital investigation processes [77], [169]–[175], mainly related to the chain of custody preservation, the growth of the data to be processed, and privacy and ethical issues when collecting such data. In addition, our research methodology identified several literature reviews which discussed the chal- lenges and limitations of forensic frameworks. For instance, in [176], the authors leveraged a summary of digital forensic frameworks and tools as well as their interrelationships by using a graph analysis methodology. In addition, they dis- cussed some challenges and limitations of privacy-preserving 25477 25477 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 11. Most well-known forensic models and guidelines. TABLE 11. Most well-known forensic models and guidelines. TABLE 11. Most well-known forensic models and guidelines. TABLE 14. High level extraction of challenges reported in forensic frameworks literature reviews. TABLE 14. High level extraction of challenges reported in forensic frameworks literature reviews. TABLE 12. Main steps in a digital forensic investigation model. TABLE 12. Main steps in a digital forensic investigation model. TABLE 13. Main features required to guarantee chain of custody preservation. various areas such as computer forensics, image, audio and video analysis, as well as facial iden- tification. As a result, ASTM E2916-19 creates a common language framework for all. TABLE 13. Main features required to guarantee chain of custody preservation. A. FORENSIC FRAMEWORKS AND PROCESS MODELS ISO 21043-2:2018: This standard specifies many requirements for the forensic processes in focus- ing on recognition, recording, collection, transport and storage of items of potential forensic value. It includes requirements for the assessment and examination of scenes but is also applicable to activ- ities that occur within the facility. This document also includes quality requirements. ISO 21043-2:2018: This standard specifies many requirements for the forensic processes in focus- ing on recognition, recording, collection, transport and storage of items of potential forensic value. It includes requirements for the assessment and examination of scenes but is also applicable to activ- ities that occur within the facility. This document also includes quality requirements. we present the most relevant standards about digital forensics investigations, which are summarised in Figure 5. ISO/IEC 27035: This is a three-part standard that provides organisations with a structured and planned approach to the management of security incident management covering a range of incident response phases ISO/IEC 27035: This is a three-part standard that provides organisations with a structured and planned approach to the management of security incident management covering a range of incident response phases ISO/IEC 17025:2017: In some terms, this standard can be considered an ‘‘infrastructure’’ standard for forensic labs. It defines the managerial and techni- cal requirements that testing and calibration labora- tories must conform to ensure technical competence and guarantee that their test are calibration results are acceptable by the corresponding suppliers and regulatory authorities. ISO/IEC 27037:2012: This standard provides gen- eral guidelines about the handling of the evidence of the most common digital devices and the cir- cumstances including devices that exist in vari- ous forms, giving the example of an automotive system [181]. ISO/IEC 27037:2012: This standard provides gen- eral guidelines about the handling of the evidence of the most common digital devices and the cir- cumstances including devices that exist in vari- ous forms, giving the example of an automotive system [181]. ASTM E2916-19: The goal of this standard is to assemble the necessary technical, scientific and legal terms and the corresponding definitions in the context of the examination of digital and multi- media evidence. Therefore, the standard spans to ISO/IEC 27038:2014: Describes the digital redac- tion of information that must not be disclosed, taking extreme care to ensure that removed infor- mation is permanently unrecoverable. 25478 VOLUME 10, 2022 F. A. FORENSIC FRAMEWORKS AND PROCESS MODELS Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews FIGURE 5. Applicability of standards and guidelines to the investigation process classes and activities. FIGURE 5. Applicability of standards and guidelines to the investigation process classes and activities. ISO/IEC 27040:2015: Provides detailed technical guidance on how organisations can define an appro- priate level of risk mitigation by employing a well- proven and consistent approach to the planning, design, documentation, and implementation of data storage security. ISO/IEC 27043:2015: It defines the key common principles and processes underlying the investiga- tion of incidents and provides a framework model for all stages of investigations. ISO/IEC 27050: This recently revised stan- dard guides non-technical and technical person- nel to handle evidence on electronically stored information (ESI). ISO/IEC 27050: This recently revised stan- dard guides non-technical and technical person- nel to handle evidence on electronically stored information (ESI). ISO/IEC 27041:2015: Describes other standards and documents to provide guidance, setting the fundamental principles ensuring that tools, tech- niques and methods, appropriately selected for the investigation. ISO/IEC 30121:2015: Provides a framework for organizations to strategically prepare for a digital investigation before an incident occurs, to maximise the effectiveness of the investigation. ISO/IEC 30121:2015: Provides a framework for organizations to strategically prepare for a digital investigation before an incident occurs, to maximise the effectiveness of the investigation. ISO/IEC 27042:2015: This standard describes how methods and processes to be used during an investi- gation can be designed and implemented to allow correct evaluation of potential digital evidence, interpretation of digital evidence, and effective reporting of findings. ETSI is a European Standards Organization that produces standards for ICT systems and services used worldwide, collaborating with numerous organisations. In 2020, ETSI published TS 103 643 V1.1.1 (2020-01) [182], a set of tech- niques for assurance of digital material in a legal proceeding, 25479 25479 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 15. High level extraction of challenges reported in forensic readiness literature reviews. TABLE 15. High level extraction of challenges reported in forensic readiness literature reviews. A. FORENSIC FRAMEWORKS AND PROCESS MODELS to provide a set of tools to assist the legitimate presentation of digital evidence.5 In the meantime, the National Institute of Standards and Technology (NIST) has released guide- lines for organisations to develop forensic capability (see also Table 11), based on the principles of forensic science in the aspect of the application of science to the law. Still, it should not be used on digital forensic investigations due to subjection to different laws and regulations, as clearly stated in their manual. The scope of NIST guidelines is incorpo- rating forensics into the information system life cycle of an organisation. The most relevant guidelines are 800-86 [183] for Integrating Forensic Techniques into Incident Response and 800-101 [184] for Mobile Device Forensics. The Scientific Working Group on Digital Evidence (SWGDE) is an organisation engaged in the field of digital and multimedia evidence to foster communication and coop- eration as well as to ensure quality and consistency within the forensic community. SWGDE has released several documents to provide the current best practices on a large variety of state of the art forensics subjects. Nonetheless, none of them is targeting or addressing drone forensics’s particularities. Finally, a review of the international development of forensic standards can be found in [185]. discussed in the literature. Nevertheless, as previously stated in the article, one of the main challenges is that cybercrime evolves faster than countermeasures and legislations, and thus, investigators are always one step behind. 5https://www.swgde.org/documents/published C. FORENSIC READABILITY AND REPORTING The continuous appearance of novel ICT technologies, paired with discovering new vulnerabilities and attacks that threaten them, dramatically increases the amount of information col- lected during forensic investigations. The latter refers to the amount of data collected from devices and systems, as well as the heterogeneous data structures required in each case and the specific forensic methodologies developed to detect such threats. In this context, creating interoperable and auditable forensic procedures is a hard task, especially due to the lack of standardised reporting mechanisms. Moreover, qualitative aspects such as the outcomes and conclusions supported by the forensic analysis are often not reported accurately in an attempt to balance between technicality and comprehensibil- ity, hindering the robustness of the findings [14], [198], [199]. Of particular relevance is the communication and readability of such reports, especially if these are to be interpreted by law practitioners, judges, and other stakeholders who do not always have the necessary technical background about the forensic tools nor the underlying technologies anal- ysed [200], [201]. The latter issue has been extensively anal- ysed according to different approaches, from lexical density and complexity [202]–[208], to cognitive and psychological features [209], [210], showcasing the need to improve the reporting mechanisms and the possible benefits of a common, standardised framework. In addition to such a framework, it is crucial to develop the corresponding training procedures for its adoption [211]. 1) PRIVACY IN EUROPE States have numerous responsibilities concerning the protec- tion of their citizens. Although the protection of privacy (in its various forms) is important, it represents but one of the duties states should fulfil [217]. Other prominent duties relate to the need to protect the life and property of citizens, to prevent disorder, to ensure that justice occurs where individuals have been the victim of criminal activity and to protect national security both offline and online [218]. In modern western societies, it is often impossible to guarantee the exercise and protect such rights and in an absolute manner to all individuals all of the time due to competing interests of stakeholder groups. Respectively, privacy is only one of such values next to, e.g., security and the need for public order. To ensure security, the state likely has to take measures that may infringe upon the privacy of individuals [219]. This entails the acquisition of data or the conduct of surveillance to prevent inter alia acts of terrorism or crime. These activities clearly interfere with and limit the privacy of citizens but do so for desirable reasons. However, interference with such competing interests should be balanced, and the rights and freedoms of all groups in society should be respected to the greatest extent [217]. Respectively, the need to balance the privacy and security interests implies that security measures that infringe upon individual privacy are not acceptable unless they really are intended to meet a need that is relating to the protection the rights and interests of others. Where such After analysing the previous literature of forensic reporting procedures and studying the technical level of the data to be included [214], [215], as well as analysing existing investiga- tion models such as ISO/IEC 27043:2015 [216], we identified a set of key points and structural features that such document should include. In parallel, we analysed the technical level associated with each characteristic as reported in the liter- ature and created a reporting guideline document, which is represented in Table 17. As it can be observed, summaries, overview descriptions and listings should be performed in a comprehensive, non-technical way. In the case of tool descriptions, as well as proofs guaranteeing the outcomes, the report should contain some technical yet understandable descriptions. Finally, the scientific aspects and details behind the analysis and the corresponding methodologies require descriptions that should be provided by qualified experts. B. FORENSIC READINESS In the past, forensic investigations leveraged a post-event approach, mainly focusing on the analysis of data related to a past incident. In this regard, forensic readiness in terms of pro-active techniques and protocols appeared to minimise the cost and the impact of incidents and are widely used nowadays [15], [186]–[188]. We can find different research approaches, such as the review conducted in [189], in which authors discussed how to achieve forensic readiness by collecting the opinion of experts to elaborate a readiness framework with which improve forensic investigations from an organizational per- spective. In the case of [190], authors discussed forensic readiness and several procedures to achieve it, such as fos- tering the use of Trusted Platform Modules (TPM). Other authors reviewed measures to achieve forensic readiness in a holistic way [15], [191]–[194], as well as recalling the relevance to include and expand the actual guidelines towards incident response readiness (e.g., as in the drafts of the ISO/IEC JTC 1/SC 27 working groups, and the ISO/IEC 27035), training and collaboration between stake- holders involved in forensic investigations and prosecution, and effective reporting readability and complexity. Table 15 describes the main forensic readiness challenges identified by the authors in the literature. It is necessary to recall that the admissibility of a piece of evidence and the forensic validation in court is mandatory to the proper prosecution of perpetrators and constitute the culminating point of an investigation [212], [213]. Therefore, several authors collected the challenges and issues related to the acceptance of evidence in court [196], [197], [212]. More- over, region-focused studies can be found in [213] and [197] for the United Kingdom and Australia, respectively. Finally, in Table 16 we provide a qualitative summary of the literature reviewed in IV according to the topics discussed in each article. From Table 16 we can see that topics such as privacy and ethics and the suitability of frameworks that are being proposed to fight novel cybercrime need to be further 25480 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews TABLE 16. Qualitative analysis of the literature reviews related with digital forensic guidelines, frameworks, tools, and readiness. Notation: ✓denotes that this topic is analysed, while ◦denotes that its only partially discussed or just named. TABLE 16. Qualitative analysis of the literature reviews related with digital forensic guidelines, frameworks, tools, and readiness. B. FORENSIC READINESS Notation: ✓denotes that this topic is analysed, while ◦denotes that its only partially discussed or just named. TABLE 16. Qualitative analysis of the literature reviews related with digital forensic guidelines, frameworks, tools, and readiness. Notation: ✓denotes that this topic is analysed, while ◦denotes that its only partially discussed or just named. TABLE 17. Proposed representation of the content of a forensic report according to the inputs collected from the literature. TABLE 17. Proposed representation of the content of a forensic report according to the inputs collected from the literature. considered as well. As seen in [195], integrating digital foren- sic readiness as a component in data protection legislation could improve actual practices across different sectors and countries. In particular, this section highlights the regulatory require- ments of working with data in Europe and in the European Union. To facilitate digital forensic readiness, tools should be developed and used in line with legal requirements, with special attention to the individual’s privacy. D. DATA MANAGEMENT AND ETHICS When discussing digital forensics and respective technology readiness, the applicable regulatory frameworks should be 25481 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews output of analogue devices or data in digital format) of poten- tial probative value that is manipulated, generated through, stored on or communicated by any electronic device’’ [228]. Respectively, to use such data, specific rules concerning the gathering and use of (digital) evidence should be adhered to as well. Electronic evidence is admissible in courts when the following sets of rules are adhered to: 1) general rules and principles concerning due process in criminal proceedings; 2) general rules of evidence in criminal proceedings and; 3) specific rules relating to electronic evidence in criminal proceedings [229]. justification does not exist, infringement of individual privacy would not be acceptable. V. DISCUSSION In Section III, we provided a topic-based taxonomy of the digital forensics literature. In what follows, we recall the chal- lenges identified in each category and provide some strategies to overcome them. In European Union Art. 4 (2) of the Treaty on the Euro- pean Union (TEU) states that national security is the sole responsibility of each Member State. To facilitate a harmo- nized approach to national security, the EU adopted several Directives and other legislative pieces in connection with criminal matters such as: 1) Charter of Fundamental Rights of the European Union, art 7 and 8. 2) 2016/679 General Data Protection Regulation 3) Statement of the Article 29 Working Party, Data protection and privacy aspects of cross- border access to electronic evidence, Brussels, 29 November 2017. 4) 2016/680/EU Law Enforcement Directive [224] 5) 2014/41/EU European Investigation Order Directive 6) EU 2000 Convention on mutual assistance in criminal matters 7) 910/2014 eIDAS Regulation [225] 8) Electronic evidence - a basic guide for First Responders Good prac- tice material for CERT first responders by ENISA, and 9) E-evidence package [226] 2) DATA PROTECTION IN EUROPE In consonance with the individual’s data protection inter- est and society’s own protective endeavours toward fighting crime and securing national security, the Council of Europe and European Union developed a common framework to be observed by technology developers, security agencies, including Police, and criminal justice system. The most rel- evant instruments of the Council of Europe relating to the processing of data as evidence are: 1) the European Conven- tion for the Protection of Human Rights and Fundamental Freedoms (ECHR) in particular with reference to the protec- tion of the rights to privacy and due process, 2) the Council of Europe Convention on Cybercrime, as this Convention remains the main and only international treaty which defines the substantive elements of cybercrimes [220], 3) the Council of Europe Convention on Mutual Assistance in Criminal Matters, and its 1978 Protocol [221], and 4) the Electronic Evidence Guide [222]. There are both current, and to-be adopted elements of the applicable legal framework, but it must be underlined that as of now, there is no comprehensive international or European legal framework providing rules relating to evidence [230]. From these documents, five overarching principles can be deducted concerning the acquisition and use of electronic evi- dence. These are: data integrity, audit trail, specialist support, appropriate training, and legality [231]. National criminal procedure codes (referred above) contain further, specific provisions regarding the record and applicability of digital evidence in criminal procedures. A second protocol concerning the ‘‘Enhanced international cooperation on cybercrime and electronic evidence’’ is also in development [223]. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS Thus, the efforts should focus on big data analysis and data mining techniques to extract the relevant investigation data from the vast amount of unrelated or redun- dant digital objects. Another issue is the case of distributed filesystem and databases or data stores, or when the foren- sic analysis should be conducted on the cloud. In the latter case, besides the specialised tools and methods, it also chal- lenges collaboration and cooperation with the cloud service providers. Finally, most research works and tools are bound to specific system architecture, OS, or hardware implemen- tation, so they have the drawback of becoming cumbersome to adjust existing solutions to new use cases and problems. In this context, more generic approaches that allow tool reuse in different cases are necessary. y g As seen in Section III-G, multimedia forensics is one of the most explored topics, according to the number of publications. Overall, while most authors focus on image forgery detection, anti-forensics is one of the most challeng- ing problems. In this regard, more efforts should be devoted to counter anti-forensic mechanisms (i.e., as part of a global digital forensics concern) and methodologies to capture novel criminal trends with the help of sophisticated real-time object detection and classification systems. In addition, multi-layer systems and ontologies should be designed to cope with mul- tiple threats at once, paired with the appropriate benchmarks to evaluate them. In parallel, the issues related to the vast amount of data to be processed should be minimised by proposing more efficient data storage and indexing mech- anisms and introducing algorithms that can process, e.g., compressed data. Following such research paths and com- bining them with the proper legislation and standardisation mechanisms will improve the success of multimedia digital forensics investigations. The recovery of digital evidence from portable and/or mobile devices is the focus of mobile forensics (MF), a sub-branch of digital forensics. Seizure, acquisition, and examination/analysis are the three categories that mobile forensics processes fall into. Several challenges exist con- cerning mobile forensics, as presented in III-C. In the MF domain, the variety of embedded OSs with shorter prod- uct life cycles and the numerous smartphone manufacturers worldwide present significant challenges for applying sound forensics approaches. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews security features built into modern mobile devices, an inves- tigator must break into the device using an exploit that will almost certainly alter the data. the internet grows exponentially, the network traffic size to be analysed to conduct a forensics investigation rises. Methods that can efficiently analyse voluminous traces of network traffic are in high demand. Additionally, the heterogeneity of network traffic protocols increases the effort required to collect evidence from all available sources. While the widespread adoption of IoT devices and IoT- related applications has improved data availability and oper- ational excellence, it has also introduced new security and forensics challenges. As presented in Section III-D, several challenges exist concerning IoT forensics. Such challenges include managing multiple streams of data sources, the com- plicated three-tier architecture of IoT and the lack of stan- dardized systems for capturing real-time logs and storing them in a valid uniform form. The preparation of highly detailed reports of all information gathered, its correspond- ing representation, and the lack of standardized systems for capturing real-time logs also serve as barriers to establishing sound IoT-related forensics mechanisms. Data encryption trends are also posing new challenges for IoT forensic inves- tigators, and cryptographically protected storage systems are arguably one of the most significant roadblocks to effec- tive digital forensic analysis. Interoperability and availability issues related to the vast number of connected IoT devices, the Big Data nature of IoT forensics evidence, and IoT forensics evidence’s various security storage challenges also represent significant IoT-related forensics challenges. Finally, the IoT forensics domain faces several regulatory challenges, partic- ularly those relating to data ownership in the cloud as defined by regional laws. Last but not least, the main challenge that network foren- sics research faces nowadays is encrypted traffic. When dig- ital forensic evidence acquisition happens at an intermediate node of the communication path, it is expected for the traffic payload to be encrypted, and methods capable of extracting information under such conditions are required. Filesystems, Memory, and Data Storage forensics have attracted the research community’s attention, as they are an abundant source of digital evidence. As discussed in Section III-E, the main challenge of these domains lies in the fact that there exist a large number of files and data contained in them. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS After revising the challenges collected in cloud forensics, most of them are closely related to data management. More concretely, data acquisition, logging, limited access to foren- sic data, cross-border data access and exchange are vital parameters in cloud forensics. In terms of log management, Marty [232] proposed using log management architecture and the guidelines for application logging in SaaS service model using technologies such as Django, Javascript, Apache, and MySQL. A centralised logging scheme was proposed by Trenwith and Venter [233] to accelerate the investigation process and provide forensic readiness. Patrascu and Patri- ciu [234] proposed a scheme to monitor various parallel activities in a cloud environment. In addition to the pre- vious works, several authors have devoted efforts towards efficient and secure evidence management in the cloud [235]–[237], including the use of blockchain such as seen in [238]. We believe that efficient evidence and logging col- lection mechanisms paired with secure and verifiable man- agement of such evidence are crucial to guarantee sound cloud forensic investigations. To rationalize the functioning and limit the increasing num- ber of legal provisions, Regulation 2016/95 repealed certain acts in the field of police cooperation and judicial cooperation in criminal matters [227]. LEAs performing digital forensics have confidentiality case levels depending on the severity of the crime. The forensic examiners sign a special con- fidentiality agreement regarding data protection upon their employment. There are policies regarding data protection, all the case relevant data is kept only to the internal network, which is protected with the use of all the necessary measures (Secure Connections, encryption, controlled access at the physical location). The forensic examination equipment is not connected to the internet when examinations are conducted. The data in question in digital forensics is referred to as elec- tronic evidence, defined as ‘‘any information (comprising the Network traffic forensics is a long-standing domain with numerous research efforts and tools. The main gaps that currently exist and on which future efforts shall be focused are related to the volume of the traffic, the different protocols that emerge mainly due to the IoT rise, and the fact that traffic is encrypted in most cases. As the use of computer systems and 25482 VOLUME 10, 2022 VOLUME 10, 2022 F. 3) DECENTRALISATION AND IMMUTABILITY The wide adoption of distributed platforms, e.g. blockchain solutions [80] and distributed storage and filesystems, imply significant challenges for digital forensics [239], [240]. Some of these structures have strong privacy guarantees and can be leveraged to exfiltrate data, orchestrate malicious cam- paigns [241]–[244], or siphon fraudulent payments [245]. Traditional logging mechanisms and access control systems allow an investigator to assess who, when, how or even from where are not relevant for many of these technologies. As a result, they are continuously abused by threat actors. These huge obstacles for digital forensics require further research on the field and the development of more targeted tools to extend the capabilities of digital investigators. In this regard, while the use of distributed platforms is not exempt from potential issues [240], they can also be potentially used to leverage community-based intelligence against threats and to leverage auditable forensic investigations [82], [246]–[248]. Following such an idea and in order to accelerate the response towards sophisticated threats and international campaigns, the community is devoting research efforts towards federated learning models [249], [250], and other emerging topics such as cognitive security [251], [252]. Given the continuous evolution of cybercrime and its harm- ful capacities, preventive strategies are paramount to fight criminal activities. The latter implies the need to reinforce digital forensic strategies at different levels, including guide- lines, regulations, research and training to implement forensic readiness holistically. According to our literature analysis, one of the key points to reinforce the actual state of practice is the definition of interoperable and easy-to-adopt legislations since current ones cannot cope with the increasing sophis- tication and the ubiquitous nature of cybercrime. Therefore, it is crucial to devote efforts towards, e.g., interoperable cross- border models with their corresponding dissemination and training procedures, which all practitioners may adopt to accelerate investigations. It is also relevant to stress the neces- sity of appropriate forensic readability and reporting. First, effective communication between all the actors involved in a forensic investigation is essential to maximise the guarantees in court. Second, the proper documentation of investigations provides valuable feedback for future investigations, enhanc- ing forensic readiness strategies. Third, the definition of a common reporting framework can accelerate investigations in which sometimes speed is crucial due to, e.g., the pos- sible volatility of evidence or to reduce harm. A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS MF, in general, present a variety of challenges such as problems with data (anonymity-enforced browsing and other anonymity services, and the considerable volume of data acquired during an investigation), availability of forensic tools (MF research approaches have long focused on acquisition techniques, while minor importance was given to the other phases of MF investigative process) and security- oriented concerns (development of new and more sophisti- cated anti-forensic methods from mobile manufacturers). It is worth noting that MF is confronted with significant chal- lenges regarding the overall MF processes’ focus. For exam- ple, it is unclear whether investigation procedures should be model-specific for each device or generic enough to form a standardized set of forensics procedures guidelines. Another critical issue is the requirement to perform live forensics (mobile devices should be powered on). Finally, due to the Blockchain forensics is a relatively new domain since blockchain technology accounts for a decade. In general, it has to be understood that the need for blockchain foren- sics methods is expected to grow in the coming years. As discussed in Section III-F current efforts focus on the examination of available data on public blockchain systems. One of the main challenges encountered is to provide effi- cient methods to conduct such analysis. The data on public ledgers continuously grows, while the storage structure dif- fers amongst different implementations. Developing methods 25483 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews to foster a culture of forensic preparedness. Essentially, every organisation and resource provider must understand that its products and services are expected to suffer a successful cyber attack. Therefore, despite the countermeasures, recov- ery methods, and mitigation strategies, they need to imple- ment policies and mechanisms to facilitate digital forensics. If the latter are not well-placed, while business continuity may not be severely harmed, one may not understand why and how the security event occurred, what needs to change, or miss even important evidence of the threat actor. and tools that can efficiently analyse data across commonly used blockchain platforms is required. Moreover, forensic analysis methods for blockchain systems’ nodes will enable more thorough investigations with more detailed results for public and private blockchain systems. 3) DECENTRALISATION AND IMMUTABILITY To this end, we proposed a forensic reporting content representation by following the common denominators found in the literature in Section III. We argue that the devotion of more efforts on this final part of the forensic flow will enrich investigations with valuable feedback and successful prosecution guarantees. 7https://www.justice.gov/opa/speech/deputy-attorney-general-rod-j- rosenstein-delivers-remarks-encryption-united-states-naval A. THE ROAD AHEAD IN DIGITAL FORENSICS’ TOPICS Finally, given the ris- ing popularity of privacy enabled blockchain systems such as Monero or ZCash, additional effort will be required to support forensic investigations on cases that include interactions on such systems. 6https://formobile-project.eu/ 4) DATA PROTECTION AND ETHICS IN CRIMINAL INVESTIGATIONS Ransomware may be regarded as the most obvious case of exploiting cryptographic primitives for malicious acts; nev- ertheless, this is not by any chance the only. Threat actors and cybercriminals, for instance, use encrypted and even covert channels to communicate, further hindering investi- gations. The latter has sparked a huge debates as many are promoting concepts such as responsible encryption7 with the adoption of, e.g., weakened encryption, cryptographic schemes such as key escrow, backdooring of cryptographic primitives etc. [253]–[256]. While they may facilitate dig- ital investigations, essentially, they undermine the scope of cryptography and security, opening the door for many inter- pretations on what lawful interception is, when it can be performed, by whom, let alone the exploitation of the mech- anisms by already malicious actors as the backdoor would be already implanted. The debate is undergoing and spans 2) FORENSIC PREPAREDNESS AND STANDARDS While in Section IV we provided an overview of digital forensics standards, unfortunately, they do not suffice current needs. To name just two which are standing out on the tip of the iceberg, cloud and mobile related investigations need to have some standards on how to be performed. Addressing the need for mobile forensics, FORMOBILE6 has initiated a broad dialogue and is developing a draft CENELEC Work- shop Agreement to fill in this gap. However, due to the specificities of cloud, IoT, drones, etc., similar actions are expected in the near future. Beyond standards and methods, there is a definite need from industry players, developers, system administrators etc., 25484 25484 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews ransomware, malware, and threats against data availability and veracity, affect digital forensics in different dimensions, regardless of the topic. NIST recently published a state of the art analysis of cloud-related challenges [34], which is aligned with the claims collected by in the cloud-based digital forensics literature reviews state in Section III-A. In the case of networks, ENISA elaborated an extensive set of security objectives and discussed them along with their corresponding recommendation measures in the topics of electronic commu- nications [259] as well as 5G networks [260]. NIST provides security guidelines for managing mobile devices in their draft SP 800-124 (rev2) [261]. The recommendations include scenarios from organization-provided to personally-owned devices and describes technologies and strategies that can be used as countermeasures and mitigations. In the context of IoT, NIST released a set of documents related to IoT device cybersecurity, covering aspects from the design and manu- facturing of the components to their disposal [262]. In par- allel, ENISA also proposed a comprehensive set of security guidelines targeting all the entities involved in the supply chain of IoT to improve security decisions when designing, building, deploying, and assessing IoT technologies [263]. Concerning data storage and data processing, several guide- lines have been proposed during the past years to reduce data breaches [264], and the proper deployment of data storage mechanisms that enable privacy by design [265]–[267], and forensic readiness [268]. Finally, despite the existence of such guidelines, forensic frameworks accommodating procedures adapted to novel types of cybercrime such as in e.g. 2) FORENSIC PREPAREDNESS AND STANDARDS social networks [269], and the proper review and evaluation of an investigation process, are necessary to assess the quality of forensic investigations [270]. multiple sectors beyond digital forensics. While fostering such approaches may greatly benefit digital forensics, the ethical and legal implications hinder such adoption and are received by the security community with scepticism. As discussed, anti-forensics methods are a challenge for almost all domains of digital forensics. Nevertheless, with the growing adoption of TPM and TEE, these challenges can be significantly augmented. For instance, as illustrated by Dunn et al. [257] ransomware can exploit these technologies to render decryption key extraction impossible. Nevertheless, it is clear that these technologies introduce significant chal- lenges for digital investigators since they may deprive them of access to critical information. In this regard, it is essential to study methods for, e.g. live forensics in the presence of TPM and TEE and to explore how the missing information can be compensated. VI. CONCLUSION AND FINAL REMARKS VI. CONCLUSION AND FINAL REMARKS The digitisation of our daily lives is a double-edged sword as beyond the myriad of advantages and comforts it pro- vides, it introduces security and privacy issues. Motivated by the lack of a general view of the digital forensics ecosys- tem, mainly because different topics are explored in an isolated way and aiming to answer several research ques- tions/concerns, this manuscript seeks to fill a literature gap by proposing a review of reviews in the field of digital forensics. Following a thorough research methodology, we identified the main digital forensics topics. We performed a taxonomy by documenting the current state of the art and practice and the main challenges in each of them. Moreover, we anal- ysed these challenges with a cross-domain perspective to highlight their relevance according to the times they were discussed in the literature. According to the outcomes (i.e. see Section III-I), such analysis provided us with enough evi- dence to prove that the digital forensics community could benefit from closer collaborations and cross-topic research since it appears that researchers are trying to find solutions to the same problems in parallel, sometimes without noticing it. 5) AUTOMATION AND EXPLAINABILITY The continuous increase in reported cybercrimes apart from the impact on the victims implies a lot of effort from investi- gators to analyse the cases. Therefore, automation of digital forensics inevitably becomes a need. While automated meth- ods for collecting log files and algorithms to identify anoma- lies or even correlating some events may exist, this does not practically translate to automated digital forensics. Even if one does not consider APT attacks, one must understand that each case has particularities differentiating it from the others. Moreover, a digital investigator has to fill in the gaps of missing information that the attacker managed to cover, including those that security mechanisms failed to record or those erroneously reported. The above implies the develop- ment of advanced machine learning and AI algorithms and tools that will underpin future digital forensics investigations. An important part of these systems is undoubtedly under- standing the scope of the investigation and the explainability of the results [258], which is critical to assess the impact of current investigations and quantify their effectiveness [14], a critical step to ensure the implementation of the proper mea- sures. The latter is a crucial part of AI and machine learning modules that have to be introduced as in order for a piece of evidence to be admissible in a court of law, one has to justify not only how and from where it has been collected but to also prove the relevance to the case, how it was used, and why it is linked with the rest of the evidence. In essence, future digital forensics systems would have to argue and reason on the collected information in a human-readable manner. The latter is a huge step forward compared to the existing state where systems prioritise log events and present the analysts with known malicious patterns in the logs, malicious binaries, or connections that deviate specific norms. 6) FORENSIC GUIDELINES AND BEST PRACTICES One of the main strategies to reduce the impact of cybercrime is to implement the recommendations of the security guide- lines and directives developed by agencies such as ENISA and NIST. The current threat landscape [6], which includes By merging the information of Table 10 and Figure 4, we extracted the amount of cross-domain challenges that 25485 VOLUME 10, 2022 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews each topic has in each forensic phase, and reported them in Table 18. As it can be observed, data acquisition along with investigation and forensic analysis are the phases that entail more challenges, according to the research community. If we analyse the data at a topic level, we can observe that IoT has many challenges to overcome in such phases. The same applies to Multimedia and Mobile forensics. Since we focus on the extracted challenges as collected in our literature review, the fact that some challenges have not been high- lighted either at topic or forensic phase level may indicate that researchers and practitioners have not devoted enough effort to them, or perhaps highlights lack of discussion towards them. Such interesting domains include value chain and financial forensics. Like other domains, the business sector’s ongoing digitisation means that sound value chain forensics mechanisms will be almost necessary within any corporate strategy for the years to come. Therefore, the potentially unexplored issues in such cases require proactive initiatives before they become obstacles in the near future. usually, literature reviews are mature and long term works not likely to be published in conferences as they do not require a fast positioning. By discussing the open issues and future trends in digital forensics, and after observing that many of the challenges raised years ago are still not solved, we believe that our literature analysis reflects with high fidelity the cur- rent state of practice and the potential challenges that may arise in the years to come, providing a fruitful ground of research. The inherent cross-jurisdiction nature of modern cyber- crime paired with the abuse of cutting edge technologies man- dates more coordinated efforts from the security and research community. With the continuously increasing amount of data that have to be analysed, it is straightforward that man- ual analysis is almost at its limits. 6) FORENSIC GUIDELINES AND BEST PRACTICES The use of fine-grained IoCs may significantly reduce the effort of the investigator. However, as already discussed, this is not always possible, especially when non-traditional computing devices are used, e.g. IoT, mobile, cloud. As a result, machine learning and arti- ficial intelligence are gradually being integrated into the logic of many tools and methods. Nevertheless, the reasoning of the results in an understandable human manner is a cross-domain challenge. Moreover, the standardisation of digital forensics processes for cloud, mobile, IoT, drones, etc., is becoming a high priority since they are an indispensable part of almost all modern digital investigations. Finally, the consensus on developing these standards and the coordinated efforts made over the past few years for countering cybercrime must be leveraged to homogenise the legislation across jurisdictions and facilitate digital investigations. A common answer to the problem and using the same measures would create a strong response against cybercrime and improve response time to security incidents and their analysis. TABLE 18. Limitations per topic according to each phase as depicted in Figure 4. TABLE 18. Limitations per topic according to each phase as depicted in Figure 4. Further to merely listing the state of practice and proposing research directions according to the identified challenges, we analysed crucial aspects of digital forensics such as stan- dards, forensic readiness, forensic reporting, and discussed the ethical and legal aspects of data management in Europe in Section IV. The insights gathered from such analysis, which were represented in the form of structured tables, qualitative literature analysis, and a proposed representation of forensic report content, successfully answered the research questions presented in Table 1. ACKNOWLEDGMENT Th f hi i The content of this article does not reflect the official opinion of the European Union. Responsibility for the information and views expressed therein lies entirely with the authors. REFERENCES W. A. Wahab, M. Shiraz, and I. Ahmad, ‘‘Network forensics: Review, taxonomy, and open challenges,’’ J. Netw. Comput. Appl., vol. 66, pp. 214–235, May 2016. [17] H. Hunt, A. Pollock, P. Campbell, L. Estcourt, and G. Brunton, ‘‘An intro- duction to overviews of reviews: Planning a relevant research question and objective for an overview,’’ Systematic Rev., vol. 7, no. 1, pp. 1–9, Dec. 2018. [41] A. Nisioti, A. Mylonas, P. D. Yoo, and V. 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[Online]. Available: https://locard.eu [223] (2018). C. of Europe. Towards a Protocol to the Budapest Con- vention. [Online]. Available: https://rm.coe.int/t-cy-pd-pubsummary- v6/1680795713 [248] L. Zarpala and F. Casino, ‘‘A blockchain-based forensic model for finan- cial crime investigation: The embezzlement scenario,’’ Digit. Finance, vol. 3, no. 3, pp. 1–32, 2021. [224] (2016). E. Union. Directive (EU) 2016/680 of the European Parliament and of the Council. [Online]. Available: https://eur-lex.europa.eu/legal- content/EN/TXT/?uri=CELEX%3A32016L0680 [249] T. Li, A. K. Sahu, A. Talwalkar, and V. Smith, ‘‘Federated learning: Challenges, methods, and future directions,’’ IEEE Signal Process. Mag., vol. 37, no. 3, pp. 50–60, May 2020. [225] E. Union. (2014). Regulation (EU) no 910/2014, of the European Parliament and of the Council. [Online]. Available: https://eur- lex.europa.eu/legal-content/EN/TXT/?uri=uriserv%3AOJ.L_.201% 4.257.01.0073.01.ENG [250] Q. Yang, Y. Liu, T. Chen, and Y. Tong, ‘‘Federated machine learning: Concept and applications,’’ ACM Trans. Intell. Syst. Technol., vol. 10, no. 2, pp. 1–19, 2019. 25491 25491 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews [251] L. Ogiela and M. R. Ogiela, ‘‘Cognitive security paradigm for cloud computing applications,’’ Concurrency Comput., Pract. Exper., vol. 32, no. 8, p. e5316, Apr. 2020. FRAN CASINO (Member, IEEE) received the B.Sc. degree in computer science, the M.Sc. degree in computer security and intelligent sys- tems, and the Ph.D. MARIOS ANAGNOSTOPOULOS AGUSTI SOLANAS (Senior Member, IEEE) received the M.Sc. degree (Hons.) in com- puter engineering from Rovira i Virgili Univer- sity (URV), in 2004, the Diploma degree in advanced studies from the Polytechnic Univer- sity of Catalonia, in 2005, and the Ph.D. degree from the Department of Telematics Engineering, Polytechnic University of Catalonia, in 2007. He is currently a Professor with the Department of Com- puter Engineering and Mathematics and the Head of the Smart Technologies Research Group, URV. He serves as a Scientific Coordinator for APWG.EU. His current research interests include smart technologies, health informatics, behavior analysis, multivariate analysis, privacy protection, and computer security. MARIOS ANAGNOSTOPOULOS received the master’s degree in information and communica- tion systems security and the Ph.D. degree in infor- mation and communication systems engineering from the University of the Aegean, Greece. He has worked as a Postdoctoral Research Fellow in cyber security at the Norwegian University of Science and Technology (NTNU) and the Singapore Uni- versity of Technology and Design (SUTD). He has joined the Department of Electronic Systems, Aalborg University, Copenhagen, as an Assistant Professor at the Commu- nication, Media and Information Technologies Section, Aalborg University, and is a member of the Cyber-Security Research Group. He is the coauthor of more than 20 publications in peer-reviewed international conferences and journals. His research interests include the area of networks and computer security, and specifically DNS security, denial of service attacks, malware analysis, and forensics. He has also served as a Program Committee Member for international conferences and has taken part in both national and interna- tional research programs. MAURO CONTI (Fellow, IEEE) received the Ph.D. degree from the Sapienza University of Rome, Italy, in 2009. After his Ph.D. degree, he was a Postdoctoral Researcher at Vrije Uni- versiteit Amsterdam, The Netherlands. In 2011, he joined as an Assistant Professor at the Univer- sity of Padua, Italy, where he became an Associate Professor, in 2015, and a Full Professor, in 2018. He has been a Visiting Researcher with GMU, UCLA, UCI, TU Darmstadt, UF, and FIU. He is a Full Professor with the University of Padua. He is also affiliated with the Delft University of Technology (TU Delft) and the University of Washington, Seattle. His research is funded by companies, including Cisco, Intel, and Huawei. His main research interests include security and privacy. MARIOS ANAGNOSTOPOULOS In these areas, he has published more than 400 papers in topmost international peer- reviewed journals and conferences. He is a Senior Member of the ACM and a fellow of the Young Academy of Europe. He has been awarded with a Marie Curie Fellowship by the European Commission, in 2012, and with a Fellowship by the German DAAD, in 2013. He was the Program Chair of TRUST 2015, ICISS 2016, WiSec 2017, ACNS 2020, and CANS 2021, and the General Chair for SecureComm 2012, SACMAT 2013, NSS 2021, and ACNS 2022. He is the Editor-in-Chief of IEEE TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, the Area Editor-in-Chief of IEEE COMMUNICATIONS SURVEYS AND TUTORIALS, and he has been an Associate Editor of several journals, including IEEE COMMUNICATIONS SURVEYS AND TUTORIALS, IEEE TRANSACTIONS ON DEPENDABLE AND SECURE COMPUTING, IEEE TRANSACTIONS ON INFORMATION FORENSICS AND SECURITY, and IEEE TRANSACTIONS ON NETWORK AND SERVICE MANAGEMENT. AMRITA GHOSAL received the Ph.D. degree in computer science and engineering from the Indian Institute of Engineering Science and Technology, India, in 2015. After her Ph.D. degree, she worked as a Postdoctoral Researcher at the Department of Mathematics, University of Padua, Italy. She is currently a Marie Skłodowska-Curie Fellow with the Department of Electronic and Computer Engineering, University of Limerick, Ireland. She has coauthored a number of book chapters. Her research interests include in the areas of security and privacy for mobile and wireless networks. Particularly, she is interested in detection, prevention, and mitigation of different DoS style attacks for smart grid, v2x, connected vehicle, cyber-physical systems, and the IoT. In these areas, she has pub- lished more than 35 papers in high quality journals and refereed conference proceedings. ISTVÁN BO ¨RO ¨CZ received the Law (JD) and postgraduate specialist Diploma degrees in infor- mation and communication technology law from the University of Pécs, in 2013 and 2015, respec- tively, and the LLM degree in law and technol- ogy from Tilburg University, in 2016. He is a Data Protection Officer at Ion Beam Applications SA (IBA) and a Researcher at the Research Group on Law, Science, Technology and Society (LSTS). He is also a member of the Health and Ageing Law Laboratory (HALL), a spinoff group within LSTS. He is involved and provides legal assistance in several EU co-funded research projects, such as ARC, LOCARD, PERSONA, STAR, INTREPID, MaTHiSiS, FORENSOR, HR-Recycler, and SUCCESS or PARENT. s_103643v010101p.pdf He is the coauthor of more than 30 publications in peer reviewed journals and conference proceedings. His research interests include related to networks security, privacy preserving techniques, and blockchain technology. He has also served as the Program Committee Member for international conferences and has taken part in both national and international research programs. [266] (2019). National Institute of Standards and Technology. Recommen- dations on Shaping technology According to GDPR Provisions— Exploring the Notion of Data Protection by Default. [Online]. Available: https://www.enisa.europa.eu/publications/recommendations- on-shaping-tec% hnology-according-to-gdpr-provisions-part-2 6 i i i l d ki [267] A. Zigomitros, F. Casino, A. Solanas, and C. Patsakis, ‘‘A survey on privacy properties for data publishing of relational data,’’ IEEE Access, vol. 8, pp. 51071–51099, 2020. [268] (2019). The European Union Agency for Cybersecurity (ENISA). Towards a Framework for Policy Development in Cybersecurity— Security and Privacy Considerations in Autonomous Agents. [Online]. Available: https://www.enisa.europa.eu/publications/considerations-in- autonomous-a% gents [269] H. Arshad, E. Omlara, I. O. Abiodun, and A. Aminu, ‘‘A semi-automated forensic investigation model for online social networks,’’ Comput. Secur., vol. 97, Oct. 2020, Art. no. 101946. [270] N. Sunde and G. Horsman, ‘‘Part 2: The phase-oriented advice and review structure (PARS) for digital forensic investigations,’’ Forensic Sci. International: Digit. Invest., vol. 36, Mar. 2021, Art. no. 301074. 25492 VOLUME 10, 2022 F. Casino et al.: Research Trends, Challenges, and Emerging Topics in Digital Forensics: A Review of Reviews MARIOS ANAGNOSTOPOULOS These projects target a range of areas, such as law enforcement, technology-induced education, human–robot interaction, smart cities, or helping the work of data protection authorities. He is a member of the Ethical Advisory Board of the Horizon2020 Project CUIDAR. His research interests include the notion of the privacy of the mind along with the legal, theoretical, and practical issues of human enhancement technologies, with special focus on cognitive enhancement. In particular, he focuses on technologies which passively read and actively affect the human brain and the mind both within and outside the field of health care. He is also an Editor of the World Law Dictionary, developed by TransLegal Sweden AB. CONSTANTINOS PATSAKIS received the B.Sc. degree in mathematics from the University of Athens, Greece, the M.Sc. degree in information security from the Royal Holloway, University of London, and the Ph.D. degree in cryptography and malware from the Department of Informatics, University of Piraeus. He has participated in sev- eral national (Greek, Spanish, Catalan, and Irish) and European research and development projects (e.g., TACTICS, MITIGATE, OPERANDO, SAURON, PRACTICIES, and YAKSHA). He worked as a Researcher at the UNESCO Chair in data privacy and as a Research Fellow at the Trinity College Dublin, Dublin, Ireland. His main research interests include cryptography, malware, security, privacy, and data anonymization. Currently, he is Assistant Professor at University of Piraeus and adjunct researcher of Athena Research and Innovation Center. 25493 VOLUME 10, 2022
https://openalex.org/W3035763495
https://escholarship.org/content/qt19v4c4wp/qt19v4c4wp.pdf?t=rdkcn7
English
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A rare presentation of hepatolithiasis in an adolescent patient: A case report
International journal of surgery case reports
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2,837
a r t i c l e i n f o Article history: Received 22 April 2020 Accepted 7 June 2020 Available online 12 June 2020 Keywords: Adolescent Case report Hepatic duct Hepatolithiasis Intrahepatic stone Article history: Received 22 April 2020 Accepted 7 June 2020 Available online 12 June 2020 INTRODUCTION: Hepatolithiasis (intrahepatic stones) is rare in adolescent patients and requires complex management strategies to prevent recurrent infections and progression to hepatic fibrosis. Surgical man- agement is often required. In cases of unclear etiology, further work-up is indicated to provide insight into future management. In this report we describe an extensive stone analysis. PRESENTATION OF CASE: A 20-year-old Caucasian female presented with known hepatolithiasis and multiple prior recurrent bouts of abdominal pain requiring hospitalization. Magnetic resonance cholangiopancreatography (MRCP) demonstrated an abnormal left-sided hepatic biliary ductal system dilatation. She was treated surgically with a formal left hepatectomy and preservation of the caudate lobe. The right ductal system had no stones or evidence of inflammation, and her bile and stones cultures were negative for organism growth. An extensive analysis demonstrated stone composition primarily of cholesterol. Keywords: Adolescent Case report Hepatic duct Hepatolithiasis Intrahepatic stone DISCUSSION: Adolescent presentations of hepatolithiasis are rare and considerations in the differential diagnosis include primary sclerosing cholangitis, bile acid transporter defects, and other known genetic diseases. This case is unique because only the left half of the intrahepatic ductal system had evidence of stone disease and the bile was sterile. A detailed stone analysis demonstrating cholesterol supersaturation provides additional context though the etiology remains unclear in this case and will require lifelong follow-up. p CONCLUSION: Early-onset hepatolithiasis is rare and requires expert management, and in some cases definitive surgical management with life-long follow-up. Extensive stone analysis and genetic testing can be performed to help identify disease etiology in unique cases. © 2020 The Author(s). Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). A rare presentation of hepatolithiasis in an adolescent patient: A case report Jonathan Freise a, Jorge Mena a, Kwun Wah Wen b, Marshall Stoller c, Sunita Ho c,d, Carlos Corvera e,∗ a School of Medicine, University of California San Francisco, San Francisco, CA, USA b Department of Pathology, University of California San Francisco, San Francisco, CA, USA c Department of Urology, University of California San Francisco, San Francisco, CA, USA d Department of Preventive and Restorative Dental Sciences, Division of Biomaterials and Bioengineering, University of California San Francisco, S Francisco, CA, USA e D f S Di i i f S i l O l U i i f C lif i S F i S F i CA USA a School of Medicine, University of California San Francisco, San Francisco, CA, USA b Department of Pathology, University of California San Francisco, San Francisco, CA, USA c Department of Urology, University of California San Francisco, San Francisco, CA, USA d Department of Preventive and Restorative Dental Sciences, Division of Biomaterials and Bioengineering, University of California San Francisco, San Francisco, CA, USA a School of Medicine, University of California San Francisco, San Francisco, CA, USA f y f f b Department of Pathology, University of California San Francisco, San Francisco, CA, USA d Department of Preventive and Restorative Dental Sciences, Division of Biomaterials and Bioengineering, University of California S Francisco, CA, USA , , e Department of Surgery, Division of Surgical Oncology, University of California San Francisco, San Francisco, CA, USA Abbreviations: EDX, Electron Dispersive X-Ray; Micro-XCT, Micro X-Ray Com- puterized Tomography; MRCP, magnetic resonance cholangiopancreatography; PSC, primary sclerosing cholangitis; SEM, Scanning Electron Microscopy. ∗Corresponding author at: UCSF Helen Diller Family Comprehensive Cancer Cen- ter, 1825 Fourth Street, 4th Floor, San Francisco, CA, 94158, USA. E-mail address: Carlos.corvera@ucsf.edu (C. Corvera). CASE REPORT – OPEN ACCESS International Journal of Surgery Case Reports 72 (2020) 343–345 https://doi.org/10.1016/j.ijscr.2020.06.017 2210-2612/© 2020 The Author(s). Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY license (http:/ creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.ijscr.2020.06.017 2210-2612/© 2020 The Author(s). Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY license (http:// creativecommons org/licenses/by/4 0/) https://doi.org/10.1016/j.ijscr.2020.06.017 2210-2612/© 2020 The Author(s). Published by Elsevier Ltd on behalf of IJS Publishing Gr creativecommons.org/licenses/by/4.0/). ed by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY license (http:// Powered by the California Digital Library University of California Powered by the California Digital Library University of California eScholarship.org 3. Discussion This case report illustrates a unique presentation of hepatolithi- asis for several reasons. While most patients are diagnosed with hepatolithiasis in the 5th or 6th decade of life, presentation in ado- lescence is rare. Primary considerations in the differential diagnosis include primary sclerosing cholangitis (PSC), recurrent pyogenic cholangitis, bile acid transporter defect, and Caroli disease. PSC is a chronic cholestatic liver disease of unknown etiology character- ized by inflammation and fibrosis of intra and extrahepatic bile ducts leading to progressive stricturing and dilatation of the biliary ducts. Given the extensive stone disease and diffuse involvement of the liver specimen, this diagnosis is unlikely. In addition, PSC generally affects the entire biliary tract and is not isolated to one side of the liver as in our patient. Recurrent pyogenic cholangitis, which is endemic in East Asia, remains unlikely given the patient’s lack of travels to that area and Caucasian descent. Moreover, her frequent hospitalizations occurred without systemic signs of infec- tions and her bile was sterile. A bile acid transporter defect, such as ABCB4/MDR3, can present with hepatolithiasis at a younger age, but the absence of significant disease in the right hepatic duct sys- tem argues against this as a cause [6]. Caroli disease, a congenital disorder characterized by multifocal, segmental dilatation of large intrahepatic ducts, is a possibility in cases of early-onset hepa- tolithiasis. However, the patients’ history of MRCP reports did not strongly favor this diagnosis because the biliary dilatation appeared more fusiform than saccular, which would be an abnormal pre- sentation. Moreover, this diagnosis was not supported by final histological examination. Ultimately, we are left with an unknown cause of this patient’s development of early-onset hepatolithiasis. We attribute her problem to a yet undescribed or undiscovered genetic cause given the patient’s strong family history of stone dis- ease. Her history was notable for early gallbladder disease in her An MRCP showed isolated and dilated biliary ducts from the cau- date lobe with eventual resolution of her symptoms. Bile culture samples from the operative procedure were negative for organ- ism growth. Pathology demonstrated numerous (up to 0.5 mm) yellow tan calculi filling the entire dilated left biliary ductal sys- tem and fibrosis and inflammation of the bile duct walls, as seen in the section of left liver lobe (Fig. 1). The hepatic parenchyma showed marked sinusoidal dilatation, congestion, and hepatic plate atrophy. No tumor or parasite was found. 2. Presentation of case A 20-year-old Caucasian female with a strong family history of early onset gallbladder disease first presented to our center with known hepatolithiasis and recurrent bouts of severe abdominal pain requiring multiple hospitalizations. She was first hospital- ized at the age of 13 for right upper quadrant abdominal pain found to have choledocholithiasis with intrahepatic and extrahep- atic biliary duct dilatation. She underwent endoscopic retrograde cholangiopancreatography during her admission had a subsequent cholecystectomy with no evidence of cholelithiasis. In the following years, she experienced recurrent bouts of severe abdominal pain without any fever or chills and multiple MRCP studies that demon- strated abnormal left hepatic biliary ductal system dilatation. At the time of her presentation to our center, she was noted to have a dominant stone impacted at the origin of her left main hepatic duct with a left sided dilated ductal system. She was taken to the operating room for a formal left hepatectomy. Intraoperatively, she was found to have palpable stone disease throughout her left liver with a large stone aggregate at the origin of the left hepatic duct. Choledochoscopy of the right sided ductal system demonstrated normal biliary epithelium without inflammation or stones. She was treated by formal left hepatectomy and preservation of her extra- hepatic biliary tract. Her preoperative symptom of abdominal pain completely resolved, however, her recovery was complicated by the development of pruritus two weeks after discharge. specimens and of the core of fractured specimens demonstrated the stones were made almost entirely of smooth plates (Fig. 2). EDX of the plates revealed presence of three major elements: carbon, nitrogen, and oxygen, with carbon being the predomi- nant element, consistent with a similar make-up to cholesterol based stones. No calcium was detected from EDX. Stone specimens were subsequently placed in different solvents: ethanol, DI water, DI water + detergent, and trichloromethane. Dissolution was only achieved in the trichloromethane solution. J. Freise et al. / International Journal of Surgery Case Reports 72 (2020) 343–345 344 Fig. 2. SEM of the surface of whole specimens and of the core of fractured specimens demonstrating stones composed almost entirely of smooth plates. Fig. 1. Hemisection of left liver lobe demonstrating yellow tan intrahepatic stones filling the dilated left biliary ductal system and fibrosis and inflammation of the bile duct walls. Fig. 1. Hemisection of left liver lobe demonstrating yellow tan intrahepatic stones filling the dilated left biliary ductal system and fibrosis and inflammation of the bile duct walls. Fig. 2. SEM of the surface of whole specimens and of the core of fractured specimens demonstrating stones composed almost entirely of smooth plates. 1. Introduction symptoms of the intrahepatic type are more frequent in younger age groups [2]. Etiology remains unknown in most cases, although genetic and environmental factors are thought to contribute. Stones are classified by composition into calcium bilirubinate stones and cholesterol stones, with 75% of cases being calcium bilirubinate [3]. Treatment goals focus on resolving recurrent infection and preventing subsequent hepatic fibrosis and progression to cholan- giocarcinoma. The most efficacious treatment to date has been surgery, including removal of affected liver segment(s) to pre- vent recurrence of stone formation and progressive hepatocellular injury. In this case report, we discuss a unique presentation of hep- atolithiasis at our academic center with a comprehensive work-up of the patient’s stone disease to better characterize etiology. This work is reported according to the SCARE and PROCESS criteria [4,5]. Hepatolithiasis is defined as the presence of gallstones in the bile ducts proximal to the confluence of the right and left hepatic ducts, irrespective of the co-existence of stone disease in the common bile duct and gallbladder. The prevalence of hepatolithiasis is highest in East Asia and rare in Western countries [1]. Moreover, inci- dence occurs most commonly in the fifth and sixth decades, though j Author(s). Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY license (http enses/by/4.0/). CASE REPORT – OPEN ACCESS J. Freise et al. / International Journal of Surgery Case Reports 72 (2020) 343–345 Declaration of Competing Interest [3] I. Endo, R. Matsuyama, R. Mori, H. Shimada, Chapter 39 - intrahepatic stones, in: W.R. Jarnagin (Ed.), Blumgart’s Surgery of the Liver, Biliary Tract and Pancreas, 2-Volume Set (Sixth Edition), Content Repository Only, Philadelphia, 2017, http://dx.doi.org/10.1016/B978-0-323-34062-5.00039-X, 642-655.e3. The authors declare that they have no conflicts of interest. Sources of funding [4] R.A. Agha, M.R. Borrelli, R. Farwana, et al., The SCARE 2018 statement: updating consensus Surgical CAse REport (SCARE) guidelines, Int. J. Surg. Lond. Engl. 60 (2018) 132–136, http://dx.doi.org/10.1016/j.ijsu.2018.10.028. Jorge Mena received the UCSF PROF PATH Pre-Doctoral Grant to support his research. [5] R.A. Agha, M.R. Borrelli, R. Farwana, et al., The PROCESS 2018 statement: updating consensus Preferred Reporting of CasE Series in Surgery (PROCESS) guidelines, Int. J. Surg. Lond. Engl. 60 (2018) 279–282, http://dx.doi.org/10. 1016/j.ijsu.2018.10.031. 3. Discussion Calculi composition was noted to be primarily of cholesterol with some calcium bilirubi- nate, bile salts and pigments consistent with a bile stone. Given the unique presentation of the patient’s disease, a more extensive analysis was performed on the stones. Material analysis of the stones was performed using Micro X-Ray Computerized Tomography (Micro-XCT) for density and internal structure, and Scanning Electron Microscopy (SEM) for ultrastructure. Elemental analysis was performed using Electron Dispersive X-Ray (EDX) Spectroscopy. Micro-XCT of stone cross section (not shown) demonstrated low mineral density through- out the specimen and a hollow core. SEM of the surface of whole References [1] A. Pausawasdi, P. Watanapa, Hepatolithiasis: epidemiology and classification, Hepatogastroenterology 44 (14) (1997) 314–316. [1] A. Pausawasdi, P. Watanapa, Hepatolithiasis: epidemiology and classification, Hepatogastroenterology 44 (14) (1997) 314–316. [2] S. Tazuma, Gallstone disease: epidemiology, pathogenesis, and classification of biliary stones (common bile duct and intrahepatic), Best Pract. Res. Clin. Gastroenterol. 20 (6) (2006) 1075–1083, http://dx.doi.org/10.1016/j.bpg.2006. 05.009. [2] S. Tazuma, Gallstone disease: epidemiology, pathogenesis, and classification of biliary stones (common bile duct and intrahepatic), Best Pract. Res. Clin. Gastroenterol. 20 (6) (2006) 1075–1083, http://dx.doi.org/10.1016/j.bpg.2006. 05.009. J. Freise et al. / International Journal of Surgery Case Reports 72 (2020) 343–345 345 Provenance and peer review Presentations of early-onset hepatolithiasis is rare, particularly in Western countries. Definitive surgical management is required in complex cases. In cases with unknown etiology, further work- up is indicated and can include stone analysis and genetic testing. If indicated, life-long follow-up should be incorporated, as is the recommended management in this case. A detailed family history, genetic testing, and extensive stone analysis may be performed to help characterize disease etiology in unique presentations. Author contribution father and paternal cousin, aunt and grandmother, which would argue for a genetic component. Genetic testing of family members may help better characterize a cause for this patient’s rare case. father and paternal cousin, aunt and grandmother, which would argue for a genetic component. Genetic testing of family members may help better characterize a cause for this patient’s rare case. Jonathan Freise: data collection, manuscript writing, design of study. The stone analysis, while not revealing of a definite diagnosis, might help point in the direction of the etiology. The stones col- lected from this patient were composed primarily of cholesterol. Cholesterol is commonly found as a major component of stones in the extrahepatic ducts and gallbladder but it is rarely found as the main component in intrahepatic stones [7]. In the absence of precipitating factors such as infection, cholestasis, or structural abnormalities which were not apparent in this patient, defects of bile metabolism are likely candidates for relative cholesterol super- saturation and nucleation within the liver ducts [8]. Jorge Mena: performed stone analysis, manuscript writing and revision. Kwun Wah Wen: evidence collection, manuscript revision. Marshall Stoller: supervision of stone analysis, design of study. shall Stoller: supervision of stone analysis, design of Sunita Ho: supervision of stone analysis, design of study, manuscript revision. Sunita Ho: supervision of stone analysis, design of study, manuscript revision. Carlos Corvera: managed patient and performed the surgery, design of study, manuscript revision, corresponding author. Carlos Corvera: managed patient and performed the surgery, design of study, manuscript revision, corresponding author. Registration of research studies Our patient will need close clinical follow-up as she remains at high risk of developing intrahepatic stone formation in her remant right liver. A life-time of interval liver function tests, and cross-sectional imaging is strongly recommended to detect early recurrent disease to prevent progressive indolent liver injury and ultimately liver failure. Indeed, occasionally in patients with recur- rent pyogenic cholangitis, liver transplantation as a salvage therapy is required [9]. 1. Name of the registry: n/a. 2. Unique Identifying number or registration ID: n/a. 3. Hyperlink to your specific registration (must be publicly acces- sible and will be checked). 3. Hyperlink to your specific registration (must be publicly acces- sible and will be checked). 4. Conclusion Carlos Corvera. Ethical approval [6] J. Benzimra, S. Derhy, O. Rosmorduc, Y. Menu, R. Poupon, L. Arrivé, Hepatobiliary anomalies associated with ABCB4/MDR3 deficiency in adults: a pictorial essay, Insights Imaging 4 (3) (2013) 331–338, http://dx.doi.org/10. 1007/s13244-013-0243-y. Ethical approval exempted by our institution. Not commissioned, externally peer-reviewed. Not commissioned, externally peer-reviewed. Consent [7] S.D. Strichartz, M.Z. Abedin, A.F. Ippoliti, M. Derezin, J.J. Roslyn, Intrahepatic cholesterol stones: a rationale for dissolution therapy, Gastroenterology 100 (1) (1991) 228–232, http://dx.doi.org/10.1016/0016-5085(91)90605-k. Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request. ( ) ( ) p // g/ / ( ) [8] X. Ran, B. Yin, B. Ma, Four major factors contributing to intrahepatic stones, Gastroenterol. Res. Pract. (2017), http://dx.doi.org/10.1155/2017/7213043. [9] You M. su, S.H. Lee, J. Kang, et al., Natural course and risk of cholangiocarcinoma in patients with recurrent pyogenic cholangitis: a retrospective cohort study, Gut Liver 13 (3) (2019) 373–379, http://dx.doi.org/10.5009/gnl18339. Open Access This article is published Open Access at sciencedirect.com. It is distributed under the IJSCR Supplemental terms and conditions, which permits unrestricted non commercial use, distribution, and reproduction in any medium, provided the original authors and source are credited.
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https://digibug.ugr.es/bitstream/10481/61827/1/remotesensing-11-00643-v2.pdf
English
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Detection of Fir Trees (Abies sibirica) Damaged by the Bark Beetle in Unmanned Aerial Vehicle Images with Deep Learning
Remote sensing
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cc-by
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Received: 4 February 2019; Accepted: 12 March 2019; Published: 16 March 2019 Abstract: Invasion of the Polygraphus proximus Blandford bark beetle causes catastrophic damage to forests with firs (Abies sibirica Ledeb) in Russia, especially in Central Siberia. Determining tree damage stage based on the shape, texture and colour of tree crown in unmanned aerial vehicle (UAV) images could help to assess forest health in a faster and cheaper way. However, this task is challenging since (i) fir trees at different damage stages coexist and overlap in the canopy, (ii) the distribution of fir trees in nature is irregular and hence distinguishing between different crowns is hard, even for the human eye. Motivated by the latest advances in computer vision and machine learning, this work proposes a two-stage solution: In a first stage, we built a detection strategy that finds the regions of the input UAV image that are more likely to contain a crown, in the second stage, we developed a new convolutional neural network (CNN) architecture that predicts the fir tree damage stage in each candidate region. Our experiments show that the proposed approach shows satisfactory results on UAV Red, Green, Blue (RGB) images of forest areas in the state nature reserve “Stolby” (Krasnoyarsk, Russia). Keywords: multi-class classification; drone; aerial photography; Siberian fir; Siberia; deep-learning; convolutional neural networks; forest health remote sensing remote sensing remote sensing Article Anastasiia Safonova 1,2,3,4,* , Siham Tabik 3 , Domingo Alcaraz-Segura 4,5,6 , Anastasiia Safonova 1,2,3,4,* , Siham Tabik 3 , Domingo Alcaraz-Segura 4,5,6 , Alexey Rubtsov 1 , Yuriy Maglinets 1 and Francisco Herrera 3 1 Institute of Space and Information Technologies, Siberian Federal University, 660074 Krasnoyarsk, Russia; alexeyruss@gmail.com (A.R.); maglinets@mail.ru (Y.M.) 1 Institute of Space and Information Technologies, Siberian Federal University, 660074 Krasnoyarsk, Russia; alexeyruss@gmail.com (A.R.); maglinets@mail.ru (Y.M.) 2 The Earth Science Museum of M.V. Lomonosov Moscow State University, 119991 Moscow, Russia 3 Andalusian Research Institute in Data Science and Computational Intelligence, University of Granada, 18071 Granada, Spain; siham@ugr.es (S.T.); herrera@decsai.ugr.es (F.H.) p g g 4 Department of Botany, University of Granada, 18071 Granada, Spain; dalcaraz@ugr.es 5 5 Andalusian Center for the Assessment and Monitoring of Global Change (CAESCG), University of Almería, 04120 Almería, Spain p 6 iEcolab., Interuniversity Institute for Earth System Research in Andalusia (IISTA), University of Granada, 18006 Granada, Spain p * Correspondence: safonova.nastya1@gmail.com   1. Introduction Taiga and Boreal forests play an important role in the global climate through the carbon, water and energy balance (Bonan [1]). In Russia, fir forests provide multiple provisioning, regulating and cultural ecosystem services and are considered a national asset of the country. Despite forests are a renewable resource, in vast areas of the world, forest degradation is too high and not compensated by regeneration (Hansen et al. [2]). One of the main problems of Russian forest degradation is the spatial spread acceleration of the four-eyed fir bark beetle (Polygraphus proximus Blandford) invasion that causes fast death of fir trees (Abies sibirica Ledeb) in various forest ecosystems. The geographic origin of P. proximus propagation comes from its natural habitat in Japan, the Korean Peninsula, Remote Sens. 2019, 11, 643; doi:10.3390/rs11060643 www.mdpi.com/journal/remotesensing 2 of 19 Remote Sens. 2019, 11, 643 Eastern China and the Russian Far East (Khabarovsk and Primorskii krai, Sakhalin and Kuril Islands) (Kuznetsov et al. [3]. In Siberia, the first beetle occurrence was registered in 2008 (Kerchev [4]) and within the last 10 years, massive outbreaks of P. proximus occurred on large forested areas in the south-eastern part of the West Siberian Plain: Tomsk, Kemerovo and Novosibirsk oblasts, Altai region, in the Altai Republic as well as in Krasnoyarsk region (Pashenova et al. and Baranchikov et al. [5,6]), where the problem became catastrophic and got out of control. The invasion of this type of beetle can lead not only to degradation of fir forests but also to create a threat to fir existence as a forest type species, with the subsequent broad implications for the regional and global climate (Helbig et al., Ma [7,8]). Death of fir trees due to P. proximus outbreaks occurs after several stages. The beetle usually attacks trunks of weakened trees, fallen deadwood and newly harvested wood. In case of massive outbreaks, P. proximus also attacks healthy trees, which can resist against the beetle attack during 2–3 years. Penetration of P. proximus under tree bark results in pervasion and reproduction of ophiostomatoid fungi (different species) and their phytopathogenic activity leads to the gradual weakening of fir trees. As the beetle further colonizes the tree, it begins to dry out. Increase of beetles quantity in a local forest stand leads to a massive death of fir trees. 1. Introduction Usually, fir trees die within 2–4 years from the moment of first beetle attack, so the task of the beetle invasion monitoring is really important and poses a challenge to develop a remote survey and early-warning system for precise estimation of forest damage states. Advances in Earth remote sensing techniques, particularly very high resolution satellite and airborne imagery, open the possibility to develop tools for regional mapping of consequences of forest pest activities. Nowadays, application of unmanned aerial vehicles (UAV) tends to be more popular in local scale forestry research because of better spatial resolution (Lehmann et al. [9]). Also, UAV data provides the basis for development of new methods in data analysis which can be applied later at larger scales (satellite data). Recently, application of deep learning, in particular convolutional neural networks (CNN), in processing of colour (RGB) images of the Earth surface from various data sources have provided high accuracy results in recognition of different plant species [10–14]. However, there are still no published studies on the application of CNN methods to very high resolution imagery for detection of forest health decline caused by pest invasions. The goal of this study was to test the possibilities of neural networks as a new approach to detect bark beetle outbreaks in fir forests. In particular, our aim was to develop and test a CNN method to automatically detect individual fir trees disturbed by P. proximus in very high resolution imagery of mixed forests. The main contributions of this paper can be listed as follows: • As far as we know, this is the first work in addressing the problem of forest damage detection caused by the P. proximus beetle in very high resolution images from UAVs with deep learning. W b ilt l b ll d d t t f th i ith f t i f d t i fit • We built a new labelled dataset of orthoimages with four categories of damage stages in fir tr g g g g • We designed a new CNN architecture to accurately classify trees in UAV images into different damage categories of trees and compared it to the most powerful CNN models in the stat. • We designed a new CNN architecture to accurately classify trees in UAV images into different damage categories of trees and compared it to the most powerful CNN models in the stat. 1. Introduction • We developed the detection model as follows. First, a new detection method selects the candidate regions that contain trees in UAV images. Then, these candidate regions are processed by the multiclass model to finally predict the category of tree damage. • We developed the detection model as follows. First, a new detection method selects the candidate regions that contain trees in UAV images. Then, these candidate regions are processed by the multiclass model to finally predict the category of tree damage. • We provide a complete description of the used methodology and the source code so that it can be replicated by other researchers to detect and classify tree damage by bark beetle in other images. Our source code can be found at https://github.com/ansafo/OurCNN. The rest of the paper is organized as follows. An introduction to Deep learning and CNNs is presented in Section 2, which includes a description of the CNN models and of the auxiliary techniques of transfer learning and data augmentation. The related works on tree classification are provided in Section 3. The study area and data acquisition are presented in Section 4. The proposed methodology is presented in Section 5, which includes a definition of fir trees damage categories, pre-processing dataset 3 of 19 Remote Sens. 2019, 11, 643 and data augmentation techniques of sample patches for training CNN models, description of the development of the classification model algorithm and creation of a data subset for independent model verification. Section 6 presents the obtained results and data analysis. Discussion and conclusions are provided in Section 7. 2. Introduction to Deep learning and CNNs Conceptually, this operation is applied to increase the abstraction of the extracted features and it usually follows the convolution layer. • A fully connected layer, which is used as a classifier of the previously calculated high level features to derive scores for each target class. • A fully connected layer, which is used as a classifier of the previously calculated high level features to derive scores for each target class. Deep CNNs are built by alternating several convolutional layers with pooling layers. At the end of the sequence, the obtained convolutions and pooling layers are connected to one or more fully connected layers. Softmax function is used to convert the scores of the output of the final fully connected layer into a set of probabilities between 0 and 1, which represent the final output layer. Before starting the training process, the weights of the CNN are initialized with random values. The CNN is then trained iteratively until convergence during a number of epochs. The loss function is used to measure how good the training process is by quantifying the distance between the prediction and the expected value. To find the best weights that correctly assign the input to the correct answer, the training process is reformulated into an optimization problem that aims to minimize the loss function. This minimization can be achieved using several optimization algorithms depending on the problem. One example is the ADAptive Moment estimation (ADAM) optimizer that computes the best weights. 2. Introduction to Deep learning and CNNs In this Section, we make a brief introduction to deep learning and particularly to CNN models (Section 2.1), including the two main approaches used to improve the learning of CNN: that is, transfer learning and data augmentation (Section 2.2). Deep Neural Networks (DNNs) are a subset of machine learning algorithms that learn from a first set of data to make predictions on new data. Unlike most machine learning algorithms, DNNs are capable of extracting the existent patterns from data automatically, without the need of external hand-crafted features. Their computation is organized into layers; each layer is composed of a number of artificial neurons. To introduce non-linearity in the network, non-linear functions are introduced at the neuron level, such as the Rectified Linear Unit (ReLU) function. Deep networks are built by stacking a very large number of layers. They provide a powerful framework for supervised learning when trained on a large number of labelled samples (Goodfellow et al. [15]). A DNN includes an input layer, an output layer and hidden layers between them where the calculations take place. One of the most powerful types of DNNs are Convolutional Neural Networks (CNNs), which have shown impressive results in image classification (given an input image, the CNN produces a label that indicates the visual content of that image) and object detection in images (given an input image the CNN produces a label together with a bounding box that indicates the region where the object-class is located in the image). There are three main types of hidden layers in a CNN: • A convolution layer, the main building block of a CNN. It is based on a fundamental operation in image processing called convolution, which consists of filtering a 2D input image with a small 2D-matrix called filter or kernel. • A convolution layer, the main building block of a CNN. It is based on a fundamental operation in image processing called convolution, which consists of filtering a 2D input image with a small 2D-matrix called filter or kernel. • A pooling layer, which reduces the input matrix in general by half. Conceptually, this operation is applied to increase the abstraction of the extracted features and it usually follows the convolution layer. • A pooling layer, which reduces the input matrix in general by half. 2.1. CNNs Models In Appendix B, we provide a brief description of the CNNs that are compared against the CNN developed in this work, that is, VGG, ResNet, Inception-V3, InceptionResNet-V2, Xception and DensNet. Remote Sens. 2019, 11, 643 4 of 19 2.2. Transfer Learning and Data Augmentation CNNs need a large volume of data to achieve good results, however, this is not always possible since building new labelled dataset is costly and time consuming. To overcome this limitation in practice, CNNs are seldom trained from scratch. Instead, their weights are initialized by the pre-trained weights on a large dataset such as, the massive ImageNet. This approach is called transfer learning. In addition, to address a new problem, not all the weights are re-trained but only the weights of the last layers, for example, the fully connected layers. This process is called fine-tuning. Data-augmentation technique consists of creating new training examples by applying geometrical transformations such as rotation and translation to the original samples. Its objective is artificially increasing the volume of the dataset. In fact, it was demonstrated in several works (Tabik et al. [16]). that increasing the volume of the dataset improves the learning of CNNs and reduces overfitting. In this paper we used the transformations explained in Table 1. Table 1. The stages followed for pre-processing the input UAV imagery and data augmentation methods used for increasing the amount of sample patches. Table 1. The stages followed for pre-processing the input UAV imagery and data augmentation methods used for increasing the amount of sample patches. Table 1. The stages followed for pre-processing the input UAV imagery and data augmentation methods used for increasing the amount of sample patches. Steps The Pre-Processing Stages of Input UAV Imagery Step 1 Selection of image patches—a set of pictures manually cropped from orthophotomosaics using QGIS 7.2.2. Step 2 Resizing of patches to 150 × 200 RGB pixels using cubic interpolation. Step 3 Manual assignment of each patch to the appropriate tree damage category. Methods for Increasing the Amount of Data Step 1 Change the saturation of RGB channels. Step 2 Remove Gaussian blur filter with a blur value of 5% and a width and height of a kernel 0.5. Step 3 Pixel averaging by collapsing an image with a normalized 4 × 4 pixel window filter. Step 4 Image rotation relatively to its centre with 5◦, 15◦, 50◦, 90◦, 180◦rotation angles. Step 5 Cropping the central rectangle of each image to half distance from each edge (discarding the framing borders) and resizing back the central rectangle to 150 × 200 RGB pixels using cubic interpolation. 3. Classification of Trees in High Resolution Imagery and Related Works [12] detected coffee crops in high-resolution SPOT images (2.5 m/pix). They used a binary classification model and presented a comparison of the accuracy of various CNN and OBIA methods. In addition, the authors used a large number of methods of preparation and preliminary processing of the image, which in the final result leads to a high accuracy of more than 95%. Very few woks have used CNNs to detect plants in UAV images. Noteworthy is the work by Ferreira et al. [21], where weed was detected in soybean cultures using CNN based on the CaffeNet architecture. The authors used a set of data acquired with an UAV in manual mode at a height of 4 m above the Earth level using an RGB camera. The accuracy of the classification was 99.5%. Though, according to the authors, the accuracy may be lower in operational circumstances due to the heterogeneity in types of soils and weeds, since testing was performed on an experimental field. In 2018, Onishi and Ise [22] classified 7 types of trees (deciduous broad-leaved tree, deciduous coniferous tree, evergreen broad-leaved tree, Chamaecyparis obtuse, pinus, others) on UAV images using supervised deep learning (GoogLeNet) with an accuracy of 89%. A similar problem to ours, which is the detection of damaged trees, is revised in the next series of works, though they mainly used classical image classification methods. In 2009, Abdullah et al. [23] identified healthy and infested trees in satellite imagery (Sentinel-2, WorldView-2 and 3 (up to 0.5 m/pix)) using partial least squares regression (PLSR). Heurich et al. [24] presented semi-automatic detection of dead trees following a spruce bark beetle outbreak in CIR aerial photographs using OBIA with accuracy up to 91%. In this work, authors used several classes, as deadwood areas, vital vegetation, non-woodland and assistant classes (shadows among dead trees and shadows among vital trees). In 2013, Ortiz et al. [25] detected a bark beetle attack with TerraSAR-X and RapidEye Data (up to 1.18 m/pix) using generalized linear models (GLM), maximum entropy (ME) and random forest (RF), with accuracies up to 74%. Meddens et al. [26] used multi-temporal disturbance detection methods to detect bark beetle-caused tree mortality and to map the subsequent forest disturbance on Landsat images with different classes (green trees, red damage stage, grey damage stage, herbaceous vegetation, bare soil, shadows, water). Kussul et al. 3. Classification of Trees in High Resolution Imagery and Related Works 3. Classification of Trees in High Resolution Imagery and Related Works In this section, we consider related works devoted to the problems of classification and detection of objects (from land-covers to organisms) on Earth remote sensing (ERS) data. Scientists from around the world are trying to solve this problem by using various ERS information with different methods and algorithms. In general, few works have addressed the detection of damaged trees using satellite or UAV images, in general they have only considered few health classes (e.g., healthy and dead trees) and did not apply CNNs but classical image classification methods. For example, Deli et al. [17] developed a new CNN-based method for classifying four land-cover classes (crops, houses, soil and forests) in land surface images. They trained the algorithm with 100 images per class. Längkvist et al. [18] proposed the classification and segmentation of satellite orthoimagery (including five classes: vegetation, ground, road, parking, railroad, building and water) using CNNs in a small city for a full, fast and accurate per-pixel classification. They selected the parameters and analysed their influence on the neural network model architecture. They also found a better performance of the CNN model compared to object-oriented methods of classification, reaching a maximum CNN classification accuracy of 94.49%. Few papers have used neural network classifiers to recognize plant species and plant growth using images from a digital camera and a cell phone camera. Dyrmann et al. [19] created a CNN capable of recognizing 22 plant species on colour images with an accuracy of 86.2%. For this, the authors used different data sets, depending on the lighting, image resolution and soil type. Razavi and Yalcin [20] proposed a CNN architecture to classify types of plants growing in agro stations. To evaluate the effectiveness of their approach, the results of the created CNN model were compared to Support Vector Remote Sens. 2019, 11, 643 5 of 19 Machine (SVM; RBF kernel and polynomial kernel) classifiers. The accuracy of the CNN classification was greater than the SVM, reaching 97.47%. Few studies have also used CNN on satellite imagery. Li et al. used deep learning models [10] to detect oil palm trees in QiuckBird images (0.6 m/pix). Guirado et al. in Reference [11] detected Ziziphus lotus shrubs on RGB satellite images from Google Earth using TensorFlow and got better accuracies than using object-based image analysis (OBIA). Baeta et al. 4. Study Area and Data Acquisition The study area is located in the territory of the “Stolby” State Nature Reserve, near Krasnoyarsk city in Central Siberia of the Russian Federation. Most of the territory (80%) constitutes the mid-mountain belt (500–800 m a.s.l.), mainly covered by mixed forests composed by seven tree species in different proportions: conifers such as pines (Pinus sylvestris, Pinus sibirica), larch (Larix sibirica), fir (Abies sibirica), spruces (Picea abies, Picea obovata) and parvifoliate trees such as birch (Betula pendula, Betula pubescens) and aspen (Populus tremula) (Ryabovol [30]). Pine trees dominate among other species and occupy 41% of the total forested area, mainly in low-mountains (200–500 m a.s.l.). Distribution of Siberian fir expands on 25% of the Stolby forest. R t S 2019 1 FOR PEER REVIEW 6 f 21 First appearance of P. proximus in the Stolby nature reserve was registered in 2011 and nowadays the roughly estimated forest damage by the beetle is about 25–30% of the fir area. Remote Sens. 2019, 1, x FOR PEER REVIEW 6 of 21 First appearance of P. proximus in the Stolby nature reserve was registered in 2011 and nowadays Four plots with different rates of the P. proximus invasion were chosen for our study at Stolby (Figure 1). the roughly estimated forest damage by the beetle is about 25–30% of the fir area. Four plots with different rates of the P. proximus invasion were chosen for our study at Stolby (Figure 1). Figure 1. Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots are fragments from the orthophotos used to build the testing dataset used for external validation or independent testing. A set of RGB images with ultra-high spatial resolution (≈5–10 cm/pix) were obtained for the Figure 1. Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots are fragments from the orthophotos used to build the testing dataset used for external validation or independent testing. Figure 1. 3. Classification of Trees in High Resolution Imagery and Related Works [13] classified tree species and different levels of ash tree (Fraxinus sp.) mortality in WorldView-2 images with resolution of 1.85 m/pix using classical methods, in particular, the linear discriminant analysis (LDA), principal component analysis (PCA), stepwise regression and OBIA methods. Species diversity and the magnitude of ash loss were assessed. The authors needed to use a wide set of remote sensing indices to obtain good accuracies. The overall accuracy varied between 83% for the seven tree species and 77% for the four different levels of ash damage. In 2015, Näsi et al. [27] also identified damaged trees (with three classes: healthy, infected and dead) using UAV images and OBIA methods. In 2017, Dash et al. [28] used a non-parametric approach based on classification trees on UAV images to monitor forest health for disease outbreak in mature Pinus radiata trees. In 2018, Näsi et al. [29] published a paper that most closely relates to ours. They identified of bark beetle infestation at the individual tree level (healthy, infested and dead) in urban forests on UAV images using support vector machine (SVM), with accuracies up to 93%. As far as we know, our work is the first in addressing the detection of damaged fir trees caused by the bark beetle using CNN methods on images acquired by UAVs (resolution below 0.1 m/pix). We considered four tree-health categories, which is higher than in previous works and allows to better assess the stage of the infestation. We also showed that it is possible to achieve good results using a relatively small training set of data. Remote Sens. 2019, 11, 643 6 of 19 4. Study Area and Data Acquisition Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots are fragments from the orthophotos used to build the testing dataset used for external validation or independent testing. A set of RGB images with ultra-high spatial resolution (≈5–10 cm/pix) were obtained for the Figure 1. Location of the four plots in the nature reserve “Stolby,” Krasnoyarsk city (Russia), where A, B plots are fragments from the orthophotos used to build the training dataset; and C, D plots are fragments from the orthophotos used to build the testing dataset used for external validation or independent testing. research sites during multiple flights of the DJI Phantom 3 Pro quadcopter (with standard built-in camera) in July 2016 (plot A and C) and of the Yuneec Typhoon H hexacopter (with CGO3+ camera) in May 2016 (plot B) and August 2018 (plot D). Imagery for research plots A and C were recorded in cloudy weather conditions at 670 m (A) and 700 m (C) altitudes (120–150 m elevation above ground), plot B and D was surveyed in sunny weather at 120 m height. Default camera settings (auto white balance, ISO 100) were applied in all aerial shots. Image composites (orthophotomosaics) were created for each plot from a set of overlapping geolocated images (300–400 partially overlapping images per plot) using the Agisoft Photoscan Professional v1.4.0 (64-bit) software (www.agisoft.com, Agisoft LLC, St. Petersburg, Russia). This software provides photogrammetric and 3D-model A set of RGB images with ultra-high spatial resolution (≈5–10 cm/pix) were obtained for the research sites during multiple flights of the DJI Phantom 3 Pro quadcopter (with standard built-in camera) in July 2016 (plot A and C) and of the Yuneec Typhoon H hexacopter (with CGO3+ camera) in May 2016 (plot B) and August 2018 (plot D). Imagery for research plots A and C were recorded in cloudy weather conditions at 670 m (A) and 700 m (C) altitudes (120–150 m elevation above ground), plot B and D was surveyed in sunny weather at 120 m height. Default camera settings (auto white balance, ISO 100) were applied in all aerial shots. Image composites (orthophotomosaics) 7 of 19 Remote Sens. 5.1. Definition of Fir Trees Damage Categories presents the classification model (Section 5 especially designed for detecting bark beet 5.1. Definition of Fir Trees Damage Categories p ( especially designed for detecting bark bee To define the categories of health status of fir trees, we followed the entomological approach by Krivets et al. [32], which differentiates six categories according to the level P. proximus invasion into the trunk and its influence on the canopy: I—healthy trees; II—weakened trees; III—heavily weakened trees; IV—dying trees; V—recently died trees; VI—old deadwood. Since the first, second and third categories can only be recognized in-situ by trunk signs that do not visibly translate into the crown, we reclassified them into four categories (skipping the second and the third one). Hence, the final categories in our classification were: a—completely healthy tree or recently attacked by beetles, b—tree colonized by beetles, c—recently died tree and d—deadwood (Figure 2). 5.1. Definition of Fir Trees Damage Categories To define the categories of health status of fir trees, we followed the entomological approach by Krivets et al. [32], which differentiates six categories according to the level P. proximus invasion into the trunk and its influence on the canopy: I—healthy trees; II—weakened trees; III—heavily weakened trees; IV—dying trees; V—recently died trees; VI—old deadwood. Since the first, second and third categories can only be recognized in-situ by trunk signs that do not visibly translate into the crown, we reclassified them into four categories (skipping the second and the third one). Hence, the final categories in our classification were: a—completely healthy tree or recently attacked by beetles, b—tree colonized by beetles, c—recently died tree and d—deadwood (Figure 2). Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. [32]): (a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently died tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the bottom horizontal pictures. (a) (b) (c) (d) Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. [32]): (a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently died tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the bottom horizontal pictures. (d) (c) (b) (a) (a) (d) (b) (c) Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. 4. Study Area and Data Acquisition 2019, 11, 643 were created for each plot from a set of overlapping geolocated images (300–400 partially overlapping images per plot) using the Agisoft Photoscan Professional v1.4.0 (64-bit) software (www.agisoft. com, Agisoft LLC, St. Petersburg, Russia). This software provides photogrammetric and 3D-model reconstruction tools [31]. The data processing workflow in Photoscan consisted of the following steps: alignment of each image location, generation of dense point cloud (3D-model), creation of digital elevation model (DEM) and eventually production of a georeferenced orthophotomosaic (orthophotos or orthoimages from now). The workflow was run several times with varying settings in order to achieve better stich of images over the complex mountain landscape of the study sites. As a result, we got four orthophotos for each one of the four sites (A, B, C and D for 2016 and 2018). Remote Sens. 2019, 1, x FOR PEER REVIEW 7 of 21 elevation model (DEM) and eventually production of a georeferenced orthophotomosaic ( h h h i f ) Th kfl l i i h i i 5. Methods (orth in ord As we have mentioned, our aim is to evaluate the possibilities of convolutional neural networks as a new approach to detect bark beetle outbreaks in fir trees in very high resolution imagery in fir and mixed forests. Thus, this section includes a definition of fir-tree damage categories (Section 5.1), describes the data pre-processing techniques we used for training the CNN models (Section 5.2), presents the classification model (Section 5.3) and finally presents the proposed detection technique especially designed for detecting bark beetle outbreaks in fir forests (Section 5.4). a result, we got four orthophotos for each one of the four sites (A, B, C and D for 2016 and 2018). 5. Methods As we have mentioned, our aim is to evaluate the possibilities of convolutional neural networks as a new approach to detect bark beetle outbreaks in fir trees in very high resolution imagery in fir and mixed forests. Thus, this section includes a definition of fir-tree damage categories (Section 5.1), describes the data pre-processing techniques we used for training the CNN models (Section 5.2), 5.1. Definition of Fir Trees Damage Categories presents the classification model (Section 5 especially designed for detecting bark bee 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models The design of the training dataset is the key to the performance of a good CNN classification model. For labelling the training dataset, we identified 50 image patches containing 4 categories of trees. In particular, we built two different datasets: • First, the dataset for training, validation and testing consisted in 50 manually sampled image-patches of single trees per each tree damage category, resulting in 200 image-patches from the plot A and B. To train the CNN model we used 80% of this selection. The remaining 20% of images were used for model internal validation. • First, the dataset for training, validation and testing consisted in 50 manually sampled image-patches of single trees per each tree damage category, resulting in 200 image-patches from the plot A and B. To train the CNN model we used 80% of this selection. The remaining 20% of images were used for model internal validation. • Second, the dataset for external testing or external validation consisted in 88 patch-images generated by our proposed detection technique from the areas C and D. • Second, the dataset for external testing or external validation consisted in 88 patch-images generated by our proposed detection technique from the areas C and D. Table 2. Four fir tree categories training and testing datasets. Each image has 150 × 200-pixels. Table 2. Four fir tree categories training and testing datasets. Each image has 150 × 200-pixels. Categories of Trees Training Dataset (Plots A and B) Testing Dataset for External Test Without Data Augmentation With Data Augmentation Training Internal Validation Training Internal Validation Test Area C Test Area D 1 40 10 880 220 5 13 2 40 10 880 220 36 14 3 40 10 880 220 5 7 4 40 10 880 220 2 6 Total: 160 40 3520 880 48 40 The pre-processing method used for preparing the input UAV images is described in Table 1 To improve the robustness and accuracy of the CNN classification models, we increased the volume of samples using data augmentation techniques. 5.2. Dataset Preprocessing and Data Augmentation Techniques of Sample patches for Training the CNN Models In particular, we increased the amount of sample patches for training the CNN models using the methods presented in Table 1. The images of the training dataset were used for training the parameters of the neural network. The internal validation set is a set of examples used to tune the parameters of the neural network and determine a stopping point for the back-propagation algorithm. The testing dataset is a set of independent external examples used to assess or validate the performance of the final classification model. 5.1. Definition of Fir Trees Damage Categories presents the classification model (Section 5 especially designed for detecting bark beet [32]): (a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently died tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the bottom horizontal pictures. Figure 2. Damage categories of Siberian fir trees used in this study (adapted from Krivets et al. [32]): (a) completely healthy tree or recently attacked by beetles, (b) tree colonized by beetles, (c) recently died tree and (d) deadwood. Top figures illustrate the vertical orthoimages corresponding to the bottom horizontal pictures. 8 of 19 Remote Sens. 2019, 11, 643 5.3. Classification Model A deep neural network was developed and trained on the prepared image samples to adapt its weights to the task of tree-damage recognition. To create a CNN and improve the quality of its training, we manually tuned the hyperparameters in the network. Several series of experiments were performed. For each experiment, the hyperparameters were altered and the consequent network’s operation quality change was estimated. The loss function, which should be minimized in the CNN (see Section 1), was established by a categorical cross-entropy loss between the input and the output classification of the images. The ADAM’s optimization was chosen among existing optimization algorithms since it was the most efficient one due to the possibility of initial calibration of the CNN. When exploring the effect of changing the learning rate parameter with the ADAM’s optimizer, the highest accuracy for CNN on the test data was achieved at a value of 0.0001. The total number of the network layers was determined by creating cascades of convolutional layers and a pooling layer. To assess the impact of the number of training epochs on the CNN accuracy, training was conducted and compared in the range from 1 to 50 epochs (see Section 6.2). Remote Sens. 2019, 11, 643 (each includes one included maximum p 9 of 19 blocks ers and The overall architecture of the CNN (Table 3, Figure 3) consisted of six convolutional blocks (each includes one convolutional layer). Additionally, the first and third convolutional blocks included maximum pooling layers. At the top of the CNN, there are two fully connected layers and one output layer. The ReLU activation function was used in the last four convolutional blocks and the Softmax function for the output layer. In order to keep the overtraining of the network under control, we used the Dropout regularization method which reduced the complexity of the model, saving the number of its parameters low. It is very important to choose an appropriate regularization coefficient, so it was set to 0.25 after the second, fourth and fifth layers and it was set to 0.5 before the output layer. Our network with various activation functions is presented in Table 3. the Softmax function for the output layer. In order to keep the overtraining of the network under control, we used the Dropout regularization method which reduced the complexity of the model, saving the number of its parameters low. 5.3. Classification Model It is very important to choose an appropriate regularization coefficient, so it was set to 0.25 after the second, fourth and fifth layers and it was set to 0.5 before the output layer. Our network with various activation functions is presented in Table 3. Table 3. The CNN network developed in this study, with different activation functions, where layers, output size and networks parameters are represented. Layers Output Size, pix Network y Our network with various activation functions is presented in Table 3. Layers Output Size, pix Network Table 3. The CNN network developed in this study, with different activation functions, where layers, output size and networks parameters are represented. Layers Output Size, pix Network Convolution 150 × 200 3 × 3, 96 Max pooling 75 × 100 2 × 2, stride 2 Convolution 75 × 100 5 × 5, 128 Dropout 75 × 100 0.25 Convolution 75 × 100 3 × 3, 128 Max pooling 38 × 50 2 × 2, stride 2 Convolution 38 × 50 3 × 3, 128 Dropout 38 × 50 0.25 Convolution 38 × 50 5 × 5, 128 Dropout 38 × 50 0.5 Convolution 38 × 50 5 × 5, 512 Global Average pooling 1 × 972,800 stride 1 Dense 1 × 972,800 ReLu Dropout 1 × 400 0.5 Dense 1 × 100 ReLu Dense 1 × 4 Softmax y p p Convolution 150 × 200 3 × 3, 96 Max pooling 75 × 100 2 × 2, stride 2 Convolution 75 × 100 5 × 5, 128 Dropout 75 × 100 0.25 Convolution 75 × 100 3 × 3, 128 Max pooling 38 × 50 2 × 2, stride 2 Convolution 38 × 50 3 × 3, 128 Dropout 38 × 50 0.25 Convolution 38 × 50 5 × 5, 128 Dropout 38 × 50 0.5 Convolution 38 × 50 5 × 5, 512 Global Average pooling 1 × 972,800 stride 1 Dense 1 × 972,800 ReLu Dropout 1 × 400 0.5 Dense 1 × 100 ReLu Dense 1 × 4 Softmax Consequently, our CNN model has the following form, presented in Figure 3. Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. Table 3. The CNN network developed in this study, with different activation functions, where layers, output size and networks parameters are represented. 5.3. Classification Model Layers Output Size, pix Network Convolution 150 × 200 3 × 3, 96 Max pooling 75 × 100 2 × 2, stride 2 Convolution 75 × 100 5 × 5, 128 Dropout 75 × 100 0.25 Convolution 75 × 100 3 × 3, 128 Max pooling 38 × 50 2 × 2, stride 2 Convolution 38 × 50 3 × 3, 128 Dropout 38 × 50 0.25 Convolution 38 × 50 5 × 5, 128 Dropout 38 × 50 0.5 Convolution 38 × 50 5 × 5, 512 Global Average pooling 1 × 972,800 stride 1 Dense 1 × 972,800 ReLu Dropout 1 × 400 0.5 Dense 1 × 100 ReLu Dense 1 × 4 Softmax Convolution 150 × 200 3 × 3, 96 Max pooling 75 × 100 2 × 2, stride 2 Convolution 75 × 100 5 × 5, 128 Dropout 75 × 100 0.25 Convolution 75 × 100 3 × 3, 128 Max pooling 38 × 50 2 × 2, stride 2 Convolution 38 × 50 3 × 3, 128 Dropout 38 × 50 0.25 Convolution 38 × 50 5 × 5, 128 Dropout 38 × 50 0.5 Convolution 38 × 50 5 × 5, 512 Global Average pooling 1 × 972,800 stride 1 Dense 1 × 972,800 ReLu Dropout 1 × 400 0.5 Dense 1 × 100 ReLu Dense 1 × 4 Softmax Consequently, our CNN model has the following form, presented in Figure 3. Figure 3. The architecture of our CNN model of deep machine learning. 5 4 P d D t ti P Figure 3. The architecture of our CNN model of deep machine learning. C tl CNN d l h th f ll i f t d i Fi 3 Table 3. The CNN network developed in this study, with different activation functions, where layers, output size and networks parameters are represented. Convolution 150 × 200 3 × 3, 96 Max pooling 75 × 100 2 × 2, stride 2 Convolution 75 × 100 5 × 5 128 Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. Figure 3. The architecture of our CNN model of deep machine learning. . Proposed Detection Process Consequently, our CNN model has the following form, presented in Figure 3. We developed a candidate lik l t b t Th 5.4. Proposed Detection Process are more likely to be a tree. Then we analysed each one of these candidate regions using our CNN- classification model. The proposed candidate selection technique is a data processing algorithm that includes a sequence of the steps presented in the Table 4. We developed a candidate selection technique to find the potential regions in the test image that are more likely to be a tree. Then we analysed each one of these candidate regions using our CNN-classification model. The proposed candidate selection technique is a data processing algorithm that includes a sequence of the steps presented in the Table 4. 10 of 19 10 of 21 Remote Sens. 2019, 11, 643 Remote Sens. 2019, 1, x FOR Table 4. The proposed candidate selection technique in the test areas cropped from test areas C and D. Steps Data Processing Algorithm Step 1 Conversion of RGB images to a grayscale colour palette Step 2 Blurring of grayscale images using the Gaussian high-pass filter in order to reduce Gaussian noise with the following function parameters: kernel size of 11 × 11 pixels and the standard deviation equal to 0 Step 3 Creation of binary images from the grayscale blurred images by application of a threshold function with the optimal brightness threshold value of input image pixels equal to 100 Step 4 Structuring of picture elements outlines by application of two successive functions (erosion and dilation) with several iterations for the binary pictures in order to distinguish individual tree crown contours and to minimize the effect of their confluence (fusion) in one object Step 5 Detection of image patches was implemented using a contour area calculation function based on the Green formula (Dawkins [33]), object size for the function was set in the range between 50 × 50 and 200 × 200 pixels D. We developed a candidate lik l t b t Th 5.4. Proposed Detection Process Steps Data Processing Algorithm Step 1 Conversion of RGB images to a grayscale colour palette Step 2 Blurring of grayscale images using the Gaussian high-pass filter in order to reduce Gaussian noise with the following function parameters: kernel size of 11 × 11 pixels an the standard deviation equal to 0 Step 3 Creation of binary images from the grayscale blurred images by application of a threshold function with the optimal brightness threshold value of input image pixels equal to 100 Step 4 Structuring of picture elements outlines by application of two successive functions (erosion and dilation) with several iterations for the binary pictures in order to distinguish individual tree crown contours and to minimize the effect of their confluence (fusion) in one object Step 5 Detection of image patches was implemented using a contour area calculation functio based on the Green formula (Dawkins [33]), object size for the function was set in the range between 50 × 50 and 200 × 200 pixels The output of this process are the candidate regions indicated by red bounding boxes in Figure 4. Finally, all the bounding boxes were analysed by the classification model. The output of this process are the candidate regions indicated by red bounding boxes in Figure 4. Finally, all the bounding boxes were analysed by the classification model. The output of this process are the candidate regions indicated by red bounding boxes in Figure 4. Finally, all the bounding boxes were analysed by the classification model. The output of this process are the candidate regions indicated by red bounding boxes in Figure 4. Finally, all the bounding boxes were analysed by the classification model. Figure 4. Pre-processing consisted of the following steps: first, converting the three band image into one grey-scale band image (PAN); second, converting grey-scale band image into blurred image; third, converting the blurred image into a binary image based on a 100 over 256 digital value threshold; and fourth, detecting categories of trees on RGB images. The 48 candidate patches identified in test area C and 40 candidate patches identified in test area D are labelled with red contour in the right panel. Figure 4. We developed a candidate lik l t b t Th 5.4. Proposed Detection Process Pre-processing consisted of the following steps: first, converting the three band image into one grey-scale band image (PAN); second, converting grey-scale band image into blurred image; third, converting the blurred image into a binary image based on a 100 over 256 digital value threshold; and fourth, detecting categories of trees on RGB images. The 48 candidate patches identified in test area C and 40 candidate patches identified in test area D are labelled with red contour in the right panel. Figure 4. Pre-processing consisted of the following steps: first, converting the three band image into one grey-scale band image (PAN); second, converting grey-scale band image into blurred image; third, converting the blurred image into a binary image based on a 100 over 256 digital value threshold; and fourth, detecting categories of trees on RGB images. The 48 candidate patches identified in test area C and 40 candidate patches identified in test area D are labelled with red contour i th i ht l Figure 4. Pre-processing consisted of the following steps: first, converting the three band image into one grey-scale band image (PAN); second, converting grey-scale band image into blurred image; third, converting the blurred image into a binary image based on a 100 over 256 digital value threshold; and fourth, detecting categories of trees on RGB images. The 48 candidate patches identified in test area C and 40 candidate patches identified in test area D are labelled with red contour in the right panel. in the right panel. 6. Results and Analysis 6. Results and Analysis In this section, we will first describe the metrics used for the evaluation of the results of our proposed model, we will then show the performance of the training and internal test process (sites A and B) and finally we will show the results of the detection on two external test datasets of UAV In this section, we will first describe the metrics used for the evaluation of the results of our proposed model, we will then show the performance of the training and internal test process (sites A and B) and finally we will show the results of the detection on two external test datasets of UAV images (sites C and D). 6. Results and Analysis In this section, we will first describe the metrics used for the evaluation of the results of our proposed model, we will then show the performance of the training and internal test process (sites A and B) and finally we will show the results of the detection on two external test datasets of UAV In this section, we will first describe the metrics used for the evaluation of the results of our proposed model, we will then show the performance of the training and internal test process (sites A and B) and finally we will show the results of the detection on two external test datasets of UAV images (sites C and D). 6.2. Evaluation of the Training Process and Comparison 6 2 Evaluation of the Training Process and Comparison To evaluate the training process of our new CNN, we used Python, Keras and TensorFlow framework with different pre-processing data augmentation techniques. Keras is a high-level API to ease the process of building and training deep learning models [35]. TensorFlow is open source software library for high performance numerical computation, which runs on various heterogeneous systems, including distributed graphics processing unit (GPU) clusters (Murray [36]). The used hardware environment in this study was Intel Xeon E5-2630v4 CPU accelerated with NVIDIA Titan Xp GPU as a platform for learning and testing. 6.2. Evaluation of the Training Process and Comparison To evaluate the training process of our new CNN, we used Python, Keras and TensorFlow framework with different pre-processing data augmentation techniques. Keras is a high-level API to ease the process of building and training deep learning models [35]. TensorFlow is open source software library for high performance numerical computation, which runs on various heterogeneous systems, including distributed graphics processing unit (GPU) clusters (Murray [36]). The used hardware environment in this study was Intel Xeon E5-2630v4 CPU accelerated with NVIDIA Titan X GPU l f f l i d i The application of data augmentation techniques to the sample patches for training the CNN models resulted in the creation of 4400 image-patches from the first data subset, which had 200 patches (Section 5.2). Xp GPU as a platform for learning and testing. The application of data augmentation techniques to the sample patches for training the CNN models resulted in the creation of 4400 image-patches from the first data subset, which had 200 h (S i 5 2) The maximum performance of our CNN model with augmented dataset was achieved at the 23th training epoch, providing an internal test accuracy of 99.7% and a minimum training loss lower than 0.01. After the 23th epoch, the validation loss was stabilized and the difference between the training and validation loss increased (Figure 5). patches (Section 5.2). The maximum performance of our CNN model with augmented dataset was achieved at the 23th training epoch, providing an internal test accuracy of 99.7% and a minimum training loss lower than 0.01. After the 23th epoch, the validation loss was stabilized and the difference between the training and validation loss increased (Figure 5) g ( g ) Figure 5. Loss and accuracy for each epoch of the CNN model training with data augmentation. Figure 5. images (sites C and D) 6.1. Evaluation Metrics 6.1. Evaluation Metrics To evaluate and compare the results of the proposed CNN model with other CNN models, we used the performance metrics based on the confusion matrix. The confusion matrix is four by four To evaluate and compare the results of the proposed CNN model with other CNN models, we used the performance metrics based on the confusion matrix. The confusion matrix is four by four (four categories) and contains the results of multi-class classifier (one class against the rest) in terms of Remote Sens. 2019, 11, 643 Remote Sens. 2019, 1, x FOR 11 of 19 11 of 21 number of true positive (TP) predictions, true negative (TN) predictions, false positive (FP) predictions and false negative (FN) predictions [34]. of number of true positive (TP) predictions, true negative (TN) predictions, false positive (FP) predictions and false negative (FN) predictions [34]. The main metrics we can extract from the matrix of confusion are: accuracy (1) precision (2) The main metrics we can extract from the matrix of confusion are: accuracy (1), precision (2), recall (3) and F-score (4). Accuracy is calculated as the total number of the correct predictions (TP + TN) divided by the total number of examples in the test set. Precision is calculated as the number of correct positive predictions (TP) divided by the total number of positive predictions (TP + FP). Recall is calculated as the number of correct positive predictions (TP) divided by the number of true positives and false negatives. F-score indicates the balance between precision and recall. The highest and best value of all these metrics is 1.0 and the worst is 0.0. The main metrics we can extract from the matrix of confusion are: accuracy (1), precision (2), recall (3) and F-score (4). Accuracy is calculated as the total number of the correct predictions (TP + TN) divided by the total number of examples in the test set. Precision is calculated as the number of correct positive predictions (TP) divided by the total number of positive predictions (TP + FP). Recall is calculated as the number of correct positive predictions (TP) divided by the number of true positives and false negatives. F-score indicates the balance between precision and recall. The highest and best value of all these metrics is 1.0 and the worst is 0.0. images (sites C and D) 6.1. Evaluation Metrics ܶܲ൅ܶܰ Accuracy = TP + TN TP + TN + FN + FP (1) Precision = TP TP + FP (2) Recall = TP TP + FN (3) F_score = 2 × Precision × Recall Precision + Recall (4) ܣܿܿݑݎܽܿݕൌ ܶܲ൅ܶܰ ܶܲ൅ܶܰ൅ܨܰ൅ܨܲ (1) ܲݎ݁ܿ݅ݏ݅݋݊ൌ ܶܲ ܶܲ൅ܨܲ (2) ܴ݈݈݁ܿܽൌ ܶܲ ܶܲ൅ܨܰ (3) ܨ_ݏܿ݋ݎ݁ൌ 2 ൈܲݎ݁ܿ݅ݏ݅݋݊ൈܴ݈݈݁ܿܽ ܲݎ݁ܿ݅ݏ݅݋݊൅ܴ݈݈݁ܿܽ (4) Accuracy = TP + TN TP + TN + FN + FP (1) Precision = TP TP + FP (2) Recall = TP TP + FN (3) F_score = 2 × Precision × Recall Precision + Recall (4) ܣܿܿݑݎܽܿݕൌ ܶܲ൅ܶܰ ܶܲ൅ܶܰ൅ܨܰ൅ܨܲ (1) ܲݎ݁ܿ݅ݏ݅݋݊ൌ ܶܲ ܶܲ൅ܨܲ (2) ܴ݈݈݁ܿܽൌ ܶܲ ܶܲ൅ܨܰ (3) ܨ_ݏܿ݋ݎ݁ൌ 2 ൈܲݎ݁ܿ݅ݏ݅݋݊ൈܴ݈݈݁ܿܽ ܲݎ݁ܿ݅ݏ݅݋݊൅ܴ݈݈݁ܿܽ (4) (1) (1) (2) (2) (4) (4) 6.2. Evaluation of the Training Process and Comparison 6 2 Evaluation of the Training Process and Comparison 6.2. Evaluation of the Training Process and Comparison 6 2 Evaluation of the Training Process and Comparison 6.3. External Test Detection Results 6.3. External Test Detection Results The confusion matrix of our CNN model in the four categories with data-augmentation are shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown is Figure 6. The confusion matrix of our CNN model in the four categories with data-augmentation are shown is Figure 6. g g Figure 6. hown is Figure 6. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate regions obtained from test areas C and D. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate regions obtained from test areas C and D. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate regions obtained from test areas C and D. Figure 6. Confusion matrix of the proposed CNN model with data augmentation on the candidate regions obtained from test areas C and D. The results based on the confusion matrix of our CNN model on each category with and without data augmentation are shown in the Table 5 The results based on the confusion matrix of our CNN model on each category with and without data augmentation are shown in the Table 5. Table 5. The performance of our CNN model with and without data augmentation on the test set for each categories of trees. The performance is expressed in terms of true positives (TP), true negatives Table 5. The performance of our CNN model with and without data augmentation on the test set for each categories of trees. The performance is expressed in terms of true positives (TP), true negatives (TN), false positives (FP), false negatives (FN), precision, recall and F-score. (TN), false positives (FP), false negatives (FN), precision, recall and F-score. 6.2. Evaluation of the Training Process and Comparison 6 2 Evaluation of the Training Process and Comparison Loss and accuracy for each epoch of the CNN model training with data augmentation. Figure 5. Loss and accuracy for each epoch of the CNN model training with data augmentation. Figure 5. Loss and accuracy for each epoch of the CNN model training with data augmentation. 12 of 19 12 of 19 Remote Sens. 2019, 11, 643 For comparison with our CNN model, we have also tested on the same input data the most powerful CNN models such as Xception, VGG-16, VGG-19, ResNet-50, Inception-V3, InceptionResNet-V2, DenseNet-121, DenseNet-169 and DenseNet-201 (Jordan [37]). A brief explanation of each model is provided in Appendix B. We adapted the last layer of all these models to the 4 classes of our problem. The obtained results of all these models after few training epochs are shown in Appendix A (Table A1). As it can be seen from Table A1, all models provide high training accuracies. InceptionResNet-V2, DenseNet-121, DenseNet-169 and DenseNet-201 (Jordan [37]). A brief explanation of each model is provided in Appendix B. We adapted the last layer of all these models to the 4 classes of our problem. The obtained results of all these models after few training epochs are shown in Appendix A (Table A1). As it can be seen from Table A1, all models provide high training accuracies. 6.3. External Test Detection Results 6.3. External Test Detection Results 2019, 1, x FOR PEER REVIEW 13 of 21 ResNet50, Inception V3, InceptionResNetV2, DenseNet121, DenseNet169 and DenseNet201 are powerful and computationally intensive models, our less computationally intensive architecture provides much better results in the damaged tree categories problem. On the other hand, the proposed model with data-augmentation achieves high F1_score on g The results of the detection on the input test areas C and D are shown in Figures 7 and 8. The red boxes present the selected areas produced by our candidate selection technique (see Section 5.4). Each red box has two numbers. The first number is the tree category estimated by the CNN model; C1, C2, C3 and C4 refer to the tree damage categories 1, 2, 3 and 4 respectively. The second number indicates the probability calculated by our classification CNN model. Actually, this probability indicates the level of confidence of the model. damage class 1, 2, 3 and 4 with 92.75%, 89.86%, 89.66% and 88.89 respectively. This can be explained by the fact that the model correctly distinguishes the colour, shape and texture of each one of the four damage classes. The results of the detection on the input test areas C and D are shown in Figures 7 and 8. The red boxes present the selected areas produced by our candidate selection technique (see Section 5.4). Each red box has two numbers. The first number is the tree category estimated by the CNN model; C1, C2, C3 and C4 refer to the tree damage categories 1, 2, 3 and 4 respectively. The second number indicates the probability calculated by our classification CNN model. Actually, this probability indicates the level of confidence of the model. Figure 7. Results of the detection of damaged tree categories on test area C. C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. Figure 7. Results of the detection of damaged tree categories on test area C. C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. Figure 7. Results of the detection of damaged tree categories on test area C. 6.3. External Test Detection Results 6.3. External Test Detection Results Categories of Trees TP TN FP FN Accuracy (%) Precision (%) Recall (%) F-Score (%) Without Augmentation 1 18 44 3 14 78.48 85.71 56.25 67.92 2 30 32 15 6 74.7 66.67 83.33 74.07 3 10 52 0 6 91.18 100 65.5 76.92 4 4 58 8 0 88.57 33.33 100 50 With Augmentation 1 32 48 5 0 94.12 86.49 100 92.75 2 31 49 2 5 91.95 93.94 86.11 89.86 Categories of Trees TP TN FP FN Accuracy (%) Precision (%) Recall (%) F-Score (%) Without Augmentation 1 18 44 3 14 78.48 85.71 56.25 67.92 2 30 32 15 6 74.7 66.67 83.33 74.07 3 10 52 0 6 91.18 100 65.5 76.92 4 4 58 8 0 88.57 33.33 100 50 With Augmentation 1 32 48 5 0 94.12 86.49 100 92.75 2 31 49 2 5 91.95 93.94 86.11 89.86 3 13 67 0 3 96.39 100 81.25 89.66 4 4 76 1 0 98.77 80 100 88.89 3 13 67 0 3 96.39 100 81.25 89.66 4 4 76 1 0 98.77 80 100 88.89 As it can be seen from Table 5, the trained CNN with data-augmentation provides the best test accuracy, recall and F1_score. The average accuracy, recall and F-score improved by 14.5%, 20.4% and 34.3% respectively. A comparison between the results obtained by our model and by more As it can be seen from Table 5, the trained CNN with data-augmentation provides the best test accuracy, recall and F1_score. The average accuracy, recall and F-score improved by 14.5%, 20.4% and 34.3% respectively. A comparison between the results obtained by our model and by more complex models with augmentation on the 88 candidate areas from images C and D is presented in Appendix A (Table A2). As it can be seen from this Table, although Xception, VGG16, VGG19, ResNet50, Remote Sens. 2019, 11, 643 13 of 19 Inception V3, InceptionResNetV2, DenseNet121, DenseNet169 and DenseNet201 are powerful and computationally intensive models, our less computationally intensive architecture provides much better results in the damaged tree categories problem. On the other hand, the proposed model with data-augmentation achieves high F1_score on damage class 1, 2, 3 and 4 with 92.75%, 89.86%, 89.66% and 88.89 respectively. This can be explained by the fact that the model correctly distinguishes the colour, shape and texture of each one of the four damage classes. Remote Sens. in cr 7. Conclusions to detect infected trees, if a box has more than one tree and at least one of them is unhealthy and the model considers the entire box as unhealthy tree class, this answer is considered as valid answer for the purpose of finding unhealthy trees. On the other hand, detecting individual trees in dense forests using only RGB bands information is a very complex task. In fact, previous studies (such as Mohan et al. [38]) that aimed to detect individual tree crowns in dense forests had to include several sources of data (high resolution images together with 3D LIDAR point cloud data), to achieve similar accuracies to our results. Actually, our results demonstrate that the proposed CNN based approach has a high potential and can be further improved in the future using more information, such as multispectral bands, NDVI or other spectral indices and 3D LIDAR data. 7. Conclusions Damage to fir trees caused by the attacks of bark beetles is a major problem in the Taiga and Boreal Forests, particularly in Central Siberia of Russia. An efficient method for recognizing categories of tree damage using remote sensing imagery could substantially help as an early warning system that allows to eliminate fir trees colonized by the P. proximus beetle, to reduce its spread to new territories. The presented results are of great interest, both for scientific and practical purposes, since such work in the task of classifying categories of tree damage caused by beetle attacks to fir trees using CNN methods on UAV images has not yet been encountered. Recognizing the categories of damage to fir trees is a difficult task due to the fact that the forest canopy is very dense and diverse. Damage to fir trees caused by the attacks of bark beetles is a major problem in the Taiga and Boreal Forests, particularly in Central Siberia of Russia. An efficient method for recognizing In this article, we developed a model based on CNNs to detect in RGB UAV images the damage to fir trees caused by bark beetles. Our network consists of six convolutional blocks. The network can recognize four damage categories in Siberian firs, from healthy to dead, with high prediction accuracy on images from different survey data. We have shown that our model provides better performance than the most powerful state-of-the-art CNN models, such as Xception, VGG, ResNet, Inception, InceptionResNet, DenseNet. 6.3. External Test Detection Results 6.3. External Test Detection Results C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. Figure 7. Results of the detection of damaged tree categories on test area C. C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. As it can be seen from Figures 7 and 8, our model correctly recognizes the tree damage categories in most candidate regions (red boxes). In the few cases where the boxes include more than one tree crown, the model correctly predicts the existence of infected trees. Since the objective of this work is to detect infected trees, if a box has more than one tree and at least one of them is unhealthy and the model considers the entire box as unhealthy tree class, this answer is considered as valid answer for the purpose of finding unhealthy trees. p p g y On the other hand, detecting individual trees in dense forests using only RGB bands information is a very complex task. In fact, previous studies (such as Mohan et al. [38]) that aimed to detect individual tree crowns in dense forests had to include several sources of data (high resolution images together with 3D LIDAR point cloud data), to achieve similar accuracies to our results. Actually, our results demonstrate that the proposed CNN based approach has a high potential and can be further improved in the future using more information, such as multispectral bands, NDVI or other spectral indices and 3D LIDAR data. 14 of 19 Remote Sens. 2019, 11, 643 Figure 8. Results of the detection of damaged tree categories on test area D. C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. As it can be seen from Figures 7 and 8, our model correctly recognizes the tree damage categories in most candidate regions (red boxes) In the few cases where the boxes include more than one tree Figure 8. Results of the detection of damaged tree categories on test area D. 6.3. External Test Detection Results 6.3. External Test Detection Results C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. Figure 8. Results of the detection of damaged tree categories on test area D. C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “–” indicate respectively correct and incorrect class estimation by our model. As it can be seen from Figures 7 and 8, our model correctly recognizes the tree damage categories Figure 8. Results of the detection of damaged tree categories on test area D. C1, C2, C3 and C4 indicate the estimated class by our CNN classification model together with the corresponding probability. The symbols “+” and “−” indicate respectively correct and incorrect class estimation by our model. in cr 7. Conclusions We showed that data augmentation substantially increased the performance of our CNN model. Our model, trained with data augmentation, showed up to 98.77% accuracy for damage categories one, three and four. The second category may have the lowest recognition accuracy due to the fact that we had to merge three categories traditionally assessed in the field (weakened trees, heavily weakened trees and dying trees) into one category—trees colonized by the beetle, since it was not possible to visually observe any differences between these three categories in the UAV imagery. Regarding alternative models, the VGG-16 model, on average, showed higher results, among other models considered in the paper. VGG-16 model recognized the first, second and fourth categories with an accuracy of 85.9%, 79.76 and 94.37%, respectively. Recognition of the third category with an Remote Sens. 2019, 11, 643 15 of 19 accuracy higher than 88.89% is achieved by ResNet-50 model. The lowest accuracies were reached by InceptionResNet-V2. In future works, we will focus on improving the segmentation of tree categories, introducing time series of data and trying to implement the three missing classes: weakened trees, heavily weakened trees and dying trees. It would also be interesting to explore if we can transfer our model to a new area with the same species and to other species, by only changing the last output layer in the network. Author Contributions: A.S. and S.T. conceived and conducted the experiments, developed the CNN model and wrote the manuscript. A.R. made a survey of the research plots and a field exploration of the territory. A.S. prepared the training and testing datasets. D.A.-S. and A.R. made changes, revised and edited the first submitted version of the manuscript. A.S., S.T., D.A.-S., A.R., Y.M. and F.H. made changes, revised and edited the final version of the manuscript. Funding: A.S. was supported by the grant of the Russian Science Foundation No. 16-11-00007. S.T. was supported by the Ramón y Cajal Programme (No. RYC-2015-18136). S.T. and F.H. received funding from the Spanish Ministry of Science and Technology under the project TIN2017-89517-P. D.A.-S. received support from project ECOPOTENTIAL, which received funding from the European Union Horizon 2020 Research and Innovation Programme under grant agreement No. 641762, from the European LIFE Project ADAPTAMED LIFE14 CCA/ES/000612 and from project 80NSSC18K0446 of the NASA’s Group on Earth Observations Work Programme 2016. A.R. was supported by the grant of the Russian Science Foundation No. 18-74-10048. Y. in cr 7. Conclusions M. was supported by the grant of Russian Foundation for Basic Research No. 18-47-242002, Government of Krasnoyarsk Territory and Krasnoyarsk Regional Fund of Science. Acknowledgments: We are very grateful to the reviewers for their valuable comments that helped to improve the paper. We appreciate the support of a vice-director of the “Stolby” State Nature Reserve, Anastasia Knorre. We also thank two Ph.D. students Egor Trukhanov and Anton Perunov from Siberian Federal University for their help in data acquisition (aerial photography from UAV) on two research plots in 2016 and raw imagery processing. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript and in the decision to publish the results. Table A1. Performance comparison of the training by proposed CNN model with other models. Table A1. Performance comparison of the training by proposed CNN model with other models. Model Without Augmentation With Augmentation Loss Accuracy Loss Accuracy Our CNN model 0.05 1 0.001 1 Xception 0.3 0.89 0.002 1 VGG-16 0.03 1 0.03 1 VGG-19 0.07 1 0.01 0.99 ResNet-50 + 4 0.21 0.94 0.01 1 Inception-V3 + 4 0.34 0.89 0.003 1 InceptionResNet-V2 + 4 0.13 0.94 0.04 0.99 DenseNet-121 + 4 0.17 0.94 0.02 0.99 DenseNet-169 + 4 0.08 1 0.004 0.99 DenseNet-201 + 4 0.12 0.94 0.01 1 Table A2. The results of testing alternative models for each class that were trained on data set with augmentation. Table A2. The results of testing alternative models for each class that were trained on data set with augmentation. Abbreviations The following abbreviations or terms are used in this manuscript: CNN Convolutional neural networks UAV Unmanned aerial vehicle P. proximus Polygraphus proximus RGB Red, green, blue ERS Earth remote sensing SVM Support vector machine RBF Radial basis function EPSG Google Earth in European Petroleum Survey Group OBIA Object based image analysis LDA Linear discriminant analysis PCA Principal component analysis RSI Remote sensing indices DJI Dajiang Innovation Technology QGIS Quantum Geographic Information System ADAM ADAptive Moment estimation ReLU Rectified linear unit TP True positives TN True negatives FP False positives FN False negatives API Application programming interface GPU Graphics processing unit CPU Central processing unit 16 of 19 Remote Sens. 2019, 11, 643 Table A1. Performance comparison of the training by proposed CNN model with other models. Categories of Trees TP TN FP FN Acc (%) Precision (%) Recall (%) F-Score (%) Xception 1 30 26 19 2 72.73 61.22 93.75 74.07 2 15 41 8 21 65.88 65.22 41.67 50.85 3 7 49 1 9 84.85 87.5 53.75 58.33 4 4 52 4 0 93.33 50 100 66.67 VGG-16 1 24 43 3 8 85.90 88.89 75 81.36 2 33 34 14 3 79.76 70.21 91.67 79.52 3 6 61 0 10 87.01 100 37.5 54.55 4 4 63 4 0 94.37 50 100 66.67 VGG-19 1 26 38 6 6 84.21 81.25 81.25 81.25 2 29 35 11 7 78.05 72.5 80.56 76.32 3 5 59 0 11 85.33 100 31.25 47.62 4 4 60 7 0 90.14 36.36 100 53.33 ResNet-50 1 32 32 16 0 80 66.67 100 80 2 20 44 1 16 79.01 95.24 55.56 70.18 3 9 55 1 7 88.89 90 56.25 69.23 4 3 61 6 1 90.14 33.33 75 46.15 Inception-V3 1 30 25 21 2 70.51 58.82 93.75 72.29 2 16 39 8 20 66.27 66.67 44.44 53.33 3 6 49 1 10 83.33 85.71 37.50 52.17 4 3 52 3 1 93.22 50 75 60 InceptionResNet-V2 1 23 27 14 9 68.49 62.16 71.88 66.67 2 18 32 11 18 63.29 62.07 50 55.38 3 6 44 6 10 75.76 50 37.5 42.86 4 3 47 7 1 86.21 30 75 42.86 DenseNet-121 1 26 33 10 6 78.67 72.22 81.25 76.47 2 24 35 8 12 74.68 75 66.67 70.59 3 6 53 1 10 84.29 85.71 37.50 52.17 4 3 56 10 1 84.29 23.08 75 35.29 DenseNet-169 1 32 26 21 0 73.42 60.38 100 75.29 2 15 43 5 21 69.05 75 41.67 53.57 3 8 50 1 8 86.57 88.89 50 64 4 3 55 3 1 93.55 50 75 60 Dense Net-201 1 32 22 22 0 71.05 59.26 100 74.42 2 15 39 7 21 65.85 68.18 41.67 51.72 3 4 50 1 12 80.6 80 25 38.1 4 3 51 4 1 91.53 42.86 75 54.55 17 of 19 17 of 19 Remote Sens. 2019, 11, 643 Appendix B. Table A1. Performance comparison of the training by proposed CNN model with other models. Brief Description of the CNNs That Are Compared against the CNN Developed in This Work, That Is, VGG, ResNet, Inception-V3, InceptionResNet-V2, Xception and DenseNet Visual Geometry Group (VGG) is a neural network architecture that has four variants, VGG-11, VGG-13, VGG-16 and VGG-19, where the number 13, 16 and 19 indicate the number of layers in the network. VGG-16 was designed by the University of Oxford to recognize objects in images. VGG-16 network is the 1st runner-up on the ImageNet Large Scale Visual Recognition Challenge (ILSVRC) comparison in 2014 with an obtained accuracy of 93.3% [39]. A distinctive feature of the architecture is a small convolution kernel of 3 × 3 pixels. The neural network architecture consists of two parts. The first part consists of alternating convolution cascades and a pooling layer: two convolution-convolution-pooling cascades and then three convolution-convolution-convolution-pooling cascades. On the pooling layer, Max Pooling is selected with a 2 × 2 square core. This part highlights the characteristic features in the image. The second part is responsible for the classification of the object in the image according to the features selected at the previous stage and includes three fully connected layers. Thus, the VGG-16 network receives images with a size of 224 × 224 pixels in three colour channels (red, green and blue) and the output represents the probability of belonging to a particular class in one hot encoding format. In our experiments, we compared our CNN to the following types of networks: VGG-16 and VGG-19. p g yp ResNet was developed by Microsoft using residual learning for recognition, localization and detection of objects in images. It was the winner of the ILSVRC 2015 competition with 150 layers and the winner of Microsoft Common Objects in Context 2015 (MS COCO) detection and segmentation [40]. The authors of ResNet used the two-layer traversal approach and applied it on a large scale, which is considered as a small classifier in the network. The network also uses a bottleneck layer, which allows the reduction of the number of features in each layer, using a 1 × 1 pixel convolution with a lower yield of features and then a 3 × 3 pixel convolution layer and again a 1 × 1 pixel convolution layer with more features. The bottleneck layer reduces computational resources while preserves features combinations. The output layer is the pooling layer with the Softmax function. Table A1. Performance comparison of the training by proposed CNN model with other models. In our experiments, we compared our CNN to ResNet-50. p p Inception-V3 is Google’s convolutional neural network for recognizing objects in an image. In December 2015, the third version of Inception was introduced with the inclusion of Batch-normalized Inception. Batch-normalization calculates the mean and standard deviation of all feature distribution maps in the output layer and then normalizes their responses with these values [20]. The last layer of the Inception network is the pooling layer with the Softmax function. To evaluate Inception-V3 on our data, we had to resize the input images from 150 × 200 to 299 × 299 pixels using cubic interpolation. p g p InceptionResNet-V2 is the union of two neural networks: Inception and ResNet. This allowed the authors to improve the accuracy of image classification. The idea of residual blocks was preserved from the Inception network and a combination of convolutional blocks from the ResNet network. Despite the fact that InceptionResNet-V2 demonstrates high accuracy compared to other existing models, it has two drawbacks: low speed and a high amount of memory used. Xception was implemented in Keras by François Chollet and it is a modification of the Inception network (Chollet [41]). The network architecture is similar to ResNet-34 but the model and code is simpler than in Inception. A distinctive feature of the network architecture is SeparableConv, which is a modifiable, separable convolution that is located at the top of the network architecture. In the network there are residual (or shortcut/skip) connections, taken from the network ResNet. In addition, the network contains residual connections, which significantly increase the accuracy of classifying objects in images. g y y y g j g DenseNet is the result of the development of the ResNet network and is based on its residual blocks [21]. The basic idea is that connections have all the possible combinations within each block, which represents a gradient of more paths and the network becomes more resistant to learning. 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English
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Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae
PLOS genetics
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14,147
Introduction encodes an integral transmembrane protein, for which the use of an alternative 59-splice site changes the number of passes through the membrane and ultimately the location of the C-terminal end of Src1p [16,17]. Alternative 39-splice site selection has been shown to regulate expression of the APE2 gene according to a temperature-dependent secondary structure in the transcript [18]. A few other alternative 39-splice sites have been described, and the use of some of these sites produces transcripts that are degraded by NMD [19]. Recent work analyzing alternative splicing across fungal species has shown that S. cerevisiae has lost some of the alternative splicing events through gene duplication and sub-functionalization of the duplicated genes, which are otherwise produced by alternative splicing in other species [20]. Nonsense-mediated mRNA decay (NMD) is an RNA degrada- tion system that degrades RNAs containing premature termination codons [1,2]. In mammalian cells and higher eukaryotes, NMD can be used to regulate gene expression, for instance by reducing the level of alternatively spliced isoforms containing premature termination codons [3,4,5,6,7,8]. This interplay between alterna- tive splicing and NMD is involved in the autoregulation of SR proteins [3,4,5]. In addition to its function in regulating non- productively spliced isoforms, NMD is also used in a variety of eukaryotes to degrade unspliced pre-mRNAs that have escaped the splicing machinery [9,10,11,12,13]. Thus, NMD is widely involved in the proofreading of splicing efficiency and accuracy. Using RNA-Seq analysis of strains mutated for NMD factors, we identify here a large number of alternative splice sites in S. cerevisiae. However, we show that splicing at these sites is generally non-productive because it introduces premature termination codons (PTC), leading to degradation of the transcripts by NMD. Non-productive splicing can be increased during environ- mental stress to contribute to a global regulatory mechanism that down-regulates transcripts levels in response to environmental cues. These results show that non-productive splice sites are widely used in S.cerevisiae, but that transcripts spliced at these sites are The yeast Saccharomyces cerevisiae has long been used as a model system to investigate the mechanisms of pre-mRNA splicing, as many components of the splicing machinery were identified through genetic screens in S. cerevisiae [14], and most splicing factors are highly conserved from yeast to mammalian cells [15]. Despite the presence of c.a. 330 intron-containing genes in S. Abstract * E-mail: guillom@chem.ucla.edu ¤ Current address: Applied BioCode Inc., Santa Fe Springs, California, United States of America. ¤ Current address: Applied BioCode Inc., Santa Fe Springs, California, United States of America. ¤ Current address: Applied BioCode Inc., Santa Fe Springs, California, United States of Am Tadashi Kawashima1¤, Stephen Douglass2, Jason Gabunilas1, Matteo Pellegrini3, Guillaume F. Chanfreau1* 1 Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California, United States of America, 2 Bioinformatics Interdepartmental Program, UCLA, Los Angeles, California, United States of America, 3 Department of Molecular, Cellular and Developmental Biology, UCLA, Los Angeles, California, United States of America April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org Abstract Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense- mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 39 splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 39-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity. Editor: Hiten D. Madhani, University of California San Francisco, United States of America Editor: Hiten D. Madhani, University of California San Francisco, United States of America Editor: Hiten D. Madhani, University of California San Francisco, United States of America Received October 8, 2013; Accepted February 4, 2014; Published April 10, 2014 Received October 8, 2013; Accepted February 4, 2014; Published April 10, 2014 Received October 8, 2013; Accepted February 4, 2014; Published April 10, 2014 Copyright:  2014 Kawashima et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by grant GM061518 to GFC. TK was supported by USPHS National Research Service Award GM07104 and by a UCLA Graduate Division Dissertation Year Fellowship. JG was supported by a Ruth L. Kirschstein National Research Service Award GM007185. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. RNA-Seq reveals the accumulation of a large number of non-productive splice variants in NMD mutants We previously showed that NMD degrades unspliced transcripts arising from a large fraction of intron-containing genes in S. cerevisiae, due to suboptimal splice sites [12,13], or upon splicing factor inactivation [21]. In addition, recent data showed that transcripts generated by the use of alternative 39-splice sites can be degraded by NMD [19]. To gain further insights into the function of NMD in the proofreading of spliced isoforms, we performed RNA sequencing of mRNAs from wild-type and isogenic upf1D, upf2D and upf3D strains defective for NMD. To identify transcripts spliced at alternative splice sites, we performed gapped alignment analysis of the RNA sequences (Table S1) using BLAT [22]. This analysis revealed numerous occurrences of spliced transcripts arising from previously unknown splice sites, in both WT and the NMD mutants. We will refer to these new splicing events as alternative splicing events, even if these are found in wild-type cells, and to the annotated splicing events as the normal or canonical splicing events. Alternative splicing events were detected more frequently in RNA samples obtained from the NMD mutants (Fig. 1A and 1B; Table S2), consistent with the fact that most of these alternative splicing events result in the introduction of a PTC, either by inducing a translational frameshift or by inserting an intronic PTC-containing sequence (Table S2). After adjusting for sequencing depth, upf1D, upf2D and upf3D showed a 1.67, 1.72, and 1.90-fold enrichment in alternative splicing events and 1.59, 1.70, and 1.79-fold enrichment in PTC-generating alternative splicing events, respectively, versus wild-type (Table S2). NMD mutants showed an approximately 1.7-fold increase in unspliced mRNAs compared to the wild-type (Table S3) when considering reads that map to intronic and exon-intron regions, confirming our previous results from tiling arrays showing the involvement of NMD in eliminating unspliced transcripts genome- wide [12]. This enrichment for unspliced RNAs in NMD mutants is probably underestimated. Although there were 4-fold more reads that mapped only to intronic regions in NMD mutants compared to wild-type (Table S3), we observed an unanticipated high number of reads that mapped to exon-intron junctions in the wild-type strain (Table S3), which lowered the overall enrichment for unspliced RNAs in NMD mutants. The consensus sequences derived from the alternative splicing events identified in wild-type and all three mutants exhibited differences from the consensus sequences derived from the canonical (normal) splicing events (Fig. 1E). Non-productive Alternative Splicing in Yeast There was limited overlap in the alternative splicing events identified in the three UPF mutants (Fig. 1C), suggesting that the depth of our sequencing analysis was not sufficient to saturate identification of all alternative splicing events, particularly those occurring at low frequencies. The list of intron containing genes (ICG) for which we did not find the use of alternative splice sites is provided in Table S4, and the list of genes for which alternative splicing events were detected is shown in Table S5. 97 out of 304 intron containing genes analyzed did not exhibit alternative splicing (Table S4). Whether this reflects the absence of competing alternative sites or the lack of depth of our sequencing analysis remains to be determined. Author Summary Accurate gene expression requires the transfer of gene information from DNA to RNA. When DNA is transcribed into RNA, part of the RNA needs to be removed (spliced) to generate a proper copy of the genetic information. This process needs to be very accurate to preserve the genetic information that will be transferred into proteins. Our study shows that in baker’s yeast, the splicing process does not always produce the correctly spliced products, as RNA splicing events frequently utilize incorrect splice sites. However, these deficient RNA molecules are eliminated from cells by a quality control mechanism to preserve the integrity of the genetic information. However, incorrect splicing is not useless, as it can be used to regulate the quantity of RNA that is generated. To investigate if alternative splicing events are due to rare events or to splicing errors that occur randomly during transcript expression, we examined the abundance of ICG mRNAs that exhibited alternative splicing events and that of ICG mRNAs for which no alternative splicing events were detected (Fig. 1D). This analysis showed that some low abundance transcripts exhibited alternative splicing, while some high abundance transcripts did not (Fig. 1D). In addition, the median abundance of genes that showed alternative splicing was 117 RPKM, while the median abundance for genes with no alternative splicing events detected was 136 RPKM. Thus, even if the most highly expressed ICG (.2200 RPKM) all exhibited alternative splicing (Fig. 1D), genes with no alternative splicing were in general expressed at higher level than genes for which alternative splicing events were detected, showing no clear correlation between transcript abundance and the detection of alternative splicing events. We conclude that the detection of alternative splicing events in our RNA-Seq analysis is not an indirect consequence of the higher number of reads for highly-expressed transcripts. eliminated by RNA quality control mechanisms. Thus, while alternative splicing is frequently utilized in higher eukaryotes to generate proteome diversity, it is mainly used in S.cerevisiae as a means to regulate transcript levels. Introduction cerevisiae, the prevalence of alternative splicing in this organism remains largely unexplored, as only a few examples of alternative splice site selection have been documented. The SRC1 gene April 2014 | Volume 10 | Issue 4 | e1004249 1 PLOS Genetics | www.plosgenetics.org RNA-Seq reveals the accumulation of a large number of non-productive splice variants in NMD mutants Alternative 59-splice sites showed a relaxation of the conserved sequences, especially at positions 4 and 6 compared to the consensus obtained from the canonical splicing events. The 39-splice sites used in alternative splicing events also showed a decrease in conservation of the polypyrimidine sequence preceding the conserved YAG, as well as a weaker conservation of the pyrimidine preceding the conserved AG dinucleotide (Fig. 1E). Thus, alternative splice sites identified by RNA sequencing showed a relaxed conservation, suggesting that these might correspond to lower efficiency splice sites, and possibly to regulated splicing events. Finally, we identified a number of alternative splicing events in either wild-type or NMD mutants that do not introduce a PTC and would potentially result in the production of proteins that differ from the SGD annotations. The list of these potential alternative proteins is presented in Table S6. However, we did not investigate these alternative protein forms further because most of the RNAs that would result in the production of these proteins were found in low abundance compared to those resulting in the production of the annotated proteins. Strategy for validation of alternative splicing events gy p g The previous RNA-Seq analysis revealed the potential wide- spread usage of alternative splice sites (SS). Figure 2 depicts specific mRNAs that were chosen for validation and further characterization. These transcripts were classified into three classes: those with 1) alternative 59-SS; 2) alternative 39-SS; and 3) a combination of both. Transcripts from class 1 included RPL22B as well as the previously reported SRC1 [16]. Class 2 transcripts included genes encoding the RNA Polymerase III April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 2 Non-productive Alternative Splicing in Yeast Figure 1. Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants. A. Venn diagram showing the overlap of alternative splice site usage between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events (both PTC-generating and non-PTC-generating). B. Venn diagram showing the overlap of alternative splicing events between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events resulting in a potential PTC. C. Venn diagram showing the overlap of alternative splicing events between the upf1D, upf2D, and upf3D strains for PTC-generating splicing events. D. Distributions of intron-containing gene transcripts showing alternative splicing events (red) or no alternative splicing events (blue) according to their overall abundance in RPKM. Transcripts for which the abundance was higher than 2,300 RPKM were grouped in the final bin. E. Sequence logo analysis of 59- and 39- splice sites for all normal and alternative splicing events detected by RNA-Seq in wild-type and NMD mutant strains. doi:10.1371/journal.pgen.1004249.g001 Figure 1. Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants. A. Venn Figure 1. Bioinformatics analysis of alternative splice site usage in wild-type and NMD mutants. A. Venn diagram showing the overlap of alternative splice site usage between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events (both PTC-generating and non-PTC-generating). B. Venn diagram showing the overlap of alternative splicing events between the wild-type and three NMD mutants pooled for all unique non-canonical splicing events resulting in a potential PTC. C. Venn diagram showing the overlap of alternative splicing events between the upf1D, upf2D, and upf3D strains for PTC-generating splicing events. D. Distributions of intron-containing gene transcripts showing alternative splicing events (red) or no alternative splicing events (blue) according to their overall abundance in RPKM. Strategy for validation of alternative splicing events Transcripts for which the abundance was higher than 2,300 RPKM were grouped in the final bin. E. Sequence logo analysis of 59- and 39- splice sites for all normal and alternative splicing events detected by RNA-Seq in wild-type and NMD mutant strains. doi:10.1371/journal.pgen.1004249.g001 labeling with an appropriate 32P-labelled ladder (data not shown) to confirm the sizes of all RT-PCR products and correlate the data back to gels obtained with Cy3-labeled primers. In addition to the wild-type and NMD-deficient upf1D strains, we analyzed the phenotypes of a number of S. cerevisiae splicing mutants. Knockout mutants of genes encoding Mud1p and Nam8p were chosen for their association with the U1 snRNP and role in 59-SS selection [23,24,25,26]. The HUB1 knockout was also included, as Hub1p was recently implicated in 59-SS selection for SRC1 [17]. Prp17p and Prp18p were selected for their involvement in the second step of splicing and potential effects on 39-SS selection [27,28]. Finally, Isy1p was also included as a potential splicing fidelity factor [29]. The splicing profiles were analyzed for each of the genes transcription factor TFC3 with a downstream alternative 39-SS, and the adenosine deaminase TAN1 with two alternative 39-SS flanking the normal 39-SS. For the third class, we examined genes encoding the glycosylphosphatidylinositol biosynthetic enzyme GPI15 and the transcriptional regulator GCR1. GPI15 exhibited the use of an alternative 59-SS with the normal 39-SS, as well as the normal 59-SS with an alternative 39-SS (Fig. 2). GCR1 showed a more complex splicing pattern with multiple combinations of 59 and 39-SS (Fig. 2). transcription factor TFC3 with a downstream alternative 39-SS, and the adenosine deaminase TAN1 with two alternative 39-SS flanking the normal 39-SS. For the third class, we examined genes encoding the glycosylphosphatidylinositol biosynthetic enzyme GPI15 and the transcriptional regulator GCR1. GPI15 exhibited the use of an alternative 59-SS with the normal 39-SS, as well as the normal 59-SS with an alternative 39-SS (Fig. 2). GCR1 showed a more complex splicing pattern with multiple combinations of 59 and 39-SS (Fig. 2). We analyzed alternative splicing events by RT-PCR using Cy3- end labeled primers, which allowed for relative comparison of the abundance of spliced and unspliced species, regardless of their size. Non-productive Alternative Splicing in Yeast Non-productive Alternative Splicing in Yeast mentioned above in each of these mutant strains by fractionation of the RT-PCR products on polyacrylamide gels (Fig. 3). For the splicing mutants for which the splicing pattern differed from the wild-type, additional RT-PCR experiments were performed in triplicate from three independent cultures and quantitated, as shown in Supporting Figures S1, S2, S3, S4 and S6. previous studies have demonstrated the roles of various splicing factors [16,17,30]. RT-PCR analysis of SRC1 splice variants confirmed the use of these two alternative 59-SS (Fig. 3). Wild-type samples showed a 60/40 ratio of SRC1-S/SRC1-L (Fig. S1), consistent with previous reports [16,17,30]. Samples from the upf1D mutant showed a pattern similar to wild-type (Fig. 3; Fig. S1), indicating that both variants are stable and not targeted by NMD. This result is consistent with our RNA-Seq analysis, which showed high sequence counts for both forms in all strains. Samples from the nam8D strain showed a slight increase in the level of unspliced transcripts (Fig. 3) due to reduced splicing efficiency [31]. The prp17D and prp18D mutants both showed a slight RT-PCR analysis confirms the involvement of Prp17p and Hub1p in SRC1 alternative splicing RT-PCR analysis confirms the involvement of Prp17p and Hub1p in SRC1 alternative splicing As a first step in validating our RT-PCR strategy, we focused on SRC1, which exhibits two possible 59-SS (Fig. 2) and for which SRC1, which exhibits two possible 59-SS (Fig. 2) and for which [31]. The prp17D and prp18D mutants both showed a slight Figure 2. Spliced species produced from the SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 genes. Species labeled with an asterisk are subject to NMD. Species labeled with two asterisks are predicted to be subject to NMD but were not observed to do so in subsequent experiments. The alternative 39-SS of SRC1 is located 4 nt upstream from the annotated 39-SS. The alternative 39-SS of RPL22B is located 64 nt downstream from the annotated 39-SS. The alternative 39-SS of TAN1 are located 6 nt upstream and 7 nt downstream from the annotated 39-SS. The alternative 39-SS of TFC3 is located 17 nt downstream from the annotated 39-SS. The alternative 59 and 39-SS of GPI15 are located 36 nt downstream and 14 nt upstream, respectively, from the annotated 59 and 39-SS. The alternative 39-SS of GCR1 are located 5 nt upstream (GUAUGG); 51 nt downstream (GUAUGG) and 627 nt downstream from the annotated 59SS. The alternative 39-SS of GCR1 are located 40 nt upstream (AUG) and 17 nt downstream (CAG) from the annotated 39-SS. doi:10.1371/journal.pgen.1004249.g002 Figure 2. Spliced species produced from the SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 genes. Species labeled with an asterisk are subject to NMD. Species labeled with two asterisks are predicted to be subject to NMD but were not observed to do so in subsequent experiments. The alternative 39-SS of SRC1 is located 4 nt upstream from the annotated 39-SS. The alternative 39-SS of RPL22B is located 64 nt downstream from the annotated 39-SS. The alternative 39-SS of TAN1 are located 6 nt upstream and 7 nt downstream from the annotated 39-SS. The alternative 39-SS of TFC3 is located 17 nt downstream from the annotated 39-SS. The alternative 59 and 39-SS of GPI15 are located 36 nt downstream and 14 nt upstream, respectively, from the annotated 59 and 39-SS. The alternative 39-SS of GCR1 are located 5 nt upstream (GUAUGG); 51 nt downstream (GUAUGG) and 627 nt downstream from the annotated 59SS. The alternative 39-SS of GCR1 are located 40 nt upstream (AUG) and 17 nt downstream (CAG) from the annotated 39-SS. Strategy for validation of alternative splicing events Because we lacked an adequate size marker for Cy3 detection, the same RT-PCR analyses were initially performed with 32P-end We analyzed alternative splicing events by RT-PCR using Cy3- end labeled primers, which allowed for relative comparison of the abundance of spliced and unspliced species, regardless of their size. Because we lacked an adequate size marker for Cy3 detection, the same RT-PCR analyses were initially performed with 32P-end April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 3 PLOS Genetics | www.plosgenetics.org RT-PCR analysis confirms the involvement of Prp17p and Hub1p in SRC1 alternative splicing doi:10.1371/journal.pgen.1004249.g002 April 2014 | Volume 10 | Issue 4 | e1004249 4 Non-productive Alternative Splicing in Yeast mutants on SRC1 splicing patterns and showed that our RT-PCR strategy is effective in analyzing the impact of specific splicing factors on splice site usage.. increase in the usage of SRC1-L 59-splice site relative to the SRC-S 59-splice site (1.4 and 1.3 fold, respectively), as suggested previously for the prp17D mutant at the protein level [17], and the prp18D mutant also exhibited an increase in unspliced precursors accumulation, consistent with previous results for other transcripts [21]. The isy1D mutant strain exhibited a clear accumulation of unspliced pre-mRNAs (Fig. 3), in agreement with the documented role of Isy1p in maintaining the proper conformation needed for the 1st step of splicing [29]. Hub1p inactivation resulted in a 3-fold reduction in the amount of SRC1-S, coinciding with an increase in SRC1-L (Fig. S1, Fig. 3), consistent with previous reports [17,30]. This reduction was also observed in the context of the upf1D mutant (Fig. S1, Fig. 3). Thus, the results described above confirmed the previously described effects of various splicing Alternative splicing patterns of GPI15 and GCR1 reveal the production of alternative non-functional protein products and the use of a non-canonical AUG 39-splice site repressed by Prp18p p y p p GPI15 in an interesting case where the two alternatively spliced products identified by our RNA-Seq analysis are not targeted by NMD. The use of an alternative GUACGU 59-splice site results in the deletion of 30 nucleotides from the 39 end of exon 1 (Fig. 2), which maintains the open-reading frame but generates a truncated protein. However, the protein product resulting from translation of this alternatively spliced product is likely to be non-functional, as this truncation removes a stretch of 10 amino acids at positions 187–197 in the most highly conserved region of this protein [35]. This transcript can be detected in samples from the wild-type and the splicing factor mutants, and does not vary in intensity in the context of upf1D, indicating that it is not targeted by NMD (AS 59, Fig. 3). In contrast, the alternatively spliced transcript generated by use of a downstream CAG 39-SS results in a PTC. However, this PTC-containing transcript would exhibit a short 85 nt 39-UTR, which might render it insensitive to NMD as suggested by the faux 39 UTR model [36,37]. Indeed, the abundance of this transcript was not increased in the upf1D mutant (Fig. 3). In addition, this transcript is expected to yield a non-functional protein due to C- terminal truncation and deletion of amino acids within the most conserved region of the protein [35]. Analysis of the pattern of selection of these two alternatively spliced transcripts in the various splicing mutants did not reveal any major effect of these mutants (Fig. 3) in contrast to the effects described above for RPL22B, TAN1 or TFC3. However, there was a slight increase in the use of the downstream alternative 39-SS in the prp17Dupf1D strain, consistent with the role of Prp17p in favoring the upstream 39-SS, as described above for TAN1 and TFC3. TAN1 exhibits a more complex alternative 39-SS pattern, where a canonical UAG 39-SS is flanked by two alternative 39 AAG sequences (Fig. 2). The use of either of these sites would generate PTC-containing transcripts. The upstream AAG (AS 39 #1) is only 6 nt away from the canonical 39-SS. The retention of 6 nt of intronic sequence would maintain the proper reading frame but would result in a PTC because the UAG sequence of the normal 39-splice site corresponds to an in-frame stop codon [32]. components Nam8p and Mud1p RPL22B showed the presence of an RPL22B showed the presence of an alternative 59-SS in the intronic sequence, which unlike SRC1, yields a PTC-containing transcript potentially targeted to NMD (Fig. 2). This alternatively spliced transcript was almost 10-fold more abundant in the upf1D mutant (Fig. 3; Fig. S2), further suggesting that it is targeted by NMD. We also detected a large accumulation of unspliced species Figure 3. RT-PCR analysis of alternatively spliced products for SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 in wild-type, NMD and various splicing mutants. The unspliced (US) species is also shown on top. The middle portions of the gel where no species were visible have been removed. In all cases, RT-PCR was performed with a Cy3-labeled primer. The labeling of the different alternatively spliced forms is according to the nomenclature shown in Figure 2. doi:10.1371/journal.pgen.1004249.g003 Figure 3. RT-PCR analysis of alternatively spliced products for SRC1, RPL22B, TAN1, TFC3, GPI15 and GCR1 in wild-type, NMD and various splicing mutants. The unspliced (US) species is also shown on top. The middle portions of the gel where no species were visible have been removed. In all cases, RT-PCR was performed with a Cy3-labeled primer. The labeling of the different alternatively spliced forms is according to the nomenclature shown in Figure 2. doi:10.1371/journal.pgen.1004249.g003 April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 5 Non-productive Alternative Splicing in Yeast in the upf1D mutant, indicating inefficient recognition of this splicing substrate. This may be the result of both the normal (GUACGU) and alternative (GUUUGU) 59-SS having non- consensus sequences (see below). Interestingly, the abundance of the alternatively spliced product was found to decrease by two to three folds when Nam8p or Mud1p were inactivated in the context of the upf1D deletion (Fig. 3; Fig. S2). The deletion of either one of these two factors might hinder the ability of the U1 snRNP to bind to the alternative suboptimal 59 GUUUGU splice site of RPL22B, resulting in decreased usage. This is consistent with the known roles of Mud1p and Nam8p in the first step of splicing [25], and suggest their direct involvement in modulating 59-SS selection of RPL22B. By contrast, no major changes were observed in the prp17D, prp18D, isy1D, hub1D mutants, either alone or in combination with the upf1D deletion (Fig. 3), showing the specificity of the effects detected with Nam8p and Mud1p. components Nam8p and Mud1p RPL22B showed the presence of an Thus, RPL22B exhibits two competing suboptimal 59-SS, one of which is highly sensitive to perturbations in the U1 snRNP. The functional significance of the alternative 59-SS of RPL22B in regulating transcript levels is investigated further below. most proximal to the branch point sequence (AS 39 #1; Fig. 3; Fig. S4). This product is barely detectable in the wild-type strain but can be observed in the prp18D strain (Fig. 3), and inactivation of Prp18p in the context of the upf1D deletion resulted in a 3-fold increase in the abundance of this species (Fig. S4). The effect of Prp18p on this 39-SS might be due to the identity of the sequences immediately following the 39-SS, which have been shown to influence 39-SS selection in the absence of a functional Prp18p [33]. Isy1p inactivation resulted in an increase of unspliced species in a similar fashion to SRC1 discussed above; however there was no effect of Isy1p, Hub1p, Mud1p and Nam8p on alternative 39- SS selection of TAN1 (Fig. 3), showing the specificity of the effects observed with Prp17p and Prp18p. Finally, unspliced TAN1 transcripts were generally not affected by NMD, except in the context of a mud1D mutant strain (Fig. 3). This observation is consistent with a recent report showing that TAN1 unspliced transcripts are retained in the nucleus by the RES complex, and are subject to NMD only when the RES complex is inactivated [34]. Overall, analysis of TFC3 and TAN1 alternative 39-SS patterns show that Prp17p and Prp18p have antagonistic roles in the selection of upstream and downstream 39-SS of TAN1, and highlight the importance of Prp17p in enhancing the use of 39-SS located closer to the branchpoint. A novel role for Prp17p in promoting the use of branchpoint proximal alternative 39-splice sites Gapped sequence alignment showed that TFC3 exhibits an alternative CAG 39-SS 17 nt downstream of the annotated AAG (Fig. 2). This product can be detected in samples from the wild- type and splicing mutants, but is 4.5-fold more abundant in the context of the upf1D deletion, showing that a large fraction of this product is degraded by NMD (Fig. 3; Fig. S3). This non- productive isoform amounts to 27% of all spliced products (Fig. S3), showing that a significant fraction of splicing generates NMD- targeted, non-productive transcripts. We observed a slight accumulation (1.7 fold) of the downstream alternative 39-splice product in the prp17D mutant (Fig. 3; Fig. S3), showing that this second step splicing factor contributes to reducing the use of this alternative 39-SS. As expected, inactivation of the first step splicing factors Mud1p or Nam8p had no effect on the pattern of 39-SS selected (Fig. 3). Alternatively spliced species of RPL22B and GCR1 increase during stress conditions Significantly, amino acid starvation did not change the levels of the alternatively spliced species of TAN1 and TFC3 that are normally subject to NMD (Fig. S11). This observation provides further evidence that the increase in the amount of alternatively spliced RPL22B transcript observed during amino acid starvation is due to a switch in splice site selection and not to an inhibition in NMD, since the level of alternatively spliced species of TAN1 and TFC3 that are normally degraded by NMD is unaffected in the same conditions. In addition to this major spliced product, we also detected a series of alternatively spliced products degraded by NMD (as denoted by asterisks in Fig. 2 and 3). Splicing from the annotated GUAUGA 59 splice site combined with the upstream CAG 39 splice site resulted in a PTC-containing transcript labeled as *A in Figs. 2 & 3. This transcript is degraded by NMD, as higher amounts are observed in all the strains containing a upf1D deletion, and it is the most abundant of all GCR1 alternatively spliced products subject to NMD (Fig. 3; Fig. S6). Another product is generated from combining the upstream GUAUGG 59-SS with the most downstream UAG 39-SS (*C in Fig. 2). This splicing event results in a PTC, as it introduces a translational frameshift, which is not detected until the 43rd amino acid is translated. The corresponding transcript accumulates at low abundance in all samples and appears to be targeted by NMD, as its abundance increases slightly in all upf1D strains. In addition, the use of this most downstream 39-SS increases almost 4-fold in the prp17Dupf1D strain when compared to the upf1D control (Fig. 3; Fig. S6). Because the 39-SS used to generate this transcript corresponds to the most downstream one, this observation provides another example of the importance of Prp17p in favoring the selection of upstream 39- SS, as shown above for TFC3, TAN1 and to a lesser extent GPI15. Another PTC-containing transcript that is degraded by NMD results from splicing of the downstream intronic GUAUGG 59-SS with the CAG 39-SS, (labeled *B in Fig. 2 and 3). This product is faint, but detectable in all cases of NMD deactivation, except in combination with nam8D or mud1D, most likely because this 59-SS has a higher sensitivity to U1 snRNP perturbations, as described above for RPL22B. Alternatively spliced species of RPL22B and GCR1 increase during stress conditions g The previous results validated our prediction that transcripts generated from the use of alternative non-productive splice sites are degraded by NMD and revealed the role of specific splicing factors in governing the choice between alternative sites. Strikingly, the sequence of some of these non-productive splice sites was found to be conserved across closely related yeast species (Fig. S8, RPL22B and Fig. S9, TAN1). Because the level of sequence conservation in intronic sequences is usually very low (Fig. S8, S9), these peaks in sequence conservations for intronic alternative splice sites might reflect their functional importance. We hypothesized that the use of some of these alternative splice sites which lead to degradation by NMD might be favored under certain conditions to down-regulate gene expression. To test this hypothesis, we monitored changes in the splicing patterns of RPL22B, TAN1, and TFC3 under stress conditions such as amino acid starvation, heat shock, LiCl-mediated hyperosmotic stress, and rapamycin treatment, as these have been reported to elicit diverse responses in the expression of intron containing genes [43,44]. In addition, various stresses cause down-regulation in ribosomal protein gene expression (many of which contain introns), presumably to relieve the cell of massive energy requirements of ribosome biogenesis and focus those resources into regulations that are the most appropriate in response to the current stress condition [45,46,47]. After 10 minutes of amino acid depletion, RPL22B showed an increase in unspliced species as well as well as a 4.5-fold increase in the level of the alternatively spliced product when compared to the SDC or YPD media controls (Fig. 4A; Fig. S10). In the upf1D strain shifted to amino acid starvation conditions, the levels of the alternatively spliced product increased compared to the wild-type strain grown in the same conditions, as would be expected when NMD transcripts are no longer degraded (Fig. 4A lanes 2 and 4). The fact that the level of the alternatively spliced transcript is 2.5-fold higher in the upf1D sample than in the wild-type sample under amino acid starvation conditions (Fig. S10) argues that the increase in the abundance of these species in the wild-type strain in these conditions is not due to NMD inhibition in these conditions, but that a change in splice site selection occurs that favors the use of the alternative splice site. Non-productive Alternative Splicing in Yeast Alternatively spliced species of RPL22B and GCR1 increase during stress conditions Non-productive Alternative Splicing in Yeast 23 nt upstream of the other alternative 39-SS (Fig. 2). The use of all of these sites was confirmed by RT-PCR, cloning and Sanger sequencing (see below and Fig. S5). The fact that some alternative splice sites escaped identification by mRNA sequencing indicates that a greater depth of coverage has the potential to identify even more alternative splice sites. Prp22p during the late stages of splicing [42], it is possible that the absence of Prp18p might indirectly hinder the function of Prp22p in proofreading 39-SS selection, and that the use of this unusual 39- SS might be the consequence of a reduced Prp22p function in the absence of Prp18p. To test this hypothesis, we analyzed GCR1 splicing in a prp22-1 mutant. RT-PCR analysis showed that the spliced product generated from the use of the AUG 39-SS did not accumulate in a prp22-1 splicing mutant (Fig. S7). Thus, the accumulation of species resulting from the use of this unusual 39- SS in the prp18Dupf1D samples is not an indirect consequence of hindered Prp22p function. The discovery of the splicing at this unusual 39-SS sequence reveals the importance of Prp18p in ensuring proper 39-SS selection for GCR1 and in repressing the use of non-canonical 39-SS sequences. Based on GCR1 annotation, the canonical spliced mRNA would use the GUAUGA 59-SS along with the most downstream UAG 39-SS (Fig. 2). This product (labeled as S(annot.) in Fig. 2 and 3), however was detected at very low levels (Fig. 3). The major spliced product observed resulted from the use of the most upstream GUAUGG 59-SS and of an upstream CAG 39-SS (labeled ‘‘S’’ in Fig. 2 and 3). This splicing event does not introduce a PTC and results in a protein that is very similar to the translation product of the annotated spliced transcript S(annot.). The annotated amino acid sequence of GCR1 from position 2 to 4 is VCT. In the major spliced product S, this sequence is replaced by QTSVDST. Thus, most of the protein is identical, except for a few N-terminal amino acids which are not expected to affect Gcr1p function, as all GCR1 mutations with phenotypic effects have been mapped to a region downstream of this short sequence stretch [38,39,40]. Based on the relative abundances of S and S(annot.), it is clear that S, and not S(annot.) is the main spliced product for the GCR1 gene. Alternative splicing patterns of GPI15 and GCR1 reveal the production of alternative non-functional protein products and the use of a non-canonical AUG 39-splice site repressed by Prp18p The downstream AAG (AS 39 #2) is 7 nt downstream of the normal 39-SS, resulting in a frameshift-induced PTC. RT-PCR analysis of the wild-type and upf1D strains confirmed the RNA-Seq data by showing that these two alternative splice products are detected at extremely low levels, unless NMD is inhibited (Fig. 3; Fig. S4). In samples from the upf1D strain, the two alternatively spliced products accumulate to similar amounts, and both species are detected at lower levels than the normal spliced product (20% of all spliced products; Fig. S4), possibly because these two suboptimal AAG sites do not compete well with the consensus canonical UAG site. Strikingly, the usage of these alternative 39 splice sites was dramatically altered when Prp17p or Prp18p were inactivated. Inactivating Prp17p resulted in an increase in the use of the downstream alternative 39-SS (AS 39#2), while the upstream alternative 39-SS (AS 39#1) was no longer used (Fig. 3; Fig. S4), showing a role of Prp17p in enhancing the use of upstream, branchpoint proximal 39-SS. By contrast, Prp18p inactivation resulted in increased usage of the alternative 39-SS GCR1 showed the most complex splicing pattern of all transcripts analyzed. Gapped alignments identified an intronic GUAUGG alternative 59-SS as well as an upstream CAG alternative 39-SS (Fig. 2). In addition to these alternative splice sites identified by RNA-Seq, RT-PCR revealed the use of an additional GUAUGG alternative 59-SS staggered 5-nt upstream of the normal 59-SS and of a non-canonical AUG alternative 39-SS April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 6 Non-productive Alternative Splicing in Yeast Non-productive Alternative Splicing in Yeast This analysis showed that cells treated in heat shock conditions resulted in much weaker signal than in the samples obtained from cells grown at 25uC, consistent with a general down-regulation of ribosomal protein genes under stress [43,44,45,46,47]. Upon NMD inactivation, we observed a rescue of transcript levels, which mostly corresponded to unspliced RNAs and to some alternatively spliced transcripts (Fig. 5B). However, changing the alternative 59-SS to a consensus sequence in the context of NMD inactivation was sufficient to recover a large amount of spliced transcripts (Fig. 5B, lane 11, lower panel). To investigate if this effect was specific to heat shock or is also observed during other stresses, we analyzed the expression of wild- type and mutated forms of RPL22B during amino acid starvation (Fig. 5C). The results observed during amino acid starvation were similar to those described during heat shock, with a large increase in the level of spliced transcripts upon changing the alternative 59- splice site to the consensus sequence. We also observed an increase in the use of the alternative 59-SS under amino acid starvation (Fig. S13). Interestingly, shifting the Upf1p-inactivated strain with the alternative 59-splice site consensus sequence from SDC to amino acid starvation conditions resulted in only a minor increase We next investigated the effect of a 20 minute heat shock at 42uC on splicing patterns. Under these conditions and in the wild-type strain, RPL22B showed an increase in unspliced as well as a decrease in the relative amount of the normal spliced product (Fig. 4A lane 5 vs. 7). More importantly, the NMD defective strain upf1D showed an even larger increase in unspliced pre-mRNAs, as well a large accumulation of the alternatively spliced product that coincides with a decreased amount of canonical spliced product (Fig. 4A lane 6 vs. 8). In these conditions, the alternatively spliced product now corresponds to more than half of all spliced species (Fig. S10). Under heat shock, this alternatively spliced product is 4-fold more abundant in the upf1D strain than in the wild-type strain. These higher levels upon NMD inactivation show that the increased accumulation of these species under heat shock is not due to a decrease in NMD efficiency. Rather, this result shows that the use of the alternative splice site is being favored in heat shock conditions. Non-productive Alternative Splicing in Yeast By contrast, TFC3 and TAN1 exhibited an accumulation of unspliced species, but decreased levels of both the canonical and alternatively spliced species (Fig. S11), consistent with a general inhibition of pre-mRNA splicing under heat shock [48,49]. Thus the pattern of alternatively spliced species of TFC3 and TAN1 that are subject to NMD is very different from that of RPL22B, further proving that the accumulation of the alternatively spliced RPL22B transcript under heat shock conditions described above is not due to a general stabilization of spliced forms degraded by NMD. Like heat shock, rapamycin treatment was shown to result in an inhibition of ribosomal proteins mRNA splicing based on microarray experiments [43]. Within 20 minutes of rapamycin treatment, RPL22B indeed showed trends similar to those observed in heat shock, but to a lesser degree, with an increase of unspliced species and of alternatively spliced RPL22B species (Fig. 4A), but no effect on the alternatively spliced TAN1 and TFC3 transcripts (Fig. S11). Hyperosmotic shock (300 mM LiCl expo- sure for 10 min) only resulted in minimal effects; there were no changes observed for TFC3 and TAN1 targets under these stress conditions (Fig. S11), and RPL22B showed only a slight increase in unspliced but the levels of spliced transcripts remained similar. Thus, RPL22B exhibits regulated use of its alternative 59-splice site, mostly under amino acid starvation and heat shock conditions, while other transcripts such as TFC3 and TAN1 did not exhibit any change in their alternative splicing profiles. Because GCR1 exhibited a very complex splicing pattern, especially in the absence of Prp18p, and because heat shock conditions resulted in the most dramatic changes in splicing for RPL22B, we next investigated the effect of heat shock on GCR1 splicing in the wild-type, upf1D, prp18D and prp18Dupf1D mutants (Fig. 4B). Under heat-shock, we detected a general inhibition of splicing, consistent with the data described above. However, we also observed an increase of the abundance of the A* form relative to the normal spliced product S, indicative of a switch from the normal GUAUGG site to the GUAUGA site. The absence of Prp18p resulted in an increase of the use of the non-canonical AUG site (*D species) in heat shock conditions, and this product now constituted one third of all spliced species. Non-productive Alternative Splicing in Yeast Thus we conclude that GCR1, like RPL22B, exhibits a switch in splice site selection during heat shock, and that Prp18p limits splicing at this non- canonical AUG site under stress conditions. Non-productive Alternative Splicing in Yeast mutations of this alternative 59-SS. The suboptimal GUUUGU alternative 59-splice site was either deleted or mutated to the consensus GUAUGU sequence at the endogenous chromosomal locus (CS, consensus mutation and D, deletion, Fig. 5A). Changing the alternative 59-SS to the consensus GUAUGU sequence resulted in detectable amounts of alternatively spliced products at 25uC, even in a functional NMD background (Fig. 5B), suggesting that the suboptimal GUUUGU sequence contributes to the low usage of this alternative site in normal conditions. Inactivation of Upf1p in this context showed that 70% of all spliced species were now being produced by splicing from the alternative consensus site (Fig. 5B; lane 5; Fig. S12 for quantitation), and that splicing efficiency was improved, as shown by a decrease in unspliced species. By contrast, deleting the alternative splice site resulted in higher amount of unspliced transcripts, especially in the upf1D background. Thus, deleting the alternative 59-splice site of RPL22B is not sufficient to improve splicing at the normal splice site, possibly because of the suboptimal sequence of the normal RPL22B 59-splice site. In addition to RT-PCR, the same strains were analyzed by northern blot (Fig. 5B, bottom panel), which yielded results similar to those obtained by RT-PCR. These results show that increasing the strength of the alternative 59-SS of RPL22B is sufficient to enhance the overall splicing efficiency of this transcript, while deleting this site results in an overall increase of unspliced RNAs. Under heat shock and NMD inactivation, this effect was even more prominent, as mutation of the alternative splice site to the consensus resulted in the alternatively spliced product being the major spliced species (Fig. 5B, lane 11). Thus, under heat shock conditions, RPL22B transcripts bearing the consensus alternative splice site mutation are now spliced almost exclusively at this site. Analysis of the mutant with a deletion of the alternative 59-SS under heat shock conditions showed that the use of the normal 59- SS is not increased at elevated temperatures when the competing alternative 59-SS has been eliminated (Fig. 5A and 5B lane 12). This mutant shows a larger accumulation of unspliced RPL22B transcript, hinting that the normal process of spliceosome assembly is perturbed on this transcript during heat shock, possibly due to the suboptimal 59-SS. To obtain a more quantitative assessment of transcript levels, rather than just assessing the ratio between the different spliced forms, we analyzed the same samples by northern blot. Alternatively spliced species of RPL22B and GCR1 increase during stress conditions Analysis of other mutants did not reveal any major influence on the pattern of 59- or 39-SS selection. Like SRC1, GCR1 exhibits two staggered 59 splice sites. However, unlike for SRC1, Hub1p has no influence on their selection (Fig. 3). A final set of NMD targets are produced by the use of the two most upstream 59-SS with a highly unusual alternative AUG 39-SS in the intronic sequence (labeled *D and *E in Figs. 2 &3). Interestingly, these products were only detected in the absence of Prp18p, suggesting that this factor is essential in preventing the use of this non-canonical 39-SS. The use of this highly unusual AUG 39 splice site was unambiguously confirmed through sequencing and RT-PCR analysis of RNAs derived from prp18Dupf1D samples (Fig. S5). The ATPase Prp22p has been implicated in the fidelity of 39-SS selection [41]. Because Prp18p functions upstream from April 2014 | Volume 10 | Issue 4 | e1004249 7 PLOS Genetics | www.plosgenetics.org Non-productive Alternative Splicing in Yeast The alternative, suboptimal 59-splice site of RPL22B contributes to the global down-regulation of RPL22B in stress conditions The fact that the usage of the alternative splice site of RPL22B is reduced in this strain while transcript levels are higher overall argues against the hypothesis that alternative splice site usage is the result of splicing errors occurring at low frequencies, as if this were the case, one would expect higher levels of alternatively spliced RPL22B transcripts upon its overexpression in the strain in which the natural RPL22B promoter was swapped for the GAL promoter. Under heat shock conditions, the use of the alternative splice site was reduced 8.1 fold in the upf1D strain expressing RPL22B under the control of the GAL promoter compared to the upf1D strain expressing RPL22B from its natural promoter and grown in galactose medium (Fig. 6, lanes 10 and 12). Thus, alternative splicing regulation of RPL22B upon heat shock is tightly linked to the identity of the RPL22B promoter, as switching the identity of the promoter is sufficient to favor the use of the normal 59-splice site. The mechanism by which the identity of the strain in normal conditions (75%; Fig. S13). In conclusion, these results show that low splicing efficiency due to the suboptimal normal and alternative 59 splice sites of RPL22B, combined with NMD degradation of the unspliced and alternatively spliced forms contribute to the general decrease in RPL22B levels as a means to rapidly halt production of this ribosomal protein under various stress conditions. The alternative, suboptimal 59-splice site of RPL22B contributes to the global down-regulation of RPL22B in stress conditions To further analyze the importance of the alternative 59-splice site of RPL22B on its splicing patterns and expression during normal and stress conditions, we investigated the effect of April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 8 Non-productive Alternative Splicing in Yeast Figure 4. RT-PCR analysis of alternatively spliced products under stress conditions. A. Analysis of RPL22B in various stress conditions. Shown are the RT-PCR products obtained from the wild-type or upf1D mutant strain after growth in the following conditions: SDC, synthetic define complete medium at 30uC; -AA, 10 minutes in SDC medium at 30uC lacking amino acid (-AA); 25uC, log phase at 25uC in YPD; H.S, 20 minutes at 42uC in YPD; YPD: log phase at 30uC in YPD; LiCl, incubation with 300 mM LiCl in YPD at 30uC for 10 minutes; RAP control, see Materials and Methods; RAP, treatment with Rapamycin for 20 minutes. B. RT-PCR analysis of GCR1 alternative splicing in heat-shock conditions. Labeling of the different species is similar to that of Figures 2 and 3. doi:10.1371/journal.pgen.1004249.g004 Figure 4. RT-PCR analysis of alternatively spliced products under stress conditions. A. Analysis of RPL22B in various stress conditions. Shown are the RT-PCR products obtained from the wild-type or upf1D mutant strain after growth in the following conditions: SDC, synthetic define complete medium at 30uC; -AA, 10 minutes in SDC medium at 30uC lacking amino acid (-AA); 25uC, log phase at 25uC in YPD; H.S, 20 minutes at 42uC in YPD; YPD: log phase at 30uC in YPD; LiCl, incubation with 300 mM LiCl in YPD at 30uC for 10 minutes; RAP control, see Materials and Methods; RAP, treatment with Rapamycin for 20 minutes. B. RT-PCR analysis of GCR1 alternative splicing in heat-shock conditions. Labeling of the different species is similar to that of Figures 2 and 3. doi:10.1371/journal.pgen.1004249.g004 replacement of the normal RPL22B promoter by the GAL promoter resulted in an increase in overall RPL22B transcript levels, but also in a decrease in the use of the alternative 59-SS (Fig. 6). Usage of the alternative 59-splice site of RPL22B is influenced by promoter identity Ribosomal protein genes are known to be transcriptionally regulated in stress conditions. To investigate the use of RPL22B 59- SS selection independently from transcriptional inhibition under heat shock, we replaced the natural RPL22B promoter with a galactose-inducible promoter. The wild-type and upf1D strains containing the natural RPL22B promoter showed no detectable difference in RPL22B splicing patterns or expression when grown in galactose containing medium (YPGal) compared to glucose- containing medium (YPD) at 25uC (Fig. 6 lanes 1–4), either by RT-PCR (top panel) or northern blot (bottom panel). Strikingly, April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 9 Non-productive Alternative Splicing in Yeast Figure 5. Effects of mutations of the RPL22B alternative 59 splice site on RPL22B splicing and expression in normal and stress conditions. A. Organization of the RPL22B precursor, with the normal and alternative 59-splice sites. Shown are the mutations to the consensus sequence (CS) GUAUGU or the deletion that entirely removes the GUUUGU sequence. B. Analysis of the effect of these mutations on RPL22B splicing and expression at normal temperatures (25uC) or after a 20 min heat shock at 42uC. N, natural 59-splice site (GUUUGU); CS, consensus sequence (GUAUGU); D = deletion of the alternative 59-splice site. Top panel: RT-PCR analysis. Bottom panel: northern blot analysis. US, *AS-59, and S indicate the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. For the northern blot, SCR1 was used as a loading control. C. Analysis of the effect of the RPL22B alternative splice site consensus mutation on RPL22B expression during amino acid starvation. Shown is a northern blot of RNA samples extracted from the indicated strains grown at 30uC in normal synthetic define complete (SDC) medium with amino acid (+) or in SDC medium lacking amino acid (2) for 10 minutes. Strains contained either the natural GUUUGU sequence at the alternative 59-splice site of RPL22B, or the consensus GUaUGU sequence. The nucleotide mutated is highlighted in lower case. Labeling of the different species is similar to that of panel B. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g005 Non productive Alternative Splicing in Yeast Figure 5. Effects of mutations of the RPL22B alternative 59 splice site on RPL22B splicing and expression in normal and stress conditions. A. Organization of the RPL22B precursor, with the normal and alternative 59-splice sites. Usage of the alternative 59-splice site of RPL22B is influenced by promoter identity Shown are the mutations to the consensus sequence (CS) GUAUGU or the deletion that entirely removes the GUUUGU sequence. B. Analysis of the effect of these mutations on RPL22B splicing and expression at normal temperatures (25uC) or after a 20 min heat shock at 42uC. N, natural 59-splice site (GUUUGU); CS, consensus sequence (GUAUGU); D = deletion of the alternative 59-splice site. Top panel: RT-PCR analysis. Bottom panel: northern blot analysis. US, *AS-59, and S indicate the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. For the northern blot, SCR1 was used as a loading control. C. Analysis of the effect of the RPL22B alternative splice site consensus mutation on RPL22B expression during amino acid starvation. Shown is a northern blot of RNA samples extracted from the indicated strains grown at 30uC in normal synthetic define complete (SDC) medium with amino acid (+) or in SDC medium lacking amino acid (2) for 10 minutes. Strains contained either the natural GUUUGU sequence at the alternative 59-splice site of RPL22B, or the consensus GUaUGU sequence. The nucleotide mutated is highlighted in lower case. Labeling of the different species is similar to that of panel B. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g005 Discussion promoter influences alternative splice site selection is unclear, but could be linked to the influence of the promoter on the speed of transcription. Nevertheless, we can conclude from these results that transcriptional down-regulation and the increased use of the alternative 59-SS provide synergistic mechanisms to limit the expression of RPL22B during stress, consistent with the global down-regulation of ribosome biogenesis during stress conditions. promoter influences alternative splice site selection is unclear, but could be linked to the influence of the promoter on the speed of transcription. Nevertheless, we can conclude from these results that transcriptional down-regulation and the increased use of the alternative 59-SS provide synergistic mechanisms to limit the expression of RPL22B during stress, consistent with the global down-regulation of ribosome biogenesis during stress conditions. Contribution of splicing factors to alternative splice site selection and splice site fidelity Contribution of splicing factors to alternative splice site selection and splice site fidelity y The analysis of double mutants in which splicing factor mutations were combined with NMD inactivation revealed some important and unexpected functions for these factors on alterna- tive splice site selection. We found that the Nam8p and Mud1p components are important for the selection of some, but not all of the alternative 59-splice sites described here. In the case of RPL22B, this requirement was likely due to the fact that the alternative 59-SS possesses a suboptimal splicing sequence, and therefore exhibits a weaker affinity for U1 binding, and a stronger requirement for Mud1p and Nam8p that impact the efficiency of U1 snRNP assembly on the alternative splice site. Strikingly, we identified a new role for Prp17p in favoring the use of upstream, branchpoint-proximal 39-SS. In all cases that we have analyzed, Prp17p inactivation resulted in an increase in the use of the downstream 39-SS. The mechanistic basis for this novel function that we describe here for Prp17p in promoting branchpoint proximal 39-SS is not fully understood. Because 39-SS close to the branchpoint are often the first ones that are being used, this novel function for Prp17p could be linked to promoting the ability of the spliceosome to scan and recognize 39-SS close to the branchpoint, or to unwind secondary structures that mask branchpoint- proximal 39-SS. The absence of Prp17p would result in a higher rate of misrecognition of 39-SS and in the use of more distal 39-SS. In addition, we found that the absence of Prp18p resulted in the selection of a non-canonical AUG 39-SS in GCR1, and that this atypical 39-SS was utilized to a greater extent during heat-shock, revealing a unique function for Prp18p in suppressing usage of a Figure 6. Replacement of RPL22B promoter by the GAL promoter results in a decrease in alternative 59-splice site usage. Shown are the products generated when growing the indicated strains (wild-type or upf1D that contained the natural RPL22B promoter or the GAL promoter upstream RPL22B) in glucose (YPD) or galactose (YPGal)-containing media. Top panel, RT-PCR analysis. US, *AS 59, and S indicate the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. Bottom Panel, Northern blot analysis. The labeling of the different species is similar to that of the top panel. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g006 Figure 6. A significant fraction of splicing events in S. cerevisiae generates non-functional RNA or protein products In this study we show that the ensemble of transcripts generated by splicing from the S. cerevisiae genome is highly complex. Most of the splicing events that we have characterized in this study are April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 10 Non-productive Alternative Splicing in Yeast non-productive, either because they result in transcripts that are targeted by NMD, or because the protein products generated from these transcripts are predicted to be non-functional (e.g. GPI15). The large number of additional splice sites identified, and their relaxed conservation (Fig. 1D) imply that the rules governing splice selection are intrinsically more flexible than previously thought. This is further illustrated by the finding that a non-canonical AUG sequence in GCR1 can be used as a 39-SS in the absence of Prp18p (Fig. 3). In some cases, non-productive alternatively spliced transcripts accumulate only at low levels (e.g. GCR1, GPI15, Fig. 3). However, for other genes such as TFC3, the alternatively spliced non-productive transcripts represent a significant fraction (close to 30%) of all RNAs generated from this locus. Thus, non-productive splicing can significantly limit the expression of these genes. This was further demonstrated by mutagenesis of the non-productive splice site of RPL22B, as changing this site to a consensus sequence was sufficient to increase the splicing efficiency and the expression of this gene (Fig. 5B). Thus, the presence of alternative and sometimes sub- optimal splice sites that compete with the normal splice site contributes to an overall decrease in the amount of productively- spliced transcripts. Because the overlap in the alternative splicing events detected in all three NMD-deficient strains was limited (Fig. 1C), and because we detected by RT-PCR some alternative splicing events that escaped detection by RNA-Seq (e.g. GCR1), it is likely that we have not exhaustively identified the ensemble of splice sites that can be used by S. cerevisiae, and that additional splice sites will be identified by deeper sequencing or systematic RT-PCR analysis. Spliceosome errors or bona-fide regulations? The widespread occurrence of non-productive splice site usage described in this study begs the question of whether the use of these splice sites is the result of mistakes by the spliceosome, which occur at low frequency (as one might suggest based on their weaker consensus sequences) or whether they correspond to sites that have been selected throughout evolution for regulatory purposes. The sequence of some of these intronic, non-productive splice sites is conserved across closely related yeast species (Fig. S8 and S9), which, given the low conservation of intronic sequences in general, argues that this might reflect some degree of functional relevance. In addition, there is no obvious correlation between transcript levels and the occurrence of alternative splicing events (Fig. 1D), which argues against the suggestion that most of the alternative splicing events that we have mapped arise from low fidelity splicing events or errors that occur randomly, and which would be expected to be more frequently detected in highly abundant transcripts. Also, replacement of the RPL22B gene promoter results in higher transcript levels but reduces the usage of the alternative 59-splice site of RPL22B (Fig. 6), providing another independent argument to suggest that the level of usage of alternative splice sites is not solely a reflection of overall transcript abundance. Finally, we show that the use of some of these alternative splice sites can be up-regulated during stress conditions (RPL22B, GCR1), and that this increased use participates in the down-regulation of RPL22B in stress conditions. Thus, this phylogenetically conserved, alternative, non-productive 59-SS of RPL22B is functionally important because it contributes to the down-regulation of RPL22B during stress. This is shown by the fact that changing this sequence to a consensus sequence results in a significant increase in transcript levels upon NMD inactivation during stress (Fig. 5). The transcriptional down-regulation of ribosomal proteins during stress has been documented previously [45]. We show here that the promoter of the RPL22B gene is essential not only because it drives transcriptional repression during stress, but also because it controls the switch in 59-SS selection that contributes to the overall repression of RPL22B during heat-shock. Thus, a combination of transcriptional and post-transcriptional regulations, through splicing inhibition [43,44], degradation of unspliced RNAs by NMD [12,47] and use of non-productive splice sites (this study) contributes to the repression of ribosomal protein production during stress. Spliceosome errors or bona-fide regulations? While several non-RPG transcripts analyzed in these stress conditions did not shown any changes, GCR1 did exhibit a change in the use of alternative splice sites during stress (Fig. 4B). This result raises the possibility that other intron-containing genes may be regulated similarly by alternative splicing as a function of different environmental growth conditions. Overall our study has revealed that the pattern of splicing events in the model eukaryote S. cerevisiae is highly complex, but masked by NMD-mediated degradation. Given the recent report that another single cell eukaryote, S.pombe shows alternative splicing patterns conserved in higher eukaryotes [50], these observations suggest that alternative splicing provides an important contribution to genetic regulations and adaptations to environmental changes in unicellular eukary- H i h f S i i h f l i li Contribution of splicing factors to alternative splice site selection and splice site fidelity Replacement of RPL22B promoter by the GAL promoter results in a decrease in alternative 59-splice site usage. Shown are the products generated when growing the indicated strains (wild-type or upf1D that contained the natural RPL22B promoter or the GAL promoter upstream RPL22B) in glucose (YPD) or galactose (YPGal)-containing media. Top panel, RT-PCR analysis. US, *AS 59, and S indicate the location of the products corresponding to the unspliced, alternatively spliced and normal spliced products, respectively. Bottom Panel, Northern blot analysis. The labeling of the different species is similar to that of the top panel. SCR1 was used as a loading control. doi:10.1371/journal.pgen.1004249.g006 April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 11 Non-productive Alternative Splicing in Yeast sites have evolved towards fine tuning transcript levels, rather than generating proteome diversity as shown in higher eukaryotes. non-canonical 39-SS. This function for Prp18p is independent from Prp22p’s function in proofreading 39-SS [41], but might complement its role to ensure the overall proper fidelity of 39-SS selection. While we have demonstrated this function for GCR1 only, a full genomic analysis of 39-SS usage in the absence of Prp18p might reveal further examples of non-canonical 39-SS being used. Yeast culture and RNA analysis y Yeast strains were grown at 25uC in YPD medium, unless indicated otherwise in the figures. For heat shock treatment, strains were pre-grown in YPD at 25uC, spun down in 50 mL Falcon tubes, resuspended in pre-warmed YPD medium and heat shocked for 20 min before harvesting. For LiCl treatment, yeast strains were grown to mid-log phase in YPD rich media at 30uC, harvested by centrifugation in 50 mL Falcon tubes, washed once with pre-warmed 50 mL of YPD+300 mM LiCl before being resuspended in pre-warmed YPD with 300 mM LiCl for 10 minutes. For Rapamycin treatment, cells were grown to mid- log phase in rich media (at 30uC), and rapamycin from a stock solution of 1 mg/mL in 90% ethanol, 10% Tween-20 was added to a final concentration of 200 ng/mL and cells were incubated for 20 minutes. The same volume of 90% ethanol, 10% Tween-20 solution used for the rapamycin treatment was added to the negative control. Sample preparation and RNA sequencing was performed by Illumina. RT-PCR analysis and northern blot was performed as described [21]. Mapping reads High throughput sequencing data have been deposited in the GEO database (accession GSE55213). All sequence files were aligned against the 2008 SGD assembly of the Saccharomyces cerevisiae genome. The novoalign software package (www. novocraft.com) and the BLAT alignment tool [22] were used to align 75 base pair reads in two steps. In the first step, sequences were aligned with novoalign allowing for up to four mismatches and no gaps. In the second step, sequences that failed to align in the first step were aligned with BLAT allowing three mismatches and gaps up to 20000 nucleotides in length. A sequence was kept for further analysis if it mapped with equal score to at most two genomic locations and did not contain a gap smaller than ten nucleotides. Intronic sequences counts Intronic sequence expression representative of unspliced RNAs was quantified for each ICG by summing reads that aligned to introns and exon-intron boundaries. Values between samples were normalized by total mapped reads to account for lane effects. p- values were computed by modeling each ICG wild-type count as a poisson random variable and calculating the probability of observing each mutant count if it were drawn from the same distribution. Supporting Information Figure S1 Quantification of the SRC1-L and SRC1-S isoforms in wild-type, upf1D and splicing mutants. Shown is the percentage of the SRC1-L and SRC1-S transcripts in various strains. Values shown are the average and standard deviations obtained from RT- PCR experiments of three independent cultures for each strain. (TIF) Figure S2 Quantification of the usage of the normal and alternative 59-splice sites of RPL22B in wild-type, upf1D and splicing mutants. Shown is the percentage of transcripts spliced at the normal 59-splice site (red) and at the alternative 59-splice site (blue). Values shown are the average and standard deviations obtained from RT-PCR experiments of three independent cultures for each strain. (TIF) Figure S9 Conservation of the intronic alternative 39-SS in TAN1. A. Screen capture of the web browser showing the RNA- Seq reads mapped for TAN1 that use the intronic alternative 39-SS in black, and the sequence conservation in closely related yeast species as blue peaks. B. Zoomed in view of the conservation of the sequence of the alternative intronic AAG 39-SS. (PDF) Figure S3 Quantification of the usage of the normal and alternative 39-splice sites of TFC3 in wild-type, upf1D and splicing mutants. Shown is the percentage of transcripts spliced at the normal 39-splice site (blue) and at the alternative 59-splice site (red). Values shown are the average and standard deviations obtained from RT-PCR experiments of three independent cultures for each strain. (TIF) Figure S10 Quantification of the usage of the alternative 59- splice sites of RPL22B in various normal media (YPD, SDC) or in stress conditions (Heat shock, amino acid starvation). Values shown are the average and standard deviations obtained from RT- PCR experiments of three independent cultures for each strain. (TIF) Figure S11 RT-PCR analysis of the spliced products of TAN1 and TFC3 under stress conditions. Shown are the products for the unspliced (US), normal spliced product (S), and the alternatively spliced species (AS) described in Figure 2. (TIF) Figure S4 Quantification of the usage of the two alternative 39- splice sites of TAN1 in wild-type, upf1D and splicing mutants. Shown is the percentage of transcripts spliced at the alternative 39- splice site #1 (blue) or #2 (red) compared to all the spliced transcripts. Values shown are the average and standard deviations obtained from RT-PCR experiments of three independent cultures for each strain. Splice site consensus sequence Figure S7 RT-PCR analysis of GCR1 splicing in the prp18 and prp22-1 mutant strains. The identity of the different spliced products is labeled according to Figure 2. (TIF) Consensus sequences for 59 and 39 ends of both canonical splice sites and alternative splice sites were represented as sequence logos. Sequence logos were constructed using the MATLAB (MathWorks) seqlogo function. Figure S8 Conservation of the intronic alternative 59-SS in RPL22B. A. Screen capture of the web browser showing the RNA- Seq reads mapped for RPL22B that use the alternative 59-SS in black, and the sequence conservation in closely related yeast species as blue peaks. The peak showing conservation of the intronic alternative 59splice site is shown on the right, since the gene is encoded on the Crick strand. B. Zoomed in view of the conservation of the sequence of the alternative 59-SS (ACAAC sequence because of the Crick Strand). (PDF) Quantification of alternative splicing events Alternative splicing events were defined as splicing events that are within ICGs and are supported by sequencing but that are not annotated in the Saccharomyces Genome Database (SGD). Counts of total alternative splicing events and PTC-generating alternative splicing events were quantified by summing all unique alternative splicing events in each sample. To determine if an alternative splicing event is PTC-generating we constructed the splice product’s sequence using the novel splicing event in the otherwise canonical transcript sequence. Counts between samples were normalized by sequencing depth. p-values were calculated by modeling the wild-type count as a poisson random variable and calculating the probability of observing each mutant’s count for both total alternative splicing events and PTC-generating April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 12 Non-productive Alternative Splicing in Yeast alternative splicing events. Venn diagrams of agreement between samples were generated using BioVenn [51]. standard deviations obtained from RT-PCR experiments of three independent cultures for each strain. (TIF) standard deviations obtained from RT-PCR experiments of three independent cultures for each strain. (TIF) Supporting Information (TIF) Figure S12 Quantitation of the use of the alternative 59-splice site of RPL22B under normal growth conditions (25uC) and after a 20 min heat shock at 42uC. Plotted are the amount of transcript spliced at the alternative splice site divided by the values obtained for all spliced species for the indicated strains. Shown are the average of 4 to 5 independent experiments with the standard deviations. Figure S5 Validation of the use of the AUG alternative 39 splice site of GCR1 by RT-PCR. Sequencing of the cloned *D and *E cDNAs determined the location of the splice junction, while sequencing of unspliced cDNAs was used to confirm that this unusual alternative 39-SS was indeed AUG, and not a SNP or other mutation of the GCR1 gene that would have converted it into an AAG. RT-PCR confirmation of the use of this AUG 39-SS was performed using reverse primers spanning the splice junction to specifically amplify distinct splicing events; either associated with *D, *E, or unspliced. The use of the AUG 39 SS was also confirmed using an intronic reverse primer just downstream of the AUG sequence and detected *D, *E, and unspliced products, as predicted (Fig. S1). A. RT-PCR strategy. All PCR include the same forward primer For, and various reverse primers that hybridize to the indicated regions of GCR1. B. RT-PCR data. Shown are the PCR products obtained from the different reverse primers shown in A. (TIF) References 17. Mishra SK, Ammon T, Popowicz GM, Krajewski M, Nagel RJ, et al. (2011) Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing. Nature 474: 173–178. 1. Isken O, Maquat LE (2007) Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev 21: 1833–1856. 2. Kervestin S, Jacobson A (2012) NMD: a multifaceted response to premature translational termination. Nat Rev Mol Cell Biol 13: 700–712. 18. Meyer M, Plass M, Perez-Valle J, Eyras E, Vilardell J (2011) Deciphering 39ss selection in the yeast genome reveals an RNA thermosensor that mediates alternative splicing. Mol Cell 43: 1033–1039. translational termination. Nat Rev Mol Cell Biol 13: 700–712. 3. Green RE, Lewis BP, Hillman RT, Blanchette M, Lareau LF, et al. (2003) Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced transcripts of human normal and disease genes. Bioinformatics 19 Suppl 1: i118– 121. 19. Plass M, Codony-Servat C, Ferreira PG, Vilardell J, Eyras E (2012) RNA secondary structure mediates alternative 39ss selection in Saccharomyces cerevisiae. RNA 18: 1103–1115. 4. Lareau LF, Inada M, Green RE, Wengrod JC, Brenner SE (2007) Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature 446: 926–929. 20. Marshall AN, Montealegre MC, Jimenez-Lopez C, Lorenz MC, van Hoof A (2013) Alternative splicing and subfunctionalization generates functional diversity in fungal proteomes. PLoS Genet 9: e1003376. 5. Ni JZ, Grate L, Donohue JP, Preston C, Nobida N, et al. (2007) Ultraconserved elements are associated with homeostatic control of splicing regulators by alternative splicing and nonsense-mediated decay. Genes Dev 21: 708–718. 21. Kawashima T, Pellegrini M, Chanfreau GF (2009) Nonsense-mediated mRNA decay mutes the splicing defects of spliceosome component mutations. RNA 15: 2236–2247. 6. Mendell JT, Sharifi NA, Meyers JL, Martinez-Murillo F, Dietz HC (2004) Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat Genet 36: 1073–1078. 22. Kent WJ (2002) BLAT–the BLAST-like alignment tool. Genome Res 12: 656– 664. 23. Liao XC, Tang J, Rosbash M (1993) An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing. Genes Dev 7: 419–428. 7. Chan WK, Huang L, Gudikote JP, Chang YF, Imam JS, et al. (2007) An alternative branch of the nonsense-mediated decay pathway. EMBO J 26: 1820– 1830. 24. Neubauer G, Gottschalk A, Fabrizio P, Seraphin B, Luhrmann R, et al. g (XLSX) Table S5 List of intron-containing genes for which alternative splicing events were detected. Shown is the list of intron- containing genes for which alternative splicing junctions were detected in at least one of the strains sequenced. Shown is the number of splice junction sequences counts for the normal (blue) and alternative (green) splicing events. For the alternative splicing events, the 59 and/or 39 splice sites which differ from the normal splice sites are marked by an asterisk. The position of these splice sites on each chromosome is also indicated. The adjusted number of reads obtained from each strain (RPKM) and the sequence of the normal and alternative 59- and 39-splice sites is shown for each of these genes. Acknowledgments We thank J. Staley for insights, discussions and comments on the manuscript and K. Roy for critical reading of the manuscript. References (1997) Identification of the proteins of the yeast U1 small nuclear ribonucleoprotein complex by mass spectrometry. Proc Natl Acad Sci U S A 94: 385–390. 8. Weischenfeldt J, Damgaard I, Bryder D, Theilgaard-Monch K, Thoren LA, et al. (2008) NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements. Genes Dev 22: 1381–1396. 25. Puig O, Gottschalk A, Fabrizio P, Seraphin B (1999) Interaction of the U1 snRNP with nonconserved intronic sequences affects 59 splice site selection. Genes Dev 13: 569–580. 9. He F, Peltz SW, Donahue JL, Rosbash M, Jacobson A (1993) Stabilization and ribosome association of unspliced pre-mRNAs in a yeast upf1- mutant. Proc Natl Acad Sci U S A 90: 7034–7038. 26. Gottschalk A, Tang J, Puig O, Salgado J, Neubauer G, et al. (1998) A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins. RNA 4: 374–393. 10. Mitrovich QM, Anderson P (2000) Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans. Genes Dev 14: 2173–2184. 27. Umen JG, Guthrie C (1995) Prp16p, Slu7p, and Prp8p interact with the 39 splice site in two distinct stages during the second catalytic step of pre-mRNA splicing. RNA 1: 584–597. 11. Jaillon O, Bouhouche K, Gout JF, Aury JM, Noel B, et al. (2008) Translational control of intron splicing in eukaryotes. Nature 451: 359–362. 28. Aronova A, Bacı´kova´ D, Crotti LB, Horowitz DS, Schwer B (2007) Functional interactions between Prp8, Prp18, Slu7, and U5 snRNA during the second step of pre-mRNA splicing. RNA 13: 1437–1444. 12. Sayani S, Janis M, Lee CY, Toesca I, Chanfreau GF (2008) Widespread impact of nonsense-mediated mRNA decay on the yeast intronome. Mol Cell 31: 360– 370. 13. Sayani S, Chanfreau GF (2012) Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae. RNA 18: 1563–1572. 29. Villa T, Guthrie C (2005) The Isy1p component of the NineTeen complex interacts with the ATPase Prp16p to regulate the fidelity of pre-mRNA splicing. Genes Dev 19: 1894–1904. 14. Vijayraghavan U, Company M, Abelson J(1989) Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev 3: 1206–1216. 30. Saha D, Banerjee S, Bashir S, Vijayraghavan U (2012) Context dependent splicing functions of Bud31/Ycr063w define its role in budding and cell cycle progression. Biochem Biophys Res Commun 424: 579–585. 15. Author Contributions Conceived and designed the experiments: TK SD JG MP GFC. Performed the experiments: TK JG. Analyzed the data: TK SD JG MP GFC. Wrote the paper: TK GFC. g (XLSX) (TIF) Figure S13 Quantitation of the use of the normal and alternative 59-splice site of RPL22B under normal growth conditions in minimal medium (SDC) and after amino acid starvation (-AA) for the strains expressing the natural (N) GUUUGU sequence at the alternative 59 splice site of RPL22B, or the consensus (CS) GUAUGU sequence in the context of wild- type UPF1 (WT) or when UPF1 has been deleted (D). Plotted are the amount of transcript spliced at the normal and alternative splice sites divided by the values obtained for all spliced species. Shown are the average of 3 independent experiments with the standard deviations. Table S1 Statistics of RNA-Seq analysis sequence alignments. (XLSX) Figure S6 Quantification of the abundance of the major alternatively spliced forms of GCR1 in wild-type, upf1D and splicing mutants. Shown is the percentage of the *D (blue), *A (red) or *C (green) spliced forms. Values shown are the average and Figure S6 Quantification of the abundance of the major alternatively spliced forms of GCR1 in wild-type, upf1D and splicing mutants. Shown is the percentage of the *D (blue), *A (red) or *C (green) spliced forms. Values shown are the average and Table S2 Number of alternative splicing events detected in wild- type and NMD-deficient strains. (XLSX) Table S2 Number of alternative splicing events detected in wild- type and NMD-deficient strains. (XLSX) April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 13 Non-productive Alternative Splicing in Yeast Table S3 Mapping of RNA-Seq reads in wild-type and NMD- deficient strains in various genomic elements and in intron- containing genes. (XLSX) Table S6 List of alternative proteins potentially generated by alternative splicing in wild-type or NMD mutants. For each open reading frame, a portion of the normal protein sequence is shown on the first line, and the sequence that differs upon the alternative splicing event is shown below. Amino acids maintained between the two forms are indicated in red. Amino acids that differ between the two forms are highlighted in bold and black. In the case of splicing events inducing a deletion, a delta sign has been added with a number corresponding to the number of amino acid deleted. The numbers of the first and last amino acids shown is indicated before and after each protein sequence, respectively. The numbers in brackets that follow each protein sequence correspond to the number of reads for the splice junctions in the wild-type strain, upf1D, upf2D and upf3D mutants. (DOCX) Table S4 List of intron-containing genes for which no alternative splicing events were detected. Shown is the list of intron-containing genes for which no alternative splicing junctions were detected in any of the strains sequenced. The adjusted number of reads obtained from each strain (RPKM) and the sequence of the 59- and 39-splice sites is shown for each of these genes. Non-productive Alternative Splicing in Yeast 33. Crotti LB, Horowitz DS (2009) Exon sequences at the splice junctions affect splicing fidelity and alternative splicing. Proc Natl Acad Sci U S A 106: 18954– 18959. 43. Bergkessel M, Whitworth GB, Guthrie C (2011) Diverse environmental stresses elicit distinct responses at the level of pre-mRNA processing in yeast. RNA 17: 1461–1478. 34. Zhou Y, Chen C, Johansson MJ (2013) The pre-mRNA retention and splicing complex controls tRNA maturation by promoting TAN1 expression. Nucleic Acids Res 41: 5669–5678. 44. Pleiss JA, Whitworth GB, Bergkessel M, Guthrie C (2007) Rapid, transcript- specific changes in splicing in response to environmental stress. Mol Cell 27: 928–937. 35. Yan BC, Westfall BA, Orlean P (2001) Ynl038wp (Gpi15p) is the Saccharomyces cerevisiae homologue of human Pig-Hp and participates in the first step in glycosylphosphatidylinositol assembly. Yeast 18: 1383–1389. 45. Li B, Nierras CR, Warner JR (1999) Transcriptional elements involved in the repression of ribosomal protein synthesis. Mol Cell Biol 19: 5393–5404. 46. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, et al. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11: 4241–4257. g y y p p y y 36. Muhlrad D, Parker R (1999) Aberrant mRNAs with extended 39 UTRs are substrates for rapid degradation by mRNA surveillance. RNA 5: 1299–1307. 37. Amrani N, Ganesan R, Kervestin S, Mangus DA, Ghosh S, et al. (2004) A faux 39-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay. Nature 432: 112–118. 47. Garre E, Romero-Santacreu L, Barneo-Munoz M, Miguel A, Perez-Ortin JE, et al. (2013) Nonsense-mediated mRNA decay controls the changes in yeast ribosomal protein pre-mRNAs levels upon osmotic stress. PLoS One 8: e61240. 38. Uemura H, Jigami Y (1995) Mutations in GCR1, a transcriptional activator of Saccharomyces cerevisiae glycolytic genes, function as suppressors of gcr2 mutations. Genetics 139: 511–521. 48. Yost HJ, Lindquist S (1991) Heat shock proteins affect RNA processing during the heat shock response of Saccharomyces cerevisiae. Mol Cell Biol 11: 1062– 1068. 39. Holland MJ, Yokoi T, Holland JP, Myambo K, Innis MA (1987) The GCR1 gene encodes a positive transcriptional regulator of the enolase and glyceraldehyde-3-phosphate dehydrogenase gene families in Saccharomyces cerevisiae. Mol Cell Biol 7: 813–820. 49. Vogel JL, Parsell DA, Lindquist S (1995) Heat-shock proteins Hsp104 and Hsp70 reactivate mRNA splicing after heat inactivation. Curr Biol 5: 306–317. 50. References Wahl MC, Will CL, Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136: 701–718. 31. Rodriguez-Navarro S, Igual JC, Perez-Ortin JE (2002) SRC1: an intron- containing yeast gene involved in sister chromatid segregation. Yeast 19: 43– 54. 16. Grund SE, Fischer T, Cabal GG, Antunez O, Perez-Ortin JE, et al. (2008) The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression. J Cell Biol 182: 897–910. 32. Chanfreau GF (2010) A dual role for RNA splicing signals. EMBO Rep 11: 720– 721. April 2014 | Volume 10 | Issue 4 | e1004249 April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org April 2014 | Volume 10 | Issue 4 | e1004249 14 Non-productive Alternative Splicing in Yeast Awan AR, Manfredo A, Pleiss JA (2013) Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans. Proc Natl Acad Sci U S A 110: 12762–12767 40. Clifton D, Fraenkel DG (1981) The gcr (glycolysis regulation) mutation of Saccharomyces cerevisiae. J Biol Chem 256: 13074–13078. with humans. Proc Natl Acad Sci U S A 110: 12762–12767 51. Hulsen T, de Vlieg J, Alkema W (2008) BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genomics 9: 488. y J 41. Mayas RM, Maita H, Staley JP (2006) Exon ligation is proofread by the DExD/ H-box ATPase Prp22p. Nat Struct Mol Biol 13: 482–490. 41. Mayas RM, Maita H, Staley JP (2006) Exon ligation is proofr H-box ATPase Prp22p. Nat Struct Mol Biol 13: 482–490. 42. James SA, Turner W, Schwer B (2002) How Slu7 and Prp18 cooperate in the second step of yeast pre-mRNA splicing. RNA 8: 1068–1077. April 2014 | Volume 10 | Issue 4 | e1004249 PLOS Genetics | www.plosgenetics.org 15
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Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis
Endocrinology
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Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Andrew M. N. Walker,1,* Nele Warmke,1,* Ben Mercer,1,* Nicole T. Watt,1 Romana Mughal,1 Jessica Smith,1 Stacey Galloway,1Natalie J. Haywood,1 Taha  Soomro,1,2 Kathryn  J. Griffin,1 Stephen  B. Wheatcroft,1 Nadira Y. Yuldasheva,1 David J. Beech,1 Peter Carmeliet,3 Mark T. Kearney,1 and Richard M. Cubbon1 Andrew M. N. Walker,1,* Nele Warmke,1,* Ben Mercer,1,* Nicole T. Watt,1 Romana Mughal,1 Jessica Smith,1 Stacey Galloway,1Natalie J. Haywood,1 Taha  Soomro,1,2 Kathryn  J. Griffin,1 Stephen  B. Wheatcroft,1 Nadira Y. Yuldasheva,1 David J. Beech,1 Peter Carmeliet,3 Mark T. Kearney,1 and Richard M. Cubbon1 1Leeds Institute of Cardiovascular and Metabolic Medicine, The University of Leeds, Leeds LS2 9JT, UK; 2Imperial College Ophthalmology Research Group, Western Eye Hospital, London NW1 5QH, UK; and 3Laboratory of Angiogenesis and Vascular Metabolism, Center for Cancer Biology, Vlaams Instituut voor Biotechnologie (VIB), Department of Oncology, University of Leuven, Leuven 3000, Belgium ORCiD numbers: 0000-0002-6741-9012 (S. B. Wheatcroft); 0000-0001-7844-3600 (R. M. Cubbon). *These authors contributed equally. Abbreviations: Con, control; EC, endothelial cell; EdU, 5-ethynyl-2′-deoxyuridine; eNOS, endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; PEC, pulmonary endothelial cell; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2; WT, wild-type Received: 7 April 2021; Editorial Decision: 20 May 2021; First Published Online: 25 May 2021; Corrected and Typeset: 24 June 2021. ISSN Online 1945-7170 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. © The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. https://academic.oup.com/endo Endocrinology, 2021, Vol. 162, No. 8, 1–15 doi:10.1210/endocr/bqab104 Research Article Endocrinology, 2021, Vol. 162, No. 8, 1–15 doi:10.1210/endocr/bqab104 Research Article Research Article Research Article Abstract Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the under- pinning cellular and molecular mechanisms are unknown. Comparing mice with whole- body insulin receptor haploinsufficiency (Insr+/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the proangiogenic growth factor Vegfa. Insr+/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to vascular endo- thelial growth factor A  (VEGF-A), including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr+/- mice. Human umbilical vein endothelial cells with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and endothelial ni- tric oxide synthase was intact, but downstream signaling to extracellular signal-reduced https://academic.oup.com/endo Endocrinology, 2021, Vol. 162, No. 8 2 kinase 1/2 (ERK1/2) was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin recep- tors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2. Key Words: angiogenesis, endothelial, ERK, insulin, vascular, VEGF Key Words: angiogenesis, endothelial, ERK, insulin, vascular, VEGF Insulin receptor (Insr) haploinsufficient (Insr+/-) mice As we have previously described (15), Insr+/- mice (also known as IRKO) were obtained from the Medical Research Council Mammalian Genetics Unit (Harwell, Oxfordshire, UK), and were maintained as heterozygotes on a C57BL/6J background. Insr+/- were compared with age-matched wild- type (WT) littermates. Insulin is a primary regulator of systemic carbohydrate and lipid metabolism (1), but also has an important role in vascular function, for example promoting vasodilation and tissue perfusion (2). Indeed, loss of endothelial insulin receptors, or perturbation of their signaling function, in- duces endothelial dysfunction, hypertension, and athero- sclerosis (3-5). Sprouting angiogenesis, the phenomenon of new capillary formation, is another fundamental ele- ment of vascular biology that is intrinsically linked to metabolism (6). In this highly orchestrated and conserved process, endothelial “tip cells” emerge from existing ves- sels, followed by proliferating stalk cells that extend the sprout and form a lumenized vessel; these neo-vessels then anastomose into an immature network that remodels to meet local demands for oxygen and metabolite transport (7). Tissue collection and processing Retinal angiogenesis was assessed in postnatal day 5 pups by precisely following the protocol of Pitulescu et al (18). In brief, all pups from at least 3 litters were included in each experi- ment, with analysis blinded to the results of genotyping data. Both eyes were processed identically with a mean value from these to represent that pup. Vascular endothelium was stained with Isolectin B4 conjugated with Alexa Fluor 488 (I21411; Thermo Fisher Scientific, Warrington, UK). Costaining with a rabbit anti-mouse anti-Collagen IV antibody (19) followed by an Alexa Fluor-647 conjugated goat anti-rabbit antibody (20) was used to visualize the vascular basement membrane. To define cell proliferation, pups were injected with 125 μg 5-ethynyl-2′-deoxyuridine (EdU) 2 hours before tissue col- lection; this was stained with Alexa Fluor 647 azide using Click-iT technology (C10640; Thermo Fisher Scientific). Endothelial cell-specific Insr haploinsufficient (ECInsr+/-) mice ECInsr+/- mice were generated by crossing mice that have loxP sites flanking exon 4 of the insulin receptor (Line 006955, The Jackson Laboratory, Bar Harbor, ME) (16) with mice possessing a Cre transgene driven by the Tie2 promoter/enhancer (Line 004128, The Jackson Laboratory) (17) and were maintained on a C57BL/6J background. ECInsr+/- were compared with age-matched Cre-negative Insrlox/+ littermates, which are referred to as WT. Abstract Insulin has been reported to promote angiogenesis in vitro and in vivo; (8-12) these studies found pro- angiogenic effects in nanomolar concentrations in vitro, but did not explore more physiological picomolar concen- trations. In vivo, insulin receptor expression is known to be enriched in human tumor endothelial tip cells (13), and loss of endothelial insulin receptors has been shown to impair angiogenesis in murine retinopathy (14). However, it remains unclear how endothelial insulin receptors influ- ence the cellular and molecular processes of angiogenesis and so we set out to define this. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Materials and Methods Animal models All experimental mice were kept in a conventional animal facility with a 12-hour light/dark cycle and received a standard chow diet. Genotyping was per- formed using PCR of ear notch (or tail-tip for pups) genomic DNA by Transnetyx (Cordova, TN). All pro- cedures were approved by the Animal Welfare and Ethical Review Committee at the University of Leeds and were conducted in accordance with The Animals (Scientific Procedures) Act of 1986 Amendment Regulations 2012 (SI 2012/3039) under United Kingdom Home Office project licenses PL40/3523 and P144DD0D6. Confocal microscopy and image analysis Microscopy was performed using a Zeiss LSM880 up- right confocal microscope with 10×/0.3NA, 20×/0.8NA, Endocrinology, 2021, Vol. 162, No. 8 3 and 40×/1.4NA objectives and Zen software (Carl Zeiss Microscopy Ltd, Cambridge, UK). Tile scanning was used to image entire retinal segments with the 20× objective and maximum intensity projection of 5 consecutive 1 Airy unit thickness z-slices was used with the 40× to define tip cells and filopodia. Image analysis used ImageJ (NIH, Bethesda, MD). Radial outgrowth was defined as the distance from the optic disc periphery to the emerging vascular front measured at 12 points in each retina. Vascular area was de- fined by binary thresholding of the Isolectin B4 signal and expressed as a percentage of the region of interest, bounded either by the peripheral or central half of the vascularized area. Vascular branching was quantified in multiple 200 × 200-μm regions of interest placed between arteries and veins, in the peripheral or central vascular plexus. Tip cell abundance was normalized to the perimeter of the con- tiguous vascular front in each image and filopodia were normalized to tip cell number. Capillary regression was de- fined as Collagen IV staining without colocalized Isolectin B4 staining, and expressed as total length per mm2 in com- plete retinal segments, as per the method of Franco et al (21). Endothelial proliferation, defined by EdU+ nuclei costaining with Isolectin B4, was quantified in multiple 200 × 200-μm regions of interest placed between arteries and veins, in the peripheral vascular plexus. embedded in optimal cutting temperature media (Tissue- Tek OCT compound, Sakura, Netherlands) before snap freezing in liquid nitrogen and cryosectioning at 10-µm thickness. Vascular endothelium was stained with Isolectin B4 conjugated with Alexa Fluor 488 (I21411; Thermo Fisher Scientific) and slides were mounted with DAPI- Fluoromount-G (Southern Biotech, AL) to define nuclei. RT-PCR Snap frozen ischemic adductor muscle was mechanic- ally lysed in Trizol reagent (Sigma Aldrich, Gillingham, UK) to isolate RNA. After reverse transcription to gen- erate cDNA (kit), quantitative PCR was performed (ABI Prism 7900HT, Applied Biosystems) using Taqman probes against murine vegfa (Mm01281449-m1), murine insr (Mm00439688_m1), murine actb (Mm00607939_s1), 18s (Mm01281449-m1); 18s or actb were used to normalize gene expression using the equation Surgical procedure Following the protocol we have published (22), 9- to 13-week-old male Insr+/- mice were anesthetized with isoflurane before dissecting the left femoral artery, ligating it proximally at the inguinal ligament and distally at the bi- furcation to saphenous and popliteal vessels, and excising the intervening segment. Ex vivo aortic ring angiogenesis Aortae were harvested from 8- to 12-week-old Insr+/- mice under terminal isoflurane anesthesia and then processed according to the protocol of Baker et  al (23). In brief, after dissection of perivascular fat and overnight storage in serum free OptiMEM media (Thermo Fisher Scientific), aortae were cut in to 1-mm-thick rings that were then em- bedded in rat type I  collagen. Rings were incubated for 5 days at 37°C in 5% CO2 in Opti-MEM media containing 2.5% fetal calf serum (FCS), 50 ng/mL VEGF-A165 (R&D Systems, Abingdon, UK) and penicillin-streptomycin, with a media change on day 3. Rings were then fixed with 4% paraformaldehyde, stained with BS-1 lectin-fluorescein (Sigma Aldrich) to define endothelium, and then imaged with an inverted confocal microscope (LSM700, Carl Zeiss Microscopy Ltd.); tiled images were collected using a 10×/0.2NA objective and stitched using Zen software. Image analysis was performed with Image J (NIH), defining the number of fluorescein staining sprouts per ring and the mean length of these sprouts; mean data were then pro- duced for each experimental animal from at least 4 rings. Laser Doppler perfusion imaging Laser Doppler analysis (Moor LDI2-HR, Moor Systems, UK) of ischemic and nonischemic limbs was performed postoperatively in a temperature-controlled environment, to confirm induction of ischemia, and repeated weekly until day 21. Images were analyzed (MoorLDI software, Version 5.3, Moor Systems, UK) to derive an ischemic to nonischemic limb perfusion ratio, based upon flux below the level of the inguinal ligament. Materials and Methods Animal models Confocal microscopy and image analysis Microscopy was performed using a Zeiss LSM880 up- right confocal microscope with 20×/0.8NA objective and Zen software (Carl Zeiss Microscopy Ltd). Image analysis used ImageJ (NIH). Vascular area was defined by binary thresholding of the Isolectin B4 signal and expressed as a percentage of the image area. Assessment of pathological angiogenesis after hind-limb ischemia 2−deltaCT× 100. 2−deltaCT× 100. Isolation and functional analysis Pulmonary endothelial cells (PECs) were isolated from both lungs of 8- to 12-week-old Insr+/- mice, precisely following the protocol of Sobczak et al (24). This uses immuno-magnetic selection of CD31+ cells, which are then cultured in EGM2 media (Lonza, Slough, UK) for 10 to 14  days before a second round of immuno- magnetic selection from ICAM2+ endothelial cells that were cultured for a further 5 to 7 days in EGM2 before functional assays. Human umbilical vein endothelial cells (HUVECs; PromoCell, Heidelberg, Germany) were cultured at 37°C in 5% CO2 in EGM2 media on 1% gelatin-coated plasticware and used between passages 3 and 6. Silencing of the in- sulin receptor was induced using insulin receptor shRNA introduced by lentiviral particles (SHCLNV-NM_00208, TRCN0000196786; MISSION, Sigma Aldrich), with GFP- targeting shRNA lentivirus particles (SHC002H; MISSION, Sigma Aldrich) serving as control. Both lentiviruses were applied at 15 multiplicity of infection and HUVECs were used in downstream experiments 4 days after transduction. Adhesion assay y HUVECs were seeded on to 1% gelatin-coated 24-well plates in EBM2 media with 1% FCS, with or without 50 ng/ mL VEGA-A165, at a density of 4 × 104 cells per well and left for 1 hour before washing 3 times with PBS and fixing with 4% paraformaldehyde. Cells were counterstained with Hoechst and Phalloidin Alexa Fluor 488 conjugate and imaged with confocal microscopy (LSM700, Carl Zeiss Microscopy Ltd.) to count adherent cells per mm2. Scratch wound assay PECs were grown to confluence in EGM2 media on 1% gelatin coated 96-well plates before forming a scratch wound using the WoundMaker tool (Essen Bioscience, Royston, UK) and imaging wound closure hourly in a live cell imaging system (Incucyte, Essen Bioscience) to define residual wound area. Scratch wound assay HUVECs were grown to confluence in EGM2 media on 1% gelatin-coated 96-well plates before forming a scratch wound using the WoundMaker tool (Essen Bioscience) and imaging wound closure 8 hours later to define percentage wound closure from baseline. Bead sprouting assay Following the protocol of Nakatsu et  al (26), HUVECs were coated on to the surface of Cytodex-3 microcarrier beads (Sigma Aldrich) and then embedded in a fibrin matrix that was overlaid with EGM2 media (Lonza) without the supplemental bullet kit, but containing 50 ng/mL human VEGF-A165 (PeproTech, NJ) and 5ng/ml human basic FGF (PeproTech). After 48 hours of incubation at 37°C in 5% CO2, 25 beads per condition were imaged with phase con- trast microscopy (Olympus CX41, Olympus Life Sciences, Southend-On-Sea, UK) and analyzed with Image J (NIH), defining sprouts per bead and the mean length of these sprouts; mean data were then produced for each experi- mental condition. Matrigel sprouting assay Twenty-four well plates were coated with growth factor reduced Matrigel (BD Biosciences, Wokingham, UK) be- fore seeding each well with 2  × 105 PEC suspended in EBM2 media (Lonza) containing 1% FCS and 50 ng/mL of VEGF-A165. After 24 hours, phase contrast microscopy was used to image each well and count tubule-like structures according to our published protocol (22). Each sample was run in triplicate, with a single mean datapoint calculated for each experimental animal. Cell proliferation Sparsely seeded PECs on 1% gelatin coated plastic, cul- tured in EGM2 media, were exposed to 10  µM EdU 2 hours before fixation with 4% paraformaldehyde and processing with the Click-iT EdU cell proliferation assay (Thermo Fisher Scientific) to label nuclei containing ac- tively forming DNA with Alexa Fluor 488 and a Hoechst nuclear counterstain. Confocal microscopy (LSM700, Carl Zeiss Microscopy Ltd.) was used to define the proportion of EdU+ nuclei. Boyden chamber Following our published protocol (25), 5  × 104 PECs were seeded in 1% gelatin coated Boyden chamber ap- paratus to define migration toward 50 ng/mL VEGF-A165. The number of migrating cells per microscopic field was counted using standard light microscopy and pre- sented as net migration by subtracting the number of cells migrating in paired control experiments without VEGF-A165 gradient. Tissue collection and processing Ischemic and contralateral gastrocnemius muscle was har- vested and fixed in 4% paraformaldehyde for 48 hours, whereas adductor muscles were snap frozen with liquid nitrogen for RNA isolation. Fixed muscle specimens were Endocrinology, 2021, Vol. 162, No. 8 4 Western blotting Western blotting HUVECs were lysed in cell extraction buffer (FNN0011; Thermo Fisher Scientific; containing, in mmol/L, 10 mM Endocrinology, 2021, Vol. 162, No. 8 5 Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 2 sodium orthovanadate, 0.5  μg/mL leupeptin, 0.2 PMSF, 0.5 μg/mL aprotinin). Cell extracts were centrifuged for 15 minutes before protein measurement using the biocinochinic acid assay (Thermo Fisher Scientific). Equal amounts of protein were resolved on SDS-polyacrylamide gels (Thermo Fisher Scientific) and transferred to polyvinyldine difluoride membranes. Immunoblotting was carried out with primary antibodies against beta-actin (27), insulin receptor-beta (28), Akt (29), phospho-S473 Akt (30), endo- thelial nitric oxide synthase (eNOS) (31), phospho-S1177 eNOS (32), extracellular signal-regulated kinase 1/2 (ERK1/2) (33), phospho-T202/Y204 ERK1/2 (34), vas- cular endothelial growth factor receptor 2 (VEGFR2) (35), and phospho-Y951 VEGFR2 (36). Blots were incubated with appropriate peroxidase-conjugated secondary anti- bodies and developed with enhanced chemiluminescence (37, 38), and imaged with SynGene chemiluminescence imaging system (SynGene, Cambridge, UK). Densitometry of phospho-proteins was normalized to respective total proteins from the same sample and then these data were normalized to the value of unstimulated control shRNA- transduced cells in each experiment (which included paired control and insulin receptor shRNA-transduced cells, with samples run on 1 membrane). VEGF-A induced signaling was calculated by subtracting the data from unstimulated (control or insulin receptor shRNA transduced) cells from their respective VEGF-A–exposed cells in each experiment. were washed with radio-immunoprecipitation assay buffer to remove any unbound antibody before incubating with cell lysate for 1 hour with rotation at room temperature to allow the biotinylated antigen-antibody complexes to form. At the end of the pull-down period, the beads were washed 5 times with radio-immunoprecipitation assay buffer (50 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, 0.5% [w/v] sodium deoxycholate, 0.1% [w/v] SDS, and 1% [v/v] Igepal) to remove any nonspecific binding. The immunoprecipitated, biotinylated VEGFR2 complexes were mixed with dissociation buffer and boiled to release the complexes from the beads. The proteins were resolved by electrophoresis through 4% to 12% polyacrylamide gels and then transferred to nitrocellulose membrane. Results Surface VEGFR2 biotinylation, immunoprecipitation, and VEGFR2 Western blotting were performed according to our previously published protocol (39). Briefly, HUVECs were incubated for 1 hour at 4°C with 0.5 mg/mL biotin sulfo-NHS (Sigma-Aldrich, Gillingham, UK), before being stimulated with 50 ng/mL VEGF at 37°C for either 5 or 15 minutes. At the end of the exposure period, the cells were washed 3 times with PBS with calcium and magne- sium before either: immediate lysis with cell extraction buffer to enable the measurement of total VEGFR2 in the sample; or, treated with 0.5 mL of 0.05% trypsin/EDTA to cleave and remove any remaining biotin-labelled, cell sur- face VEGFR2, meaning detected biotin-labelled VEFGR2 would define only internalized protein. The trypsinized cell pellet was lysed using cell extraction buffer as before. Immunoprecipitation of VEGFR2 was carried out using protein A  Dynabeads (Thermo Fisher Scientific) loaded with anti-VEGFR2 antibody (diluted 1:100) (35) for 30 minutes with rotation at room temperature. The beads Statistics All data are presented as mean (SEM). Comparison be- tween groups was performed using Student t tests, or 2-way ANOVA for time series data. All tests were 2-sided and statistical significance was defined as P < 0.05. Western blotting The membrane was blocked for 1 hour in PBS (1.5  mmol/L KH2PO4, 2.7 mmol/L Na2HPO4, 150 mmol/L NaCl [pH 7.4]) containing 5% (w/v) dried milk powder and 0.1% (v/v) Tween-20, followed by incubation with peroxidase- conjugated streptavidin (1:1000 dilution in PBS containing 0.1% [v/v] Tween-20) for 1 hour. Bound peroxidase conju- gates were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences). Quantification of immunoblots was performed using ImageJ software. Pathological angiogenesis is impaired in Insr+/- mice and is associated with impaired responsiveness to VEGF To study pathological angiogenesis, we induced hind- limb ischemia in Insr+/- mice and quantified limb perfu- sion recovery every 7 days using laser Doppler imaging. This revealed that, in spite of similar reductions in limb perfusion immediately postoperatively, Insr+/- exhibited lower ischemic limb perfusion at all timepoints there- after (Fig. 1A), being 82% (5.6) in WT and 62% (7.1) in Insr+/- (P < 0.05) at day 21. Histological analysis of gastrocnemius muscle also revealed a lower capillary density in the ischemic limb of Insr+/- at day 21 (vas- cular area 12% [0.5] vs 8% [0.6] in WT and Insr+/-, re- spectively, P < 0.05 Fig. 1B), in spite of greater VEGFA mRNA in the ischemic limb adductor muscle of Insr+/- (31.4% [6.7] of 18S mRNA in Insr+/- vs 9.8% [2.5] WT P  <  0.05; Fig. 1C). Notably, Vegfa mRNA was similar 6  Endocrinology, 2021, Vol. 162, No. 8 gure 1. Angiogenesis is impaired in Insr+/- mice with hindlimb ischemia. (A) Ischemic to nonischemic limb perfusion defined by laser Doppler maging, with representative day 21 images, showing impaired recovery from hindlimb ischemia in Insr+/- vs WT (n = 14, 13). (B) Confocal immuno-l uorescence of ischemic and nonischemic gastrocnemius muscle reveals reduced capillary density in ischemic Insr+/- vs WT muscle. Representative mages of ischemic muscle show isolectin B4 stained capillaries in red and nuclei in blue. Scale bars denote 50 μm. (n = 8, 7). (C) Vegfa mRNA ormalized to 18S mRNA is higher in the ischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). D) Vegfa mRNA normalized to 18S mRNA is higher the nonischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). *P < 0.05. Insr, insulin receptor; WT, wild-type. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6 Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Figure 1. Angiogenesis is impaired in Insr+/- mice with hindlimb ischemia. (A) Ischemic to nonischemic limb perfusion defined by laser Doppler imaging, with representative day 21 images, showing impaired recovery from hindlimb ischemia in Insr+/- vs WT (n = 14, 13). (B) Confocal immuno- fluorescence of ischemic and nonischemic gastrocnemius muscle reveals reduced capillary density in ischemic Insr+/- vs WT muscle. Representative images of ischemic muscle show isolectin B4 stained capillaries in red and nuclei in blue. Scale bars denote 50 μm. (n = 8, 7). Pathological angiogenesis is impaired in Insr+/- mice and is associated with impaired responsiveness to VEGF This revealed fewer capillary sprouts emerging from Insr+/- aortae in the presence of VEGF (30 [2.2] in WT vs 22 [2.4] in Insr+/-; P < 0.05; Fig. 2A), and the length of sprouts was also reduced (1037 µm [39] WT vs 847 µm [38] Insr+/-; P < 0.05; Fig. 2B). Next, we isolated PECs for functional studies; these exhibited appropriate reduc- tion in Insr mRNA (85% [5] of 18S in WT vs 49% [4] in Insr+/-; P < 0.05; Fig. 2C). We then conducted a Matrigel in vitro angiogenesis assay, which demonstrated reduced tubule formation in Insr+/- (28 [2] tubules per microscopic field in WT vs 14.5 [2.4] in Insr+/-; P < 0.05; Fig. 2D). Similarly, a scratch wound assay performed on confluent PECs revealed significantly slower closure of the wound formed in Insr+/- PECs (area under curve 4,430,670 arbi- trary units WT [154,516] vs 5,085,825 [126,748] arbi- trary units Insr+/-; P < 0.05; Fig. 2E). Because the Matrigel and scratch wound assays define responses to VEGF-A with other stimulatory factors, we then performed as- says to more specifically define functional responses to VEGF-A. First, we conducted a migration assay using Boyden chamber apparatus and found fewer Insr+/- PECs migrated toward VEGF-A165 (7.5 [1.7] WT vs 2.2 [1.1] Insr+/- net cell migration to VEGF per microscopic field; P  <  0.05; Fig. 2F). Second, we studied VEGF-A165–in- duced PEC proliferation using EdU incorporation and elicited no difference between Insr+/- and WT (10.2% [3.5] WT vs 8.9% [2.6] Insr+/- EdU+ cells; P = 0.77; Fig. 2G). Collectively, these data imply that Insr+/- endothe- lial cells have selectively impaired migratory responses to VEGF-A. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 To discern whether loss of insulin receptors expressed by ECs contribute to the retinal vascular phenotype of Insr+/-, we then studied mice with ECInsr+/-. Again, the ra- dial outgrowth of their retinal vascular plexus was similar to controls (1377 µm [32] WT vs 1375 µm [40] ECInsr+/-; P  =  0.96; Fig. 4A), although there was reduced vascular area (front 46.5% [0.5] WT vs 44.0 [0.5] ECInsr+/-, P < 0.05; center 36.7% [0.4] WT vs 33.9% [1.0] ECInsr+/-, P  <  0.05; Fig. 4B) and branching complexity (front 1192.5 [7.0] WT vs 1043.3 [20.4] ECInsr+/- branch points per mm2, P  <  0.05; center 1083.5 [14.3] WT vs 1000.0 [30.5] ECInsr+/-, P < 0.05; Fig. Pathological angiogenesis is impaired in Insr+/- mice and is associated with impaired responsiveness to VEGF 4C) in both the peripheral and central vascular plexuses of ECInsr+/-. Also mirroring Insr+/- phenotype, there was a reduction in the number of sprouting tip cells of ECInsr+/- (21.5 [0.2] WT vs 18.7 [0.3] ECInsr+/- tip cells/mm; P < 0.05; Fig. 4D), along with fewer filopodia per length of vascular forefront (21.2 [0.4] WT vs 18.7 [0.3] ECInsr+/- filopodia per 100 µm; P < 0.05), but with no difference in filopodia per tip cell between geno- types (21.8 [0.7] WT vs 22.2 [0.5] ECInsr+/- filopodia per tip cell; P = 0.65; Fig. 4E). Collectively, these data suggest that endothelial cell insulin receptor expression is important in the generation of vascular sprouts, and the branching struc- ture of the nascent vasculature. Pathological angiogenesis is impaired in Insr+/- mice and is associated with impaired responsiveness to VEGF (C) Vegfa mRNA normalized to 18S mRNA is higher in the ischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). D) Vegfa mRNA normalized to 18S mRNA is higher in the nonischemic limb adductor muscle of Insr+/- vs WT (n = 4, 4). *P < 0.05. Insr, insulin receptor; WT, wild-type. 7 Endocrinology, 2021, Vol. 162, No. 8 P < 0.05; Fig. 3B) and reduced vascular branching com- plexity (front 48.0 [0.4] WT vs 41.9 [0.7] Insr+/- branches per microscopic field, P < 0.05; center 52.0 [1.3] WT vs 46.4 [1.1] Insr+/- branches per microscopic field, P < 0.05; Fig. 3C) in both central and peripheral vascular plex- uses of Insr+/-. High-resolution images of the emerging vascular plexus revealed a reduction in the number of sprouting tip cells of Insr+/- (21.2 [0.3] WT vs 18.3 [0.2] Insr+/- tip cells per mm, P  <  0.05; Fig. 3D), fewer filo- podia per length of vascular forefront (22.0 [0.5] WT vs 18.1 [0.3] Insr+/- filopodia per 100  µm, P  <  0.05), and fewer filipodia per tip cell (22.1 [0.3] WT vs 20.6 [0.4] Insr+/- filopodia per tip cell, P < 0.05; Fig. 3E). Because reduced vascularity could also be explained by increased vessel regression, we quantified the number of “empty” collagen IV sleeves (ie, collagen IV basement membrane without overlying Isolectin B4, linking 2 regions of estab- lished vasculature) in the retinal periphery; we observed fewer in Insr+/- vs WT, indicating regression was not ex- aggerated (0.46 [0.02] WT vs 0.40 [0.02] Insr+/- regressed vessels/100 µm2; P = 0.028; Fig. 3F). Next, we asked if endothelial cell (EC) proliferation was reduced in Insr+/-, but found similar numbers of EdU+ ECs in the peripheral retinal vasculature of both genotypes (781 [44] WT vs 814 [24] Insr+/- EdU+ EC per mm2; P = 0.53; Fig. 3G). Overall, these data are compatible with a reduction in vascular sprouting in the emerging vasculature of Insr+/-, resulting in a less branched neovasculature. in the nonischemic limb adductor muscle or WT and Insr+/- (1.7% [0.3] of 18S mRNA in WT vs. 1.4% [0.3] in Insr+/-; Fig. 1D). The increased ischemic muscle expres- sion of Vegfa is indicative of significant residual ischemia and could also imply an inadequate functional response to this central regulator of angiogenesis. Pathological angiogenesis is impaired in Insr+/- mice and is associated with impaired responsiveness to VEGF To address this possibility, we explanted aortae from a separate group of Insr+/- mice to embed rings in a collagen matrix con- taining VEGF-A165, which induces sprouting angiogen- esis. This revealed fewer capillary sprouts emerging from Insr+/- aortae in the presence of VEGF (30 [2.2] in WT vs 22 [2.4] in Insr+/-; P < 0.05; Fig. 2A), and the length of sprouts was also reduced (1037 µm [39] WT vs 847 µm [38] Insr+/-; P < 0.05; Fig. 2B). Next, we isolated PECs for functional studies; these exhibited appropriate reduc- tion in Insr mRNA (85% [5] of 18S in WT vs 49% [4] in Insr+/-; P < 0.05; Fig. 2C). We then conducted a Matrigel in vitro angiogenesis assay, which demonstrated reduced tubule formation in Insr+/- (28 [2] tubules per microscopic field in WT vs 14.5 [2.4] in Insr+/-; P < 0.05; Fig. 2D). Similarly, a scratch wound assay performed on confluent PECs revealed significantly slower closure of the wound formed in Insr+/- PECs (area under curve 4,430,670 arbi- trary units WT [154,516] vs 5,085,825 [126,748] arbi- trary units Insr+/-; P < 0.05; Fig. 2E). Because the Matrigel and scratch wound assays define responses to VEGF-A with other stimulatory factors, we then performed as- says to more specifically define functional responses to VEGF-A. First, we conducted a migration assay using Boyden chamber apparatus and found fewer Insr+/- PECs migrated toward VEGF-A165 (7.5 [1.7] WT vs 2.2 [1.1] Insr+/- net cell migration to VEGF per microscopic field; P  <  0.05; Fig. 2F). Second, we studied VEGF-A165–in- duced PEC proliferation using EdU incorporation and elicited no difference between Insr+/- and WT (10.2% [3.5] WT vs 8.9% [2.6] Insr+/- EdU+ cells; P = 0.77; Fig. 2G). Collectively, these data imply that Insr+/- endothe- lial cells have selectively impaired migratory responses to VEGF-A. in the nonischemic limb adductor muscle or WT and Insr+/- (1.7% [0.3] of 18S mRNA in WT vs. 1.4% [0.3] in Insr+/-; Fig. 1D). The increased ischemic muscle expres- sion of Vegfa is indicative of significant residual ischemia and could also imply an inadequate functional response to this central regulator of angiogenesis. To address this possibility, we explanted aortae from a separate group of Insr+/- mice to embed rings in a collagen matrix con- taining VEGF-A165, which induces sprouting angiogen- esis. Developmental angiogenesis is impaired in Insr+/- and ECInsr+/- mice Next, we asked whether the abnormalities of patho- logical angiogenesis in Insr+/- were recapitulated during developmental angiogenesis, which we assessed using whole-mounted retinas, at P5 when the vasculature is still developing. The radial outgrowth of the retinal vascular plexus was similar in Insr+/- and WT (1367 µm [50] WT vs 1354 µm [56] Insr+/-, P = 0.87; Fig. 3A), although there was reduced vascular area in the peripheral vascular plexus of Insr+/- (48.8% [0.6] WT vs 46.0% [0.7] Insr+/-, Endocrinology, 2021, Vol. 162, No. 8 8 Figure 2. Insr+/- exhibits impaired in vitro functional responses to VEGF. Capillary sprouting from aortic rings embedded in a collagen matrix with VEGF-A165 is reduced in Insr+/- vs WT (A), as is mean sprout length (B); representative images show isolectin B4 staining of endothelium in green, with scale bars denoting 1000 μm (n = 5, 5). (C) Insulin receptor (Insr) mRNA normalized to 18S mRNA is reduced in Insr+/- vs WT PEC (n = 5, 5). (D) In vitro angiogenesis in Matrigel is impaired in Insr+/- vs WT PEC (n = 7, 4). (E) Scratch wound closure is impaired in Insr+/- vs WT PEC (n = 5, 6). (F) Migration toward VEGF-A165 in Boyden chamber apparatus is impaired in Insr+/- vs WT PEC (n = 5, 4). (G) Proliferation defined by nuclear EdU incorporation is similar in Insr+/- and WT PEC (n = 11, 7). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; PEC, pulmonary endothelial cell; VEGF, vas- cular endothelial growth factor; WT, wild-type. Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Figure 2. Insr+/- exhibits impaired in vitro functional responses to VEGF. Capillary sprouting from aortic rings embedded in a collagen matrix with VEGF-A165 is reduced in Insr+/- vs WT (A), as is mean sprout length (B); representative images show isolectin B4 staining of endothelium in green, with scale bars denoting 1000 μm (n = 5, 5). (C) Insulin receptor (Insr) mRNA normalized to 18S mRNA is reduced in Insr+/- vs WT PEC (n = 5, 5). (D) In vitro angiogenesis in Matrigel is impaired in Insr+/- vs WT PEC (n = 7, 4). (E) Scratch wound closure is impaired in Insr+/- vs WT PEC (n = 5, 6). (F) Migration toward VEGF-A165 in Boyden chamber apparatus is impaired in Insr+/- vs WT PEC (n = 5, 4). Insulin receptor silencing impairs human EC functional responsiveness to VEGF shRNA vs 95.4 [9.1] Insr shRNA cells per microscopic field; P < 0.05; Fig. 5E). Overall, these data suggest that the insulin receptor is also important for VEGF-A–induced angiogenic sprouting and cell motility in human EC. To explore the relevance of these data in human ECs, we transduced HUVECs with lentivirus particles to de- liver insulin receptor targeting shRNA (referred to as Insr shRNA), or control GFP-targeting shRNA (referred to as control [Con] shRNA), reducing insulin receptor pro- tein by 40% (Fig. 5A). Transduced HUVECs were then coated onto Cytodex-3 carrier beads and embedded in a fibrin matrix to study VEGF-A165–induced sprouting angiogenesis in vitro. Mirroring data from Insr+/- aortic ring sprouting experiments (Fig. 2A-C), we observed that Insr shRNA HUVECs produced fewer sprouts (6.5 [1.2] Con shRNA vs 3.4 [1.0] Insr shRNA sprouts per bead; P < 0.05; Fig. 5B), although the mean length of sprouts was unaffected (78.3 µm [10.7] Con shRNA vs 65.0 µm [14.0] Insr shRNA, P > 0.05; Fig. 5C). Insr shRNA HUVECs ex- hibited impaired scratch wound closure (84.7% [4.7] Con shRNA vs 74.0% [3.7] Insr shRNA; P < 0.05; Fig. 5D) and impaired adhesion to gelatin coated plates, which was more marked in the presence of VEGF-A165 (vehicle 138.7 [5.5] Con shRNA vs 85.6 [6.9] Insr shRNA cells per microscopic field; P  <  0.05; VEGF-A165 184.3 [6.6] Con Developmental angiogenesis is impaired in Insr+/- and ECInsr+/- mice (G) Proliferation defined by nuclear EdU incorporation is similar in Insr+/- and WT PEC (n = 11, 7). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; PEC, pulmonary endothelial cell; VEGF, vas- cular endothelial growth factor; WT, wild-type. Endothelial insulin receptors are required for VEGFR2 internalization and subsequent ERK signaling Because we had demonstrated that insulin receptor expres- sion influenced functional responses to VEGF-A in human and murine ECs, we then asked if this was associated with altered VEGF-A signaling. VEGF-A promotes angiogen- esis by binding to VEGFR2, a cell membrane-bound re- ceptor tyrosine kinase that initiates a complex intracellular signaling cascade. We therefore stimulated Insr shRNA and control shRNA HUVECs with 50 ng/mL VEGF-A165 and studied major VEGF-A signaling nodes 5 and 15 minutes later, along with unstimulated cells (Fig. 6A). Insr shRNA HUVECs exhibited unaffected activation of VEGFR2 (measured by phosphorylation at Y951), or the down- stream nodes Akt (measured by phosphorylation at S473) and eNOS (measured by phosphorylation at S1177) (data 9 Endocrinology, 2021, Vol. 162, No. 8 ure 3. Developmental angiogenesis is impaired in the neonatal P5 retina of Insr+/- mice. (A) Radial outgrowth of the developing retinal vascula- is comparable in Insr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm 7, 13). (B) Vascular endothelial area is reduced in the peripheral half of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (C) Vascular branching educed in the peripheral and central zones of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (D) Emerging tip cells per millimeter of vascular t perimeter are reduced in Insr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting m (n = 7, 13). (E) The number of filopodia per tip cell is similar in Insr+/- vs WT (n = 7, 13). (F) The number of regressed vessels, defined as Collagen eeves (red) without overlying isolectin B4 (green) in representative images, is lower in Insr+/- than WT (n = 11, 10). (G) The number of proliferating othelial cells, defined as EdU+ nuclei (red) overlying isolectin B4 (white) in representative images, is similar in Insr+/- and WT (n = 13, 5). *P < 0.05. 5 th l 2′ d idi I i li t WT ild t Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Downloaded from https://academic.oup.com/endo/article/162/8 Downloaded from https://academic.oup.com/endo/article/162/8/bqab104/6284300 by guest on 01 March 2023 Figure 3. Developmental angiogenesis is impaired in the neonatal P5 retina of Insr+/- mice. Endothelial insulin receptors are required for VEGFR2 internalization and subsequent ERK signaling (A) Radial outgrowth of the developing retinal vascula- ture is comparable in Insr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm (n = 7, 13). (B) Vascular endothelial area is reduced in the peripheral half of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in Insr+/- vs WT (n = 7, 13). (D) Emerging tip cells per millimeter of vascular front perimeter are reduced in Insr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 50 μm (n = 7, 13). (E) The number of filopodia per tip cell is similar in Insr+/- vs WT (n = 7, 13). (F) The number of regressed vessels, defined as Collagen IV sleeves (red) without overlying isolectin B4 (green) in representative images, is lower in Insr+/- than WT (n = 11, 10). (G) The number of proliferating endothelial cells, defined as EdU+ nuclei (red) overlying isolectin B4 (white) in representative images, is similar in Insr+/- and WT (n = 13, 5). *P < 0.05. EdU, 5-ethynyl-2′-deoxyuridine; Insr, insulin receptor; WT, wild-type. Endocrinology, 2021, Vol. 162, No. 8 10 Figure 4. Developmental angiogenesis is impaired in the neonatal P5 retina of ECInsr+/- mice. (A) Radial outgrowth of the developing retinal vascu- lature is comparable in ECInsr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm (n = 7, 6). (B) Vascular endothelial area is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (D) Emerging tip cells per millimeter of vascular front perimeter are reduced in ECInsr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 50 μm (n = 7, 6). (E) The number of filopodia per tip cell is similar in ECInsr+/- vs WT (n = 7, 6). ECInsr, endothelial cell insulin receptor; WT, wild-type. Figure 4. Developmental angiogenesis is impaired in the neonatal P5 retina of ECInsr+/- mice. Endothelial insulin receptors are required for VEGFR2 internalization and subsequent ERK signaling (A) Radial outgrowth of the developing retinal vascu- lature is comparable in ECInsr+/- and WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 500 μm (n = 7, 6). (B) Vascular endothelial area is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (C) Vascular branching is reduced in the peripheral and central zones of the retinal vasculature in ECInsr+/- vs WT (n = 7, 6). (D) Emerging tip cells per millimeter of vascular front perimeter are reduced in ECInsr+/- vs WT, with representative images showing white isolectin B4 staining of endothelium and scale bars denoting 50 μm (n = 7, 6). (E) The number of filopodia per tip cell is similar in ECInsr+/- vs WT (n = 7, 6). ECInsr, endothelial cell insulin receptor; WT, wild-type. [15.6] Con shRNA vs 87.4% [3.8] Insr shRNA surface- retained VEFGR2; P  <  0.05; 15 minutes: 20.6% [12.9] Con shRNA vs 89.8% [0.9] Insr shRNA surface-retained VEFGR2; P < 0.05; Fig. 6D,E), which is known to select- ively impede ERK1/2 activation. Overall, these data reveal a selective deficit in VEGF-A signal transduction in Insr shRNA HUVECs, which is likely to result from impaired internalization of activated VEGFR2. not shown). However, downstream activation of ERK1/2 (measured by phosphorylation at T202/Y204) was im- paired in Insr shRNA HUVECs at 5 and 15 minutes after stimulation (5-minute change: 3.6 [0.95] Con shRNA vs 1.9 [0.53] Insr shRNA pERK/ERK ratio; P < 0.05; 15-minute change: 4.9 [0.98] Con shRNA vs 2.5 [1.02] Insr shRNA pERK/ERK ratio; P < 0.05; Fig. 6B). Control experiments defining the signaling response to insulin revealed clear re- duction in Akt activation after 15 minutes, confirming that Insr shRNA induced the expected changes in activation of this major insulin signaling node (Fig. 6C). Importantly, the activation of Akt and ERK1/2 downstream of VEGFR2 follows highly distinct pathways, with internalization of VEGFR2 being essential for only the latter (40); more- over, integrins are known to influence this process (41). Hence, we then asked whether VEGF-A induced internal- ization of VEGFR2 was impaired in Insr shRNA HUVEC by performing a surface biotinylation assay to quantify surface-resident and internalized VEGFR2 protein. This revealed that, although baseline surface located VEGFR2 was similar in both groups, there was reduced VEGFR2 internalization in Insr shRNA HUVECs (5 minutes: 41.6% Major findings and implications Our data reveal for the first time that endothelial insulin receptors are required for appropriate migration and angiogenic sprouting in response to VEGF-A, both in vitro and in vivo. At a molecular level, we found that insulin re- ceptor expression promotes the internalization of VEGF-A– activated VEGFR2, allowing signaling to ERK1/2. Our data suggest that the proangiogenic effects of insulin receptors relate to crosstalk with VEGF-A signaling, although the na- ture of this interaction, and whether insulin participates in the process, requires further study (Fig. 7). This previously Endocrinology, 2021, Vol. 162, No. 8 11 Figure 5. Insr knockdown in HUVECs impairs functional responses to VEGF. (A) Insulin receptor protein knockdown of 40% was achieved in Insr shRNA HUVECs vs control shRNA HUVECs, with representative gel (n = 7, 7). (B) Angiogenic sprout numbers were reduced from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs, with representative microscopy images (n = 3, 3). (C) Angiogenic sprout length was similar from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs (n = 3, 3). (D) Scratch wound closure was impaired in Insr shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). (E) Adhesion to gelatin was impaired in Insr shRNA HUVECs vs control shRNA HUVECs, espe- cially in context of media supplemented with VEGF-A165; representative microscopy images show DAPI-defined nuclei in blue and phalloidin-defined filamentous actin in red, with scale bars denoting 250 μm (n = 4, 4). *P < 0.05. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; VEGF, vascular endothelial growth factor. Figure 5. Insr knockdown in HUVECs impairs functional responses to VEGF. (A) Insulin receptor protein knockdown of 40% was achieved in Insr shRNA HUVECs vs control shRNA HUVECs, with representative gel (n = 7, 7). (B) Angiogenic sprout numbers were reduced from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs, with representative microscopy images (n = 3, 3). (C) Angiogenic sprout length was similar from Cytodex beads coated with Insr shRNA HUVECs vs control shRNA HUVECs (n = 3, 3). (D) Scratch wound closure was impaired in Insr shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). Major findings and implications (E) Adhesion to gelatin was impaired in Insr shRNA HUVECs vs control shRNA HUVECs, espe- cially in context of media supplemented with VEGF-A165; representative microscopy images show DAPI-defined nuclei in blue and phalloidin-defined filamentous actin in red, with scale bars denoting 250 μm (n = 4, 4). *P < 0.05. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; VEGF, vascular endothelial growth factor. unappreciated crosstalk establishes a further link between systems regulating metabolism and angiogenesis. Ronald Kahn’s group indicate a ligand-independent role of insulin receptors in the membrane trafficking of brown pre-adipocytes (46), potentially aligning with our findings. However, although the concentrations of in- sulin experienced by sprouting endothelial cells in vivo are unknown, our in vitro data are likely to reflect low picomolar concentrations of insulin because of its pres- ence in fetal calf serum. This may suggest a role for in- sulin in promoting VEGF-induced ERK signaling, as has been shown for epidermal growth factor signaling (47). Another explanation may be that insulin regulates a common endocytic mechanism for its own receptor and VEGFR2, as discussed later. Insulin and angiogenesis A number of studies have shown insulin exerts proangiogenic effects, although they did not dissect the role of endothelial insulin receptors (8-12). These mainly in vitro studies revealed proangiogenic effects of nanomolar range insulin, but did not explore more physiological picomolar concentrations. The extent to which picomolar insulin augments ERK signaling in endothelial cells is a source of disagreement in the litera- ture (11, 42-44), probably reflecting known heterogen- eity between endothelial populations, including in their insulin receptor expression. Notably, insulin receptor expression is reported to be enriched in endothelial tip cells of human tumors (13), yet tip cells generally lack a lumen (45), so may be exposed to lower concentrations of insulin than other endothelial cells. Hence, one pos- sible explanation for our data is a ligand-independent role of tip cell insulin receptors. Indeed, recent data from The only existing data describing the role of endothe- lial insulin receptors in angiogenesis were published by Kahn’s group in 2003. Using mice with complete deletion of endothelial insulin receptors, they found a 57% reduc- tion in retinal neovascularization during oxygen-induced retinopathy (14). However, they did not study the indi- vidual cellular processes contributing to angiogenesis, or examine VEGF signaling. Our in vivo data suggest that insulin receptors regulate endothelial tip cell emergence 12 Endocrinology, 2021, Vol. 162, No. 8 Figure 6. Insr knockdown in HUVECs impairs signaling responses to VEGF. (A) Representative blots illustrating major VEGF signaling nodes in Insr shRNA HUVECs and control shRNA HUVECs grown on gelatin at baseline, 5 minutes, and 15 minutes after stimulation with VEGF-A165 50 ng/ mL, (B) with quantification of VEGF-induced phosphorylation of ERK1/2 at 5 and 15 minutes (n = 4, 4). (C) Representative blot illustrating impaired insulin-stimulated Akt phosphorylation in Insr shRNA HUVECs vs control shRNA HUVECs after 15 minutes’ exposure to 10 and 100 nm insulin. (D) Representative blots and (E) quantification from surface biotinylation experiment illustrating impaired internalization of VEGFR2 in Insr shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). (D) Detected biotin-labelled VEGFR2 in the presence or absence of TE and so is not directly represented in €, which presents derived internalization data. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; TE, trypsin exposure; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. Figure 6. Insr knockdown in HUVECs impairs signaling responses to VEGF. Insulin and angiogenesis (A) Representative blots illustrating major VEGF signaling nodes in Insr shRNA HUVECs and control shRNA HUVECs grown on gelatin at baseline, 5 minutes, and 15 minutes after stimulation with VEGF-A165 50 ng/ mL, (B) with quantification of VEGF-induced phosphorylation of ERK1/2 at 5 and 15 minutes (n = 4, 4). (C) Representative blot illustrating impaired insulin-stimulated Akt phosphorylation in Insr shRNA HUVECs vs control shRNA HUVECs after 15 minutes’ exposure to 10 and 100 nm insulin. (D) Representative blots and (E) quantification from surface biotinylation experiment illustrating impaired internalization of VEGFR2 in Insr shRNA HUVECs vs control shRNA HUVECs (n = 4, 4). (D) Detected biotin-labelled VEGFR2 in the presence or absence of TE and so is not directly represented in €, which presents derived internalization data. HUVEC, human umbilical vein endothelial cell; Insr, insulin receptor; TE, trypsin exposure; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. studied, ERK1/2 activation has recently emerged as regu- lating endothelial tip cell function and sprouting angiogen- esis; (54, 55) importantly, tip cells are exposed to the highest VEGF-A concentrations during angiogenesis, and tip cell ERK phosphorylation is prevented in vivo by VEGF inhib- ition (7, 55). There are many known interacting partners of VEGFR2, which can modify its signaling and internal- ization (41, 56). The insulin receptor signaling adaptor, in- sulin receptor substrate-1, has been implicated in receptor endocytosis (57), and is reported to interact with VEGFR2 (58). Interestingly, recent data show that insulin signaling to ERK1/2 (and Src homology phosphatase 2) feeds back via serine phosphorylation of insulin receptor substrate-1 to induce insulin receptor internalization, which aug- ments ERK1/2 signaling (59). Therefore, it is possible that VEGFR2 internalization is similarly affected, and this pu- tative insulin-dependent mechanism also warrants further exploration. Integrins are also well established to modulate the propagation of ERK1/2 signals downstream of many growth factor receptors, including during angiogenesis, so and migration, although with no major impact on endo- thelial cell proliferation. Notably, we found no reduction in nonischemic muscle tissue vascularity of Insr+/- mice, implying that impaired vascularization ultimately catches up; this is seen in many published examples of impaired angiogenesis (48-50), presumably reflecting persistent acti- vation of proangiogenic programmes. Limitations Although we show that internalization of activated VEGFR2 is impaired, the underlying mechanism of this requires further investigation. It will also be interesting to explore signal transduction downstream of other re- ceptor tyrosine kinases to assess the generalizability of this phenomenon. As alluded to earlier, it is also im- portant to acknowledge that impaired VEGFR2 intern- alization may not be the only mechanism by which Insr silencing impairs VEGF-A responses; indeed, intracellular signaling networks are highly complex, as is their per- turbation. Moreover, our work only sought to describe the fundamental links between insulin receptors and VEGF signaling during angiogenesis, and so we cannot comment on disease relevance. However, obesity-induced insulin resistance is associated with reduced vascular in- sulin receptor expression and impaired angiogenesis (63), so it would be interesting to explore endothelial VEGFR2 internalization and ERK signal transduction Conclusions We show that endothelial insulin receptors are required for appropriate migration and angiogenic sprouting in re- sponse to VEGF-A, along with internalization of activated VEGFR2 and downstream signaling to ERK1/2. This novel link between major regulators of systemic metabolism and angiogenesis warrants further mechanistic exploration to understand its wider relevance. VEGFR2 signaling VEGF-A binding to VEGFR2 induces a complex intracel- lular signaling cascade (51), a crucial element of which is internalization (endocytosis) of ligand-bound VEGFR2. This moves the phosphorylated receptor to a domain where it is less susceptible to the phosphatase PTP1B, hence sustaining signal transmission, which is particularly important for ERK1/2 signaling (40). VEGFR2 internaliza- tion, and subsequent ERK1/2 signaling, is known to be cru- cial to vascular biology (eg, during arterial morphogenesis and lymphatic specification) (52, 53). Although less well Endocrinology, 2021, Vol. 162, No. 8 13 Figure 7. Schematic illustration of the proposed role of Insr during sprouting angiogenesis. Insr expression is known to be enriched in tip ECs, which migrate along VEGF gradients, leading emerging sprouts during angiogenesis. Knockdown of Insr in ECs impairs VEGF signaling to ERK1/2 as a result of impaired VEGFR2 internalization, which manifests as diminished sprout formation and EC migration. EC, endothelial cell; ERK, extracellular signal- regulated kinase; Insr, insulin receptor; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. Figure 7. Schematic illustration of the proposed role of Insr during sprouting angiogenesis. Insr expression is known to be enriched in tip ECs, which migrate along VEGF gradients, leading emerging sprouts during angiogenesis. Knockdown of Insr in ECs impairs VEGF signaling to ERK1/2 as a result of impaired VEGFR2 internalization, which manifests as diminished sprout formation and EC migration. EC, endothelial cell; ERK, extracellular signal- regulated kinase; Insr, insulin receptor; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2. in this setting. 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https://openalex.org/W4385207978
https://jurnal.unimed.ac.id/2012/index.php/handayani/article/download/6528/9534
Indonesian
null
UPAYA MENINGKATKAN BELAJAR PKN MELALUI PENERAPAN MODEL NHT DI KELAS V SDN 050676 KEBUN BALOK
Jurnal handayani
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cc-by-sa
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UPAYA MENINGKATKAN BELAJAR PKN MELALUI PENERAPAN MODEL NHT DI KELAS V SDN 050676 KEBUN BALOK Zaitun Guru Kelas di SD Negeri 050676 Kebun Balok Surel : tesiaite@yahoo.com Abstract: Efforts To Improve Student Learning Outcomes Pkn Through Cooperative Learning Model Application Type NHT In Class V SDN 050676 Garden State Beam.This study aims to improve students' understanding of the material. The research subject of sixth grade students by the number of students as many as 13 people. Student activity data according to the second observation observer in the cycle I and II are Activities writing / reading (37.1%), working LKS (25.9%), asked their peers (13.5%), to ask the teacher (11.8 %), and are not relevant to KBM (11.8%). Activities of students in the second cycle of observation by both observers, among others: writing / reading (24.7%), Working LKS (49.9%), asked their peers (16.5%), ask the teacher (10.6%), and are not relevant to KBM (2.4% ). Keywords: Cooperative Learning type Numbered Heads Together, Learning Outcomes, Learning Activities Keywords: Cooperative Learning type Numbered Heads Together, Learning Outcomes, Learning Activities Abstrak: Upaya Meningkatkan Belajar Pkn Melalui Penerapan Model Pembelajaran Kooperatif Tipe NHT Di Kelas V SDN 050676 Kebun Balok. Penelitian ini bertujuan untuk meningkatkan pemahaman siswa terhadap materi. Subjek penelitian siswa kelas VI dengan jumlah siswa sebanyak 13 orang. Data aktivitas siswa menurut kedua pengamatan pengamat pada siklus I dan II adalah Aktivitas menulis/membaca (37,1%), mengerjakan LKS (25,9% ), bertanya sesama teman (13,5%), bertanya kepada guru (11,8%), dan yang tidak relevan dengan KBM (11,8%). Aktivitas siswa pada Siklus II menurut pengamatan kedua pengamat antara lain: menulis/membaca (24,7%), Mengerjakan LKS (49,9%), bertanya sesama teman (16,5%), bertanya kepada guru (10,6%), dan yang tidak relevan dengan KBM (2,4%). Kata Kunci : Pembelajaran Kooperatif tipe Numbered Heads Together, Hasil Belajar Siswa, Aktivitas Belajar Kata Kunci : Pembelajaran Kooperatif tipe Numbered Heads Together, Hasil Belajar Siswa, Aktivitas Belajar PENDAHULUAN oleh ombak dan tergulung oleh gelombang, serta mudah kehilangan arah dalam melangkah, kecuali bagi yang memiliki daya tahan dan daya-suai yang tinggi serta pedoman dan pegangan hidup yang kuat. Oleh karena itu pembentukan karakter siswa yang aktif, kreatif dan terampil sangat diperlukan, agar kelak siswa mampu bersaing di era globalisasi yang juga mencerminkan kualiatas bangsa dan negaranya. Dalam kehidupan suatu negara, pendidikan memegang peranan yang amat penting untuk menjamin kelangsungan hidup bangsa dan negara, karena pendidikan merupakan wahana untuk meningkatkan dan mengembangkan kualitas sumber daya manusia. Pendidikan adalah usaha sadar dan terencana untuk mewujudkan suasana belajar dan proses pembelajaran agar peserta didik secara aktif mengembangkan potensi dirinya untuk memiliki kekuatan spiritiual keagamaan, pengendalian diri, kepribadian, kecerdasan, akhlak mulia, serta keterampilan yang diperlukan dirinya, masyarakat, bangsa dan negara. Berdasarkan uraian di atas, penulis sebagai tenaga pendidik terdorong agar secara langsung ikut dalam mempersiapkan siswa/siswi yang aktif, kreatif dan terampil demi kelangsungan bangsa dan negara sesuai dengan tujuan pendidikan nasional Indonesia. Hal yang paling nyata penulis lakukan yakni dengan cara meningkatkan kualitas pembelajaran di kelas agar kiranya pembelajaran di sekolah menjadi Di era globalisasi dan pasar bebas sekarang ini manusia dihadapkan pada perubahan-perubahan besar yang tidak menentu dan sulit diprediksi. Manusia ibarat buih di lautan lepas yang mudah terseret p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 146 Jurnal Handayani (JH).Vol 6 (2) Desember 2016, Halm 146- 152 pembelajaran yang menyenangkan bagi siswa yang mampu meningkatkan motivasi belajar siswa, aktivitas belajar siswa dan hasil belajar siswa. (NHT) Numbered Heads Together digunakan untuk mengajarkan isi akademik atau untuk mengetahui sejauh mana pemahaman siswa terhadap suatu materi pelajaran yang diajarkan. Guru menciptakan interaksi yang mendorong rasa ingin tahu, ingin mencoba, bersikap mandiri dan ingin maju. Guru memberikan suatu informasi yang mendasar saja sebagai dasar pemikiran bagi anak didik dalam mencari dan menemukan sendiri informasi lainnya. j Berdasarkan pengalaman penulis selama mengajar, masalah belajar pasti selalu di temukan pada setiap kelas yang penulis ajar. Pada umumnya masalah belajar siswa tergolong pada tiga bagian yakni rendahnya motivasi dan minat belajar siswa, rendahnya aktivitas belajar siswa dan rendahnya hasil belajar siswa. Pembelajaran yang telah dilakukan selama dua bulan setelah memasuki ajaran baru tampak masalah yang dominan dalam pembelajaran adalah aktivitas belajar dan hasil belajar yang rendah. PENDAHULUAN Dalam proses pembelajaran dari 13 siswa yang ada di kelas V hanya 3 orang yang aktif dalam proses pembelajaran, 10 lainnya tampak pasif dan ada seorang siswa yang tidak perduli sama sekali tentang materi pelajaran dan tugas-tugas serta tidak ada interaksi di dalam kelas. Siswa tersebut jarang sekali berbicara dan tugas di sekolah tidak dikerjakan dengan baik seperti teman- teman yang lain. Disamping itu akibat rendahnya aktivitas belajar siswa berdampak besar terhadap hasil belajar siswa. Dari 11 orang hanya 45,5% yang nilai sehari-hari dan ujian bulanan yang mencapai KKM dan sisanya harus mengulang pelajaran untuk memperbaiki nilai. Hal tersebut membuat proses pelajaran terhambat. Penulis telah mengupayakan beberapa inovasi untuk lebih meningkatakan kualitas pembelajaran seperti penerapan model pembelajaran dan pemanfaatan media ajar, namun upaya tersebut belum dapat semaksimal mungkin meningkatakn kualiatas pembelajaran yang akan berimbas pada peningkatan hasil belajar siswa. Keberhasilan penelitian sebelumya yang menerapkan model pembelajaran Inkuiri mendapat pengaruh positif terhadap peningkatan hasil belajar siswa, selanjutnya akan dilakukan penelitin lanjutan pada kelas yang sama dengan model yang berbeda. Oleh karena itu perlu kiranya di lakukan sebuah penelitian untuk melihat efektivitas dari inovasi yang penulis terapkan agar dapat dilihat letak kesalahannya sehingga dapat ditemukan tindakan perbaikan yang tepat agar inovasi yang dilakukan benar-benar optimal. Adapun alternatif pemecahan masalah belajar siswa dalam penelitian ini Dari uraian latar belakang, maka penulis dapat merumuskan permasalahan yang akan dijawab yaitu 1) Apakah penerapan model pembelajaran Numbered Heads Together (NHT) dapat meningkatkan hasil belajar siswa pada bidang studi PKn di kelas VI SD Negeri 050676 Kebun Balok?;2) Apakah penerapan model pembelajaran Numbered Heads Together (NHT) dapat meningkatkan aktivitas belajar siswa pada bidang studi PKn di kelas V SD Negeri 050676 Kebun Balok?. Sehingga berdasarkan rumusan masalah tersebut, penelitian ini bertujuan untuk; 1) Mengetahuui apakah penerapan model pembelajaran Numbered Heads Together (NHT) dapat meningkatkan hasil belajar siswa pada bidang studi PKn di kelas V SD Negeri 050676 Kebun Balok; 2) Mengetahui apakah penerapan model pembelajaran Numbered Heads Together (NHT) dapat meningkatkan aktivitas belajar siswa pada bidang studi PKn di kelas V SD Negeri 050676 Kebun Balok. Model Pembelajaran Kooperatif Tipe Numbered Heads Together (NHT) atau penomoran berpikir bersama adalah merupakan jenis pembelajaran kooperatif yang dirancang untuk mempengaruhi pola interaksi siswa dan sebagai alternatif terhadap struktur kelas. PEMBAHASAN Pembelajaran menggunakan model pembelajaran kooperatif pendekatan struktural Numbered Head Together memberikan kesempatan kepada siswa untuk saling membagikan ide-ide dan mempertimbangkan jawaban yang paling tepat sehingga secara tidak langsung menuntut siswa untuk mau dan mampu mengkonstruksi pengetahuan melalui proses kelompok maupun individu melalui setiap tahapan model pembelajaran di setiap Siklus. Penelitian ini dilaksanakan dalam dua siklus. Masing-masing siklus dilaksanakan dua kali pertemuan, pertemuan digunakan untuk pembahasan materi dengan alokasi waktu 2x35 menit. Hal ini disesuaikan dengan jadwal pelajaran PKn di kelas V SD Negeri 050676 Kebun Balok. Alat pengumpul data dalam penelitian ini adalah: tes hasil belajar untuk mengetahui kemampuan kognitif dan lembar observasi untuk mengamati aktivitas belajar siswa. Penelitian ini berbentuk Penelitian Tindakan Kelas (PTK). PTK pertama kali diperkenalkanoleh psikologi sosial Amerika yang bernama Kurt Lewin pada tahun 1946 (Aqib, 2006 :13). Menurut Lewin dalam Aqib (2006 : 21) menyatakan bahwa dalam satu Siklus terdiri atas empat langkah, yaitu perencanaan (planning), tindakan (acting), observasi (observing) dan refleksi (reflecting). Langkah-langkah pengolahan data sebagai berikut: Sebelum dilaksanakan Siklus I dilakukan uji awal untuk menjajaki kemampuan awal siswa. Hasil pretes menunjukkan nilai rata-rata 39,2 dengan nilai terendah 30 dan tertinggi 60. Dengan ketuntasan minimal (KKM) sebesar 70 maka ketuntasan klasikal hanya sebesar 0%. 1. Merekapitulasi nilai pretes sebelum tindakan dan nilai tes akhir Siklus I dan Siklus II. 2. Menghitung nilai rerata atau persentase hasil belajar siswa sebelum dilakukan tindakan dengan hasil belajar setelah dilakukan tindakan pada Siklus I dan Siklus II untuk mengetahui adanya peningkatan hasil belajar. 2. Menghitung nilai rerata atau persentase hasil belajar siswa sebelum dilakukan tindakan dengan hasil belajar setelah dilakukan tindakan pada Siklus I dan Siklus II untuk mengetahui adanya peningkatan hasil belajar. Nilai hasil Formatif dalam Siklus I disajikan dalam tabel. Tabel Deskripsi Data Formatif I Nilai Frekuensi Rata-rata 40 3 61,5 60 6 80 4 Jumlah 13 3. Untuk penilaian aktivitas digunakan rumus sebagai berikut: 3. Untuk penilaian aktivitas digunakan rumus sebagai berikut: % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠 𝑗𝑢𝑚𝑙𝑎ℎ𝑠𝑘𝑜𝑟𝑦𝑎𝑛𝑔𝑑𝑖𝑝 % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠 % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ % 𝑃𝑟𝑜𝑝𝑜𝑟𝑠𝑖 𝐴𝑘𝑡𝑖𝑣𝑖𝑡𝑎𝑠 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ𝑥 = 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑦𝑎𝑛𝑔 𝑑𝑖𝑝𝑒𝑟𝑜𝑙𝑒ℎ = 𝑗 𝑦 𝑔 𝑝 𝑗𝑢𝑚𝑙𝑎ℎ 𝑠𝑘𝑜𝑟 𝑖𝑑𝑒𝑎𝑙 𝑥 100% (Majid, 2009:268) 4. Ketentuan persentase ketuntasan belajar kelas: 4. Ketentuan persentase ketuntasan belajar kelas: Merujuk pada tabel di atas, nilai terendah formatif I adalah 40 berjumlah 3 orang siswa dan nilai tertinggi 80 berjumlah 4 orang siswa. Zaitun, Upaya Meningkatkan Belajar ... Zaitun, Upaya Meningkatkan Belajar ... demikian, dalam kelompok siswa diberi nomor masing-masing sesuai urutannya. Berkaitan dengan indikator kinerja Suwandi dan Madyo Eko Susilo (2007:36) menyatakan bahwa ”Indikator kinerja merupakan rumusan kinerja yang akan dijadikan dalam menentukan keberhasilan atau keefektifan penelitian. Dalam penelitian ini indikator pencapaian apabila nilai siswa secara individu mencapai KKM Bahasa Indonesia yaitu 70 yang ditetapkan sekolah dan secara klasikal ≥ 85% siswa mencapai KKM tersebut. METODE Pengambilan data untuk penelitian ini dilakukan di SD Negeri 050676 Kebun Balok di Jalam Pendidikan Desa Kebun Balok Kecamatan Wampu Kabupaten Langkat. Waktu penelitian dilakukan selama 5 bulan terhitung mulai bulan Februari sampai bulan Juni tahun 2016. Subjek penelitian adalah siswa-siswi Kelas V SD Negeri 050676 Kebun Balok yang berjumlah 13 orang Tahun Pelajaran 2015/2016. PEMBAHASAN 9 orang siswa mendapat nilai di bawah kriteria ketuntasan minimal dan ketuntasan klasikal sebesar 40,7%. Nilai ini berada di bawah kriteria keberhasilan sehingga dapat dikatakan KBM siklus I kurang berhasil memberi ketuntasan belajar. PENDAHULUAN Numbered Heads Together (NHT) pertama kali dikembangkan oleh Spenser Kagen (1993) untuk melibatkan lebih banyak siswa dalam menelaah materi yang tercakup dalam suatu pelajaran dan mengecek pemahaman mereka terhadap isi pelajaran tersebut (Trianto, 2007: 62). Pengertian lain dari model ini (Istarani, 2011: 12) merupakan rangkaian penyampaian materi dengan menggunakan kelompok sebagai wadah dalam menyatakan persepsi/pikiran siswa terhadap pertanyaan yang dilontarkan atau diajukan guru, yang kemudian akan dipertanggungjawabkan oleh siswa sesuai dengan nomor permintaan guru dari masing-masing kelompok. Dengan Adapun alternatif pemecahan masalah belajar siswa dalam penelitian ini yakni dengan menerapan model pembelajaran Numbered Heads Together (NHT). Model pembelajaran kooperatif tipe p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 147 kelas belajar Ketuntasan % 100   K Sb ΣSb = Jumlah siswa yang mendapat nilai ≥ KKM ΣK = Jumlah siswa % 100   K Sb ΣSb = Jumlah siswa yang mendapat nilai ≥ KKM ΣK J l h i K ΣSb = Jumlah siswa yang mendapat nilai ≥ KKM ΣK = Jumlah siswa 148 Jurnal Handayani (JH).Vol 6 (2) Desember 2016, Halm 146-152 Data hasil formatif I ini dapat disajikan kembali dalam grafik histogram berikut ini. Data hasil formatif I ini dapat disajikan kembali dalam grafik histogram berikut ini. Merujuk pada hasil belajar dan aktivitas belajar siswa serta dokumentasi penelitian, belum tercapainya hasil belajar siswa secara klasikal 85% seperti indikator ketercapaian pada penelitian ini disebabkan oleh : Grafik Data Hasil Formatif I Grafik Data Hasil Formatif I 1. Masih terjadi kesalahan-kesalahan dalam penarikan kesimpulan oleh siswa yang menandakan terjadi kesalahpahaman dalam diskusi kelompok. p 2. Jawaban siswa yang sama dengan jawaban temannya masih banyak hal ini menunjukkan bahwa siswa cenderung untuk menyalin jawaban temannya. Grafik Data Hasil Formatif I Penilaian aktivitas diperoleh dari lembar observasi aktivitas yang dilakukan pada saat siswa bekerja kelompok. Pengamatan dilakukan oleh dua pengamat selama 20 menit kerja kelompok dalam setiap kegiatan belajar-mengajar (KBM). 3. Sebagian siswa masih belum terbiasa dengan alur pembelajaran atau masih bingung diperlihatkan dengan besarnya aktivitas menulis dan membaca yang baru dilakukan di sekolah (42%) yang seharusnya sudah dilakukan di rumah. p g j g j Tabel Aktivitas Belajar Siswa Siklus I Tabel Aktivitas Belajar Siswa Siklus I Tabel Aktivitas Belajar Siswa Siklus I No Aktivitas Jumlah Pro- porsi 1 Menulis/membaca 63 37,1% 2 Mengerjakan LKS 44 25,9% 3 Bertanya pada teman 23 13,5% 4 Bertanya pada guru 20 11,8% 5 Yang tidak relevan dengan KBM 20 11,8% JUMLAH 170 100% 4. Sebagian siswa belum memiliki rasa tanggung jawab terhadap tugasnya dalam kelompok diperlihatkan dengan aktivitas mengerjakan LKS yang hanya mencapai 25,9%. 5. Ada siswa yang lebih suka mengerjakan soal sendiri. 6. Pada tahap pengajuan pertanyaan sebagian besar siswa tidak memusatkan perhatian dalam mengerjakan LKS, terbukti dari tingginya persentase aktivitas yang tidak relevan dengan KBM yaitu 11,8%. Dengan pengamatan setiap dua menit, maka nilai maksimum yang mungkin teramati untuk satu kategori aktivitas selama 20 menit tersebut adalah 10 kali. Nilai aktivitas untuk setiap KBM adalah rata-rata dari nilai aktivitas kedua pengamat. Karena dalam satu siklus terdapat dua KBM, maka nilai aktivitas tiap kategori untuk satu siklus adalah rata-rata dari aktivitas kedua KBM. (5) Pengelolaan waktu diharapkan lebih efisien. Data hasil belajar Siklus II disajikan dalam tabel berikut ini. P d Sikl II t l h k Tabel Deskripsi Data Hasil Formatif II Nilai Frekuensi Rata-rata 80 10 84,6 100 3 Jumlah 13 Tabel Deskripsi Data Hasil Formatif II Tabel Deskripsi Data Hasil Formatif II Nilai Frekuensi Rata-rata 80 10 84,6 100 3 Jumlah 13 Data hasil formatif II ini dapat disajikan kembali dalam bentuk grafik histogram dan dapat dilihat pada grafik berikut ini Data hasil formatif II ini dapat disajikan kembali dalam bentuk grafik histogram dan dapat dilihat pada grafik berikut ini. Pada Siklus II guru telah menerapkan model pembelajaran kooperatit tipe tutor sebaya dengan baik dan dilihat dari aktivitas siswa serta hasil belajar siswa pelaksanaan proses belajar mengajar sudah berjalan dengan baik. Maka tidak diperlukan revisi terlalu banyak, tetapi yang perlu diperhatikan untuk tindakah selanjutnya adalah memaksimalkan dan mepertahankan apa yang telah ada dengan tujuan agar pada pelaksanaan proses belajar mengajar selanjutnya penerapan pembelajaran tuntas dapat meningkatkan proses belajar mengajar sehingga tujuan pembelajaran dapat tercapai. Akan tetapi karena keterbatasan biaya dan waktu dalam desain penelitian maka penelitian direncanakan dalam dua siklus saja. Grafik Hasil Formatif Siklus II Nilai rata-rata tes siswa sebelum menerapkan model pembelajaran kooperatif tipe Numbered Head Together adalah 39,2 dan setelah diterapkan model pembelajaran kooperatif tipe Numbered Head Together meningkat menjadi 61,5 pada Siklus I dan 84,8 pada Siklus II. Ketuntasan belajar secara klasikal juga mengalami peningkatan, sebelumnya hanya ada lima siswa tuntas 70 (ketuntasan secara klasikal 30,7%) pada Siklus I dan pada siklus II menjadi 100%. Dapat disimpulkan KBM Siklus I gagal memberikan ketuntasan klasikal pada siswa sedangkan KBM Siklus II berhasil memberikan ketuntasan klasikal. j Pembelajaran pertemuan pertama pada Siklus I diawali dengan pengelompokkan siswa menjadi 3 kelompok, masing-masing kelompok berjumlah 4-5 orang siswa. Kemudian dari masing-masing kelompok tersebut diberi nomor identitas dalam kelompok dari 1-4. Saat pembelajaran berlangsung masih terlihat siswa belum biasa menangkap alur dan konsep yang diberikan guru saat pembelajaran. Hal ini terlihat dari selama proses pembelajaran sebagian siswa hanya sibuk dengan kegiatan mereka masing- masing meskipun guru sudah menegur mereka. Saat presentasi kelompok, masih banyak siswa yang salah dalam menarik kesimpulan dan ada siswa yang tidak mengikuti alur jalannya diskusi sehingga siswa tersebut tidak dapat memahami isi pelajaran yang sedang berlangsung. Sedangkan, data hasil aktivitas siswa dapat dilihat pada tabel berikut ini. kelas belajar Ketuntasan 7. Waktu yang digunakan dalam kegiatan pembelajaran tidak sesuai dengan waktu yang direncanakan sebelumnya. Perbaikan yang akan dilakukan pada sebelum melakukan Siklus II adalah sebagai berikut: Perbaikan yang akan dilakukan pada sebelum melakukan Siklus II adalah sebagai berikut: (1) Penjelasan dan penekanan kepada siswa tentang pembelajaran kooperatif di antaranya kerja sama dalam menyelesaikan tugas, saling membantu, dan berdiskusi harus diperhatikan. Data aktivitas belajar siswa ditampilkan dalam bentuk diagram dapat dilihat pada grafik berikut ini. Grafik Aktivitas Belajar Siswa Siklus I (2) Pada pelaksanaan siklus II penomoran siswa pada masing-masing kelompok sama dengan pelaksanaan siklus I untuk menghindari siswa saling berebut dalam penentuan nomor. (3) Guru harus lebih sering mengunjungi kelompok pada tahap berpikir bersama berlangsung untuk menghindari siswa ngobrol sendiri di luar materi pelajaran. (4) Guru harus memberikan penghargaan kepada siswa/kelompok yang mendapatkan skor tertinggi. Grafik Aktivitas Belajar Siswa Siklus I p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 149 Zaitun, Upaya Meningkatkan Belajar ... (5) Pengelolaan waktu diharapkan lebih efisien. Data hasil belajar Siklus II disajikan dalam tabel berikut ini. Tabel Deskripsi Data Hasil Formatif II Nilai Frekuensi Rata-rata 80 10 84,6 100 3 Jumlah 13 Data hasil formatif II ini dapat disajikan kembali dalam bentuk grafik histogram dan dapat dilihat pada grafik berikut ini. Grafik Hasil Formatif Siklus II Nilai rata-rata tes siswa sebelum menerapkan model pembelajaran kooperatif tipe Numbered Head Together adalah 39,2 dan setelah diterapkan model pembelajaran kooperatif tipe Numbered Head Together meningkat menjadi 61,5 pada Siklus I dan 84,8 pada Siklus II. Ketuntasan belajar secara klasikal juga mengalami peningkatan, sebelumnya hanya ada lima siswa tuntas 70 (ketuntasan secara klasikal 30,7%) pada Siklus I dan pada siklus II menjadi 100%. Dapat disimpulkan KBM Siklus I gagal memberikan ketuntasan klasikal pada siswa sedangkan KBM Siklus II berhasil memberikan ketuntasan klasikal. Sedangkan, data hasil aktivitas siswa dapat dilihat pada tabel berikut ini. Tabel Data Aktivitas Belajar Siswa Siklus II No Aktivitas Jumlah Pro- porsi 1 Menulis/membaca 42 24,7% 2 Mengerjakan LKS 78 45,9% 3 Bertanya pada teman 28 16,5% 4 Bertanya pada guru 18 10,6% Yang tidak relevan dengan (5) Pengelolaan waktu diharapkan lebih efisien. Data hasil belajar Siklus II disajikan dalam tabel berikut ini. Tabel di atas dapat dikonversi menjadi diagram histogram seperti berikut ini. (5) Pengelolaan waktu diharapkan lebih efisien. Data hasil belajar Siklus II disajikan dalam tabel berikut ini. (5) Pengelolaan waktu diharapkan lebih efisien. Tabel Data Aktivitas Belajar Siswa Siklus Tabel Data Aktivitas Belajar Siswa Siklus II No Aktivitas Jumlah Pro- porsi 1 Menulis/membaca 42 24,7% 2 Mengerjakan LKS 78 45,9% 3 Bertanya pada teman 28 16,5% 4 Bertanya pada guru 18 10,6% 5 Yang tidak relevan dengan KBM 4 2,4% JUMLAH 170 100% Pertemuan kedua pada Siklus I, kegiatan pembelajaran dilaksanakan sama seperti pada pertemuan pertama. Mulai dari pengelompokkan, pemberian nomor pada masing-masing siswa. Pertemuan kedua ini siswa sudah mulai beradaptasi dan aktif saat p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 150 Jurnal Handayani (JH).Vol 6 (2) Desember 2016, Halm 146-152 menyelesaikan tugas, saling membantu, dan berdiskusi harus diperhatikan. menyelesaikan tugas, saling membantu, dan berdiskusi harus diperhatikan. pembelajaran berlangsung. Guru juga ikut memberikan masukkan dalam kerja kelompok jika terdapat perselisihan atau perbedaan pendapat saat diskusi kelompok berlangsung. Kerja sama antarkelompok sudah mulai terlihat. Presentasi kelompok juga berlangsung dengan baik. Namun demikian perolehan nilai rata-rata kelas 61,5 dengan ketuntasan kelas hanya mencapai 30,7%. Sehingga dapat dikatakan ketuntasan klasikal belum tercapai. Hal ini karena pembelajaran Siklus I masih terkendala pada (2) Pada pelaksanaan siklus II, penomoran siswa pada masing-masing kelompok sama dengan pelaksanaan siklus I untuk menghindari siswa saling berebut dalam penentuan nomor. (3) Guru harus lebih sering mengunjungi kelompok pada tahap berpikir bersama berlangsung untuk menghindari siswa bercerita di luar materi pelajaran. (3) Guru harus lebih sering mengunjungi kelompok pada tahap berpikir bersama berlangsung untuk menghindari siswa bercerita di luar materi pelajaran. (3) Guru harus lebih sering mengunjungi kelompok pada tahap berpikir bersama berlangsung untuk menghindari siswa bercerita di luar materi pelajaran. (4) Guru harus memberikan penghargaan kepada siswa/kelompok yang mendapatkan skor tertinggi. (1) Masih terjadi kesalahan-kesalahan dalam penarikan kesimpulan oleh siswa yang menandakan terjadi kesalahpahaman dalam diskusi kelompok. (5) Pengelolaan waktu diharapkan lebih efisien. Hasil yang diperoleh setelah melakukan Siklus II yaitu adanya peningkatan aktivitas belajar siswa. Aktivitas menulis dan membaca menurun sedikit dari 31,7% menjadi 24,7%. Aktivitas mengerjakan LKS dari siklus I dan II memiliki proporsi yang berbeda yaitu 25,9% meningkat pada siklus II 45,9%. Sementara aktivitas bertanya pada teman mengalami peningkatan dari 13,5% menjadi 16,5%. Bertanya pada guru mengalami penurunan dari 11,8% menjadi 10,6%. Dan aktivitas tidak relevan mengalami penurunan dari 11,8% menjadi 2,4%. (2) Jawaban siswa yang sama dengan jawaban temannya masih banyak. Hal ini menunjukkan bahwa siswa cenderung untuk menyalin jawaban temannya. (5) Pengelolaan waktu diharapkan lebih efisien. (3) Sebagian siswa masih belum terbiasa dengan alur pembelajaran atau masih bingung, diperlihatkan dengan besarnya aktivitas menulis dan membaca yang baru dilakukan di sekolah (37,1%) yang seharusnya sudah dilakukan di rumah. (4) Sebagian siswa belum memiliki rasa tanggung jawab terhadap tugasnya dalam kelompok diperlihatkan dengan aktivitas mengerjakan LKS yang hanya mencapai 25,9%. Nilai rata-rata tes siswa sebelum menerapkan model pembelajaran kooperatif tipe Numbered Head Together adalah 39,2 dan setelah diterapkan model pembelajaran kooperatif tipe Numbered Head Together meningkat menjadi 61,5 pada Siklus I dan 84,6 pada Siklus II. Ketuntasan belajar secara klasikal juga mengalami peningkatan, ketuntasan secara klasikal Pretest 0% menjadi 61,5% pada Siklus I dan pada siklus II menjadi 84,6%. Dapat disimpulkan KBM Siklus I gagal memberikan ketuntasan klasikal sedangkan KBM Siklus II berhasil memberikan ketuntasan klasikal. (5) Ada siswa yang lebih suka mengerjakan soal sendiri. (5) Ada siswa yang lebih suka mengerjakan soal sendiri. (6) Pada tahap pengajuan pertanyaan sebagian besar siswa tidak memusatkan perhatian dalam mengerjakan LKS, terbukti dari tingginya persentase aktivitas yang tidak relevan dengan KBM yaitu 12,67%. (7) Waktu yang digunakan dalam kegiatan pembelajaran tidak sesuai dengan waktu yang direncanakan sebelumnya. Dengan demikian dapat dikatakan bahwa proses pembelajaran menggunakan model pembelajaran kooperatif tipe Numbered Heads Together (NHT) dapat meningkatkan aktivitas belajar dan ketuntasan pembelajaran siswa pada pembelajaran PKn di Kelas V SD Negeri 050676 Kebun Balok. Namun, dapat juga peneliti sampaikan bahwa terjadinya peningkatan aktivitas belajar dan hasil belajar siswa ini dimungkinkan karenakan siklus yang berulang. Dengan demikian, hal ini juga dapat dijadikan sebagai bahan kajian lanjutan. Sehingga pada penelitian ini masih dilanjutkan pada Siklus II untuk mencapai ketuntasan kelas minimal 85%. Pembelajaran pertemuan pertama pada Siklus II dikondisikan sama seperti pada Siklus I, namun ada beberapa perbaikan pada kelemahan-kelemahan yang terjadi saat pembelajaran pada Siklus I, diantaranya : (1) Penjelasan dan penekanan kepada siswa tentang pembelajaran kooperatif di antaranya kerja sama dalam p-ISSN: 2355 - 1739 e-ISSN: 2407 - 6295 151 Zaitun, Upaya Meningkatkan Belajar ... Suwandi, Sarwiji dan Madyo Ekosusilo, 2007, Pendidikan dan Latihan Profesi Guru (PLPG), Panitia Sertifikasi Guru Rayon 13, Surakarta. Zaitun, (2016), Implementasi Model Pembelajaran Kooperatif Tipe Numbered Heads Together (NHT) Untuk Meningkatkan Aktivitas Belajar Pkn Di Kelas V Sd Negeri 050676 Kebun Balok Semester Genap T.P. 2015/2016, UD Toma, Medan. Data aktivitas siswa menurut kedua pengamatan pengamat pada siklus I dan II adalah Aktivitas menulis/membaca (37,1%), mengerjakan LKS (25,9% ), bertanya sesama teman (13,5%), bertanya kepada guru (11,8%), dan yang tidak relevan dengan KBM (11,8%). Aktivitas siswa pada Siklus II menurut pengamatan kedua pengamat antara lain: menulis/membaca (24,7%), Mengerjakan LKS (49,9%), bertanya sesama teman (16,5%), bertanya kepada guru (10,6%), dan yang tidak relevan dengan KBM (2,4%). Dengan menerapkan model pembelajaran Kooperatif tipe numbered heads together, aktivitas belajar siswa dari Siklus I ke Siklus berikutnya mengalami peningkatan. KESIMPULAN Berdasarkan analisis data dan pembahasan maka dapat disimpulkan hasil penerapan model pembelajaran kooperatif tipe Numbered Heads Together dalam pembelajaran PKn di Kelas V SD Negeri 050676 Kebun Balok tahun pembelajaran 2015/2016 berikut ini: Trianto, (2010), Mendesain Model Pembelajaran Inovatif-Progresif, Penerbit Kencana Prenada Media Group, Jakarta. Hasil penelitian menunjukkan bahwa pembelajaran dengan menggunakan model pembelajaran Numbered Heads Together dapat meningkatkan hasil belajar PKn siswa kelas V SDN. Melihat hasil analisis data penelitian menunjukkan bahawa hasil tes hasil belajar siswa pada Siklus I meningkat siknifikan dbandingkan dengan siklus II. Dari hasil tes hasil belajar pada Siklus I yang tuntas sebanyak 4 orang (30,7%), sedangkan secara kelas belum tuntas. Dari hasil tes hasil belajar pada Siklus I yang tuntas sebanyak 13 orang (100%) secara kelas dikatakan tuntas karena jumlah siswa yang memperoleh nilai diatatas nilai KKM (70) lebih dari 85%. DAFTAR RUJUKAN Aqib, Zainal, 2006, Penelitian Tindakan Kelas, Penerbit Yrama Widya, Bandung. Istarani,(2012).58 Model Pembelajaran Inovatif, Media Persada, Medan. Istarani,(2012).58 Model Pembelajaran Inovatif, Media Persada, Medan. Majid, Abdul, 2009, Perencanaan Pembelajaran Mengembangkan Standar Kompetensi Guru, Penerbit PT. Remaja Rosdakarya Offset, Bandung. 152
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Healthcare workers’ compliance with COVID-19 preventive and control measures at De Martino Hospital, Mogadishu, Somalia: a cross-sectional study
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Healthcare workers’ compliance with COVID-19 preventive and control measures at De Martino Hospital, Mogadishu, Somalia: a cross-sectional study Abdullahi Ibrahim Janay Dokuz Eylül University Bulent Kilic  Dokuz Eylül University Belgin Unal  Dokuz Eylül University DOI: https://doi.org/10.21203/rs.3.rs-4232936/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Page 1/19 Page 1/19 Research Article Keywords: Compliance, Healthcare workers, COVID-19 prevention and control, Hospital Posted Date: April 19th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4232936/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Results In total, 58.3% of the participants had good compliance with IPC. There were significant associations between IPC compliance and the type of healthcare worker (doctors and doctor assistants: 72.3%, nurses and paramedical staff: 67.3%, non-clinical staff: 5.7%, p < 0.01). After adjusting for potential confounding factors, compared to non-clinical staff, doctors and doctor assistants (OR: 12.11, 95% CI: 2.23–65.84) and nurses and paramedical staff (OR: 21.38, 95% CI: 4.23–108.01) had higher compliance with infection prevention and control measures. There were no significant associations between compliance and sex, marital status, vaccination status, or smoking (p > 0.05 for all). Conclusions Inadequate compliance with COVID-19 IPC measures was observed among hospital workers. Prioritizing awareness campaigns and behavior change interventions, especially among non-clinical staff, is crucial for effective COVID-19 infection prevention and control within hospitals. Methods A cross-sectional study was conducted at De Martino Public Hospital, Mogadishu, Somalia from August to October 2022, with the participation of 204 healthcare workers (response rate = 97%). Compliance was assessed using responses to 25 questions on a five-point Likert-type scale, and a median score of 20 was used to dichotomize compliance scores. A chi-square test and logistic regression analysis were performed to check the associations between healthcare workers’ sociodemographic information, related factors to IPC, work conditions and practices on COVID-19, and IPC compliance during healthcare interventions using SPSS 23 version. Background Healthcare workers are a high-risk group for COVID-19 and protecting them is crucial for healthcare delivery. Limited studies have explored compliance with infection prevention and control (IPC) practices among Somali healthcare workers. This study aimed to determine compliance with IPC practices among healthcare workers in De Martino Public Hospital, Somalia. Introduction All age groups are susceptible to the virus, but elderly people with underlying health conditions are at greater risk of severe illness [4]. Healthcare workers (HCWs) are also vulnerable, emphasizing the importance of infection prevention and control (IPC) practices and personal protective equipment (PPE) use [5]. IPC strategies include non-pharmaceutical interventions such as school and workplace closures, event bans, stay-at-home orders, and movement restrictions, which have reduced transmission [6]. Personal protective measures are also an important component of COVID-19 infection prevention and control [7]. Vaccines have been developed, but their effectiveness varies by variant, with better protection against severe outcomes. Vaccine effectiveness against symptomatic disease was greater for the delta variant than for the omicron variant. With previous variants, vaccine effectiveness against severe disease, including hospitalization and death, has been higher and retained for longer than effectiveness against mild disease [8]. The World Health Organization (WHO) declared that COVID-19 is an emergency public health problem of international concern that posed a high risk to countries with vulnerable health systems on January 30, 2020 [9]. Although the Director General of the WHO declared the end of COVID-19 as a public health emergency and is no longer a global threat on May 5th 2023, he said that COVID-19 is still killing and warned of the risks from the new emerging variants [10]. In fragile settings like Somalia where the number of HCWs is significantly lower than that required for Sustainable Development Goals, the protection of health workers is vital [11]. To protect HCWs, Enough supplies of PPE should be ensured and HCWs should be trained in using it. The environmental hygiene of hospitals and the personal hygiene of HCWs should be maintained [2]. HCWs should maintain contact reduction to reduce the number of infections [7]. It was reported from China that the potential risk of COVID-19 has largely improved the IPC behaviors of HCWs working in hospitals [12]. Determination of the compliance of hospital workers with the COVID-19 IPC practices and the factors affecting this compliance is important for the protection of hospital workers during the pandemic. Studies assessing COVID-19 IPC practices in Somali hospitals and HCWs are limited. This cross-sectional study assessed COVID-19 IPC compliance among HCWs at De Martino Public Hospital during healthcare interventions and determined the factors affecting compliance to improve hospital workers’ compliance with COVID-19 IPC practices. Introduction The COVID-19 pandemic was the most recent and worst pandemic in the 21st century, and resulted in 765 million confirmed cases and 6.9 million deaths worldwide as of May 3, 2023 [1]. Page 2/19 Page 2/19 This highly infectious virus can be transmitted by asymptomatic carr making isolation until testing negative crucial [2]. Quarantine measur endemic areas or with confirmed contact and for mild cases not requ groups are susceptible to the virus, but elderly people with underlying severe illness [4]. Healthcare workers (HCWs) are also vulnerable, em prevention and control (IPC) practices and personal protective equip IPC strategies include non-pharmaceutical interventions such as sch bans, stay-at-home orders, and movement restrictions, which have re protective measures are also an important component of COVID-19 i Vaccines have been developed, but their effectiveness varies by varia severe outcomes. Vaccine effectiveness against symptomatic diseas for the omicron variant. With previous variants, vaccine effectiveness hospitalization and death, has been higher and retained for longer th [8]. The World Health Organization (WHO) declared that COVID-19 is an e international concern that posed a high risk to countries with vulnera 2020 [9]. Although the Director General of the WHO declared the end emergency and is no longer a global threat on May 5th 2023, he said warned of the risks from the new emerging variants [10]. In fragile se of HCWs is significantly lower than that required for Sustainable Dev health workers is vital [11]. To protect HCWs, Enough supplies of PPE be trained in using it. The environmental hygiene of hospitals and the maintained [2]. HCWs should maintain contact reduction to reduce th It was reported from China that the potential risk of COVID-19 has lar HCWs working in hospitals [12]. Determination of the compliance of practices and the factors affecting this compliance is important for t during the pandemic. Studies assessing COVID-19 IPC practices in S This cross-sectional study assessed COVID-19 IPC compliance amon during healthcare interventions and determined the factors affecting workers’ compliance with COVID-19 IPC practices. Methods This highly infectious virus can be transmitted by asymptomatic carriers and those in the incubation period, making isolation until testing negative crucial [2]. Quarantine measures are essential for persons from endemic areas or with confirmed contact and for mild cases not requiring medical attention [3]. Study population and sample size The study population is 210 HCWs working at De Martino Hospital between August and November 2022. HCWs include all hospital staff who provide health services directly or indirectly, such as managers, secretaries, doctors, nurses, laboratory technicians, radiologists, pharmacists, cleaners, security personel, or other personnel. All HCWs working in the hospital were included in the study. In total, 204 HCWs were reached during data collection in the study and their informed consent was obtained using a written form. Methods This hospital-based cross-sectional study aimed to assess COVID-19 IPC compliance among HCWs at De Martino Public Hospital during healthcare interventions and to determine the factors affecting their compliance. De Martino Public Hospital is located in Mogadishu, Somalia’s capital. It was built during Italy’s colonial era and is currently under the control of the Ministry of Health and Human Services of the Federal Government of Somalia. It provides free medical care, especially for inpatients. During the COVID-19 Page 3/19 Page 3/19 pandemic, the hospital exclusively served COVID-19 patients. The hospital has 115 beds and offers a range of services, including polyclinics, inpatient care, emergency services, and intensive care. In 2019, there were a total of 61 health facilities run by the government in the Benadir region, which encompasses the capital city [13]. De Martino Hospital in this region serves as a secondary referral hospital (RH) indicating its role in providing specialized care. It’s important to note that while RHs like De Martino Hospital offer valuable specialist services, they may face challenges in meeting the extensive demand due to limited capacity and resources [14]. During the COVID-19 pandemic, De Martino Hospital emerged as a key center for managing COVID-19 cases, playing a vital role in the national response. This underscores its importance in addressing public health challenges, even during unprecedented events like a global pandemic. In 2019, there were a total of 61 health facilities run by the government in the Benadir region, which encompasses the capital city [13]. De Martino Hospital in this region serves as a secondary referral hospital (RH) indicating its role in providing specialized care. It’s important to note that while RHs like De Martino Hospital offer valuable specialist services, they may face challenges in meeting the extensive demand due to limited capacity and resources [14]. During the COVID-19 pandemic, De Martino Hospital emerged as a key center for managing COVID-19 cases, playing a vital role in the national response. This underscores its importance in addressing public health challenges, even during unprecedented events like a global pandemic. The study variables To assess HCWs’ compliance with COVID-19 prevention and control practices (IPC), the use of personal practices during their healthcare interactions in the hospital was measured using a self-report questionnaire. Accordingly, the questionnaire was created under three main headings; personal protective equipment (PPE), hand washing and hygiene (HH) and other COVID-19 IPC practices. The data on personal characteristics, related factors to IPC, work conditions and practices on COVID-19 were also included in the questionnaire. The questionnaire was adapted from the WHO’s risk assessment tool for healthcare workers in the context of COVID-19 [15], with some modifications according to the suitability of the hospital facility and using literature guidelines. The questionnaire is attached as "Supplementary file 1". The anonymity and confidentiality of the participants were ensured; a specific number was given to every questionnaire, and there was no information that could identify individual participants during or after data collection. A pilot study was conducted initially; then, the questionnaire was distributed to the hospital staff and the data were collected between August and November 2022. There were 25 questions measuring the compliance of HCWs with COVID-19; 12 questions for PPE, 7 questions for HH and 6 questions for other COVID-19 IPC IPC on the questionnaire. Responses to each question were measured using a 5-point Likert scale: "always (5 points), often (4 points), sometimes (3 points), rarely (2 points), or never (1 point)". The scores of each person’s responses were categorized into two categories and the answers "always" and "most of the time" were taken as compliance. Then, the compliance responses of each participant were summed and the median was taken into account as the cut-off point; scores above the median were considered “high compliance”. Data analysis Page 4/19 For descriptive analyses, the variables measured by scale values were converted into categorical ones, and all independent variables are presented as percentages. To determine the association between independent variables and high compliance with the COVID-19 IPC measures, the chi-square test was used by converting the scores of compliance with the COVID-19 IPC measures into two categories, and the answers "always" and "most of the time" were taken as “compliance”. Then, the compliance responses of each participant were collected and the median was considered the cut-off point. Scores above the median were considered “high compliance” and presented by percentage for descriptive analysis. To identify the key independent factors associated with a high level of compliance with COVID-19 IPC measures, variables that showed significant associations with high compliance in univariate analyses were included in logistic regression models. The backward LR method was used to identify predictive variables associated with compliance with IPC measures and the association was presented with odds ratio and 95% confidence interval. The Statistical Package for Social Sciences (SPSS 23) was used for the data analysis. P values lower than 0.05 were considered significant. Results In total, data from 204 HCWs were analyzed, achieving a response rate of 97%. Among the participants, 51% were male (Table 1, column 4). The majority of HCWs (46.1%) fell in the 20 to 29 age group, while only 15.7% were aged 40 and above (Table 1, column 5). Approximately, 77.8% held bachelor’s degrees or higher (Table 1, column 6). Nurses and paramedical staff constituted the largest group (51%), followed by (31.9%) doctors and doctor assistants (Table 2, column 4). Outpatient workers accounted for 20.1% of the participants, while inpatient workers made up 26.5%. HCWs in other clinical departments comprised 29.9% of the total (Table 2, column 5). Compliance with COVID-19 IPC practices among healthcare workers The total IPC compliance rate was 58.3% among the participants. HCWs demonstrated 55.9% compliance with PPE during healthcare interactions, 55.4% compliance with HH, and 52% compliance other COVID-19 IPC measures during healthcare interactions (Fig. 1). Figure 1. COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % Results Page 5/19 Table 1 COVID-19 IPC compliance in HCWs during healthcare provision by sociodemographic characteristics and related factors to IPC Independent variables n % COVID-19 IPC compliance Total IPC% PPE use% HH% Other IPC% High p* High p* High p* High p* Sex Male Female 105 98 51.7 48.3 60 56.1 0.58 61 51 0.15 55.2 55.1 0.98 52.4 51.0 0.85 Age group 20–29 30–39 40 and above 94 78 32 46.1 38.2 15.7 62.6 61.5 37.5 0.03 59.6 59 37.5 0.07 62.8 55.1 34.4 0.02 57.4 44.9 53.1 0.26 Education level -Secondary degree and below -Associate degree -Undergraduate and graduate degrees 23 22 158 11.3 10.8 77.8 8.7 54.5 66.5 <  0.01 13 50 63.3 <  0.01 8.7 45.5 63.9 <  0.01 26.1 54.5 55.7 0.03 Experience years 1–5 6–10 11 and above 118 59 20 59.9 29.9 10.2 58.5 64.4 50 0.50 57.6 59.3 45 0.52 58.5 57.6 45 0.53 55.9 45.8 50 0.43 Marital status Married Unmarried Divorced/Widow 102 16 84 50.5 7.9 41.6 57.8 56.3 59.5 0.96 55.1 50 59.5 0.66 53.9 64.3 57.1 0.91 50 31.3 58.3 0.12 Having a child Yes No 107 94 53.2 46.8 57.9 58.5 0.94 55.1 57.4 0.74 54.2 57.4 0.64 47.7 57.4 0.17 Training on COVID-19 171 83.8 61.4 0.04 59.1 0.04 56.9 0.63 53.2 0.41 Table 1 Independent variables n % COVID-19 IPC compliance Total IPC% PPE use% HH% Other IPC% High p* High p* High p* High p* Yes No 33 16.2 42.4 39.5 51.5 45.5 COVID-19 information From official sources From social media 85 113 42.9 57.1 48.2 67.3 0.01 41.2 69 <  0.01 40 69 <  0.01 43.5 60.2 0.02 COVID-19 infection Yes No Unknown 95 46 63 46.6 22.5 30.9 66.3 41.3 58.7 0.02 62.1 26.2 60.3 0.01 58.9 39.1 61.9 0.04 50.5 47.8 57.1 0.59 COVID-19 Vaccination Yes No 180 24 88.2 11.8 59.4 50 0.38 57.2 45.8 0.29 56.7 45.8 0.32 50.6 62.5 0.27 Vaccination doses 0 1 2 and above 24 56 124 11.8 27.5 60.8 50 48.2 64.5 0.08 45.8 55.3 58.1 0.54 45.8 55.4 57.3 0.59 62.5 42.9 54 0.21 Smoking cigarette Yes No 33 168 16.4 83.6 48.5 60.1 0.22 48.5 57.1 0.36 39.4 58.3 0.05 36.4 54.8 0.05 An old /chronic patient in the family Yes No 103 96 50.8 48.2 52.5 63.5 0.11 52.4 58.3 0.40 50.5 59.4 0.21 50.5 54.2 0.60 *Chi t t Page 7/19 Table 2 COVID-19 IPC compliance in HCWs during healthcare provision by working conditions and practices on COVID-19 Independent variables n % COVID-19 IPC compliance Total IPC% PPE use% HH% Other IPC% High p* High p* High p* High p* Profession Doctors and doctor assistants Nurses and Paramedical staff Non-clinical staff 65 104 35 31.9 51 17.2 72.3 67.3 5.7 <  0.01 66.2 66.3 5.7 <  0.01 70.8 60.2 11.4 <  0.01 46.2 61.5 34.3 0.01 Department Outpatient Inpatient Other clinical departments Non-clinical departments 41 54 61 48 20.1 26.5 29.9 23.5 68.3 70.4 70.5 20.8 <  0.01 65.9 64.8 70.5 18.8 <  0.01 65.9 62.9 70.5 18.8 <  0.01 46.9 53.7 59 45.8 0.47 Direct COVID-19 care Yes No Unknown 109 56 39 53.4 27.5 19.1 67.9 37.5 61.5 <  0.01 66.1 30.4 64.1 <  0.01 62.4 35.7 64.1 <  0.01 49.5 51.8 59 0.60 Face-to-face contact with COVID-19 patient Yes No Unknown 109 63 30 53.9 31.2 14.9 75.2 31.7 53.3 <  0.01 72.5 28.6 53.3 <  0.01 68.8 33.3 53.3 <  0.01 54.1 50.8 50 0.88 Direct contact with COVİD-19 environment Yes No 95 66 41 47 32.7 25.3 69.5 41 58.5 <  0.01 70.5 33.3 56.1 <  0.01 57.9 45.5 63.4 <  0.14 52.6 47 58.5 0.50 h Page 8/19 Independent variables n % COVID-19 IPC compliance Total IPC% PPE use% HH% Other IPC% High p* High p* High p* High p* Unknown Presence at AGP performance Yes No Unknown 112 76 13 55.7 37.8 6.5 82.1 27.6 30.8 <  0.01 77.7 26.3 38.5 <  0.01 78.6 31.6 7.7 <  0.01 56.3 44.7 53.8 0.30 Type of AGP procedure -Not applicable -Open airway aspiration +Sputum collection +Tracheotomy -Tracheal intubation +Cardiopulmonary resuscitation -Nebulizer treatment -More than one 89 12 25 42 32 44.5 6 12.5 21 16 28.1 83.3 84 73.8 93.8 <  0.01 28.1 83.3 80 69 84.4 <  0.01 28.1 83.3 80 69 90.6 <  0.01 46.1 50 60 50 65.6 0.36 *Chi square test Note: N: Number of participants, IPC: COVID-19 Infection Prevention and Control, PPE: Personal Protective Note IPC: infection prevention and control, PPE: personal protective equipment, HH: hand hygiene. Page 9/19 Page 9/19 Age and educational status were significantly associated with overall COVID-19 IPC and HH compliance (P  < 0.05 for all). Educational status was associated with compliance with PPE (p < 0.01) and other COVID-19 IPC measures (p = 0.03). Younger age groups and higher education level groups expressed better compliance (Table 1). There was no association between any of the IPC, PPE, HH compliance and marital status, having a child at home, having an old/chronic disease patient at home, or having COVID-19 vaccination. There was no significant association between the IPC dimensions and work experience years (Table 1). Participants who received training on COVID-19 reported higher compliance with total IPC and PPE than those who did not receive training (p < 0.05). There was a significant association between the type of HCW and overall compliance with COVID-19 IPC, PPE and HH; non-clinical staff members showed significantly lower levels of compliance for all three dimensions (p < 0.01 for all). The same pattern was observed in the departments where HCWs worked; non- clinical departments showed lower compliance (p < 0.01 for all). HCWs not providing direct care to COVID- 19 patients, not having face-to-face contact, and not being present during the AGP performance reported lower compliance (p < 0.01 for all). HCWs not having direct contact with the COVID-19 environment reported lower compliance in total IPC and PPE use (p < 0.01 for all). There was no significant association with any of the working conditions or practices on COVID-19 and compliance with other IPC measures except the type of HCW (p = 0.01) (Table 2). In the logistic regression model, profession, having training on COVID-19, providing direct care to COVID-19 patients and presence when performing AGP were independent factors associated with total IPC compliance among HCWs. Doctors and doctor assistants and nurses and paramedical staff expressed a higher level of compliance with overall IPC compared to non-clinical staff (Doctors OR: 12.11, 95% CI: 2.23– 65.84, nurses and paramedical staff OR: 21.38, 95% CI: 4.23–108.01). HCWs who received training on COVID-19 exhibited higher COVID-19 compliance than those who did not (OR: 3.48, 95% CI: 1.06–11.35). HCWs who were present when AGPs were performed exhibited higher compliance compared to those uncertain about that (OR: 12.45, 95% CI: 12.16–71.76) (Table 3). Page 9/19 In the logistic regression model, profession, having training on COVID-19, providing direct care to COVID-19 patients and presence when performing AGP were independent factors associated with total IPC compliance among HCWs. Doctors and doctor assistants and nurses and paramedical staff expressed a higher level of compliance with overall IPC compared to non-clinical staff (Doctors OR: 12.11, 95% CI: 2.23– 65.84, nurses and paramedical staff OR: 21.38, 95% CI: 4.23–108.01). HCWs who received training on COVID-19 exhibited higher COVID-19 compliance than those who did not (OR: 3.48, 95% CI: 1.06–11.35). HCWs who were present when AGPs were performed exhibited higher compliance compared to those uncertain about that (OR: 12.45, 95% CI: 12.16–71.76) (Table 3). Page 10/19 Page 10/19 Page 10/19 Table 3 Independent determinants of COVID-19 IPC compliance in HCWs during healthcare provision: Results of multivariate logistic regression analysis* (Backward elimination method)   COVID-19 IPC compliance Variables in the model Total IPC OR (95% CI) PPE use OR (95% CI) HH OR (95% CI) Other IPC OR (95% CI) Profession         Doctors & doctor assistants 12.11 (2.23– 65.84) 11.59 (1.94– 69.01) - 1.35 (0.56– 3.24) Nurses & paramedical staff 21.38 (4.23– 108.01) 17.91 (3.22– 99.64) - 2.59 (1.13– 5.93) Non-clinical staff Ref. Ref. - Ref. Department         Inpatient - - 4.97 (1.62– 15.21) - Other clinical departments - - 6.2 (2.1– 18.35) - Outpatient - - 4.21 (1.34– 13.23) - Non-clinical departments - - Ref. - COVID-19 material         From official sources - 0.38 (0.17– 0.83) 0.45 (0.22– 0.92) - From social media - Ref. Ref. - Direct COVID-19 care         Provide 0.26 (0.07– 0.98) 1.02 (0.38– 2.76) - - Unknown 0.9 (0.2–3.99) 4.10 (1.12– 15.03) - - Does not provide Ref. Ref. - - Face-to-face contact with COVID-19 patient         Have 1.5 (0.27–8.07) - 1.06 (0.32– 3.51) - Does not have 0 37 (0 07– 0 4 (0 12– Table 3 Independent determinants of COVID-19 IPC compliance in HCWs during healthcare provision: Results of multivariate logistic regression analysis* (Backward elimination method) of COVID-19 IPC compliance in HCWs during healthcare provision: Results of logistic regression analysis* (Backward elimination method) Independent determinants of COVID-19 IPC compliance in HCWs during heal multivariate logistic regression analysis* (Backward eliminati COVID-19 IPC compliance COVID-19 IPC compliance Direct contact with COVID-19 environment         Have - 2.59 (0.88– 7.6) - - Does not have - 1.08 (0.36– 3.23) - - Unknown - Ref. Page 9/19 Nurses and paramedical staff expressed higher compliance compared to non-clinical staff (OR: 2.59, 95% CI: 1.13–5.93) (Table 3). Page 9/19 - - Presence at AGP performance         Present 12.45 (2.16– 71.76) 4.36 (0.8– 23.79) - - Not present 1.59 (0.3–8.21) 0.7 (0.13– 3.52) - - Unknown Ref. Ref. - - Training on COVID-19         Received 3.48 (1.06– 11.35) 2.66 (0.93– 7.66) - - Did not receive Ref. Ref. - - Education level         Undergraduate and graduate degrees - - 8.48 (1.64– 43.94) - Associate degree - - 3.67 (0.56– 24.04) - Secondary degree and below - - Ref. - *Variables included in the logistic regression model For the total COVID-19 IPC: age group, education, receiving training, source of COVID-19 information, covid infection, type of HCW, type of department, providing direct COVID-19 care, Having face-to-face contact with COVID-19 patients, having direct contact with the COVID-19 environment, presence at AGP performance and type of AGP. For PPE: education, training, source of COVID-19 information, covid infection, Type of HCW, Type of department, providing direct COVID-19 care, having face-to-face contact with COVID-19 patients, having direct contact with the COVID-19 environment, presence at AGP performance and type of AGP. For HH: age group, education, source of COVID-19 information, COVID-19 infection, type of HCW, type of department, providing direct COVID-19 care, having face-to-face contact with COVID-19 patients. For Other COVID-19 IPC: education, source of COVID-19 information, Type of HCW. Note: IPC: Infection Prevention and Control, OR: Odds Ratio, CI: Confidence Interval, PPE: Personal Protective Equipment HH: Hand Hygiene AGP: Aerosol Generating Procedure Page 12/19 Page 12/19 For PPE; profession, the source of COVID-19 material and providing direct care to COVID-19 patient were independent factors. Compliance with PPE showed significant differences among HCWs in various roles. Doctors and doctor assistants (OR: 11.59, 95% CI: 1.94–69.01) and nurses and paramedical staff (OR: 17.91, 95% CI: 3.22–99.64) exhibited higher compliance compared to non-clinical staff. HCWs who accessed COVID-19 information from official sources expressed lower compliance compared to those who accessed information from social media (OR: 0.38, 95% CI: 0.17–0.83) (Table 3). For HH, departments where HCWs worked, source of COVID-19 material and education level were independent factors. Clinical departments showed higher compliance compared to non-clinical departments (inpatient OR: 4.97, %95 CI: 1.62–15.21, outpatient OR: 4.21, %95 GA: 1.34–13.23, other clinical departments OR: 6.2, %95 GA: 2.1–18.35). HCW participants who read COVID-19 related material from Official sources reported lower compliance than those who read from social media (OR: 0.45, 95% CI: 0.22–0.92) (Table 3). For Other IPC compliance, profession was the only predictor. Discussion This study was designed to assess COVID-19 IPC compliance among HCWs during healthcare interventions and to determine the factors affecting compliance. The study showed that 58.3% of HCWs had high compliance with COVID-19 IPC measures during healthcare interventions. The compliance was over 50% in all domains (PPE use, HH, and other IPC). Non-clinical staff had lower compliance compared with clinical staff. There are several studies that reported high compliance with COVID-19 IPC among HCWs. In Ghana, a study with 424 HCWs in COVID-19 treatment centers reported high compliance with hand hygiene (88.4%) and PPE usage (90.6%) [16]. Two Ethiopian studies, involving 403 and 422 participants, reported good COVID-19 infection prevention practices in 64.3% and 63.5% of healthcare workers, respectively. The first study revealed 96.1% compliance with hand hygiene but only 45.2% compliance with PPE usage, possibly due to PPE availability, comfort, negligence, or education [17, 18]. Compared to previous studies, our findings indicated lower IPC compliance rates. This variance may be attributed to differences in study methods and the timing of data collection. While our study used compliance scores above the median for each domain, the referenced studies used either above-average scores or cutoff points of 60% or 75% of total compliance scores. It is also possible that healthcare workers’ adherence to IPC measures decreased over time since the pandemic’s onset. A study on healthcare worker HH practices revealed a 13.7% increase upon room exit during the initial COVID-19 wave. Compliance decreased by 9.9% post-lockdown but rebounded by 2.8% in the second wave [19]. Some studies have reported low HCW compliance with COVID-19 IPC measures. For instance, in a study involving 422 HCWs at COVID-19 referral hospitals in Ethiopia, overall compliance with COVID-19 prevention practices was only 22% [20]. In this study, only 63.4% of the participating HCWs received training on COVID- Page 13/19 Page 13/19 19, 58.2% read COVID-19 materials and 83.2% of the HCWs felt a shortage of appropriate PPE in the hospital. 19, 58.2% read COVID-19 materials and 83.2% of the HCWs felt a shortage of appropriate PPE in the hospital. A review identified various barriers to HCWs’ compliance with IPC guidelines for respiratory infectious diseases, including the availability of training programs, PPE supply, and individual factors such as knowledge, attitudes, beliefs, and PPE discomfort [21]. Discussion Additionally, a study conducted in Uganda; involving 657 HCWs at community hospitals, revealed that only 37.0% of participants had good COVID-19 IPC practices, despite high rates of mask usage and hand washing [22]. In our study, doctors, doctor assistants, nurses, and paramedical staff demonstrated higher COVID-19 IPC compliance than non-clinical staff, except in HH practices. This aligns with prior research. For instance, a study performed in private-not-for-profit community hospitals in Uganda revealed a significant association between clinical HCWs and good COVID-19 IPC practices [22]. Similarly, a study in COVID-19 treatment centers in Ghana revealed that non-clinical staff exhibited significantly lower compliance with hand hygiene and PPE usage compared to clinical staff [16]. The difference in compliance may be due to the higher risk faced by clinical healthcare workers in close contact with COVID-19 patients. A study from Somalia, reported that ancillary staff, including security workers and cleaners, have a higher infection risk due to lower knowledge and adherence to infection control measures when handling suspected COVID-19 patients. Healthcare assistants are often informally employed and receive less attention than formal employees such as doctors, nurses, and technologists [11]. Our study showed that HCWs who were present during AGPs exhibited higher compliance with overall IPC measures compared to those who were uncertain about that. Our study aligns with Ashinyo ME et al.’s study that reported high compliance with COVID-19 IPC protocols during AGPs [16]. Healthcare workers performing AGPs face a higher risk of COVID-19 infection, possibly explaining their heightened compliance [23]. Our study also showed that HCWs who had received training on COVID-19 exhibited higher COVID-19 compliance with overall IPC than those who did not. This is in line with 2 studies on 422 HCWs in Ethiopia; Etafa W. et al [20] and Arsemahagn MA [24], and a review study by Cooper S. et al. [25]. Our study also showed that HCWs who had received training on COVID-19 exhibited higher COVID-19 compliance with overall IPC than those who did not. This is in line with 2 studies on 422 HCWs in Ethiopia; Etafa W. et al [20] and Arsemahagn MA [24], and a review study by Cooper S. et al. [25]. Interestingly, our study revealed that HCWs who obtained COVID-19 information from official sources showed lower compliance with PPE and HH compared to those who accessed information from social media. Discussion In contrast, a study on the Somali population reported the opposite, where HCWs who relied on social media for COVID-19 information exhibited lower compliance, likely due to misinformation [26]. However, De Martino HCWs may follow specific social media pages they trust for COVID-19 information. To improve compliance, HCWs must receive continuous awareness and training in COVID-19 IPC guidelines. Policymakers should develop comprehensive programs to increase awareness among HCWs at all levels and provide the necessary equipment and supplies for effective IPC practices in healthcare settings. This study has limitations. Firstly, there could be recall bias; because participants were asked about their compliance with COVID-19 IPC measures during the late stages of the pandemic when no COVID-19 Page 14/19 Page 14/19 patients were likely admitted, and some PPE like respirators, gowns, and face shields might not have been used. To minimize this bias, we asked about daily IPC practices and included specific questions for suspected or confirmed COVID-19 patients. Since the study was a single-center study, De Martino Public Hospital may not fully represent other pandemic hospitals in Somalia, although it plays a significant role in treating COVID-19 patients. Nonetheless, the inclusion of all staff members and the high response rate enhances the generalizability of the results within the hospital. Furthermore, the limited number of participants resulted in a wide confidence interval for the odds ratio derived from the model. Abbreviations IPC: infection prevention and control HCWs: healthcare workers PPE: personal protective equipment WHO: World Health Organization RH: referral hospital HH: Hand washing and Hygiene Conclusions A notable portion of hospital workers reported inadequate compliance with COVID-19 IPC measures, with particularly low adherence among non-clinical staff. This lower compliance might be linked to their perception of low risk and a lack of awareness regarding COVID-19. It is essential to recognize that all HCWs hospitals are susceptible to COVID-19 infection. Therefore, prioritizing awareness campaigns and behavior change interventions, especially among non-clinical staff, is crucial for effective COVID-19 prevention and control within hospitals. Consent for publication Not applicable Data availability All data generated or analyzed during this study are included in this published article and its supplementary information files. Funding There is no funding support for this research. Competing interests The authors declare that they have no competing interests. Author contributions All authors (AIJ, BK, BU) contributed equally to the conception and design of the research, data collection, data analysis, data interpretation and creating the manuscript. All authors revised the manuscript and approved the final version of the article. Ethics approval and consent to participate The study received ethical approval from Dokuz Eylul University’s Non-Interventional Research Ethics Committee (approval date: 17.08.2022, decision number: 2022/26-08) and adhered to the principles of the Declaration of Helsinki and local institutional guidelines. Official permission was granted by De Martino Public Hospital in Mogadishu, Somalia. Research participants were informed about the study’s purpose and methodology, and their consent was obtained during the data collection process. Declarations Supplementary information Supplementary file 1: Questionnaire for compliance of healthcare workers with COVID-19 prevention and control measures.docx: is the questionnaire used for data collection in English. Acknowledgments First, we would like to express our sincere gratitude to the managers of De Martino Hospital for allowing us to conduct our study at the hospital. Second, we thank Dr. Lul Ahmed Abdi, Maternity Department, De Martino hospital who was the link person between us and the hospital administration. She also supported us in delivering the questionnaire papers to Page 15/19 the hospital workers and collecting it after filling during the data collection process, since she works at the hospital and knows the hospital well. the hospital workers and collecting it after filling during the data collection process, since she works at the hospital and knows the hospital well. 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World Health Organization. Risk assessment and management of exposure of health care workers in the context of COVID-19: Interim Guidance March 19, 2020. Pp.1-6. Available from: Page 17/19 https://www.who.int/publications/i/item/WHO-2019-nCov-HCW_risk_assessment-2020.2 https://www.who.int/publications/i/item/WHO-2019-nCov-HCW_risk_assessment-2020.2 https://www.who.int/publications/i/item/WHO-2019-nCov-HCW_risk_assessment-2020.2 16. Ashinyo ME, Dubik SD, Duti V, Amegah KE, Ashinyo A, Asare BA, et al. Infection prevention and control compliance among exposed healthcare workers in COVID-19 treatment centres in Ghana: A descriptive cross-sectional study. PLOS ONE, 2021;16(3):16. https://doi.org/10.1371/journal.pone.0248282 17. Gebremeskel TG, Kiros K, Gesesew HA, Ward PR. Assessment of Knowledge and Practices Toward COVID-19 Prevention Among Healthcare Workers in Tigray, North Ethiopia. Frontiers of Public Health, 2021;9:1-8. doi: 10.3389/fpubh.2021.614321 16. Ashinyo ME, Dubik SD, Duti V, Amegah KE, Ashinyo A, Asare BA, et al. Infection prevention and control compliance among exposed healthcare workers in COVID-19 treatment centres in Ghana: A descriptive cross-sectional study. PLOS ONE, 2021;16(3):16. https://doi.org/10.1371/journal.pone.0248282 17. Gebremeskel TG, Kiros K, Gesesew HA, Ward PR. Assessment of Knowledge and Practices Toward COVID-19 Prevention Among Healthcare Workers in Tigray, North Ethiopia. Frontiers of Public Health, 2021;9:1-8. doi: 10.3389/fpubh.2021.614321 18. Jemal B, Aweke Z, Mola S, Hailu S, Abiy S, Dendir G. Knowledge, attitude, and practice of healthcare workers toward COVID-19 and its prevention in Ethiopia: A multicenter study. SAGE Open Medicine, 2021;9:4. https://doi.org/10.1177/20503121211034389 19. Huang F, Armando M, Dufau S, Florea O, Brouqui P, Boudjema S. COVID-19 outbreak and healthcare worker behavioural change toward hand hygiene practices. Journal of Hospital Infection, 2021;111:27- 31. https://doi.org/10.1016/j.jhin.2021.03.004 20. Etafa W, Gadisal G, Jabessa S, Takele T. Healthcare workers’ compliance and its potential determinants to prevent COVID-19 in public hospitals in Western Ethiopia. BMC Infectious Diseases, 2021;21(1):1-7. https://doi.org/10.1186/s12879-021-06149-w 21. Houghton C, Meskell P, Delaney H, Smalle M, Glenton C, Booth A, et al. Barriers and facilitators to healthcare workers’ adherence with infection prevention and control (IPC) guidelines for respiratory infectious diseases: a rapid qualitative evidence synthesis. Cochrane Database of Systematic Reviews, 2020;4(4):8-15. DOI: 10.1002/14651858.CD013582. 21. Houghton C, Meskell P, Delaney H, Smalle M, Glenton C, Booth A, et al. References Barriers and facilitators to healthcare workers’ adherence with infection prevention and control (IPC) guidelines for respiratory infectious diseases: a rapid qualitative evidence synthesis. Cochrane Database of Systematic Reviews, 2020;4(4):8-15. DOI: 10.1002/14651858.CD013582. 22. Kamacooko O, Kitonsa J, Bahemuka UM, Kibengo FM, Wajja A, Basajja V, et al. Knowledge, Attitudes, and Practices Regarding COVID-19 among Healthcare Workers in Uganda: A Cross-Sectional Survey. International Journal of Environtal Research and Public Health, 2021;18(13):1-10. https://doi.org/10.3390/ijerph18137004. 22. Kamacooko O, Kitonsa J, Bahemuka UM, Kibengo FM, Wajja A, Basajja V, et al. Knowledge, Attitudes, and Practices Regarding COVID-19 among Healthcare Workers in Uganda: A Cross-Sectional Survey. International Journal of Environtal Research and Public Health, 2021;18(13):1-10. https://doi.org/10.3390/ijerph18137004. 23. Harrison D, Muradalia K, El Sahlyb H, Bozkurtc B, Jneid H. Impact of the SARS-CoV-2 pandemic on health-care workers. Hospital Practice, 2020;48(4):162. DOI: 10.1080/21548331.2020.1771010 23. Harrison D, Muradalia K, El Sahlyb H, Bozkurtc B, Jneid H. Impact of the SARS-CoV-2 pandemic on health-care workers. Hospital Practice, 2020;48(4):162. DOI: 10.1080/21548331.2020.1771010 24. Asemahagn MA. Factors determining the knowledge and prevention practice of healthcare workers towards COVID-19 in Amhara region, Ethiopia: a cross-sectional survey. Tropical Medicine and Health, 2020;48:1-9. https://doi.org/10.1186/s41182-020-00254-3 24. Asemahagn MA. Factors determining the knowledge and prevention practice of healthcare workers towards COVID-19 in Amhara region, Ethiopia: a cross-sectional survey. Tropical Medicine and Health, 2020;48:1-9. https://doi.org/10.1186/s41182-020-00254-3 25. Cooper S, Wiyeh A, Schmidt B, Wiysonge CS. Cochrane corner: factors that influence compliance by healthcare workers with infection prevention and control guidelines for COVID-19 and other respiratory infections. Pan African Medical Journal,2020;35(2):2. DOI: 10.11604/pamj.supp.2020.35.2.23012 25. Cooper S, Wiyeh A, Schmidt B, Wiysonge CS. Cochrane corner: factors that influence compliance by healthcare workers with infection prevention and control guidelines for COVID-19 and other respiratory infections. Pan African Medical Journal,2020;35(2):2. DOI: 10.11604/pamj.supp.2020.35.2.23012 26. Ahmed M, Fodjo J, Gele A, Farah A, Osman S, Guled I, et al. COVID-19 in Somalia: Adherence to Preventive Measures and Evolution of the Disease Burden. Pathogens, 2020;9(9):10. doi:10.3390/pathogens9090735 26. Ahmed M, Fodjo J, Gele A, Farah A, Osman S, Guled I, et al. COVID-19 in Somalia: Adherence to Preventive Measures and Evolution of the Disease Burden. Pathogens, 2020;9(9):10. doi:10.3390/pathogens9090735 COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % Note: IPC: infection prevention and control, PPE: personal protective equipm Figures Page 18/19 Fi 1 Figure 1 COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % COVID-19 IPC compliance in the HCWs (total IPC, PPE use, HH, other IPC), % Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. This is a list of supplementary files associated with this preprint. Click to download. BMCPublicHealthSupplementaryfile1Questionnaire.docx.docx BMCPublicHealthSupplementaryfile1Questionnaire.docx.docx Page 19/19
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DISSEMINATED INTRAVASCULAR COAGULATION SYNDROME IN SURGICAL TREATMENT OF PATIENTS WITH LIVER MALIGNANCIES
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Синдром диссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Сомонова О.В.1 • Елизарова А.Л.1 • Матвеева И.И.1 Сомонова О.В.1 • Елизарова А.Л.1 • Матвеева И.И.1 Исследования системы гемостаза проводились до операции и  на 1–20-е  сутки после опера- ции на автоматическом анализаторе гемостаза “STA-R Evolution” и на агрегометре “Chrono-log”. Результаты. После хирургического вмеша- тельства на печени подострый синдром дис- семинированного внутрисосудистого сверты- вания (ДВС) выявлен у  34  больных. Наиболее часто (65%) он развивался после правосто- ронней гемигепатэктомии и  характеризовал- ся снижением концентрации фибриногена до 121  мг/дл (p < 0,001), факторов протромбино- вого комплекса до 45%  (р < 0,05), антитромби- на III до 48%  (р < 0,05) на фоне значительного повышения D-димера до 14,5  мкг/мл (р < 0,05). У  12  больных с  подострой формой синдрома ДВС развились тромбозы вен нижних конеч- ностей, у  9  – тяжелая печеночная недоста- точность. У  пациентов с  тяжелой печеночной недостаточностью наблюдалось статистически значимое (p < 0,05) снижение протромбиновой активности до 45%, антитромбина  III до  44%, плазминогена ниже 50%  при высоком уровне D-димера (> 20 мкл/мл) и фактора Виллебранда. Заключение. Хирургические вмешатель- ства у  больных злокачественными опухолями печени вызывают развитие синдрома ДВС. Своевременная диагностика и коррекция гемо- стазиологических факторов риска развития тя- желой печеночной недостаточности позволяют улучшить результаты хирургического лечения больных вторичными злокачественными опу- холями печени. Актуальность. В структуре хирургических вме- шательств у больных злокачественными опухо- лями печени преобладают обширные резекции или расширенные гемигепатэктомии, сопрово- ждающиеся серьезными послеоперационными осложнениями. Цель  – выяснить роль нару- шений системы гемостаза в патогенезе после- операционной печеночной недостаточности, а  также тромбогеморрагических осложнений у  больных злокачественными опухолями пе- чени. Материал и методы. Под наблюдением находились 120  больных злокачественными опухолями печени (20 пациентов с первичными опухолями печени и  100  – с  метастатическим колоректальным раком печени). Обширные резекции печени (право- и  левосторонняя гемигепатэктомия простая и  расширенная) были выполнены у  100  (84%) больных, резек- ция нескольких сегментов печени – у 20 (16%). Ключевые слова: резекция печени, система гемостаза, печеночная недостаточность doi: 10.18786/2072-0505-2016-44-5-640-646 З Такие вмешательства характеризуются высокой травматичностью, длительностью, обширной ра- невой поверхностью, развитием острой порталь- ной гипертензии в  раннем послеоперационном периоде [6–8]. З аболеваемость колоректальным раком в России составляет порядка 50 тыс. слу- чаев в  год, при этом от  20 до  50%  паци- ентов при первичном обращении имеют метастазы в печень [1, 2]. Резекция печени оста- ется единственным методом, позволяющим зна- чительно увеличить продолжительность жизни (до уровня 5-летней выживаемости 74%) [3, 4]. Частота послеоперационных осложнений у  больных, перенесших расширенные гемиге- патэктомии, варьирует от  14,4 до  45%  [9–11]. Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 ренная) были выполнены у 100 (84%) больных, ре- зекция нескольких сегментов печени – у 20 (16%). При гистологическом исследовании первич- ных опухолей установлен низкодифференци- рованный или высокодифференцированный гепатоцеллюлярный рак (10 больных) и холангио­ целлюлярный рак (10  больных). При гистологи- ческом исследовании метастатических очагов обнаружена аденокарцинома кишечного типа с различной степенью дифференцировки. ренная) были выполнены у 100 (84%) больных, ре- зекция нескольких сегментов печени – у 20 (16%). Сомонова Оксана Васильевна – д-р мед. наук, вед. науч. сотр., клинико- диагностическая лаборатория1 * * 115478, г. Москва, Каширское шоссе, 24, Российская Федерация. Тел.: +7 (499) 324 28 90. E-mail: somonova@mail.ru недостаточность характеризуется протромбино- вой активностью по Квику < 50% (соответствует международному нормализованному отноше- нию > 1,7) и  повышением уровня билирубина > 50 ммоль/л (2,9 мг/дл) на 5-е сутки после опера- ции [14]. Если состояние пациента отвечает кри- териям «50/50», риск смерти составляет 59%, если нет – то только 1,2% [15]. При гистологическом исследовании первич- ных опухолей установлен низкодифференци- рованный или высокодифференцированный гепатоцеллюлярный рак (10 больных) и холангио­ целлюлярный рак (10  больных). При гистологи- ческом исследовании метастатических очагов обнаружена аденокарцинома кишечного типа с различной степенью дифференцировки. К другим тяжелым осложнениям резекции печени относят острые кровотечения, тромбоз воротной вены, тромбоз глубоких вен нижних конечностей, тромбоэмболию легочной артерии, в  патогенезе которых немаловажное значение принадлежит нарушениям в  системе гемостаза [15, 16]. Причины активации свертывания крови у  больных злокачественными новообразовани- ями многообразны. Среди них следует отметить выделение опухолевыми клетками высокоактив- ного тканевого фактора и ракового прокоагулян- та, а  также прокоагулянтную активность моно- цитов, тромбоцитов и  эндотелиальных клеток в  ответ на опухоль. Активация прокоагулянт- ного и тромбоцитарного звеньев гемостаза ведет к появлению тромбина и локальному отложению фибрина вокруг опухолевых клеток. Это форми- рует матрицу для опухолевого роста и  ангиоге- неза, способствует развитию венозного тромбоза и  синдрома диссеминированного свертывания крови (ДВС) [17, 18]. Елизарова Анна Львовна – канд. биол. наук, ст. науч. сотр., клинико- диагностическая лаборатория1 Елизарова Анна Львовна – канд. биол. наук, ст. науч. сотр., клинико- диагностическая лаборатория1 Матвеева Ирина Ивановна – д-р мед. наук, заведующая клинико- диагностической лабораторией1 Сопутствующая патология диагностирована в 58 (48%) наблюдениях, в том числе сахарный ди- абет (у 10 больных), гепатит В (7), гепатит С (4), ишемическая болезнь сердца, артериальная ги- пертония (20), варикозная болезнь, хроническая венозная недостаточность (12). Неоадъювантная химиотерапия была проведена у 20 больных. Показатели системы гемостаза оценивали по 10 параметрам, наиболее точно отражающим со- стояние ее основных звеньев: прокоагулянтного, тромбоцитарного, антикоагулянтного, фибри- нолитического с  маркерами внутрисосудистого свертывания крови. Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Исследования системы гемо- стаза проводились на автоматических коагулоло- гических анализаторах “STA-R Evolution” и агре- гометре фирмы “Chrono-log” до операции и  на 1–20-е сутки после операции. На автоматическом анализаторе “STA-R Evolution” определяли акти- вированное частичное тромбопластиновое время (АЧТВ), протромбиновую активность по Квику, концентрацию фибриногена по Клауссу, актив- ность антитромбина  III, протеина  С, плазмино- гена, α2-антиплазмина, концентрацию D-димера, фактор Виллебранда. Агрегацию тромбоцитов исследовали на агрегометре фирмы “Chrono-log”. Печень играет ключевую роль в регуляции ге- мостаза. Учитывая, что в  печени синтезируется большинство факторов свертывания крови и ин- гибиторов, а  также белков, участвующих в  фи- бринолизе, своевременная диагностика и  кор- рекция нарушений в системе гемостаза позволят снизить частоту осложнений и тем самым улуч- шить результаты хирургического лечения боль- ных злокачественными опухолями печени. При статистической обработке результатов рассчитывали средние арифметические значения и  стандартное отклонение (M ± SD). Сравнение количественных данных в  двух несвязанных группах проводилось с  использованием t-кри- терия Стьюдента для независимых выборок. Достоверными считались результаты с ошибкой менее 5% (p < 0,05). Цель исследования – выяснить роль наруше- ний системы гемостаза в патогенезе послеопера- ционной печеночной недостаточности, а  также тромбогеморрагических осложнений у  больных злокачественными опухолями печени. Исследования выполнялись в  соответствии с принципами Хельсинкской декларации, разра- ботанной Всемирной медицинской ассоциацией. Материал и методы Под нашим наблюдением находились 120  боль- ных злокачественными опухолями печени (муж- чин – 56, женщин – 64). Из них 20 пациентов были с первичными опухолями печени и 100 пациен- тов с  метастатическим колоректальным раком печени. Контрольная группа состояла из 40 прак- тически здоровых людей. 1 ФГБУ «Российский он- кологический научный центр им. Н.Н. Блохина» Минздрава России; 115478, г. Москва, Каширское шоссе, 24, Российская Федерация Сомонова О.В., Елизарова А.Л., Матвеева И.И. Синдром диссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Сомонова О.В., Елизарова А.Л., Матвеева И.И. Синдром диссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени В  структуре послеоперационных осложнений и  причин летальности (3–14%) ведущее место занимает печеночная недостаточность разной степени выраженности [12, 13]. Однозначных и  общепринятых критериев пострезекционной печеночной недостаточности в настоящее время нет. Обычно под этим термином понимают нару- шение одной или нескольких синтетических или экскреторных функций печени и/или печеноч- ную энцефалопатию разной степени. Достаточно точным представляется критерий «50/50», со- гласно которому пострезекционная печеночная ур В мире ежегодно выявляют около 1  млн новых наблюдений первичного рака печени. Гепатоцеллюлярный рак по частоте составляет около 85–90%  всех первичных злокачественных опухолей печени. Резекции печени может быть подвергнуто от  13 до  30%  пациентов, 5-летняя выживаемость после этой операции достигает 14–61% [5]. ур В мире ежегодно выявляют около 1  млн новых наблюдений первичного рака печени. Гепатоцеллюлярный рак по частоте составляет около 85–90%  всех первичных злокачественных опухолей печени. Резекции печени может быть подвергнуто от  13 до  30%  пациентов, 5-летняя выживаемость после этой операции достигает 14–61% [5]. В последние 20  лет хирургическое лечение больных злокачественными опухолями печени представлено в  основном обширными резекци- ями или расширенными гемигепатэктомиями. 640 Оригинальные статьи Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Результаты Как видно из данных табл. 1, при изучении ис- ходного состояния у больных первичными и вто- ричными злокачественными опухолями печени установлено достоверное укорочение АЧТВ и уве- личение концентрации фибриногена в  1,3  раза, что указывает на активацию прокоагулянтного Обширные резекции печени (право- и  лево- сторонняя гемигепатэктомия простая и  расши- 641 еминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 Таблица 1. Показатели системы гемостаза у больных первичным и метастатическим раком печени до начала лечения Показатель Здоровые доноры (n = 40) Больные раком печени (n = 120) Активированное частичное тромбопластиновое время, с 38 ± 0,8 34 ± 0,9* Протромбиновая активность по Квику, % 92 ± 2 91 ± 1,3 Концентрация фибриногена, мг/дл 300 ± 8,5 384 ± 14* Агрегация тромбоцитов: степень агрегации МА, % 65 ± 1,9 80 ± 1* Антитромбин III, % 87 ± 1,7 81 ± 0,9* Протеин С, % 130 ± 2,5 75 ± 2,3* Плазминоген, % 110 ± 1,9 90 ± 1,8* α2-антиплазмин, % 93 ± 3,8 99 ± 2 D-димер, мкг/мл 0,4 ± 0,2 1,6 ± 0,4* Фактор Виллебранда, % 110 ± 10 234 ± 10* Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD) * Различия статистически значимы (p<0 05) по сравнению со здоровыми донорами Таблица 1. Показатели системы гемостаза у больных первичным и метастатическим раком печени до начала лечения преимущественно в виде подострых и хрониче- ских форм синдрома ДВС. Подострый синдром ДВС выявлен у  34  больных; наиболее часто (65%) он развивался после правосторонней ге- мигепатэктомии и характеризовался существен- ным и  статистически значимым повышением уровня D-димера (до  12  мкг/мл на 3–4-е  сутки после операции, до 14  мкг/мл на 9–10-е  сутки) и  фактора Виллебранда (до  415% на 3–4-е  сут- ки), что свидетельствовало о выраженной акти- вации внутрисосудистого свертывания крови (табл. 2). Наблюдалось умеренное укорочение АЧТВ с  1-х  суток послеоперационного пери- ода, максимально  – на 3–6-е сутки (p < 0,05). Одновременно выявлено существенное сниже- ние активности факторов протромбинового ком- плекса: протромбиновая активность по Квику снижалась до 53% на 2-е  сутки после операции и  оставалась резко сниженной до 9–10-х  суток (в 2 раза на 7–8-е сутки). Концентрация фибрино- гена также существенно снижалась после резек- ции печени (до 121 мг/дл на 7–8-е сутки после опе- рации) (p < 0,001). Результаты В  послеоперационный период обращало на себя внимание значительное умень- шение уровня естественных антикоагулянтов (антитромбина  III до 48%, протеина  С  до  50%), а также компонентов фибринолитической систе- мы (плазминогена до  52%), защищающих орга- низм от тромбообразования. * Различия статистически значимы (p < 0,05) по сравнению со здоровыми донорами звена свертывающей системы крови. Отмечены изменения со стороны тромбоцитарного зве- на в  виде усиления агрегационной способно- сти тромбоцитов. Активация прокоагулянтного и  тромбоцитарного звеньев системы гемостаза ведет к  появлению тромбина, повышенному от- ложению фибрина с  последующим его лизисом, о чем свидетельствует увеличение маркеров вну- трисосудистого свертывания крови. У  больных метастатическим колоректальным раком печени концентрация D-димера, одного из надежных и чувствительных маркеров тромбообразования, была увеличена в  4  раза. Что касается фактора Виллебранда  – показателя повреждения стенки сосуда и  активации системы гемостаза, он был повышен в 2,1 раза. В ответ на усиленное внутри- сосудистое свертывание крови происходит рас- ходование естественных ингибиторов тромбина и  других активных сериновых протеаз: у  боль- ных раком печени выявлено снижение уровня ан- титромбина III и протеина С (в 1,7 раза). Таким образом, можно говорить о развитии у больных с  первичными и  метастатическими опухолями печени гиперкоагуляции с признаками хрониче- ского внутрисосудистого свертывания крови. У 12 больных с подострой формой синдрома ДВС развились тромбоэмболические осложне- ния. По локализации они распределились следу- ющим образом: тромбоз поверхностных и глубо- ких вен нижних конечностей зарегистрирован в 10 наблюдениях, тромбозы воротной вены пе- чени и  левой печеночной вены  – по 1  случаю. Несмотря на снижение факторов свертывания крови, применение низкомолекулярных гепа- ринов в сочетании с концентратом антитромби- на III и свежезамороженной плазмой у больных с  подострой формой синдрома ДВС восстанав- ливало факторы свертывания крови – по данным ультразвукового допплеровского сканирования, венозные тромбозы не определялись либо наблю- далась реканализация просвета сосуда. У остальных пациентов (преимущественно больные с резекцией нескольких сегментов пече- ни) отмечались менее выраженные изменения си- стемы гемостаза, характерные для хронической формы синдрома ДВС. У больных с  подострым диссеминирован- ным внутрисосудистым свертыванием крови, оперированных на печени, в  раннем послеопе- рационном периоде отмечались клинические Установлено, что хирургические вмеша- тельства на печени вызывали глубокие измене- ния системы гемостаза, которые развивались 642 Оригинальные статьи Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 а у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого Таблица 2. Результаты Показатели системы гемостаза у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого свертывания (n = 34) после операции Показатель До операции Послеоперационный период, сутки 1-е 2-е 3–4-е 5–6-е 7–8-е 9–10-е Активированное частичное тромбопластиновое время, с 33 ± 0,3 29 ± 0,5* 28 ± 0,6* 26 ± 0,7* 26 ± 1* 27 ± 0,9* 30 ± 1,4* Протромбиновая активность по Квику, % 92 ± 2,4 69 ± 1,4* 53 ± 2,4* 52 ± 2,1* 50 ± 3,6* 45 ± 2,6* 48 ± 2,9* Концентрация фибриногена, мг/дл 418 ± 9 207 ± 12* 197 ± 15,1* 145 ± 10,2* 151 ± 11* 121 ± 17* 125 ± 16* Агрегация тромбоцитов: степень агрегации МА, % 80 ± 2,3 68 ± 1,9* 60 ± 2* 67 ± 2,5* 53 ± 3* 54 ± 2,4* 56 ± 3,4* Антитромбин III, % 82 ± 1,9 61 ± 2,1* 58 ± 1,9* 50 ± 2* 48 ± 1,8* 51 ± 2,3* 54 ± 2,4* Протеин С, % 76 ± 2,2 58 ± 2,5* 48 ± 2,8* 50 ± 2,8* 52 ± 2,9* 57 ± 2,4* 54 ± 3* Плазминоген, % 91 ± 2,1 69 ± 2,7* 52 ± 2,7* 55 ± 3* 52 ± 2,8* 55 ± 2,4* 68 ± 2,9* D-димер, мкг/мл 1,6 ± 0,2 4 ± 0,4* 11 ± 0,5* 12,1 ± 0,9* 13,2 ± 0,7* 12,3 ± 0,6* 14,5 ± 0,8* Фактор Виллебранда, % 234 ± 6,2 243 ± 8,1 380 ± 13* 415 ± 11* 393 ± 10* 340 ± 12* 300 ± 13,3* Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD) * Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом Таблица 2. Показатели системы гемостаза у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого свертывания (n = 34) после операции достоверно отличаясь от показателей пациентов без тяжелой печеночной недостаточности (табл. 4). Одновременно в этой группе больных отмечалось резкое снижение активности факторов протром- бинового комплекса, уровня антитромбина  III и  плазминогена по сравнению с  аналогичными показателями больных без тяжелой печеночной недостаточности (p < 0,05). Следовательно, у  па- циентов с тяжелой печеночной недостаточностью наблюдалось снижение протромбиновой актив- ности до 45%, антитромбина III до 44%, плазми- ногена ниже 50% при высоком уровне D-димера (> 20 мкл/мл) и фактора Виллебранда. Эти пока- затели можно отнести к  гемостазиологическим факторам риска развития тяжелой печеночной недостаточности. Результаты признаки печеночной недостаточности  – сла- бость, сонливость, легкие формы желтухи и энце- фалопатии, возникшие вследствие недостаточно- го объема оставшейся части печени. Активность патологического процесса в  печени характери- зовалась выраженным цитолитическим син- дромом: при биохимическом исследовании крови (табл. 3) выявлено повышение активно- сти аланинаминотрансферазы (АЛТ) в  среднем в  8,6 ± 1,5  раза и  аспартатаминотрансферазы (АСТ) – в 8,8 ± 1,9 раза. Снижение соотношения АСТ/АЛТ до 0,65 ± 0,13 на 3-и и 5-е сутки (в нор- ме коэффициент де  Ритиса равен  1,33) может говорить о  тяжести поражения клеток печени. Информативным показателем степени поврежде- ния паренхимы печени было повышение билиру- бина в 2 раза на 1–5-е сутки послеоперационного периода. После расширенных резекций печени достоверно снизилось содержание в крови обще- го белка и альбумина (в 1,5 раза на 1–5-е сутки по- сле операции) (см. табл. 3). Сомонова О.В., Елизарова А.Л., Матвеева И.И. Синдром диссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печен Сомонова О.В., Елизарова А.Л., Матвеева И.И. Заключение Показатели системы гемостаза у больных злокачественными опухолями печени с тяжелой печеночной недостаточностью (n = 9) Показатель До операции Послеоперационный период, сутки 1-е 3–4-е 5–6-е Концентрация фибриногена, мг/дл 422 ± 8 177 ± 12*, † 140 ± 15,1* 144 ± 10,2* Протромбиновая активность по Квику, % 82 ± 4 49 ± 1*, † 45 ± 2,1*, † 47 ± 2,9* Антитромбин III, % 78 ± 2,3 49 ± 2,3*, † 44 ± 1,8*, † 48 ± 2,6* Плазминоген, % 85 ± 3 39 ± 2,7*, † 46 ± 2,6*, † 52 ± 3,1* D-димер, мкг/мл 2,1 ± 0,9 20 ± 1,8*, † 20 ± 2,8*, † 20 ± 3*, † Фактор Виллебранда, % 240 ± 6,9 425 ± 8,1*, † 442 ± 18,2* 540 ± 14,2*, † * Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом † Различия статистически значимы (p < 0,05) по сравнению с показателями пациентов без тяжелой печеночной недостаточности Таблица 3. Показатели биохимического исследования крови у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого свертывания (n = 34) после операции Показатель Норма До операции Послеоперационный период, сутки 1-е 3–4-е 5–6-е АЛТ, ед/мл < 40 22,9 ± 2,1 344,7 ± 62,2* 198,6 ± 39,8* 88,2 ± 22,5* ACT, ед/мл < 37 27,1 ± 2,07 358 ± 67* 109,1 ± 12,9* 50,8 ± 9,3* АСТ/АЛТ 1,33 0,87 ± 0,07 1,06 ± 0,06 0,67 ± 0,09 0,65 ± 0,13 Общий билирубин, мкмоль/л 0–20,5 20,3 ± 1,8 49,8 ± 8* 42 ± 10,5* 39,1 ± 4,08* Общий белок, г/л 60–80 73,8 ± 1,8 49,9 ± 1,8* 51,2 ± 1,02* 52,1 ± 3* Альбумин, г/л 35–50 41,2 ± 1,1 31,3 ± 1,97* 31 ± 1,3* 27,6 ± 2,9* АЛТ – аланинаминотрансфераза, АСТ – аспартатаминотрансфераза Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD) * Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом Таблица 3. Показатели биохимического исследования крови у больных злокачественными опухолями печени с подострым синдромом диссеминированного нутрисосудистого свертывания (n = 34) после операции Таблица 4. Конфликт интересов Конфликт интересов Авторы сообщают об отсутствии конфликта интересов и финансовой заинтересованности в ходе написания данной статьи и не несут ответственности за наличие и содержание рекламных материалов, размещенных на страни- цах журнала. кова  ОВ, Шишкина  НА. Резекция печени: современные технологии при опухолевом поражении. Анналы хирургической гепато- логии. 2010;15(2):9–17. рака. Онкологическая колопроктология. 2014;(1):14–20. 2. Патютко ЮИ, Сагайдак ИВ, Котельников АГ, Поляков  АН, Чучуев  ЕС,  Пылев  АЛ, Чистя- рака. Онкологическая колопроктология. 2014;(1):14–20. 2. Патютко ЮИ, Сагайдак ИВ, Котельников АГ, Поляков  АН, Чучуев  ЕС,  Пылев  АЛ, Чистя- Заключение Показатели системы гемостаза у больных злокачественными опухолями печени с тяжелой печеночной недостаточностью (n = 9) Показатель До операции Послеоперационный период, сутки 1-е 3–4-е 5–6-е Концентрация фибриногена, мг/дл 422 ± 8 177 ± 12*, † 140 ± 15,1* 144 ± 10,2* Протромбиновая активность по Квику, % 82 ± 4 49 ± 1*, † 45 ± 2,1*, † 47 ± 2,9* Антитромбин III, % 78 ± 2,3 49 ± 2,3*, † 44 ± 1,8*, † 48 ± 2,6* Плазминоген, % 85 ± 3 39 ± 2,7*, † 46 ± 2,6*, † 52 ± 3,1* D-димер, мкг/мл 2,1 ± 0,9 20 ± 1,8*, † 20 ± 2,8*, † 20 ± 3*, † Фактор Виллебранда, % 240 ± 6,9 425 ± 8,1*, † 442 ± 18,2* 540 ± 14,2*, † * Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом † Различия статистически значимы (p < 0,05) по сравнению с показателями пациентов без тяжелой печеночной недостаточности таза у больных злокачественными опухолями печени с тяжелой печеночной недостаточностью (n = 9) Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом † Различия статистически значимы (p < 0,05) по сравнению с показателями пациентов без тяжелой печеночной недостаточност эффективным и  адекватным. К  гемостазиологи- ческим факторам риска развития тяжелой пече- ночной недостаточности можно отнести повы- шение уровня D-димера, фактора Виллебранда на фоне резкого снижения протромбиновой ак- тивности, содержания антитромбина III и уров- ня плазминогена. Их своевременная диагностика и коррекция позволяют улучшить результаты хи- рургического лечения больных вторичными зло- качественными опухолями печени. защищающих организм от тромбообразования, что согласуется с  данными литературы [19, 20]. Поскольку применение низкомолекулярных ге- паринов в сочетании с концентратом антитром- бина  III и  свежезамороженной плазмой у  боль- ных с  тромбозами вен снижает интенсивность внутрисосудистого свертывания крови, приво- дит к  восстановлению факторов свертывания, этот метод лечения тромботических осложне- ний у  онкологических больных представляется Заключение Проведенные исследования показали, что хирур- гические вмешательства у больных злокачествен- ными опухолями печени вызывают развитие различных форм синдрома ДВС со значитель- ным повышением маркеров внутрисосудистого свертывания крови на фоне резкого снижения активности факторов протромбинового ком- плекса, уровня естественных антикоагулянтов и  компонентов фибринолитической системы, Выделена группа больных (9  человек) с  раз- витием тяжелой печеночной недостаточности в послеоперационном периоде. При изучении си- стемы гемостаза у этих пациентов было установ- лено, что уровень D-димера (20 мкг/мл) и фактор Виллебранда повышались в  большей степени, 643 иссеминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 защищающих организм от тромбообразования, [19 20] эффективным и  адекватным. К  гемостазиологи- ф й Конфликт интересов Таблица 3. Показатели биохимического исследования крови у больных злокачественными опухолями печени с подострым синдромом диссеминированного внутрисосудистого свертывания (n = 34) после операции Показатель Норма До операции Послеоперационный период, сутки 1-е 3–4-е 5–6-е АЛТ, ед/мл < 40 22,9 ± 2,1 344,7 ± 62,2* 198,6 ± 39,8* 88,2 ± 22,5* ACT, ед/мл < 37 27,1 ± 2,07 358 ± 67* 109,1 ± 12,9* 50,8 ± 9,3* АСТ/АЛТ 1,33 0,87 ± 0,07 1,06 ± 0,06 0,67 ± 0,09 0,65 ± 0,13 Общий билирубин, мкмоль/л 0–20,5 20,3 ± 1,8 49,8 ± 8* 42 ± 10,5* 39,1 ± 4,08* Общий белок, г/л 60–80 73,8 ± 1,8 49,9 ± 1,8* 51,2 ± 1,02* 52,1 ± 3* Альбумин, г/л 35–50 41,2 ± 1,1 31,3 ± 1,97* 31 ± 1,3* 27,6 ± 2,9* АЛТ – аланинаминотрансфераза, АСТ – аспартатаминотрансфераза Данные представлены в виде среднего арифметического значения и стандартного отклонения (M ± SD) * Различия статистически значимы (p < 0,05) по сравнению с дооперационным периодом Таблица 4. Литература кова  ОВ, Шишкина  НА. Резекция печени: современные технологии при опухолевом поражении. Анналы хирургической гепато- логии. 2010;15(2):9–17. 1. Патютко ЮИ, Котельников АГ, Мамонтов КГ, Пономаренко  АА, Лазарев  АФ. Непосред- ственные результаты резекций печени по поводу метастазов колоректального 1. Патютко ЮИ, Котельников АГ, Мамонтов КГ, Пономаренко  АА, Лазарев  АФ. Непосред- ственные результаты резекций печени по поводу метастазов колоректального 644 Оригинальные статьи Альманах клинической медицины. 2016 Июнь-июль; 44 (5): 640–646 treatment. Liver Int. 2008;28(6):767–80. doi: 10.1111/j.1478-3231.2008.01777.x. 3. Kanas GP, Taylor A, Primrose JN, Langeberg WJ, Kelsh MA, Mowat FS, Alexander DD, Choti MA, Poston G. Survival after liver resection in meta- static colorectal cancer: review and meta-anal- ysis of prognostic factors. Clin Epidemiol. 2012;4:283–301. doi: 10.2147/CLEP.S34285. 9. Huang ZQ, Xu LN, Yang T, Zhang WZ, Huang XQ, Cai SW, Zhang AQ, Feng YQ, Zhou NX, Dong JH. Hepatic resection: an analysis of the impact of operative and perioperative factors on mor- bidity and mortality rates in 2008 consecu- tive hepatectomy cases. Chin Med J  (Engl). 2009;122(19):2268–77. 16. Meijer  C, Wiezer  MJ, Hack  CE, Boelens  PG, Wedel  NI, Meijer  S, Nijveldt  RJ, Statius Muller  MG, Wiggers  T, Zoetmulder  FA, Borel Rinkes IH, Cuesta MA, Gouma DJ, van de Vel- de CJ, Tilanus HW, Scotté M, Thijs LG, van Leeu- wen  PA. Coagulopathy following major liver resection: the effect of rBPI21 and the role of decreased synthesis of regulating proteins by the liver. Shock. 2001;15(4):261–71. 4. Патютко  ЮИ, Пылев  АЛ, Сагайдак  ИВ, Ко- тельников АГ, Подлужный ДВ, Поляков АН, Абгарян  МГ, Чучуев  ЕС,  Гахраманов  АД, Шишкина НА, Агафонова МГ. Десятилетняя выживаемость больных злокачественными опухолями печени после хирургического лечения. Анналы хирургической гепатоло- гии. 2010;15(3):39–47. 10. Вишневский  ВА, Кубышкин  ВА, Чжао  АВ, Икрамов  РЗ. Операции на печени. М.: Миклош; 2003. 157 с. 11. Патютко ЮИ. Хирургическое лечение злока- чественных опухолей печени. М.: Практиче- ская медицина; 2005. 312 c. 17. Amin  C, Mackman  N, Key  NS. Microparticles and cancer. Pathophysiol Haemost Thromb. 2008;36(3–4):177–83. doi: 10.1159/000175155. 12. Jarnagin  WR, Gonen  M, Fong  Y, DeMat- teo RP, Ben-Porat L, Little S, Corvera C, Weber S, Blumgart  LH. Improvement in perioperative outcome after hepatic resection: analysis of 1,803 consecutive cases over the past de- cade. Ann Surg. 2002;236(4):397–406. doi: 10.1097/01.SLA.0000029003.66466.B3. 5. Чиссов ВИ, Бутенко АВ, Вашакмадзе ЛА, Си- доров  ДВ, Гришин  НА, Ложкин  МВ, Степа- нов СО, Хомяков ВМ, Швейкин АО, Гуц ОВ. Хирургическое лечение первичного и  ме- тастатического рака печени. Российский онкологический журнал. 2010;(5):8–12. 18. References Shishkina NA, Agafonova MG. Desyatiletnyaya vyzhivaemost' bol'nykh zlokachestvennymi opukholyami pecheni posle khirurgichesko- go lecheniya [Ten-years survival after surgical management of the liver malignancies]. Annaly khirurgicheskoy gepatologii [Annals of Surgical Hepatology]. 2010;15(3):39–47 (in Russian). Shishkina NA, Agafonova MG. Desyatiletnyaya vyzhivaemost' bol'nykh zlokachestvennymi opukholyami pecheni posle khirurgichesko- go lecheniya [Ten-years survival after surgical management of the liver malignancies]. Annaly khirurgicheskoy gepatologii [Annals of Surgical Hepatology]. 2010;15(3):39–47 (in Russian). 8. Kin Y, Nimura Y, Hayakawa N, Kamiya J, Kondo S, Nagino M, Miyachi M, Kanai M. Doppler analysis of hepatic blood flow predicts liver dysfunc- tion after major hepatectomy. World J  Surg. 1994;18(1):143–9. 1. Patyutko YuI, Kotel'nikov AG, Mamontov  KG, Ponomarenko AA, Lazarev AF. Neposredstven- nye rezul'taty rezektsiy pecheni po povodu metastazov kolorektal'nogo raka [Immediate results of hepatectomy for metastatic colorec- tal cancer]. Onkologicheskaya koloproktologiya [Oncological Coloproctology]. 2014;(1):14–20 (in Russian). 9. Huang ZQ, Xu LN, Yang T, Zhang WZ, Huang XQ, Cai SW, Zhang AQ, Feng YQ, Zhou NX, Dong JH. Hepatic resection: an analysis of the impact of operative and perioperative factors on mor- bidity and mortality rates in 2008 consecu- tive hepatectomy cases. Chin Med J  (Engl). 2009;122(19):2268–77. 5. C 5. Chissov VI, Butenko AV, Vashakmadze LA, Sidor- ov  DV, Grishin  NA, Lozhkin  MV, Stepanov  SO, Khomyakov VM, Shveykin AO, Guts OV. Khiru- rgicheskoe lechenie pervichnogo i  metasta- ticheskogo raka pecheni [Surgical treatment for primary and metastatic tumors of the liver]. Rossiyskiy Onkologicheskiy Zhurnal [Russian Journal of Oncology]. 2010;(5):8–12 (in Russian). 2. Patyutko YuI, Sagaydak IV, Kotel'nikov AG, Polya- kov AN, Chuchuev ES, Pylev AL, Chistyakova OV, Shishkina NA. Rezektsiya pecheni: sovremennye tekhnologii pri opukholevom porazhenii [Liver resection: current technologies for cancer]. An- naly khirurgicheskoy gepatologii [Annals of Sur- gical Hepatology]. 2010;15(2):9–17 (in Russian). 10. Vishnevskiy  VA, Kubyshkin  VA, Chzhao  AV, Ikramov  RZ. Operatsii na pecheni [Liver sur- gery]. Moscow: Miklosh; 2003. 157 p. (in Rus- sian). 3. Kanas GP, Taylor A, Primrose JN, Langeberg WJ, Kelsh MA, Mowat FS, Alexander DD, Choti MA, Poston G. Survival after liver resection in meta- static colorectal cancer: review and meta-anal- ysis of prognostic factors. Clin Epidemiol. 2012;4:283–301. doi: 10.2147/CLEP.S34285. 6. Sato T, Yasui A, Kondo S, Kato M, Kameoka N, Nimura  Y. Changes in liver blood flow after hepatectomy in conscious dogs. Surg Today. 1996;26(9):688–93. 11. Patyutko YuI. Khirurgicheskoe lechenie zlok- achestvennykh opukholey pecheni [Surgical treatment for hepatic malignancies]. Moscow: Prakticheskaya meditsina; 2005. 312 p. (in Rus- sian). 7. Литература Magnus  N, D'Asti  E, Meehan  B, Garnier  D, Rak  J. Oncogenes and the coagulation sys- tem  – forces that modulate dormant and aggressive states in cancer. Thromb Res. 2014;133 Suppl  2:S1–9. doi: 10.1016/S0049- 3848(14)50001-1. 6. Sato T, Yasui A, Kondo S, Kato M, Kameoka N, Nimura  Y. Changes in liver blood flow after hepatectomy in conscious dogs. Surg Today. 1996;26(9):688–93. 19. Umemura  Y, Yamakawa  K, Kiguchi  T, Yoshi- kawa  Y, Ogura  H, Shimazu  T, Hamasaki  T, Fujimi  S. Design and evaluation of New Uni- fied Criteria for Disseminated Intravascular Coagulation based on the Japanese Associ- ation for Acute Medicine Criteria. Clin Appl Thromb Hemost. 2016;22(2):153–60. doi: 10.1177/1076029615591169. 13. Герок  В, Блюм  ХЕ. Заболевания печени и  желчевыводящей системы. Пер. с  нем. М.: МЕДпресс-информ; 2009. 200 с. 14. Balzan  S, Belghiti  J, Farges  O, Ogata  S, Sau- vanet  A, Delefosse  D, Durand  F. The "50-50 criteria" on postoperative day 5: an accurate predictor of liver failure and death after hepa- tectomy. Ann Surg. 2005;242(6):824–8. doi: 10.1097/01.sla.0000189131.90876.9e. 7. Sato Y, Koyama  S, Tsukada  K, Hatakeyama  K. Acute portal hypertension reflecting shear stress as a  trigger of liver regeneration fol- lowing partial hepatectomy. Surg Today. 1997;27(6):518–26. 20. Wada H, Matsumoto T, Yamashita Y. Diagnosis and treatment of disseminated intravascu- lar coagulation (DIC) according to four DIC guidelines. J Intensive Care. 2014;2(1):15. doi: 10.1186/2052-0492-2-15. 8. Kin Y, Nimura Y, Hayakawa N, Kamiya J, Kon- do S, Nagino M, Miyachi M, Kanai M. Doppler analysis of hepatic blood flow predicts liver dysfunction after major hepatectomy. World J Surg. 1994;18(1):143–9. 15. van den Broek  MA, Olde Damink  SW, De- jong CH, Lang H, Malagó M, Jalan R, Saner FH. Liver failure after partial hepatic resection: definition, pathophysiology, risk factors and References Sato  Y, Koyama  S, Tsukada  K, Hatakeyama  K. Acute portal hypertension reflecting shear stress as a  trigger of liver regeneration fol- lowing partial hepatectomy. Surg Today. 1997;27(6):518–26. 12. Jarnagin  WR, Gonen  M, Fong  Y, DeMat- teo RP, Ben-Porat L, Little S, Corvera C, Weber S, Blumgart  LH. Improvement in perioperative 4. Patyutko YuI, Pylev  AL, Sagaydak  IV, Ko- tel'nikov AG, Podluzhnyy DV, Polyakov AN, Ab- garyan  MG, Chuchuev  ES, Gakhramanov  AD, 645 р еминированного внутрисосудистого свертывания при хирургическом лечении больных злокачественными опухолями печени Almanac of Clinical Medicine. 2016 June-July; 44 (5): 640–646 treatment. Liver Int. 2008;28(6):767–80. doi: 10.1111/j.1478-3231.2008.01777.x. outcome after hepatic resection: analysis of 1,803 consecutive cases over the past de- cade. Ann Surg. 2002;236(4):397–406. doi: 10.1097/01.SLA.0000029003.66466.B3. that modulate dormant and aggressive states in cancer. Thromb Res. 2014;133 Suppl 2:S1–9. doi: 10.1016/S0049-3848(14)50001-1. outcome after hepatic resection: analysis of 1,803 consecutive cases over the past de- cade. Ann Surg. 2002;236(4):397–406. doi: 10.1097/01.SLA.0000029003.66466.B3. 16. Meijer  C, Wiezer  MJ, Hack  CE, Boelens  PG, Wedel  NI, Meijer  S, Nijveldt  RJ, Statius Muller  MG, Wiggers  T, Zoetmulder  FA, Borel Rinkes IH, Cuesta MA, Gouma DJ, van de Vel- de CJ, Tilanus HW, Scotté M, Thijs LG, van Leeu- wen  PA. Coagulopathy following major liver resection: the effect of rBPI21 and the role of decreased synthesis of regulating proteins by the liver. Shock. 2001;15(4):261–71. 19. Umemura  Y, Yamakawa  K, Kiguchi  T, Yoshi- kawa  Y, Ogura  H, Shimazu  T, Hamasaki  T, Fujimi  S. Design and evaluation of New Uni- fied Criteria for Disseminated Intravascular Coagulation based on the Japanese Associ- ation for Acute Medicine Criteria. Clin Appl Thromb Hemost. 2016;22(2):153–60. doi: 10.1177/1076029615591169. 13. Gerok W, Blum HE. Krankheiten der Leber und des biliären Systems. Stuttgart  – New York: Schattauer Verlag; 2007 (in German). 14. Balzan  S, Belghiti  J, Farges  O, Ogata  S, Sau- vanet A, Delefosse D, Durand F. The "50-50 crite- ria" on postoperative day 5: an accurate predic- tor of liver failure and death after hepatectomy. Ann Surg. 2005;242(6):824–8. doi: 10.1097/01. sla.0000189131.90876.9e. 17. Amin  C, Mackman  N, Key  NS. Microparticles and cancer. Pathophysiol Haemost Thromb. 2008;36(3–4):177–83. doi: 10.1159/000175155. 20. Wada  H, Matsumoto  T, Yamashita  Y. Diagno- sis and treatment of disseminated intravas- cular coagulation (DIC) according to four DIC guidelines. J  Intensive Care. 2014;2(1):15. doi: 10.1186/2052-0492-2-15. 15. van den Broek  MA, Olde Damink  SW, De- jong CH, Lang H, Malagó M, Jalan R, Saner FH. 1 N.N. Blokhin Russian Cancer Research Center; 24 Kashirskoe shosse, Moscow, 115478, Russian Federation References Liver failure after partial hepatic resection: definition, pathophysiology, risk factors and 18. Magnus N, D'Asti E, Meehan B, Garnier D, Rak J. Oncogenes and the coagulation system – forces Somonova O.V.1 • Elizarova A.L.1 • Matveeva I.I.1 Somonova O.V.1 • Elizarova A.L.1 • Matveeva I.I.1 Rationale: Extended resections or extended lo- bectomies are the most common types of surgical interventions in patients with liver malignancies, and they are associated with serious post-opera- tive complications. Aim: To characterize the role of hemostasis abnormalities in the pathophys- iology of post-operative hepatic insufficiency, as well as that of thrombotic and hemorrhagic complications in patients with liver malignancies. Materials and methods: One hundred and twen- ty patients with liver malignancies were recruited into the study (20  patients with primary hepatic tumors and 100  with colorectal cancer and liver metastases). Extended liver resections (right and left simple and extended lobectomies, both sim- ple and extended) were performed in 100 (84%) of patients; multi-segmental liver resections, in 20  (16%). Assessment of hemostasis was done pre-operatively and at days 1 to 20 after surgery (hemostasis analyzer system STA-R Evolution and Chrono-log aggregometer). Results: After surgi- cal intervention in the liver, subacute disseminat- ed intravascular coagulation (DIC) was found in 34 patients. It was most common (65%) after the right lobectomy and was associated with a  de- crease in fibrinogen levels to 121 mg/dL (p < 0.01), prothrombin complex factors, to 45%  (р < 0.05), antithrombin III to 48%  (р < 0.05), with a  sig- nificant increase in D dimmer levels of up to 14.5 mcg/mL (р < 0.05). Twelve patients with sub- acute DIC developed deep venous thrombosis of the lower extremities, and 9 patients had severe hepatic insufficiency. Patients with severe hepat- ic insufficiency had a  statistically significant de- crease in prothrombin activity to 45%  (p < 0.05), antithrombin III to 44%, plasminogen < 50%, with high D dimer (> 20  mcl/mL) and von Willebrand factor levels. Conclusion: Surgical interventions in patients with liver malignancy may lead to the development of DIC. Early diagnosis and correc- tion of hemostasis-related risk factors of hepatic insufficiency allows for improvement of the results of surgery in patients with secondary hepatic ma- lignancies. Somonova Oksana V. – MD, PhD, Leading Research Fellow, Clinical Diagnostic Laboratory1 * * 24 Kashirskoe shosse, Moscow, 115478, Russian Federation. Tel.: +7 (499) 324 28 90. E-mail: somonova@mail.ru Elizarova Anna L. – PhD (in Biol.), Senior Research Fellow, Clinical Diagnostic Laboratory1 Matveeva Irina I. – MD, PhD, Head of Clinical Diag- nostic Laboratory1 Matveeva Irina I. Somonova O.V.1 • Elizarova A.L.1 • Matveeva I.I.1 – MD, PhD, Head of Clinical Diag- nostic Laboratory1 Key words: liver resection, hemostasis system, he- patic insufficiency Key words: liver resection, hemostasis system, he- patic insufficiency doi: 10.18786/2072-0505-2016-44-5-640-646 doi: 10.18786/2072-0505-2016-44-5-640-646 646
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English
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Outpatient decolonization after recurrent skin infection with Panton-Valentine leukocidin (PVL)-producing S. aureus—The importance of treatment repetition
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PLOS ONE PLOS ONE RESEARCH ARTICLE OPEN ACCESS Citation: Hanitsch LG, Kru¨ger R, Hoppe P-A, Humme D, Pokrywka A, Niebank M, et al. (2020) Outpatient decolonization after recurrent skin infection with Panton-Valentine leukocidin (PVL)- producing S. aureus—The importance of treatment repetition. PLoS ONE 15(4): e0231772. https://doi. org/10.1371/journal.pone.0231772 Editor: Bok-Luel Lee, Pusan National University, REPUBLIC OF KOREA Editor: Bok-Luel Lee, Pusan National University, REPUBLIC OF KOREA Editor: Bok-Luel Lee, Pusan National University, REPUBLIC OF KOREA Received: November 1, 2019 Accepted: March 31, 2020 Published: April 21, 2020 Received: November 1, 2019 Accepted: March 31, 2020 Published: April 21, 2020 Copyright: © 2020 Hanitsch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Background Recurrent skin abscesses are often associated with Panton-Valentine leukocidin-producing strains of S. aureus (PVL-SA). Decolonization measures are required along with treatment of active infections to prevent re-infection and spreading. Even though most PVL-SA patients are treated as outpatients, there are few studies that assess the effectiveness of outpatient topical decolonization in PVL-SA patients. Outpatient decolonization after recurrent skin infection with Panton-Valentine leukocidin (PVL)-producing S. aureus—The importance of treatment repetition Leif G. Hanitsch1,2, Renate Kru¨ger2,3, Pia-Alice Hoppe2,3, Daniel Humme2,4, Anna Pokrywka2,4, Michaela Niebank2,5, Miriam Stegemann2,5, Axel Kola6, Rasmus LeistnerID2,6* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Institute of Medical Immunology, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 2 Interdisciplinary workgroup on PVL-positive S. aureus, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 3 Department of Pediatric Pulmonology and Immunology, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 4 Department of Dermatology and Allergy, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany, 5 Department of Internal Medicine, Infectious Diseases and Pulmonary Medicine, Charite´ Universita¨tsmedizin, Berlin, Germany, 6 Institute of Hygiene and Environmental Medicine, Charite´ Universita¨tsmedizin Berlin, Berlin, Germany * rasmus.leistner@charite.de Methods Copyright: © 2020 Hanitsch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. We assessed the results of topical decolonization of PVL-SA in a retrospective review of patient files and personal interviews. Successful decolonization was defined as the absence of any skin abscesses for at least 6 months after completion of the final decolonization treat- ment. Clinical and demographic data was assessed. An intention-to-treat protocol was used. Conclusion In our cohort, topical decolonization was a successful preventive measure for reducing the risk of PVL-SA skin abscesses in the outpatient setting. Special attention should be given to patients living in multiple person households because these settings could confer a risk that decolonization will not be successful. Results Our cohort consisted of 115 symptomatic patients, 66% from PVL-positive MSSA and 19% from PVL-positive MRSA. The remaining 16% consisted of symptomatic patients with close contact to PVL-SA positive index patients but without detection of PVL-SA. The majority of patients were female (66%). The median age was 29.87% of the patients lived in multiple person households. Our results showed a 48% reduction in symptomatic PVL-SA cases after the first decolonization treatment. The results also showed that the decrease continued with each repeated decolonization treatment and reached 89% following the 5th treatment. Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Abbreviations: IQR, interquartile range; MRSA, Methicillin resistant Staphylococcus aureus; PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 1 / 9 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization A built multivariable Cox proportional-hazards model showed that the absence of PVL-SA detection (OR 2.0) and living in single person households (OR 2.4) were associated with an independently increased chance of successful decolonization. A built multivariable Cox proportional-hazards model showed that the absence of PVL-SA detection (OR 2.0) and living in single person households (OR 2.4) were associated with an independently increased chance of successful decolonization. MSSA, Methicillin susceptible Staphylococcus aureus; PCR, polymerase chain reaction; PVL, Panton-Valentine leucocidin; SA, Staphylococcus aureus. Introduction Recurrent skin abscesses in patients who do not have a predisposing condition are often associated with Panton-Valentine leukocidin-producing strains of S. aureus (PVL-SA) [1–3]. Although in North America PVL-SA is predominantly associated with community-acquired MRSA related primarily to the clone USA 300 [3, 4], in Europe the vast majority of PVL-posi- tive S. aureus (PVL-SA) strains are methicillin-susceptible (MSSA) [5]. The verification of PVL-SA colonization or infection requires microbiological screening for S. aureus and addi- tional PCR testing for the production of Panton-Valentine leukocidin by amplifying the encoding genes LukS/LukF [1]. There can be a considerable delay in diagnosis despite its typical clinical presentation with non-immunocompromised patients who suffer from recurrent skin abscesses. This results in multiple episodes of skin infections and often leads to the transmission of pathogens to close contacts [1, 2, 6, 7]. The diagnostic confirmation of a PVL-SA skin infection can be challenging for several reasons. Although microbiological screening has a high sensitivity for monocloncal PVL-positive MRSA, it is not clear how well nasal screening performs in cases of polyclonal MSSA colonization [8–10]. Moreover, often many members of a family or individuals in the same household can be affected. But the ping-pong-like transmission and retransmission events of S. aureus can make PVL-SA colonization time-dependent [10, 11]. This makes it difficult to eradicate PVL-SA colonization in all potential carriers. After the primary treatment of PVL-SA-related infections, secondary prevention requires topical decolonization [6, 12–14]. Despite the fact that many patients show relevant skin infec- tions, most cases can be treated in an outpatient setting. This includes decolonization proce- dures. However, there is a dearth of studies regarding outpatient decolonization and its specific hurdles in the outpatient setting for patients with recurrent PVL-SA skin infections. Hence, in this article we will focus on factors for the success of PVL-SA topical decolonization that are relevant to the outpatient setting. Methods Our cohort consisted of patients with recurrent skin infections combined with the detection of PVL-positive S. aureus (PVL-SA) as well as their close contacts who sometimes also displayed symptoms. All cohort members were seen in our outpatient clinic between December 2010 and August 2017. Recurrence of an abscess was defined as more than one abscess that required surgical drainage within a two-month period. Patients with a (predisposing) chronic skin con- dition, e.g. Acne inversa or severe atopic dermatitis, were excluded from the present study. In 2 / 9 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization clusters with more than one symptomatic and / or PVL-SA-positive patient, the topical decolo- nization protocol was followed for all household members. In case of active PVL-SA skin infections, patients received a rifampicin-based dual antibiotic regime combined with TMP/ SMX or as determined by antibiogram results. In addition, patients were advised to start a decolonization treatment at home after the resolution of the infection. Decolonization involved a 5-day standard decolonization protocol that included daily antiseptic washing of the whole body and hair with a commercially available octenidin-based product, antiseptic treatment of the oral cavity (octenidin-based), intranasal application of mupirocin, and extended hygiene measures including the washing of clothes at more than 60˚C and disposing of personal care products [2]. Success of the decolonization procedure was defined as a period of at least six months without a skin abscess or other SSTI following completion of the decolonization treatment. Decolonization was repeated in cases when skin abscesses recurred. The following variables were systematically evaluated in interviews to assess the factors that reduced the success of decolonization: age, gender, number of decolonization treatments, household size, number of close contacts to the index case, abscess localization, and phenotype of PVL-SA (MRSA or MSSA). A cluster was defined as individuals in the index patient’s immediate environment (e.g. family or friends with frequent close physical contact) who were also colonized or infected with PVL-SA. Household size was defined as the number of individuals living together in a household with the index patient. All patients were screened for PVL-SA in nares, throat and wounds, if applicable. Swabs were cultivated on Columbia Agar with 5% sheep blood. Species identification and antimicro- bial susceptibility testing were performed using a Vitek 2 system and applying EUCAST break- points. Methods The detection of PVL LukS/LukF was performed using PCR [15]. In order to assess parameters influencing the length of time needed for successful decoloni- zation, we performed a univariate Kaplan-Meier analysis and a multivariable Cox regression of all symptomatic patients, applying an intention-to-treat protocol. All analyses were per- formed using SPSS (IBM SPSS statistics, Somer, NY, USA). Ethics approval and consent to participate: The study was conducted in accordance with the Declaration of Helsinki, national, and institutional standards. The study was approved by the local Ethics Committee (Charite´, Berlin, Germany, EA2/190/17). The study is based on sec- ondary data that was generated for routine clinical care. Within this scope, written consent for treatment purposes and secondary analysis was provided by each patient. Consent for minors under age 18 was obtained from parents or guardians. The Ethics Committee waived the requirement for additional consent for this study. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 Results Sixty seven index patients with PVL-SA-positive recurrent skin abscesses presented to our out- patient clinics between December 2010 and August 2017. Altogether, they reported 128 close contacts (accounting for n: 195). 48 of 128 contacts reported recurrent skin abscesses. Of the remaining 80 asymptomatic contacts, 40 contacts participated in PVL-SA screening, revealing an additional 21 PVL-SA positive cases. The remaining 40 asymptomatic contact patients were not available for examination of PVL-SA (Fig 1). Taken together, of 155 patients and contacts, PVL-SA was verified in 118 cases (76%). One patient was colonized with both phenotypes, hence 27 MRSA (23%), 92 MSSA (78%) were detected. By the time of diagnosis (skin abscess due to PVL-SA), the patients’ medical histories showed a median of 4 episodes of skin abscesses (IQR 2–10) and a median delay of 5 months PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 3 / 9 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Fig 1. Flow chart of all enrolled patients. https://doi.org/10.1371/journal.pone.0231772.g001 Fig 1. Flow chart of all enrolled patients. htt //d i /10 1371/j l 0231772 001 Fig 1. Flow chart of all enrolled patients. Fig 1. Flow chart of all enrolled patients. https://doi.org/10.1371/journal.pone.0231772.g001 between first episode and final diagnosis (IQR 1–14). We found it helpful to calculate the aver- age number of abscesses per month as an estimator of the severity of the infection and the psy- chological strain on patients. The result was a median of 1.0 (IQR 0.3–2.8). Common abscess sites included extremities (50%), trunk (47%), and head or face (42%), without any specific dis- tribution pattern. Few patients (4%) showed invasive infections, such as necrotizing fasciitis, necrotizing pneumonia or bloodstream infection. Based on the patients’ past medical histories, many reported that they had undergone multi- ple antibiotic treatments and multiple episodes of surgical intervention before being diagnosed with skin abscesses from PVL-SA. 38 (33%) patients reported hospital stays for the treatment of their recurrent abscesses with a length of stay between 1 and 42 days (median 3 days). Table 1 provides an overview of all assessed parameters. Following an intention-to-treat protocol, we analyzed all 115 symptomatic patients for suc- cessful topical decolonization. 77 (67%) received systemic antimicrobial treatment beforehand without relevant reduction in infection incidence. The median follow-up time was 15 months after the final decolonization treatment. Results After the first decolonization treatment, the number of symptomatic patients was reduced by 48% (56 patients) (Fig 2). 13 patients were lost to fol- low up (11%). Altogether, this amounted to a success rate of 89% after 5 decolonization treatments. The following parameters were considered in the Cox regression analysis: age, gender, number of decolonization treatments, single vs. multiple person household and phenotype of PVL-SA (MSSA/MRSA). Factors independently associated with a successful decolonization were living in a single household (in contrast to a multiple person household) and undetected PVL-SA in symptomatic patients (Table 2 and Fig 3). Discussion Skin and soft tissue infection caused by PVL-positive S. aureus are frequently associated with recurrent episodes of infection, delayed diagnoses, and eventually an increased risk of trans- mission often resulting in clusters of affected patients in close personal proximity [1–3, 6, 16]. Although these infections are often community-acquired, there is a lack of literature that ana- lyzes the effectiveness of outpatient decolonization [17, 18]. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 4 / 9 (PVL)-producing S. aureus—The importance of repeting decolonization MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining clinically asymptomatic for at least 6 months after final decolonization treatment. PLOS ONE PLOS ONE PLOS ONE Table 1. Overview on the analyzed PVL-SA patients. Parameter Total cohort (n = 155) Clinically symptomatic (n = 115) Clinically asymptomatic (n = 40) Median (range) / % (number) Median (range) / % (number) Median (range) / % (number) Age (years) 29 (0–75) 28 (0–65) 31 (0–75) Male sex 44% (68) 43% (49) 48% (19) Months before diagnosis not applicable 5 (0–158) not applicable Number of abscesses before diagnosis not applicable 4 (1–100) - Number of antimicrobial treatments before diagnosis 1 (0–15) 1 (1–15) 0 (0–3) Number of surgical treatments before diagnosis not applicable 1 (1–13) - Detected pathogen PVL-positive S. aureus MRSA 17% (27) 19% (22) 13% (5) MSSA 59% (92) 66% (76) 40% (16) Not detected 24% (37) 16% (18) 48% (19) Successful decolonization Yes 88% (137) 89% (102) not applicable Lost to follow up 8% (13) 11% (13) - Number of decolonization treatments 1 (1–5) 1 (1–5) 1 (1–3) Household size Single person 10% (15) 13% (15) - Multiple persons 90% (140) 87% (100) 100% (40) Abscess Localization Extremities 37% (58) 50% (58) - Trunk 35% (54) 47% (54) - Head/Face 31% (48) 42% (48) - Gluteal 22% (34) 30% (34) - Axilla 13% (20) 17% (20) - Genital 11% (17) 15% (17) - Inguinal 5% (8) 7% (8) - Invasive 3% (4) 4% (4) - MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining clinically asymptomatic for at least 6 months after final decolonization treatment. MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining clinically asymptomatic for at least 6 months after final decolonization treatment. MRSA, methicillin-resistant S. aureus. MSSA, methicillin-susceptible S. aureus. PVL, Panton-Valentine leukodicin. Successful decolonization was defined as remaining clinically asymptomatic for at least 6 months after final decolonization treatment. https://doi.org/10.1371/journal.pone.0231772.t001 Fig 2. Kaplan-Meier curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine leukocidin-positive Staphylococcus aureus. https://doi.org/10.1371/journal.pone.0231772.g002 Fig 2. Kaplan-Meier curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine leukocidin-positive Staphylococcus aureus. Fig 2. Kaplan-Meier curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine leukocidin-positive Staphylococcus aureus. 5 / 9 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Table 2. Multivariable Cox regression analysis of parameters associated with successful decolonization. Parameter P-value OR 95% Confidence Interval Lower Upper PVL-S. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 PLOS ONE aureus phenotype MSSA Reference MRSA 0.615 0.873 0.516 1.479 Undetected 0.012 2.030 1.170 3.523 Single household 0.006 2.372 1.285 4.381 PVL, pantone-valentine leukocidine. MSSA, Methicillin-susceptible S. aureus. MRSA, methicillin-resistant S. aureus. OR, odds ratio. https://doi.org/10.1371/journal.pone.0231772.t002 Table 2. Multivariable Cox regression analysis of parameters associated with successful decolonization. Pathogen transfer within households has been described repeatedly as the dominant trans- mission route for community-acquired S. aureus in the past [7, 11, 19, 20]. Our data under- scores this observation for PVL-SA, as we found that 69/88 (78%) contacts screened were also clinically symptomatic and / or PVL-positive (Fig 1). The clinical relevance of household con- tact is further emphasized by our observation that living in a multiple person household was a relevant risk factor for requiring repeated decolonization as compared to living alone. Knox et al. stated that the degree of physical contact among household members and the amount of time spent at home determine the risk of transmission [11]. This could result in epidemic S. aureus clones ‘ping-ponging’ between family members [19–22]. These observa- tions support the need for the simultaneous decolonization of close physical contacts, at least in cases where patients remain PVL-SA colonized despite decolonization treatment. Fig 3. Multivariable Cox regression curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine leukocidin-positive Staphylococcus aureus. https://doi org/10 1371/journal pone 0231772 g003 Fig 3. Multivariable Cox regression curve of PVL-SA decolonization in 115 symptomatic patients. PVL-SA, Panton-Valentine leukocidin-positive Staphylococcus aureus. https://doi.org/10.1371/journal.pone.0231772.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 6 / 9 PLOS ONE (PVL)-producing S. aureus—The importance of repeting decolonization Our results show that symptomatic patients in which PVL-SA could not be detected have a higher chance of successful decolonization. Hence, this observation could be an indication that a low-level colonization with PVL-SA might facilitate the eradication process. Earlier stud- ies of MRSA decolonization have shown that the success of eradication can be dependent on the localization of the colonization [23–25]. In our cohort, testing for PVL-SA was not per- formed separately, using different swabs for nose and throat. In the present study many patients reported a past medical history typical of PVL-SA patients–recurrent skin abscess that often required incision and drainage and reporting other affected patients in their immediate environment. PVL-SA-positive patients are often symp- tomatic for several months, before they are finally diagnosed and treated for PVL-SA [16, 26– 29]. PLOS ONE Our results thus underscore earlier work that shows that a diagnosis can be challenging in areas with low PVL-SA prevalence [6, 30]. The patients in our cohort were on average 29 years old, confirming earlier studies on patients with community-onset PVL-SA-associated skin abscess [30, 31]. Whether there are socio-cultural or medico-biological factors driving this observation is unclear. Our study has several limitations. This is a retrospective study on PVL-SA patients from a single institution. It represents the patient population from our area and mirrors the results of our locally established treatment procedures. All patients with an active infection were treated with an oral antimicrobial agent—at least once—parallel to topical treatment. This makes it impossible to determine the effect of the antimicrobial therapy on the decolonization results. However, considering the fact that 2/3 of our patients had received antibiotics without any topical decolonization and continued to suffer from recurrent skin abscesses argues against a major protective role of isolated antibiotic use in these patients. Patients in our cohort had a mean total of four skin abscesses in the five months prior to topical decolonization; after suc- cessful decolonization, all patients were asymptomatic for at least 6 months with a mean fol- low-up of 15.6 months. Conclusion Our data shows that patients with skin abscesses associated with PVL-SA can be successfully treated with decolonization as outpatients, but they often require multiple attempts at decolo- nization. The delayed success of treatment of patients living in multiple person households suggests that decolonization treatment should be conducted simultaneously on all close contacts. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 References 1. Shallcross LJ, Fragaszy E, Johnson AM, Hayward AC. The role of the Panton-Valentine leucocidin toxin in staphylococcal disease: a systematic review and meta-analysis. The Lancet Infectious dis- eases. 2013; 13:43–54. https://doi.org/10.1016/S1473-3099(12)70238-4 PMID: 23103172 2. 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Writing – original draft: Leif G. Hanitsch, Rasmus Leistner. Writing – review & editing: Leif G. Hanitsch, Renate Kru¨ger, Pia-Alice Hoppe, Daniel Humme, Anna Pokrywka, Michaela Niebank, Miriam Stegemann, Axel Kola, Rasmus Leistner. Resources: Axel Kola, Rasmus Leistner. Supervision: Renate Kru¨ger, Pia-Alice Hoppe, Daniel Humme, Anna Pokrywka, Michaela Niebank, Miriam Stegemann, Axel Kola. Validation: Leif G. Hanitsch, Daniel Humme, Axel Kola, Rasmus Leistner. Visualization: Rasmus Leistner. Visualization: Rasmus Leistner. PLOS ONE | https://doi.org/10.1371/journal.pone.0231772 April 21, 2020 Author Contributions Conceptualization: Leif G. Hanitsch, Pia-Alice Hoppe, Anna Pokrywka, Rasmus Leistner. Data curation: Leif G. Hanitsch, Rasmus Leistner. Formal analysis: Axel Kola, Rasmus Leistner. Investigation: Leif G. Hanitsch, Rasmus Leistner. Methodology: Leif G. Hanitsch, Rasmus Leistner. Project administration: Leif G. Hanitsch, Rasmus Leistner. Conceptualization: Leif G. 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Pulmonary Mucormycosis as the Leading Clinical Type of Mucormycosis in Western China
Frontiers in cellular and infection microbiology
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ORIGINAL RESEARCH published: 22 November 2021 doi: 10.3389/fcimb.2021.770551 Pulmonary Mucormycosis as the Leading Clinical Type of Mucormycosis in Western China Junyan Qu 1, Xijiao Liu 2 and Xiaoju Lv 1* 1 Center of Infectious Disease, West China Hospital, Sichuan University, Chengdu, China, 2 Radiology Department, West China Hospital, Sichuan University, Chengdu, China Edited by: Kai Huang, University of Texas Medical Branch at Galveston, United States Reviewed by: Muhammad Usman Tariq, Abu Dhabi School of Management, United Arab Emirates Valentina Giudice, University of Salerno, Italy Dora Edith Corzo-Leon, University of Aberdeen, United Kingdom *Correspondence: Xiaoju Lv lvxj33966@126.com Specialty section: This article was submitted to Clinical Microbiology, a section of the journal Frontiers in Cellular and Infection Microbiology Received: 04 September 2021 Accepted: 04 November 2021 Published: 22 November 2021 Citation: Qu J, Liu X and Lv X (2021) Pulmonary Mucormycosis as the Leading Clinical Type of Mucormycosis in Western China. Front. Cell. Infect. Microbiol. 11:770551. doi: 10.3389/fcimb.2021.770551 The aim was to better understand the clinical characteristics of patients with mucormycosis in western China. We retrospectively investigated the clinical, laboratory, radiological and treatment profiles of mucormycosis patients during a 10-year period (2010–2019). As a result, 59 proven mucormycosis were enrolled in this study. It was found that 52.5% of patients had worse clinical outcomes. Pulmonary mucormycosis (PM) was the most common clinical manifestation. The most frequent risk factor was diabetes mellitus (38, 64.4%) for mucormycosis patients. Cough (43, 93.5%), fever (24, 52.2%) and hemoptysis/bloody phlegm (21, 45.7%) were the most common manifestations of PM. There were no differences in clinical manifestations, risk factors and laboratory tests between different clinical outcome groups (P>0.05). Lymph node enlargement (30, 65.2%), patchy shadows (28, 60.9%), cavitation (25, 53.3%) and bilateral lobe involvement (39, 84.8%) were the most common on chest CT. Nodule was more common in good outcome group (P <0.05). A total of 48 cases (81.4%) were confirmed by histopathological examination, 22 cases (37.3%) were confirmed by direct microscopy. PM patients were treated with amphotericin B/amphotericin B liposome or posaconazale had better clinical outcomes (P <0.05). In conclusion, PM was the most common clinical type of mucormycosis in China. Diabetes mellitus was the most common risk factor. PM has diverse imaging manifestations and was prone to bilateral lobes involvement. Early diagnosis and effective anti-mucor treatment contribute to successful treatment. Keywords: mucormycosis, pulmonary mucormycosis, diabetes, clinical analysis, clinical outcome INTRODUCTION Mucormycosis is a rare, emerging and opportunistic fungal infection with high morbidity and mortality caused by filamentous fungi of the Mucoraceae family, order Mucorales. Mucorales fungi are ubiquitous in nature. Humans are infected mainly by inhaling sporangiospores, occasionally through the ingestion of contaminated food or traumatic inoculation (Prakash and Chakrabarti, 2019; Shariati et al., 2020). The prevalence of mucormycosis varies between developing and developed countries, ranging from 0.01 to 14 per 100 000 population in Europe and India (Bitar et al., 2009; Chakrabarti and Singh, 2014; Ruhnke et al., 2015). With the growth of the number of Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis information, underlying diseases, use of corticosteroid or immunosuppressive agent, clinical manifestations, laboratory data, radiologic findings, diagnostic procedures, therapeutic strategies and clinical outcomes at 90 days were collected. The study was approved by the Ethics Committee of West China Hospital, Sichuan University. Because all the data in this study were routinely obtained, written informed consent was waived. immunocompromised patients, increased awareness and development of diagnostic techniques, the incidence of mucormycosis is rising (Prakash et al., 2019). According to clinical presentations, mucormycosis is mainly classified as rhino-orbito-cerebral, pulmonary, cutaneous, gastrointestinal and disseminated types (Skiada et al., 2020). The common risk factors of mucormycosis are diabetes, hematological malignancy, use of corticosteroids or immunosuppressants, and trauma (Petrikkos et al., 2014; Jeong et al., 2019). However, the main cause of mucormycosis also varies in different countries. Hematological malignancies are the main cause in countries with high income, while diabetes mellitus (DM) or trauma are the main cause in developing countries (Chakrabarti and Singh, 2014). Diagnosis of mucormycosis is challenging because of the low sensitivity and specificity of clinical diagnostic methods (Skiada et al., 2018). The mortality of mucormycosis remains high, it may be related to delayed diagnosis, high cost of managing mucormycosis and limited treatment options (Skiada et al., 2018). Previous studies on the characteristics of mucormycosis have been conducted mainly in America, Europe and India (Prakash and Chakrabarti, 2019). Data about mucormycosis from China is sparse. The causative agents of mucormycosis vary with different geographical locations (Prakash and Chakrabarti, 2019), and the epidemiology, the clinical disease pattern of mucormycosis vary from country to country. To better understand the clinical characteristics of patients with mucormycosis in China, in this retrospective study, we compared the demographic features, clinical presentations, laboratory data, radiographic findings and therapeutic strategies in mucormycosis patients with different clinical outcomes who were admitted to a university hospital from Jan 2010 to Dec 2019 in western China. Laboratory Studies Laboratory tests including complete blood count, blood biochemistry, procalcitionin (PCT), C-reactive protein, T lymphocyte subset, HIV testing, serum (1,3)-beta-D-glucan test (BDG test), and galactomannan test (GM test) were performed. Clinical samples (including blood, sputum, secretions, urine) were aseptically collected and cultured under aerobic or anaerobic conditions. Bacterial species were isolated and identified using MicroScan WalkAway-96 System (Siemens, USA). Fungal culture was performed on Sabouraud dextrose agar (SDA) and incubated at 30°C. All the items were performed in the Department of Laboratory Medicine of our hospital. Radiological Assessment Imaging examinations such as chest computed tomography (CT), abdominal CT, fibroptic bronchoscopy were performed at the discretion of the treating physicians. The CT scans were performed using 64-row multi-slice spiral CT scanner (SOMATOM definition AS+, Siemens) in our hospital. All images were reviewed independently by two experienced radiologists. For patients with PM, chest CT findings including nodule, mass, cavity, patchy consolidation, ground glass opacity, reversed halo sign, lymph node enlargement, pleural effusion and the distribution of the lesion in the lungs were recorded. Treatment Strategies and Clinical Outcomes The patients with mucormycosis were treated with amphotericin B (AmB, 0.5-1 mg/kg per day) (North China Pharmaceutical Co., Ltd., China) or amphotericin B liposome (LAmB, 3-6 mg/kg per day) in accordance with the drug instructions, guidelines (Skiada et al., 2013) and patient tolerance. Posaconazole oral suspension (40mg/ml, 10ml, twice daily) (Patheon Inc, Whitby, ON, Canada) was used in patients who have contraindications to amphotericin B or who cannot tolerate the side effects of amphotericin B like reduced kidney function, electrolyte imbalances, nausea and vomiting. The treatment duration was determined by the treating physicians according to the patient’s treatment response and the size of the focus. Management of patients with diabetes included dietary guidance, tight glucose control with insulin and/or hypoglycemic drugs, multiple flash glucose monitoring daily and treatment of complications. A multidisciplinary consulting team, including diabetes, infectious disease, nutrition, wound therapy, surgery, clinical microbiology, and clinical pharmacy, that offered specialist advice, on-going management. Other underlying diseases of these patients such as immune system disease, chronic lung disease were treated normally and systematically. For bacterial MATERIALS AND METHODS Patients From Jan 2010 to Dec 2019, the patients with a diagnosis of mucormycosis at hospital discharge were retrospectively reviewed in West China Hospital, Sichuan University, a 4,300-bed academic tertiary hospital in Chengdu, China. According to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria and previous references (De Pauw et al., 2008; Petrikkos et al., 2014; Skiada et al., 2018), inclusion in the final study group required the diagnosis of proven mucormycosis as defined as follows: (1) age ≥ 14 years; (2) clinical manifestations and radiographic findings consistent with mucormycosis; and (3) histological presence of mucormycosis in tissue specimens, and/or broad-based, ribbon-like, non-septate hyphae with right-angle branching filamentous fungi on direct microscopy of clinical specimens, and/or Mucorales species cultured from clinical specimens. Based on clinical presentation and the involvement of the body sites, rhino-orbital-cerebral, pulmonary, cutaneous and disseminated mucormycosis were classified (Jeong et al., 2019). The following data of demographic Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis These patients were classified as pulmonary type and other type mucormycosis based on clinical type. The demographic and clinical outcomes of these patients were summarized in Table 1. The ages of patients with PM was higher than that of patients with non-pulmonary mucormycosis (P=0.015). The most frequent underlying disease was diabetes mellitus (38, 64.4%) for mucormycosis patients. More patients with PM had diabetes mellitus than patients with non-pulmonary mucormycosis (P=0.027). A total of 31 mucormycosis patients (52.5%) had worse clinical outcomes, clinical outcome was not obviously different between the two groups (P>0.05). co-infection patients, empirical or targeted antimicrobial therapy were given. Clinical outcomes of patients with mucormycosis were evaluated at 90 days after diagnosis. According to clinical manifestations, laboratory findings and image changes, their clinical outcomes were divided into good and worse outcome. Death or disease progression or persistence were classified as worse outcome. The continuous improvement of clinical symptoms and imaging findings were classified as good outcome. Statistical Analysis Statistical analyses were performed using IBM SPSS Statistics for Windows v.26.0 (IBM Corp., Armonk, NY, USA). The ShapiroWilk normality test was used to test the normality of all quantitative variables. Continuous variables with normal distribution were presented as mean± standard deviation (SD) and compared by Student’s t-test. The relationship between categorical variables was assessed using Chi-square test or Fisher’s exact test. A two-tailed P value lower than 0.05 was considered statistically significant. Demographic and Clinical Characteristics in Patients With PM Demographic and clinical features of PM patients with different clinical outcomes were shown in Table 2. The most common clinical symptoms of PM patients were cough (43, 93.5%), fever (24, 52.2%) and hemoptysis/bloody phlegm (21, 45.7%). Diabetes mellitus (33, 71.7%) was the most frequent underlying disease. There were no significant difference in the gender, age, clinical symptoms and underlying diseases beween good and worse outcome group (P>0.05). For laboratory results, there was no significant difference between the two groups (P > 0.05). RESULTS Demographic Characteristics and Clinical Outcomes of Mucormycosis Patients Findings of CT Scan in Patients With PM A total of 68 patients with mucormycosis were admitted to our hospital from January 2010 to December 2019, 59 of whom with proven mucormycosis (mean age 54.75 ± 14.72 years; 44 males) were enrolled in this retrospective study. Pulmonary mucormycosis (PM) was the most commonly observed manifestation (46/59, 78.0%), followed by rhino-orbitalcerebral (9/59, 15.3%) and disseminated mucormycosis (3/59, 5.0%). There was only one case of cutaneous mucormycosis. The chest CT features of patients with PM were diverse, as shown in Figure 1. The detail of chest CT findings in these patients were shown in Table 3. The most common manifestations were lymph node enlargement (30, 65.2%), patchy shadows (28, 60.9%), cavitation (25, 53.3%) and nodules (24, 52.2%). Nodule was more common in good outcome group than in worse outcome group (P <0.05). Lymph node enlargement were more common in patients with worse outcome (P<0.05). Bilateral TABLE 1 | Demographic and clinical outcomes in patients with mucormycosis. Variables Male Age (years) (mean ± SD) Underlying diseases or risk factors Diabetes mellitus Immune system disease Corticosteroid medication or immunosuppressive drugs Hematological malignancy Solid tumor Chronic kidney diseases Chronic lung disease Chronic liver disease Truma None Co-infection of other pathogens Bacteria Other fungi Clinical outcome Good outcome Worse outcome Clinical type P-value All (N = 59) Pulmonary type (N = 46) Other type (N = 13) 44 (74.6) 54.75 ± 14.72 35 (76.1) 57.41 ± 12.40 9 (69.2) 45.31 ± 18.64 0.616 0.015* 38 (64.4) 2 (3.4) 4 (6.8) 3 (5.1) 2 (3.4) 3 (5.1) 8 (13.6) 2 (1.7) 2 (3.4) 8 (13.6) 33 (71.7) 1 (2.2) 3 (6.5) 1 (2.2) 2 (4.3) 3 (6.5) 7 (15.2) 1 (2.2) 1 (2.2) 6 (13.0) 5 (38.5) 1 (7.7) 1 (7.7) 2 (15.4) 0 (0.0) 0 (0.0) 1 (7.7) 1 (7.7) 1 (7.7) 2 (15.4) 0.027* 0.395 1.000 0.119 1.000 1.000 0.671 0.395 0.395 1.000 42 (71.2) 16 (23.7) 32 (69.6) 14 (30.4) 10 (76.9) 2 (15.4) 0.738 0.481 28 (47.5) 31 (52.5) 23 (50.0) 23 (50.0) 5 (38.5) 8 (61.5) 0.462 *P < 0.05. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 3 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis TABLE 2 | Demographic and clinical characteristics in patients with pulmonary mucormycosis. Variables Male Age Presenting symptoms and signs Fever Cough hemoptysis or bloody phlegm Chest pain Shortness of breath Underlying diseases or risk factors Diabetes mellitus Immune system disease Corticosteroid medication or immunosuppressive drugs Hematological malignancy Solid tumor Chronic kidney diseases Chronic lung disease Chronic liver disease Truma None Laboratory data White blood cell (× 109/L) Neutrophil (%) Lymphocyte (× 109/L) Eosinophil (× 109/L) Mononuclear cell (× 109/L) Procalcitionin (ng/mL) C-reactive protein (mg/L) Eerythrocyte sedimentation rate (mm/h) CD4+ T lymphocyte (%) CD8+ T lymphocyte (%) CD4/CD8 ratio CD4+ T lymphocyte (cells/ml) CD8+ T lymphocyte (cells/ml) Co-infecting agent Bacteria Virus Fungi All (N = 46) (n,%) Good outcome (N = 23) (n,%) Worse outcome (N = 23) (n,%) P-value 35 (76.1) 57.41 ± 12.40 5 (21.7) 56.00 ± 13.44 6 (26.1) 58.82 ± 11.38 0.288 0.446 24 (52.2) 43 (93.5) 21 (45.7) 7 (15.2) 14 (30.4) 13 (56.5) 21 (91.3) 9 (39.1) 2 (8.7) 9 (39.1) 11 (47.8) 22 (95.7) 12 (52.2) 5 (21.7) 5 (21.7) 0.768 1.000 0.554 0.414 0.200 33 (71.7) 1 (2.2) 3 (6.5) 1 (2.2) 2 (4.3) 3 (6.5) 7 (15.2) 1 (2.2) 1 (2.2) 6 (13.0) 16 (69.6) 0 (0.0) 2 (8.7) 1 (4.3) 0 (0.0) 2 (8.7) 4 (17.4) 1 (4.3) 1 (4.3) 4 (17.4) 17 (73.9) 1 (4.3) 1 (4.3) 0 (0.0) 2 (8.7) 1 (4.3) 3 (13.0) 0 (0.0) 0 (0.0) 2 (8.7) 1.000 1.000 1.000 1.000 0.489 1.000 1.000 1.000 1.000 0.665 8.69 ± 3.76 69.38 ± 12.10 1.62 ± 0.73 0.19 ± 0.26 0.50 ± 0.20 0.12 ± 0.13 73.17 ± 82.08 60.42 ± 23.31 36.55 ± 12.54 30.30 ± 10.80 1.48 ± 1.01 740.2 ± 357.47 466.80 ± 253.69 0.665 0.170 0.180 0.397 0.539 0.289 0.831 0.278 0.771 0.475 0.409 0.062 0.250 13 (56.5) 1 (4.3) 8 (34.8) 1.000 1.000 0.763 9.13 ± 6.84 71.96 ± 12.24 1.48 ± 0.67 0.33 ± 1.17 0.49 ± 0.23 0.21 ± 0.49 70.31 ± 87.94 55.23 ± 24.06 36.94 ± 10.33 31.61 ± 9.49 1.36 ± 0.77 565.45 ± 288.69 396.82 ± 176.78 9.58 ± 74.42 ± 1.35 ± 0.48 ± 0.47 ± 0.29 ± 66.75 ± 49.00 ± 37.32 ± 32.92 ± 1.23 ± 419.83 ± 338.50 ± 25 (54.3) 2 (4.3) 18 (39.1) 12 (52.2) 1 (4.3) 10 (43.5) were treated with amphotericin B/LAmB. More patients with PM were treated with AmB/LAmB (P=0.006) or posaconazale (P=0.004) in good outcome group than in worse outcome group. For patients with PM, only 3 patients were treated with surgery and LAmB, but there was no statistical difference in clinical outcome (P>0.05). The most common adverse events of AmB/LAmB included renal insufficiency (16/42, 38.1%) and hypokalemia (9/42, 21.4%) in this study. The severity of all adverse events were mild to moderate (grade 1-2) according to the US National Cancer Institute Common Toxicity Criteria (NCI-CTC) (US, National Cancer Institute, 2017). The adverse reaction ratio of AmB/LAmB has not statistical difference between the two groups (P>0.05). As shown in Table 4. lungs were involved in 84.8% (39/46) of patients, with the left lower lobe (20, 43.5%) and right lower lobe (18, 39.1%) most commonly involved. There was no significant difference in the lobe of lesion distribution between the two groups (P>0.05). There were 13 patients with Mucor and Aspergillus co-infection and 1 patient with lung cancer. Based on imaging findings, 12 patients were suspected to be tumors, and 2 patients were suspected to be aspergillosis in the absence of concomitant Aspergillus. Figure 2 showed the changes of chest CT lesions during the follow-up of a patient with pulmonary fungal infection (Mucor with Aspergillus). Diagnostic Procedures and Treatment Strategies Of the 59 proven patients with mucormycosis, 48 cases (81.4%) were confirmed by histopathological examination, 22 cases (37.3%) were confirmed by direct microscopy. There was no significant difference in the diagnostic procedures between the two groups (P>0.05). A total of 47 patients (79.7%) received treatment. Treatment strategies for these patients with mucormycosis include antifungal drugs, surgery, or both. Most patients (38/59, 64.4%) Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9.02 12.11 0.60 1.46 0.26 0.66 97.40 24.64 8.00 8.17 0.40 92.04 45.12 DISCUSSION Mucormycosis is rare, neglected, and associated with high mortality rates. The study found that 52.5% of patients with mucormycosis had poor clinical outcomes. Which is similar to previous research results (Jeong et al., 2019). PM was the most 4 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis A B C D E F G H FIGURE 1 | The findings of chest computed tomography of pulmonary mucormycosis (patients with good outcome: A, B; patients with worse outcome: C–H). (A) Multiple pleomorphic lesions in both lungs; (B) cavity with muralnodule (Mucor co-infecting with Aspergillus); (C) cavity in left lung; (D) thick-walled cavity shadow with gas-fluid level (Mucor co-infecting with Klebsiella pneumoniae); (E) mass shadow in the upper lobe of the left lung; (F) patchy shadow, nodules and consolidation in right lung; (G) cavity and ground-glass opacity; (H) reversed halo sign. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 5 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis TABLE 3 | Chest CT findings of proven patients with pulmonary mucormycosis . Morphology Consolidation Nodule Ground-glass opacity Mass Cavitation Patchy shadow Fibrosis Pleural effusion Pleural thickening Lymph node enlargement Reversed halo sign Lobe of lesion distribution Left upper lobe Left lower lobe Right upper lobe Right middle lobe Right lower lobe Bilateral involvement All (N = 46) (n,%) Good outcome (N = 23) (n,%) Worse outcome (N = 23) (n,%) P-value 20 (43.5) 24 (52.2) 7 (15.2) 15 (32.6) 25 (53.3) 28 (60.9) 17 (37.0) 22 (47.8) 17 (37.0) 30 (65.2) 5 (10.9) 9 (39.1) 16 (69.6) 5 (21.7) 8 (34.8) 10 (43.5) 14 (60.9) 8 (34.8) 11 (47.8) 6 (26.1) 11 (47.8) 2 (8.7) 11 (47.8) 8 (34.8) 2 (8.7) 7 (30.4) 15 (65.2) 14 (60.9) 9 (39.1) 10 (43.5) 11 (47.8) 19 (82.6) 3 (13.0) 0.767 0.038* 0.414 1.000 0.139 1.000 1.000 1.000 0.221 0.029* 1.000 12 (26.1) 20 (43.5) 12 (26.1) 11 (23.9) 18 (39.1) 39 (84.8) 5 (21.7) 11 (47.8) 7 (30.4) 8 (34.8) 8 (34.8) 20 (87.0) 7 (30.4) 9 (39.1) 5 (21.7) 3 (13.0) 10 (43.5) 19 (82.6) 0.738 0.767 0.738 0.165 0.763 1.000 *P < 0.05. increased mortality (Spellberg et al., 2012). Neutropenia was not found in our patients with PM, it might not be an important risk factor for mortality in these patients. The most frequent imaging findings of PM were patchy shadows, cavitation and pulmonary nodules in this study. According to the imaging findings, PM might be misdiagnosed as tumor or pulmonary aspergillosis. Nodule was more common in good outcome group. Lymph node enlargement were more common in patients with worse outcome. Many previous studies have found multiple nodules, reversed halo sign, and cavities associated with PM (McAdams et al., 1997; Marom and Kontoyiannis, 2011). Pulmonary nodules, halo sign and cavitation was also found in patients pulmonary invasive aspergillosis (Althoff Souza et al., 2006; Muldoon et al., 2016). The reversed halo sign has been considered an important clue to the diagnosis of PM, however, it has also been described in other pulmonary diseases, including invasive pulmonary aspergillosis, tuberculosis, organising pneumonia and malignancy (Sullivan and Rana, 2019). Therefore, early biopsy to establish the underlying cause is very important in patients with suspected pulmonary fungal infections. Diagnosis of mucormycosis is challenging. In clinical practice, laboratory diagnosis of mucormycosis includes histopathology, direct examination and cultures (Skiada et al., 2020). Most patients in this study were diagnosed by histopathology. Many previous studies have also reported that histopathology was the main diagnostic method of mucormycosis (Prakash and Chakrabarti, 2019). In this study, Mucorales culture positive was found only in 13 patients. It was previously reported that the positive rate of Mucorales culture could reach 79% (Prakash and Chakrabarti, 2019). Which may be related to the low vigilance of doctors to invasive pulmonary mycosis and the absence of fungal culture for puncture specimens. More needs to be done to raise awareness of pulmonary mycosis among doctors. In this study, twelve patients did not receive treatment and left the hospital against medical advice after diagnosis because of their critical condition and financial constraints. PM patients were treated common form of mucormycosis, accounting for 78.0% of all the patients with mucormycosis. Although previous studies have reported that rhino-orbital mucormycosis was the most common clinical type (Patel et al., 2020), PM maybe more common in China (Peng et al., 2019). This study also found the most common underlying disease was diabetes mellitus. Previous studies have shown that PM mainly occurred in patients with hematological malignancies, while rhino-orbital mucormycosis mostly occurred in patients with diabetes mellitus (Skiada et al., 2011; Chakrabarti and Singh, 2014). However, our results were consistent with those from China (Peng et al., 2019). Perhaps because the number of diabetes patients in China is much larger than that of hematological malignancies (Liu et al., 2019; Khan et al., 2020), the underlying disease of more mucormycosis patients is diabetes. The high prevalence of diabetes in China and endemic mucor species different from other countries may also be the reasons for the high incidence of PM in diabetic patients in China. More detailed etiological studies of mucormycosis need to be done. We found the incidence of mucormycosis was tended to rise, which may be owing to the improvement of fungal diagnostic technology, health awareness and the visiting rate. In addition, diabetes incidence is on the rise worldwide, especially in China and India (Khan et al., 2020), which may be the reason for the increase in the incidence of mucormycosis in China. For PM, the most common clinical symptoms were cough, fever, and hemoptysis/bloody phlegm. This result was similar to some previous studies (Skiada et al., 2011; Peng et al., 2019). Molecular-based assays can help to identify different fungal species and it can be used as a supplement to conventional diagnostic methods (Machouart et al., 2006; Springer et al., 2016). The study also found that laboratory results were not associated with patient outcomes. Most previous studies have not found a correlation between laboratory results and clinical outcomes in patients with mucormycosis (Patel et al., 2020; Son et al., 2020), and only a few studies have found that neutropenia may be associated with Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 6 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis A B C D E F FIGURE 2 | Pulmonary fungal infection (Mucor with Aspergillus) in a 69-year-old man with uncontrolled diabetes, chronic obstructive pulmonary disease and liver cirrhosis. CT imaging showed thick-walled cavity shadow with gas-fluid level in the right upper lobe and left lower lobe of the lung (A). The lesion was significantly reduced after half a month of treatment with posaconazole (B). The lesion continued to shrink after 40 days (C), three months (D), five months (E) and 1 year (F) of treatment. alone was observed. Bilateral lobe involvement occurred in 84.8% of patients in this study, which was similar to previous studies (Peng et al., 2019). Multifocal involvement limited the surgical options, early and effective antifungal therapy may be more important for patients with PM. The recommended combination of antifungal agents and surgical treatment as a treatment option for mucormycosis may be based on the presence of more rhinoorbito-cerebral mucormycosis in other countries. More researches may be needed to optimize treatment options for different types of mucormycosis. New treatment drugs or methods, such as combination of lipid amphotericin B and caspofungin or posaconazole, VT-1161, deferasirox in combination with a polyene, hyperbaric oxygen and so on, still deserve to be expected to improve clinical outcome (Skiada et al., 2018). with amphotericin B or posaconazole had better clinical outcomes, while untreated patients had poor outcomes. For rhino-orbitocerebral and disseminated mucormycosis patients, there was no statistically significant difference in clinical outcome between different treatment strategies, which maybe related to the small number of cases. In addition, patients with rhino-orbito-cerebral mucormycosis who underwent surgery and antifungal therapy had better clinical outcomes. At present, treatment options for mucormycosis remain very limited. LAmB combined with surgery are strongly recommend as first-line therapy. Isavuconazole and posaconazole are also options as second-line agents (Cornely et al., 2019). For patients with PM, only 3 patients in this study were received pulmonary lobectomy, so no benefit of antifungal combined surgical treatment over antifungal therapy Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 7 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis TABLE 4 | Diagnostic procedures and treatment strategies for proven patients with mucormycosis . Diagnostic method Cluture Direct microscopy Histology Cluture positive only Direct microscopy positive only Histology positive only Cluture+histology Direct microscopy+histology Cluture+Direct microscopy BDG test GM test Therapeutic strategies Pulmonary AmB/LAmB Posaconazale LAmB+Surgery untreated Rhino-orbito-cerebral AmB/LAmB Posaconazale AmB+Surgery Disseminated AmB untreated Cutaneous AmB+Surgery Adverse reactions of AmB/LAmB Pulmonary Hypokalemia Renal insufficiency Phlebophlogosis Allergic reaction Gastrointestinal toxicity Cardiotoxicity Rhino-orbito-cerebral Hypokalemia Renal insufficiency Disseminated Hypokalemia Renal insufficiency Gastrointestinal toxicity Agranulocytosis All (N = 59) (n,%) Good outcome (N = 28) (n,%) Worse outcome (N = 31) (n,%) P-value 13 (22.0) 22 (37.3) 48 (81.4) 8 (13.6) 2 (3.4) 25 (52.4) 4 (6.8) 19 (32.2) 1 (1.7) 3 (5.1) 3 (5.1) 7 (25.0) 7 (25.0) 23 (82.1) 5 (17.9) 1 (3.6) 14 (50.1) 2 (7.1) 6 (21.4) 0 (0.0) 1/24 (4.2) 2/20 (10.0) 6 (19.4) 15 (48.4) 25 (80.6) 3 (9.7) 1 (3.2) 11 (35.5) 2 (6.5) 13 (41.9) 1 (3.2) 2/25 (8.0) 1/20 (5.0) 0.755 0.105 1.000 0.458 1.000 0.260 1.000 0.105 1.000 1.000 1.000 All (N=46) (n,%) 28 (60.9) 11 (23.9) 3 (6.5) 11 (23.9) All (N=9) (n,%) 7 (77.8) 1 (11.1) 1 (22.2) All (N=3) (n,%) 2 (66.7) 1 (33.3) All (N=1) (n,%) 1 (100.0) Good outcome (N=23) (n,%) 19 (82.6) 10 (43.5) 1 (4.3) 0 (0.0) Good outcome (N=3) (n,%) 2 (66.7) 0 (0.0) 1 (33.3) Good outcome (N=1) (n,%) 1 (100.0) 0 (0.0) Good outcome (N=1) (n,%) 0 (100.0) Worse outcome (N=23) (n,%) 9 (39.1) 1 (4.3) 2 (8.7) 11 (47.8) Worse outcome (N=6) (n,%) 5 (83.3) 1 (16.7) 0 (0.0) Worse outcome (N=2) (n,%) 1 (50.0) 1 (50.0) Worse outcome (N=0) (n,%) 0 (0.0) P-value All (N=31) (n,%) 7 (22.6) 11 (35.5) 1 (3.2) 1 (3.2) 4 (12.9) 3 (10.7) All (N=8) (n,%) 1 (1.3) 4 (50.0) All (N=2) (n,%) 1 (50.0) 1 (50.0) 1 (50.0) 1 (50.0) Good outcome (N=20) (n,%) 5 (25.0) 8 (40.0) 1 (5.0) 1 (5.0) 2 (10.0) 2 (10.0) Good outcome (N=3) (n,%) 0 (0.0) 3 (100.0) Good outcome (N=1) (n,%) 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0) Worse outcome (N=11) (n,%) 2 (18.2) 3 (27.3) 0 (0.0) 0 (0.0) 2 (18.2) 1 (9.1) Worse outcome (N=5) (n,%) 1 (20.0) 1 (20.0) Worse outcome (N=1) (n,%) 1 (100.0) 0 (0.0) 1 (100.0) 1 (100.0) P-value 0.006* 0.004* 1.000 0.000* P-value 1.000 1.000 0.333 P-value NA NA P-value NA 1.000 0.698 1.000 1.000 0.601 1.000 P-value NA NA P-value NA NA NA NA AmB, amphotericin B; BDG test, (1,3)-beta-D-glucan test; GM test, galactomannan test; LAmB, liposomal amphotericin B. *P < 0.05; NA, Not Applicable. study was limited. More larger-scale, multicenter studies of mucormycosis in the real world should be done. This study has some limitations. First, it was a retrospective observational study. Only some patients have complete followup data in our hospital, especially imaging data, and the followup of some patients could only be completed by telephone. Second, the detection rate of tissue culture was low, the pathological diagnosis was determined mainly by morphology and special staining. It is important to raise the awareness of doctors about fungal disease and the importance of tissue culture. Third, because mucormycosis is a relatively rare fungal disease in China (Liao et al., 2013), the number of cases in this Frontiers in Cellular and Infection Microbiology | www.frontiersin.org CONCLUSIONS The most common clinical type of mucormycosis in China was PM. The most common risk factor was diabetes mellitus. Diabetic patients with clinical manifestations of febrile and 8 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis hemoptysis, CT findings of nodules, cavities and bilateral lung involvement should be vigilant against PM. Early diagnosis and effective anti-mucor treatment are very important to improve the prognosis of patients with mucormycosis. required for this study in accordance with the national legislation and the institutional requirements. AUTHOR CONTRIBUTIONS XL and JQ conceived of and designed the study. JQ and XL collected, analysed or interpreted data. JQ wrote the draft. All authors read, revised and approved the final manuscript. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author. FUNDING This study was supported by Sichuan Province Science and Technology Support Program of China (grant number: 2021YFS0170), 1•3•5 project for disciplines of excellenceClinical Research Incubation Project, West China Hospital, Sichuan University (grant number: 2021HXFH032). ETHICS STATEMENT The studies involving human participants were reviewed and approved by Ethics Committee of West China Hospital, Sichuan University. Written informed consent for participation was not Marom, E. M., and Kontoyiannis, D. P. (2011). Imaging Studies for Diagnosing Invasive Fungal Pneumonia in Immunocompromised Patients. Curr. Opin. Infect. Dis. 24 (4), 309–314. doi: 10.1097/QCO.0b013e328348b2e1 McAdams, H. P., Rosado de Christenson, M., Strollo, D. C., and Patz, E. F. Jr. (1997). Pulmonary Mucormycosis: Radiologic Findings in 32 Cases. AJR Am. J. Roentgenol 168 (6), 1541–1548. doi: 10.2214/ajr.168.6.9168721 Muldoon, E. G., Sharman, A., Page, I., Bishop, P., and Denning, D. W. (2016). Aspergillus Nodules; Another Presentation of Chronic Pulmonary Aspergillosis. BMC Pulm Med. 16 (1), 123. doi: 10.1186/s12890-0160276-3 Patel, A., Kaur, H., Xess, I., Michael, J. S., Savio, J., Rudramurthy, S., et al. (2020). A Multicentre Observational Study on the Epidemiology, Risk Factors, Management and Outcomes of Mucormycosis in India. Clin. Microbiol. Infect. 26 (7), 944 e949–944.e915. doi: 10.1016/j.cmi.2019.11.021 Peng, M., Meng, H., Sun, Y., Xiao, Y., Zhang, H., Lv, K., et al. (2019). Clinical Features of Pulmonary Mucormycosis in Patients With Different Immune Status. J. Thorac. Dis. 11 (12), 5042–5052. doi: 10.21037/jtd.2019.12.53 Petrikkos, G., Skiada, A., and Drogari-Apiranthitou, M. (2014). Epidemiology of Mucormycosis in Europe. Clin. Microbiol. Infect. 20 (Suppl 6), 67–73. doi: 10.1111/1469-0691.12563 Prakash, H., and Chakrabarti, A. (2019). Global Epidemiology of Mucormycosis. J. Fungi (Basel) 5 (1), 26. doi: 10.3390/jof5010026 Prakash, H., Ghosh, A. K., Rudramurthy, S. M., Singh, P., Xess, I., Savio, J., et al. (2019). A Prospective Multicenter Study on Mucormycosis in India: Epidemiology, Diagnosis, and Treatment. Med. Mycol 57 (4), 395–402. doi: 10.1093/mmy/myy060 Ruhnke, M., Groll, A. H., Mayser, P., Ullmann, A. J., Mendling, W., Hof, H., et al. (2015). University of Manchester in Association With the Lp: Estimated Burden of Fungal Infections in Germany. Mycoses 58 (Suppl 5), 22–28. doi: 10.1111/ myc.12392 Shariati, A., Moradabadi, A., Chegini, Z., Khoshbayan, A., and Didehdar, M. (2020). An Overview of the Management of the Most Important Invasive Fungal Infections in Patients With Blood Malignancies. Infect. Drug Resist. 13, 2329–2354. doi: 10.2147/IDR.S254478 Skiada, A., Lanternier, F., Groll, A. H., Pagano, L., Zimmerli, S., Herbrecht, R., et al. (2013). Diagnosis and Treatment of Mucormycosis in Patients With Hematological Malignancies: Guidelines From the 3rd European Conference on Infections in Leukemia (ECIL 3). Haematologica 98 (4), 492–504. doi: 10.3324/haematol.2012.065110 Skiada, A., Lass-Floerl, C., Klimko, N., Ibrahim, A., Roilides, E., and Petrikkos, G. (2018). Challenges in the Diagnosis and Treatment of Mucormycosis. Med. Mycol 56 (suppl_1), 93–101. doi: 10.1093/mmy/myx101 REFERENCES Althoff Souza, C., Muller, N. L., Marchiori, E., Escuissato, D. L., and Franquet, T. ( 2006 ). Pulmon ary In vasiv e Asperg illosi s an d Can di diasis in Immunocompromised Patients: A Comparative Study of the HighResolution CT Findings. J. Thorac. Imaging 21 (3), 184–189. doi: 10.1097/ 01.rti.0000213552.16011.ad Bitar, D., Van Cauteren, D., Lanternier, F., Dannaoui, E., Che, D., Dromer, F., et al. (2009). Increasing Incidence of Zygomycosis (Mucormycosis), France, 19972006. Emerg. Infect. Dis. 15 (9), 1395–1401. doi: 10.3201/eid1509.090334 Chakrabarti, A., and Singh, R. (2014). Mucormycosis in India: Unique Features. Mycoses 57 (Suppl 3), 85–90. doi: 10.1111/myc.12243 Cornely, O. A., Alastruey-Izquierdo, A., Arenz, D., Chen, S. C. A., Dannaoui, E., Hochhegger, B., et al. (2019). Global Guideline for the Diagnosis and Management of Mucormycosis: An Initiative of the European Confederation of Medical Mycology in Cooperation With the Mycoses Study Group Education and Research Consortium. Lancet Infect. Dis. 19 (12), e405–e421. doi: 10.1016/S1473-3099(19)30312-3 De Pauw, B., Walsh, T. J., Donnelly, J. P., Stevens, D. A., Edwards, J. E., Calandra, T., et al. (2008). Revised Definitions of Invasive Fungal Disease From the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin. Infect. Dis. 46 (12), 1813–1821. doi: 10.1086/588660 Jeong, W., Keighley, C., Wolfe, R., Lee, W. L., Slavin, M. A., Kong, D. C. M., et al. (2019). The Epidemiology and Clinical Manifestations of Mucormycosis: A Systematic Review and Meta-Analysis of Case Reports. Clin. Microbiol. Infect. 25 (1), 26–34. doi: 10.1016/j.cmi.2018.07.011 Khan, M. A. B., Hashim, M. J., King, J. K., Govender, R. D., Mustafa, H., and Al Kaabi, J. (2020). Epidemiology of Type 2 Diabetes - Global Burden of Disease and Forecasted Trends. J. Epidemiol. Glob Health 10 (1), 107–111. doi: 10.2991/ jegh.k.191028.001 Liao, Y., Chen, M., Hartmann, T., Yang, R. Y., and Liao, W. Q. (2013). Epidemiology of Opportunistic Invasive Fungal Infections in China: Review of Literature. Chin. Med. J. (Engl) 126 (2), 361–368. Liu, W., Liu, J., Song, Y., Zeng, X., Wang, X., Mi, L., et al. (2019). Burden of Lymphoma in China, 2006-2016: An Analysis of the Global Burden of Disease Study 2016. J. Hematol. Oncol. 12 (1), 115. doi: 10.1186/s13045-019-0785-7 Machouart, M., Larché , J., Burton, K., Collomb, J., Maurer, P., Cintrat, A., et al. (2006). Genetic Identification of the Main Opportunistic Mucorales by PCRRestriction Fragment Length Polymorphism. J. Clin. Microbiol. 44, 805–810. doi: 10.1128/JCM.44.3.805-810.2006 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9 November 2021 | Volume 11 | Article 770551 Qu et al. Clinical Analysis of Mucormycosis US, National Cancer Institute (2017). Common Terminology Criteria for Adverse Events (CTCAE): Version 5.0. Available from: https://ctep.cancer.gov/ protocoldevelopment/electronic_applications/docs/CTCAE_v5_Quick_ Reference_8.5x11.pdf. (Accessed December13, 2021). Skiada, A., Pagano, L., Groll, A., Zimmerli, S., Dupont, B., Lagrou, K., et al. (2011). Gaustad P Et Al: Zygomycosis in Europe: Analysis of 230 Cases Accrued by the Registry of the European Confederation of Medical Mycology (ECMM) Working Group on Zygomycosis Between 2005 and 2007. Clin. Microbiol. 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The Reversed Halo Sign and Mucormycosis. Lancet Infect. Dis. 19 (12), 1379. doi: 10.1016/S1473-3099(19)30548-1 Frontiers in Cellular and Infection Microbiology | www.frontiersin.org Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2021 Qu, Liu and Lv. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). 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Financial Statements Are About To Get A New Look
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Financial Statements are about to get a New Look Financial Statements are about to get a New Look Clemense Ehoff Jr. Kean University, ehoffc@cwu.edu Clemense Ehoff Jr. Kean University, ehoffc@cwu.edu Follow this and additional works at: https://digitalcommons.cwu.edu/cobfac Part of the Corporate Finance Commons Follow this and additional works at: https://digitalcommons.cwu.edu/cobfac Follow this and additional works at: https://digitalcommons.cwu.edu/cobfac Part of the Corporate Finance Commons College of Business College of Business College of Business Recommended Citation Recommended Citation Recommended Citation Recommended Citation Ehoff, Jr., C. (2010). Financial statements are about to get a new look. Journal of Business and Economics Research 8(12), 69-76. DOI: 10.19030/jber.v8i12.783 This Article is brought to you for free and open access by the College of Business at ScholarWorks@CWU. It has been accepted for inclusion in All Faculty Scholarship for the College of Business by an authorized administrator of ScholarWorks@CWU. For more information, please contact scholarworks@cwu.edu. Volume 8, Number 12 Journal of Business & Economics Research – December, 2010 ABSTRACT In October 2008, The FASB and the IASB issued a discussion letter for comment proposing a complete redesign of financial statements. This was the result of a project that began for both boards in 2001. More than 200 comment letters were received from individuals, accounting firms, professional societies, corporations, and others from the business community. The FASB and IASB have analyzed the input, and are currently preparing an exposure draft scheduled for release in early 2011. This paper reviews the proposed changes to the financial statements, summarizes the favorable and unfavorable responses contained in the comment letters, and examines the implications that the changes will have on the business community, the accounting profession, educators, and investors. Keywords: Financial Statements; Comment letters; FASB; IASB Keywords: Financial Statements; Comment letters; FASB; IASB Keywords: Financial Statements; Comment letters; FASB; IASB INTRODUCTION n October 16, 2008, the Financial Accounting Standards Board (FASB) and the International Accounting Standards Board (IASB) issued a discussion paper “Preliminary Views on Financial Statement Presentation,” the result of a project that began for both boards in 2001. The paper sought public comment, to be used as input in the development of an exposure draft. The comment period ended on April 14, 2009. 227 comment letters were received from individuals, accounting firms, professional societies, corporations, academics, and others from the business community. A comment letter summary was presented at the July 14, 2009 IASB/FASB meeting. In July 2010, the IASB and the FASB posted a draft of the Exposure Draft, a working document that expresses the current and tentative decision made so far on this project. O bliO The changes to financial statement presentation proposed by the IASB and FASB are far reaching and will have an enormous impact on the entire business community. This paper reviews the proposed changes to the financial statements, summarizes the favorable and unfavorable responses contained in the comment letters, and examines the implications that the changes will have on the business community, the accounting profession, educators, and investors. Financial Statements Are About To Get A New Look Clemense Ehoff Jr., Kean University, USA Clemense Ehoff Jr., Kean University, USA THE DISCUSSION PAPER The October 16, 2008 Discussion Paper (Preliminary Views, 2008) was the result of a project that began in 2001. In undertaking the financial statements presentation project, the FASB and IASB „s intention was to establish a new standard for presenting information in the financial statements. In doing so, they set a goal of improving the usefulness of financial statement information to help users make more informed decisions as capital providers. Three broad objectives for financial statement presentation were developed: 1. Cohesiveness: There is a clear relationship between items across financial statements. 1. Cohesiveness: There is a clear relationship between items across financial statements. regation of Information: Financial information should be disaggregated into reasonably nous groups of items so that it is useful in predicting an entity‟s future cash flows. 2. Disaggregation of Information: Financial information should be disaggregated into reasonably homogenous groups of items so that it is useful in predicting an entity‟s future cash flows. 2. Disaggregation of Information: Financial information should be disaggregat homogenous groups of items so that it is useful in predicting an entity‟s future cash flow g g p p g y 3. Liquidity and Flexibility: Liquidity information helps users asses the entity‟s ability to meet financial commitments as they become due. Information flexibility helps users assess the entity‟s ability to invest in opportunities and react to unexpected situations. g g p p g y 3. Liquidity and Flexibility: Liquidity information helps users asses the entity‟s ability to meet financial commitments as they become due. Information flexibility helps users assess the entity‟s ability to invest in opportunities and react to unexpected situations. 69 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 In comparing these structures, several significant changes are noticed: 1. Assets and liabilities are still divided into current and long-term categories, but are further divided into business (and divided again into operating and investing components) and financing categories. 2. In the new Statement of Financial Position, subtotals for current assets, current liabilities, total assets and total liabilities have been included and placed at the bottom of the statement. 3. The new Statement of Income goes beyond traditional Net Income and includes components of other comprehensive income in arriving at total comprehensive income. 4. The new Statement of Income does not contain a separate line item for extraordinary items. 5. The new Statement of Cash Flows must use the direct method. 1. Assets and liabilities are still divided into current and long-term categories, but are further divided into business (and divided again into operating and investing components) and financing categories. g p g g p g g 2. In the new Statement of Financial Position, subtotals for current assets, current liabilities, total assets and total liabilities have been included and placed at the bottom of the statement. 3. The new Statement of Income goes beyond traditional Net Income and includes components of other comprehensive income in arriving at total comprehensive income. p g p The new Statement of Income does not contain a separate line item for extraordinary items. g p w Statement of Income does not contain a separate line item for extraordinary items. The new Statement of Cash Flows must use the direct method. The fourth statement in the proposed model is the statement of changes in equity. The structure proposed for this statement resembles a reconciliation of beginning balances, ending balances, and how each amount changed during the period. Each component of equity is presented in the statement. Currently, a reconciliation of only Retained Earnings (or Owners‟ Equity) is shown. The fourth statement in the proposed model is the statement of changes in equity. The structure proposed for this statement resembles a reconciliation of beginning balances, ending balances, and how each amount changed during the period. Each component of equity is presented in the statement. Currently, a reconciliation of only Retained Earnings (or Owners‟ Equity) is shown. The statement of financial position, the income statement, the statement of cash flows, and the statement of changes in equity comprise a complete set of financial statements. Samples of each financial statement are presented in the Appendix. In comparing these structures, several significant changes are noticed: There are several additional important aspects of the proposed presentation model worth mentioning. First, the model relies on a management approach to classify assets and liabilities in the business and financing sections in a manner that best reflects the way the asset or liability is used within the entity. Second, the Boards have concluded that use of the direct method for the statement of cash flows is more consistent with the objectives of coherency and disaggregation of information. Finally, the proposed presentation model includes a new schedule that reconciles cash flows to comprehensive income. This schedule will be included in the notes to the financial statements. Table 3 illustrates the structure of the schedule: Table 3: Reconciliation Schedule Statement of Cash Flows Cash Flows Accruals, Allocations and Other Recurring Valuation Adjustment All Other Comprehensive Income Statement of Comprehensive Income Cash from wholesale Customers 1,928,798 662,602 2,991,400 Sales-Wholesale Cash from retail customers 643,275 4,575 647,850 Sales-retail Total cash from customers 2,572,073 667,177 3,219,250 Total Revenue Note: Adapted from Discussion Paper (Preliminary Views, 2008). The discussion paper included an invitation for comments on the proposals included in the paper. Twenty- six questions were included in the paper. Respondents were asked to respond in writing by April 14, 2009. The discussion paper included an invitation for comments on the proposals included in the paper. Twenty- six questions were included in the paper. Respondents were asked to respond in writing by April 14, 2009. Journal of Business & Economics Research – December, 2010 The proposed financial statement model divides the statement of financial position (balance sheet), the income statement, and the statement of cash flows into two sections: business activities, and financing activities. The business section is further subdivided into operating activities and investing activities. The financing section describes how the entity‟s business activities are financed, segregating owner and non-owner sources. Discontinued operations are shown separately from the entity‟s business and financing activities. Income taxes are shown separately in the statement of financial position and the statement of cash flows. In the income statement, income tax information is shown separately for 1) income from continuing operations, 2) discontinued operations, and 3) other comprehensive income items. Table 1 illustrates the proposed classification scheme. Table 1: Proposed Financial Statement Classification Statement of Financial Position Statement of Comprehensive Income Statement of Cash Flows Business  Operating assets and liabilities  Investing assets and liabilities Business  Operating income and expenses  Investment income and expenses Business  Operating cash flows  Investing cash flows Financing  Financing assets  Financing liabilities Financing  Financing asset income Financing  Financing asset cash flows  Financing liability cash flows Income taxes Income taxes on continuing operations (business and financing) Income taxes Discontinued operations Discontinued operations net of tax Discontinued operations Other comprehensive income, net of tax Equity Equity Note: Adapted from Discussion Paper (Preliminary Views, 2008). This structure is substantially different from the current financial statement structure as shown in Table 2: structure is substantially different from the current financial statement structure as shown in Table 2 Table 2: Current Financial Statement Classification Balance Sheet Income Statement Statement of Cash Flows (Direct or Indirect Method) Assets  Current Assets  Property, Plant & Equipment  Other Assets Operating Income  Sales  Cost of Goods Sold  Gross Profit  Operation Expenses  Operating Income Operating Activities  Operating cash flows  Investing cash flows Liabilities  Current Liabilities  Long-term Liabilities Other  Other Revenue  Other Expense Investing Activities Equity Continuing Operations  Income Before Taxes  Income Taxes  Income from Continuing Operations Financing Activities Discontinued operations net of tax Change in Cash Extraordinary Item (net of tax) Net Income Note: Adapted from Intermediate Accounting (6th ed.) by D. Spiceland, J. Sepe, & M. Nelson. New York: McGraw-Hill Irwin. (2011). 70 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 THE COMMENT LETTERS Respondents who opposed the direct method claimed that management was not currently using operating cash receipts and payments information to run its business and financial statement users were not asking for it. These respondents also pointed to a complete retooling of their accounting and financial reporting systems, suggesting that the costs of such an endeavor would far outweigh the benefits. 7. Direct method statement of cash flows – Two-thirds of the respondents did not agree that the direct method would provide more decision-useful information than the indirect method, and a majority of the respondents did not favor requiring all entities to use only the direct method. Respondents who opposed the direct method claimed that management was not currently using operating cash receipts and payments information to run its business and financial statement users were not asking for it. These respondents also pointed to a complete retooling of their accounting and financial reporting systems, suggesting that the costs of such an endeavor would far outweigh the benefits. 8. g Reconciliation schedule – Most respondents did not favor the proposed schedule that reconciles the statement of comprehensive income with the cash flows statement. They thought that 1) the reconciliation schedule was rather long and might be too complex for most financial statement users to grasp, and 2) the cost of preparing the schedule by far outweighed the benefits. Reconciliation schedule – Most respondents did not favor the proposed schedule that reconciles the statement of comprehensive income with the cash flows statement. They thought that 1) the reconciliation schedule was rather long and might be too complex for most financial statement users to grasp, and 2) the cost of preparing the schedule by far outweighed the benefits. 9. Application to nonpublic entities – Although the board had not considered whether the proposed financial statement presentation model should apply to nonpublic companies, respondents were asked for their views on this issue. Respondents‟ views were mixed. Those that favored inclusion cited two reasons: 1) if the proposed financial statement presentation model proved useful for public entities, then it should prove useful for nonpublic entities, and 2) one presentation model eliminates possible confusion among users. THE COMMENT LETTERS g 3. A separate equity section - There was majority support for a separate equity section. 4. A separate discontinued operations section – There was nearly unanimous support for a separate discontinued operations section. 3. A separate equity section - There was majority support for a separate equity section. 3. A separate equity section - There was majority support for a separate equity section. 4. A separate discontinued operations section – There was nearly unanimous support for a separate discontinued operations section. 5. Management approach to classification – Respondents‟ views were mixed on this issue. Respondents who favored the management approach think that classifying assets and liabilities in a manner that reflects their use provides relevant information for financial statement users. Respondents who did not support this approach pointed to reduced comparability among entities. 5. Management approach to classification – Respondents‟ views were mixed on this issue. Respondents who favored the management approach think that classifying assets and liabilities in a manner that reflects their use provides relevant information for financial statement users. Respondents who did not support this approach pointed to reduced comparability among entities. 6. Statement of comprehensive income – Respondents were split on whether an entity should include all components of comprehensive income in a single statement or two separate statements. Respondents who favored the approach pointed to greater transparency, consistency and comparability. Respondents who opposed the single statement approach argued that operating income and net income were the primary focus of most investors, and that the inclusion of other comprehensive items within a single statement might lead to confusion. 6. Statement of comprehensive income – Respondents were split on whether an entity should include all components of comprehensive income in a single statement or two separate statements. Respondents who favored the approach pointed to greater transparency, consistency and comparability. Respondents who opposed the single statement approach argued that operating income and net income were the primary focus of most investors, and that the inclusion of other comprehensive items within a single statement might lead to confusion. 7. g t ead to co us o . Direct method statement of cash flows – Two-thirds of the respondents did not agree that the direct method would provide more decision-useful information than the indirect method, and a majority of the respondents did not favor requiring all entities to use only the direct method. THE COMMENT LETTERS The IASB/FASB boards received a total of 227 comment letters. The comments were reviewed and summarized by the technical staff. A comment letter summary report was presented for discussion at the IASB/FASB July 14, 2009 meeting. A summary of the respondents is presented below: A summary of the respondents is presented below: 71 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 Table 4: Comment Letter Summary Total IFRS U.S. GAAP Preparers 98 73 25 Auditors 38 29 9 Users 24 17 7 Standard setters/Regulators 22 22 0 Academics 18 7 11 Others 27 22 5 Total 227 170 57 Note: Adapted from Comment Letter Summary (2009) Some of the main points highlighted in the technical staff summary are as follows: Some of the main points highlighted in the technical staff summary are as follow 1. Objectives - There was general support for the Boards‟ objectives of cohesiveness, disaggregation, liquidity, and financial flexibility. However, most respondents took issue with the concept of line-item cohesiveness, and felt that cohesiveness might be more appropriate if applied at a higher level. Some respondents felt that presenting disaggregated information in the notes to financial statements might be more appropriate than on the face of the financial statements. Lastly, many respondents suggested that the proposed financial statement changes were not designed to meet the needs of a broad range of users, but seemed to be designed more for analysts who use financial statement information for valuation purposes. 1. Objectives - There was general support for the Boards‟ objectives of cohesiveness, disaggregation, liquidity, and financial flexibility. However, most respondents took issue with the concept of line-item cohesiveness, and felt that cohesiveness might be more appropriate if applied at a higher level. Some respondents felt that presenting disaggregated information in the notes to financial statements might be more appropriate than on the face of the financial statements. Lastly, many respondents suggested that the proposed financial statement changes were not designed to meet the needs of a broad range of users, but seemed to be designed more for analysts who use financial statement information for valuation purposes. 2. Separation of business activities from financing activities – Most respondents supported the separation of business activities from financing activities, although several respondents pointed out that separation might prove to be difficult in actual practice, suggesting that the distinction might be arbitrary, thereby reducing the information‟s usefulness. Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 ies, and 2) the proposed presentation model might be too complex for small nonpublic companies. companies, and 2) the proposed presentation model might be too complex for small nonpublic com Clearly, the most significant area of concern from the respondents was the mandate requiring companies to use the direct method in presenting the statement of cash flows. Over two-thirds of the respondents (including such companies as McDonald Corporation (Comment Letter # 120), Intel Corporation (Comment Letter # 25), Microsoft Corporation (Comment Letter # 134), and Bayer (Comment Letter #57)) have raised serious objections, claiming that the cost far outweighs the benefits. Intel‟s controller estimated that implementation costs for his company would exceed $5 million, and ongoing costs would approximate $2million per year (Comment Letter #25). THE STAFF DRAFT OF THE EXPOSURE DRAFT On July 1, 2010, the FASB and the IASB posted a draft copy of an exposure draft on financial statement presentation (Staff Draft, 2010). Although similar to the Discussion Paper issued in October 2008, this 151- page document takes on the style and structure of a typical FASB statement, focusing more on the “nuts and bolts” of implementation and less on justification. The basic financial statement presentation model has changed little between the two documents. The significant changes are as follows: 1. The Boards have decided that the proposed financial statement presentation standard should apply to all business entities, public and private. 1. The Boards have decided that the proposed financial statement presentation standard should apply to all business entities, public and private. 1. The Boards have decided that the proposed financial statement presentation standard sh b i titi bli d i t business entities, public and private. p p 2. The Boards have dropped the reconciliation statement (reconciling the statement of comprehensive income with the statement of cash flows on a line-by-line basis. Instead, a reconciliation of significant asset and liability accounts will be included in the notes to the financial statements. Despite the opposition heard from more than two-thirds of the respondents, the Boards have remained steadfast in requiring the companies use the direct method in presenting the statement of cash flows. The exposure draft of the financial statement presentation model is scheduled for release during 1st quarter of 2011. Given the similarity in scope between the Discussion Paper and the Staff draft, it is reasonable to assume that little will change between the Staff Draft and the final Exposure Draft. THE COMMENT LETTERS Those who opposed inclusion cited two reasons: 1) the cost doesn‟t justify the benefit for nonpublic Application to nonpublic entities – Although the board had not considered whether the proposed financial statement presentation model should apply to nonpublic companies, respondents were asked for their views on this issue. Respondents‟ views were mixed. Those that favored inclusion cited two reasons: 1) if the proposed financial statement presentation model proved useful for public entities, then it should prove useful for nonpublic entities, and 2) one presentation model eliminates possible confusion among users. Those who opposed inclusion cited two reasons: 1) the cost doesn‟t justify the benefit for nonpublic 72 Journal of Business & Economics Research – December, 2010 IMPLICATIONS The proposed financial statement presentation model has enormous and far- reaching implications. It is worthwhile to examine the implications that the changes are likely to have on the business community, the accounting profession, educators, and investors. First, there is the cost associated with implementing the new financial statement presentation model. As already mentioned, Intel‟s controller had estimated implementation costs of more than $5 million, with ongoing costs approximating $2million per year. Even assuming that his estimate is somewhat exaggerated, it is safe to conclude that the cost to Corporate America for retooling its accounting software to accommodate this new financial statement presentation model is enormous. Every accounting system, every software package (both custom and off- the-shelf), and nearly every financial model will require a significant update. Given the increase in financial information, audit fees will undoubtedly rise. Corporate America (and corporate stockholders indirectly) will be paying the bill for the new financial statement presentation model. In terms of winners and losers, the accounting and finance software producers are clear winners, while financial statement preparers (the companies and their stockholders) are the losers. It only takes a quick perusal of Table 1 and Table 2 to conclude that financial statements and the accompanying notes to the financial statements will be more complex and contain more information. Accounting firms will be the winners, increase their fess to cover the additional work. The financial statement preparers (the companies and their stockholders) are the losers, paying a higher price to the public accounting firms. Educators should fare nicely as a result of the new financial statement presentation model, especially in the continuing professional education segment. Much of Corporate America has not even seen the proposed financial 73 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 statement presentation model; it may come as quite an initial shock. Educators come across as winners, with potentially plenty to do for a long while. Every accounting and finance book and profession publication will require an update. Book sales should soar. There are no losers here. statement presentation model; it may come as quite an initial shock. Educators come across as winners, with potentially plenty to do for a long while. Every accounting and finance book and profession publication will require an update. Book sales should soar. There are no losers here. CONCLUSION The IASB/FASB boards have spent nearly 10 years on designing a new financial statement presentation model. Having gone through countless meetings and absorbing an enormous amount of commentary from accountants, educators, financial analysts, businessmen and investors, the boards are close to bringing this project to its conclusion. This project and its implications are too big to escape controversy. The objection by many respondents to elimination of the indirect method approach to the statement of cash flows appears lost for now, but the new financial statement presentation model must go through the exposure draft phase before becoming “cast in stone.” Since December 2009, an 18-member panel formed by the AICPA, the Financial Accounting Foundation (the FASB‟s parent organization), and the National Association of State Boards of Accountancy have been working on models that are based on current U.S. GAAP that would result in different standards for private companies (Private Company Financial Reporting, September 2010). So, as the IASB/FASB financial statement presentation model project turns the corner and heads “into the home stretch,” a “relative” group puts forth an effort suggesting that financial standards (including financial statement presentation models) be split into two groups: one for public companies, and one for private companies. What effect the work of this group will have on the IASB/FASB financial statement presentation model is not clear at this time. What is clear is that financial statements, as we know them, are about to change. A working draft of the financial statement presentation model can be viewed on the FASB‟s website. And although significant changes to the model are still possible, it is now time to start preparing for a new set of financial statements. IMPLICATIONS Finally, investors and analysts will have a new set of financial statements and notes to analyze, containing substantially more data. For the sophisticated investor and analyst, the new financial statement model should satisfy their seemingly inexhaustible appetite for more data. For the unsophisticated investor, the new financial statement presentation model might give them heartburn. The level of sophistical seems to be the deciding factor as to which investor or analyst is a winner or a loser. AUTHOR INFORMATION Dr. Clemense Ehoff Jr., CPA is Assistant Professor of Accounting, Kean University, Union New Jersey. He holds a Ph. D in Business Administration from San Francisco‟s Golden Gate University. He has more than 30 years professional business experience and has held full-time faculty and adjunct positions at universities predominantly in the Eastern United States. Over the last ten years, Dr. Ehoff has been involved in teaching accounting and tax courses in an online platform. He operates a consulting and tax practice. He has published articles in Elevator World, and other journals. 1. DeFelice, A. (2010, September). Blue-Ribbon Panel Narrows Field for Private Company Financial Reporting. Journal of Accountancy, 24-28. 2. Financial Accounting Standards Board (FASB) (2008) Discussion Paper: Preliminary Views on Financial Statement Presentation. Retrieved February 15, 2009 from http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2FDocument_C%2FDocument Page&cid=1175801986226http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2F Document_C%2FDocumentPage&cid=1175801986226. 3. Financial Accounting Standards Board (FASB)(2009) Comment Letters. Retrieved October 12, 2009 from http://www.fasb.org/jsp/FASB/CommentLetter_C/CommentLetterPage&cid=1218220137090&project_id= 1630-100. 4. International Accounting Standards Board (IASB), & Financial Accounting Standards Board (FASB) (2009) Comment Letter Summary presented at the July 14, 2009 Boards meeting. Retrieved August 5, 2010 from http://www.iasb.org/NR/rdonlyres/791DE489-E887-4652-9DD1- 79789C36F032/0/FSP0907b17Cobs.pdf. p 5. Financial Accounting Standards Board (FASB)(2010) Staff Draft of an Exposure Draft on Financial Statement Presentation. Retrieved July 20, 2010 from http://www.fasb.org/cs/BlobServer?blobcol=urldata&blobtable=MungoBlobs&blobkey=id&blobwhere=11 75820952978&blobheader=application%2Fpdf. 6. Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill Irwin. g g (2009) Comment Letter Summary presented at the July 14, 2009 Boards meeting. Retrieved August 5, 2010 from http://www.iasb.org/NR/rdonlyres/791DE489-E887-4652-9DD1- 79789C36F032/0/FSP0907b17Cobs.pdf. 5. Financial Accounting Standards Board (FASB)(2010) Staff Draft of an Exposure Draft on Financial Statement Presentation. Retrieved July 20, 2010 from http://www.fasb.org/cs/BlobServer?blobcol=urldata&blobtable=MungoBlobs&blobkey=id&blobwhere=11 75820952978&blobheader=application%2Fpdf. 6. Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill Irwin. REFERENCES 1. DeFelice, A. (2010, September). Blue-Ribbon Panel Narrows Field for Private Company Financial Reporting. Journal of Accountancy, 24-28. 2. Financial Accounting Standards Board (FASB) (2008) Discussion Paper: Preliminary Views on Financial Statement Presentation. Retrieved February 15, 2009 from http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2FDocument_C%2FDocument Page&cid=1175801986226http://www.fasb.org/cs/ContentServer?c=Document_C&pagename=FASB%2F Document_C%2FDocumentPage&cid=1175801986226. _ g 3. Financial Accounting Standards Board (FASB)(2009) Comment Letters. Retrieved October 12, 2009 from http://www.fasb.org/jsp/FASB/CommentLetter_C/CommentLetterPage&cid=1218220137090&project_id= 1630-100. g 3. Financial Accounting Standards Board (FASB)(2009) Comment Letters. Retrieved October 12, 2009 from http://www.fasb.org/jsp/FASB/CommentLetter_C/CommentLetterPage&cid=1218220137090&project_id= 1630-100. 74 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 4. International Accounting Standards Board (IASB), & Financial Accounting Standards Board (FASB) (2009) Comment Letter Summary presented at the July 14, 2009 Boards meeting. Retrieved August 5, 2010 from http://www.iasb.org/NR/rdonlyres/791DE489-E887-4652-9DD1- 79789C36F032/0/FSP0907b17Cobs.pdf. 5. Financial Accounting Standards Board (FASB)(2010) Staff Draft of an Exposure Draft on Financial Statement Presentation. Retrieved July 20, 2010 from http://www.fasb.org/cs/BlobServer?blobcol=urldata&blobtable=MungoBlobs&blobkey=id&blobwhere=11 75820952978&blobheader=application%2Fpdf. 6. Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill Irwin. pp p 6. Spiceland, D., Sepe, J., & Nelson, M. (2011). Intermediate Accounting (6th ed.). New York: McGraw-Hill Irwin. 75 75 Journal of Business & Economics Research – December, 2010 Journal of Business & Economics Research – December, 2010 Volume 8, Number 12 NOTES Volume 8, Number 12 Volume 8, Number 12 Volume 8, Number 12 76
https://openalex.org/W1978244119
https://link.springer.com/content/pdf/10.1007/s00259-012-2176-y.pdf
English
null
Pharmacokinetic modelling of N-(4-[18F]fluorobenzoyl)interleukin-2 binding to activated lymphocytes in an xenograft model of inflammation
European journal of nuclear medicine and molecular imaging
2,012
cc-by
6,612
Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 DOI 10.1007/s00259-012-2176-y Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 DOI 10.1007/s00259-012-2176-y ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract de Vries Department of Nuclear Medicine & Molecular Imaging, University Medical Center Groningen, Groningen, The Netherlands V. Di Gialleonardo: A. Signore: A. T. M. Willemsen: J. W. A. Sijbesma: R. A. J. O. Dierckx: E. F. J. de Vries Department of Nuclear Medicine & Molecular Imaging, University Medical Center Groningen, Groningen, The Netherlands V. Di Gialleonardo: A. Signore: A. T. M. Willemsen: J. W. A. Sijbesma: R. A. J. O. Dierckx: E. F. J. de Vries Department of Nuclear Medicine & Molecular Imaging, University Medical Center Groningen, Groningen, The Netherlands Keywords Pharmacokinetics . [18F]FB-IL2 . Molecular imaging . Inflammation V. Di Gialleonardo: A. Signore Nuclear Medicine Unit, Faculty of Medicine and Psychology, “Sapienza” University, Rome, Italy Pharmacokinetic modelling of N-(4-[18F]fluorobenzoyl) interleukin-2 binding to activated lymphocytes in an xenograft model of inflammation Valentina Di Gialleonardo & Alberto Signore & Antoon T. M. Willemsen & Jurgen W. A. Sijbesma & Rudi A. J. O. Dierckx & Erik F. J. de Vries Received: 30 January 2012 /Accepted: 6 June 2012 /Published online: 10 July 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract the Akaike information criterion (AIC) and correlation between PET measurements and the number of CD25- positive cells were evaluated. Purpose N-(4-[18F]Fluorobenzoyl)interleukin-2 ([18F]FB- IL2) specifically binds to interleukin-2 receptors (IL-2R) and thus may be used to detect inflammation processes using positron emission tomography (PET). We now vali- dated whether [18F]FB-IL2 can be used to quantify activated human peripheral blood mononuclear cells (hPBMC) in rats by pharmacokinetic modelling. p Results A high correlation between ex vivo tracer uptake (standardized uptake value) in the xenograft and the number of inoculated CD25-positive cells was observed (R200.90). Plasma time-activity curves showed a rapid washout of the radiopharmaceutical from blood, while the time-activity curves of the inflammatory lesions showed slower washout. Time-activity curves could be fitted well by the Logan analysis method, indicating that the binding between [18F] FB-IL2 and CD25 is reversible. AIC indicated that data could be modelled best by a two-tissue reversible compart- ment model. A high correlation was observed between the binding potential and the number of CD25-positive cells (R200.876, p<0.0001). Based on binding potential mea- sured by PET, the limit of detection was about 160,000 CD25-positive cells per 200 μl lesion (95 % confidence). Conclusion [18F]FB-IL2 kinetics in this animal model of inflammation could be best described by a reversible two- tissue compartment model. The [18F]FB-IL2 binding poten- tial is a suitable measure for accurate quantification of lym- phocytic infiltration in pathological conditions with PET. Methods Eleven Wistar rats were subcutaneously inoculated in the shoulder with different amounts of phytohaemaggluti- nin (PHA) activated hPBMC 15 min before i.v. injection of [18F]FB-IL2. A 60-min dynamic PET scan was acquired and arterial blood sampling and metabolite analysis were per- formed. At the end of the scan, animals were terminated and the inflammatory lesion dissected. PET data were analysed using Logan and Patlak analysis as well as one-tissue and two- tissue compartment models. Model preferences according to Conclusion [18F]FB-IL2 kinetics in this animal model of inflammation could be best described by a reversible two- tissue compartment model. The [18F]FB-IL2 binding poten- tial is a suitable measure for accurate quantification of lym- phocytic infiltration in pathological conditions with PET. Electronic supplementary material The online version of this article (doi:10.1007/s00259-012-2176-y) contains supplementary material, which is available to authorized users. V. Di Gialleonardo: A. Signore: A. T. M. Willemsen: J. W. A. Sijbesma: R. A. J. O. Dierckx: E. F. J. hPBMC activation and FACS analysis Human blood was obtained from the local blood bank. hPBMC were isolated from human peripheral blood by density gradient medium centrifugation (Lymphoprep, Axis-Shield) using the rapid centrifugation procedure devel- oped by Bøyum [23, 24]. Cells were kept in RPMI 1640 supplemented with L-glutamine, 10 % fetal calf serum (FCS), 100 IU penicillin/ml and 100 μg streptomycin/ml (all from GIBCO). Isolated hPBMC were incubated for 48 h with 5 μg/ml of phytohaemagglutinin (PHA-P, Sigma- Aldrich) at 37 °C and 5 % CO2 for cell activation. At the day of the microPET scan, overexpression of CD25 was determined by fluorescence-activated cell sorting (FACS) analysis. For this purpose, the cultured activated hPBMC were washed once with 3 ml of cold phosphate-buffered saline (PBS) and resuspended in PBS at a concentration of 106 cells/ml. Aliquots (0.1 ml) of the cell suspension were incubated for 45 min in ice with PE-CD25 antibody or PE- IgG antibody (E-Bioscience) as controls. After being washed twice in 3 ml of cold PBS, the cells were resuspended in 0.5 ml of FACS solution (PBS supplemented with 5 % FCS) immediately prior to analysis using a FACS Calibur (BD Biosciences). FACS data were analysed using Winlist 5.0 software (Verity Software House) in order to determine the percentage of CD25-expressing cells in each sample. Since IL-2 is the natural ligand of the IL-2R, this glycoprotein was selected as a potential radiopharma- ceutical for imaging activated T lymphocytes [16]. In the past, IL-2 was labelled with various isotopes for single proton emission computed tomography (SPECT) imaging [17–21]. The labelled IL-2 derivatives showed good results in terms of localization of the inflammation within the limitations of gamma camera imaging. Label- ling with a suitable positron emission tomography (PET) isotope may improve sensitivity of this radiopharmaceu- tical and therefore N-(4-[18F]fluorobenzoyl)interleukin-2 ([18F]FB-IL2) was developed by our group. In our pre- vious studies, we demonstrated the ability of [18F]FB- IL2 to selectively detect activated human peripheral blood mononuclear cells (hPBMC) in rodent models of inflammation by noninvasive PET imaging [22]. In ad- dition, [18F]FB-IL2 was able to detect insulitis, a chron- ic inflammatory process that precedes the clinical onset of type 1 diabetes, in diabetes-prone rats and mice (data submitted for publication). The detection of chronic inflammation is important for diagnostic, prognostic purposes and for monitoring the disease progression and the efficacy of treatment. hPBMC activation and FACS analysis To this end, quantitative measurement of the extent of lymphocytic infiltration is required. In the present study we aimed to investigate whether [18F]FB-IL2 PET is able to quantify the amount of activated lymphocytes in infiltrated tissue. For this purpose, pharmacokinetic modelling was performed. Wistar rats were subcutane- ously inoculated with activated hPBMC and subsequent- ly studied with [18F]FB-IL2 microPET. Dynamic PET data were analysed using various kinetics modelling approaches and correlated to the number of CD25- positive cells administered. Synthesis of [18F]FB-IL2 [18F]FB-IL2 was synthesized as described elsewhere [22]. [18F]FB-IL2 was obtained in 25 % radiochemical yield based on succinimidyl 4-[18F]fluorobenzoic acid ([18F] SFB) (corrected for the decay), with a specific activity of 117±6 GBq/μmol at the end of synthesis and with a radio- chemical purity >95 %. Introduction A. Signore (*) Medicina Nucleare, Ospedale S.Andrea, “Sapienza” University, Via di Grottarossa 1035, 00189 Rome, Italy e-mail: alberto.signore@uniroma1.it High levels of interleukin-2 receptors (IL-2R) can be found mainly on the surface of activated T lymphocytes (both CD4+ and CD8+, mainly Th1 lymphocytes) [1–3] after endogenous stimulation. T lymphocytes are activated by inflammatory 1552 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 Materials and methods processes, such as tissue degeneration [4–6], autoimmune diseases [7, 8], viral, fungal and mycobacterial infections, graft rejection and in tumour infiltrates [9–15]. These disor- ders are characterized by the activation of the immune system and slow recruitment of immune cells (peripher- al blood mononuclear cell, PBMC) in the lesion. The infiltration of PBMC in the inflammatory lesion is the histopathological hallmark of chronic inflammation, in contrast to the granulocyte infiltration found in acute inflammatory conditions. During activation, immune cells overexpress multimeric IL-2R, which are able to sustain immune cell proliferation and cell survival. The IL-2R consists of three subunits (α, β and γ chains, called CD25, CD122 and CD132, respectively). CD25 contains the main binding site for IL-2 and can be present as a transmembrane or soluble receptor. Because IL-2R expression is low in resting immune cells, the receptor might be a suitable biomarker to study active inflammation in chronic inflammatory diseases. PET acquisition protocol and arterial blood sampling Rats were anaesthetized with 2 % isoflurane in medical air and a cannula for blood sampling was surgically inserted in the femoral artery. The animals were placed in a transaxial posi- tion in the PET camera (microPET Focus 220, Siemens/Con- corde) with the shoulders of the rats in the field of view. Rats were injected trough the penile vein with 17.1±1.2 MBq of [18F]FB-IL2 and a dynamic PET scan was immediately ac- quired for 60 min. After the emission scan, a transmission scan of 515 s with a 57Co point source was obtained for the correction of tissue attenuation. Two animals were scanned simultaneously in each scan session. Arterial blood was sampled during the course of the scan at 15, 30, 45, 60, 75, 90, 120, 180, 300, 450, 600, 900, 1,800 and 3,600 s after tracer injection. When a blood sample was collected, 0.1 ml of heparinized saline was injected via the artery cannula to prevent large changes in blood pressure. Blood samples were used to measure the plasma radioactivity concentration. Blood was centrifuged (5 min at 13,000 rpm) to separate cells from plasma. Then, 50 μl of each plasma sample was collected and the activity in these plasma samples was measured with a gamma counter (LKB Wallac, Turku, Finland). Plasma activ- ity was corrected for decay. Animals All animal experiments were carried out according to the Dutch Regulations for Animal Welfare. The protocol was approved by the Ethics Committee of the University of Groningen (protocol number: 5705 C). After arrival, 11 Wistar rats (Charles River, The Nether- lands) were acclimatized for at least 7 days. The animals were housed in Makrolon cages on a layer of wood shavings in a room with constant temperature (21±2 °C) and 12-h light-dark regime. Commercial chow and tap water were available ad libitum. All rats were subcutaneously implanted with an increasing number of activated hPBMC in 100 μl PBS mixed with 100 μl of Matrigel (Becton Dickinson, The 1553 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 identification of the radioactive metabolites. HPLC analyses were carried out with Elite LaChrom Merck Hitachi L-7100 pump system using Luna C18-column (5 μm, 4.6×250 mm) equipped with both UV (Elite LaChrom VWR Hitachi L-2400 UV detector set at 254 nm) and a Bicron radioactivity monitor. Gradient elution was performed using a mixture of 0.1 % aqueous trifluoroacetic acid (solvent A) and 0.1 % trifluoro- acetic acid in acetonitrile (solvent B). The following gradient profile was used: 0–5 min 0 % B, 5–10 min 40 % B, 10– 35 min 65 % B, 35–45 min 100 % B and 45–47 min 0 % B at flow rate of 1 ml/min. Retention times were: 24 min for [18F] FB-IL2, 26 min for [18F]FBA and 30 min for [18F]SFB. The same HPLC procedure was applied to analyse urine samples for the formation of radioactive metabolites, using samples collected 60 min after tracer injection. Netherlands). The activated cells were inoculated in the right shoulder of the animal 15 min before the PET experiment. PET acquisition protocol and arterial blood sampling Image reconstruction All emission scans were normalized and corrected for random coincidences, dead time, scatter, attenuation and decay. Emis- sion sinograms were reconstructed using an ordered subset expectation maximization (OSEM) algorithm with 4 iterations and 16 subsets. Three-dimensional regions of interest (3-D ROIs) were generated automatically based on an intensity threshold method using Inveon Research Workplace software (Inveon, Siemens, USA). Briefly, all frames (0-60 min) were summed and an ROI of the inflammatory region was gener- ated automatically with a 50 % threshold using a region growing method, i.e. only pixels were included with tracer uptake greater than 50 % of the maximum value within the lesion. The resulting ROIs were used on the original data set to create the corresponding time-activity curves (TACs) using standard software (Inveon, Siemens, USA). Radiometabolite analysis of rat plasma was performed in a separate study in rats without hPBMC lesion. Arterial blood samples were collected at 1, 10, 20, 30, 40 and 60 min after administration of 32.1±9.8 MBq [18F]FB-IL2. Blood was centrifuged for 5 min at 13,000 rpm to separate the cells from the plasma fraction. After centrifugation, 5 μl of each plasma sample was spotted onto a thin-layer chromatography (TLC) plate (Merck F-254 silica gel strip). The strip was eluted at room temperature with ethyl acetate/n-hexane (3:1). [18F]SFB migrated with the solvent front (Rf01), and [18F]FB-IL2 remained at the origin (Rf00). The degradation product 4- [18F]fluorobenzoic acid ([18F]FBA) has an Rf00.7. Detection of the reference compounds on the TLC plates was performed by UV light (254 nm). For radiolabelled compounds, the detection on the TLC was performed by phosphor storage imaging (multisensitive screens, Packard). These screens were exposed to the TLC strips for a few minutes and subsequently read out using a Cyclone phosphor storage imager (PerkinElmer, USA) and analysed with OptiQuant software. Ex vivo biodistribution After the PET scan, Wistar rats were sacrificed by extirpa- tion of the heart while under deep anaesthesia. The xeno- graft consisting of Matrigel mixed with inoculated cells was dissected and weighed. As a reference tissue muscle from the contralateral shoulder was isolated. Radioactivity in each sample was measured by an automated gamma counter (LKB Wallac, Turku, Finland). Radioactivity accumulation in the target lesion was expressed as standardized uptake value (SUV), using the formula: [(tissue activity concentra- tion (MBq/g)]/[(injected dose (MBq)/body weight (g)]. Ex vivo measurement of tracer accumulation For comparison with the PET studies, ex vivo measurement of radioactivity accumulated in the inflammatory lesion was performed. The radiopharmaceutical uptake of the dissected Matrigel + hPBMC was calculated in ten animals. In one animal, the ex vivo tracer accumulation could not be deter- mined due to an experimental error during dissection and only kinetic modeling was performed in this animal. Muscle in the shoulder contralateral with respect to the site of hPBMC inoculation was used as a control tissue. As shown in Fig. 1, there is a strong correlation between [18F]FB-IL2 uptake and the number of inoculated activated CD25+ cells (R200.90, p value <0.0001). As expected, no correlation was found between muscle uptake and number of inoculated cells (R200.13, p value00.31). [18F]FB-IL2 kinetics Figure 4a shows the blood clearance of [18F]FB-IL2. Biex- ponential curve-fitting analysis of [18F]FB-IL2 blood Statistical analysis All data are expressed as mean ± standard deviation. Corre- lations were calculated with the linear regression algorithm in Sigma Plot and were considered statistically significant whenever R2>0.5 and p<0.05. Fig. 1 Correlation between [18F]FB-IL2 uptake (ex vivo SUV) and the number of CD25+ cells in the inflammatory lesion (closed symbols) and in muscle as a reference tissue (open symbols). R200.90, p<0.0001, y00.14x−0.006 and R200.13, p00.31, y00.005x+0.013 for the inflammatory lesion and muscle, respectively Kinetic analysis Pharmacokinetic modelling of the tissue TACs was performed using standard software (Inveon, Siemens, USA). Graphical Logan analysis and Patlak analysis were used to determine the volume of distribution (Vd). Two compartmental models were An additional 0.2 ml plasma sample obtained at 60 min was filtered through a Vivaspin filter with a 30 kDa cutoff; 0.1 ml of this filtrate was analysed by analytical HPLC for the Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1554 used to fit ROI data. These were a reversible one-tissue com- partment model (1TCMR, one tissue compartment with two kinetic rate constants) and a reversible two-tissue compartment model (2TCMR, two tissue compartments with four kinetic rate constants). The blood pool compartment was considered as the input function using the present terminology [25]. Best- fit analysis was used to calculate the Vd and with the latter model also the binding potential (BP). Vd is defined as K1/k2 for the 1TCMR and (K1/k2)(1+BP) for the 2TCMR, with the BP being equal to k3/k4. The optimal model was selected based on the Akaike information criterion (AIC) values generated by the analysis software. AIC selects the model with the best fit of the data, taking into account the number of fitted data points and the number of fitted parameters [26]. Small animal PET imaging At 60 min after intravenous injection of [18F]FB-IL2, acti- vated hPBMC could be clearly visualized in the right shoul- der of all animals (Fig. 2). By applying 50 % of SUVmax as the threshold, an ROI was obtained for the inflammatory lesions. SUV values of [18F]FB-IL2 were calculated for each animal and were correlated with the number of CD25-positive cells in the inflamed region. As shown in Fig. 3, there was a moderate correlation between the cell number and the SUV of [18F]FB-IL2 PET (R200.56, p00.008). Limit of detection To assess the sensitivity of the PET method, the limit of detection (LOD) for CD25-positive cells was determined by measuring the BP of [18F]FB-IL2 in the contralateral shoul- der (control). For this purpose, the ROI that was generated for the inflammatory lesion at the injection site was copied to the contralateral unaffected shoulder. The LOD was defined as the mean of the BP in the control tissue + 2 times the standard deviation in order to assure with 95 % confi- dence that a signal at the inflammatory lesion was not due to statistical variability. Subsequently, the LOD was applied to calculate the minimum number of CD25-positive cells that can be detected with [18F]FB-IL2, using the correlation between the number of CD25-positive cells and the BP (Fig. 7). FACS analysis FACS analysis FACS analysis The percentage of CD25-positive cells was calculated on the day of the microPET experiment. PHA-P caused variable stimulation of overexpression of CD25 on the cell surface of hPBMC. The fraction of activated cells ranged between 5.5 and 9.9 %. The number of the inoculated hPBMC was corrected for the fraction of CD25-positive cells before correlation with the PET results. The number of CD25- positive cells injected in the shoulder of the rats ranged from 0.17×106 to 1.9×106. Fig. 1 Correlation between [18F]FB-IL2 uptake (ex vivo SUV) and the number of CD25+ cells in the inflammatory lesion (closed symbols) and in muscle as a reference tissue (open symbols). R200.90, p<0.0001, y00.14x−0.006 and R200.13, p00.31, y00.005x+0.013 for the inflammatory lesion and muscle, respectively Pharmacokinetic modelling Pharmacokinetic modelling using the [18F]FB-IL2 TACs at the inoculation site from 0 to 60 min after tracer injection showed a significantly better fit (p<0.0001) using Logan graphical analysis (R200.97±0.02) than with Patlak analysis (R200.81±0.12). In addition, Logan analysis required a delay time of only 3 min, whereas for Patlak analysis a delay time of 15 min had to be applied to obtain a reasonable fit (Fig. 5). This suggests that the binding between the fluorinated IL-2 and CD25 is better described by a reversible model. 1555 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 Fig. 2 Small animal PET images of [18F]FB-IL2 in the inflammatory lesion of a rat inoculated with 0.99×106 CD25+ cells. Coronal (a), sagittal (b) and transaxial (c) views of the thorax/abdomen of the rat. The inflammatory lesion is indicated by a red arrow. The image represents uptake in the inflammatory lesion from 0 to 60 min after injection of [18F]FB-IL2. In the transverse image, the submandibular glands are also visible Fig. 2 Small animal PET images of [18F]FB-IL2 in the inflammatory lesion of a rat inoculated with 0.99×106 CD25+ cells. Coronal (a), sagittal (b) and transaxial (c) views of the thorax/abdomen of the rat. The inflammatory lesion is indicated by a red arrow. The image represents uptake in the inflammatory lesion from 0 to 60 min after injection of [18F]FB-IL2. In the transverse image, the submandibular glands are also visible represents uptake in the inflammatory lesion from 0 to 60 min after injection of [18F]FB-IL2. In the transverse image, the submandibular glands are also visible FB-IL2 with a retention time of 22 min. [18F]FBA and [18F] FB-IL2 (26 and 24 min, respectively) were not found in the urine. Because of the high stability of the tracer in plasma, the total activity in plasma without correction for metabo- lites was used as input function for the pharmacokinetic modelling study. clearance showed a two-phase blood clearance with 25± 11 % of the injected activity having a half-clearance time of 0.71±0.29 min and 75±11 % of the injected activity having a half-clearance time of 8.4±2.6 min. Figure 4b shows the average TAC for the inflammatory lesion in the animals inoculated with approximately 106 CD25-positive hPBMC. In contrast to plasma, the clearance from the cell inoculation site is slower and it can be described by a one-phase curve (0–60 min) with a half-clearance time of 37±4 min. clearance showed a two-phase blood clearance with 25± 11 % of the injected activity having a half-clearance time of 0.71±0.29 min and 75±11 % of the injected activity having a half-clearance time of 8.4±2.6 min. Figure 4b shows the average TAC for the inflammatory lesion in the animals inoculated with approximately 106 CD25-positive hPBMC. In contrast to plasma, the clearance from the cell inoculation site is slower and it can be described by a one-phase curve (0–60 min) with a half-clearance time of 37±4 min. Metabolite analysis Metabolites in plasma were analysed by TLC and by HPLC. TLC analysis did not show any formation of hydrophobic metabolites or formation of [18F]FBA (hydrolysis product). HPLC analysis confirmed that at 60 min post-injection (p.i.), the fraction of intact [18F]FB-IL2 in plasma was still 97.7±1.2 %. Urine samples were analysed as well. After 60 min of tracer distribution, the main excretion prod- uct is an unknown hydrophilic degradation product of [18F] Next, the distribution volume was calculated by Logan graphical analysis and compartment analysis using a 1TCMR and a 2TCMR model. Correlation between the number of CD25+ cells and the distribution volume are displayed in Fig. 6. The distribution volume showed a moderate correlation with the number of CD25+ cells when either Logan analysis or compartment analysis with a 1TCMR or 2TCMR model was used (R200.59, 0.59 and 0.52, respectively). Fig. 3 Correlation between PET imaging-derived [18F]FB-IL2 uptake expressed as SUV and the number of CD25+ cells present in the inflamed region. (R200.56, p00.008, y00.07x+0.17) The model of preference for compartment modelling was selected based on AIC values. The AIC values are 273±16 and 241±23 for 1TCMR and 2TCMR, respectively, with a highly significant difference between those models (p00.0011). Thus, TACs could be fit best using a 2TCMR model. In contrast to the distribution volume and the SUV, the BP is only dependent on the receptor binding characteristics of the radiopharmaceutical. To exclude any influence of differences in perfusion between ani- mals, BP were calculated using the 2TCMR model. The number of CD25-positive cells and the corresponding BP values are plotted in Fig. 7. The BP is 0.45 for the Fig. 3 Correlation between PET imaging-derived [18F]FB-IL2 uptake expressed as SUV and the number of CD25+ cells present in the inflamed region. (R200.56, p00.008, y00.07x+0.17) Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1556 Fig. 4 [18F]FB-IL2 time-activity curves a for plasma and b for the inflamed region (mean ± standard deviation of animals injected with 106 CD25+ human activated PBMC) Fig. 4 [18F]FB-IL2 time-activity curves a for plasma and b for the inflamed region (mean ± standard deviation of animals injected with 106 CD25+ human activated PBMC) lesion with 0.17×106 CD25-positive cells and gradually increases up to 7.44 for the lesion with 1.9×106 CD25+ cells. The BP increases by a factor of 4 for each million cells preset in the inflammatory lesion. Metabolite analysis There is a strong correlation between the BP and the number of CD25- positive cells (R2 0.88, p<0.0001). . 5 Representative Logan and Patlak graphical analysis for the inflamed region. a Logan graphical analysis showing a good fit of the inflamed ion from 3 min after [18F]FB-IL2 injection, whereas b Patlak graphical analysis shows a delay time of 15 min Fig. 5 Representative Logan and Patlak graphical analysis for the inflamed region. a Logan graphical analysis showing a good fit of the inflamed region from 3 min after [18F]FB-IL2 injection, whereas b Patlak graphical analysis shows a delay time of 15 min Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1557 Fig. 6 Correlation between the volume of distribution (Vd) determined by a Logan analysis, b 1TCMR and c 2TCMR and the number of CD25-positive cells. R2 was 0.59, p00.006, y00.32x+0.49 for Logan analysis. R2 was 0.59, p00.005, y00.34x+0.48 for 1TCMR, and R2 was 0.52, p00.013, y00.32 x+0.56 for 2TCMR analysis. R2 was 0.59, p00.005, y00.34x+0.48 for 1TCMR, and R2 was 0.52, p00.013, y00.32 x+0.56 for 2TCMR analysis. R2 was 0.59, p00.005, y00.34x+0.48 for 1TCMR, and R2 was 0.52, p00.013, y00.32 x+0.56 for 2TCMR Fig. 6 Correlation between the volume of distribution (Vd) determined by a Logan analysis, b 1TCMR and c 2TCMR and the number of CD25-positive cells. R2 was 0.59, p00.006, y00.32x+0.49 for Logan Limit of detection untreated shoulder. The average BP of [18F]FB-IL2 in the contralateral shoulder was 0.42±0.20. Using a 95 % confi- dence interval, the LOD can be defined as the average back- ground signal plus 2 standard deviations. In this model, the LOD of [18F]FB-IL2 would therefore be equal to 0.82. Using the equation for the correlation between the BP and the number of CD25-positive hPBMC (Fig. 7), we calculated that the lowest number of CD25-positive cells that can be reliably (95 % confidence) detected by this methods is approximately 160,000 cells in a lesion with a volume of 200 μl. To assess the sensitivity of [18F]FB-IL2, the LOD was deter- mined by measuring the background signal in the contralateral Fig. 7 Correlation between the binding potential and the number of CD25-positive cells. R2 0.88, p<0.0001, y04.06x+0.17 Discussion The present investigation aims to demonstrate that [18F]FB- IL2 PET imaging is a suitable tool to quantify lymphocyte infiltration in tissues. In this study, pharmacokinetic analysis of [18F]FB-IL2 was carried out in a rat xenograft model of inflammation. Different parametric methods were used to investigate which method was best to quantitatively mea- sure activated immune cell infiltration. Fig. 7 Correlation between the binding potential and the number of CD25-positive cells. R2 0.88, p<0.0001, y04.06x+0.17 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 1558 injected cells (165,000 cells). A moderate correlation was found between the number of CD25-positive cells and the SUV determined from PET data (R200.56). This correlation obtained from the imaging study was substantially lower than the one from the ex vivo studies. This discrepancy might be explained by partial volume effects, which can underestimate the uptake in small or heterogeneous target regions. In this study, inflammatory lesions were relatively small (injection volume 0.2 ml), when compared to the spatial resolution of the PET camera (1.35 mm full-width at half-maximum). In addition, inflammatory lesions could be heterogeneous, because of the inhomogeneous presence of the Matrigel in the subcutaneous layer where the hPBMC are trapped. Furthermore, the inflammatory lesions are ir- regularly shaped. The inhomogeneity and irregular shape of the lesion could lead to differences in influence of the partial volume effect between lesions. Moreover, inflammation is a process that involves several changes in the inflammatory lesion, such as change in tissue perfusion and vessel perme- ability, which can reduce the usefulness of SUVs. Therefore, pharmacokinetic modelling might be required for accurate quantification of lymphocyte infiltration. Wistar rats inoculated with different numbers of human activated PBMC were selected as the animal model of choice. The big advantage of this animal model is the possibility to determine the exact number of inoculated CD25-positive tar- get cells by measuring PHA-P-induced CD25 expression using FACS analysis. PHA-P is a strong activator of hPBMC. One of the most important markers of this activation process is the alpha subunit of the IL-2R (CD25). PHA-P-mediated cell activation depends on several factors such as time of incuba- tion and amount of hPBMC in culture. Moreover, hPBMC derived from different healthy subjects show different predis- position to the activation. In this study, hPBMC were isolated from blood obtained from the local blood bank. Discussion hPBMC in these samples are less prone to activation by PHA-P than freshly isolated hPBMC from peripheral blood. Consequently, the fraction of CD25-expressing cells in the cell cultures was relatively low in this study, but the experimental design of this study allowed us to correct the data for the exact number of CD25-positive cells inoculated in the inflammatory lesion. Ideally, an internal control could have been added to this model by injection of an equal number of non-activated hPBMCs or Matrigel alone in the contralateral shoulder of the animal. In a previous study in mice, however, we observed that inoculation of the Matrigel without cells al- ready induced a strong migration of the activated hPBMC to the contralateral shoulder [22]. This cell migration caused a variable decrease of the activated PBMC in the inoculation site. Consequently, injection of a control xenograft would not allow us to have an exact estimation of the number of CD25-positive cells present in the inflammatory lesion and thus would hamper correlation of tracer accumulation with the number of CD25-positive cells. For the pharmacokinetic modelling, we used the total [18F] FB-IL2 plasma TAC without correction for tracer metabolites as the input function, since [18F]FB-IL2 was highly stable in plasma. More than 97 % of the tracer remained intact in plasma 60 min p.i. In contrast, only radioactive metabolites were found in urine. This suggests that [18F]FB-IL2 is metab- olized by the kidneys and subsequently secreted into the urine. This observation is in agreement with the fate of intravenously injected native IL-2, which is also cleared into the urine after renal tubular catabolism [27]. Although the model used in this study does not represent all aspects of inflammation, it is an ideal model for the validation of the quantitative analysis of activated T lym- phocytes using [18F]FB-IL2 PET. Other animal models of inflammation may better mimic disease processes, but do not allow exact quantification of the number of CD25- positive cells and can only give an indirect and inaccurate estimation of the number of cells present in the inflamma- tory lesion. Therefore, these more physiological animal models do not allow accurate correlation of the PET signal with the number of CD25 cells. A complicating factor in our xenograft animal model could be the involvement of host immune cells. Discussion Quantification of the tracer uptake as SUVor distribution volume is less accurate, as these parameters are not only dependent on receptor binding characteristics, but also on perfusion effects. The results of this study indicate that [18F]FB-IL2 PET could provide hitherto unavailable opportu- nities to assess lymphocytic infiltration in inflammatory dis- eases in a non invasive and quantitative manner. The technique is likely sensitive enough to detect even low numbers of infiltrating lymphocytes or small changes in the inflammatory response. Despite the fact that we did not include a control lesion in the contralateral side, we were still able to estimate the amount of nonspecific uptake at the inflammatory lesion from the correlations of the tracer uptake with the number of CD25-positive cells. In fact, Figs. 3 and 6 show that both the curves of the SUV and the distribution volume as func- tions of the number of CD25-positive cells do not cross the y-axis at the origin, indicating that an SUVof approximately 0.17 and a distribution volume of 0.5 are due to nonspecific uptake (i.e. in case the number of CD25-positive cells is 0). However, when the tracer uptake is expressed as the BP the nonspecific binding is negligible, as the curve in Fig. 7 almost crosses the y-axis at the origin. This again stresses the importance of pharmacokinetic modelling to compen- sate for nonspecific effects on tracer uptake. 18 Discussion To avoid interference of the host immune response, the interval between inoculation and PET imaging was therefore kept as short as possible. Because the tracer is directly injected into the systemic circulation, the peak of the activity concentration in plasma of [18F]FB-IL2 reaches its maximum value within 15 s after tracer injection and then drops progressively. The plasma [18F]FB-IL2 concentration initially declines quite rapidly with an α phase of 0.71±0.29 min due to the elimination from the plasma and the distribution of the tracer in tissue. The elimination of tracer from plasma is described by a β phase with a half-life of 8.4±2.6 min. In contrast to the plasma curves, the TACs of the inflammatory lesion show a clearance half-life of 37±4 %. These data suggest that after an initial nonspecific distribution of the tracer to the entire body of the animal [18F]FB-IL2 is retained in the inflamma- tory lesion because of the presence of target cells that over- express the IL-2R. Uptake of [18F]FB-IL2 in the region of inoculated acti- vated hPBMC, measured ex vivo as SUV, increased with the number of inoculated CD25-positive cells and an excellent correlation between these parameters was found (R200.90). Logan and Patlak analysis are simple graphical methods to estimate reversible and irreversible binding of a tracer to the target, respectively. We used these methods to study the binding characteristics of [18F]FB-IL2. The TACs of the hPBMC xenografts showed a better fit with Logan analysis than with the Patlak analysis method, indicating that binding MicroPET images showed clear [18F]FB-IL2 uptake at the cell inoculation site, even for the smallest number of 1559 Eur J Nucl Med Mol Imaging (2012) 39:1551–1560 between [18F]FB-IL2 and the IL-2R could be reversible. Compartment modelling showed that the kinetics of [18F] FB-IL2 are best described by a 2TCMR. However, the volume of distribution derived from Logan, 1TCMR or 2TCMR showed only moderate correlations with the cell number present in the inflammatory lesion. In contrast, BPs are strongly correlated with the number of CD25-positive cells present in the inflammatory lesion (R200.88). The BP increases proportionally with the increase of the number of activated hPBMC in the inflammatory region, suggesting that with this method changes in the number of activated hPBMC should be easily detectable. activated hPBMC can be best quantified by measuring the BP with [18F]FB-IL2 PET. Conflicts of interest None. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. References 1. Benczik M, Gaffen SL. The interleukin (IL)-2 family cytokines: survival and proliferation signaling pathways in T lymphocytes. Immunol Invest 2004;33(2):109–42. 2. Gaffen SL, Liu KD. Overview of interleukin-2 function, produc- tion and clinical applications. Cytokine 2004;28(3):109–23. Taken together, our data clearly demonstrate that [18F] FB-IL2 BP is a better measure of activated hPBMC in the inflammatory lesion than the distribution volume or SUV. Both the distribution volume and the SUV depend not only on the binding characteristics of the tracer to IL-2R, but also on the perfusion of the tracer into the lesion. In contrast, BP is independent of tracer influx (K1) and efflux (k2), but only depends on the ratio of receptor binding and release. 3. Malek TR, Castro I. Interleukin-2 receptor signaling: at the interface between tolerance and immunity. Immunity 2010;33 (2):153–65. 4. Meyer KC. The role of immunity and inflammation in lung senes- cence and susceptibility to infection in the elderly. Semin Respir Crit Care Med 2010;31(5):561–74. 4. Meyer KC. The role of immunity and inflammation in lung senes- cence and susceptibility to infection in the elderly. Semin Respir Crit Care Med 2010;31(5):561–74. 5. Singh T, Newman AB. 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Cancer-associated stroke
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Cancer-associated stroke Dubenko O.Ye.1,2, ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com 1State Organization «Grigoriev Institute for Medical Radiology and Oncology o O.Ye.1,2, ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com 1State Organization «Grigoriev Institute for Medical Radiology and Oncology of the National Academy of Medical Sciences of Ukraine», Kharkiv, Ukraine 2Kharkiv Medical Academy of Postgraduate Education of the Ministry of Health of Ukraine, Kharkiv, Ukraine Dubenko O.Ye.1,2, ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com 1State Organization «Grigoriev Institute for Medical Radiology and Oncology of the National Academy of Medical Sciences of Ukraine», Kharkiv, Ukraine 2Kharkiv Medical Academy of Postgraduate Education of the Ministry of Health of Ukraine, Kharkiv, Ukraine РЕЗЮМЕ Ключові слова: Актуальність. Системний рак і мозковий інсульт є найбільш розповсюдженими захворюваннями і найчастішою причиною смерті у людей старшого віку в усьому світі. Взаємозв’язок між раком та інсультом складний, оскільки рак призводить до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. Мета роботи. Проаналізувати можливі механізми розвитку рак-асоційованого інсульту, підходи до діагностики та лікування. інсульт, рак, рак-асоційований інсульт, коагулопатія. DOI: https://doi.org/10.46879/ukroj.4.2022.52-61 УДК: 61.616-006.04:616-005.755 DOI: https://doi.org/10.46879/ukroj.4.2022.52-61 УДК: 61.616-006.04:616-005.755 Рак-асоційований інсульт Дубенко О.Є.1,2,ORCID: 0000-0002-4911-5613, e-mail: olgadubenko05@gmail.com 1Державна установа «Інститут медичної радіології та онкології ім. С.П. Григор’єва Національної академії медичних наук України», Харків, Україна 2Харківська медична академія післядипломної освіти Міністерства охорони здоров’я України, Харків, Україна DOI: https://doi.org/10.46879/ukroj.4.2022.52-61 УДК: 61.616-006.04:616-005.755 © Дубенко О.Є., 2022 Для цитування: Для цитування: Дубенко О.Є. Рак-асоційований інсульт. Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61. DOI: https://doi.org/10.46879/ukroj.4.2022.52-61 Огляд літератури Literature review Огляд літератури Literature review Огляд літератури Literature review Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Для кореспонденції: Дубенко Ольга Євгеніївна Харківська медична академія після- дипломної освіти, кафедра неврології; вул. Амосова, буд. 58, Харків, Україна, 61176; il l d b k 05@ il Матеріали та методи. Підбір публікацій виконано за базами даних PubMed (https://pubmed.ncbi.nlm.nih.gov/), ClinicalKeyElsevier (https://www.clinicalkey.com), Cochrane Library (https://www.cochranelibrary.com/) та Google Scholar (https://scholar.google.com/) опублікованих у 2012–2022 рр., у яких висвітлювались відомості про рак-асоційований інсульт. Результати та їх обговорення. Відомі декілька основних патофізіологічних механізмів рак-асоційованого інсульту, що можуть бути безпосередньо пов’язані з раком, або викликані ускладненнями раку. Найбільш частими є внутрішньо- судинна ракова коагулопатія, небактеріальний тромботичний ендокардит, прямий вплив пухлини на судинну систему або терапевтичні та діагностичні втручання для лікування раку – ускладнення хіміотерапії, променева терапія, а також активація звичайних механізмів інсульту, насамперед атеросклерозу. Ризик інсульту також пов’язаний з агресивністю раку. Характерними ознаками рак-асоційованого інсульту, пов’язаного з раковою коагулопатією, є множинні ураження в різних артеріальних ділянках, а також лабораторні дані, що вказують на коагулопатію у вигляді підвищення рівня D-димеру, підвищеного С-реактивного протеїну, низького гематокриту. ; e-mail: olgadubenko05@gmail.com Результати та їх обговорення. Відомі декілька основних патофізіологічних механізмів рак-асоційованого інсульту, що можуть бути безпосередньо пов’язані з раком, або викликані ускладненнями раку. Найбільш частими є внутрішньо- судинна ракова коагулопатія, небактеріальний тромботичний ендокардит, прямий вплив пухлини на судинну систему або терапевтичні та діагностичні втручання для лікування раку – ускладнення хіміотерапії, променева терапія, а також активація звичайних механізмів інсульту, насамперед атеросклерозу. Ризик інсульту також пов’язаний з агресивністю раку. Характерними ознаками рак-асоційованого інсульту, пов’язаного з раковою коагулопатією, є множинні ураження в різних артеріальних ділянках, а також лабораторні дані, що вказують на коагулопатію у вигляді підвищення рівня D-димеру, підвищеного С-реактивного протеїну, низького гематокриту. Висновки. Рак призводить до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. Ці механізми є множинними і складними, однак найбільш відомим та поширеним є ракова внутрішньосудинна коагулопатія, яка має специфічні лабораторні біомаркери. Численні дослідження продовжують спрямовуватись на вивчення факторів, пов’язаних з коагулопатією у пацієнтів, які перенесли інсульт, особливості раку та оцінки біологічних маркерів внутрішньо- судинної коагулопатії, що викликає інсульт, а також моніторинг ефектів антикоагулянтної терапії у пацієнтів із рак-асоційованим інсультом. © Дубенко О.Є., 2022 Висновки. Рак призводить до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. Ці механізми є множинними і складними, однак найбільш відомим та поширеним є ракова внутрішньосудинна коагулопатія, яка має специфічні лабораторні біомаркери. Численні дослідження продовжують спрямовуватись на вивчення факторів, пов’язаних з коагулопатією у пацієнтів, які перенесли інсульт, особливості раку та оцінки біологічних маркерів внутрішньо- судинної коагулопатії, що викликає інсульт, а також моніторинг ефектів антикоагулянтної терапії у пацієнтів із рак-асоційованим інсультом. INTRODUCTION Найбільш значними причинами тяжкості захворю- вань у людей старшого віку залишаються серцево- судинні захворювання та злоякісні новоутворення [1]. Поєднання раку та інсульту призводить до погіршення дієздатності та збільшення інвалідизації. Але, поєд- нання раку та інсульту може бути не просто випадко- вою комбінацією двох найбільш розповсюджених захворювань. Рак призводить до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. На теперішній час накопичено чимало наукових знань про асоціацію між раком та інсуль- том, і можливі механізми рак-асоційованого інсульту. Взаємозв’язок між раком та інсультом відомий в усьому світі, і викликає значний науковий та практичний інтерес. Цей взаємозв’язок складний та багатобічний. У низці досліджень науковці намагалися з’ясувати, які саме види раку мають сильніший зв’язок із ви- никненням інсульту. У хворих з діагностованим раком легенів, підшлункової залози, колоректальним раком, молочної залози та простати існує більш висока частота інсульту. Ризик інсульту також пов’язаний Найбільш значними причинами тяжкості захворю- вань у людей старшого віку залишаються серцево- судинні захворювання та злоякісні новоутворення [1]. Поєднання раку та інсульту призводить до погіршення дієздатності та збільшення інвалідизації. Але, поєд- нання раку та інсульту може бути не просто випадко- вою комбінацією двох найбільш розповсюджених захворювань. Рак призводить до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. На теперішній час накопичено чимало наукових знань про асоціацію між раком та інсуль- том, і можливі механізми рак-асоційованого інсульту. Взаємозв’язок між раком та інсультом відомий в усьому світі, і викликає значний науковий та практичний інтерес. Цей взаємозв’язок складний та багатобічний. The most substantial causes of disease severity in older people are still cardiovascular disorders and malignancies [1]. The combination of cancer and stroke leads to impaired functional capacity and increased disability. However, the combination of cancer and stroke may not just be a random one of the two most common diseases. Cancer causes an increased risk of stroke due to specific pathophysiological mechanisms. At pre- sent, there is a comprehensive scientific expertise in the relation between cancer and stroke, and possible mechanisms of cancer-associated stroke. The relation- ship between cancer and stroke is known worldwide and is of a considerable scientific and practical interest. It is complex and diverse. In a number of studies, scientists were trying to figure out what kinds of cancer were mostly associated with stroke. The patients diagnosed with lung, pancreas, colon and rectum, breast and prostate cancer have a higher incidence of stroke. The risk of stroke is also related to cancer aggressiveness. Зв’язок роботи з науковими програмами, планами і темами The article is a fragment of the planned research project of State Organization «Grigoriev Institute for Medical Radiology and Oncology of the National Academy of Medical Sciences of Ukraine» «Development of the individual approaches to antiblastomic therapy in patients recovered from COVID-19», code: NAMN 04.22, state registration number: 0121U112052, period for perfor- mance: 2022–2024; led by Director of State Organization «Grigoriev Institute for Medical Radiology and Oncology of the National Academy of Medical Sciences of Ukraine», Doctor of Medical Sciences, Professor M.V. Krasnoselskyi; Head of Radiation Therapy Department, Doctor of Medical Sciences, Professor V.P. Starenkyi. Стаття є фрагментом планової науково-дослідної роботи Державної установи «Інститут медичної радіоло- гії та онкології ім. С.П. Григор’єва Національної акаде- мії медичних наук України», «Розроблення індивіду- альних підходів до проведення антибластомної терапії у пацієнтів, які перенесли COVID-19» НАМН.04.22., номер державної реєстрації 0121U112052, термін виконання 2022–2024 рр., керівники: директор Держав- ної установи «Інститут медичної радіології та онкології ім. С.П. Григор’єва Національної академії медичних наук України», доктор медичних наук, професор М.В. Красносельський, завідувач Відділу радіології, доктор медичних наук, професор В.П. Старенький. For correspondence: Dubenko Olga Evgeniyvna Kharkiv Medical Academy of Postgraduate Education of the Ministry of Health of Ukraine, department of neurology; 58, Amosova Str., Kharkiv, Ukraine, 61176; e-mail: olgadubenko05@gmail.com Results and discussion. There are several underlying pathophysiological mechanisms of cancer-associated stroke, which may be directly related to cancer or caused by cancer complications. The most common ones are intravascular cancer coagulopathy, non-bacterial thrombotic endocarditis, a direct impact of the tumor on the vascular system or therapeutic and diagnostic interventions for the treatment of cancer that is chemotherapy, radiation therapy complications, as well as activation of the usual mechanisms of stroke, atherosclerosis in particular. The risk of stroke is also related to cancer aggressiveness. The characteristic features of cancer-associated stroke which is related to cancer coagulopathy are multiple lesions in various arterial areas, as well as laboratory data indicating coagulopathy in the form of increased level of D-dimer, elevated C-reactive protein, low hematocrit.i © Dubenko O.E., 2022 © Dubenko O.E., 2022 Conclusions. Cancer leads to an increased risk of stroke due to specific patho- physiological mechanisms. These mechanisms are multiple and complex, but the most well-known and widespread is cancer intravascular coagulopathy, which has specific laboratory biomarkers. Numerous studies continue investigating the factors associated with coagulopathy in stroke patients, cancer characteristics, and evaluation of biological markers of stroke-causing intravascular coagulopathy, as well as monitoring the effects of anticoagulation therapy in patients with cancer-associated stroke. For citation: Key words: Background. Systemic cancer and stroke are the most common diseases and the frequent death factors in older people worldwide. The relationship between cancer and stroke is complex, since cancer leads to an increased risk of stroke through specific pathophysiological mechanisms. stroke, cancer, cancer-associated stroke, coagulopathy. Purpose. Analyzing possible mechanisms of development of cancer-associated stroke, strategies of diagnosis and treatment. Materials and methods. The contributions were selected based on PubMed (https://pubmed.ncbi.nlm.nih.gov/), Clinical Key Elsevier (https://www.clinicalkey.com), Literature review 52 ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Cochrane Library (https://www.cochranelibrary.com/) and GoogleScholar (https://scholar.google.com/) databases published from 2012 to 2022, dealing with cancer-associated stroke. Dubenko Olga Evgeniyvna Kharkiv Medical Academy of Postgraduate Education of the Ministry of Health of Ukraine, department of neurology; 58, Amosova Str., Kharkiv, Ukraine, 61176; e-mail: olgadubenko05@gmail.com INTRODUCTION Lung, pancreas, colon and rectum cancer, representing a highest stroke risk, У низці досліджень науковці намагалися з’ясувати, які саме види раку мають сильніший зв’язок із ви- никненням інсульту. У хворих з діагностованим раком легенів, підшлункової залози, колоректальним раком, молочної залози та простати існує більш висока частота інсульту. Ризик інсульту також пов’язаний Literature review 53 Огляд літератури ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 із агресивністю раку. Рак легенів, підшлункової залози та колоректальний рак, які представляють найвищий ризик інсульту, як правило, діагностується на більш пізній стадії, ніж рак грудей і простати [2, 3]. Крім того, хворі на рак та інсульт мають спільні фактори серцево- судинного ризику, такі як артеріальна гіпертонія, куріння, цукровий діабет та метаболічний синдром. is usually diagnosed at a later stage, compared to breast and prostate cancer [2, 3]. In addition, cancer and stroke patients have the common cardiovascular risk factors, such as arterial hypertension, smoking, diabetes mellitus and metabolic syndrome. The nature of stroke in cancer patients can be ischemic or hemorrhagic, while ischemic stroke is observed more often than hemorrhagic and the risk is higher within the first 6 months after cancer was diagnosed [4]. The types of cancer in hemorrhagic stroke are somewhat different. A high risk of cancer of the small intestine, liver, kidney, nervous system, thyroid gland, endocrine system, Non- Hodgkin lymphoma, myeloma and leukemia was reported. The mechanism of hemorrhage is multifactorial including enhanced formation of dilated thin-walled intratumoral vessels, rupture of these newly formed vessels, tumor invasion into existing vessels, and tumor necrosis [5, 6]. Характер інсульту у хворих на рак може бути ішемічним або геморагічним, при цьому ішемічний інсульт спостерігається частіше, ніж геморагічний і ри- зик був вищим в перші 6 місяців після встановлення діагнозу раку [4]. Типи раку при геморагічному інсульті дещо інші. Повідомлялося про високий ризик раку тонкої кишки, печінки, нирок, нервової системи, щито- подібної залози, ендокринної системи, неходжкінської лімфоми, мієломи і лейкемії. Механізм геморагії є багатофакторним і включає посилене утворення розширених тонкостінних внутрішньопухлинних судин, розрив цих новоутворених судин, інвазію пухлини у вже існуючі судини та некроз пухлини [5, 6]. МАТЕРІАЛИ ТА МЕТОДИ ДОСЛІДЖЕННЯ The contributions were selected based on Pub Med (https://pubmed.ncbi.nlm.nih.gov/), Clinical Key Elsevier (https://www.clinicalkey.com), Cochrane Library (https://www.cochranelibrary.com/) and GoogleScholar (https://scholar.google.com/) databases published from 2012 to 2022, dealing with cancer-associated stroke. Підбір публікацій виконано за базами даних Pub Med (https://pubmed.ncbi.nlm.nih.gov/), Clinical Key Elsevier (https://www.clinicalkey.com), Cochrane Library (https://www.cochranelibrary.com/) та GoogleScholar (https://scholar.google.com/), опублікованих у 2012– 2022 рр., у яких висвітлювались відомості про рак-асоційований інсульт. INTRODUCTION Stroke can develop in patients diagnosed with cancer, or precede cancer [7]. In the United States, one out of 10 patients hospitalized with stroke has been diag- nosed with cancer [8]. Інсульт може розвинутись у пацієнтів з діагностова- ним раком, або передувати діагнозу раку [7]. У Сполу- чених Штатах у кожного з 10 пацієнтів, госпіталізованих з інсультом, був виявлений рак [8]. Purpose. Analyzing possible mechanisms of deve- lopment of cancer-associated stroke, strategies of diagnosis and treatment. Мета роботи. Проаналізувати можливі механізми розвитку рак-асоційованого інсульту, підходи до діагностики та лікування. Огляд літератури Механізми ракової коагулопатії та їх роль у розвитку рак-асоційованого інсульту One of the main causes of stroke in cancer patients is coagulopathy. Disrupted blood coagulation, that is disseminated intravascular coagulopathy, is the most common cause of cardiovascular diseases in cancer patients. The first postmortem characteristics of cerebral intravascular coagulation were described in 1975 in pati- ents with breast cancer, leukemia, and lymphoma in the setting of widespread metastases and sepsis. In cancer patients, the most common cause of cerebrovascular thrombosis is a hypercoagulable state that accompa- nies cancer resulting in systemic and cerebral arterial or venous thrombosis [12, 13]. Однією з основних причин інсульту у хворих на рак є коагулопатія. Порушення згортання крові – дисемі- нована внутрішньосудинна коагулопатія – це най- поширеніша причина серцево-судинних захворювань в онкологічних хворих. Перші посмертні характеристики церебрального внутрішньосудинного згортання крові були описані ще в 1975 р. у хворих на рак молочної залози, лейкемію та лімфому, на тлі поширених метастазів і сепсису. У хворих на рак найпоширенішою причиною цереброваскулярного тромбозу є гіпер- коагуляційний стан, який супроводжує рак, внаслідок чого розвиваються системні і церебральні артеріальні або венозні тромбози [12, 13]. Intravascular coagulopathy can be caused by the procoagulant activity of the tumor and the proinflamma- tory response. Tumor cells express procoagulants, tissue factor (which binds to factor VII) and cancer procoagulant, and release inflammatory cytokines and vascular endo- thelial growth factor, mediators that enhance procoagulant activity and angiogenesis. They also overexpress cytokines that attract leukocytes, possibly causing an inflammatory response with a prothrombotic effect. The most common types of cancer causing coagulopathy are lung and pancreatic cancer [14]. It has been recently shown that the so-called neutrophil extracellular traps are important in occurring thrombosis in cancer coagulopathy, which are formed due to the fact that activated neutro- phils secrete decondensed chromatin, which creates a framework for the adhesion of platelets, erythrocytes and platelet adhesion molecules [15].i Внутрішньосудинна коагулопатія може бути викли- кана прокоагулянтною активністю пухлини та проза- пальною відповіддю. Пухлинні клітини експресують прокоагулянти, тканинний фактор (який зв’язується із фактором VII) та прокоагулянт раку, і вивільняють запальні цитокіни та фактор росту ендотелію судин, медіатори, які посилюють прокоагулянтну активність і ангіогенез. Вони також надлишково експресують цито- кіни, які приваблюють лейкоцити, можливо, викликаючи запальну реакцію із протромботичним ефектом. Най- більш поширені типи раку, що викликають коагуло- патію – це рак легенів і підшлункової залози [14]. Mechanisms of cancer coagulopathy and their role in cancer-associated stroke development Mechanisms of cancer coagulopathy and their role in cancer-associated stroke development RESULTS AND DISCUSSION Патофізіологія рак-асоційованого інсульту Pathophysiology of cancer-associated stroke There are several essential pathophysiological mechanisms of stroke in cancer patients, which may directly be related to cancer, or caused by cancer compli- cations like coagulopathy, infections, a direct impact of the tumor on the vascular system, or therapeutic and diag- nostic interventions for the treatment of cancer that is chemotherapy complications, radiation therapy, hemato- poietic stem cell transplantation occuring during treatment or even years after [9].l Існують кілька основних патофізіологічних механіз- мів інсульту у хворих на рак, які можуть бути безпосе- редньо пов’язаними з раком, або викликані ускладнен- нями раку, такі як коагулопатія, інфекції, прямий вплив пухлини на судинну систему, або терапевтичні та діаг- ностичні втручання для лікування раку – ускладнення хіміотерапії, променева терапія, трансплантація гемо- поетичних стовбурових клітин, які можуть відбуватися під час лікування або навіть через роки після нього [9]. Прямий вплив пухлини, або безпосередньо пов’я- заний з пухлиною інсульт, у клінічній практиці зустріча- ється рідко і його важко визначити. Прямі пухлинні ефекти значно відрізняються і включають інвазію пухлинною масою або лептоменінгеальними інфільтра- тами в артеріальний і венозний синус, пухлинні емболії, здавлення кровоносної судини пухлинним ростом, набряк ложа пухлини та внутрішньопухлин- ний крововилив [10]. A direct influence of the tumor, or stroke directly related to the tumor, is rare in clinical practice and it is difficult for identifying. Direct tumor effects vary consi- derably and include invasion by the tumor mass or leptomeningeal infiltrates into the arterial and venous sinuses, tumor emboli, compression of a blood vessel by tumor growth, tumor bed edema and intratumoral hemorrhage [10]. Non-bacterial thrombotic endocarditis, also known as marantic endocarditis, is a recognized cause of cardio- embolic stroke in cancer patients. In non-bacterial thrombotic endocarditis, sterile non-infectious platelet- thrombin vegetations develop on the heart valves (almost exclusively in the left heart valves, mitral and aortic) associated with widespread systemic and cerebral Небактеріальний тромботичний ендокардит, також відомий як марантичний ендокардит, є визнаною причиною кардіоемболічного інсульту у хворих на рак. При небактеріальному тромботичному ендокардиті на клапанах серця розвиваються стерильні неінфек- ційні тромбоцитарно-тромбінові вегетації (майже Literature review 54 Огляд літератури ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. RESULTS AND DISCUSSION 2022;30(4):52–61 виключно в лівих клапанах серця, мітрального та аортального) в асоціації з поширеним системним та церебральним тромбозом, і це найчастіше спричи- нено основною злоякісною пухлиною. Зазвичай він проявляється системним і легеневим емболізмом, причому найпоширенішим неврологічним усклад- ненням є ішемічний інсульт. Точної поширеності не- бактеріального тромботичного ендокардиту у хворих на рак не встановлено, для його скринінгу використо- вуються ехокардіографічні дослідження. Патогенез його не досить відомий, але припускається наявність гіперкоагуляційного стану. Рак у таких випадках частіше поширений, а інфаркт мозку є пізнім ускладненням, однак у рідкісних випадках небактеріальний тромбо- тичний ендокардит при церебральному інфаркті є ознакою раку. В одному оригінальному дослідженні повідомляється про небактеріальний тромботичний ендокардит у 9,3% хворих на рак, і рак у 59% пацієнтів з небактеріальним тромботичним ендокардитом (дослідження проводилися при розтині) [11]. thrombosis, and this is most often caused by an underlying malignanсy. It is usually manifested by systemic and pulmonary embolism, while the most common neuro- logical complication is ischemic stroke. The exact preva- lence of non-bacterial thrombotic endocarditis in cancer patients has not been established, echocardiographic studies are used for its screening. Its pathogenesis is not well known, but the presence of a hypercoagulable state is assumed. Cancer is more common in such cases, and cerebral infarction is a late complication, however, in rare cases, non-bacterial thrombotic endocarditis in cerebral infarction is a cancer sign. One original study reported non-bacterial thrombotic endocarditis in 9.3% of cancer patients, and cancer in 59% of patients with non-bacterial thrombotic endocarditis detected at autopsy [11]. Механізми ракової коагулопатії та їх роль у розвитку рак-асоційованого інсульту Нещодавно було показано, що у виникненні тромбозів при раковій коагулопатії мають значення так звані нейтрофільні екстрацелюлярні пастки, які формуються внаслідок того, що активовані нейтрофіли виділяють деконденсований хроматин, який створює каркас для адгезії тромбоцитів, еритроцитів і тромбоцитарних молекул адгезії [15]. Stroke, due to cancer-related coagulopathy, is a defi- ned disease. Studies of different populations have repor- ted unique characteristics of this condition. Thus, neuro- imaging on diffusion-weighted images (DWI) shows multiple lesions involving numerous different arterial sites. Laboratory data also indicate coagulopathy in the form of increased D-dimer levels, elevated C-reactive protein, and low hematocrit. The factors associated with coagulopathy in stroke patients, cancer characteristics and assessment of biological markers of stroke-causing intravascular coagulopathy, as well as monitoring the effects of anticoagulant therapy in patients with active cancer and stroke were studied in the OASIS-Cancer trial [16, 17]. Cancer-associated stroke patients had Інсульт внаслідок коагулопатії, пов’язаної з раком, є визначеною хворобою. В дослідженнях різних популяцій повідомлялося про унікальні характерис- тики цього стану. Так, при нейровізуалізації на дифу- зійно-зважених зображеннях (DWI) спостерігаються множинні ураження, що охоплюють численні різні артеріальні ділянки. Лабораторні дані також вказують на коагулопатію у вигляді підвищення рівня D-димеру, підвищеного С-реактивного протеїну, низького гемато- криту. Вивчення факторів, пов’язаних із коагулопатією у пацієнтів, які перенесли інсульт, особливості раку Literature review Literature review 55 Огляд літератури Огляд літератури ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 increased levels of extracellular vesicles obtained from the cancer cells, correlating with D-dimer levels. The levels of circulating extracellular vesicles were shown to be higher in patients with lung adenocarcinoma, a well-known type of cancer with cancer coagulopathy, than in other types of cancer (such as squamous cell). When comparing two groups of patients with ischemic stroke, one of which had cancer and the other did not, it was found that in the group of patients who had cancer, there was a significantly higher level of the inflammatory marker C-reactive protein, as well as fibrinogen, D-dimer, deep vein and pulmonary artery thrombosis. Ischemic stroke in cancer patients was accompanied by a signi- ficantly higher mortality rate and greater disability, determined by the modified Rankin scale. Therefore, the pathophysiology and mechanism of ischemic stroke in cancer patients were determined by different risk factors, stroke biomarkers and subtypes compared to non-cancer cases. Механізми ракової коагулопатії та їх роль у розвитку рак-асоційованого інсульту It was concluded that the stroke pathogenesis in such cases was most likely due to the state of hyper- coagulation or side effects of cancer treatment [18]. та оцінки біологічних маркерів внутрішньосудинної коагулопатії, що викликає інсульт, а також моніторинг ефектів антикоагулянтної терапії у пацієнтів з активним раком та інсультом вивчались у дослідженні OASIS-Cancer [16, 17]. У пацієнтів з інсультом, пов’яза- ним з раком, були підвищені рівні екстрацелюлярних везикул, отриманих від ракових клітин, які корелювали з рівнями D-димеру. Показано, що рівні циркулюючих екстрацелюлярних везикул були вищими у пацієнтів з аденокарциномою легенів, яка є добре відомим типом раку з розвитком ракової коагулопатії, ніж при інших типах раку (таким як плоскоклітинний). При порівнянні двох груп пацієнтів з ішемічним інсуль- том, в одних з яких був рак, а в інших раку не було, виявлено, що в групі пацієнтів, які хворіли на рак, був достовірно вищий рівень маркера запалення С-реактивного протеїну, а також фібриногену, D-димеру, тромбозів глибоких вен та легеневої арте- рії. Ішемічний інсульт в онкологічних хворих супро- воджувався значно вищим рівнем смертності та більшою інвалідністю, визначеною за модифікованою шкалою Ренкін. Отже, патофізіологія та механізм ішемічного інсульту у пацієнтів з онкологічними за- хворюваннями були зумовлені різними факторами ризику, біомаркерами інсульту та підтипами порівняно з нераковими випадками. Було зроблено висновок, що патогенез інсульту в таких випадках швидше за все зумовлений станом гіперкоагуляції або побіч- ними ефектами лікування раку [18]. A better understanding of the molecular targets underlying stroke-related coagulopathy will help determine stroke risk and monitor the effectiveness of cancer-asso- ciated stroke therapy. Patients with occult cancer may have the characteristic symptoms and neuroimaging pattern of cerebral infarction suggesting cancer-related stroke and demonstrating elevated D-dimer levels. Thus, screening for occult malignancy (using serologic or radiologic studies) should be considered for patients who have had charac- teristic patterns of infarction, elevated D-dimer levels, and unobvious etiology or pathogenetic subtype of stroke. Thoracoabdominal-pelvic computed tomography or positron emission tomography should be performed first, since lung, pancreas, genitourinary tract (prostate, ovary, or kidney), gastrointestinal tract (colorectal and stomach) or breast cancer are the most common types (in order of frequency) of known as well as occult cancers in patients with cancer-related stroke. Labora- tory predictors of malignant neoplasm in patients with cryptogenic stroke can also be increased levels of fibrinogen and C-reactive protein [19, 20]. Краще розуміння молекулярних мішеней, що лежать в основі коагулопатії, пов’язаної з інсультом, допоможе визначити ризик інсульту і моніторувати ефективність терапії при рак-асоційованому інсульті. Огляд літератури Механізми ракової коагулопатії та їх роль у розвитку рак-асоційованого інсульту Пацієнти з прихованим раком можуть мати характерні симптоми та нейровізуалізаційний паттерн інфаркту мозку, що вказують на інсульт, пов’язаний з раком, і демонструвати підвищені рівні D-димеру. Таким чином, у пацієнтів, які мали характерні картини інфаркту, підвищений рівень D-димеру та відсутність очевидної етіології та патогенетичного підтипу інсульту, слід розглянути можливість скринінгу на приховане зло- якісне новоутворення (за допомогою серологічних або радіологічних досліджень). Торакоабдомінально- тазову комп’ютерну томографію або позитронну емісійну томографію слід проводити у першу чергу, оскільки рак легенів, підшлункової залози, сечостате- вих шляхів (простати, яєчників або нирок), шлунково- кишкового тракту (колоректальний і шлунок) або молоч- ної залози, є найбільш частими типами (у порядку частоти) як відомих, так і прихованих ракових захво- рювань у пацієнтів з інсультом, пов’язаним з раком. Лабораторними предикторами злоякісного новоутво- рення у пацієнтів з криптогенним інсультом також можуть бути підвищений рівень фібриногену та С-реак- тивного протеїну [19, 20]. i g The stroke risk can also increase due to activation of normal stroke mechanisms, primarily atherosclerosis. Cardiovascular diseases primarily result from athero- sclerosis complications, which is defined as a chronic and progressive inflammatory condition caused by uncontrolled proliferation of vascular smooth muscle cells, endothelial cells, and macrophages in situ associated with the immune response. The progression of the disease at different stages usually ends with a thrombotic process that can lead to myocardial infarction or stroke. There are lots of similarities with the progression of neoplastic processes in cancer and atherogenesis. Deregulation of cell proliferation and, therefore, cell cycle progression, changes in the synthesis of important transcription factors as well as adhesion molecules, changes in the control of angiogenesis, and molecular similarities that occur after chronic inflammation are just some of the processes that become a part of the phenomena that atherosclerosis and cancer are closely related. Thus, de- spite the fact that these two pathologies were previously considered to be unrelated, a careful analysis of the molecular manifestations of both diseases has revealed important similarities, suggesting that they are closely related. One of the most important peculiarities of both р у [ ] Ризик інсульту може збільшитися також внаслідок активації звичайних механізмів інсульту, насамперед атеросклерозу. Серцево-судинні захворювання є, передусім, результатом ускладнень атеросклерозу, який визначається як хронічний і прогресуючий запаль- ний стан, спричинений неконтрольованою проліфе- рацією гладком’язових клітин судин, ендотеліальних клітин і макрофагів insitu, пов’язаних з імунною відповіддю. Механізми ракової коагулопатії та їх роль у розвитку рак-асоційованого інсульту Прогресування захворювання на різних стадіях зазвичай закінчується тромботичним проце- сом, який може призвести до інфаркту міокарда або Literature review 56 Literature review Огляд літератури Огляд літератури ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 diseases is uncontrolled cell proliferation, which contribu- tes to the appearance and severity of lesions at the late stages of both diseases. Deregulation of cell prolife- ration caused by a state of oxidative stress facilitates in many cases the development of atherosclerotic plaque, as well as various types of cancer.l інсульту. Відомо багато подібностей з прогресуванням неопластичних процесів при раку та атерогенезі. Дерегуляція проліферації клітин і, отже, прогресування клітинного циклу, зміни в синтезі важливих факторів транскрипції, а також молекул адгезії, зміна в контролі ангіогенезу та молекулярні подібності, які виникають після хронічного запалення, – це лише деякі з процесів, що стають частиною явищ, які тісно пов’язують атеросклероз і рак. Таким чином, незважаючи на те, що раніше ці дві патології вважалися непов’язаними, завдяки ретельному аналізу молекулярних проявів обох захворювань було виявлено важливу подібність, що свідчить про їх тісний зв’язок. Однією з найваж- ливіших характеристик обох захворювань є неконтро- льована проліферація клітин, що сприяє виникненню та тяжкості уражень на пізніх стадіях обох захво- рювань. Дерегуляція проліферації клітин, спричинена станом окисного стресу, у багатьох випадках сприяє розвитку атеросклеротичної бляшки, а також різних видів раку. Запальний процес під час атерогенезу опосередковується міграцією моноцитів до стінки судини, що є ключовою подією у зростанні атеро- склеротичного ураження. Завдяки диференціації моноцити утверджуються як макрофаги та, зрештою, як збагачені ліпідами пінисті клітини. Макрофаги, отримані з моноцитів, розпізнають та інтерналізують окиснені ліпопротеїни через рецептори-переймачі, де збагачені ліпідами пінисті клітини сприяють роз- витку некротичного ядра, ключового елемента враз- ливої атеросклеротичної бляшки. На молекулярному рівні присутність кристалів холестерину також активує запалення, вивільняючи цитокіни IL-1β, які вважаються важливими медіаторами запалення [21, 22]. З іншого боку, моноцитарні макрофаги, що часто зустрічаються як клітини-господарі в пухлинах, функціонують як ком- поненти запальної відповіді, що створює підтримуючу строму, яка бере участь у зростанні пухлини. Однак багато хворих на рак і серцево-судинні захворювання мають загальний субстрат, який пов’язує ці два захво- рювання. Наприклад, у хворих на рак часто виявляють метаболічні та судинні розлади, серед яких абдомі- нальне ожиріння, змінений метаболізм глюкози, аномалії ліпопротеїдів і гіпертонія [23, 24]. Механізми ракової коагулопатії та їх роль у розвитку рак-асоційованого інсульту yp The inflammatory process during atherogenesis is mediated by monocyte migration to the vessel wall, which is a key event in the growth of atherosclerotic lesions. Due to differentiation, monocytes become established as macrophages and eventually as lipid-enriched foam cells. Monocyte-derived macrophages recognize and internalize oxidized lipoproteins via receptors that receive signals, where lipid-enriched foam cells promote the development of the necrotic core, a key element of a vulnerable atherosclerotic plaque. At the molecular level, cholesterol crystals also activate inflammation by releasing cytokines IL-1β, which are considered to be important inflammation mediators [21, 22]. On the other hand, monocytic macrophages, often found as host cells in tumors, function as inflammatory response сomponents creating a supportive stroma that participates in tumor growth. However, a lot of cancer and cardiovascular disease patients share a common substrate that links these two diseases. For example, cancer patients often have metabolic and vascular disorders, including abdominal obesity, altered glucose metabolism, lipo- protein abnormalities, and hypertension [23, 24]. Огляд літератури Cancer treatment and stroke risk Пошкод- ження ендотелію зі змінами його проникності, призво- дить до додаткового фіброзу та неоваскуляризації в стінках судин у пацієнтів з наявними раніше атеро- склеротичними бляшками, що ще більше сприяє нестабільності бляшок. Пацієнти з наявною ішемічною хворобою серця особливо вразливі. (наприклад, IL-6 та IL-8), міжклітинних молекул адгезії (наприклад, ICAM-1), фактора росту фібробластів і стимулює реакцію нейтрофілів із вторинним вивіль- ненням цитокінів. Крім того, радіація може індукувати апоптоз через p53 відповідь на пошкодження ДНК або апоптоз, активований мітохондріями. Пошкод- ження ендотелію зі змінами його проникності, призво- дить до додаткового фіброзу та неоваскуляризації в стінках судин у пацієнтів з наявними раніше атеро- склеротичними бляшками, що ще більше сприяє нестабільності бляшок. Пацієнти з наявною ішемічною хворобою серця особливо вразливі. a neutrophil response with secondary cytokine release. Besides, radiation can induce apoptosis through the p53 DNA damage response or mitochondria-activated apoptosis. Damage to the endothelium with changes in its permeability leads to additional fibrosis and neovas- cularization in the walls of vessels in patients with previously existing atherosclerotic plaques, which further contributes to plaque instability. Patients with existing coronary heart disease are particularly vulnerable. Chemotherapy-induced vascular toxicity is associated with the onset and progression of atherosclerosis. Vascular damage associated with chemotherapy is often mani- fested by endothelial dysfunction with loss of vasodilation effect and inhibition of anti-inflammatory and vascular reparative function. Platelets are the connecting link between atherosclerosis and cancer. Activated platelets contribute to the severity of inflammation in atherosclerosis by releasing inflammatory mediators. Platelets are able to oxidize low-density lipoproteins, which are the main factor in the formation of atherosclerotic plaques, and also interact with neutrophils and eosinophils and contri- bute to the formation, expansion and thrombosis of atherosclerotic plaques. Platelets attract inflammatory cells to tumors, which promotes tumor growth. In addition, the procoagulant effect of cancer itself increases the activity of platelets and reduces the production of nitric oxide in the endothelium, which increases the formation of atherosclerotic plaque and leads to an increased risk of cardiovascular events [25–27]. Індукована хіміотерапією судинна токсичність пов’язана з початком і прогресуванням атеросклерозу. Пошкодження судин, пов’язані з хіміотерапією, часто проявляються ендотеліальною дисфункцією із втратою ефекту вазодилатації та пригніченням протизапаль- ної та судиннорепаративної функції. Тромбоцити є зв’язуючою ланкою між атеросклерозом і раком. Активовані тромбоцити сприяють запальній тяжкості при атеросклерозі, вивільняючи медіатори запалення. Діагностичні та терапевтичні підходи при рак-асоційованому інсульті The time interval from the diagnosis of cancer to the development of a stroke varies considerably and also depends on cancer type. Usually, in cases of solid tumors, longer periods of time pass in comparison with hemato- logical malignant tumors. If a cancer patient has had stroke, the clinician must determine the exact cause by carefully examining the clinical setting, taking into account the type of cancer and its treatment. Інтервал часу від діагностики раку до розвитку інсульту значно відрізняється і залежить також від типу раку. Зазвичай при солідних пухлинах проходять більш тривалі періоди часу, ніж при гематологічних злоякісних пухлинах. Якщо у хворого на рак стався інсульт, клініцист повинен точно визначити причину, уважно вивчивши клінічну обстановку, враховуючи тип раку та його лікування. У разі криптогенного інсульту у хворих на рак важливе значення можуть мати лабораторні показ- ники, важливішими з яких є високий рівень D-димеру, низький рівень тромбоцитів, порушені тести коагуля- ційної функції, високий рівень фібриногену та збіль- шення часткового тромбопластинового часу [28]. In the case of cryptogenic stroke in cancer patients, laboratory indicators can be important, the most important of which are a high level of D-dimer, a low level of platelets, impaired coagulation function tests, a high level of fibrino- gen, and an increase in partial thromboplastin time [28]. g p p [ ] Recanalization therapy, such as thrombolysis with recombinant tissue plasminogen activator (rtPA) and mechanical thrombectomy, are methods with proven efficacy in treatment of ischemic stroke, however their efficacy and safety in cancer patients remain controversial. Still there is a growing number of research showing that acute stroke in cancer patients can be treated with rtPA, and active cancer should not be considered as an absolute contraindication for rtPA use. In a number of studies, it has been shown that intravenous thrombo- lysis is not associated with a higher risk of bleeding in cancer patients, and the neurological condition of such patients improves faster [29, 30]. Current guidelines for the treatment of acute stroke indicate that the use of recanalization approaches, such as thrombolysis and endovascular therapy, are not contraindicated to cancer patients, but the response to recanalization р у [ ] Реканалізаційна терапія, така як тромболізис реком- бінантним тканинним активатором плазміногену (rTPA) та механічна тромбектомія, є методами з доведеною ефективністю в лікуванні ішемічного інсульту, але їх ефективність та безпека хворих на рак залишаються суперечливими. Cancer treatment and stroke risk Тромбоцити здатні окиснювати ліпопротеїни низької щільності, які є головним фактором утворення атеро- склеротичних бляшок, а також взаємодіють з нейтро- філами і еозинофілами та сприяють утворенню, роз- ширенню та тромбозу атеросклеротичних бляшок. Тромбоцити залучають запальні клітини до пухлин, що сприяє зростанню пухлини. Крім того, прокоагулянт- ний ефект раку сам по собі підвищує активність тромбоцитів і знижує вироблення оксиду азоту в ендо- телії, що посилює формування атеросклеротичної бляшки та призводить до підвищення ризику серцево- судинних подій [25–27]. Cancer treatment and stroke risk Cancer treatment and stroke risk Although cancer can cause atherosclerosis through various mechanisms, the most common are the effects of anticancer drugs and radiation therapy. Cancer chemo- therapy and radiation therapy are associated with an increased risk of developing atherosclerosis and cardiovascular disease.f Хоча рак може викликати атеросклероз через різні механізми, найбільш поширеними є наслідки використання протипухлинних препаратів і променевої терапії. Хіміотерапія і променева терапія раку асоціюються з підвищенням ризику розвитку атеро- склерозу та серцево-судинних захворювань. Ionizing radiation affects not only cancerous, but also non-cancerous cells, especially those that proli- ferate rapidly, such as endothelial and bone marrow cells, as well as local parenchymal cells within the irradiated area. Cell cycle arrest, senescence, and apoptosis are induced as a consequence of DNA damage. At high doses, ionizing radiation can lead to depletion of paren- chymal and vascular endothelial cells with both macro- and microvascular effects. Radiotherapy has a direct effect on cells in the pathogenesis of vascular damage involving endothelial cells, which is an early sign of radiation-induced vascular damage. This causes endo- thelial damage with increased expression of interleukins (eg, IL-6 and IL-8), intercellular adhesion molecules (eg, ICAM-1), fibroblast growth factor, and stimulates Іонізуюче випромінювання впливає не лише на ракові, а й на неракові клітини, особливо на ті, що швидко проліферують, наприклад, клітини ендо- телію та кісткового мозку, а також локальні парен- хіматозні клітини в межах опроміненої ділянки. Зупинка клітинного циклу, старіння та апоптоз індукуються як наслідок пошкодження ДНК. У високих дозах іонізуюче випромінювання може призвести до висна- ження паренхіматозних і судинних ендотеліальних клітин як з макро-, так і мікросудинними ефектами. Радіотерапія надає прямий вплив на клітини в пато- генезі пошкодження судин із залученням ендотелі- альних клітин, що є ранньою ознакою радіаційно- індукованого ураження судин. Це викликає пошкод- ження ендотелію з посиленням експресії інтерлейкінів Literature review 57 Огляд літератури Literature review ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 (наприклад, IL-6 та IL-8), міжклітинних молекул адгезії (наприклад, ICAM-1), фактора росту фібробластів і стимулює реакцію нейтрофілів із вторинним вивіль- ненням цитокінів. Крім того, радіація може індукувати апоптоз через p53 відповідь на пошкодження ДНК або апоптоз, активований мітохондріями. Огляд літератури Діагностичні та терапевтичні підходи при рак-асоційованому інсульті З’являється все більше досліджень, які доводять, що гострий інсульт у хворих на рак можна лікувати rTPA, і активний рак не слід розглядати як абсолютне протипоказання для використання rTPA. В низці досліджень було продемонстровано, що внутрішньовенний тромболізис не пов’язаний з вищим ризиком кровотечі у хворих на рак, а неврологічний стан таких пацієнтів покращується швидше [29, 30]. Сучасні рекомендації щодо терапії гострого інсульту свідчать, що застосування реканалізаційних підходів, таких як тромболізис та ендоваскулярна терапія, Literature review Literature review Огляд літератури Огляд літератури 58 ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 не протипоказані для пацієнтів з раком, однак відпо- відь на реканалізаційну терапію може відрізнятися у хворих на рак, та у хворих без раку [31]. У систем- ному огляді та мета-аналізі також було підтверджено, що системний рак не є абсолютним протипоказанням для внутрішньовенного тромболітичного лікування, а ефективність та безпека не відрізняються порівняно з пацієнтами без раку [32]. Ефективність механічної тромбектомії при рак-асоційованому інсульті зали- шається не досить відомою. Однак дослідження продемонструвало, що механічна тромбектомія є придатним методом його лікування, однак підвищен- ня D-димеру і грубий неврологічний дефіцит можуть бути предикторами несприятливого виходу [33]. therapy may differ between cancer patients and non- cancer patients [31]. A systematic review and meta- analysis also confirmed that systemic cancer is not an absolute contraindication to intravenous thrombolytic treatment, and efficacy and safety are not different compared to non-cancer patients [32]. The effectiveness of mechanical thrombectomy in cancer-associated stroke remains poorly known. However, the study has demonstrated that mechanical thrombectomy is a suitable method of its treatment, however the elevated D-dimer and gross neurological deficit may be predictors of adverse outcome [33]. In addition to revascularization therapy, low molecular weight heparins are usually used to treat ischemic stroke in cancer patients. When comparing low-molecular- weight heparins with new oral anticoagulants, similar results were obtained in terms of clinical outcome, mortality, and the risk of hemorrhagic complications [34]. Крім реваскуляризаційної терапії для лікування ішемічного інсульту у хворих на рак зазвичай застосо- вуються низькомолекулярні гепарини. При порівнянні низькомолекулярних гепаринів з новими перораль- ними антикоагулянтами отримані схожі результати відносно клінічного виходу, смертності та ризику геморагічних ускладнень [34]. REFERENCES 1. Global, regional, and national burden of diseases and injuries for adults 70 years and older: systematic analysis for the Global Burden of Disease 2019 Study. British medical journal. 2022;376. DOI: https://doi.org/10.1136/bmj-2021-068208 1. Global, regional, and national burden of diseases and injuries for adults 70 years and older: systematic analysis for the Global Burden of Disease 2019 Study. British medical journal. 2022;376. (In English). DOI: https://doi.org/10.1136/bmj-2021-068208 p g j 2. Kuan A.S., Teng C.J., Wu H.H., Su V.Y., Chen Y.T. et al. Risk of ischemic stroke in patients with ovarian cancer: A nation wide population based study. BMC medicine. 2014. Vol. 12. 53 р. DOI: https://doi.org/10.1186/1741-7015-12-53 2. Kuan A.S., Teng C.J., Wu H.H., Su V.Y., Chen Y.T. et al. Risk of ischemic stroke in patients with ovarian cancer: A nation wide population based study. BMC medicine. 2014. Vol. 12. 53 р. DOI: https://doi.org/10.1186/1741-7015-12-53 2. Kuan AS, Teng CJ, Wu HH, Su VY, Chen YT et al. Risk of ischemic stroke in patients with ovarian cancer: A nationwide population-based study. BMC medicine. 2014;12:53. (In English). DOI: https://doi.org/ 10.1186/1741-7015-12-53 p g 3. Navi B.B., Reiner A.S., Kamel H., Iadecola C., Elkind M.S., Panageas K.S., DeAngelis L.M. Association between incident cancer and subsequent stroke. Annals of neurology. 2015. Vol. 77(2). Р. 291–300. DOI: https://doi.org/10.1002/ana.24325 3. Navi BB, Reiner AS, Kamel H, Iadecola C, Elkind MS, Panageas KS, DeAngelis LM. Association between incident cancer and subsequent stroke. Annals of neurology. 2015;77(2):291–300. (In English). DOI: https://doi.org/10.1002/ana.24325 4. Zöller B, Sundquist J, Sundquist K. 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Blood. 2015;126(4):494–9. (In English). DOI: https://doi.org/10.1182/blood- 2015-02-626788 6. p g 6. CONCLUSIONS Поєднання раку і мозкового інсульту є актуальною проблемою клінічної медицини в усьому світі. Рак призводить до збільшення ризику інсульту внаслідок специфічних патофізіологічних механізмів. Ці меха- нізми є множинними і складними, однак найбільш відомим та поширеним є ракова внутрішньосудинна коагулопатія, яка має специфічні лабораторні біо- маркери. Численні дослідження продовжують спрямо- вуватись на вивчення факторів, пов’язаних з коагуло- патією у пацієнтів, які перенесли інсульт, особливості раку та оцінки біологічних маркерів внутрішньосу- динної коагулопатії, що викликає інсульт, а також моніторинг ефектів антикоагулянтної терапії у пацієнтів із рак-асоційованим інсультом. The combination of cancer and cerebral stroke is an urgent problem of clinical medicine all over the world. Cancer leads to an increased risk of stroke due to specific pathophysiological mechanisms. These mechanisms are multiple and complex, but the most well-known and widespread is cancer intravascular coagulopathy, which has specific laboratory biomarkers. Numerous studies continue investigating the factors associated with coagulo- pathy in stroke patients, cancer characteristics, and evaluation of biological markers of stroke-causing intravascular coagulopathy, as well as monitoring the effects of anticoagulation therapy in patients with cancer-associated stroke. REFERENCES Schwarzbach C.J., Schaefer A., Ebert A., Held V., Bolognese M., Kablau M., Hennerici M.G. Stroke and cancer: The importance of cancer-associated hypercoagulation as a possible stroke etiology. Stroke. 2012. Vol. 43(11). Р. 3029–3034. DOI: https://doi.org/10.1161/ STROKEAHA.112.658625 12. Schwarzbach CJ, Schaefer A, Ebert A, Held V, Bolognese M, Kablau M, Hennerici MG. 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Guideline for Healthcare Professionals from the American Heart Association/ American Stroke Association. Stroke. 2018;49(3):e46–234. (In English). DOI: https://doi.org/10.1161/STR.0000000000000158fi 32. Huang S., Lu X., Tang L. V., Hu Y. Efficacy and safety of intravenous thrombolysis for acute ischemic stroke in cancer patients: a systemic review and meta-analysis. American Journal of Translational Research. 2020. Vol. 12(8). Р. 4795–4806. 32. Huang S, Lu X, Tang LV, Hu Y. Efficacy and safety of intravenous thrombolysis for acute ischemic stroke in cancer patients: a systemic review and meta-analysis. American Journal of Translational Research. 2020;12(8):4795–806. (In English).f 33. ISSN 2708-7166 (Print) ISSN 2708-7174 (Online) Lee E-J, Bae J, Jeong H-B, Lee EJ, Jeong HY, Yoon B-W. Effectiveness of mechanical thrombectomy in cancer-related stroke and associated factors with unfavorable outcome. BMC Neurology. 2021;21:57. (In English). 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DOI: https://doi.org/10.1016/ j.jstrokecerebrovasdis.2017.07.029 Конфлікт інтересів Автор заявляє про відсутність конфлікту інтересів та власної фінансової зацікавленості в підготуванні даної статті. The author states no conflict of interest or own financial incentive associated with publishing the paper. Перспективи подальших досліджень Further studying the multiple mechanisms that associate cancer and stroke will make it possible to intentionally develop the strategies of the treatment and prevention of cancer- associated stroke. Подальше вивчення множинних механізмів, які поєднують рак та мозковой інсульт дозволить цілеспрямовано розроб- ляти підходи до лікування та профілактики рак-асоційова- ного інсульту. Conflict of interest REFERENCES Darby SC, Ewertz M, McGale P et al. Risk of ischemic heart disease in women after radiotherapy for breast cancer. New England Journal of Medicine. 2013;368:987–98. (In English). DOI: https://doi.org/10.1056/ NEJMoa1209825 24. 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DOI: https://doi.org/10.1097/HCO.0000000000000404f 26. Lysov Z, Dwivedi DJ, Gould TJ, Liaw PC. Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA. Blood Coagul Fibrinolysis. 2017;28(1):72–82. (In English). DOI: https://doi.org/10.1097/MBC.0000000000000546 26. Lysov Z., Dwivedi D.J., Gould T.J., Liaw P.C. Procoagulant effects of lung cancer chemotherapy: impact on microparticles and cell-free DNA. Blood Coagul Fibrinolysis. 2017. Vol. 28(1). Р. 72–82. DOI: https://doi.org/10.1097/MBC.0000000000000546 27. Nardi E., Prastaro M., Santoro C. Vascular Damage Induced by Chemotherapy and Radio therapy in Oncological Patients. American Journal of Biomedical Science & Research. 2022. Vol. 15(5). DOI: https://doi.org/10.34297/AJBSR.2022.15.002146 27. Nardi E, Prastaro M, Santoro C. Vascular Damage Induced by Chemotherapy and Radiotherapy in Oncological Patients. American Journal of Biomedical Science & Research. 2022;15(5). (In English). DOI: https://doi.org/10.34297/AJBSR.2022.15.002146 Literature review 60 Огляд літератури Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 Український радіологічний та онкологічний журнал. 2022. Т. 30. № 4. С. 52–61 Ukrainian journal of radiology and oncology. 2022;30(4):52–61 INFORMATION ABOUT AUTHORS Dubenko Olha Yevheniivna – Doctor of Medical Sciences, Professor, Senior Scientist of Radiation Pathology and Palliative Care Department of State Organization «Grigoriev Institute for Medical Radiology and Oncology of the National Academy of Medical Sciences of Ukraine»; Professor of Neurology Department of Kharkiv Medical Academy of Postgraduate Education; 58, Amosov Str., Kharkiv, Ukraine, 61176; Дубенко Ольга Євгеніївна – доктор медичних наук, професор, старший науковий співробітник відділення проме- невої патології та паліативної медицини Державної установи «Інститут медичної радіології та онкології ім. С.П. Григор’єва Національної академії медичних наук України»; професор кафедри неврології Харківської медичної академії після- дипломної освіти, вул. Амосова, буд. 58, Харків , Україна, 61176 e-mail: olgadubenko05@gmail.com e-mail: olgadubenko05@gmail.com g @g Phone: +38 (050) 660-14-42 Внесок автора: інформаційний пошук та аналіз наукових публікацій за темою статті, написання тексту статті. Author contributions: information search and analysis of scientific publications on the subject of the article, writing the article. Прийнято до друку Accepted for printing 08.12.2022 Опубліковано Published 10.12.2022 Прийнято до друку Accepted for printing 08.12.2022 Опубліковано Published 10.12.2022 Отримано після рецензування Received after review 27.10.2022 Отримано після рецензування Received after review 27.10.2022 61 Literature review Огляд літератури
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https://www.frontiersin.org/articles/10.3389/fnut.2023.1103303/pdf
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Investigating the causal association between branched-chain amino acids and Alzheimer's disease: A bidirectional Mendelian randomized study
Frontiers in nutrition
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TYPE Original Research PUBLISHED 31 March 2023 DOI 10.3389/fnut.2023.1103303 TYPE Original Research PUBLISHED 31 March 2023 DOI 10.3389/fnut.2023.1103303 TYPE Original Research PUBLISHED 31 March 2023 DOI 10.3389/fnut.2023.1103303 OPEN ACCESS EDITED BY Lais Bhering Martins, University of Texas Health Science Center at Houston, United States REVIEWED BY Wen Liu, Capital Medical University, China Baruh Polis, Yale University, United States Andrew C. Shin, Texas Tech University, United States *CORRESPONDENCE Gang-yu Ding jonathandin@126.com Hui-dong Tang thd10495@rjh.com.cn † Xiao-hang Qian1,2,3†, Xiao-li Liu4†, Bin Zhang4, Yuan Lin5, Jian-hua Xu6, Gang-yu Ding6* and Hui-dong Tang1,2,3* 1Department of Geriatrics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 2Medical Center on Aging of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 3Department of Neurology and Institute of Neurology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 4Department of Neurology, Shanghai University of Medicine and Health Sciences Afliated Sixth People’s Hospital South Campus, Shanghai, China, 5Department of Gastroenterology, Jiading District Central Hospital Afliated Shanghai University of Medicine and Health Sciences, Shanghai, China, 6Department of Neurology, Jiading District Central Hospital Afliated Shanghai University of Medicine and Health Sciences, Shanghai, China †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Nutrition, Psychology and Brain Health, a section of the journal Frontiers in Nutrition RECEIVED 20 November 2022 ACCEPTED 06 March 2023 PUBLISHED 31 March 2023 CITATION Qian X-h, Liu X-l, Zhang B, Lin Y, Xu J-h, Ding G-y and Tang H-d (2023) Investigating the causal association between branched-chain amino acids and Alzheimer’s disease: A bidirectional Mendelian randomized study. Front. Nutr. 10:1103303. doi: 10.3389/fnut.2023.1103303 Background: There are many metabolic pathway abnormalities in Alzheimer’s disease (AD). Several studies have linked branched-chain amino acid (BCAA) metabolism disorders with AD but have not obtained consistent results. The purpose of this study is to explore the causal association between BCAA concentration and the risk of AD. Methods: A bidirectional Mendelian randomized (MR) study was applied to explore the causal efect between BCAA level and the risk of AD. Genetic instrumental variables from the genome-wide association study (GWAS) of serum BCAA levels [total BCAAs (115,047 participants), valine (115,048 participants), leucine (115,074 participants), and isoleucine (115,075 participants)] from the UK Biobank and AD (21,982 AD cases and 41,944 controls) from the International Genomics of Alzheimer’s Project were applied to explore the causal efect through the inverse variance-weighted (IVW) method, MR-Egger, and weighted median, accompanied by multiple pluripotency and heterogeneity tests. © 2023 Qian, Liu, Zhang, Lin, Xu, Ding and Tang. Alzheimer’s disease, branched-chain amino acids, valine, leucine, isoleucine, Mendelian randomized study Frontiers in Nutrition KEYWORDS Data sources and selection of genetic instruments Branched-chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), are three types of essential amino acids in the human diet (9). They are not only involved in protein synthesis but also possess various metabolic pathways (10, 11). Altered BCAA metabolism has been shown to be associated with AD in an increasing number of studies (12). However, different studies showed controversial and mixed results. For instance, a previous study of the cerebrospinal fluid (CSF) and plasma amino acid composition has demonstrated a significant reduction of valine in AD patients compared to healthy controls (HC) (13). In a study of sporadic AD patients without receiving any medication, alterations of 23 metabolites were detected, including significantly decreased valine levels (14). In addition, a longitudinal study in APP/PS1 transgenic mice involving profiling of the brain and the plasma metabolome has found seriously disturbed polyamines and BCAA metabolism (15). Notably, the plasma levels of valine were shown to be significantly reduced in AD mice. In a more recent study, lower plasma valine level was shown to correlate with the rate of cognitive decline (16). Nonetheless, there were elevated BCAA concentrations in the serum of AD patients in a small sample study (17). Similarly, elevated isoleucine levels have been observed in patients with mild cognitive impairment (MCI) (18). These heterogeneous results may be related to the susceptibility of metabolites influenced by multiple factors, such as lifestyle and diet, immune response, genetic variations, and gut microbiota (19). These uncontrollable confounding factors make it difficult to distinguish symptoms from causes. Therefore, the use of more reliable research methods will help to elucidate the causal association between BCAA levels and the risk of AD. The genetic summary statistics of AD (21,982 AD cases and 41,944 controls) were accessed from the International Genomics of Alzheimer’s Project (IGAP), which was composed of four consortia: Cohorts for Heart and Aging Research in Genomic Epidemiology Consortium (CHARGE), the European Alzheimer’s Disease Initiative (EADI), Alzheimer’s Disease Genetics Consortium (ADGC), and Genetic and Environmental Risk in AD/Defining Genetic, Polygenic and Environmental Risk for Alzheimer’s Disease Consortium (GERAD/PERADES) (22). Study design overview Alzheimer’s disease (AD), the main type of dementia, becomes one of the most serious public health threats in the world (1). It is estimated that there are currently more than 50 million people worldwide with dementia, which is expected to triple in 2050 (2). Among them, AD accounts for 60–80% of dementia (3). Typical pathological features of AD include extracellular amyloid-β (Aβ) plaques accumulated by Aβ and intracellular neurofibrillary tangles formed by phosphorylated tau (4). However, there is still a lack of accurate explanation of the pathogenesis and effective disease-modifying treatment for AD (5). Increased evidence suggests that the disruption of various metabolic pathways is another important feature of AD (6, 7). Metabolites are the end biochemical products of various biological pathways, such as amino acids, peptides, lipids, and nucleic acids (8). They can reflect the alteration in the complex biological pathways involved in AD caused by the interaction of genetic, environmental, and behavioral factors (6). We performed a bidirectional Mendelian randomized study to assess the causal effect between BCAA levels and the risk of AD (Figure 1). The bidirectional Mendelian randomization study was built on the following three assumptions: First of all, the instrumental variables (IVs) were not related to the confounders. Second, there was a strong correlation between IVs and exposure. Third, IVs can affect outcomes (AD) only through exposure and not through other pathways. The analysis of this study was based on genome-wide association study (GWAS) data of BCAAs and Alzheimer’s disease in the public database. Therefore, ethics approval and consent are not required for this study. Frontiers in Nutrition OPEN ACCESS This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Results: The forward MR analysis showed that there was no causal efect of total BCAAs (OR: 1.067, 95% CI: 0.838–1.358; p = 0.838), valine (OR: 1.106, 95% CI: 0.917–1.333; p = 0.292), leucine (OR: 1.096, 95% CI: 0.861–1.396; p = 0.659), and isoleucine (OR: 1.457, 95% CI: 1.024–2.742; p = 0.037) levels on the risk of AD. The reverse analysis showed that AD was related to reduced levels of total BCAAs (OR: 0.979, 95% CI: 0.989–0.990; p < 0.001), valine (OR: 0.977, 95% CI: 0.963–0.991; p = 0.001), leucine (OR: 0.983, 95% CI: 0.973–0.994; p = 0.002), and isoleucine (OR: 0.982, 95% CI: 0.971–0.992; p = 0.001). Conclusion: We provide robust evidence that AD was associated with a decreased level of BCAAs, which can serve as a marker for early diagnosis of AD. Alzheimer’s disease, branched-chain amino acids, valine, leucine, isoleucine, Mendelian randomized study 01 Frontiers in Nutrition frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 frontiersin.org Mendelian randomized analysis method. In addition, MR-Egger and weighted median were applied to verify the results of the IVW method. After removing SNPs, the results showed that there were no casual effects on the levels of total BCAAs (IVW, OR: 1.067, 95% CI: 0.838–1.358; p = 0.838), valine (IVW, OR: 1.106, 95% CI: 0.917–1.333; p = 0.292), leucine (IVW, OR: 1.096, 95% CI: 0.861–1.396; p = 0.659), and isoleucine (IVW, OR: 1.457, 95% CI: 1.024–2.742; p = 0.037) on the risk of AD (Figures 2A–D, Table 1). Similarly, the results for MR-Egger and weighted median do not suggest an effect of BCAA levels on the risk of AD. Based on these results, we found no causal effect of BCAA levels on the risk of AD. Two-sample MR was applied to explore the causal effect between BCAA levels and the risk of AD. The inverse variance- weighted (IVW) method was applied to the standard MR analysis. In addition, the MR-Egger and weighted median were also performed to verify the causal effect (21, 26). Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) global test was conducted to identify the horizontal pleiotropic of IVs (26). Cochran’s Q statistic was applied to assess the heterogeneity of IVW and MR-Egger (27). The MR-Egger intercept test was also used to estimate the potential horizontal pleiotropy of the MR results. In addition, the leave-one-out analysis was also used to eliminate potential pleiotropy by assessing the effects of a single IV on causal effect by removing each IV from the IVW method (28). All analyses in this study were performed through the “two-sample MR” and the “MR-PRESSO” packages in R software (v3.60). As four types of exposures were analyzed, the statistically significant threshold was set as P < 0.0125 after the Bonferroni correction. Data sources and selection of genetic instruments For BCAAs (total BCAAs, valine, leucine, and isoleucine), the GWAS statistics were obtained from the MRC IEU OpenGWAS project (https://gwas.mrcieu.ac.uk/), including 115,047 participants for total BCAAs (GWAS ID “met-d- Total_BCAA”), 115,048 participants for valine (GWAS ID “met-d-Val”), 115,074 participants for leucine (GWAS ID “met- d-Leu”), and 115,075 participants for isoleucine (GWAS ID “met-d-Ile”) from the UK Biobank. All participants were of European ancestry. Instrumental variables (IVs) for AD and BCAAs were extracted under the same criteria. To be specific, the GWAS statistical difference threshold of all relevant SNPs from each GWAS was set to P < 5 × 10−8. The PLINK clumping algorithm was applied to prune for the independence of SNPs in linkage disequilibrium under the threshold of r2 < 0.001 in a 10,000 kb window (23). The palindromic SNPs with a minor allele frequency (MAF) of <0.01 were excluded from the aforementioned instrument SNPs. The PhenoScanner GWAS database (http://phenoscanner.medschl. cam.ac.uk) was applied to remove possible confounding of the exposure–outcome associations (24). In this study, gender, age, diabetes, and cardiovascular disease were selected as confounders. The SNPs associated with them were excluded. Furthermore, the F statistic was calculated to assess the strength of the selected genetic variants (25). The SNPs used as IVs in this study are presented in Supplementary Table S1. The F statistic is presented in Supplementary Table S2. Mendelian randomization (MR) study is a methodology that can be applied to explore the causal relationships between exposures (risk factors) and outcomes (diseases) by using genetic variants (20). The genetic variants are only related to the risk factors but are not affected by any confounders (21). In this study, a bidirectional Mendelian randomized approach was applied to explore the causal effects between BCAA levels and the risk of AD. 02 frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 FIGURE 1 Summary of this bidirectional MR study between the level of BCAAs and the risk of AD in this study. SNP, single-nucleotide polymorphism; BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. FIGURE 1 Summary of this bidirectional MR study between the level of BCAAs and the risk of AD in this study. SNP, single-nucleotide polymorphism; BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. FIGURE 1 Summary of this bidirectional MR study between the level of BCAAs and the risk of AD in this study. SNP, single-nucleotide polymorphism; BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Genetically predicted AD on the BCAA levels In the reverse MR analysis, we explored the causal effect of AD on the BCAA levels through IVW, MR-Egger, and weighted median. Our investigation revealed that AD was related to a decreased level of total BCAAs (IVW, OR: 0.979, 95% CI: 0.989– 0.990, p < 0.001), valine (IVW, OR: 0.977, 95% CI: 0.963– 0.991; p = 0.001), leucine (IVW, OR: 0.983, 95% CI: 0.973- −0.994, p = 0.002), and isoleucine (IVW, OR: 0.982, 95% CI: 0.971–0.992, p = 0.001) (Figures 2E–H, Table 2). Similarly, the results of MR-Egger and weighted median were consistent with the results of the IVW method. Taken together, these results revealed that AD was obviously associated with decreased BCAA levels. Frontiers in Nutrition frontiersin.org Sensitivity analyses to the risk of AD. Conversely, the reverse MR analysis suggests that AD is significantly associated with decreased BCAA levels, suggesting that AD is the cause but not the result of changed BCAA levels. There were no obvious heterogeneities in the causal effect between AD and BCAAs (Tables 1, 2). The intercept of the MR-Egger regression and MR-PRESSO test revealed no obvious horizontal pleiotropy (Tables 1, 2). In addition, the included instrumental variables show apparent symmetry in the funnel plot to exclude the directional pleiotropy (Supplementary Figure S1). Meanwhile, the leave-one-out analysis showed all SNPs contributing to consistent causal estimates (Supplementary Figure S2). The aforementioned analysis proves the reliability of the results of the study. Several previous observational cohort studies have linked BCAAs to AD. An earlier study found that there was reduced levels of leucine and valine in the CSF of patients with AD, but no statistically significant differences in plasma (13). In Alzheimer’s Disease Neuroimaging Initiative-1 (ADNI-1) cohort, there was a decreased serum valine level in AD patients compared to HC and stable mild cognitive impairment (sMCI) patients (32). Similarly, serum valine levels were lower in patients with progressive MCI (pMCI) than in HC (32). More importantly, serum valine levels in pMCI patients were negatively correlated with Tau and pTau levels in CSF and could be used to predict the risk of progression from MCI to AD (32). In a prospective study of eight cohorts, BCAAs were found to be negatively associated with dementia and the risk of AD (31). However, in another observational study, serum isoleucine levels were elevated in dementia, compared with HC (33). Similarly, elevated blood levels of BCAAs in patients with AD were reported in a small sample study cohort (17). In APP/PS1 mice model, there was an increased plasma BCAA level compared with wide-type mice (34). After high-fat feeding, the plasma BCAA level was significantly upregulated in the WT group, which was not observed in the APP/PS1 group (34). A high saturated fat/glycemic index diet significantly increased CSF BCAA levels in MCI patients but had no significant effect on the HC group (35). Combined with the results of this study, we speculate Genetically predicted BCAA levels on the risk of AD The causal effect of BCAA levels on the risk of AD was conducted through the inverse variance-weighted (IVW) MR 03 frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 FIGURE 2 Scatterplot of the genetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Scatterplot of the genetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucin on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; A enetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine otal BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Scatterplot of the genetic causal efect sbetween BCAAs level the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. Frontiers in Nutrition frontiersin.org P ’s dise al ef P M − − M − − − − M − − Discussion Alzheimer’s disease, the most common type of dementia, currently lacks disease-modifying treatment strategies. To make matters worse, the current understanding of the pathogenesis of AD is limited. As a type of essential amino acid, BCAAs have been proven to be closely associated with AD (3, 29). The alteration of BCAA concentration in AD patients is correlated with disease progression and AD-related pathological features (30, 31). However, a causal association between AD and BCAA has not been established. In this study, we attempt to explore the causal effect between AD and BCAAs (total BCAAs, valine, leucine, and isoleucine) using a bidirectional two-sample MR study. The results do not show evidence that BCAA levels are causally related 04 frontiersin.org MR-egger intercept (p value) MR-PRESSO global test (p value) Cochran’s Q (p value) −0.011 (0.474) 0.135 10.312 (0.244) 0.001 (0.900) 0.451 8.131 (0.701) −0.011 (0.482) 0.164 10.311 (0.325) 0.031 (0.435) 0.199 8.888 (0.113) MR-egger intercept (p value) MR-PRESSO global test (p value) Cochran’s Q (p value) 0.001 (0.632) 0.495 18.464 (0.425) 0.002 (0.385) 0.022 31.089 (0.028) −0.0004 (0.816) 0.905 10.835 (0.901) −0.0001 (0.910) 0.707 12.588 (0.815) ue ue 1 MR analysis β value OR 95% CI P value 0.065 1.067 0.838–1.358 0.838 0.177 1.193 0.918–1.550 0.918 0.225 1.252 0.773–2.028 0.772 0.100 1.106 0.917–1.333 0.292 0.119 1.126 0.894–1.419 0.315 0.081 1.085 0.767–1.535 0.656 0.092 1.096 0.861–1.396 0.659 0.216 1.241 0.944–1.634 0.869 0.275 1.316 0.763–2.269 0.352 0.376 1.457 1.024–2.742 0.037 0.361 1.434 1.014–2.029 0.414 −0.150 0.860 0.248–2.986 0.824 sease; OR, odd ratio; CI, confidence interval. efects of AD on the level of BCAAs. MR analysis β value OR 95% CI P value −0.021 0.979 0.989–0.990 <0.001 −0.020 0.980 0.964–0.994 0.008 −0.024 0.976 0.961–0.991 0.007 −0.023 0.977 0.963–0.991 0.001 −0.020 0.980 0.964–0.995 0.011 −0.029 0.971 0.952–0.990 0.009 −0.017 0.983 0.973–0.994 0.002 −0.019 0.982 0.968–0.995 0.010 −0.016 0.984 0.970–0.999 0.050 −0.019 0.982 0.971–0.992 0.001 −0.019 0.981 0.966–0.996 0.013 −0.018 0.982 0.967–0.997 0.031 sease; OR, odd ratio; CI, confidence interval. Qian et al. 10.3389/fnut.2023.1103303 diagnosis of AD. Future studies need to further clarify how AD leads to lower levels of BCAAs. diagnosis of AD. Future studies need to further clarify how AD leads to lower levels of BCAAs. that these inconsistent results occurred in ordinary observational studies due to the limited sample size and the susceptibility of BCAAs to various confounding factors such as diet. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments We sincerely appreciated the data provided by the MRC IEU OpenGWAS project. Funding Branched-chain amino acids (BCAAs) are essential amino acids and account for one-third of the total amino acid content of the human body (30). After being absorbed by the gut, BCAAs are widely distributed in many organs for metabolism, mainly including the muscle, brain, liver, and adipose tissue (37). Among them, the muscle is the main metabolic place of BCAAs, accounting for approximately 50% (30, 38). In addition, the brain also absorbs a proportion of BCAAs in the blood (30). In the mammalian brain, BCAAs are involved in multiple functions, including neurotransmitter synthesis, protein synthesis, and energy production (39). In this study, AD patients are found to have reduced BCAA levels, which may be related to BCAA intake and metabolic processes. Circulating BCAAs are derived from diet, proteolysis, and gut microbiota (40). First, changes in gastrointestinal function have been observed in the AD mice model (41, 42). This may lead to reduced absorption of BCAAs from the diet in AD. Second, AD or MCI patients usually experience subclinical malnutrition (43). This resulted in reduced protein reserve and proteolysis levels in AD. Third, a series of studies in recent years have demonstrated the imbalance of gut microbiota in AD (12, 41). This may lead to changes in the abundance of gut microbiota related to BCAAs metabolism and ultimately reduce the level of circulating BCAAs. However, these are just some possible reasons why AD has reduced BCAA levels. Further studies are needed to clarify the specific mechanism. In addition, supplementing seven amino acids (leucine, phenylalanine, lysine hydrochloride, isoleucine, histidine hydrochloride, valine, and tryptophan) can improve cognitive, psychological, and social functions in middle-aged and older adults (44). Furthermore, supplementing L-norvaline (an isoform of valine) for 2 months can reverse cognitive decline and synaptic loss in triple-transgenic (3 × Tg) mice at the age of 4 months (45). Therefore, the supplementation of BCAAs may be one of the strategies to delay cognitive decline in AD patients. This study was supported by the National Natural Science Foundation of China [81971014], the Hundred Faculty Talent Pool Program at Shanghai University of Medicine & Health Sciences in China, and the Shanghai Municipal Commission of Health and Family Planning [20184Y0056]. Discussion The results of our study explain the causal relationship between AD and BCAAs from the root. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnut.2023. 1103303/full#supplementary-material Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Author contributions H-dT and G-yD designed the study and prepared the manuscript. X-hQ and X-lL developed the methodology and analyzed the data. All authors discussed the results and approved the manuscript. Data availability statement In 2017, Larsson and Markus also reported the results of a study using Mendelian randomization to explore the causal association of BCAAs with the risk of AD (36). The results showed that only higher isoleucine levels were associated with the risk of AD but not leucine and valine (36). Our study differs from other studies in several ways. In this study, the GWAS datasets related to BCAAs are selected with a larger sample size, containing 115,047 participants. From the perspective of methodology, Larsson and Markus (36) only used a single methodology to illustrate the effect of BCAAs on the risk of AD. In our study, IVW, MR-Egger, and weighted median are adopted in the analysis for Mendelian randomization, which provides an important guarantee for the reliability of our research results. The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors. Frontiers in Nutrition SUPPLEMENTARY FIGURE S1 Funnel plot of the causal efect between BCAAs level and the risk of AD. 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Increased susceptibility to metabolic dysregulation in a mouse model of Alzheimer’s disease is associated with impaired hypothalamic insulin signaling and elevated BCAA levels. Alzheimer’s Demen. (2016) 12:851–61. doi: 10.1016/j.jalz.2016.01.008 17. Li H, Ye D, Xie W, Hua F, Yang Y, Wu J, et al. Defect of branched-chain amino acid metabolism promotes the development of Alzheimer’s disease by targeting the mTOR signaling. Biosci Rep. (2018) 38:BSR20180127. doi: 10.1042/BSR201 80127 17. Li H, Ye D, Xie W, Hua F, Yang Y, Wu J, et al. Defect of branched-chain amino acid metabolism promotes the development of Alzheimer’s disease by targeting the mTOR signaling. Biosci Rep. (2018) 38:BSR20180127. doi: 10.1042/BSR201 80127 35. Russin KJ, Nair KS, Montine TJ, Baker LD, Craft S. Diet effects on cerebrospinal fluid amino acids levels in adults with normal cognition and mild cognitive impairment. J Alzheimer’s Dis. (2021) 84:843–53. doi: 10.3233/JAD-210471 18. Wang X, Sun G, Feng T, Zhang J, Huang X, Wang T, et al. SUPPLEMENTARY FIGURE S1 This bidirectional MR study indicates that AD is associated with decreased BCAA levels, which can serve as a marker for early 06 frontiersin.org 10.3389/fnut.2023.1103303 Qian et al. isoleucine. Each instrumental SNP was represented by a black dot. SNPs, single-nucleotide polymorphisms; BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. (H) AD on isoleucine. BCAAs, branched-chain amino acids; AD, Alzheimer’s disease. SUPPLEMENTARY TABLE S1 Characteristics of selected SNPs for analysis of the causal efect between BCAA level and the risk of AD from IGAP. Frontiers in Nutrition SUPPLEMENTARY FIGURE S2 Leave-one-out analysis of the causal efect between BCAAs level and the risk of AD. (A) Total BCAAs on AD, (B) valine on AD, (C) leucine on AD, (D) isoleucine on AD, (E) AD on total BCAAs, (F) AD on valine, (G) AD on leucine, 45. Polis B, Srikanth KD, Elliott E, Gil-Henn H, Samson AO. L-Norvaline reverses cognitive decline and synaptic loss in a murine model of Alzheimer’s disease. Neurotherapeutics. (2018) 15:1036–54. doi: 10.1007/s13311-018-0669-5 41. Qian XH, Liu XL, Chen G, Chen SD, Tang HD. Injection of amyloid-β to lateral ventricle induces gut microbiota dysbiosis in association with inhibition of cholinergic anti-inflammatory pathways in Alzheimer’s disease. J Neuroinflam. (2022) 19:236. doi: 10.1186/s12974-022-02599-4 42. Pellegrini C, Fornai M, D’Antongiovanni V, Antonioli L, Bernardini N, Derkinderen P. The intestinal barrier in disorders of the central nervous system. Lancet Gastroenterol Hepatol. (2023) 8:66–80. doi: 10.1016/S2468-1253(22)00241-2 37. Brosnan JT, Brosnan ME. Branched-chain amino acids: enzyme and substrate regulation. J Nutr. (2006) 136:207s–11s. doi: 10.1093/jn/136.1.207S 38. Trautman ME, Richardson NE, Lamming DW. Protein restriction and branched-chain amino acid restriction promote geroprotective shifts in metabolism. Aging Cell. (2022) 21:e13626. doi: 10.1111/acel.13626 37. Brosnan JT, Brosnan ME. Branched-chain amino acids: enzyme and substrate regulation. J Nutr. (2006) 136:207s–11s. doi: 10.1093/jn/136.1.207S 38. Trautman ME, Richardson NE, Lamming DW. Protein restriction and branched-chain amino acid restriction promote geroprotective shifts in metabolism. Aging Cell. (2022) 21:e13626. doi: 10.1111/acel.13626 39. J.D. Fernstrom. Branched-chain amino acids and brain function. J Nutr. (2005) 135:1539s–46s. doi: 10.1093/jn/135.6.1539S 40. McGarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease. Nat Rev Cardiol. (2023) 20:77–89. doi: 10.1038/s41569-022-00760-3 41. Qian XH, Liu XL, Chen G, Chen SD, Tang HD. Injection of amyloid-β to lateral ventricle induces gut microbiota dysbiosis in association with inhibition of cholinergic anti-inflammatory pathways in Alzheimer’s disease. J Neuroinflam. (2022) 19:236. doi: 10.1186/s12974-022-02599-4 43. S. Ogawa. Nutritional management of older adults with cognitive decline and dementia. Geriatr Gerontol Int. (2014) 14:17–22. doi: 10.1111/ggi.12252 40. McGarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease. Nat Rev Cardiol. (2023) 20:77–89. doi: 10.1038/s41569-022-00760-3 39. J.D. Fernstrom. Branched-chain amino acids and brain function. J Nutr. (2005) 135:1539s–46s. doi: 10.1093/jn/135.6.1539S Frontiers in Nutrition 42. Pellegrini C, Fornai M, D’Antongiovanni V, Antonioli L, Bernardini N, Derkinderen P. The intestinal barrier in disorders of the central nervous system. Lancet Gastroenterol Hepatol. (2023) 8:66–80. doi: 10.1016/S2468-1253(22)00241-2 43. S. Ogawa. Nutritional management of older adults with cognitive decline and dementia. Geriatr Gerontol Int. (2014) 14:17–22. doi: 10.1111/ggi.12252 44. Suzuki H, Yamashiro D, Ogawa S, Kobayashi M, Cho D, Iizuka A, et al. Intake of seven essential amino acids improves cognitive function and psychological and social function in middle-aged and older adults: A double-blind, randomized, placebo-controlled trial. Front Nutr. (2020) 7:586166. doi: 10.3389/fnut.2020. 586166 45. Polis B, Srikanth KD, Elliott E, Gil-Henn H, Samson AO. L-Norvaline reverses cognitive decline and synaptic loss in a murine model of Alzheimer’s disease. Neurotherapeutics. (2018) 15:1036–54. doi: 10.1007/s13311-018-0669-5 References Sodium oligomannate therapeutically remodels gut microbiota and suppresses gut bacterial amino acids- shaped neuroinflammation to inhibit Alzheimer’s disease progression. Cell Res. (2019) 29:787–803. doi: 10.1038/s41422-019-0216-x 36. Larsson SC, Markus HS. Branched-chain amino acids and Alzheimer’s disease: a Mendelian randomization analysis. Sci Rep. (2017) 7:13604. doi: 10.1038/s41598-017-12931-1 07 frontiersin.org Qian et al. 10.3389/fnut.2023.1103303 10.3389/fnut.2023.1103303 42. Pellegrini C, Fornai M, D’Antongiovanni V, Antonioli L, Bernardini N, Derkinderen P. The intestinal barrier in disorders of the central nervous system. Lancet Gastroenterol Hepatol. (2023) 8:66–80. doi: 10.1016/S2468-1253(22)00241-2 38. Trautman ME, Richardson NE, Lamming DW. Protein restriction and branched-chain amino acid restriction promote geroprotective shifts in metabolism. Aging Cell. (2022) 21:e13626. doi: 10.1111/acel.13626 43. S. Ogawa. Nutritional management of older adults with cognitive decline and dementia. Geriatr Gerontol Int. (2014) 14:17–22. doi: 10.1111/ggi.12252 39. J.D. Fernstrom. Branched-chain amino acids and brain function. J Nutr. (2005) 135:1539s–46s. doi: 10.1093/jn/135.6.1539S 44. Suzuki H, Yamashiro D, Ogawa S, Kobayashi M, Cho D, Iizuka A, et al. Intake of seven essential amino acids improves cognitive function and psychological and social function in middle-aged and older adults: A double-blind, randomized, placebo-controlled trial. Front Nutr. (2020) 7:586166. doi: 10.3389/fnut.2020. 586166 40. McGarrah RW, White PJ. Branched-chain amino acids in cardiovascular disease. Nat Rev Cardiol. (2023) 20:77–89. doi: 10.1038/s41569-022-00760-3 08 Frontiers in Nutrition frontiersin.org
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Domestic value chains as stepping stones to global value chain integration
World economy
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Domestic value chains as stepping stones to global value chain integration∗ Cosimo Beverelli† Victor Stolzenburg†‡ Robert B. Koopman†‡ Cosimo Beverelli† Simon Neumueller§ January 12, 2019 Abstract We study the role of Domestic Value Chains (DVCs) for Global Value Chain (GVC) inte- gration. In the presence of industry specific fixed costs of fragmenting production and of switching across input suppliers, DVCs can either be stepping stones or stumbling blocks for GVCs. Focusing on backward linkages, that is the sourcing of intermediates, we provide robust empirical evidence in favour of the stepping stone hypothesis. In our benchmark specification a one standard deviation increase in DVC integration raises subsequent GVC integration by about 0.4%. To identify the mechanisms at work, we exploit two dimensions of industry level heterogeneity: product differentiation (a proxy of fragmentation costs) and relationship specificity (a proxy of the costs of switching between suppliers). We find that DVC integration is less conducive to GVC integration in industries that are characterized by relatively high switching costs and relatively low fragmentation costs. ted K A 1 In the remainder, a distinction will be drawn between ‘industries’, ‘broad industries’ and ‘sectors’. Industries will refer to the ISIC Rev. 3 two digit codes (or aggregation thereof) listed in the first column of Table A-2. Broad industries will refer to the following six aggregates of industries, listed in the last column of Table A-2: Primary, Raw material processing, Light manufacturing, Heavy manufacturing, Electronics and Services. Sectors will be the three macro aggregates of economic activity: primary (ISIC codes 01-14), manufacturing (ISIC codes 15-37), and services (ISIC codes 40-95). Keywords: Domestic value chains; global value chains; input-output linkages. p JEL Classification: F14; F15; F63. p JEL Classification: F14; F15; F63. ce ∗The opinions expressed in this paper should be attributed to its authors. They are not meant to represent the positions or opinions of the WTO and its Members and are without prejudice to Members’ rights and obligations under the WTO. Any errors are attributable to the authors. This paper builds upon background research for the Global Value Chain Development Report 2017, a joint project by IDE-JETRO, OECD, RCGVC, World Bank, and WTO. Without implicating them, we thank seminar participants at the GVC Development Report 2016 Background Paper Conference, the Geneva Trade and Development Workshop, the University of Trento, the 16th RIEF meetings, and the Graduate Institute, Geneva for useful comments. c g , , †Economic Research Division, World Trade Organization. Rue de Lausanne 154, 1202 Geneva, Switzerland. E-mail: cosimo.beverelli@wto.org; robert.koopman@wto.org; victor.stolzenburg@wto.org (corresponding author). ‡Graduate Institute of International and Development Studies, Chemin Eug`ene-Rigot 2, 1202 Geneva, Switzer- c g , , †Economic Research Division, World Trade Organization. Rue de Lausanne 154, 1202 Geneva, Switzerland. E-mail: cosimo.beverelli@wto.org; robert.koopman@wto.org; victor.stolzenburg@wto.org (corresponding author). c E-mail: cosimo.beverelli@wto.org; robert.koopman@wto.org; victor.stolzenburg@wto.org (corresponding author). ‡Graduate Institute of International and Development Studies, Chemin Eug`ene-Rigot 2, 1202 Geneva, Switzer- land. c ‡Graduate Institute of International and Development Studies, Chemin Eug`ene-Rigot 2, 1202 Geneva, Switzer- land. §Office for West Africa, United Nations Economic Commission for Africa, P.O. BOX 744 Niamey, Niger. E-mail: NSimon@uneca.org. §Office for West Africa, United Nations Economic Commission for Africa, P.O. BOX 744 Niamey, Niger. E-mail: NSimon@uneca.org. A This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/twec.12779 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. 1 Introduction Article Global value chains (GVCs) are an important phenomenon of 21st century trade. Not only final goods or services get traded, but intermediate products and stages get outsourced and production becomes more and more fragmented. GVCs are often developed by large firms that coordinate input sourcing and assembly decisions, establishing industry linkages across borders.1 Seminal work by Hummels et al. (2001) unveiled the growing importance of this international production sharing. In more recent work, Johnson and Noguera (2012a, 2012b) characterize the difference between value added trade and gross trade, showing that the GVC revolution, as measured by trade in value added, is ongoing. In a similar fashion, Timmer et al. (2014) show that global fragmentation, proxied by the foreign value added content of production, has rapidly increased since the early 1990s. pted This development is of considerable interest for policy makers since both theory and em- pirics suggest that integrating in GVCs can lead to higher productivity and GDP. For instance, Baldwin and Robert-Nicoud (2014) show how GVCs cause productivity improvements akin to technological change by embedding the prominent features of the Grossman and Rossi-Hansberg (2008) model into a general equilibrium setting. Empirically, Kummritz (2016) finds that at the industry level higher GVC integration results in higher labor productivity and value added. ccep A central question that arises in this context is what determines GVC integration. Hummels et al. (2001), Johnson and Noguera (2012a), and Baldwin and Lopez Gonzalez (2015) find that structural factors such as country size, industrial structure, and location play a key role. In particular, small economies that are located close to GVC hubs such as Germany, Japan, and the United States exhibit strong linkages into GVCs. Kowalski et al. (2015) show that in addition policy matters. Countries with open trade and investment policies and sound institutions tend to integrate into GVCs more easily. to integrate into GVCs more easily. Article A substantial part of the variation in GVC integration, however, remains unexplained. To provide an illustration, Figure 1 plots the levels of GVC integration (measured as foreign value added in exports as a share of total exports) of three country pairs: Sweden and Belgium; Switzerland and Singapore; Romania and Bulgaria. Within each pair, countries are similar in terms of the structural and policy factors used in the literature to explain GVC participation. This article is protected by copyright. All rights reserved. 1 Introduction One can still see significant differences in levels of GVC integration within pairs of matched countries, especially in the last two pairs. Such differences cannot be explained by the drivers of GVC integration identified in the literature. @ Figure 1 about here A y y ( ) 4 See, for example, Antr`as and Helpman (2004) and Grossman and Helpman (2005). @ Figure 1 about here A Accepted A recent and growing strand of research, revisiting earlier work by Hirschman (1958), dis- cusses the positive implications of domestic linkages within and across industries.2 Building on this literature, in this paper we investigate whether such domestic linkages are stepping stones or stumbling blocks for GVCs, and the mechanisms behind the relationship between GVCs and domestic value chains (DVCs). Various papers have modeled firms’ sourcing choices as either one between global offshoring and in-house production,3 or a simultaneous decision between global offshoring, domestic outsourcing and in-house production.4 Our interest is to empirically assess a sequential development, in which the amount of globally sourced intermediates is dependent on the pre-existing level of domestic outsourcing because fragmenting further and replacing do- mestic for foreign suppliers may be costly. To identify the sequential relationship, we exploit the fact that the international fragmentation of production only started to expand at a global scale in the 1990s, as a result of a dramatic fall in cross-border trade and communication costs. Domestic fragmentation, in turn, has been available to firms much longer. Therefore, initial 2 See, among others, Jones (2011), Bartelme and Gorodnichenko (2015), Bernard et al. (2015), and Dhyne and Rub´ınov´a (2016). 3 See, for example, Jones and Kierzkowski (1990, 2001), Antr`as (2003), Grossman and Helpman (2003), and Fally and Hillberry (2015). 4 See, for example, Antr`as and Helpman (2004) and Grossman and Helpman (2005). This article is protected by copyright. All rights reserved. levels of DVC integration are unlikely to be affected by current levels of GVC integration. d Article The mechanism linking GVC and DVC integration that we focus on relates to the frag- mentation costs associated with slicing up production, and to the costs of switching suppliers. We refer to fragmentation costs as all the costs paid by a firm deciding to source inputs ex- ternally, rather than producing them in-house.5 Here, we focus on that part of fragmentation costs that can be considered as one-offfixed costs. Such fixed costs entail, among other things, the necessary codifying of tacit knowledge, the downsizing of plants and workforce, the pro- tection of intellectual property, the adaptation of the firm’s structure, and learning about the fragmentation process.6 Once these costs have been paid for domestic fragmentation, they are not incurred again. A 6 This is analogous to the literature on learning to export where firms start their internationalization by exporting small amounts to easily accessible markets to learn about the export process and their export potential before expanding their activities (see for instance Schmeiser, 2012). @ Figure 1 about here A This implies that DVC integration should serve as a stepping stone to subse- quent GVC integration, particularly in industries characterized by relatively large fragmentation costs. Other things being equal, this would be reflected in a positive relationship between DVC and GVC integration. epted Conversely, we refer to switching costs as the costs paid by a firm when it decides to stop sourcing an input from a supplier and starts sourcing the same input from another supplier, either located in the same country or abroad. Examples are contract termination fees and supplier searching and training costs. For a given level of domestic fragmentation, high switching costs should reduce subsequent international fragmentation making DVCs a stumbling block to GVC integration. Other things being equal, this would be reflected in a negative relationship between DVC and GVC integration. Acc Thus, in the presence of both fragmentation costs and switching costs, the sign of the rela- tionship between DVCs and GVCs is dependent on the relative importance of switching vis-`a-vis fragmentation costs and needs to be determined empirically. To that effect, we propose a novel measure of DVCs, equal to the share of domestically sourced inputs in domestic output, where This article is protected by copyright. All rights reserved. rticle the latter is computed excluding foreign sourced inputs. We are able to compute this measure for up to 61 countries and 34 industries.7 We show that higher levels of domestic fragmentation at the beginning of our sample (mid 1990s) positively affect GVC integration, as measured by foreign value added in exports, at the end of the sample (late 2000s). In the benchmark specifi- cation, a one unit increase in DVC integration raises subsequent GVC integration by 2.4%. This suggests that DVC integration is a stepping stone for GVC integration, and that, overall, the fragmentation cost channel dominates the switching cost channel. Moreover, by taking DVCs into account, we are able to reduce the unexplained variation in GVC integration by about 30%. Accepted Ar To investigate the hypothesized role of fragmentation and switching costs for the relation- ship between DVCs and GVCs, we exploit manufacturing industry-level heterogeneity along two dimensions: the degree of product differentiation and contract intensity (i.e. relationship specificity). p g g 9 The case of high fragmentation and low switching costs is not observed in the data. A complete list of countries and industries is available in Appendix A, respectively in Tables A-1 and A- A p pp , p y 8 Since Nunn (2007)’s contract intensity measure employs Rauch (1999)’s classification, we show in the robustness section that our results hold when alternative proxies for switching and fragmentation costs are used. 9 This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. 9 The case of high fragmentation and low switching costs is not observed in the data. @ Figure 1 about here A Fixed fragmentation costs are likely to be higher in industries producing a high share of differentiated goods since these industries tend to be more innovation- and skill- in- tensive, therefore they have higher costs of codifying tacit knowledge or downsizing as shown by Voigtl¨ander (2014). Switching suppliers, in turn, is costlier in industries characterized by a high degree of contract intensity because it involves the payment of contract termination fees and similar costs that tend to increase with the relationship-specificity of inputs. Accordingly, we use Rauch (1999)’s classification for differentiated goods to proxy for fragmentation costs, and Nunn (2007)’s contract intensity measure to proxy for switching costs.8 The 26 manufac- turing industries are then grouped into three categories: industries with high fragmentation and high switching costs, industries with low fragmentation and low switching costs, and industries with low fragmentation and high switching costs.9 In line with the suggested mechanism, we find that the positive role of DVCs for subsequent GVC integration is driven by the first two groups of industries, suggesting that fragmentation costs tend to be more relevant than switch- This article is protected by copyright. All rights reserved. ing costs. For the third group of industries, which is characterized by high switching costs and low fragmentation costs, the positive effect of DVCs disappears entirely. icle The remainder of the paper is organized as follows. Section 2 provides details on the measures employed to proxy for DVC and GVC, describes the data used and the identification strategy. Section 3 presents the empirical results. Section 4 concludes and suggests some policy implications. This article is protected by copyright. All rights reserved. ti 2.1 Measuring value chain integration and industry level characteristics cepted Ar The literature has developed several measures for GVC participation that can be split up into backward and forward linkages. The former are related to intermediates sourced from abroad. The latter are related to domestic intermediates exported abroad. Since our channel between DVCs and GVCs relates to the costs of sourcing intermediate goods, we focus on backward linkages measures. These measures are typically based on Inter Country Input Output (ICIO) tables, which are jl  ik matrices that represent supply and demand relationships within and across industries (i,j) and countries (k,l). A generic element mji lk of an ICIO matrix gives the value of intermediate goods supplied by industry j of country l to industry i of country k. ICIOs indicate not only cross country linkages but also within country linkages. This implies that the same data source can be used to build both our DVC and GVC integration indicators, limiting the scope for measurement error. In the next subsections, we first discuss the standard measures of GVC integration proposed in the literature and also used in this study. Next, we move to our novel measure of DVC integration. rt 2 Empirical methodology Ar In this section we discuss the methodology to examine the role of DVCs for subsequently linking into GVCs. To do so, we postulate the following reduced form model at the country-industry level: gvcikt βdvciks  λœι  γœxikt  εikt, (2.1) (2.1) pted where i indexes industries; k indexes countries; t and s @ t index time; the scalar β is the coefficient of interest, to be estimated; γ is a column vector of coefficients to be estimated; x is a column vector of controls; λ is a column vector of fixed effects; ι is a column vector of 1’s; and ε is a random error term. The variables in x, and the elements in and dimension of λ, are specified in Section 2.3, where the identification strategy is discussed. Accep While equation (2.1) can give the net effect of DVCs for GVC integration, it does not provide information on the mechanism driving the relationship. Therefore, in a second step we proceed estimating a variant of equation (2.1) that exploits the industry-level variation in the data to analyze the suggested role of fragmentation and switching costs. For this purpose, we build proxies for industries’ fragmentation and switching costs and then classify industries based on the relative magnitude of the costs into three groups: industries with high fragmentation and high switching costs (HFHS industries), industries with low fragmentation and low switching costs (LFLS industries), and industries with low fragmentation and high switching costs (LFHS This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. industries). We postulate the following reduced form model with interactions between DVCs and these industry-level characteristics: industries). We postulate the following reduced form model with interactions between DVCs and these industry-level characteristics: gvcikt 䜈dvciks  cœ i  λœι  γœxikt  εikt, (2.2) (2.2) c where δ is a column vector of coefficients to be estimated and ci  ˆHFHS i LFLS i LFHS i).10 c where δ is a column vector of coefficients to be estimated and ci  ˆHFHS i LFLS i LF ic where δ is a column vector of coefficients to be estimated and ci  ˆHFHS i LFLS i LFHS i).10 ic where δ is a column vector of coefficients to be estimated and ci  ˆHFHS i LFLS i LFHS i).10 c 2.1.1 GVC integration Ac The recent literature on GVCs has used various indicators to capture the rise of global production networks. Recently, Hummels et al. (2001)’s Vertical Specialization measure and its refinements by Wang et al. (2013) and Koopman et al. (2014) have emerged as standard indicators, also 10 A 10 The c vector does not contain a HFLS dummy since, as mentioned in footnote 9, the case of high fragmen- tation and low switching costs is not observed. A 10 The c vector does not contain a HFLS dummy since, as mentioned in footnote 9, the case of high fragmen- tation and low switching costs is not observed. This article is protected by copyright. All rights reserved. adopted in the present analysis. adopted in the present analysis. icle The baseline GVC indicator is fvax, the foreign value added content in the production of exports. To construct this indicator, value added needs to cross at least two borders to be counted towards GVC trade. To obtain the required value added flows, it is necessary to decompose gross exports using information from ICIOs.11 In the simplest case with two countries and two industries, the decomposition can be illustrated as follows: V ˆI  A1E ’ ––––––––––––– ” vaeii kk vaeij kk vaeii kl vaeij kl vaeji kk vaejj kk vaeji kl vaejj kl vaeii lk vaeij lk vaeii ll vaeij ll vaeji lk vaejj lk vaeji ll vaejj ll “ ————————————— • , V ˆI  A1E ’ ––––––––––––– ” vaeii kk vaeij kk vaeii kl vaeij kl vaeji kk vaejj kk vaeji kl vaejj kl vaeii lk vaeij lk vaeii ll vaeij ll vaeji lk vaejj lk vaeji ll vaejj ll “ ————————————— • , cepted where E is a 4  4 matrix in which the diagonal elements give each industry’s gross exports for N industries and G countries, V is a 4  4 matrix in which the diagonal elements give each industry’s value added to output ratio, and A is the 4  4 Input Output coefficient matrix, i.e. each element of A gives the intermediates that each industry supplies for $1 of output in every other industry.12 The intuition behind the decomposition becomes clear by recalling that these intermediate flows are the output of other industries, which means that they consist of value added and other intermediates themselves. 13A more exhaustive explanation of the approach can be found in Wang et al. (2013). 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015). 12 In the general case of G countries and N industries, the dimension of the E, V , A (and, conseque 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015). 12 In the general case of G countries and N industries, the dimension of the E, V , A (and, conseque matrices is GN  GN. 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015). 12 This article is protected by copyright. All rights reserved. c 2.1.1 GVC integration ˆI  A1 approximates these indirect links between industries and when combined with the values in the V matrix gives the actual value added flows so that the elements of the vae matrix are estimates of the industry-level value added origins of each industry’s exports.13 Our benchmark GVC indicator fvax for industry i in country k is then given by: Our benchmark GVC indicator fvax for industry i in country k is then given by: Our benchmark GVC indicator fvax for industry i in country k is then given by: fvax ik  lnˆFVAX ik lnˆQ l Q j vaeji lk (2.3) (2.3) 11 This is technically implemented using the R package decompr by Quast and Kummritz (2015). 12 In the general case of G countries and N industries, the dimension of the E, V , A (and, consequently, vae) matrices is GN  GN 13A more exhaustive explanation of the approach can be found in Wang et al. (2013). This article is protected by copyright. All rights reserved. where l x k. Thus, fvax ik is equal to the sum of value added from all industries j of all foreign countries l in the exports of industry i in country k.14 where l x k. Thus, fvax ik is equal to the sum of value added from all industries j of all foreign countries l in the exports of industry i in country k.14 e cle As a simple alternative indicator, used in robustness exercises, we additionally calculate the log of imported inputs, i2p, following Baldwin and Lopez Gonzalez (2015). This measure implies a broader definition of GVC and can easily be derived from ICIOs as follows: i2pik  lnˆI2Pik lnˆQ l Q j mji lk, (2.4) (2.4) (2.4) r where l x k.15 A 15 In this case, too, sourcing from ISIC Rev. 3 group C (mining industry) is excluded. 16 Note that, as for GVC measures, we exclude intermediates sourced from the mining industry (ISIC Rev. 3 group C). This article is protected by copyright. All rights reserved. 14 Sourcing from ISIC Rev. 3 group C (mining industry) is excluded to avoid effects stemming fr variations in commodities. In Section 3.3 we use further strategies to deal with the mining industry. 15 g g y In this case, too, sourcing from ISIC Rev. 3 group C (mining industry) is excluded. 15 In this case, too, sourcing from ISIC Rev. 3 group C (mining industry) is excluded. 16 Note that, as for GVC measures, we exclude intermediates sourced from the mining indu cc 18 A number of recent papers that measure gains from trade in the presence of intermediates consider the relationship between foreign input use and domestic input use. Blaum et al. (2015) combine domestic and foreign inputs in a constant elasticity of substitution (CES) function, with elasticity of substitution ε ranging between 1.4 and 2.4. Similarly, in Tintelnot et al. (2017), firms use a CES input bundle of workers and domestic and foreign inputs, with elasticity of substitution ρ A 1. The authors use values of ρ ranging from 1.5 to 2. Antr`as et al. (2017) assume that, from the perspective of firms, inputs are differentiated by country of origin, with an elasticity of substitution across inputs from any two locations equal to 1  θ. Their benchmark estimate of θ – which corresponds to the shape parameter of the Fr´echet distribution of firm-specific intermediate input efficiencies – is equal to 1.789. A 19 A complete list by country and industry is available in the Appendix. See Tables A-3 and A-4. 20 The Rauch classification treats goods as differentiated if they are neither reference priced nor traded on organized exchanges. A 2.1.2 DVC integration pted A For DVC integration (the explanatory variable of interest in equation (2.1)) we suggest a novel indicator, applying the following approach. First, we decompose an industry’s output into three parts based on the different inputs required to produce it: i) its own value added; ii) domestically sourced intermediate goods; and iii) foreign sourced intermediate goods. We refer to the combination of i) and ii) as ‘virtual domestic output’, since it is the virtual part of an industry’s output that is produced with domestic content only. With this distinction at hand, we proceed to define our DVC indicator as the share of domestically sourced intermediate goods in virtual domestic output: dvcik  Pj mji kk Pj mji kk  vaik . (2.5) (2.5) c In other words, dvc is the share of domestic inputs in total output excluding foreign inputs. c In other words, dvc is the share of domestic inputs in total output excluding foreign inputs. c In other words, dvc is the share of domestic inputs in total output excluding foreign inputs. cc That is, it captures how much of an establishment’s domestically produced output is supplied by other establishments instead of being produced in-house.16 cc That is, it captures how much of an establishment’s domestically produced output is supplied by other establishments instead of being produced in-house.16 by other establishments instead of being produced in-house.16 The reason for calculating the DVC indicator in this particular way is as follows. W A The reason for calculating the DVC indicator in this particular way is as follows. We only 14 S i f ISIC R 3 C ( i i i d t ) i l d d t id ff t t i f i A , , g g p ( g y) 16 Note that, as for GVC measures, we exclude intermediates sourced from the mining industry (ISIC Rev. 3 group C). ed Article look at virtual domestic output instead of total output to avoid a mechanical correlation with the dependent variable which, as discussed in Section 2.1.1 above, is a subset of foreign sourced intermediate goods. These, in turn, are a subset of total output (recall that we decompose total output into three parts: own value added, domestically sourced intermediate goods, and foreign sourced intermediate goods). Hence, using total output as denominator of our dvc measure would correlate dependent and independent variable. This article is protected by copyright. All rights reserved. omplete list by country and industry is available in the Appendix. See Tables A-3 and A-4. Rauch classification treats goods as differentiated if they are neither reference priced nor traded on exchanges. The difference between total output and virtual domestic output in 1995 was in any case minimal an ore, as shown in Section 3.3, the results are not sensitive to the choice of denominator of the dvc variable A 2.1.2 DVC integration By considering only virtual domestic content, we break this mechanical link.17 An alternative solution to the problem of mechanical correlation between the main explanatory variable (dvc) and the dependent variable (gvc) would be the use of levels, rather than shares, of the dvc variable. This approach, however, would also create a positive (negative) mechanical correlation between the two variables in the presence of any complementarity (substitutability) between foreign and domestic inputs in production.18 To summarize, we avoid a mechanical correlation between our GVC and DVC indicators by constructing the former in levels and the latter in shares, using in its denominator only virtual domestic content. Table 1 shows the top and bottom ten countries in terms of the dvc variable in 1995 (averaged across all industries for each country).19 ed Article look at virtual domestic output instead of total output to avoid a mechanical correlation with the dependent variable which, as discussed in Section 2.1.1 above, is a subset of foreign sourced intermediate goods. These, in turn, are a subset of total output (recall that we decompose total output into three parts: own value added, domestically sourced intermediate goods, and foreign sourced intermediate goods). Hence, using total output as denominator of our dvc measure would correlate dependent and independent variable. By considering only virtual domestic content, we break this mechanical link.17 An alternative solution to the problem of mechanical correlation between the main explanatory variable (dvc) and the dependent variable (gvc) would be the use of levels, rather than shares, of the dvc variable. This approach, however, would also create a positive (negative) mechanical correlation between the two variables in the presence of any complementarity (substitutability) between foreign and domestic inputs in production.18 To summarize, we avoid a mechanical correlation between our GVC and DVC indicators by constructing the former in levels and the latter in shares, using in its denominator only virtual domestic content. Table 1 shows the top and bottom ten countries in terms of the dvc variable in 1995 (averaged across all industries for each country).19 @ Table 1 about here A q 19 A complete list by country and industry is available in the Appendix. See Tables A-3 and A-4. 20 The Rauch classification treats goods as differentiated if they are neither reference priced nor i d h A ( ) 22 A detailed explanation of the classifications of industries is available in Appendix B. Note that Nunn uses the Rauch classification to determine if an intermediate is sourced on a contractual basis. Thus, our switching and fragmentation cost proxies depend indirectly and directly on the Rauch classification. However, since the Nunn classification looks at an industry’s intermediates and the Rauch classification at an industry’s output, there is no strong mechanical correlation between the two. We show in Section 3.3 that our results are robust to employing alternative proxies for switching and fragmentation costs such as Costinot (2009)’s complexity classification. p 2.1.3 Industry fragmentation costs and switching costs e As benchmark proxy for fragmentation costs, we use Rauch (1999)’s classification for differenti- ated goods concorded to our industry-level data.20 The reason for this choice is that industries ated goods concorded to our industry-level data.20 The reason for this choice is that in e with a high share of differentiated goods tend to be more innovation and skill intensive.21 As a result, we can assume that codifying tacit knowledge, downsizing, and other fragmentation related activities are more costly for these industries. Article Regarding switching costs, Nunn (2007)’s contract intensity (or relationship specificity) measure is a straightforward proxy. Different industries require different amounts of relation- ship specific inputs. When these inputs are supplied at arm’s length, complex contracts are needed to address the resulting hold up problem. Nunn (2007) shows that, as a result, in- dustries differ in their contract intensity. A substantive share of fixed switching costs, such as contract cancellation fees, only occur when intermediates cannot be sourced from organized markets, but are sourced using contract-based relations with suppliers. Nunn (2007)’s indicator measures the share of intermediates that are sourced through such contract-based relations in total intermediates, and is thus a natural proxy for our exercise.22 epted Table 2 lists the industries separated into the three categories and shows that the selected proxies create a sensible allocation. LFLS industries like ‘Food and beverages’ typically source inputs from organized markets and, thus, should find it easy to switch between suppliers. At the same time, their production tends to be neither skill nor innovation intensive and so frag- mentation should be equally simple. In contrast, the identified HFHS industries such as ‘Motor vehicles’ or ‘Electronics’ are dependent on a highly skilled workforce and complex intermediates. Lastly, LFHS industries such as ‘Electrical machinery’ (e.g. cables) require complex intermedi- ates but production is homogeneous and non complex. p Lastly, LFHS industries such as ‘Electrical machinery’ (e.g. cables) require complex intermedi- ates but production is homogeneous and non complex. 21 See, for example, Voigtl¨ander (2014) for evidence on this relationship. This article is protected by copyright. All rights reserved. for example, Voigtl¨ander (2014) for evidence on this @ Figure 2 about here A ted Ar Similarly, when we look at our DVC integration indicator by country (Table 1), we see that countries that are well integrated into GVCs also had strong domestic linkages in 1995. Interestingly, however, while the top ten is dominated by countries traditionally highly involved into GVCs, such as China, Slovakia, or Korea, it also contains countries like Croatia and New Zealand, which do not exhibit strong linkages into GVCs. The same pattern holds when exam- ining the bottom ten. While Cambodia and Greece lag behind in GVC participation, Mexico is highly integrated. This suggests heterogeneity in the effects of DVCs, which will be taken into account in the empirical analysis. @ Table 2 about here A This article is protected by copyright. All rights reserved. 2.2 Descriptive evidence ticle When taking our GVC and DVC indicators to the data, we find preliminary evidence that DVC integration is a stepping stone for GVC integration. Figure 2 unconditionally correlates GVCs in 2008 (in logs) with the measure of DVCs in 1995. The slope coefficient is equal to 0.47. The stepping-stone effect of DVC integration for GVC integrations also constitutes suggestive evidence that the fragmentation cost channel dominates the switching cost channel. @ Figure 2 about here A p 2.3 Identification Accep Estimation results of the reduced form equations (2.1) and (2.2) could in particular be affected by omitted variable bias. For instance, high levels of GVC integration could be driven by a coun- try’s institutions, which might also affect domestic production fragmentation. To deal with this concern, we use a combination of fixed effects and control variables that account for structural and policy determinants of GVC participation identified in the literature. More specifically, we use industry fixed effects to control for structural differences across industries that might correlate at once with GVC and DVC integration. Heavy manufactures and downstream indus- tries, for instance, are more likely to develop backward linkages than agriculture. In addition, This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. le we include country-broad industry fixed effects to take care of (non time-varying) policy and structural differences across countries – in terms of country size, economic development, remote- ness, institutions, endowments as well as the industrial structure of the country – that may vary across broadly defined industries in each country.23 le we include country-broad industry fixed effects to take care of (non time-varying) policy and structural differences across countries – in terms of country size, economic development, remote- ness, institutions, endowments as well as the industrial structure of the country – that may vary across broadly defined industries in each country.23 pted Artic Industry and country-broad industry fixed effects fail to control for any potentially omitted variable that varies across countries-industries and might be correlated with DVC integration, even within broad industries. We include two such variables: i) a dummy equal to one for comparative advantage industries, ca; and ii) industry level ‘composite’ import tariffs, ctau. The comparative advantage dummy synthetically accounts for all those determinants of patterns of specialization of a given country in a given industry (e.g., country labour abundance in a labour- intensive industry). It is calculated using Balassa (1965)’s revealed comparative advantage index, but on the basis of value added exports.24 The ‘composite’ import tariffs variable captures differences in the trade policy environment at the country-industry level. For each industry in a given country, it is calculated as the weighted average of tariffs that the inputs into the industry face, with weights being given by the input output coefficient. Ac 23 As the dependent variable is at the country-industry level, we cannot use country-industry fixed effects to control for a country’s industrial structure. To be nevertheless as conservative as possible, we form country-broad industry fixed effects by interacting country fixed effects with fixed effects for the six ‘broad industries’ defined in the last column of Table A-2. These country-broad industry fixed effects control for the fact that larger industries within a country have more scope for fragmentation. A y p g 24 The Balassa index is given by eik~ek Pk eik~ Pk ek . It compares an industry’s share in a country’s exports, ek, to that industry’s share in global exports. An index value larger than 1 indicates comparative advantage industries. This article is protected by copyright. All rights reserved. y g p 25 See, for instance, Hsieh and Klenow (2009). A y g 24 The Balassa index is given by eik~ek Pk eik~ Pk ek . It compares an industry’s share in a country’s exports, ek, to that industry’s share in global exports. An index value larger than 1 indicates comparative advantage industries. 25 See, for instance, Hsieh and Klenow (2009). that industry’s share in global exports. An index value larger than 1 indicates comparative advantage industries. 25 See, for instance, Hsieh and Klenow (2009). 25 See, for instance, Hsieh and Klenow (2009). p 2.3 Identification We follow the literature and use United States’ input output coefficients under the assumption that they are determined by technological factors as opposed to market distortions.25 ccep Considering that the aim is to examine how initial patterns in DVCs have affected subse- quent GVC integration, we use dvc values for 1995 and fvax values for 2008. This also addresses potential concerns about a reverse causality bias since the rapid expansion of GVCs only started around 1995 (Wang et al., 2017 show that in 1995 GVCs only accounted for 3% of global pro- duction). This limits any potential reverse effect on DVC integration values in 1995. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. e The benchmark reduced form model for the effect of DVCs on GVC integration (equation (2.1)) is then estimated as: gvcik2008 βdvcik1995  γ1caik2008  γ2ctauik2008  λ  λi  λbk  εik2008, (2.6) gvcik2008 βdvcik1995  γ1caik2008  γ2ctauik2008  λ  λi  λbk  εik2008, (2.6) (2.6) rtic where β is the coefficient of interest; λ is a constant; and λi and λbk are respectively industry and broad industry-country fixed effects. Since the mechanism determining the role of DVCs for GVC integration is ambiguous as outlined in the introduction, we do not have a prior on the sign of β. A The benchmark reduced form model for testing the mechanism behind the DVC-GVC link (equation (2.2)), in turn, is estimated as: The benchmark reduced form model for testing the mechanism behind the DVC-GVC link (equation (2.2)), in turn, is estimated as: gvcik2008 δ1dvcik1995  LFLSi  δ2dvcik1995  HFHSi  δ3dvcik1995  LFHSi  γ1caik2008 γ2ctauik2008  λ  λi  λbk  εik2008. (2.7) gvcik2008 δ1dvcik1995  LFLSi  δ2dvcik1995  HFHSi  δ3dvcik1995  LFHSi  γ1caik2008 γ2ctauik2008  λ  λi  λbk  εik2008. (2.7) (2.7) γ2ctauik2008  λ  λi  λbk  εik2008. γ2ctauik2008  λ  λi  λbk  εik2008. te In equation (2.7), δ1 gives the effect of DVCs on subsequent GVC integration in LFLS industries; δ2 gives the effect for HFHS industries; and δ3 gives the effect for LFHS industries. Accept A positive δ1 would suggest that fragmentation costs play a larger role than switching costs, while a negative δ1 would imply the opposite. p 2.3 Identification We include the HFHS dummy as falsification test, since we expect δ2 not to be significantly different from δ1 since in both industry categories the relative level of fragmentation vis-`a-vis switching costs is identical. In other words, if the channel we propose is at work, there is no reason to believe that the effect of DVCs on GVCs should be different in HFHS industries as compared to LFLS industries. The coefficient of interest in equation (2.7) is δ3, which is expected to be significantly smaller than δ1 and δ2. This is because in LFHS industries the relative level of switching costs, as compared to fragmentation costs, is higher than in the benchmark LFLS industries. Accordingly, DVC integration in these industries is less likely to lead to GVC integration. This article is protected by copyright. All rights reserved. Article The proposed strategy of adding industry and broad-industry country fixed effects, as well as the ca and ctau controls, does not guarantee that the empirical correlation between dvc (alone or interacted with industry fragmentation/switching costs dummies) and gvc is free of any omitted variable bias. Therefore, as described in Section 3.3, panel estimations using data for the years 2000 and 2005 in addition to 1995 and 2008 are presented. These estimations allow to include a rich structure of fixed effects, namely country-year, industry-year, and industry- country, controlling for any unobserved factor that might be correlated with GVCs and DVCs and varies across two of the three dimensions (countries, industries and time) of the data. This should minimize concerns related to omitted variable bias. pted A Moreover, the approach of using 1995 values for the dvc variable and 2008 values for the gvc variable does not automatically exclude any reverse causality. As also detailed in Section 3.3, we thus estimate regressions with an interaction term between gvc and a high income dummy as a way of addressing these concerns. The reason is that if there is reverse causality flowing from GVCs to DVCs, we would like our dvc values to be from a point in time where the extent of GVCs was negligible, so that the estimates would be unbiased even in the presence of reverse causality. In 1995 the extent of GVC integration might not have been negligible in developed countries, but was certainly so in many developing countries. A 26 The instrument for dvcikt is, therefore, the variable dvcIV ikt  Plxk dvcilt  gdplt, where gdplt is the share of country l in total GDP of all countries in the sample at time t. The weights sum up to one. This article is protected by copyright. All rights reserved. p 2.3 Identification Accep Killing two birds (omitted variables and reverse causality) with one stone, we further propose an instrumental variable (IV) strategy that exploits exogenous variation in the DVC variable. To obtain such exogenous variation, we regress dvc in the first stage on the weighted average of other countries’ dvc values, with the weights given by country size (as proxied by GDP).26 The intuition is the following. Think of DVC values as having three components: a country-specific, an industry-specific and a country-industry component. The first two components are absorbed by fixed effects, and are not a source of concern. The third component itself can further be decomposed in two parts: i) an idiosyncratic part specific to each country-industry (e.g. man- This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. ted Article agerial ability, reputation); and ii) a part correlated across countries due to the interaction of structural industry characteristics and country characteristics (e.g. market size matters more for domestic fragmentation in complex industries than in non-complex industries). The idiosyn- cratic part might introduce an omitted variable bias – for instance if high managerial ability specific to an industry in a country might lead to both domestic and foreign fragmentation. In contrast, the correlated part should be free of such bias, because the individual contribution of the industry- and country-specific components is controlled for by fixed effects, so that identi- fication is obtained only through the exogenous interaction of these variables. By using other countries’ DVC variables as an instrument, the estimates are only identified by this exogenous part, which is by definition uncorrelated with the endogenous idiosyncratic part. Therefore, the average value of dvc in other countries is a legitimate instrument for dvc, since it both meets the relevance and the exclusion requirements. We additionally weight the instrument by country size since we assume that the DVC values of larger countries in our sample are closest to a true optimal undistorted DVC value because large countries have a larger set of available suppliers, which means that their DVC values are not artificially restricted by the absence of adequate domestic outsourcing opportunities. This article is protected by copyright. All rights reserved. p 2.4 Data p 2 F Accep For the calculation of the DVC and GVC indicators, as well as the revealed comparative ad- vantage measure, we employ the most recent and most advanced release of OECD ICIO tables, covering 61 countries and 34 2 digit ISIC Rev. 3 industries for the years 1995, 2000, 2005, and 2008 to 2011. To create ICIOs, the OECD combines national IO tables with international trade data. As OECD countries have a harmonized construction methodology, potential discrepancies between national IO tables should be minor. Furthermore, the advanced harmonization across countries reduces to a minimum the use of proportionality assumptions to derive the ratio of imported intermediates in an industry’s demand. The OECD has used elaborate techniques This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. to deal with processing trade. Due to the outstanding role of processing trade in GVCs, this implies a significant improvement for the reliability of the database.27 Article For the tariffmeasure, we additionally take HS 2 digit MFN tariffs from the UNCTAD TRAINS database and concord them to the ICIOs’ ISIC Rev. 3 classification employing a concordance table provided by WITS. The Rauch classification is available at the 4 digit SITC Rev. 2 level. To concord it to ISIC Rev. 3, we need to apply a crosswalk via SITC Rev. 3. The necessary concordance tables are provided by Eurostat. In cases where ISIC industries are concorded to both homogeneous and differentiated SITC commodities, we use trade weights from COMTRADE to determine if an ISIC industry is differentiated or homogeneous.28 Accepted A For additional specifications we employ as supplementary controls the distance of country k to the closest GVC hub (China, Germany, Japan, and the United States) from CEPII and GDP data from the World Development Indicators. We also perform several robustness checks, some of which necessitate additional data. Firstly, in one robustness exercise we replace the Rauch classification for fragmentation costs with Costinot (2009)’s measure of industrial complexity. It assesses how many days of training an average worker in a given industry needs to complete a job, and is hence a direct measure of an industry’s skill content. The data is available for a subset of 3 digit SIC codes at the 1972 revision. To match these with ISIC codes, we first concord them to the 1987 SIC revision using concordance tables by the NBER. A pp 29 Details are provided in Appendix B. All concordances and classifications are available from the authors upon request. This article is protected by copyright. All rights reserved. 27 See Koopman et al. (2012) for an analysis of China’s processing trade. 28 This article is protected by copyright. All rights reserved. 27 See Koopman et al. (2012) for an analysis of China’s processing trade. 28 28 See Appendix B for details. Koopman et al. (2012) for an analysis of China’s processing trade. A di B f d t il S K t l (2012) f l i f Chi ’ i t d 28 See Appendix B for details. 29 te 3 Results cept OThis section is structured in three parts. We start by discussing the outcome of equation (2.6), which estimates the net effect that higher values of DVC integration have for subsequent GVC integration. In the second part, we assess the mechanism behind this effect by disentangling the role of fragmentation and switching costs as identified by equation (2.7). In the last part of the section we present a series of robustness checks. p 2.4 Data We then use a concordance table between SIC87 and ISIC Rev. 3 built by Statistics Canada.29 Secondly, in a separate exercise we replace the Nunn’s proxy for contract intensity with US capital intensity data from the OECD. In capital intensive industries, suppliers are often required to make large sunk investments. Consequently, they are likely to demand contracts with high cancellation fees. This makes capital intensity a likely proxy for switching costs. This data is readily available at the ISIC level. d Article The country coverage comprises developing and developed economies, allowing to examine whether the level of development plays any role in shaping the relationship between domestic and global value chains. In addition, the industry coverage includes primary, manufacturing, and services sectors. The latter play an increasingly dominant role in value chains. However, there are no tariffs nor Rauch measures available for services. Therefore, we use estimates based only on the manufacturing and primary sectors as benchmark but perform robustness checks that include services without the tariffcontrol, assuming that all services industries are differentiated and excluding non-tradeable services industries such as education, health, or construction. Concerning the time period examined, we use 1995 and 2008 because these are the earliest and latest available data points before the start of the prolonged global financial crisis. Finally, we exclude in the benchmark estimations the oil exporting countries Saudi Arabia and Brunei; Chinese Taipei, for which no tariffdata is available; and the United States, to avoid endogeneity stemming from the construction of the weighted tariffand capital intensity variables.30 3.1 The net effect of DVCs on GVCs A Table 3 reports the results for the first part of this exercise. The coefficient on dvc is positive and statistically significant in all specifications, indicating that domestic value chains facilitate 30 Exclusion of the United States from the sample is standard practice in the literature that uses US IO weights – see for instance Beverelli et al. (2017). A Table 3 reports the results for the first part of this exercise. The coefficient on dvc is positive and statistically significant in all specifications, indicating that domestic value chains facilitate A 30 Exclusion of the United States from the sample is standard practice in the literature that uses US IO weights – see for instance Beverelli et al. (2017). A 30 Exclusion of the United States from the sample is standard practice in the literature that uses US IO weights – see for instance Beverelli et al. (2017). This article is protected by copyright. All rights reserved. subsequent GVC integration. @ Table 3 about here A @ Table 3 about here A This article is protected by copyright. All rights reserved. @ Table 3 about here A rticle Columns (1) and (2) give the results for primary and manufacturing sectors only, while columns (3) and (4) include industries in the services sector, but at the cost of not controlling for tariffs. While all estimates are similar in magnitude and significance, we take the point estimate in column (2) as benchmark because it includes the full set of fixed effects described in equation (2.6) and it is comparable to the analysis in Section 3.2. d Ar The benchmark specification suggests that a one unit increase in initial DVC integration leads to 2.4% higher subsequent GVC integration. This implies that a one standard deviation increase in DVC integration raises GVC integration by approximately 0.2 standard deviations or 0.4%. In addition, back of the envelope calculations comparing R squared of the benchmark regression with and without the dvc measure suggest that DVCs can reduce the unexplained variation in contemporaneous GVC integration by about 30%. Accepte Since our benchmark estimates only allow for within-broad-industry-country variation, they might hide gains that stem from variation in DVC integration across countries which are empha- sized by some studies such as Bartelme and Gorodnichenko (2015). Therefore, we additionally report in columns (1) and (3) results that only control for industry fixed effects, including in the vector of controls the following three variables: log of per capita GDP at constant 2005 prices; log of GDP at constant 2005 prices; and log distance to the closest GVC hub (CHN, DEU, JPN, or USA). These variables capture basic country characteristics such as size, development status, and remoteness. Since the estimates in columns (1) and (3) are potentially subject to an omitted variable bias due to the exclusion of country fixed effects, we interpret them as upper bounds of the DVC effect. Table 3 shows that the coefficients are indeed larger in magnitude than the benchmark estimates, although the difference is not large. This article is protected by copyright. All rights reserved. icle In summary, these findings highlight a further advantage of comprehensive domestic linkages through their contribution to GVC participation. In the light of the literature that has unveiled productivity enhancing effects of GVC integration (Kummritz, 2016), our results can help explain why efficient DVCs facilitate economic development. Our findings also speak to the literature on the determinants of GVC integration. We show that differences in pre-existing domestic linkages can account for differences in observed GVC linkages. @ Table 3 about here A Arti The descriptive evidence discussed in Section 2.1 has however illustrated that there are countries with strong DVC linkages which are not well integrated into GVCs. To unveil the heterogeneity in the GVC-DVC relationship, we now empirically assess the relevance of the mechanism related to the industry-specific costs discussed above. A 3.2 The role of fragmentation and switching costs ed In this section, we estimate equation (2.7), which separates the effects of DVCs on GVCs in LFHS (low fragmentation, high switching costs) industries as opposed to HFHS (high fragmentation, high switching costs) industries, and LFLS (low fragmentation, low switching costs) industries. cepte As a reminder, we expect the coefficient on the LFHS dummy to be significantly smaller than the coefficients on the HFHS and the LFLS dummies. This is due to the fact that the former dummy captures industries in which switching costs tend to be high, which makes a change from domestic to foreign suppliers costly, but fragmentation costs low, which shuts down the stepping stone channel of DVCs for GVCs. In addition, we expect the coefficients on the HFHS and the LFLS dummies to be not significantly different, since the relative incidence of fragmentation and switching costs should be identical in the two cases. Ac Table 4 reports the corresponding results. As in Table 3, columns (1) and (2) exclude industries in the services sector, while columns (3) and (4) include them. We replicate in columns (1) and (3) the basic results presented in Table 3 for comparison, while columns (2) and (4) report the new estimates from equation (2.7). This article is protected by copyright. All rights reserved. @ Table 4 about here A icle case of Singapore, ‘Textiles, leather and footwear – TEX’ in the case of Bulgaria).31 This means that in these three under-performing countries the structure of the economy causes domestic linkages to prevent GVC integration, despite the fact that other factors, such as location, would facilitate GVC integration. For policy makers this implies that they need to take into account both the strength of domestic linkages and the structure of their economies when they develop GVC strategies. @ Table 4 about here A t c e The results are in line with our expectations. The coefficients on the interaction of the dvc variable with the HFHS and the LFLS dummies (δ1 and δ2 in equation (2.7)) are positive and statistically significant in both samples, while the coefficients on the interaction with the LFHS dummy (δ3 in equation (2.7)) is smaller and, in fact, not statistically different from zero. This is strong evidence in favour of the proposed channel which links initial DVC patterns to subsequent GVC integration by taking into account the varying importance of fragmentation and switching costs across industries. Ar Our preferred specification in column (2) suggests that in HFHS and LFLS industries, a one percentage point increase in initial DVC linkages increases contemporaneous GVC integration by 2.3 to 3.1%. However, this positive effect of DVCs is not observed in LFHS industries. pted In terms of our proposed channel, these findings are further evidence that fragmentation costs are more important than switching costs for the DVC-GVC link. This is because in industries where both costs are high (low), the positive effect of DVCs prevails. Similarly, even when switching costs are high but fragmentation costs are low, the net effect is around zero instead of turning negative. Thus, the net effect that we found in Section 3.1 is confirmed and can be explained by the fact that fragmentation costs are more relevant than switching costs for moving from DVCs to GVCs. Acce These findings can also explain the observed heterogeneity in the DVC-GVC link as pre- sented in the introduction. In Figure 3 we revisit the matched country pairs from Figure 1. There, we grouped countries based on the known structural and policy determinants of GVC integration and showed that there remains substantial unexplained variation in the data. Here, we add leading export industries to the chart. It is striking that all the leading export industries of the under-performers (respectively, ‘Paper – PAP’ for Sweden, and ‘Chemicals – CHM’ for Switzerland and for Romania) fall into the LFHS category whereas the comparison group is more specialized in HFHS industries (‘Computer, electronic and optical products – CEQ’ in the This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. A 31 An exception is Belgium, which is also specialized in ‘Chemicals’. However, its DVC integration levels in 1995 were fairly low in this industry while Switzerland, Sweden and Romania had high values of DVC integration in this industry. 32 Column (1) of Table 5 reproduces the estimates of the preferred specification of column (2) of Table 3 for convenience. A in this industry. 32 Column (1) of Table 5 reproduces the estimates of the preferred specification of column (2) of Table 3 for convenience. This article is protected by copyright. All rights reserved. Ar 3.3 Robustness tests and IV regressions ccepted A We expose our results to a battery of robustness checks starting with the identification assump- tions. While we have argued that in 1995 reverse causality from GVCs to DVCs should have been minor, since only 3% of global production was related to GVCs, there is evidence that in high income countries GVCs did already play a role. We augment equation (2.6) with an interaction term between the DVC measure and a high income dummy, equal to one if a country was classified as high income in the World Bank classification in 1995. This allows us to see if the effect is also present in developing economies, for which the assumption of the absence of reverse causality is not questionable. This estimation can also provide insights on a possi- ble development dimension to our findings. The results are in column (2) of Table 5.32 The high income interaction term is not significant. We interpret this result (which is robust to several alternative constructions of country groups) as an indication that the effect of DVC on GVC integration is not driven by reverse causality, since the effect is also present in developing economies, where GVCs linkages only developed in relatively recent years. @ Table 5 about here A A 31 An exception is Belgium, which is also specialized in ‘Chemicals’. However, its DVC integration levels in 1995 were fairly low in this industry while Switzerland, Sweden and Romania had high values of DVC integration in this industry. 32 Column (1) of Table 5 reproduces the estimates of the preferred specification of column (2) of Table 3 for convenience. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Article Next, we further address concerns about omitted variable bias by fully exploiting the time variation in our data. More specifically, we additionally use data for the years 2000 and 2005 and estimate a panel version of equation (2.6). The specification includes a one period lagged DVC variable (following Bartelme and Gorodnichenko, 2015) and country-year, industry-year, and industry-country fixed effects. The specification without the time dimension remains our preferred model since the objective is to assess the effect of initial DVC conditions unaffected by the rise of GVCs. However, the advantage of this robustness check is that we can add a rich structure of fixed effects, controlling for any unobserved factor that might be correlated with GVC and DVC and varies across two of the three dimensions (countries, industries and time) of the data. Column (3) of Table 5 shows that the coefficient on dvc remains positive and statistically significant. Its magnitude decreases by 75%. This can be explained by a potential linear accumulation of the effect over time. cepted The results of the instrumental variable regression using the variable dvcIV described in Section 2.3 as an instrument for dvc are in column (4) of Table 5. All coefficients are in line, in terms of sign, magnitude and statistical significance, with the benchmark coefficients reported in column (1). The Cragg-Donald Wald F statistic of 176.6 is well above the corresponding critical values tabulated in Stock and Yogo (2005), suggesting that the instrument is relevant. Note further that the first-stage correlation between the instrument and dvc is statistically significant. Taken together, the results of columns (1)-(3) of Table 5 allow to conclude that domestic linkages causally affect GVC participation, in other words they are stepping stones for integration in global value chains. Acc The stepping-stone result is subject to five further robustness checks addressing measure- ment and sample selection issues. The results of these tests are displayed in columns (5)-(9) of Table 5. @ Table 5 about here A First, we test if our results are dependent on the selected GVC measure. For this purpose, we replace fvax with the i2p indicator of Baldwin and Lopez Gonzalez (2015), which is simply the value of intermediate imports (see equation 2.4 and Section 2.1.1). We find in This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. column (5) of Table 5 that this variation has no relevant impact on our estimates. column (5) of Table 5 that this variation has no relevant impact on our estimates. icle We then proceed to vary the sample composition by including the previously excluded countries Saudi Arabia, Brunei, and the United States and, in a separate exercise, non tradeable services industries to examine if the results are sensitive to specific sample changes (see Table A-2 for the list of non-tradeable industries). Columns (6) and (7) of Table 5 show that the results are not affected in any meaningful way. pted Arti In a next step, we replace the GVC value of 2008 with the values of 2005 (in column (8)) or 2011 (in column (9)). This allows assessing whether the results are dependent on a specific time period, in particular whether the global financial crisis is relevant for the DVC-GVC relationship. The estimates in columns (8) and (9) of Table 5 suggest that this is not the case. In addition, the latter result that uses GVC values for 2011, combined with the estimates from the robustness exercise that uses a panel setup (column (3)) reveals suggestive evidence on the role of DVCs in the current slowdown in GVC expansion.33 The panel estimates suggest that the effect of DVCs accumulates over time until 2008. However, the coefficient for 2008 is not statistically different from the coefficient for 2011, implying that the accumulation has ended. This would be in line with the fact that the boost that initial DVC integration provided to GVC expansion has been fully exploited which, in turn, would contribute to explaining the observed slowdown in GVC expansion. Accep We now turn to a set of robustness checks concerning the mechanism behind the DVC- GVC link of Table 4, starting with the results of IV regressions. A ( ) 34 The benchmark coefficients of column (1) of Table 4 are the ones of column (2) of Table 4, and are reported for convenience. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. A 33 See, for instance, Degain et al. (2017) for recent evidence that the GVC expansion has stalled since 2008. @ Table 5 about here A The IV results, presented in column (2) of Table 6, are similar, in terms of sign, magnitude and statistical significance, to the benchmark coefficients of column (1).34 Namely, the coefficients on the interaction of the dvc variable with the HFHS and the LFLS dummies are positive and statistically significant, and the coefficient on the interaction with the LFHS dummy is smaller and not statistically ticle different from zero. The value of the Cragg-Donald Wald F statistic of 54.65 again suggests that the instrument is relevant, and the first-stage correlation between the instruments (interaction terms between dvcIV and industry dummies) and the variables to be instrumented (interaction terms between dvc and industry dummies) – presented in columns (2.a, 2.b and 2.c) – are statistically significant. We therefore confirm that the mechanisms behind the causal effect of initial DVC patterns on subsequent GVC integration are related to the varying importance of fragmentation and switching costs across industries, as discussed in Section 3. @ Table 6 about here A @ Table 6 about here A cepted A The next three columns of Table 6 present the results of three robustness checks that concern the measuring of industry dummies that proxy for fragmentation and switching costs. Firstly, we vary the cutoffcondition which classifies industries as differentiated and, indirectly, as contract intensive. For this, it is necessary to recall that the Rauch classification is at the 4 digit SITC level, while our industry classification used to construct the GVC and DVC variables is the 2 digit ISIC. As a results, several SITC industries are assigned to one ISIC industry. As explained in detail in Appendix B, in the benchmark estimation, we define a 2 digit ISIC industry as differentiated if the trade share of differentiated 4 digit SITC industries matched with the 2 digit ISIC industry exceeds the mean value across all ISIC industries. We now use the median as cutoffinstead. This reduces our LFHS industries to ‘Pulp and paper products’, ‘Chemicals’, and ‘Mining and quarrying’, with the other two in the last column of Table 2 being now classified as HFHS industries. Acc Column (3) of Table 4 shows the estimates for this slightly stricter cutoff. Qualitatively, the results hold while the magnitude of the LFHS coefficient increases at the expense of the coefficient on the HFHS interaction. @ Table 5 about here A Since we increased the cutoff, the ratio of switching to frag- mentation costs in LFHS industries has increased causing a reclassification of LFHS industries as HFHS industries. Therefore, the change in magnitudes is sensible and confirms our channel. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. rticle In the two next robustness exercises, we change the measures used to classify the industries into their respective categories. First, we use Costinot (2009)’s industrial complexity indicator to proxy for fragmentation costs. As discussed in Section 2.4, it is a proxy for an industry’s average skill intensity and can accordingly capture the costs of downsizing and protecting intellectual property well. The results in column (4) of Table 6 show that this change has no relevant impact on our estimates. As a second variation, we use capital intensity as an alternative proxy for switching costs and equally find no meaningful impact on our results, as can be seen in column (5) of Table 6. d Ar Overall, the two main results concerning the net effect of DVCs for GVC integration, and the mechanisms behind this link, are robust to variations in the identification assumptions, the sample composition, and to the employed indicators and proxies. Therefore, we conclude that DVCs and their interaction with a country’s industrial structure are an important driving factor for integration in GVCs. e 4 Conclusions cept GVCs have become a dominant factor in international trade. Recent theoretical and empirical research has shown that integration in GVCs can lead to gains in productivity and welfare. This begs the question of what drives GVC participation. While previous research has revealed a set of important structural factors and policies, there remains substantial unexplained variation in GVC integration patterns across countries. Acc This paper has shed light on a new determinant of these patterns by providing evidence that initial levels of DVC integration affect contemporaneous GVC integration. In other words, levels of domestic fragmentation established before the rise of GVCs can explain a relevant share of current variation in GVC integration. The results hold for countries at varying stages of development, over varying time periods, and when changing the sample composition and identifying assumptions. In our preferred specification, a one standard deviation increase in This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. DVC integration raises subsequent GVC integration by 0.4%. Besides, we are able to decrease the unexplained variation in GVC integration by about 30%. Article The mechanisms behind the DVC-GVC link investigated in this paper are related to industry- specific fixed costs of fragmentation and of switching suppliers. On the one hand, high fixed fragmentation costs allow, due their sunk nature, DVCs to act as stepping stones for GVCs. On the other hand, high fixed costs of switching suppliers should lead to a negative association between DVCs and GVCs. Our findings on the net effect suggest that the fragmentation cost channel is more important than the switching cost channel. Exploiting the industry variation in fragmentation and switching costs in the data, we have confirmed that this is indeed the case. The facilitating effect of DVCs is absent in industries characterized by low fragmentation costs, but high switching costs. ed The policy implications are relevant and straightforward. Key barriers to GVC integration might stem from the interplay of domestic linkages and industrial structure. These linkages should be taken into account when designing and evaluating GVC integration strategies. This article is protected by copyright. All rights reserved. References e Antr`as, P., 2003, “Firms, Contracts, and Trade Structure,” Quarterly Journal of Economics, 118, 1375–1418. c Antr`as, P., T. C. Fort, and F. Tintelnot, 2017, “The Margins of Global Sourcing: Theory and Evidence from U.S. Firms,” American Economic Review, 107, 2514–64. rti Antr`as, P., and E. Helpman, 2004, “Global Sourcing,” Journal of Political Economy, 112, 552– 580. This article is protected by copyright. All rights reserved. 580. Ar Balassa, B., 1965, “Trade Liberalisation and “Revealed” Comparative Advantage,” The Manch- ester School, 33, 99–123. A Baldwin, R., and J. Lopez Gonzalez, 2015, “Supply-chain Trade: A Portrait of Global Patterns d Baldwin, R., and J. 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All rights reserved. Article Figures Figure 1: GVC integration of matched countries 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! Notes: OECD data for 2008. Data are averaged across industries. GVC in- tegration is measured as foreign value added in exports (normalized by total exports). Country names are displayed in Table A-1. Figures Figure 1: GVC integration of matched countries 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! Notes: OECD data for 2008. Data are averaged across industries. GVC in- tegration is measured as foreign value added in exports (normalized by total exports). Country names are displayed in Table A-1. 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! Notes: OECD data for 2008. Data are averaged across industries. GVC in- tegration is measured as foreign value added in exports (normalized by total exports). Country names are displayed in Table A-1. Notes: OECD data for 2008. Data are averaged across industries. GVC in- tegration is measured as foreign value added in exports (normalized by total exports). Country names are displayed in Table A-1. Figure 2: Correlation scatterplot between GVC in 2008 and DVC in 1995 Notes: OECD data. Each point is a country-industry combination. The GVC measure is from equation (2.3). The DVC measure is from equation (2.5). Notes: OECD data. Each point is a country-industry combination. The GVC measure is from equation (2.3). The DVC measure is from equation (2.5). Notes: OECD data. Each point is a country-industry combination. The GVC measure is from equation (2.3). The DVC measure is from equation (2.5). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Figure 3: Differences in GVC integration of matched countries – revisited 0! 5! 10! 15! 20! 25! 30! 35! 40! 45! 50! SWE! BEL! CHE! SGP! ROU! BGR! CHM! CHM! PAP! CHM! CEQ! TEX! Notes: OECD data for 2008. Data are averaged across industries. GVC in- tegration is measured as foreign value added in exports (normalized by total exports). Country names are displayed in Table A-1. Notes: OECD data for 2008. Data are averaged across industries. GVC in- tegration is measured as foreign value added in exports (normalized by total exports). Country names are displayed in Table A-1. This article is protected by copyright. This article is protected by copyright. All rights reserved. All rights reserved. es Table 1: DVC integration by country in 1995 Top 10 Bottom 10 Country DVC Country DVC China 57.73% Austria 38.68% Singapore 56.03% Israel 38.11% Czech Republic 53.59% Canada 37.92% Slovakia 53.14% Greece 37.56% Bulgaria 51.37% Hong Kong 36.85% Korea 50.14% Malta 35.92% New Zealand 49.91% Mexico 34.69% Estonia 48.52% Luxembourg 29.76% Hungary 48.07% Cyprus 28.84% Croatia 47.95% Cambodia 23.50% Notes: OECD data for 1995. Data are averaged across industries. The DVC measure is from equation (2.5). Tables Tables Table 1: DVC integration by country in 1995 Table 1: DVC integration by country in 1995 Top 10 Bottom 10 Country DVC Country DVC China 57.73% Austria 38.68% Singapore 56.03% Israel 38.11% Czech Republic 53.59% Canada 37.92% Slovakia 53.14% Greece 37.56% Bulgaria 51.37% Hong Kong 36.85% Korea 50.14% Malta 35.92% New Zealand 49.91% Mexico 34.69% Estonia 48.52% Luxembourg 29.76% Hungary 48.07% Cyprus 28.84% Croatia 47.95% Cambodia 23.50% Notes: OECD data for 1995. Data are averaged across industries. The DVC measure is from equation (2.5). Top 10 Bottom 10 Table 2: Industries by category Table 2: Industries by category LFLS HFHS LFHS Agriculture Textile and leather products Mining and quarrying Food and beverages Wood products Pulp and paper products Coke and refined petroleum Non metallic mineral products Chemicals Basic metals Fabricated metals Rubber and plastics Machinery n.e.c. Electrical machinery n.e.c. Electronic and optical products Motor vehicles Other transport equipment Manufacturing n.e.c. Notes: Based on the Rauch and Nunn classifications. L - low, H - high, F - fragmentation costs, S - switching costs. Industries are defined using the ISIC Rev. 3, 2 digit classification (see Table A-2). Notes: Based on the Rauch and Nunn classifications. L - low, H - high, F - fragmentation costs, S - switching costs. Industries are defined using the ISIC Rev. 3, 2 digit classification (see Table A-2). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 3: The net effect of DVC integration on GVC integration (1) (2) (3) (4) Primary & Manufacturing All sectors dvc 2.566*** 2.275*** 2.900*** 2.422*** [0.451] [0.484] [0.379] [0.373] ca 1.519*** 1.099*** 1.472*** 1.025*** [0.074] [0.073] [0.065] [0.062] ctau -0.002 -0.014 [0.013] [0.019] Observations 1,025 1,025 1,449 1,449 R-squared 0.705 0.877 0.704 0.872 Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)). This article is protected by copyright. All rights reserved. Columns (1) and (3) include industry fixed effects and log of GDP, log of per capita GDP, and log of hub distance as controls. Columns (2) and (4) include industry and broad industry-country fixed effects. All sectors include primary, manufacturing and services. Table 3: The net effect of DVC integration on GVC integration Table 4: Industrial heterogeneity in the effect of DVC integration on GVC integration ble 4: Industrial heterogeneity in the effect of DVC integration on GVC integration ble 4: Industrial heterogeneity in the effect of DVC integration on GVC integrat (1) (2) (3) (4) Primary & Manufacturing All sectors dvc 2.275*** 2.422*** [0.484] [0.373] dvc  LFLS 3.068*** 3.081*** [0.706] [0.690] dvc  HFHS 2.336*** 2.552*** [0.788] [0.484] dvc  LFHS 0.196 0.123 [0.845] [0.818] ca 1.099*** 1.085*** 1.025*** 1.014*** [0.073] [0.074] [0.062] [0.062] ctau -0.014 -0.010 [0.019] [0.018] Observations 1,025 1,025 1,449 1,449 R-squared 0.877 0.879 0.872 0.873 Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)). Columns (1) and (3) report the benchmark estimations of columns (2) and (4) of Table 3. All columns include industry and broad industry-country fixed effects.All sectors include primary, manufacturing and services. Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)). Columns (1) and (3) report the benchmark estimations of columns (2) and (4) of Table 3. All columns include industry and broad industry-country fixed effects.All sectors include primary, manufacturing and services. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table 5: The net effect of DVC integration on GVC integration – Robustness (1) (2) (3) (4) (5) (6) (7) (8) (9) Benchmark High income Panel IV GVC measure All countries All sectors 2005 2011 Dep. Variable: fvax fvax fvax i2p fvax fvax fvax fvax fvax dvc 2.275*** 1.892*** 0.568** 2.744*** 2.008*** 1.717*** 2.598*** 2.127*** 2.448*** [0.484] [0.716] [0.222] [0.630] [0.449] [0.506] [0.346] [0.462] [0.530] dvc  high income 0.770 [0.867] ca 1.099*** 1.098*** 0.240*** 1.103*** 0.749*** 1.167*** 1.090*** 1.210*** 1.157*** [0.073] [0.072] [0.032] [0.063] [0.060] [0.081] [0.063] [0.071] [0.085] ctau -0.014 -0.014 0.002 -0.015 0.020 0.009 0.001 -0.014 [0.019] [0.019] [0.006] [0.016] [0.016] [0.024] [0.016] [0.019] Cragg-Donald Wald F statistic 176.6 First stage (dep. This article is protected by copyright. All rights reserved. variable: dvc) dvcIV -19.173*** [3.761] Observations 1,025 1,025 2,896 1,025 1,026 1,078 1,883 1,025 989 R-squared 0.877 0.877 0.748 0.877 0.884 0.881 0.858 0.881 0.873 Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)) in columns (1)-(3) and (5)-(9); i2p (see equation (2.4)) in column (4). All columns excluding column (3) include industry and broad industry-country fixed effects. Column (3) includes country-year, industry-year, and industry-country fixed effects. All columns excluding column (7) exclude services industries. Number of observations in first stage regression: 1,025. dvcIV defined in footnote 26. protected by copyright. All rights reserved. e net effect of DVC integration on GVC integration – Robustness This article is protected by copyright. All rights reserved. Table 6: Industrial heterogeneity in the effect of DVC integration on GVC integration – Robustness Table 6: Industrial heterogeneity in the effect of DVC integration on GVC integration – Robustness (1) (2) (3) (4) (5) Benchmark IV Cutoff Complexity Capital intensity dvc  LFLS 3.068*** 2.271*** 3.106*** 3.050*** 3.093*** [0.706] [0.637] [0.766] [0.841] [0.776] dvc  HFHS 2.336*** 4.603*** 1.962*** 2.472*** 1.929*** [0.788] [1.025] [0.694] [0.684] [0.694] dvc  LFHS 0.196 1.169 0.701 1.236 1.621 [0.845] [0.863] [0.930] [0.976] [1.082] ca 1.085*** 1.074*** 1.093*** 1.100*** 1.100*** [0.074] [0.064] [0.075] [0.075] [0.074] ctau -0.010 -0.012 -0.012 -0.020 -0.014 [0.018] [0.016] [0.018] [0.020] [0.019] (2.a) (2.b) (2.c) Cragg-Donald Wald F statistic 54.65 First stage dep. variable: dvc  LFLS dvc  HFHS dvc  LFHS dvcIV  LFLS -18.462*** 0.026 1.065 [4.864] [0.710] [0.655] dvcIV  HFHS 1.533* -25.414*** 0.186 [0.917] [4.819] [0.769] dvcIV  LFHS 3.607*** -0.146 -22.595*** [1.308] [1.366] [3.089] Observations 1,025 1,025 1,025 911 1,025 R-squared 0.879 0.878 0.878 0.879 0.878 Notes: *** p@0.01, ** p@0.05, * p@0.1. Robust standard errors in parentheses. Dependent variable: fvax (see equation (2.3)). All columns include industry and broad industry-country fixed effects. All columns exclude services industries. Number of observations in first stage regressions: 1,025. dvcIV defined in footnote 26. Table 6: Industrial heterogeneity in the effect of DVC integration o This article is protected by copyright. All rights reserved. Note: Countries in bold excluded in benchmark estimations (Tables 3 and 4). This article is protected by copyright. All rights reserved. pted Article Appendices A Data Table A-1: OECD ICIO country coverage ISO3 Country ISO3 Country AUS Australia ITA Italy ARG Argentina JPN Japan AUT Austria KHM Cambodia BEL Belgium KOR Korea BGR Bulgaria LTU Lithuania BRA Brazil LUX Luxembourg BRN Brunei LVA Latvia CAN Canada MEX Mexico CHE Switzerland MLT Malta CHL Chile MYS Malaysia CHN China NLD Netherlands COL Colombia NOR Norway CRI Costa Rica NZL New Zealand CYP Cyprus PHL Philippines CZE Czech Republic POL Poland DEU Germany PRT Portugal DNK Denmark ROU Romania ESP Spain RUS Russia EST Estonia SAU Saudi Arabia FIN Finland SGP Singapore FRA France SVK Slovak Republic GBR United Kingdom SVN Slovenia GRC Greece SWE Sweden HKG Hong Kong, China THA Thailand HRV Croatia TUN Tunisia HUN Hungary TUR Turkey IDN Indonesia TWN Chinese Taipei IND India USA United States IRL Ireland VNM Viet Nam ISL Iceland ZAF South Africa ISR Israel Note: Countries in bold excluded in benchmark estimations (Tables 3 and 4). Table A-1: OECD ICIO country coverage ISO3 Country ISO3 Country AUS Australia ITA Italy ARG Argentina JPN Japan AUT Austria KHM Cambodia BEL Belgium KOR Korea BGR Bulgaria LTU Lithuania BRA Brazil LUX Luxembourg BRN Brunei LVA Latvia CAN Canada MEX Mexico CHE Switzerland MLT Malta CHL Chile MYS Malaysia CHN China NLD Netherlands COL Colombia NOR Norway CRI Costa Rica NZL New Zealand CYP Cyprus PHL Philippines CZE Czech Republic POL Poland DEU Germany PRT Portugal DNK Denmark ROU Romania ESP Spain RUS Russia EST Estonia SAU Saudi Arabia FIN Finland SGP Singapore FRA France SVK Slovak Republic GBR United Kingdom SVN Slovenia GRC Greece SWE Sweden HKG Hong Kong, China THA Thailand HRV Croatia TUN Tunisia HUN Hungary TUR Turkey IDN Indonesia TWN Chinese Taipei IND India USA United States IRL Ireland VNM Viet Nam ISL Iceland ZAF South Africa ISR Israel Note: Countries in bold excluded in benchmark estimations (Tables 3 and 4). This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table A-2: OECD ICIO industry coverage pted Article ISIC Industry Industry Broad Rev. pt Notes: Industries in bold are classified as non-tradeable. Benchmark estimations (Tables 3 and 4) only include industries up to ISIC 37. This article is protected by copyright. All rights reserved. 3 code description industry 01T05 AGR Agriculture Primary 10T14 MIN Mining and quarrying Primary 15T16 FOD Food products, beverages, and tobacco Light manufacturing 17T19 TEX Textiles, leather and footwear Light manufacturing 20 WOD Wood and products of wood and cork Light manufacturing 21T22 PAP Pulp, paper, paper products, printing and publishing Light manufacturing 23 PET Coke, refined petroleum products and nuclear fuel Raw material processing 24 CHM Chemicals and chemical products Raw material processing 25 RBP Rubber and plastics products Raw material processing 26 NMM Other non-metallic mineral products Raw material processing 27 MET Basic metals Heavy manufacturing 28 FBM Fabricated metal products Heavy manufacturing 29 MEQ Machinery and equipment n.e.c Heavy manufacturing 30,32,33 CEQ Computer, electronic and optical products Electronics 31 ELQ Electrical machinery and apparatus n.e.c Electronics 34 MTR Motor vehicles, trailers and semi-trailers Heavy manufacturing 35 TRQ Other transport equipment Heavy manufacturing 36T37 OTM Manufacturing n.e.c; recycling Light manufacturing 40T41 EGW Electricity, gas and water supply Services 45 CON Construction Services 50T52 WRT Wholesale and retail trade Services 55 HTR Hotels and restaurants Services 60T63 TRN Transport and storage Services 64 PTL Post and telecommunications Services 65T67 FIN Finance and insurance Services 70 REA Real estate activities Services 71 RMQ Renting of machinery and equipment Services 72 ITS Computer and related activities Services 73T74 BZS Research and development and other business services Services 75 GOV Public administration and defence Services 80 EDU Education Services 85 HTH Health and social work Services 90T93 OTS Other community, social and personal services Services 95 PVH Private households with employed persons Services Notes: Industries in bold are classified as non-tradeable. Benchmark estimations (Tables 3 and 4) only include industries up to ISIC 37. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Notes: Industries are ranked by decreasing DVC integration. Only industries included in benchmark estimations (Tables 3 and 4) are reported. The DVC measure is defined in equation (2.5). Industry descriptions are displayed in Table A-2. ti B Construction of cost proxies rt B.1 Fragmentation costs Table A-5: Summary statistics for main variables Table A-5: Summary statistics for main variables Variable Observations Mean Std. Dev. Min Max dvc 1025 0.54 0.13 0.06 0.94 fvax 1025 6.28 2.13 -2.65 12.23 ca 1025 0.36 0.48 0.00 1.00 ctau 1025 4.92 4.48 0.00 30.54 Notes: In-sample statistics based on benchmark estimations (column (2) of Table 3). The variable dvc (see equation (2.5)) is for the year 1995. The variable fvax (see equation (2.3)) is for the year 2008. This article is protected by copyright. All rights reserved. Table A-3: DVC integration, country average across industries Table A-3: DVC integration, country average across industries ISO3 DVC ISO3 DVC CHN 0.58 CHL 0.44 SGP 0.56 TUR 0.43 CZE 0.54 SVN 0.43 SVK 0.53 RUS 0.43 BGR 0.51 IDN 0.43 KOR 0.50 USA 0.43 NZL 0.50 FRA 0.42 EST 0.49 PHL 0.42 TWN 0.48 DEU 0.42 HUN 0.48 CHE 0.41 HRV 0.48 ISL 0.41 ROU 0.48 MYS 0.41 POL 0.48 CRI 0.40 AUS 0.47 TUN 0.39 VNM 0.47 DNK 0.39 BEL 0.47 COL 0.39 IRL 0.47 NOR 0.39 ESP 0.47 ARG 0.39 PRT 0.46 AUT 0.39 ITA 0.46 ISR 0.38 LVA 0.46 CAN 0.38 FIN 0.45 GRC 0.38 IND 0.45 HKG 0.37 LTU 0.45 MLT 0.36 BRA 0.44 MEX 0.35 JPN 0.44 LUX 0.30 ZAF 0.44 CYP 0.29 GBR 0.44 SAU 0.28 NLD 0.44 BRN 0.27 THA 0.44 KHM 0.24 SWE 0.44 Notes: Countries are ranked by decreasing DVC integration. Only countries included in benchmark estimations (Tables 3 and 4) are reported. The DVC measure is defined in equation (2.5). Country names are displayed in Table A-1. Notes: Countries are ranked by decreasing DVC integration. Only countries included in benchmark estimations (Tables 3 and 4) are reported. The DVC measure is defined in equation (2.5). Country names are displayed in Table A-1. Notes: Countries are ranked by decreasing DVC integration. Only countries included in benchmark estimations (Tables 3 and 4) are reported. The DVC measure is defined in equation (2.5). Country names are displayed in Table A-1. This article is protected by copyright. All rights reserved. Table A-4: DVC integration, industry average across countries Table A-4: DVC integration, industry average across countries ISIC Rev. 3 Industry code DVC 34 MTR 0.70 15T16 FOD 0.69 27 MET 0.66 17T19 TEX 0.64 20 WOD 0.63 25 RBP 0.62 35 TRQ 0.61 24 CHM 0.60 36T37 OTM 0.60 30,32,33 CEQ 0.59 29 MEQ 0.59 31 ELQ 0.58 21T22 PAP 0.58 28 FBM 0.54 26 NMM 0.52 23 PET 0.49 60T63 TRN 0.44 01T05 AGR 0.41 65T67 FIN 0.38 73T74 BZS 0.36 50T52 WRT 0.35 72 ITS 0.35 10T14 MIN 0.32 71 RMQ 0.31 64 PTL 0.31 Notes: Industries are ranked by decreasing DVC integration. Only industries included in benchmark estimations (Tables 3 and 4) are reported. The DVC measure is defined in equation (2.5). Industry descriptions are displayed in Table A-2. This article is protected by copyright. All rights reserved. Ar Rauch (1999)’s product differentiation d A As discussed in Section 2, we use the Rauch classification as benchmark measure for fragmen- tation costs. The classification is available in two variants (liberal and conservative version) at the 4 digit SITC Rev. 2 level. We choose the liberal version, which we concord to ISIC Rev. 3 using a crosswalk via SITC Rev. 3 based on concordance tables provided by Eurostat. pte For the analysis, this creates two problems. First, the classification is only available for the primary and manufacturing sectors. Second, due to the higher disaggregation several SITC categories are matched with each individual ISIC industry. We take the first issue into account by excluding industries in the services sector from the benchmark estimates and, in addition, by including services in robustness checks in which we label them as differentiated. Acce The second point is only problematic when the SITC categories concorded to ISIC industries differ in their categorization. To determine the ISIC industry’s category in this case, we use gobal trade data from WITS at the SITC 4 digit level. We classify ISIC industries as differentiated if the trade share of differentiated SITC industries matched with them exceeds the mean value across all ISIC industries. Alternatively, we use the median value for robustness checks. A Costinot (2009)’s complexity A Costinot (2009)’s complexity In cases where there are several complexity observations matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting industry level values of the complexity variable. ed A categories. This means several SIC industries have identical complexity values. We match these with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables by the NBER and Statistics Canada. In cases where there are several complexity observations matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting industry level values of the complexity variable. pt B.2 Switching costs p Nunn (2007)’s contract intensity p Nunn (2007)’s contract intensity A Costinot (2009)’s complexity A Costinot’s measure is available for 41 unique observations covering 85 3 digit manufacturing SIC A Costinot’s measure is available for 41 unique observations covering 85 3 digit manufacturing SIC This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. Table B-1: Industry complexity Table B-1: Industry complexity ISIC Rev. 3 Industry code Complexity 35 TRQ 28.23 24 CHM 26.32 30,32,33 CEQ 25.36 29 MEQ 21.98 34 MTR 21.77 28 FBM 19.42 27 MET 18.29 21T22 PAP 17.29 31 ELQ 15.84 26 NMM 15.59 20 WOD 12.47 15T16 FOD 12.38 36T37 OTM 11.41 25 RBP 10.10 17T19 TEX 5.04 Notes: Industries are ranked by decreasing value of complexity. Complexity is the average number of training days a new workers requires to be prepared for a job in a given industry. The data are taken from Costinot (2009) and concorded to ISIC Rev. 3. industry descriptions displayed in Table A-2. Notes: Industries are ranked by decreasing value of complexity. Complexity is the average number of training days a new workers requires to be prepared for a job in a given industry. The data are taken from Costinot (2009) and concorded to ISIC Rev. 3. industry descriptions displayed in Table A-2. ed A categories. This means several SIC industries have identical complexity values. We match these with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables by the NBER and Statistics Canada. In cases where there are several complexity observations matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting industry level values of the complexity variable. ed A categories. This means several SIC industries have identical complexity values. We match these with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables by the NBER and Statistics Canada. In cases where there are several complexity observations matched with one ISIC industry, we use the simple mean. Table B-1 presents the resulting industry level values of the complexity variable. ed A categories. This means several SIC industries have identical complexity values. We match these with our ISIC industries using a crosswalk via the 1987 SIC revision using concordance tables by the NBER and Statistics Canada. This article is protected by copyright. All rights reserved. p Nunn (2007)’s contract intensity p Nunn (2007)’s contract intensity ce Nunn’s contract intensity z is given by the share of differentiated intermediates in total inter- mediates that an industry sources as follows: zi Q j θjiDifj, A where θji  mji~Pj mji, and Dif is a dummy equal to one if industry j is differentiated and m’s are generic elements of an ICIO matrix. Thus, the measure is built based on the Rauch classification and we can apply the values calculated using the methodology described above. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. le To simplify the empirical analysis we then transform this continuous variable into a dummy by defining each industry j as having high switching costs if zi is at least equal to the median z of non-differentiated industries. For robustness exercises we replace this measure with capital intensity data readily available from the OECD. This article is protected by copyright. All rights reserved.
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Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota
Antibiotics
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cc-by
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antibiotics Article Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota Pedro Miguela-Villoldo 1,2 , Miguel A. Moreno 1,2 , Agustín Rebollada-Merino 1,3 , Antonio Rodríguez-Bertos 1,3 , Marta Hernández 4 , David Rodríguez-Lázaro 5 , Alejandro Gallardo 6 , Alberto Quesada 6,7 , Joaquín Goyache 1,2 , Lucas Domínguez 1,2 and María Ugarte-Ruiz 1, * 1 2 3 4 5 6   Citation: Miguela-Villoldo, P.; Moreno, M.A.; Rebollada-Merino, A.; 7 * VISAVET Health Surveillance Centre, Universidad Complutense de Madrid, Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain; pedromig@ucm.es (P.M.-V.); mamoreno@ucm.es (M.A.M.); agusrebo@ucm.es (A.R.-M.); arbertos@ucm.es (A.R.-B.); jgoyache@ucm.es (J.G.); lucasdo@visavet.ucm.es (L.D.) Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain Departamento de Medicina y Cirugía Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain Laboratorio de Biología Molecular y Microbiología, Instituto Tecnológico Agrario de Castilla y León, Ctra. Burgos Km. 119, 47071 Valladolid, Spain; ita-HerPerMa@itacyl.es Área de Microbiología, Departamento de Biotecnología y Ciencia de los Alimentos, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain; drlazaro@ubu.es Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Avda. de la Universidad s/n, 10003 Caceres, Spain; alexgsoler@unex.es (A.G.); aquesada@unex.es (A.Q.) INBIO G+C, Universidad de Extremadura, 0004 Caceres, Spain Correspondence: maria.ugarte@ucm.es; Tel.: +34-913-944-097; Fax: +34-913-943-795 Rodríguez-Bertos, A.; Hernández, M.; Rodríguez-Lázaro, D.; Gallardo, A.; Quesada, A.; Goyache, J.; Domínguez, L.; et al. Colistin Selection of the Mcr-1 Gene in Broiler Chicken Intestinal Microbiota. Antibiotics 2021, 10, 677. https://doi.org/10.3390/ antibiotics10060677 Academic Editor: Mark A Holmes Received: 7 May 2021 Accepted: 3 June 2021 Published: 5 June 2021 Abstract: Colistin has a long story of safe use in animals for the treatment and prevention of certain bacterial diseases. Nevertheless, the first description of the mcr-1 gene showed that colistin resistance can spread by horizontal gene transfer and changed the landscape. This study aimed to assess the effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants. In this study, 100 one-day-old chicks were divided into four groups of 25 animals (G1, G2, G3, and G4). Animals from G3/G4 were challenged with mcr-1-carrying Salmonella (day 7), while colistin (600 mg/L) was administered daily to G2/G4 animals through drinking water (from day 8 to day 15). Two quantitative PCR assays were performed to compare the amount of Salmonella and mcr-1 that were present in the caecal samples. We observed that levels of mcr-1 were higher in G3/G4 animals, especially G4, due to the spread of mcr-1-carrying Salmonella. On day 21, Salmonella levels decreased in G4, reaching similar values as those for G3, but mcr-1 levels remained significantly higher, suggesting that colistin may accelerate the spreading process when mcr-1-carrying bacteria reach the gut. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: colistin; mcr-1; Salmonella; in vivo assay; real-time PCR quantification; microbiota; chicks published maps and institutional affiliations. 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ The gut microbiome constitutes a rich and diverse microbial ecosystem that influences host nutrition and physiology, developing important functions that are beneficial for host health [1]. The microbiota of new-born chicks, first colonised by bacteria of the facultative anaerobic Proteobacteria phylum, progressively changes during the first 19 days of life [2]. Commensal Enterobacteriaceae is a major constituent of Proteobacteria and plays an important role in the protection of the gut against colonisation by pathogens, such as Salmonella enterica (especially serovars Typhimurium and Enteritidis), obligate anaerobic spore-forming bacteria, such as Clostridia species, and facultative anaerobic 4.0/). Antibiotics 2021, 10, 677. https://doi.org/10.3390/antibiotics10060677 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 677 2 of 10 bacteria from the Streptococcus and Staphylococcus genera [3]. However, Proteobacteria, especially Enterobacteriaceae, play a critical role in the emergence and maintenance of antimicrobial resistance, as the genes responsible are more prone to be found in this phylum (and also in Firmicutes) than in other phyla of the gut microbiome [4]. For example, plasmidencoded resistance genes can move between gut bacteria via horizontal gene transfer. Thus, commensal Enterobacteriaceae become an excellent reservoir for antimicrobial resistance genes, making them accessible to other pathogenic or commensal bacteria [4,5]. Antimicrobial resistance is a public health concern worldwide and has become an increasing threat to human and animal health over the last decade, limiting the therapeutic alternatives available to treat infections caused by multidrug-resistant (MDR) pathogens. The increase of human infections due to multidrug-resistant Gram-negative bacteria, especially those producing extended-spectrum beta-lactamases and carbapenemases, has forced the reintroduction of colistin to treat infections caused by these microorganisms in human medicine, as it is often the only effective antimicrobial against them [6–8]. Consequently, colistin has been classified among the antibiotic groups with activity against “Critical Priority” or “High Priority” pathogens identified by the World Health Organisation (WHO) [9] and is considered to be the last line to treat these infections. The prevalence of colistin resistance in Enterobacteriaceae from food animals has been increasing annually and is thought to be related to the use of colistin in veterinary medicine on a global scale [10,11]. Colistin has been used for decades in animals to treat and prevent infectious diseases [6,12,13], usually administered orally in feed or drinking water [14,15]. The Committee for Medicinal Products for Veterinary Use (CVMP) of the European Medicines Agency (EMA) has restricted the administration of colistin sulphate to poultry to 3 to 7 days to treat gastrointestinal infections [16]. Until 2015, all described colistin resistance mechanisms were related to chromosomal point mutations, mainly involving the two-component regulatory system (pmrAB/phoPQ) and its negative regulator mgrB. However, a new plasmid-mediated gene, called mcr-1, was first described in that year, which meant that horizontal transfer was possible [8,17]. Thus, due to the increase of colistin resistance and the emergence of the mcr-1 gene, the EMA recommended minimising the use of colistin in animals in the European Union (EU) in 2016, aiming to limit its use to 5 mg/population correction unit (PCU) at the national level, with a desirable level of 1 mg/PCU [15]. Since then, colistin resistance levels have appeared to drop with the decrease in its use. We also observed this trend in a recent study carried out in Spain [18]. There have been no studies of the effects of colistin administration on the intestinal microbiota of the neonatal gut and its ability to select colistin-resistant bacteria. Thus, we aimed to assess the effect of colistin administration on the dispersion of resistance in the microbiota of day-old broiler chicks and how the presence of mcr-1 genes influences the spread of colistin resistance determinants using a monophasic Salmonella Typhimurium strain carrying a mcr-1 gene. 2. Results 2.1. Clinical Signs and Pathological Findings Four animals (two animals from G1, one from G3, and one from G4) died during the first week during the adjustment period. Post-mortem examination revealed fibrinous pericarditis and perihepatitis (septicaemic colibacillosis) as the cause of death. We collected samples from these animals, as their deaths occurred the day before the first sampling. Thus, none of the samples were excluded from the experiment. There were no significant changes found in any animal included in the study during the post-mortem examinations throughout the experiment. 2.2. Salmonella Counts in the Chick Samples The amount of Salmonella in the chick caecal samples was estimated by plate count. Only animals from the Salmonella-challenged groups (G3 and G4) showed growth of Antibiotics 2021, 10, x FOR PEER REVIEW Antibiotics 2021, 10, 677 3 of 11 3 of 10 The amount of Salmonella in the chick caecal samples was estimated by plate count. Only animals from the Salmonella-challenged groups (G3 and G4) showed growth of SalSalmonella SMID2 plates (Figure mean values non-statistically different monella ononSMID2 plates (Figure 1). 1). TheThe mean values werewere non-statistically different (p < 1 to 1.2 × 105 CFU/mg (mean of 2.0 × 104 CFU/mg) for G3 (p 0.05): < 0.05): from <5.4 × 10 1 5 4 from <5.4 × 10 to 1.2 × 10 CFU/mg (mean of 2.0 × 10 CFU/mg) for G3 (without 1 to 3.6 × 105 CFU/mg (mean of 2.3 × 104 CFU/mg) (without and colistin)colistin) and from <5.4from × 101<5.4 to 3.6××10 105 CFU/mg (mean of 2.3 × 104 CFU/mg) for G4 (colistin fortreated). G4 (colistin treated). Chicks from G4 had higher Salmonella counts from D9 time to D14, Chicks from G4 had higher Salmonella counts from D9 to D14, at which G4 at which time G4 reached the highest value. Finally, after the withdrawal of colistin (D14), reached the highest value. Finally, after the withdrawal of colistin (D14), both G3 and G4 both G3 and G4 showed similar CFU counts (FigureSalmonella 1). All recovered Salmonella showed similar CFU counts on D21 (Figure 1).on AllD21 recovered isolates were mcrisolates werebymcr-1-positive by conventional PCR. 1-positive conventional PCR. Figure 1. Evolution of Salmonella counts over 21 days in chick counts caecal samples: CTRL: control group; Group 1 (G1): animals Figure 1. Evolution of Salmonella over 21 days in chick caecal samples: CTRL: control Group 1 (G1): animals without colistin nor Group Salmonella challenge; Group 2 (G2): colistin adwithout colistin nor Salmonellagroup; challenge; Group 2 (G2): colistin administered; 3 (G3): Salmonella challenged; Group ministered; Group 3 (G3): Salmonella challenged; Group1 4Salmonella (G4): colistin administered Salmo4 (G4): colistin administered and Salmonella challenged. Dots represent outliers; challenge startedand at D7 for nella challenged. Dots represent outliers; 1 Salmonella challenge started at D7 for groups G3 and G4 groups G3 and G4 (sampling from D7 was performed before the challenge was started); 2 Colistin was provided to chicks (sampling from D7 was performed before the challenge was started); 2 Colistin was provided to from groups G2 and G4 from D8 to D14. chicks from groups G2 and G4 from D8 to D14. 2.3. Quantitative 2.3. QuantitativeqPCR qPCRofofthe themcr-1 mcr-1 Gene Gene InIntotal, caecal samples sampleswere wereanalysed analysed (7, 20, 20, 20, 20,and 20, 20 and 20 samtotal,87 87of of 100 100 caecal on on D7 D7 (7, 20, samples from the the control group, G1, G2, and G4,and respectively). Overall, 13Overall, samples13 were exples from control group, G1,G3, G2, G3, G4, respectively). samples cluded because it was it impossible to obtain samplessamples (at least (at 100least mg)100 for mg) DNAfor were excluded because was impossible to enough obtain enough extraction. DNA extraction. QuantitativePCR PCRdata datafor forthe the mcr-1 mcr-1 gene of group and Quantitative of the the77animals animalsfrom fromthe theCTRL CTRL group and 21 animals from G1 (neither receiving colistin nor challenged with Salmonella) showed 21 animals from G1 (neither receiving colistin nor challenged with Salmonella) showed high inter-individualvariability. variability. Two Two animals animals from the barely high inter-individual fromthe theCTRL CTRLgroup groupshowed showed the barely detectable presence of the mcr-1 gene, which remained below the quantification limit, and detectable presence of the mcr-1 gene, which remained below the quantification limit, and 33to 1.6 × 106 mcr-1 6 four animals from G1 showed quantifiable levels ranging from 6.7 × 10 four animals from G1 showed quantifiable levels ranging from 6.7 × 10 to 1.6 × 10 mcr-1 genecopies copiesper permg mgcaecal caecalcontent, content, establishing establishing aa slight 1717 gene slight background. background.The Theremaining remaining chicks showedno noqPCR qPCRsignals signalsor or showed showed mcr-1 The chicks showed mcr-1 values valuesbelow belowthe thequantitation quantitationlimit. limit. The values for the mcr-1 levels from the groups not challenged with Salmonella (G1 and G2) values for the mcr-1 levels from the groups not challenged with Salmonella (G1 and G2) remainedsimilar similarthroughout throughout most reaching their peaks on D11 (3.2 ×(3.2 105 and remained mostofofthe thestudy, study, reaching their peaks on D11 × 105 6 and 2.6 × 10 gene copies per mg caecal content, respectively) (Table 1). Concerning the Salmonella-challenged groups (G3 and G4), chicks from G3 (colistin untreated) showed an increase from less than 1.0 × 102 to 1.9 × 105 mcr-1 copies/mg from D9 to D21. The 2.6 × 106 gene copies per mg caecal content, respectively) (Table 1). Concerning the Salmonella-challenged groups (G3 and G4), chicks from G3 (colistin untreated) showed an increase from less than 1.0 × 102 to 1.9 × 105 mcr-1 copies/mg from D9 to D21. The number of Antibiotics 2021, 10, 677 of5 10 mcr-1 copies/mg for chicks from G4 (colistin treated) increased from 1.5 × 103 to 2.1 × 410 for the same period. The greatest differences between the four groups were observed on D11 and D14, coinciding with the end of colistin administration, with G4 showing significantly higher mcr-1 values, as the median of these values was outside the interquartile number of mcr-1 copies/mg for chicks from G4 (colistin treated) increased from 1.5 × 103 range of the other groups (Figure 2). In addition, chickens from G1 and G2 showed a nonto 2.1 × 105 for the same period. The greatest differences between the four groups were significant increase in the median from D9 to D21 (p = 0.23), whereas mcr-1 levels increased observed on D11 and D14, coinciding with the end of colistin administration, with G4 throughout the study in the challenged groups, G3 and G4. showing significantly higher mcr-1 values, as the median of these values was outside the interquartile range ofPCR thedata other (Figure 2). In G1 and G2 Table 1. Quantitative forgroups mcr-1 gene copies/mg peraddition, day of lifechickens (expressedfrom in Log10). showed a non-significant increase in the median from D9 to D21 (p = 0.23), whereas mcr-1 mcr-1 gene copies/mg (expressedgroups, in Log10) levels increased throughout the study in the challenged G3 and G4. 1 SC CA Day 7 Day 9 Day 11 Day 14 Day 21 Table 1. Quantitative PCR data for mcr-1 gene copies/mg per day of life (expressed in Log10). ̅ ̅ ̅ ̅ ̅ (D7) (D8) 𝒙 Me 𝒙 Me 𝒙 Me 𝒙 Me 𝒙 Me CTRL 2 G1 G2 G3 G4 SC (D7) CA (D8) No No No Yes Yes No No Yes No Yes CTRL mcr-1 Gene Copies/mg (Expressed in Log10) No No 2.07 <2.0 NA NA NA 2 Day 7 1 ¯ G1 No Me x G2 No Day 9 No¯ x Yes NA No 2.49 Yes <2.0 NA NA NA NA NA Me NA Day 11 ¯ 2.49 2.60Me5.51 x <2.0 <2.0 6.43 NA NA NA Day 14 ¯ 2.52 3.83 Me 2.47 x 2.75 3.67 <2.0 NA NA Day 21 ¯3.24 <2.0 Me x 2.60 <2.0 2.07 <2.0 NA NA NA NA NA NA NA Yes NA 5.26 2.90 5.32 5.47 5.29 NA G3 NA 2.60 5.51 <2.02.524.47 3.83 2.47 5.24 3.24 <2.0 Yes NA 4.23 5.38 6.37 5.68 5.34 NA G4 NA <2.0 6.43 3.022.755.46 3.67 <2.0 6.01 2.60 <2.0 NA <2.0 4.47(D8): Colistin 2.90 administration 5.32 5.24 5.47 5.29 SC (D7): NA Salmonella 5.26 challenge (at day 7), CA (colistin treatment NA 5.46data were 5.38not analysed 6.37 due 6.01 5.68 of animals; 5.34 started atNA day 8), 𝑥̅ :4.23 Mean, Me:3.02 median; NA: to the absence CTRL: control animals without colistin nor Salmonella challenge; G2: Me: colistin adminisSC (D7): Salmonella challenge (at day 7), CA (D8): group; ColistinG1: administration (colistin treatment started at day 8), x: Mean, median; NA: 1 the CTRL G3:ofSalmonella challenged; colistin and Salmonella challenged; data were not analysed due to thetered; absence animals; CTRL: controlG4: group; G1: administered animals without colistin nor Salmonella challenge; G2: samples wereG4: composed of a differentand number of animals due 1tothe theCTRL inability to obtain sufficient colistin administered; G3: Salmonella challenged; colistin administered Salmonella challenged; samples were composed 2 CTRL of a different number of animals due to the inability obtainFive sufficient samples from 13 chicks. Five animals group were sampled samples from 13 to chicks. animals per group were sampled for the per other days; datafor only the other days; 2 CTRL data only available for D7. After D7, chicks were considered from different groups (G1 to G4), depending on the on available for D7. After D7, chicks were considered from different groups (G1 to G4), depending experimental conditions. the experimental conditions. Figure 2. Quantitative qPCR data of mcr-1 gene copies/mg obtained per day of life of the chicks for each studied group: Group 1 (G1): animals without colistin nor Salmonella challenge; Group 2 (G2): colistin administered; Group 3 (G3): Salmonella challenged; Group 4 (G4): colistin administered and Salmonella challenged. Dots represent outliers; 1 Salmonella challenge started at D7 for groups G3 and G4 (sampling from D7 was performed before starting the challenge); 2 Colistin was provided to chicks from groups G2 and G4 from D8 to D14. Antibiotics 2021, 10, 677 5 of 10 2.4. Comparison of Salmonella Spp. and mcr-1 Quantitative qPCR Data Quantitative PCR data for Salmonella quantification for the groups, including chicks inoculated with mcr-1-carrying Salmonella, showed median values for G3 (without colistin) of <3.3 × 102 , 5.1 × 103 , 1.5 ×1 03 , 1.3 × 104 , and 1.1 × 104 copies per mg for days 7, 9, 11, 14, and 21, respectively. For G4 (colistin administered), the corresponding median values were <3.3×102 , 1.7 × 103 , 1.7 × 106 , 4.7 × 104 , and 1.6 × 104 copies/mg (Figure 3). Quantification of mcr-1 and Salmonella showed similar values on D9 (p < 0.05, Table 2), indicating that most copies of the mcr-1 genes in the samples came from the inoculated S. Typhimurium strain. The levels of mcr-1 and Salmonella increased thereafter in G4, during the colistin administration period, whereas the levels remained approximately the same in G3. After the withdrawal of colistin, Salmonella levels in G4 decreased, reaching G3-equivalent values on D21. However, mcr-1 levels of both groups remained significantly Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 11 higher (Table 2), suggesting that mcr-1 may have been transferred to other intestinal bacterial species (Figure 3). Figure 3. Comparison between qPCR3.data for Salmonella and mcr-1 throughoutand themcr-1 studyobtained for the challenged groups Figure Comparison between qPCRobtained data for Salmonella throughout the study for the challenged groups (G3 4and G4): Groupadministered 3 (G3): Salmonella Group 4 (G4): colistin (G3 and G4): Group 3 (G3): Salmonella challenged; Group (G4): colistin and challenged; Salmonella challenged. Dots administered Salmonella challenged. Dots outliers; 1 Colistin was provided tofor chicks represent outliers; 1 Colistin was provided toand chicks from group G4, from D8represent to D14; * Salmonella challenge started on D7 from group G4, from D8 to D14; * Salmonella challenge started on D7 for groups G3 and G4 (samgroups G3 and G4 (sampling from D7 was performed before starting the challenge). pling from D7 was performed before starting the challenge). Table 2. T-test p-values obtained from a comparative analysis of mcr-1 and Salmonella qPCR quantitative data per sampling day. 3. Discussion p-Values Food-producing animals have been highlighted as potential reservoirs for the dissemination of colistin-resistance determinants, especially 2015, when Liu qPCR Data Group D7 D9 D11 since lateD14 D21 et al. identified the plasmid-mediated colistin resistance gene, mcr-1, in China [17]. Horizontal mcr-1 G3 vs. G4 ND 0.41 0.03 * 0.02 * 0.86 gene transfer have a critical role 0.03 in the Salmonella G3 vs. processes G4 ND been proposed 0.69 to play0.02 * * spreading 0.69of the mcr-1 gene [19,20]. In the present study, colistin administration had a significant effect mcr-1 vs. Salmonella G3 ND 0.06 0.44 * <0.01 * <0.01 * on the spread G4 of mcr-mediated in <0.01 the chicks’ microbiota. Its effect mcr-1 vs. Salmonella ND colistin resistance 0.94 * <0.01 * <0.01 *was greater when mcr-1-carrying bacteria were introduced into the gut environment in the * Statistically significant differences (p < 0.05); G3: Salmonella challenged; G4: colistin administered and Salmonella challenged.; Sampling colistin, mcr-1 colistin resistance appeared become widedays: D7: day 7; D9: day 9; D11: presence day 11; D14:ofday 14; D21: in daywhich 21.; ND:case The t-test was not applied for data from D7 due to the different number of samples available fromspread. each group. We used a Salmonella enterica serovar Typhimurium strain because neonatal chicks are highly susceptible to colonisation by Salmonella serovars [21] and it is easily identified in chicks because it is not a component of their early microbiota. We studied the effect of colistin by administering it for seven days, as recommended by the EMA for treatment of enteric infections caused by susceptible non-invasive E. coli [15], and focused on the mcr1 gene because it is the mobile gene most frequently related to colistin resistance world- Antibiotics 2021, 10, 677 6 of 10 3. Discussion Food-producing animals have been highlighted as potential reservoirs for the dissemination of colistin-resistance determinants, especially since late 2015, when Liu et al. identified the plasmid-mediated colistin resistance gene, mcr-1, in China [17]. Horizontal gene transfer processes have been proposed to play a critical role in the spreading of the mcr-1 gene [19,20]. In the present study, colistin administration had a significant effect on the spread of mcr-mediated colistin resistance in the chicks’ microbiota. Its effect was greater when mcr-1-carrying bacteria were introduced into the gut environment in the presence of colistin, in which case mcr-1 colistin resistance appeared to become widespread. We used a Salmonella enterica serovar Typhimurium strain because neonatal chicks are highly susceptible to colonisation by Salmonella serovars [21] and it is easily identified in chicks because it is not a component of their early microbiota. We studied the effect of colistin by administering it for seven days, as recommended by the EMA for treatment of enteric infections caused by susceptible non-invasive E. coli [15], and focused on the mcr-1 gene because it is the mobile gene most frequently related to colistin resistance worldwide [17]. This scenario has been described in other studies, in which a link was proposed between colistin use and the spread of the mcr-1 gene [12,18,22]. Comparison of the two groups to which colistin was administered (G2 and G4) showed colistin treatment to significantly increase mcr-1 levels in G4 (in which the chicks were challenged with Salmonella) but not G2 (in which the chicks were not challenged with Salmonella). This study shows the importance of the presence of an intestinal bacterial population that carries the mcr-1 gene for colistin resistance for the dispersion associated with its use. This is clear from the data of the mcr-1 challenged chicks which were not administered colistin (G3). Chicks from non-challenged groups (G1 and G2) showed a baseline presence of mcr-1 from the first day of sampling, which may have been due to the pre-existence of these genes in certain bacterial groups different from Salmonella. However, chicks from these groups (G1 and G2) showed parallel progression, with no significant differences in their mcr-1 outcomes. Chicks from G3, despite not having been given colistin, showed an increase in mcr-1 levels, which became significantly different from those of the animals from G1 and G2 by D14 and D21. Similarly, in assessing the effect of colistin administration, chicks challenged with mcr-1-carrying Salmonella (G4) showed significantly more (p-value < 0.01) mcr-1 copies/mg relative to the non-challenged group (G2) (Figure 2), demonstrating that the presence of gene-carrying Salmonella in the gut is more decisive in gene dispersal than the effect of the antibiotic, as has been seen in other studies [23]. Salmonella counts in chicks from group G4 were higher than those in animals from group G3, as observed for the Salmonella qPCR results. Harbouring mcr genes usually suppose a negative fitness cost to the carrier bacteria [24] and maybe this fact was responsible for lower levels of Salmonella detected in G3 than those from G4, since Salmonella in G4 had a selective advantage because of colistin treatment. Thus, data from group G4 support the initial hypothesis about the effect of colistin use on the spread of colistin resistance caused by mcr family genes. These data are consistent with the high dispersion capacity of the mcr-1 gene, especially in the presence of colistin, as previously described [12,13]. Therefore, the mcr-1 levels of group G4 (colistin administered–Salmonella challenged) began to decline after exposure to colistin stopped, but slower than those of Salmonella, remaining similar to the values of group G3, which suggests that the reduction corresponded to mcr-1 harboured by the Salmonella and that the remaining mcr-1 quantified on D21 mainly came from other bacterial species of the microbiota that had been selected by the administration of colistin. Thus, our results highlight the possible horizontal transfer of genes from Salmonella to other intestinal bacteria, which may have allowed mcr-1 levels to remain higher for a longer period in both groups (G3 and G4). Although the clonal spread of mcr-1-carrying Salmonella is common, there is a close association between certain serotypes of Salmonella enterica, as serovar Typhimurium, and mcr-1 gene and different types of plasmid also play an important role in the conjugation phenomenon [8,25]. There- Antibiotics 2021, 10, 677 7 of 10 fore, further studies are needed to determine the type of plasmid where mcr-1 was located and confirm this hypothesis. 4. Methods 4.1. Ethical Approval Experimental procedures were approved by the University Complutense of Madrid Animal Care and Ethics Committee (date of approval: 31/07/2019; registration number: 99/107262.9/19) in compliance with the regulations of the Community of Madrid (PROEX 152/19). Animal experiments took place in the biosafety level 3 (BSL-3) facilities of the VISAVET Health Surveillance Centre and animals were housed according to the European legislation on animal welfare (Directive 2010/63/EU). 4.2. Experimental Design In total, 100 one-day-old broiler chicks (Ross 308) were obtained from a commercial hatchery and housed at the VISAVET Surveillance Centre facilities for the 21 days of the experiment under the same environmental conditions described previously by HerreroEncinas et al. [26]. On the first day, when the animals arrived, box litter samples were collected for Salmonella spp. detection following ISO 6579:2017 standards. All samples were found negative. Chicks were divided into four groups of 25 animals. All chicks were housed in different cages until day (D) 7 but under the same environmental and feeding conditions. Thus, they were considered to be a single group for data analysis (control group: CTRL). From D7, chicks from each cage were exposed to different conditions to form four groups: animals without colistin nor Salmonella challenge (G1), colistin administered (G2), Salmonella challenged (G3), and colistin administered and Salmonella challenged (G4). On D7, chicks from G3 and G4 were challenged with a Salmonella enterica subsp. enterica serovar Typhimurium (monophasic variant) strain that was positive for mcr-1 through their drinking water (3.3 × 105 CFU/mL). Starting from D8, colistin (colistin sulphate 1,025,000 IU, Acolan, Spain) was administered for seven days to chickens of groups G2 and G4 through their drinking water (600 mg/L) adjusting it to a concentration of 75,000 IU recommended in poultry by the EMA [15], replacing the water and corresponding dose of colistin every 24 h. 4.3. Sampling and Sample Preparation On days 7, 9, 11, 14, and 21, five randomly selected animals from each group (CTRL and G1 to G4) were sedated intramuscularly with diazepam and euthanised with an overdose of sodium pentobarbital by intraperitoneal injection. Caecum faeces were collected during the autopsy, and an aliquot of 1.5 g was stored at 4 ◦ C for analysis using microbiological methods in the following 24 h. The remaining caecal faeces collected from each animal was preserved at −80 ◦ C for molecular analysis. Salmonella Counting Using Selective Media A gram of each fresh aliquot was mixed with 2 mL saline (0.85% NaCl). Then, 50 µL was diluted into 9 mL brain–heart infusion broth (BHI) supplemented with a 10 µg colistin disk and incubated at 37 ◦ C for 4 h. Then, six 10-fold serial dilutions were carried out in BHI. ChromID selective medium Salmonella Agar (SMID2) (bioMérieux, Marcy-l’Étoile, France) was used for Salmonella counting. SMID2 plates were inoculated with 100 µL of −3 and −4 BHI sample dilutions and incubated at 37 ◦ C for 24 h. After incubation, all colonies suspected to be Salmonella were counted according to the manufacturer’s specifications. One colony of each SMID2 plate was streaked onto blood agar plates and incubated at 37 ◦ C for 24 h for subsequent species confirmation by mass spectrometry using a Bruker Daltonics UltrafleXtrem MALDI TOF/TOF instrument (Bruker Daltonics, Bremen, Germany). Conventional PCR was performed to confirm the presence of the mcr-1 gene [27]. Antibiotics 2021, 10, 677 8 of 10 4.4. Quantitative Assay for Mcr-1 Direct DNA extraction from chick caecal samples was carried out using a commercial kit (FASTI001-1 FavorPrep Stool DNA Isolation Mini Kit, Favorgen-Europe, Vienna, Austria) following the manufacturer’s specifications (elution volume of 200 µL), coupled with a specific SYBRGreen (Thermo Fisher Scientific, Vilnius) real-time PCR assay for quantitative detection of the mcr-1 gene (qPCR), as described previously by Li J et al. [28] and further validated in our previous work [29]. Samples were considered positive if quantitative values were >1.00 × 102 fg/µL (equivalent to 1.58 × 103 copies/mg caecal content). 4.5. Quantitative Real-Time PCR Assay for Salmonella Quantitative real-time PCR was carried out for the quantitative detection of Salmonella spp. in each sample using a commercial “Salmonella spp. DNA extraction and real-time PCR detection” kit (Kylt® Salmonella spp. (FS), Oldenburg, Germany). Two microliters of each DNA elute were also run in triplicate. A sample was considered to be positive when its cycle threshold (CT) was ≤42. 4.6. Statistical Analysis The data obtained by qPCR were analysed using a t-test for two related samples after normalisation by logarithmic transformation into Log10. A Kruskal–Wallis test was used to analyse differences in SMID2 Salmonella counts among experimental groups. A difference was considered significant when the p-value was <0.05 for both statistical tests. 4.7. Data Visualisation All figures included in this study were illustrated with R (core team 2019) [30] using the ggplot2 package (H. Wickham, 2016) [31]. 5. Conclusions The presence of mcr-1-carrying S. enterica serovar Typhimurium in the gut resulted in the spread of the mcr-1 gene, probably due to horizontal gene transfer. The administration of colistin accelerated this process by selecting the colistin-resistant bacteria present in the gut microbiota, keeping mcr-1 levels constant after the withdrawal of colistin. Author Contributions: Conceptualization, P.M.-V., M.A.M., L.D., and M.U.-R.; methodology, P.M.-V., A.R.-M., A.R.-B., M.H., and D.R.-L.; software, M.H., D.R.-L., and A.Q.; validation, M.H., D.R.-L., and A.Q.; formal analysis, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; investigation P.M-V., M.A.M., L.D., and M.U-R.; resources, P.M-V., M.A.M., L.D., and M.U-R.; data curation, P.M.-V., M.A.M., L.D., and M.U.-R.; writing—original draft preparation, P.M.-V., M.A.M., L.D., and M.U.-R.; writing—review and editing, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; visualization, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; supervision, P.M.-V., M.H., M.A.M., D.R.-L., A.G., A.Q., J.G., L.D., and M.U.-R.; project administration, P.M.-V., M.A.M., L.D., and M.U.-R.; funding acquisition, P.M.-V., M.A.M., L.D., and M.U.-R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Economy, Industry, and Competitiveness (AGL2016-74882-C3), the Spanish Ministry of Agriculture, Fishing, and Food, and the Autonomous Community of Madrid (S2013/ABI-2747). Pedro Miguela-Villoldo was supported by the FPI Programme (BES-2017-080264) from the Spanish Ministry of Science, Innovation, and Universities and Estefanía Martínez Fernández by a grant co-funded by the European Social Fund and Youth Employment Initiative (YEI) (PEJ-2017-TL/BIO- 7114). Work in the laboratory of Alberto Quesada is also funded by the Junta de Extremadura and FEDER (IB16073 and GR15075) in Spain. Institutional Review Board Statement: Experimental procedures were approved by the University Complutense of Madrid Animal Care and Ethics Committee (date of approval: 31/07/2019; registration number: 99/107262.9/19) in compliance with the regulations of the Community of Madrid (PROEX 152/19). Animal experiments took place in the biosafety level 3 (BSL-3) facilities of the VISAVET Health Surveillance Centre and animals were housed according to the European legislation on animal welfare (Directive 2010/63/EU). Antibiotics 2021, 10, 677 9 of 10 Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: The authors wish to thank the technicians María García, Estefanía Rivero, Nisrin Maasoumi, and Estefanía Martínez for their excellent technical assistance at the Foodborne Zoonoses and Antibiotic Resistance Unit, the staff of the Quality and Biosafety Unit, María Mazariegos, Laura Delgado, David Duque, and Pedro Alcubilla, for their technical support at the BSL3 facilities and Red de Investigación en Sanidad Animal (RISA). Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 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The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing maize to growing-finishing hybrid duck on growth performance, nutrient digestibility, carcass trait, and VFA
Journal of The Indonesian Tropical Animal Agriculture/Journal of the Indonesian tropical animal agriculture
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cc-by-sa
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ABSTRAK Penelitian bertujuan untuk mengetahui pengaruh penggunaan bungkil sawit yang diberi perlakuan enzim terhadap nilai nutrisi, performans produksi, dan kualitas karkass bebek hibrida. Materi dalam penelitian ini 196 DOD Itik pedaging [(Peking x Khaki Campbell)] dengan KK (410.52 ± 95.25 g). Metode penelitian yang digunakan adalah percobaan lapang dengan Rancangan Acak Lengkap dengan 5 perlakuan dan 4 ulangan. Pakan kontrol (tanpa substitusi BIS), BIS25 ( Pakan basal + substitusi Jagung dengan 25% BIS penambahan α-β-mannanase), BIS50 ( Pakan basal + substitusi Jagung dengan 50% BIS penambahan α-β-mannanase), BIS75 ( Pakan basal + substitusi Jagung dengan 75% BIS penambahan α-β-mannanase), BIS100 ( Pakan basal + substitusi Jagung dengan 100% BIS penambahan α-β-mannanase). Data hasil penelitian dianalisis variansi menggunakan rancangan acak lengkap (RAL) dengan perlakuan tingkat penggunaan BIS-enzim. Dilanjutkan dengan uji Duncan’s Multiple Range Test untuk mengetahui perbedaan rataan antar perlakuan menggunakan SAS University. Hasil penelitian menunjukan bahwa substitusi jagung dengan BIS yang di tambahakan enzim α-β-mannanase tidak berpengaruh nyata (P>0,05) terhadap penampilan produksi. Namun, berpengaruh nyata (P<0,05) terhadap kecernaan dan mampu menurunkan lemak perut pada daging bebek hibrida. Disimpulkan bahwa penggantian bungkil inti sawit sebanyak 25% dengan penambahan enzim α-β-mannanase mampu meningkatkan kecernaan dan menurunkan lemak perut bebek hibrida. Kata kunci : bebek hibrida, bungkil inti sawit, enzim α-β-mannanase, jagung, substitus The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing maize to growing-finishing hybrid duck on growth performance, nutrient digestibility, carcass trait, and VFA O. Sjofjan, D. N. Adli*, M. H. Natsir, Y. F. Nuningtyas, I. Bastomi and F. R. Amalia Department of Feed and Animal Nutrition, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java 65145 - Indonesia *Corresponding E-mail: danungnuradli1994@gmail.com O. Sjofjan, D. N. Adli*, M. H. Natsir, Y. F. Nuningtyas, I. Bastomi and F. R. Amalia Department of Feed and Animal Nutrition, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java 65145 - Indonesia *Corresponding E-mail: danungnuradli1994@gmail.com O. Sjofjan, D. N. Adli*, M. H. Natsir, Y. F. Nuningtyas, I. Bastomi and F. R. Amalia Department of Feed and Animal Nutrition, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java 65145 - Indonesia *Corresponding E-mail: danungnuradli1994@gmail.com Received October 28, 2020; Accepted December 22, 2020 Received October 28, 2020; Accepted December 22, 2020 J. Indonesian Trop. Anim. Agric. pISSN 2087-8273 eISSN 2460-6278 http://ejournal.undip.ac.id/index.php/jitaa 46(1):29-39, March 2021 DOI: 10.14710/jitaa.46.1.29-39 J. Indonesian Trop. Anim. Agric. pISSN 2087-8273 eISSN 2460-6278 http://ejournal.undip.ac.id/index.php/jitaa 46(1):29-39, March 2021 DOI: 10.14710/jitaa.46.1.29-39 J I T A A Journal of the Indonesian Tropical Animal Agriculture Accredited by Ditjen Penguatan Risbang No. 60/E/KPT/2016 INTRODUCTION in the second place (BPS, 2020). Thus, conditions produce agro-industrial waste from palm kernel meal (PKM) included in poultry diets that had a low price and nutritional content. The palm kernel meal is imported into the European land (EU), cited from (BPS-RI, 2020) the statistics show that 34,064 tons of PKM were imported in 2018/2019. The production of palm kernel meal as animal feed in Europe at the level of 0.48 tonnes in 2018- 2019 (Wilkinson and Young, 2020). The World Health Organization (WHO) announced the coronavirus novel (COVID-19) as a global pandemic outbreak as of March 11, 2020. A total of 4.87 million cases have been confirmed worldwide, with 1.66 million people recovering and 321 thousand people die. The pandemic spread over the world and faced developing countries e.g. Indonesia. COVID-19 impact to livestock sector especially poultry production. Intensive poultry production systems demand a supply of high protein- and easily available in developing countries. To fulfill the requirement, the efficiency on poultry production must be developed (Sjofjan and Adli, 2020). Duck demand increase due it ease and fulfillment in developing countries like Indonesia. The statistical data demand on the poultry market increased nationally (Adli et al., 2018). The data until the fourth quarter of 2019, broilers increased about 4.9% compared to the population in 2018 (1.4 billion heads) (BPS-RI, 2020). Generally, poultry rearing keys were dependent on breed, feed, and management to the purposes the profitable. The golden triangle was giving 86% including also feed intake, live weight, and gain/ratio for each breed of ducks. Feed costs were giving huge around 65-75% of total production in the poultry industry. According to the newest regulation Permendag/10/2020 to anticipate the COVID-19, the Indonesian government has imposed a ban on the import of live animal species and imported by- product originating from China or transiting into Indonesian territory (BPS-RI, 2020). The rules made the researchers or farmers develop an alternative to reduce the cost of maize as main feed in farming. One potential feed to replace maize was palm kernel meal since thus palm kernel meal had similar energy content and protein but high in the crude fiber (Wilkinson and Young, 2020). ABSTRACT In this experiment, we investigated the effect of increasing levels of palm kernel meal containing α- β-mannanase replacing maize to growing-finishing hybrid duck on growth performance, nutrient digestibility, and carcass trait. One hundred and ninety- six hybrid ducks [(Peking x Khaki Campbell)] with 410.52 ± 95.25 g BW) were allotted to 5 dietary treatments with 9 ducks (unsexed) per pen and 4 replicates per treatments. These dietary treatments were: NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). There was a curve linear decrease in the growing phase and finisher phase in feed intake as the level of α-β-mannanase palm kernel meal increased in the diet. Curvilinear, the result was presented no significant difference (P>0.05) on the initial body weight, growing and finishing phase body weight. The result of this study showed the used Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 29 α-β-mannanase in the palm kernel meal presented a significant difference (P < 0.05) on the nutrient digestibility of dry matter and crude protein. Furthermore, Carcass traits showed the reflection in line with the final weight and internal organs were better to compare to control 0.5% using α-β-mannanase in the palm kernel meal. In summary, the increase of the level of palm kernel meal with α-β-mannanase enzyme was had a positive effect on the hybrid ducks and the replacement of palm kernel meal by 25% with the addition of the α-β-mannanase enzyme was able to improve digestibility and reduce abdominal fat of hybrid ducks. Keywords: α-β-mannanase, carcass trait, digestibility, hybrid ducks, palm kernel meal J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 INTRODUCTION Indonesia was at the top level in the world to produce the palm kernel meal above 40.56 MT in 2019-2020, following with Malaysia Indonesia Imports large quantities of staple components of feed for broilers both maize and fishmeal (BPS, 2020), which imported 200 thousand tons in 2020 for maize and 800 tonnes/ year. Reducing imports of feed ingredients is one way of reducing greenhouse gas emissions to which sea freight is a major contributor. A potential local feed Indonesia ingredient that could be used to replace maize is PKM. Even though, the PKM had high crude fiber the breed can be adapted more to the high crude fiber that was a duck (Jang et al., 2020). Indonesia has developed meat ducks that have a fast growth rate, namely Hybrid ducks with a short maintenance period of 45 days. Hybrid ducks are the result of a cross between a Peking duck (male) and a Khaki Champbell duck (a female). A cross between Peking ducks and Khaki Champbell ducks were conducted to produce Day Old Duck (DOD) of final stock broiler duck’s quality. Peking ducks have rapid weight gain, while Khaki ducks Champbell has a high body weight and high egg production compared to other local ducks. The major non- starch polysaccharide (NSP) component in poultry were α-β-mannanase, which is linear with polysaccharide (Jang et al., 2020). However, there have some limitations to giving into duck’s diet. To combat this limitation, an exogenous enzyme of α-β-mannanase had been combined with PKM diet as replacing for maize of growing-finishing ducks. The addition of α-β-mannanase helps the palm kernel meal to stimulate the releasing of volatile fatty acid in the caecum of the ducks J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 30 facilitated. At 24 days to 38 days of age (feeding growing phase); at 38-64 days of age (feeding finishing phase). The Composition of Feed in the experiment showed in Table 2 and Table 3. (Sharmila et al., 2014). In addition, the α-β- mannanase was given to reduce the crude fiber in accordance to improve nutrient digestibility in the gut of duck. When the nutrient digestibility the palm kernel meal in feed should be increase palatability and continued to increase the feed intake and give a positive impact on the growth performance of the ducks. Preparation Palm Kernel Meal containing α-β- Mannanase The palm kernel meal was sifted to separate the meal from the remaining shells, then placed on the floor that had been coated with trash bags and sacks. Suspensions of α-β-mannanase are homogenized in a blender then added at 0.010% per 1 kg of PKM. The last step was putting the PKM in a sack with holes to allow entry of air and storing it for 12 hours at room temperature after which it was sun-dried adapted from (Adli et al., 2020). The dietary treatments were: NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β-mannanase 25%), PKM50 (maize replacement with PKM α- β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). A commercial α-β-mannanase and palm kernel meal from Pt. Wilmar Cahaya Indonesia Tbk was used for this experiment. The study was carried out to investigate the effect of increasing levels of palm kernel meal containing α-β-mannanase replacing maize to growing-finishing hybrid duck on growth performance, nutrient digestibility, and meat quality. Then, the most suitable level of treatments that able to apply in the duck farm was chosen. Ducks Rearing Condition One hundred and ninety-six hybrid ducks [(Peking x Khaki Campbell)] with 410.52 ± 95.25 g BW were allotted to 5 dietary treatments with 9 ducks (unsexed) per pen and 4 replicates per treatments with completely randomized design (CRD) for the experimental design. All ducks were kept in an environmentally controlled room with a fan and air facility. The housing relatively temperature and humidity among 29°C and 64%, respectively. The rice hull- littered floor pens with a height of 3.3 (1.9 x 1.9) m2 per pen. The lighting program was set at 23 hours light and one-hour darkness. Ducks were reared under the supervision of a veterinarian. The hybrid duck was taken from a commercial farmer from Blitar Regency, East Java, Indonesia. Growth Performances The ducks were individually weighed at the growing phase in time to determine the coefficients variation, weekly at days of 24, 31, 38, 45, and final week (64). The body weight gain (BWG) of duck were determined by weighed difference amount at the initial and previous weeks. The feed intake calculated routine by deviation feed offered to ducks and remained feed every week where areas, after calculated the dead broiler remain in the current experiment. Feed/gain determined by feed intake divided by body weight gain of the ducks during the Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 31 Feeding Treatment Programmed The nutrient composition of diet ingredients is presented in Table 1. The ducks (male and female) were ad libitum feed and watering Table 1. Nutrient Compositions of Diet Ingredients (g / kg, as-fed basis) Item Maize Soybean meal Palm kernel meal Dry Matter 93.4 92.2 93.5 Crude protein 8.7 44.5 16 Fat 4 1.5 1.5 Crude fibre 2 3.5 1.67 Ash 1.5 6 6.6 Nitrogen free extract 70.5 30 53.5 Table 1. Nutrient Compositions of Diet Ingredients (g / kg, as-fed basis) Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 31 Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 31 Table 2. Nutrient Compositions of Diet Ingredients of Growing Phase Ducks (g / kg, as-fed basis) Nutrient Compositions of Diet Ingredients of Growing Phase Ducks (g / kg, as-fed basis) Ingredients, g/kg Treatments NC PKM 25 PKM50 PKM75 PKM100 Maize 540 515 490 465 440 Rice bran 150 150 150 150 150 Soybean meal 120 120 120 120 120 MBM 50 50 50 50 50 50 F.M. 60 50 50 50 50 50 PKM - 25 50 75 100 Powder Limestone 30 30 30 30 30 Grit 41 41 41 41 41 Betaine 1 1 1 1 1 Palm oil 10 10 10 10 10 α-β-mannanase - 1 1 1 1 Premix 5 5 5 5 5 Analyses composition, g/kg Dry matter 87.05 87.18 87.30 87.43 87.55 Crude protein 16.83 17.01 17.19 17.38 17.56 Fat 6.42 6.35 6.29 6.23 6.17 Crude Fibre 3.63 3.95 4.28 4.60 4.93 Ash 6.20 6.27 6.33 6.39 6.45 Nitrogen Free extract 48.33 47.90 47.48 47.05 46.63 Metabolizable energy (Kcal/kg) 2683.48 2635.73 2587.98 2540.23 2492.48 Lysine 0.92 0.92 0.93 0.93 0.94 Methionine 0.51 0.52 0.52 0.53 0.53 Met. + Cystine 0.78 0.79 0.79 0.80 0.80 Calcium 3.71 3.72 3.72 3.73 3.73 Total Phosphorus 0.85 0.85 0.86 0.87 0.88 Av. Phosphorus 0.39 0.40 0.41 0.42 0.44 Bulk density, g / L 585 584 580 569 565 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk density = the weight of experimental feed per unit volume (g / L). Feeding Treatment Programmed 2: Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid: 80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04 g 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk density = the weight of experimental feed per unit volume (g / L). 2: Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid: 80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04 g experiment. Mortalities were recorded per pen as well in (%) from the beginning until the end of the experiment (Sjofjan et al., 2020). The 24 ducks from the group, with range nearest live BW, were separated at the days 64 weeks ducks will be sacrificed of the experiment. Ducks were slaughtered ready to cook method to obtain relatively organ weight, and scalded after. J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 32 Table 3. Nutrient Compositions of Diet Ingredients of Finishing Phase Ducks (g / kg, as-fed basis) e 3. Nutrient Compositions of Diet Ingredients of Finishing Phase Ducks (g / kg, as-fed basis) Ingredients, g/kg Treatments NC PKM 25 PKM50 PKM75 PKM100 Maize 550 515 500 475 450 Rice bran 170 170 170 170 170 Soybean meal 90 90 90 90 90 MBM 50 50 50 50 50 50 F.M. 60 50 50 50 50 50 PKM - 25 50 75 100 Powder Limestone 30 30 30 30 30 Grit 41 41 41 41 41 Betaine 1 1 1 1 1 Palm oil 10 10 10 10 10 α-β-mannanase - 1 1 1 1 Premix 5 5 5 5 5 Analyses composition, g/kg Dry matter 86.96 87.09 87.21 87.34 87.46 Crude protein 15,81 1599 16.17 16.35 16.54 Fat 6.66 6.60 6.54 6.47 6.41 Crude Fibre 3.76 4.08 4.41 4.73 5.06 Ash 6.15 6.21 6.28 6.34 6.40 Nitrogen Free extract 48.91 48.48 48.06 47.63 47.21 Metabolizable energy (Kcal/kg) 2695.68 2647.93 2600.18 2552.43 2509.68 Lysine 0.84 0.85 0.85 0.86 0.86 Methionine 0.50 0.50 0.51 0.51 0.52 Met. Feeding Treatment Programmed + Cystine 0.75 0.76 0.76 0.77 0.78 Calcium 3.71 3.71 3.71 3.72 3.72 Total Phosphorus 0.86 0.87 0.88 0.89 0.89 Av. Phosphorus 0.39 0.40 0.41 0.42 0.44 Bulk density, g / L 585 584 580 569 565 1NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk density = the weight of experimental feed per unit volume (g / L). 2Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid: 80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04 1NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Bulk density = the weight of experimental feed per unit volume (g / L). 2Vitamin A: 2,500,000 UI; Vitamin D3: 600,000 UI; Vitamin E: 4,000 mg; Vitamin K3: 400 mg; Folic acid: 80 mg; Choline: 100,000 mg; Mangan: 14 g; Zn: 40 g; Fe: 32 g; Cu: 48 g; Iodine: 0.5 g; Co: 0.28 g; Se: 0.04 Digestibility’s Analysis Where IE=ingested energy; FE=fecal energy voided by the fed birds; while FEL=fasting energy loss by the unfed birds. Where IE=ingested energy; FE=fecal energy voided by the fed birds; while FEL=fasting energy loss by the unfed birds. Excreta samples were collected and stored in plastic trays. Then, immediately placed in a combination of liquid of NA2PO4 2%; NA2H2PO4 2%, 24% Formaldehyde; and 900 ml reverse osmosis water for digestibility’s analysis. The data were used to calculate AME, AMEn, TME, and TMEn values according to the following formulae: The values corrected to zero N balance, AMEn and TMEn, are calculated as follows: The values corrected to zero N balance, AMEn and TMEn, are calculated as follows: AMEn=AME-(8.22×ANR/FI) TMEn=TME-(8.22×FNL/FI) - (8.22×ANR/FI) Where ANR=apparent N retention; FI=feed intake; and FNL=fasting N loss by the unfed bird; The factor 8.22 kcal/g for N retained in the body has been used according to Mustafa et al., (2003). TMEn=TME-(8.22×FNL/FI) - (8.22×ANR/FI) Where ANR=apparent N retention; FI=feed intake; and FNL=fasting N loss by the unfed bird; The factor 8.22 kcal/g for N retained in the body has been used according to Mustafa et al., (2003). AME=IE-FE TME=AME+FEL 33 Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) Figure 1. The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing maize on the growth performance of ducks. The symbols represent growing body weight (♦) and finishing body weight (n). Figure 1. The effect of increasing levels of palm kernel meal containing α-β-mannanase replacing maize on the growth performance of ducks. The symbols represent growing body weight (♦) and finishing body weight (n). the level of α-β-mannanase palm kernel meal increased in the diet (Table 4). Curvilinear, the result was presented no significant difference (P<0.05) on the initial body weight, growing and finishing phase body weight. The results of this study presented α-β-mannanase palm kernel meal had improvement the daily gain, feed/gain ratio both growing and finishing phase compared to control (growing phase at 3.31; 2.97; 2.96 vs. 3.34 (control)) and (finishing phase 3.21; 2.93; 2.93; 2.87 vs. 3.45 control). Compared with the study from Park et al., (2018) that study the used β- mannanase in the palm gives an improvement on the growth performance of white Pekin ducks compare to control. The trends used β-mannanase also used in other poultry to increase performance (Park et al., 2018). The use of α-β-mannanase inhibits the negative effect of NSP in the duck intestinal. α-β-mannanase is an endogenous enzyme in the cell wall that supporting-agent in breaking down the NSP in the palm kernel meal. In addition, α-β-mannanase increasing of NSP can reduce lower nutrient digestibility and mortalities. In these studies, the percentage of the mortalities were at a better level compared with the control (3.43; 0.00; 1.26; and 3.43 vs. 4.26 (control)). The previous researcher had shown the palm kernel meal must be treated by enzyme since a lot of non-starch polysaccharides (NSPs) e.g. mannan and xylene, together with anti-nutritional factors. In this study, α-β-mannanase helps degrades into shorter chains, which, more absorptive well in the nutrients. AMEn=AME-(8.22×ANR/FI) According to Wilkinson and Young VFA Analysis The VFA analyses were following procedure by (Sharmila et al., 2014) and (Adli and Sjofjan, 2020). The chyme from ileum were taken approximately one gram and diluted with distilled water (1:1 wt/vol) in a tube. Later, homogenization and centrifugation, 1 mL of clear supernatant was transferred into ampula, around 0.2 mL metaphosphoric acid solution was added and subjected to another homogenization before it was placed on ice for at least 30 min to allow the protein to settle. Then analyses it on the gas chromatography (Agilent 69890 N series: USA). Continually, the analyses of proximate of the feed sample were determined dry matter, ash, crude fiber, fat, and crude protein was used association of official analytical chemists method. On the other hand, the amino acid using HPLC (HP Agilent 1200 series, USA); crude protein were used (Kjeltech analyses, Foss Detector, Switzerland) and gross energy using (Parr Oxygen Bomb 1108; USA). Data Analysis Data of the first experiment were statistically analyzed using SAS University version 4.0 red hat (64-bit) and the differences among treatment means (p <0.05) were determined using Duncan’s multiple range test (Sjofjan et al., 2020). RESULTS AND DISCUSSION There was a curve linear decrease in the growing phase and finisher phase in feed intake as J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 34 Table 4. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize on the Growth Performance Performance 0.10% α-β-mannanase SEM P-values NC vs PKM NC PKM 25 PKM50 PKM75 PKM100 Body weight, g Initial body weight, g 415 418 417 422 433 2.22 0.31 Growing phase body weight, g 988 978 976 985 987 0.22 0.22 Finishing phase body weight, g 1,923 1,823 1,877 1,877 1,923 12.33 0.0015 Daily gain, g 56.5b 59.2a 58.7ab 57.8b 58.9ab 4.55 0.0001 F/G ratio Growing phase 3.34a 3.31a 2.97b 2.96b 2.95b 3.32 0.0001 Finishing phase 3.45a 3.21a 2.93b 2.93b 2.87b 0.88 0.0012 Mortalities % 4.26 3.43 0.00 1.26 3.43 0.23 0.0015 Growing phase feed intake, g 1,923 1,824 1,727 1,562 1,453 0.21 0.0012 Finisher phase feed intake, g 2,023 1,923 1,825 1,725 1,726 0.19 0.0023 ab Mean values in the same row without common superscript differ at P<005 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). Table 4. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize on the Growth Performance Mean values in the same row without common superscript differ at P<005 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). (2020) that palm kernel meal had low digestibility at the range of 65-75%, to help that some enzyme was added to bind the phytase. The replacement of up to 15% for maize by palm kernel meal was made balancing for digestible amino acids, total phosphorus, and metabolizable energy (Wilkinson and Young, 2020). In addition, from Natsir et al. (2018) the feed intake of untreated PKM higher compare with PKM treated by the enzyme at the level (3242.34 vs. 3097.91 gram/live bird), the results due to energy content lower compared to PKM treated by the enzyme. RESULTS AND DISCUSSION In this study, α-β- mannanase has stimulated the result of crude protein and dry matter by using methyl donor in the cycle. The methyl donor of α-β-mannanase reduce the phytase content and absorb the nutrients well. While the β-mannanase must be transformed into α-β-mannanase first before inside to mitochondria in the synthesis cycle. The latter was significantly affected (P<0.05) with Mustafa et al. (2003) in percent digestibility of palm kernel meal treated by the enzyme (44.09 vs. 41.20 (untreated)). Mustafa et al. (2003) stated the PKM treated by an enzyme found increasing it was due to adequate amino acid levels that arginine, isoleucine, valine, and methionine. In addition, from (Pasaribu et al., 2019) the increase of palm kernel meal using microbial fermented that produced mannanase enzyme were at 24- 32%. and crude protein. The α-β-mannanase help to give improvement (42.20; 41.30; 44.11; and 42.13 vs 40.20% (control)) for dry matter. In line with nutrient digestibility of crude protein were also increasing (42.20; 41.30; 44.11; 42.13 vs. 40.20% (control)) for crude protein. In this study, α-β- mannanase has stimulated the result of crude protein and dry matter by using methyl donor in the cycle. The methyl donor of α-β-mannanase reduce the phytase content and absorb the nutrients well. While the β-mannanase must be transformed into α-β-mannanase first before inside to mitochondria in the synthesis cycle. The latter was significantly affected (P<0.05) with Mustafa et al. (2003) in percent digestibility of palm kernel meal treated by the enzyme (44.09 vs. 41.20 (untreated)). Mustafa et al. (2003) stated the PKM treated by an enzyme found increasing it was due to adequate amino acid levels that arginine, isoleucine, valine, and methionine. In addition, from (Pasaribu et al., 2019) the increase of palm kernel meal using microbial fermented that produced mannanase enzyme were at 24- 32%. Carcass traits showed the reflection in line with the final weight and internal organs were better to compare to control 0.5% using α-β- mannanase in the palm kernel meal. It (Table 6) was not only affected by final weight and internal organs but also depend on exogenous factors in these conditions were using α-β-mannanase in the palm kernel meal. Moreover, the breed in these studies were hybrid ducks [(Peking x Khaki Campbell)] also one factor affected to carcass trait. RESULTS AND DISCUSSION The increased the broiler body weight during the experiment due to metabolism from the enzymatic cycles, the enzyme that absorbs nutrient content combine with metabolism to produce and grow the organs in the ducks. The curve linear of live weight gain may be impacted by feed consumption day by day. The curve linear were affected from feeding programmed, one factor when feeding method designed ad-libitum both of sex (male and female). However, relatively temperature also helps to increase feed intake during research the average temperature 1-35 days were (26.19o morning and 28.63o afternoon) with humidity (88.94o morning and 88.69o afternoon). The lower temperature at the chicken house may help increase feed intake to eat more the experimental diets. Contrast findings to a study from Stęczny and Kokoszyński (2020) interactions between enzyme and palm kernel meal were no interaction for 35 days of age (2880 experimental vs. 2886 control group). The digestibility reflected the impact of the F/G ratio (Table 5). The result of this study showed the used α-β-mannanase in the palm kernel meal presented a significant difference (P>0.05) on the nutrient digestibility of dry matter Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 35 AMEn were better compared to control (1,754; 1,756; 1765; 1777 vs. 1,752 (control)) and (1,844; 1833; 1,823; 1844 vs 1,855 (control)). Furthermore, additional study is required to confirm these issues. Compared with Mustafa et al. (2003) that the no significant difference found in the AME and TME valued to higher NDF result that ranged between 36-41%. The value of AME was 1,870 kcal/kg continued AMEn, TME, and TMEn values were line 1,743; 2053; and 1874 kcal/kg (Mustafa et al., 2003). Park et al. (2018) stated with increasing β-mannanase concentration on the nutrient digestibility also increased significantly (P<0.0001). The nutrient digestibility in line increasing due to amino acid histidine and threonine stimulated the mucin secretion. The goblet cell mucin secretion function was developed by the discharge of histamine from cell E linked with O-linked glycosylation in the intestine (Park et al., 2018). and crude protein. The α-β-mannanase help to give improvement (42.20; 41.30; 44.11; and 42.13 vs 40.20% (control)) for dry matter. In line with nutrient digestibility of crude protein were also increasing (42.20; 41.30; 44.11; 42.13 vs. 40.20% (control)) for crude protein. ab Mean values in the same row without common superscript differ at p<005 1 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). RESULTS AND DISCUSSION Relatively organ weight in this study was The PKM treated by enzyme didn’t help apparent metabolizable energy (AME) and apparent metabolizable energy n-correction due to ME value in the ducks might be higher than broiler (Mustafa et al., 2003). In this study, the AME, AMEn, TME, and TMEn might be different at growing and finishing periods. The AME and Table 5. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize on the Nutrient Digestibility Nutrient Digestibility (%) 0.10% α-β-mannanase SEM P-values NC vs PKM NC PKM 25 PKM50 PKM75 PKM100 Dry matter 40.20b 42.20a 41.30b 44.11a 42.13a 0.88 0.002 Crude protein 52.27b 53.39b 54.49a 55.56a 52.24b 1.23 0.004 AME (kcal / kg) 1,855 1,844 1,833 1,823 1,844 123.23 0.003 AMEn (kcal/kg) 1,752 1,754 1,756 1,765 1,777 111.11 0.011 TME (kcal/kg) 2,073 2,074 2,075 2,077 2,073 55.4 0.33 TMEn (kcal/kg) 1,893 1,844 1,906 1,852 1,888 54.5 0.22 ab Mean values in the same row without common superscript differ at p<005 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement e Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Nutrient Digestibility 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 36 Table 6. The Effect of Increasing Levels of Palm Kernel Meal Containing α-β-mannanase Replacing Maize on the Carcass Traits and Relatively Organ Weight Table 6. RESULTS AND DISCUSSION The Effect of Increasing Levels of Palm Kernel Meal Containing α-β-mannanase Replacing Maize on the Carcass Traits and Relatively Organ Weight 0.10% α-β-mannanase SEM P-values NC vs PKM NC PKM 25 PKM50 PKM75 PKM100 Gizzard 2.69 2.94 2.83 3.25 2.99 0.88 0.002 Heart 0.63 0.59 0.67 0.60 0.63 1.23 0.004 Liver 2.04 1.90 2.05 2.19 1.92 123.23 0.003 Spleen 0.066 0.063 0.081 0.067 0.067 111.11 0.011 Pancreas 0.32 0.26 0.25 0.30 0.26 55.4 0.33 Abdominal fat 2.2 2.0 2.0 1.6 1.5 54.5 0.22 Caeca length (cm) 15.53 16.16 16.48 17.01 16.56 2.22 0.001 Caeca width (cm) 1.88 1.70 1.56 1.86 1.75 0.11 0.002 Carcass weight (g) 1203.75 1301.50 1260 1293 1192.25 234.5 0.003 Carcass (%) 61.47 65.87 63.70 63.23 61.72 2.22 0.004 Breast meat (%) 27.09 28.82 29.60 26.45 25.14 0.22 0.0015 ab Mean values in the same row without common superscript differ at p<005 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soybean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). consistent in line with Chinajariyawong and Muangkeow (2011) and Huang et al. (2009). The abdominal fat was linearly decreasing with increasing levels of α-β-mannanase in the palm kernel meal compared to control (2.0; 2.0; 1.6;1.5 vs. 2.2 (control)). The lower abdominal fat might be consistent with increasing crude fiber content. Compared with Chinajariyawong and Muangkeow (2011) studies the palm kernel meal adjusted with β-mannanase were decrease the abdominal fat. In addition, from Barbour et al., (2006) the supplemented of palm kernel meal with enzyme were decreasing of abdominal fat pat as levels of PKM increased. The higher immune organs result was supposing to enzymatic activities either mode of action and immune stimulation. The factors affected to the significantly different of the stomach is the ability of the absorption. Wang et al. RESULTS AND DISCUSSION (2018) stated the variance of the broiler are affects the absorption and the capacities of the stomach. The growth factors correlate with age, while the uses of the broiler in the relative age cause the growth of the internal organs the same. In some studies, according to Manafi et al. (2018) it results did not increase in liver and spleen both male and female groups (P>0.05), where areas, bursa of fabricius as immune organ were increased (P<0.05). The number VFA presented no significant difference (P<0.05) (Table 7). The result of isobutyric of the treatment were not better than the control (16.5; 16.2; 16.3; 16.4 vs. 16.0 (control)) and followed by n-butyric were (12.1; 12.2; 11.2; 10.2 vs. 11.9 (control)). The production of VFAs depended on the caecal in caecum when crude fiber breakdown the NSPs in the palm kernel meal. The α-β-mannanase enzyme extent of the fermentation process depends on the microbial population in the caecum. Dunkley et al. (2007) reported that acetic acid was the primary source of VFA produced in the caecal parts followed by butyrate and propionate. The fermentation process to produce acetic acid was started with i) breakdown of the NSP to simply sugar (xylan to xylose to xylooligomers) that might escape from enzymatic digestion, later enter the caecum (Sharmila et al., 2014). Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) 37 Table 7. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize on the Volatile Fatty Acid Table 7. The Effect of Increasing Levels of Palm Kernel Meal containing α-β-mannanase Replacing Maize on the Volatile Fatty Acid VFA 0.10% α-β-mannanase P-values NC vs PKM NC PKM 25 PKM50 PKM75 PKM100 SEM Iso butyric 16.0 16.5 16.2 16.3 16.4 0.21 0.0014 n-butyric 11.9 12.1 12.2 11.2 10.2 0.22 0.0012 Iso-valeric 8.5 8.6 8.7 8.8 8.9 0.12 0.0011 ab Mean values in the same row without common superscript differ at p<005 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). 1 NC (negative control; maize-soyabean-meal based diet), PKM25 (maize replacement with PKM α-β- mannanase 25%), PKM50 (maize replacement with PKM α-β-mannanase 50%), PKM75 (maize replacement with PKM α-β-mannanase 75%), PKM100 (maize replacement with PKM α-β-mannanase 100%). ACKNOWLEDGMENTS The author appreciated to LPPM, the University of Brawijaya for Research Grant Associate professor for granted Number: 732/UN.10. F05/PN/2020 and Non-Tax Revenue (PNBP) University of Brawijaya SP DIPA- 023.17.2.677512/2020. Thank you to Pt. Wilmar Cahaya Indonesia Tbk for providing the palm kernel meal. Last but not least, thanks to Ms. Ainun Seruni Pizar [Missouri, University, and Columbia, Missouri, United States of America] for providing joint premium account Grammarly granted. Chinajariyawong, C. and N. Muangkeow, N. 2011. Carcass yield and visceral organs of broiler chickens fed palm kernel meal or Aspergillus wentii TISTR 3075 fermented palm kernel meal. WJST. 8(2):175-185. Dunkley, D. K., C.S. Dunkley, N.L. Njongmeta,T.R. Callaway, M.E. Hume,L.F. Kubena, D.J. Nisbet and S.C. Ricke. 2007. Comparison of in vitro fermentation and molecular microbial profiles of high fiber substrates incubated with chicken cecal inocula. Poult. Sci. 86(5):801-810. CONCLUSION Adli, D. N. and O. Sjofjan. 2020. Growth performance, serum blood biochemistry, and intestinal properties of Arbor Acres Broiler fed diets containing mannan-riched fraction (MRF) and probiotic-enhanced liquid acidifier. Bul. Pet. 44(2): 34-42. The increase of the level palm kernel meal with α-β-mannanase enzyme wase had a positive effect on the hybrid ducks and replacement of palm kernel meal by 25% with the addition of the α-β-mannanase enzyme was able to improve digestibility and reduce abdominal fat of hybrid ducks. BPS-RI. 2020. Livestock in figures 2020. Badan Pusat Statistik. Jakarta. Barbour, G. W., M. T. Farran, N. N. Usayran, A. H. Darwish, M.G. Uwayjan, M. G. and V.M. Ashkarian. 2006. Effect of soybean oil supplementation to low metabolizable energy diets on production parameters of broiler chickens. J. Appl. Poult. Res. 15(2):190-197. REFERENCES Adli, D.N. and O. Sjofjan. 2018. Nutrient content evaluation of dried poultry waste urea molasses block (DPW-UMB) on In-vitro analysis. Sains Pet. 16(2):50-53. Huang, Y., J.S. Yoo, H.J. Kim, Y. Wang, Y.J. Chen, J.H. Cho and I.H. Kim. 2009. Effect of bedding types and different nutrient densities on growth performance, visceral organ weight, and blood characteristics in broiler chickens. J. Appl. Poult. Res. 18(1):1-7. Adli D. N., O. Sjofjan., M. H. Natsir, Y. F. Nuningtyas, N. Sholikah and A. C. Marbun. 2020. The effect of replacing maize with fermented palm kernel meal (FPKM) on broiler performance. LRRD. 32(120). Jang, J. C., K.H. Kim, D.H. Kim, S.K. Jang, J.S. Hong, P.S. Heo and Y.Y. Kim. 2020. Effects of increasing levels of palm kernel meal J.Indonesian Trop.Anim.Agric. 46(1):29-39, March 2021 38 kernel meal-based diet. J. Anim. and Poult. Sci. 3(1):19-28. containing β-mannanase to growing- finishing pig diets on growth performance, nutrient digestibility, and pork quality. Livest. Sci. 10(1): 40-41. Sjofjan O, M.H. Natsir, D.N. Adli, D.D Adelina and L.M. Triana. 2020. Effect of symbiotic flour (lactobacillus sp. and fos) to the egg quality and performance of laying hens. IOP Conf. Series: Earth and Environmental Sci., 465, P.012-033. Manafi, M., M. Hedayati and S. Mirzaie. 2018. Probiotic bacillus species and saccharomyces boulardii improve performance, gut histology, and immunity in broiler chickens. South Afr. J. Anim. Sci 48(2):379-388. Sjofjan, O. and D.N. Adli. 2020. Effect of dietary of supplementation mannan-riched fraction (mrf) and probiotic-enhanced liquid acidifier on the growth performance, serum blood biochemistry, and intestinal properties of broilers. In IOP Conference Series: Earth and Environmental Sci., 478(1), P. 012066. Mustafa, M. F., A.R. Alimon, M.W. Zahari, I. Idris and M. H. Bejo. 2003. Nutrient digestibility of Palm kernel Cake for Muscovy ducks. Asian-Australas J. Anim. Sci. 17(4):514-517. Natsir, M. H., I. Djunaidi, O. Sjofjan, A. Suwanto, E. Puspitasari, and L.J. Virginia. 2018. The effect of corn substitution with palm kernel meal treated by enzyme on production performance and carcass quality of broiler. Bul. Pet. 42(2):103-108. Sjofjan, O., D.N. Adli, I. Djunaidi and K. Kuncoro. 2020. Utilization of Biogas Liquid Waste For Starter In The Fermentation of Rice Husk as A Potential Feed For Poultry. Anim. Prod. 22(1):24-30. Park, J., S. Jung and J. Carey. 2019. Effects of a commercial beta-mannanase product on growth performance, intestinal histomorphology, bone and body composition, and amino acid digestibility in white Pekin ducks. J. Appl. Poult. Res. 28(1): 63-71. Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.) REFERENCES Stęczny, K. and D. Kokoszyński. 2020. Effect of probiotic preparations (EM) and sex on morphometric characteristics of the digestive system and leg bones, and caecal microflora in broiler chickens. J. Appl. Anim Res. 48(1):45-50. Pasaribu, T., E.B. Laconi and I.P. Kompiang. 2019. Evaluation of the nutrient contents of palm kernel cake fermented by microbial cocktails as a potential feedstuff for poultry. J. Indonesian Trop. Anim. Agric. 44(3):295- 302. Wang, Y., Z. Dong, D. Song, H. Zhou, W. Wang, H. Miao, L. Wang and A. Li. 2018. Effects of microencapsulated probiotics and prebiotics on growth performance, antioxidative abilities, immune functions, and caeca micro flora in broiler chickens. Food and Agr. Immun 3(8):1-11. Sharmila, A., K. Azhar, M.N.Hezmee and A.A. Samsudin. 2014. Effect of xylanase and cellulase supplementation on growth performance, volatile fatty acids and caecal bacteria of broiler chickens fed with palm Wilkinson, J. M. and R.H.Young. 2020. Strategies to reduce reliance on soya bean meal and palm kernel meal in livestock nutrition. J. Appl. Anim. Nutr. 8(2):75-82. 39 Palm Kernel Meal containing α-β-mannanase Replacing Maize for Ducks (A. Sjofjan et al.)
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Assessment of protective forest plantations on gray forest and sod-podzolic soils of the Predkamye region of the Republic of Tatarstan
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1 Introduction of national and other programmes to preserve the environment, improve the efficiency of fertility, ensure environmental and food security of the country, reduce the level of discomfort in places of work and human habitation [8]. To date, agricultural science has developed and implemented the principles of adaptive- landscape farming. The most important principle is the application of a set of anti-degradation measures: organisational and economic, forest-reclamation, agrotechnical, meadow-reclamation, hydrotechnical, and others. The framework of adaptive-landscape farming is a system of protective forest plantations [5, 6, 7, 17]. Establishment of protective forest plantations provides multifunctional and long-term protection of soils from erosion. Under the influence of soil and wind erosion, unprotected soils lose fertility and are withdrawn from agricultural turnover. Due to the created plantations, species diversity is enriched, and ecological conditions of agricultural lands are improved [20, 3]. They have a lasting effect on the indicators and properties of washed away soils, restoring the lost fertility [12]. Forest strips on agricultural lands form a framework of erosion protection, and the efficiency of the complex as a whole depends to a great extent on their location in the relief [1, 2, 22]. In the presence of an interconnected system of forest ameliorative plantations, crop yields increase by 8–18%. On average, the impact of 1 ha of forest plantations extends to 10–12 ha of adjacent fields. According to A.I. Petelko, the scientifically justified need for forest plantations in the country is 14 million ha, but there are only 3.2 million ha [13]. The strategy for the development of protective afforestation in the Russian Federation until 2025 has been developed. It is aimed at creating a complete system of ameliorative protective forest plantations as a mandatory component The role of planted forests in carbon storage is exceptional. Forests located on agricultural land are of great importance for the absorption of greenhouse gases. According to Ivanov A.Y., Durmanov N.D. (2021), 1 ha of forests on the lands of the forest fund can absorb, on average, about 1 tonne of greenhouse gases per year. Protective and anti-erosion forests on agricultural lands increase this indicator 7 times up to about 7 tonnes per year. Assessment of protective forest plantations on gray forest and sod-podzolic soils of the Predkamye region of the Republic of Tatarstan Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov Kazan state agricultural university, 65, Karl Marks St., Kazan, 420015, Russia Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov K i l l i i 65 K l M k S K 420015 R i Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov Aigul Mukhametshina, Haris Musin, Nail Mirsiyapov, Salavat Usmanov* and Ildar Khalilov Kazan state agricultural university, 65, Karl Marks St., Kazan, 420015, Russia Kazan state agricultural university, 65, Karl Marks St., Kazan, 420015, Russia Abstract. Protective forest plantations increase soil fertility and fulfil an important role of soil protection from erosion. Much attention is paid to protective afforestation in the Republic of Tatarstan. In the region, in 2022, protective forest plantations were established on the area of 1801 hectares, of which ravine-beam forests make up 1763 hectares, including 645 hectares with terracing and 38 hectares of field protection strips. Protective forest plantations were created in Aznakaevsky municipal district on the area of 600 ha and in settlements of 114 ha of "compensatory" reforestation and afforestation. The article presents the results of the survey of existing protective forest plantations in the Predkamya zone of the Republic of Tatarstan on sod-podzolic and grey forest soils. The area is characterised by strong washing away, erosion and gully formation, where the area of washed away soils is 66%, erosion-hazardous lands amount to 15.5%. A sanitary condition of plantations, productivity and carbon deposition were assessed. According to silvicultural and taxation indicators, the average age of the surveyed stands is 48 years; the completeness ranges from 0.5 to 0.9, bonitet ranges from I to II. In terms of the sanitary condition, trees of the 1st and 2nd categories predominate; in some areas forest management measures are required. Under the canopy of the plantations, there is a natural regeneration of petiole oak, which is an indigenous species of the Pre- Kama region of the Republic of Tatarstan. On all surveyed sample areas, the largest amount of carbon stock is concentrated on PP7, PP8 and PP19: 786.96, 833.7 and 828.09 tonnes, respectively. These are mixed stands of pine and birch. * Corresponding author: salusmanov7@yandex.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/bioconf/202410300065 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024) BIO Web of Conferences 103, 00065 (2024) FIES 2023 d by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution commons.org/licenses/by/4.0/). * Corresponding author: salusmanov7@yandex.ru 1 Introduction At the same time, according to various estimates, from 40 to 90 million hectares of agricultural land in Russia are overgrown with forests, which are not yet accounted for in the national statistics of greenhouse gas absorption because they do not belong to managed BIO Web of Conferences 103, 00065 (2024) FIES 2023 https://doi.org/10.1051/bioconf/202410300065 years, the length of ravines has increased by more than 10 thousand kilometres. forests [4]. This is approximately 26 million tonnes of carbon accumulation annually [9, 19]. It is considered a promising direction to increase soil productivity of these lands through carbon farming, application of modern methods of biotechnology and breeding. In March 1997, the Cabinet of Ministers of the Republic of Tatarstan adopted Resolution No. 216 and approved the Comprehensive Programme of Soil Fertility Improvement and Erosion Protection in the Republic of Tatarstan for 1997–2005. It provides for a set of anti-erosion measures. In accordance with the programme, 7.8 thousand ha of protective plantations were planted [14]. To date, most of the forest belts are gradually dying off, subject to diseases and in a neglected state. There is a need to carry out a set of forestry measures to preserve and renew the systems of field protective forest belts [10, 21]. For the effective functioning of existing forest plantations, it is necessary to assess their current state and to develop measures to maintain them. In this regard, it is relevant to conduct research in the Predkamye zone of the Republic of Tatarstan. The aim of the work is to assess the condition of protective forest plantations on grey forest and sod- podzolic soils in the Predkamya zone of the Republic of Tatarstan. Special attention is paid to protective afforestation in the region in order to prevent water and wind erosion of soil, to increase the productivity of agricultural land, to protect settlements, enterprises and other objects from unfavourable natural phenomena and man-made impacts. According to the data of the Ministry of Forestry of RT, in 2022, protective forest plantations were established on the area of 1801 hectares, of which ravine-beam forests make up 1763 hectares, including 645 hectares with terracing, and field protection strips occupy 38 hectares. Protective forest plantations were created in the Aznakaevsky municipal district within the framework of climate projects on an area of 600 ha and in settlements of 114 ha of "compensatory" reforestation and afforestation. 2 Objects and methods of research Protective forest plantations in the Predkamya zone of the Republic of Tatarstan were chosen as the object of research. Sample areas were laid in accordance with OST 56-69-83 "Sample forest inventory areas. Laying method" [11]. Glasometer and instrumental measurements (an altimeter, a full-meter, a measuring fork) were performed on each sample area. When carrying out the works, we were guided by the rules of sanitary safety in forests. They were approved by the Decree of the Government of the Russian Federation of 20 May 2017, No. 607; Order of the Ministry of Natural Resources of Russia of 16.09.2016, No. 480 "On approval of the order of forest pathological surveys and the form of the act of forest pathological survey" [15, 16]. A total of 20 sampling areas (hereinafter referred to as PAs) in the Predkamya zone of the Republic of Tatarstan were investigated. Tree counting was carried out at the PPs by thickness gradation (2 cm), and each tree was assigned to a category of condition [11]. During the research, 2000 trees were measured and surveyed. On a 100-metre long trial area, trees were assessed according to the following indicators. The number of trees was counted; a trunk diameter was measured with a measuring fork at a breast height of 1.3 m; a height was determined with a Danilin altimeter, and natural vegetation was visually described. Plots of different ages were selected for these purposes. Fig. 1. Protective afforestation over the last 5 years 2470 2415 2450 3559 2515 0 500 1000 1500 2000 2500 3000 3500 4000 ϮϬϭϴ ϮϬϭϵ ϮϬϮϬ ϮϬϮϭ ϮϬϮϮ zĞĂƌƐ Линейная (zĞĂƌƐͿ >ŝŶĞĂƌ 1 Introduction The volumes of protective afforestation for the last five years are shown in Fig. 1. 3 Analysis and discussion of the results Characteristics of plantations on grey forest and sod- podzolic soils Table 1. Characteristics of plantations on grey forest and sod- podzolic soils No. n/a Composition of plantations Breed Age, years Height, m Diameter, cm Bonnitet Completeness Area, ha 1 2 3 4 5 6 7 8 9 PP1 7E3Lz European spruce, Siberian larch 30 8 10 12 14 1а 0.8 3.5 PP2 7B3E. Hanging birch, European spruce 30 16 6 18 10 1а 0.8 5.8 PP3 10С Common pine 54 20 22 1 0.6 4.5 PP4 8B1C 1E Hanging birch 60 22 26 1а 0.6 5 PP5 10Т Poplar white 50 24 32 II 0.6 3.0 PP6 10Б Hanging birch 65 23 32 1 0.6 5.3 PP7 9C1B Pine Common birch Birch overhanging 50 15 22 10 24 18 1 0.7 7.2 PP8 9C1B Pine Common birch Birch overhanging 50 15 22 10 32 16 1 0.5 7.0 PP9 10Б Hanging birch 50 22 28 1а 0.6 PP10 10Б Hanging birch 60 20 22 1а 0.6 4.0 PP11 6C4E European spruce Common pine 35 10 12 8 10 1 0.9 8.0 PP12 10Б Hanging birch 80 24 36 1а 0.6 2.7 PP13 8E1B1 C European spruce Hanging birch Common pine 40 10 12 1 0.8 2.65 PP14. 10Е European spruce 40 11 12 1 0.8 8.5 PP15. 10Б Hanging birch 60 22 36 1а 0.6 2.2 PP16 10Б Hanging birch 60 22 32 1а 0.6 7.3 PP17 10Os. Poplar trembling 60 21 28 II 0.6 1.9 PP18 10Б Hanging birch 60 21 28 1а 0.6 2.2 PP19. 5B5S Hanging birch Common pine 60 21 24 32 28 1а 0.7 7.2 PP20 8T2B Poplar Hanging birch 50 22 23 32 28 II 0.6 2.9 PP-3 is a clean composition roadside forest belt of 5 rows; a row spacing width is 2 m, in a row of 1.3 m. A composition of plantations is 10С. Taxation parameters are Cf. d =22, Cf. h =20 m. The forest belt is located at a distance of 30 m from the road. On the site, there are self-seeded pine trees of different ages (3–8 years old). In order to form a stable multi-age plantation, it is advisable to carry out measures to promote natural regeneration or mineralised strips. Weakened pine trees (2nd category) account for 35.0 %. 3 Analysis and discussion of the results Preservation and rational use of lands are the main conditions for ensuring stable development of the agro- industrial complex and economy of the Republic of Tatarstan. Over the last decade, the area of agricultural land subject to erosion has increased. A high degree of agricultural land ploughing is observed in Arsk and Baltasinsk districts: 85–86%. On average, this indicator for the region is 77.0%. The greatest increase is observed in the zone of Predvolzhye (34.7%) and Predkamye (30.6%). Fig. 1. Protective afforestation over the last 5 years For the study, sample areas were established in the protective forest plantations in the Predkamya zone of the Republic of Tatarstan (Sabinsky, Arsky, Atninsky municipal districts). Territorially, the study area is located in the catchment area of the Volga slope rivers flowing parallel to each other from north-east to south- west: Ashit, Kazanka and Meshi. The area is characterised by strong washout, erosion and gully formation, where the area of washed out soils is 66%, erosion-prone lands are 15.5%. In this zone, light grey forest soils and sod-podzol soils are most widespread on In the most pronounced form, the effect of water erosion is manifested in the processes of gully formation. The number of active gully tops is about 20 thousand, and their length is 27.4 thousand km. Over the last 40 2 2 BIO Web of Conferences 103, 00065 (2024) FIES 2023 https://doi.org/10.1051/bioconf/202410300065 watershed plateaus and upper parts of slopes. There are also grey and dark grey forest and sod soils. the sanitary condition, trees of the 1st category predominate (90%) being without signs of damage. At this site, weakened trees account for 10%; there are minor mechanical damages. The results of the survey and the silvicultural and taxonomic characterisation of the plantations at the PP are given below (Table 1). PP-2 is a mixed composition roadside forest belt of 6-row YehBhEhEhEhBhB, a row spacing width is 2 m, in a row of 1.3 m. A composition of plantations is 5E5B. Taxation indices of birch are Cf. d =18, Cf. h =16 m, spruce is Cf. d =10, Cf. h =6 m. The forest belt is located at a distance of 25 m from the road. The slow-growing species European spruce lags behind the birch. In this forest belt, the trees of the 1st category predominate (95 %), "weakened" trees of the 2nd category are 5.0 %. Table 1. PP-1 is a mixed composition roadside forest belt representing a mix in row 8Ex4Ltsx8Ex4Lts, 5 rows. A row spacing width is 2.5 m, in a row of 1.3 m. A plantation composition is 7E3Lts. Taxation parameters are an average diameter of spruce (hereafter Cf. d) Cf. of d = 12 cm, an average height (hereafter Cf. h) is Cf. h = 8 m, a larch is Cf. d = 14, Cf. h = 10 m. The introduced larch forms a clump, where the forest environment under the canopy is formed more rapidly due to needle fall. It is located at a distance of 25 m from the road. In terms of 3 Analysis and discussion of the results PP 11 is a mixed composition roadside forest belt, with row spacing 2 m wide, in a row of 1 m. A composition is 6C4E. Taxation parameters of European spruce are Cf. d = 8, Cf. h = 10 m. Taxation indices of common pine are Cf. d = 10, Cf. h = 12 m. The condition of the stands is satisfactory. Damage is localised along the road: yellowed needles, pine trees are drying out. There are trees of 5th and 6th categories. Natural regeneration is at the expense of birch. LNP is not formed; litter is in the form of fallen needles. PP 11 is a mixed composition roadside forest belt, with row spacing 2 m wide, in a row of 1 m. A composition is 6C4E. Taxation parameters of European spruce are Cf. d = 8, Cf. h = 10 m. Taxation indices of common pine are Cf. d = 10, Cf. h = 12 m. The condition of the stands is satisfactory. Damage is localised along the road: yellowed needles, pine trees are drying out. There are trees of 5th and 6th categories. Natural regeneration is at the expense of birch. LNP is not formed; litter is in the form of fallen needles. PP 9 is a clear-cut field-protective forest belt. A width of row spacing is 3 m, in a row of 3 m. A composition of plantations is 10Б. Taxation indices of common pine are Cf. d = 22, Cf. h = 28 m. The condition of stands is satisfactory, trees of the 1st category "without signs of weakening" prevail (85.0%). There is the self-seeding of common pine, birch, oak. It is necessary to take measures to promote natural regeneration. PP 12 is a pure composition of a field protective forest belt, with row spacing 3 m wide, in the row of 1.5 m. A composition is 10B. Taxation indices of birch are Cd. d = 36, Cd. h = 24 m. The condition of plantations is satisfactory; trees of the 2nd "weakened" category prevail (75.0%). The forest environment is formed. Oviposition of gypsy moths is found on almost all counted trees. Fruiting bodies of the true moth were also found on trees of the 5th and 6th categories. Natural regeneration takes place due to maple, linden, birch. Forest management measures with preservation of natural regeneration are required. 3 Analysis and discussion of the results In our opinion, the deterioration of the condition of plantations is due to their close proximity to the roadway. PP-4 is a mixed composition roadside forest belt of old-growth 6-row. A row spacing width is 3 m, in a row of 1.5 m. Taxation parameters are Cf. d = 26, Cf. h = 22 m. A composition of plantations is 8B1C1E + shrub Acacia yellow. BxExBxBxSxBxBxB is reconstructed. Trees of the 1st category "without signs of weakening" predominate (95%). PP 5 is a pure field-protective forest belt representing an 8-row old-growth; the row spacing is 3 m wide, 1.5 m in a row. A composition of plantations is 10Т. Cf. d = 32, Cf. h =24 m. A forest litter thickness is up to 25 cm. A forest environment has been formed. Natural regeneration is due to aspen, sharp-leaved maple, small- leaved linden, birch, oak, oak, poplar of shoot origin. The predominant category of trees is "without signs of weakening" (65%), and category 3 is "severely weakened" (35.0%). Sanitary cuttings with removal of faulted trees are required. PP 6 is a 6-row pure field protection forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition is 10B. Taxation parameters are Cf. d = 32, Cf. h = 26 m. Forest environment is formed; characteristic forest plants are present. The condition of plantations is satisfactory, single adult silkworms were found. Clutches were not found. Natural regeneration is due to birch, oak, oak, oak, maple. Trees of the 1st category "without signs of weakening" are 70.0%, and 30.0% of the 2nd category are "weakened". Sanitary cuttings with removal of faunal trees and promotion of natural regeneration are required. PP 7 is a mixed composition field protective forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8C2B. Taxation indices of common pine are Cf. d = 24, Cf. h = 22 m. Birch is lagging behind in growth. Taxation indices of birch are Cf. d = 18, Cf. h = 10 m. The condition of the stands is satisfactory; trees of the 1st category "without signs of 3 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024) BIO Web of Conferences 103, 00065 (2024) FIES 2023 weakening" predominate (80%). Natural regeneration is carried out by birch and oak. 3 Analysis and discussion of the results It is necessary to carry out measures to promote natural regeneration. 5th and 6th categories. The natural regeneration of trees is due to oyster maple, petiole oak and small-leaved linden. Measures to promote natural regeneration are required. Fig. 2. PP 7 Fig. 4. PP 10 Fig. 2. PP 7 Fig. 2. PP 7 Fig. 3. PP 8 Fig. 4. PP 10 Fig. 4. PP 10 Fig.5. PP 12 Fig. 3. PP 8 PP 8 is a mixed composition field-protective forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8C2B. Taxation indices of common pine are Cf. d = 32, Cf. h = 22 m. The birch lags behind in growth. Taxation parameters of birch are Cf. d = 10, Cf. h = 16 m. The condition of the stands is satisfactory; trees of the 1st category "without signs of weakening" predominate (75.0%). The natural regeneration of the forest is represented by common pine, birch, oak (Fig. 3). It is necessary to carry out measures to promote natural regeneration. PP 8 is a mixed composition field-protective forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8C2B. Taxation indices of common pine are Cf. d = 32, Cf. h = 22 m. The birch lags behind in growth. Taxation parameters of birch are Cf. d = 10, Cf. h = 16 m. The condition of the stands is satisfactory; trees of the 1st category "without signs of weakening" predominate (75.0%). The natural regeneration of the forest is represented by common pine, birch, oak (Fig. 3). It is necessary to carry out measures to promote natural regeneration. Fig.5. PP 12 Fig.5. PP 12 PP 11 is a mixed composition roadside forest belt, with row spacing 2 m wide, in a row of 1 m. A composition is 6C4E. Taxation parameters of European spruce are Cf. d = 8, Cf. h = 10 m. Taxation indices of common pine are Cf. d = 10, Cf. h = 12 m. The condition of the stands is satisfactory. Damage is localised along the road: yellowed needles, pine trees are drying out. There are trees of 5th and 6th categories. Natural regeneration is at the expense of birch. LNP is not formed; litter is in the form of fallen needles. 3 Analysis and discussion of the results The condition of plantations is satisfactory; trees of the 1st category are predominantly without signs of weakening (85.0%). g g ( ) PP 14 is a clean composition of frontage protective forest plantations. A row spacing width is 3 m, in the row of 1.5 m. A composition is 10E. Taxation indices of European spruce are Cf. d = 12, Cf. h = 11 m. There is intraspecific competition, no silvicultural measures (thinning). An insignificant number of trees damaged by bark beetle-typograph is up to 5.0%, it is of natural character. PP 15 is a pure composition field protective forest belt. A width of row spacing is 3 m, in the row of 2 m. A composition is 10B. Taxation parameters of birch are Cd. d = 36, Cd. h = 22 m. Category 1 trees predominate (85.0%) without signs of weakening. PP 16 is a clean composition of the forest fringe. A width of row spacing is 3 m, in the row of 2 m. A composition is 10 B. Taxation parameters of birch are Cf. d = 32, Cf. h = 22 m. The age is 60 years. Trees of the 1st category prevail without signs of weakening (74.0%). Single trees of category 6 are old deadwood. PP 17 is a pure composition of a field-protective forest belt. A row spacing width is 3 m, in a row of 1.5 m. A composition of plantations is 10 Os. Taxation parameters of aspen are Cf. d = 28, Cf. h = 21 m. The age is 60 years. Trees of the 2nd "weakened" category predominate (85.0%). Trees of category 6 are old deadwood; sanitary felling is necessary. PP18 is a clear field protective forest belt. A width of row spacing is 3 m, in the row of 2.0 m. A composition of plantations is 10Б. Taxation parameters of birch are Cf. d = 28, Cf. h = 21 m. The age is 60 years. Trees of the 1st category without signs of weakening prevail (85.0%). PP19 is a mixed composition field-protective forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition is 5B5C. Taxation parameters of birch are Cf. d = 32, Cf. h = 21 m. The age is 60 years. Taxation parameters of common pine are Cf. d = 28, Cf. h = 24 m. 3 Analysis and discussion of the results PP 10 is a pure field-protective forest belt, with the row spacing of 3 m in width, 1.5 m in a row. A composition of plantations is 10Б. Taxation indices of birch are Cf. d = 22, Cf. h = 20 m. The condition of the stands is satisfactory; "weakened" trees of the 2nd category predominate (92.0%). Ovipositors of gypsy moths were found on almost all counted trees. Fruiting bodies of the true moth were also found on trees of the 4 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024) FIES 2023 Table 2. Volume of deposited carbon in forest plantations Tree species Area, ha Total stock, m3 Total phytomass, tonnes Carbon stock, tonnes 1 2 3 4 5 Trial area 1 Spruce 3.5 171.5 77.17 38.59 Larch 80.5 53.13 26.57 Total 252.0 130.3 65.16 Trial square 2 Spruce 5.8 214.6 96.57 48.29 Birch 545.2 354.38 177.19 Total 759.8 450.95 225.48 Trial area 3 Pine 4.5 832.5 416.2 208.1 Trial area 4 Birch 5.0 700.0 455.0 227.5 Pine 70.0 35.0 17.5 Spruce 45.0 20.25 10.13 Total 815 510.25 255.13 Trial square 5 Poplar 3.0 705.0 317.2 158.6 Trial square 6 Birch 5.3 869.2 564.98 282.49 Trial square 7 Pine 7.2 1180.8 590.4 295.2 Birch 302.4 196.56 98.28 Total 1483,2 786.96 393.48 Trial square 8 Pine 7.0 1358.0 679 339.5 Birch 238.0 154.7 77.35 Total 1596 833.7 416.85 Trial square 9 Birch 1.0 147.0 95.55 47.77 Trial area 10 Birch 4.0 496.0 322.4 161.2 Trial square 11 Spruce 8.0 360 162.0 81.0 Pine 480.0 240 120.0 Total 992 470.4 235.2 Trial area 12 Birch 2.7 523.8 340.47 170.23 Trial square 13 Spruce 2.65 108.6 48.9 24.4 Birch 18.6 12.6 6.0 Pine 10.2 5.1 2.5 Total 137.4 66.6 32.9 Trial square 14 Spruce 8.5 459.3 206.7 103.3 Trial area 15 Birch 2.2 378.4 245.96 122.98 Trial square 16 Birch 7.3 1116.9 725.98 362.99 Trial square 17 Poplar 1.9 362.9 181.45 90.73 Trial square 18 Birch 2.2 345.4 224.51 112.26 Trial square 19 Birch 7.2 733.6 476.84 238.42 Pine 702.5 351.25 175.63 Total 1436.1 828.09 414.05 Trial area 20 Poplar 2.9 426.3 191.83 95.92 Birch 118,9 77.28 38.64 Total 545.2 269.11 134.56 PP 13 is a mixed composition field protective forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition is 8E1B1C. Taxation parameters are Cf. d=12, Cf. h=10 m. 3 Analysis and discussion of the results Category 1 trees without signs of weakening prevail (80.0%). p ( ) PP 20 is a mixed composition field-protective forest belt. A width of row spacing is 3 m, in the row of 1.5 m. A composition of plantations is 8T2B. Taxation parameters of poplar are Cf. d = 32, Cf. h = 22 m. The age is 60 years. Taxation parameters of birch are Cf. d = 28, Cf. h = 23 m. Trees of the 2nd "weakened" category predominate (85.0%). Trees of the 5th and 6th categories are present in insignificant quantities. It is necessary to carry out forest management measures. 5 https://doi.org/10.1051/bioconf/202410300065 BIO Web of Conferences 103, 00065 (2024) FIES 2023 Table 3. Annual volumes of average stemwood growth and carbon sequestration in sample plots structure. The annual change in stem wood stock was assessed to calculate the mass of carbon to be deposited in the living phytomass of forests. The obtained growth data were used to calculate phytomass fractions and the amount of carbon deposited per year [18]. In all surveyed sample plots, the largest amount of carbon stock was concentrated in PP7, PP8 and PP19: 786.96, 833.7 and 828.09 tonnes, respectively. These are mixed stands of pine and birch (Table 2). 3 Analysis and discussion of the results carbon sequestration in sample plots Tree species Increase by stock Phytomass growth Carbon sequestration m3/year m3 /ha*year tonnes per year tonnes/ha*year tonnes per year tonnes/ha*year Trial area 1 Spruce 5.72 1.63 2.57 0.73 1.29 0.37 Larch 2.68 0.77 1.77 0.51 0.89 0.25 Trial square 2 Spruce 7.15 1.23 3.22 0.56 1.61 0.28 Birch 18.17 3.13 11.81 2.04 5.91 1.02 Trial area 3 Pine 15.4 3.4 7.7 1.7 3.85 0.85 Trial area 4 Birch 11.66 2.33 7.58 1.52 3.79 0.76 Pine 1.16 0.23 0.58 0.12 0.29 0.06 Spruce 0.75 0.15 0.34 0.07 0.17 0.03 Trial square 5 Poplar 14.1 4.7 6.34 2.1 3.17 1.05 Trial square 6 Birch 13.37 2.52 8.69 1.64 4.35 0.82 Trial square 7 Pine 23.6 3.3 11.8 1.6 5.9 0.8 Birch 20.16 2.8 13.10 1.82 6.55 0.91 Trial square 8 Pine 27.16 3.39 13.58 1.94 6.79 0.97 Birch 9.2 1.31 10.31 1.47 5.16 0.74 Trial square 9 Birch 2.94 2.94 1.91 1.91 0.96 0.96 Trial area 10 Birch 8.26 2.07 5.37 1.34 2.69 0.67 Trial square 11 Spruce 10.2 1.2 4.6 0.81 0.6 0.3 Pine 19.2 2.4 9.60 1.20 4.80 0.60 Trial area 12 Birch 6.55 2.43 4.26 1.58 2.13 0.79 Trial square 13 Spruce 2.7 1.0 1.2 0.5 0.6 0.2 Birch 0.5 0.2 0.3 0.1 0.2 0.1 Pine 0.3 0.1 0.1 0.0 0.1 0.0 Trial square 14 Spruce 11.5 1.4 5.2 2.0 2.6 1.0 Trial area 15 Birch 6.31 2.86 4.10 1.86 2.05 0.93 Trial square 16 Birch 18.62 2.55 12.10 1.66 6.05 0.83 Trial square 17 Aspen 6.05 3.18 3.02 1.59 1.51 0.80 Trial square 18 Birch 5.76 2.62 3.74 1.70 1.87 0.85 Trial square 19 Birch 12.23 1.69 7.95 1.10 3.97 0.55 Pine 11.71 1.63 5.85 0.81 2.93 0.41 Trial area 20 Poplar 8.5 2.9 3.8 1.3 1.90 0.65 Birch 1.98 0.68 1.29 0.44 0.64 0.22 Carbon stocks and rates of carbon storage in forest d d f d i i h i According to literature data, a promising species in carbon sequestration is poplar (Populus) in northern and continental climates. This species is common in part of Russia and is found in many Russian cities. It has a relatively high carbon sequestration potential of 1.8– 6.35 t CO2-eq/ha/year, a high growth rate of 1.524–3.6 m/year, a short productive cycle of 10–15 years [22]. Out of all the 20 surveyed sites, two of them have PP5 and PP20 poplar in their composition. Conclusions The surveyed protective forest plantations on sod- podzolic and grey forest soils are in a satisfactory condition. According to silvicultural and taxation indicators, the average age of the surveyed stands is 48 years; the completeness is within the range from 0.5 to 0.9, and the bonitet is within the range from I to II. In terms of the sanitary condition, trees of the 1st and 2nd categories prevail; in some areas, forest management measures are required. Under the canopy of plantations, there is a natural regeneration of petiole oak, which is a native species of the Pre-Kama region of the Republic of Tatarstan, as well as birch, pine, and oak maple. Established protective forest plantations fulfil their functions, i.e. reduce erosion and increase soil fertility. In areas with viable undergrowth, it is advisable to carry out measures to promote natural regeneration and conservation. Preservation of undergrowth promotes further formation of stable mixed, mixed-aged, complex in structure, protective forest plantations. The largest volume of deposition was detected in mixed stands of birch and pine, poplar. 3 Analysis and discussion of the results According to the silvicultural and taxonomic characteristics, the plantations were classified as a II class of bonitet. At these sites, it is necessary to carry out sanitary cuttings. The volume of carbon deposition in these areas was 3.17 and 1.90 tonnes/ha per year. References 1. A.T. Barabanov, A.I. Petelko, A.V. Kulik, A.V. Vypova, New technology of placement of flow- regulating forest strips on slope lands, Izv. NV AUK 2(54) (2019) 2. A.T. Barabanov, Erosion-hydrological assessment of interaction between natural and anthropogenic factors in the formation of surface runoff of melt water and adaptive-landscape farming (FSC Agroecol. RAS, Volgograd, 2017), 188 p. Carbon stocks and rates of carbon storage in forest ecosystems depend on forest productivity, their condition, species composition, age and commodity 6 BIO Web of Conferences 103, 00065 (2024) FIES 2023 https://doi.org/10.1051/bioconf/202410300065 and dry-steppe zones of the Volga region, Niva Povolzhye 2(43) (2017) 3. V.I. Erusalimsky, V.A. Rozhkov, Multifunctional role of protective forest plantations, Bull. of Soil Inst. Pochv. inst. 88 (2017) 18. E.M. Romanov, T.V. Nureeva, T.F. Miftakhov, A.S. Puryaev, Ecological and raw material role of forests of the Republic of Tatarstan, Bull. of Volga Reg. State Technol. Univ., Ser.: Forest, Ecology, Nature Manag. 2, 5–18 (2015) 4. A.Y. Ivanov, M.P. Orlov, K.V. Piksendeev, Y.E. Rovnov et al., Battle for the climate: carbon farming as Russia's stake, Expert report, ed. by A.Y. Ivanov, N.D. Durmanov (Izd. dom Higher School of Econ., Moscow, 2021), 120 p. ISBN 978-5-7598-2519-7 (in reg.), ISBN 978-5-7598-2281-3 (e-book). 19. V.G. Sergienko, Impact of expected climate change on the carbon balance and ecosystem productivity in the forest sector of the Russian Federation, Proc. of the St. Petersburg Res. Inst. of Forestry 1, 74–90 (2018) 5. K.N. Kulik, Development of agroforestry science in Russia, Izv. NV AUC 3(35) (2014) 6. K.N. Kulik, A.T. Barabanov, A.S. Manaenkov, Forecast of the development of protective afforestation in Russia until 2020, Probl. of Forecast 4 (2015) 20. D.K. Suchkov, Role and economic efficiency of protective forest plantations in the restoration and transformation of landscapes, Sci. and Agron. J. 1(102), 20–23 (2018) 7. K.N. Kulik, Protective afforestation in the Russian Federation: problems and development strategy until 2020, Theor. and appl. probl. of agroindustry complex 1 (2009) 21. G.A. Petrova, A.R. Mukhametshina, H.G. Musin, R.H. Mukhametshina, H.G. Musin, R.H. Gafiyatov, Analysis of the Aspen Growth (Populus tremula L.) obtained by in Vitro in the Republic of Tatarstan, BIO Web Conf. 37 (2021) 8. K.N. Kulik, Strategy for the development of protective afforestation in the Russian Federation until 2025, Bull. of Agricult. Design 3, 5–11 (2015) 22. S.T. Zhang, J.Z. Zhang, Y. Liu, Y.C. Liu, Effects of farmland vegetation row direction on overland flow hydraulic characteristics, Hydrol. References Res.. 49(6), 1991– 2001 (2018) 9. P.I. Lakida, Carbon stocks in the phytomass of forest phytocenoses of the National Park “Pripyat- Stokhod”, Problems of forestry and silviculture, In: Collection of scientific papers of IL NAS of Belarus, Iss. 77 (Inst. of Forestry of the National Acad. of Sci. of Belarus, Gomel, 2017), pp. 99–107. 10. A.R. Mukhametshina, G.A. Petrova, H.G. Musin, I.R. Taziev, Analysis of the state of agroforest landscapes of the Republic of Tatarstan and ways of their restoration, Innovative technologies in agroindustrial complex in the conditions of digital transformation, Proc. of the Int. Sci. and Pract. Conf. (Volgograd, 09–11 February 2022) (Volgograd State Agrar. Univ., Volgograd, 2022), Vol. I, pp. 421–426, EDN JSIVAC 11. OST 56-69-83 Sample forest inventory areas. Method of laying 12. A.I. Petelko, Restoration of fertility of washed away soils, Prirodoobustroystvo 1 (2017) 13. A.I. Petelko, N.E. Novikov, Protective afforestation, Vest. AIC Stavropolya 3(15), 175–178 (2014) 14. On the Comprehensive Programme of Soil Fertility Improvement and Protection against Erosion in the Republic of Tatarstan for 1997–2005, Resolution of the Cabinet of Ministers of the Republic of Tatarstan of 12 March 1997, no. 216 15. Resolution of the Government of the Russian Federation, of 20 May 2017, no. 607 16. On approval of the procedure for conducting forest pathological surveys and the form of the act of forest pathological survey, Order of the Ministry of Natural Resources and Environment of Russia, dated 16.09.2016, no. 480 17. P.N. Proezdov, D.A. Mashtakov, A.V. Panfilov, Theoretical substantiation of adaptive-landscape systems of farming and agroforestry in the steppe 7
https://openalex.org/W2025840121
https://www.biodiversitylibrary.org/partpdf/211254
English
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A new blattoid from the Cretaceous formation of North America
Proceedings of the United States National Museum
1,906
public-domain
547
By Anton HANDLIRSCH, Adjunct Curator of the Royal Imperial Natural History Museum, Vienna, Austria. Adjunct Curator of the Royal Imperial Natural History Museum, Vienna, Austria. During the summer of 1903, while members of the U. 5. Geological Survey were investigating the Judith River beds of the Upper Cre- taceous of Montana, the following very interesting blattoid was discovered; | @Translated from the German by Lucy Peck Bush, librarian and assistant, geo- logical department, Yale University Museum. View This Item Online: https://www.biodiversitylibrary.org/item/53438 DOI: https://doi.org/10.5479/si.00963801.29-1439.655 Permalink: https://www.biodiversitylibrary.org/partpdf/211254 View This Item Online: https://www.biodiversitylibrary.org/item/53438 DOI: https://doi.org/10.5479/si.00963801.29-1439.655 Permalink: https://www.biodiversitylibrary.org/partpdf/211254 STANTONIA CRETACEA, new species. Description.— Front wing 20 mm. long, nearly elliptical, and three and one-third times as long as wide. Costal area reduced, reaching two-fifths the length of the wing, and without distinct veins, lancet shaped. Radius extending in an almost straight course to the tip of the wing and with its eight more or less compound branches, which STANTONIA CRETACEA. STANTONIA CRETACEA. are directed obliquely forward, taking up nearly half the surface of the wing. Parallel with the radius runs a second principal vein, from which three simple and two compound branches are sent off backward, part striking the apical border and part the inner margin. [am not in a position to determine whether these veins pertain to the media @Translated from the German by Lucy Peck Bush, librarian and assistant, geo- logical department, Yale University Museum. PROCEEDINGS U. S. NATIONAL Museum, VOL. XXIX—No. 1439. Proc. N. M. vol. xxix—05-——46 655 656 EDINGS OF THE NATIONAL MUSEUM. XXIX, and the cubitus or only to the latter; still it seems to me the most probable that the first four veinlets belong to the media and the last six to the cubitus. Or, is it possible that the media has entirely disap- peared? The anal area is long and narrow, three and one-half times as long as high, and occupies almost two-fifths the length of the wing; its veins run parallel with the posterior margin, and nearly all end on the suture. The veins are remarkably stout. I was not able to dis tinguish accessory or cross veins. This highly specialized blattoid form is the first that has been found in the Cretaceous formation, and may well be regarded as the type of a distinct family. Holotype.—Cat. No. 35389, U.S.N.M. Holotype.—Cat. No. 35389, U.S.N.M. Locality.—The genus is named in honor of Dr. T. W. Stanton, of the U. S. Geological Survey, who collected the type specimen in the Judith River beds of the Upper Cretaceous, at Willow Creek, Mon- tana, where it was found associated with the fossil plants described by Dr. F. H. Knowiton in Bulletin No. 257 of the U.S. Geological Survey. Holding Institution Smithsonian Libraries and Archives This file was generated 29 March 2024 at 07:49 UTC Handlirsch, Anton. 1906. "A new blattoid from the Cretaceous formation of North America." Proceedings of the United States National Museum 29(No. 1439), 655–656. https://doi.org/10.5479/si.00963801.29-1439.655. Copyright & Reuse Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. Visit BHL at https://www.biodiversitylibrary.org. This file was generated 29 March 2024 at 07:49 UTC
https://openalex.org/W3018455884
https://discovery.ucl.ac.uk/10133518/1/cmb.2020.0445.pdf
English
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Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores
Journal of computational biology
2,021
cc-by
13,113
ABSTRACT Some organizations such as 23andMe and the UK Biobank have large genomic databases that they re-use for multiple different genome-wide association studies. Even research studies that compile smaller genomic databases often utilize these databases to investigate many related traits. It is common for the study to report a genetic risk score (GRS) model for each trait within the publication. Here, we show that under some circumstances, these GRS models can be used to recover the genetic variants of individuals in these genomic databases—a recon- struction attack. In particular, if two GRS models are trained by using a largely overlapping set of participants, it is often possible to determine the genotype for each of the individuals who were used to train one GRS model, but not the other. We demonstrate this theoretically and experimentally by analyzing the Cornell Dog Genome database. The accuracy of our recon- struction attack depends on how accurately we can estimate the rate of co-occurrence of pairs of single nucleotide polymorphisms within the private database, so if this aggregate information is ever released, it would drastically reduce the security of a private genomic database. Caution should be applied when using the same database for multiple analysis, especially when a small number of individuals are included or excluded from one part of the study. Keywords: genetic risk scores, genomic privacy, GWAS, long-term privacy, reconstruction attack. Reconstructing Genotypes in Private Genomic Databases from Genetic Risk Scores BROOKS PAIGE,1,2 JAMES BELL,1 AURE´LIEN BELLET,3 ADRIA` GASCO´ N,1,4 and DAPHNE EZER1,4,5 JOURNAL OF COMPUTATIONAL BIOLOGY Volume 28, Number 5, 2021 Mary Ann Liebert, Inc. Pp. 435–451 DOI: 10.1089/cmb.2020.0445 JOURNAL OF COMPUTATIONAL BIOLOGY Volume 28, Number 5, 2021 Mary Ann Liebert, Inc. Pp. 435–451 DOI: 10.1089/cmb.2020.0445 JOURNAL OF COMPUTATIONAL BIOLOGY Volume 28, Number 5, 2021 Mary Ann Liebert, Inc. Pp. 435–451 DOI: 10.1089/cmb.2020.0445 1The Alan Turing Institute, London, United Kingdom. 2Department of Computer Science, University College London, London, United Kingdom. 3Inria, Parc Scientifique de la Haute Borne Park Plaza, Villeneuve d’Ascq, France. 4University of Warwick, Coventry, United Kingdom. 5Department of Biology, University of York, York, United Kingdom. Brooks Paige, et al., 2021. Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the t f th C ti C Li (htt // ti /li /b /4 0) hi h it t i t d Brooks Paige, et al., 2021. Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Keywords: genetic risk scores, genomic privacy, GWAS, long-term privacy, reconstructio PAIGE ET AL. PAIGE ET AL. research focus on long-term privacy of genomic databases rests on the longevity of the encryption scheme (Huang et al., 2015), it is also important to remember that these genomic databases are not just sitting on a server somewhere, but are also being continually utilized for making new scientific discoveries. Each time these databases are accessed and the scientific results are published, there is a risk that information will be leaked and that eventually this would enable an attacker to reconstruct private information held in the database. Genomic researchers are already aware that some forms of aggregate data from their databases should not be released publicly, because there is a risk that an attacker may be able to determine whether a particular individual is a member of the database (a membership inference attack). For instance, such attacks have already been developedforsummarystatisticsabout the frequencyofsingle nucleotidepolymorphisms(SNPs;Cai etal.,2015; Dwork et al., 2015; Simmons and Berger, 2015). Membership inference attacks have also been developed for the case where a person is allowed to repeatedly query a database to learn whether at least one individual contains a particular SNP(ShringarpureandBustamante,2015; Raisaroetal.,2017; vonThenenetal.,2018).Thesekindsof aggregate statistics about the frequency or presence/absence of a particular SNP in a database might be useful to release to the broader research community, but it is not an essential output of the research process. However, the main research findings—that is, the SNPs associated with the trait of interest and their strength of association—are essential to publish since the entire purpose of these genomic research projects is to uncover the relationship between genetic variants and phenotypic traits. Moreover, knowledge of these SNPs can lead to new diagnosis procedures or new potential drug targets, so their release is important for the public interest (Visscher et al., 2017). However, even this information can potentially leak private information about individuals in the database. For instance, Im et al. (2012) found that information about individuals in a genomic database is leaked when studies publish whether each SNP is correlated or anti- correlated to the trait of interest. It is important to quantify how much information is leaked by publishing these research findings, so that scientists can make informed decisions about when to publish their results and whether it is worth risking the privacy of the participants. PAIGE ET AL. g p y p p In this study, we demonstrate that the kind of research output that is published from genome-wide association studies (GWAS) has the potential to leak enough information to recover the SNPs of individuals in the database (a reconstruction attack), under specific circumstances. In particular, we focus on the release of genetic risk scores (GRS), a common research output for finding genetic associations with continuous traits (Qi et al., 2011; Belsky et al., 2013; Zhao et al., 2014; Chouraki et al., 2016; Day et al., 2017; Knowles and Ashley, 2018). We also focus on cases where a database is repeatedly used to perform a GWAS analysis, but not all the individuals are part of all the analyses. This could be the case because some individuals drop out of the study or skip specific survey questions. Alternatively, some databases, such as 23andMe, may grow in size over time and allow several GWAS to be performed within a short period. Under these circumstances, we demonstrate that it is possible to completely reconstruct the SNPs of an individual by using a custom expectation–maximization (EM) algorithm. We also provide suggestions for avoiding this kind of attack. To be clear, this article focuses on the simpler case in which the exact same trait is investigated in multiple GWAS studies; however, we expect that some version of this attack may be developed in the near future for the case of multiple highly correlated traits. 1. INTRODUCTION I n a survey of genomic privacy experts, the long-term privacy of genomic information was deemed both the most important and the most challenging problem to overcome (Mittos et al., 2019). If an individual’s password or ID number gets leaked, it is always possible to change it. However, it is impossible for a person to change their genetic code and they will pass part of it onto their children, so any information leaks can have long-term impacts on both the individual and their descendants. Although much of the I 435 436 1.1. Overview of scenarios that will be investigated We demonstrate a series of reconstruction attacks that enable us to infer the genotypes of individuals in private genomic databases, based on publicly released GRS. These attacks will initially be deployed in a very favorable scenario, but the scope of the attack will be subsequently expanded, building up to the scenario shown in Figure 1. It is worth noting that the reconstruction attacks that we will describe do not depend on (1) how the SNPs were initially filtered or (2) how strongly they associate with the trait of interest. We will begin by investigating a simple scenario: Two GWAS studies are performed to identify SNPs associated with the same trait, and the two studies use the same set of participants, except that the second study includes one extra individual. In addition, we will assume that we know the frequencies of each SNP and the frequencies that pairs of SNPs co-occur in the same individual. We assume that both studies publish the coefficients associated with the GRS models that they infer as part of the analysis. Next, we will consider the case in which the second study includes more than one additional participant and we demonstrate that in many circumstances this still allows us to easily reconstruct the individual genotypes of all the individuals that are found in the second study but not the first (see Section 3.2). 437 RECONSTRUCTION ATTACKS FROM GRS FIG. 1. We investigate the case where two GWAS studies are performed on two datasets that mostly contain the same individuals. We reconstruct the genotype of those individuals added to the second study, using the GRS from each study and an estimate of SNP frequencies. GRS, genetic risk score; GWAS, genome-wide association studies; SNP, single nucleotide polymorphism. FIG. 1. We investigate the case where two GWAS studies are performed on two datasets that mostly contain the same individuals. We reconstruct the genotype of those individuals added to the second study, using the GRS from each study and an estimate of SNP frequencies. GRS, genetic risk score; GWAS, genome-wide association studies; SNP, single nucleotide polymorphism. Afterward, we will demonstrate that we do not need to know the precise frequencies of SNPs and frequencies of co-occurring SNPs, as long as we have a reasonable estimate of these values from public databases (see Section 3.3). We also briefly discuss how loosening additional restrictions would impact our ability to predict individual genotypes. 1.1. Overview of scenarios that will be investigated In particular, we analyze the case where the two sets of SNPs that are used by the two studies are not identical. These results imply that if two sets of GRS are released on two genetic datasets with largely overlapping populations, it may be possible to reconstruct the genotypes of those individuals who participated in one study but not the other (Fig. 1). PAIGE ET AL. where we have defined the symmetric (N + 1) · (N + 1) matrix K as where we have defined the symmetric (N + 1) · (N + 1) matrix K as K = 1 M F> MFM: K = 1 M F> MFM: (4) (4) Now, suppose a second study is run, targeting the same phenotype, which adds a single extra individual with SNPs represented by the N length vector x0. This corresponds to adding the row /> 0 = [ x> 0 1 ] to the design matrix, and extending y with the additional phenotypic value y0 for the new individual. The updated estimator (i.e., the GRS values for the second study) is given by ^bM + 1 = (F> MFM + /0/> 0 ) - 1(F> MyM + y0/0): (5) (5) We assume that both GRS models ^bM and ^bM + 1 are released publicly. An attacker aims at using this knowledge to reconstruct /0 (the genotype of the added individual). Through algebraic re-arrangement (see Section 5.2), we find that: /0 = 1 C K(^bM + 1 - ^bM) (6) (6) where C is a scalar, specifically C = 1 M (y0 - /> 0 ^bM + 1). Eq. (6) means that /0 is a scalar multiple of K(^bM + 1 - ^bM). proach, thus, centers on the use of the vector that we define as d1, Our approach, thus, centers on the use of the vector that we define as d1, d1 = D K(^bM + 1 - ^bM) = C/0‚ (7) (7) corresponding to a rescaled copy of the input SNP data in the design matrix /0, which can be easily computed from the two parameter vectors if the matrix K is known. As seen in Section 3.1, we can use d1 to exactly reconstruct the added individual with 100% accuracy. corresponding to a rescaled copy of the input SNP data in the design matrix /0, which can be easily computed from the two parameter vectors if the matrix K is known. As seen in Section 3.1, we can use d1 to exactly reconstruct the added individual with 100% accuracy. We additionally consider the case where m additional individuals have been included in the second study, yielding a new GRS model ^bM + m including these M + m participants. PAIGE ET AL. The extra rows of the design matrix now form a matrix Fm of size m · (N + 1), where each row is an individual that was added to the second study and each column is an SNP (and the last column contains only 1). The corresponding analog to Eq. (7) for multiple individuals, which we derive in Section 5.2, is dm = D K(^bM + m - ^bM) = F> mCm‚ (8) (8) where Cm is a vector of length m. For sufficiently small m (relative to N), exact reconstruction of all m added individual genomes is also possible in this setting, following the algorithm we will introduce in Section 3.2. where Cm is a vector of length m. For sufficiently small m (relative to N), exact reconstruction of all m added individual genomes is also possible in this setting, following the algorithm we will introduce in Section 3.2. The previous examples have focused on cases in which the participants in the first study are a subset of the individuals in the second study. In Section 5.7, we consider the case in which the first study has some participants that are not found in the second study and vice versa. We show that the same strategies for reconstructing the genome can be used as in the previous scenario that we discussed, in which multiple participants are added to the second study. 2. METHODS GRS models describe the relationship between a particular phenotype of interest and particular SNPs. These models are fit in a two-stage process: First, a reduced set of SNPs is selected from a potentially very large pool of candidates; then, this reduced set is used as the independent variables in a linear regression analysis. The set of SNPs is selected by first filtering for those that significantly correlate to the trait of interest, after controlling for other covariates. These SNPs are then further filtered to ensure that they are far apart from one another, to decrease the correlation between them. In this setting, we suppose that M individuals have taken part in a study, and N SNPs have passed the filtration steps to be used in a linear model. Let yM be the vector of M real-valued phenotypes, and XM be an M · N binary matrix, where XM[i‚ j] = 1 if individual i has SNP j. To include an intercept term in the linear model, we define the design matrix FM to be the M · (N + 1) matrix FM = XM 1M ½ : (1) (1) The GRS model parameter bM is just the coefficient vector of the linear model yM = FMbM + e‚ (2) yM = FMbM + e‚ (2) where e is independent Gaussian noise. Given FM and phenotypes yM, the maximum likelihood estimate of this parameter has a closed form where e is independent Gaussian noise. Given FM and phenotypes yM, the maximum likelihood estimate of this parameter has a closed form ^bM = D 1 M K - 1(F> MyM)‚ (3) (3) 438 – For i = 1‚ . . . ‚ N: Kii estimates the probability that SNP i has value 1 (i.e., the frequency of the SNP in the population). – For i = 1‚ . . . ‚ N - 1 and j > i: Kij = Kji estimates the probability that both SNP i and SNP j are 1 simultaneously (i.e., the frequency of SNP i and SNP j co-occurring in the same individual). – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji also estimates the probability that SNP i has value 1, that is, Ki‚ N + 1 = KN + 1‚ i = Kii. – Finally, KN + 1‚ N + 1 = 1. or i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji also estimates the probability that SNP i has value 1, that i + 1 = KN + 1‚ i = Kii. 3.1. Complete reconstruction of one individual’s genotype when SNP frequency information is known 3.1. Complete reconstruction of one individual’s genotype when SNP frequency inform is known The first, most straightforward case is when only one participant is added between the first and second studies, that is, where ^bM is the GRS for the first study (containing M participants), and ^bM + 1 is the GRS for the second study as described in Eqs. (3) and (5). Both of these are vectors of length N + 1, where the first N indices correspond to the relationship between each SNP and the trait and the last element is the intercept of the linear model. For now, we also assume we are in the setting where the matrix K is known, for example, because the SNP frequency information has been publicly released. ^ ^ Given K, ^bM + 1, and ^bM, we can use d1 (a vector of length N + 1) to precisely determine the genotype of the individual who was added to the database. For each i = 1‚ . . . ‚ N, the ith entry of d1 is either equal to 0 if /0 contains a 0 (i.e., the individual does not have the SNP at that index) or to C if /0 contains a 1 (i.e., the individual has the SNP at that index). In other words, it is possible to exactly read off the SNPs of the added individual in this setting. Indeed, we tested this strategy on the Cornell Dog Database and found that we were able to reconstruct the genotype of the dog that was added to the second study with 100% accuracy, on both common and uncommon SNPs (Fig. 2A). A B C FIG. 2. (A) We have perfect accuracy in reconstructing the genotype when K is known (using 200 random SNPs to estimate average breed weight in the Cornell Dog Database). (B) We can reconstruct all the genotypes of multiple dogs that are added to the second study and (C) this works in practice by using the data from the Cornell Dog Database, as in (A). B C A C C B A FIG. 2. (A) We have perfect accuracy in reconstructing the genotype when K is known (using 200 random SNPs to estimate average breed weight in the Cornell Dog Database). RECONSTRUCTION ATTACKS FROM GRS 439 Thus, knowledge of SNP frequencies and pairwise co-frequencies from the original study are all that is required to compute K. In the following Sections 3.1 and 3.2, we consider adding one and multiple individuals at once, respectively, in the setting where this matrix K can be estimated exactly. However, although ^bM, ^bM + 1 and M are likely to be published along with the study, an attacker would often need to estimate K from other publicly available data. Most studies will report some information about the study population (such as whether the study focused on individuals from a specific continent), which can help with estimating K. From this information, we can estimate the value of K in similar populations as those used in the study using publicly available data, for example, from the HapMap project. Our additional experiments in Section 3.3 use a custom EM algorithm to find maximum likelihood estimates of /0 when the matrix ^K  K is estimated from independent public data. The derivation of this EM algorithm is given in Section 5.4.3, and a formal analysis of the reconstruction error of /0 given the error in ^K is found in Section 5.2.1. 2.1. Estimation of K As it turns out, the entries of matrix K correspond to simple population-level statistics of the SNPs, which could either be inadvertently released (under the assumption they would be safe to share), or could be estimated from another sample from the same population. In fact, the entries of K depend only on the SNP frequencies and SNP co-occurrence frequencies in the dataset: – For i = 1‚ . . . ‚ N: Kii estimates the probability that SNP i has value 1 (i.e., the frequency of the SNP in the population). – For i = 1‚ . . . ‚ N - 1 and j > i: Kij = Kji estimates the probability that both SNP i and SNP j are 1 simultaneously (i.e., the frequency of SNP i and SNP j co-occurring in the same individual). – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji also estimates the probability that SNP i has value 1, that is, Ki N + 1 = KN + 1 i = Kii. – Finally, KN + 1‚ N + 1 = 1. 3. RESULTS The key observation from the previous section is that the vectors d1 and dm, derived from the change in parameter vectors ^b from a first study to a second study, take only a finite number of values thanks to the fact that the design matrices F contain only zeros and ones. In particular, when m new individuals are added to the second study, each entry of the vector dm can only take at most 2m values, and a zero value corresponds to the setting where all individuals have the most common variant for that SNP. This section describes algorithmically how these vectors can be used to recover the genomes of the additional individuals, as well as empirical tests that use the Cornell Dog Genome dataset as a case study (Hayward et al., 2016). More details on the experimental setup can be found in Section 5.1. PAIGE ET AL. 440 3.2. Complete reconstruction of multiple individuals’ genotype when SNP frequency information is known We now consider the case where m additional individuals have been included in the second study, yielding a new GRS model ^bM + m, including these M + m participants. Consider again Eq. (8) described earlier. The ith row of Fm is a binary vector that represents the com- bination of the m individuals who have SNP i. This means that, for a fixed value of Cm, the value of the vector dm at index i is uniquely determined by the combination of individuals who have SNP i (Fig. 2B). In other words, there will be at most 2m unique values taken by entries of dm, each corresponding to a combination of the values in vector Cm (Fig. 2C). If we were to learn which values of dm are also found in Cm, then we could infer the complete genotypes of all the m individuals added to the second study. We would be able to reconstruct m complete genotype vectors, although it would be impossible to know which of the genotypes corresponded to which of the m individuals. In fact, in many cases it is extremely straightforward to determine which values in dm cor- respond to values in Cm. Here, we describe a simple algorithm for finding Cm when there are exactly 2m unique values in dm. If this is not the case, please refer to the more complete algorithm in Section 5.3. 1. First, extract all unique, nonzero values from dm. Find the sum of all pairs of values in this vector p 3. Find all values that are in Eq. (1), but not in Eq. (2). The values of Cm appear in this list. There is no way to know which value of Cm corresponds to which index, so for simplicity we can randomly assign them indices. 3. Find all values that are in Eq. (1), but not in Eq. (2). The values of Cm appear in this list. There is no way to know which value of Cm corresponds to which index, so for simplicity we can randomly assign them indices. 4. Each value in Cm corresponds to a specific individual who was added to the second study. Each value in dm can be described as a sum of a unique combination of values in Cm. PAIGE ET AL. For instance, if dm[i] = Cm[j] + Cm[k], this means that the SNP at position i is found in individual j and k, but no one else. 4. Each value in Cm corresponds to a specific individual who was added to the second study. Each value in dm can be described as a sum of a unique combination of values in Cm. For instance, if dm[i] = Cm[j] + Cm[k], this means that the SNP at position i is found in individual j and k, but no one else. We tested this approach by using the Cornell Dog Database, in a test scenario where the second study added three different dogs. We were able to uniquely identify the genotypes of all three dogs with 100% accuracy, with both common and uncommon SNPs (Fig. 2C). 3.1. Complete reconstruction of one individual’s genotype when SNP frequency information is known (B) We can reconstruct all the genotypes of multiple dogs that are added to the second study and (C) this works in practice by using the data from the Cornell Dog Database, as in (A). PAIGE ET AL. 4. DISCUSSION In this study, we demonstrate that private information is leaked when GRS models are published, specifically in the case where two sets of largely overlapping individuals are used for multiple studies. In particular, we show that we can recover SNPs from an individual in a private database—a reconstruction attack. Even though we would not have a name associated with this genotype, it may be possible to identify the individual once the genotypic data are available to the attacker. For instance, the attacker may have FIG. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ^K, as compared with a natural baseline that always predicts the most common variant at each SNP locus. We use this as a baseline, because without any additional information about bM and bM + 1, the most accurate prediction of the dog’s genotype would be to predict the most common variant at each locus. Here, we define accuracy as the proportion of SNPs that are correctly identified in the dog that was found in the second GWAS study, but not the first. Each distribution is constructed from 500 experimental test points, in which we (1) took 10 random splits of the full dog dataset, assigning dogs to either the public or private dataset; (2) for each split, we tested the reconstruction 50 times, each time adding a different randomly sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of increasing size, improving performance. EM, expectation–maximization. FIG. 4. Accuracy at reconstruction of genomes x0 using EM estimation and a noisy estimate ^K, as compared with a natural baseline that always predicts the most common variant at each SNP locus. We use this as a baseline, because without any additional information about bM and bM + 1, the most accurate prediction of the dog’s genotype would be to predict the most common variant at each locus. Here, we define accuracy as the proportion of SNPs that are correctly identified in the dog that was found in the second GWAS study, but not the first. 3.3. Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database Previously, we assumed that the attacker had access to the matrix K, which consists of population-level statistics on frequencies and co-occurrence frequencies of SNPs. Although this could be released volun- tarily by organizations that are not aware of the risk, we now consider the case where K is not directly available to the attacker but is instead estimated from a separate public database assumed to correspond to individuals from the same population. We simulated this scenario by using the Cornell Dog Database by taking one random set of dogs for building the GRS model, and a second non-overlapping set of dogs for estimating ^K. We compared the value of ^d1 = ^K(^bM + 1 - ^bM) with the known value of /0. We observe that ^d1 has significantly different values at indices where /0[i] = 0 and /0[i] = 1; examples for the cases where one and three dogs are added can be seen in Figure 3. ^ Themainchallengeisthatthevector ^d1 nowincludesadditionalnoise,sowecannotsimplyuseitsentryatindex N + 1 to estimate C, nor do the entries i with /0[i] = 0 also correspond directly to ^d1[i] = 0. Instead, we develop a custom EM algorithm to find a maximum likelihood estimate of the constant C and recover /0, that is, to determine the probability that each /0[i] = 0 or /0[i] = 1, based on the value of ^d1 (see Section 5.4.3 for details). We find that this method can successfully reconstruct the correct value of /0[i] much better than a baseline that uses the public dataset to independently estimate the most common variant for each of the SNP (Fig. 4). Crucially, we show that our approach is able to reconstruct, with relatively high accuracy, the genotypes of dogs even when they differ significantly from those in the public dataset (Fig. 5). This shows that our attack is able to extract information about the particular individuals that differ across the two studies, not merely about the general population as in the most-common-variant baseline. By definition, dogs that have genotypes that differ significantly from the general population have a higher proportion of uncommon SNPs, and the ability to recover these uncommon SNPs is particularly important from a privacy per- spective. Indeed, uncommon SNPs can be used to identify a particular individual and are also more likely to be associated with disease phenotypes, which is sensitive information. 3.4. Accurate estimation of an individuals’ genotype when different SNPs are used in each study When GRS models are constructed, the first step is to filter the set of SNPs down to a small set of SNPs that are (1) significantly correlated to the trait after covariates are considered and (2) far apart from one another along the genome. If the two studies use two different sets of SNPs to construct the GRS model, it is still possible to recover whether or not each of the SNPs in the overlap is present in the new individual. This process is highly analogous to the previous cases and is detailed in Section 5.6. 3.3. Accurate estimation of an individual’s genotype when SNP frequency information is estimated from a public database In general, we find that the larger the RECONSTRUCTION ATTACKS FROM GRS 441 FIG. 3. Example values taken by the noisy vector ^d, given the true value of the corresponding SNP in the genome. (Left) adding one new participant; (right) adding three new participants. These figures are analogous to those in Figure 2, although in the case where K is not known and instead estimated from an independent public database. FIG. 3. Example values taken by the noisy vector ^d, given the true value of the corresponding SNP in the genome. (Left) adding one new participant; (right) adding three new participants. These figures are analogous to those in Figure 2, although in the case where K is not known and instead estimated from an independent public database. public dataset available, and the more similar the dataset is to the unknown private dataset, the better we are able to reconstruct the genome of the added individual. Full details and description of the experimental setting are given in Section 5.1. We also derive theoretical error bounds for our estimate of /0 based on the error in ^K in Section 5.4.1. public dataset available, and the more similar the dataset is to the unknown private dataset, the better we are able to reconstruct the genome of the added individual. Full details and description of the experimental setting are given in Section 5.1. We also derive theoretical error bounds for our estimate of /0 based on the error in ^K in Section 5.4.1. This task becomes more challenging when multiple individuals are added simultaneously and K is unknown; an algorithm for estimating Fm for m > 1, along with additional empirical results, is given in Section 5.4. 4. DISCUSSION Each distribution is constructed from 500 experimental test points, in which we (1) took 10 random splits of the full dog dataset, assigning dogs to either the public or private dataset; (2) for each split, we tested the reconstruction 50 times, each time adding a different randomly sampled dog to the second GWAS study. The private dataset always has 1000 individuals; the public test dataset is of increasing size, improving performance. EM, expectation–maximization. 442 PAIGE ET AL. FIG. 5. Results of Figure 4 broken down by individual dogs. Here, each point represents a dog and we define atypicality as the proportion of uncommon variants that the dog has compared with the public database—for instance, if 51% or more of dogs in the public database have a G in a specific locus, but this dog has a T, then this would count toward the dog’s atypicality. In other words, dogs further to the right are less and less similar to average dogs present in the public dataset (measured by percentage of different variants). In contrast to the most-common-variant baseline, our method generalizes well even to dogs that are highly dissimilar to those in the public dataset. Larger public databases (right) provide more accurate population estimates of ^K, leading to more accurate reconstructions overall. FIG. 5. Results of Figure 4 broken down by individual dogs. Here, each point represents a dog and we define atypicality as the proportion of uncommon variants that the dog has compared with the public database—for instance, if 51% or more of dogs in the public database have a G in a specific locus, but this dog has a T, then this would count toward the dog’s atypicality. In other words, dogs further to the right are less and less similar to average dogs present in the public dataset (measured by percentage of different variants). In contrast to the most-common-variant baseline, our method generalizes well even to dogs that are highly dissimilar to those in the public dataset. Larger public databases (right) provide more accurate population estimates of ^K, leading to more accurate reconstructions overall. access to partial genotypic information of the individual and then be able to identify them. Alternatively, they could use the genotype information to predict ethnicity and other phenotypic traits that could then be used to uniquely identify the individual. 4.1. Suggestions for good practice We provide a number of simple suggestions for good practice that would help limit this attack. We provide a number of simple suggestions for good practice that would help limit this attack. 1. Aggregate statistics about the frequency of SNPs in the database or the frequency of co-occurrence of SNPs should never be released. We have shown that this information, combined with GRS, allows to precisely reconstruct individual genomes in various settings. It may be possible to release noisy versions of SNP frequency data, but this would be equivalent to releasing ^K (our estimated K from the public database). With our EM algorithm, we have demonstrated that it is still possible to do some genotypic reconstruction with a noisy ^K, but this becomes harder as the noise in ^K increases. On the other hand, providing a very noisy ^K may be of limited utility to the scientific community. 2. If a genetic dataset is intended to serve for multiple complementary analyses, it is important that all study participants are used in every analysis performed. If there are missing phenotypic data from a few individuals, they should not be included in any of the analyses performed, or their privacy may be compromised. 3. When multiple individuals are added in between two studies, then the ability to reconstruct the genomes dependsonthenumberofSNPs,beinglargerelativetothenumberofindividuals.Inparticular,ifmnewdogs are added, exact reconstruction is only possible by using the approach in Section 3.2 if the number of SNPs N> 2m. Thus, we suggest to avoid releasing multiple studies that differ by fewer than log2 N individuals. 4. DISCUSSION We also note that even an incomplete reconstruction attack (in which only a proportion of the SNPs are correctly identified) is likely to be sufficient to perform a membership inference attack. Investigating the relationship between the reconstruction attack and the membership inference attack will be a subject of future research. Importantly, if the attackers were unable to link the genomic data with a particular individual, the reconstruction attack would still be a breach in privacy that could have serious conse- quences. For instance, the patient may have only consented to have their genomic data used in particular kinds of research studies, whereas the attacker may use the reconstructed genomic data for a different (potentially unethical) purpose. 4.2. Extensions and future work Although we have analyzed the case where the genome is represented by binary values of 0 or 1, often studies instead count the number of times each allele is present, which would lead to a design matrix F containing values 0‚ 1‚ or 2. In this scenario, K no longer contains the frequencies of SNPs and their co- occurrences, but it is something slightly more complicated that we describe in Section 5.8. This does not dramatically change the approach in this study, except in that the vector dm can take 3m possible values, 443 RECONSTRUCTION ATTACKS FROM GRS rather than 2m. In practice, then, studies that use allele counts are somewhat more robust to attacks; the multiple dog reconstruction attack would likely be ambiguous if 3m > N, rather than 2m > N. p g y g A possible countermeasure to our reconstruction attack could consist of randomly perturbing the GRS models before releasing them, as done in differentially private linear regression (Wang, 2018). However, a naive application of this strategy could destroy the utility of the models. A formal and empirical analysis of the effectiveness of such protection against reconstruction attacks, as well as of the usefulness of the resulting GRS models to genomic researchers, is beyond the scope of this article and left for future work. Another countermeasure is to refrain from releasing precise information about the population structure of the study population to prevent the attacker from estimating K effectively. This would, however, limit the utility of the research study, because the researchers would not know to what populations the research applies. Our work has a number of limitations. For instance, we only test our EM algorithm on dog data. Dog populations may have different population structures than human populations due to selective breeding, so in the future we aim at investigating how properties of population structure will impact our ability to estimate K and the accuracy of our reconstruction attack. It may seem unlikely on the surface that two GWAS analyses will include nearly the same participants. One potentially common setting where this could arise is when a single study collects both genotype and phenotype data from a single set of participants, and it releases multiple models to predict multiple traits. 5.1. Experimental details 5.1.1. Cornell dog database. To experimentally test the reconstruction attacks, we used data from the Cornell Dog Genome Database, which contains data about SNPs from a wide range of dog breeds and a number of associated phenotypic traits. The two traits we focused on were average breed weight and average breed height, because these two phenotypes had the fewest number of missing values. For the initial investigation, we binarized the genotype matrix—considering all heterogenous alleles to have a value of 1. (We also repeated the analysis with the original genotype matrix.) Only common SNPs (i.e., SNPs that were found in 25%–75% of the dogs) were used, leaving 23,497 SNPs. For each linear model built, M = 1000 dogs were randomly sampled as the ‘‘private’’ dataset and N = 200 SNPs were randomly selected. To ensure that the SNPs that were sampled were spatially distributed, the SNPs were randomly sampled in a stratified way, so one SNP was selected in every 23‚ 497 200 -sized bin. 5.1.2. Experiment with imprecise K. First, two linear models were constructed to predict average breed weights: one with the M = 1000 randomly sampled dogs and another that contained one additional randomly sampled dog. This gives ^bM and ^bM + 1. To mimic the process of estimating K from a public database, we randomly sampled an additional 200, 400, or 800 dogs that were not included as part of the original set and used this to estimate K, which we denote by ^K. Now, we could calculate ^K(^bM + 1 - ^bM) and compare this with the known /0 for the additional dog from the second study. These additional dogs are taken from a third ‘‘test’’ dataset, disjoint from both the public and private data. The plots in Figures 4 and 5 are produced by re-running the algorithms across 10 random public/private/test splits, where the ‘‘test’’ dataset has 50 dogs that are each individually considered as candidates for the (M + 1)th dog added to the private dataset. 4.2. Extensions and future work In this case, there may be a small number of individuals who are used in one analysis, but not the other; for instance, there may be a small subset of participants who skip a particular survey question that was used to collect phenotype information, and this is, indeed, evident in a recent study (Jiang et al., 2019). In such settings, it could be very possible for multiple released GRS models to be computed on sets of individuals that differ by only a few participants. In future work, we aim at extending our analysis and attack to settings where multiple GRS models are released, each predicting different but highly correlated traits. 5.3. Algorithm for identifying unique genotypes of multiple dogs when K is known Although the simple approach described in the main article will work in many cases, there are a few special circumstances where a more complex algorithm may be required. In particular, it would not work if there are combinations of SNPs that are not observed among the individuals added to the database. For instance, if there is not an SNP location where the first individual has an SNP variant and the others do not, then we would miss the corresponding value in Cm. However, it is still possible to identify all the values in Cm through a more complex algorithm: 1. First, extract all unique, nonzero values from dm. 2. Find the sum of all pairs of values in Eq. (1). 3. Find all values that are in Eq. (1), but not in Eq. (2). 4. If there are exactly m values in Eq. (3) and the sum of these values equal the last value of dm (corresponding to the intercept term), then we have found the correct values of Cm. p g p 5. Otherwise, this suggests that there are one or more elements of Cm that are missing from Eq. (3) and possibly a few values in Eq. (3) that are not in Cm. 5. Otherwise, this suggests that there are one or more elements of Cm that are missing from Eq. (3) and possibly a few values in Eq. (3) that are not in Cm. 6. Begin by subtracting every pair of values in Eq. (3). These are now also potential values of C 7 S h f f l f E (3) d (6) h h l l f d Th 7. Search for a set of m values from Eqs. (3) and (6) that sum to the last element of dm. There may be more than one set of values for which this is true. 8. If this search is unsuccessful, repeat steps 6–7. Eventually, a set of m values summing to dm should be found. 8. If this search is unsuccessful, repeat steps 6–7. Eventually, a set of m values summing to dm should be found. 9. If more than one possible set of values is found for Cm in Eq. (7), it is still possible to compare these sets and identify which is the most likely to contain the true values of Cm. 5.3. Algorithm for identifying unique genotypes of multiple dogs when K is known For each possible Cm vector, a set of genotypes can be constructed for the m additional individuals. Using the frequencies of each SNP, it is possible to calculate the probability of observing each genotype. The set of values that produces the most likely genotypes for the m individuals is most likely to be the correct one. In addition, this algorithm depends on the fact that it is extremely unlikely that if someone were to sample three random continuous numbers i, j, and k, it would just so happen that i + j = k. There is an extremely small chance that a value of Cm would be un-discoverable because of a coincidence of this nature. 5.2. Adding multiple dogs Here, we explain Eqs. (7) and (8). Note that the former is a special case of the latter so we will only explain the latter in detail. First note that, by definition, ^bM = (F> MFM) - 1F> MyM = (MKM) - 1FT MyM‚ ^bM + m = (F> M + mFM + m) - 1F> M + myM + m = (MKM + mKm) - 1F> M + myM + m: ^bM = (F> MFM) - 1F> MyM = (MKM) - 1FT MyM‚ 444 PAIGE ET AL. Substituting these into the left hand side of the following equation gives the right hand side: Substituting these into the left hand side of the following equation gives the right hand side: (MKM + mKm)^bM + m - MKM ^bM = FT mym: (9) (9) (MKM + mKm)^bM + m - MKM ^bM = FT mym: (9) (MKM + mKm)^bM + m - MKM ^bM = FT mym: This equation can be rearranged to give KM(^bM + m - ^bM) = 1 M FT mym - m M Km ^bM + m = 1 M FT m(ym - Fm ^bM + m): Defining the length m vector Cm = 1 M (ym - Fm ^bM + m) yields the form used in Eq. (8). For the special case of m = 1, Cm is a scalar and we recover Eq. (7). 5.3. Algorithm for identifying unique genotypes of multiple dogs when K is known RECONSTRUCTION ATTACKS FROM GRS 5.4.1. Analytic bound on k /0 - ^/0 k. For convenience, we only consider the case of adding a single individual, though the generalization is quite straightforward. If ^K is substituted for K in our reconstruction Eq. (7), we get an approximation of /0 that we denote ^/0. We would like to bound the (relative) error between /0 and ^/0. Later, we ignore the constant factors C and ^C for simplicity, noting that these scaling factors are estimated from the resulting /0 or ^/0 anyway. We, thus, consider u0 = K(^bM + 1 - ^bM) and ^u0 = ^K(^bM + 1 - ^bM). Using k  k on vectors, and also on matrices, we denote the corresponding operator norm. The relative error between u0 and ^u0 is given by: k u0 - ^u0 k k u0 k = k ( ^K ^K - 1 - ^KK - 1)u0 k k u0 k  k ^K ^K - 1 - ^KK - 1 k = k ^K( ^K - 1 - K - 1) k : Note that ^K - 1 - K - 1 = ^K - 1(K - ^K)K - 1 and hence k ^u0 - u0 k k u0 k  k K - 1 kk K - ^K k ‚ (10) (10) This means that we can bound the error by two quantities. The term k K - 1 k is bounded earlier by 1= min (eig(K)), which is finite as soon as K is nonsingular. This is not a strong requirement, as in the case of linear regression it is required for ^bM + 1 and ^bM to exist. Note that in the case of L2-regularized linear regression (i.e., ridge regression), K is replaced by K + kI, where k is the regularization parameter, and we can directly bound this term by k. The key term in Eq. (10) is k K - ^K k, the error in estimating K by ^K. Let us assume that the public database used to obtain ^K follows the same distribution as the private database used to fit the GRS models. Denote by ^M the number of individuals used to estimate ^K. Then, under classic boundedness assumptions and leveraging matrix concentration inequalities such as matrix Bernstein Tropp (2015), we can show that E[ k K - ^K k ] = O(1= ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi min ( ^M‚ M) p ). RECONSTRUCTION ATTACKS FROM GRS This shows that the error in estimating K is small as long as the private and public databases are large enough. 5.4.2. Modeling the error in ^K. In this section, we define a model to capture the error in ^K, which leads to the EM algorithm for estimating /0, which is used in the experiments. As our estimated ^K drifts from the true K, this expression ^K(^bM + 1 - ^bM) would produce a wider range of values than just 0 and C. Let eij*N (0‚ r2) be independent noise, which we assume corrupts each element of Kij; that is, given the estimated matrix ^K suppose 5.4.2. Modeling the error in ^K. In this section, we define a model to capture the error in ^K, which leads to the EM algorithm for estimating /0, which is used in the experiments. As our estimated ^K drifts from the true K, this expression ^K(^bM + 1 - ^bM) would produce a wider range of values than just 0 and C. leads to the EM algorithm for estimating /0, which is used in the experiments. As our estimated K drifts from the true K, this expression ^K(^bM + 1 - ^bM) would produce a wider range of values than just 0 and C. Let eij*N (0‚ r2) be independent noise, which we assume corrupts each element of Kij; that is, given the estimated matrix ^K, suppose Let eij*N (0‚ r2) be independent noise, which we assume corrupts each element of Kij; that is, given the estimated matrix ^K, suppose Kij*N ( ^Kij‚ r2)‚ (11) (11) for some small r2. This is clearly an oversimplification (as we know K is e.g., bounded and symmetric), but it is a useful starting point that allows derivation of a simple estimation algorithm. For notational brevity, in this and the following section we define the vector (12) D = ^bM + m - ^bM (12) which corresponds to the difference between the two GRS model parameter vectors when m additional dogs are added. Given the true value of K, the system of equations F> mcm = KD relates the known quantity D and the Gaussian-distributed K with the matrix Fm and the unknown values in the vector cm 2 Rm. 5.4. Estimating K If the true matrix K is unknown, it can be estimated with public data. We denote this estimator by ^K. For ^K to be an accurate estimate, the data that it is generated from must be drawn from the same (or a sufficiently similar) population as that used in the private study. We will model this assuming no dis- crepancy between population distributions; however, when we discuss how to evaluate whether the esti- mate is good, that assessment should account for this systematic error as well. Later, we will be primarily concerned with the error due to the subsampling in both the private and public datasets. Also of note, the same analysis given next applies to the scenario in which the researchers do not release K, but rather release a ‘‘noisy’’ version of K, where the noise is drawn from a normal distribution. They might consider doing this if they feel that releasing information about SNP frequencies is important for the research community, but they do not wish to release the real K because this would allow for an exact reconstruction of genotype. This noisy K could still be used in a reconstruction attack in the same way as an estimate of K from a public database is used. RECONSTRUCTION ATTACKS FROM GRS 445 PAIGE ET AL. With some algebraic re-arrangement, and since for the true underlying value of K we have KD = F> mcm, we can write this as With some algebraic re-arrangement, and since for the true underlying value of K we have KD = F> mcm, we can write this as ^KD*N ( X m j = 1 Cj/j‚ r2D>DI) (13) (13) where C1‚ . . . ‚ Cm and r are parameters we need to estimate. The vector ^KD is observed ‘‘data,’’ computed from the public SNP database and the two released parameter vectors. We can model each of the entries of Fm, which are zeros and ones, as Bernoulli distributions, whose prior probabilities correspond to the public dataset estimated frequencies. This suggests a model for ^KD that is akin to a constrained mixture of Gaussians. where C1‚ . . . ‚ Cm and r are parameters we need to estimate. The vector ^KD is observed ‘‘data,’’ computed from the public SNP database and the two released parameter vectors. We can model each of the entries of Fm, which are zeros and ones, as Bernoulli distributions, whose prior probabilities correspond to the public dataset estimated frequencies. This suggests a model for ^KD that is akin to a constrained mixture of Gaussians. For the special case of m = 1, with only a single scalar C and vector /0, this reduces to ^KD*N (C/0‚ r2D>DI): (14) (14) 5.4.3. Parameter estimation with EM. We now can use this model to derive EM algorithms for finding maximum likelihood estimates of all parameters, and estimate the posterior distribution over SNP variants for the added individuals between the two studies. For notational convenience in this section, denote the entries of the m new individuals Fm 2 f0‚ 1gN + 1‚ m as zi‚ j, for i = 1‚ . . . ‚ N + 1 and j = 1‚ . . . ‚ m, and let zj denote the column vector z1‚ j‚ . . . ‚ zN + 1‚ j. Denote the prior probabilities for each i as a1‚ . . . ‚ aN + 1, where a1‚ . . . ‚ aN are the (public) population frequencies for each SNP, and aN + 1 = 1. Let x1‚ . . . PAIGE ET AL. ‚ xN + 1 denote the entries of the fixed (observed) vector x = ^KD, which in this simplified notation is distributed as p(xjcm‚ Fm‚ r2) = N (xj X m j = 1 Cjzj‚ r2D>DI): p(xjcm‚ Fm‚ r2) = N (xj X m j = 1 Cjzj‚ r2D>DI): posing we know values of C1‚ . . . ‚ Cm‚ r2, to estimate the entries of Fm we want to find p(zjx‚ C‚ r2 Supposing we know values of C1‚ . . . ‚ Cm‚ r2, to estimate the entries of Fm we want to find p(zjx‚ C‚ r2), p(zjx‚ cm‚ r2) / p(xjcm‚ Fm‚ r2)p(z): p(zjx‚ cm‚ r2) / p(xjcm‚ Fm‚ r2)p(z): An EM algorithm to estimate cm‚ r2 would proceed by alternately: An EM algorithm to estimate cm‚ r2 would proceed by alternately: 1. Given cm‚ r2, estimate the posterior distribution p = p(zjx‚ cm‚ r2); 1. Given cm‚ r2, estimate the posterior distribution p = p(zjx‚ cm‚ r2); 2. Given the posterior p, maximize L = Ep[ log p(xjcm‚ Fm‚ r2)] with respect to cm and r2. 1. Given cm‚ r2, estimate the posterior distribution p = p(zjx‚ cm‚ r2); 2. Given the posterior p, maximize L = Ep[ log p(xjcm‚ Fm‚ r2)] with respect to cm and r2. For each zi‚ j, we can analytically compute the distribution For each zi‚ j, we can analytically compute the distribution p(zi‚ j = 1jx‚ cm‚ zk6¼j‚ r2) p(zi‚ j = 1jx‚ cm‚ zk6¼j‚ r2) (15) = aiN (xijCj + P k6¼j Ckzi‚ k‚ r2D>D) aiN (xijCj + P k6¼j Ckzi‚ k‚ r2D>D) + (1 - ai)N (xij P k6¼j Ckzi‚ k‚ r2D>D) ‚ (15) the conditional probability of each particular entry taking a value of 1, rather than 0, for each zj given the values of the other zk, k 6¼ j. Note that each SNP location i can be treated independently; however, each of the individuals j = 1‚ . . . ‚ m individuals must be considered jointly. the conditional probability of each particular entry taking a value of 1, rather than 0, for each zj given the values of the other zk, k 6¼ j. Note that each SNP location i can be treated independently; however, each of the individuals j = 1‚ . . . ‚ m individuals must be considered jointly. 5.4.4. Exact EM algorithm when 1 individual is added. RECONSTRUCTION ATTACKS FROM GRS This breaks down into a sum across the entries in cm, with KD = X m j = 1 Cj/j: KD = X m j = 1 Cj/j: We need to estimate all m constants Cj‚ j = 1‚ . . . ‚ m. j If K is Gaussian [following Eq. (11)], then the linear transformation KD is Gaussian as well. We denote each of the rows of K as a vector ki, i = 1‚ . . . ‚ N; then, for each row, the scalar value k> i D*N (^k> i D‚ r2D>D)‚ meaning overall the vector KD is distributed N ( ^KD‚ r2D>DI). 446 PAIGE ET AL. RECONSTRUCTION ATTACKS FROM GRS This yields @L @C = X i pi r2DTD (xi - C)‚ @L @r2 = X N i = 1 pi @ @r2 log N (xijC‚ r2D>D) + (1 - pi) @ @r2 log N (xij0‚ r2D>D)‚ which we set equal to zero and solve to find which we set equal to zero and solve to find ^C = P i pixi P i pi ‚ (17) ^C = P i pixi P i pi ‚ (17) ^r2 = 1 ND>D X N i = 1 pi(xi - C)2 + (1 - pi)x2 i : (18) (18) These updates taken together can be used to define an EM algorithm that optimizes the values of C and r2, despite the fact that the entries of /0 are unknown; once C and r2 are then known, the vector p will give probability estimates for each entry of /0. p y y /0 The overall EM algorithm can be summarized by the following iterative updates: The overall EM algorithm can be summarized by the following iterative updates: 1. pi  p(zi = 1jx‚ ^C‚ ^r2) = aiN (xij ^C‚ ^r2D>D) aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D), 2. ^C) P i pixi P i pi , 3. ^r2) 1 ND>D PN i = 1 pi(xi - ^C)2 + (1 - pi)x2 i : 1. pi  p(zi = 1jx‚ ^C‚ ^r2) = aiN (xij ^C‚ ^r2D>D) aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D), P 1. pi  p(zi = 1jx‚ ^C‚ ^r2) = aiN (xij ^C‚ ^r2D>D) aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D), 2. ^C) P i pixi P i pi , 3. ^r2) 1 ND>D PN i = 1 pi(xi - ^C)2 + (1 - pi)x2 i : 1. pi  p(zi = 1jx‚ ^C‚ ^r2) = aiN (xij ^C‚ ^r2D>D) aiN (xij ^C‚ ^r2D>D) + (1 - ai)N (xij0‚ ^r2D>D), P i 3. ^r2) 1 ND>D PN i = 1 pi(xi - ^C)2 + (1 - pi)x2 i : To initialize the algorithm, we can set pi to some initial probabilities, and find initial values for ^C‚ ^r2; we experimented with setting both to the prior probabilities per SNP estimated from the public data and to the vector of all zeros (corresponding to a ‘‘hard’’ initialization at the value of the baseline estimate), and we found no qualitative difference in performance. PAIGE ET AL. For the special case of m = 1, this yields a tractable exact EM algorithm. Since there are no other individuals, Eq. (15) reduces to p(zjx‚ C‚ r2), with 5.4.4. Exact EM algorithm when 1 individual is added. For the special case of m = 1, this yields a tractable exact EM algorithm. Since there are no other individuals, Eq. (15) reduces to p(zjx‚ C‚ r2), with pi = p(zi = 1jx‚ C‚ r2) = aiN (xijC‚ r2D>D) aiN (xijC‚ r2D>D) + (1 - ai)N (xij0‚ r2D>D) (16) (16) the posterior probability of each particular entry taking a value of 1, rather than 0. To maximize L = Ep[ log p(xjC‚ /0‚ r2)] with respect to C and r2, we first compute the derivatives of L = X i X zi p(zij . . . ) log p(xijC‚ zi‚ r2) = X N i = 1 pi log N (xijC‚ r2D>D) + (1 - pi) log N (xij0‚ r2D>D): RECONSTRUCTION ATTACKS FROM GRS 447 RECONSTRUCTION ATTACKS FROM GRS 5.4.5. Stochastic EM (SEM) for multiple individuals. For m > 1, the exact posterior depends on all individuals and does not have a compact form. However, we can easily approximate the posterior by Gibbs sampling using Eq. (15), which describes the full conditional distribution p(zi‚ j = 1jx‚ cm‚ zk6¼j‚ r2), iteratively drawing samples for each individual j. We can use this for parameter estimation of r2 and each C1‚ . . . ‚ Cm by using the stochastic EM algorithm Celeux and Diebolt (1985), which differs from a standard EM algorithm in that the expectation step (evaluating the posterior) is replaced by Monte Carlo sampling. In this algorithm, we alternately 1. draw approximate posterior samples of zi‚ j by one or more sweeps of Gibbs sampling, following Eq. (15); 1. draw approximate posterior samples of zi‚ j by one or more sweeps of Gibbs sampling, following Eq. (15); 2 2. conditioned on the current sampled values zi‚ j, find values of r2 and C1‚ . . . ‚ Cm which maximize the likelihood N (xij Pm j = 1 Cjzi‚ j‚ r2D>DI). 2. conditioned on the current sampled values zi‚ j, find values of r2 and C1‚ . . . ‚ Cm which maximize the likelihood N (xij Pm j = 1 Cjzi‚ j‚ r2D>DI). Although this does not converge to an exact parameter value, under suitable conditions the algorithm converges in distribution to a Gaussian centered on the maximum likelihood estimate of the parameter. A point estimate can be extracted by averaging across many iterations after convergence. In contrast to the EM updates, the updates for values of Cj and r2 given actual sampled values of zj are straightforward and do not scale combinatorially in m. Optimizing cm corresponds to solving a least-squares problem, that is, min C1‚ ...‚ Cm X N + 1 i = 1 (xi - X j = 1 Cjzi‚ j)2 = min cm k x - Zcm k2 2 ‚ using the vector notation cm = [C1‚ . . . ‚ Cm]> 2 Rm, x = [x1‚ . . . ‚ xN + 1]> 2 RN + 1, and Z 2 [0‚ 1]N + 1‚ m, has the solution using the vector notation cm = [C1‚ . . . ‚ Cm]> 2 Rm, x = [x1‚ . . . 5.5. Scaling of EM algorithm with size of private dataset Figure 7 demonstrates the change in accuracy of the EM algorithm over a range of different private database sizes. For this test, a synthetic dataset with 100 SNPs and 1,000,000 individuals is generated; 10,000 are held out as a public database, and 30 individuals are taken as a fixed test dataset of new dogs to add and are used to estimate EM algorithm accuracy, across increasingly large private database sizes. The algorithm has stable performance for increasingly large private databases. PAIGE ET AL. PAIGE ET AL. The maximum likelihood estimate of r2 given this estimated ^cm is simply the mean squared error The maximum likelihood estimate of r2 given this estimated ^cm is simply the mean squared error ^r2 = 1 N + 1 X N i = 1 (xi - ^c> mzi)2: (20) (20) To address permutation invariance in the entries 1‚ . . . ‚ m, we enforce an ordering on the estimated values of Cj, with C1  C2  . . .  Cm. This breaks the symmetry across the indices of the m new indi- viduals added in the second study, and it is handled by a projection operation at each iteration, in which the estimated values are sorted in ascending order after each maximization step. g p Empirical results quantifying the performance of this algorithm are shown in Figure 6, in an experi- mental setup similar to that for evaluating EM when a single dog is added to a dataset in the main article, with unknown K. A private dataset is assumed to contain 1000 individuals, whereas a separate public dataset of 800 is available; m = 3 new individuals are added to the private dataset to produce two parameter vectors bM and bM + m. On average, the SEM algorithm predicts the correct SNP 75.5% of the time, relative to 71.5% for the ‘‘most common variant’’ baseline, a moderate improvement. RECONSTRUCTION ATTACKS FROM GRS ‚ xN + 1]> 2 RN + 1, and Z 2 [0‚ 1]N + 1‚ m, has the solution using the vector notation cm = [C1‚ . . . ‚ Cm]> 2 Rm, x = [x1‚ . . . ‚ xN + 1]> 2 RN + 1, and Z 2 [0‚ 1]N + 1‚ m, has the solution ^cm = (Z>Z) - 1Z>x: (19) ^cm = (Z>Z) - 1Z>x: (19) 448 RECONSTRUCTION ATTACKS FROM GRS 449 FIG. 7. Accuracy at reconstruction of the genome of one additional individual, using EM estimation and a noisy estimate ^K, measured as the size of the initial private database increases. For very small private databases, accuracy is very high, as changes in entries of b are clearly attributable to the new individual. Beyond a certain threshold, overall accuracy is quite stable. Error bars show mean and two standard deviations. FIG. 7. Accuracy at reconstruction of the genome of one additional individual, using EM estimation and a noisy estimate ^K, measured as the size of the initial private database increases. For very small private databases, accuracy is very high, as changes in entries of b are clearly attributable to the new individual. Beyond a certain threshold, overall accuracy is quite stable. Error bars show mean and two standard deviations. and now, from the second experiment, we have and now, from the second experiment, we have and now, from the second experiment, we have and now, from the second experiment, we have (K0 + /0T 0/0 0)^b0 = F0Ty0: (22) (22) Taking the difference between these expressions, as earlier, gives Taking the difference between these expressions, as earlier, gives K0 ^b0 - K ^b = F0Ty0 - FTy - /0T 0/0 0 ^b0: (23) (23) Restricting to the overlapping set gives that Restricting to the overlapping set gives that [K0 ^b0] - [K ^b] = [F0Ty0 - FTy - /0T 0/0 0 ^b0]: (24) Noting that [K] = [K0] and that [F0Ty0] - [FTy] = [/T 0y0] we get that [K]([^b0] - [^b]) = [/T 0](y0 0 - /0 ^b0): (25) (24) (25) Analogous to the previous cases, (y00 - /0 ^b0) is a scalar that we can label C and we get Analogous to the previous cases, (y00 - /0 ^b0) is a scalar that we can label C and we get [/T 0] = 1 C [K]([^b0] - [^b]): (26) (26) Thus, if K is known, it can be used to deduce whether the additional individual has each of the SNPs in the overlapping set. If K is not known exactly, it can be estimated from public data just as in the same SNP case. 5.6. Estimating /0 with different SNP sets Here, we analyze what can still be said in the event that the two studies do not use exactly the same set of SNPs. We will still assume that the sets of SNPs are considered to have a significant overlap. For this purpose we will need a greater variety of notation. A primed variable denotes that it corresponds to the second set of SNPs, for example, K0 is the co-occurrence matrix from the original M users for the second experiment. If a vector or matrix is surrounded by square brackets, this denotes the same object but with rows and columns corresponding to SNPs not in the overlap removed, for example, [K] denotes the co-occurrence matrix from the first experiment restricted to the overlapping SNPs. As described earlier, from the first experiment, we have As described earlier, from the first experiment, we have K ^b = FTy (21) (21) K ^b = FTy FIG. 6. Results for running the stochastic EM algorithm when estimating SNPs for three additional dogs simulta- neously. This experimental setup replicates the experiment for one additional dog, across 5 public/private/test dataset splits, with 20 different test sets of three additional dogs for each. (Left) Accuracy at predicting SNP presence relative to the ‘‘most common variant’’ baseline. On average, the SEM algorithm predicts the correct SNP 75.5% of the time, relative to 71.5% for the baseline. (Right) As in the one-dog example, we see relative improvement in the performance of our algorithm when considering more atypical dogs. SEM, stochastic EM. FIG. 6. Results for running the stochastic EM algorithm when estimating SNPs for three additional dogs simulta- neously. This experimental setup replicates the experiment for one additional dog, across 5 public/private/test dataset splits, with 20 different test sets of three additional dogs for each. (Left) Accuracy at predicting SNP presence relative to the ‘‘most common variant’’ baseline. On average, the SEM algorithm predicts the correct SNP 75.5% of the time, relative to 71.5% for the baseline. (Right) As in the one-dog example, we see relative improvement in the performance of our algorithm when considering more atypical dogs. SEM, stochastic EM. RECONSTRUCTION ATTACKS FROM GRS AUTHOR DISCLOSURE STATEMENT The authors declare they have no competing financial interests. 5.8. Description of K when the genotypes are non-binary – Finally, KN + 1‚ N + 1 = 1. PAIGE ET AL. and the following a + b length vector: ra + b = (ya - Fa ^bM + a)‚ - (yb - Fb ^bM + b) h i (28) (28) Then, this gives us: KM(^bM + a - ^bM + b) = 1 M Fa + bra + b (29) (29) This means that having two nonoverlapping participant sets is equivalent to the setting in which the first study is a subset of the second (only m is now a + b). FUNDING INFORMATION This project was funded by the Alan Turing Institute Research Fellowship under EPSRC Research grant (TU/A/000017); EPSRC/BBSRC Innovation Fellowship (EP/S001360/1), and under the EPSRC grant EP/N510129/1. It was also partly funded by a grant from CPER Nord-Pas de Calais/FEDER DATA Advanced data science and technologies 2015–2020. 5.8. Description of K when the genotypes are non-binary 5.8. Description of K when the genotypes are non-binary In many cases, GRS are calculated on genotype matrices that are non-binary. In particular, they may take on three discrete values 0, 1, and 2, where 0 indicates that the most common variant is homozygous, 1 indicates that the individual is heterozygous for the uncommon variant, and 2 indicates that the individual is homozygous for the uncommon variant. If this is the case, the description of K will change. However, it is still the case that the entries of K depend only on the SNP frequencies and SNP co-occurrence frequencies in the dataset, and that knowledge of SNP frequencies and pairwise co-frequencies from the original study, are all that is required to compute K. – For i = 1‚ . . . ‚ N: Kii = pAa + 4pAA where paa is the frequency of individuals being heterozygou uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon – For i = 1‚ . . . ‚ N: Kii = pAa + 4pAA where paa is the frequency of individuals being heterozygous for the uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon variant. uncommon variant and pAA is the frequency of individuals being homozygous for the uncommon variant. – For i = 1‚ . . . ‚ N - 1 and j > i: Kij = Kji = pAa=Bb + 2pAA=Bb + 4pAA=BB where pAa=Bb is the frequency that both SNPs are simultaneously heterogygous, pAA=Bb is the frequency that one SNP is homozygous for the rare variant and the other is heterogygous simultaneously, and pAA=BB is the frequency that that uncommon variants are found to be homozygous simultaneously. yg y – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. – Finally, KN + 1‚ N + 1 = 1. yg – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. – For i = 1‚ . . . ‚ N and j = N + 1: Kij = Kji = pAa + 2pAA. 5.7. Case in which each GWAS study adds two new sets of participants 5.7. Case in which each GWAS study adds two new sets of participants This article mostly explores the case in which one study’s participants are a subset of the other study’s participants. Here, we demonstrate that this is equivalent to the case where each of the two studies contains a small number of participants that are not found in the other study. In particular, let us say that the first study has M + a participants and the second study has M + b participants, where the first M participants are shared between the studies, but there are a participants that are found in the first study but not the second, and b participants that are found in the second study but not in the first. Following on from Eq. (9), we see that: KM(^bM + a - ^bM + b) = KM(^bM + a - ^bM) - KM(^bM + b - ^bM) = 1 M FT a(ya - Fa ^bM + a) - FT b(yb - Fb ^bM + b) h i : Let us define the following (N + 1) · (a + b) matrix obtained by concatenating the two genotype matrices: fine the following (N + 1) · (a + b) matrix obtained by concatenating the two genotype matrices: Fa + b = Fa‚ Fb ½  (27) Fa + b = Fa‚ Fb ½  (27) PAIGE ET AL. 450 PAIGE ET AL. REFERENCES Belsky, D.W., Moffitt, T.E., Sugden, K., et al. 2013. Development and evaluation of a genetic risk score for obesity. Biodemography Soc. Biol. 59, 85–100. g p y Cai, R., Hao, Z., Winslett, M., et al. 2015. Deterministic identification of specific individuals from GWAS results. Bioinformatics 31, 1701–1707. Celeux, G., and Diebolt, J. 1985. The SEM algorithm: A probabilistic teacher algorithm derived from the EM algorithm for the mixture problem. Comput. Stat. Q. 2, 73–82. Chouraki, V., Reitz, C., Maury, F., et al. 2016. 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All Supplementary Data from Novel BAFF-Receptor Antibody to Natively Folded Recombinant Protein Eliminates Drug-Resistant Human B-cell Malignancies &lt;i&gt;In Vivo&lt;/i&gt;
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Supplementary figure legends 1 1 Day 0 3 6 9 12 16 Vaccination (D2C cells, s.c. at food pad) Check Ab production In vaccinated mice boost 20 establish hybridoma clones 14 Clone screening Supplementary Figure 1 A B Clone (h)BAFF-R-expressing L cells (OD 450 nm) Parental L cell (OD 450 nm) C53 0.552 0.095 C55 1.067 0.102 C67 0.615 0.093 C90 0.645 0.116 C39 0.137 0.091 Hybridoma clones screened by ELISA for antibody production Supplementary Figure 1 Supplementary Figure 1 Supplementary Figure 1 0 3 6 9 12 16 Vaccination (D2C cells, s.c. at food pad) Check Ab production In vaccinated mice boost 20 establish hybridoma clones 14 Clone screening Check Ab production In vaccinated mice establish hybridoma clones A Vaccination (D2C cells, s.c. at food pad) 20 B B Hybridoma clones screened by ELISA for antibody production Hybridoma clones screened by ELISA for antibody production Clone (h)BAFF-R-expressing L cells (OD 450 nm) Parental L cell (OD 450 nm) C53 0.552 0.095 C55 1.067 0.102 C67 0.615 0.093 C90 0.645 0.116 C39 0.137 0.091 Hybridoma clones screened by ELISA for antibody production H. Qin, L. W. Kwak Supp. Fig. 1 Anti human BAFF-R monoclonal antibody generation and clone selection. A. L cell clone D2C, which stably expressed human (h)BAFF-R with a C-terminal GFP tag on the intracellular domain, was used to immunize BALB/c mice according to the schedule shown. Splenic tissue was harvested on day 20 and B-cell hybridoma clones were established. B. ELISA results from five hybridoma supernatants using anti-mouse IgG-HRP. Clones 53, 55, 67, and 90 produced BAFF-R- specific mAbs, whereas Clone 39 did not (representative of other negative clones). 2 2 Supplementary Figure 2 mAb C-53 mAb C-55 mAb C-67 mAb-90 Mino 293T unstained 2nd Ab only hybridoma sup at 1/200 hybridoma sup at 1/10 hybridoma sup at 1/50 Anti-mouse IgG-APC Counts mAb C-53 Mino mAb C-55 mAb C-67 mAb-90 293T Counts 293T unstained 2nd Ab only hybridoma sup at 1/200 hybridoma sup at 1/10 hybridoma sup at 1/50 H. Qin, L. W. Kwak Supp. Fig. 2 Verification that selected hybridoma clones bind MCL cells. Binding of hybridoma Clone 53, 55, 67, and 90 supernatants (1/10, 1/50, and 1/200 dilutions) to Mino (mantle cell lymphoma) and 293T (negative control) cell lines assessed by flow cytometry performed with anti-mouse IgG-APC. Supp. Fig. 2 Verification that selected hybridoma clones bind MCL cells. Binding of hybridoma Clone 53, 55, 67, and 90 supernatants (1/10, 1/50, and 1/200 dilutions) to Mino (mantle cell lymphoma) and 293T (negative control) cell lines assessed by flow cytometry performed with anti-mouse IgG-APC. 3 Supplementary Figure 3 Supplementary Figure 3 mAb C-53 mAb C-55 mAb C-67 mAb C-90 H. Qin, L. W. Kwak mAb C-53 mAb C-53 mAb C-55 mAb C-55 mAb C-90 mAb C-67 H. Qin, L. W. Kwak Supp. Fig. 3 Dose-dependent binding of purified mAbs to human BAFF-R. Mouse mAb from hybridoma Clones 53, 55, 67, 90 supernatant were purified by protein A affinity chromatography. Binding of serially diluted (1 µg/106 cells – 1.6 ng/106 cells) purified mouse mAb to Mino cells was assessed by flow cytometry with anti-mouse IgG-APC secondary antibody. Supp. Fig. 3 Dose-dependent binding of purified mAbs to human BAFF-R. Mouse mAb from hybridoma Clones 53, 55, 67, 90 supernatant were purified by protein A affinity chromatography. Binding of serially diluted (1 µg/106 cells – 1.6 ng/106 cells) purified mouse mAb to Mino cells was assessed by flow cytometry with anti-mouse IgG-APC secondary antibody. 4 Supplementary Figure 4 Supplementary Figure 4 JeKo-1 (MCL) SU-DHL-6 (DLBCL) Raji (Burkitt) RL (FL) Unstained control Anti-(m)IgG-APC only 0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC 2 ug/106 cells C90 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC 2 ug/106 cells C55 mAb + Anti-(m)IgG-APC H Qi L W K k JeKo-1 (MCL) SU-DHL-6 (DLBCL) JeKo-1 (MCL) SU-DHL-6 (DLBCL) Unstained control Anti-(m)IgG-APC only 0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC 2 ug/106 cells C90 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC 2 ug/106 cells C55 mAb + Anti-(m)IgG-APC Unstained control Anti-(m)IgG-APC only 0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC 2 ug/106 cells C90 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC 2 ug/106 cells C55 mAb + Anti-(m)IgG-APC Raji (Burkitt) RL (FL) Raji (Burkitt) H. Qin, L. W. Kwak Supp. Fig. 4 (h)BAFF-R mAbs recognized non-Hodgkin lymphoma cell lines in vitro. Mouse mAb Clones 55 and 90 bound additional cell lines at high (2 µg mAb/ 106 cells) and low (0.05 µg mAb/ 106 cells) doses: JeKo-1 (mantle cell lymphoma), SU-DHL-6 (diffuse large B cell lymphoma), Raji (Burkitt lymphoma) and RL (follicular lymphoma). Flow cytometry analysis was performed with anti-mouse IgG-APC. 5 Supplementary Figure 5 Supplementary Figure 5 mAb + anti-mouse IgG-APC unstained C90: low conc. C90 high conc. 2nd Ab only C55: low conc. C55: high conc. Mantle cell lymphoma (Pt# 10009) Diffuse large B-cell lymphoma (Pt# 10101) Follicular lymphoma (Pt# 0505383) Mantle cell lymphoma (Pt# 10009) Diffuse large B-cell lymphoma (Pt# 10101) Follicular lymphoma (Pt# 0505383) Diffuse large B-cell lymphoma (Pt# 10101) Diffuse large B-cell lymphoma (Pt# 10101) Diffuse large B-cell lymphoma (Pt# 10101) Mantle cell lymphoma (Pt# 10009) Mantle cell lymphoma (Pt# 10009) Follicular lymphoma (Pt# 0505383) Follicular lymphoma (Pt# 0505383) mAb + anti-mouse IgG-APC mAb + anti-mouse IgG-APC unstained C90: low conc. C90 high conc. 2nd Ab only C55: low conc. C55: high conc. H. Qin, L. W. Kwak Supp. Fig. 5 (h)BAFF-R mAbs recognized lymphoma patient samples. Mantle cell lymphoma, diffuse large B cell lymphoma, and follicular lymphoma patient samples were stained with mouse mAbs C55 and C90 at high (2 µg/106 cells) and low (0.05 µg/106 cells) concentrations. Flow cytometry analysis was performed with anti-mouse IgG-APC. Supp. Fig. 5 (h)BAFF-R mAbs recognized lymphoma patient samples. Mantle cell lymphoma, diffuse large B cell lymphoma, and follicular lymphoma patient samples were stained with mouse mAbs C55 and C90 at high (2 µg/106 cells) and low (0.05 µg/106 cells) concentrations. Flow cytometry analysis was performed with anti-mouse IgG-APC. 6 mentary Figure 6A Ko-1 (MCL) i (BL) un Ab un Ab (FL) (DLBCL) un Ab un Ab BAFFRBD C55 C90 BAFFR positive cells mean fluorescence intensity (n=3) Cell line BAFFRBD C55 C90 JeKo-1 (MCL) 2179±109 1249±19 6590±66 Raji (BL) 904±13 1137±18 1240±13 RL (FL) 534±43 598±14 710±20 LY3 (DLBCL) 267±32 297±8 297±7 BAFFRBD H. Qin, L. W. Kwak Supplementary Figure 6B Supplementary Figure 6B (MCL) (DLBCL) BAFFRBD C55 C90 BAFFR positive cells mean fluorescence intensity (n=5) Patient BAFFRBD C55 C90 10022 (MCL) 1008±25 1082±23 2397±190 10101 (DLBCL) 187±7 201±2 226±3 un Ab un Ab MCL) DLBCL) BAFFRBD C55 C90 un Ab un Ab C55 C90 H. Qin, L. W. Kwak Supp. Fig. 6 BAFF-R antibody specific binding was correlated to BAFF-R expression on NHL lines and primary samples. Alexa fluor 647-conjugated chimeric antibodies and commercial APC-conjugated BAFF-R antibody binding (A) a panel of NHL cell lines and (B) two NHL primary patient samples. un: unstained control; Ab: stained sample. C55, C90, and commercial antibodies were applied at minimal binding dose – 40 ng, 120 ng, and 500 ng per 106 cells, respectively. Mean fluorescence intensity (MFI) is calculated as mean ± s.d. of the integrated peaks from cell lines (performed in triplicates) and primary samples (performed in pentaplicates). 7 Supplementary Figure 7 A Raji RL A B Raji RL A Raji RL RL B H. Qin, L. W. Kwak Supp. Fig. 7 Chimeric antibodies required NK cells for cytotoxicity against tumor cells. Calculated specific ADCC lysis on NHL lines (A) Raji, RL, and (B) JeKo-1 cells (target). Cells were labeled with chromium-51 and incubated with chimeric mAb (C55, C90, or rituximab) and with or without NK cells (effector) at effector to target ratio of 20:1. 8 8 Supplementary Figure 8 Supplementary Figure 8 Kwak pplementary Figure 9 R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 U 2 6 6 S p e c ific ly s is % R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 J u rk a t S p e c ific ly s is % R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 S K N O -1 S p e c ific ly s is % R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 R S 4 ;1 1 S p e c ific ly s is % * * R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 M E C -1 S p e c ific ly s is % * * A Supplementary Figure 9 R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 R S 4 ;1 1 S p e c ific ly s is % * * A A M E C -1 R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 U 2 6 6 R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 J u rk a t S p e c ific ly s is % R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 S K N O -1 S p e c ific ly s is % B B H. Supplementary Figure 8 Unlabeled cells Labeled cells Labeled cells + 1 hr incubation at 37C Red: Unlabeled cells Blue: Labeled cells Orange: Labeled cells + 1 hr incubation at 37C Figure 8 H. Qin, L. W. Kwak Unlabeled cells igure 8 Labeled cells Labeled cells Unlabeled cells Labeled cells + 1 hr incubation at 37C Red: Unlabeled cells Blue: Labeled cells Orange: Labeled cells + 1 hr incubation at 37C H. Qin, L. W. Kwak Supp. Fig. 8 Limited internalization was observed with BAFF-R mAbs. Mino cells were incubated with mAb C90 (0.05 g/106 cells) at 4 °C for 20 min followed by incubation at 37 °C for 1 h. Flow cytometry analysis was performed with anti-mouse IgG-APC. Cells were gated for surface localized antibodies (OUT) and loss of cell surface staining (IN). 9 upplementary Figure 9 R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 R S 4 ;1 1 S p e c ific ly s is % * * R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 M E C -1 S p e c ific ly s is % * * A H. Qin, L. W. Supplementary Figure 8 Qin, L. W. Kwak Supp. Fig. 9 BAFF-R antibody induced cytotoxicity in BAFF-R expressing lines. Antibody- induced cytotoxicity measured by chromium-51 release after incubation of malignant human B- cell tumors with C55, C90, or rituximab and effectors (NK cells) at effector to target ratio (E:T) of 20:1. Percentage of cell specific lysis of target cells: (A) Acute lymphoblastic and chronic lymphoblastic leukemia lines RS4;11 and MEC-1, respectively, and (B) BAFF-R negative lines U266 (multiple myeloma), Jurkat (T cell leukemia), and SKNO-1 (acute myeloid leukemia). All experimental groups were performed in triplicates. **P < 0.01 compared with controls two-tailed Student’s t-test. 10 Supplementary Figure 10 Supplementary Figure 10 Supplementary Figure 10 H. Qin, L. W. Kwak ** ** ** ** H. Qin, L. W. Kwak Supp. Fig. 10 BAFF-R mAb induced ADCC in BAFF-R expressing primary MCL samples. ADCC effects were measured by chromium-51 release after primary MCL patient samples were incubated with antibodies and effectors (NK cells) at E:T = 20:1. Calculated percentage of cell specific lysis of target cells are shown. All experimental groups were performed in triplicates. **P < 0.01 compared with controls two-tailed Student’s t-test. 11 Supplementary Figure 11 Supplementary Figure 11 H Qin L W Kwak JeKo-1 WT CD20 KO #3 CD20 KO #18 CD20 KO #19 CD20 KO #31 CD20 KO #34 CD20 KO #49 CD20 KO #50 CD20-APC RFP CD20 KO #25 37 kDa 25 kDa 50 kDa 37 kDa A CD20 KO #31 CD20 KO #34 CD20 KO #49 CD20 KO #50 RFP CD20 KO #25 CD20 KO #31 CD20 KO #34 CD20 KO #49 CD20 KO #50 CD20 KO #25 CD20-APC 37 kDa 25 kDa 50 kDa 37 kDa B B H. Qin, L. W. Kwak Supplementary Figure 11 C Supplementary Figure 11 C D JeKo-1 WT CD20 KO #3 CD20 KO #18 CD20 KO #19 CD20 KO #25 1 WT CD20 KO #3 CD20 KO #18 CD20 KO #19 CD20 KO #25 KO #31 CD20 KO #34 CD20 KO #49 CD20 KO #50 JeKo-1 WT CD20 KO #3 CD20 KO #18 CD20 KO #19 CD20 KO #25 P RFP CD20 KO #31 CD20 KO #34 CD20 KO #49 CD20 KO #50 RF CD20 KO #31 CD20 KO #34 CD20 KO #49 CD20 KO #50 RF BAFFR-APC Supp. Fig. 11 CD20 knock out clones generated with CRISPR. CD20 knock out clones of JeKo- 1 were generated with a commercial CRISPR/HDR system substituting RFP at the CD20 locus. (A) Clones were screened and sorted by flow cytometry for CD20-/RFP+ expression. (B) Western blotting with anti-CD20 antibodies was performed on total cell lysate from CD20-/RFP+ clones. β- actin was blotted as a loading control. (C) Cell viability post-knockout was followed for 20 passages. JeKo-1 wildtype (WT) was used as a control. (D) Clones as in (A) were screened for BAFF-R/RFP+ expression to confirm that BAFF-R expression had not been affected by the CRISPR/HDR manipulation of CD20. 12 Supplementary Figure 12A Supplementary Figure 12A Supplementary Figure 12A Day 0 Day3 8 13 18 4 5 6 7 9 10 11 12 14 15 16 17 Ibrutinib 90 days 25 mg/kg ibrutinib was administered by IP injection every other day from Day3 to Day17 Day3 8 13 18 4 5 6 7 9 10 11 12 14 15 16 17 Ibrutinib 90 days 25 mg/kg ibrutinib was administered by IP injection every other day from Day3 to Day17 25 mg/kg ibrutinib was administered by IP injection every other day from Day3 to Day17 H. Qin, L. W. Kwak Supplementary Figure 12B Supplementary Figure 12B Z-138 Z-138 PBS Ibrutinib PBS Ibrutinib PBS Ibrutinib PBS Ibrutinib PBS Ibrutinib Ibrutinib D13 D13 D16 D16 D16 D16 D19 H. Qin, L. W. Kwak Supp. Fig. 12 Ibrutinib treatment failed to protected mice challenged with ibrutinib-resistant Z-138 cells. (A) Tumor challenge and treatment schedule is shown for NSG mice challenged with either ibrutinib-sensitive JeKo-1 or ibrutinib-resistant Z-138 tumors. Mice were administered 25 mg/kg ibrutinib in DMSO by IP injection according to the schedule. Tumor control groups received PBS injections. (B) Bioluminescence images of tumor cells in mice were taken throughout study; representative days are shown. 13 Supplementary Figure 13 Supplementary Figure 13 Z-138 mouse model ** P < 0.001 compared with controls Z-138 mouse model ** P < 0.001 compared with controls Z-138 mouse model ** P < 0.001 compared with controls H. Qin, L. W. Kwak Supp. Fig. 13 Chimeric antibodies targeting human BAFF-R elicited in vivo therapeutic effects against Z-138 ibrutinib-resistant tumors. Bioluminescence images of mice challenged with luciferase-expressing, ibrutinib-resistant Z-138 cells followed by antibody treatments as in Figure 4. Control group mice received PBS, NK cells alone, or rituximab on the same schedule. 80-day tumor-free and overall survival curves of the mice shown. Tumor free rate and survival differences between experimental and all control groups were analyzed by log-rank test (groups of n=5, ** P<0.01). 14 Supplementary Figure 14 A B A H. Qin, L. W. Kwak Supplementary Figure 14C Supplementary Figure 14C Purified B cells Purified T cells Purified NK cells Gated myeloid cells Unstained control Anti-(m)IgG-APC only 0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells (m)IgG + Anti-(m)IgG-APC H. Qin, L. W. Kwak Purified T cells Unstained control Anti-(m)IgG-APC only 0.05 ug/106 cells C90 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells C55 mAb + Anti-(m)IgG-APC 0.05 ug/106 cells (m)IgG + Anti-(m)IgG-APC Purified B cells Purified T cells Unstained cont Anti-(m)IgG-APC 0.05 ug/106 cell 0.05 ug/106 cell 0.05 ug/106 cell Purified T cells Gated myeloid cells Purified NK cells Gated myeloid cells H. Qin, L. W. Kwak Supp. Fig. 14 Characterization of BAFF-R binding against normal B cells. PBMC from healthy donors were co-stained with APC-conjugated C90 chimeric antibody and (A) a lymphocyte marker panel (anti-CD20-PE, anti-CD3-PacificBlue, and anti-CD56-FITC) or (B) a myeloid cell marker panel (anti-CD45-PE, anti-CD15-PerCP-Cy5.5, and anti-CD14-PacificBlue). Each specific immune cell sub-population was gated and analyzed for binding with BAFF-R antibodies. (C) B cells, T cells, and NK cells were isolated from PBMC with commercial specific cell type isolation kits and stained with C55 and C90 (0.05 µg mAb/106 cells). Flow cytometry analysis was performed with anti-mouse IgG. Myeloid cells from PBMC were gated for CD66b+ and analyzed for mAb C55 and C90 staining. 15 Supplementary Figure 15 Supplementary Figure 15 Supplementary Figure 15 H. Qin, L. W. Kwak R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 B c e lls S p e c ific ly s is % * * R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 B c e lls S p e c ific ly s is % * * R itu x im a b C 9 0 C 5 5 C o n tro l Ig G N K o n ly 0 2 0 4 0 6 0 8 0 B c e lls S p e c ific ly s is % * * H. Qin, L. W. Kwak Supp. Fig. 15 BAFF-R mAb elicited ADCC effects on normal human B cells. Antibody- dependent cell-mediated cytotoxicity (ADCC) effects were measured on purified normal B cells by chromium-51 release after incubation with rituximab, C90, C55, Control IgG, and NK cells only at E:T = 20:1. Calculated percentage of cell specific lysis of target cells are shown. All experimental groups were performed in triplicates. **P < 0.05 compared with NK cell only and Control IgG by two-tailed Student’s t-test. Control IgG by two-tailed Student’s t-test. 16
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Editorial
EURASIP Journal on Advances in Signal Processing
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Vahid Tarokh Department of EECS MIT, Cambridge, MA 02139, USA Email: vahid@mit.edu Department of EECS MIT, Cambridge, MA 02139, USA Email: vahid@mit.edu Department of EECS MIT, Cambridge, MA 02139, USA Email: vahid@mit.edu Xiang-Gen Xia They propose to directly assign channel sym- bols to transmit antennas at different states by exploiting the properties of the state transitions in the trellis. The paper by D. Tujkovic, M. Juntti, and M. Latva-aho considers space- time turbo code designs. They propose a design method for recursive space-time trellis codes and parallel-concatenated space-time turbo coded modulation that can be applied to an arbitrary existing space-time trellis code. This method en- ables a large, systematic increase in coding gain while pre- serving the maximum transmit diversity gain and bandwidth efficiency property of the space-time trellis code considered. The next six papers are on decoding methods of space- time coded transmissions. The paper by B. A. Bjerke and J. G. Proakis considers the equalization of a multiple trans- mit and receive antenna wireless system. They consider un- coded, convolutionally coded, turbo coded MIMO channels, and also precoded MIMO channels. The paper by E. Erez and M. Feder proposes new decoding techniques for diver- sity channels employing space-time codes when the chan- nel coefficients are unknown to both transmitter and re- ceiver. They propose an efficient implementation of the gen- eralized maximum-likelihood ratio test algorithm and an energy weighted decoder. The paper by S. K. Jayaweera and H. V. Poor considers low complexity multiuser detection q In space-time coding, space-time code design is one of the most important topics. Unlike the conventional sin- gle antenna AWGN channel, the multiple antenna channel transfer function is a matrix instead of a scalar, even in the simplest propagation scenario. So, the space-time code de- sign is more challenging than in single antenna systems. Concerning a multiple antenna channel, that is, a multi- input multi-output (MIMO) system, two cases can arise: the MIMO channel can be known or unknown at the receiver. Since the channel is a complex matrix, signal processing may play an even more important role than it does in a single an- tenna system. When we deal with space-time matrices, the code size may be huge and therefore fast decoding algorithms are critically important. As a result, space-time coding has become one of the most active research areas in wireless com- munications. The EURASIP JASP has dedicated two special issues to this subject. The first issue has 11 papers that spread over space-time code designs, decoding methods for space- time coded transmissions, and MIMO systems. EURASIP Journal on Applied Signal Processing 2002:3, 209–210 c⃝2002 Hindawi Publishing Corporation EURASIP Journal on Applied Signal Processing 2002:3, 209–210 c⃝2002 Hindawi Publishing Corporation Dirk Slock Mobile Communications Department, EURECOM Institute, 2229 route des Cretes, BP 193, 06904 Sophia Antipolis Cedex, France Email: dirk.slock@eurecom.fr Xiang-Gen Xia Department of ECE, University of Delaware, Newark, DE 19716, USA Email: xxia@ee.udel.edu Space-time coding for multiple transmit and multiple receive antenna systems has recently attracted considerable atten- tion in the wireless communications area. It has been shown theoretically that for certain channels the capacity of a mul- tiple antenna system increases linearly with the number of antennas, which implies that multiple antenna systems have huge potential applications in broadband wireless communi- cations. In a multiple antenna system, a key building block is multiple antenna coding and modulation, called space-time coding. In single antenna systems, the coding and modula- tion only deals with one-by-one complex symbols or symbol sequences. However, in multiple antenna systems, the space- time coding deals with complex symbol matrices or matrix sequences. The first three papers are on space-time code designs. The paper by S. M. Haas, J. H. Shapiro, and V. Tarokh considers space-time coding issues for wireless optical channels. The space-time codes are used to overcome turbulence-induced fading in an atmospheric optical heterodyne communica- tion system. They propose a new criterion for the space- time code design for such channels. The paper by Z. Safar and K. J. R. Liu considers a systematic space-time trellis code design method by jointly considering diversity and coding advantages. They propose to directly assign channel sym- bols to transmit antennas at different states by exploiting the properties of the state transitions in the trellis. The paper by D. Tujkovic, M. Juntti, and M. Latva-aho considers space- time turbo code designs. They propose a design method for recursive space-time trellis codes and parallel-concatenated space-time turbo coded modulation that can be applied to an arbitrary existing space-time trellis code. This method en- ables a large, systematic increase in coding gain while pre- serving the maximum transmit diversity gain and bandwidth efficiency property of the space-time trellis code considered. The first three papers are on space-time code designs. The paper by S. M. Haas, J. H. Shapiro, and V. Tarokh considers space-time coding issues for wireless optical channels. The space-time codes are used to overcome turbulence-induced fading in an atmospheric optical heterodyne communica- tion system. They propose a new criterion for the space- time code design for such channels. The paper by Z. Safar and K. J. R. Liu considers a systematic space-time trellis code design method by jointly considering diversity and coding advantages. Xiang-Gen Xia The next six papers are on decoding methods of space- time coded transmissions. The paper by B. A. Bjerke and J. G. Proakis considers the equalization of a multiple trans- mit and receive antenna wireless system. They consider un- coded, convolutionally coded, turbo coded MIMO channels, and also precoded MIMO channels. The paper by E. Erez and M. Feder proposes new decoding techniques for diver- sity channels employing space-time codes when the chan- nel coefficients are unknown to both transmitter and re- ceiver. They propose an efficient implementation of the gen- eralized maximum-likelihood ratio test algorithm and an energy weighted decoder. The paper by S. K. Jayaweera and H. V. Poor considers low complexity multiuser detection EURASIP Journal on Applied Signal Processing 210 existing wireless systems. He received one best journal paper award from the IEEE-SP and one from EURASIP in 1992. He is the coau- thor of two IEEE-Globecom98 best student paper awards. He has been an Associate Editor for the IEEE-SP Transactions. methods for space-time coded synchronous multiple-access systems in the presence of independent Rayleigh fading. They show that optimal space-time codes designed for sin- gle user channels can still provide full diversity in the mul- tiuser channel. The paper by J. Liu, J. Li, and E. G. Larsson considers a differential space-time block code modulation for DS-CDMA systems. They propose three demodulation schemes, namely a differential space-time block code Rake receiver, a differential space-time block code deterministic receiver, and a differential space-time block code determinis- tic de-prefix receiver. The paper by Y. Liu and X. Wang pro- poses a space-time differential decoding technique based on multiple-symbol detection and decision-feedback by making use of the second-order statistics of the fading. The paper by J. Zhang and P. M. Djuri´c considers joint estimation and de- coding of space-time trellis codes by employing a new emerg- ing tool in statistical signal processing, sequential importance sampling. Vahid Tarokh received his Ph.D. degree in Electrical Engineering from the University of Waterloo, Ontario, Canada in 1995. From August 1995 to May 1996, he was em- ployed by the Coordinated Science Labora- tory, of the University of Illinois Urbana- Champaign, as a visiting Professor. He then joined the AT&T Labs-Research, where he was employed as a Senior Member of Tech- nical Staff, Principal Member of Technical Staff, and the Head of the Department of Wireless Communica- tions and Signal Processing until August 2000. Xiang-Gen Xia In the fall of 2000, Dr. Tarokh joined the Department of Electrical Engineering and Com- puter Sciences of MIT as an Associate Professor, where he is cur- rently employed. Dr. Tarokh received numerous awards including the 1987 Gold Tablet of The Iranian Math Society, the 1995 Gov- ernor General of Canada’s Academic Gold Medal, the 1999 IEEE Information Theory Society Prize Paper Award, (jointly with A. R. Calderbank and N. Seshadri), and more recently the 2001 Alan T. Waterman Award. The final two papers are on MIMO systems. The pa- per by A. Grant considers information theoretic properties of flat fading channels with multiple antennas when per- fect channel knowledge at the receiver is assumed. Expres- sions for maximum information rates and outage probabili- ties are derived. It is shown that the use of orthogonal mod- ulating signals is asymptotically optimal in terms of infor- mation rate. The paper by T.-L. Tung and K. Yao considers the problem of channel estimation and optimal power allo- cation for a multiple-antenna OFDM system. They develop a least-squares channel estimation approach, derive a per- formance bound, investigate the optimal training sequence for initial channel acquisition, and derive the optimal power allocation solution that maximizes the bandwidth efficiency under power and QoS constraints. Xiang-Gen Xia received his B.S. degree in mathematics from Nanjing Normal Univer- sity, Nanjing, China, and his M.S. degree in mathematics from Nankai University, Tian- jin, China, and his Ph.D. degree in Electrical Engineering from the University of Southern California, Los Angeles, USA in 1983, 1986, and 1992, respectively. He was a Lecturer at Nankai University, China during 1986–1988, a Teaching Assistant at University of Cincin- nati, USA during 1988–1990, a Research Assistant at the University of Southern California, USA during 1990–1992, and a Research Sci- entist at the Air Force Institute of Technology during 1993–1994. He was a Senior/Research StaffMember at Hughes Research Labo- ratories, Malibu, California, during 1995–1996. In September 1996, he joined the Department of Electrical and Computer Engineer- ing, University of Delaware, Newark, Delaware, USA, where he is currently an Associate Professor. His current research interests in- clude communication systems including equalization and coding; SAR and ISAR imaging of moving targets, wavelet transform and multirate filterbank theory and applications; time-frequency anal- ysis and synthesis; and numerical analysis and inverse problems in signal/image processing. Dr. Xia has over 80 refereed journal articles published, and 4 U.S. patents awarded. Xiang-Gen Xia He is the author of the book “Modulated Coding for Intersymbol Interference Channels” (New York, Marcel Dekker, 2000). Dr. Xia received the National Science Foundation (NSF) Faculty Early Career Development (CAREER) Program Award in 1997, the Office of Naval Research (ONR) Young Investigator Award in 1998, and the Outstanding Overseas Young Investigator Award from the National Nature Science Foundation of China in 2001. He also received the Outstanding Junior Faculty Award of the Engineering School of the University of Delaware in 2001. He is currently an Associate Editor of the IEEE Transactions on Mobile Computing, the IEEE Transactions on Signal Processing and the EURASIP Journal of Applied Signal Processing. He is also a Member of the Signal Processing for Communications Technical Committee in the IEEE Signal Processing Society. Dirk Slock Vahid Tarokh Xiang-Gen Xia Dirk Slock received the engineer’s degree from the University of Gent, Belgium in 1982. In 1984 he was awarded a Fulbright scholarship for Stanford University, USA, where he received his M.S. in Electrical En- gineering, M.S. in Statistics, and Ph.D. in Electrical Engineering in 1986, 1989, and 1989, respectively. While at Stanford, he de- veloped new fast recursive least-squares al- gorithms for adaptive filtering. In 1989–91, he was a member of the research staffat the Philips Research Labo- ratory, Belgium. In 1991, he joined the Eurecom Institute where he is now Associate Professor. At Eurecom, he teaches statistical signal processing and speech coding for mobile communications. His research interests include DSP for mobile communications: antenna arrays for (semi-blind) equalization/interference cancel- lation and spatial division multiple access, space-time processing and audio coding. More recently, he has been focusing on receiver design, downlink antenna array processing, and speech coding for third generation systems, and introducing spatial multiplexing in
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“Challenge” or “Hindrance”? The Effect of Leader–Member Exchange on Employee Resilience in Public Sector Organizations
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“Challenge” or “Hindrance”? The Effect of Leader– Member Exchange on Employee Resilience in Public Sector Organizations Yan-Han ZHU  Southwest University Yue YUAN Research Article Keywords: leader-member exchange(LMX), employee resilience, challenge appraisals, hindrance appraisals, experiment Posted Date: March 14th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-4025326/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/26 Page 1/26 Abstract Background: In complex, volatile, and uncertain societies, employee resilience is crucial for public sector organizations to navigate crises and maintain steady growth. Methods: Whether and how leader-member exchange (LMX) influence employee resilience? The present study offers an answer to this question through a multi-method (field and experiment) research conducted with a sample of public sector organizations’ members in China. Study 1 conducted a comprehensive meta-analysis to review the available evidence and assess the influence of LMX on employee resilience. Subsequently, Study 2 conducted a questionnaire survey of 118 members in public sector organizations to investigate the practical impact of LMX on employee resilience. Finally, Study 3 used a scenario-based simulation experiment to manipulate the quality of LMX and to explore the mediating role of stress appraisals. Results:The meta-analysis conducted in Study 1, which included 42 independently extracted samples from 36 studies, explored the importance and scarcity of existing research on the influence of LMX on employee resilience in public sector organizations through two important aspects of leadership and interpersonal relationships inherent in LMX. Study 2, a questionnaire survey, showed that LMX has a significant direct positive effect on employee resilience. Furthermore, using an experimental design, Study 3 revealed that stress appraisals, both challenge and hindrance appraisals, play a mediating role in the relationship between LMX and employee resilience. Conclusions: The present research revealed that LMX has a significant and positive effect on employee resilience; challenge and hindrance appraisals significantly mediate the relationship between LMX and employee resilience. These findings have practical implications for individuals and organizations from a pragmatic perspective. 1 Introduction Currently, unforeseen events and sudden changes (such as public health crises such as COVID-19 or natural disasters such as forest fires) inundate the entire organizational milieu [1], causing unprecedented disruptions to economic activities and people’s daily lives. This is particularly challenging for public sector organizations, which are tasked with protecting the public interest and serving the needs of the community [2]. In the face of an increasingly volatile and highly uncertain environment, the social governance system itself is being challenged [3], requiring active adaptation to changing circumstances and effective responses to public needs. How to empower members of these organizations to adapt rapidly to change and respond effectively to crises has become a critical aspect of public management practice. Research has shown that resilience can enable organizations and their members to withstand risks and play a crucial role in responding to crises [1]. Consequently, resilience in organizational contexts has attracted considerable attention and debate among scholars [4]. Page 2/26 Page 2/26 Organizational member resilience refers to the ability of employees to adapt and respond positively to stress in the workplace[5, 6]. It is characterized by employees’ ability to persevere, recover quickly, grow, and achieve goals in the face of adversity such as conflict and failure at work [7, 8, 9]. It plays a crucial role in helping employees to overcome detrimental effects such as role ambiguity and turnover intentions [10, 11] while increasing job satisfaction [6, 12], well-being, and performance [13, 14], among others. Employees are central to an organization’s ability to adapt to change in uncertain and dynamic environments [15, 16]. Employees of public sector organizations are the concrete implementers of policies and national laws and regulations, the performers of state public affairs and the exercisers of administrative power, and the managers of social affairs [17]. They are the critical windows of government to the public and influence the quality of the relationship between government and citizens [18]. However, there is little research on employee resilience in public sector organizations and systematic reflection on the triggering factors and ways to improve it. Improving employee resilience to cope with the unknown is a pressing issue for them. How leaders manage, treat, and evaluate their subordinates has a strong influence on their working lives [19] and self-concept [20]. On the one hand, in Eastern cultures, leaders represent hierarchy and authority [21]. 1 Introduction Employees’ attitudes and behaviours are more likely to be influenced by their leaders, especially in public sector organizations [22, 23]. Interpersonal relationships at work, on the other hand, are important not only for their instrumental value (e.g. they are often used in business transactions) but also because of the central role they play in the self-concept of Chinese people [24, 20]. Chinese people often find themselves embedded in a network of personal relationships that define their existence and influence their thoughts and emotions [25]. While hierarchy is valued in Chinese culture, relationships are also valued [26]. Leader-member exchange (LMX) refers to the quality of the relationship between employees and their supervisors [27], and encompasses a relationship-oriented approach to leadership. Therefore, it is important to pay attention to the interpersonal relationships between employees and their leaders in the context of public sector organizations. According to LMX theory, employees who experience high-quality LMX relationships feel greater trust and respect and receive resources and support that help them overcome barriers to achieving their goals. As a result, employees develop a more positive self-concept, exhibit higher levels of effort, and demonstrate greater employee resilience [28]. Although research has begun to explore the role of LMX as a facilitator of employee resilience, the question of how LMX enhances employee resilience by influencing employees’ appraisals of stress in objective situations remains unanswered. Further research is needed to explore the role of leaders in cultivating cognitive resources to promote employee resilience. Stress appraisals (challenge and hindrance) arise from a dual perception of the individual and the environment. When encountering a stressor, the individual perceives the stimuli as harmful (already causing damage to oneself), threatening (possibly causing damage or loss), or challenging (possibly leading to mastery or benefit). This assessment considers the individual’s available resources and coping options for dealing with stressful situations [29]. As a result, the perception of resource availability interacts with challenge and hindrance appraisals to shape coping strategies and subsequent outcomes [30]. The assessment of employee stressors within an organization is primarily influenced by resources. These resources also enhance individuals’ ability to navigate stressful events, protecting them from Page 3/26 psychological dysfunctions such as anxiety and burnout [31]. According to the conservation of resources (COR) theory, individuals with sufficient resources are better equipped to acquire more resources. 1 Introduction Individuals strive to obtain, cultivate, and safeguard resources as they are more effective in mitigating the negative impact of resource loss during work [32]. LMX is a positive resource that provides members with information, support, and self-esteem [33]. It enables members to access tangible and intangible assets from their supervisors [34]. Based on the COR theory, it is argued that LMX can activate specific components of both challenge and hindrance appraisals. High-quality LMX fosters emotional and material support for employees, providing them with opportunities for growth and development. High-quality LMX motivates employees to positively evaluate the organization, which can alleviate pressures from the organizational environment and boost employee resilience. Conversely, low-quality LMX fosters a belief among employees that their efforts are inadequately reciprocated, inducing negative evaluations of the organizational environment and diminishing employee initiative. In summary, LMX can influence employees' behaviour by shaping their cognitive appraisal process. Stress appraisals play a crucial mediating role in the relationship between LMX and employee resilience. psychological dysfunctions such as anxiety and burnout [31]. According to the conservation of resources (COR) theory, individuals with sufficient resources are better equipped to acquire more resources. To gain insight into employee resilience, we utilized a meta-analytic approach and analyzed 101 independent samples from 2010 onwards (Study 1). The analysis revealed a significant positive correlation between leadership, interpersonal relationships, and employee resilience. However, there is a research gap regarding the association between LMX and employee resilience, particularly within the realm of Chinese public organizations. Our study aims to examine and enhance comprehension of this specific relationship. In light of the aforementioned findings, we conducted a questionnaire survey to investigate the correlation between LMX and employee resilience in public sector organizations (Study 2). Our hypothesis was based on LMX theory and posited a positive relationship between LMX and employee resilience. This study aims to complement and validate the field of practical public organizations in light of our meta-analytic results. To examine the mediating role of stress appraisals, an experimental study was conducted involving employees in public sector organizations (Study 3). This approach was used to reduce the impact of extraneous variables and gain a more precise understanding of the mediating effects of challenging and hindering stress appraisals in the relationship between LMX and employee resilience. This study makes several contributions. First, it sheds light on the relationship between LMX and employee resilience in the context of public sector organizations. 2 Literature Review and Theoretical Hypotheses 2.1 Study 1: Meta-Analysis Research on the relationship between LMX and employee resilience is limited. The meta-analysis of LMX on employee resilience is challenging to determine the significance of this limited research in revealing differences between public sector and other organizations. Previous studies have demonstrated that leadership and interpersonal relationships are critical factors in employee resilience. Although LMX has not been extensively explored, research on these topics has provided in-depth insights into their significance in organizations. To gain a comprehensive understanding of the impact of LMX on employee resilience, this study conducts a meta-analysis of existing research on leadership and interpersonal relationships. The review examines relevant studies and provides data support for the elicitation of LMX. We first conducted a review to identify published articles related to employee resilience at the end of January 2024. Specifically, we used “employee resilience” or “subordinate resilience” in the Web of Science, PsycARTICLES, PsycINFO, JSTOR, SAGE, Springer, Elsevier, and Google Scholar searches. Additionally, the reference lists of the included articles were thoroughly scanned to ensure comprehensive coverage of the literature. Only peer-reviewed articles published in English were considered for the analysis, ensuring a manageable dataset for examination. Research on the relationship between LMX and employee resilience is limited. The meta-analysis of LMX on employee resilience is challenging to determine the significance of this limited research in revealing differences between public sector and other organizations. Previous studies have demonstrated that leadership and interpersonal relationships are critical factors in employee resilience. Although LMX has not been extensively explored, research on these topics has provided in-depth insights into their significance in organizations. To gain a comprehensive understanding of the impact of LMX on employee resilience, this study conducts a meta-analysis of existing research on leadership and interpersonal relationships. The review examines relevant studies and provides data support for the elicitation of LMX. We first conducted a review to identify published articles related to employee resilience at the end of January 2024. Specifically we used “employee resilience” or “subordinate resilience” in the Web of Science used “employee resilience” or “subordinate resilience” in the Web of Science, PsycARTICLES, PsycINFO, JSTOR, SAGE, Springer, Elsevier, and Google Scholar searches. Additionally, the reference lists of the included articles were thoroughly scanned to ensure comprehensive coverage of the literature. Only peer-reviewed articles published in English were considered for the analysis, ensuring a manageable dataset for examination. 2 Literature Review and Theoretical Hypotheses To be eligible for inclusion in this meta-analysis, studies had to meet specific criteria. First, they needed to employ quantitative research methods and provide empirical data, excluding purely theoretical and review studies. Second, the samples across studies had to be independent. If there was any overlap or crossing between two studies, only the study with more detailed information or a larger sample size was included. The analysis should primarily focus on individual-level effects rather than unit or organizational effects. For cross-level studies, only individual-level results were considered. After a thorough screening process, 101 articles out of the initial pool of 331 articles were deemed eligible for inclusion in the meta-analysis. For more details, please refer to Appendix. To extracted relevant information from the articles, including the title, year of publication, authorship, study population, subject area (e.g., Personnel of public sector organizations), percentage of female participants, and correlation coefficients. Research suggests that employee resilience leads to positive outcomes for both the individual and the organization [35, 36]. Therefore, it is important to consider strategies that can improve employee resilience. The meta-analysis revealed that personal traits, job characteristics, and the organizational environment are all factors that influence employees’ ability to resist stress. It is worth noting that leadership and colleagues have been identified as influential factors that affect individual behaviour within the organizational context and have received considerable attention in relevant studies. 1 Introduction The findings highlight that the development of employee resilience hinges upon the dimensions of LMX quality. Secondly, the research advances our understanding of stress appraisals as an antecedent of employee resilience. The appraisal mechanisms for nurturing and cultivating employee resilience remain relatively unexplored in research. The results show a significant relationship between stress appraisals and employee resilience. High levels of these (e.g., high LMX) prompt subordinates to appraise their leader’s actions and guidance more favourably. This positive appraisal enhances subordinate motivation and promotes resilience. Finally, this study used different research methods to validate the mediating mechanisms linking LMX to employee resilience in public sector organizations. In light of these findings, leaders in public sector organizations are encouraged to develop effective communication skills that align with the valued and trusted goals of Page 4/26 Page 4/26 subordinates. By doing so, leaders can positively influence the appraisal process of subordinates, leading to increased resilience and potentially improved future performance. 2.1.1 Leadership and Employee Resilience Page 5/26 “Leader” is a critical factor closely intertwined with and exerting influence on organizational members and goals. It encompasses the process through which leaders utilize their motivated employees and guide them towards achieving organizational objectives [37]. Leadership embodies both the behaviours exhibited by leaders and the personal characteristics that are perceived by organizational members as influencing others [38]. In management, the selection of an appropriate leader can enhance communication and interaction between leaders and employees, ultimately enhancing leadership effectiveness and organizational performance. By aggregating relevant analyses from existing studies on leader relationships, 23 studies were included in this meta-analysis, comprising a total sample size of 7,170 individuals (refer to Appendix for details). “Leader” is a critical factor closely intertwined with and exerting influence on organizational members and goals. It encompasses the process through which leaders utilize their motivated employees and guide them towards achieving organizational objectives [37]. Leadership embodies both the behaviours exhibite by leaders and the personal characteristics that are perceived by organizational members as influencing others [38]. In management, the selection of an appropriate leader can enhance communication and interaction between leaders and employees, ultimately enhancing leadership effectiveness and organizational performance. By aggregating relevant analyses from existing studies on leader relationships, 23 studies were included in this meta-analysis, comprising a total sample size of 7,170 individuals (refer to Appendix for details). A random effects model was used to examine the overall correlation between leadership and employee resilience, after taking into account factors such as publication bias, heterogeneity, and the treatment of previous studies. The findings showed a correlation coefficient of 0.21, with a 95% confidence interval of [0.079, 0.334]. As per established guidelines, correlation coefficients of r = 0.1, r = 0.2, and r = 0.3 indicate low, moderate, and strong correlations, respectively. Therefore, it can be concluded that there is a moderate positive correlation between leadership and employee resilience. Although individual studies showed inconsistent correlation coefficients for different aspects of leadership and employee resilience, the overall outcome of the 23 relevant studies indicated a moderate positive correlation. This research examines the impact of different facets of leadership on employee resilience. The study highlights the importance of considering other factors that may influence how leaders affect employee behaviour. Employee perception can alter the mechanisms through which leadership operates, so it is important to include additional influential factors when examining the relationship between leadership and employee resilience. 2.1.3 Discussion The meta-analysis revealed a significant relationship between leadership, interpersonal relationships at work and employee resilience. Studies on employee resilience at work have mainly examined the effect of leadership on resilience (e.g. paradoxical leadership [43]). However, studies explaining how leadership affects employee resilience are not very consistent. On the other hand, limited research has examined the influence of interpersonal relationships at work on employee resilience. These studies claim that workplace relationships can serve as important sources of support that contribute to increased employee resilience. For example, factors such as support from others [35] and relationships with one's supervisor [44] have been examined in this regard. Thus, it is crucial to consider the combination of leadership and interpersonal relationships within organisations, with particular emphasis on the important role of LMX. By focusing on the interplay between leadership and interpersonal relationships, we can further elucidate how these dynamics influence employee resilience. The interpersonal dynamics within an organization play an essential role in shaping organizational performance and behaviour. Employees who experience strong support and positive relationships exhibit different attitudes and behaviours when faced with challenges [45, 46]. At the same time, the leader, as a central contextual influence within the organization, has the power to profoundly affect the psychological states of employees and is seen as an essential catalyst for fostering employee resilience [19]. Combined with relational leadership theory, the quality of LMX determines the work experience of employees [47]. Compared to employee characteristics and personal perceptions, leader-member interactions have a more pronounced effect on work outcomes. LMX is a critical construct for exploring these relationships. When leaders and employees strive to cultivate better relationships, they foster positive perceptions of each other, which in turn leads to mutually beneficial outcomes [48]. Therefore, exploring LMX, particularly within traditional Chinese culture, helps to unravel central aspects of how leaders shape employee resilience and shed light on the significant implications of leader influence in different cultural contexts. Study 1 found a positive relationship between leadership, interpersonal relationships at work, and employee resilience. Few studies in the literature have focused on the critical dyadic relationships that are pervasive in public sector organizations. Most of the limited current research has focused on business organizations. Therefore, in order to test generalisability, this study was further explored using quantitative research methods to investigate whether there is a positive relationship between LMX and employee resilience in public sector organizations. 2.1.2 Interpersonal relationships and employ Interpersonal relationships enable individuals to access or exchange resources through social interactions, which can help facilitate positive coping in the face of difficulty. Employees work in teams where frequent interpersonal interactions play a crucial role and may influence work attitudes, behaviours, and well-being [39]. Specifically, relational resources in the workplace include relationships with coworkers, customers, leaders, etc. Conducted by aggregating the relevant analyses of existing interpersonal relationships, 13 relevant pieces of literature were included in the meta-analysis, with a total of 3,338 people (see Appendix). After accounting for publication bias, heterogeneity, and the treatment of previous studies, a random effects model was used to explore the degree of overall correlation between interpersonal relationships at work and employee resilience. The results showed a correlation coefficient of 0.36 and a 95% confidence interval of [0.260,0.452] for the effect size. According to previous suggestions, r = 0.1, r = 0.2 and r = 0.3 are weak, moderate, and strong correlations respectively. Therefore, it can be concluded that there is a strong positive correlation between interpersonal relationships at work and employee resilience. This result is consistent with the findings of most current studies. Interpersonal relationships at work create a sense of community, trust, and shared values among employees. It is associated with intimacy, open communication, access to information, and timely help [40]. Workplace relationships help individuals cope communication, access to information, and timely help [40]. Workplace relationships help individuals cope Page 6/26 Page 6/26 with stressful work situations by providing social and emotional support [41], and they play a role in achieving positive organizational outcomes [42]. 2.2 Leader-Member Exchange(LMX) P 7/26 LMX was posited by Graen and Uhl-Bien (1995)[27] as an alternative to behavioural theories of leadership that attempt to explain leadership as a function of personal characteristics, dispositions, and attitudes. LMX theory posits that the quality of leader–member relationships is instrumental in determining member Page 7/26 Page 7/26 behaviour in the workplace [49]. It differs between employees, and this variability may be responsible for differences in member behaviours and work outcomes [50]. LMX is distinct in its usage of a dyadic level of analysis that proposes that a leader shares differential, unique relationships with each member, and workplace outcomes are determined by the quality of this relationship [50]. In contrast, traditional leadership studies implicitly assume that the leader’s style is perceived similarly by different subordinates and gives rise to similar behaviours across subordinates. However, LMX theory posits that “leadership” is not a homogenous concept and that each subordinate may view the leader’s style differently [51]. In real organizational situations, it is often difficult for employees to discern the “style” of the leader [50]. However, subordinates experience a certain degree of comfort/discomfort with the leader, which informs them about their relationship with the leader. LMX is a more exhaustive construct than leadership because it conceives of leadership as a two-way relationship consisting of leader expectations and subordinate reciprocation [52]. For example, Dulebohn et al. (2012) [51] posit that LMX is a better indicator of subordinate performance than leadership, as the perception of leadership develops from the quality of the leader- subordinate relationship. Employees do not learn and imitate leaders unconditionally. The process by which different types of leadership come into play has an important relationship with employees’ perceptions of their working relationships with leaders at work. 2.3 LMX and Employee Resilience According to LMX theory, limited by resources and energy, leaders establish different relationships with subordinates and adopt different management styles and strategies. The quality of LMX plays a significant role in employee behaviours [28, 49]. A relationship that is high in trust, clarity, and reciprocity provides a sense of comfort and is termed high quality. Relationships that are low in trust, transparency, and exchange are termed low-quality LMX relationships [27]. With high-quality LMX, leaders can motivate subordinates and encourage positive evaluations of situations. Such LMX, with high trust, reciprocity, and clarity of communication, makes subordinates faithful to the mission and committed to the organization [57]. High-quality LMX encourages employees to adopt thoughtful, creative, and adaptive solutions to stress rather than disorganized, defensive and maladaptive keys [58]. It helps employees actively cope with changes and setbacks in the workplace, promotes the growth and development of subordinates [59], and plays a positive role in the development of employee resilience [58]. Therefore, we propose the following hypothesis: Hypothesis 1 LMX positively associated with employee resilience. 2.4 LMX and Employee Resilience via Stress Appraisals (Challenge and Hindrance) Lazarus & Folkman (1984)[29] define “stress” as a process initiated when the demands of the environment impose or exceed the resources of the individual. Stress does not exist in the individual or the environment alone but is a combination of the individual and the environment. Evaluating the individual and the environment combines two critical evaluation processes: primary and secondary appraisal [53]. A preliminary appraisal assigns meaning to any event or situation to determine whether it threatens oneself. Page 8/26 Secondary appraisal refers to what one can do and is how individuals assess internal and external coping resources. Primary and secondary appraisals converge to determine whether a person’s interaction with the environment is perceived as meaningful to the individual and, if so, whether it is primarily disruptive, threatening, or challenging. The appraisal process is implicit in the challenge-hindrance stressor framework. Validation studies have generally supported the assumption that challenge stressors are appraised as challenging and hindrance stressors are appraised as hindering [54, 55, 56]. Therefore, while challenge stressors refer to the presence of challenge demands, challenge appraisals refer to an individual’s subjective interpretation that the demands have potential for personal gain, growth, development, and well-being. Hindrance stressors refer to the presence of hindrance demands, while hindrance appraisals refer to an individual’s subjective interpretation that the potential to result in personal loss, constraints, or harm is demanded. Secondary appraisal refers to what one can do and is how individuals assess internal and external coping resources. Primary and secondary appraisals converge to determine whether a person’s interaction with the environment is perceived as meaningful to the individual and, if so, whether it is primarily disruptive, threatening, or challenging. The appraisal process is implicit in the challenge-hindrance stressor framework. Validation studies have generally supported the assumption that challenge stressors are appraised as challenging and hindrance stressors are appraised as hindering [54, 55, 56]. Therefore, while challenge stressors refer to the presence of challenge demands, challenge appraisals refer to an individual’s subjective interpretation that the demands have potential for personal gain, growth, development, and well-being. Hindrance stressors refer to the presence of hindrance demands, while hindrance appraisals refer to an individual’s subjective interpretation that the potential to result in personal loss, constraints, or harm is demanded. Lazarus’ (1966) [60] transactional theory of stress suggests that the environment influences and alters individuals’ cognitive process information about objective stressors. 2.4 LMX and Employee Resilience via Stress Appraisals (Challenge and Hindrance) Integrating Lazarus’ arguments with our theoretical foundation, we found that certain environmental factors may influence employees’ experience of stress in objective settings. Secondary appraisals of stressors refer to the process by which individuals appraise stressors as the process by which individuals appraise internal and external coping resources [29]. Thus, an individual’s perceived resources in an organization can influence the stress they experience, which further influences employee attitudes and behaviours. In the workplace, leaders determine the allocation of resources and serve as essential triggers for employee resilience [61]. LMX is an important organizational context in an individual’s work life and reflects the support of the organization and leaders for the individual [62]. High-quality LMX can buffer the drain on employee resources from environmental stressors and help individuals focus on potential growth opportunities and positive stress evaluations, resulting in positive behaviours [63]. Individuals who make different stress appraisals show significant differences in attitudes and behaviours [64]. Specifically, the greater the quality of LMX is, the more subordinates will have adequate resources to cope with the demands of the stressors [27], actively assess internal and external coping resources in secondary appraisals, and produce challenging stress appraisals while reducing hindering appraisals. Hypothesis a: LMX is positively associated with challenge appraisals. Hypothesis b: LMX is negatively associated with hindrance appraisals Hypothesis b: LMX is negatively associated with hindrance appraisals. Based on the COR theory, LMX can be considered an environmental condition, i.e., a critical contextual factor that ensures the transmission of other resources [65]. Furthermore, in the framework of COR theory, in addition to environmental resources, resources can be divided into three categories, such as objects, individual characteristics, and energy, and the degree of matching of these four categories of resources affects the accumulation status of resources [66]. According to the resource gain spiral path, employees with larger support pools have more resources to invest and will focus on maintaining or expanding their networks [31]. Well-supported employees are encouraged to demonstrate network-oriented resilience behaviours (e.g., engaging in collaborative work, seeking support, etc.) and develop supportive networks to meet challenges [13, 45]. Thus, the conditioned resource of high-quality LMX allows us to perceive more Page 9/26 Page 9/26 support from the organization, view difficulties as opportunities and challenges, and reduce our perceptions of negative barriers, thus increasing employee resilience as an energy resource. High-quality LMX influences employees to make more challenging appraisals and enhances employee initiative at work. Thus, when employees are faced with risks, challenges, or setbacks at work, they focus on how to improve the organization's current situation to improve efficiency and solve problems, proactively seek solutions, choose positive coping strategies, and work to change the current situation to achieve their goals, increasing employee resilience in the process [13]. High-quality LMX also prevents employees from making hindrance appraisals and decreases their withdrawal behaviour in the face of difficulties, thus increasing employee resilience. High-quality LMX influences employees to make more challenging appraisals and enhances employee initiative at work. Thus, when employees are faced with risks, challenges, or setbacks at work, they focus on how to improve the organization's current situation to improve efficiency and solve problems, proactively seek solutions, choose positive coping strategies, and work to change the current situation to achieve their goals, increasing employee resilience in the process [13]. High-quality LMX also prevents employees from making hindrance appraisals and decreases their withdrawal behaviour in the face of difficulties, thus increasing employee resilience. High-quality LMX emphasizes that employees and leaders have close relationships, know each other well, can identify each other’s strengths, and can build relatively strong relationships of trust based on emotions and interests. They receive more frequent job coaching, training opportunities, and advancement opportunities. 3 Method To evaluate the impact of LMX on employee resilience in public sector organizations and its mechanism, a contextual experiment and questionnaire were designed and conducted. In Study 2, the questionnaire method was used to examine the main effect between LMX and employee resilience, which initially confirmed its validity in public sector organizations. Study 3 explored the underlying mechanisms through a between-subjects contextual experiment. This experimental method enhances the internal validity of the research model and complements the strengths of the survey research method. The experimental study enhances the internal validity of the research model by providing more reliable inferences about the causal relationships between the variables, particularly in clarifying the direction of causality and circumventing the endogeneity problem of reciprocal causality (e.g., whether LMX and employee resilience are interactive causalities). It also allows for repeated validation of the research model. An experimental study can increase the reliability and persuasiveness of research findings from a survey study by testing their applicability at a specific time and in a specific context. 3.1 Study 2: Questionnaire Study Hypothesis b: LMX is negatively associated with hindrance appraisals. This makes employees feel more resourceful as members of the leader's “inner circle”. Employees compare resources to stress when making stress appraisals. When resources are adequate, employees believe that they can perform their jobs and will evaluate the organizational environment positively, generating challenging stress appraisals. In addition, employees who make challenging appraisals have more significant work initiative. When faced with risks, challenges, or setbacks at work, they focus on how to improve the organization’s current situation to improve efficiency and solve problems, proactively seek solutions, choose proactive coping strategies, and strive to change the status quo to achieve goals, increasing employee resilience in the process [13]. Therefore, we hypothesized that LMX is positively associated with challenge appraisals and negatively associated with hindrance appraisals. Low-quality LMX makes it difficult for employees to form an emotional connection with their leaders beyond the subordinate relationship. Compared to the privileges of “insiders”, this group of staff believes that they are being unfairly treated by the leadership and are unlikely to receive sufficient leadership support, fair promotion opportunities, and a lack of interpersonal and material resources necessary to solve problems. A low level of LMX is also detrimental. With low-quality LMX, more employees perceive resource scarcity and risk, and they are more likely to make hindrance appraisals. Employees who make hindrance appraisals are hindered in their pursuit of self-relevant goals, which may lead them to adopt retreating behaviours and exhibit avoidance tendencies in the face of difficulties rather than proactively seeking solutions [67], demonstrating lower employee resilience. Therefore, high-quality LMX can motivate employees to take a more positive attitude toward the environment and stress in the organization. By increasing employees’ challenge appraisals and decreasing employees’ hindrance appraisals, stress in the organization can be transformed into motivation, which can enhance employees’ motivation to take initiative, maintain openness and self-confidence, and take a positive approach to challenging difficulties and break through bottlenecks. Thus, we mobilize individual and work resources in multiple ways so that employees can fully engage in their work and cope with the organizational challenge. In other words, we hypothesize the following: Page 10/26 Hypothesis a: Challenge appraisals mediate the relationship between LMX and employee resilience. Hypothesis b: Hindrance appraisals mediate the relationship between LMX and employee resilience. The theoretical framework of this study is summarized in Fig. 1 Research Model. 3.1 Study 2: Questionnaire Study Measure Leader-member exchange (LMX) : LMX was measured using the seven-item LMX-7 scale developed by Graen and Uhl-Bien (1995) [27]. Respondents rated the extent to which they considered the LMX score to be an appropriate description of their work on a five-point scale (1 = strongly disagree, 5 = strongly agree). A sample item was “How would you characterize your working relationship with your leader?” The scale had a Cronbach’s α of 0.87. Employee resilience: The researchers measured the construct using a 9-item scale developed by Näswall et al. (2013) and Näswall et al. (2019) [6]. The scale consists of a 7-point rating system (ranging from 1 = strongly disagree to 7 = strongly agree) and measures employee resilience based on the extent to which respondents consider it an accurate description of their work. An example item is “I effectively collaborate with others to handle challenges at work.” The scale demonstrated high internal consistency with a Cronbach’s α of 0.92. Sample and Procedures The survey was conducted online using the platform wjx.cn. In Study 2, we distributed a questionnaire to full-time staff members of public sector organizations enrolled in the MPA program at a university in China. The questionnaire explained the study’s relevance and emphasized the respondents’ anonymity and privacy. Respondents could discontinue answering questions at any point during the survey. The research team's contact information was provided for any questions or concerns regarding the questionnaire. Respondents provided informed consent by initiating the questionnaire. To ensure method consistency and improve data validity, LMX and employee resilience were measured at two different time points. Respondents’ personal information was collected during the first time point (T1), followed by the measurement of LMX. Employee resilience was measured at the second time point (T2). The respondents’ last four digits of student IDs were separated to ensure effective data matching for the aforementioned variables. The survey received responses from 118 participants, with 112 full-time staff (95% response rate) included in the analysis after listwise deletion. Of these valid samples, 59.8% were female and 64.3% were married. All participants held a bachelor’s degree. In terms of age, 60.7% of the participants were younger than 30 Page 11/26 Page 11/26 years, 32.1% were between 31 and 35 years, 6.3% were between 36 and 40 years, and only 0.9% were older than 41 years. The tenure distribution was as follows: 33.0% had less than 5 years, 52.7% had 6 to 10 years, and 14.3% had 11 or more years. years, 32.1% were between 31 and 35 years, 6.3% were between 36 and 40 years, and only 0.9% were older than 41 years. The tenure distribution was as follows: 33.0% had less than 5 years, 52.7% had 6 to 10 years, and 14.3% had 11 or more years. 3.1.2 Results Table 1 presents the means and standard deviations of the variables analysed in this study, along with their correlations. The LMX was found to have a positive correlation with employee resilience (r = 0.18, p < 0.01). The demographic data collected indicated a positive association between gender and age with tenure, and between job type and position level. However, none of the demographic variables showed any correlation with LMX or employee resilience, and therefore were not considered as covariates. To test Hypothesis 1, a simple linear regression analysis was conducted. The analysis revealed a statistically significant relationship between LMX and employee resilience (β=0.21, t = 2.15, R2=0.11, p < 0.05), thus supporting Hypothesis 1. Page 12/26 Page 12/26 Page 12/26 Page 12/26 Table 1  Descriptive Statistics and Correlations   Variable Mean SD 1 2 3 4 5 6 7 8 9 1.Gender 1.60 0.49 1                 2.Age 1.47 0.66 0.175 1               3.Education 3.12 0.32 0.07 -0.01 1             4.Tenure 1.81 0.67 0.24* 0.70** 0.10 1           5.Job Type 1.66 0.88 0.12 0.17 0.01 0.04 1         6.Position 1.23 0.42 -0.11 0.06 -0.13 0.12 -0.20* 1       7.Marital Status 1.70 0.52 0.012 0.29** 0.11 0.44** -0.07 0.12 1     8.LMX 3.54 0.61 -0.14 -0.05 -0.07 -0.12 0.021 0.01 -0.06 1   9.Employee Resilience 5.66 1.05 -0.04 0.13 -0.04 0.05 0.123 0.03 -0.10 0.18* 1 Note. N = 112. * p < 0.05; ** p <0 .01. Gender:1=male, 2=famale; Age: 1=30 years or below, 2=31-35 years, 3=36-40 years, 4= 41 years or above; Education:1=High school or below, 2=junior college, 3=Bachelor degree, 4=Master degree or above; Job Type:1= general management, 2= administrative law enforcement, 3= professional technology; Position:1= No managerial responsibility, 2= grass-roots cadres, 3= middle-level cadres, 4= higher-level cadres; Marital Status:1= unmarried, 2= married, 3= other. Table 1  Descriptive Statistics and Correlations Participants and Procedures The study included 472 participants who were full-time staff working in public sector organizations in China. The participants were selected using the exponential, non-discriminative ‘snowball sampling’ method, which included MPA students enrolled in a part-time MPA Program at a large university in the southwestern region of China, as well as other working samples. After list-wise deletion, the effective sample consisted of 448 full-time staff, resulting in a 94.9% response rate. Of these valid samples, 55.9% were female, and the mean age was 30.6 years (SD=3.2). Scenarios and experimental manipulations Scenarios were created to manipulate LMX quality to resemble encounters in real workplace contexts. The initial versions of these scenarios underwent pre-testing for realism and clarity by experts, Ph.D. students, and full-time staff of public sector organizations to validate the textual material. Minor revisions were Page 13/26 made based on their feedback. LMX was manipulated in the scenarios, resulting in a between-subjects design. Participants were instructed to imagine themselves in the described situation and make their own decisions. The material provides information about LMX quality in two conditions: low-quality LMX and high-quality LMX. In the low-quality LMX condition, the working relationship between you and the leader is limited. In daily work, the leader only provides instructions and refrains from making subjective evaluations. This approach may limit their understanding of your work potential and disregard your obstacles at work and urgent needs in life. In a high-quality LMX condition, you feel that your leader is entirely satisfied with your work and provides valuable information that other colleagues may not know. This fosters a closer relationship between you and your leader compared to your colleagues. Additionally, the leader provides support outside of work and assists in problem-solving, even with personal issues. Measures Challenge Appraisals. Three items from the Employee Stress Scale developed by LePine et al. (2016) [64] were used to measure challenge appraisals. The items were rated on a Likert scale of 1 (strongly disagree) to 5 (strongly agree). Such as “Working on your job requirements contributes to your personal growth and well-being”. The Cronbach’s α was 0.91. Hindrance Appraisals. To measure employees’ hindrance appraisals, we utilised three items from LePine et al. (2016) Employee Stress Scale [64]. The items were rated on a Likert scale ranging from 1 (strongly disagree) to 5 (strongly agree). Such as “Your job requirements limit your ability to achieve your personal goals and development”. The Cronbach’s α was  0.94. Employee Resilience. We measured employee resilience using the Employee Resilience Scale developed by Näswall et al. (2019) [6]. The scale comprises nine questions that were rated on a Likert Scale ranging from 1 (strongly disagree) to 7 (strongly agree). Such as “You work effectively with others to solve work challenges” . The Cronbach’s alpha was  was 0.95. Attention test. One general question was designed to test whether respondents knew the information and were careful to fill in the answers in the context. Specifically, participants were asked to answer a conventional attention test entry, such as “Please select strongly agree for this option” . Descriptive Statistical Analysis Table 2 provides descriptive statistical results for each research variable. Page 14/26 Page 14/26 Table 2  Descriptive statistical analysis for each research variable. Gender Age CA HA ER Total Mean 0.43 30.55 3.26 2.70 4.87 SD 0.50 3.30 1.07 1.03 1.30 Scenario 1 (N=227) Mean 0.43 30.56 2.63 3.16 4.15 SD 0.50 3.28 0.85 0.97 1.23 Scenario 2 (N=221) Mean 0.43 30.54 3.91 2.12 5.60 SD 0.50 3.31 0.86 0.81 0.91 Note: CA = Challenge Appraisals; HA = Hindrance Appraisals; ER = Employee Resilience Table 2  Descriptive statistical analysis for each research variable. To test the hypotheses of the experimental study, ANOVA was used to examine the main effects of challenge appraisals, hindrance appraisals, and employee resilience, with LMX quality (high vs. low) as the independent variable. The study also tested the mediating effects, with challenge appraisals, hindrance appraisals, and employee resilience as dependent variables. Analysis of Variance The results of the analysis of variance (ANOVA) indicate a significant main effect of LMX on employee resilience (F(1,448) = 199.56, p < 0.001, partial η2 = 0.31). Specifically, employees with a high level of LMX demonstrated significantly greater resilience (M = 5.60, SD = 0.91) than those with a low level of LMX (M = 4.15, SD = 1.23). Additionally, there was a significant main effect of LMX on challenge appraisals (F(1,448) = 251.41, p < 0.001, partial η2 = 0.36). The study discovered that high-quality LMX was linked to significantly greater challenge appraisals (M = 3.91, SD = 0.86) in comparison to low-quality LMX (M = 2.63, SD = 0.85). A significant main effect of LMX quality on hindrance appraisals was observed, F(1, 448) = 154.01, p < 0.001, partial η2 = 0.26. Hypotheses 1 and 2 were supported. Regression Analysis To test hypothesis 3, we used the bootstrapping method (bootstrap sample=5000) recommended by Preacher et al. (2007) to examine the indirect effects of LMX on employee resilience through challenge appraisals and hindrance appraisals. The results, shown in Figure 2 and Table 3, indicate that LMX significantly affects employee resilience through both challenge and hindrance appraisals. Therefore, hypothesis 3 was supported. Page 15/26 Table 3  Pathway and effect decomposition of LMX impact ER 4 Di i Table 3  Pathway and effect decomposition of LMX impact ER Path Effect Boot SE 95% Boot LLCI ULCI 1.Total effect of LMX on ER 0.54 0.04 0.46 0.62 2.Direct effect of LMX ON ER 0.41 0.05 0.31 0.51 3.Indirect effect(s) of LMX ON ER Total 0.77 0.06 0.66 0.89 CA 0.61 0.07 0.48 0.76 HA 0.16 0.06 0.05 0.28 CA minus HA 0.46 0.11 0.22 0.68 Note: LMX = Leader-Member Exchange; CA = Challenge Appraisals; HA = Hindrance Appraisals; ER = Employee Resilience Note: LMX = Leader-Member Exchange; CA = Challenge Appraisals; HA = Hindrance Appraisals; ER = Employee Resilience 4 Discussion Note: LMX = Leader-Member Exchange; CA = Challenge Appraisals; HA = Hindrance Appraisals; ER = Employee Resilience 4 Discussion The questionnaire results showed a significant positive correlation between LMX and employee resilience in public sector organizations in China. Public sector organizations in China are characterized by hierarchical rankings, positional disparities, and role obligations [68]. Society is known for its 'relationship orientation' and 'authority orientation' [20]. Examining the relationship between LMX and employee resilience in public sector organizations can enhance overall organizational resilience at both the leadership and workplace levels. This can enable public sector organizations to effectively navigate challenges. The results support the hypotheses of previous studies and align with the findings from the meta-analysis conducted in Study 1. Meng et al. (2019) suggested that a healthy supervisor-subordinate relationship can empower employees to handle demanding responsibilities, leading to organizational success. The nature of the LMX has been found to influence the appraisal process (28). Therefore, leaders should frame goals in a way that fosters subordinates' positive view of the situation, generating psychological engagement with the job and the organization. Therefore, individuals invest their time and energy in understanding a problem and typically view it as a challenge rather than a threat [9]. LMX also impacts employee resilience by influencing their perception of stressors to some extent, which requires further investigation. Study 3 used a contextual simulation approach based on COR theory to examine the effect of LMX on employee resilience and the mediating role of stress appraisals. The results indicated a positive association between LMX and employee resilience. This finding is consistent with Kakkars’ (2019) research, which indicates that LMX has a more positive impact on employee attitudes and behaviours than studies on leadership style and workplace interpersonal relationships in terms of employee resilience [28]. In the context of Chinese public sector organizations, employees who perceive a higher LMX can effectively reduce power distance. This leads to increased communication and exchange between leaders and employees, ultimately enhancing employee resilience. Additionally, the effect of LMX on employee resilience is mediated by stress appraisal. Employees may perceive LMX as a challenge, which plays a Page 16/26 Page 16/26 crucial role in demonstrating their resilience. High-quality LMX provides individuals with additional resources and attention, enabling them to perceive it as a challenging opportunity for growth, development, and happiness. Thus, when employees encounter risks, challenges, or setbacks at work, they concentrate on improving the organization's efficiency and problem-solving. 4 Discussion They proactively seek solutions, choose positive coping strategies, and work to change the status quo to achieve their own goals, thereby increasing their resilience. This study has significant theoretical and practical implications. The study contributes to the literature on interpersonal relationships and leadership in the workplace by clarifying the specific dimensions of leader relationships that influence employee resilience. Previous research has predominantly focused on the relationship between leadership style and employee resilience [19, 68], with less emphasis on the positive effects of LMX on employee resilience [28]. This study suggests that the nature of the relationship between leaders and subordinates, as opposed to simple leadership styles and interpersonal relationships, plays an essential role in developing employee resilience [28]. Especially in China, “relationships” are the linchpin of interpersonal harmony, and employees in organizations not only want to be satisfied at the material level but also need to be satisfied at the social and spiritual levels. We need to pay attention to grassroots officials as the source of employee resilience. In the practice of human resource management in public sector organizations, departmental leaders should pay attention to communication with subordinates, establish good relationships between subordinates and subordinates, cultivate a higher level of care and trust between leaders and subordinates, and increase the frequency of communication between leaders and subordinates. Another crucial contribution of this study is identifying stress appraisals as mediating mechanisms that promote resilience, thus enriching the literature on resilience and stress appraisals. Until now, resilience has mainly been associated with positive emotion regulation and focus [28]. However, this study highlights the significant influence of stress appraisals, which include emotional perceptions, on employee resilience. The perceived gains and losses of resources by employees can influence their stress appraisals, which in turn affects their ability to cope with distress, also known as employee resilience. Cognitive appraisals play a critical mediating role in the relationship between LMX and employee resilience. This finding suggests that LMX has dual effects on the process of cultivating employee resilience. On one hand, it directly influences individual employee resilience through its enhancing or diminishing effects. However, an individual's resilience can be affected by their perception of the situation. LMX is a resource that can lead to an overestimation of an individual's ability to handle situational demands, resulting in a challenging assessment. 4 Discussion This adaptive perception further enhances the sense of self-control, leading individuals to adopt positive coping strategies and achieve positive task completion. Ultimately, this can increase employee resilience in the face of adversity. Conversely, low LMX triggers an underestimation of personal resources, leading to appraisals of inadequacy in meeting situational demands. This tendency to maladapt reduces the sense of self-control, generates negative coping styles and reduces the resilience of employees. Consequently, in practice, individuals with limited resources should actively reorient their perceptions towards challenges, as this reorientation serves to enhance resilience in stress-coping scenarios. Page 17/26 Limitations and future directions In addition, there are certain limitations in this study. First, despite efforts to control variables and processes in contextual simulation experiments, there are inherent limitations in simulating real-world LMX situations. Contextual simulations serve as scientific abstractions, deliberately aiming to isolate and control the influence of other factors for research purposes. Therefore, it is essential to supplement and validate the experimental findings with field studies or alternative methods to establish greater consistency between the results obtained in the experimental context and real-world outcomes. Second, this study did not consider the potential impact of certain variables beyond cognitive appraisals on the research findings. There may be additional mediating variables and moderating effects in the relationship between LMX effectiveness and employee resilience. Thus, future studies should delve more deeply into the mechanisms through which LMX influences employee resilience and incorporate factors such as employee personality traits and organizational culture. This will enable a more comprehensive investigation of the nuanced relationships between LMX and employee resilience. 5 Conclusion Based on the COR theory, the present research explores the relationship between LMX and employee resilience in public sector organizations in China Mainland. It investigates the importance of LMX as a key factor in developing resilience using questionnaires and experimental studies. The research also highlights the crucial mediating role of stress appraisals, including both challenges and hindrances. Ethics approval and consent to participate Participants were informed about the purpose, procedures, potential risks, and benefits of the study prior to their participation. They were made aware that their participation was voluntary and that they had the right to withdraw their consent at any time without any negative consequences. Additionally, participants were assured of anonymity and confidentiality throughout the study and that their data would be used solely for research purposes. Participants were asked any questions or concerns that were addressed by the researchers before providing informed consent. The Ethics Committee of the Department of Psychology at Southwest University approved this study (H23223), and the study was conducted according to the Declaration of Helsinki. Informed consent was obtained from the participants. Acknowledgements Not applicable Competing interests The authors declare that they have no competing interests. Authors’ contributions YY wrote the manuscript and analysed the data under the guidance of ZYH. ZYH contributed to the study design, data collection and critical revisions. All authors contributed to the article and approved the submitted version. Consent for publication Not applicable Availability of data and materials Page 18/26 The data were not available due to privacy or ethical restrictions. The data that support the findings of this study are available upon request from the corresponding author. Funding The National Natural Science Foundation of China(No. 72374174). References 1. Linnenluecke M K. 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LePine J A, Podsakoff N P, LePine M A. A meta-analytic test of the challenge stressor–hindrance stressor framework: An explanation for inconsistent relationships among stressors and performance. Academy of management journal. 2005; 48(5): 764-775. https://doi.org/10.5465/amj.2005.18803921 56. Rodell J B, Judge T A. Can “good” stressors spark “bad” behaviors? The mediating role of emotions in links of challenge and hindrance stressors with citizenship and counterproductive behaviors. 7941.12033 Journal of Applied Psychology. 2009; 94(6): 1438. https://doi.org/10.1037/a0016752 57. Kuvaas B, Buch R. Leader‐member exchange relationships and follower outcomes: The mediating role of perceiving goals as invariable. Human Resource Management. 2018; 57(1): 235-248. https://doi.org/10.1002/hrm.21826 57. Kuvaas B, Buch R. Leader‐member exchange relationships and follower outcomes: The mediating role of perceiving goals as invariable. 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Frontiers in Psychology. 2016; 7: 924. https://doi.org/10.3389/fpsyg.2016.00924 69. Gaddy J W, Gonzalez S P, Lathan C A, Graham P K. The perception of authentic leadership on subordinate resilience. Military Behavioral Health. 2017; 5(1): 64-72. https://doi.org/10.1080/21635781.2016.1243495 Figures Figure 1 Figure 2 Figures Page 24/26 Figure 1 Research Model Figure 2 The meditation model with unstandardized coefficients Figure 1 Research Model Figure 2 The meditation model with unstandardized coefficients. Page 25/26 Page 25/26 Note: Standard errors and 95% confidence intervals for 5000 samples are in parentheses below.***p < 0.001. Appendix.docx Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 26/26
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Difficulties of caregivers of cow's milk allergy patients in understanding the labeling of processed products
˜The œWorld Allergy Organization journal
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cc-by
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Amoroso et al. World Allergy Organization Journal 2015, 8(Suppl 1):A62 http://www.waojournal.org/content/8/S1/A62 MEETING ABSTRACT Open Access Difficulties of caregivers of cow’s milk allergy patients in understanding the labeling of processed products Marina Amoroso1*, Mariana Forti1, Cleonir Beck1, Glauce Yonamine2, Andrea Gushken1, Mayra Dorna1, Ana Paula Moschione Castro1, Antonio Carlos Pastorino1, Cristina Jacob1 From 3rd WAO International Scientific Conference (WISC) 2014 Rio de Janeiro, Brazil. 6-9 December 2014 Background Cow’s Milk Allergy (CMA) is the main food allergy (FA) in children and its treatment includes restricted milk diet. The correct identification of terms that mean milk ensures the efficacy of treatment.The aim of this study was to identify the main factors involved in the caregivers’ ability to recognize the presence of milk in products labels. Methods Cross-sectional and descriptive study was carried out with CMA patient’s caregivers in follow-up at a pediatric reference center for FA. All of them were previously instructed about labels. This study included a questionnaire about the knowledge regarding the labels reading. Caregivers evaluated 20 labels (15 of foods, 3 of medicines and 2 of cosmetics) and should decide if the product was safe and the reason it can or not be offered to the patients. 15/20 contained words meaning milk protein. Results were expressed in number of labels reading (20 labels/caregivers) Results Twenty-eight caregivers fulfilled the questionnaire. The caregivers were 78.5% mothers and 21.5% fathers and about their schooling years 78.2% finished high school or college degree. The median of patient’s follow-up was 12 months (0.03-144). Twenty caregivers deal with patients that presented at least one episode of anaphylaxis. All caregivers referred label reading: 25 read every time they buy the product and only 9 read the labels after buying, before storing and offering.Labels were correctly read in 75.7 % being lactose, casein and whey protein the most common correctly identified terms. The most common mistakes were related to the terms lactil and caramel color. About medicines the most difficulties was to localize de terms related to milk in the package leaflet. Main factors regarding wrong comprehension were small or unclear printing, difficult localization and ingredients in foreign language Conclusions Labels reading is part of food allergy treatment and demands constant reinforcements stressing the necessity of continuous label reading. Labels quality must improve in many aspects in order to help caregivers understanding of milk labels and avoid patient’s accidental intake. Authors’ details 1 Unit of Allergy and Immunology, Brazil. 2Nutritionist of Division of Nutrition, Brazil. Published: 8 April 2015 doi:10.1186/1939-4551-8-S1-A62 Cite this article as: Amoroso et al.: Difficulties of caregivers of cow’s milk allergy patients in understanding the labeling of processed products. World Allergy Organization Journal 2015 8(Suppl 1):A62. 1 Unit of Allergy and Immunology, Brazil Full list of author information is available at the end of the article © 2015 Amoroso et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W4378715871
https://raco.cat/index.php/Hipertext/article/download/409648/511223
es
Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior
Hipertext.net
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ISSN 1695-5498 HIPERTEXT.NET · REVISTA ACADÉMICA SOBRE DOCUMENTACIÓN DIGITAL Y COMUNICACIÓN INTERACTIVA Número 26 · Mayo de 2023 Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior Terese Mendiguren Galdospin Koldobika Meso Ayerdi Jesús Ángel Pérez Dasilva María Ganzabal Learreta Universidad del País Vasco/ Euskal Herriko Unibertsitatea Universidad del País Vasco/ Euskal Herriko Unibertsitatea Universidad del País Vasco/ Euskal Herriko Unibertsitatea Universidad del País Vasco/ Euskal Herriko Unibertsitatea terese.mendiguren@ehu.eus https://orcid.org/0000-0003-3092-6608 koldo.meso@ehu.eus https://orcid.org/0000-0002-0400-133X jesusangel.perez@ehu.eus https://orcid.org/0000-0002-3383-4859 maria.ganzabal@ehu.eus https://orcid.org/0000-0002-4231-402X Bimodal teaching in times of pandemic. Interim solution and challenge for the future in higher education RESUMEN Esta investigación pone el foco en las experiencias y reflexiones del profesorado de comunicación de la Universidad del País Vasco en torno a la enseñanza bimodal, diseñada para el curso 2020/21 en el marco de la pandemia. Esta estrategia docente tiene por objetivo reducir el flujo del alumnado en las aulas, al mismo tiempo que asegura la oferta de clases presenciales. La mitad de las personas matriculadas se conectaban a clase desde sus hogares, mientras la otra mitad asistía al aula presencialmente. Estos turnos se iban alternando semanalmente. El objetivo del estudio es analizar las dificultades, los errores y los aciertos en la adaptación de la docencia al contexto de la enseñanza bimodal. Se ha optado por una metodología cualitativa, dividida en dos fases de actuación. En primer lugar, se realizó Focus Group con profesorado de los tres grados de comunicación y posteriormente se confeccionó una encuesta que fue remitida quienes imparten sus clases de forma bimodal. Este tipo de docencia pretende ser un híbrido entre la estrategia presencial y la virtual, y obtener los beneficios de ambas. Sin embargo, mantener los dos focos de atención y satisfacer las necesidades que surgen en ambos contextos es un reto difícil. Existe un alto grado de preocupación por cuestiones relacionadas con la adaptación de las asignaturas a este tipo de enseñanza, y principalmente, por la pérdida de interacción con el alumnado. PALABRAS CLAVE Enseñanza bimodal; Educación superior; Pandemia; Profesorado; Covid-19; Enseñanza digital. ABSTRACT This research focuses on the communication lecturers´experiences about bimodal education in the University of the Basque Country. This teaching strategy aims to reduce the flow of students in classrooms, while ensuring the supply of face-to-face classes. Half of the people enrolled connect to class from their homes, while the other half attend the classroom in person. These shifts alternate weekly. The objective of the study is to analyze the difficulties, errors and successes in adapting teaching to the context of bimodal teaching. A qualitative methodology has been chosen, divided into two phases of action. In the first place, a Focus Group was carried out with teachers of the three communication degrees and later a survey was made that was sent by those who teach their classes in a bimodal way. This type of teaching aims to be a hybrid between the face-to-face and the virtual strategy, and to obtain the benefits of both. However, maintaining the two focuses of attention and meeting the needs that arise in both contexts is a difficult challenge. There is a high degree of concern for issues related to the adaptation of subjects to this type of teaching, and mainly, for the loss of interaction with students. KEYWORDS Bimodal education; Higher education; Pandemic; Teachers; Covid-19; Digital learning. Mendiguren-Galdospin, T., Meso-Ayerdi, K., Pérez-Dasilva, J. A. y Ganzabal-Learreta, M. (2023). Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior. Hipertext.net, (26), 83-92. https://doi.org/10.31009/hipertext.net.2023.i26.13 Esta obra está bajo la licencia Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional (CC BY-NC-ND 4.0) Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta 1. Introducción A comienzos del 2020, la propagación del Covid-19 llevó a la OMS a declarar el brote de coronavirus como pandemia global. Ante el descontrol y la muerte de miles de personas, prácticamente todos los gobiernos del mundo decretaron el confinamiento de la población y sellaron sus fronteras, clausuraron los negocios, las actividades culturales, las competiciones deportivas y cerraron los colegios para contener su propagación (Pérez-Dasilva, Meso-Ayerdi y Mendiguren-Galdospin, 2020). La pandemia del coronavirus afectó a más de 1500 millones de estudiantes en el mundo, según la UNESCO1. En este contexto, las universidades se vieron obligadas a suspender, de un día para otro, las clases presenciales y a continuar con su docencia en formato virtual (Sahu, 2020). La suspensión de las actividades docentes presenciales para dar paso a la virtualización supuso una presión elevada para el profesorado y el alumnado en su adaptación al nuevo escenario (Tejedor et al., 2020). Fue un proceso no planificado (Corell y García-Peñalvo, 2021) que generó inquietud en la comunidad universitaria (de la Calle et al., 2021) y que diversos autores calificaron como “enseñanza remota de emergencia” (Ferri, Grifoni y Guzzo, 2020; Hodges et al., 2020; Pérez-López, Vázquez-Atochero y Cambero-Rivero, 2020; Rahiem, 2020; Toquero, 2020). Los profesores convirtieron sus hogares en aulas improvisadas (Rapanta et al., 2020) y aprendieron a utilizar software especializado para impartir sus clases de forma virtual (Bao, 2020; Ebner et al., 2020; Mishra, Gupta y Shree, 2020; Motala y Menon, 2020). El sistema universitario tuvo que sobrevivir a la primera oleada de la pandemia sin apenas margen de maniobra y esto dejó patentes las brechas digitales existentes: de acceso a Internet y de competencias tecnológicas, básicamente. Fue en las universidades presenciales en las que se dio la mayor brecha competencial (García-Peñalvo, 2020). Varios estudios analizaron las soluciones adoptadas en las diferentes universidades (Osman, 2020; Radha et al., 2020; Rapanta et al., 2020; Sobaih, Hasanein y Abu Elnasr, 2020) y la calidad de la enseñanza recibida (Lee et al., 2021; Novikov, 2020; Pérez-López, Vázquez-Atochero y Cambero-Rivero, 2020; Rahiem, 2020; Xhelili et al., 2021; Xiong, Jiang y Mok, 2020). En este sentido, diversos trabajos coincidieron en señalar que la pandemia agrandó las desigualdades en el caso de familias con bajo poder adquisitivo o nivel cultural, por lo que el debate sobre la necesidad de alfabetización mediática de los jóvenes se volvió indispensable (Frau-Meigs, 2020). Fue el caso de países subdesarrollados como Pakistán o Sri Lanka, donde la gran mayoría de los estudiantes no pudieron acceder a Internet por cuestiones técnicas y económicas. (Adnan y Anwar, 2020; Olaganwatte, 2020; Sobaih, Hasanein y Abu Elnasr, 2020). Esta situación también se vivió en los países desarrollados entre los estudiantes de estratos de población con menos recursos (Drane, Vernon y O’Shea, 2020; Van Lanc84 Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext ker y Parolin, 2020). Otros autores se centraron en los efectos de la enseñanza remota en la salud de los universitarios como la ansiedad (Ozamiz-Etxebarria et al., 2020; Wang y Zhao, 2020), la salud mental (Dodd et al., 2021; Haider y Al-Salman, 2020; Sahu, 2020) o la depresión (Fawaz y Samaha, 2021). La rápida reacción de las instituciones de enseñanza superior ante este “cisne negro” (Wind et al., 2020) permitió finalizar el curso académico 2019-2020 sin demasiados problemas. Pero sin tiempo para descansar, las universidades tuvieron que prepararse para el siguiente año en un contexto marcado por la incertidumbre y donde la improvisación ya no servía de excusa. 2. Cambio de paradigma en la enseñanza universitaria Las universidades se vieron en la necesidad de resolver en poco tiempo y con escasas herramientas una situación inesperada (Hodges et al., 2020). Las reacciones iniciales a la decisión de estas instituciones fueron variadas: algunos observadores la alabaron como un paso necesario en la lucha contra la difusión del Covid-19 y otros la criticaron como una reacción exagerada (Murphy, 2020). Lo cierto es que la irrupción de la pandemia provocó un cambio súbito en la universidad, desde un entorno presencial a uno virtual, generando disfunciones y dudas (Bartolomé-Pina, 2020). En ese sentido, algunos autores plantearon la necesidad de ser cautos a la hora de sacar conclusiones, ya que aún era pronto para poder valorar los resultados obtenidos ante estas primeras medidas adoptadas y también la precipitación a la hora de actuar podía llevar a falsos éxitos que tan solo podrían ser aparentes (Toquero, 2020). Otros autores, a su vez, pusieron de manifiesto la necesidad de repensar las instituciones educativas para que no perdieran su razón de ser (Tarabini, 2020), lo que se dio en llamar “educación pos pandemia” (Iglesias et al., 2020; Esteban-Guitart et al., 2020). Poco antes del verano de ese mismo año, muchas instituciones se plantearon sí podrían reanudar la enseñanza presencial y cómo hacerlo (Weeden y Cornwell, 2020). En esta dirección, por ejemplo, la UNESCO y el IESALC emitieron un conjunto de principios rectores para planear la salida de la crisis del Covid-19 en la educación superior (Giannini, 2020), entre los que se encontraban la necesidad prepararse con tiempo para la reanudación de las clases presenciales y rediseñar los procesos de enseñanza y aprendizaje. Parece necesario analizar con cuidado los problemas y alcances que la educación a distancia evidenció durante el confinamiento (Ordorika, 2020; Chipia y Santiago, 2020). Los educadores sintieron que el aprendizaje virtual era una buena alternativa, pero echaban de menos la interacción con el alumnado (Chellathurai, 2020). De hecho, la virtualidad puede Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior ser asociada con un aumento de la carga de trabajo, lo que establece la necesidad de revisar los procesos formativos “con el objetivo de no saturar al alumnado de actividades extracurriculares que pueden ocasionar la deserción del mismo” (Tejedor et al., 2021b, s.n.). Para algunos, los contactos directos cara a cara entre los estudiantes y el profesor, así como los estudiantes entre sí en grupos, son elementos importantes en la impartición de las clases (Ożadowicz, 2020). La solución parecía estar en combinar la docencia en el aula con la formación en línea. Tubagus, Muslim, y Suriani (2020) abogan por una integración óptima entre la enseñanza presencial y el aprendizaje basado en la tecnología. En lo que respecta a los aspectos tecnológicos, la pandemia sirvió para poner a prueba la solvencia de herramientas propias de las universidades que, por norma general, habían quedado relegadas a un segundo o tercer nivel de importancia (Tejedor et al., 2021a). En términos parecidos de una educación híbrida, algunos autores defienden la idea de superar las barreras tradicionales entre la formación presencial y virtual. Pardo y Cobo (2020) proponen transitar hacia experiencias de aprendizaje expandidas, líquidas y distribuidas en una línea de tiempo narrativa multimedia a través de la cual los docentes y estudiantes puedan servirse de modo colaborativo de lo mejor de la presencialidad y la cultura digital. De cara al curso 2020/2021, muchas universidades adoptaron la enseñanza presencial tradicional con el aprendizaje digital en línea. Es lo que se conoce como blended learning o aprendizaje mixto (Garrison y Kanuka, 2004; Bartolomé-Pina, 2008, Castro, 2019), si bien recibe otras denominaciones como hybrid learning o aprendizaje híbrido (Cochrane et al., 2020), formación bimodal (Yábar y Barbarà, 1999; Espinoza-Guzmán y Zermeño, 2017; Steiman y Luna, 2020), B-learning (Bartolomé-Pina, 2004; Núñez-Barriopedro, Monclúz, y Ravina-Ripoll, 2019; Sánchez-Ruiz et al., 2021), o incluso educación flexible (Salinas, 2000) y enseñanza semipresencial (Leão y Bartolome, 2003). Las instituciones se impusieron la necesidad de repensar el nuevo escenario educativo ante una situación de pos pandemia que requería imponer normas sanitarias en particular relacionadas con el contacto interpersonal. 3. La enseñanza bimodal En el periodo de “nueva normalidad”, cuya duración parecía difícil de predecir, la obligatoriedad de cumplir las medidas de prevención y de protección impuestas por las autoridades sanitarias llevó a las instituciones académicas a diseñar una serie de pautas de actuación con el objetivo de mantener el mayor grado posible de presencialidad que fuera compatible y respetuoso con dichas medidas. Las orientaciones que se establecieron pretendían preservar, en la medida de lo posible, el carácter presencial de la docencia de la Universidad, pero adaptándola a escenarios de distanciamiento social, aforo limitado y adopción de medidas higiénico-sanitarias. En un primer momento, las orientaciones para planificar el curso 2020-2021 continuaban planteando una perspectiva de formación presencial adaptada, es decir, preferentemente presencial, pero que se complementara con clases telemáticas y sesiones asíncronas. Se creía que la experiencia obtenida durante el confinamiento del curso 2019-20 debía ser aprovechada para utilizar formas digitales de interacción en aquellas actividades que pudieran beneficiarse de dichas metodologías, pero se recomendó que no se impartieran de manera exclusivamente telemática más de un número concreto de asignaturas por grupo y cuatrimestre (que variaba en función de cada universidad), lo que implicaba descartar que pudiera impartirse un curso entero en modalidad no presencial. Cada titulación seleccionó aquellas actividades que requirieran presencialidad para asegurar que el alumnado adquiriera, de forma adecuada, las competencias recogidas en los correspondientes planes de estudio. Como posibles criterios de selección, se utilizaron, entre otros, dar prioridad presencial en modalidades prácticas frente a las de carácter magistral y expositivo, a los cursos iniciales frente a los cursos superiores y organizar la docencia por turnos. Se partía de la idea de utilizar la metodología de trabajo habitual de las asignaturas en su formato presencial, pero parte del alumnado las seguiría a distancia, a través de la herramienta para videoconferencias que se pusiera a disposición de la comunidad universitaria, preferentemente de forma síncrona, lo que le daría la posibilidad de interactuar con el docente y con sus compañeros. En el caso de las actividades formativas asíncronas la recomendación fue que se mantuviera un nivel de interacción alto con el alumnado. Ante esta formación presencial adaptada, se contemplaron distintas alternativas: 1. Combinar la presencialidad física y la telepresencialidad con el grupo al completo. Se trataba de que el profesorado y una parte del alumnado compartieran una misma aula física y el resto participara vía videoconferencia. En este caso se organizó la alternancia entre ambos grupos de manera equitativa para el alumnado (p. ej. por semanas: la mitad del grupo recibía docencia presencial durante una semana y en la siguiente no acudía al centro y trabajaba de forma telepresencial). 2. Impartir determinadas modalidades docentes de forma mayoritariamente a distancia, esto es, determinadas modalidades docentes de una asignatura se impartieran a través de videoconferencia sin presencia física del alumnado en el centro. A la hora de planificar aquellas asignaturas o actividades con un alto contenido práctico se tuvieron en cuenta una serie de alternativas, como las de optimizar el tiempo de estancia en 85 Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta Figura 1. ¿Qué grado de dificultad o preocupación le generan las siguientes cuestiones? Fuente: Elaboración propia laboratorios o priorizar aquello que no pudiera ser trabajado a distancia. Por lo que respecta a la evaluación, los centros planificaron ésta de forma presencial, siempre que el cumplimiento de las medidas establecidas en la normativa vigente lo permitiera. Ahora bien, dadas las circunstancias excepcionales e imprevisibles en las que todavía continuaba España antes del inicio del curso 2020-2021, se recomendó que todas las asignaturas hicieran un planteamiento de evaluación continua además de una propuesta alternativa de evaluación no presencial. En lo que respecta a la Universidad del País Vasco, se optó combinar la presencialidad física y la telepresencialidad para las clases magistrales con los grupos que superaran los 50 alumnos, tratando de garantizar la presencialidad total en los subgrupos de las clases prácticas de cada asignatura. Por tanto, los grupos numerosos (más de 50 alumnos) fueron organizados para recibir las clases de este modo, que se registró con la denominación de docencia bimodal. 4. Metodología El estudio de la percepción y la experiencia del profesorado en torno a la enseñanza bimodal durante el curso 2020/21 se ha realizado en la Facultad de Ciencias Sociales y de la Comunicación del País Vasco. Se ha optado por una metodología cualitativa, dividida en dos fases de actuación. En la primera de ellas, se decidió realizar un Focus Group integrado por cuatro docentes de los distintos grados de Comunicación2. La información obtenida gracias al Focus Group fue clave para la confección de un cuestionario, diseñado ad hoc para este estudio, que en una segunda fase de actuación fue remitida al profesorado de la Facultad. A través preguntas cerradas y semiabiertas, se pretendía conocer más exhaustivamente el grado de preocupación, cansancio, aprendizaje o crítica que podía generar la estrategia docente híbrida por parte del profesorado. Se registró una participación activa de 61 docentes en la encuesta, que tuvo lugar durante la última semana del mes de abril y la primera del mes de mayo (2021). Para la 86 Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext selección de la muestra hubo que hacer un registro del profesorado que imparte docencia bimodal y evitar así un envío masivo e indiscriminado de la encuesta al personal docente de la Facultad. Se recurrió a un tipo de muestra no probabilística, que se suele emplear en los análisis con gran peso cualitativo y cuando la finalidad del estudio no es generalizar los resultados a toda la población, sino recopilar datos comparativos y descriptivos. Se trata de determinar la muestra bajo el criterio del equipo investigador tomando como referencia los objetivos del estudio, lo que Babbie (1999) denomina muestreo deliberado o crítico. La muestra no probabilística intencional, que suele ser pequeña y heterogénea, se selecciona procurando que esta sea representativa y depende de la intención del investigador (Visauta, 1989). Partíamos de la hipótesis de que el profesorado sentía un nivel de agotamiento superior al de años anteriores y que había una general animadversión a la estrategia docente bimodal. Existía además el objetivo de conocer los motivos que contribuían a ello. Para el análisis de los resultados y su cálculo estadístico se ha empleado el lenguaje de programación no procedimental que ofrece Microsoft Excel. Así, se ha optado por estudiar al profesorado de Comunicación, ya que precisamente representa una muestra que hibrida la intención docente con la intención comunicativa, que es, al fin y al cabo, el objetivo de la bimodalidad: tratar de garantizar el proceso de aprendizaje del alumnado, empleando las herramientas necesarias que faciliten la comunicación en los entornos presencial y online. 5. Resultados 5.1. Aspectos relacionados con la tecnología Ante la pregunta realizada en este estudio sobre el grado de preocupación que ha supuesto la dependencia de la tecnología para impartir las clases, algunos docentes sí han reconocido en este aspecto un quebradero de cabeza, ya que ha sido respondido con un alto grado de preocupación por el 15% de las personas encuestadas y con algo de preocupación por un 48% (Figura 1). Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior Respecto a la formación recibida para conocer el funcionamiento de la tecnología instalada en las aulas, más de la mitad de las respuestas consideran que no ha sido escasa, si bien es cierto que algunas voces apuntan el hecho de que, a la hora de la verdad, las cosas son más difíciles en la puesta en escena real que en los cursos de formación. “El problema”, apuntan en algunos casos concretos, “no es no saber manejarse con tres pantallas, es utilizarlas a la vez que se expone un Power Point, porque había que estar pendiente de lo que ven los de clase y lo que ven los de casa”. Además, existen aulas “en las que el cañón, el ordenador con la cámara, y el ordenador principal no están colocados de modo que sea fácil colocarse en un lugar en el que me vean, escuchen y se pueda manejar todo”. Pero es algo que apenas ocurre y señalan que “la universidad ha hecho un esfuerzo en preparar las aulas con la tecnología necesaria para impartir docencia bimodal” (Figura 2). Por otro lado, se ha aprendido mucho a lo largo de este curso porque ahora se sabe que “los micros captan todo el ruido del aula” y se puede tener en cuenta en los ritmos y las rutinas; por ejemplo, el docente ahora sabe que no debe empezar a hablar hasta que haya silencio absoluto en el aula o debe evitar los debates con varias voces al mismo tiempo. Una gran mayoría reconoce que el alumnado no le ha trasladado su parecer en lo que respecta a la docencia bimodal. Entre las opiniones recibidas sí se registra una preocupación por los problemas técnicos (29,5%). La casuística es muy grande. “Desde el que tiene problemas para conectarse, hasta el que tiene que compartir ordenador con sus padres, el que tiene que venir a la universidad para conectarse… o el que está viendo la clase desde debajo de las mantas con la Tablet”. Pese a que el 59% de los encuestados consideran que la bimodalidad es una mera solución pasajera ante la situación de la pandemia (Figura 3), algunas personas opinan que esto ha llegado para quedarse, quizá no tal y como está introducido ahora, pero creen que no desaparecerá. 5.2. Aspectos relacionados con el contenido de las materias El 36% de los profesores y profesoras encuestados afirman que la adaptación de su asignatura a la bimodalidad le ha supuesto mucha más carga de trabajo de la habitual. Más de la Figura 3. ¿Ofrece alguna oportunidad/ventaja esta modalidad respecto a la presencialidad? Fuente: Elaboración propia mitad, un 52% reconoce que le ha supuesto algo más de carga de trabajo y únicamente el 12% no lo considera en absoluto. De hecho, existe a posteriori un aprendizaje y una experiencia que va a suponer quizá una segunda readaptación de la estrategia docente. Mientras un 5% de los docentes afirma que cambiará su estrategia totalmente para el año siguiente, un 72% reconoce que tendrá que adaptarla en cierta medida. De hecho, la sensación es que la docencia en la forma bimodal no consiste únicamente en ser consciente de que se está impartiendo materia en dos espacios distintos a la vez. Así lo atestiguan testimonios como “ni el contenido, ni la forma de impartir me valía. Quizá sí para un aula presencial o para una situación de confinamiento, pero no para una bimodal”. Únicamente un 10% reconocía que no tendría que readaptar su estrategia de cara al siguiente curso. En lo que respecta a la participación del alumno en el proceso formativo, solamente un 6,5% reconoce que este tiene un rol activo. Respecto al ritmo a la hora de impartir la materia, algunos de los testimonios recogidos tanto en el Focus Group como en las preguntas semiabiertas de la encuesta, afirman que es inevitable hacer pequeños parones continuos a la hora de mostrar, entre otras cosas, ejemplos que hay que compartir de modo presencial y de modo online. “Hay que cambiar la asignatura para adaptarla a los ritmos de clase, que no son iguales, y para captar la atención de los que están en casa a la vez que los que están en clase”. Además, en algunos casos en los que la práctica también ha sido bimodal, la sustitución de prácticas grupales por individuales ha supuesto un incremento notable del tiempo dedicado a las correcciones de ejercicios. Por otro lado, el 36% de profesorado encuestado asegura que la formación y el aprendizaje recibidos por parte del alumnado se están viendo muy mermados este año. Un 54% está bastante o algo de acuerdo con esta afirmación, mientras que solo un 10% no lo considera en absoluto. Otro problema detectado entre los testimonios recogidos es la evasión general del alumnado incluso si está en clase: “Hay que subir los materiales completos a Egela3 por causa de Figura 2. El alumnado le ha trasladado su opinión sobre la bimodal en forma de:... Fuente: Elaboración propia la bimodalidad, y eso supone la puntilla para su atención. La palabra hablada les parece redundante”. 87 Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta 5.3. La atención y el Feed back con el alumnado Según recoge este estudio, el mayor quebradero de cabeza al que se ha enfrentado el profesorado es la dificultad de mantener la interrelación y el feed-back con el alumnado presente y el grupo que se conecta online (Figura 1). No es lo mismo enfocar la estrategia y los esfuerzos en garantizar ese feed-back cuando toda la audiencia está conectada online, como ocurrió durante el confinamiento riguroso, que cuando se tiene la mitad del grupo en el aula de forma presencial y la otra mitad de forma online. El problema no ha estado tanto en jugar con pantallas, sino en interaccionar realmente con todo el grupo. “Cuando están en casa, no se sienten parte de la clase. Es muy difícil interaccionar con la gente que está en el aula y con la gente que está fuera. Da la sensación de que el mismo mecanismo, las mismas preguntas, las mismas sugerencias, no se pueden canalizar de la misma manera”. Existen situaciones, reconocen algunos testimonios de este estudio, en las que el docente ve inevitable centrarse en los que están en el aula, pero otros no se lo plantean así: “En el aula pongo el chat abierto para los que están en casa puedan interactuar”. El 16% de los encuestados aseguran que el chat es una buena herramienta para interaccionar y que, en ciertos momentos, es un recurso más empleado que el de alzar la mano presencialmente en el aula. Sin embargo, el alumnado dice que cuando está en casa le cuesta mucho conectar con el grupo en clase. También se ha observado un peligro de absentismo, ya que “los que están en casa se han permitido, según avanzaba el cuatrimestre, tomarse la clase como opcional”. El soporte a través del que siguen las clases también influye. “Mantener la atención en un móvil donde el encuadre abierto que tienen de la clase hace que te vean muy pequeño. No es fácil mantener la atención así”. 5.4. Aspectos emocionales Una gran mayoría, el 84% de las personas participantes en la encuesta ha reconocido que se encuentra más cansada a estas alturas del curso, en comparación con años anteriores. Las casuísticas son variadas, por ejemplo, “en el caso de la enseñanza cooperativa activa la bimodalidad se convierte en un handicap emocional y pedagógico. El seguimiento continuo al alumnado se hace tremendamente difícil y las dinámicas de trabajo se rompen cuando están en casa. Es un volver a empezar cada 15 días”. Algunas personas esperaban que esta situación fuera más sencilla tras pasar por la etapa del confinamiento y haber tenido que adaptar la docencia a un modo de proceder distinto. Pero reconocen que aquella lógica no vale de igual modo. Se nota más cansancio, dicen, “no solo por las circunstancias de la enseñanza bimodal, sino por hablar con la mascarilla y encima a través de una pantalla”. También encuentran un problema en la pérdida de intimidad que implica el hecho de que todas las clases puedan ser grabadas por el alumnado. “Nada les impide grabar lo que se emite por 88 Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext BBC. Esto implica que una frase del docente fuera de contexto puede acabar siendo viral”. Este tipo de reflexiones ya forman parte de la conversación habitual entre el profesorado. La enseñanza bimodal es un tema recurrente entre compañeros para el 64% de las personas encuestadas. Únicamente un 3% opina que no es un tema habitual de conversación. En lo que respecta a la asistencia, se temía que hubiera fuga de presencial a virtual. Esto es, que el alumnado con turno presencial optara por quedarse en casa y conectarse online. Pero no ha sido así. “Les gusta la semana que les toca venir presencialmente”. Según el informe del Focus Group, lo que más le gusta al alumnado y cuando más aprende es en los debates que surgen en clase”. 6. Discusión La comunidad universitaria ha estado forzada a hacer frente a una situación de incertidumbre por motivo de la pandemia. La idea general es que la enseñanza bimodal ofrece la posibilidad de cursar los estudios de forma semipresencial pero se pierde la interacción personal, las dinámicas de grupo y los debates en el aula. Existe una dicotomía entre la ventaja que supone poder llegar a más alumnado y la calidad en la manera de llegar a este. Hay más opciones para llegar, pero menos calidad de feedback real, de conexión humana y emocional, cuya importancia es de sobra reconocida. Entre las ventajas, se ha fomentado el uso de diferentes plataformas digitales y se ha reflexionado sobre los materiales que se emplean para impartir docencia y nuevas estrategias de enseñanza. Sin embargo, el foco del debate hay que ponerlo en el nivel formativo que encuentra el alumnado en este contexto. Bolonia introdujo la idea del aprendizaje frente a la idea enseñanza y la del rol activo del alumnado frente al pasivo. Durante los últimos años se ha tratado de enfocar los esfuerzos en garantizar la puesta en marcha de estrategias que garanticen este tipo de aprendizaje. El peligro de la bimodal es enfocar los esfuerzos en el medio para llegar, más que en el método. Está latente el temor a volver a las clases magistrales tradicionales a través de una pantalla y sin la participación real del alumnado. Por otro lado, no se debe confundir la bimodalidad con la docencia online, en la que el profesorado puede enfocar sus esfuerzos de otro modo y conseguir una participación activa. En la docencia bimodal, existe un público presente ocupando el mismo espacio que el docente, al que también se debe prestar atención. La tecnología ofrece la oportunidad de poner en contacto a las personas que se encuentran a distancia geográfica, pero ese contacto no garantiza un acercamiento per sé. Para que llegue, el mensaje debe estar muy focalizado en quién está detrás de una pantalla o, en su caso, en quien está presente. Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior Esta tarea es difícil para el profesorado, que en general ha sentido frustración y cierta sensación de que no está llegando a ambos espacios a la vez. Para que la bimodalidad funcione, toca reflexionar y repensar la forma de impartir las clases. Por ejemplo, fragmentar y alternar los tiempos de atención al alumnado de los dos espacios. El reto es encontrar una estrategia más eficaz para el alumnado online y presencial. El objetivo debería ser conseguir que la docencia bimodal, aparte de ser una solución para hacer frente a esta situación, se convierta en una opción altamente eficaz. Actualmente, la sensación general es que de momento no se están aprovechando las ventajas de la presencialidad, ni las de la modalidad online. 7. Conclusiones La universidad se ha adaptado a una circunstancia difícil. Quizá esta modalidad docente no vino para quedarse, pero lo cierto es que la estrategia bimodal salvó el curso 2020-21 y posibilitó cierta presencialidad en el ejercicio de la docencia, garantizando un entorno seguro en aquellas facultades que contaban con una elevada tasa de matriculación. Lo cierto es que se confirma el cansancio y un alto grado de frustración en el profesorado. De esta experiencia se puede además extraer un aprendizaje. Se han conocido las necesidades reales y cotidianas que requiere intentar abarcar al mismo tiempo los dos espacios en los que trabajan los docentes: el presencial y el virtual. Aunar estos lugares, hibridando dos estrategias distintas en una, no es tarea fácil. Son dos realidades que precisan necesidades diversas. Se ha demostrado que las estrategias enfocadas únicamente al alumnado que está conectado no tienen cabida cuando a la vez un grupo asiste de manera presencial. Existe, por tanto, la necesidad de buscar nuevas alternativas y estrategias docentes adaptables a la bimodalidad, que conjuguen la capacidad de formar sin olvidar la emoción y la conexión; encontrar alternativas más creativas e innovadoras que apelen a la participación; y fomentar más que nunca el rol activo del alumnado en su proceso de aprendizaje. La adaptación a la estrategia bimodal requiere un cambio de paradigma y exigirá esfuerzo y tiempo. Si se entiende la bimodalidad como una solución temporal y de urgencia ante una situación extraordinaria y caduca, la experiencia docente durante el curso 2020-21 puede quedar en una mera reflexión. Pero lo cierto es que no se puede predecir si en un futuro volverá a ser necesario hacer frente a otra crisis sanitaria que requiera tener interiorizados modelos educativos y estrategias docentes adaptables. Financiación Este trabajo forma parte del proyecto “Noticias, redes y usuarios en el sistema híbrido de medios” (RTI2018-095775-B-C41)”, financiado por el Plan Nacional del I+D+i, del Ministerio de Economía y Competitividad, y por el Fondo Europeo de Desarrollo Regional (FEDER) y del Grupo Consolidado IT1112-16 del Gobierno Vasco. Notas al final 1. https://news.un.org/es/story/2020/08/1478302 2. Un profesor de Comunicación Audiovisual, una profesora de Publicidad, y dos profesoras de Periodismo, por tratarse del grado con mayor número de alumnado. 3. Egela es el nombre de la plataforma que se emplea en la UPV/ EHU para la relación con el alumnado y la compartición de materiales y prácticas, estrategias evaluadoras, foros, etc. Referencias Adnan, M. y Anwar, K. (2020). Online Learning amid the COVID-19 Pandemic: Students’ Perspectives. Journal of Pedagogical Sociology and Psychology, 2(1), 45-51. https://doi.org/10.33902/ JPSP.2020261309 Babbie, E. (1999). 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International Journal of Web-Based Learning and Teaching Technologies (IJWLTT), 12(1), 19-31. https://doi. org/10.4018/IJWLTT.2017010102 Esteban-Guitart, M., Iglesias, E., González-Patiño, J. y González-Ceballos, I. (2020). La personalización educativa en tiempos de cambio e innovación educativa. Un ejemplo ilustrativo. Aula Abierta, 49(4). 395-404. https://doi.org/10.17811/rifie.49.4.2020 Fawaz, M. y Samaha, A. (2021). E-learning: Depression, anxiety, and stress symptomatology among Lebanese university students during COVID-19 quarantine. Nursing Forum, 56(1), 52-57. https://doi. org/10.1111/nuf.12521 Ferri, F., Grifoni, P. y Guzzo, T. (2020). Online Learning and Emergency Remote Teaching: Opportunities and Challenges in Emergency Situations. Societies, 10(4), 86. https://doi.org/10.3390/soc10040086 Frau-Meigs, D. (2020). Pédagogie à distance: Les enseignements du e-confinement. The Conversation. http://theconversation.com/pedagogie-a-distance-les-enseignements-du-e-confinement-137327 García-Peñalvo, F. J. (12 de mayo de 2020). El sistema universitario ante la COVID-19: Corto, medio y largo plazo. Universidad sí, https:// www.universidadsi.es/sistema-universitario-covid-19/ Garrison, D.R. y Kanuka, H. (2004). Blended learning: Uncovering its transformative potential in higher education. The Internet and Higher Education, 7(2), 95-105. https://doi.org/10.1016/j.iheduc.2004.02.001 Giannini, S. (2020). COVID-19 y educación superior: de los efectos inmediatos al día después. Revista Latinoamericana de Educación Comparada: RELEC, 11(17), 1-57. Haider, A. S. y Al-Salman, S. (2020). Dataset of Jordanian university students’ psychological health impacted by using e-learning tools during COVID-19. Data in brief, 32, 106104. https://doi.org/10.1016/j. dib.2020.106104 Hodges, Ch., Moore, S., Lockee, B., Trust, T. y Bond, A. (2020). The Difference Between Emergency Remote Teaching and Online Learning. Educause Review. https://er.educause.edu/articles/2020/3/the-difference-between-emergency-remote-teaching-and-online-learning Murphy, M.P.A. (2020). COVID-19 and emergency eLearning: Consequences of the securitization of higher education for post-pandemic pedagogy. Contemporary Security Policy, 41(3), 492-505. https://doi. org/10.1080/13523260.2020.1761749 Novikov, P. (2020). Impact of COVID-19 emergency transition to on-line learning onto the international students’ perceptions of educational process at Russian university. Journal of Social Studies Education Research, 11(3), 270-302. Núñez-Barriopedro, E., Monclúz, I. M. y Ravina-Ripoll, R. (2019). El impacto de la utilización de la modalidad B-Learning en la educación superior. Alteridad. Revista de Educación, 14(1), 26-39. Olaganwatte, Ch. (2020). The Covid-19: Impact on education. Journal of Asian and African Social Science and Humanities, 5, 13-14. Osman, M. E. (2020). Global impact of COVID-19 on education systems: The emergency remote teaching at Sultan Qaboos University. Journal of Education for Teaching, 46(4), 463-471. https://doi.org/ 10.1080/02607476.2020.1802583 Ordorika, I. (2020). Pandemia y educación superior. Revista de la Educación Superior, 194(49), 1-8. https://www.scielo.org.mx/pdf/ resu/v49n194/0185-2760-resu-49-194-1.pdf. Ożadowicz, A. (2020). Modified Blended Learning in Engineering Higher Education during the COVID-19 Lockdown—Building Automation Courses Case Study. Educacion Sciences, 10, 292. https://doi. org/10.3390/educsci10100292 Ozamiz-Etxebarria, N., Santa-María, M. D., Munitis, A. E. y Gorrotxategi, M. P. (2020). Reduction of COVID-19 anxiety levels through relaxation techniques: A study carried out in northern Spain on a sample of young university students. Frontiers in Psychology, 11, 2038. https:// doi.org/10.3389/fpsyg.2020.02038 Pardo, H. y Cobo, C. (2020). 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Multiambiente de aprendizagem: a integração da sala de aula com os laboratorios experimentais e de multimeios. Revista Brasileira de Tecnologia Educacional, 159(160), 75-80. Rahiem, M. D. H. (2020). The Emergency Remote Learning Experience of University Students in Indonesia amidst the COVID-19 Crisis. International Journal of Learning, Teaching and Educational Research, 19(6), 1-26. https://doi.org/10.26803/ijlter.19.6.1 90 Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext Enseñanza bimodal en época de pandemia. Solución provisoria y reto para el futuro en la educación superior Rapanta, C., Botturi, L., Goodyear, P., Guàrdia, L. y Koole, M. (2020). Online University Teaching During and After the Covid-19 Crisis: Refocusing Teacher Presence and Learning Activity. Postdigital Science and Education, 2(3), 923-945. https://doi.org/10.1007/s42438-02000155-y Sahu, P. (2020). 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Responses to COVID-19 in Higher Education: Social Media Usage for Sustaining Formal Academic Communication in Developing Countries. Sustainability, 12(16), 6520. https://doi.org/10.3390/su12166520 Steiman, B. y Luna, A. (2020). La educación bimodal como práctica de enseñanza innovadora. Hologramática, 33(3), 201-213. https://www. cienciared.com.ar/ra/doc.php?n=2285 Tarabini, A. (2020). ¿Para qué sirve la escuela? Reflexiones sociológicas en tiempos de pandemia global. Revista de Sociología de la Educación, 13(2), 145-155. https://doi.org/10.7203/RASE.13.2.17135 Tejedor, S., Cervi, L., Tusa, F. y Parola, A. (2020). Educación en tiempos de pandemia: reflexiones de alumnos y profesores sobre la enseñanza virtual universitaria en España, Italia y Ecuador. Revista Latina, (78), 1-21. https://doi.org/10.4185/RLCS-2020-1466 Weeden, K. y Cornwell, B. (2020). TheSmall-World Network of CollegeClasses: Implications forEpidemic Spread on a University Campus. Sociological Science, 7, 222-241. https://doi.org/10.15195/ v7.a9 Wind, T. R., Rijkeboer, M., Andersson, G. y Riper, H. (2020). The COVID-19 pandemic: The ‘black swan’ for mental health care and a turning point for e-health. Internet Interventions, 20, 100317. https:// doi.org/10.1016/j.invent.2020.100317 Xhelili, P., Ibrahimi, E., Rruci, E. y Sheme, K. (2021). Adaptation and perception of online learning during COVID-19 pandemic by Albanian university students. International Journal on Studies in Education, 3(2), 103-111. https://doi.org/10.46328/ijonse.49 Xiong, W., Jiang, J. y Mok, K. H. (2020). Hong Kong university students’ online learning experiences under the Covid-19 pandemic. Higher Education Policy Institute–Blog. https://www.hepi. ac.uk/2020/08/03/hong-kong-university-students-online-learning-experiences-under-the-covid-19-pandemic Yábar, J. M. y Barbarà, P. L. (1999). La Universitat Autònoma de Barcelona: el camino hacia una universidad bimodal en el marco de las Tecnologías de la Información y la Comunicación. Educar, (25), 113-118. https://raco.cat/index.php/Educar/article/view/20718 CV Terese Mendiguren Galdospin. Es Profesora Agregada de la Universidad del País Vasco UPV/EHU, Departamento de Periodismo. Es Doctora en Ciencias de la Información por la Universidad del País Vasco. Como investigadora, ha participado en 20 proyectos de investigación y ha publicado más de 30 artículos científicos en revistas de prestigio incluidas en bases de datos como JCR, SCOPUS o Dice-Cindoc. Actual- Tejedor, S.; Cervi, L.; Escoda, A.; Parola, A.; Tusa, F. (2021a). Higher Education response in time of coronavirus: perceptions of teachers and students, and open innovation. Journal of Open Innovation: Technology, Market, and Complexity, 7(43), 1-15. https://doi.org/10.3390/ joitmc7010043 mente, es miembro del proyecto “Noticias, redes y usuarios Tejedor, S.; Cervi,L; Tusa, F.; Parola, A. (2021b). Los docentes universitarios frente al cambio a la educación virtual impuesta por el coronavirus. Revista Sociedade e Estado, 36(3). https://doi.org/10.1590/ s0102-6992-202136030004 compagina con la gestión como Vicedecana de Comunicación Toquero, C.M. (2020). Challenges and Opportunities for Higher Education amid the COVID-19 Pandemic: The Philippine Context. Pedagogical Research, 5(4), em0063. https://doi.org/10.29333/pr/7947 Tubagus, M., Muslim, S. y Suriani, S. (2020). Development of learning management system-based blended learning model using claroline in higher education. International Journal of Interactive Mobile Technologies, 14, 186-194. https://doi.org/10.3991/ijim.v14i06.13399 Van Lancker, W. y Parolin, Z. (2020). COVID-19, school closures, and child poverty: A social crisis in the making. The Lancet Public Health, 5(5), e243-e244. https://doi.org/10.1016/S2468-2667(20)30084-0 Visauta, B. (1989). Técnicas de investigación Social. I: Recogida de datos. Promociones y Publicaciones Universitarias SA. Wang, C. y Zhao, H. (2020). The impact of COVID-19 on anxiety in Chinese university students. Frontiers in psychology, 11, 1168. https:// doi.org/10.3389/fpsyg.2020.01168 en el sistema híbrido de medios” (RTI2018-095775-B-C41) y del Grupo consolidado de investigación Gureiker (IT111216). Sus líneas de investigación son Periodismo en Internet e Innovación Educativa y Nuevas Metodologías Docentes, que de su Facultad. Koldobika Meso Ayerdi. Es profesor titular y director del Departamento de Periodismo de la UPV/EHU. Doctor en Periodismo. Como profesor, ha impartido docencia en Grado y Posgrado. Ha publicado más de un centenar de trabajos en forma capítulos, libros y artículos en revistas académicas de calidad. Ha presentado más de cien ponencias y comunicaciones en congresos nacionales e internacionales. Su labor investigadora ha sido desarrollada en el marco de su participación continuada en más de una decena de proyectos de investigación financiados en convocatorias públicas competitivas. También ha dirigido Proyectos de Innovación Educativa y ha participado en otros como investigador, en el marco de las actividades del Grupo Especializado en Innovación Educativa KZBerri. Igualmente, ha participado en diversos contratos de investigación y transferencia relevantes con entidades públi91 Terese Mendiguren Galdospin, Koldobika Meso Ayerdi, Jesús Ángel Pérez Dasilva y María Ganzabal Learreta cas (Euskal Irrati Telebista/Radiotelevisión Pública Vasca). Tiene reconocidos 4 sexenios de investigación. Jesús Ángel Pérez Dasilva. Es profesor Pleno en el Departamento de Periodismo de la UPV/EHU. Ha sido vicedecano de la Facultad de Ciencias Sociales y de la Comunicación (20092012), director del Máster de Comunicación Social (2016-2018) y miembro de la Comisión Universitaria de Evaluación Docente (Docentiaz, 2016-2022). Forma parte del grupo de investigación Gureiker. Ha sido profesor visitante en la Universidad de Cambridge (2012) y ha participado en el programa Erasmus+ de movilidad docente en las siguientes universidades: Trieste (2011), Beira interior (2013), Oporto (2015), Braga (2016), Wroclaw (2017 y 2019). Ha participado en 27 actividades de investigación en convocatorias competitivas siendo en 5 de ellas el investigador principal. Es coautor de 65 artículos científicos (13 Q1 y Q2 en SJR/WOS), 27 capítulos de libros y 100 contribuciones en congresos. Tiene 3 sexenios. Sus líneas de investigación se centran en el periodismo digital, la innovación educativa y las redes sociales. María Ganzabal Learreta. Es profesora agregada del Departamento de Periodismo de la Universidad del País Vasco. Es doctora en Periodismo y máster en Documentación Digital por la Universidad Pompeu Fabra. Ha trabajado en diversas agencias de comunicación y en el Departamento de Arte de la revista americana New Woman. La docencia en materias de Grado está relacionada con asignaturas como Periodismo Social y Participativo y Narrativas Transmedia. Asimismo, imparte docencia en Posgrado, en el Máster de Estudios Feministas y de Género y en el Máster de Comunicación Social donde imparte materias relacionadas con el género y el periodismo. Ha publicado múltiples trabajos en forma capítulos, libros y artículos en revistas académicas de calidad, También ha presentado ponencias y comunicaciones en congresos nacionales e internacionales. Estas publicaciones se han desarrollado dentro del marco de investigación del que forma parte en más de una decena de proyectos de investigación financiados en convocatorias públicas competitivas, tanto a nivel universitario, autonómico y del Plan Nacional I+D+I. PUBLICIDAD El Observatorio de Cibermedios es una producción del Grupo de Investigación en Documentación Digital y Comunicación Interactiva (DigiDoc) del Departamento de Comunicación de la Universitat Pompeu Fabra. https://observatoriocibermedios.upf.edu/ 92 Hipertext.net, n. 26. 2023 · https://raco.cat/index.php/Hipertext El Observatorio de Cibermedios (OCM) forma parte del proyecto del Plan Nacional “Parámetros y estrategias para incrementar la relevancia de los medios y la comunicación digital en la sociedad: curación, visualización y visibilidad (CUVICOM)”. PID2021-123579OB-I00 (MICINN), Ministerio de Ciencia e Innovación ( España).
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ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ﺍﻟﺪﻛﺘﻮﺭ ﻓﺎﺿﻞ ﻛﺎﻣﻞ ﺍﳌﻮﺳﻮﻱ ﺟﺎﻣﻌﺔ ﺍﻟﻜﻮﻓﺔ ـ ﻛﻠﻴﺔ ﺍﻟﻔﻘﻪ ﺍﳌﻘﺪﻣﺔ ل ا  ا او ُ ام  اَ ّإن ٌ ز ُم ؛ا  ا ادر ا  َولرَ ا اَُ ي ا اب ا  ،  ذت و واة وا ؛   ا اكُ ا الل وأ و أ ا ا  ِدات ا أن او إذْ ار اوإ ؛ وء اع أو ا ِا ا ط  ا  َ اُ إ ل مآن اّ ان ؛ ا ا أ    ن ا وإذا َْَ ِ  َاج إ ي ابُ ا أ ا  و ُبُ ا    ٍ اع أمِ بِ اَ ا ُ ، و رم ِِم    ا   ء، و وا وا وا ا ا ن  يي وا ٌَُ  ٌء ا ا ن  ،  واا ل . ء ا إنا  ول اول ا َ ؛ ِ  َن أن ا او  ِا َ آراء ت وذ ا و ا  وا ءا  ن أر تء ذ ا ان م ان ا . ووا ا  ا اّء، و أ اوء ، واع اّغ ، وأمء اء وا ،  ة  ءد وا ا ءَ اّ ء ا ا ان ن  ل ا . أ إ  ا ا أ ت ا ا ام اوأ ا . ﻱ ﺟﺎﻣﻌﺔ ﺍﻟﻜﻮﻓﺔ ـ ﻛﻠﻴﺔ ﺍﻟﻔﻘﻪ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ......................................................... ....... ) ٥٦٣ ( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ﺍﳌﻘﺪﻣﺔ ﺍﻟﺪﻛﺘﻮﺭ ﻓﺎﺿﻞ ﻛﺎﻣﻞ ﺍﳌﻮﺳﻮﻱ ﺟﺎﻣﻌﺔ ﺍﻟﻜﻮﻓﺔ ـ ﻛﻠﻴﺔ ﺍﻟﻔﻘﻪ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ . ) ٥٦٣ ( ﺍﳌﺒﺤﺚ ﺍﻷﻭﻝ ﻭﺍﺻﻄﻼﺣﺎً ً ﺗﻌﺮﻳﻒ ﺍﻻﺳﺘﺜﻨﺎء ﻟﻐﺔ . ﺍﻻﺳﺘﺜﻨﺎء ﻟﻐﺔ : ة : ّ، وا    : ردَ ء اَ)) : ر ل ا ءا (() ١   رد ، و ا ذ))ء ادي أنوز آ( ورأى ا (()  ء إا ٢ ل :  ،ر ء( وا  ، ًء ، ا ، ا .اج ، ا ، جل ا ﺍﳌﻘﺪﻣﺔ ل ا  ا او ُ ام  اَ ّإن ٌ ز ُم ؛ا  ا ادر ا  َولرَ ا اَُ ي ا اب ا  ،  ذت و واة وا ؛   ا اكُ ا الل وأ و أ ا ا  ِدات ا أن او إذْ ار اوإ ؛ وء اع أو ا ِا ا ط  ا  َ اُ إ ل مآن اّ ان ؛ ا ا أ    ن ا وإذا َْَ ِ  َاج إ ي ابُ ا أ ا  و ُبُ ا    ٍ اع أمِ بِ اَ ا ُ ، و رم ِِم    ا   ء، و وا وا وا ا ا ن  يي وا ٌَُ  ٌء ا ا ن  ،  واا ل .ء اإنا  ول اول ا َ ؛ ِ  َن أن ا او  ِا َ آراء ت وذ ا و ا  وا ءا  ن أر تء ذ ا ان م ان ا . ووا ا  ا اّء، و أ اوء ، واع اّغ ، وأمء اء وا ،  ة  ءد وا ا ءَ اّ ء ا ا ان ن  ل ا . أ إ  ا ا أ ت ا ا ام اوأ ا . ) ٦٦٣ ( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻷﺻﻮﻟﻴﲔ : ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻷﺻﻮﻟﻴﲔ :  ا ا و واظ ا  ء ا إ ا م )ت  ا  ، ي ا٨٧٤ ،  ا لُ )) ام ( :لُ ل، واذاف ا أط ُ و ب إذا ا و ء إذا ا )((٧ ا ون اي، وآ ا  ا ا ا ( وزاد )ت ا ا ذ   ا٥٠٥ ل :))  ، إذ ا ( ذ ل ذو ُَه أمَو ل  َدُ   ر ا أن رةٍ دالٍ  ٍ  )(( ولا ٨ ) تمّّ ا( و ٣٠٤ ) ذ إ (٩  ا ( .وا ا وأ إ َء اُَ    ّ أن إ ا وا اأم )ت  ى ا ،٥٦٤ ،   ء ا   ء: )) ا( أن ا دوا أن ا ا أن ، إ آ   أدّِ  ء اجأو إ   ا ن  ا ، .... وان ، و :  ذ ن  ء  ، ً ا ودع ز ا : ا  ا ء ، وا  ا  )(( اء٠١ اج  واوء ادوات ا اج ا وى لا ا( ، و ن   ض امّ ا .    ف رأي ا  و  أ ء ان فل ال: ))و اذء إا ، أو   دا  ج ، ٌ  ، وذ    و م ّم ؛ ؛ ن دا  )((   د ن  ب ا َُجْ اّ أم١١  انُ إ   .( ا رؤ ّ وإماْ وا  ِء َ ا ء ِا ّ . ار ا إظ  وظ د و ﺍﻻﺳﺘﺜﻨﺎ ء ﺍﺻﻄﻼﺣﺎً ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ : )ت   ء   أ٠٨١ د)ت  ا( و٥٨٢ (، و ي )تا ٨٣٥  ا أدوات ودر ا ا ا درم ( ؛  "    ُتَ )) : طل ا  ا و  ، ت ب ان ء ه   ة ا ف " ا ل، دون ل وا اب ا  ى  ، ء ا ،  أوا (( واا )٣ )ت   ا (٢٩٣ ل  ( ء : أن ا:)) و ه(()    أ  ه ، أو  َ أد  َجُ٤ ل ا(و )ت ٣٤٦  ، إذا  ا ه  لء" ا " ا أن(:)) ا   ف ا ء ،  ، وّل او أن اج ا  )((ء ا   ، و ءّ ا ،    و ( ٥ )ت   اّ. ا ٢٧٦  : ))و ف ا  ءف ا  (  أو وك ر أو  ا أو جا ة (() ا ٦ ( م ي ن ا ا  ا اج ، و ا ء  ا اّأن  ى أدواتل إ د د  اجا ا  أن أ، واوا ، و ا إ  ا ن دا َجْ أ َ أنُء وا  أن  .ء ا  اج إ  ، و  ء أدوات ) ٧٦٣ ( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ... ﺃﺭﻛﺎﻥ ﲨﻠﺔ ﺍﻻﺳﺘﺜﻨﺎء ء  وأداة ا  ا ن أر  ة ا ء ا  ا  ء أداة ا دة ن   ا؛أوا ا ب إ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ٨٦٣ ( ، وإذا  ء و أداة ار وإ ا و  ا ب ان    اٌ أ  إ  و ء ا ا ت ة ا اّ ِّغ ، وء ا ُ ا ؛ و اء إا ء إ  هّ ء ا اوا إ  ،  ٌغُ ٌءٌ وا ٌء . ام .   ا ءا ن ا  ،   ، و    ب أي ا  طا ر ان ا اب اَ ا  ط ن  ء ان ظ ن  ٌ ط   ب ، و ا   . ءَ ا إن ا ق اى ا و  :ﻍ ﱠ ﺍﻻﺳﺘﺜﻨﺎء ﺍﳌﻔﺮ    ا   اْُ   غءَ ا اُ أن ذ   M C D E F G H I J K L M N O P Q R S T U V W X Y Z [ \ ] ^ _ ` a L )(. ٢١   و  أو  ن عا ا  أن ن ااو : ل . ( أو ا )اا ْْ ٌََ َا إَ ْَ  )٣١ ( ،  إ  ُم َناّ ، وأ ا  اء إ ا  .وا  ا ً ؛  إ أذ وأم  ء    وع ا . ءاع ارجَ أم ُُْَو    ا   اْُ   غءَ ا اُ أن ذ   M C D E F G H I J K L M N O P Q R S T U V W X Y Z [ \ ] ^ _ ` a L )(. ٢١ ﺃﻧﻮﺍﻉ ﺍﻻﺳﺘﺜﻨﺎء:  ء اء ا َّ    وا ا ر ء ام ء ا اع ا؛ وا  ا   ن ا  : و ت وار ا ه ، و  ا    ن ا  : و ا  و  وإ  و  إ ء و ا  ا  ا اّ  أم ءال ا  ء  ا   زا   ج   وا  جُ ولا ا ن  واا ٤١ )  وزا وه إذ ن ا (، و ت  ا ه ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .... ) ٩٦٣ (  ان ؛ء ال ا  ا ا( وا ا )ا  ل أو  أو ز   وا ا  ا  ء ا ا  أم( ، و ا ) رة  ا، وا ب)إ   ، وإذا ورد ذ ا  ء ا ))  ز ا  ،( )((  ز ، و٥١ ط    ءء اازي: ))ال ا( ، و ز (()  ا   ، و ا ٦١  أ ن ل( . إنّ ا   ،  إدر    ن اّ، أ ًءة ا ا ): الأ ( ٧١ ١راك و ا  ، وإذا و ز ، و  ا  ء إنّ ا ). ًءا ٨١آن ا ا و ا ء   أيا ا بوَلَ أ(و   ء ا بر اوأ : ط ١ راك و ا  ، وإذا و ز ، و  ا  ء إنّ ا ). ًءا ٨١ آن ا ا و ا ء   أيا ا بوَلَ أ(و   ء ا بر اوأ : ط ،   ا  م  أ ا إ ذ م( ، و)  ()إ .أ )  إ  (ر )إ اي ما ( ٩١ :     ا     ْب. ان M Í Î Ï Ð Ñ Ò Ó Ô ! " # $ % & ' ( ) L )٠٢ ( . ﺃﻧﻮﺍﻉ ﺍﻻﺳﺘﺜﻨﺎء:  ء د ر ا  ء ا ،  ا  ء إ ا :   ز ، و ا  ء ا ،   د ر و M G H I J K L M N O P Q R L )١٢   ا   (   . و ا : M ° ± ² ³ ´ µ ¶ L )٢٢ (، ا : ))   .و ا    و ا  إِ ْِ ِِ ُ َ عَ اَا ((M Y Z [ \ ] ^ _ ` a e L )٣٢    ّ( ، وا :   . و  ا ، واM § ¨ © ª « ¬ ® ¯ ° ± ² ³ L )٤٢     ا ر ، ( ):م ال ا ( ٥٢ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ......................................................... ....... ) ٠٧٣ (   أن  و اع ا  ل ّ ي)تل ا٢٥٥ ، ء ا  ءا اٌ و زه )) : ( ز ا  إ ذ  و ر (()وا ٦٢ (. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .....   أن  و اع ا  ل ّ ي)تل ا٢٥٥ ،ء ا  ءا اٌ و زه )) : ( ز ا  إ ذ  و ر (()وا ٦٢ (.   در أ ن )) : ل ا : إذا ا ا  فة او ،  ا ط ء واُ درب أ ّول امأي ا ا ، (( إ ن  وَ ا  إ درب ا  و ء اأي ا او أرا ا   ا   ا  إ درن أ ل إن د أن .ا ٢ ) آن ا ا و ا  وا ء ا .٧٢ ء ن ا (.)) و ه (()  ا  م  ن ا ا  وارداا ٨٢ ( . ٢ ) آن ا ا و ا  وا ء ا .٧٢ ء ن ا (.)) و ٢ ) آن ا ا و ا  وا ء ا .٧٢ ء ن ا (.)) و ه (()  ا  م  ن ا ا  وارداا ٨٢ ( . ٣ آن ا  و . أم )ا ٩٢ (. ه (()  ا  م  ن ا ا  وارداا ٨٢ ( . ﺃﻧﻮﺍﻉ ﺍﻻﺳﺘﺜﻨﺎء:  نِ ؛ِ ا ا ِدِهةِ و  َءَ اَ اُ ا أَكَ ٌز ء از، وا ا  ا ا ُ ْ أن  اا ُ ّون أم ن اّ . وأٍ  ء اي  ُُ ، ٌ  ٌءا  ا ونَ ا ن وا از ا ء ا ا ب  ا)) : ل لر ا   وب ا   ا را ز و  ا ا ول وع ا م  َ ان ؛ ا ر  ذ ول  ا ا ا ا أن ورا  و  ... را  ا ا   رٌ( أرادوا  ا ا ) : ن   ّ ...وأ م ّم  ل ا  آد  أنُ ن ا ا ا ذر، و  إ )((٠٣ ( ، ُ،  لنَ ا ل ال إ م   ا أن  ا فة او  و  م َب ان ٌ ؛  ا  ٌ  ّبُ إ ا :    ء اّم  اّ ، وأ () لا ا  ب ا  ا  )   ل وا ا ق ))اّح ا   ل ا ،    ر ا و ا ا   اذا م( ام زا ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ١٧٣ ( ٍ و ب ا إ ا ن  ُَ ، وإذا أ ل ا ن ذ ) (( ط ولا ١٣   ا ء نّ ا ا ن د ال ، (  اَ و  طا ّ أن ُل ٌ،  ب إ ا   ن ؛  أو أي أو ط ن ان ا ، ا ب إا      وا ا ق ا ،ب ا ًا أو  آ ) عُ ا َاب ا أن  ، وا ٢٣ را دة إ   :ٌل أ .(  ة ار إ ٌر دل : إ ر ، و  ومار إ، ّن ؛ ء   ( أنّ )إ  د ، وا اء ا ا ٌَ دا ر،  د ة ّم  ة  ن ا و ()  ءت وا ة . )  ا ًء اة و ٌ و ان٣٣  ء. ( ِارإ .   ِوم و :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء   ط أ ا أنء إ اّِ وط ا وإنّ ا ء إب ا أ ا    أم   ا أن، وا ء ؛ أداة ا ا ن اذا ، ا َ أرب و ا : رأ ًءا بَ  ِا : أ  و م وا : إ   ُ بَ اا ا : أ ُ ، و  ا ا  ا ،ة ق ا ، وطا ا و ك   ءا ءن ا و : ))   ، وا  ر ا   َ إَ ااِ أُ ْا (() ٤٣   و وّقَ، وا ْ إن ا: ا ( وا  ءً و اء و اد  وء ، وود ا  :ءاّوطنا ١ ) ه ْَ ، و  ءل اب ا. و ( ٥٣ ء ى أدواتِ ا إ  اع أي انم ا ل اد اإن   و   ن وا لُ من ا إذام و   ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ... ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ٢٧٣ ( . )   أولَُ٦٣ ى  ا ا  ا َب ا : أ. ( . ا ز إ و   ا ف ل  ، و ا و لون ا  ، أع امة ا  ءة أراد ا  و و  اذا ذ اي: إن    رواون إ  ة ؟ وه ا ن  ء ا ذ س   ل  ،  ا ن ، و  ء ا ز ).  ا  ء ا ، ءَ ا ِْ إن ا ا ٧٣  َْُ ( ، إذ (() إ و أن    ا : ))إذا س٨٣ َ ُ م(، و )ف ذ ٩٣  ّم ؛ د ه ا  س  ا ق ا(. :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء ز ا  ا: ))وم   ا ذ ذ( ، و (( ) ا ُاوا ٧٤  ك ً   ا  ء ا ( . و ذ     :ل   ال: ))وأو   ا ره ا در ا  ا م ،را إ ا :  ، ول ا لا  ل أ ،وا   ،  را  إ ت؛ ا ءز ا :ا  ا  د أن و ، و ا    ن  ا أن : وارل ا ...   ، وإ  إذا  ا  ءز ا ّ، وإم     ، أورُ ا   ا  نرك ا   ل ا أ نر ا وا :  M G H I J K L M N O P Q R L ) ( ٨٤ ٣. ر  ن ا ا ، و  ق  ن ا أن :  ى اء .و ا  ان يا . أ ا  و  ا  أن ا ان ب  ط  ل م  .  ارا ب . ا  ، و ب ا ط  :    اق ا أ (()ا وط ن ء اق ا ))إذا ا ن اوا ٩٤ ( . اج إُ ار ا أ ، و  م ا؛  ا  ن اّوا ٢ .)   ا  ن ا ْ.أن ٤٤ ي : )) ال ا. ( ،  ، و ب أّزه أ ،  ا  ء ا  ءا ة وا ا  ، و  أوا ون(() ا ، و ٥٤ ( .   ن  ا  ء أنّ ا وأه ا  ا  إذا أّ ،را : ا، وارا ّٌ إء أ )) :ل ْ( إذ)أ  ، ن ا أ  ا ، ل ا   إ  ، لن ازا  أ ل ّا: إم ، و أ  رع اوا و ء ، وا او (() ٦٤ . ز ا  ا: ))وم   ا ذ ذ( ، و (( ) ا ُاوا ٧٤  ك ً   ا  ء ا ( . :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء وإط ة ،  َ ا  ، و ا  ه ذء واع ا  ذ )((  إذا مّ ر : )) واذ ا اد ذ ن ُّّم٠٤  م ، ( )ي ذا ١٤  ذْ إِ( .أ ن  ،ت ا ِ ا ادي ا ُ  أن ف ا ، و ا   ، و ءا ْا إن     :ل ريرواه ا با ا  ُأت  َب أ(.ووط  نة ) ا  ا       ا  ، ة  أ ل (ج )ا ل ا ر   داود ن ل)) :ل   أة ا   اط  ، ءَ ا ْ إن  ل ،  ا  ُ رس  ً   و أو ة أة وا ا إ   ، ءَ ا ان  ُي م ، وا ر ءت )(( ن أم  ا  وا ، ءَ ا ل إن ، ه ٍ٢٤ لُ : (. أ ج   ا   أة م ا   ا  ُ ا ب  وٍ و دي أي ْ دون أنا    ا نم ُ ان ر ة ا ا    َٍ أ و  وءَ ا ْلَ : إن ْ أن    ؟ ون م إذا ديٍ   م ؛ ا ي اا ا جن م ( ر ) . وا )ء ا ان ( ٣٤ل ا .  مو .  دُ ان ا ء ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ٣٧٣ ( ٢ .)   ا  ن ا ْ.أن ٤٤ ي : )) ال ا. ( ،  ، و ب أّزه أ ،  ا  ء ا  ءا ة وا ا  ، و  أوا ون(() ا ، و ٥٤ ( .   ن  ا  ء أنّ ا وأه ا  ا  إذا أّ ،را : ا، وارا ّٌ إء أ )) :ل ْ( إذ)أ  ، ن ا أ  ا ، ل ا   إ  ، لن ازا  أ ل ّا: إم ، و أ  رع اوا و ء ، وا او (() ٦٤ . :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء و ذ     :ل   ال: ))وأو   ا ره ا در ا  ا م ،را إ ا :  ، ول ا لا  ل أ ،وا   ،  را  إ ت؛ ا ءز ا :ا  ا  د أن و ، و ا    ن  ا أن : وارل ا ...   ، وإ  إذا  ا  ءز ا ّ، وإم     ، أورُ ا   ا  نرك ا   ل ا أ نر ا وا :  M G H I J K L M N O P Q R L ) ( ٨٤ :  M G H I J K L M N O P Q R L ) ( ٨٤ ٣. ر  ن ا ا ، و  ق  ن ا أن :  ى اء .و ا  ان يا . أ ا  و  ا  أن ا ان ب  ط  ل م  .  ارا ب . ا  ، و ب ا ط  :    اق ا أ (()ا وط ن ء اق ا ))إذا ا ن اوا ٩٤ ( . اج إُ ار ا أ ، و  م ا؛  ا  ن اّوا ا ،  زٌ إَ زَ : أ  ّل : ))إم ة اذ ا ء ا   ا ب . ا  ، و ب ا ط  :    اق ا أ (()ا وط ن ء اق ا ))إذا ا ن اوا ٩٤ ( .     اج إُ ار ا أ ، و  م ا؛  ا  ن اّوا ا ،  زٌ إَ زَ : أ  ّل : ))إم ة اذ ا ء ا   ا ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .... ) ٤٧٣ ( (( )   ٍ وا ا وا ّم ؛ ٠٥ ل    أن . (  ، ار اُ أراد رّّم ة ؛ ا ان ة ، و  إمة دم   ). :ﺷﺮﻭﻁ ﺻﺤﺔ ﺍﻻﺳﺘﺜﻨﺎء ُُز ر ُاروا ١٥   اف ا)) ا ارنّ ا ( ؛ (()٢٥ أو  أم اء ،  ء ا ))  ان ا (.وا (()أ ٣٥ ل   ،(  ،  ٍا ار ا  ا إ ة إ   ٌا ء ق ا م ء ا ا   إ ة إ ّ  ل  اق ا اول وء ا ا  ا ا ء ا أي اا  . وط  و   وذم ار ا   إ ة إ ل م ا ا  وا ة ا  م اف ا   م ) ( ٤٥ ﻣﻮﺍﺿﻊ ﰲ ﺍﻻﺳﺘﺜﻨﺎء  واع آراءِ ا وا ف ا ء ا ا ه  َم  ء و  ا ا ءا : :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ  واع آراءِ ا وا ف ا ء ا ا ه  َم  ء و  ا ا ءا : :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ د ،  ا ن ان د ا ءِ اِ ن إ اذ  ام ن اّة .ا ون ا ة و ن إ : َل ْ أن :  د اذا ا ءُ اْَ .أ ل   اد ا  وا ن ا      وان ا إذاء ا ا  ّ : )) إما (() ةا ( ٥٥  د اذا ا ءُ اْَ .أ ل   اد ا  وا ن ا      وان ا إذاء ا ا  ّ : )) إما (() ةا ( ٥٥ ن ) ّد ا ا ء ا .ب ٦٥  :ل ا أ أن ( ، أي  .  امة دم : ل ي ا  ا ذ ذ . وإ  ، و : وا  ، و و و   : أل ا  ق)) ، ت ا  ا و : أ  ، و و : ل ا  ق ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ... ) ٥٧٣ ( ، ن ذ واذا وا  ظ أو   ء ا ق  م ؛ ا ا  و و ء ا ، و ا ّم ؛أ ق (()  و ا ( ٧٥ ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﻋﺪﺓ ﲨﻞ : :  ل M ! " # $ % & ' ( ) * + , - . / 0 1 2 3 4 5 6 7 8 9 : ; < = > ? @ A B C D E F G H I J K L M N O P Q R S T U V W X Y Z [ \ ] ^ _ ` L )٨٥ (. :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ  ب  )ء ا ( ف ا  ) ا ا ا ءا د  ْَ  ا  او ا ( ؟ و  ام   )  ( أو  ل،(د  ء ) ا ءا ا ؟ () ءف ا م :  M \ ] ^ _ ` a b c d e f g h i j k l m n o p q r s t u v w x y z { | L )٩٥ ل  ا ،( د ل   ء و ا   ا ا ءإنّ ا  ، ود ل   ء و ا ا ء ا ان،و ل ا إ ): ٠٦ ١ (.  اء إع ا ر  ظ. ا   ،  ون ةا : M ! " # $ % & ' ( ) * + , - . / 0 1 2 3 4 5 6 7 8 9 : ; < = > ? @ A B C D E F G H I J K L M N O P Q R S T U V W X Y Z [ \ ] ^ _ ` a b L ).١٦ (ً ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ... ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ٦٧٣ ( ٢ ) ا ء اع ا. ر ٢٦ : .( M K L M N O P Q R S T U V W X Y Z [ \ ] ^ _ ` a b c d e f g h i j k l m n o p q r s t u v w x y z { | } ~ L ٣٦ )ﱠ (. ، وذ ف ا      ا  اء و  إ أ ءار ا ج ا  ا اع وان ا اذا ان   ء را .ا إا  واس واا س ا ال ا  اا  وا اس ا ا واا )ا ( ٤٦ ٣ ل ا ،ول اء إع ا. ر ه ، و   ا )) : )(( ا  إ ٥٦ ن ول ا د   وا ، ( . نم ا ٤ ء ا . :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ أن :  ا M \ ] ^ _ ` a b c d e f g h i j k l m n o p q r s t u v w x y z { | L )٦٦  دةل ا  و ا (ء )ا( .  ل ا   دو    ل  ا ،  ا إ وا ا دق    او دة   ة   ا ن ا ؛   وام  اء إا  ره ا ة اء إدة ا  ، واذا ءن ان وا ن  ن أ و ق ا ُّمء ؛ ا  ا ا ا ء)٧٦ (  واآن ا ا دة ا ا ءد ا و  او ؛  وا اُ   أنّ ا إ ا ة ا  ا ا   و     ذ اب ، و ا ي ٌ أ  ّم ه ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ... ) ٧٧٣ ( ه ا : )) و ن  أ ط ا م ، و ام   او ة (( ) ا  ض  ٨٦  : )) وا ا ا طَ اّ ا( و ف ، ر ا ن م  أ ذ ، ول أ ا  ان ، ل   )((    أأ ٩٦ )   ا ذ( و ٠٧ ( ، وا دي ) ا ١٧ ) من ا ( ، وا ٢٧ ن ) (، وا ٣٧ ن   انّ ا ، ( )(( ل ا ا  ر ف ، و ه ا ل: ))و٤٧ ( .  وان أو أ ُ: )) إذا ذ ل ا  ء ، وا   ،   ن إ ن و   : ا ٌم   إن  ، ه ِُ ؛   ، ص  ؛ إذ ا   ُ ) ة ا آ ا  وا زل إ اء وث ا    ، و ( وِ)أ ، وم    (( ذ  )إ( إا ،   ن إذا ا   ذ ، إ َّ   و م  وا  ، وان أو ا  ن إن ء أ او  )(( ا ٥٧ ( . :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ ) ٨٧٣ (  ة وا   : ا ،  اد ام   ، اء او، وا (()م ن إا ٦٧ ( ل : ))واذا  ن  أّوأ  ت وا ءات ا ن   إء ران ا ،   ن إ ن و   : ا ،وا ن إ  ن ، وا  : ا ا ر ا ا إذات ، وا   ل وا وا ان ا ، ن   (ا  ا : ) ا )((تف ا آ ٧٧ . ( ) ا   ُ  ا أنطى و ٨٧ ( : ى  و ارادة ا ٌ د    ا    د ّ : أمأ :   انM \ ] ^ L ((ا  ا : )) إ  إ .  د ل  ، و د إ ، ام ا : ا ، ض وا ّ ا ؛ إن د ّ : أمما   ا  وا ، وإ   ، إ ، وزرت ا وا  داركء ، وا ا: ا .  ا إ ا ، وار ا  د ُ ء أو ن ا ، واذا داو ن ا : اذاا  ة وا   : ا ،  اد ام   ، اء او، وا (()م ن إا ٦٧ ( ل : ))واذا  ن  أّوأ  ت وا ءات ا ن   إء ران ا ،   ن إ ن و   : ا ،وا ن إ  ن ، وا  : ا ا ر ا ا إذات ، وا   ل وا وا ان ا ، ن   (ا  ا : ) ا )((تف ا آ ٧٧ . ( )((تف ا آ ٧٧ . ( ) ا   ُ  ا أنطى و ٨٧ ( : ى  و ارادة ا ٌ د    ا    د ّ : أمأ :   انM \ ] ^ L ((ا  ا : )) إ  إ . :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ ه ا : )) و ن  أ ط ا م ، و ام   او ة (( ) ا  ض  ٨٦  : )) وا ا ا طَ اّ ا( و ف ، ر ا ن م  أ ذ ، ول أ ا  ان ، ل   )((    أأ ٩٦ )   ا ذ( و ٠٧ ( ، وا دي ) ا ١٧ ) من ا ( ، وا ٢٧ ن ) (، وا ٣٧ ن   انّ ا ، ( )(( ل ا ا  ر ف ، و ه ا ل: ))و٤٧ ( .  وان أو أ ُ: )) إذا ذ ل ا  ء ، وا   ،   ن إ ن و   : ا ٌم   إن  ، ه ِُ ؛   ، ص  ؛ إذ ا   ُ ) ة ا آ ا  وا زل إ اء وث ا    ، و ( وِ)أ ، وم    (( ذ  )إ( إا ،   ن إذا ا   ذ ، إ َّ   و م  وا  ، وان أو ا  ن إن ء أ او  )(( ا ٥٧ ( . ل  ا ، و ا  ا ي وأ  وُ ا اّوأ ل ،  ا  وذ ا ع ا  ، ل ا ل ا إ إذ  ذ ا ا رك ، اح ا دي اء ا و ال : )) أن ء ا ا او ، إذا   ُ َف ا :    M \ ] ^ _ ` a b c d e f g h i j k l m n o p L   ة أنّ ا ا  ، ان   ا  ( ، أو أنّ ار ) ا (ون)   ا ،ل وا ا زع ا ب  ن ،   ة او اا    وال ا  ، ن ا و  ا ، و أو أ  م ة ا ام إن ، واات ا ا ا  وم ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ . :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ : ا ا و ا اه ا، وادمل وا ١. ا  ن ا، إذ ا ا   وا ا ا ء  دون دا ا و  اام .  ٢.  ر ا   ء ات ا د   ٌ  ن .  ود اا  ٢.  ر ا   ء ات ا د   ٌ  ن .  ود اا .٣ء ان ارس ا   ّوه ْ ، ءع ا    ّوه ين ا ا  ءب ا ا  و اا ء .ب اا .٣ء ان ارس ا   ّوه ْ ، ءع ا    ّوه ين ا ا  ءب ا ا  و اا ء .ب اا ٤.  ا ،  ا ا  واا  ا ا  ءا  ا ا دون د ا ا وا  ّوا رات وا ا ر ا . نه ا  و  ا تظ ٤.  ا ،  ا ا  واا  ا ا  ءا  ا ا دون د ا ا وا  ّوا رات وا ا ر ا . نه ا  و  ا تظ ٥.  ا ن ات ودر ا ءن ادرس ا اد . ا ار  ا    ق  او .٦ ال وع ا  ن ال ااط  ا  ا ء ،  ا   ، وُ ذ  ا ء ا   ام ، وارواوه واا ورس وااو ٥.  ا ن ات ودر ا ءن ادرس ا اد . ا ار  ا    ق  او .٦ ال وع ا  ن ال ااط  ا  ا ء ،  ا   ، وُ ذ  ا ء ا   ام ،  و ا رواوه واا و ر س    وا او ق .ا .٦ ال وع ا  ن ال ااط  ا  ا ء ، ا   ، وُ ذ  ا ء ا   ام ،  و ا رواوه واا و ر س    وا او ق .ا ٧. ا  واط ا أو إ ا اد  اد ا  ءا ، ا .  ذ   ك    اده ا ا وا :ﺍﻻﺳﺘﺜﻨﺎء ﻣﻦ ﺍﻟﻌﺪﺩ  د ل  ، و د إ ، ام ا : ا ، ض وا ّ ا ؛ إن د ّ : أمما   ا  وا ، وإ   ، إ ، وزرت ا وا  داركء ، وا ا: ا .  ا إ ا ، وار ا  د ُ ء أو ن ا ، واذا داو ن ا : اذاا ) ا   ُ  ا أنطى و ٨٧ ( : ى و ارادة ا ٌ د    ا    د ّ : أمأ :   انM \ ] ^ L ((ا  ا : )) إ  إ .  د ل  ، و د إ    ، ام ا : ا ، ض وا ّ ا ؛ إن د ّ : أمما   ا  وا ، وإ   ، إ ، وزرت ا وا  داركء ، وا ا: ا .  ا إ ا ، وار ا  د ُ ء أو ن ا ، واذا داو ن ا : اذاا   .  د : اا ا  ؛ إذ   ، وإن ا  ا : إن اا    اا . ل وا ن اذاّ، و ا  ءن ا إذا راءه ا ة ن و ن   و  و   : ا  ا ء د  ءا )   ا ء ا . ءْ اَ إ ٩٧ ( . ﺍﳋﺎﲤــﺔ  ا رب ا  ا وآ  ا ا ا ة واوا ن(.ل و  ) ءة وا ا ا ن اذاّ، و ا  ءن ا إذا راءه ا ة ن و ن   و  و   : ا  ا ء د  ءا )   ا ء ا . ءْ اَ إ ٩٧ ( .  ا رب ا  ا وآ  ا ا ا ة واوا ن(.ل و  ) ءة وا ا ا ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .... ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ٩٧٣ ( دم وأن ورد ٍ و ز   أن ل ا أ ا  : ا ا و ا اه ا، وادمل وا ١. ا  ن ا، إذ ا ا   وا ا ا ء  دون دا ا و  اام . Abstract Praise be to Allah, peace and blessings be the best all the people Mohammed Secretary and Haddat right and the leaders of all creatures and after: Looks at the knowledge of the language being one of the terms of the diligent worker with assets of Islamic jurisprudence to ete necessary ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ . ٠٨٣ ( to know the holy book of God, which is the first source of Islamic law, which touches the daily conduct practical for every Muslim such as prayer, fasting, transactions, etc., it is known that worships rely on verbal text and acts and statements and approve the infallible up to taxpayers by speech inaudible or illegible after the era of the infallible; and if so, must know the Arab speech styles; because the Koran was revealed. It is the manner of allocation methods and means some of what covered the speech with being compared with his output falls within the style allocation among other types, including its relevant wording of Commons, such as the condition and character and purpose exception including Mainvsal for the term of Commons such as mental and auditory, when a speech exception is devoted to the language, which will be the area of research, God Almighty. Find the three sections was taking exception to the first part, and idiomatically definition language when linguists and fundamentalists when it reported the views of some linguists and fundamentalists. The second section, entitled exception to the Staff stated inter exception clause exception vacuum, and the types of exception, the terms of the exception. The third Almiges was titled Placements in exception ensures the exception of the number and the exception of a few sentences, and finally was the finale, which contained the most important findings. Alla ask the Almighty to reconcile ١ ب ن ا (/ ٤١ ٥١١ ٢ : س ا( ا ٤١٢ /٢ ٣ ا ا (/٢ ٥٨١ ٤ ( ا ٨٣ ٥ ح ا (/ ١ ٤١٤ ٦ ح ا (/٢ ٢٨١ ٧ ن ( ا / ١ ٧٣١ ز ا اح ا ، و / ٣ ١٨٢ .  م. Abstract ا ٨ ( ا / ٢ ٩٧١ ١ ب ن ا (/ ٤١ ٥١١ ٢ : س ا( ا ٤١٢ /٢ ٣ ا ا (/٢ ٥٨١ ٤ ( ا ٨٣ ٥ ح ا (/ ١ ٤١٤ ٦ ح ا (/٢ ٢٨١ ٧ ن ( ا / ١ ٧٣١ ز ا اح ا ، و / ٣ ١٨٢ .  م. ا ٨ ( ا / ٢ ٩٧١ ) ١٨٣ ( ٩ د ر وا ا : ( ام ٦٢ / ٣ ٠١   ( ا ١٤٥ /٢ ١١ د ر وا( ا / ٣ ٧٢١ ٢١ ان/  ( ال ٤٤١ ٣١ ا /ءم( ا ٣ ٤١  ا ر: ا( ام ١/ ٢٤٥ ل / رج ا :، وام ٣٤ ديء :،وام ل / ا ل إا ٨٣١ . ٥١ ذا ( ا . ٥٨ /٢ ٦١ ل ( ا ٠٣ /٣ ٧١ ل  ا( ام ١٣ /٣ . ا ، . ٨١ ر ،  ا: ا( ام ٣/ ٩٧٢ ٩١ ل ا أ ة: ا( ام ٢/ ٣٧٦ ( ٠٢ /ا ٠٣١٣ ١٢ / ا( ا ٥٢ ٦٢ ء/  ا. و ٢٩ ، ٩٢ ، ٧٥١ ٢٢ اء/( ا ٧٧ ٣٢ / ا ء( ا ٧٥١ ٤٢ د/ (٣٤ ٥٢ ص ا م :ح د . م اان ا( د . ٣٣ ٦٢ ل  ا ل ا :( ام ١١٢ . ٧٢ ازي  ل: ا( ام ١/ ٨٠٤ اح ا، و ٣/ ٦٨٢ ٨٢    . ي ا ا ا ، . د ا يء ا( ا د داب ، ا ٨٢ ٦٠٠٢ .  ٩٢ . ر  ا : ا( ام / ٣ ٩٧٢ ٠٣ جا ا ، . د ام : ( ام ٢ ص ٤٨٦  ا . اد  ، ٧٨٩١ ر   .وم بره ا ر ا ا و/ ١ ٣٦٢ . ١٣  ( ا / ٢ . ٧٨ ٢٣ ا ا . .د ام :  ام (/ ٢ ٧٨٦  ٧٢ ازي  ل: ا( ام ١/ ٨٠٤ اح ا، و ٣/ ٦٨٢ ٨٢    . ي ا ا ا ، . د ا يء ا( ا د داب ، ا ٨٢ ٦٠٠٢ .  ٩٢ . ر  ا : ا( ام / ٣ ٩٧٢ ٠٣ جا ا ، . د ام : ( ام ٢ ص ٤٨٦  ا . Abstract اد  ، ٧٨٩١ ر   .وم بره ا ر ا ا و/ ١ ٣٦٢ . ٢٣ ا ا . .د ام :  ام (/ ٢ ٧٨٦ ) ٢٨٣ ( ٣٣  ا :( ام ٠٧ / ١١ ٤٣  ا / ة( ا ٧٨١ ٥٣ ل : اام : ام / ١ ٣٨٣ ٦٣ ي  . ل ا : م( ام / ٢ ٠١٤ ٧٣( م ة /  ا : زمس : ا  ا٣٦١ ل  ا : م، و ٢/ ، ٧١١ ن وا / ١ ٥٨٣ :  وا، وا / ١ ٤٠٣ ل :د ا، وار ٨٤١ ٨٣ ى ا ا  ا( أ ٨٤ / ٠١ ه   ي ا، وا / ٥١ ١٥١ ّ. و ء  ا ا :  /٦ ٥٨٣ . ٩٣ وا ا:  (/٤ ٢٨١ . ٠٤  ا ( ا ٤٢ . ١٤ ي  ا (٥١ / ٥٨٢ . ٢٤ آواب . ص/  ام ان م:   ب ، ءم ادب ا .ري ا ( /٦ ، ٠٣ ٦٦٥ ) ٤٢٤٣ ب  ، ورن واب ا (ويح ا)  (. وام ء :ا ١١ / ٤٢١ ) ٥٦٢٤ اة . ا  ( ٣٤ ري(  ) ار ا( ا ٦/ ٠٧٥ / ،/ ١١ ٢٢١ ٤٤ ا  . اظ ا وظ ا : رو( ام / ٢ ٧٤٧ ٥٤ ي  ( ا ١/ ٦٩٤ د ااط: ، وام ٩٤٣ ا ٢/ ١٨١ ل وا ٠٣ /٣ ٦٤  . ن( ا / ١ ٨٣١ ٩٣١ ٧٤ ح ( ة ا وة ا/١ ٩٧٣ ٨٤  ل ا أ ا( ا / ٣ ٧٧٢ ٨٧٢ /ارة ا   ا،وا ٥٢ ٦٢ ٩٤ ن( ا ١/ ٣٤١ا  اوام ١٠٥ /٢ ي وا ٩٨٣ ٠٥ . ا ا . . د : اج ا :  ا ل( ا ١/ ٧٩٢ ١٥  : ا( ام ٢/ ٣٨١ ٢٥ م  ، ل ات أ  ( . ٨٧ ٣٥ .م ا . ا اا   إ ا (٩٦ / ١١ ٤٥ ،  : ا( ام ٣٧ ٥٥ ه  وو جح ا ج( ا ٢/ ٩٤١ ٣٣  ا :( ام ٠٧ / ١١ ٤٣  ا / ة( ا ٧٨١ ٥٣ ل : اام : ام / ١ ٣٨٣ ٦٣ ي  . Abstract ل ا : م( ام / ٢ ٠١٤ ٧٣( م ة /  ا : زمس : ا  ا٣٦١ ل  ا : م، و ٢/ ، ٧١١ ن وا / ١ ٥٨٣ :  وا، وا / ١ ٤٠٣ ل :د ا، وار ٨٤١ ٨٣ ى ا ا  ا( أ ٨٤ / ٠١ ه   ي ا، وا / ٥١ ١٥١ ّ. و ء  ا ا :  /٦ ٥٨٣ . ٩٣ وا ا:  (/٤ ٢٨١ . ١٤ ي  ا (٥١ / ٥٨٢ . ٢٤ آواب . ص/  ام ان م:   ب ، ءم ادب ا .ري ا ( /٦ ، ٠٣ ٦٦٥ ) ٤٢٤٣ ب  ، ورن واب ا (ويح ا)  (. وام ء :ا ١١ / ٤٢١ ) ٥٦٢٤ اة . ا  ( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ......................................................... ....... ) ٣٨٣ ( ) ٣٨٣ ( ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ......................................................... ....... ) ٣٨٣ ( ٦٥ ي  : ا( ام ٠٨٣ ٧٥   ( ا / ٢ ٩٤٥ ٨٥ ة / ( ا ٩٤٢ ٩٥ ر /( ا ٤٥ ٠٦ . ا ر  :ال ا :أ( ام ١/ ٢١٢ ١٦ ء/( ا ٢٩ ٢٦ ل ا ا . ام ، وا ا ا ذ ا( ذ ١/ ٢١٢ ح  :.وام  اا ٣/ ٣١٣ ٣٦ ة /( ا ٣٣- ٤٣ ٤٦ : ا( ام ١/ ٦٤٢ ٥٦ ا( ا ) و ا ام ٧٨٨٥ ( . ٠٩ /٦ ٢٥ ر/ ( ا ٤ ٥ ٦٦ ر /( ا ٤ ٧٦ ا:( ام ٧٨ / ١١ : ااو ٨٨ ٣٢٣ روس ا. ا ١٤٧ /٣ ، .ا ٠١ /٣ ا، ا ٤٠٥ /٢ ٨٦ . ا ا (/ ٢ ٦٩١ ب.َف ا ار  ا  ا. و ٩٦ ، ( ا / ٢ ٦٩١ ٠٧ . ح ا (/ ٢ ٤١٢ ١٧ ح ا (/٢ ٢٥١ ٢٧ َب ف ا( ار / ٣ ١٢٥١ ٣٧ ن / ا (/ ٣ ٦٢٢ ٤٧ ب / َف ا( ار ٣ / ٢٢٥١ ٥٧ ح ا (٤١٢ /٢ /وا ٣ ة .رة ا  ٦٧ . ح ا (/٢ ١٥١  ا ٤ ررة ا  ٧٧ َب َف ا( ار ٣/ ١٢٥١ ، و ا ٤و٥ ررة ا  ٨٧ . Abstract ا ا (/ ٢ ٦٩١ ٧٩١ ٩٧ ن  ا : ( ام / ٢ ٦٢٢ ٧٢٢ ٦٥ ي  : ا( ام ٠٨٣ ٧٥   ( ا / ٢ ٩٤٥ ٨٥ ة / ( ا ٩٤٢ ٩٥ ر /( ا ٤٥ ٠٦ . ا ر  :ال ا :أ( ام ١/ ٢١٢ ١٦ ء/( ا ٢٩ ٢٦ ل ا ا . ام ، وا ا ا ذ ا( ذ ١/ ٢١٢ ح  :.وام  اا ٣/ ٣١٣ ٣٦ ة /( ا ٣٣- ٤٣ ٤٦ : ا( ام ١/ ٦٤٢ ٥٦ ا( ا ) و ا ام ٧٨٨٥ ( . ٠٩ /٦ ٢٥ ر/ ( ا ٤ ٥ ٦٦ ر /( ا ٤ ٧٦ ا:( ام ٧٨ / ١١ : ااو ٨٨ ٣٢٣ روس ا. ا ١٤٧ /٣ ، .ا ٠١ /٣ ا، ا ٤٠٥ /٢ ٨٦ . ا ا (/ ٢ ٦٩١ ب.َف ا ار  ا  ا. و ٩٦ ، ( ا / ٢ ٦٩١ ٠٧ . ح ا (/ ٢ ٤١٢ ١٧ ح ا (/٢ ٢٥١ ٢٧ َب ف ا( ار / ٣ ١٢٥١ ٣٧ ن / ا (/ ٣ ٦٢٢ ٤٧ ب / َف ا( ار ٣ / ٢٢٥١ ٥٧ ح ا (٤١٢ /٢ /وا ٣ ة .رة ا  ٦٧ . ح ا (/٢ ١٥١  ا ٤ ررة ا  ٧٧ َب َف ا( ار ٣/ ١٢٥١ ، و ا ٤و٥ ررة ا  ٨٧ . ا ا (/ ٢ ٦٩١ ٧٩١ ٩٧ ن  ا : ( ام / ٢ ٦٢٢ ٧٢٢ ٦٥ ي  : ا( ام ٠٨٣ ٧٥   ( ا / ٢ ٩٤٥ ٨٥ ة / ( ا ٩٤٢ ٩٥ ر /( ا ٤٥ ٠٦ . ا ر  :ال ا :أ( ام ١/ ٢١٢ ١٦ ء/( ا ٢٩ ٢٦ ل ا ا . ام ، وا ا ا ذ ا( ذ ١/ ٢١٢ ح  :.وام  اا ٣/ ٣١٣ ٣٦ ة /( ا ٣٣- ٤٣ ٤٦ : ا( ام ١/ ٦٤٢ ٥٦ ا( ا ) و ا ام ٧٨٨٥ ( . ٠٩ /٦ ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ .. ) ٤٨٣ ( ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ ﻊ ١. .آن اا ٢. وي )ت  ل ا ل إج ا  جح ا  جا ٥٨٦ (   ا    )تا ٦٥٧ )ت ج ا ه(وو ١٧٧. وت .  اء .دار ا ا  : . (. ١ ٤٨٩١ .  ٣. )ت    أ  . ل ا أ ا ٦٥٤ . دار  : أ( .م  وت . ةق اا . ١ ٠٨٩١ . ٣. )ت    أ  . ل ا أ ا ٦٥٤ . دار  : أ( .م  وت . ةق اا . ١ ٠٨٩١ . ٤.  ا ل ا أ ا ي) ا  ١٣٦ زاق  ا : .(   ض ودار ا . ا . دار ا١ وت  .٣٠٠٢ . ٤.  ا ل ا أ ا ي) ا  ١٣٦ زاق ا : .(   ض ودار ا . ا . دار ا١ وت  .٣٠٠٢ . ٥.    . ي ا أ ا.. د ا يء اا دداب . ا ٨٢٦٠٠٢ . ٦. )ت ام ا أ ن (و ا) اوا ٠٦٣ ( ض  رق: ط .( ة )د.ت . ا . دار اا ٦. )ت ام ا أ ن (و ا) اوا ٠٦٣ ( ض  رق: ط .( ة )د.ت . ا . دار اا ٧. اج( )ت  ا ) ا وفي اي ا ا     ا لا ٦١٣ ن د.ت. . وت .  ا .  ا ا ، : د. ( ٧. اج( )ت  ا ) ا وفي اي ا ا     ا لا ٦١٣ ن د.ت. . وت .  ا .  ا ا ، : د. ( ٨.  ،من ا  ،بن ا  َبَف اإر .  ن : ر .  ا، ةا ٨٩٩١ . ٩. )ت م ا   ،ل ا   ا ل إد اإر ٥٥٢١ (.   وت  ق دار ا   :. ١ ٠٠٠٢ . ٩. )ت م ا   ،ل ا   ا ل إد اإر ٥٥٢١ (.   وت  ق دار ا   :. ١ ٠٠٠٢ . . ٠١ )ت ا ر  .ال اا ٨٨٣١ وت  .رف( دار ا٥٢٤١ . . ١١ اا ) ر ار ا ل ا أ ٤٩٧ . ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ ﻊ  ا  :.( ف اووزارة ا ٢٩٩١ . . ٢١ ي )ت  ا ا  ء ا . ل ال ا٢٥٥ : د . . (  ة اث .دار ا ا ز. ١ ٢٩٩١ .  ﺍﻻﺳﺘﺜﻨﺎء ﻋﻨﺪ ﺍﻟﻠﻐﻮﻳﲔ ﻭﺍﻷﺻﻮﻟﻴﲔ ..... ) ٥٨٣ ( . ٤١ )ت  ا ا   ا .ل ا أ نا ٨٧٤ ا.:د( م   . ء . دار اا . ٤ ٧٩٩١ . . ٥١  ا  اق ا ا . ل ا أ ةا ازي)ت دي اوز ٦٧٤  .د . دار ا   : (. ١ ٣٨٩١ . . ٥١  ا  اق ا ا . ل ا أ ةا ازي)ت دي اوز ٦٧٤  .د . دار ا   : (. ١ ٣٨٩١ .   . ٦١ ي )ت  ا آن (. أ آي ا ن ا)ي ا٠١٣   . (  وت .اث اء ا . دار ا د ١ وت .  .١٠٠٢ .  . ٦١ ي )ت  ا آن (. أ آي ا ن ا)ي ا٠١٣   . (  وت .اث اء ا . دار ا د ١ وت .  .١٠٠٢ . . ٧١ ا  د . ار واا )ت م ا٣٠٤   ا . :د ( وت .  ا .  زم أ٨٩٩١ . . ٨١  اح ا .  وا ا ا ل، ا ا  واا ج)ت ا ٩٧٨ )ت  ا ا ا  (١٦٨  وت / ( . دار ا. ١ ٦٩٩١ .   ا . ٩١ ي) ا ا. ل ا وع ا  ٢٧٧  : د.( .( )د. تا ا . ٠٢ ) ت ذاما أ ظ ، ل ا أ ا ٠١٥  أ .: د ( .  ا .ا ٢ وت ..٠٠٠٢ . . ٠٢ ) ت ذاما أ ظ ، ل ا أ ا ٠١٥  أ .: د ( .  ا .ا ٢ وت ..٠٠٠٢ . . ١٢ ا ا . اا اج )ت١٨٤ . دري ا: إ. (، ١ ١١٤١ . .ان . ا . ر ا .  اا . ٢٢ ن )ت  ا . مح ا  ن ا٦٠٢١  د : ( ة ، ا ا . ا ٢٠٠٢ . . ٣٢ ل ا  . ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ ﻊ  ا  روس اا ول(  ا )ا ا  )ت ٦٨٧ . ااث وا .  ا  ا ا. ( . ٣٢ ل ا  .  ا  روس اا ول(  ا )ا ا  )ت ٦٨٧ . ااث وا .  ا  ا ا. (  . ٤٢  ار .دار ا  : ، م اان اد ١ وت .  .٣٩٩١ . . ٤٢  ار .دار ا  : ، م اان اد ١ وت .  .٣٩٩١ . . ٥٢ : أ.  ا ا ا   ا .ال ا أ إرا ه  ، دار م اا ٨٤٣١ . . ٥٢ : أ.  ا ا ا   ا .ال ا أ إرا ه  ، دار م اا ٨٤٣١ .  . ٦٢ )ت  ا ا  ا. ل ا أظ ا وظ ارو ٠٢٦ :  (  وت،  . دار ا ا ا ،د . ٦ ٨٩٩١ .  ) ٦٨٣ ( . ٧٢ ر    ا . وي ا وي( ااب ا )اا اد )ت  ار ٨٩٥ ان .  . ا . ر ا .  ا ا. ( . ٨٢ )ت  ا   ا ى. أ اا ٨٥٤ در ا  : . ( . ن . وت .  ا. دار ا ٣ . ٣٠٠٢ . ٩٢ )ت ن ا    ا .ء ا ا .٨٤٧  (. ١ ٣١٤١ .  ن ، وت .  ا . ٩٢ )ت ن ا    ا .ء ا ا .٨٤٧  (. ١ ٣١٤١ .  ن ، وت .  ا . ٠٣ وت ، . ا رق وط در ا  :.  . ح ا ،  ان: دار ا١٠٠٢.  . ١ . ٠٣ وت ، . ا رق وط در ا  :.  . ح ا ،  ان: دار ا١٠٠٢.  . ١ . ١٣ )ت    ل ا، ة ا وة ا ح٢٧٦ ن م : .( ورين ا ا . اد . م ا .٨٧٩١ . . ١٣ )ت    ل ا، ة ا وة ا ح٢٧٦ ن م : .( ورين ا ا . اد . م ا .٨٧٩١ . . ٢٣ دي )ت ا ا ا   ا . ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ ﻊ  ا ا ح٦٨٦ : د  (   اب ، دار ا  أ ١ وت  ،٨٩٩١ . . ٣٣ ر )ت ا وف ا ا أ  . اح ا٢٧٩ د(. ا ،  ا ى ،و ا ا .٧٨٩١ . . ٤٣ )ت    ا ،ح ا٦٤٦  ، دار ا  ا: أ.(  ا . ١ ٣١٠٢ .  د . ٥٣ ر ا ا ح   ي)ت ٦٧٦ . ن . دار ا  : (  وت، ٥١ ٨٠٠٢ . . ٦٣ )ت  ا ا  . ل ا ا ةا ٠٦٤  ر:. ( . ريما ١ ان  .ا . رة .٧١٤١ . . ٦٣ )ت  ا ا  . ل ا ا ةا ٠٦٤  ر:. ( . ريما ١ ان  .ا . رة .٧١٤١ . . ٧٣ )ت م ا  ري ا ح ري ا٢٥٨  ا :،(    ا. دار ا ا اد ز و، ١ ٩٨٩١ . . ٨٣ )ت م ا ا . (د)ار وا ا٣٠٤  ا : ( .  ز ا  ١  ا٨٩٩١ . ٨٣ )ت م ا ا . (د)ار وا ا٣٠٤  ا : ( .  ز ا  ١  ا٨٩٩١ . ٩٣ دي )ت وز آ ا ا. س اا ٧١٨  . وت .  ا ( ٨ ، ٥٠٠٢ . . ٩٣ دي )ت وز آ ا ا. س اا ٧١٨  . وت .  ا ( ٨ ، ٥٠٠٢ . ) ٧٨٣ ( . ٠٤ )ت ما  ر . ل ا د ااط٩٨٤   : . ( وت .  ا . دار اا ٧٩٩١ .  . ٠٤ )ت ما  ر . ل ا د ااط٩٨٤   : . ( وت .  ا . دار اا ٧٩٩١ .  . ١٤ )ت زا ا . ل اام٠٣٢١ ان . . ط ا ا( . ا ٨٧٣١ .  . ٢٤ ر) ت  ل ا ،بن ا١١٧ ن وت. در (. دار٥٥٩١ . . ٢٤ ر) ت  ل ا ،بن ا١١٧ ن وت. در (. دار٥٥٩١ . . ٣٤ ازي)ت  ا    ا ،إل ا أ ا ٦٨٤ .  ا( ا . ةا ٧٥٩١ .  . ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ ﻊ ٣٤ ازي)ت  ا    ا ،إل ا أ ا ٦٨٤ .  ا( ا . ةا ٧٥٩١ .  . ٤٤ ) ا ال ا،ل ا ل ادئ ا٦٢٧  ا: ،( ، لا ٣  ا، ا ٤٠٤١ . ٤٤ ) ا ال ا،ل ا ل ادئ ا٦٢٧  ا: ،( ، لا ٣  ا، ا ٤٠٤١ . ٥٤ ا  ا )ت  ا ا    ا . ا٠٦٤  : ( . ن . وت . ت ي ا.  ا. ١ ٢٩٩١ . . ٥٤ ا  ا )ت  ا ا    ا . ا٠٦٤  : ( . ن . وت . ت ي ا.  ا. ١ ٢٩٩١ . . ٦٤ )ت  ا  ر ا م ا . ا ا ووا ا٧٠٨ : (   ا ، دار ا در ا ا . ١ ١٠٠٢ . ٧٤  ل ا .  ا لل وا آل ا ل إرج ا . دي اظ  : .  ا ١ . اء ا ا. . ٨٤ . ا . اد  ،ا ا ..د ام٧٨٩١ .. . ٩٤ )ت ي ا ا  ا . ل ا أ ا ٦٣٤  : (  وت  ا، دار اا . ١ ٣٨٩١ .  . ٠٥ ،  ا دار ا م   .، دل ات أ ١ وت  ، ود ٠٠٠٢ . . ٠٥ ،  ا دار ا م   .، دل ات أ ١ وت  ، ود ٠٠٠٢ . . ١٥ ازي  ا ا .ل ا أ  لا ) ٦٠٦  .ام ا: د.ط. (، ٢ وت ،  ا٢١٤١ . . ١٥ ازي  ا ا .ل ا أ  لا ) ٦٠٦  .ام ا: د.ط. (، ٢ وت ،  ا٢١٤١ . . ٢٥  ) ا ا   ا .ز اا ى إ ا  رف ا ا:.(ما . ١ ٧١٤١ ان .ارا.   . ٣٥ ) ا ا . ل ا  ا ٥٠٥ :  ( . ن ا   وت/  ا، ١ ٧٩٩١ .   . ٣٥ ) ا ا . ل ا  ا ٥٠٥ :  ( . ن ا   وت/  ا، ١ ٧٩٩١ . ) ٨٨٣ ( . ٤٥ وي )ت  ا ال . ﻗﺎﺋﻤﺔ ﺍﳌﺼﺎﺩﺭ ﻭﺍﳌﺮﺍﺟ ﻊ م ا ل إج ا ح  ل ام ٥٨٦ ي)ت  ا ا ل ا . (٢٧٧ .  ا ن . :د. (  وت.  دار ا ٩٩٩١,١ .  . ٥٥  ح  ا ا )تط ا ا  . ا ا١١٩ (  ، ا : ة، ا  ل ا:،٠٠٠٢ . 
https://openalex.org/W2118541630
https://zenodo.org/record/1091/files/Manuscript-Sinoxygen-2012-004.pdf
English
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A Versatile Synthesis of Meyers’ Bicyclic Lactams from Furans: Singlet‐Oxygen‐Initiated Reaction Cascade
Angewandte Chemie
2,012
cc-by
3,850
A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts with furan substrates to initiate a remarkable cascade reaction sequence** Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* Dedicated to Professor K. C. Nicolaou range from harnessing microwave energy to accomplish the desired reaction[6a] or employing Lewis acid catalysis,[6b] to activating the acid.[6c] A second commonly employed,[7] but stepwise, route to these bicyclic lactams, also originally emanating from the Meyers’ group,[2a] relies heavily on N-acyliminium chemistry extensively elucidated by Speckamp.[8] In this case, a succinimide intermediate is substituted at the C5 position (usually by addition of a Grignard reagent) and then encouraged to undergo intramolecular cyclisation under acidic conditions. Several domino reactions affording specific bicyclic lactams have also been reported recently,[9] as well as, other stepwise approaches to the scaffold.[10] This unceasing interest in finding new syntheses for the Meyer’s bicyclic lactams in itself provides testament to the usefulness of the scaffold and the diversity of potential applications there are for it. Herein, we introduce a new and mild method for the synthesis of a wide variety of Meyers’ bicyclic lactams. This entirely novel approach, which uses a singlet oxygen-mediated cascade reaction sequence, is made particularly powerful because the one-pot reaction begins from furan substrates which can be multiply-substituted (A, Scheme 1) at will and with ease, thus allowing access to highly substituted scaffolds, frequently with excellent stereoselectivity, as products from the initial reaction without extra manipulations. In this way, the new methodology also exhibits a very high degree of step-[11] and atom-economy[12] and utilises the selective green reagent, singlet oxygen, to achieve these very rapid increases in molecular complexity with precision and minimum waste, thereby attaining many of the recently established criteria for an ideal synthesis.[13] A new and powerful method for the synthesis Meyers’ bicyclic lactams beginning from furans is reported herein that uses a remarkable one-pot singlet oxygen-initiated cascade reaction sequence to deliver the corresponding bicyclic lactams in high yield. Ever since they were pioneered Meyers’ homochiral bicyclic lactams[1, 2] (B, Scheme 1) have been exceedingly popular and versatile scaffolds for the enantioselective construction of new stereogenic centres, including the most challenging type - quaternary carbon centres. DOI: 10.1002/anie.200((will be filled in by the editorial staff)) DOI: 10.1002/anie.200((will be filled in by the editorial staff)) (Powerful new method for synthesizing bicyclic lactams ) [] The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007- 2013)/ERC grant agreement no. 277588. We also thank Prof. Robert Stockman and Mr. George Procopiou for their help in taking HRMS. [] Prof. Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou Department of Chemistry University of Crete Vasilika Vouton, 71003, Iraklion Crete (Greece) Fax: +30 2810 545166 E-mail: vasil@chemistry.uoc.gr A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts with furan substrates to initiate a remarkable cascade reaction sequence** Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* They have been utilised in myriad different ways to target a wide variety of natural products,[2, 3] non- natural molecules possessing interesting biological activity,[2, 4] and, also in a diverse array of other synthetic endeavours.[2, 5] The most general method for their synthesis, introduced in the seminal work of Meyers’,[1, 2] wherein a γ-ketoacid is condensed with an aminoalcohol under dehydrating conditions in refluxing toluene, is still, by far, the most commonly employed means of accessing the bicyclic scaffold B. Modifications have been made with the aim of introducing milder variants[6] to the Meyers’ lactamisation; these Scheme 1. Generalised representation of the transformation achieved with this new methodology. Scheme 1. Generalised representation of the transformation achieved with this new methodology. The idea for this new methodology was born from our experience in the field of furan photooxygenations[14] which has taught us to regard the furan motif as a readily accessible and easy to manipulate, 1,4-dienone equivalent (or precursor).[15] Once this fact was acknowledged, it seemed potentially very interesting to ask whether the aforementioned intermediate (C, Scheme 2) could be intercepted by an amino-alcohol, and, from thence, rearrange and cyclise (under acid catalysis) to afford the Meyer’s bicyclic lactams, without resorting to the dehydrating conditions, or high temperatures, which have traditionally been employed. The highly ambitious concept is summarised, in mechanistic terms, in Scheme 2 (note: only selected steps are shown here). Thus, when a furan is oxidised, upon exposure to singlet oxygen in MeOH, intermediates of type C are known to form easily after the in situ reduction (with Me2S) of a hydroperoxy functionality that remains after the solvent induced collapse of the initially formed endoperoxide adduct has occurred.[14,15] We hypothesised that introduction of an 1,2- aminoalcohol into the flask at this stage should regioselectively afford the aminal D (via the more stable, more highly substituted, [] Prof. Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou Department of Chemistry University of Crete Vasilika Vouton, 71003, Iraklion Crete (Greece) Fax: +30 2810 545166 E-mail: vasil@chemistry.uoc.gr [] The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007- 2013)/ERC grant agreement no. 277588. We also thank Prof. Robert Stockman and Mr. George Procopiou for their help in taking HRMS. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. [] Prof. A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts with furan substrates to initiate a remarkable cascade reaction sequence** Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou Department of Chemistry University of Crete Vasilika Vouton, 71003, Iraklion Crete (Greece) Fax: +30 2810 545166 E-mail: vasil@chemistry.uoc.gr [] Prof. Dr. G. Vassilikogiannakis, Dr. D. Kalaitzakis, Dr. T. Montagnon, Ms. I. Alexopoulou Department of Chemistry University of Crete Vasilika Vouton, 71003, Iraklion Crete (Greece) Fax: +30 2810 545166 E-mail: vasil@chemistry.uoc.gr [] The research leading to these results has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007- 2013)/ERC grant agreement no. 277588. We also thank Prof. Robert Stockman and Mr. George Procopiou for their help in taking HRMS. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. 1 Table 1. One-pot synthesis of various bicyclic lactams starting from furan substrates. Scheme 2. Mechanistic summary for the one-pot cascade reaction sequence that transforms furans A into bicyclic lactams B. Scheme 2. Mechanistic summary for the one-pot cascade reaction sequence that transforms furans A into bicyclic lactams B. oxonium cation) that would open to imino enal E, as shown in Scheme 2, and then close again to afford 2-pyrrolidinone G, via 2H- pyrrol-2-ol F. It was hoped that addition of an acid at this point, would affect the desired hydroxy-acyl imminium cyclisation to give the corresponding bicyclic lactam (H  B) as the final product of a highly ambitious one-pot cascade reaction sequence. Gratifyingly, and, despite the extraordinary complexity of the designed reaction cascade, we were able to successfully transfer the concept from a hypothetical “on paper” idea to a working laboratory protocol, as the results detailed in Table 1 clearly show. Thus, in the simplest case where 2-methylfuran was oxidised with singlet oxygen,[16] followed by in situ reduction (Me2S) of the resultant peroxide, and then by the addition of 2-aminoethanol, and later catalytic TFA (30 mol%), to the same flask, the desired corresponding Meyers’ bicyclic lactam was obtained (Entry 1, Table 1). With this proof of principle in hand, we set out to systematically investigate the effect of varying both the furan and 1,2-aminoalcohol substitution patterns, and, concomitantly, delineate the precise stereochemical outcomes of the cascade reaction sequence (Table 1). Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones De Clercq, R. Bouillon, A. Verstuyf, Bioorg. Med. Chem. Lett. 2004, 14, 3885-3888; d) T. D. Aicher, B. Balkan, P. A. Bell, L. J. Brand, S. H. Cheon, R. O. Deems, J. B. Fell, W. S. Fillers, J. D Fraser, J. Gao, D. C. Knorr, G. G. Kahle, C. L. Leone, J. Nadelson, R. Simpson, H. C. Smith, J. Med. Chem. 1998, 41, 4556-4566; e) M. D. Ennis, R. L. Hoffman, N. B. Ghazal, D. W. Old, P. A. Mooney, J. Org. Chem. 1996, 61, 5813-5817; f) A. I. Meyers, L. Snyder, J. Org. Chem. 1993, 58, 36-42. l d i l ) S S i [5] y , y , g , , For selected representative examples, see: a) S. Sen, V. R. Potti, R. Surakanti, Y. L. N. Murthy, R. Pallepogu, Org. Biomol. Chem. 2011, 9, 358-360; b) J. E. Resek, J. Org. Chem. 2008, 73, 9792-9794; c) D. Enders, O. Niemeier, T. Balensiefer, Angew. Chem. 2006, 118, 1491- 1495; Angew. Chem. Int. Ed. 2006, 45, 1463-1467; d) H. C. Shen, F.- X. Ding, S. L. Colletti, Org. Lett. 2006, 8, 1447-1450; e) T. E. Nielsen, S. Le Quement, M. Meldal, Org. Lett. 2005, 7, 3601-3604; f) M. D. Groaning, G. P. Brengel, A. I. Meyers, Tetrahedron 2001, 57, 2635- 2642; g) J. A. Nieman, M. D. Ennis, Org. Lett. 2000, 2, 1395-1397. In the case of Entry 8, the two stereoisomeric final products could be separated by column chromatography, but subsequently proved to be highly susceptible to racemisation (even on brief standing in CDCl3 solution) via opening up of the bicyclic lactam to the 2-pyrrolidinone (analogous to intermediate G, Scheme 2) and subsequent reclosure. Finally, and again related to intermediate G; when 2-benzylfuran was photooxidised and reacted with 2- aminoethanol, the yield was a mere 30% (Entry 2, Table 1) and the product recovered was in equilibrium with an open form;[20] however, when 2-aminoethanol is replaced with either S-serine ethyl ester (Entry 4, Table 1), or R-phenylglycinol (Entry 6, Table 1), the product yield improves dramatically to 59 and 58%, respectively, presumably due to the Thorpe-Ingold angle compression effect favouring ring closure. 2642; g) J. A. Nieman, M. D. Ennis, Org. Lett. 2000, 2, 1395-13 [6] g [6] a) M. Jida, R. Deprez-Poulain, S. Malaquin, P. Roussel, F. Agbossou- Niedercorn, B. Deprez, G. Laconde, Green Chem. 2010, 12, 961-964; b) A. N. Cayley, R. J. Cox, C. Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones the final bicyclic lactam products with no significant stereoselectivity (dr = 55:45, R1/R3 anti: R1/R3 syn, Entry 7; dr = 55:45, R1/R2 syn: R1/R2 anti, Entry 8), except in the case of menthofuran (Entry 9) which is unique in its fused bicyclic nature. [1] For first report, see: A. I. Meyers, M. Harre, R. Garland, J. Am. Chem. Soc. 1984, 106, 1146-1148. [2] For reviews of the field, see: a) D. Romo, A. I. Meyers, Tetrahedron 1991, 47, 9503-9569; b) M. D. Groaning, A. I. Meyers, Tetrahedron 2000, 56, 9843-9873. The following observations are also worthy of note. It has been reported in the literature[19] that the product of the reaction shown in Entry 1, readily opens and reacts intermolecularly to form the centrosymmetric dimer under aqueous acidic conditions (Scheme 3). We found this to be true (indeed, it also applied to other more heavily substituted products, although to a lesser extent), but we also found that this undesired dimerisation could be avoided by moderating the amount of TFA employed in the cyclisation step, and, in the most vulnerable cases by neutralising this TFA with NEt3 prior to concentration of the sample. In general, TFA 20 mol% was initially added and further additions (up to a maximum of 60 mol%) were only made when deemed necessary according to tlc analysis of the reaction mixture. The TFA cyclisation step took between 1 to 9 hours to reach completion. This tendency to dimerise may be the reason that, somewhat counter intuitively, it would appear that the bulkier the R1 ring junction substitution is the higher the yields obtained are (Entries 7 and 9). Likewise, it has been reported in other methods[10a] that mechanistically converge with ours by going through intermediates of type G (Scheme 2) that these intermediates were fleeting and could not be isolated and characterised; however, though they cannot be described as stable, we have been able to isolate and take 1H and 13C NMR spectra of some representative intermediates (see Supporting Info.). , , [3] For recent representative examples, see: a) M. E. Jung, J. J. Chang, Org. Lett. 2010, 12, 2962-2965; b) M. Saito, S. Kuwahara, Biosci. Biotechnol. Biochem. 2005, 69, 374-381; c) D. L. J. Clive, J. Wang, J. Org. Chem. 2004, 69, 2773-2784; d) R. C. Hughes, C. A. Dvorak, A. Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones I. Meyers, J. Org. Chem. 2001, 66, 5545-5551; e) M. W. Carson, G. Kim, M. F. Hentemann, D. Trauner, S. J. Danishefsky, Angew. Chem. 2001, 113, 4582-4584; Angew. Chem. Int. Ed. 2001, 40, 4450-4452; f) D. Trauner, J. B. Schwarz, S. J. Danishefsky, Angew. Chem. 1999, 111, 3756-3758; Angew. Chem. Int. Ed. 1999, 38, 3542-3544. [4] 4] For some representative examples, see: a) J. D. Hansen, B. J. Newhouse, S. Allen, A. Anderson, T. Eary, J. Schiro, J. Gaudino, E. Laird, A. C. Allen, D. Chantry, C. Eberhardt, L. E. Burgess, Tetrahedron Lett. 2006, 47, 69-72; b) D. P. Becker, D. L. Flynn, C. I. Villamil, Bioorg. Med. Chem. Lett. 2004, 14, 3073-3075; c) S. Demin, D. Van Haver, M. Vandewalle, P. J. De Clercq, R. Bouillon, A. Verstuyf, Bioorg. Med. Chem. Lett. 2004, 14, 3885-3888; d) T. D. Aicher, B. Balkan, P. A. Bell, L. J. Brand, S. H. Cheon, R. O. Deems, J. B. Fell, W. S. Fillers, J. D Fraser, J. Gao, D. C. Knorr, G. G. Kahle, C. L. Leone, J. Nadelson, R. Simpson, H. C. Smith, J. Med. Chem. 1998, 41, 4556-4566; e) M. D. Ennis, R. L. Hoffman, N. B. Ghazal, D. W. Old, P. A. Mooney, J. Org. Chem. 1996, 61, 5813-5817; f) A. I. Meyers, L. Snyder, J. Org. Chem. 1993, 58, 36-42. 5] For selected representative examples, see: a) S. Sen, V. R. Potti, R. Surakanti, Y. L. N. Murthy, R. Pallepogu, Org. Biomol. Chem. 2011, 9, 358-360; b) J. E. Resek, J. Org. Chem. 2008, 73, 9792-9794; c) D. Enders, O. Niemeier, T. Balensiefer, Angew. Chem. 2006, 118, 1491- 1495; Angew. Chem. Int. Ed. 2006, 45, 1463-1467; d) H. C. Shen, F.- X. Ding, S. L. Colletti, Org. Lett. 2006, 8, 1447-1450; e) T. E. Nielsen, S. Le Quement, M. Meldal, Org. Lett. 2005, 7, 3601-3604; f) M. D. Groaning, G. P. Brengel, A. I. Meyers, Tetrahedron 2001, 57, 2635- 2642; g) J A Nieman M D Ennis Org Lett 2000 2 1395-1397 For some representative examples, see: a) J. D. Hansen, B. J. Newhouse, S. Allen, A. Anderson, T. Eary, J. Schiro, J. Gaudino, E. Laird, A. C. Allen, D. Chantry, C. Eberhardt, L. E. Burgess, Tetrahedron Lett. 2006, 47, 69-72; b) D. P. Becker, D. L. Flynn, C. I. Villamil, Bioorg. Med. Chem. Lett. 2004, 14, 3073-3075; c) S. Demin, D. Van Haver, M. Vandewalle, P. J. A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts with furan substrates to initiate a remarkable cascade reaction sequence** Dimitris Kalaitzakis Tamsyn Montagnon Ioanna Alexopoulou and Georgios Vassilikogiannakis* Commercially available furan starting materials were used throughout except in the cases of 2-benzylfuran (Entries 2, 4, 6) and the furan shown in Entry 7, where the requisite substrates were synthesised using known procedures.[17] In general, the yields are remarkably high when the large increase in molecular complexity that has been achieved in this one-pot procedure is taken into consideration, and given the intricate nature of the cascade reaction sequence that has been successfully implemented. Regarding the stereochemical outcomes (confirmed by NOE studies),[18] it was found that having a defined stereogenic centre adjacent to the amino group (R4 attachment position) on the 1,2-aminoalcohol induces highly stereoselective formation of the product’s ring junction (position R1 in the product, Scheme 1), as can be seen from Entries 3 – 10 (Table 1). In contrast, having a defined stereogenic centre adjacent to the alcohol (R5 attachment position) on the 1,2-amino- alcohol leads to no significant stereoselectivity being achieved for the overall reaction, with both ring junction isomers being formed (dr = 55:45, R1/R5 anti: R1/R5 syn, Entry 11). Similarly, substituents at the R2 and R3 positions of the furan substrate are incorporated in 2 Scheme 3. Acid catalysed dimerisation of bicyclic lactams. that this method will prove to be a powerful addition to the synthetic chemist’s armoury. Received: ((will be filled in by the editorial staff)) Published online on ((will be filled in by the editorial staff)) Received: ((will be filled in by the editorial staff)) Published online on ((will be filled in by the editorial staff)) Scheme 3. Acid catalysed dimerisation of bicyclic lactams. Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones Ménard-Moyon, J. P. Schmidt, R. J. K. Taylor, Tetrahedron Lett. 2007, 48, 6556-6560; c) M. Penhoat, S. Leleu, G. Dupas, C. Papamicaël, F. Marsais, V. Levacher, Tetrahedron Lett. 2005, 46, 8385-8389; d) R. Deprez-Poulain, N. Willand, C. Boutillon, G. Nowogrocki, N. Azaroual, B. Deprez, Tetrahedron Lett. 2004, 45, 5287-5290. [7] For a recent example, see: A. A. Bahajaj, M. H. Moore, J. M. Vernon, Tetrahedron 2004, 60, 1235-1246. In summary, we have introduced a new and versatile method for the synthesis of Meyers’ bicyclic lactams using a green, step- and atom-economic singlet-oxygen-initiated cascade reaction sequence. The mild method’s broad synthetic potential derives from the fact that it begins from simple furan substrates which can be readily synthesised and/or substituted to give many possible variations, as desired; a feature that contrasts with the limitations (both in terms of their synthesis and subsequent handling and purification) imposed by the γ-ketoacids precursors used in the traditional Meyers’ synthesis.[1,2] Furthermore, the cascade initiator, singlet oxygen is an extremely selective reagent conferring on its reactions very broad functional group tolerance and a complete lack of need for protecting groups.[14] For all these reasons it is believed [8] W. N. Speckamp, H. Hiemstra, Tetrahedron 1985, 41, 4367-4416. [9] a) S. Comesse, A. Martel, A. Daïch, Org. Lett. 2011, 13, 4004-4007; b) R. Medimagh, S. Marque, D. Prim, J. Marrot, S. Chatti, Org. Lett. 2009, 11, 1817-1820. [10] a) E. Ascic, J. F. Jensen, T. E. Nielsen, Angew. Chem. 2011, 123, 5294-5297; Angew. Chem. Int. Ed. 2011, 50, 5188-5191; b) I. Kanizsai, Z. Szakonyi, R. Sillanpää, M. D’hooghe, N. De Kimpe, F. Fülöp, Tetrahedron Asymmetry 2006, 17, 2857-2863. [11] P. A. Wender, B. L. Miller Nature 2009, 460, 197-201. [12] B. M. Trost, Science 1991, 254, 1471-1477. [13] a) I. S. Young, P. S. Baran, Nature Chem. 2009, 1, 193-205; b) T. Gaich, P. S. Baran, J. Org. Chem. 2010, 75, 4657-4673; c) T. Newhouse, P. S. Baran, R. W. Hoffmann, Chem. Soc. Rev. 2009, 38, 3 3010-3021; (d) For the first introduction to the "ideal synthesis" concept, see: Hendrickson, J. B. J. Am. Chem. Soc. 1975, 97, 5784. 3010-3021; (d) For the first introduction to the "ideal synthesis" concept, see: Hendrickson, J. B. J. Am. Chem. Soc. 1975, 97, 5784. [16] The furan substrate in MeOH at 0 ºC with rose bengal (10-4 M), added as photosensitiser, was exposed to irradiation from a xenon Variac Eimac Cermax 300W visible spectrum light for a period of 8 mins whilst oxygen was gently bubbled through the reaction mixture. [17] F h i f b lf S i I f i f Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones Entry for the Table of Contents (Please choose one layout) Entry for the Table of Contents (Please choose one layout) Layout 2: (Powerful new method for synthesizing bicyclic lactams ) Dimitris Kalaitzakis, Tamsyn Montagnon, Ioanna Alexopoulou and Georgios Vassilikogiannakis* __________ Page – Page A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts with furan substrates to initiate a remarkable cascade reaction sequence A new and powerful method for the synthesis Meyers’ bicyclic lactams beginning from furans is reported that uses a remarkable one-pot singlet oxygen-initiated cascade reaction sequence to deliver the corresponding bicyclic lactams in high yield. Layout 2: Keywords: Meyers’ bicyclic lactams · singlet oxygen · cascade reaction · furan oxidation · 2-pyrrolidinones [16] The furan substrate in MeOH at 0 ºC with rose bengal (10-4 M), added as photosensitiser, was exposed to irradiation from a xenon Variac Eimac Cermax 300W visible spectrum light for a period of 8 mins whilst oxygen was gently bubbled through the reaction mixture. 1 h i f b lf S i f i f [14] For selected examples see: a) D. Noutsias, I. Alexopouou, T. Montagnon, G. Vassilikogiannakis, Green Chem. 2012, 14, 601-604; b) A. Kouridaki, T. Montagnon, M. Tofi, G. Vassilikogiannakis, Org. Lett. 2012, 14, 2374-2377; c) G. Vassilikogiannakis, I. Alexopoulou, M. Tofi, T. Montagnon, Chem. Commun. 2011, 47, 259-261; d) T. Montagnon, D. Noutsias, I. Alexopoulou, M. Tofi, G. Vassilikogiannakis, Org. Biomol. Chem. 2011, 9, 2031-2039. e) M. Tofi, K. Koltsida, G. Vassilikogiannakis, Org. Lett. 2009, 11, 313- 316; f) T. Montagnon, M. Tofi, G. Vassilikogiannakis, Acc. Chem. Res. 2008, 41, 1001-1011; g) T. Georgiou, M. Tofi, T. Montagnon, G. Vassilikogiannakis, Org. Lett. 2006, 8, 1945-1948; h) G. Vassilikogiannakis, M. Stratakis, Angew. Chem. 2003, 115, 5623- 5626; Angew. Chem. Int. Ed. 2003, 42, 5465-5468; [17] For synthesis of benzylfuran, see: Supporting Information for procedure; For synthesis of the furan substrate shown in Entry 7 (Table 1), see: A. Aponick, C.-Y. Li, J. Malinge, E. F. Marques, Org. Lett. 2009, 11, 4624-4627. [18] Full details of NOE studies are included in the Supporting Information. [19] C. Wedler, H. Schick, D. Scharfenberg-Pfeiffer, G. Reck, Liebigs Ann. Chem. 1992, 29-32. [20] The open form corresponds to intermediate G except that the preferred 2-pyrrolidinone, in this instance, is that which places the double bond exo from the five-membered ring and in conjugation with the Ph group, namely, 5-benzylidene-1-(2-hydroxyethyl)pyrrolidin-2-one. The exact structure is given in the supporting information. [15] For pioneering works on furan photooxygenations, see: a) C. S. Foote, M. T. Wuesthoff, S. Wexler, I. G. Burstain, R. Denny, G. O. Schenck, K. H. Schulte-Elte, Tetrahedron 1967, 23, 2583-2599; b) K. Gollnick, A. Griesbeck, Tetrahedron 1985, 41, 2057-2068; c) B. L. Feringa, Recl. Trav. Chim. Pays-Bas 1987, 106, 469-488. (Powerful new method for synthesizing bicyclic lactams ) Dimitris Kalaitzakis, Tamsyn Montagnon, Ioanna Alexopoulou and Georgios Vassilikogiannakis* __________ Page – Page Dimitris Kalaitzakis, Tamsyn Montagnon, Ioanna Alexopoulou and Georgios Vassilikogiannakis* __________ Page – Page A versatile new synthesis of Meyers’ bicyclic lactams: singlet oxygen reacts with furan substrates to initiate a remarkable cascade reaction sequence A new and powerful method for the synthesis Meyers’ bicyclic lactams beginning from furans is reported that uses a remarkable one-pot singlet oxygen-initiated cascade reaction sequence to deliver the corresponding bicyclic lactams in high yield. 5
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Cognitive function among older adults
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http://www.rsp.fsp.usp.br/ Rev Saude Publica. 2018;52 Suppl 2:4s Rev Saude Publica. 2018;52 Suppl 2:4s Supplement ELSI-Brazil Original Article Supplement ELSI-Brazil Original Article Correspondence: Erico Castro-Costa Av. Augusto de Lima, 1715 30190-002 Belo Horizonte, MG, Brasil E-mail: ericocastrocosta@gmail.com How to cite: Castro-Costa E, Lima- Costa MF, Bof de Andrade F, Souza- Junior PRB, Ferri CP. Cognitive function among older adults: ELSI- Brazil results. Rev Saude Publica. 2018;52 Suppl 2:4s. Cognitive function among older adults: ELSI-Brazil results Erico Castro-CostaI, Maria Fernanda Lima-CostaI,II, Fabíola Bof de AndradeI,II, Paulo Roberto Borges de Souza JuniorIII, Cleusa Pinheiro FerriIV I Fundação Oswaldo Cruz. Instituto René Rachou. Núcleo de Estudos em Saúde Pública e Envelhecimento. Belo Horizonte, MG, Brasil , , II Fundação Oswaldo Cruz. Instituto René Rachou. Programa de Pós-Graduação em Saúde Coletiva. Belo Horizonte, MG, Brasil , III Fundação Oswaldo Cruz. Instituto de Comunicação e Informação Científica e Tecnológica em Saúde. Rio de Janeiro, RJ, Brasil , III Fundação Oswaldo Cruz. Instituto de Comunicação e Informação Científica e Tecnológica em Saúde. Rio de Janeiro, RJ, Brasil J , J, IV Universidade Federal de São Paulo. Faculdade de Medicina. Departamento de Psicobiologia. São Paulo, SP, Brasil J , J, IV Universidade Federal de São Paulo. Faculdade de Medicina. Departamento de Psicobiologia. São Paulo, SP, Brasil Data Source The ELSI-Brazil is a population-based cohort study, designed to represent the Brazilian population aged 50 years or older, and with the objective of investigating the dynamics of aging in the Brazilian population and its determinants. The baseline was established between the years 2015/2016. More details can be found on the research’s homepagea and in another publication21. ELSI-Brazil baseline data collection included: 1) a household interview; 2) an individual interview with the participant; 3) physical measurements; 4) laboratory tests; 3) storage of blood aliquots for future analysis. a Fundação Oswaldo Cruz. Brazilian Longitudinal Study of Aging. Rio de Janeiro; c2015 [cited 2017 Nov 28]. Available from: http://elsi.cpqrr.fiocruz.br INTRODUCTION Cognitive function is an important determinant of independence and better quality of life among older adults1. According to recent projections, the elderly population will triple in Brazil and will increase from 19.6 million in 2010 to 66.5 million in 20502, making it the sixth largest elderly population in the world3. Aging is a complex phenomenon and its impact on health conditions and functionality occurs heterogeneously among older adults. These differences are associated with genetic4, environmental and social determinants5-8, and individual characteristics of the elderly9,10. Previous population-based studies with national samples from several countries have shown that socio-demographic factors11,12 and differences between rural and urban residence areas13,14 are related to alterations in cognitive function among older adults, showing important cultural and geographic variations11,12, 15,16. Brazil is a country of continental proportions, has one of the highest levels of inequality17, occupies the 75th position in the Human Development Index (HDI)18 and presents differences for this index among its macroregions19. There has been an increasing number of studies on the cognitive function of Brazilian older adults20. However, it was observed that the majority (72%) was done in the Southeast and none of these studies had a design that allowed the comparison of cognitive function among Brazilian macroregions20. The Brazilian Longitudinal Study of Aging (ELSI-Brazil) used a national sample representative of individuals aged 50 years or older and allows us to investigate the cognitive function of older Brazilian adults. Thus, the objective of the present study was: 1) to investigate the cognitive function of older adults stratified by the Brazilian macroregions; 2) to compare the association between sociodemographic factors and the place of residence with cognitive function and the variations between the macroregions; 3) to investigate whether the variations in the sociodemographic structure and the place of residence observed are totally or partially responsible for the potential differences in cognitive function among macroregions. ABSTRACT ABSTRACT OBJECTIVE: To investigate macroregional variations in cognitive function in a national sample representative of the Brazilian population aged 50 years and older. METHODS: Data from the baseline of the Longitudinal Study of Brazilian Elderly (ELSI-Brazil), collected between 2015 and 2016, were used. Memory was measured by means of a 10-word list and executive function, by semantic verbal fluency, based on the naming of animals. Gender, age, education, and rural or urban residence were potentially confounding RESULTS: Among the 9,412 ELSI-Brazil participants, 9,085 were included in the analysis; 53.9% were women and the average age was 63.0 (0.42) years. After adjusting for potential confounding variables, average scores for memory and verbal fluency were lower in the Northeast region and higher in the Midwest and Southeast, respectively. In the South region, higher scores were found for immediate and combined memory. In all regions, older participants and those with lower schooling had worse scores for memory and verbal fluency. CONCLUSIONS: There are differences in cognitive function among older adults in the different macroregions, independent of age, gender, schooling, and rural or urban residence. Correspondence: Erico Castro-Costa Av. Augusto de Lima, 1715 30190-002 Belo Horizonte, MG, Brasil E-mail: ericocastrocosta@gmail.com DESCRIPTORS: Aged. Cognition. Memory. Socioeconomic factors. Received: Dec 18, 2017 Approved: Mar 18, 2018 How to cite: Castro-Costa E, Lima- Costa MF, Bof de Andrade F, Souza- Junior PRB, Ferri CP. Cognitive function among older adults: ELSI- Brazil results. Rev Saude Publica. 2018;52 Suppl 2:4s. Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided that the original author and source are credited. 1s Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. Ethical Aspects The ELSI-Brazil Project was approved by the Ethics Commission of Fiocruz, Minas Gerais (CAAE 34649814.3.0000.5091). All participants signed separate informed consent forms for all research procedures. Cognitive Function The study adopts a conceptual framework common to other large-scale longitudinal studies of aging in the world, such as the Health and Retirement Study21. This specific concept of the cognitive function module in ELSI-Brazil allows direct comparison of Brazilian results with results found in other countries, such as: China [the China Health and Retirement Longitudinal Study (https://g2aging.org/?section=study&studyid=4)], the United States [the US Health and Retirement Study (http://hrsonline. isr.umich.edu)], England [the English Longitudinal Study of Ageing (http://www.natcen.ac.uk/elsa)], Mexico [Mexican Health and Aging Study(mhasweb.org/Resources.aspx)] etc. Cognitive function among older Brazilian adults Castro-Costa E et al. In the present analysis, we selected two variables that represent three well-established domains of cognitive function: memory, language and executive function. Memory was evaluated through the word list learning test, in which 10 words were read to the participants. Immediate memory was defined as the repetition of the words immediately after their reading. Late memory (delayed recall) was their repetition five minutes after presentation. The participant was kept busy filling out the questionnaire during the interval between the two tasks. Combined memory was obtained by the sum of the scores for immediate and late memory. Language and executive function were measured by the semantic verbal fluency test (animal category), in which participants were asked to say the name of as many animals as they could in a one-minute period. Statistical Analyses Statistical analyzes were conducted in the Stata 14.1 program. Due to the complex design of the sample with stratification, all analyzes were done using weighting factors for each stratum, thus obtaining in the final results the contribution of each stratum according to its actual weight, and not through its participation in the sample. The use of Stata’s svy command allowed the output of robust standardized error. To compare the characteristics of the participants among the macroregions, the analysis of variance (ANOVA) was weighted for continuous variables and the Pearson’s chi-squared test was weighted for the categorical variables. Multivariate linear regressions with a 95% confidence interval were performed to estimate the relationship between memory (immediate, late and combined) and verbal fluency with gender, age, schooling and place of residence for each macroregion. Finally, the average of the variables memory (immediate, late and combined) and verbal fluency was standardized separately and then simultaneously, by gender, age, schooling and place of residence, using the direct method22 for each macroregion. Other Variables Also considered were the variables gender, age (50-59, 60-69, 70-79, 80 years or older), schooling (< 4, 4-7, 8-11, > 11) and place of residence (rural or urban). Ethical Aspects RESULTS Of the 9,412 ELSI-Brazil baseline participants, 9,085 (96.5%) presented complete information for all variables and were included in this analysis. The main characteristics of the participants by region are presented in Table 1. In the total sample, the average age was 63.0 years (SE = 0.42) and the majority were female (53.9%) and had less than 11 years of schooling (76.2%). Table 2 shows the average scores and multivariate analyzes of the association between memory and verbal fluency scores stratified by macroregions. Residents in the Southeast, South and Midwest regions had the best performances, both in the memory and in the verbal fluency evaluation. Older adults in the Southern macroregion had the best performance in the variables immediate memory and combined memory, while the best performance in late memory and verbal fluency occurred among residents of the Midwest and Southeast, respectively. Older residents with low schooling had worse memory performance (immediate, late and combined) and worse verbal fluency in all macroregions. On the other hand, rural participants showed worse performance in immediate and Cognitive function among older Brazilian adults Castro-Costa E et al. combined memory in the Northeast and Southeast, and in late memory in the Southeast. In the Midwest, rural residents performed better in late and combined memory. Regarding verbal fluency, participants from the rural area of the South and Midwest macroregion, and women from the North and Midwest, presented worse performance of this function. Table 1. Characteristics of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. RESULTS Characteristic Total North Northeast Southeast South Midwest p n = 9,085 n = 713 n = 2,416 n = 3,825 n = 1,234 n = 897 Gender: female (%) 53.9 49.9 54.6 54.5 53.6 52.1 0.934 Age – average (SE) 63.0 (0.42) 62.1 (0.67) 63.3 (0.75) 63.0 (0.72) 63.0 (0.92) 62.3 (1.1) 0.945 Education (years) (%) < 0.0001 > 11 23.8 26.3 18.3 26.3 24.2 23.7 8–10 11.9 15.6 8.9 12.9 12.2 11.3 4–7 31.4 25.4 23.2 34.4 36.9 31.8 < 4 32.8 32.7 49.5 26.4 26.7 33.2 Place of residence: Rural (%) 15.3 17.7 30.3 6.5 21.6 5.6 0.006 Memory - mean (SE) Immediate 4.3 (0.05) 4.2 (0.11) 3.8 (0.08) 4.5 (0.06) 4.6 (0.07) 4.3 (0.07) < 0.0001 Late 2.9 (0.06) 2.7 (0.15) 2.4 (0.09) 3.0 (0.07) 3.0 (0.09) 3.1 (0.13) < 0.0001 Combined 7.3 (0.10) 7.0 (0.24) 6.2 (0.17) 7.5 (0.13) 7.7 (0.16) 7.6 (0.19) < 0.0001 Verbal fluency - mean (SE) 12.6 (0.29) 11.4 (0.37) 11.4 (0.85) 13.2 (0.41) 12.6 (0.40) 12.9 (0.38) 0.012 All estimates were weighted by the sample parameters and sample weights of the subjects. SE: standard error. For continuous and categorical variables, F tests were used, and the weighted chi-square, corrected for the study design, was used as a complex sample. All estimates were weighted by the sample parameters and sample weights of the subjects. SE: standard error. For continuous and categorical variables, F tests were used, and the weighted chi-square, corrected for the study design, was used as a complex sample. Table 2. Average scores and multivariate analyzes of the association between the cognitive function scores an chooling and place of residence of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI Table 2. Average scores and multivariate analyzes of the association between the cognitive function scores and the variables gender, age, schooling and place of residence of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. F: female; M: male All multivariate regressions were adjusted for gender, age, schooling and place of residence. RESULTS 4s Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. Standardized average memory (immediate, late and combined) and verbal fluency scores are arranged and compared between the macroregions in Table 3. The direct standardization of memory and verbal fluency averages by the individual effect of gender, age, schooling and place of residence and by the simultaneous effect of these four variables did not alter the pattern of difference between the regions. The values of the averages observed and simultaneously standardized by the four variables are shown in the Figure. s of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), Table 3. Standardized average scores of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. RESULTS Cognitive function Standardized by gender Standardized by age Standardized by schooling Standardized by place of residence Standardized by the 4 variables Average Score 95%CI Average Score 95%CI Average Score 95%CI Average Score 95%CI Average Score 95%CI Immediate memory North 4.24 4.00–4.47 4.03 3.85–4.20 4.11 3.91–4.31 4.23 4.03–4.43 4.08 3.92–4.24 Northeast 3.83 3.65–4.00 3.74 3.60–3.88 3.97 3.87–4.07 3.89 3.71–4.08 3.82 3.72–3.93 Southeast 4.51 4.40–4.63 4.44 4.34–4.55 4.37 4.30–4.44 4.45 4.36–4.54 4.38 4.31–4.45 South 4.60 4.45–4.74 4.50 4.32–4.68 4.46 4.35–4.57 4.62 4.50–4.74 4.50 4.38–4.61 Midwest 4.43 4.29–4.57 4.29 4.18–4.40 4.37 4.33–4.42 4.41 4.29–4.54 4.33 4.25–4.40 Late Memory North 2.76 2.45–3.07 2.56 2.24–2.87 2.65 2.26–3.05 2.76 2.44–3.09 2.62 2.25–2.99 Northeast 2.42 2.24–2.61 2.34 2.20–2.48 2.54 2.41–2.68 2.48 2.31–2.66 2.42 2.32–2.52 Southeast 3.05 2.91–3.20 2.97 2.86–3.08 2.89 2.80–2.99 2.98 2.86–3.11 2.90 2.81–2.99 South 3.10 2.92–3.28 2.99 2.81–3.17 2.96 2.81–3.11 3.12 2.98–3.27 3.01 2.86–3.16 Midwest 3.15 2.90–3.42 2.99 2.81–3.18 3.10 2.98–3.22 3.18 2.94–3.41 3.09 2.96–3.22 Combined Memory North 7.01 6.55–7.48 6.60 6.17–7.03 6.78 6.18–7.37 7.01 6.52–7.49 6.72 6.21–7.22 Northeast 6.26 5.92–6.61 6.09 5.83–6.35 6.52 6.30–6.74 6.38 6.04–6.73 6.25 6.06–6.43 Southeast 7.58 7.33–7.83 7.42 7.22–7.62 7.28 7.13–7.43 7.44 7.24–7.65 7.29 7.14–7.44 South 7.70 7.39–8.01 7.50 7.16–7.84 7.43 7.18–7.67 7.75 7.50–7.99 7.52 7.27–7.76 Midwest 7.60 7.22–7.99 7.29 7.02–7.57 7.49 7.35–7.62 7.62 7.28–7.96 7.46 7.28–7.64 Verbal fluency North 11.44 10.66–12.23 11.23 10.73–11.73 11.30 10.98–11.71 11.53 10.83–12.24 11.30 10.70–11.89 Northeast 11.41 9.69–13.13 11.23 9.79–12.66 11.79 9.88–13.71 11.60 9.66–13.54 11.54 9.53–13.55 Southeast 13.21 12.41–14.01 13.33 12.34–14.32 13.26 12.10–14.41 13.26 12.31–14.21 13.41 11.94–14.89 South 12.62 11.83–13.40 12.48 11.69–13.26 12.39 11.85–12.92 12.74 12.23–13.26 12.41 11.91–12.91 Midwest 12.97 12.22–13.71 12.75 12.07–13.44 12.86 12.32–13.40 12.87 12.14–13.60 12.77 12.11–13.44 Table 3. Standardized average scores of the cognitive function of the 9,085 sample participants. Brazilian Longitu 2015-2016. 16 14 12 10 18 16 14 12 0 North observed immediate memory Northeast Southeast South Midwest standardized late memory observed verbal fluency standardized immediate memory observed combined memory observed late memory standardized combined memory standardized verbal fluency Figure. Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. 16 14 12 10 18 16 14 12 0 North observed immediate memory Northeast Southeast South Midwest standardized late memory observed verbal fluency standardized immediate memory observed combined memory observed late memory standardized combined memory standardized verbal fluency Figure. RESULTS Cognitive function North Northeast Southeast South Midwest Average 95%CI Average 95%CI Average 95%CI Average 95%CI Average 95%CI Memory Immediate 4.22 3.99–4.45 3.83 3.65–4.00 4.51 4.40–4.63 4.60 4.45–4.75 4.43 4.30–4.56 Late 2.76 2.45–3.07 2.42 2.23–2.60 3.05 2.90–3.20 3.09 2.92–3.27 3.15 2.89–3.41 Combined 6.99 6.53–7.46 6.26 5.92–6.60 7.58 7.32–7.83 7.70 7.39–8.01 7.60 7.22–7.99 Verbal fluency 11.52 10.78–12.27 11.41 9.73–13.09 13.24 12.42–14.06 12.64 11.84–13.44 13.01 12.25–13.77 β 95%CI β 95%CI β 95%CI β 95%CI β 95%CI Immediate Memory Gender (F versus M) 0.22 -0.15–0.59 0.01 -0.10–0.13 0.07 -0.07–0.21 0.04 -0.17–0.27 0.03 -0.13–0.19 Age (≥ 75 versus < 75) -1.95 -2.33– -1.57 -1.31 -1.45– -1.18 -1.20 -1.36– -1.03 -1.55 -1.90– -1.19 -1.49 -1.16– -1.36 Education (< 11 versus ≥ 11) -0.96 -1.28– -0.64 -1.17 -1.36– -0.97 -1.02 -1.13– -0.91 -1.01 -1.19– -0.82 -1.07 -1.49– -0.66 Place of residence (rural versus urban) -0.06 -0.30–0.16 -0.26 -0.39– -0.13 -0.51 -0.66– -0.36 -0.04 -0.32–0.24 0.01 -0.07–0.09 Late Memory Gender (F versus M) 0.09 -0.17–0.35 0.07 -0.07–0.21 0.07 -0.08–0.23 0.26 0.06–0.46 0.00 -0.44–0.44 Age (≥ 75 versus < 75) -1.76 -2.23– -1.30 -1.13 -1.30– -0.96 -1.52 -1.70– -1.33 -1.54 -1.76– -1.32 -1.45 -1.70– -1.20 Education (< 11 versus ≥ 11) -1.02 -1.27– -0.76 -1.22 -1.41– -1.03 -1.21 -1.35– -1.06 -1.11 -1.32– -0.89 -1.12 -1.57– -0.67 Place of residence (rural versus urban) 0.13 -0.25–0.53 -0.24 -0.53–0.05 -0.54 -0.77– -0.30 -0.22 -0.45–0.02 0.39 0.25–0.52 Combined Memory Gender (F versus M) 0.31 -0.28–0.90 0.08 -0.14–0.31 0.14 -0.12–0.42 0.31 -0.11–0.73 0.03 -0.55–0.61 Age (≥ 75 versus < 7 5) -3.72 -4.52– -2.92 -2.44 -2.70– -2.19 -2.72 -3.04– -2.40 -3.09 -3.66– -2.53 -2.94 -3.25– -2.62 Education (< 11 versus ≥ 11) -1.99 -2.51– -1.46 -2.39 -2.73– -2.05 -2.23 -2.47– -1.99 -2.12 -2.46– -1.78 -2.20 -3.06– -1.34 Place of residence (rural versus urban) 0.06 -0.50–0.63 -0.50 -0.86– -0.15 -1.05 -1.34– -0.76 -0.26 -0.74–0.22 0.40 0.22–0.58 Verbal Fluency Gender (F versus M) -1.24 -2.04– -0.44 0.02 -2.07–2.13 -1.44 -3.34–0.45 -0.46 -0.96–0.03 -0.99 -1.72– -0.27 Age (≥ 75 versus < 75) -1.84 -2.66– -1.03 -2.33 -4.35– -0.31 3.74 -2.23–9.72 -2.04 -2.71– -1.38 -2.09 -2.76– -1.42 Education (< 11 versus ≥ 11) -1.57 -2.44– -0.70 -1.75 -4.04–0.52 -2.71 -3.53– -1.88 -3.60 -4.24– -2.97 -2.45 -4.15– -0.75 Place of residence (rural versus urban) -0.20 -1.18–0.77 -1.07 -3.53–1.39 0.90 -3.97–5.78 -0.73 -1.46– -0.01 -1.10 -1.84– -0.36 F: female; M: male All multivariate regressions were adjusted for gender, age, schooling and place of residence. DISCUSSION DISCUSSION This is the first national study with a representative sample of the population aged 50 years or older that compared cognitive function in the five Brazilian macroregions, where the same tests were applied in the same period. Residents of the Southeast, South and Midwest regions presented better cognitive function in all the applied tests. However, there were variations on the ranking, depending on the ability evaluated. In all regions, there was a worsening of the three types of memory and verbal fluency correlated with the oldest age and low level of schooling, except for the Southeast macroregion, where the worsening of verbal fluency was not correlated with older age. Regarding the place of residence, we observed, inconsistently, that residents of the rural area exhibited lower scores for tests that evaluate memory and verbal fluency. However, residents of the rural area of the Midwest performed better in late memory than those in the urban area. The female gender presented the worst performance in verbal fluency tests in the North and Midwest macroregions. Finally, the standardized averages of the three types of memory and of verbal fluency by the effect of gender, age, schooling and place of residence showed that these variables did not explain all the differences found among the Brazilian macroregions. This study has several advantages such as: 1) the use of a representative sample of Brazilian adults aged 50 and older; 2) the direct evaluation of cognitive function through validated and applied tests in the same period, which allows comparisons between the Brazilian macroregions and with other countries; 3) training and certification of the interviewers according to the protocols developed for the study; 4) quality assurance and control of data collection by conducting previous pilot studies in order to identify and correct potential problems in procedures and interviews. However, the study also has limitations that must be considered when interpreting the results. The use of a weighted sample may underestimate the averages for cognitive function due to its design effect. However, the use of specific analyzes for weighting is likely to overcome this limitation. Although all dimensions of cognitive function were not evaluated in this study, scores of our measures (10-word list and verbal fluency test) were normally distributed with no evidence of floor or ceiling effect23, commonly found in low education populations. RESULTS Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. Figure. Observed and standardized average scores by gender, age, schooling and place of residence of the cognitive function of the 9,085 sample participants. Brazilian Longitudinal Study of Aging (ELSI-Brazil), 2015-2016. Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. DISCUSSION The results of the present study are directly comparable with studies conducted in high- income countries (Health Retirement Study [HRS], English Longitudinal Study of Ageing [ELSA], the Irish Longitudinal Study on Ageing [TILDA])11,12 and in middle income countries, such as (Mexican Health and Aging Study [MHAS] and the Chinese Health and Retirement Longitudinal Study [CHARLS])15,16, in which cognitive function was assessed using the same measure for memory (a list of 10 words, with the exception of the MHAS study, which used a list of eight words) and verbal fluency (animal category semantic fluency test). ELSI-Brazil, even with a younger sample due to the inclusion of middle-aged adults, presented lower averages for all abilities evaluated (immediate, late and combined memory) when compared to the three studies in high-income countries (HRS, ELSA, TILDA)11,12 which used a sample of elderly individuals aged 60 years or older. Probably, this occurred due to the great difference in schooling between these samples, since this factor plays an important role in cognitive function24. In ELSI-Brazil, 76.2% of the sample of Brazilians aged 50 and older have less than 12 years of schooling; in HRS, ELSA and TILDA, rates are 56%, 48% and 44% for those aged 65 years and older, and 39%, 28%, 28% for those aged 57-64 years, respectively11,12. Compared with studies in middle- and low-income countries, where low schooling was similar between ELSI-Brazil, MHAS and CHARLS15,16. It was observed that in the Mexican study, in spite of presenting an older population (adults 60 years old or older), performance in all three skills was better compared to our findings (immediate memory: 4.8 versus 4.3; late memory: 4.4 versus 2.9, and verbal fluency: 15.3 versus 12.6). This was because the participants in the MHAS sample were healthier than those in the ELSI-Brazil since the tests for cognitive evaluation were applied only to those who had not had a stroke or did Cognitive function among older Brazilian adults Castro-Costa E et al. Cognitive function among older Brazilian adults Castro-Costa E et al. not present depressive symptoms16. In addition, the memory was tested by a list of eight words, which probably influenced the results obtained with this version, overestimating the performance of the participants16. Regarding the Chinese study15, combined memory performance was worse than in ELSI-Brazil (3.3 versus 7.3), probably because the majority of participants were from the rural area13,14. DISCUSSION Comparisons with other Brazilian studies are limited due to the differences between the age groups and the outcomes studied. In a study with employees aged 35-74 at six universities in the Northeast, Southeast and South macroregions, memory was evaluated by a list of 10 words applied at three different occasions (immediate, late, recognition), while semantic verbal fluency was evaluated by the animal category test25. The averages for late memory ranged from four to eight in men and five to eight in women, while the average values for verbal fluency ranged from 12 to 21 among men and 13 to 21 among women according to participants’ schooling25. Like the Longitudinal Study on Adult Health (ELSA-Brazil), the present study also demonstrated that older participants with low educational level had worse performance in memory and verbal fluency. However, the scores were lower in all tests when compared to those found in ELSA-Brazil. This discrepancy probably occurred because ELSA-Brazil was conducted exclusively in the urban area (six capitals), while ELSI-Brazil was conducted both with participants living in the urban area and with those living in rural areas. Memory and Verbal Fluency assessments, in the present study, are part of the cognitive assessment of the Consortium to Establish the Registry for Alzheimer’s Disease (CERAD)26, translated and validated to Brazilian Portuguese for carrying out in Brazilian elderly population27. Previous studies have shown that older people with lower levels of schooling and those coming from rural areas have worse cognitive performance11-16,27; data for the gender variable are inconsistent11,12,15,16,28. Our findings replicated the associations previously demonstrated for memory and verbal fluency in other studies with population-based samples11- 16,26,27, with the exception of results for late and combined memory, which showed that participants from rural areas in the Midwest had the best performance. This is likely to have happened because the main activity in this macroregion is farming, which possibly favors better socioeconomic conditions, which in turn may have contributed to this performance. Finally, the results of the present study showed that the performance of adults aged 50 years or older on cognitive function tests is worse than that found in high-income countries (United States, England and Ireland) and inconsistent with that of middle-income countries (worse than that found in Mexico and better than that found in China). We observed that the association between sociodemographic, settlement factors and cognitive function had consistent variation patterns in the Brazilian macroregions. DISCUSSION However, as these factors do not fully explain the differences in cognitive function in adults aged 50 years or older observed between regions, other contextual and cultural factors not investigated here may play a relevant role in the differences found. REFERENCES 1. Cigolle CT, Langa KM, Kabeto MU, Tian Z, Blaum CS. Geriatric conditions and disability: the Health and Retirement Study. 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Health Psychol. 2011;30(4):377-85. https://doi.org/101037/a0022826 8. Iliffe S, Kharicha K, Harari D, Swift C, Gillmann G, Stuck AE. Health risk appraisal in older people 2: the implications for clinicians and commissioners of social isolation risk in older people. Br J Gen Pract. 2007;57(537):277-82. 9. World Health Organization. Closing the gap in a generation: health equity through action on social determinants of health: final report of the Commission on Social Determinants of Health. Geneva: WHO; 2008 [cited 2017 Nov 23]. Available from: http://whqlibdoc.who.int/ publications/2008/9789241563703_eng.pdf 10. Dannefer D. Cumulative advantage/disadvantage and the life course: cross-fertilizing age and social science theory. J Gerontol B Psychol Sci Soc Sci. 2003;58(6):S327-37. https://doi.org/10.1093/geronb/58.6.s327 11. Langa KM, Llewellyn DJ, Lang IA, Weir DR, Wallace RB, Kabeto MU, et al. REFERENCES Cognitive health among older adults in the United States and in England. BMC Geriatr. 2009;9:23. https://doi.org/10.1186/1471-2318-9-23 12. Savva GM, Maty SC, Setti A, Feeney J. Cognitive and physical health of the older populations of England, the United States, and Ireland: international comparability of the Irish Longitudinal Study on Ageing. J Am Geriatr Soc. 2013;61 Suppl 2:s291-8. https://doi.org/10.111/jgs12196 13. Saenz JL, Downer B, Garcia MAG, Wong R. Cognition and context: rural-urban differences in cognitive aging among older Mexican adults. J Aging Health. 2017 Apr 1::898264317703560. https://doi.org/10.1177/0898264317703560 14. Cassarino M, O’Sullivan V, Kenny RA, Setti A. Environment and cognitive aging: a cross-sectional study of place residence and cognitive performance in the Irish Longitudinal Study on Aging. Neuropsychology. 2016;30(5):543-57. https://doi.org/10.1037/neu0000253 15. Lei X, Smith JP, Sun X, Zhao Y. Gender differences in cognition in China and reasons for change over time: evidence from CHARLS. J Econ Aging. 2014:4:46-55. https://doi.org/10.1016/j.jeoa.2013.11.001 16. Mejía-Arango S, Wong R, Michaels-Obregón A. Normative and standardized data for cognitive measures in the Mexican Health and Aging Study. Salud Publica Mex. 2015;57 Suppl 1:S90-6. 17. World Bank. Gini Index (World Bank estimate) 2011-2015. Washington (DC); 2015. [cited 2017 Nov 23]. Available from: https://data.worldbank.org/indicator/SI.POV.GINI 18. United Nations Development Programme. Human Development Reports. New York: UNDP [cited 2017 Nov 23]. Available from: http://hdr.undp.org/en 19. Programa das Nações Unidas para o Desenvolvimento. Desenvolvimento humano nas macrorregiões brasileiras: 2016. Brasília (DF): PNUD, IPEA, FJP; 2016. 20. Martins NIM, Caldas PR, Cabral ED, Lins CCSA, Coriolano MGWS. Instrumentos de avaliação cognitiva utilizados nos últimos 5 anos em idosos brasileiros. Cienc Saude Coletiva. 2017 out [cited 2017 Nov 23]:0402. Available from: http://www.cienciaesaudecoletiva.com.br/artigos/instrumentos- de-avaliacao-cognitiva-utilizados-nos-ultimos-5-anos-em-idosos-brasileiros/16454?id=16454 21. Lima-Costa MF, Andrade FB, Souza Jr PRB, Neri AL, Duarte YAO, Castro-Costa E, de Oliveira C. The Brazilian Longitudinal Study of Aging (ELSI-Brazil): objectives and design. Am J Epidemiol. 2018 Jan 31. https://doi.org/10.1093/aje/kwx387 22. National Institute on Aging; World Health Organization. Global Health and Aging. Bethesda (MD): National Institute of Health; 2011 [cited 2017 Nov 23]. (NIH publication, 11-773). Available from: http://www.who.int/ageing/publications/global_health.pdf 23. Batty GD, Deary IJ, Zaninotto P. Association of cognitive function with cause-specific mortality in middle and older age: follow-up of participants in the English Longitudinal Study of Ageing. Am J Epidemiol. 2016;183(3):183-90. https://doi.org/10.1093/aje/kwv139 24. Park DC. The basic mechanism accounting for age-related decline in cognitive function. In: Park DC, Schwarz N, editors. Cognitive aging: a primer. New York: Psychology Press; 2000. p.3-21. 25. Cognitive function among older Brazilian adults Castro-Costa E et al. 28. Ferreira L, Ferreira Santos-Galduróz R, Ferri CP, Fernandes Galduróz JC. Rate of cognitive decline in relation to sex after 60 years-of-age: a systematic review. Geriatr Gerontol Int. 2014;14(1):23- 31. https://doi.org/10.1111/ggi.12093 26. Morris JC, Heyman A, Mohs RC, Hughs JP, Van Belle G, Fillenbaum G, et al. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part I. Clinical and neuropsychological assessment of Alzheirmer’s disease. Neurology. 1989;39(9):1159-65. https://doi.org/10.1212/WNL.39.9.1159 27. Bertolucci PHF, Okamoto IH, Toniolo Neto J, Ramos LR, Brucki SMD. Desempenho da população brasileira na bateria neuropsicológica do Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Rev Psiq Clin (São Paulo). 1998;25(2):80-3. Cognitive function among older Brazilian adults Castro-Costa E et al. REFERENCES Passos VMA, Giatti L, Bensenor I, Tiemeier H, Ikram MA, Figueiredo RC, et al. Education plays a greater role than age in cognitive test performance among participants of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil). BMC Neurol. 2015;15:191. https://doi.org/10.1186/s12883-015-0454-6 8 https://doi.org/10.11606/S1518-8787.2018052000629 8s Funding: The ELSI-Brazil baseline study was supported by the Brazilian Ministry of Health (DECIT/SCTIE – Department of Science and Technology from the Secretariat of Science, Technology and Strategic Inputs (Grant 404965/2012-1); COSAPI/DAPES/SAS – Healthcare Coordination of Older Adults, Department of Strategic and Programmatic Actions from the Secretariat of Health Care) (Grants 20836, 22566, and 23700); and the Brazilian Ministry of Science, Technology, Innovation and Communication. Authors’ Contribution: Data analysis and interpretation: ECC, MFLC, FBA, PRBSJ, CPF. Manuscript writing: ECC, MFLC, FBA, PRBSJ, CPF. Manuscript critical review: ECC, MFLC, FBA, PRBSJ, CPF. All authors have approved the final version of the manuscript and assume public responsibility for its content. Conflict of Interest: The authors declare no conflict of interest. 9s
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Recent Trends in Inequality and Poverty in Developing Countries
Handbook of income distribution
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www.cedlas.econo.unlp.edu.ar *  This  paper  corresponds  to  chapter  10  of  the  Handbook  of  Income  Distribution,  volume  2,  edited  by  A.   Atkinson  and  F.  Bourguignon.   **   Facundo   Alvaredo   is   with   CONICET,   EMod/OMI-­‐Oxford   University   and   Paris   School   of   Economics;   Leonardo  Gasparini  is  with  CEDLAS,  Facultad  de  Ciencias  Económicas,  Universidad  Nacional  de  La  Plata   and   CONICET.   This   paper   was   completed   while   Leonardo   Gasparini   was   visiting   professor   at   the   University  of  British  Columbia.  We  are  grateful  to  participants  to  the  conference  “Recent  Advances  in   the  Economics  of  Income  Distribution”  (Paris),  the  AAEP  Meetings  (Rosario),  and  especially  to  Francois   Bourguignon  and  Tony  Atkinson  for  valuable  comments  and  suggestions.  We  are  also  grateful  to  David   Jaume,  Darío  Tortarolo,  Carolina  López,  Julian  Amendolaggine,  Santiago  Garganta,  Florencia  Pinto,  Pablo   Gluzmann,  Leopoldo  Tornarolli,  Javier  Alejo,  Juan  Zoloa,  and  Carolina  García  Domench  (all  at  CEDLAS)  for   outstanding  research  assistance.  We  alone  are  responsible  for  any  errors.  Financial  support  from  the   ESRC-­‐DFID  joint  fund  is  gratefully  acknowledged. Centro de Estudios Distributivos, Laborales y Sociales Maestría en Economía Facultad de Ciencias Económicas Recent Trends in Inequality and Poverty in Developing Countries Facundo Alvaredo y Leonardo Gasparini Documento de Trabajo Nro. 151 Noviembre, 2013 ISSN 1853-0168 This  version:  November  4,  2013 Recent  Trends  in  Inequality  and  Poverty in  Developing  Countries    * Facundo  Alvaredo   Leonardo  Gasparini** Abstract This  chapter  reviews  the  empirical  evidence  on  the  levels  and  trends  in   income/consumption  inequality  and  poverty  in  developing  countries. It   includes  a  discussion  of  data  sources  and  measurement  issues,  evidence   on  the  levels  of  inequality  and  poverty  across  countries  and  regions,  an   assessment   of   trends   in   these   variables   since   the   early   1980s,   and   a   general   discussion   of   their   determinants. There   has   been   tremendous   progress   in   the   measurement   of   inequality   and   poverty   in   the   developing   world,   although   serious   problems   of   consistency   and   comparability   still   remain. The   available   evidence   suggests   that   on   average  the  levels  of  national  income  inequality  in  the  developing  world   increased  in  the  1980s  and  1990s,  and  declined  in  the  2000s. There  was   a  remarkable  fall  in  income  poverty  since  the  early  1980s,  driven  by  the   exceptional   performance   of   China   over   the   whole   period,   and   the   generalized   improvement   in   living   standards   in   all   the   regions   of   the   developing  world  in  the  2000s. JEL  Codes:  D31,  I32 JEL  Codes:  D31,  I32 Alvaredo-Gasparini Alvaredo-Gasparini 1. Introduction Poverty and inequality are certainly among the main concerns in the developing world. A typical developing country is characterized by high levels of material deprivation, and large dispersion in individual wellbeing, at least when compared to a typical developed economy. Fighting poverty and minimizing the unjust inequalities are top priorities in the developing world. The United Nations, in the famous declaration of the Millennium Development Goals (MDGs), proposed as target number 1 to halve income poverty in the developing world from 1990 to 2015. The reduction of inequality does not occupy the same privileged position in the agenda, but few would not list it as a central social concern. While chapter 9 of this Handbook deals with poverty and inequality in the developed world, this chapter documents patterns and changes in the developing countries. There is no need to argue about the relevance of including a separate chapter in the Handbook: the developing world is home of 85% of total world population, and bears levels of poverty and inequality far higher than in the rich countries. While in a typical developing economy the share of people striving to survive with less than 2 dollars a day is more than 30%, that share is close to zero in the industrialized countries. In fact, on this basis poverty is an issue exclusively of the developing world. The differences in income inequality are presumably also large, although the comparisons are hindered by the fact that national household surveys typically capture income in developed countries and consumption expenditures in developing ones. High poverty and inequality are pervasive characteristics of the developing world; however, they are not immutable features of these economies. There is convincing evidence pointing to a robust decline in the levels of absolute income poverty in the developing world over the last decades, and substantial progress in the reduction of deprivation in various non-monetary dimensions - education, health, sanitation, access to infrastructure. Changes in income inequality have been much less clear, as relative inequality has risen in some countries and fallen in others. In fact, the evidence suggests that on average the developing countries are today (2013) somewhat more unequal than three decades ago. This chapter reviews the empirical evidence on the levels and trends in income inequality and poverty in developing countries. INDEX 1. INTRODUCTION ............................................................................................................................ 3 2. THE DEVELOPING WORLD: CHARACTERIZATION AND DATA ..................................... 4 3. INEQUALITY: LEVELS ................................................................................................................. 9 4. INEQUALITY: TRENDS .............................................................................................................. 23 5. POVERTY: LEVELS ..................................................................................................................... 56 6. POVERTY: TRENDS .................................................................................................................... 66 7. CONCLUDING REMARKS ......................................................................................................... 90 2 2 Alvaredo-Gasparini Alvaredo-Gasparini 1. Introduction We focus the analysis on the income/consumption approximations to welfare; in particular the chapter deals mainly with relative inequality across individuals in household consumption expenditures per capita, and with absolute poverty defined over that welfare variable, and considering alternative international lines defined in US dollars adjusted for purchasing power parity (PPP). This choice is restricted by space limitations and does not imply ignoring that a general assessment of poverty and inequality should also include other non- monetary dimensions (e.g. health, education) and other monetary variables (e.g. wealth). Other chapters in the Handbook contribute to fill those gaps. 3 Alvaredo-Gasparini Alvaredo-Gasparini The analysis in this chapter is mostly focused on inequality and poverty within countries and not within supra-national regions or in the world.1 Although issues of global inequality are increasingly relevant, inequality is still primarily a national concern. People are generally worried about inequality mainly in their countries, and public policies are typically aimed at reducing disparities among individuals within national boundaries. The analysis in this chapter is mostly focused on inequality and poverty within countries and not within supra-national regions or in the world.1 Although issues of global inequality are increasingly relevant, inequality is still primarily a national concern. People are generally worried about inequality mainly in their countries, and public policies are typically aimed at reducing disparities among individuals within national boundaries. The empirical evidence shown in this chapter is drawn from the academic literature, regional and country papers, and open-access databases, in particular the PovcalNet project developed in the World Bank. Although most of the evidence is based on statistics obtained from national household surveys, we also report results from tax records (the World Top Incomes Database, WTID) and international surveys (the Gallup World Poll) to illustrate some issues. Even though the main purpose of the chapter is presenting basic evidence on levels and trends, we also briefly review the main discussions on determinants of recent changes in inequality and poverty. The rest of the chapter is organized as follows. In section 2 we briefly characterize the economies in the developing world, and discuss the data sources and some measurement issues. The following two sections are assigned to the main topic in this volume – inequality. In section 3 we document the levels of income inequality in the developing world, while in section 4 we summarize the evidence on trends since the early 1980s. 1. Introduction The next two sections repeat the sequence for poverty: section 5 compares levels across countries, and section 6 summarizes trends and discusses the evidence at the regional level.2 Section 7 closes with a summary and some final remarks. 2 The separate treatment of inequality and poverty is somewhat artificial, as they are just two characteristics of the same income distribution. However, and despite some possible overlapping and duplications, we prefer to follow most of the literature and discuss both concepts separately. 2. The developing world: characterization and data In this section we briefly characterize the economies of the developing world, and review the sources of data to measure and analyze income poverty and inequality. 1 Global inequality is analyzed in chapter 12 of this volume. 2 2.1. Developing countries The division between developed and developing countries is a helpful simplification that can be done in different arbitrary ways. In this chapter we follow the World Bank’s main criterion based on gross national income (GNI) per capita: developing countries are those with per capita GNI below a certain nominal threshold (US$ 12,276 in 2011). These nations are usually classified into six geographical regions: East Asia and Pacific (EAP), Eastern Europe and Central Asia (ECA), Latin America and the Caribbean (LAC), Middle East and North Africa (MENA), South Asia (SA) and Sub-Saharan Africa (SSA). 4 Alvaredo-Gasparini The Appendix includes a list of all the developing countries in each region with their populations.3 The developing countries cover almost 75% of the total land area in the world and represent 85% of the total population. Table 2.1 summarizes some basic demographic and economic statistics. Table 2.1 Population, GNI per capita and Human Development Index, 2010 Developing countries by region Table 2.1 Population, GNI per capita and Human Development Index, 2010 Developing countries, by region Source: population is taken from the United Nations Demographic Yearbook. Gross National Income (GNI) per capita in international dollars adjusted for purchasing power parity (PPP) and in current US$ (Atlas method) are taken from World Development Indicators. The Human Development Index (HDI) is from the UNDP Human Development Report. GNI and HDI are unweighted averages across countries. Countries Population (millions) PPP Atlas method HDI Developing countries 153 5,840 7,023 4,291 0.608 East Asia and Pacific 24 1,961 4,911 2,992 0.619 Eastern Europe and Central Asia 30 478 12,558 7,815 0.751 Latin America and the Caribbean 31 584 9,789 6,433 0.706 Middle East and North Africa 13 331 6,462 3,647 0.636 South Asia 8 1,633 3,429 1,704 0.535 Sub-Saharan Africa 47 853 3,288 1,798 0.450 Developed countries 62 1,055 37,303 38,818 0.857 00 Total 216 6,894 15,682 14,181 0.663 GNI per capita Table 2.1 Population, GNI per capita and Human Development Index, 2010 Developing countries, by region Source: population is taken from the United Nations Demographic Yearbook. Gross National Income (GNI) per capita in international dollars adjusted for purchasing power parity (PPP) and in current US$ (Atlas method) are taken from World Development Indicators. The Human Development Index (HDI) is from the UNDP Human Development Report. GNI and HDI are unweighted averages across countries. 3 In this chapter we include emerging economies as part of the developing world, a decision that implies some overlapping with chapter 9. In the period under analysis some countries graduated from the set of developing countries; to avoid selection bias we do not drop them from the analysis. 2.1. Developing countries According to these indicators Eastern Europe and Central Asia is the most developed region in the group: per capita GNI is almost twice the mean for the developing world, and the Human Development Index (HDI) is significantly higher. Latin American and the Caribbean ranks second, and Middle East and North Africa third. Although economic growth in Asia has been remarkable in the last decades, per capita GNI and other development indicators are on average still below the mean of the developing world. South Asia is significantly less developed than East Asia and the Pacific. Sub- Saharan Africa is the poorest and least developed region of the world. The mean of the national per capita GNIs in that region is less than 50% of the developing world mean, and less than 10% of the mean of the industrialized economies. 2.2. Data sources National household surveys are the main source of information for distributive analysis. Since one of the central goals of these surveys is measuring living standards, they typically include questions to construct a monetary proxy for wellbeing: income 5 Alvaredo-Gasparini and/or expenditures on consumption goods. Although some developing countries started to implement national household surveys after World War II, it is only recently that governments engaged in programs of regularly collecting information through household surveys, often with the help of some international organization. Distributive statistics for the developing world are rare before the 1970s, and reasonably robust only from the 1990s on. There has been a remarkable increase in the availability of national household surveys over the last decades. A chapter like this one, that includes a broad assessment of income inequality and poverty in developing countries, could hardly have been written two decades ago, and is a sign of the huge progress made on data collection. However, as we discuss below, data limitations are still stringent, and allow only a still blurred picture of inequality and poverty. The databases for international distributive analysis can be classified into two groups: those that produce statistics with microdata from surveys or administrative records, and those that collect, organize and report statistics from other sources. The former group includes the World Bank´s PovcalNet, the Luxembourg Income Study, the World Income Distribution database, the World Top Incomes Database and some regional initiatives. The second one includes the seminal work by Deininger and Squire (1996) and its follow-up - the WIDER´s World Income Inequality Database, the All the Ginis database, and some other projects. The main source of information for poverty and inequality analysis at a large international scale in the developing world is the World Bank´s PovcalNet, a compilation of distributive data built up from national household surveys, generally fielded by national statistical offices. PovcalNet, used for the World Bank’s World Development Indicators, includes statistics constructed mostly from household survey microdata, and in some few countries from grouped tabulations. At the moment of writing this database includes more than 850 surveys from almost 130 countries, representing more than 90% of the population of the developing world, spanning the period 1979-2011. 4 Statistics are derived from the estimation of a general quadratic and a beta Lorenz curves from grouped data. Shorrocks and Wan (2008) propose an algorithm that reproduces individual data from grouped statistics with a higher degree of accuracy. 2.2. Data sources The website of PovcalNet provides public access to data to generate estimates for selected countries and alternative poverty lines from grouped data.4 Martin Ravallion and Shaohua Chen, the developers of PovcalNet, have produced several papers exploiting the dataset (Ravallion and Chen, 1997; Chen and Ravallion, 2001, 2010, 2012). This project has been increasingly influential in shaping the assessment of inequality, and in particular poverty, in the developing world by researchers and policy practitioners. It is, for instance, the source used to monitor the poverty-reduction goal of the MDGs. This chapter draws heavily on statistics computed by the PovcalNet project. Some regional initiatives aimed at computing social statistics from household survey microdata in a standardized way are useful to study distributive issues in specific 6 Alvaredo-Gasparini geographic areas, and as sources of information for world databases. For instance, the Socioeconomic Database for Latin America and the Caribbean (SEDLAC), jointly developed by CEDLAS at the Universidad Nacional de La Plata (Argentina) and the World Bank’s LAC poverty group (LCSPP), includes distributive and labor statistics for LAC constructed using consistent criteria across countries and years. BADEINSO, developed by the United Nations´ ECLAC, is also a large and good-quality database on economic and social variables in LAC. In Eastern and Central Europe the World Bank ECA database includes statistics for 28 countries since 1990 computed from direct access to household surveys. The Household Expenditure and Income Data for Transitional Economies developed by Branko Milanovic in the World Bank is the predecessor of that database. Milanovic has also built the World Income Distribution (WYD) database, which includes data for five benchmark years (1988, 1993, 1998, 2002 and 2005) for 146 countries, 75% obtained from direct access to household surveys. The dataset has been used in several studies to compute global inequality (Milanovic, 2002, 2005, 2012). The Luxembourg Income Study (LIS), described in chapter 9 of this volume, includes distributive information computed from household survey microdata for developed countries. LIS also reports statistics for several transitional economies in Eastern Europe and recently has added some developing countries in Latin America (Brazil, Colombia, Guatemala, Mexico, Peru and Uruguay). The growth in the availability of distributive statistics stimulated efforts to gather and organize them. Deininger and Squire (1996) put together a large dataset of quintile shares and Gini coefficients for most countries since World War II taken from different studies and national reports. pp 7 SWIID should also be reviewed critically: in many cases it requires a case-by-case analysis, which is simply a sign that much effort is still needed in putting together comparable statistics. As it is based on secondary datasets, external problems are inadvertently incorporated. 5 WIID was initially compiled over 1997-1999 for the UNU/WIDER-UNDP project "Rising Income Inequality and Poverty Reduction: Are They Compatible?" directed by Giovanni Andrea Cornia. Tabatabai (1996) at International Labour Organization also made an independent effort to put together distributive statistics for many countries in the world. 6 Analyzing the Deininger and Squire (DS) dataset, Atkinson and Brandolini (2001) conclude that “users could be seriously misled if they simply download the accept series (i.e., the “high quality” subset)”. Although WIID implies a significant improvement from the original DS dataset, a similar word of caution applies. distributive statistics for many countries in the world. 6 Analyzing the Deininger and Squire (DS) dataset, Atkinson and Brandolini (2001) conclude that “users could be seriously misled if they simply download the accept series (i.e., the “high quality” subset)”. Although WIID implies a significant improvement from the original DS dataset, a similar word of caution applies. 7 SWIID should also be reviewed critically: in many cases it requires a case-by-case analysis, which is simply a sign that much effort is still needed in putting together comparable statistics As it is based on 5 WIID was initially compiled over 1997-1999 for the UNU/WIDER-UNDP project "Rising Income Inequality and Poverty Reduction: Are They Compatible?" directed by Giovanni Andrea Cornia. Tabatabai (1996) at International Labour Organization also made an independent effort to put together distributive statistics for many countries in the world. 6 Analyzing the Deininger and Squire (DS) dataset, Atkinson and Brandolini (2001) conclude that “users could be seriously misled if they simply download the accept series (i.e., the “high quality” subset)”. Although WIID implies a significant improvement from the original DS dataset, a similar word of caution applies. 7 SWIID should also be reviewed critically: in many cases it requires a case-by-case analysis, which is simply a sign that much effort is still needed in putting together comparable statistics. As it is based on secondary datasets, external problems are inadvertently incorporated. 2.2. Data sources This panel database, which greatly promoted the empirical study of the links between inequality and other economic variables, was updated and extended by the UNU/WIDER-UNDP World Income Inequality Database (WIID) (WIDER, 2008).5 The WIID database includes Gini coefficients, quintile and decile shares, and the income shares of the top 5% and bottom 5%. The information is drawn from very different sources, which raises comparability concerns.6 To provide guidance in the use of the database, ratings are given to the observations, based on the survey quality, the coverage, and the quality of the information provided by the original source. The SWIID database is an effort to identify reasonably comparable information in WIID (Solt, 2009).7 7 Alvaredo-Gasparini The All the Ginis database, assembled also by Branko Milanovic, is a compilation and adaptation of Gini coefficients retrieved from five datasets: LIS, SEDLAC, WYD, the World Bank ECA database, and WIID. Besides gathering all the information in a single file, the All the Ginis database is useful as it provides information on the welfare concept and recipient unit to which the reported Gini refers, facilitating the comparisons. The Chartbook of Economic Inequality, assembled by Atkinson and Morelli (2012), presents a summary of evidence about changes in economic inequality (income/consumption, earnings and wealth) in the period from 1911 to 2010 for 25 countries. The information drawn from household surveys for the seven countries in the developing world included in the database (Argentina, Brazil, India, Indonesia, Malaysia, Mauritius and South Africa) starts in the 1950s. All the datasets mentioned above are based on data from national household surveys.8 Even when they are the best available source of information for distributive analysis, household surveys are plagued with problems for international comparative studies, because, among other reasons, the questionnaires and the procedures to compute income/consumption variables differ among countries, and frequently also within a country over time. Some surveys inquire about income and others about consumption, some capture net income and some gross income, in some cases variables are reported on a weekly basis and in others on a monthly basis, items as the imputed rent for owner occupied housing are included in some surveys and ignored in others.9 Even in those projects that made explicit efforts to reduce these differences, comparability issues persist, as problems rooted in differences in questionnaires are difficult to be completely overcome. These problems are well recognized in the literature. 8 The exception is the Chartbook of Economic Inequality, that uses a range of sources. 9 In addition, the typical problems of under-reporting and selective compliance are negligible in some cases and endemic in others. See Deaton (2003, 2005) and Korinek et al. (2006). 8 The exception is the Chartbook of Economic Inequality, that uses a range of sources. 9 2.2. Data sources For instance, Chen and Ravallion (2012) state that “…there are problems that we cannot deal with. For example, it is known that differences in survey methods (such as questionnaire design) can create non-negligible differences in the estimates obtained for consumption or income”. In a survey of global income inequality, Anand and Segal (2008) share those concerns. There are some alternatives to reduce the comparability problems, although they all come at a price. Gallup conducts a survey in nearly all nations in the world with almost exactly the same questionnaire. The Gallup World Poll is particularly rich in self- reported measures of quality of life, opinions, and perceptions, but it also includes basic questions on demographics, education, and employment, and a question on household income. In principle, the Gallup World Poll allows a distributive analysis in nearly all the countries in the world based on the same income question. The downside is that measurement errors may be very large when reported income is 8 Alvaredo-Gasparini based only on one question and with sample sizes of just around 1000 observations per country.10 The Estimated Household Income Inequality (EHII) data set produced by the University of Texas Inequality Project (UTIP) is based on UTIP-UNIDO, a global data set that calculates industrial pay-inequality measures for 156 countries from 1963 to 2003, using the between-groups component of a Theil index, measured across industrial categories in the manufacturing sector (Galbraith and Kum, 2005).11 Although in principle the use of industrial pay information could lend some homogeneity into the comparisons, the technique employed may not provide an accurate measure of inequality over the whole income distribution. 10 Gasparini and Gluzmann (2012) compare basic demographic statistics drawn from the Gallup Poll with those computed from the national household surveys of the LAC countries for year 2006, and conclude that in most countries statistics from the Gallup Poll, including income poverty and inequality, are roughly consistent with those from national household surveys. 11 Specifically, EHII consists on estimates of gross household income inequality, computed from an OLS regression between the Deininger and Squire inequality measures and the UTIP-UNIDO manufacturing pay inequality measures, controlling for the source of information in the inequality data (income/expenditure, gross/net, and household/per capita measures) and for the share of manufacturing employment in total employment. 12 The drawbacks of computing inequality in the distribution of consumption or income per capita to measure distributive justice have been widely acknowledged. Among other limitations, it is a one- dimensional approach, it is focused on results not opportunities, it ignores the value of publicly provided goods such as education and health services, and it adopts a simple adjustment for demographics ignoring intra-household inequality, economies of scale and differences in needs (Ferreira and Ravallion, 2009). However, extending inequality measurement to alleviate these limitations in a way that keeps international comparisons feasible has been proved difficult. 12 The drawbacks of computing inequality in the distribution of consumption or income per capita to measure distributive justice have been widely acknowledged. Among other limitations, it is a one- dimensional approach, it is focused on results not opportunities, it ignores the value of publicly provided goods such as education and health services, and it adopts a simple adjustment for demographics ignoring intra-household inequality, economies of scale and differences in needs (Ferreira and Ravallion, 2009). However, extending inequality measurement to alleviate these limitations in a way that keeps international comparisons feasible has been proved difficult. 10 Gasparini and Gluzmann (2012) compare basic demographic statistics drawn from the Gallup Poll with those computed from the national household surveys of the LAC countries for year 2006, and conclude that in most countries statistics from the Gallup Poll, including income poverty and inequality, are roughly consistent with those from national household surveys. Specifically, EHII consists on estimates of gross household income inequality, computed from an OLS gression between the Deininger and Squire inequality measures and the UTIP-UNIDO manufacturing y inequality measures, controlling for the source of information in the inequality data come/expenditure, gross/net, and household/per capita measures) and for the share of anufacturing employment in total employment. 15 We decided to apply the same coefficient to all LAC countries after failing to find significant regularities between the ratio consumption/income Ginis and other observable variables for the seven countries in the sample. WDR (2006) reports consumption and income Ginis in four Latin American countries; the mean ratio of the Ginis is 0.81. The value is somewhat lower (0.77) for the eight non-LA countries in the sample. 3. Inequality: levels In this section we present results regarding the level of inequality in the developing countries, deferring to the next section the discussion of the trends. In most of the section we measure inequality computed over the distribution of household consumption per capita, using data from PovcalNet.12 Consumption is usually regarded as a better measure of current welfare than income on both theoretical and practical grounds, especially in developing countries (Deaton and Zaidi, 2002). As it is usual in this literature, we frequently refer to income inequality, despite the fact that statistics are constructed over the distribution of consumption expenditures. As discussed above, this chapter is mainly focused on within-country inequality, so welfare disparities are measured among individuals living within the national boundaries. Although globalization is increasingly raising global inequality concerns, inequality remains mainly a national matter. This view also leads us to mostly document unweighted statistics of inequality measures across countries, a practice that is consistent with the typical cross-country approach in the development literature. Weighting by population would imply an assessment of inequality in a region or in the world strongly affected by some highly-populated countries, such as 9 Alvaredo-Gasparini China, India and Indonesia in Asia, or Brazil and Mexico in Latin America, and almost ignoring the situation in other less-populated nations. Having said that, since the decision of taking each political entity as a unit in the analysis is certainly debatable; we show some results using both unweighted and population-weighted statistics.13 13 See some arguments on this debate in Bourguignon et al. (2004). g g g ( ) 14 The countries are Argentina, Costa Rica, Ecuador, Mexico, Nicaragua, Panama and Peru. 13 See some arguments on this debate in Bourguignon et al. (2004). 3.1. Inequality in the developing countries We start by comparing inequality levels across developing countries based on the Gini coefficient for the distribution of household consumption per capita for year 2010, computed in PovcalNet mostly from household survey microdata. Other inequality measures are highly correlated with the Gini coefficient. For instance, in PovcalNet and WIID datasets the Pearson and Spearman correlations of the Gini and several extreme inequality measures (e.g. the 90/10 and 80/20 income-share ratios) exceed 0.9. PovcalNet includes information for the distribution of per capita consumption expenditures, except in almost all Latin American and a few Caribbean countries, for which income inequality statistics are reported. In the analysis that follows we adjust the income Gini coefficients in that region to reflect the gap between income and consumption inequality estimates. Specifically, we selected seven Latin American countries with household surveys that include reasonably good consumption and income data in several years:14 on average the ratio of the consumption/income Ginis is 0.861 (standard deviation of 0.046). We apply that coefficient to the 22 Latin American and Caribbean countries with income data to approximate their consumption Ginis.15 In most cases the observations correspond to year 2010, or adjacent years. However, some countries are lacking a recent household survey (or it was dropped due to quality concerns). In fact, in 24 countries the survey used to estimate inequality in 2010 was carried out between 2000 and 2005, while in 6 cases (5 of them in the Caribbean) the observation corresponds to the 1990s. With that caveat in mind, the PovcalNet dataset has relatively recent distributive information for 82% of the countries in the developing world, representing 97% of its total population (see Table A.1 in the Appendix). The country coverage across regions is heterogeneous. In East Asia and Pacific PovcalNet includes 12 out of the 24 developing countries, which nonetheless represent 96% of the total population of the area. The coverage in Eastern Europe and Central Asia is almost complete, lacking information only for the small Kosovo. In LAC the coverage is complete in continental Latin America, but weak in the Caribbean. Anyway, countries with information represent 98% of the total population in LAC (the 10 Alvaredo-Gasparini main missing country in terms of population is Cuba). The dataset in Middle East and North Africa does not contain information only for Lebanon and Libya, which represent 3% of the MENA population. 16 PovcalNet reports Ginis above 63.1 for Comoros and Seychelles, two small island countries in the Indian Ocean. However, the results are not well established. For instance, the reported Gini in Seychelles is 42.7 in 2000 and 65.8 in 2007, a highly implausible change in just seven years. 17 The harmonic mean is similar in SSA than in LAC. 3.1. Inequality in the developing countries In South Asia the only country missing is Afghanistan, while in Sub-Saharan Africa there is information for 42 out of the 47 countries, representing 95% of the population. Figure 3.1 displays the range of Gini coefficients for 122 countries around year 2010, ranking from the least unequal (Ukraine, 25.6) to the most unequal economy (South Africa, 63.1).16 The mean value is 39.8, while the median is 39.2. More than half of the observations are in the range [35, 45]. Only seven Eastern Europe countries have Ginis below 30, and five Sub-Saharan African countries have Ginis higher than 55. The population-weighted mean is less than one point lower than the simple mean (39.1), a result affected by the relatively low level of inequality in populous India and Indonesia (China has a Gini somewhat higher than the world mean). Figure 3.1 shows the position of some of the most populated countries: Brazil has high inequality levels, China and Russia intermediate values, and India and Indonesia relatively low levels in the context of the developing world. Figure 3.1 Gini coefficients for the distribution of household consumption per capita Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: countries sorted by their Gini coefficients. 20 25 30 35 40 45 50 55 60 65 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Gini coefficient Brazil China Indonesia India Russia Gini coefficients for the distribution of household consumption per capita Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: countries sorted by their Gini coefficients. The variability of Gini coefficients across countries is large compared to the changes within countries over time, at least for the period for which we have more robust information (since the early 1980s). Li, Squire and Zou (1998) find in the Deininger and 11 Alvaredo-Gasparini Squire dataset that 90% of the total variance in the Gini coefficient is explained by variation across countries, while only a small percentage is accounted by variation over time. From this observation Li et al. (1998) conclude that inequality should be mainly determined by factors which differ substantially across countries, but tend to be relatively stable within countries over time. We find a similar result in a panel of developing countries from 1981 to 2010 (PovcalNet data): 88.5% of the variance in that panel is accounted by variation across countries. 3.1. Inequality in the developing countries Squire dataset that 90% of the total variance in the Gini coefficient is explained by variation across countries, while only a small percentage is accounted by variation over time. From this observation Li et al. (1998) conclude that inequality should be mainly determined by factors which differ substantially across countries, but tend to be relatively stable within countries over time. We find a similar result in a panel of developing countries from 1981 to 2010 (PovcalNet data): 88.5% of the variance in that panel is accounted by variation across countries. The inequality rankings are relatively stable over time. The Spearman-rank correlation coefficient for the Ginis in 1981 and 2010 is 0.68, while it rises to 0.74 for 1990 and 2010, both significant at 1%. The last decades witnessed enormous economic, social and political changes in the developing world, but, although the income distributions have been affected with various intensities, the world inequality ranking has not changed much, a fact that suggests the existence of some underlying factors that are stronger determinants of the level of inequality. In Figure 3.2 developing countries are grouped in regions. Sub-Saharan Africa is the geographic area that includes countries with the highest inequality levels, but it is also the region with the highest dispersion, possibly in part due to measurement errors (Table 3.1). Although eight out of the ten highest Gini coefficients belong to Sub- Saharan African countries, and the arithmetic mean of the Gini coefficient is the highest in the world, the median is lower than in Latin America.17 Figure 3.2 Gini coefficients for the distribution of household consumption per capita Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: each bar represents a country in a given geographic region of the developing world. 20 25 30 35 40 45 50 55 60 65 Gini coefficient East Asia & Pacific Eastern Europe & Central Asia Latin America & the Caribbean Middle East & North Africa South Asia Sub-Saharan Africa Gini coefficients for the distribution of household consumption per capita Developing countries, 2010 Source: own calculations based on PovcalNet (2013). ( ) Note: each bar represents a country in a given geographic region of the developing world. Note: each bar represents a country in a given geographic region of the developing world. 3.1. Inequality in the developing countries 12 Alvaredo-Gasparini Table 3.1 Gini coefficients for the distribution of household consumption per capita Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: unweighted statistics. Mean Median Coef. Var. Min. Max. East Asia and Pacific 38.1 36.7 0.101 31.9 43.5 Eastern Europe and Central Asia 33.6 33.7 0.144 25.6 43.6 Latin America and the Caribbean 43.8 44.8 0.104 34.7 52.8 Middle East and North Africa 36.0 36.1 0.091 30.8 40.9 South Asia 35.0 36.3 0.081 30.0 38.1 Sub-Saharan Africa 44.4 42.1 0.175 33.3 63.1 Developing countries 39.8 39.2 0.181 25.6 63.1 Table 3.1 Gini coefficients for the distribution of household consumption per capita Developing countries, 2010 Latin America and the Caribbean has been typically pointed out as the most unequal region in the world. Deininger and Squire (1996), for instance, state that their dataset confirm the “familiar fact that inequality in Latin America is considerably higher than in the rest of the world”.18 This type of assessment however is usually made combining income Ginis for LAC with consumption Ginis for other regions, and/or ignoring Sub- Saharan Africa. With the adjustment mentioned above to take the consumption/income gap into consideration (factor 0.861), we find that the mean Gini for LAC is 43.8, slightly lower than in SSA (44.4), but the median is higher (44.8 in LAC and 42.1 in SSA). To reach the result of a higher mean Gini in LAC than in SSA we would need an adjustment parameter higher than 0.92; such value is larger than what we estimated in all LA countries in the sample, except Mexico. The rest of the regions in the developing world have Ginis mostly below 40. The arithmetic mean is 38.1 in East Asia and Pacific, 36.0 in Middle East and North Africa, and 35.0 in South Asia. Inequality is likely to be higher in MENA, since several oil- producing countries are excluded for being high-income economies (and also for lack of information).19 Eastern Europe and Central Asia is the region with the lowest inequality levels, with a mean Gini coefficient of 33.6. Interestingly, the dispersion measured by the coefficient of variation is higher than in the rest of the regions, except SSA. Almost all very highly unequal countries (Gini coefficients above 50) are in Sub-Saharan Africa (Table 3.2). This region, however, has a similar share of countries in the high and middle categories. 19 Bahrain, Kuwait, Oman, Qatar, Saudi Arabia, United Arab Emirates are in that group. Malta and Isra are also ignored for being developed and Lebanon and Libya are excluded for lack of information. 18 See also Lopez Calva and Lustig (2010) and Chen and Ravallion (2012). 19 3.1. Inequality in the developing countries In contrast, in LAC most countries have high levels of inequality, while in EAP, MENA and SA most countries are in the middle-inequality group. Only ECA has economies with low inequality (Gini coefficients below 30). 13 13 Alvaredo-Gasparini Alvaredo-Gasparini Table 3.2 Classification of countries by level of inequality and by region Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: countries are classified according to the value of the Gini coefficient for the distribution of household consumption per capita. Very high High Middle Low Total [50-70] [40-50) [30-40) [20-30) East Asia and Pacific 0 3 8 0 11 Eastern Europe and Central Asia 0 5 16 7 28 Latin America and the Caribbean 2 17 6 0 25 Middle East and North Africa 0 1 10 0 11 South Asia 0 0 7 0 7 Sub-Saharan Africa 10 14 16 0 40 Total 12 40 63 7 122 Inequality Table 3.2 Classification of countries by level of inequality and by region Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: countries are classified according to the value of the Gini coefficient for the distribution of household consumption per capita. Very high High Middle Low Total [50-70] [40-50) [30-40) [20-30) East Asia and Pacific 0 3 8 0 11 Eastern Europe and Central Asia 0 5 16 7 28 Latin America and the Caribbean 2 17 6 0 25 Middle East and North Africa 0 1 10 0 11 South Asia 0 0 7 0 7 Sub-Saharan Africa 10 14 16 0 40 Total 12 40 63 7 122 Inequality Table 3.2 Classification of countries by level of inequality and by region Developing countries, 2010 Note: countries are classified according to the value of the Gini coefficient for the distribution of household consumption per capita. The All the Ginis dataset (ATG) includes Gini coefficients from LIS, SEDLAC, WYD, the World Bank ECA database and WIID. We selected consumption Ginis from ATG for year 2005 or close, and applied a similar adjustment as described above for those countries in LAC with only income Ginis. The basic results are similar to the ones obtained with PovcalNet data. The linear correlation coefficient for the Gini between both data sources is 0.763, while the Spearman rank correlation is 0.771, both significant at 1%. The Gini coefficients in ATG go from 23.1 (Czech Republic) to 62.9 (Comoros). 20 The OECD high-income countries rank as the least unequal in the world with a mean income Gini of 32.8. 3.1. Inequality in the developing countries The mean and median coincide in 40.1. Again, more than half of the observations are in the range [35, 45]. Only several Eastern European countries have Ginis below 30, while only four Sub-Saharan African countries have Ginis higher than 55. The evidence on inequality levels in the developing world drawn from WIID is similar. For instance, based on a sample of income Ginis for around 2005, Gasparini et al. (2013) find that the mean Gini for the six Sub-Saharan African countries in the dataset is 56.5, followed by Latin America (52.9), Asia (44.7) and Eastern Europe and Central Asia (34.7).20 We find that the linear correlation coefficient for year 2005 for the Gini coefficient in PovcalNet and WIID is 0.871, and the Spearman coefficient is 0.820. The Luxembourg Income Study database (see chapter 9 of this volume) covers 36 countries, including 6 in Latin America, which occupy the top places in all the income inequality rankings. The LA Ginis go from 50.6 in Colombia to 43.9 in Uruguay; the most unequal non-LA country is Russia with a value of 40.8, while the rest of the countries in LIS go from 37 (USA) to 22.8 (Denmark). The mean Gini for the Eastern European countries in LIS is slightly higher than the mean for the high-income economies. Data from the World Development Indicators also suggest that inequality in the developing world is significantly higher than in the OECD high-income countries. The mean income Gini for the latter group is 32.2, which is lower than in any other region in the world. 14 14 Alvaredo-Gasparini The EHII database confirms the high inequality levels of Sub-Saharan Africa and Latin America, but perhaps surprisingly, it records similar levels in South Asia and Middle East and North Africa.21 The mean Gini for the period 1998-2002 is 47.3 in SSA, 46.4 in LAC, 46.4 in MENA and 47.2 in SA. According to this dataset inequality is relatively lower in East Asia and Pacific (44.9) and Eastern Europe and Central Asia (44.0). The estimated level of the Gini coefficient is substantially lower in the developed economies; the mean is equal to 36.5.22 The Pearson (Spearman) correlation coefficient between EHII and PovcalNet Ginis is 0.642 (0.603), lower than the resulting value when comparing PovcalNet with WIID or ATG, but still significant at 1%. 22 This mean excludes the oil-rich Arab countries. When including these countries in the sample th Gi i j t 39 21 See also Galbraith and Kum (2005). 22 This mean excludes the oil-rich Arab countries. When including these countries in the sample the mean Gini jumps to 39. 21 See also Galbraith and Kum (2005). 22 This mean excludes the oil-rich Arab countries. When including these countries in the sample the 3.1. Inequality in the developing countries Most international databases do not provide confidence intervals for the point estimates of the distributive measures, making impossible the assessment of the statistical significance of the differences among countries. However, given that the indicators are calculated from large national household surveys, the confidence intervals are typically relatively narrow. SEDLAC provides the confidence intervals for all the Gini coefficients in Latin America: for instance, the 95% confidence interval for the income Gini was [43.9, 44.7] in Argentina 2010, [53.5, 54.0] in Brazil 2009; and [47.0, 47.9] in Mexico 2010. Differences in the point estimates of more than 1 Gini point are always statistically significant (Figure 3.3). Figure 3.3 Gini coefficient and confidence intervals (95%) Distribution of household income per capita Latin American countries, 2010 Figure 3.3 Gini coefficient and confidence intervals (95%) Distribution of household income per capita Latin American countries, 2010 Source: own calculations based on SEDLAC (CEDLAS and the World Bank). 43 48 53 58 Argentina Brazil Chile Colombia Costa Rica Dominican R. Ecuador Honduras Mexico Panama Paraguay Peru Uruguay Gini coefficient Source: own calculations based on SEDLAC (CEDLAS and the World Bank). 15 15 Alvaredo-Gasparini 24 Take the distribution of the shares of deciles 7 to 9 in total consumption across countries. While the percentile 10 of that distribution is 34.7, the percentile 90 is just 38.3. The corresponding intervals for the distribution of the share accruing to the poorest 60% [25.4, 38.2] and the richest 10% [24.3, 39.3] are much wider. 23 Again, figures for Latin American (and a few Caribbean) countries are estimated based on the comparison of income and consumption microdata of seven countries in that region. 23 Again, figures for Latin American (and a few Caribbean) countries are estimated based on the comparison of income and consumption microdata of seven countries in that region. 24 Take the distribution of the shares of deciles 7 to 9 in total consumption across countries. While the percentile 10 of that distribution is 34.7, the percentile 90 is just 38.3. The corresponding intervals for the distribution of the share accruing to the poorest 60% [25.4, 38.2] and the richest 10% [24.3, 39.3] are much wider. 23 Again, figures for Latin American (and a few Caribbean) countries are estimated based on the comparison of income and consumption microdata of seven countries in that region. 24 Take the distribution of the shares of deciles 7 to 9 in total consumption across countries. While the percentile 10 of that distribution is 34.7, the percentile 90 is just 38.3. The corresponding intervals for the distribution of the share accruing to the poorest 60% [25 4 38 2] and the richest 10% [24 3 39 3] 3.2. Inequality beyond the Gini coefficient The international databases usually allow a closer look at the distributions in the world beyond a single parameter, such as the Gini coefficient. Table 3.3 reports some basic statistics of the decile shares in 120 countries around 2010.23 On average (unweighted) the poorest 10% of the population in a country accrues 2.6% of total consumption reported in the survey: that share climbs to 31.5% for the top 10%. In a typical developing country the aggregate consumption of the poorest 60% of the population is similar than the consumption of the top 10%. It is interesting to notice that the coefficient of variation of the decile consumption shares across countries is decreasing up to the top decile, when it strongly rises: countries in the world seem substantially different in the consumption share of the poor and the rich, but not in the share of the middle strata, in particular the upper- middle strata. This fact is naturally related to the typical shape of the real-world income distributions. For instance, under log-normality, the derivative of the decile shares with respect to the standard deviation of log incomes is U-shaped. Table 3.4 Table 3.4 Correlation coefficients across countries of decile consumption shares Developing countries, 2010 Source: own calculations based on PovcalNet (2013). *=significant at 1%. d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d1 1 d2 0.9355* 1 d3 0.8930* 0.9883* 1 d4 0.8421* 0.9624* 0.9910* 1 d5 0.8042* 0.9273* 0.9647* 0.9787* 1 d6 0.7336* 0.8739* 0.9291* 0.9623* 0.9847* 1 d7 0.6310* 0.7734* 0.8436* 0.8950* 0.9378* 0.9736* 1 d8 0.3127* 0.4711* 0.5624* 0.6446* 0.7253* 0.8085* 0.8982* 1 d9 -0.5793* -0.4905* -0.4112* -0.3258* -0.2389* -0.1232 0.0527 0.4390* 1 d10 -0.7844* -0.9032* -0.9452* -0.9689* -0.9844* -0.9891* -0.9650* -0.7962* 0.118 1 Table 3.4 Correlation coefficients across countries of decile consumption shares Developing countries, 2010 Source: own calculations based on PovcalNet (2013). *=significant at 1%. d1 d2 d3 d4 d5 d6 d7 d8 d9 d10 d1 1 d2 0.9355* 1 d3 0.8930* 0.9883* 1 d4 0.8421* 0.9624* 0.9910* 1 d5 0.8042* 0.9273* 0.9647* 0.9787* 1 d6 0.7336* 0.8739* 0.9291* 0.9623* 0.9847* 1 d7 0.6310* 0.7734* 0.8436* 0.8950* 0.9378* 0.9736* 1 d8 0.3127* 0.4711* 0.5624* 0.6446* 0.7253* 0.8085* 0.8982* 1 d9 -0.5793* -0.4905* -0.4112* -0.3258* -0.2389* -0.1232 0.0527 0.4390* 1 d10 -0.7844* -0.9032* -0.9452* -0.9689* -0.9844* -0.9891* -0.9650* -0.7962* 0.118 1 Correlation coefficients across countries of decile consumption shares Developing countries, 2010 Correlation coefficients across countries of decile consumption shares Source: own calculations based on PovcalNet (2013). *=significant at 1%. Table 3.3 Table 3.3 Deciles shares, distribution of household consumption per ca Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: unweighted statistics. Deciles Mean Std. Dev. Coef.Var. Min. Max. 1 2.6 0.81 0.31 1.0 4.4 2 3.8 0.86 0.23 1.5 5.8 3 4.8 0.90 0.19 2.0 6.8 4 5.8 0.92 0.16 2.6 7.8 5 6.8 0.92 0.13 3.5 8.8 6 8.1 0.87 0.11 4.7 9.9 7 9.6 0.80 0.08 6.6 11.0 8 11.7 0.65 0.06 9.0 12.7 9 15.3 0.84 0.05 12.7 17.6 10 31.5 6.12 0.19 19.5 51.7 Deciles shares, distribution of household consumption per capita Developing countries, 2010 The aggregate consumption share of deciles 5 to 9 is on average around 50%, and it is very stable across countries. Palma (2011) has labeled this phenomenon the homogeneous middle. Variability across countries is actually smaller in the upper- middle deciles (deciles 7 to 9).24 The proportion of total consumption accruing to that group is quite similar in all geographic regions of the world: it ranges from 35.9% in SSA to 37.3% in ECA. The main difference across regions lies in the share of the bottom 16 Alvaredo-Gasparini 60% compared to those in the upper 10%. For example, while the share of deciles 7 to 9 in total consumption is almost the same in ECA (37.3%) and LAC (37.1%), the share of the bottom 60% is more than 7 points higher in the former (36.4% and 29.1%). The correlation coefficients for the decile shares in total consumption provide information about the structure of the distributions across countries (Table 3.4). In a cross-country perspective, gains are highly positively correlated in the first 8 deciles; on the other hand, for decile 10 correlations are all negative and large, except with decile 9, for which the correlation is non-significant. Gains in the participation of the richest 10% are tightly linked to losses in the share of the poorest 80% of the population. The table suggests that when we move up in the ladder of countries according to the share of the bottom deciles, we expect to see gains in the lowest strata obtained mostly against the share of the upper 10% of the population (and not for instance against the middle strata, and in alliance with the rich). 3.3. Inequality in the Gallup World Poll The Gallup World Poll provides new evidence on the international comparisons of income inequality, as it includes identical income and household structure questions applied to national samples in 132 countries. Of course, the reliability of the national inequality estimates in Gallup is lower than those obtained with household surveys, since only one income question is used to approximate well-being, and the sample sizes are considerably smaller. However, Gluzmann (2012) finds that the correlation coefficient between the Gini coefficients computed with Gallup microdata and those reported in the World Development Indicators (WDI) that are based on per capita income is high (0.85). Interestingly, the relationship with the consumption Ginis in WDI is much weaker; the linear correlation coefficient is 0.21, non-significant at 10%. International surveys with similar questionnaires across countries, such as the Gallup World Poll, could hardly be a substitute for household surveys as the main source for distributive analysis at the country level, but they may have a great potential for 17 17 Alvaredo-Gasparini international comparisons of social variables. Future improvements in the quality of these surveys could turn them into a very valuable source for comparative international research. Gasparini and Gluzmann (2012) use microdata from the Gallup World Poll 2006 to compute inequality in each region of the world. According to the unweighted mean of the national income Gini coefficients, Latin America is the most unequal region in the world (excluding Africa, which is not in the sample). The cross-country Gini in Latin America is 49.9, slightly larger than in South Asia (48.9), and Eastern Asia and Pacific (47.1). Countries in Eastern Europe and Central Asia (41.8), North America (39.2) and especially Western Europe (34.0) are the least unequal. Alternatively, regional inequality can be measured by considering each region as a single unit, and computing inequality among all individuals in that unit, after translating their incomes to a common currency - a concept usually labeled global inequality (see chapter 12 of this Handbook). The global Gini in Latin America is 52.5, a value higher than in Western Europe (40.2), North America (43.8) and Eastern Europe and Central Asia (49.8), but lower than in South Asia (53.2) and Eastern Asia and Pacific (59.4). The change in the rankings between the two concepts of inequality is driven by the differences across regions in the heterogeneity among countries in terms of mean income. 3.3. Inequality in the Gallup World Poll Gasparini and Gluzmann (2012) report that the between component in a Theil decomposition accounts for 8% of total regional inequality in Latin America and 32.4% in East Asia and Pacific. 3.4. Top incomes Until the recent developments in the literature of top incomes from tax records (Atkinson and Piketty, 2007, 2010; see also chapter 16 in this volume), inequality research has been mostly based on household surveys, which suffer from several limitations when focusing on the upper end of the distribution. Household surveys are all but ideal for studying top shares: the rich are usually missing from surveys, either for sampling reasons or because they refuse to cooperate with the time-consuming task of completing or answering to a long form. Because extreme observations are sometimes regarded as data “contamination”, the rich may be intentionally excluded or top coded so as to minimize bias problems generated by presumably less-reliable outliers, or to preserve anonymity. Additionally, survey data present severe under- reporting at the top: the richest individuals are more reluctant to disclose their incomes, or have diversified portfolios with income flows that are difficult to value. Székely and Hilgert (1999) look at surveys from eighteen Latin American household surveys and confirm that the ten highest incomes reported are often not much larger than the salary of an average manager in the given country at the time of the survey. In general, the profile of the average individual in the top 10% of the distribution is closer to the prototype of highly educated professionals earning labor incomes, rather than capital owners. Consequently, the inequality that we are able to measure with 18 Alvaredo-Gasparini household surveys can be severely affected, regarding both levels and dynamics, in those cases or periods in which an important part of the story takes place at the top. Tax and register data are being increasingly preferred over surveys in studying distributive issues at the top. In fact, under certain conditions registry data can provide valuable information to improve survey-based estimates. Typically, incomes reported to the surveys are checked against the registers, or incomes are directly taken from administrative sources for the individuals in the sample. Even if the combination of survey and administrative data can be seen as an improvement, there remains the issue of the sampling framework for the top of the distribution.25 In any case, statistics offices in the developing world are not exploiting register data to complement surveys yet. The use of tax statistics is not without drawbacks. 26 The reasons for which the rich and wealthy may be particularly dissuaded from disclosing their fortunes and incomes to authorities in the developing world may go beyond tax concerns, lest the information revealed fall into the wrong hands. Alvaredo and Londoño (2013) report that in Colombia, until recently plagued by high insecurity, anecdotal evidence suggests that during the intense political violence of the 1990s leaked personal tax returns were used by criminal groups to target victims and kidnap for ransom. 25 If high-income individuals are not properly identified in the sample framework, comparing the incomes reported to the surveys against those in the registers one by one is only a partial improvement. In the UK, for example, the ONS scales up the surveys’ incomes so that the surveys’ averages match the average income in tax data. 27 The National Accounts-based measure of household disposable income has been defined as: balance of households’ primary incomes + social benefits other than social transfers in kind − employers’ actual social contributions − imputed social contributions − attributed property income of insurance policyholders − imputed rentals for owner occupied housing − fixed capital consumption – employees’ social security contributions – taxes on income and wealth paid by households. 3.4. Top incomes First, since only a fraction of the population files a tax return, studies using tax data are restricted to measuring top shares, which are silent about changes in the lower and middle part of the distribution. Second, tax data are collected as part of an administrative process and do not seek to address research needs; both income and tax units are defined by the tax laws and vary considerably across time and countries. Third and most important, estimates are affected by tax avoidance and tax evasion; the rich, in particular, have a strong incentive to understate their taxable incomes. These elements, which are common to all countries, become critical in the developing world, characterized by tax systems with low enforcement and multiple legal ways to avoid the tax.26 A number of researchers have addressed the differences in the ability of tax and survey data to represent income inequality, trying to reconcile the evidence using the two sources (see Alvaredo, 2011; Burkhauser et al., 2012 for the US). Unfortunately, and despite multiple requests, at the moment of writing only a few developing countries have made available microdata from the income tax (namely Colombia, Ecuador and Uruguay). Alvaredo and Londoño (2013), and Alvaredo and Cano (forthcoming) show that, in contrast to survey-based results, high-income individuals are, in essence, rentiers and capital owners. This feature differs from the pattern found in several developed countries in recent decades, where it has been shown that the large increase in the share of income going to the top groups has been mainly due to spectacular increases in executive compensation and high salaries, and to a lesser 26 The reasons for which the rich and wealthy may be particularly dissuaded from disclosing their fortunes and incomes to authorities in the developing world may go beyond tax concerns, lest the information revealed fall into the wrong hands. Alvaredo and Londoño (2013) report that in Colombia, until recently plagued by high insecurity, anecdotal evidence suggests that during the intense political violence of the 1990s leaked personal tax returns were used by criminal groups to target victims and kidnap for ransom. 19 19 Alvaredo-Gasparini extent to a partial restoration of capital incomes. 3.4. Top incomes While the working rich have joined capital owners at the top of the income hierarchy in the United States and other English-speaking countries, Colombia and Ecuador remain more traditional societies where the top income recipients are still the owners of the capital stock. Results confirm that incomes reported to the tax authorities are considerably higher than those recorded in the surveys at the top. A natural question, which has received much attention lately, is the extent to which tax data can complement household surveys in examining the level of inequality in developing countries. Alvaredo and Londoño (2013) compare the Colombian household survey with the tax micro-data over the years 2007-2010. The total household income from the survey is 60-65% of the NAS measure of disposable income.27 Such gap cannot be seen as an accurate measure of missing income in household surveys, because both sources are different, but a partial explanation may well be at the top of the distribution. As a simple exercise, these authors replace all the incomes above the percentile 99 in the survey with those from tax data (net of taxes and social security contributions to render both sources comparable), under the assumption that the top 1% is poorly captured in the survey. Two elements are worth mentioning. First, the difference between the adjusted survey’s total income (that is, the survey’s incomes of the bottom 99% plus the net-of-tax incomes from the tax data above the percentile 99) and the NAS figure goes down to 75-80%. Second, the Gini coefficient of individual incomes goes up from 55 to 61 in 2010.28 These findings challenge the general skepticism regarding the use of tax data from developing countries to study inequality. Such estimates should be regarded as a lower bound, to take into account the effects of evasion and under reporting. Nevertheless, they show that incomes reported to tax authorities can be a valuable source of information, under certain conditions that require a case-by-case analysis. 28 These results are still approximations, as defining individual actual incomes from the Colombian tax records is not always straightforward. 29 The Gini for the developed countries is computed over the distribution of income per capita, and not consumption per capita, a fact that probably underestimates the slope of the curve. 30 It is also confirmed estimating GDP with the Atlas method, and using the All the Ginis database. 31 31 The Latin American “excess inequality” is documented in Londoño and Székely (2000); Gasparin Cruces and Tornarolli (2011), and others. 3.5. Inequality and development Is the level of inequality in a country associated to its development stage? In this section we take advantage of a cross-section of national Gini coefficients for year 2010 to take a look at this issue. Of course, this topic is related to the long-lasting debate initiated with the seminal contributions by Lewis (1954) and Kuznets (1955), who argued that the process of industrialization would imply an inverse U pattern for inequality. However, the empirical test for the Kuznets curve requires time-series or 28 These results are still approximations, as defining individual actual incomes from the Colombian tax records is not always straightforward. 20 20 Alvaredo-Gasparini panel data, and not just a cross-section, since it is a hypothesis about the dynamics of an economy over its development process. The causal relationship between development and inequality is the subject of a large literature that has to face numerous empirical challenges, and hence it is far from settled (see Anand and Kanbur, 1993; Fields, 2002; Banerjee and Duflo, 2003; Dominics et al., 2008; and Voitchovsky, 2009 for assessments). In this section we simply document the empirical relationship between these two variables across countries in a recent point in time without exploring the difficult issue of causality. The first panel in Figure 3.4 plots the Gini coefficient for the distribution of consumption per capita against per capita gross national income (GNI).29 The figure seems to reveal a decreasing relationship between inequality and development. The linear correlation coefficient between the Gini coefficient and per capita GNI is -0.56 (statistically significant at the 1% level). An inverse-U shape shows up in the second panel of Figure 3.4, when per capita GNI is presented in logs. However, the increasing segment of the curve covers only very poor Sub-Saharan African countries. The relationship Gini-GNI is decreasing in the range of GNI of most countries in the world. 21 21 Alvaredo-Gasparini Figure 3.4 Inequality and development Per capita gross national income (GNI) and Gini coefficient, 2010 Source: own calculations based on WDI and PovcalNet (2013). 32 In order to compute changes we discard countries in PovcalNet with less than four observations over the period 1981-2010, or with observations concentrated in a narrow time-period. The sample we use for the calculations on trends include 76 countries that represent 88% of the developing world 3.5. Inequality and development Lind and Mehlum test: H0: monotone or U shape; H1: inverse U shape. Lind and Mehlum test: H0: monotone or U shape; H1 4. Inequality: trends In this section we report the recent trends in income inequality in the developing countries. We start laying out the general patterns, and then deep into the evidence for each region. Although most of the section deals with relative inequality, we devote a section to explore patterns for absolute inequality, and a section to document aggregate welfare changes. We end with a brief summary of the methodologies and main issues in the debate on inequality determinants in the developing world. 3.5. Inequality and development 10 20 30 40 50 60 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 50000 Gini coefficient GNI per capita (PPP) Developed EAP ECA LAC MENA SA SSA 10 20 30 40 50 60 5 6 7 8 9 10 11 Gini coefficient log GNI per capita (PPP) Developed EAP ECA LAC MENA SA SSA Inequality and development GNI per capita (PPP) Source: own calculations based on WDI and PovcalNet (2013). The results of the regressions in Table 3.5 and the Lind and Mehlum (2010) test confirm an inverse U shape for the relationship between the Gini coefficient and log GNI per capita in a cross-section of countries.30 The result seems also valid, although becomes considerable weaker, when restricting the sample to developing economies. The turning points implicit in the regressions correspond to around US$ 1800, a value that is lower than the per capita GNI of most developing countries, except for some economies in Sub-Saharan Africa. The inclusion of regional dummies reveals that East Asian, and especially Latin American and Sub-Saharan African countries are particularly unequal, even when controlling for their levels of economic development.31 22 Alvaredo-Gasparini Table 3.5 Relationship between Gini coefficient and GNI per capita Table 3.5 Relationship between Gini coefficient and GNI per capita Note: robust cluster standard errors in brackets. * significant at 10%; ** significant at 5%; *** significant at 1%. Omitted category: Eastern Europe and Central Asia. (i) (ii) (iii) (iv) log GNIpc 24.24 24.44 18.01 26.54 (9.52)** (4.48)*** (8.23)* (6.58)** log GNIpc squared -1.606 -1.409 -1.202 -1.541 (0.552)** (0.34)*** (0.53)* (0.48)** Developed countries -1.416 (2.76) East Asia & Pacific 7.352 7.170 (1.43)*** (1.62)*** Latin America & Caribbean 10.238 10.157 (0.53)*** (0.62)*** Middle East & North Africa 2.334 2.144 (1.28) (1.48) South Asia 1.705 1.515 (1.79) (1.97) Sub-Saharan Africa 13.749 13.660 (2.33)*** (2.34)*** Constant -49.34 -72.10 -61.67 -80.27 (38.69) (13.17)*** (28.64)** (20.06)** Observations 146 146 121 121 R-squared 0.31 0.58 0.07 0.45 Lind and Mehlum test for inverse U shape | t | 2.72 2.31 1.35 2.0 p-value 0.004 0.011 0.089 0.024 All countries Only developing countries Table 3.5 Relationship between Gini coefficient and GNI per capita Relationship between Gini coefficient and GNI per capita * significant at 10%; ** significant at 5%; *** significant at 1%. Omitted category: Eastern Europe and Central Asia. Lind and Mehlum test: H0: monotone or U shape; H1: inverse U shape. population. In order to build a constant sample in a few cases Gini coefficients are imputed assuming constant inequality. Income Ginis in LAC are adjusted as explained in the previous section. 33 This result is in part driven by the lack of information on changes in inequality over this period for several countries in the developing world. See below. 4.1. General changes The available evidence suggests that on average the levels of national income inequality in the developing world increased in the 1980s and 1990s, and declined in the 2000s. Using data from PovcalNet, the mean Gini for the distribution of per capita consumption expenditures increased from 37.2 in 1981 to 39.4 in 2010 (Figure 4.1).32 23 Alvaredo-Gasparini The mean was basically unchanged between 1981 and 1987,33 then increased more than three points to reach a value of 40.5 in 1999, and from 2002 it started to fall, although slowly (from 40.6 in 2002 to 39.4 in 2010). The mean was basically unchanged between 1981 and 1987,33 then increased more than three points to reach a value of 40.5 in 1999, and from 2002 it started to fall, although slowly (from 40.6 in 2002 to 39.4 in 2010). Figure 4.1 Gini coefficient Unweighted mean for developing countries, 1981-2010 Figure 4.1 Gini coefficient Unweighted mean for developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: the national Gini coefficients are computed over the distribution of household consumption per capita. 33 35 37 39 41 43 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Note: the national Gini coefficients are computed over the distribution of household consumption per capita. Figure 4.2 adds to the picture the changes at different percentiles of the distribution of national Ginis. The figure makes clear that on average the changes in the last decades have not been large, compared to the range over which the Gini varies across countries. The picture also reveals that the growth in the mean Gini in the late 1980s and 1990s was mainly due to the substantial increase in the low-inequality countries, in particular Eastern Europe and Central Asia economies after the fall of communism, and also some Asian economies in the early stage of economic take-off. Instead, the fall in the 2000s was widespread, although more intense in those countries above the median, such as those in Latin America. This observation suggests convergence in the levels of inequality in the developing economies. In fact, the standard deviation for the distribution of Gini coefficients substantially fell over time: 11.2 in 1981, 10.1 in 1990, 7.4 in 1999 and 7.2 in 2010. Countries in the developing world are still very different in terms of income inequality but differences have become considerably smaller over the last three decades. 4.1. General changes Figure 4.2 adds to the picture the changes at different percentiles of the distribution of national Ginis. The figure makes clear that on average the changes in the last decades have not been large, compared to the range over which the Gini varies across countries. The picture also reveals that the growth in the mean Gini in the late 1980s and 1990s was mainly due to the substantial increase in the low-inequality countries, in particular Eastern Europe and Central Asia economies after the fall of communism, and also some Asian economies in the early stage of economic take-off. Instead, the fall in the 2000s was widespread, although more intense in those countries above the median, such as those in Latin America. This observation suggests convergence in the levels of inequality in the developing economies. In fact, the standard deviation for the distribution of Gini coefficients substantially fell over time: 11.2 in 1981, 10.1 in 1990, 7.4 in 1999 and 7.2 in 2010. Countries in the developing world are still very different in terms of income inequality but differences have become considerably smaller over the last three decades. 24 Alvaredo-Gasparini Figure 4.2 Gini coefficient g Gini coefficient Unweighted statistics for developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: the national Gini coefficients are computed over the distribution of household consumption per capita. 10 15 20 25 30 35 40 45 50 55 60 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 percentile 10% percentile 90% percentile 25% percentile 75% median mean Gini coefficient Gini coefficient Unweighted statistics for developing countries, 1981-2010 Unweighted statistics for developing countries, 1981-2010 10 15 20 25 30 35 40 45 50 55 60 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 percentile 10% percentile 90% percentile 25% percentile 75% median mean Source: own calculations based on PovcalNet (2013). Note: the national Gini coefficients are computed over the distribution of household consumption per capita. Note: the national Gini coefficients are computed over the distribution of household consumption per capita. 34 We classify countries in groups according to whether the Gini went up or down by more or less than 2.5% in a period. That threshold is certainly arbitrary. A change of 2.5% applied to the mean Gini in the developing world - which is around 40- represents 1 Gini point. A change of 1 point in the Gini coefficient is typically statistically significant, given the sample sizes of the national household surveys. 4.1. General changes A closer inspection of the data reveals that the result of a stable mean Gini in most of the 1980s is driven by the lack of information for several countries, and by a substantial heterogeneity in the changes of those with information (Table 4.1).34 The strong rise in the mean Gini in the 1990s is associated to a large proportion of countries with growing inequality in a framework of much improved information. The tide seems to have turned in the 2000s, when most of the countries in the sample experienced a fall in inequality. But even in this decade of widespread social improvement, the country performances in terms of inequality reduction were quite heterogeneous. In fact, in 20% of the economies of the developing world the Gini coefficient increased between 2002 and 2010, while in 15% of the countries the changes were smaller than 2.5%. 25 Alvaredo-Gasparini Alvaredo-Gasparini Table 4.1 Proportion of countries classified in groups according to the change in the Gini coefficient Source: own calculations based on PovcalNet (2013). Note: “Fall” includes countries where the Gini fell more than 2.5% in the period, “Increase” include countries where the Gini rose more than 2.5%, “No change” includes countries where the Gini changed less than 2.5%, “No information” includes countries without two independent observations in each period. 1981-1990 1990-2002 2002-2010 Fall 14.7 22.7 65.3 No change 21.3 16.0 14.7 Increase 34.7 60.0 20.0 No information 29.3 1.3 0.0 Total 100.0 100.0 100.0 Proportion of countries classified in groups according to the change in the Gini coefficient Source: own calculations based on PovcalNet (2013). 1981-1990 1990-2002 2002-2010 Fall 14.7 22.7 65.3 No change 21.3 16.0 14.7 Increase 34.7 60.0 20.0 No information 29.3 1.3 0.0 Total 100.0 100.0 100.0 ( ) Note: “Fall” includes countries where the Gini fell more than 2.5% in the period, “Increase” include countries where the Gini rose more than 2.5%, “No change” includes countries where the Gini changed less than 2.5%, “No information” includes countries without two independent observations in each period. The bulk of the countries in the sample (64%) experienced a change in the pattern of inequality around the turn of the century, from non-falling to decreasing inequality, while only a few experienced a pattern of continuous increasing (12%) or decreasing (15%) disparities over the two decades. 4.1. General changes The inverse-U shape for the inequality pattern observed for many economies in the developing world could be consistent with the Kuznets story of economic growth for countries located close to the curve turning point. However, we fail to find any significant correlation between the type of the inequality pattern and different measures of development and growth. The inverse-U pattern in the period 1981-2010 appears to have been common to a wide range of economies (more on this below). The growth in the population-weighted mean of the Gini coefficient across developing countries was stronger than the increase in the unweighted mean (Figure 4.3). While the latter increased 2.2 points in the period 1981-2010, the former jumped 7.5 points. The gap between the two means shrunk from 5.4 points in the early 1980s to almost zero in the late 2000s. This pattern is mainly accounted by the dramatic surge in income inequality in China over the period. Interestingly, the fall in the unweighted mean Gini in the 2000s does not show up in the weighted mean, mainly because of the increase in inequality in China (also in India and Indonesia, but to a lesser extent). Although the Gini coefficient for a typical developing country significantly decreased in the 2000s, the national Gini for a typical person in the developing world did not fall. 26 Alvaredo-Gasparini Figure 4.3 Gini coefficient Weighted and unweighted means Developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: the Gini coefficients are computed over the distribution of household consumption per capita. 30 32 34 36 38 40 42 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 unweighted weighted Figure 4.3 Gini coefficient Weighted and unweighted means Developing countries, 1981-2010 Figure 4.3 Gini coefficient Weighted and unweighted means Developing countries, 1981-2010 ( ) Note: the Gini coefficients are computed over the distribution of household consumption per capita. In the rest of this section we go beyond the Gini coefficient and track changes along the distribution. In Figure 4.4 each point in a growth-incidence curve (GIC) indicates the unweighted mean across countries in the annual rate of growth of real consumption per capita (in PPP US$) for a given decile of the national distributions.35 There is a stark contrast in the GIC corresponding to the 1990s and the 2000s. 35 The GIC depicted in Figure 4.4 is not the world growth-incidence curve, where for instance decile 1 would include the poorest 10% of the world population. 4.1. General changes The first one is clearly increasing, suggesting growing inequalities, while the second is decreasing (and flatter), indicating a fall in well-being disparities in the 2000s. On average, in that decade consumption per capita grew by more than annual 4% in the three bottom deciles of the national distributions and by 3% in the top decile. 27 27 Alvaredo-Gasparini Figure 4.4 Growth-incidence curves Annualized growth rate in consumption per capita by decile Unweighted mean for developing countries Source: own estimates based on PovcalNet (2013). Note: annual change in consumption per capita (PPP US$). 1990s=1990-2002, 2000s=2002-2010. -3 -2 -1 0 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 Deciles 1990s 2000s Figure 4.4 Growth-incidence curves Annualized growth rate in consumption per capita by decile Unweighted mean for developing countries 9 10 Deciles Deciles Source: own estimates based on PovcalNet (2013). Note: annual change in consumption per capita (PPP US$). 1990s=1990-2002, 2000s=2002-2010 annual change in consumption per capita (PPP US$). 1990s=1990-2002, 2000s=2002-2010. Naturally, the contrast between decades is also evident when looking at income shares. The results are summarized in Figure 4.5: while the share of the bottom 60% fell 2 points in the 1990s and increased 0.9 points in the 2000s, the performance of the top 10% was almost the exact mirror: increased 2.4 points in the 1990s and fell 1 point in the following decade. The share of the “middle” (deciles 7 to 9) has remained quite stable over the two last decades (36.9 in 1990, 36.5 in 1999 and 36.6 in 2010). This stratum seems not only quite homogeneous across countries but also over time (Palma, 2011). Figure 4.5 Decile shares Unweighted mean for developing countries, 1990-2010 Source: own estimates based on PovcalNet (2013). Note: the decile shares are computed over the distribution of household consumption per capita. 28 30 32 34 1990 1993 1996 1999 2002 2005 2008 2010 Deciles 1 to 6 Decile 10 Figure 4.5 Decile shares Unweighted mean for developing countries, 1990-2010 Unweighted mean for developing countries, 1990 2010 S i b d P lN (2013) 28 30 32 34 1990 1993 1996 1999 2002 2005 2008 2010 Deciles 1 to 6 Decile 10 Source: own estimates based on PovcalNet (2013). ( ) Note: the decile shares are computed over the distribution of household consumption per capita. 36 We prefer not to report the regional patterns before 1990 since the number of observations is small in several regions. The numbers of countries by region in the sample we use to assess inequality trends are: 8 in EAP, 20 in ECA, 19 in LAC (all in Latin America, none from the Caribbean), 5 in MENA, 4 in SA and 20 in SSA. 4.1. General changes ( ) Note: the decile shares are computed over the distribution of household consumption per capit 28 28 Alvaredo-Gasparini Alvaredo-Gasparini 4.2. Changes by region Changes in inequality have been heterogeneous across the six geographical regions of the developing world (Figure 4.6).36 The mean Gini coefficient in Latin America increased more than two points in the 1990s, and then dropped in the 2000s by a larger amount. The data reveals almost no change in inequality in Sub-Saharan Africa over the two last decades and some decline in the five MENA countries included in the sample. Instead, the Gini coefficient increased more than two points in Asia, and more than six points in Eastern Europe and Central Asia. Figure 4.6 suggests again some pattern toward convergence: the gaps in inequality among regions in the developing world are smaller now than two decades ago. For instance, while the gap in the Gini coefficient between Latin America and ECA was 18 points in the early 1990s, it shrank to 11 points in the late 2000s. Figure 4.6 Gini coefficients Unweighted means by region, 1990-2010 Figure 4.6 Gini coefficients Unweighted means by region, 1990-2010 Source: own estimates based on PovcalNet (2013). Note: the Gini coefficients are computed over the distribution of household consumption per capita. 25 30 35 40 45 50 1990 1993 1996 1999 2002 2005 2008 Gini coefficient Latin America Middle East & North Africa East Asia & Pacific South Asia Eastern Europe & Central Asia Sub-Saharan Africa Latin America Sub-Saharan Africa Middle East & North Africa Eastern Europe & Central Asia Source: own estimates based on PovcalNet (2013). Note: the Gini coefficients are computed over the distribution of household consumption per capita. Note: the Gini coefficients are computed over the distribution of household consumption per ca In all regions the share of countries with falling inequality rose in the 2000s, as compared to the 1990s. The two most remarkable changes in the pattern occurred in Latin America, and in Eastern Europe and Central Asia. While the Gini went down in 26% of the LA economies in the 1990s, that share increased to 95% in the 2000s. In ECA while the growth in inequality was generalized in the 1990s, more than half of the countries experienced reductions in the 2000s. 36 We prefer not to report the regional patterns before 1990 since the number of observations is small in several regions. 4.2. Changes by region The numbers of countries by region in the sample we use to assess inequality trends are: 8 in EAP, 20 in ECA, 19 in LAC (all in Latin America, none from the Caribbean), 5 in MENA, 4 in SA and 20 in SSA. 29 Alvaredo-Gasparini Using data from PovcalNet, Chen and Ravallion (2012) report changes in the within component of the global mean-log deviation between 1981 and 2008. This within component is a population-weighted measure of the national inequalities. They find substantial increases in East Asia and Pacific (from 0.125 to 0.256) and Eastern Europe and Central Asia (from 0.128 to 0.225), smaller increases in South Asia (from 0.156 to 0.181), Latin America and the Caribbean (from 0.541 to 0.561) and sub-Saharan Africa (from 0.338 to 0.347) and a fall in MENA (from 0.256 to 0.215). Bastagli et al. (2012) report similar patterns using data from PovcalNet, SEDLAC and LIS. The picture of national inequalities in the developing world is similar when using other databases. For instance, the unweighted mean Gini in the All the Ginis database assembled by Milanovic grew from 36.2 in 1990 to 40.7 in 1999 and then dropped to 39.7 by 2005. While in the 1990s inequality rose in 63% of the economies in the ATG database, that share dropped to 35% in the 2000s. The recorded increase in the 1990s was generalized across regions, but especially intense in Eastern Europe and Central Asia (9 Gini points), while the fall in the 2000s was larger in MENA and Latin America. Cornia and Kiiski (2001), Cornia (2011) and Dhongde and Miao (2013) document similar results using WIID data. We find that the linear (rank) correlation coefficient for the change in the Gini coefficient between 1990 and 2005 recorded in PovcalNet and WIID is 0.776 (0.868), significant at 1%. The corresponding values for the comparison between PovcalNet and ATG are 0.721 and 0.765. The evidence drawn from the EHII database is also roughly consistent with the patterns discussed above. The mean Gini for the developing world remained almost unchanged in the 1980s, increased in the 1990s from 42.5 in 1990 to 47.0 in 1999, and dropped to 46.5 in 2002 (the latest available date).37 While in 62% of the countries inequality increased in the early 1990s, that share dropped to 55% between 1993 and 1999, and to 49% between 1999 and 2002. 37 These estimates are computed dropping countries with few observations in the period. 4.2. Changes by region The regional patterns are roughly consistent with those described above. The main difference is that EHII reveals a dramatic increase in inequality in the Middle East and North Africa (7 Gini points) that is not present in the evidence drawn from household surveys. In the rest of this section we briefly review the literature on inequality changes in each geographic region of the developing world, while we take a closer look to the story of some particular cases: Brazil, China, India, Indonesia and South Africa. East Asia and Pacific The inequality patterns in East Asia and Pacific can be traced based on information from only 8 out of the 24 countries in the region, which nonetheless represent 96% of its total population. This set includes Cambodia, China, Indonesia, Lao PDR, Malaysia, Philippines, Thailand and Vietnam. There is scattered evidence for Fiji, Micronesia, Mongolia and Timor-Leste, while information is either lacking or too scarce for 30 30 Alvaredo-Gasparini Alvaredo-Gasparini American Samoa, Kiribati, Korea, Dem. Rep., Marshall Islands, Myanmar, Palau, Papua New Guinea, Samoa, Solomon Islands, Tonga, Tuvalu and Vanuatu. American Samoa, Kiribati, Korea, Dem. Rep., Marshall Islands, Myanmar, Palau, Papua New Guinea, Samoa, Solomon Islands, Tonga, Tuvalu and Vanuatu. The slightly increasing pattern showed in Figure 4.6 for the unweighted mean of the consumption Gini in EAP hides important differences across countries (ADB, 2012; Chusseau and Hellier, 2012; Ravallion and Chen, 2007; Sharma et al., 2011; Solt, 2009; Zin, 2005). Consumption inequality increased in most economies in the region during the 1990s, with the exception of Thailand and Malaysia. The increase was particularly strong in China, where the consumption Gini climbed around 7 points in that decade. The performance in the 2000s was more heterogeneous: inequality continued increasing in China, Lao PDR and Indonesia, and also went up in Malaysia, while there is evidence pointing to a fall in consumption inequality in Cambodia, Philippines, Thailand and Vietnam. Overall, considering the two decades, EAP combines countries with systematic increases in inequality (China, Indonesia, Lao PDR), several cases in which inequality had a cyclical pattern, ending in 2010 at similar levels than in 1990 (Cambodia, Malaysia, Philippines and Vietnam), and only one successful story of consistent reduction in consumption inequality: Thailand, for which the estimated reduction in the Gini coefficient exceeded five points; from 45.3 to 39.4 over the last two decades. Universal social policies, including basic education and health, have been stressed by many authors as significant drivers of that fall (Jomo and Baudot 2007). Probably the most striking phenomenon regarding inequality in EAP was the strong rise that took place in the two most populous countries of the region, China and Indonesia: the Gini coefficient went up around five points in Indonesia and more than ten points in China over the last two decades. Such dynamics happened in a context of high growth and falling poverty, most notably in China. Sharma et al. East Asia and Pacific (2011) summarize the main factors behind these changes: (i) the realignment of activity away from agriculture and towards industry and services; (ii) the skill premium rose due to the unmatched growing demand for skills, and even the emigration of skilled workers; (iii) inequalities in educational attainment in secondary and tertiary schooling have become an increasingly important source of unequal incomes; (iv) a lack of infrastructure linking urban areas with rural areas and other barriers to labor mobility have further accentuated inequality; (v) in China most of income inequality (40 per cent) is now accounted for by rural-urban differences in income levels, and the concentration of growth in urban areas is creating a rural-urban divide in employment and earning possibilities, exacerbated by the much more rapid development that occurred in coastal areas. The interested reader is referred to the boxes on China and Indonesia. China China China Since 1978, when China started the pro-market reforms, GDP has increased at an average rate close to 10 per cent, and household per capita income has grown more Since 1978, when China started the pro-market reforms, GDP has increased at an average rate close to 10 per cent, and household per capita income has grown more 31 31 Alvaredo-Gasparini than 7 per cent per year. Such remarkable economic transformation has been accompanied by important changes in inequality and poverty. China is a successful story of reduction in absolute poverty (World Bank, 2009; Ravallion and Chen, 2007, 2008; Minoiu and Reddy, 2008). Chen and Ravallion (2008) find that between 1981 and 2005 the number of poor people (with a poverty line of $1.25) went down from 839 to 204 million in terms of consumption, and from 816 to 135 million in terms of income. However, it is argued that it will be harder for China to maintain its past rate of progress against absolute poverty without addressing the problem of rising inequality. The increase in income inequality in China over the last three decades has been widely documented. Ravallion and Chen (2007) and World Bank (2009) show that income inequality rose from the mid-1980s through 1994, dipping a bit in the late 1990s, and then edging upward thereafter. The Gini coefficient rose from 25 in 1981 to 39 in 1999. Li et al. (2013), among others, document an additional increase in the 2000s, a pattern explained by the widening of the rural-urban income gap, and the increase in income from property and assets, driven by the development of urban residential real-estate markets, the expansion of stock and capital markets, the growth of private enterprises, and other property rights. In marked contrast to most developing countries, relative inequality is higher in China’s rural areas than in urban areas. However, there has been convergence over time with a steeper increase in inequality in urban areas Absolute inequality has increased appreciably, both between and within both urban and rural areas. Indonesia During the thirty years before the Asian crisis of 1997-1998, which coincided with the New Order under Suharto’s dictatorship, Indonesia GDP grew at an average rate of 7% per year, while per capita GDP increased by 3.5 times. The process was not smooth and went through different phases that implied immense structural change. Agriculture was the main economic sector in the 1970s and benefited from the introduction of high yielding variety seeds and other technical progress; the oil commodity boom (1974-1981) generated a windfall income that allowed for the subsidization of rice production and the improvement of the country’s infrastructure, with positive effects on life expectancy at birth and school enrollment rates. The period of deregulation and external oriented policies after 1985 set the basis for the development of the manufacturing sector. Despite problems with the data, scholars agree in that there was a systematic drop in poverty rates between 1976 and 1997. At the same time, overall consumption inequality in Indonesia did not change markedly with development until the late 1980s. On the contrary, the household per capita consumption Gini displayed a falling trend between 1964 and 1987 (from 35 to 32). Subsequently, although urban inequality increased, this was partly offset by declines in rural inequality. Cameron (2002) argues that Indonesia can be considered to be ‘lucky’ in the sense that its 32 Alvaredo-Gasparini industrial center happens to be close to rural Java where many of the country’s poorest families make their home. These households benefited from the off-farm employment opportunities that industrialization offered. Alatas and Bourguignon (2000) found that the returns to land size decreased between 1980 and 1996; opportunities for off-farm earnings for rural households also contributed to falling rural inequality. Inequality (but not poverty) started to rise seven years before the crisis. Alatas and Bourguignon (2000) decompose such increase between components associated with changes in the structure of earnings, changes in occupational choice and changes in the socio-demographic structure of the population. They find as main explanations the migration from rural to urban areas and the increase in non-farm self-employed work. Indonesia was severely hit by a financial crisis: in 1997-1998 GDP dropped by 15%. This turned into a sharp decrease in inequality and an increase in poverty. Skoufias and Suryahadi (2000) find that this pattern seems to have arisen from a decrease in regional inequality. Indonesia Urban areas (which tend to be wealthier than neighboring rural areas) were hit harder, and the urban middle class, who lost their formal sector jobs, was harshly affected. As the crisis reduced the per capita expenditure of households, the percentage reduction was probably less among the poorer population than among the less poor population. Since 2001, and along with the process of decentralization of powers to local authorities, a general pattern of rising consumption inequality has been observed: the Gini increased by 5 points from 33 in 2002 to 38 in 2010. Miranti et al. (2013) suggest that the recent increases in inequality may be linked to the higher share of workers employed in the informal sector (70 per cent), hence not covered by minimum wage legislation or employment protections. 38 Much information also existed about pre-1990 earnings and household incomes in (former) Czechoslovakia, Hungary and Poland (Atkinson and Micklewright, 1992) 39 Milanovic and Ersado (2010); Ivaschenko (2002); Ferreira (1999); Milanovic (1998); Cornia (1996); Cornia and Reddy (2001); Mitra and Yemtsov (2006). 39 Milanovic and Ersado (2010); Ivaschenko (2002); Ferreira (1999); Milanovic (1998); Cornia (199 Cornia and Reddy (2001); Mitra and Yemtsov (2006). 38 Much information also existed about pre-1990 earnings and household incomes in (forme Czechoslovakia, Hungary and Poland (Atkinson and Micklewright, 1992) 39 Eastern Europe and Central Asia The transition from central planning to market system in the countries of Eastern Europe (EE) and those belonging to the former Soviet Union (FSU) had profound socioeconomic impacts, which could be better documented (compared to the situation in previous decades) due to the improvements in the production and release of information by the new administrations (Milanovic, 1998).38 The fall of the communist regimes was followed by a substantial increase in inequality in almost all countries.39 According to PovcalNet data the mean Gini for the distribution of per capita consumption expenditures grew from 26.4 in 1990 to 31.9 in 1996. The increase in the first half of the 1990s was particularly strong in those countries belonging to the FSU and in Southeast Europe, and somewhat milder in those economies that joined the European Union. Such developments have been linked to the process of privatization, 33 Alvaredo-Gasparini which implied an increase in earnings dispersion in comparison to the more compressed wage structure of the state-owned firms. One key characteristic of the planned economies was the imposition of wage “grids” that forced a wage compression; the fast transition from wage setting under the wage grids toward a less regulated labor market provoked a rise in the returns to education, and hence a surge in inequality.4041 The economic liberalization also triggered changes in the sectorial structure of the economy; in particular the ensuing de-industrialization during the transition is linked to an increase in inequality (Ferreira, 1999, Milanovic, 1999, Ivaschenko, 2002; Birdsall and Nellis 2003). Milanovic and Ersado (2010) highlight the role played by the inception or increase of tariffs for utilities, while Standing and Vaughan-Whitehead (1995) point to the weakening of the minimum wage as key factors behind the increase in inequality. After the initial surge in the early 1990s, inequality continued growing in the region in the second half of the 1990s although at slower rates. The patterns were more heterogeneous in the 2000s; inequality increased in some economies, but went down in most countries, especially those in the FSU (World Bank, 2005). The mean Gini for Eastern Europe and Central Asia in the late 2000s was lower than in the late 1990s but still significantly higher (around 7 Gini points) than before the transition.42 40 See Fleisher, Sabirianova and Wang (2005) for a study of 10 transition economies and Gorodnichenko and Sabirianova (2005) for the cases of Russia and Ukraine. 41 In Hungary, for instance, the income share accrued by the top 1% almost doubled between 1992 and 2009, from 6-7% to 12%. Half of the increase was due to capital income, while the other half to increased earnings (Mavridis and Mosberger, forthcoming). 42 The reader is referred to chapter 20 for a survey of cross-country studies on the multiple causes of inequality in OECD, including many countries in Eastern Europe. 40 See Fleisher, Sabirianova and Wang (2005) for a study of 10 transition economies and Gorodnichenko and Sabirianova (2005) for the cases of Russia and Ukraine. 41 In Hungary, for instance, the income share accrued by the top 1% almost doubled between 1992 an 2009, from 6-7% to 12%. Half of the increase was due to capital income, while the other half increased earnings (Mavridis and Mosberger, forthcoming). 42 The reader is referred to chapter 20 for a survey of cross-country studies on the multiple causes inequality in OECD, including many countries in Eastern Europe. and Sabirianova (2005) for the cases of Russia and Ukraine. 41 In Hungary, for instance, the income share accrued by the top 1% almost doubled between 1992 and 2009, from 6-7% to 12%. Half of the increase was due to capital income, while the other half to increased earnings (Mavridis and Mosberger, forthcoming). 42 The reader is referred to chapter 20 for a survey of cross-country studies on the multiple causes of inequality in OECD including many countries in Eastern Europe Latin America and the Caribbean All Latin American countries carry out household surveys that provide information to obtain estimates of poverty and inequality. All regular surveys in the region include income questions; still in a few of them consumption expenditures are reported, as well. The increasing availability of surveys in Latin America allowed the creation of databases that make efforts to standardize the generation of poverty and inequality statistics, favoring a close monitoring of the social and labor situation in the region (SEDLAC by CEDLAS and the World Bank, and BADEINSO by UN´s ECLAC). In contrast, the situation in the Caribbean is much less favorable, as surveys are isolated and information is not easy to access. In fact, trends shown in the literature and in this section are restricted to Latin America, which represents 94% of total LAC population. Latin America experienced two distinct distributive patterns in the last three decades (IDB, 1999; de Ferranti et al., 2004; Lopez Calva and Lustig, 2010; Gasparini et al., 2011). During the 1980s, 1990s, and the crises at the turn of the century, income inequality soared in most countries for which comparable data are available. The mean Gini for the distribution of household per capita income crawled from 50.1 in 1980 to 34 34 Alvaredo-Gasparini 51.5 in 1986, 51.9 in 1992, 53.0 in 1998 and 53.4 in 2002 (Gasparini et al., 2013). The frequent macroeconomic crises that hit the region in that period were unequalizing because the poor were less able to protect themselves from high and runaway inflation, and adjustments programs frequently hurt the poor and the middle-ranges disproportionately (Lustig, 1995). The market-oriented reforms that started in Chile in the 1970s and became widespread in the region in the 1990s were associated with rising inequality, although this pattern had a notable exception in the case of Brazil (López Calva and Lustig, 2010). In most countries employment reallocations brought about by trade liberalization and the skilled-biased technical change associated to the modernization of the economy implied a sizeable reduction in the demand for unskilled labor, which led to higher inequality. In some countries adjustments that led to a contraction in the demand for labor affected unskilled workers disproportionately. All these changes took place in a framework of weak labor institutions and safety nets, and hence their consequences made a full impact on the social situation (Gasparini and Lustig, 2011). 43 See Barros et al. (2010), Ferreira et al. (2007), Foguel and Azevedo (2007), Hoffmann (2006) and Langoni (2005). Latin America and the Caribbean Starting in the late 1990s in a few countries and in the early 2000s for the rest, inequality began to decline. The mean Gini for the distribution of household per capita income dropped from 53.4 in 2002 to 50.9 in 2008 (Gasparini et al., 2013). Updated SEDLAC and BADEINSO statistics suggest that the downward trend continued, and that at the time of writing (2013) the mean income Gini in the region would be again at around the value of the early 1980s. The evidence, in fact, indicates that between 2002 and 2012, income inequality went down in all Latin American economies. This remarkable decline appears to be driven by a large set of factors, including the improved macroeconomic conditions that fostered employment, the petering out of the unequalizing effects of the reforms in the 1990s, the expansion of coverage in basic education, stronger labor institutions, the recovery of some countries from severe unequalizing crises and a more progressive allocation of government spending, in particular monetary transfers. The empirical evidence on the driving factors of the recent fall in inequality is, however, still scarce and fragmentary (López Calva and Lustig, 2010; Gasparini and Lustig, 2011; Cornia, 2011). Brazil For decades Brazil was singled out as the most unequal economy in Latin America, and in some rankings even the most unequal in the world. In the late 1980s the Gini coefficient for the distribution of household per capita income reached values higher than 60. But from that point on inequality started to decrease, first slowly in the 1990s, and then more dramatically in the 2000s.43 By 2011 the Gini reached an unprecedented low value of 52.7, several points below the level of some other Latin 44 These values are taken from SEDLAC (2013). All sources confirm the strong decreasing pattern in inequality in Brazil. 45 For evidence and discussion on inequality in the 1990s see Ferreira and Paes de Barros (1999), and Ferreira et al. (2006). Brazil For decades Brazil was singled out as the most unequal economy in Latin America, and in some rankings even the most unequal in the world. In the late 1980s the Gini coefficient for the distribution of household per capita income reached values higher than 60. But from that point on inequality started to decrease, first slowly in the 1990s, and then more dramatically in the 2000s.43 By 2011 the Gini reached an unprecedented low value of 52.7, several points below the level of some other Latin 35 Alvaredo-Gasparini American economies (e.g. Honduras, Colombia, Bolivia).44 Brazil - the fifth most populous nation in the world - is still a high-inequality country, but it stands out as a successful case of consistent reduction of income disparities. American economies (e.g. Honduras, Colombia, Bolivia).44 Brazil - the fifth most populous nation in the world - is still a high-inequality country, but it stands out as a successful case of consistent reduction of income disparities. Data from the Brazil´s national household survey (PNAD) reveals a drop in the Gini of 2 percentage points in the late 1970s, and no systematic changes for most of the 1980s, until a deep macroeconomic crisis hit the country, pulling inequality to unprecedented levels. The Gini went up from 59.2 in 1986 to 62.8 in 1989 and returned to 59.9 in 1993. During the 1990s the Gini moved down very slowly, decreasing by just 1 percentage point between 1993 and 2001. That pace drastically increased in the 2000s: the Gini went down from 58.8 in 2001 to 52.7 in 2011, averaging a fall of 0.6 percentage points a year. During ten years per capita income of the poorest 10% of the Brazilian population grew at an average annual rate of 7%, almost three times the national average. In an in-depth study of the determinants of inequality changes in Brazil, Barros et al. (2010) highlight the role played by the sharp fall in earnings inequality and the substantial increase in public transfers as the two main direct determinants of the decline in income disparities since the early 2000s.45 They find that half of the reduction of inequality in labor incomes was associated to the educational progress that took place over the previous decade, that significantly increased the ratio between skilled and unskilled workers. Brazil The average years of education for the adult population grew 22% in the 2000s and the Gini coefficient computed over the distribution of that variable fell 23%, values well above the mean for Latin America (Cruces et al., 2013). Using different decomposition techniques, Barros et al. (2010) and Azevedo et al. (2011) find a sizeable impact of the fall in the returns to education on earnings inequality. Several authors have also found a reduction in spatial and sectorial labor market segmentation. Although it is not yet clear which factors are behind these trends, the evidence seem to point to a relatively faster expansion of some productive sectors in the Brazilian agricultural ‘hinterland’ as opposed to the metropolitan/industrial areas (Lustig et al., 2012). The substantial increase in the minimum wage -68% in real terms between 2002 and 2010 (CEPAL, 2011)- is also underlined as one important force behind the fall in household income inequality, given that the minimum wage sets the floor for both unskilled workers earnings and for social security benefits. The strong expansion of public transfers accounts to a large share of the fall in income inequality in Brazil (Azevedo et al., 2011; Barros et al., 2010; Bérgolo et al., 2011; Lustig et al., 2012). The main force was the rapid expansion in the coverage of government cash transfers targeted to the poor, mainly a transfer to the elderly and disabled 36 36 Alvaredo-Gasparini (Benefício de Prestação Continuada) and Brazil’s signature conditional cash transfer program Bolsa Família.46 y ( ) ( ) 47 See Acar and Dogruel (2012), Adams and Page (2003), Bibi and Nabli (2010), Iqbal (2006), Page (2007), and Salehi-Isfahani (2010). 48 According to Iqbal (2006) reliable pre-1985 household surveys are only available for Tunisia and Egypt. 47 See Acar and Dogruel (2012), Adams and Page (2003), Bibi and Nabli (2010), Iqbal (2006), Page (2007 and Salehi-Isfahani (2010). 46 See Fiszbein and Schady (2009) and Veras Soares (2007). 47 46 See Fiszbein and Schady (2009) and Veras Soares (2007). 47 ( ) 48 According to Iqbal (2006) reliable pre-1985 household surveys are only available for Tunisia and Egyp 49 “In many MENA countries, from the Maghreb to the Arabian peninsula, power is wielded by rather narrow groups… Seen from this perspective, the most puzzling thing about inequality in the Middle East is how low it is” (Robinson, 2009). Middle East and North Africa These values place MENA as a region of moderate inequality within the developing world, a fact that have puzzled some authors, that would predict higher 37 Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo-Gasparini income disparities given the political process and the balance of political power in those societies.49 income disparities given the political process and the balance of political power in those societies.49 Middle East and North Africa Data constraints are particularly limiting when analyzing distributive issues in Middle East and North Africa. The lack of accessible and comparable household surveys makes it difficult even to identify the extent of poverty and inequality in most MENA countries. The oil-rich economies (Bahrain, Kuwait, Oman, Qatar, United Arab Emirates, and Saudi Arabia) enjoy high levels of per capita income, and are usually not included into the analysis of the developing world. In any case, distributive data is rarely available for these economies. A second group, by far the largest in terms of population, consists of middle income countries. Within this group there are no public accessible information for Lebanon and Libya, just one data point for Djibouti, Iraq, Syrian Arab Republic, West Bank and Gaza, and only a few for Algeria and Yemen. In sum, the only MENA countries for which it is possible to track changes in poverty and inequality over time are Egypt, Iran, Jordan, Morocco and Tunisia, but even in these cases data is scattered and often of low-quality. MENA has a long way to go to build a reliable, comparable and sustainable system of household surveys and distributive statistics. Despite this constraint, several studies shed some light about the trends in inequality in this region.47 Authors coincide in dividing the last four decades into three periods. The first one, spanning until 1985, was characterized by rapid economic growth. Page (2007) reports a substantial reduction in income inequality between the mid-1970s and the early 1990s.48 Data from PovcalNet confirms that fall, although the magnitude is more modest. “Middle Eastern economies entered their rapid growth period with income distributions that were becoming more egalitarian, reflecting the political ideology and policies of post-colonial governments” (Page, 2007). The second period covers the late 1980s and most of the 1990s, and is characterized by low economic growth, and meager or no social gains: real per capita incomes increased by less than 1.5% per year, while income distributions were rather stable. The downward pattern in inequality appeared to have resumed in the 2000s, although at a slow pace. According to our estimates based on PovcalNet the mean Gini fell from 38.7 in 2002 to 36.8 in 2010. South Asia South Asia has been a region of low inequality for developing world standards, though rising since the early 1990s. In India, further discussed in a separate box, the consumption Gini moved from 30.8 in 1993 to 33.9 in 2010. Bangladesh displayed relatively low inequality throughout the 1980s (Gini equal to 26.1 in 1984), but the situation worsened since the beginning of the 1990s: the Gini climbed to 32.1 by 2010. Khan (2008) argues that incomes from non-farm sources and the high concentration of land tenure have all been disequalizing forces, while the positive effects of the more- evenly distributed farm income were offset partly by its declining share in total income. Scholars do not always agree about the distributive changes in Pakistan; PovcalNet helps defining the picture by providing consumption Ginis of 33.2 for 1990, 28.7 for 1996 and 30.0 for 2008. The high economic growth during the 1980s contributed to a sharp decline in poverty, but accompanied by a mild increase in inequality. The fall in economic growth during the 1990s resulted in a rise in poverty while inequality decreased modestly. According with Akmal (2008) in Pakistan there is an institutional structure that excludes a large proportion of the population from the process of economic growth as well as governance. Sri Lanka experienced rising inequalities between 1985 (Gini of 32.5) and 2007 (Gini 40.3) –among the highest increase in the region during the period of free market reforms, integration to the world markets and high growth–, with a reversal of the trend towards 2010 (Gini of 38.3) and persistent regional disparities due to conflict. Nepal presents similar dynamics. Gosh (2012) notes that rising inequality reflects two components: first, growing vertical inequality within the modern industrial sector driven by the returns to skill; and second, increasing disparities between the industrial fast-growing sector and the traditional agricultural activities. India Chakravarty (1987) argues that even if policymakers in India adopted a development strategy based on central planning over the forty years following independence, “there was a tolerance towards income inequality, provided it was not excessive and could be seen to result in a higher rate of growth than would be possible otherwise.” One of the explicit goals of the socialist program was to limit the economic power of the elite in the context of a mixed economy. From the mid-1980s, however, India gradually Chakravarty (1987) argues that even if policymakers in India adopted a development strategy based on central planning over the forty years following independence, “there was a tolerance towards income inequality, provided it was not excessive and could be seen to result in a higher rate of growth than would be possible otherwise.” One of the explicit goals of the socialist program was to limit the economic power of the elite in the context of a mixed economy. From the mid-1980s, however, India gradually 38 Alvaredo-Gasparini adopted market-oriented economic reforms. Initially, these were accompanied by an expansionist fiscal policy involving allocations to rural areas, in order to counterbalance the negative redistributive effects of the liberalization. The speed of reforms accelerated during the early 1990s; as in many developing countries influenced by the Washington consensus, the focus shifted away from state intervention towards liberalization, privatization and globalization. Most analysis on inequality in India over the last three decades are based on the observations from the expenditure surveys conducted in 1983, 1987/8, 1993/94, 2004/05 and 2009/10 for urban and rural areas, which have allowed for an analysis pre and post reforms (see Figure 4.10 below). Inequality has increased significantly in the post-liberalization years, and this was much more pronounced in the urban areas; on the contrary, estimates of absolute poverty measures have systematically fallen since 1983. 50 According to Mazundar (2012) the reasons for this pattern of growth are to be found not in the tertiary sector but in the Indian manufacturing sector, which has been characterized by a dualistic size distribution where middle-size firms are missing. Sub-Saharan Africa Although recently there have been many improvements, the lack of a consistent body of household surveys undermine the assessment of income inequality in Sub-Saharan Africa. Time series data on inequality is severely lacking in most SSA countries, hindering the inferences about trends in the region. For example, in PovcalNet some SSA countries are missing (Equatorial Guinea, Eritrea, Mauritius, Somalia, Zimbabwe), while for thirteen of them there is only one observation in the database for the whole period 1981-2010. In fact, very few countries have reliable surveys in the 1980s, and it is not until the mid-1990s when inequality could be really traced with some confidence in the region. Regional studies typically report a mixed picture, with both increases and decreases in inequality (e.g. ECA, 2004), a fact that could reflect the heterogeneity in the region, but also could be caused by noise in the country estimations (Christiansen et al., 2002; Okojie and Shimeles, 2006). Bigsten and Shimeles (2003) for instance report that for 17 African countries the trend in inequality shows significant variations over short periods, causing concern about measurement problems. The available evidence seems to support some few broad facts about consumption inequality in the Sub-Saharan African countries. First, inequality is very high on average, possibly the highest in the world. This result is in stark contrast with the presumption of low inequality in SSA, held for a long time based on the predictions of Kuznets-like models and the absence of reliable data.51 Second, on average inequality does not seem to have changed much in the 1990s and 2000s. Data from PovcalNet and other sources suggest a slow downward pattern; but in any case the evidence is mixed and weak. Third, the heterogeneity among countries in terms of inequality levels and patterns is large, partly possibly due to various measurement errors. It is hard to identify a prototype of an inequality pattern in SSA, as in other regions such as LAC or ECA. The scarce literature on inequality in SSA is consistent with these observations. Go, Nikitin, Wang and Zou (2007) report that high income inequality levels in SSA have remained more or less constant over the last four decades. Okojie and Shimeles (2006) underline the fact that SSA is one of the most unequal regions in the world, and that disparities have remained persistent over time. India Mazundar (2012) summarizes the main drivers of these changes: (i) the lead in employment and output growth has been taken not by manufacturing but by the tertiary sector, which displays higher inequality in pay;50 (ii) much of the labor reallocated from agriculture is absorbed in the informal sector, where earnings are only slightly higher than the poverty line; (iii) although numerous social insurance schemes have been established, their actual impact has been limited and regressive as they have disproportionately benefited workers in the small formal sector; (iv) the modest and selective increase in social sector spending is constantly threatened by the budget deficit; (v) the education polices implemented over the years have been biased towards the promotion of tertiary education and have neglected basic primary and lower secondary education. From a different perspective, Banerjee and Piketty (2010) looked at the tax-based shares of top incomes. Their results suggests that the gradual liberalization of the Indian economy made it possible for the top 1% to substantially increase their share of total income, from 4.7% in 1980 to 8.9% in 1999. While in the 1980s the gains were shared by everyone in the top percentile, in the 1990s it was only those in the top 0.1% who benefited the most. The authors stress that these results could be linked to the debate around the Indian growth paradox of the 1990s. According to the household expenditure survey conducted by the National Sample Survey (NSS), real per capital growth during the 1990s was fairly limited, in sharp contrast with the fast growth measured by national accounts. It was suggested that much of such growth could have gone to the rich, absent from surveys. Banerjee and Piketty (2010) conclude that top incomes could explain between 20 and 40% of the puzzle, which still leaves the bulk of the difference unaccounted for. 39 Alvaredo-Gasparini Alvaredo-Gasparini 51 Several studies have sought to explain the unexpected result of high inequality in Sub-Saharan Africa (Milanovic, 2003; Okojie and Shimeles, 2006; Moradi and Baten, 2005; and van de Walle, 2008). Sub-Saharan Africa In contrast, Sala-i- Martin and Pinkovskiy (2010) picture a more optimistic scenario, reporting a significant downward pattern for inequality during the period of growth (1995 to 2006). 52 See, for example, McGrath (1983); McGrath and Whiteford (1994); Klasen (1997 and 2005); Nattrass and Seekings (1997); Terreblanche (2002); Dollery (2003); van der Berg and Louw (2004); Leibbrandt et al., (2009); Leibbrandt et al., (2010); and Aron et al., (2009). South Africa Over the last thirty years there have been important studies of overall inequality and poverty in South Africa, and a heated debate about trends in post-apartheid 40 Alvaredo-Gasparini transition.52 South Africa has long been regarded as having one of the most unequal societies in the world. Consistent with this view, the country has the highest Gini coefficient of household consumption per capita (63.1 in 2010). During the early 1970s, the previously constant racial shares of income started to change in favour of the blacks, at the expense of the whites, in a context of declining per capita incomes (McGrath, 1983; McGrath and Whiteford, 1994). But while inter-racial inequality fell throughout the eighties and nineties, inequality within race groups increased (Simkins 1991; Whiteford and Van Seventer 2000). Leibbrandt et al. (2010) provide evidence from comparable households’ surveys conducted in 1993, 2000 and 2008. These authors find that since the fall of apartheid inequality continued to increase steadily, both for the whole population and within each racial group. The high level of overall income inequality accentuated between 1993 and 2008, incomes becoming increasingly concentrated in the top decile. The overall Gini coefficient increased by four percentage points, from 66 to 70. The Gini coefficient for the African and Asian/Indian population rose more sharply. Van der Berg and Louw (2004) also conclude that rising black per capita incomes over the past three decades have narrowed the interracial income gap, although increasing inequality within the black and Asian/Indian population seems to have prevented any decline in aggregate inequality. In explaining these changes, scholars agree in that the labor market played a dominant role, where a rise in the number of blacks employed in skilled jobs (including civil service and other high-pay government positions) coupled with increasing mean wages for this group of workers. Leibbrandt et al. (2010) indicate that in the initial post- apartheid period participation rates increased faster than absorption rates with a consequent increase in unemployment across all deciles. Since 2000 the aggregate unemployment rate declined marginally driven by increased absorption of those individuals in the top deciles. In the lower deciles the early post-apartheid trend continued to 2008. Indeed, this lack of successful integration into the labor market is the reason that many of these households find themselves at the bottom of the income distribution. South Africa State transfers have increased their importance as an income source but not in a way that has narrowed the distribution of per capita income. They have, however compensated for the decreasing share of remittance income. Since 1993 poverty has remained virtually constant, or fallen slightly. Leibbrandt et al. (2010) argue that while their impact on poverty incidence remains negligible overall, government social assistance grants (mainly the child support grant, the disability grant and the old-age pension) have been crucial in reducing poverty among the poorest households and the elderly. Two-thirds of income to the bottom quintile now comes from social assistance, even when many eligible individuals do not receive any 41 41 Alvaredo-Gasparini grant for instrumental reasons. Despite the increase in educational attainment, younger age cohorts have the highest incidence and shares of poverty and this has not improved notably over time, suggesting that the labor market has not been playing a strongly virtuous role as a driver of poverty alleviation over the post-apartheid period. Increasing inequality and stable poverty are consistent with the rising trend in top income shares recorded between 2002 and 2010 by Alvaredo and Atkinson (2010), which could be associated to the favorable conditions in the world market for agricultural commodities, the increase in the value of minerals other than gold, and the developments in the financial sector. 4.3. Inequality convergence As suggested above, there are signs of inequality convergence among countries in the developing world. As an example, the mean Gini coefficient for the 20 most unequal countries in our PovcalNet sample in 1981 fell 11% in the following three decades, while it increased 58% for the 20 most egalitarian economies. Bénabou (1996) was the first to present empirical evidence for cross-country convergence in income inequality with data from 1970 to 1990 drawn from the Deininger and Squire dataset. He found evidence consistent with the predictions of a neoclassical growth model that yields convergence of the entire income distribution and not just the first moment. Evidence on inequality convergence was also found in studies that used improved data: Ravallion (2003) based on PovcalNet, Bleaney and Nishiyama (2003) based on WIID, and Dhongde and Miao (2013) using both datasets. With variations, a typical inequality convergence study estimates α β it i i it G G G t e 1 1 - = ( + )( -1)+ for t = 2, …, T ; i = 1, …, N, α β it i i it G G G t e 1 1 - = ( + )( -1)+ for t = 2, …, T ; i = 1, …, N, where Git is the Gini coefficient for country i in year t and eit is an heteroscedastic error term. The parameter β measures the link between the change and the initial Gini, and therefore β<0 indicates inequality convergence. The intercept adds a notion of the time required to attain convergence: a higher value for α is associated to a slower convergence process. Models could be estimated with the Gini coefficient in levels or logs. In his early study Bénabou (1996) found a β coefficient of -0.039 for a small sample of around 30 countries. Naturally, estimates of β vary according to the data used, the period covered, the time horizons considered, and the regression model applied. Ravallion (2003) estimated a value of -0.028 in the 1990s, Bleany and Nishiyama (2003) a value of -0.0125 between 1965 and 1990, and Dhongde and Miao (2013) a value of -0.022 from 1980 to 2005. This literature has also found that the impact of the initial Gini coefficient on the inequality change diminishes over longer time horizons, and that the speed of inequality convergence is higher than the speed of convergence in per capita income. 53 Caselli et al. (1996) and Dhongde and Miao (2013) discuss biases that may arise in an OLS model. 54 See, as an example, the analysis of Atkinson and Lugo (2010) for Tanzania. 4.3. Inequality convergence 42 42 Alvaredo-Gasparini We add to this literature our own estimates, taking advantage of the PovcalNet panel of 76 countries from 1981 to 2010 used in this section. Table 4.2 shows the OLS and IVE estimates of α and β for different initial years.53 The parameter β is negative and significant in all the specifications, suggesting evidence for inequality convergence. The estimated coefficients are in the range of those estimated in the literature. Table 4.2 Inequality convergence Models of the change in the Gini coefficient Note: robust t statistics in parentheses; *significant at 5%; **significant at 1%; the heteroskedasticity- consistent covariance matrix estimator is used (HC1). IVE estimates use the initial value as the instrument for the inequality measure in the second survey. The number of observations is 456 in the first panel, and 281 in the second. Gini Index OLS 1.098 -0.026 0.49 0.908 -0.023 0.35 (18.90)** (20.38)** (11.20)** (10.97)** IVE 1.271 -0.031 0.47 0.855 -0.021 0.35 (17.91)** (17.61)** (9.83)** (9.90)** Difference 0.173 -0.005 -0.053 0.002 Hausman Test (4.26)** (4.03)** (1.69)* (2.61)** Log Gini Index OLS 0.118 -0.032 0.65 0.105 -0.029 0.27 (28.41)** (28.11)** (15.53)** (15.14)** IVE 0.135 -0.037 0.63 0.104 -0.028 0.27 (25.79)** (24.75)** (14.07)** (13.66)** Difference 0.017 -0.005 -0.001 0.001 Hausman Test (5.30)** (5.01)** (0.49) (0.48) R 2 Initial year 1981 Initial year 1990 R 2 Intercept (α) Slope (β) Intercept (α) Slope (β) Inequality convergence Models of the change in the Gini coefficient Note: robust t statistics in parentheses; significant at 5%; significant at 1%; the heteroskedasticity consistent covariance matrix estimator is used (HC1). IVE estimates use the initial value as the instrument for the inequality measure in the second survey. The number of observations is 456 in the first panel, and 281 in the second. While the evidence for inequality convergence in the last decades seems well- established, the reasons driven that pattern are not clear. As mentioned before, Bénabou (1996) finds the evidence on convergence consistent within the framework of a neoclassical growth model. In contrast, the evidence for unconditional inequality convergence is interpreted by Ravallion (2003) as the result of policy and institutional convergence since around 1990, when socialist planned economies became more market-oriented, and non-socialist economies adopted market reforms. 54 See, as an example, the analysis of Atkinson and Lugo (2010) for Tanzania. 4.4. Absolute inequality While relative inequality has been the preferred concept in empirical work in development economics, absolute views of inequality certainly have some intuitive appeal (Amiel and Cowell, 1999; Atkinson and Brandolini, 2004). Interestingly, the trends in the two concepts over the last decades have been different in the developing world.54 The fact that most countries experienced economic growth, while at the same time relative inequality did not fall, implied widening absolute income differences. On 43 Alvaredo-Gasparini average, the absolute difference in monthly consumption per capita between the top and bottom 10% of each country increased over the two decades from US$415 (PPP adjusted) in 1990, to US$497 in 2002, and US$646 in 2010. In more than 90% of the countries in the sample that absolute difference was higher in 2010 than in 1990. The contrast between the recent trends in absolute and relative inequality in the developing countries is illustrated in Figure 4.7. While relative inequality rose in the late 1980s and early 1990s, absolute inequality declined, driven by a reduction in mean income. The strong growth in the developing world since mid-1990s is reflected in the substantial hike in the degree of absolute inequality. Although some equalizing forces operated in the 2000s that reduced the relative gaps, they were not enough to narrow the absolute gaps in a context of economic growth. Based on these facts, Ravallion (2004) argues that the disagreements over whether inequality in the world has gone up or down may partly be due to differing views about the importance of absolute versus relative conceptions of inequality. Figure 4.7 Absolute and relative Gini coefficients Unweighted means, developing countries, 1981-2010 Source: own estimates based on PovcalNet (2013). Note: normalized to 100=mean over the period 1981-2010.The Gini coefficients are computed over the distribution of household consumption per capita. 80 90 100 110 120 130 140 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Relative Absolute g Absolute and relative Gini coefficients Unweighted means, developing countries, 1981-2010 Note: normalized to 100=mean over the period 1981-2010.The Gini coefficients are computed over the distribution of household consumption per capita. 4.5. Aggregate welfare The typical way of assessing the economic performance of a country is by means of its per capita income or output. However, this practice is valid only when the evaluator’s welfare function is utilitarian. Except in this extreme case, measuring aggregate welfare involves not only knowing the mean but also other elements of the income distribution, in particular the degree of inequality. Although social welfare functions are naturally arbitrary, since they depend on the analyst’s value judgments, it is 44 44 Alvaredo-Gasparini common in the literature to work with anonymous, Paretian, symmetric and quasiconcave functions. For simplicity, here we consider the abbreviated welfare function proposed by Sen (1976), WS=µ.(1-G), where µ is the mean of the distribution and G is the Gini coefficient. Figure 4.8 shows the unweighted mean of WS for the developing countries in the period 1990-2010 computed from household survey data. In general, aggregate welfare has followed changes in per capita consumption. The fall in mean consumption in the early 1990s (mostly due to the negative performance in ECA) was reinforced by the increase in inequality, driving welfare down by around 15%. Between 1993 and 2002 mean consumption went up but the change was counterbalanced by a similar increase in the Gini, keeping welfare roughly constant. The 2000s witnessed a robust increase in mean consumption, along with some fall in inequality, implying a 40% increase in aggregate welfare between 2002 and 2010. According to these calculations the mean aggregate welfare in the developing countries was 22% higher in 2010 than in 1990, implying an annual growth rate of around 1%. Figure 4.8 Aggregate welfare Sen welfare function, unweighted mean, developing countries, 1990-2010 Source: own estimates based on PovcalNet (2013). Note: normalized to 100=value in 1990. 60 70 80 90 100 110 120 130 140 1990 1993 1996 1999 2002 2005 2008 2010 Mean Gini Welfare Figure 4.8 Aggregate welfare Sen welfare function, unweighted mean, developing countries, 1990-2010 Source: own estimates based on PovcalNet (2013). Note: normalized to 100=value in 1990. In order to calculate welfare it is necessary to have estimates of the mean income and some inequality measure. Ideally, both parameters should be estimated from the same source, typically a household survey, as we have done so far. Some authors have taken a different approach, anchoring the mean to a variable from National Accounts, such as per capita GDP or aggregate household consumption expenditures. 4.5. Aggregate welfare For several reasons changes in mean income from household surveys tend to differ significantly from changes in per capita GDP (Deaton, 2003, 2005; Anand and Segal, 2008). Some of these differences are natural since per capita income and GDP are different concepts, but some are rooted in measurement errors both in household surveys and in National 60 70 80 90 100 110 120 130 140 1990 1993 1996 1999 2002 2005 2008 2010 Mean Gini Welfare gg g Sen welfare function, unweighted mean, developing countries, 1990-2010 In order to calculate welfare it is necessary to have estimates of the mean income and some inequality measure. Ideally, both parameters should be estimated from the same source, typically a household survey, as we have done so far. Some authors have taken a different approach, anchoring the mean to a variable from National Accounts, such as per capita GDP or aggregate household consumption expenditures. For several reasons changes in mean income from household surveys tend to differ significantly from changes in per capita GDP (Deaton, 2003, 2005; Anand and Segal, 2008). Some of these differences are natural since per capita income and GDP are different concepts, but some are rooted in measurement errors both in household surveys and in National Accounts. Some authors pay the price of the potential inconsistency of using two 45 Alvaredo-Gasparini different data sources (i) in order to avoid departing from the typical growth and development literature that is based on National Accounts data, (ii) as a way to alleviate the underreporting issue in household surveys, and (iii) to avoid problems related to the unavailability of surveys for many years in several countries (Ahluwalia et al., 1979; Bourguignon and Morrison, 2002; Sala-i-Martin, 2006; Bhalla, 2002). In Figure 4.9 we report the results of computing the unweighted average of aggregate welfare across developing countries using alternative mean income variables. According to these estimates mean welfare in the developing world grew at an annual 1% from 1990 to 2010 using mean consumption per capita from household surveys, 1.6% using per capita GNI from WDI, and 2.1% using per capita GDP from Penn World Tables (PWT) (Heston et al., 2012).55 These discrepancies are worrying and call for increasing efforts to understand and reduce the gaps among data sources. 55 In fact, this difference comes from the combination of higher growth recorded in the National Accounts in the 1990s compared to household surveys, and the opposite result in the 2000s. For instance, while per capita GDP slightly fell between 2008 and 2010, mean consumption in household surveys increased at annual 2%. y 56 Some authors have computed global welfare, ignoring the division of the world in countries. The evidence suggest an increase in aggregate welfare in the developing world in the last decades (Pinkovskiy and Sala-i-Martin, 2009; Atkinson and Brandolini, 2010; Pinkovskiy, 2013). 55 In fact, this difference comes from the combination of higher growth recorded in the National Accounts in the 1990s compared to household surveys, and the opposite result in the 2000s. For instance, while per capita GDP slightly fell between 2008 and 2010, mean consumption in household surveys increased at annual 2%. 56 Some authors have computed global welfare, ignoring the division of the world in countries. The evidence suggest an increase in aggregate welfare in the developing world in the last decades (Pinkovskiy and Sala-i-Martin, 2009; Atkinson and Brandolini, 2010; Pinkovskiy, 2013). 57 The results for China (1986-2003) and Indonesia (1982-2004) in the WTID are based on household surveys and not on tax records. The fact that top income share estimates are lower than in the most egalitarian developed countries shows that they are likely to be underestimated. In the case of China, the rising trend is robust and can be taken as an indicator of the true dynamics of concentration at the top. Piketty and Qian (2009) show that top income shares increased at a very high rate during the period, which is consistent with the evidence discussed in section 4. The top decile share rose from about 17 per cent in 1986 to almost 28 per cent in 2003—that is, by more than 60 per cent. The top 1 per cent income share more than doubled between 1986 and 2001, from slightly more than 2.6 per cent in 1986 to 5.9 per cent in 2003. 58 4.5. Aggregate welfare Figure 4.9 Aggregate welfare for alternative mean variables Sen welfare function, unweighted mean, developing countries, 1990-2010 Source: own estimates based on PovcalNet (2013), WDI and PWT. Note: mean anchored to per capita consumption (PovcalNet), GNI per capita (WDI) and GDP per capita (PWT). Normalized to 100=value in 1990. 60 80 100 120 140 160 1990 1993 1996 1999 2002 2005 2008 2010 consumption (surveys) GNI (WDI) GDP (PWT) Aggregate welfare for alternative mean variables Sen welfare function, unweighted mean, developing countries, 1990-2010 ( ), Note: mean anchored to per capita consumption (PovcalNet), GNI per capita (WDI) and GDP per capi (PWT). Normalized to 100=value in 1990. Note: mean anchored to per capita consumption (PovcalNet), GNI per capita (WDI) and GDP per capita (PWT). Normalized to 100=value in 1990. The population-weighted mean of the welfare measure grew at a much higher rate, due to the positive performance of several large countries.56 The growth rate between 1990 and 2010 was 2.3% using household survey data, 3% anchoring mean income to per capita GNI from WDI, and 3.3% when using the Penn World Tables. 46 46 Alvaredo-Gasparini Alvaredo-Gasparini 58 We do not review the results for former colonial territories being prepared as part of an on-going project carried out by Atkinson (British colonies), and Alvaredo, Cogneau and Piketty (French colonies). This project has assembled data for some forty colonies covering the periods before and after independence. Unfortunately, the series stop before 1970 in most cases due to unavailability of recent data. 4.6. Trends from tax records At the moment of writing (2013), the WTID offers estimates of the tax-based shares of top incomes for a small number of developing countries: Argentina, Colombia, India, Mauritius and South Africa.57 Malaysia is available from Atkinson (forthcoming), and will be included in the WTID as soon as the proper documentation is finished. Preliminary results for Ecuador are available from Alvaredo and Cano (forthcoming).58 Ongoing research analyzes the cases of Brazil, Chile, Ecuador and Uruguay. Consequently, evidence in this respect is still fragmentary, not only because this particular research program is rather recent in what concerns the developing world, but also because of the unavailability of tax data. The results for the top 1% income share are presented in Figure 4.10, together with survey-based Gini coefficients. Several elements are worth mentioning. First, both sources are not directly comparable for the reasons discussed in section 3. The top share estimates are in general before taxes, while survey Ginis are net of taxes; in addition the units of analysis usually do not match. Second, there is substantial heterogeneity in this group, both in levels and dynamics, compared to the evidence discussed in chapter 16 for developed countries. It should be borne in mind that the differences in the tax systems across countries imply different income concepts, so that top share levels should be read with this caveat in mind. Third, Leigh (2007), who analyzes 13 developed countries, and find that there is a strong and significant relationship between top income shares and broader inequality measures, concludes that “panel data on top income shares may be a useful substitute for other measures of inequality over periods when alternative income distribution measures are of low quality, or unavailable”. Figure 4.10 seems to agree with the results in Leigh (2007) in some cases but not in others. For example, the evolutions of the Gini and the top 1% share in Colombia are particularly diverging, while the dynamics in Mauritius is remarkably similar. Top shares and broader, synthetic inequality measures can very well display different trends. The problem arises when the top group plays a major role in the changes in inequality and survey data fail to capture high incomes. 58 We do not review the results for former colonial territories being prepared as part of an on-going project carried out by Atkinson (British colonies), and Alvaredo, Cogneau and Piketty (French colonies). 59 Bourguignon et al. (2008) and Ferreira (2010) are excellent references for methodological issues in recent research on inequality determinants. 4.6. Trends from tax records Note: Gini coefficient for Malaysia 2004 identified as outlier. 4.6. Trends from tax records This project has assembled data for some forty colonies covering the periods before and after independence. Unfortunately, the series stop before 1970 in most cases due to unavailability of recent data. 47 47 Alvaredo-Gasparini Figure 4.10 Top 1% income share and Gini coefficients Top 1% income share and Gini coefficients Argentina, Colombia, Malaysia, Mauritius, India and South Africa Source: own estimates based on WTID. Note: Gini coefficient for Malaysia 2004 identified as outlier. 4.6. Trends from tax records 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Argentina top 1% income share Gini-Urban HH per capita income (SEDLAC) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Colombia top 1% income share Gini-HH per capita income (Povcalnet) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Malaysia top 1% income share Gini-HH per capita income (Povcalnet) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Mauritius top 1% income share Gini-HH disposable income (Atkinson and Morelli, 2012) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) India top 1% income share Gini-Urban HH per capita consumption (Povcalnet) Gini-Rural HH per capita consumption (Povcalnet) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) South Africa top 1% income share Gini-HH per capita income (Leibbrandt et al., 2010) Argentina, Colombia, Malaysia, Mauritius, India and South Africa Argentina, Colombia, Malaysia, Mauritius, India and South Africa 35 45 55 65 75 5 10 15 20 25 Gini coefficient Top 1% income share (%) Argentina top 1% income share Gini-Urban HH per capita income (SEDLAC) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Colombia top 1% income share Gini-HH per capita income (Povcalnet) Top 1% income share (%) 25 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 25 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Mauritius top 1% income share Gini-HH disposable income (Atkinson and Morelli, 2012) 25 35 45 55 65 75 0 5 10 15 20 25 Gini coefficient Top 1% income share (%) Malaysia top 1% income share Gini-HH per capita income (Povcalnet) 25 35 45 55 65 0 5 10 15 20 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) Gini-HH per capita income (Povcalnet) 25 35 45 55 65 75 0 5 10 15 20 25 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Gini coefficient Top 1% income share (%) India top 1% income share Gini-Urban HH per capita consumption (Povcalnet) Gini-Rural HH per capita consumption (Povcalnet) Top 1% income share (%) 25 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 25 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 South Africa 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 25 0 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 2012 Source: own estimates based on WTID. 4.7. Exploring inequality changes Explaining changes in the income distribution is a very difficult and challenging task that lies well beyond the objectives of this chapter. In this section we briefly review some methodologies to study the determinants of the income distribution changes and lay out some of the main results regarding developing countries.59 Certainly, there has been sustained progress in our understanding of the factors that shape the income distributions, but yet the image that emerges from reviewing the literature is still that of a patchwork of numerous hypotheses without conclusive empirical support. 48 48 Alvaredo-Gasparini Decompositions are one of the most widely used techniques to characterize income distribution changes. Typically, an income model is estimated, and a counterfactual distribution is simulated modifying some elements of the estimated income model (e.g. parameters or the distribution of observable factors), while keeping the rest fixed. The difference between the actual and the simulated distribution captures the first- round partial-equilibrium effect of the change under study.60 The method generates entire counterfactual distributions, and hence can capture the heterogeneity of impacts throughout the distribution. The decompositions have been typically used to shed light on the impact of changes in the returns to education, in the demographic, sectorial, occupational and educational composition of the population, and in labor and social policies. The decompositions do not allow for the identification of causal effects, and suffer from the usual problems of equilibrium-inconsistency and path dependence. Nevertheless, these types of exercises are informative about the relative strength of several direct determinants that may be driving the distributive changes, and therefore could be useful in identifying areas in which to focus the research efforts. Ideally, income distribution changes should be studied in a general equilibrium framework, since they are the result of complex processes that involve all sorts of effects and interactions throughout the economy. Computable general equilibrium (CGE) models have been applied to study changes in the income distributions around the developing world. These exercises, however, depend critically on parameters and functions that are difficult to estimate, and rely on many simplifying assumptions. The more recent macro-micro approach combines a CGE model (the macro component) with a microsimulation (the micro component). CGE models provide a framework to assess consistency of policy alternatives, but lack the necessary disaggregation for the analysis of distributional issues, which is provided by the microsimulations. 60 See Bourguignon et al. (2005); Barros et al. (2006) and Bourguignon et al. (2008), for methodological proposals; Bourguignon et al. (2005) for applications to Asia and Latin America, and Inchauste et al. (2012) for a recent application to poverty reduction in Bangladesh, Peru and Thailand. Fortin et al. (2011) and Essama Nssah (2012) are useful surveys of the economic literature on decompositions. 61 Ferreira et al. (2010) use this approach to estimate the effect of a trade liberalization episode on the distributions of wages and household incomes in Brazil. 61 Ferreira et al. (2010) use this approach to estimate the effect of a trade liberalization episode on the distributions of wages and household incomes in Brazil. 4.7. Exploring inequality changes The macro and micro components of this methodology communicate through aggregate variables such as employment levels and wage rates that are generated by the CGE model and used as inputs in the microsimulations (Bourguignon et al., 2008; Bourguignon and Bussolo, 2012). In a related approach, rather than building a full general equilibrium model of the economy, researchers rely on a reduced-form relationship between a set of observed exogenous variables (such as changes in tariff rates) and a set of sector- level variables (such as industry-skill wage premia), that serve as inputs in the microsimulations.61 A very different strand of the literature involves the estimation of cross-country regressions, typically with panel data, where an aggregate measure of overall inequality, such as the Gini coefficient, is linked to various potential causal factors (e.g. 49 49 Alvaredo-Gasparini Li et al., 1998; Anderson, 2005). Naturally, endogeneity problems are endemic to this approach, which is useful to characterize the structure of correlations among variables, but less successful in identifying causal links. Most of the literature takes a less ambitious but probably more productive road, and focus on the partial-equilibrium impact of specific shocks and policy changes, using different identification strategies depending on the characteristics of the shock/policy and the data available. Examples of these methodologies include (i) a typical supply and demand approach, where the impact of indicators of trade, technology or other factors on the relative wage between skilled and unskilled workers is estimated, controlling for relative supply; (ii) the cost function approach, where the impact of several indicators on the share of skilled wages in total wage bill is estimated, using flexible cost functions (usually a translog cost production function); and (iii) mandated wage regressions.62 When experiments with random assignment are available, causal links are more clearly identified. For instance, the conditional cash transfer program Progresa in Mexico was initially implemented with a random assignment of treated and control rural villages, which allowed a rigorous impact evaluation. ( ) 63 See also Parker and Skoufias (2001) and Gertler (2004) for impact evaluations studies of the Mexican conditional cash transfer program. 64 For instance, according to SEDLAC data, on average in Latin America in 2010 the share of labor income in total household income was 82.3%, the share of transfers (including pensions) 13.9%, and the rest of the sources just 3.8%. 65 62 See Anderson (2005). 63 See also Parker and Skoufias (2001) and Gertler (2004) for impact evaluations studies of the Mexican conditional cash transfer program. 64 For instance, according to SEDLAC data, on average in Latin America in 2010 the share of labor income in total household income was 82.3%, the share of transfers (including pensions) 13.9%, and the rest of the sources just 3.8%. 65 See Caselli and Michaels (2013). 62 See Anderson (2005). ( ) 63 See also Parker and Skoufias (2001) and Gertler (2004) for impact evaluations studies of the Mexica conditional cash transfer program. 64 65 See Caselli and Michaels (2013). 64 For instance, according to SEDLAC data, on average in Latin America in 2010 the share of labor incom in total household income was 82.3%, the share of transfers (including pensions) 13.9%, and the rest the sources just 3.8%. 4.7. Exploring inequality changes Taking advantage of that design, Todd and Wolpin (2006) estimate a full structural model of behavior, including education, fertility and labor supply decisions, a model that can be used to simulate the distributive impact of policies and shocks.63 The bulk of the distributive analysis in developing countries has focused mainly on the labor market and on public and private transfers, while largely setting aside the role played by other sources of income, such as capital, land rents and business profits. The neglect of these other factors is essentially due to the fact that household surveys fail to capture these income sources properly.64 This shortcoming has, for instance, severely limited the study of the impact of the natural resources exploitation on inequality, a relevant topic in several developing countries.65 Most studies narrow the analysis to particular indicators of the labor market, such as the wage gap between skilled and unskilled labor (the wage premium) or the returns to education. For instance, Bourguignon et al. (2005) show that increases in returns to schooling were large contributors to increasing inequality in East Asia and Latin America in the 1990s. In what follows we review some general debates on the determinants of recent inequality changes. Growth and development. As shown in Section 3.5 there is a significant negative relationship between inequality and measures of development, such as GNI per capita, in a cross-section of countries. From this evidence Ferreira and Ravallion (2009) 50 50 Alvaredo-Gasparini conclude that “high inequality is a feature of underdevelopment”. However, the short or medium run relationship between inequality and development has proved to be elusive. There appears to be no evidence in the last decades of a significant correlation between the growth rate of an economy and the change in the inequality level (Ravallion and Chen, 1997; Ravallion, 2001; Dollar and Kraay, 2002; Ferreira and Ravallion, 2009).66 Ravallion (2007), for example, analyzed 290 episodes in 80 countries in 1980-2000 and found a correlation coefficient non-significant at the 10% level between the changes in the log of the Gini coefficient and changes in the log of mean income in real terms between successive household surveys. The analysis of more recent data from PovcalNet leads to the same conclusion. Using 473 spells in the period 1981-2010 we find a non-significant coefficient of -0.0094. 68 Ravallion (2004) argues that, on average, growth is not associated with increases in relative inequality but absolute inequality, and it is these higher absolute gaps between "rich" and "poor" that generate the perception of an unequal growth processes. 66 A related literature finds no support for a Kuznets curve with longitudinal data (Bruno, 1998; Field 2002; Hellier and Lambrecht, 2012). 67 67 The relationship becomes negative, although just slightly significant, when using the change in log re per capita GDP (Penn World Tables) or per capita GNI (WDI) as measures of growth. 68 pp g 2002; Hellier and Lambrecht, 2012). 67 The relationship becomes negative, although just slightly significant, when using the change in log real per capita GDP (Penn World Tables) or per capita GNI (WDI) as measures of growth. 68 Ravallion (2004) argues that, on average, growth is not associated with increases in relative inequality but absolute inequality, and it is these higher absolute gaps between "rich" and "poor" that generate the perception of an unequal growth processes. 67 The relationship becomes negative, although just slightly significant, when using the change in log real per capita GDP (Penn World Tables) or per capita GNI (WDI) as measures of growth. 68 Ravallion (2004) argues that, on average, growth is not associated with increases in relative inequality b t b l t i lit d it i th hi h b l t b t " i h" d " " th t t 4.7. Exploring inequality changes A similar result applies when restricting the sample to observations after 1990 or 2000, or when considering longer spells.67 The data suggests that among both growing and contracting economies, inequality increased about as often as it fell. In the last decades economic growth has been distribution-neutral on average in the developing countries.68 Globalization. Much of the recent public and academic debate on inequality changes has been related to the rise in globalization. In the latest decades most developing countries have experienced increasing openness to international trade, capital markets flows and foreign direct investment. The theoretical channels linking these changes to inequality are multiple and complex, which accounts for the lack of conclusive empirical results (Wood, 1997; Rama, 2003; Winters et al., 2004; Anderson, 2005; Goldberg and Pavcnik, 2007; Harrison et al., 2011). While studies in cross sections of developing countries are inconclusive in relation to the impact of globalization upon inequality, several longitudinal estimates concerning countries taken separately or in small groups reveal a positive correlation between openness and the relative demand for skilled labor (Anderson, 2005; Goldberg and Pavnick, 2007; Harrison et al., 2011; Chusseau and Hellier, 2012). Trade openness may affect the income distribution through various channels. The traditional Stolper-Samuelson effect predicts a reduction in the skill premium in unskilled-labor-abundant developing countries, a prediction that does not appear to be confirmed by the facts (Goldberg and Pavnick, 2007; Feenstra, 2008). While some of the research has pointed then to non-trade factors - such as skill-biased technological change and labor institutions - to explain rising wage gaps, in recent years new mechanisms have been explored through which trade can increase income inequality. These mechanisms include heterogeneous firms 51 Alvaredo-Gasparini and bargaining, trade in tasks, labor frictions and incomplete contracts (Harrison et al., 2011). In addition, competition among developing countries may increase inequality in middle-income countries (e.g. Latin America) competing with low-income economies.69 Also, the growing size of the developing world, as new countries enter the world markets, may foster inequality by augmenting the world endowment of unskilled labor. The literature finds that the mechanisms through which globalization affects income distribution are country, time, and case specific, and therefore the impacts of trade liberalization need to be examined in conjunction with other concurrent policy reforms (Goldberg and Pavnick, 2007). 69 The increase in inequality in Latin America has also been explained arguing that it is a region relatively abundant in natural resources, while in the onset of liberalization Asian countries were relatively abundant in unskilled labor (Wood, 1997). 70 According to Tinbergen (1975) changes in earnings inequality are the outcome of a “race” between technological progress raising the demand for skills, and the expansion of education raising the supply of skills. 71 See Gasparini et al. (2011) for Latin America. 71 See Gasparini et al. (2011) for Latin America. 4.7. Exploring inequality changes In addition, and due to various limitations, the literature is mostly focused on the static link between globalization and income distribution that typically operates through changes in relative prices and wages, rather than on the dynamic, more indirect link from trade to growth, and then to poverty and inequality. Technology and education. Skill-biased technological change has been a popular explanation for the rise in inequality in the developed countries. Changes in technology, such as the use of computers, increase the relative demand for skilled workers driving the skill premium up. This hypothesis is also plausible in the developing world, where globalization increased the transfers of more skill-intensive technologies from the North, and fostered imports of capital goods, typically complementary of skilled labor. Several studies find that openness-driven technological transfers tend to increase inequality in emerging countries (Conte and Vivarelli, 2007). The increase in the wage skill premium may be temporary, as the introduction of new technologies requires a transitional period during which skilled workers are employed to adapt the firm to the new technology (Pissarides, 1997; Helpman and Trajtenberg, 1998). The empirical applications usually show evidence on the short and medium-run effects of the reforms, failing to capture the long-run impact. The generalized fall in inequality in the 2000s in the developing world might be in part attributed to the petering out of the unequalizing initial impact of the liberalizing reforms and technological shocks experienced by many countries in the 1990s. The increase in education may counteract the effect of skill-biased technological change in the Tinbergen´s race between education and technology.70 In fact, education has expanded in the developing world at high rates during the last decades, mitigating the impact of other factors that tend to increase the wage premium.71 However, the link between education and income inequality may not be that straightforward. Given 52 52 Alvaredo-Gasparini the convexities in the returns to education, even an equalizing increase in schooling may generate an unequalizing change in the distribution of earnings. Bourguignon, Ferreira and Lustig (2005) have labeled this phenomenon “the paradox of progress”, a situation where an educational expansion is associated to higher inequality.72 Market reforms. Several developing countries have implemented market-oriented reforms in the last decades, reducing regulations and privatizing firms. 74 For instance, Lustig (2012) finds that in Latin America when indirect taxes are taken into account, the net income of the poor and the near poor can be lower than it was before taxes and cash transfers. 75 ILO (2010) reports that in the early 2000s the share of the population above the legal retirement age receiving a pension in developing economies was, on average, around 40 per cent, as compared to 90 per cent in European economies. 73 See Cornia (1996), Milanovic (1998), Ferreira (1999), Cornia and Reddy (2001), and Milanovic an Ersado (2010). 74 p p 75 ILO (2010) reports that in the early 2000s the share of the population above the legal retirement age receiving a pension in developing economies was, on average, around 40 per cent, as compared to 90 per cent in European economies. 74 For instance, Lustig (2012) finds that in Latin America when indirect taxes are taken into account, th net income of the poor and the near poor can be lower than it was before taxes and cash transfers. 72 Inequality may also increase after an education expansion, given that wage dispersion is larger higher educational levels (Alejo, 2012). 73 Inequality may also increase after an education expansion, given that wage dispersion is larger at higher educational levels (Alejo, 2012). 73 See Cornia (1996), Milanovic (1998), Ferreira (1999), Cornia and Reddy (2001), and Milanovic and Ersado (2010). 74 4.7. Exploring inequality changes The paradigmatic case includes the former socialist planned economies in ECA, but the transition from centrally planned to market-oriented economies was also experienced by several African and Asian countries, including China. The evidence suggests a significant increase in inequality over the transition period. That surge has been linked to the process of privatizations, that implied an increase in the earnings dispersion in comparison to the more compressed wage structure of the state-own firms, and the institutional and regulatory reforms that have increased competition in product and factor markets and decreased the bargaining power of labor.73 Other non-socialist economies also adopted market-friendly reforms; Ravallion (2003) argues that in some cases (e.g. Brazil) pre-reform controls benefited the rich and kept inequality high, and then reforms help lowering inequality, while in some others (e.g. India) the controls (and the reforms) had the opposite effect. Fiscal and social policy. Developing countries are characterized by relatively low levels of taxation, heavy reliance on regressive revenue instruments, and low coverage and benefit levels of transfer programs (World Bank, 2006). This structure limits the redistributive potential of fiscal policy and in some cases even exacerbates the market income disparities.74 While average tax ratios for advanced economies exceed 30 per cent of GDP, ratios in developing economies (excluding emerging Europe) generally fall in the range of 15–20 per cent of GDP (Baltagi et al., 2012). Tax collection is not only lower but also more regressive than in developed countries. The difficulties in collecting more progressive taxes are related to the high levels of self-employment and sizeable informal sectors, which limit the capacity of the tax authorities to verified taxpayers´ income and assets. On the spending side, in most developing economies social spending is relatively low, and participation in social insurance schemes is restricted to high-income workers in the formal sector and to public sector employees.75 All these factors combine for a low redistributive impact of the fiscal 53 53 Alvaredo-Gasparini policy. For instance, Goñi et al. (2008) and Lustig (2012) find that the tax and transfer system in Latin America decreased the market Gini by only 2 percentage points, a meager impact compared to the 20-points impact estimated in 15 European economies. Since the mid-1990s there have been some encouraging signs of improvement, especially in terms of increasing coverage and better targeting of social policies. 77 A collapse is identified as a cumulative percentage drop in per capita GDP (from peak to trough) of at least 9.5% for 1911 to 1950 and of 5% for the post-1950 period. Their results are somewhat different when they look at banking crisis and consumption collapses, but not more conclusive. 76 In the case of the Gini coefficient, a change is considered significant when it is higher than 0.7 percentage points (that is, 1/3 of 2 Gini points). 77 4.7. Exploring inequality changes The recent expansion of conditional cash transfer programs (CCTs) implies a promising approach for enhancing the distributive impact of public spending in developing economies. CCTs typically transfer income to poor households, conditional on households making certain investments on their children's human capital – education, health and nutrition. Such programs have been adopted in many developing economies, including some Sub-Saharan African countries, although on a smaller scale (Fiszbein and Shady, 2009; Garcia and Moore, 2012). CCTs became particularly popular in LAC: by 2010 there were 18 countries in Latin America and the Caribbean applying CCTs, covering 20% of total LAC population, and spending on average 0.40% of GDP (Cruces and Gasparini, 2012). Soares et al. (2009) estimate that the CCTs in Brazil and Mexico reduced the Gini for disposable income by 2.7 percentage points, accounting for about a fifth of the decrease in that index between the mid-1990s and the mid- 2000s. Macroeconomic crises. The scale of the recent crisis has placed the distributional impact of macroeconomic shocks back on the agenda. Banking crises, crashes in stock and real estate markets and GDP collapses are events with potential large effects on the income distribution. Atkinson and Morelli (2011) were the first in addressing this issue from an empirical, historical and global perspective. They investigated the effect of crisis on inequality as well as the impact of inequality on the probability of economic crises, by analyzing the history of banking, consumption and GDP collapses over a 100- year period in 25 countries, out of which only 6 are developing economies. These authors observed the variation in distributional variables taking a 5-year window before and after the crisis date, and classified each one according to whether inequality was increasing, constant or decreasing before and afterwards.76 Table 4.3, panel A, reproduces their results specifically regarding GDP collapses.77 They identified 103 crises, but for only one third there is information on inequality changes. The shadowed diagonal shows combinations where the trajectory was unchanged; above the diagonal are cases where the trajectory “bent” downward; below the diagonal are cases where the trajectory “bent” upward. 4.7. Exploring inequality changes As it is readily apparent, one cannot draw firm conclusions: (i) the raw totals show that most crisis did not involve changes in 54 54 Alvaredo-Gasparini inequality ex-post; (ii) the number of cases above the diagonal is low and not very different from the cases below the diagonal, which means that GDP crisis are not necessarily associated with a specific direction in the change of inequality; (iii) the inverted V shape (inequality increasing and then decreasing) is not prevalent. Atkinson and Morelli (2011) conclude that “economic crises differ a great deal in whether or not they were preceded by rising inequality, and, in any case, where there was such a rise, causality is not easy to establish.” When banking crisis are analyzed instead of GDP drops, the cases in which inequality tend to increase following a crisis are in majority. We replicated their methodology for the years 1980-2010 to take into account the set of developing countries, and the results are shown in panel B of Table 4.3.78 Even if our list is not exhaustive and could be considerably improved, we identified at least 67 crises episodes. As in general they occurred during the 1980s or early 1990s, it is not surprising that in most cases inequality changes before the crises remain unknown due to data unavailability. There is a tendency for inequality to rise after a GDP collapse (10 cases), but again the numbers are too small to draw conclusions, and this could just be the continuation of a previous tendency. This is not necessarily in contradiction with Atkinson and Morelli (2011) due to at least two reasons: (i) GDP crisis may well be more correlated with financial crisis in the developing world, and (ii) such conclusion is highly influenced by the experience of the transition economies after the fall of the Berlin Wall. It should also be noted that several of the canonical Latin American exchange rate crisis of the 1980s and 1990s, with the exception of Argentina and Brazil (included in Atkinson and Morelli, 2011) do not fall within our classification of a collapse. In this sense, there is much work to be done about the magnitude of a crisis and its sensitivity on the two-way relationship with inequality. 79 The case studies are numerous; see for example Forbes (2011) and Lustig (1990). 78 Given the higher volatility of per capita GDP in developing countries, we have kept the threshold of 9.5% drop to identify crisis for 1980-2010. The data come from the World Bank Development Indicators. p y Changes in Gini coefficient are taken from PovcalNet. 78 Given the higher volatility of per capita GDP in developing countries, we have kept the threshold of 9.5% drop to identify crisis for 1980-2010. The data come from the World Bank Development Indicators. Changes in Gini coefficient are taken from PovcalNet. 79 The case studies are numerous; see for example Forbes (2011) and Lustig (1990). 4.7. Exploring inequality changes Ferreira and Ravallion (2009), for instance, report that in Indonesia a large share of the increase in inequality was associated with migration from wage employment in agriculture to urban self-employment. Milanovic (2012) argues about the key role of migration in global inequality. 4.7. Exploring inequality changes Of course this brief review does not exhaust the multiple factors behind distributive changes in the developing world; in fact, arguably any shock or policy could affect the income distribution. For instance, demographic factors, such as the decline in fertility, the rise in life expectancy and the growing importance of assortative mating and single-parent households have been identified as relevant sources of inequality changes. Labor policies are a key target for research, as well. Several studies find that the weakening of labor institutions such as unions and the declining real value of minimum wages were responsible for the increase in earnings inequality in several developing countries, especially in the 1990s, while more ambitious labor policies contributed to the reduction in inequality in the 2000s. Migration and sector changes are also determinants of inequality changes, studied at least since the seminal contributions by Lewis (1954) and Kuznets (1955). Changes in inequality are associated to the geographic and sectorial pattern of growth (Loayza and Raddatz, 2010). Ferreira and Ravallion (2009), for instance, report that in Indonesia a large share of the increase in inequality was associated with migration from wage employment in agriculture to urban self-employment. Milanovic (2012) argues about the key role of migration in global inequality. Others. Of course this brief review does not exhaust the multiple factors behind distributive changes in the developing world; in fact, arguably any shock or policy could affect the income distribution. For instance, demographic factors, such as the decline in fertility, the rise in life expectancy and the growing importance of assortative mating and single-parent households have been identified as relevant sources of inequality changes. Labor policies are a key target for research, as well. Several studies find that the weakening of labor institutions such as unions and the declining real value of minimum wages were responsible for the increase in earnings inequality in several developing countries, especially in the 1990s, while more ambitious labor policies contributed to the reduction in inequality in the 2000s. Migration and sector changes are also determinants of inequality changes, studied at least since the seminal contributions by Lewis (1954) and Kuznets (1955). Changes in inequality are associated to the geographic and sectorial pattern of growth (Loayza and Raddatz, 2010). 4.7. Exploring inequality changes In any case, the pattern in Latin America points to an increase in inequality before the crashes (regressive inflation tax, rise in unemployment due to openness to trade and loss of competitiveness from exchange rate mismanagement), then followed by short-term reductions after stabilization programs.79 55 Alvaredo-Gasparini Table 4.3 Inequality and GDP collapses Source: own calculations based on PovcalNet (2013) and WDI. Panel A (Atkinson and Morelli, 2011) Panel B (developing countries 1980-2010) Table 4.3 Inequality and GDP collapses Source: own calculations based on PovcalNet (2013) and WDI. Others. Of course this brief review does not exhaust the multiple factors behind distributive changes in the developing world; in fact, arguably any shock or policy could affect the income distribution. For instance, demographic factors, such as the decline in fertility, the rise in life expectancy and the growing importance of assortative mating and single-parent households have been identified as relevant sources of inequality changes. Labor policies are a key target for research, as well. Several studies find that the weakening of labor institutions such as unions and the declining real value of minimum wages were responsible for the increase in earnings inequality in several developing countries, especially in the 1990s, while more ambitious labor policies contributed to the reduction in inequality in the 2000s. Migration and sector changes are also determinants of inequality changes, studied at least since the seminal contributions by Lewis (1954) and Kuznets (1955). Changes in inequality are associated to the geographic and sectorial pattern of growth (Loayza and Raddatz, 2010). Ferreira and Ravallion (2009), for instance, report that in Indonesia a large share of the increase in inequality was associated with migration from wage employment in agriculture to urban self-employment. Milanovic (2012) argues about the key role of migration in global inequality. Panel A (Atkinson and Morelli, 2011) Panel B (developing countries 1980-2010) Table 4.3 Inequality and GDP collapses Source: own calculations based on PovcalNet (2013) and WDI. Panel A (Atkinson and Morelli, 2011) Panel B (developing countries 1980-2010) Inequality and GDP collapses Inequality and GDP collapses Inequality and GDP collapses and WDI. Panel B (developing countries 1980-2010) Panel B (developing countries 1980-2010) Panel B (developing countries 1980-2010) Panel A (Atkinson and Morelli, 2011) Panel A (Atkinson and Morelli, 2011) ( , ) Source: own calculations based on PovcalNet (2013) and WDI. Others. 5. Poverty: levels The vast literature on poverty measurement suggests that there are neither normative nor objective arguments to set an unambiguous threshold below which everybody is poor and above which everyone is non-poor (Deaton, 1997). Despite this central conceptual ambiguity, reducing poverty is a deliberate policy objective for governments around the world. The international community has embraced this goal as reflected in the first Millennium Development Goal of halving poverty by 2015. In this section we focus on measures of poverty in the income/consumption space using international poverty lines in terms of US dollars adjusted for purchasing power parity (PPP). This choice implies taking a one-dimensional, monetary, static, absolute view of poverty, that certainly has many limitations and drawbacks, but it is still the best available paradigm to summarize deprivations in the world. 56 Alvaredo-Gasparini The $1-a-day per person at PPP is a poverty standard meant to define an international norm to gauge at the inability to pay for food needs.80 The $1 line, proposed in Ravallion et al. (1991) and used in World Bank (1990), was chosen as being representative of the national poverty lines found among low-income countries. The line was recalculated in 1993 PPP terms at $1.0763 a day (Chen and Ravallion, 2001), and more recently in 2005 PPP at $1.25 a day (Ravallion et al., 2009). The PPP adjustment is based on the 2005 round of the International Comparison Program (World Bank, 2008).81 The $1.25 line is usually deemed too low for middle-income countries; for that reason it is typical to compute poverty with the $2-a-day standard, which is close to the median of the official poverty lines chosen by the developing countries. Although these international lines have been criticized, their simplicity and the lack of reasonable and easy-to-implement alternatives have made them the standard for international poverty comparisons.82 While the measurement of poverty with national lines takes into consideration that societies differ in the criteria used to identify the poor, the international lines are unavoidable instruments to compare absolute poverty levels and trends across countries, and provide regional and world poverty counts. The World Bank is the main institution that regularly produces information on poverty measurement in the developing world drawn from original microdata from household surveys.83 In 2013, the World Bank released an update of the developing world's poverty estimates for 1981-2010. p y 83 Ahulwalia et al. (1979) was an early attempt to measure poverty in the developing countries. 81 See the discussions on the PPP adjustments in Deaton and Dupriez (2008), Deaton and Heston (201 and Ravallion (2010). 82 82 For the debate on the international measurement of poverty, see Reddy and Pogge (2010), Deaton (2010) and chapter 12 by Anand and Segal in this volume. Gentilini and Sumner (2012) compute global poverty using the national poverty lines officially set in each country instead of using international poverty standards. 80 For simplicity, the sign $ is used to refer to US dollars. 81 80 For simplicity, the sign $ is used to refer to US dollars. 81 80 For simplicity, the sign $ is used to refer to US dollars. 81 See the discussions on the PPP adjustments in Deaton and Dupriez (2008), Deaton and Heston (2010) and Ravallion (2010). 82 For the debate on the international measurement of poverty, see Reddy and Pogge (2010), Deaton (2010) and chapter 12 by Anand and Segal in this volume. Gentilini and Sumner (2012) compute global poverty using the national poverty lines officially set in each country instead of using international poverty standards. 83 Ahulwalia et al. (1979) was an early attempt to measure poverty in the developing countries. 5. Poverty: levels The new poverty estimates combine the PPP exchange rates for household consumption from the 2005 International Comparison Program with data from more than 850 household surveys across 127 developing countries. In this section we rely heavily on that dataset (PovcalNet). The problem of the choice of the welfare variable discussed for inequality in section 3 applies to the measurement of poverty, as well. While poverty estimates in PovcalNet refer to consumption deprivation, in most countries in Latin America and a few others in the rest of the world they are constructed from income data. After computing consumption and income poverty in 22 household surveys of 7 Latin American countries using the $2 standard, we find that on average the ratio consumption/income poverty is 0.97 with only small differences across countries. Given this piece of evidence we decided not to perform an adjustment for income poverty figures in the analysis that follows. 57 57 Alvaredo-Gasparini Alvaredo-Gasparini 5.1. Income poverty in the developing world Although poverty is a ubiquitous characteristic of the developing economies, its severity widely varies across countries. Figure 5.1 shows the poverty headcount ratio in most of the developing countries in the world, using the $2-a-day poverty line. The figure reveals the enormous differences among developing nations in terms of monetary deprivation. While there are economies where the proportion of the population living with less than 2 dollars a day is below 2%, in several countries that proportion exceeds 80%. The problem of absolute income poverty has a radically different scale in some countries compared to others, even in the developing world. Figure 5.1 Poverty headcount ratio Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Poverty headcount China Indonesia Brazil India Source: own calculations based on PovcalNet (2013). Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste $2-a-day line. In 2010, 41 per cent of the population in the developing world lived with less than $2 a day. The unweighted mean headcount ratio was significantly lower: in a typical developing country 33 per cent of the population was poor according to that criterion. The difference between the weighted and unweighted mean is not determined by China, as the incidence of poverty in that country is similar to the developing world mean, but by India (and to a lesser extent Indonesia and Pakistan), where the deprivation measures are substantially higher. In fact, when ignoring India both the weighted and unweighted headcount ratios become very close (33.3 and 32.7). The median poverty rate is also lower than the mean (23.5 for the $2 line). Table 5.1 reports these results for other indices and poverty lines. Interestingly, when using the $1.25 line the weighted mean is lower than the unweighted mean for the poverty gap 58 Alvaredo-Gasparini and the squared poverty gap, a result driven by the relatively low value of these indicators in China and Indonesia. 84 Rwanda, Mozambique, Malawi, Nigeria, Tanzania, Zambia, Madagascar, Burundi, Congo Dem. Rep. and Liberia. 85 The share of India and China in the world poor is 47% with the $1.25 line, 52% with the $2 line and 51% with the $4 line. 85 The share of India and China in the world poor is 47% with the $1.25 line, 52% with the $2 line an 51% with the $4 line. 5.1. Income poverty in the developing world and the squared poverty gap, a result driven by the relatively low value of these indicators in China and Indonesia. Table 5.1 Poverty measures Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. weighted mean std.dev median $ 1.25 poverty line Headcount 21.4 19.6 22.9 9.3 Poverty gap 6.5 7.5 10.7 2.9 Squared poverty gap 3.0 4.0 6.5 1.0 $ 2 poverty line Headcount 41.0 33.0 30.1 23.5 Poverty gap 15.8 14.5 16.4 7.1 Squared poverty gap 8.1 8.3 10.9 4.0 $ 4 poverty line Headcount 66.7 54.8 33.6 59.3 Poverty gap 35.9 29.6 24.0 25.4 Squared poverty gap 22.9 19.5 18.5 13.2 unweighted Note: poverty computed over the distribution of consumption/income per capita. The picture of poverty in the developing world is not significantly affected by changing the poverty indicator or the poverty line. The correlations across countries when using alternatively the headcount (H), the poverty gap (PG) and the squared poverty gap (SPG) with a given poverty line are all higher than 0.9. For a fixed indicator the correlations are higher than 0.95 when changing the poverty line. The correlations are only slightly lower when changing both the indicator and the line (e.g. 0.85 for SPG with the $1 line and H with the $2 line). The top ten steps in the poverty ladder, using the headcount ratio with the $2 line, are all occupied by Sub-Saharan African countries.84 The following ten features also eight SSA economies, in addition to a Caribbean country (Haiti), and a South Asian nation (Bangladesh). However, given its size, India is the country with the largest number of poor people. While around 840 million people in that country live with less than $2 a day, the number in the second nation in that ranking, China, is less than a half (359). Both countries are home of 52% of the poor in the world, while the following four countries - Nigeria, Bangladesh, Indonesia and Pakistan – represent 19%. Of course, these exact figures are valid only for a specific definition of income poverty, but the main results are robust to changes in indices and poverty lines.85 59 59 Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo-Gasparini As expected, the relationship between mean consumption and poverty is very tight (Figure 5.2). 5.1. Income poverty in the developing world A simple model of the headcount ratio ($2 line) on log mean consumption per capita estimated in a cross section of developing countries for 2010 accounts for more than 70% of the variation in the data. Figure 5.2 Poverty headcount ratio and log consumption per capita Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted 2$-a-day line. 0 10 20 30 40 50 60 70 80 90 100 3 3.5 4 4.5 5 5.5 6 6.5 7 headcount ratio ln consumption per capita (PPP US$) Figure 5.2 Poverty headcount ratio and log consumption per capita Developing countries, 2010 ln consumption per capita (PPP US$) Source: own calculations based on PovcalNet (2013). Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted 2$-a-day line. ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted 2$-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste 2$-a-day line. The cross-country relationship between poverty and inequality is much looser (Figure 5.3). The correlation coefficient between the headcount ($2 line) and the Gini is 0.17 (barely significant at 5%). The relationship is somewhat tighter with other poverty indices, but still in all cases the correlation coefficients are lower than 0.3. The magnitude of the correlations is similar when considering different income shares as measures of inequality. 60 60 Alvaredo-Gasparini Alvaredo-Gasparini Alvaredo Gasparini Figure 5.3 Poverty headcount ratio and Gini coefficient Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. 0 10 20 30 40 50 60 70 80 90 100 20 25 30 35 40 45 50 55 60 65 70 headcount ratio Gini coefficient Figure 5.3 Poverty headcount ratio and Gini coefficient Developing countries, 2010 l l b d l ( ) 0 10 20 30 40 50 60 70 80 90 100 20 25 30 35 40 45 50 55 60 65 70 headcount ratio Gini coefficient Figure 5.3 Poverty headcount ratio and Gini coefficient Developing countries, 2010 Source: own calculations based on PovcalNet (2013). ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. 5.1. Income poverty in the developing world ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste $2-a-day line. Table 5.2 shows some simple regressions aimed at characterizing the relationship between poverty, mean income and inequality in a cross-section of developing countries. The results of course do not have any causal implication, and then are not helpful to orient policy, but nonetheless are illustrative of the empirical relationship among these three variables. An increase (cross-country) of 1% in mean consumption is associated to a fall of around 2% in the headcount ratio, while a drop of 1% in the Gini coefficient is associated to a reduction of around 3.5% in poverty measured by the headcount. The results are similar when measuring deprivation with the squared poverty gap. Table 5.2 Regressions of poverty measures Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 in the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles 1 to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) ln y -2.0 -2.0 -2.1 -2.0 -2.1 -2.0 -2.2 -2.1 (0.114)*** (0.085)*** (0.086)*** (0.086)*** (0.127)*** (0.092)*** (0.084)*** (0.091)*** ln Gini 3.32 3.76 (0.329)*** (0.349)*** Share d1 -0.74 -0.91 (0.073)*** (0.071)*** Share d1-d6 -0.12 -0.13 (0.011)*** (0.012)*** Constant 12.6 0.1 14.8 16.1 11.0 -3.1 13.8 15.1 (0.577)*** (1.30) (0.484)*** (0.561)*** (0.64)*** (1.39) (0.473)*** (0.586)*** R2 0.72 0.85 0.85 0.85 0.68 0.84 0.87 0.85 log headcount ratio log squared poverty gap Table 5.2 Regressions of poverty measures Developing countries, 2010 Regressions of poverty measures Developing countries, 2010 ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste $2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 in the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles 1 to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. ln y=log mean household consumption/income per capita; share d1= share of decile 1 in the household consumption/income per capita distribution; share d1-d6=cumulative share of deciles 1 to 6. Robust standard deviations are shown under the coefficients. *** significant at 1% level. 61 61 Alvaredo-Gasparini 5.2. Poverty by region Poverty has a clear regional component: Table 5.3 reveals that Eastern Europe and Central Asia is always the region with the lowest income poverty, followed by Middle East and North Africa and Latin America and the Caribbean. Poverty in South Asia is substantially larger than in Eastern Asia when weighting by population, but roughly similar when ignoring weights. All income poverty measures are substantially higher in Sub-Saharan Africa than in the rest of the developing world. Table 5.3 Poverty indicators by region Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. East Asia & Pacific Eastern Europe & Central Asia Latin America & Caribbean Middle East & North Africa South Asia Sub-Saharan Africa Weighted $1.25 line Headcount 12.5 1.0 5.5 2.4 31.0 48.5 Poverty gap 2.8 0.3 2.9 0.6 7.1 20.9 Squared poverty gap 0.9 0.2 2.1 0.2 2.4 11.8 $2 line Headcount 29.7 2.4 10.4 12.0 66.7 69.9 Poverty gap 9.7 0.7 4.7 2.8 23.4 35.7 Squared poverty gap 4.2 0.3 3.2 1.0 10.5 22.4 Unweighted $1.25 line Headcount 17.9 1.6 8.6 3.5 17.6 41.5 Poverty gap 5.1 0.5 4.0 0.9 3.9 16.6 Squared poverty gap 2.3 0.2 2.6 0.3 1.3 8.8 $2 line Headcount 38.4 5.2 15.6 13.5 43.5 62.2 Poverty gap 13.8 1.5 7.0 3.5 14.1 30.2 Squared poverty gap 6.7 0.6 4.4 1.4 6.1 18.1 ( ) Note: poverty computed over the distribution of consumption/income per capita. Figure 5.4 unveils the considerable heterogeneity within each geographic region. When using the $2 line, the poverty headcount ratio ranges in EAP from 1.4 (Malaysia) to 70.6 (Timor-Leste), in ECA from 0.1 (Slovenia) to 35.6 (Georgia), in LAC from 1.2 (Uruguay) to 80.1 (Haiti), in MENA from 1.6 (Jordan) to 45.6 (Yemen), in SA from 6.8 (Maldives) to 76.5 (Bangladesh), and in SSA from 1.5 (Seychelles) to 94.5 (Liberia). Figure 5.5 displays a map of the poverty levels in the world that illustrates the regional differences, as well as the within-region heterogeneities. 62 62 Alvaredo-Gasparini Figure 5.4 Poverty headcount ratio Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. 5.2. Poverty by region 0 10 20 30 40 50 60 70 80 90 100 Latin America & the Caribbean Sub-Saharan Africa Eastern Europe & Central Asia East Asia & Pacific Middle East & North Africa South Asia Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste $2-a-day line. There is a considerable degree of spatial correlation of poverty measures across countries. The Moran scatterplot is a way to illustrate that spatial correlation (Figure 5.6). The horizontal axis shows the normalized headcount ratio of a country ($2 line), while the vertical axis depicts a weighted average of its neighbors´ normalized poverty rates, where neighborhood is defined in terms of geographical proximity. The graph suggests a strong positive correlation between a country poverty incidence rate and that of its neighbors (the Moran correlation coefficient is 0.507, significant at 1%). Almost 80% of the countries are either in the HH cells (high poverty for the country and its neighbors) or in the LL cells. 63 63 Alvaredo-Gasparini Figure 5.6 Spatial correlation of poverty rates Moran´s scatterplot Developing countries, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. z is the normalized poverty headcount ratio (the value minus the mean, divided by the standard deviation), Wz is the weighted average of the normalized poverty headcount ratios of a country´s neighbors, where the weights W are defined in terms of contiguity. HH LH HL LL -1 0 1 2 3 Wz -1 0 1 2 3 z Moran scatterplot (Moran's I = 0.507) , Moran scatterplot (Moran's I = 0.507) Source: own calculations based on PovcalNet (2013). ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. z is the normalized poverty headcount ratio (the value minus the mean, divided by the standard deviation), Wz is the weighted average of the normalized poverty headcount ratios of a country´s neighbors, where the weights W are defined in terms of contiguity. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. z is the normalized poverty headcount ratio (the value minus the mean, divided by the standard deviation), Wz is the weighted average of the normalized poverty headcount ratios of a country´s neighbors, where the weights W are defined in terms of contiguity. 5.2. Poverty by region The poverty gap indicator has an intuitive-appealing interpretation: when normalized by the poverty line and the total population of a country, it gives the total cost needed to end poverty, in the particular case in which cash transfers could be perfectly targeted to poor people in the amount just needed to reach the poverty line, and no changes in behavior take place. Table 5.4 shows the unweighted mean across countries of the cost of eliminating poverty as percentage of GDP under this scenario in each region. Although the context is clearly unrealistic, the figures give a rough idea of the magnitude of the task of fighting poverty in each region of the developing world in relation to the available economic resources. While eliminating poverty with the $2 line in this scenario would require on average less than 1 GDP point in the economies of ECA and between 1 and 2 points in MENA and LAC, the size of the effort is larger in Asia and orders of magnitude greater in Sub-Saharan Africa. 64 64 Alvaredo-Gasparini Table 5.4 The cost of eliminating poverty Total poverty gap as percentage of GDP Unweighted means by region, 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. Region $ 1.25 $ 2 East Asia and Pacific 0.6 2.9 Eastern Europe and Central Asia 0.1 0.3 Latin America and the Caribbean 0.7 1.7 Middle East and North Africa 0.2 1.0 South Asia 1.2 6.6 Sub-Saharan Africa 8.1 23.0 Poverty lines Note: poverty computed over the distribution of consumption/income per capita. International surveys, such as the Gallup Poll, provide an opportunity to alleviate some of the typical comparability problems of household surveys, since survey design and questionnaires are identical across countries. However, as discussed above, these surveys have still small samples, and measurement errors are presumably large, given that only one income question is included. The correlation between headcount ratios computed with the Gallup Poll and PovcalNet is 0.32, significant at 2%, while the rank Spearman correlation is 0.61, significant at 1%. Figure 5.7 shows the cumulative density function in some regions of the world, based on Gallup data. There is first-order stochastic dominance of the Western Europe distribution over the rest, while the South Asian distribution is dominated by the rest. The curves of ECA and EAP cross each other, although they do so at high-income levels. 86 Chapter 12 in this volume also covers the issue of global poverty in the developing world. In our chapter the interest is more focused on the country trends, and then we make more use of unweighted statistics. 6. Poverty: trends In the last decades the developing world has made undeniable progress toward the goal of ending absolute poverty. The evidence suggests that the first goal of the Millennium Development Goals – to halve extreme poverty from 1990 to 2015 – was already met in 2010 for the aggregate of developing countries. However, the strong decline in global absolute poverty hides substantial heterogeneities across economies and over time. In this section we document and characterize trends in income poverty in the countries of the developing world since the early 1980s to 2010, and trace those changes to economic growth and distributional changes.86 The literature on international poverty trends can be divided into two main strands. The first one makes comparisons based exclusively on household survey microdata. This ambitious road that requires access to surveys for many countries over time has been taken by several initiatives at the regional level, and by the World Bank at a global scale, mainly through the work of Martin Ravallion and Shaohua Chen. The second strand combines estimates of the national income distributions with GDP or aggregate consumption data drawn from National Accounts to anchor the mean. Bourguignon and Morrison (2002), Bhalla (2002), Karshenas (2003) and Sala-i-Martin (2006) are examples of this literature. In this section we mainly document the results of the first approach. 5.2. Poverty by region Figure 5.7 Distribution functions Source: own estimates based on microdata from Gallup World Poll 2006. Note: cumulative distribution functions of per capita household income. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0 5000 10000 15000 20000 25000 30000 South Asia East Asia & Pacific East Europe & Central Asia Western Europe Figure 5.7 Distribution functions Source: own estimates based on microdata from Gallup World Poll 2006. Note: cumulative distribution functions of per capita household income. 65 65 Alvaredo-Gasparini Alvaredo-Gasparini 6. Poverty: trends 87 The cumulative distribution function of 2010 lies always below the functions corresponding to all previous years. The first-order stochastic dominance assures that the result of lower poverty in 2010 is robust to the choice of poverty line and valid for a broad class of poverty measures (Atkinson, 1987). 6.1. Trends in income poverty Data from PovcalNet reveals a widespread fall in absolute income poverty in the developing countries over the last three decades (Figure 6.1). Only for a few nations poverty in 2010 was not lower than in 1981; that set includes some economies in Eastern Europe, Central Asia and Sub-Saharan Africa, and only a few in Latin America and the Caribbean. The poverty decline in the 2000s was even more generalized: in only 8 out of 121 countries did poverty increase between 1999 and 2010 (5 in Sub- Saharan Africa). 66 66 Alvaredo-Gasparini Figure 6.1 Poverty headcount ratio Developing countries Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Poverty 1981-2010 Poverty 1999-2010 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Headcount ratio 2010 Headcount ratio 1981 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Headcount ratio 2010 Headcount ratio 1999 Figure 6.1 Poverty headcount ratio Developing countries Poverty 1981-2010 Figure 6.1 Poverty headcount ratio Developing countries Poverty 1981-2010 Figure 6.1 Poverty headcount ratio Developing countries Poverty 1981-2010 Poverty 1981-2010 Pover 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 Headcount ratio 2010 Headcount ratio 1981 0 10 20 30 40 50 60 70 80 90 100 0 Headcount ratio 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Source: own calculations based on PovcalNet (2013). ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste $2-a-day line. The proportion of the population in the developing world living with less than $1.25 per day decreased from 52% in 1981 to 20.8% in 2010, which implies a trend decline of around one point per year (Figure 6.2).87 This is a remarkable achievement that should not be overlooked. It would be hard to find other episodes in history where the extent of extreme poverty was reduced so massively in such a short period of time. Poverty headcount ratio y Weighted and unweighted means, developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). 87 The cumulative distribution function of 2010 lies always below the functions corresponding to all previous years. The first-order stochastic dominance assures that the result of lower poverty in 2010 is robust to the choice of poverty line and valid for a broad class of poverty measures (Atkinson, 1987). 0 10 20 30 40 50 60 70 80 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Unweighted - $ 1.25 line Unweighted - $ 2 line Weighted - $ 1.25 line Weighted - $ 2 line y Weighted and unweighted means, developing countries, 1981-2010 y Weighted and unweighted means, developing countries, 1981-2010 Weighted and unweighted means, developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). 0 10 20 30 40 50 60 70 80 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Unweighted - $ 1.25 line Unweighted - $ 2 line Weighted - $ 1.25 line Weighted - $ 2 line Source: own calculations based on PovcalNet (2013). 0 10 20 30 40 50 60 70 80 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Unweighted - $ 1.25 line Unweighted - $ 2 line Weighted - $ 1.25 line Weighted - $ 2 line Source: own calculations based on PovcalNet (2013). 67 Alvaredo-Gasparini Note: poverty computed over the distribution of consumption/income per capita. However, this extraordinary result should be put in perspective. First, even after this global social improvement one of every five persons in the developing world still lives in extremely deprived conditions (less than $1.25 a day), while four out of ten people have household per capita consumption levels lower than a frugal $2 per day. However, this extraordinary result should be put in perspective. First, even after this global social improvement one of every five persons in the developing world still lives in extremely deprived conditions (less than $1.25 a day), while four out of ten people have household per capita consumption levels lower than a frugal $2 per day. Second, the positive performance of China is key for the global result. Ignoring China, the poverty decline is less impressive: the headcount ratio fell 16 instead of 31 points in three decades (Table 6.1).88 In fact, when ignoring China it is not clear that the developing world could meet the MDG for poverty reduction in 2015. While the population-weighted mean of the poverty incidence rate dropped 31 points between 1981 and 2010, the unweighted mean declined about 10 points, and the median went down just 8 points. The extreme poverty rate ($1.25) of a typical developing country was reduced from 29.5% in 1981 to 19% in 2010, which represents a fall of around a third of a point per year, a figure less impressive than the corresponding one for the global poverty rate (one point a year). In fact, this decline took place only since the late-1990s. 88 When ignoring China the distribution of 2010 still dominates (first-order stochastic dominance) 1981 although the distance between the two cumulative distributions is smaller. The curve for 1999 lies below 1981 and 1990 but only for poverty lines lower than $3 a day. y Weighted and unweighted means, developing countries, 1981-2010 A typical developing country did not experience any improvement for almost two decades: the unweighted poverty rate for the developing world was 29.5% in 1981, 29.8% in 1990 and 28.8% in 1996. From then on poverty declined more consistently, especially between 2002 and 2008, when for a typical developing economy the rate of poverty reduction was almost one point a year. 68 68 Alvaredo-Gasparini Table 6.1 Change in poverty measures (points) Developing countries, 1981-2010 Table 6.1 Change in poverty measures (points) Developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita. Poverty measure 1981- 1990 1990- 1999 1999- 2010 1981- 2010 Poverty measure 1981- 1990 1990- 1999 1999- 2010 1981- 2010 Headcount - $1.25 line Poverty gap - $1.25 line Unweighted Unweighted Mean 0.3 -1.9 -8.9 -10.5 Mean 0.2 -1.6 -4.2 -5.7 Median 4.9 0.0 -12.9 -8.0 Median 0.3 0.3 -4.0 -3.4 Weighted Weighted Mean -9.0 -8.9 -13.4 -31.2 Mean -6.4 -3.7 -4.8 -14.9 Without China -2.8 -3.1 -10.0 -15.9 Without China -1.6 -1.5 -3.7 -6.8 Headcount - $2 line Poverty gap - $2 line Unweighted Unweighted Mean 0.7 -0.6 -11.0 -11.0 Mean 0.3 -1.5 -6.5 -7.7 Median -0.3 -3.4 -17.1 -20.7 Median 2.3 0.8 -9.2 -6.1 Weighted Weighted Mean -4.9 -7.1 -16.6 -28.5 Mean -6.6 -5.4 -8.8 -20.8 Without China -1.2 -1.1 -10.9 -13.1 Without China -1.7 -1.8 -6.4 -9.9 Headcount - $4 line Poverty gap - $4 line Unweighted Unweighted Mean 0.9 2.2 -11.4 -8.3 Mean 0.4 -0.4 -9.0 -9.0 Median 2.1 -2.1 -16.3 -16.3 Median -0.4 -1.4 -12.3 -14.1 Weighted Weighted Mean -1.7 -1.3 -14.0 -17.0 Mean -8.4 -4.6 -12.1 -25.1 Without China -1.2 1.7 -8.3 -7.8 Without China -1.4 -0.5 -7.8 -9.7 ( ) Note: poverty computed over the distribution of consumption/income per capita. The fall in poverty appears less startling when using higher poverty lines. While the headcount ratio with the $1.25 line fell 60% from 1981 to 2010, it declined 41% when measured with the $2 line and 20% with the $4 line. In fact, while the MDG goal of halving $1-a-day poverty from the value in 1990 was already met in 2010, the assessment is different when using the $2 line: the headcount ratio in 2010 was about 2/3 of the value in 1990. 89 See Reddy and Pogge (2010), Chen and Ravallion (2012) and Cockburn et al. (2012). y Weighted and unweighted means, developing countries, 1981-2010 The performance of the developing world in terms of poverty reduction also looks much less spectacular when considering poverty counts, instead of the typical measures that are invariant to the size of the population. In fact, for the majority of the developing countries (63%) the number of poor people was higher in 2010 than in 1981. Even during the booming 2000s the poverty count increased in 30% of the nations. The number of poor people was in the late 2000s only slightly lower than in the early 1980s (2,585 million in 1981 and 2,394 million in 2010); ignoring China the poverty count actually increased in 422 million (from 1,613 to 2,035). Some authors have suggested that the difference in the assessment of world poverty when alternatively using the headcount ratio or the number of poor people may be behind some of the controversies in the public debate about globalization and the social performance of the world in the last decades.89 Since the calculation of global poverty with microdata is very cumbersome, and requires having a large number of comparable household surveys, some authors 69 69 Alvaredo-Gasparini estimate the changes in poverty with National Accounts data, anchoring the mean of the income distribution to output or domestic consumption taken from National Accounts, using secondary distributive data and making assumptions about the functional form of the income distribution, typically the lognormal parameterization (Ahluwalia et al., 1979; Chotikapanich et al., 1997; Bhalla, 2002; Sala-i-Martin, 2006; Pinkovskiy and Sala-i-Martin, 2009).90 This methodology allows ambitious calculations in terms of coverage, but it faces some obvious caveats as changes in National Accounts aggregates may be misleading proxies for changes in household per capita income, and the secondary distributive data in which the estimates are usually based are mined with several comparability problems.91 According to the estimates by Pinkovskiy and Sala-i-Martin (2009) (PSM) using a sample of 191 countries, and distributive data from the WIID, world poverty rates ($2 line) went down from 45.2% in 1970 to 37.8% in 1981, 24.9% in 1990, 16.8% in 1999, and 13% in 2006. y Weighted and unweighted means, developing countries, 1981-2010 That pattern implies a fall substantially faster than what is estimated with household surveys data alone in the 1980s and 1990s, but slower in the 2000s.92 For instance, while the magnitude of the poverty incidence rates in PSM for the $3 line is similar than in PovcalNet with the $1.25 line, the declines have been different: in PSM the headcount ratio fell at annual 2.6% and 3.5% in the 1980s and 1990s, respectively, while the rates were 2.1% and 2.5% with PovcalNet data. Instead, in the 2000s poverty fell at annual 3.1% in PSM and at annual 4.4% in PovcalNet. 90 It should be noticed that the World Bank poverty estimates also use National Accounts (NAS) data, although to a limited extent. For instance, for countries with only one household survey, poverty is estimated by applying the NAS consumption estimates to the available distribution data, assuming the Lorenz curve remains fixed. 91 An intermediate alternative is proposed by Karshenas (2003), who calibrates survey means using National Accounts statistics. Calibrated survey means are read off the fitted curve for the mean of household survey consumption conditional on the NAS mean. 93 See Datt and Ravallion (1992), Kakwani and Subbarao (1990), Kakwani (2000) and Mahmoudi (200 for different proposals. 92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallio (2010) and Pinkovskiy and Sala-i-Martin (2009). 93 90 It should be noticed that the World Bank poverty estimates also use National Accounts (NAS) data, although to a limited extent. For instance, for countries with only one household survey, poverty is estimated by applying the NAS consumption estimates to the available distribution data, assuming the Lorenz curve remains fixed. 91 An intermediate alternative is proposed by Karshenas (2003), who calibrates survey means using National Accounts statistics. Calibrated survey means are read off the fitted curve for the mean of household survey consumption conditional on the NAS mean. 92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallion (2010) and Pinkovskiy and Sala-i-Martin (2009). 93 See Datt and Ravallion (1992), Kakwani and Subbarao (1990), Kakwani (2000) and Mahmoudi (2001) for different proposals. y pp y g p , g Lorenz curve remains fixed. 91 An intermediate alternative is proposed by Karshenas (2003), who calibrates survey means using National Accounts statistics. Calibrated survey means are read off the fitted curve for the mean of household survey consumption conditional on the NAS mean. 92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallion 92 Dhongde and Minoiu (2010) explore several factors behind the different results in Chen and Ravallion (2010) and Pinkovskiy and Sala-i-Martin (2009). 93 See Datt and Ravallion (1992), Kakwani and Subbarao (1990), Kakwani (2000) and Mahmoudi (2001) 94 Since the decompositions could be carried out changing the base year, the table shows the averages over the two exercises. To obtain the results we use lineal approximations to the Pen curve based on information on mean consumption per capita by decile from PovcalNet. Table 6.2 Decomposition of poverty changes Developing countries, 1990-2010 Decomposition of poverty changes Developing countries, 1990-2010 Note: the columns show unweighted means across a sample of 76 developing countries of the change in poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. Note: the columns show unweighted means across a sample of 76 developing countries of the change in poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. 6.2. Decomposing poverty changes Researchers frequently use decompositions of changes in poverty into growth and redistribution effects in order to characterize poverty trends.93 The growth effect refers to the poverty change between two years that would have occurred if the mean income had changed as it did, but the shape of the distribution had stayed fixed. On the other hand, the redistribution effect records the poverty change between two years that would have occurred if the shape of the distribution had changed in the way it did, but the mean had remained fixed. Of course, this is just a mechanical exercise, as indicators of economic growth, and changes in inequality and poverty are just three different ways of aggregate information on income dynamics, and therefore they are all jointly determined by the general equilibrium of the economy. In that sense the decompositions are helpful to illustrate the way incomes have changed and affected 70 70 Alvaredo-Gasparini poverty, but they are silent on the fundamental factors underlying poverty changes, and on the policies recommended to reduce deprivations more effectively. The change in the growth-inequality-poverty triangle was very different in the 2000s compared to the previous decade. Table 6.2 illustrates this difference showing the unweighted mean of the growth and redistribution effects of changes in poverty for a sample of 76 developing countries with information on deciles shares in PovcalNet.94 The mild fall in the poverty headcount ratio ($2 line) in the 1990s can be decomposed into a poverty-decreasing growth effect that outweighed a poverty-increasing redistribution effect. On average (unweighted) mean consumption grew at an annual 0.2 per cent, and the Gini increased about 0.3 points per year, implying a very modest decline in poverty. In contrast, in the 2000s both effects contributed to a falling poverty. A robust increase of mean consumption (more than 3 per cent a year) and a slow fall in inequality (around 0.1 Gini points a year) combined to yield a substantial reduction in indicators of material deprivation. The growth effect was particularly large, accounting for 90 per cent of the fall in the headcount ratio ($2 line). This result does not mean that distributional changes are not important, but instead that they have not been the main drivers of poverty reduction in the past. Table 6.2 Decomposition of poverty changes Developing countries, 1990-2010 Source: own calculations based on data from PovcalNet. 6.2. Decomposing poverty changes Note: the columns show unweighted means across a sample of 76 developing countries of the change in poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. Actual change Growth Redistribution 1990-1999 -0.3 -1.5 1.2 1999-2010 -10.1 -9.0 -1.1 1990-2010 -10.5 -10.4 -0.1 Effects Table 6.2 Decomposition of poverty changes Developing countries, 1990-2010 Source: own calculations based on data from PovcalNet. Note: the columns show unweighted means across a sample of 76 developing countries of the change in poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. Actual change Growth Redistribution 1990-1999 -0.3 -1.5 1.2 1999-2010 -10.1 -9.0 -1.1 1990-2010 -10.5 -10.4 -0.1 Effects 6.3. The cost of closing the poverty gap Although still a challenging problem, eliminating absolute extreme income poverty is an increasingly affordable target. Based on PovcalNet data we computed the population-weighted poverty gap index in the developing world as a share of global GDP. This fraction, that indicates the fiscal effort required to end poverty in a scenario of perfect targeting, absent behavioral responses, has been substantially falling over time as poverty decreased and global GDP went up. The resources needed to close the poverty gap with the $1.25 line as a share of global GDP declined from 1.3% in 1981 to 71 Alvaredo-Gasparini 0.2% in 2010 (the corresponding values for the $2 line are 3.6% and 1%).95 As a share of the GDP in the developing world the cost of closing the poverty gap fell from 1.9% in 1981 to 0.4% in 2010 (5.4% and 1.8% for the $2 line). Kanbur and Sumner (2011) highlight the fact that while in 1990 over 90 per cent of the world’s extremely poor people ($1.25) lived in countries classified as low-income countries (LICs), by the late 2000s this share dropped to less than 30 per cent. From the fact that most of the world’s poor live in middle-income countries with the domestic financial capacity to end at least extreme poverty, Sumner (2012) concludes that poverty reduction is increasingly becoming a domestic issue of national distribution and domestic political economy, rather than primarily an aid and international issue. Table 6.3 suggests than on average (unweighted) across developing countries the redistributive national effort to end extreme poverty fell from 8.2% of GDP in 1981 to 4% in 2010. The median value is much lower, and has fallen from 1.9% in 1981 to 0.5% in 2010. The third column shows the share of countries where the cost of eliminating extreme income poverty is less than 1 GDP point. That share substantially increased in the 2000s, from 41.3% in 1999 to 55.4% in 2010. Similarly, the share of countries for which closing the poverty gap is fiscally very burdensome (in the table more than 3 GDP points) fell from more than 50% in 1990 to about 30% in 2010. Sumner (2012) reports similar trends, although a lower proportion of countries with poverty gap/GDP less than 1%. Ravallion (2010) also notes that most middle- income countries would require very small additional taxation to end poverty. 95 This computation assumes zero poverty in the high-income countries, when deprivation is measured with the international lines. Note: poverty computed over the distribution of consumption/income per capita. Unweighted statistic Source: own calculations based on PovcalNet (2013). Source: own calculations based on PovcalNet (2013). 96 The contrast between East Asia and the rest of the developing world is evident in terms of progress towards the first goal of the MDGs. EAP already met the goal in the early 2000s, far ahead of the rest. 6.3. The cost of closing the poverty gap Table 6.3 Poverty gap as share of GDP Mean, median, share of countries with gap/GDP less than 1% and greater than 3% Developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Mean Median % less 1 GDP point % more 3 GDP points Mean Median % less 1 GDP point % more 3 GDP points 1981 8.2 1.9 42.4 45.7 19.9 5.4 22.9 60.4 1984 8.4 2.2 41.3 45.7 20.5 6.6 19.8 60.4 1987 8.2 2.3 39.1 46.7 19.9 7.1 20.8 60.4 1990 8.1 3.3 39.1 51.1 19.6 7.0 24.0 59.4 1993 9.2 2.4 40.2 47.8 22.0 7.5 25.0 59.4 1996 9.2 2.0 40.2 42.4 21.6 7.3 24.0 60.4 1999 6.9 2.0 41.3 39.1 17.9 5.9 25.0 60.4 2002 5.9 1.4 41.3 40.2 15.7 4.8 27.1 58.3 2005 5.2 1.0 48.9 35.9 14.0 3.7 32.3 55.2 2008 4.3 0.7 50.0 33.7 11.6 3.0 40.6 50.0 2010 4.0 0.5 55.4 31.5 11.1 3.0 42.7 50.0 Poverty line $1.25 Poverty line $2 Poverty gap as share of GDP Mean, median, share of countries with gap/GDP less than 1% and greater than 3% Developing countries, 1981-2010 72 Alvaredo-Gasparini Alvaredo-Gasparini 6.4. Regional trends The patterns in income poverty over time have been heterogeneous across the geographic regions of the developing world (Table 6.4). At least three facts are worth stressing: (i) the remarkable decline in poverty in Asia over the last three decades, (ii) the lack of significant progress in the rest of the regions in the 1980s and 1990s, and (iii) the generalized fall in poverty in the 2000s. Arguably, the main fact about poverty dynamics in the last three decades has been the noteworthy decline in Asia. The share of people living with less than $2 a day went down from 92.4% to 29.7% between 1981 and 2010 in East Asia and Pacific, and from 87.2% to 66.7% in South Asia.96 The performance of EAP is enhanced by the presence of China, but also the unweighted mean dramatically fell in this region (from 70.4% in 1981 to 38.4% in 2010). The fall in the unweighted mean was similar in South Asia (from 80.3% to 43.5%). Almost all countries in Asia experienced drops in income poverty over the period 1981-2010. The reductions were on average larger in the 2000s than in the previous decades. For instance, in South Asia the unweighted mean fell 5.6 points in the 1980s, 10.9 in the 1990s and 20.4 in the 2000s. In the rest of the developing world the performance was weak and even negative in the 1980s and 1990s. Income poverty rose in Latin America in the 1980s, in Eastern Europe and Central Asia in the 1990s, and in Sub-Saharan Africa in both decades. In contrast, all regions experienced falling poverty in the 2000s. The reductions were large, and in general outweighed the weak performance of the previous decades. For instance, on average (unweighted) income poverty ($2 line) went down 72% in ECA, 43% in MENA, 34% in LAC and 12% in SSA. The proportion of countries where the headcount ratio fell more than 5% in the 2000s is above 90% in all these regions, with the exception of SSA, where the corresponding proportion is 66%. 73 73 Alvaredo-Gasparini Table 6.4 Poverty headcount ratio, developing world 1981-2010 Weighted and unweighted means Source: own calculations based on PovcalNet (2013). Note: the columns show unweighted means across a sample of 76 developing countries of the change in the poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. 6.4. Regional trends 1981 1990 1999 2010 Unweighted mean; $1.25 line East Asia and Pacific 53.4 44.3 33.3 17.9 Eastern Europe and Central Asia 4.0 4.5 7.5 1.4 Latin America and the Caribbean 13.2 14.4 13.7 8.6 Middle East and North Africa 10.3 7.2 6.8 3.5 South Asia 59.7 51.3 37.8 17.6 Sub-Saharan Africa 49.3 54.0 52.2 41.5 Unweighted mean; $2 line East Asia and Pacific 70.4 65.8 56.2 38.4 Eastern Europe and Central Asia 10.1 10.2 17.0 4.8 Latin America and the Caribbean 24.3 25.9 23.8 15.6 Middle East and North Africa 28.6 24.0 23.5 13.5 South Asia 80.3 74.7 63.9 43.5 Sub-Saharan Africa 67.1 71.5 71.0 62.2 Population-weighted mean; $1.25 line East Asia and Pacific 77.2 56.2 35.6 12.5 Eastern Europe and Central Asia 1.9 1.9 3.8 0.7 Latin America and the Caribbean 11.9 12.3 11.9 5.5 Middle East and North Africa 9.6 5.7 5.0 2.4 South Asia 61.1 53.8 45.1 31.0 Sub-Saharan Africa 51.4 56.5 57.9 48.5 Population-weighted mean; $2 line East Asia and Pacific 92.4 81.0 61.7 29.7 Eastern Europe and Central Asia 8.3 6.9 12.1 2.3 Latin America and the Caribbean 23.7 22.5 22.0 10.4 Middle East and North Africa 30.0 23.4 21.9 12.0 South Asia 87.2 83.6 77.8 66.7 Sub-Saharan Africa 72.2 75.9 77.5 69.9 The contrast between Asia and the rest of the developing world in terms of poverty reduction is even more dramatic when the calculations are carried out anchoring the mean of the income distribution to GDP from National Accounts. Figure 6.3 shows regional estimates taken from Pinkovskiy and Sala-i-Martin (2009), where the sharp declines in poverty in East and South Asia stand out. According to these estimates there would be poverty convergence across the regions of the developing world, with the exception of Sub-Saharan Africa. 74 74 Alvaredo-Gasparini Figure 6.3 Poverty headcount ratio Developing countries, 1970-2006 Source: Pinkovskiy and Sala-i-Martin (2009). Note: poverty line $2 a day. 6.4. Regional trends 0 10 20 30 40 50 60 70 80 90 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 East Asia South Asia Sub-Saharan Africa Latin America Middle East and North Africa Figure 6.3 Poverty headcount ratio Developing countries, 1970-2006 Figure 6.3 Poverty headcount ratio Developing countries, 1970-2006 Figure 6.3 Poverty headcount ratio Developing countries, 1970-2006 0 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 Source: Pinkovskiy and Sala-i-Martin (2009). Source: Pinkovskiy and Sala-i-Martin (2009). Note: poverty line $2 a day. Regions have been different in terms of the growth-inequality-poverty triangle (Table 6.5). The growth effect was strong in Asia dwarfing a much smaller (and sometimes poverty-increasing) redistribution effect. The increase in poverty in ECA in the 1990s is associated with both negative growth and inequality rise, while the fall in poverty in the following decade is mostly accounted by neutral positive growth. In Latin America growth contributed in both decades, but only in the 2000s did the redistribution effect become poverty-reducing. Finally, in Africa (MENA and SSA) the growth effect in the 2000s was the major contributing factor to the fall in poverty. Table 6.5 Decomposition of poverty changes Developing countries Source: own calculations based on data from PovcalNet. Note: the columns show unweighted means across a sample of 76 developing countries of the change in the poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. Actual Actual change Growth Redistribution change Growth Redistribution EAP MENA 1990-1999 -10.5 -10.7 0.2 1990-1999 -1.5 -0.6 -1.0 1999-2010 -19.4 -19.2 -0.2 1999-2010 -10.1 -8.0 -2.1 1990-2010 -30.0 -30.3 0.3 1990-2010 -11.6 -8.4 -3.3 ECA SA 1990-1999 8.6 6.1 2.5 1990-1999 -9.0 -10.9 2.0 1999-2010 -11.5 -10.5 -1.0 1999-2010 -13.2 -12.0 -1.1 1990-2010 -2.9 -3.9 1.0 1990-2010 -22.1 -23.3 1.2 LAC SSA 1990-1999 -3.2 -5.3 2.1 1990-1999 -1.8 -1.4 -0.4 1999-2010 -7.9 -5.3 -2.6 1999-2010 -5.9 -6.0 0.0 1990-2010 -11.1 -10.2 -0.9 1990-2010 -7.7 -7.8 0.0 Effects Effects Table 6.5 Decomposition of poverty changes Developing countries Note: the columns show unweighted means across a sample of 76 developing countries of the change in the poverty headcount ratio ($2 line), and the growth and redistribution effects from the poverty change decomposition. 97 See Ahuja et al. (1997) and Nissanke and Thorbecke (2010) as examples of a vast literature. 98 p p g $ / y p y 99 During the early and mid-1990s poverty declined substantially, but then in the late 1990s to the early 2000s the downward trend stalled. Li et al. (2013) argue that further reductions in poverty became more challenging due to several factors, for example, the fact that a high proportion of the remaining poverty was geographically dispersed and transient, and also because poverty had become less responsive to macroeconomic growth (World Bank, 2009). Policies adopted after 2002, such as the minimum living guarantee program, the new rural cooperative medical system, and the new rural pension system, have addressed some of these factors. 6.4. Regional trends 75 75 Alvaredo-Gasparini In the rest of this section we take a closer look at the changes in poverty reported by the literature in each geographic region of the developing world. In the rest of this section we take a closer look at the changes in poverty reported by the literature in each geographic region of the developing world. 98 According to World Bank (2012), poverty in East Asia and Pacific “will continue to decline, and th share of people living on $2/day will reach 24.5 per cent by the end of 2013.” 97 See Ahuja et al. (1997) and Nissanke and Thorbecke (2010) as examples of a vast literature. 98 According to World Bank (2012), poverty in East Asia and Pacific “will continue to decline, and the share of people living on $2/day will reach 24.5 per cent by the end of 2013.” 99 During the early and mid-1990s poverty declined substantially, but then in the late 1990s to the early 2000s the downward trend stalled. Li et al. (2013) argue that further reductions in poverty became more challenging due to several factors, for example, the fact that a high proportion of the remaining poverty was geographically dispersed and transient, and also because poverty had become less responsive to macroeconomic growth (World Bank, 2009). Policies adopted after 2002, such as the minimum living guarantee program, the new rural cooperative medical system, and the new rural pension system, have addressed some of these factors. East Asia and Pacific As mentioned above, East Asia and Pacific has achieved an impressive record in terms of poverty reduction. The fall in the indicators of material deprivation has been strong, sustained over the two last decades and widespread across countries.97 The poverty headcount ratio with the $2 line fell from 92.4% in 1981 to 81% in 1990, 61.7% in 1999 and 29.7% in 2010.98 China’s progress against absolute poverty was a key factor behind this dynamics (World Bank, 2009; Ravallion and Chen, 2007; Minoiu and Reddy, 2008). Rural areas accounted for the bulk of the gains to the poor, although migration to urban areas helped; rural economic growth was far more important to national poverty reduction than urban economic growth; agriculture played a far more important role than the secondary or tertiary sources of GDP, mainly from the efficiency gains after the decollectivization of agriculture. Ravallion and Chen (2007) claim that, though they cannot offer a rigorous test against alternative explanations, “the halving of the national poverty rate in the first few years of the 1980s was largely attributable to picking these blow-lying fruits of agrarian reform”. Provinces starting with relatively high inequality saw slower progress against poverty, due both to lower growth and a lower growth elasticity of poverty reduction. While in 1990 the incidence of poverty in China was roughly 25 points higher than in the rest of the developing world, by the end of the 2000s, it had fallen more than 10 points below the average.99 Between 1990 and 2010 the headcount ratio fell from 60% to 12% in China ($1.25 line), and from 54% to 18% in Indonesia, the two most populated countries in the region. The rest of the economies experienced similar patterns. The headcount ratio dropped from 58% to 15% in Cambodia, from 12% to 0.4% in Thailand, and from 73% to 14% in Vietnam. The reductions were less spectacular, but anyway significant in the Pacific countries (Micronesia, Philippines, Papua New Guinea, Timor-Leste). According to Asian Development Bank (2012) using the national poverty lines, poverty declined in all economies except Mongolia, Micronesia, Samoa, Timor-Leste, Tonga and Tuvalu. With 76 Alvaredo-Gasparini Alvaredo-Gasparini respect to the poverty reduction target of the Millennium Development Goals, of the ten economies for which data are available, 6 have achieved the poverty target, and Cambodia is very close to reaching it by 2015. 100 For an analysis of the growth elasticities of poverty reduction, see World Bank (2005), chapter 2. 101 In the Caribbean poverty is estimated based on consumption expenditures, but surveys are still scarce, so poverty changes are difficult to monitor. Eastern Europe and Central Asia The evidence clearly suggests that poverty in Eastern Europe and Central Asia increased during the 1990s and decreased during the 2000s. The collapse of output in many of these countries following the dismantling of the Soviet Union, along with hyperinflation that wiped out savings, resulted in a dramatic drop in living standards for the majority of people and the emergence of poverty as a major issue. However, according to Simai (2006) poverty in the region was not a new phenomenon: most of the countries began their transformation with extensive hidden unemployment and at least one-tenth of its population below the subsistence level. The transition also involved setbacks in non-monetary dimensions of wellbeing. Past achievements in social welfare came under pressure, the most striking being the reduction in life expectancy: in 1995, life expectancy for males in the Russian Federation was just 58 years, 10 years less than that of men in China (UN, 2004). Table 6.4 reminds the reader that poverty rates in these countries have always been much lower than in the other developing regions, irrespective of the line considered and of the weighted or unweighted averages, and despite the fact that low-income CIS (Georgia, Uzbekistan, Moldova, Armenia, Kygryz Republic, Tajikistan) display figures well above the regional mean. When growth resumed after the Russian crisis in 1998, poverty started to fall.100 It was not until 2004 that the region as a whole returned to the level of GDP recorded in 1990 (World Bank, 2005). Much of the poverty reduction initially occurred in the populous middle-income countries (Kazakhstan, Russian Federation, Ukraine), but eventually it spread almost everywhere. Scholars have identified four main explanations for the poverty dynamics: (i) the positive growth in output and wages; (ii) the decline in inequality, with incomes of poorer households increasing faster than average in some countries (CIS) but not in others (Poland, Romania); (iii) the increasing role of public transfers, with benefits improving in coverage and adequacy; and (iv) private remittances, which in many cases far exceeded public funding. East Asia and Pacific Lao PDR, Philippines, and Timor-Leste are progressing more slowly toward that goal. Latin America and the Caribbean Poverty estimates in Latin America, available since the 1970s, were always mostly based on income, not consumption.101 Despite the fact that statistics were initially 77 Alvaredo-Gasparini weak, there is agreement among researchers that during the 1970s economic growth pushed poverty down in the region (Altimir, 1979, 1996). In contrast, the “lost decade" of the 1980s was characterized by recurrent crises and output stagnation, that brought about a weak social performance. Latin American economic growth resumed in the 1990s, but in the context of growing inequalities, a combination that resulted in a modest decline in poverty (Londoño and Székely, 2000). The exceptional economic conditions in the 2000s and the consolidation of more ambitious social policies implied a sharp drop in poverty indicators in that decade. Gasparini et al. (2013) estimate that the income poverty headcount ratio ($2.5 line) fell from 36% in the early 1970s to 27.3% in 1980, slowly increased to reach 27.8% in 1992, mildly decreased to 24.9% in 2003, and was reduced dramatically to 16.3% by 2009.102 The number of poor people in the region is estimated to have fallen from 119 million in 1992 to 89 million in 2009 (Gasparini et al., 2013). Other indicators of income poverty and of various types of material deprivation are consistent with these results. Data from SEDLAC confirms that the reduction in poverty continued in the period 2009-2013, despite a deceleration in the GDP growth rates. The improvements in social indicators in the 2000s have been linked to at least two factors: on the one hand, most of the region’s economies experienced robust growth together with upswings in employment and labor income; on the other, all countries boosted social spending and put ambitious social protection systems into place or expanded the scope of their existing systems (López Calva and Lustig, 2010; Cruces and Gasparini, 2012). The performance in terms of poverty reduction has been heterogeneous across the regions in Latin America. While Central America experienced a slow decline in income deprivation over the last two decades, in the rest of the regions the rapid poverty fall of the 2000s contrasts with the sluggish or even negative performance of the 1990s. SEDLAC data indicate that using the international poverty line of $2.5, the average (unweighted) poverty rate in Southern South America increased from 17.7% in 1992 to 18.5% in 2003, and then dropped to 9.1% by 2010. 102 Estimates are based on SEDLAC statistics, Londoño and Székely (2000), Wodon et al. (2001) and official poverty estimates from all countries in Latin America. Latin America and the Caribbean In the same period the average poverty rate in the Andean region first rose from 30.2% to 33%, and then declined to 17.5%. In contrast, poverty in Central America fell more uniformly over the two decades: 33.3% in 1992, 28.6% in 2003 and 23.1% in 2010. More than half of the population in Latin America lives in Brazil or Mexico. After a decline in the early 1990s, income poverty in Brazil remained stable for about a decade: the poverty incidence rate with the $2.5 line was 27.8% in 1995 and 27.1% in 2003. After that plateau, the country experienced a fast and sustained reduction in income poverty, reaching 13.9% in 2010. The Mexican economy was hit by a serious crisis in the mid-1990s (the so-called Tequila crisis) that raised income poverty from 17.8% in 1992 to 43.4% in 1995 (SEDLAC data, $2.5 line). From that peak, income poverty initiated a consistent decline to reach a value of 12% in 2006, with no gains in the second half of the 2000s. 78 78 Alvaredo-Gasparini Middle East and North Africa As discussed in section 4 it is useful to divide the last four decades in MENA into three periods. The first one, spanning until the mid-1980s, was characterized by strong economic growth: the average per capita income growth for the region between 1975 and 1984 was over 4.5%. Adams and Page (2003) argue that given MENA´s relatively equal income distribution, this economic growth had a powerful impact on reducing poverty in the region. However, assessing that progress is difficult since pre-1985 household surveys are only available for Tunisia and Egypt. Iqbal (2006) reports that in Tunisia poverty fell from 51% in 1965 to 16% in 1985, while Egypt’s achievement was also impressive, with poverty declining from 82% to 53% between 1975 and 1985. Page (2007) estimates a fall in the incidence of poverty ($2 line) in the region from 57% in the late 1970s to 22% in the early 1990s. The second period, covering the late 1980s and most of the 1990s, was characterized by a sluggish economic performance, in part due to low prices for hydrocarbons, declining remittances and aid flows, as well as a low payoff to the reforms that were implemented. On average for the developing MENA countries, real per capita incomes went up by less than 1.5% per year. The proportion of those living with less than $2 per day stayed roughly unchanged around 22% for a decade (PovcalNet). Iqbal (2006) reports that “by 2001, approximately 52 million people were poor, an increase in absolute numbers of approximately 11.5 million people, compared with the situation in 1987”. The 1990s were a lost decade for economic growth and poverty reduction in the developing economies of the MENA region (Page, 2007). The downward pattern in poverty appeared to have resumed in the 2000s. The incidence of poverty ($2 line) fell from 22% in 1999 to 12% in 2010. According to PovcalNet data the number of people living with less than $2 a day increased by 7 million in the 1990s, and then was reduced by 20 million in the 2000s (from 60 to 40 million). While in the 1990s poverty went down in a third of the MENA countries, in the 2000s it went down in all economies, with the possible exception of Yemen. South Asia South Asia achieved impressive economic growth in the past fifteen years. Since 1996 until the recent global crisis, GDP growth in the region exceeded 5 percent per year. As a consequence, poverty rates were considerably reduced. In Bangladesh, India and Nepal, absolute poverty (headcount ratio, $1.25 line) fell by 18, 15 and 43 percentage points, respectively, between 1996 and 2010. In Pakistan, it fell by 22 percentage points between 2002 and 2010 (yet, one in 10 children still dies before his fifth birthday and only 57 percent of children complete primary school). Devarajan and Nabi (2006) optimistically expected the region to have single-digit poverty rates in 2015 if growth rates accelerated to 10 percent per year. This has indeed happened already in Sri Lanka (where poverty dropped from 17% in 1996 to 4% in 2010) as well as in 79 Alvaredo-Gasparini Maldives (where, according to the 2006 census, about 1 per cent of the population was living on less than $1 a day). Maldives (where, according to the 2006 census, about 1 per cent of the population was living on less than $1 a day). Growth has been instrumental in reducing poverty rates, but, as Ghani (2010) pointed out from a gloomier perspective, poverty rates were not falling fast enough to reduce the total number of poor people. Those living on less than $1.25 a day increased from 549 million in 1981 to 595 million in 2005. In India, where around three-quarters of these poor live, the number increased from 420 million in 1981 to 455 million in 2005 (Ravallion et al. 2009). The situation seems to have slightly improved according to the most recent observations, but the question remains: why has India not performed as well as China in this dimension? One explanation may be related to the already mentioned Indian paradox: survey-based households’ income growth has been much lower than National Accounts-based GDP growth. 103 According to the $4 line, more than 90 per cent of the SSA population is actually considered poor. Sub-Saharan Africa The economic and social performance of Sub-Saharan African countries has been frustrating. Five decades after most nations gained independence, poverty is still at very high levels in SSA, in fact the highest in the world. Fortunately, it seems that finally, after years of impoverished economic performance, the last decade shows some signs of improvement. Based on figures from PovcalNet, the incidence of poverty increased between 1981 and 1999 from 51% to 58% for the $1.25 per day line, and from 72% to 77% for the $2 line (the unweighted statistics are not very different).103 Because of the increase in population the number of people living with less than $1.25 a day almost doubled in Sub-Saharan Africa during those years, from 205 million to 377 million. These results are even more disappointing when compared to the rest of the developing world. The share of the world’s poor living in Sub-Saharan Africa rose from 11% in 1981 to 22% in 1999. Artadi and Sala-i-Martin (2003) find that the SSA poverty rate was stable in the 1970s, around a value of 49%, and soared in the 1980s and early 1990s reaching 60% in 1995, following the deterioration of the aggregate per capita income of Sub-Saharan economies. They report a nearly 50% reduction in the worldwide poverty rate between 1980 and 2000, that contrast with a 27% increase in SSA. Following the dismal performance of the 1980s and early 1990s, SSA witnesses some economic and social progress. Poverty declined considerably from their heights of the mid-1990s; in fact the reduction in extreme poverty from that date to the late 2000s was similar than in the rest of the developing world, excluding China. Chen and Ravallion (2012) stressed the fact that for the first time since 1981 the share of people in Sub-Saharan Africa living below $1.25 a day was less than 50%. Changes in poverty have been closely linked to economic growth. According to Fosu (2010), poverty has declined in SSA by about 0.5 percentage points per year since the mid-1990s, quite 80 Alvaredo-Gasparini comparable with South Asia’s record. Sala-i-Martin and Pinkovskiy (2010) estimate that African poverty has been falling steadily since 1995. According to these authors the African poverty rate in 2006 was 31.8%, 30 per cent lower than in 1995, and 28 per cent lower than in 1990. Sub-Saharan Africa The decline in poverty in the 2000s has been quite extended: as reported above, 2/3 of the SSA countries in PovcalNet have experienced reductions larger than 5%. Sala-i-Martin and Pinkovskiy (2010) find that “poverty fell for both landlocked as well as coastal countries; for mineral-rich as well as mineral-poor countries; for countries with favorable or with unfavorable agriculture; for countries regardless of colonial origin; and for countries with below or above median slave exports per capita during the African slave trade. Hence, the substantial decline in poverty is not driven by any particular country or set of countries”. However, Fosu (2009) highlights the considerable heterogeneity across country experiences. For example, while high economic growth in Botswana has been transformed to only a minimal decline in poverty, Ghana has succeeded in translating its relatively modest growth to considerable poverty reduction. Fosu (2009) attributes this contrast to the difference in the levels of income inequality between the two countries. Despite the encouraging signs of progress in the fight against poverty in Sub-Saharan Africa, Chen and Ravallion (2012) alert that the lags in survey data availability and problems of comparability and coverage, rise up concerns about how robust this positive trend will prove to be. 104 The analysis is restricted to countries with initial poverty above 5%. 105 The first panel in Figure 6.4 suggests an increase in the dispersion of absolute changes at higher values of the initial poverty level. A quantile regression analysis confirms that the slope of the regression line is negative and significant for the lower quantiles (the high-performers in terms of poverty reduction), and statistically non-significant for the higher quantiles. The systematic difference in the slopes across quantiles suggests that poverty convergence depends on factors beyond the initial poverty level. *=significant at 10%, **=significant at 5%, ***=significant at 1%. Note: standard errors in parenthesis. Note: standard errors in parenthesis. Source: own calculations based on PovcalNet (2013). 6.5. Poverty convergence Given the heterogeneity among economies in terms of social improvement, a natural question is whether countries starting out with a high incidence of material deprivation tend to be the ones with higher rates of poverty reduction. Figure 6.4 suggests some signs of poverty convergence in absolute terms but not in proportionate terms.104 The 1981 poverty level ($2 line) is negatively correlated with the annualized change over the period 1981-2010, but not with the proportionate change (annualized log difference). In the first panel the regression line has a slope of -0.009 with a t-ratio of -4.51, based on a robust (White) standard error. This result means that on average the absolute fall in poverty has been larger in countries with higher poverty incidence, but since the difference across countries is small, there is absence of poverty convergence in proportionate terms. 81 81 Alvaredo-Gasparini Figure 6.4 Poverty convergence among developing countries 1981-2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day per person line. Absolute changes Proportionate changes -.1 -.05 0 .05 annual growth rate in headcount ratio 1981-2010 0 20 40 60 80 100 headcount ratio 1981 -3 -2 -1 0 1 annual change in headcount ratio 1981-2010 0 20 40 60 80 100 headcount ratio 1981 Poverty convergence among developing co 1981-2010 Absolute changes -3 -2 -1 0 1 annual change in headcount ratio 1981-2010 0 20 40 60 80 100 headcount ratio 1981 Proportionate changes -.1 -.05 0 .05 annual growth rate in headcount ratio 1981-2010 0 20 40 60 80 100 headcount ratio 1981 Source: own calculations based on PovcalNet (2013). ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day per person line. ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste $2-a-day per person line. The results for other poverty indices, lines and time periods are similar (Table 6.6). The coefficients for the absolute change in poverty are negative but small, while the coefficients for the proportionate change are in general positive, but in most cases non-significant.105 Source: own calculations based on PovcalNet (2013). 106 See Chen and Ravallion (1997), World Bank (2000), Ravallion (2001, 2007, 2012), Fields (2002), Bourguignon (2003), Besley and Burguess (2003), Kraay (2006), Fosu (2011) as examples of a large literature. Table 6.6 Poverty convergence among developing countries Coefficients of initial poverty on a poverty change regression Table 6.6 Poverty convergence among developing countries Coefficients of initial poverty on a poverty change regression Source: own calculations based on PovcalNet (2013) Index Poverty line Absolute changes Proportionate changes Absolute changes Proportionate changes Absolute changes Proportionate changes Headcount $ 1.25 -0.017 0.001 -0.018 0.010 -0.016 0.023 (0.002)*** (0.004) (0.003)*** (0.004)* (0.004)*** (0.006)*** Headcount $ 2 -0.009 0.006 -0.008 0.016 -0.008 0.028 (0.002)*** (0.004) (0.002)*** (0.004)** (0.004)** (0.007)*** Poverty gap $ 1.25 -0.021 -0.007 -0.026 -0.001 -0.024 0.014 (0.002)*** (0.004) (0.003)*** (0.005) (0.005)*** (0.007)** Poverty gap $ 2 -0.016 0.001 -0.018 0.010 -0.016 0.024 (0.002)*** (0.004) (0.003)*** (0.005)* (0.004)*** (0.008)*** Squared poverty gap $ 1.25 -0.024 -0.013 -0.032 -0.008 -0.030 0.006 (0.002)*** (0.004)** (0.004)*** (0.006) (0.007)*** (0.006) Squared poverty gap $ 2 -0.020 -0.005 -0.024 0.002 -0.022 0.014 (0.002)*** (0.004) (0.003)*** (0.004) (0.005)*** (0.005)*** 1990-2010 1981-2010 1999-2010 *=significant at 10%, **=significant at 5%, ***=significant at 1%. 82 Alvaredo-Gasparini Ravallion (2012) argues that the lack of poverty convergence (in proportionate terms), despite evidence for mean convergence and for the poverty-reduction impact of growth, suggests that the dynamic processes for growth and poverty reduction depend directly on the initial level of poverty. He shows evidence on two adverse effects of being a country with high poverty levels: first, these countries tend to grow at a slower pace, controlling for the initial mean, and second a high poverty rate implies lower “productivity” of economic growth in terms of poverty reduction (lower poverty- growth elasticity). 6.6. Poverty and growth The dynamics of poverty are closely related to income growth. The economic literature has gathered abundant evidence supporting the fact that absolute poverty tends to fall with economic growth.106 Moreover, the longer the growth spells under consideration, the larger the share of the variance in poverty that is accounted for by the growth component (Ferreira, 2010). Figure 6.5 illustrates this relationship by showing poverty along with two alternatives measures of mean income: per capita gross national income (GNI) constructed from National Accounts, and per capita consumption obtained from household surveys. The figure shows the unweighted mean of these variables among developing countries in the period 1981-2010. On average, the economic performance of the developing countries was weak in the 1980s and early 1990s, hindering the perspectives of poverty reduction. Since the mid-1990s economic growth resumed in most countries in the developing world, a process that accelerated in the 2000s, leading to a sustained decrease in all measures of income poverty. At the end of the decade that pattern was slowed down, but not halted, by the poor economic performance of several developing countries associated to the international crisis. 83 83 Alvaredo-Gasparini Figure 6.5 Per capita GNI, per capita consumption and poverty headcount ratio Unweighted mean, developing countries, 1981-2010 Figure 6.5 Per capita GNI, per capita consumption and poverty headcount ratio Unweighted mean, developing countries, 1981-2010 Figure 6.5 Per capita GNI, per capita consumption and poverty headcount ratio Unweighted mean, developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: per capita gross national income in constant 2005 PPP dollars, per capita consumption from household surveys in constant 2005 dollars. Unweighted averages across developing countries, series displayed with mean=100. Headcount poverty ratio $2-a-day line, unweighted averages across developing countries. 35 37 39 41 43 45 47 49 51 53 55 80 90 100 110 120 130 140 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Headcount ratio GNI and consumption GNI pc Consumption pc Poverty headcount ($2 line) Per capita GNI, per capita consumption and poverty headcount ratio Unweighted mean, developing countries, 1981-2010 35 37 39 41 43 45 47 49 51 53 55 80 90 100 110 120 130 140 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 2010 Headcount ratio GNI and consumption GNI pc Consumption pc Poverty headcount ($2 line) Source: own calculations based on PovcalNet (2013). 6.6. Poverty and growth Source: own calculations based on PovcalNet (2013). Note: per capita gross national income in constant 2005 PPP dollars, per capita consumption from household surveys in constant 2005 dollars. Unweighted averages across developing countries, series displayed with mean=100. Headcount poverty ratio $2-a-day line, unweighted averages across developing countries. Figure 6.6 is another illustration of the relationship between poverty reduction and economic growth. The figure shows that the change in poverty is closely negatively related to economic growth, either in per capita consumption expenditures drawn from household surveys or in GNI from NAS. Notice that in both panels the fitted line approximately crosses the (0,0) point. Figure 6.6 is another illustration of the relationship between poverty reduction and economic growth. The figure shows that the change in poverty is closely negatively related to economic growth, either in per capita consumption expenditures drawn from household surveys or in GNI from NAS. Notice that in both panels the fitted line approximately crosses the (0,0) point. Figure 6.6 Figure 6.6 Growth and poverty reduction in the developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: the horizontal axis shows the annual growth rate between 1981 and 2010 in per capita consumption from household surveys in constant 2005 dollars (panel 1) and in per capita gross national income in constant 2005 PPP dollars (panel 2); the vertical axis shows the annual growth change in the poverty headcount ratio ($1.25 line) in the same period. The size of each circle is proportional to the country population in 2010. 1. Poverty reduction and growth in per capita consumption 2. Poverty reduction and growth in per capita GNI -.2 -.1 0 .1 annual growth rate in poverty headcount ratio -.05 0 .05 .1 per capita GNI annual growth rate -.2 -.1 0 .1 annual growth rate in poverty headcount ratio -.05 0 .05 .1 per capita consumption annual growth rate Growth and poverty reduction in the developing countries, 1981-2010 1. Poverty reduction and growth in per capita consumption 2. Poverty reduction and growth in per capita GNI 1. Poverty reduction and growth in per capita consumption 2. Poverty reduction and growth in per capita GNI 1. Poverty reduction and growth in per capita consumption -.2 -.1 0 .1 annual growth rate in poverty headcount ratio -.05 0 .05 .1 per capita consumption annual growth rate Source: own calculations based on PovcalNet (2013). Note: the horizontal axis shows the annual growth rate between 1981 and 2010 in per capita consumption from household surveys in constant 2005 dollars (panel 1) and in per capita gross national income in constant 2005 PPP dollars (panel 2); the vertical axis shows the annual growth change in the poverty headcount ratio ($1.25 line) in the same period. The size of each circle is proportional to the country population in 2010. 84 84 Alvaredo-Gasparini Since the positive correlation between poverty reduction and growth is a well- established result, research in this area is mainly focused on estimating the magnitude of the corresponding elasticity, an issue that may have relevant policy implications. “If (the elasticity) is reasonably high, then poverty reduction strategies almost exclusively relying on economic growth are probably justified. If it is low, however, ambitious poverty reduction strategies might have to combine both economic growth and some redistribution” (Bourguignon, 2003). 107 The sample includes 725 spells in 76 countries for which consistent information for the whole period is available. Results do not significantly vary when we restrict the analysis to more recent periods which allows a larger sample of countries. Figure 6.6 The growth elasticity of poverty reduction is typically estimated by regressing the annualized proportional change in a poverty indicator against the annualized growth rate of mean income (per capita income or consumption from surveys, or per capita GDP, GNI, or aggregate private consumption from NAS) in a sample of growth spells. In a regression without controls the resulting coefficient is the total elasticity, as opposed to a partial elasticity that can be obtained by holding inequality and other factors constant. Based on a dataset for 67 countries over the period from 1981 to 1994, and using the $1 line, Ravallion and Chen (1997) find a central estimate for the poverty-growth elasticity of -3.1. Values estimated by other authors are somewhat lower (in absolute terms): around -2 in World Bank (2000), -1.6 in Bourguignon (2003), -2.6 in Adams (2004), and -1.8 in Ferreira and Ravallion (2009). More recently, Ravallion (2012) reports an elasticity of −1.4 for the $1.25 line. The elasticity is lower when using the growth rate of consumption per capita from NAS (−0.8), and also lower when using a higher poverty line. The confidence intervals around the regression coefficient are typically wide. Ravallion (2007) reports that the 95% confidence interval implies that for a poverty level of 40%, an annual growth rate of 2% is consistent with poverty reductions ranging from 1% to 7%. Estimates of the total growth elasticity of poverty reduction over the period 1981-2010 obtained with the latest version of PovcalNet data are shown in Table 6.7.107 All the elasticities are negative and significant at 1%. The growth elasticity of poverty reduction, as measured by the proportion of individuals below $1.25 a day, is around - 1.5. The results in Table 6.7 confirm that elasticities are lower in absolute value when considering a higher poverty line. The result of lower elasticities using GNI from National Accounts holds but only until the 2000s, suggesting a change in the relative trends between consumption reported in household surveys and output estimated in NAS. In general, the absolute value of the elasticities estimated with both sources have increased in the 2000s compared to previous decades, suggesting that growth was better translated to the poor in that period of falling inequality. For instance, while the elasticity, using the $1.25 line and consumption per capita, was -1.53 for the period 1981-2010, it amounted to -1.83 when restricting the analysis to the 2000s. Figure 6.6 85 Alvaredo-Gasparini Table 6.7 Total poverty-growth elasticities Developing countries Source: own calculations based on PovcalNet (2013). Note: all elasticities are significant at 1% level. 1981 - 2010 1990-2010 1999-2010 1981 - 2010 1990-2010 1999-2010 1.25 -1.53 -1.56 -1.83 -1.46 -1.51 -2.16 2 -1.39 -1.41 -1.68 -1.17 -1.19 -1.91 4 -1.30 -1.38 -1.10 -0.78 -0.81 -1.21 1.25 -1.75 -1.77 -1.87 -1.44 -1.47 -2.09 2 -1.50 -1.47 -1.85 -1.35 -1.30 -1.83 4 -1.41 -1.47 -1.39 -0.95 -0.98 -1.54 1.25 -2.18 -2.25 -1.87 -1.50 -1.52 -1.87 2 -1.60 -1.52 -1.86 -1.44 -1.29 -1.91 4 -1.40 -1.43 -1.54 -1.04 -1.04 -1.71 Consumption per capita GNI per capita Index Poverty line Headcount Poverty gap Squared PG Table 6.7 Total poverty-growth elasticities Developing countries So far, we have reported total elasticities, which can be seen as summary measures of the co-movements of poverty and growth. The literature has tried to improve this characterization by adding other variables into the analysis, typically measures of inequality and development. The empirical evidence supports the intuition that higher inequality tends to reduce the absolute value of the elasticity, by shifting the gains from growth away from the poor (Ravallion, 1997, 2001; Kraay, 2006).108 For example, based on a sample of 65 countries during 1981-2005, and using the $1 poverty line, World Bank (2005) reports that the poverty-growth elasticity is highest among low- inequality countries (with a value of approximately -4.0 for countries with Ginis in the mid-20s) and lowest among high-inequality countries (close to -1.0 for countries with a Gini coefficient around 60). The change in inequality is also found as a significant direct determinant of the elasticity. For instance, Ravallion (2001) finds that the median rate of reduction in the poverty headcount ratio ($1 line) among growing economies was 10% per year among countries with falling inequality, and 1% per year for those countries with rising inequality. Using a sample of 114 growth spells from the mid-1980s to the mid-1990s in 26 developing countries Bourguignon (2003) finds an elasticity of -1.6 in the model without controls and -2 when controlling for the change in the Gini. The latter specification increases the R2 from 0.266 to 0.419, suggesting that the heterogeneity in distributional changes is as much responsible for the variation in poverty changes across growth spells as the heterogeneity in the speed of growth. 108 Although the intuition is compelling, the result is theoretically ambiguous. See Ravallion (2007) for a proof, and Bourguignon (2003) for a case in which assuming log-normality yields an unambiguous result. Figure 6.6 Bourguignon (2003) also finds positive and significant coefficients for the interaction terms between the growth rate and both (i) the initial level of inequality, and (ii) the level of development (proxied by the poverty line over mean income). He also reports negative coefficients for the interaction terms between the change in the Gini and those two variables. The 86 Alvaredo-Gasparini first four columns in Table 6.8 in general confirm the results in Bourguignon (2003), using a larger dataset spanning three decades.109 Table 6.8 Regressions for annualized proportional change in the poverty headcount ratio Developing countries, 1981-2010 Table 6.8 Regressions for annualized proportional change in the poverty headcount ratio Developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: sample of 76 countries with information for the period 1981-2010. Poverty indicator=headcount ratio, $2-a-day line. Growth rates computed from per capita consumption in household surveys. Unconditional (1) (2) (3) (4) (5) (6) g = growth rate in per capita consumption -1.391 -1.467 -2.879 -2.910 -2.111 -2.073 (0.147)*** (0.144)*** (0.570)*** (0.590)*** (0.206)*** (0.188)*** dGini = change in Gini coefficient 1.488 1.566 3.195 (0.265)*** (0.279)*** (1.206)*** g * initial Gini coeficient 0.022 0.023 (0.013)* (0.014)* g * (poverty line/mean consumption) 0.903 0.879 (0.094)*** (0.101)*** dGini* initial Gini coefficient -0.019 (0.027) dGini * (poverty line/mean consumption) -1.666 (0.345)*** g * initial poverty rate 0.021 0.021 (0.003)*** (0.003)*** log initial poverty rate -1.559 (0.927)* Intercept 0.150 -0.008 0.187 0.064 -0.255 4.108 (0.225) (0.203) (0.154) (0.208) (0.122)** (2.655) Observations 725 695 695 695 725 725 R2 0.414 0.502 0.583 0.610 0.508 0.516 Conditional on Inequality and development Poverty Table 6.8 Regressions for annualized proportional change in the poverty headcount ratio Developing countries, 1981-2010 Table 6.8 Regressions for annualized proportional change in the poverty headcount ratio Developing countries, 1981-2010 Source: own calculations based on PovcalNet (2013). Note: sample of 76 countries with information for the period 1981-2010. Poverty indicator=headcount ratio, $2-a-day line. Growth rates computed from per capita consumption in household surveys. 109 Instead, unlike Bourguignon (2003) we fail to find a coefficient close to -1 for the interaction between the growth rate and the theoretical value of the poverty-growth elasticity under the lognormal assumption. 6.7. Relative poverty So far we have presented evidence on absolute poverty, an option that requires keeping the real value of the poverty line fixed over time. The alternative is relative poverty, where the line is adjusted to reflect changes in a general measure of economic well-being. Although the measurement of relative poverty has not been common in the developing world, sustained economic growth have triggered the update of absolute poverty lines in some countries (e.g. China and India), and foster discussions on relative poverty. The measurement of relative, instead of absolute, poverty, can be justified on two grounds (Sen, 1983; Chen and Ravallion, 2008). First, the very concept of poverty may depend on social norms that are likely different across countries and over the development process. Second, even for a fixed norm, an absolute line in the space of welfare may require a relative line in the space of consumption if individual welfare also depends on relative consumption with respect to the rest of the society where the person lives. The typical relative poverty line is set as a constant proportion of the mean (or median) of the distribution, implying that poverty does not fall (increase) when all incomes grow (fall) at the same rate. This result is considered problematic by many authors who prefer weakly versions in which the cost of inclusion is not a constant proportion of mean income. For instance, following Atkinson and Bourguignon (2001), Ravallion and Chen (2012) propose a poverty line that is fixed up to a critical value of the mean, where it rises with elasticity less than one. In particular, Ravallion and Chen (2012) set the line for country i at time t as zit=max[$1.25, ($1.25+Mit)/2], where Mit is the country and date specific mean. Figure 6.7 reveals an overall trend decline in the incidence of weakly relative poverty in the developing world. The fall has been relatively meager, so that the number of poor by this measure actually increased from 2.3 billion in 1981 to 2.7 billion in 2008. While the coefficient in an OLS regression between the proportionate rate of poverty reduction (annualized differences in the logs) and the rate of growth in the mean is -1.89 (standard error=0.23) for the absolute measure of poverty, it becomes -0.38 (standard error=0.08) for the relative measure. Figure 6.6 Unconditional (1) (2) (3) (4) (5) (6) g = growth rate in per capita consumption -1.391 -1.467 -2.879 -2.910 -2.111 -2.073 (0.147)*** (0.144)*** (0.570)*** (0.590)*** (0.206)*** (0.188)*** dGini = change in Gini coefficient 1.488 1.566 3.195 (0.265)*** (0.279)*** (1.206)*** g * initial Gini coeficient 0.022 0.023 (0.013)* (0.014)* g * (poverty line/mean consumption) 0.903 0.879 (0.094)*** (0.101)*** dGini* initial Gini coefficient -0.019 (0.027) dGini * (poverty line/mean consumption) -1.666 (0.345)*** g * initial poverty rate 0.021 0.021 (0.003)*** (0.003)*** log initial poverty rate -1.559 (0.927)* Intercept 0.150 -0.008 0.187 0.064 -0.255 4.108 (0.225) (0.203) (0.154) (0.208) (0.122)** (2.655) Observations 725 695 695 695 725 725 R2 0.414 0.502 0.583 0.610 0.508 0.516 Conditional on Inequality and development Poverty Regressions for annualized proportional change in the poverty headcount ratio Developing countries, 1981-2010 ( ) Note: sample of 76 countries with information for the period 1981-2010. Poverty indicator=headcount ratio, $2-a-day line. Growth rates computed from per capita consumption in household surveys. The results suggest the possibility of a double dividend from reducing inequality: first, given a growth rate, lower inequality is associated directly with less poverty, and second, less inequality means more poverty reaction to a given growth rate, that is an acceleration of poverty reduction for a given rate of economic growth. Ravallion (2007) illustrates the relevance of lower inequality for the perspectives of poverty reduction assuming a country with a poverty incidence rate of 40% and a growth rate of 2% per year: with an initial Gini coefficient of 30 it would take 11 years to reduce poverty by half, while with a Gini of 60 it would take 35 years. In a more recent study Ravallion (2012) finds that the (absolute) growth elasticity of poverty reduction tends to be lower in countries with a higher initial poverty rate (see also the two last columns in Table 6.8). Ravallion (2012) finds a large attenuating effect of a higher initial poverty rate on the elasticity: at an initial poverty rate of 10% the elasticity is −2.2, while it falls to −0.5 at a poverty rate of 80%. The results are robust to 87 Alvaredo-Gasparini the inclusion of additional interaction effects with the initial Gini coefficient, the partial elasticity of poverty reduction holding the Lorenz curve constant, the primary school enrollment rate, life expectancy, the price of investment goods, and regional dummies. Figure 6.6 Unfortunately, while the characterizations of the growth elasticity of poverty reduction discussed in this section are useful, the literature is still far from being able to clearly identify the structural factors that are behind the differences in the elasticities, and therefore is relatively silent about the specific policies that could foster a larger impact of economic growth on poverty. 6.7. Relative poverty Only East Asia has experienced a marked reduction in the incidence of relative poverty: from 81% in 1981 to 42% in 2008. In fact it is the only region in which the poverty 88 Alvaredo-Gasparini count was reduced according to this measure. Weakly relative poverty measures rose or stayed unchanged in most regions in the 1990s, and slowly went down in the 2000s. Figure 6.7 Poverty headcount ratio with absolute and weakly relative poverty lines Developing countries, 1981-2008 Source: Ravallion and Chen (2012). In Table 6.9 we extend the estimates of Ravallion and Chen (2012) to 2010, and add estimates of strongly relative poverty (50 per cent of mean income) and anchored poverty, computed by “anchoring” the relative lines (50 per cent of mean income) to the values in 1990, updating them only by domestic price changes (see chapter 9 in this volume). Our view of poverty trends widely varies across methodologies: while the unweighted anchored poverty rate fell 32% between 1990 and 2010, the strongly relative poverty rate went up 2%. The differences are even larger when considering the population-weighted statistics. 0 10 20 30 40 50 60 70 80 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 Absolute ($ 2 per day) Weakly relative Figure 6.7 Poverty headcount ratio with absolute and weakly relative poverty lines Developing countries, 1981-2008 Source: Ravallion and Chen (2012). 0 10 20 30 40 50 60 70 80 1981 1984 1987 1990 1993 1996 1999 2002 2005 2008 Absolute ($ 2 per day) Weakly relative Figure 6.7 Poverty headcount ratio with absolute and weakly relative poverty lines Developing countries, 1981-2008 g Poverty headcount ratio with absolute and weakly relative poverty lines Developing countries, 1981-2008 Absolute ($ 2 per day) Weakly relative Source: Ravallion and Chen (2012). In Table 6.9 we extend the estimates of Ravallion and Chen (2012) to 2010, and add estimates of strongly relative poverty (50 per cent of mean income) and anchored poverty, computed by “anchoring” the relative lines (50 per cent of mean income) to the values in 1990, updating them only by domestic price changes (see chapter 9 in this volume). Our view of poverty trends widely varies across methodologies: while the unweighted anchored poverty rate fell 32% between 1990 and 2010, the strongly relative poverty rate went up 2%. The differences are even larger when considering the population-weighted statistics. Note: weakly relative poverty computed according to Ravallion and Chen (2012). Strongly relative lines set at 50% of mean income. Anchored poverty computed by “anchoring” the relative lines (50% of mean income) to the values in 1990, and updating them only by domestic price changes. 7. Concluding remarks There has been a remarkable improvement in the availability of information for distributive analysis in the last decades due to increasing efforts by researchers, national governments and international organizations. To be sure, the picture of inequality and poverty in the developing world is substantially sharper now than in the late 1990s, when the first volume of this Handbook was written. There remain, however, enormous data limitations that make that picture only a very rough approximation of reality. Household surveys are lacking in some countries and are carried out very occasionally in others. Changes in methodology over time are frequent; a fact that generally implies improvement in the data collection, but that at the same time introduces comparability issues with previous surveys that are difficult to deal with. Household surveys have endemic problems in capturing some income and consumption items and in dealing with selective compliance and under-reporting issues. The gaps with National Accounts aggregates, usually variable over time, are a disturbing sign of measurement errors and conceptual issues. Comparability across countries is another big problematic issue, as there are few efforts among national agencies to standardize surveys or at least some criteria to gather and process information. Another issue of concern is the difficulty in obtaining statistical confidence intervals for the distributive statistics, either because agencies do not report them, do not provide information on sampling issues, or do not release the microdata. In sum, in order to be able to characterize and track distributive changes in the developing world with more accuracy we need more efforts to extend the coverage and frequency of household surveys, and improve their reliability and comparability across countries. There is still a long way to go to get an accurate assessment of poverty and inequality in the developing world. From the data available, some general facts emerge. High poverty and inequality are pervasive characteristics of the developing world, but are not immutable features of these economies. The evidence suggests a robust decline in the levels of income absolute poverty in the developing world, driven mainly by East Asia in the 1980s and 1990s, but generalized to the rest of the developing countries in the 2000s. Income poverty has been reduced in most countries and in the world as whole, making the achievement of the first MDG possible. Despite these positive results there are reasons for concern. 6.7. Relative poverty Table 6.9 Absolute and relative poverty Developing countries, 1990-2010 Source: own calculations based on data from PovcalNet (sample of 76 countries). N kl l i d di R lli d Ch (2012) S l l i li absolute ($2) weakly relative (RC) strongly relative anchored absolute ($2) weakly relative (RC) strongly relative anchored 1990 40.6 46.5 28.0 28.0 64.7 54.1 19.7 19.7 1993 39.8 47.9 29.0 32.9 62.7 53.7 21.1 19.1 1996 38.6 47.4 29.1 32.1 58.0 51.4 22.0 17.1 1999 38.3 47.5 30.1 31.9 56.7 50.2 22.9 16.9 2002 36.7 46.9 30.1 29.6 52.5 49.5 23.7 15.2 2005 32.3 45.0 29.5 24.8 45.6 46.5 23.7 12.3 2008 28.7 43.2 29.0 19.9 42.1 46.1 24.9 9.9 2010 27.7 42.4 28.6 18.9 39.4 45.6 26.6 9.5 weighted unweighted Table 6.9 Absolute and relative poverty Developing countries, 1990-2010 Note: weakly relative poverty computed according to Ravallion and Chen (2012). Strongly relative lines set at 50% of mean income. Anchored poverty computed by “anchoring” the relative lines (50% of mean income) to the values in 1990, and updating them only by domestic price changes. Note: weakly relative poverty computed according to Ravallion and Chen (2012). Strongly relative lines set at 50% of mean income. Anchored poverty computed by “anchoring” the relative lines (50% of mean income) to the values in 1990, and updating them only by domestic price changes. 89 Alvaredo-Gasparini Alvaredo-Gasparini 7. Concluding remarks The task of fighting poverty continues to be very challenging: around 1.2 billion people survive with less than $1.25 a day, an extremely low standard. Also, most of the people who succeeded in jumping the $1.25 line in the last decades are still poor by the standards of middle-income developing countries, and remain highly vulnerable if economic conditions worsen. Moreover, due to economic growth, concerns about relative poverty may be increasingly important, and the evidence in that sense is less optimistic. The evidence on income relative inequality suggests that on average the developing countries are somewhat more unequal than three decades ago. The patterns have been different by period and region. Relative income inequality rose in the late 1980s 90 90 Alvaredo-Gasparini and in the 1990s. The changes were larger in Eastern Europe and Central Asia, probably as a result of the transition from a central-planned to a more market- oriented economy; in East Asia, likely as a consequence of the economic take-off; and in Latin America probably as the result of recurrent macroeconomic crises and some structural transformations. Distributive changes in the 2000s become more equalizing in most of the developing world, but the changes were rather moderate and with considerable heterogeneity across countries. 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Data American Samoa 0.066 Albania 3.181 X Antigua and Barbuda 0.087 Algeria 34.428 X Afghanistan 32.518 Angola 18.038 X Nigeria 150.666 X Cambodia 13.823 X Armenia 3.079 X Argentina 39.714 X Djibouti 0.856 X Bangladesh 145.478 X Benin 8.356 X Rwanda 10.004 X China 1324.655 X Azerbaijan 8.763 X Belize 0.322 X Egypt 78.323 X Bhutan 0.701 X Botswana 1.955 X São Tomé & P. 0.160 X Fiji 0.844 X Belarus 9.602 X Bolivia 9.618 X Iran 72.289 X India 1190.864 X Burkina Faso 15.515 X Senegal 11.787 X Indonesia 234.951 X Bosnia and Herzegovina 3.774 X Brazil 191.543 X Iraq 30.178 X Maldives 0.308 X Burundi 7.943 X Seychelles 0.087 X Kiribati 0.097 Bulgaria 7.623 X Chile 16.796 X Jordan 5.787 X Nepal 28.905 X Cameroon 18.759 X Sierra Leone 5.612 X Korea, Dem. Rep. 24.126 Croatia 4.434 X Colombia 45.006 X Lebanon 4.167 Pakistan 167.442 X Cape Verde 0.487 X Somalia 8.922 Lao 6.022 X Czech Republic 10.424 X Costa Rica 4.522 X Libya 6.150 Sri Lanka 20.217 X Central African R. 4.238 X South Africa 48.793 X Malaysia 27.502 X Estonia 1.341 X Cuba 11.267 Morocco 31.321 X Chad 10.654 X South Sudan 8.977 Marshall Islands 0.053 Georgia 4.384 X Dominica 0.068 Syria 19.638 X Comoros 0.697 X Sudan 32.438 X Micronesia 0.110 X Hungary 10.038 X Dominican Republic 9.665 X Tunisia 10.329 X Congo, Dem. R. 62.475 X Swaziland 1.032 X Mongolia 2.667 X Kazakhstan 15.674 X Ecuador 14.057 X West Bank and Gaza 3.937 X Congo, Rep. Note: an “X” in the Data column marks that the country has distributive information in PovcalNet. Source: population (in million) is taken from the United Nations Demographic Yearbook. References Assessing the impact of a school subsidy program in Mexico: Using a social experiment to validate a dynamic behavioral model of child schooling and fertility. American Economic Association 96(5), pp. 1384-1417. United Nations-UNECE (2004) Economic Survey of Europe, 2004 No. 1, chapter 7. United Nations-UNECE (2004) Economic Survey of Europe, 2004 No. 1, chapter 7. van der Berg, S., and Louw, M. (2004). Changing patterns of South African income distribution: towards time series estimates of distribution and poverty. South African Journal of Economics, 72(3), pp. 546-572. Van de Walle, N. (2008). The institutional origins of inequality in Sub-Saharan Africa. Cornell University Mario Einaudi Center for International Studies Working Paper 6-08. Voitchovsky, S. (2009). Inequality, Growth and Sectoral Change. Chapter 22 in Nolan, Salverda and Smeeding (eds.), Oxford Handbook of Economic Inequality. Whiteford, A. and Van Seventer, D. (2000). South Africa’s changing income distribution in the 1990s. Studies in Economics & Econometrics 24(3), pp. 7-30. Winters, A., McCulloch, N. and McKay, A. (2004). Trade liberalization and poverty: The Evidence so far. Journal of Economic Literature 42, pp. 72-115. Wodon, Q., Castro, R. and Lee, K. (2001). Poverty in Latin America: Trends (1986-1998) and determinants. Latin American Journal of Economics - formerly Cuadernos de Economía 38(114). Wood, A. (1997). Openness and wage inequality in developing countries: The Latin American challenge to East Asian conventional wisdom. World Bank Economic Review 11(1), pp. 33-57. World Bank (2000). World Development Report: Attacking Poverty. New York: Oxford University Press. World Bank (2005). ECAPOV II. Growth, Poverty and Inequality in Eastern Europe and Former Soviet Union. Washington, DC: The World Bank. World Bank (2006). World development report: Equity and development. World Bank and Oxford University Press. World Bank (2006). Can South Asia reduce poverty in a generation? http://go.worldbank.org/NF9FFJTM20 World Bank (2008). Global Purchasing Power Parities and Real Expenditures. 2005 International Comparison Program. Washington, DC: The World Bank. World Bank (2009). The World Development Report 2009: Reshaping Economic Geography. Washington, D.C.: The World Bank. World Bank (2012). East Asia and Pacific Data Monitor, October. World Bank (2013). The East Asian Miracle: Economic Growth and Public Policy. Oxford University Press. Zin, R.H.M. (2005). Income distribution in East Asian developing countries: recent trends. Asian-Pacific Economic Literature, 19(2), 36-54. Zin, R.H.M. (2005). Income distribution in East Asian developing countries: recent trends. Asian-Pacific Economic Literature, 19(2), 36-54. References 3.836 X Tanzania 42.268 X Myanmar 47.250 Kosovo 1.747 El Salvador 6.130 X Yemen 22.627 X Côte d'Ivoire 18.987 X Togo 5.777 X Palau 0.020 Kyrgyz Republic 5.319 X Grenada 0.104 Eritrea 4.948 Uganda 31.339 X Papua New Guinea 6.549 Latvia 2.266 X Guatemala 13.691 X Ethiopia 79.446 X Zambia 12.380 X Philippines 90.173 X Lithuania 3.358 X Guyana 0.752 X Gabon 1.450 X Zimbabwe 12.452 Samoa 0.182 Macedonia 2.053 X Haiti 9.736 X Gambia 1.636 X Solomon Islands 0.510 Moldova 3.570 X Honduras 7.303 X Ghana 23.264 X Thailand 68.268 X Montenegro 0.629 X Jamaica 2.687 X Guinea 9.559 X Timor-Leste 1.078 X Poland 38.126 X Mexico 110.627 X Guinea-Bissau 1.454 X Tonga 0.103 Romania 21.514 X Nicaragua 5.636 X Kenya 38.455 X Tuvalu 0.010 Russian Federation 141.950 X Panama 3.406 X Lesotho 2.127 X Vanuatu 0.228 Serbia 7.350 X Paraguay 6.230 X Liberia 3.658 X Vietnam 85.122 X Slovak Republic 5.407 X Peru 28.463 X Madagascar 19.546 X Slovenia 2.021 X St. Kitts and Nevis 0.051 Malawi 14.005 X Tajikistan 6.691 X St. Lucia 0.170 X Mali 14.460 X Turkey 70.924 X St. Vincent and G. 0.109 Mauritania 3.295 X Turkmenistan 4.918 X Suriname 0.515 X Mauritius 1.269 Ukraine 46.258 X Trinidad and Tobago 1.331 X Mozambique 22.333 X Uzbekistan 27.303 X Uruguay 3.334 X Namibia 2.200 X Venezuela 27.935 X Niger 14.450 X Sub-Saharan Africa South Asia East Asia and Pacific Alvaredo-Gasparini Figure 5.5 Figure 5.5 Map of poverty in the developing world, circa 2010 Map of poverty in the developing world, circa 2010 Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. < 5 5 - 10 10 - 15 15 - 20 20 - 30 30 - 40 40 - 50 50 - 60 60 - 70 70 - 80 > 80 High Incomes No Data Poverty headcount (%) Source: own calculations based on PovcalNet (2013). No Data Source: own calculations based on PovcalNet (2013). Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. ( ) Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjusted $2-a-day line. Note: poverty computed over the distribution of consumption/income per capita with the PPP-adjuste 107
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Tomruk piyasası aktörlerinin dikili ağaç satış yönetmeliklerinde yapılan değişikliklere ilişkin algıları
Turkish Journal of Forestry
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Turkish Journal of Forestry | Türkiye Ormancılık Dergisi 2023, 24(4): 378-389 | Research article (Araştırma makalesi) Turkish Journal of Forestry Perceptions of log market actors on revisions to the regulations of the sale of standing tree Abstract: The standing tree sales method is a sales method used in Türkiye as well as all over the world. The Standing Sales Regulation, which first came into force in 1996, has been amended several times. However, the effects of these amendments are not clear. Although the amendments made with the enactment of Decree No. 6877 were aimed at solving the existing problems, it is unclear whether this amendment really solved the problems or not. This study sought to determine how the changes made by identifying the differences between Decree No. 6521 and Decree No. 6877 affect all stakeholders in the standing sale mechanism and whether the problems have been resolved. For this purpose, the opinions of the stakeholders were obtained by means of a questionnaire survey conducted among forest cooperatives, forest villagers, employees of forest enterprises and enterprises processing wood-based products in the Kastamonu Regional Forestry Directorate, which was defined as the study area. Face-to- face interviews with the stakeholders were used to collect the data. Percentage and tabular methods were used to express the opinions of the interest groups, and the Kruskal-Wallis analysis and Mann-Whitney U test were used to determine whether their opinions on the changes differed. The study's results indicate that the recent regulatory changes have influenced interest groups in varying ways. The yield percentage calculation has improved, but transportation issues remain continuous. The compulsory employment of Forest Engineers in standing sales is not accepted except by forest enterprises. In addition, in the production of forest products, the employment of workers from outside the region where production takes place is not accepted by other interest groups except the forestry administration, and the production periods should not be fixed but should be determined according to the quantity to be produced. q y p Keywords: Log market, Standing tree sales, Regulation no. 6521, Regulation no. 6877, Kastamonu, Türkiye Tomruk piyasası aktörlerinin dikili ağaç satış yönetmeliklerinde yapılan değişikliklere ilişkin algıları Alper Aközlüa , Gökhan Şena,* Özet: Dikili ağaç satış metodu tüm dünyada olduğu gibi Türkiye’de de uygulanan bir satış yöntemidir. İlk kez 1996’da yürürlüğe giren dikili satış yönetmeliği birçok kez değişikliğe uğramıştır. Buna karşın bu değişikliklerin etkilerinin neler olduğu belirli değildir. 6877 Sayılı Tamim’in yürürlüğe girmesi ile oluşan değişiklikler ile mevcut sorunların çözümü hedeflenmişse de bu değişimin gerçekten sorunlara çözüm bulup bulmadığı bir muallaktır. Bu çalışmada 6521 sayılı tamim ile 6877 sayılı tamim arasındaki farklılıklar belirlenerek yapılan değişikliklerin dikili satış mekanizmasındaki tüm ilgi gruplarını nasıl etkilediği ve sorunların çözüme kavuşup kavuşmadığı belirlenmeye çalışılmıştır. Bu amaçla çalışma alanı olarak belirlenen Kastamonu Orman Bölge Müdürlüğü’nde orman kooperatifleri, orman köylüleri, orman işletmesi çalışanları ve odun kökenli ürün işleyen işletmeler ile yapılan anket çalışması ile ilgi gruplarının görüşleri belirlenmiştir. Verilerin elde edilmesinde ise yüz yüze anket yöntemi uygulanmıştır. İlgili grupların görüşlerinin ifade edilmesinde yüzde ve tablo yöntemleri kullanılırken, değişimler hakkındaki görüşlerinin farklılık gösterip göstermediği aşamasında Kruskal-Wallis analizi, Mann-Whitney U testinden yararlanılmıştır. Çalışma sonuçları, tamimlerde yapılan değişimlerin ilgi gruplarını farklı yönde etkilediğini göstermektedir. Bu değişikliklerden verim yüzdesi hesabının iyileştiği, transportta yaşanan sorunların halen tam olarak giderilemediği, dikili satışlarda zorunlu Orman Mühendisi çalıştırılmasının orman işletmeleri hariç pek kabul görmediği belirtilmektedir. Bununla birlikte paydaşların iş güvenliği konusundaki farkındalığının halen çok yüksek olmadığı, işletmeler hariç diğer paydaşlar tarafından peyderpey satış ve vadeli ödeme yöntemlerinin olumlu karşılandığı, orman ürünü üretimlerinde yerel alan dışından işçi çalıştırılmasının pek kabul görmediği ve üretim sürelerinin işin miktarına göre düzenlenmesi gerektiği paydaşlar tarafından belirtilmektedir. A ht k li l T k i Dikili ğ t 6521 l t i 6877 l t i K t Tü ki ş g g ğ p y ş Anahtar kelimeler: Tomruk piyasası, Dikili ağaç satışı, 6521 sayılı tamim, 6877 sayılı tamim, Kastamonu, Türkiye @ * Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr ✓ ✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023  a Kastamonu Üniversitesi, Orman Fakültesi, Orman Mühendisliği Bölümü, 37150, Merkez, Kastamonu @  a Kastamonu Üniversitesi, Orman Fakültesi, Orman Mühendisliği Bölümü, 37150, Merkez, Kastamonu @ * Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr ✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023 Citation (Atıf): Aközlü, A., Şen, G., 2023. Tomruk piyasası aktörlerinin dikili ağaç satış yönetmeliklerinde yapılan değişikliklere ilişkin algıları. Turkish Journal of Forestry, 24(4): 378- 389. DOI: 10.18182/tjf.1395550 1. Giriş New York eyaletinde özel orman sahipleri yaptıkları bireysel kontratlar ile satışlarını gerçekleştirirken devlet ormanlarında bu işlem halka açık bir ihale süreci ile gerçekleştirilmektedir (DEC, 2023). İrlanda’da ise 3 farklı satış yöntemi kullanılmaktadır. Bunlar dikili satış, rampada/yol kenarında satış ve kapı satışıdır (satılan ürün satıcının adresine kadar götürülür) (Teagasc, 2023). Yüzde 80 oranında özel ormana sahip olan Avusturya’da (Schenker, 1995) ise yapılan orman ürünü satışlarının büyük çoğunluğu (%85) alivreli satış yöntemi ile gerçekleştirilmektedir. Ancak 200 hektar ve üzerindeki ormanlardan yapılacak satışlarda ise dikili satış yöntemi kullanılmaktadır (Puwein, 2007). Kongo’da genelde tüm hakları ile ormanlar yüklenicilere verilerek satış işlemi yapılmakta (Counsell, 2006), Almanya, Rusya ve İngiltere gibi ülkelerde ise dikili satış yöntemi kullanılmaktadır (Ünal ve Karakaya, 2002). İngiltere’de ayrıca, depolardan ihale ile ve sunta, kağıt vb. üretimi yapan şirketlere uzun vadeli sözleşmeler ile de satışlar gerçekleştirilebilmektedir (FE, 2023). Fransa’da ise doğrudan satışlar (%26,8), tedarik sözleşmeleri (18,7) ve açık artırmalı satışlar (%54,5) ile orman ürünlerinin satışları gerçekleştirilmektedir (Elyakime ve Cabanettes, 2009). Türkiye’de işletme ve üretim bakımından ormancılık sektöründe tekel bir yapının olması beraberinde bazı sorunları da getirmektedir. Bu durum da OGM’nin halen ne üretirsem onu satarım anlayışı ile pazarlama faaliyetlerini sürdürmesine neden olmaktadır (Demirel, 2006). Buna karşın uluslararası ticaret boyut değiştirmiş ülkeler ekonomik çıkarları için gümrük vergi ve fonlarının kaldırılması gibi yeni kararlar almıştır (Türker ve Yazıcı, 1998). Bunun yanında Avrupa Birliği (AB) ile gümrük birliği anlaşmasının yapılması, Sovyetler Birliği’nin dağılması ve bu ülkelerden Türkiye’ye bol miktarda düşük fiyatlı odun hammaddesi girişinin gerçekleşmesi (Daşdemir, 2003) OGM’nin tekel özelliğini sekteye uğratmaya başlamıştır. Bu gelişmeler ormancılık sektöründe üretimde kalitenin artırılması, maliyetlerin düşürülmesi, tüketici istek ve taleplerinin dikkate alınması ve yeni pazarlama anlayışlarının oluşturulması gerekliliğini ortaya çıkarmıştır (Demirel, 2006). Değişen piyasa yapısının ve satışların artırılması amacı ile tomruk satışlarında dikili satış uygulamasına geçilmesi öngörülmüştür. Maliyetlerin düşürülecek olması, alıcının istediği gibi ürünü işleyebilmesine olanak sağlaması, üretim sürecindeki ekonomik kayıpları en aza indirmesi, işçilerin sigortalanmasının sağlanması gibi nedenler bu yöntemin uygulamaya başlamasında etkili olmuştur (Daşdemir, 2011). Dikili ağaç satışı uygulaması ilk defa 1996 yılında çıkarılan 5038 sayılı yönetmelikle başlamıştır. Ancak orman idaresinin koruma, takip ve kontrol bakımından endişelerinin bulunması, köylülerin sosyo-ekonomik konularda itilafa düşme endişesi, dikili satış fiyatı ve verim yüzdelerinin doğru belirlenememe ihtimali, alınan dikili üretiminin köylüye yaptırılıp yaptırılmayacağının belirli olmaması, kooperatifleri başvuracaklar listesine almaması gibi nedenlerden dolayı dikili satış yöntemi yaygınlaşamamıştır (Kaplan, 2005). Görülen bu eksikliklerin giderilmesi amacıyla 6057 sayılı tamim, ilk tamimin yayınlanmasından iki yıl sonra yürürlüğe girmiştir. 1. Giriş kanunlar ile odun ve diğer orman ürünlerinin üretimi, hasadı ve ticareti belirli kurallara ve izinlere bağlı hale gelmiştir. Ormanlar, geçmişte insanların faydalanmasına açık bir şekilde varlığını sürdürmekte idi. Çıkarılan çeşitli yasalar sonrasında ormanlardan yapılan faydalanmalar sınırlandırılmış ve bazı kurallara bağlı hale getirilmiştir. Bu Orman ürünlerinin üretim ve satışı ile ilgili olarak dünya üzerinde çeşitli sistemler uygulanmaktadır. Bu sistemlerin temelinde de ormanların sahipliliği oldukça büyük etki göstermektedir. Özellikle özel orman sahipleri pazarlama ve satış konusunda daha esnek iken, devlete ait ormanlarda ve  a Kastamonu Üniversitesi, Orman Fakültesi, Orman Mühendisliği Bölümü, 37150, Merkez, Kastamonu @ * Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr ✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023 Citation (Atıf): Aközlü, A., Şen, G., 2023. Tomruk piyasası aktörlerinin dikili ağaç satış yönetmeliklerinde yapılan değişikliklere ilişkin algıları. Turkish Journal of Forestry, 24(4): 378- 389. DOI: 10.18182/tjf.1395550 @ * Corresponding author (İletişim yazarı): gsen@kastamonu.edu.tr ✓ ✓ Received (Geliş tarihi): 24.10.2023, Accepted (Kabul tarihi): 18.11.2023 Turkish Journal of Forestry 2023, 24(4): 378-389 379 tarafından işletileceği ve devlet ormanlarına ait her çeşit işlerin Orman Genel Müdürlüğü (OGM) tarafından yapılacağı” ifade edilmiştir (Daşdemir, 2011). Günümüzde Türkiye’de özel orman sahipleri ya da tapulu arazilerinde yetiştirdikleri orman ürünlerini kişiler istedikleri şekilde satma hakkına sahiptir. Devlet ormanlarında üretilen odun ürünlerinin satış yöntemleri ise üretim öncesi ve sonrası döneme göre yapılmaktadır. Üretimden sonra yapılan satışlar açık arttırmalı satış, yükselterek açık artırmalı satış, eksilterek açık artırmalı satış, teklif usulü gizli arttırmalı satış (kapalı zarf), pazarlıklı satış ve tahsisli satış şeklinde yapılırken, üretimden önce ise dikili ağaç satışları ve alivreli satışlar şeklinde uygulanmaktadır (Miraboğlu, 1983; Daşdemir, 2011). devlet işletmeciliğinde yönetilen orman alanlarından yapılacak üretim ve satış faaliyetleri daha sıkı bir denetim altında ve belirli yasa ve yönetmeliklere bağlı kalınarak yapılmaktadır. Orman ürünleri satış yöntemlerinde özel orman sahipleri direkt olarak yüklenici ile yapacakları kontratlar ile satışlarını gerçekleştirebilirler. Buna karşın devlet ormanlarında satışların kesim yapıldıktan sonra mı yoksa ağaçlar halen dikili halde iken mi satış yapılacağı önem kazanmaktadır. Bu kapsamda ülkeler farklı yöntemler kullanmaktadır. Finlandiya’da kullanılan odunların %9’u devlet orman işletmelerinden, %85’i özel orman işletmelerinden, %6’sı endüstriyel ormancılık yapan özel işletmelerden karşılanmaktadır. Finlandiya’daki yuvarlak odun satışlarının %14,5’i dikili satış ve geri kalanı da alivreli satış olarak gerçekleşmektedir (Melkas ve Oy, 2022). Litvanya’da hem devlet hem de özel şahıslara ait ormanlar bulunmaktadır. Ancak üretim ve satış işlerinin tamamına yakını özel işletmeler tarafından gerçekleştirilmektedir. Devlet ormanlarından yapılan tomruk satışlarının %10’u dikili satışlar, geri kalanı ise alivreli satış şeklinde pazarlanmaktadır (Gaizutis, 2005; Toppinen vd., 2005). 1. Giriş Bu tamimde tahsisli dikili ağaç satışlarının kooperatifler tarafından da alınabilmesinin önü açılmıştır. Böylece orman köylülerinin dikili halde ağaçları alıp, üretimini yapıp pazara sunma hakları oluşmuştur (Ünal ve Karakaya, 2002). Ancak dikili ağaç satışındaki aksaklıklar tam olarak yine çözülememiş ve bu tamimlerin devamında 2004 yılında 6350 sayılı tamim, 2007 yılında 6521 sayılı tamim, 2013 yılında ise 6877 sayılı tamim yürürlüğe girmiştir (OGM, 2013). Türkiye’de de diğer ülkelere benzer şekilde birden çok satış yöntemi uygulanmaktadır. Osmanlı döneminde halk ormanlardan istediği şekilde yararlanırken (Cibal-i Mübaha), 1870 yılında çıkarılan Orman Nizannamesi ile bu serbest kullanım durumu sınırlandırılmıştır (Özdönmez vd., 1989). Satışlar ile alakalı olarak getirdiği yenilik ise kesilecek ürünlerin damgalanma şartı ve üretilen tomrukların idarece belirlenecek olan bir bedel üzerinden satılmasıdır (Diker, 1947). Ayrıca üretim işlerinin yükleniciler tarafından yapılacağı dikili satışlarda uygulanmaya başlamıştır. 3116 Sayılı Orman Kanunu çıkarıldığında “devlet ormanlarının devlet tarafından işletilmesi” hükmü getirilerek (31. Madde) özel firmaların işlettiği ormanlar devlet işletmesine alınmıştır (Daşdemir, 2011). 1945 yılında çıkartılan 4785 sayılı yasa ile ormanların büyük bir bölümü devlet mülkiyeti altına alınmış, devlet ormanı dışındaki mülkiyet hakkı neredeyse bitmiş ve orman sınırı içine giren alan miktarı da artırılmıştır (Gümüş, 2004). Daha sonra 1956 yılında 6831 sayılı Orman Kanunu çıkarılmış ve bu kanuna göre “devlet ormanlarının devlet Yürürlüğe giren her bir tamim bir öncekinin aksaklıklarını ve eksikliklerini gidermek amacı ile yapılmaktadır. Buna karşın her bir değişiklik tomruk piyasasındaki tüm paydaşları etkilemekte ve onlar için olumlu ya da olumsuz yeni durumlar oluşturmaktadır. Bu Turkish Journal of Forestry 2023, 24(4): 378-389 380 nedenle bu değişimlerin etkilerinin belirlenmesi yapılacak yeni çalışmalar içinde önemli olmaktadır. Dikili satışlar ile ilgili çeşitli çalışmalar yapılsa da yasa veya yönetmeliklerin değişimlerinin etkilerinin analiz edildiği başka bir çalışmaya rastlanılamamıştır. Bu çalışmada, 6521 sayılı dikili satış tamiminin yürürlükten kaldırılıp 6877 sayılı dikili satış tamiminin yürürlüğe girmesi ile oluşan değişikliklerin dikili ağaç satışı ile alakalı ilgi grupları üzerindeki etkilerinin incelenmesi amaçlanmıştır. Çalışma sonuçlarının hem literatürdeki eksikliğin doldurulmasında hem de karar verici ve kanun yapıcılara önemli bir referans bilgileri sağlayacağı düşünülmektedir. Şekil 1. Kastamonu coğrafi konumu 2.2.1. Örnek alan büyüklüğünün belirlenmesi Çalışmada öncelikle para, zaman, araç, gereç ve uzman yetersizliği vb. sebeplerle örnek alan belirlemesi gerçekleştirilerek sınırlı sayıdaki örnek/grup birimde örneklem araştırmaları yapılmaktadır (Özdamar, 2002). Bu kapsamda çalışmada öncelikle anket uygulaması yapılacak Orman İşletme Müdürlükleri belirlenmiştir. Samatlar, Kastamonu sınırları içerisinde bulunan Orman İşletme Müdürlükleri’nden işletmesinin bazı dönemlerde Araç işletmesine bağlı olmasından dolayı Araçsam adı ile değerlendirilmiştir. Karadere Orman işletme Müdürlüğü sınırları içinde yerleşim yeri olmadığından dolayı da Kastamonu Orman İşletme Müdürlüğü ile değerlendirilerek araştırmada Kaskar olarak isimlendirilmiştir. KOBM’ne bağlı Orman İşletme Müdürlükleri dikili satışta etkili olabilecek belirlenen bazı değişkenlere göre (Çizelge 1) Ranking metodu ile sıralanmıştır (Çizelge 2). Ranking (sıralama) tekniğinde, karar elemanları karar verici tarafından göreceli önem derecelerine göre sıralanmaktadır (Yılmaz, 2006). Bu teknik kapsamında orman işletme müdürlüklerinin göreceli öncelik derecelerine göre hüküm verilmekte ve sıralanmaktadır. Bu sıralama işlemi, “dokuz dereceli ölçek” vasıtasıyla yapılmaktadır. Bu ölçekte; 1- Zayıf oranda önemli, 3- Daha az önemli, 5- Orta derecede önemli, 7- Daha çok önemli, 9- Aşırı derecede önemli olarak kabul edilmektedir. Bunlar yanında “2, 4, 6 ve 8 değerleri” de orta değerler olarak kullanılabilmektedir (Güngör ve Şen, 2018). Ranking’de en yüksek değerleri alan 3 Orman İşletme Müdürlüğü örnek alan olarak belirlenmiş ve bu alanları temsil edebilecek istatistiksel olarak anlamlı en küçük anket sayıları belirlenmiştir. 2.1. Materyal 2.1. Materyal Bu çalışmanın materyalini, 2007 tarihli 6521 sayılı tamim ile 2013 yılında yürürlüğe giren 6877 sayılı tamim arasındaki değişikliklerin ilgi grupları üzerindeki etkilerini belirlemek amacıyla Kastamonu ilinde yapılan anketlerden elde edilen veriler, konu ile ilgili daha önce yapılmış çalışmalar ve diğer ikincil veriler oluşturmaktadır. Şekil 1. Kastamonu coğrafi konumu Çalışma Kastamonu ilinde gerçekleştirilmiştir. Kastamonu ili, Karadeniz bölgesinin batı kesiminde 41-42° kuzey enlemleri ile 33-46° doğu boylamları arasında yer almaktadır (Şekil 1). Yüzölçümü 13.108 km2 olan il, Türkiye yüzölçümünün yaklaşık %1,7’sini oluşturmaktadır (Kravkaz- Kuscu, 2018; Şen vd., 2022; TOB, 2023). İl genelinde en yüksek sıcaklık ortalaması 16°C, en düşük sıcaklık ortalaması ise 4,2°C’dir ve yıllık ortalama yağış miktarı 486,4 mm’dir (Kurnaz ve Güneş Şen, 2023). Kastamonu ili tarımsal faaliyetlerin yüksek olduğu ve il yüzölçümünün %64’ünün ormanlarla kaplı olduğu bir ildir. 695.763 hektarı normal kapalı, 180.551 hektarı boşluklu kapalı olmak üzere toplam orman alanı 876.314 hektardır. Bu ormanların yönetimi 16 işletme müdürlüğü olan Kastamonu Orman Bölge Müdürlüğü (KOBM) tarafından gerçekleştirilmektedir (KOBM, 2023). Kastamonu ilinde Küre Dağları Milli Parkı ve Ilgaz Dağı Milli Parkı olmak üzere iki adet milli park bulunmaktadır (Ayan vd., 2007). Küre Dağları Milli Parkı Platin Yaban Hayatı Sertifikasına sahiptir (Meydan ve Öztürk, 2023). Kastamonu iline bağlı 20 ilçe ve 1.054 köy bulunmaktadır (TÜİK, 2020). 2.2. Yöntem Bu çalışmada, 6521 sayılı dikili satış tamiminin yürürlükten kaldırılıp 6877 sayılı dikili satış tamiminin yürürlüğe girmesi ile oluşan değişikliklerin dikili ağaç satışı ile alakalı ilgi gruplarının görüşlerinin belirlenmesi amacıyla oluşturulan araştırma hipotezi aşağıdaki gibidir; H0a: Tomruk piyasası aktörlerinin dikili satış yönetmeliklerinde yapılan değişiklikler hakkındaki bilgi düzeyleri ve görüşleri arasında fark yoktur. Turkish Journal of Forestry 2023, 24(4): 378-389 381 Çizelge 1. Anket yapılacak Orman İşletme Müdürlüğü seçiminde kullanılan değişkenler Dikili satışa konu orman ürünü miktarı/Toplam üretilen orman ürünü miktarı (m3) Kooperatif sayısı (adet) Odun kökenli ürün işleyen işletme sayısı (adet) Toplam köy nüfusu Dikili satışa konu ürün miktarı (m3) Verimli orman alanı (ha) Değişken 1 2 3 4 5 6 Azdavay 0,12 5,00 5,00 6096 13770,00 29079,00 Cide 0,42 4,00 24,00 18657 32209,00 35643,50 Çatalzeytin 0,18 7,00 10,00 4075 10778,00 19309,00 Daday 0,18 35,00 5,00 7501 27518,00 52422,00 İhsangazi 0,13 9,00 3,00 3530 8025,00 19420,50 İnebolu 0,17 6,00 12,00 25543 14423,00 40341,10 Küre 0,13 14,00 21,00 14401 14891,00 28549,50 Taşköprü 0,08 61,00 25,00 19451 22018,00 82469,50 Tosya 0,40 24,00 350,00 10934 47050,00 55503,00 Pınarbaşı 0,11 3,00 5,00 3463 9376,00 28992,00 Bozkurt 0,27 7,00 10,00 4640 16520,00 21892,00 Hanönü 0,00 16,00 0,00 2270 347,00 19247,00 Araçsam 0,26 2,00 7,00 14661 31422,00 61997,00 Kaskar 0,17 68,00 150,00 31443 23840,50 66873,00 Çizelge 2. Anket yapılacak Orman İşletme Müdürlüğü seçimi için yapılan Normalizasyon sonuçları Normalize -1 Normalize -2 Normalize -3 Normalize -4 Normalize -5 Normalize -6 Toplam normalize Genel oranlama Azdavay 0,05 0,02 0,01 0,04 0,05 0,05 0,21 0,04 Cide 0,16 0,02 0,04 0,11 0,12 0,06 0,51 0,08 Çatalzeytin 0,07 0,03 0,02 0,02 0,04 0,03 0,21 0,03 Daday 0,07 0,13 0,01 0,05 0,10 0,09 0,45 0,08 İhsangazi 0,05 0,03 0,00 0,02 0,03 0,03 0,17 0,03 İnebolu 0,06 0,02 0,02 0,15 0,05 0,07 0,39 0,06 Küre 0,05 0,05 0,03 0,09 0,05 0,05 0,33 0,05 Taşköprü 0,03 0,23 0,04 0,12 0,08 0,15 0,65 0,11 Tosya 0,15 0,09 0,56 0,07 0,17 0,10 1,14 0,19 Pınarbaşı 0,04 0,01 0,01 0,02 0,03 0,05 0,17 0,03 Bozkurt 0,10 0,03 0,02 0,03 0,06 0,04 0,27 0,05 Hanönü 0,00 0,06 0,00 0,01 0,00 0,03 0,11 0,02 Araçsam 0,10 0,01 0,01 0,09 0,12 0,11 0,43 0,07 Kaskar 0,06 0,26 0,24 0,19 0,09 0,12 0,96 0,16 1,00 1,00 1,00 1,00 1,00 1,00 6,00 1,00 elge 1. Anket yapılacak Orman İşletme Müdürlüğü seçiminde kullanılan değişkenler Belirlenen işletme müdürlüklerindeki 4 ilgi grubu ile anket yapılması kararlaştırılmıştır. 2.2. Yöntem Aşağıda bu değişiklikler maddeler halinde gösterilmiştir. alınmıştır. Aşağıda bu değişiklikler maddeler halinde gösterilmiştir. 2.2. Yöntem Bunlar; orman kooperatifleri (ORKOP), orman köylüsü (OK), orman işletme müdürlüğü çalışanları (OİMÇ) ve odun kökenli ürün işleyen işletmeler (OKÜİ)’dir. Yapılacak anket sayıları eşitlik 1 ile belirlenmiştir. anketler hazırlanmıştır. Oluşturulan anket formları yapılan ön uygulama ile denenmiş ve yenilenmiştir. Hazırlanan anketler belirlenen ilgi gruplarına yüz yüze anket yöntemi ile uygulanmış olup, bu anketlerde ilgi gruplarına dair tanımlayıcı özellikleri ve değişen dikili ağaç satışı tamimlerinin paydaşlar üzerine etkilerini belirlemeye yönelik sorulara yer verilmiştir. n = F∗t2∗P∗Q (F∗m2)+(t2∗P∗Q) (1) Çalışmadaki analizler SPSS 21 istatistik paket programı ile gerçekleştirilmiştir. Gruplar arası denek sayılarının eşit olmaması ve verilerin sıralı olması nedeniyle ilgi grupları arasında farkların olup olmadığı tek yönlü varyans analizinin parametrik olmayan karşılığı olan Kruskal-Wallis Analizi ile incelenmiştir (Özdamar, 2002). İstatistiksel olarak gruplar arasındaki farkın anlamlı olduğu durumlarda hangi gruplar arasında farkın olduğunu bulmak için Mann-Whitney U testi yapılmış ve sonrasında Bonferroni düzeltmesi yapılarak grupların etki büyüklüğü hesaplanmıştır. Bonferroni düzeltmesi 3 ve daha fazla grup için yapılmaktadır. 3 grup olduğunda her birinin kendi alfa yanılma yüzdesi olduğundan 0,05 üçe bölünür ve anlamlılık 0,0167’ye göre değerlendirilir. 3’den fazla gruplarda ise bu 0,01 olarak kabul edilir. Z değeri için etki büyüklüğü r = Z / √N formülüyle bulunur (Cevahir, 2020). (1) Formülde n= Örnek büyüklüğü, F= Anakütle, P= Tahmini oran, Q= 1-P, t= Güven düzeyi %95 (Tablo değeri: 1,96) ve m= Hata payı %10’dır. Formüle göre belirlenen örneklem büyüklükleri Çizelge 3’te gösterilmiştir. Anket güvenilirliğini artırmak için ulaşılabilen daha fazla kişiyle anket yapılmıştır. 2.2.2 Anket formlarının geliştirilmesi ve değerlendirme yöntemleri Anket formlarının belirlemesinde öncelikle 6521 ve 6877 sayılı dikili satış yönetmelikleri incelenerek farklılıkları ortaya çıkarılmıştır. Devamında çalışmanın amacında belirlenen hipotezlerin test edilmesine imkân verecek şekilde Turkish Journal of Forestry 2023, 24(4): 378-389 382 Çizelge 3. Çalışmadaki ana kütle, örneklem ve uygulanan anket sayıları Anket yapılan işletme müdürlükleri OİMÇ OK OKÜİ ORKOP Ana kütle (Kişi) Örnek sayısı Anket sayısı (Adet) Ana kütle (Kişi) Örnek sayısı Anket sayısı (Adet) Ana kütle (Kişi) Örnek sayısı Anket sayısı (Adet) Ana kütle (Kişi) Örnek sayısı Anket sayısı (Adet) Tosya 11 25 8 4.922 96 66 350 81 54 24 59 18 Taşköprü 14 11 8.158 67 25 18 61 32 Kaskar 17 11 11.460 60 150 63 68 20 Toplam 42 30 24.540 193 525 135 153 70 ORKOP: Orman kooperatifleri, OK: Orman Köylüsü, OİMÇ: Orman İşletme M0üdürlüğü Çalışanları, OKÜİ: Odun Kökenli Ürün İşleyen İşletmeler Çizelge 3. Çalışmadaki ana kütle, örneklem ve uygulanan anket sayıları OİMÇ OK alınmıştır. Değişiklik 1. ORKOP’ların %18,7’sinin ortak sayısı 10-20, %34,3’ünün 21-40, %18,6’sının 41-60 ve %28,4’ünün de 61- 371 arasında değiştiği belirlenmiştir. ORKOP katılımcılarının %90’ı kooperatiflerindeki üye sayısının yeterli olduğunu ifade etmişlerdir. Katılımcıların faaliyet alanları tamamıyla ormancılıktır ancak ek olarak sulama, tarım, akaryakıt ve taşıma işleriyle de ilgilendiklerini belirtmişlerdir. Değişiklik 2. 3.1. İlgi gruplarının bazı özellikleri Değişiklik 1. Değişiklik 1. Anket uygulaması yapılan ilgi gruplarının bazı tanımlayıcı özellikleri her bir grup için ayrı ayrı verilmiştir. OK’deki katılımcıların %23,8’i (46 kişi) 20-40 yaş aralığında, %66,3’ü (128 kişi) 41-60 yaş aralığında ve %9,9’u da 61-80 yaş aralığındadır. OK katılımcılarının %20,7’si okuryazar, %68,9’u ilköğretim mezunu, %9,8 oranında lise ve dengi okul mezunu ve %0,5 oranında ise yüksekokul- üniversite mezunudur. Katılımcıların %92,5’i en az üç iş kolundan diğerleri ise iki iş kolundan gelir elde etmektedirler. Gelir elde edilen iş kollarının %17,9’unu tarım, %26,1’ini hayvancılık, %35,9’unu ormancılık, %12,6’sı ticaret oluşturmaktadır. %7,5’i ise emekli maaşı almakta olduğunu belirtmişlerdir. OK katılımcılarının %43’ü (83 kişi) yıllık 9.000-15.000 TL, %31,1’i 16.000-20.000 TL, %13’ü 21.000- 29.000 TL, %9,3’ü 30.000-40.000 TL ve %3,6’sı ise %41.000-50.000 TL yıllık gelir elde ettiklerini belirtmişlerdir. Gelirlerinin tamamını ormancılıktan kazanan kişilerin oranı sadece %3,6, %50’ye kadar olan kısmını ormancılıktan sağlayanların oranı ise %72,6 olarak belirtilmiştir. 6521/2- “Dikili ağaç satışında, dikili ağacın m3 satış bedeli, satışa konu bölme veya bölmecikte pazarlama imkânları da göz önüne alınarak üretilmesi öngörülen emvale göre her ağaç türü için verim yüzdelerinin tespit edilmesi ve bu yüzdelere göre çıkması gereken ürünlerin fiyatlandırılması yapılarak belirlenmektedir. Verim yüzdeleri gerçeğe ne kadar yakın belirlenirse alıcı ve satıcı yönünden doğru sonuca ulaşılacak ve dikili satışın yaygınlaşmasına olumlu katkı sağlayacaktır. Verim yüzdesi tespitinde ayrıca alt tabakadaki fertler ile şüceyrat odununun dikili damgaya ve verim yüzdesine dâhil etmede özellikle dikkatli olunarak, deneme ağaçları seçilirken objektif kriterler ve aşağıdaki hususlara uyulacaktır.” 6877/A-2- “Dikili ağaç satışında maliyet bedeli, muhammen bedel ve satış fiyatına esas teşkil etmesi nedeniyle; satışa konu bölme veya bölmecikte ki her ağaç türü için üretilmesi öngörülen ürünlerin verim yüzdesi hassasiyetle belirlenir. Ancak, yapılan dikili damgada toplam miktarın %5'inin altında olup piyasa değeri açısından özel kıymet ifade etmeyen ağaç türlerinde ayrıca verim yüzdesi tespit edilmeyebilir. Bu durumda söz konusu hacim benzer ağaç türü hacmine ilave edilmek suretiyle değerlendirilir. Ayrıca verim yüzdesi tespitinde alt tabakadaki fertler ile şüceyrat odununu dikili damgaya ve verim yüzdesine dâhil etmede özellikle dikkatli olunarak, deneme ağaçları seçiminde objektif kriterlere ve aşağıdaki hususlara uyulur.” ORKOP’ların ortak sayıları 10 ile 371 kişi arasında değişmektedir. ORKOP’ların %18,7’sinin ortak sayısı 10-20, %34,3’ünün 21-40, %18,6’sının 41-60 ve %28,4’ünün de 61- 371 arasında değiştiği belirlenmiştir. ORKOP katılımcılarının %90’ı kooperatiflerindeki üye sayısının yeterli olduğunu ifade etmişlerdir. Katılımcıların faaliyet alanları tamamıyla ormancılıktır ancak ek olarak sulama, tarım, akaryakıt ve taşıma işleriyle de ilgilendiklerini belirtmişlerdir. ORKOP’ların ortak sayıları 10 ile 371 kişi arasında değişmektedir. Değişiklik 2. Anket yapılan orman idaresi personelinin %60’ının meslekteki süreleri 2-5 yıl, %30’unun 6-8 yıl, %10’ununda 21-40 yıldır. 6521/2.1.1- “Satışa konu bölme veya bölmecikteki ağaçların Dikili Kabuklu Gövde Hacmi (DKGH) ve çap kademelerine dağılımı göz önüne alınarak DENEME AĞACI HACİM BÜYÜKLÜĞÜ tespit edilir. Hacim büyüklüğü satışa konu ağaçların temsiline imkan verecek şekilde 1 m3, 3 m3, 5 m3, 8 m3 gibi belirlenecektir. Burada dikkat edilmesi gereken husus D.K.G.H miktarı arttıkça ve ağırlıklı çap grubu kalınlaştıkça daha iyi temsil için deneme ağacı hacim büyüklüğünün artması gerekmektedir.” 6521/2.1.1- “Satışa konu bölme veya bölmecikteki ağaçların Dikili Kabuklu Gövde Hacmi (DKGH) ve çap kademelerine dağılımı göz önüne alınarak DENEME AĞACI HACİM BÜYÜKLÜĞÜ tespit edilir. Hacim büyüklüğü satışa konu ağaçların temsiline imkan verecek şekilde 1 m3, 3 m3, 5 m3, 8 m3 gibi belirlenecektir. Burada dikkat edilmesi gereken husus D.K.G.H miktarı arttıkça ve ağırlıklı çap grubu kalınlaştıkça daha iyi temsil için deneme ağacı hacim büyüklüğünün artması gerekmektedir.” OKÜİ katılımcılarının faaliyet alanları kereste ticareti, mobilya üretimi ve marangozlardan oluşmaktadır. Katılımcıların %34,8’i 50-68, %31,9’u 42-49 ve %33,3’ü de 25-37 yaş aralığındadır. Bu kişilerin %45,9´u ilköğretim, %43,0’ı lise ve dengi ve %8,1’i de yüksekokul-üniversite mezunudur. %3’ü ise sadece okuryazardır. Bu kişilerin %31,1’inin yıllık geliri 32.000-50.000 TL, %16,3’ünün 52.000-75.000 TL aralığında, %10,4’ ünün 80.000-94.000 TL aralığında olduğu belirlenmiştir. Gelirleri hakkında bilgi vermeyenlerin oranı ise %42,2’dir. 6877/1.2.1.1. “Satışa konu bölme veya bölmecikteki ağaçların DKGH ve çap kademelerine dağılımı göz önüne alınarak deneme ağacı hacim büyüklüğü tespit edilir. Hacim büyüklüğü, satışa konu ağaçların temsiline imkan verecek şekilde 1 m3, 3 m3, 5 m3, 8 m3 gibi belirlenir. Burada dikkat edilmesi gereken husus DKGH miktarı arttıkça ve ağırlıklı çap grubu kalınlaştıkça daha iyi temsil için deneme ağacı hacim büyüklüğünün artması gerekmektedir. Hacim büyüklüğü tespit edilirken (deneme alanı alınanlar hariç) alınacak olan deneme ağacı hacmi o ağaç türünün damgadaki toplam miktarının %2’sinden aşağı olamaz. Ancak; kızılçam 6877/1.2.1.1. “Satışa konu bölme veya bölmecikteki ağaçların DKGH ve çap kademelerine dağılımı göz önüne alınarak deneme ağacı hacim büyüklüğü tespit edilir. Hacim büyüklüğü, satışa konu ağaçların temsiline imkan verecek şekilde 1 m3, 3 m3, 5 m3, 8 m3 gibi belirlenir. Burada dikkat edilmesi gereken husus DKGH miktarı arttıkça ve ağırlıklı çap grubu kalınlaştıkça daha iyi temsil için deneme ağacı hacim büyüklüğünün artması gerekmektedir. Hacim büyüklüğü tespit edilirken (deneme alanı alınanlar hariç) alınacak olan deneme ağacı hacmi o ağaç türünün damgadaki toplam miktarının %2’sinden aşağı olamaz. Ancak; kızılçam 3. Bulgular 3.1. İlgi gruplarının bazı özellikleri Değişiklik 5. Değişiklik 5. Değişiklik 5. 3.2. 6521 ve 6877 sayılı dikili ağaç satış usul ve esasları hakkındaki tamimler arasındaki farklılıklar 3.2. 6521 ve 6877 sayılı dikili ağaç satış usul ve esasları hakkındaki tamimler arasındaki farklılıklar 3.2. 6521 ve 6877 sayılı dikili ağaç satış usul ve esasları hakkındaki tamimler arasındaki farklılıklar Dikili satış uygulamasında yapılan değişiklikler hakkında ilgi gruplarının görüşlerini belirlemek için öncelikle 6521 ve 6877 sayılı tamimler arasındaki değişiklikler belirlenmiştir. Tamimlerdeki farklılıklar belirlenirken uygulamada ilgi gruplarına etki edebilecek seviyedeki değişiklikler dikkate Turkish Journal of Forestry 2023, 24(4): 378-389 383 Orman Kalkındırma Kooperatiflerinin tahsisli dikili ağaç satışlarında üretim işçiliği üyelerince yapılacağı yazılı beyan edilmesi halinde sigorta mevzuatı yönünden sorumlulukları kendilerine ait olmak üzere Sosyal Sigortalar Kurumu’na bildirimde bulunulmayacaktır.” Orman Kalkındırma Kooperatiflerinin tahsisli dikili ağaç satışlarında üretim işçiliği üyelerince yapılacağı yazılı beyan edilmesi halinde sigorta mevzuatı yönünden sorumlulukları kendilerine ait olmak üzere Sosyal Sigortalar Kurumu’na bildirimde bulunulmayacaktır.” Orman Kalkındırma Kooperatiflerinin tahsisli dikili ağaç satışlarında üretim işçiliği üyelerince yapılacağı yazılı beyan edilmesi halinde sigorta mevzuatı yönünden sorumlulukları kendilerine ait olmak üzere Sosyal Sigortalar Kurumu’na bildirimde bulunulmayacaktır.” tensil sahası ve endüstriyel plantasyon sahalarında, meşcerenin homojen yapıya sahip olması halinde %1’den az olmamak şartıyla belirlenebilir.” Değişiklik 3. Değişiklik 4. 6521/ (Tamimde yok) 6877/1.17. “Orman yön 6521/ (Tamimde yok) 6877/1.17. “Orman yönetiminin belgelendirildiği (Forest Stewardship Council –FSC- ve Programme for the Endorsement of Forest Certification -PEFC-) ormanlarda yapılacak dikili ağaç satışlarında belgelendirmenin zorunlu gerekleri şartnamelerin özel hükümler bölümünde belirtilir.” 6877/1.17. “Orman yönetiminin belgelendirildiği (Forest Stewardship Council –FSC- ve Programme for the Endorsement of Forest Certification -PEFC-) ormanlarda yapılacak dikili ağaç satışlarında belgelendirmenin zorunlu gerekleri şartnamelerin özel hükümler bölümünde belirtilir.” 6521/2.4-“Alıcı elde ettiği ürünleri nakletmek istediğinde Orman İşletme Şefliğinin ölçü ve tespitine göre nakline izin verilecektir. Peyderpey satış veya parçalı ölçüm yapılması halinde; satışı yapılan dikili gövde hacminden çıkabilecek ürün miktarı gözetilerek toplam dikili miktarın %75’ne kadarının nakline izin verilecektir. Dikili miktarın %25’inden az olmamak üzere kalan miktar öncekilerle birlikte işletme şefliğince değerlendirilerek dikili kabuklu gövde hacminden veya yüzde yüzün üzerinde verim yüzdesi tespiti yapılmış ise bu miktardan az olması durumunda işletme şefliğince nakline müsaade edilecektir. Dikili satış işleminin her aşaması idarece kontrol edilerek, tüm ölçü tespit tutanaklarının birer nüshası İşletme Müdürlüğüne gönderilecektir. Elde edilen toplam ürün miktarı; dikili kabuklu gövde hacminden veya yüzde yüzün üzerinde verim yüzdesi tespiti yapılmış ise bundan fazla olması durumunda, son %25’lik kısma ait bölüm nakledilmeden durum İşletme Müdürlüğünce Bölge Müdürlüğüne bildirilir. Bölge Müdürlüğünce kurulacak komisyon marifetiyle alıcının da katılabileceği şekilde en geç bir hafta içerisinde durum incelenerek herhangi bir usulsüzlük olup olmadığını tespitle işletmesine bildirilecektir. Usulsüzlük yoksa ürünün nakline izin verilecek olup aksi durumda şartname hükümlerine göre işlem yapılır.” 6521/ (Tamimde yok) 6521/ (Tamimde yok) 6877/1.18. “Baltalık uygulamaları, sıklık bakımı gibi önceden DKGH'nin sağlıklı olarak tespit edilemediği çalışmalarda dikili ağaç satışı söz konusu olması durumunda genel sahayı temsil edecek büyüklükte ve özellikte alınacak deneme alanı ölçümleri genel sahaya uyarlanarak işlem yapılır.” 6877/1.18. “Baltalık uygulamaları, sıklık bakımı gibi önceden DKGH'nin sağlıklı olarak tespit edilemediği çalışmalarda dikili ağaç satışı söz konusu olması durumunda genel sahayı temsil edecek büyüklükte ve özellikte alınacak deneme alanı ölçümleri genel sahaya uyarlanarak işlem yapılır.” Değişiklik 7. Değişiklik 6. Değişiklik 6. Değişiklik 3. 6877/1.15. “Bölge Müdürlüğünün dikili ağaç satışlarına ait, ihale onay yazısı veya tahsis fiyatına ait onay yazısının İşletme Müdürlüğü kaydına girdiği tarihten itibaren 15 gün içinde, İşletme Müdürlüklerince satışın konusu, alıcısı, yeri ve mahiyeti hakkında SSGK bildirimde bulunulur. Ayrıca müşterinin fiilen işe başladığı tarih de SGK’ya bildirilir. Ancak; tahsisli dikili ağaç satışlarında orman köylerini kalkındırma kooperatiflerinin üretim işçiliğini kendi üyelerince yapılacağını yazılı beyan etmesi halinde sigorta mevzuatı yönünden sorumlulukları kendilerine ait olmak üzere SGK'ya bildirimde bulunulmaz.” 6521/2.4- “Dikili satışa konu bölme ve bölmecikteki verim yüzdesinin tespiti için gerekli iş ve işlemlere ait işçilik giderleri 730/1000 ölçme ve diğer giderler hesabından karşılanacaktır.” 6521/2.4- “Dikili satışa konu bölme ve bölmecikteki verim yüzdesinin tespiti için gerekli iş ve işlemlere ait işçilik giderleri 730/1000 ölçme ve diğer giderler hesabından karşılanacaktır.” 6877/1.2.4. “Dikili ağaç satışına konu bölme ve bölmecikteki verim yüzdesinin tespiti için gerekli iş ve işlemlere ait işçilikler Genel Yönetim Giderleri-İşçi Ücret Giderleri Hesabından (770.04 nolu hesap) idareye ait işçiler tarafından yapılır. İdareye ait işçilerin yeterli olmaması durumunda, OGM tarafından gönderilen talimata uygun olarak verim yüzdelerinin tespiti konusunda hizmet alımı yapılabilir.” Değişiklik 8. 3.3. İlgi gruplarının dikili satış tamim değişikliği hakkındaki görüşleri 6521/ (Tamimde yok) Ü 6521/ (Tamimde yok) Ü ( y ) 6877/4.8.2. “Orman Ürünleri Satış Usul ve Esasları Hakkında Yönetmeliğin 9 uncu maddesinin (1) inci fıkrasının (ö) bendine göre “Yıllık odun hammadde işleme kapasitesi yirmibeşbin metreküp ve üzeri olan her türlü fabrika ve tesislere, yıllık kapasite kullanım miktarlarına göre Genel Müdürlükçe belirlenen oranlarda odun ve dikili ağaçlar” tahsisli satış usulüyle satılabilir. Yıllık odun hammadde işleme kapasitesi 25 bin metreküp ve üzeri olan her türlü fabrika ve tesislere yapılacak tahsisli satışlarda, dikili ağaç satışı yaptıran gerçek ve tüzel kişiler üretim işçiliğini kendileri yapabileceği gibi dışarıdan hizmet alımıyla da yaptırabilirler. Dikili ağaçlardan elde edilen ürünlerin tahsis yapılan fabrika veya tesislerde değerlendirilemeyecek olanları hariç, tahsise konu fabrika ve tesislerin dışında bir yere nakline müsaade edilmeyeceği gibi bu ürünlerin işlenmeden devrine veya satışına da izin verilmez. Tahsisli Dikili Ağaç Satış Esasları Bu madde kapsamındaki fabrika ve tesislere yapılacak tahsisli satışlarda, gerçek dışı beyanda bulunulması, sahte evrak ibraz edilmesi, dikili ağaç satışı yaptıran gerçek ve tüzel kişilerin dikili ağaç satışından elde ettiği ürünlerden kendi fabrika ve tesislerinde işlenebilecek olanları tahsise konu fabrika ve tesislerde işlemeksizin devretmesi veya satması durumunda kalan tahsisleri iptal edilerek, 5 yıl süre ile tahsis verilmez. Ayrıca bu fabrika ve tesislerle İdaremiz arasında protokol imzalanacak olup, bu y p 6877/1.6. “Müşteri elde ettiği ürünleri nakletmek istediğinde; işletme şefliğince düzenlenen ölçü ve tespit tutanakları esas alınarak ürünlerin nakliye tezkeresi ile nakline izin verilir. Dikili ağaç satış işleminin her aşaması idarece kontrol edilerek, tüm ölçü tespit tutanaklarının birer nüshası son ölçü tespit tutanağı ile birlikte İşletme Müdürlüğüne gönderilir. Yapılan ölçü tespitleri sırasında, dikili ağaç satışından elde edilen toplam ürün miktarı; verim yüzdesinin %10'u üzerinde gerçekleşmesi halinde durum İşletme Müdürlüğünce Bölge Müdürlüğüne bildirilir. Bölge Müdürlüğünce kurulacak komisyon marifetiyle müşterinin da katılabileceği şekilde en geç bir hafta içerisinde durum incelenerek herhangi bir usulsüzlük olup olmadığını tespitle İşletme Müdürlüğüne bildirilecektir. Usulsüzlük olması durumunda mevzuat gereğince işlem yapılacaktır. Usulsüzlük yoksa ürünün nakline izin verilecektir.” y 6521/14- “Satışın yapılmasını müteakip, İşletme Müdürlüklerince satışın konusu, yeri ve mahiyeti hakkında ilgili Sosyal Sigortalar Kurumu’na bilgi verilecektir. Ancak Turkish Journal of Forestry 2023, 24(4): 378-389 384 protokol ve şartnamelerdeki hükümlere göre işlemler gerçekleştirilecektir.” belirtilen gün sayıları dikkate alınarak idareyle belirledikleri iş takvimine göre iş mahallinde bulundurmak zorundadır.” 3.3. İlgi gruplarının dikili satış tamim değişikliği hakkındaki görüşleri Değişiklik 8. 6521/ (Tamimde yok) Tomruk piyasası aktörlerinin dikili satış yönetmeliklerinde yapılan değişiklikler hakkındaki bilgi düzeyleri ve görüşleri arasında fark olup olmadığının belirlenmesine yönelik oluşturulan araştırma hipotezinin test edilmesine yönelik sorulan dokuz soruya alınan cevaplar Kruskal-Wallis ve Man-Whitney U testleri ile analiz edilerek grupların görüşleri arasında istatistiksel olarak anlamlı farklılıklar olup olmadığı belirlenmeye çalışılmıştır. İlgi gruplarının görüşleri ve analiz sonuçlarında gruplar arasında istatistiksel olarak anlamlı farklılık bulunanlar Çizelge 4- Çizelge 13’de gösterilmiştir. 6877/4.8.2. “Orman köylerini kalkındırma kooperatifleri ile köylerdeki gerçek kişilere yapılacak tahsisli satışlarda; gerçek dışı beyanda bulunulması, sahte evrak ibraz edilmesi, dikili ağaç satışı alanların üretim işini yapmaksızın aldıkları dikili ağaçları başkalarına devretmesi veya satması durumunda tahsisleri iptal edilerek teminatları irad kaydedilir. Bu durumdaki orman köylerini kalkındırma kooperatifleri ile köylerdeki gerçek kişilere 5 yıl tahsisen dikili ağaç satışı yapılmaz. Tahsis talebinde bulunduğu halde aynı yıl içerisinde iki defa satışını yaptırmayan talep sahiplerine bir yıl tahsisli dikili ağaç satışı yapılmaz.” Değişiklik 9. “6875 sayılı tamimle verim yüzdesi tespiti için hacim büyüklüğünün DKGH’nin %2 ye çıkarılması sizce verim yüzdesi hesabının daha sağlıklı yapılmasını sağlar mı?” (S1) sorusuna ORKOP katılımcılarının %87,5’i ve OKÜİ’lerin %93,3’ü alanın daha iyi temsil edileceği ve daha doğru sonuç alınacağı düşüncesi ile evet derken, ORKOP’un %14,3’ü ve OKÜİ’nin %6,7’si ise verim yüzdesinin hali hazırda yanlış hesaplandığını ve değişme olmayacağını belirterek hayır demiştir. İki grubun görüşleri arasında anlamlı fark olup olmadığına dair yapılan Mann-Whitney U testi sonuçları Çizelge 4’te gösterilmiştir. 6521/ (Tamimde yok) 6521/ (Tamimde yok) 6877/5.1. “Dikili ağaç satışı yaptırmak isteyen gerçek ve tüzel kişiler, Bölge Müdürlüğü dâhilinde 60 günlük üretim periyodunda 500 m3 ve üzeri satışlar için ihalenin kesinleşmesini müteakip satış yaptırmadan önce, 5531 sayılı Orman Mühendisliği, Orman Endüstri Mühendisliği ve Ağaç İşleri Endüstri Mühendisliği Hakkında Kanun uyarınca orman ürünleri üretimi alanında yetkili orman mühendisi veya orman yüksek mühendisi çalıştırmak zorunda olup, müşteri oda belgesi ile birlikte; 5.1.1. Bölge Müdürlüğü dâhilinde 60 günlük üretim periyodunda, işletme müdürlüklerinden 12.000 m³ ve katları dikili ağaç satın alanlar; her 12.000 m³ için en az 6 ay süreli hizmet akdiyle, orman mühendisi veya orman yüksek mühendisi çalıştırdığına dair noterden düzenlenmiş sözleşmeyi, beşinci bölüm 5.1.2. Bölge Müdürlüğü dâhilinde 60 günlük üretim periyodunda, İşletme Müdürlüklerinden 12.000 m³’e kadar dikili ağaç satın alanlar; en az 6 ay süreli hizmet akdiyle orman mühendisi veya orman yüksek mühendisi çalıştırdığına dair noterden düzenlenmiş sözleşmeyi veya dikili ağaç satış süresini kapsayacak şekilde düzenlenmiş olmak şartıyla, serbest ormancılık bürosu ya da ormancılık şirketiyle yapılmış danışmanlık hizmet alımı sözleşmesini ibraz etmek zorundadır. Bir orman mühendisi veya orman yüksek mühendisi 2 aylık üretim periyodunda 6.000 m³’e kadar dikili ağaç satışında danışmanlık hizmeti verebilir. Müşteri, orman mühendisi ya da orman yüksek mühendisini yukarıda yapılan açıklama çerçevesinde Ek-7'deki örnekte Çizelge 4’te görüldüğü üzere ORKOP ve OKÜİ’nin S1 hakkındaki görüşleri açısından istatistiksel olarak anlamlı bir farklılık olmadığı tespit edilmiştir. “2013 öncesindeki ve sonrasındaki 6521 ve 6877 sayılı tamim dönemlerinde dikili satışa verilen alanlarda transport sorunu yaşadınız mı ve yaşıyor musunuz? (S2) sorusuna ORKOP’un %57,1’i, OK’nin %7,3’ü ve OKÜİ’lerin % 10,4’ü 6521 sayılı tamimde transportta sorunlar yaşadıklarını, ORKOP’un %15,7’si, OK’nin %1’i ve OKÜİ’ninde %4,4’ü de herhangi bir transport sorunu yaşamadıklarını belirtmişlerdir. 6877 sayılı tamimle transport sorunlarının halledildiğini söyleyenlerin oranı ise ORKOP’ta %58,6, OK’de 56,2 ve OKÜİ’de %14,12dir. Sorunların tamim değişikliğine rağmen devam ettiğini söyleyenlerin oranı ise ORKOP’ta %14,2, OK’de %2,1 ve OKÜİ’de %0,7 olarak belirtilmiştir. Gruplardaki katılımcıların geri kalanları ise bu konu hakkında fikir beyan etmemişlerdir. Üç grubun S2 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 5’te gösterilmiştir. Çizelge 4. 6521/ (Tamimde yok) İlgi gruplarının S1 hakkındaki görüşlerine dair farklılık analizi Mann-Whitney U Testi Gruplar N Sıra ortalaması Mann-Whitney U Wilcoxon W Z Anlamlılık düzeyi (p) S1 ORKOP 57 106,09 4042,0 15068,0 -0,921 0,357 OKÜİ 148 101,81 S1-N ORKOP 57 106,09 4042,0 15068,0 -0,921 0,357 OKÜİ 148 101,81 Anlamlılık düzeyi: p<0,05 ise anlamlı Turkish Journal of Forestry 2023, 24(4): 378-389 385 Çizelge 5. İlgi gruplarının S2 hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Gruplar N Sıra ortalaması Ki-Kare sd Anlamlılık düzeyi (p) S2-6521 sayılı tamim ORKOP 51 43,88 1,582 2 0,453 OK 16 39,94 OKUİ 20 47,55 S2-6877 sayılı tamim KOOP 51 42,07 2,108 2 0,349 OK 16 44,72 OKÜİ 20 48,35 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 5. İlgi gruplarının S2 hakkındaki görüşlerine dair farklılık analizi Çizelge 5’te görüldüğü üzere ORKOP, OK ve OKÜİ’nin S2 hakkındaki görüşleri açısından istatistiksel olarak anlamlı bir farklılık olmadığı tespit edilmiştir. “Dikili satış ödemelerinin vadeli olarak yapılması dikili satış alımlarının artmasında rol oynamakta mıdır?” (S5) sorusuna ORKOP’un %98,6’sı, OK’un %73’ü, OİMÇ’nin %73,3’ü ve OKÜİ’nin %94,8’i dikili satışı artıracağı, ORKOP’un %1,4’ü, OK’un %25,4’ü, OİMÇ’nin %10’u ve OKÜİ’nin %5,2’si dikili satışları artırmayacağı şeklinde görüş bildirmiştir. ğ p ş “2013 yılında yapılan değişiklikle birlikte 6877 sayılı tamimde, 60 günlük üretim periyodunda, orman işletmelerinden 6.000 m³ ve katları dikili ağaç satın alanlar için; her 6.000 m³ için en az 6 ay süreli hizmet akdiyle, orman mühendisi veya orman yüksek mühendisi çalıştırma zorunluluğu getirilmiş olmasını nasıl değerlendirirsiniz ve nedenleri nelerdir?” (S3) sorusuna ORKOP’un %88,6’sı, OK’nin %93,8’i, OİMÇ’nin %82,8’i ve OKÜİ’nin %62,2’si gerekli ve faydalı bulduğunu belirtirken, ORKOP’un %11,4’ü, OK’nin %6,2’si, OİMÇ’nin %17,2’si ve OKÜİ’nin %37,8’i ise gereksiz bulduğunu belirtmiştir. Orman mühendisliği çalıştırma zorunluluğunu gerekli bulanlar içerisinden ORKOP’ta %11,4, OK’de %6,2, OİMÇ’de %82,8 ve OKÜİ’de %37,8 oranında bunun nedenini teknik takip ve iş imkanı oluşturması olarak belirtmişlerdir. Gereksiz bulanlar içerisinde ORKOP’un %88,6’sı, OK’de %93,8’i OİMÇ’de %17,2’si ve OKÜİ’de %62,2’si ise mühendislerin işe gitmediğini sadece çalışma belgelerini kiraladıklarını gereksiz bulma nedenleri olarak ifade etmişlerdir. g İlgi gruplarının S5 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 8’de gösterilmiştir. y ç Ç g g ş Çizelge 8’de görüldüğü üzere ORKOP, OK, OKÜİ ve OİMÇ’nin S5 hakkındaki görüşleri açısından istatistiksel olarak anlamlı farklılıkların olduğu tespit edilmiştir. “2013 yılında değişen (6877) tamim ile gelen “peyderpey satış kaldırılmıştır” maddesini destekliyor musunuz?” (S6) sorusuna ORKOP’un %14,3’ü, OK’nin %5,2’si, OİMÇ’nin %100’ü desteklediğini, ORKOP’un %85,2’si, OK’nin %94,8’i ve OKÜİ’nin tamamı desteklemediğini belirtmiştir. 6521/ (Tamimde yok) İlgi gruplarının peyderpey satışın kaldırılmasını destekleme ve desteklememe nedenleri Çizelge 9’da gösterilmektedir. Çizelge 9’da görüldüğü üzere ORKOP’un %87,1’i maddi yetersizlikler ve ihaleye girerken gereken nakit bulma zorluğundan dolayı desteklememektedir. OK’un %72,5’i kooperatiflerin dikili satış alamadığı ve işçilerin sigorta sorunundan dolayı yükleniciler ile çalışılmak zorunda kalınmasından dolayı büyük oranda desteklememektedir. OİMÇ’lerin tamamı işin kontrolünün sağlanması ve işlerin zamanında bitirilebilmesi nedenleriyle peyderpey satışın kaldırılmasını desteklediğini belirtmiştir. OKÜİ’lerin tamamı da bu uygulamanın küçük işletmeler için zararlı olduğundan dolayı eski uygulamanın devamının gerekli olduğunu belirtmektedirler. g İlgi gruplarının S3 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 6’te gösterilmiştir. Çizelge 6’te görüldüğü üzere ORKOP, OK, OKÜİ ve OİMÇ’nin S3 ve nedenleri hakkındaki görüşleri açısından istatistiksel olarak anlamlı farklılıkların olduğu tespit edilmiştir. 6877 sayılı tamimle birlikte iş güvenliği ve çevreye daha duyarlı dikili satışın yapılmasını nasıl değerlendiriyorsunuz?” (S4) sorusuna ORKOP’un %25,7’si, OK’nin %20,2’si, OİMÇ’nin %73,3’ü ve OKÜİ’nin %59,3’ü çevre ve işçilerin sağlığı açısından olumlu olduğunu, ORKOP’un %47,2’si OK’nin %41,5’i, OİMÇ’nin %26,7’si ve OKÜİ’nin %34,1’i işleri yavaşlattığını belirtmişlerdir. Buna karşın ORKOP’un %27,1’i, OK’nin %38,3’ü ve OKÜİ’nin de %6,6’sı da değişen bir şey olmadığını ifade etmişlerdir. İlgi gruplarının S6 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 10’de gösterilmiştir. y g g Çizelge 10’da görüldüğü üzere ORKOP, OK, OKÜİ ve OİMÇ’nin S6 hakkındaki görüşleri açısından istatistiksel olarak anlamlı farklılıkların olduğu tespit edilmiştir. “Köylerin çevrelerindeki ormanlarda yapılacak üretimlerde yöre dışından şahısların ihaleye katılmalarını nasıl değerlendiriyorsunuz?” (S7) sorusuna ORKOP’un tamamı, OİMÇ’lerin ise %50’si olumsuz bir durum olarak gördüklerini belirtmiştir. OK’nin %94,5’i ve OKÜİ’nin %89,6’sı ise bu durumu olumlu karşıladıklarını beyan etmişlerdir. Bu düşüncelerinin nedenleri Çizelge 11’de gösterilmiştir. İlgi gruplarının S4 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 7’de gösterilmiştir. Çizelge 7’de görüldüğü üzere ORKOP, OK, OKÜİ ve OİMÇ’nin S4 hakkındaki görüşleri açısından istatistiksel olarak anlamlı farklılıkların olduğu tespit edilmiştir. Turkish Journal of Forestry 2023, 24(4): 378-389 386 Çizelge 6. İlgi gruplarının S3 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki-Kare sd Anlam. düzeyi (p) Gruplar Mann- Whitney U Wilcoxon W Z Anlam. 6521/ (Tamimde yok) düzeyi (p) S3 ORKOP 70 236,77 108,161 3 0,000 ORKOP-OİMÇ 291,0 726,0 -6,871 0,000 OK 193 248,19 ORKOP-OKÜİ 3480,0 12660,0 -3,942 0,000 OİMÇ 29 85,55 OK-OİMÇ 678,0 1113,0 -9,769 0,000 OKÜİ 135 180,91 OK-OKÜİ 8925,0 18105,0 -6,905 0,000 OİMÇ-OKÜİ 1077,0 1512,0 -4,398 0,000 S3-N ORKOP 70 236,16 116,071 3 0,000 ORKOP-OİMÇ 291,0 726,0 -6,871 0,000 OK 193 249,22 ORKOP-OKÜİ 3480,0 12660,0 -3,942 0,000 OİMÇ 29 84,22 OK-OİMÇ 639,5 1074,5 -10,481 0,000 OKÜİ 135 180,03 OK-OKÜİ 8806,5 17986,5 -7,317 0,000 OİMÇ-OKÜİ 1077,0 1512,0 -4,398 0,000 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 6. İlgi gruplarının S3 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Ma Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 7. İlgi gruplarının S4 ve nedenleri hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki-Kare sd Anlam. düzeyi (p) Gruplar Mann- Whitney U Wilcoxon W Z Anlam. düzeyi (p) S4 ORKOP 70 232,92 75,603 3 0,000 ORKOP-OKÜİ 2787,5 11967,5 -5,007 0,000 OK 193 257,34 ORKOP-OİM 607,5 1042,5 -3,183 0,001 OİMÇ 29 149,88 OK-İM 1510,5 1945,5 -4,235 0,000 OKÜİ 135 153,14 OK-OKÜİ 6584,5 15764,5 -8,121 0,000 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 7. İlgi gruplarının S4 ve nedenleri hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Ma Çizelge 8. İlgi gruplarının S5 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki-Kare sd Anlam. düzeyi (p) Gruplar Mann- Whitney U Wilcoxon W Z Anlam. düzeyi (p) S5 ORKOP 70 232,92 43,603 3 0,000 ORKOP-OK 50285,0 7513,5 -4,456 0,000 OK 193 257,34 ORKOP-OKÜİ 4547,5 7032,5 -1.314 0,000 OİMÇ 29 149,88 ORKOP-OİM 747,0 3232,0 -4,133 0,000 OKÜİ 135 153,14 OK-OKÜİ 10172,0 19352,0 -5,069 0,000 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 8. İlgi gruplarının S5 ve nedenler hakkındaki görüşlerine dair farklılık analizi K k l W lli M Çizelge 9. İlgi gruplarının peyderpey satış usulün 6877 sayılı tamim ile kaldırılması hakkındaki düşüncelerinin nedenleri ORKOP (%) OK (%) OİMÇ (%) OKÜİ (%) Maddi yetersizlikler ve nakit bulma zorluğu 87,1 22,3 - - Alanın tamamına girilmesinden dolayı iş imkânının artması 12,9 5,2 - - Kooperatifler dikili satışları alamadığı için ve sigorta sorunlarından dolayı yüklenici ile birlikte çalışılması gerekiyor - 72,5 - - İşin zamanında bitmesi için - - 46,4 - İşin kontrolünün sağlanması ve karışıklıkların önlenebilmesi için - - 53,3 - Küçük işletmeler için zararlı olduğundan kaldırılmasını ve eski uygulamanın devam etmesi daha uygun olacaktır - - - 100 Çizelge 9. 6521/ (Tamimde yok) İlgi gruplarının peyderpey satış usulün 6877 sayılı tamim ile kaldırılması hakkındaki düşüncelerinin nedenleri ORKOP (%) OK (%) OİMÇ (%) OKÜİ (%) Maddi yetersizlikler ve nakit bulma zorluğu 87,1 22,3 - - Alanın tamamına girilmesinden dolayı iş imkânının artması 12,9 5,2 - - Kooperatifler dikili satışları alamadığı için ve sigorta sorunlarından dolayı yüklenici ile birlikte çalışılması gerekiyor - 72,5 - - İşin zamanında bitmesi için - - 46,4 - İşin kontrolünün sağlanması ve karışıklıkların önlenebilmesi için - - 53,3 - Küçük işletmeler için zararlı olduğundan kaldırılmasını ve eski uygulamanın devam etmesi daha uygun olacaktır - - - 100 Çizelge 10. İlgi gruplarının S6 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki-Kare sd Anlam. düzeyi (p) Gruplar Mann- Whitney U Wilcoxon W Z Anlam. düzeyi (p) S6 ORKOP 70 208,50 242,208 3 0,000 ORKOP-OİMÇ 145,0 580,0 -7,903 0,000 OK 193 227,94 ORKOP-OKÜİ 4085,0 6570,0 -4,071 0,000 OİMÇ 29 25,50 OK-OİMÇ 145,0 580,0 -12,483 0,000 OKÜİ 135 2337,42 OK-OKÜİ 12449,0 31170,0 -2,195 0,028 OİMÇ-OKÜİ 14,5 449,5 -12,506 0,000 ORKOP-OK 1743,5 4228,5 -10,362 0,000 S6-N ORKOP 70 60,97 336,770 3 0,000 ORKOP-OİMÇ ,000 2485,0 -8,953 0,000 OK 193 159,58 ORKOP-OKÜİ 39,5 2524,5 -13,975 0,000 OİMÇ 29 274,17 OK-OİMÇ 140,0 18861,0 -9,645 0,000 OKÜİ 135 358,22 OK-OKÜİ 171,5 18892,5 -16,483 0,000 OİMÇ-OKÜİ 29,0 464,0 -12,346 0,000 Anlamlılık düzeyi: p<0,05 ise anlamlı Maddi yetersizlikler ve nakit bulma zorluğu Maddi yetersizlikler ve nakit bulma zorluğu Maddi yetersizlikler ve nakit bulma zorluğu Çizelge 10. İlgi gruplarının S6 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki-Kare sd Anlam. düzeyi (p) Gruplar Mann- Whitney U Wilcoxon W Z Anlam. düzeyi (p) S6 ORKOP 70 208,50 242,208 3 0,000 ORKOP-OİMÇ 145,0 580,0 -7,903 0,000 OK 193 227,94 ORKOP-OKÜİ 4085,0 6570,0 -4,071 0,000 OİMÇ 29 25,50 OK-OİMÇ 145,0 580,0 -12,483 0,000 OKÜİ 135 2337,42 OK-OKÜİ 12449,0 31170,0 -2,195 0,028 OİMÇ-OKÜİ 14,5 449,5 -12,506 0,000 ORKOP-OK 1743,5 4228,5 -10,362 0,000 S6-N ORKOP 70 60,97 336,770 3 0,000 ORKOP-OİMÇ ,000 2485,0 -8,953 0,000 OK 193 159,58 ORKOP-OKÜİ 39,5 2524,5 -13,975 0,000 OİMÇ 29 274,17 OK-OİMÇ 140,0 18861,0 -9,645 0,000 OKÜİ 135 358,22 OK-OKÜİ 171,5 18892,5 -16,483 0,000 OİMÇ-OKÜİ 29,0 464,0 -12,346 0,000 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 10. İlgi gruplarının S6 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Ma Turkish Journal of Forestry 2023, 24(4): 378-389 387 şartları beğenmeyebileceğini ve ihaleye girmekten vazgeçebileceğini belirtmişlerdir. Çizelge 11’den de görüldüğü üzere ORKOP, OK ve OKÜİ büyük oranda aynı fikirdedir ve sadece köylülerin kendi köy sınırları içerisinde yapılacak işlerde çalışmasının gerekli olduğunu belirtmektedirler. İlgi gruplarının S8 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 13’te gösterilmiştir. İlgi gruplarının S7 hakkındaki görüşleri arasında anlamlı fark olup olmadığına dair yapılan Kruskal-Wallis ve Mann- Whitney U testi sonuçları Çizelge 12’de gösterilmiştir. Çizelge 13’de görüldüğü üzere ORKOP, OK, OKÜİ ve OİMÇ’nin S8 hakkındaki görüşleri açısından istatistiksel olarak anlamlı farklılıkların olduğu tespit edilmiştir. y g g Çizelge 12’de görüldüğü üzere ORKOP, OK, OKÜİ ve OİMÇ’nin S7 hakkındaki görüşleri açısından istatistiksel olarak anlamlı farklılıkların olduğu tespit edilmiştir. “Üretim pazarlama periyodunda süre yetiyor mu? (S9) sorusuna ORKOP’un %35,7’si, OK’nin %6,7’si ve OKÜİ’nin %12,6’sı evet yanıtını vermiştir. Bazen diyenlerin oranı ORKOP’ta %17,i, OK’de %3,1 ve OKÜİ’de %8,12dir. ORKOP’un %20’si ve OK’nin %1,6’sı ise hayır cevabını vermişlerdir. Geri kalan katılımcılar ise konu hakkında fikirlerinin olmadığını belirtmişlerdir. Yapılan Kruskal- Wallis analizi sonuçlarında ilgi gruplarının üretim ve pazarlama periyodunda sürenin yeterliliği hakkındaki görüşleri arasında istatistiksel olarak anlamlı bir fark tespit edilememiştir. “Aynı yıl içerisinde iki defa tahsis talebinde bulunduğu halde satış anlaşmasını imzalamayanların yeni bir ihaleye katılımının iptal edilme yetkisini nasıl değerlendiriyorsunuz?” (S8) sorusuna ORKOP’un %82,92u OK’nin %93,8’i, OİMÇ’nin tamamı ve OKÜİ’nin %98,5’i olumlu bir değişim olduğunu beyan etmişlerdir. Bu kararların nedeni olarakta değişikliğin suistimallerin engellenmesine fayda sağlayacağını düşünmeleri olarak belirtmişlerdir. Olumsuz olarak değerlendirenler ise alıcının gerektiğinde Çizelge 11. 4. Tartışma ve sonuç Ormancılık mesleği iş kazalarının yoğun olarak yaşandığı bir iş koludur. Ancak bunun dışında aşırı fiziki strese neden olan aşırı hava koşulları, gürültü, vibrasyon, gibi nedenlerle duyma bozukluğu vb. çeşitli meslek hastalıklarına da neden olmaktadır. Yeni Zelanda’da yapılan bir çalışmada da on beş yıl tomruk üretiminde çalışan orman işçilerinin yarısında duyma sorunları göründüğü belirtilmektedir. Bu kapsamda yeni tamimde iş güvenliği ile ilgili değişiklikler olumlu gözükmektedir. 15 yıl odun üretim işinde çalışan işçilerin yarısında duyma bozukluğu meydana geldiği belirlenmiştir (Poschen, 1993). Bu çalışma tamime eklenen maddeyi destekler niteliktedir. Buna karşın özellikle orman köylülerinin büyük çoğunluğunun yapılan çalışmaların bir işe yaramayacağını ve işleri yavaşlatacağını düşünmesi üretim işinde çalışan ve tehlikelerle yüz yüze olan kişilerin bu durumu anlamamış ve içselleştirememiş olduğunu göstermektedir. Bu durumun özellikle eğitimsizlikten kaynaklandığı düşünülmektedir. Buna karşın orman işçileri iş güvenliği donanımlarının pahalı olmasından dolayı da iş güvenliği çalışmalarına sıcak bakmadıklarını görüşmeler esansında belirtmişlerdir. Oysaki yapılan çalışmalar başlık, göz koruyucu, güvenlik eldiveni, pantolon ve bot gibi kişisel koruyucu donanım kullanımının yaralanmaları yıllık bazda %20 azalttığını göstermektedir. Finlandiya’da yapılan bu çalışma da özellikle botların bu yaralanmaların azaltılmasında çok önemli olduğunu belirtmektedir (Klen ve Vayrynen, 1984). Belirtilmesi gereken bir husus da ergonomik olmayan iş güvenliği ekipmanlarının kaza riskini artırmasıdır. Burada önemli olan bir diğer önemli hususta orman idarecilerinin yaklaşık 527’sinin bu uygulamaların kazaların önüne geçmede etkili olmadığını düşünmeleridir. Bu işleri koordine edecek ve uygulamasını yönetecek kişilerin bu işe inanmıyor oluşu iş güvenliği çalışmalarının henüz idareciler tarafından bile tam olarak öneminin anlaşılamamış olduğunu göstermektedir. Araştırmada dikili satışla alakalı olan dört farklı ilgi grubuna (orman kooperatifleri, orman köylüleri, orman işletmesi çalışanları ve odun kökenli ürün işleyen işletmeler) anket uygulaması yapılmıştır. Anket, gözlem ve mülakatlar sonucunda elde edilen bulgular neticesinde elde edilen sonuçlar aşağıda verilmiştir. ç ş ğ ş Dikili satış uygulamasının en önemli aşaması olan verim yüzdesi tespitine göre ihaleye çıkılmakta ve bölmeyi teslim edecek ölçümler yeterince dikkatli ve hassas bir şekilde yapılmadığında alıcı ya da orman işletmeleri maddi zarara uğrayabilmektedir. Verim yüzdesinin hesabı ile ilgili değişikliği odun kökenli ürün işleyen işletmelerin ve kooperatiflerin büyük çoğunluğu olumlu karşılamıştır. Doğu Karadeniz’de yapılan bir çalışmada çalışma sonuçlarını destekler niteliktedir. Odun kökenli ürün işleyen işletmelerin yaklaşık %75’i verim yüzdesi hesabından memnun olmadıklarını belirtmişlerdir. Bunun nedeni olarak ta %44,8’i seçilen ağaçların verim yüzdesini yansıtmadığını, %33,3’ü ise orman işletmesinin verim yüzdesini hesaplamada yanlışlıklar yaptığı şeklinde görüş bildirmişlerdir. 4. Tartışma ve sonuç Kooperatiflerin ise neredeyse yarıya yakını (%42,8) verim yüzdesinden memnun olmadıklarını belirtmiş ve bu kişilerde verim yüzdesi hesabındaki ağaç seçiminin yanlış yapıldığını ve arazi şartlarından kaynaklanan değer kayıplarının, bölmecikler bazında hesaplanan verim yüzdesi değerine eklenmediğini belirtmişlerdir (İslamoğlu, 2010). Ayrıca ilgi gruplarının %10-15’inin verim yüzdesi hesabının yanlış yapıldığını ve değişikliklerin bunu değiştirmeyeceğini düşünmeleri orman idaresine olan güvensizliği göstermesi bakımından önemlidir. Üretim işlerinin daha çok kooperatifler yolu ile yapılmasından dolayı en fazla sorununda kooperatifler tarafından yaşadığını görmekteyiz. Odun kökenli ürün işleyen işletmeler müşteri pozisyonunda olduklarından onların da yüksek oranda sorun yaşadığı görülmektedir. Boylama ve bölmeden çıkarma dikili satış uygulamasının en önemli aşamalarından biri olduğundan bu kısımda çıkan olumsuzluklar ve sorunlar paydaşların dikili satış üzerindeki görüşlerini olumsuz etkilemektedir. Mevcut transport sorunları bir nebzede olsa çözülmüş görünse de halen devam eden sorunlar bulunmaktadır. Dikili satışların vadeli yapılmasının satışları artırıcı etkide bulunduğu ilgi grupları tarafından dile getirilmiş olsa da ipotek olarak gösterilebilen gayrimenkullerin yerine değerli kâğıtların geçmesi gelir durumu çok düşük olan orman köylüsü için huzursuzluk yaratan bir durum oluşturmaktadır. Bunun sonucunda orman köylüleri ve kooperatifler sadece yüklenicilerin taşeronluğunu yapan, onlar için aracı kurum gibi çalışan bir yapı haline bürünmüşlerdir. Bunun sonucunda da elde edilecek gelir yörede kalmayacağından kırsal kalkınmaya olan desteği de azalmaktadır. Dikili satışlardaki usulsüzlük ve diğer aksamaların giderilmesi noktasında orman mühendisi çalıştırma zorunluluğunun getirilmesi her ne kadar olumlu bir adım gibi görünse de uygulamadaki aksaklıklar kooperatiflerin ve orman köylülerinin büyük çoğunluğunun bu durumu istememesine yol açmaktadır. Ancak tamimde belirtildiği üzere orman mühendislerinin danışmanlık açısından ve alıcı ile orman idaresi arasındaki teknik takibin sağlanması açısından önemli oldukları bir gerçektir. Buna karşın bu değişikliğin yapılmasının nedenlerinden biri usulsüzlüklerin önlenmesidir. Bu kapsamda çalışanların başlarında bir denetim görevlisinin bulunmasını istememeleri ve bunu olumsuz olarak değerlendirmeleri de anlaşılabilir bir durumdur. Benzer şekilde Yener (2014) çalışmasında, dikili üretiminde çalışan orman mühendislerinin her ne kadar orman işletme şeflerinin sorumluluklarını azaltmamış olsa da çıkabilecek sorunlarda da muhataplarının yükleniciler yerine bölmede çalışan orman mühendislerinin olmasının teknik elemanların sorunlar karşısında işlerini kolaylaştırdığını belirtmektedir. Peyderpey satışın kaldırılması işletmeler haricinde pek destek bulmamıştır. Bu durumla birlikte üretimini yapıp sattığı malların parası ile farklı işlere de girebilirken bu durumda alanın tamamını almak gerektiğinden orman köylüleri ve kooperatifler düşük gelirleri nedeni ile oldukça zorlanmaktadırlar. Sonucunda da köylü ve kooperatifleri müteahhitlere muhtaç eden bir durum oluşmaktadır. Yeni tamimde belirtilen altmış günlük üretim süresinin yeterli olup olmadığı tam olarak belirli değildir. Ancak, dikili satış sürecinde kesme, boylama, sürütme, transport ve pazarlama faaliyetleri ile birlikte bu sürenin kısa olduğu düşünülmektedir. Maddi yetersizlikler ve nakit bulma zorluğu İlgi gruplarının S7’ye dair görüşlerinin nedenleri ORKOP % OK % OİMÇ % OKÜİ % Kendi sınırları içerisindeki köylülerle çalışmalı 100 93.2 3.3 89.6 Çalışabilir (işgücü yetersizse) 0 6.2 13,3 10.4 Rekabet getirecektir - - 3.3 - İşin yavaşlaması davaya konu olması - - 26.7 - Daha düşük fiyattan çalışılacağı için devlete kazanç sağlabır - - 30 - İhaleyi alan şahıs kendi işçilerini çalıştırabilir - - 23.5 - Çizelge 11. İlgi gruplarının S7’ye dair görüşlerinin nedenleri Çizelge 12. İlgi gruplarının S7 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki-Kare sd Anlam. düzeyi (p) Gruplar Mann-Whitney U Wilcoxon W Z Anlam. düzeyi (p) S7 ORKOP 70 234 71,556 3 0,000 ORKOP-OİMÇ 490,0 925,0 -6,499 0,000 OK 193 221,83 ORKOP-OKÜİ 4235,0 4235.0 -2,784 0,005 OİMÇ 29 123,57 OK-OİMÇ 1510,5 1945,5 -7,171 0,000 OKÜİ 135 211,56 OİMÇ-OKÜİ 1148,0 1583 -5,280 0,000 S7- N ORKOP 70 212,50 242,812 3 0,000 ORKOP-OİMÇ ,000 2485,0 -9,718 0,000 OK 193 200,13 ORKOP-OKÜİ 4235 4235 -2,784 0,005 OİMÇ 29 413,00 OK-OİMÇ ,000 18721,50 -12,826 0,000 OKÜİ 135 191,86 OİMÇ-OKÜİ ,000 9180 -10,986 0,000 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 12. İlgi gruplarının S7 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann- Mann-Whitney U testi Çizelge 13. İlgi gruplarının S8 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann-Whitney U testi Gruplar N Sıra ort. Ki- Kare sd Anlam. düzeyi (p) Gruplar Mann-Whitney U Wilcoxon W Z Anlam. düzeyi (p) S8 ORKOP 70 237,01 21,047 3 0,000 ORKOP-OİMÇ 841 1276 -2,366 0,018 OK 193 214,92 ORKOP-OKÜİ 3985 13165 -4,205 0,000 OİMÇ 29 200,50 OKÜİ 135 203,66 S8- N ORKOP 70 237,60 21,804 3 0,000 ORKOP-OKÜİ 6017 24738 -2,714 0,007 OK 193 214,27 ORKOP-OKÜİ 3985 13165 -4,205 0,000 OİMÇ 29 201,00 OKÜİ 135 204,16 Anlamlılık düzeyi: p<0,05 ise anlamlı Çizelge 13. İlgi gruplarının S8 ve nedenler hakkındaki görüşlerine dair farklılık analizi Kruskal-Wallis Mann Turkish Journal of Forestry 2023, 24(4): 378-389 388 Kaynaklar Meydan, K., Öztürk, S., 2023. Küre Dağları Milli Parkı’nın Hızlı Kırsal Değerlendirme Yöntemi ile Ekoturizm Potansiyelinin Belirlenmesi. İksad Yayınevi, Ankara DOI: https://dx.doi.org/10.5281/zenodo.10057177 Ayan, S., Öztürk, S., Yiğit, 2007. Karadeniz Bölgesi Milli Parklarının PAN Parks Sertifikasyon Sistemine Uygunlukları. 38. ICANAS Uluslararası Asya ve Kuzey Afrika Çalışmaları Kongresi, Ankara, Türkiye, 105-129. g Miraboğlu, M., 1983. Ormancılık İşletme İktisadı. İ.Ü. Orman Fakültesi, İstanbul. Cevahir, E., 2020. SPSS ile nicel veri analizi rehberi. İzmir, Kibele. Orman Genel müdürlüğü (OGM), 2013. Dikili ağaç satışı, T.C. Orman ve Su İşleri Bakanlığı Orman Genel Müdürlüğü İşletme ve Pazarlama Dairesi Başkanlığı, http://www.ogm.gov.tr/ ekutuphane/Tamimler/Dikili%20A%C4%9Fa%C3%A7%20Sat %C4%B1%C5%9F%C4%B1%20(30.04.2013%20de%C4%9F %C5%9F.i%C5%9Fl.).pdf, Erişim: 11.05.2015. Ö İ İ Counsell, S., 2006. Forest governance in the Democratic Republic of Congo. An NGO perspective. Recommendations for a Voluntary Partnership Agreement with the EU. Moreton in Marsh, FERN. Daşdemir, İ., 2003. Asli Orman Ürünlerinde Fiyat Analizi (Zonguldak Orman Bölgen Müdürlüğü Örneği). ZKÜ Bartın Orman Fakültesi Yayınları, Bartın. Özdamar, K., 2002. Paket Programlar İle İstatistiksel Veri Analizi-2 (Çok Değişkenli Analizler) SPSS-MİNİTAB, 4. Baskı. Kaan Kitabevi, Eskişehir. Ö İ y Daşdemir, İ., 2011. Ormancılık İşletme Ekonomisi. BÜ Orman Fakültesi Yayınları, Bartın. Özdönmez, M., İstanbullu, T., Akesen, A., 1989. Ormancılık Politikası. İÜ Orman Fakültesi Yayın No: 401, İstanbul. Demirel, E., 2006. Açık arttırmalı tomruk satışlarında fiyat oluşum sürecinin incelenmesi (Kahramanmaraş Orman Bölge Müdürlüğü örneği). Yüksek Lisans, ZKÜ Fen Bilimleri Enstitüsü, Zonguldak. Poschen, P., 1993. Forestry, A Safe and Healthy Profession, Unasylva 44 (l), 172-185 Puwein, W., 2007. Wood market in Poland towards stabilisation Price Setting Systems in Austria. International and interindustry conference, 4 December, Poznan. pp. 25-36 Department of Environmental Conservtion (DEC), 2023. Timber sales on State Forests. https://www.dec.ny.gov/nature/forests- trees/state-forests/timber-sales, Erişim: 09.09.2023 Schenker, S., 1995. Private forest companies in Austrıa: Conflicts and compensation. https://www.fao.org/3/w3722E/w3722e12. htm#TopOfPage, Erişim: 12.10.2023. Diker, M., 1947. Türkiye’de Ormancılık, Dün- Bugün- Yarın. Tarım Bakanlığı, Orman Genel Müdürlüğü Yayınları, Sayı: 61, Akın Matbaası, Ankara. Şen, G., Akbulut, S., Karaköse, M., 2022. Ethnopharmacological study of medicinal plants in Kastamonu province (Türkiye). Open Chemistry, 20(1): 873-911. Elyakime, B., Cabanettes, A., 2009. How to improve the marketing of timber in France?.Forest Policy and Economics, 11(3): 169- 173. Teagasc, 2023. Watch: Timber categories and sales methods - conifer forest management, https://www.teagasc.ie/news--events/ daily/forestry/watch-timber-categories-and-sales-methods--- conifer-forest-management-.php, Erişim. 12.09.2023. Gaizutis, A., 2005. Gaining a Position For Lithuanian Small-Scale Forestry Through Creation Of Marketing, Network For Wood Trade And Services. Unasylva, 228 (58) 48-52. https://www.fao.org/3/a1346e/a1346e13.pdf Erişim: 06.08.2023. Toppinen, A, Viitanen, J., Leskinen, P., Toivonen, R. 2005. 4. Tartışma ve sonuç Özellikle Doğu Karadeniz Bölgesi gibi coğrafi şartlarının ve hava koşullarının zor olabildiği alanlarda her gün arazide çalışma yapılamaması gibi durumlar üretim süresinin az olduğunun düşünülmesini haklı çıkarmaktadır. Turkish Journal of Forestry 2023, 24(4): 378-389 389 Açıklama Kurnaz, S., Güneş Şen, S., 2023. İbreli ve yapraklı meşcere türlerinde toprak özellikleri. 3rd International Conference on Engineering and Applied Natural Sciences, 14-17 Ocak, Konya, Türkiye, pp.507-513. Bu çalışma, “Ormancılıkta Dikili Satış Yöntemindeki Değişim ve Uygulamadaki Sorunların Tüm Paydaşlar Açısından İncelenmesi (Kastamonu Orman Bölge Müdürlüğü Örneği)” başlıklı yüksek lisans tezinden üretilmiş olup, çalışmanın geniş özeti IV. Ormancılıkta Sosyo-Ekonomik Sorunlar Kongresi’nde sunulmuştur. Melkas, R., Oy, M. 2022. Wood measuring methods used in Finland 2021. https://www.metsateho.fi/wp-content/uploads/ Tuloskalvosarja-2022-7-EN-Wood-measuring-methods-used- in-Finland-2021.pdf, Erişim: 06.08.2023. Kaynaklar Dynamics of roundwood prices in Estonia, Finland and Lithuania. Baltic Forestry, 11(1), 88-96. Gümüş, C., 2004. Ormancılık Politikası, Cilt 1. KTÜ Orman Fakültesi Yayın No:34.444S, Trabzon. Türker, M.F., Yazıcı, K., 1998. Üçüncü Sınıf Normal Boy Ladin Tomruk Açık Artırmalı Satışlarında Fiyat Oluşumu Sürecinin Araştırılması (Trabzon Orman Bölge Müdürlüğü Örneği). KTÜ Orman Fakültesi Seminer Kitabı, Trabzon. Güngör, E., Şen, G., 2018. Selecting suitable forest areas for honey production using the AHP: A case study in Turkey. CERNE, 24(1): 67-79. TÜİK, 2023. Nüfus ve Demografi 2020, https://data. tuik.gov.tr/Kategori/GetKategori?p=Nufus-ve-Demografi-109 Erişim: 15.09.2023 ( ) İslamoğlu, N., 2010. Devlet ormanlarında yapılan dikili ağaç satışının paydaşlar açısından irdelenmesi (Trabzon Orman Bölge Müdürlüğü Örneği). Yüksek Lisans Tezi, KTÜ Fen Bilimleri Enstitüsü, Trabzon. Forestry England (FE), 2023. Timber Sales. https://www. forestryengland.uk/timber/timbersales Erişim: 13.10.2023 Kaplan, E., 2005. Orman Ürünlerinin Değerlendirilmesinde Dikili Ağaç Satışı. 1.Çevre ve Ormancılık Şurası, Cilt 3, Antalya. TOB, 2023. Kastamonu Coğrafi Yapısı, https://kastamonu. tarimorman.gov.tr/Menu/46/Cografi-Yapisi, Erişim: 01.09.2023 Ü Kastamonu Orman Bölge Müdürlüğü (KOBM), 2023. Orman varlığı. https://www.ogm.gov.tr/kastamonuobm/ormanlarimiz/ orman-varligi, Erişim: 22.08.2023. Ünal, S., Karakaya, A., 2002. Orman ürünlerinin üretiminde dikili ağaç satışı ve uygulamada kooperatiflerin yeri. I.Ulusal Ormancılık Kooperatifleri Sempozyumu Bildiriler Kitabı, 22-23 Mart, Kastamonu, Türkiye, pp. 51-58. Klen, T., Vayrynen, S., 1984. The role of personal protection in the prevention of accidental injuries in logging work. Journal of Occupational Accidents, 6(4): 263-275. Yener, A., 2014. Kahramanmaraş Orman İşletme Müdürlüğü’nde üretim işlerinde vahidi fiyat ve dikili satış yöntemlerinin karşılaştırılması üzerine araştırmalar. Yüksek Lisans Tezi, Kahramanmaraş Sütçü İmam Üniversitesi, Fen Bilimleri Enstitüsü, Kahramanmaraş. Kravkaz-Kuscu, I.S., Sariyildiz, T., Cetin, M., Yigit, N., Sevik, H., Savaci, G., 2018. Evaluation of the soil properties and primary forest tree species in Taskopru (Kastamonu) district. Fresenius Environmental Bulletin, 27(3): 1613-1617. Yılmaz, E., 2006. R’WOT Technique: an Example Application with Participatory Approach in Beekeeping Industry. Doğu Akdeniz Ormancılık Araştırma Enstitüsü Yayını, Doğu Akdeniz Ormancılık Araştırma Müdürlüğü Matbaası, Tarsus.
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DEVELOPMENT OF METHODS FOR ASSESSMENT AND SELECTION OF UNMANNED AERIAL VEHICLE FOR MINE RECONNAISSANCE
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Vadym Neroba1 y 1Department of General Military Disciplines, National Academy of the State Border Guard Service of Ukraine named after Bohdan Khmelnytskyi, Khmelnytskyi, Ukraine vadym.rostislavovich@gmail.com ORCID: http://orcid.org/0000-0002-7232-3285 AB ST R AC T Object of research: comparative assessment and selection of an unmanned aerial vehicle for mine reconnaissance sample. Investigated problem: substantiation of the methodological apparatus for comparative as- sessment and selection of an unmanned aerial vehicle for mine reconnaissance sample, tak- ing into consideration the presence of both quantitative and qualitative indicators. Main scientific results: the methods of comparative assessment and selection of an unmanned aerial vehicle for mine reconnaissance sample is developed. The technique is based on an ex- pert method, which allows a drone sample to be evaluated and selected objectively, taking into consideration the presence of both quantitative and qualitative indicators. At the same time, group interaction and discussion of experts are realized. When the judgments do not coincide, an artificial consensus is not imposed. The number of experts is not limited. The experts are not linked in any way. The need to ensure transitive consistency (10–12 %) makes it possible to record attempts by an expert (experts) to artificially overestimate the indicators of one of the drone samples (or the one being evaluated), therefore, the indicators of another sample will au- tomatically deteriorate. The principle of impartiality and fairness is maintained. A well-trained objective coordinator is not required, and the reality is that the absence of the disrupting the problem solution possibility is due to a change in the psychological situation among the experts. Area of practical use of research results: humanitarian demining in the interests of ensur- ing the detection of mines for various purposes by sappers from a safe distance. At the same time, an increase within the probability of mines detecting is ensured due to special equip- ment installed onboard the drone. Innovative technological product: a technique has been developed that allows not only assessing the drone samples for mine reconnaissance objectively, but making an objective choice of a sample for specific requirements also. Scope of application of the innovative technological product: clearance of the terrain re- maining after the end of hostilities. With the help of unmanned aerial vehicles, a significant acceleration of the demining process is possible, especially in those territories where mines are installed and being for a sufficiently long time. (2020). Development of methods for assessment and selection of unmanned aerial vehicle for mine reconnaissance. ScienceRise, 5, 44–50. doi: http://doi.org/10.21303/2313-8416.2020.001496 Object of research: comparative assessment and selection of an unmanned aerial vehicle for mine reconnaissance sample. Investigated problem: substantiation of the methodological apparatus for comparative as- sessment and selection of an unmanned aerial vehicle for mine reconnaissance sample, tak- ing into consideration the presence of both quantitative and qualitative indicators. Main scientific results: the methods of comparative assessment and selection of an unmanned aerial vehicle for mine reconnaissance sample is developed. The technique is based on an ex- pert method, which allows a drone sample to be evaluated and selected objectively, taking into consideration the presence of both quantitative and qualitative indicators. At the same time, group interaction and discussion of experts are realized. When the judgments do not coincide, an artificial consensus is not imposed. The number of experts is not limited. The experts are not linked in any way. The need to ensure transitive consistency (10–12 %) makes it possible to record attempts by an expert (experts) to artificially overestimate the indicators of one of the drone samples (or the one being evaluated), therefore, the indicators of another sample will au- tomatically deteriorate. The principle of impartiality and fairness is maintained. A well-trained objective coordinator is not required, and the reality is that the absence of the disrupting the problem solution possibility is due to a change in the psychological situation among the experts. Area of practical use of research results: humanitarian demining in the interests of ensur- ing the detection of mines for various purposes by sappers from a safe distance. At the same time, an increase within the probability of mines detecting is ensured due to special equip- ment installed onboard the drone. Innovative technological product: a technique has been developed that allows not only assessing the drone samples for mine reconnaissance objectively, but making an objective choice of a sample for specific requirements also. Scope of application of the innovative technological product: clearance of the terrain re- maining after the end of hostilities. With the help of unmanned aerial vehicles, a significant acceleration of the demining process is possible, especially in those territories where mines are installed and being for a sufficiently long time. © The Author(s) 2020. This is an open access article under the CC BY license http://creativecommons.org/licenses/by/4.0). 1. 1. The object of research 1. 1. The object of research The object of research is a comparative assessment and selection of an unmanned aerial vehicle for mine reconnaissance sample while using quantitative and qualitative indicators. No. 5 (70), 2020 No. 5 (70), 2020 ScienceRise. Special issue ISSN 2313-8416 Vadym Neroba1 A RT I C L E I N F O Article history: Received date 14.09.2020 Accepted date 22.10.2020 Published date 30.10.2020 Section: Control systems D O I 10.21303/2313-8416.2020.001496 K E Y W O R D S mines reconnaissance unmanned aerial vehicle methods of system analysis expert review technique for a drone assessing and sampling ScienceRise, 5, 44 50. doi: http://doi.org/10.21303/2313 8416.2020.001496 © The Author(s) 2020. This is an open access article under the CC BY license http://creativecommons.org/licenses/by/4.0). AB ST R AC T AB ST R AC A RT I C L E I N F O A RT I C L E I N F O DEVELOPMENT OF METHODS FOR ASSESSMENT AND SELECTION OF UNMANNED AERIAL VEHICLE FOR MINE RECONNAISSANCE Vadym Neroba1 1Department of General Military Disciplines, National Academy of the State Border Guard Service of Ukraine named after Bohdan Khmelnytskyi, Khmelnytskyi, Ukraine vadym.rostislavovich@gmail.com ORCID: http://orcid.org/0000-0002-7232-3285 1. 3. Suggested solution to the problem A number of Ukrainian and foreign specialists and scientists were involved in various issues related to research on the use of unmanned aircraft for solving the problems of military facilities reconnaissance: A. Ananin, L. Artiushin, M. Dougherty, V. Kirilenko, V. Kolesnikov, M. Mitrak- hovich, S. Mosov, S. Saliy, A. Samkov, A. Selyukov, V. Silkov, A. Feschenko and others. S. Mosov and V. Kolesnikov in their work [1] gave the system requirements for the selection of unmanned aerial systems for performing reconnaissance and surveillance tasks. At the same time, these requirements are of a general nature, although they can be used to determine quantita- tive indicators for evaluating UAV samples. A team of authors in [2] presented the results of research related to unmanned aerial systems from the standpoint of a comparative assessment of their combat capabilities. The methodological approaches proposed in [2] do not consider the issue of assessment and sampling of UAVs with specified equipment for reconnaissance of mines. A team of authors in [3] analyzed the use of unmanned aircraft in military conflicts of our time, highlighting the features of the use of unmanned aircraft systems of various purposes. At the same time, the issues of using UAVs for detecting mines are not covered in the monograph. A team of authors in [3] analyzed the use of unmanned aircraft in military conflicts of our time, highlighting the features of the use of unmanned aircraft systems of various purposes. At the same time, the issues of using UAVs for detecting mines are not covered in the monograph. A feature of work [4] is the mathematical apparatus of multi-criteria choice of reconnais- sance unmanned aircraft systems. At the same time, the given mathematical apparatus uses only quantitative indicators. A. Ananiev in [5] proposed a set of tactical and technical requirements for unmanned air- craft systems and defined their tasks in the system of protecting the state border. Among the tasks, the detection of mines is not defined, which today is important for the protection of the state border on the south-eastern border of Ukraine. A. Ananiev in [5] proposed a set of tactical and technical requirements for unmanned air- craft systems and defined their tasks in the system of protecting the state border. Among the tasks, the detection of mines is not defined, which today is important for the protection of the state border on the south-eastern border of Ukraine. 1. 2. Problem description The territory of Ukraine, which is considered to be contaminated with explosives, is ap- proximately 16,000 km2. By 2019 almost 300 military and more than 2,500 civilians died since the beginning of hostilities due to the explosion of mines and explosive objects. The threat of a mine situation to troops, border guards and the civilians indicates the exis- tence of a discrepancy. On the one hand, these are requirements for the relevant level of technical means for area engineering reconnaissance conducting for the mines presence, taking into consid- eration the achievements of modern scientific and technological progress. On the other hand – the 44 Innovative technologies in industry moral obsolescence of ground-based technical means of mines searching left over from the times of the Soviet Union. moral obsolescence of ground-based technical means of mines searching left over from the times of the Soviet Union. Elimination of this discrepancy is possible through the development of more effective tech- nical means of engineering reconnaissance of mines. New approaches are needed for timely and safe remote detection of mines, taking into consideration the latest achievements of scientific and technological progress. One of these innovative approaches was the idea of using an unmanned aerial vehicle (hereinafter – UAV) for mine reconnaissance. The payload of the drone is a view (with image formation) technical means of mine reconnaissance. With the help of such reconnaissance drones, it is possible to accelerate the demining process significantly, especially in those territories where mines have been installed and be- ing for a sufficiently long time. Such studies are currently being carried out by the scientists of a number of leading countries of the world: the USA, Great Britain, Israel, Russia, China, Switzerland, etc. The use of specific technical means of UAVs for mine reconnaissance is an urgent di- mension of scientific research. This requires the development of the methods for comparative assessment and selection of UAVs based on the results of the study of system analysis. 3. Research results One of the specific features of the task of comparative assessment and selection of a specific UAV sample from a certain set of existing is a significant number of possible alternative solutions based on the indicators of their technical improvement. To compare several samples, methods of expert assessments are most often used, as well as theoretical methods associated with solving single-criterion or multi-criteria problems. Objectification of the selection of UAVs for mine reconnaissance is aimed, firstly, the ap- plication of a systematic approach in the process of assessing various UAVs, and, secondly, the introduction or development of new effective methods for comparing and selecting UAVs. To ensure objectivity in the assessment and selection of UAVs for mine reconnaissance, it is advisable to develop a method of comparative assessment and selection based on the results of the study of system analysis methods. One of these methods is the rather well-known Delphi method, which is a method of estab- lishing expert judgments on the basis of the anonymity of experts and their physical separation, as well as the presence of controlled feedback [9]. To maintain anonymity and physical separation, this method aims to avoid some of the potential pitfalls of group decision making, and the purpose of feedback is to allow each expert to read the average opinion of other experts. If there are sufficient positive aspects, the method has corresponding disadvantages. The first drawback should be attributed to the fact that the separation of experts practically excludes the possi- bility of the emergence of new approaches to solving the problem, which can be developed and tested in the course of discussions. Another disadvantage lies in the way of constructing the questionnaire, according to which the survey of experts is carried out. If the questionnaires are not constructed suf- ficiently, then the conclusions of the experts who answer the question will also be not objective due to the fact that the conclusions are actually determined by the questions that are asked. Another well-known systemic method of assessment and selection is the Strategic Assumption Surfacing and Testing (hereinafter – SAST) method, which is based on preliminary “polishing” of as- sumptions (elimination of inconsistency), which are subsequently used to solve the problem, as well as their ranking [10]. The method is well adapted to the analysis of poorly structured problems, in which the development of a solution is based on acutely conflicting assumptions. 1. 3. Suggested solution to the problem In collective work [6] the authors provide approximate calculations of the main parameters and characteristics of unmanned aerial vehicles, which do not take into consideration the tasks of conducting aerial reconnaissance (observation). V. Neroba in [7] systematized the conditions and factors that would affect the specific tech- nical means of UAV in terms of mines reconnaissance, allowed to determine the quantitative pa- rameters for assessing the samples of UAVs. V. Neroba in [7] systematized the conditions and factors that would affect the specific tech- nical means of UAV in terms of mines reconnaissance, allowed to determine the quantitative pa- rameters for assessing the samples of UAVs. M. Dougherty [8] cited the features of the use of unmanned aircraft in local wars and con- flicts in the civilian sphere, without parameters systematizing, which can be used to assess and select samples of unmanned aerial vehicles. M. Dougherty [8] cited the features of the use of unmanned aircraft in local wars and con- flicts in the civilian sphere, without parameters systematizing, which can be used to assess and select samples of unmanned aerial vehicles. The analysis results show that the issue of using the specific technical means of reconnais- sance (hereinafter – STMR) installed on UAVs to solve the tasks of mines reconnaissance as objects of aerial reconnaissance remains in the topical arsenal. Only quantitative indicators are used to assess the UAV. The issues of assessment and selection of UAVs for mine reconnaissance require research. This requires the development of a comparative assessment methods and the choice of a 45 ScienceRise. Special issue ISSN 2313-8416 No. 5 (70), 2020 UAV model, on which specific technical means of mine reconnaissance are installed, while taking into consideration quantitative and qualitative indicators. UAV model, on which specific technical means of mine reconnaissance are installed, while taking into consideration quantitative and qualitative indicators. The aim of the article. To develop a methods for comparative assessment and selection of an UAV sample, on which STMR are installed for mine reconnaissance, while taking into consid- eration quantitative and qualitative indicators. 2. Materials and methods The development of the methods for the comparative assessment and selection of an UAV sample for mine reconnaissance based on the results of the study of system analysis methods. 3. Research results However, it requires such an objective arbiter, proficient in the art of interpersonal dialogue and experienced in group dynamics theory. Otherwise, the application of the SAST method is doomed to failure. Moreover, the use of the method requires the involvement of a sufficiently large number of experienced qualified experts. Another systematic procedure is the hierarchy analysis method (hereinafter – HAM) [11]. In contrast to the Delphi method, HAM supports group interaction and discussions. Thus, during explo- ration of the assumptions underlying individual decisions, new and important knowledge emerges. The expediency of this approach is confirmed by the experience of conducting business games during the USSR period [12]. In case of disagreement, the HAM does not impose an artifi- cial consensus, since it does not withdraw, but takes into consideration the opinions that fall out of the general channel in the calculations (the value of the discrepancy is allowed). Comparison of HAM with SAST allows to conclude that they are similar at the stage of the problematic task structuring. At the same time, a well-trained objective coordinator is not required, and it is real that there is no possibility of disrupting the solution of the problem due to a change in the psychological situation among experts. Thus, according to the results of the analysis, it can be concluded that the HAM is the most well-known of the acceptable system methods, which is advisable to use in terms of methods for 46 Innovative technologies in industry assessing and sampling of UAVs for mines reconnaissance developing. The theoretical work [2] confirms this conclusion. The software implementation of the HAM is available online on the Internet resource [13]. The first step of the HAM is to decompose the problem of assessing and selecting a UAV model for mine reconnaissance and presenting it in the form of a hierarchy (Fig. 1). At the first (highest) level, there is a common target z – the best UAV model for mine reconnaissance. At the second level, there are indicators K={ki}, which clarify the goal, and at the third (lower) level, there is a set of applicants A={dj} from among the UAV samples that must be evaluated. Level 1 Level 2 Level 3 Fig. 1. Scheme of decomposition of tasks, estimation and visualization of UAVs Fig. 1. 3. Research results Special issue ISSN 2313-8416 No. 5 (70), 2020 No. 5 (70), 2020 Table 2 The scale for assessing the relative importance w w 1 3 5 7 9 2, 4, 6, 8 Advantage degree Equal importance Moderate impor- tance Essential Significant Very strong Intermediate Table 3 Pairwise comparison matrices k1 d1d2...dm Priority vector k2 d1d2...dm Priority vector d1 1 1 2 d d w w ... 1 m d d w w 1 1 k d u d1 1 1 2 d d w w ... 1 m d d w w 2 1 k d u d2 2 1 d d w w 1 ... 2 m d d w w 1 2 k d u d2 2 1 d d w w 1 ... 2 m d d w w 2 2 k d u ... ... ... ... dm 1 m d d w w 2 m d d w w ... 1 1 m k d u dm 1 m d d w w 2 m d d w w ... 1 2 m k d u ... d1d2...dm Priority vector kn d1d2...dm Priority vector d1 1 ... ... ... ... d1 1 1 2 d d w w ... 1 m d d w w 1 n k d u d2 ... 1 ... ... ... d2 2 1 d d w w 1 ... 2 m d d w w 2 n k d u ... ... ... 1 ... ... ... ... dm ... dm 1 m d d w w 2 m d d w w ... 1 n m k d u Pairwise comparison matrices In the process of pairwise comparisons, to identify and eliminate possible violations of transitive consistency, the value of the consistency index r should be determined to calculate the consistency ratio χ: max , 1 λ − = − s r s 0 100 %, χ = r r (2) (2) r – the index of random consistency [14]: 1.0808 0 1.67 , − = s r e (3) (3) λmax – the maximum eigenvalue of the matrix: λmax – the maximum eigenvalue of the matrix: λmax – the maximum eigenvalue of the matrix: max 1 1 . 3. Research results Scheme of decomposition of tasks, estimation and visualization of UAVs At the second step, it is necessary to establish the local priorities of the kj indicators by con- cluding square matrices of i pairwise comparisons (Table 1) and conducting expert comparisons using the scale for assessing the relative importance w (Table 2). Square matrices are inversely symmetric, that is, the matrix element bji=wj/wi=1/bij. Similar matrices should be constructed for paired comparisons of each dj sample of the UAV at the third level in relation to the indicators ki of the second level (Table 3). Sets of local priorities are formed from the groups of matrices of pairwise comparisons, indicating the relative influence of a set of elements on an element of the level adjacent from above. The process of forming local priorities is based on calculating eigenvectors for each matrix and normalizing the result to one: 1 1 1 1 , s i s i s s j j i s j i j w u w w w = = = = ∏ ∑∏ (1) (1) ui – the local priority of the i-th element of the column of the matrix of pairwise comparisons; s – the number of elements of the matrix of pairwise comparisons in the column. ui – the local priority of the i-th element of the column of the matrix of pairwise comparisons; s – the number of elements of the matrix of pairwise comparisons in the column. Table 1 Setting local priorities for indicators kj Indicators k1 k2 . . . k(n–1) kn Local priority k1 1 1k w 2 k w . . . 1k w ( 1) − n k w 1k w n k w 1ku k2 2 k w 1k w 1 . . . 2 k w ( 1) − n k w 2 k w n k w 2 ku . . . . . . . . . . . . . . . . . . . . . k(n–1) ( 1) − n k w 1k w ( 1) − n k w 2 k w . . . 1 ( 1) − n k w n k w ( 1) − n ku kn n k w 1k w n k w 2 k w . . . n k w ( 1) − n k w 1 n ku 47 ScienceRise. 4. Discussion of research results The proposed technique allows, firstly, not only to evaluate the UAV samples for mine reconnaissance objectively, but to carry out an objective selection of the UAV sample also. Secondly, the need to ensure transitive consistency makes it possible to record attempts to artificially overestimate the indicators of one of the UAV samples (or the one being evaluated), therefore, the indicators of another UAV sample will automatically deteriorate and the consis- tency ratio will go beyond the acceptable limits. This allows the principle of impartiality and fairness to be maintained. When applying the methods, it is proposed to add the following to the composition of indicators K={ki}, which clarify the goal – the choice of a UAV model for mines reconnais- sance: the presence of a stabilized platform on board the UAV sample for placing a STMR; maximum payload weight of the UAV sample; operating temperature (winter, summer) of the UAV sample; time spent in the air of the UAV sample; controllability of the UAV sample by the external pilot (pilot-operator); purchase cost of the UAV sample; the cost of operating a UAV sample; the maneuverability of the UAV sample; resistance of the UAV sample to wind gusts; maintainability of the UAV sample; operational reliability of the UAV sample; diagnostic abil- ity to the state of a UAV sample. y p The composition of indicators K={ki} may vary depending on the goal being achieved. The technique can be applied in the context of comparative assessment and selection of UAVs for mines reconnaissance, which are equipped with STMR: RGB– cameras, infrared camer- as, multi- and hyper spectral cameras. To ensure the objectivity of obtaining the results of applying the methods, the value of the agreement ratio should not exceed 10–12 %. The technique can be used in conditions when it is necessary to evaluate each (or one) UAV sample. In this case, a “normative” UAV model should be used as an additional UAV sample. Af- ter determining the global priority of the “normative” UAV sample, the level of the UAV sample is compared, evaluated relative to the normative by calculating the difference between the global priorities of the “normative” UAV sample and the UAV sample estimated, or calculating the per- centage between them. The direction of further research should be considered the development of recommenda- tions for organizing the training of external pilots and UAV operators for mine reconnaissance. 3. Research results = = λ = ∑∑ s s j i i j i w u w (4) (4) The value χ should not exceed 10–12 %, otherwise it will be necessary to additionally check the judgments of experts [11]. In the case where experts do not have sufficient experience in making judgments or a suf- ficient level of professional training to carry out the process of setting local priorities, the criteria should be pre-ranked. The penultimate step in determining z is the implementation of the principle of synthesis. The priorities are synthesized starting from the second level down. Local priorities i j k d u are multi- plied by the priority iku of the corresponding indicator at the highest level and summed up for each element in accordance with the criteria that this element affects: 48 Innovative technologies in industry ( ) ( ) 1 1 1 1 2 1 2 2 2 1 2 1 2 1 2 ... ... , ,... , ,... . ... ... ... ...       =       d k d k dm k d k d k dm d d dm k k kn knd knd kndm u u u u u u l l l u u u u u u (5) (5) The procedure for synthesizing local priorities is carried out to the lowest level, as a result of which the global priorities lj of each UAV sample – dj are determined. The ranking of the obtained priorities allows to determine the UAV sample with the highest priority value, which will be the most pleasant among the UAV samples that are evaluated. 4. Discussion of research results Another promising area may be the development of recommendations for technical operation of an unmanned aircraft complex designed organizing to carry out mine reconnaissance tasks. References [1] Mosov, S. P., Kolesnikov, V. O. (2016). Vymohy do vyboru bezpilotnykh aviatsiinykh kompleksiv dlia vykonannia zavdan roz- vidky ta sposterezhennia. Zbirnyk naukovykh prats Tsentru voienno-stratehichnykh doslidzhen Natsionalnoho universytetu oborony Ukrainy imeni Ivana Cherniakhovskoho, 1 (56), 24–28. [2] Mitrakhovich, M. M., Silkov, V. I., Samkov, A. V., Burshtynskaia, Kh. V. et. al.; Silkov, V. I. (Ed.) (2012). Bespilotnye aviat- sionnye kompleksy: metodika sravnitelnoi otsenki boevykh vozmozhnostei. Kyiv: TSNII VVT VS Ukrainy, 288. [3] Mosov, S. P., Pohoretskyi, M. V., Salii, S. M., Sieliukov, O. V., Feshchenko, A. L.; Mosov, S. P. (Ed.) (2019). Bezpilotna aviatsiia u viiskovii spravi. Kyiv: Interservis, 324. [4] Kyrylenko, V., Artiushyn, L., Steshenko, P. (2018). Matematychnyi aparat bahatokryterialnoho vyboru rozviduvalnykh bezpi- lotnykh aviatsiinykh kompleksiv. Zbirnyk naukovykh prats Natsionalnoi akademii Derzhavnoi prykordonnoi Ukrainy. Seriia: viiskovi ta tekhnichni nauky, 1 (75), 115–133. [5] Ananin, O. (2016). Taktyko-tekhnichni vymohy do bezpilotnykh aviatsiinykh kompleksiv ta yikh zavdannia v systemi ok- horony derzhavnoho kordonu. Zbirnyk naukovykh prats Natsionalnoi akademii Derzhavnoi prykordonnoi Ukrainy. Seriia: viiskovi ta tekhnichni nauky, 2 (68), 181–194. [6] Iliushko, V. M., Mitrakhovich, M. M., Samkov, A. V., Silkov, V. I. et. al. Silkov, V. I. (Ed.) (2009). Bespilotnye letatelnye apparaty: metodiki priblizhennykh raschetov osnovnykh parametrov i kharakteristik. Kyiv: TSNII VVT VS Ukrainy, 302. [7] Neroba, V. R. (2019). Systematyzatsiia umov i faktoriv, shcho vplyvatymut na vydovi tekhnichni zasoby rozvidky min bezpi- lotnoho litalnoho aparatu. Zbirnyk naukovykh prats Viiskovoi akademii, 2 (12 (1)), 48–54. [8] Dogerti, M. (2017). Drony: pervii illiustrirovannii putevoditel po BPLA. Moscow: Izd-vo «E», 224. [9] Pill, J. (1971). The Delphi method: Substance, context, a critique and an annotated bibliography. Socio-Economic Planning Sciences, 5 (1), 57–71. doi: http://doi.org/10.1016/0038-0121(71)90041-3 [10] Mitroff, I. I., Sagasti, F. (1973). Epistemology as General Systems Theory: An Approach to the Design of Complex Deci- sion-Making Experiments. Philosophy of the Social Sciences, 3 (2), 117–134. doi: http://doi.org/10.1177/004839317300300202  [11] Saati, T. (1993). Priniatie reshenii: metod analiza ierarkhii. Moscow: Radio i sviaz, 320. sion-Making Experiments. Philosophy of the Social Sciences, 3 (2), 117–134. doi: http://doi.org/10.1177/004839317300300202  [11] Saati T (1993) Priniatie reshenii: metod analiza ierarkhii Moscow: Radio i sviaz 320 [11] Saati, T. (1993). Priniatie reshenii: metod analiza ierarkhii. Moscow: Radio i sviaz, 320. [12] Schedrovitskii, G. P. (1995). Izbrannye trudy. Moscow: Shk. Kult. Polit., 800. [13] Metod analiza ierarkhii onlain. Available at: https://axd.semestr.ru/upr/hierarchies.php Last accessed: 20.09.2019 [14] Zahorka, O. M., Mosov, S. P., Sbitniev, A. I., Stuzhuk, P. I. (2005). 5. Conclusions A methods of comparative assessment and selection of a UAV sample for mine reconnais- sance have been developed, based on the well-known expert method for analyzing hierarchies. The methods have several advantages. The first is to support group interaction and discussion. Second, if the judgments do not coincide, an artificial consensus is not imposed, because opinions that fall out of the general channel in the calculations are not withdrawn, but taken into consideration. Third, a well-trained objective coordinator is not required, and the reality is that the lack of the possibility of disrupting the solution of the problem is due to a change in the psychological environ- ment among experts. Fourth, both quantitative and qualitative indicators are compared simultane- ously through quantitative formalization. 49 ScienceRise. Special issue ISSN 2313-8416 No. 5 (70), 2020 References Elementy doslidzhennia skladnykh system viiskovoho pryznachennia. Kyiv: NAOU, 100. 50
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Retrofitting Sense-Specific Word Vectors Using Parallel Text
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Proceedings of NAACL-HLT 2016, pages 1378–1383, San Diego, California, June 12-17, 2016. c⃝2016 Association for Computational Linguistics Abstract create a graph structure comprising two classes of relations: form-based relations between each word form and its respective senses, and meaning-based relations between word senses with similar mean- ings. This graph structure is then used to transform a traditional VSM into an enriched VSM, where each point in the space represents a word sense, rather than a word form. This approach is appealing as, un- like with prior sense-aware representations, senses are defined categories in a semantic lexicon, rather than clusters induced from raw text (Reisinger and Mooney, 2010; Huang et al., 2012; Neelakantan et al., 2015; Tian et al., 2014), and the method does not require performing word sense disambiguation (Guo et al., 2014). Jauhar et al. (2015) recently proposed to learn sense-specific word representations by “retrofitting” standard distributional word rep- resentations to an existing ontology. We ob- serve that this approach does not require an ontology, and can be generalized to any graph defining word senses and relations between them. We create such a graph using transla- tions learned from parallel corpora. On a set of lexical semantic tasks, representations learned using parallel text perform roughly as well as those derived from WordNet, and combining the two representation types significantly im- proves performance. In this paper, we observe that the crucial mean- ing relationships in the Jauhar et al. retrofitting process—the word sense graph—can be inferred based on another widely available resource: bilin- gual parallel text. This observation is grounded in a well-established tradition of using cross-language correspondences as a form of sense annotation (Gale et al., 1992; Diab and Resnik, 2002; Ng et al., 2003; Carpuat and Wu, 2007; Lefever and Hoste, 2010, and others). Using parallel text to define sense dis- tinctions sidesteps the persistent difficulty of identi- fying a single correct sense partitioning based on hu- man intuition, and avoids large investments in man- ual curation or annotation. 1 Introduction Vector space models (VSMs) provide a powerful tool for representing word meanings and modeling the relations between them. While these models have demonstrated impressive success in capturing some aspects of word meaning (Landauer and Du- mais, 1997; Turney et al., 2010; Mikolov et al., 2013; Baroni et al., 2014; Levy et al., 2014), they generally fail to capture the fact that single word forms often have multiple meanings. This can lead to counterintuitive results—for example, it should be possible for the nearest word to rock to be stone in everyday usage, punk in discussions of music, and crack (cocaine) in discussions about drugs. We use parallel text and word alignment to in- fer both word sense identities and inter-sense rela- tions required for the sense graph, and apply the approach of Jauhar et al. to retrofit existing word vector representations and create a sense-based vec- In a recent paper, Jauhar et al. (2015) introduce a method for “retrofitting” generic word vectors to create sense-specific vectors using the WordNet se- mantic lexicon (Miller, 1995). From WordNet, they 1378 1378 types of relations between senses: tor space, using bilingual correspondences to define word senses. When evaluated on semantic judgment tasks, the vector spaces derived from this graph per- form comparably to and sometimes better than the WordNet-based space of Jauhar et al., indicating that parallel text is a viable alternative to WordNet for defining graph structure. Combining the output of parallel-data-based and WordNet-based retrofitted VSMs consistently improves performance, suggest- ing that the different sense graph methods make complementary contributions to this sense-specific retrofitting process. C(V ) = arg min V X i−ij α∥ˆui −vij∥2 + X ij−i′j′ βr∥vij −vi′j′∥2 (1) (1) The objective encourages similarity between a word’s vector and its senses’ vectors (first term), as well as similarity between the vectors for senses that are related in the sense graph (second term). Defining a sense graph from parallel text. Our key observation is that, although Jauhar et al. (2015) assume their sense graph to be an ontology, this graph can be based on any inventory of word-sense and sense-sense relationships. In particular, given a parallel corpus, we can follow the tradition of translation-as-sense-annotation: the senses of an En- glish word type can be defined by different possible translations of that word in another language. 2 Model Retrofitting. The technique introduced by Jauhar et al. (2015) is based on what we will call a sense graph, which we formulate as follows. Nodes in the sense graph comprise the words wi in a vocabu- lary W together with the senses sij for those words. Labeled, undirected edges include word-sense edges ⟨wi, si,j⟩, which connect each word to all of its pos- sible senses, and sense-sense edges ⟨sij, si′j′⟩la- beled with a meaning relationship r that holds be- tween the two senses. Operationalizing this observation is straightfor- ward, given a word-aligned parallel corpus. If En- glish word form ei is aligned with Chinese word form cj, then ei(cj) is a sense of ei in the sense graph, and there is a word-sense edge ⟨ei, ei(cj)⟩. Edges signifying a meaning relation are drawn be- tween sense nodes if those senses are defined by the same translation word. For instance, English senses swear(发誓) and vow(发誓) both arise via align- ment to 发誓(fashi), so a sense-sense edge will be drawn between these two sense nodes. See Figure 2 for illustration. Jauhar et al. use WordNet to define their sense graph. Synsets in the WordNet ontology define the sense nodes, a word-sense edge exists between any word and every synset to which it belongs, and WordNet’s synset-to-synset relations of synonymy, hypernymy, and hyponymy define the sense-sense edges. Figure 1 illustrates a fragment of a WordNet- based sense graph, suppressing edge labels. 3 Evaluation Tasks. We evaluate on both the synonym selection and word similarity rating tasks used by Jauhar et al. Synonym selection nicely demonstrates the advan- tages afforded by sense partitioning: if we believe that spin means “make up a story”, then we are not likely to perform well on a question in which the correct synonym is twirl. Word similarity rating, on the other hand, is a classic test of the extent to which vector representations simulate human intuitions of word relations in general. Adopting Jauhar et al.’s notation, the original vec- tor space to be retrofitted is defined by the original word-form vectors ˆui for each wi ∈W, and the goal is to infer a set V of sense-specific vectors vij cor- responding to each sense sij. Jauhar et al. use the sense graph to define a Markov network with vari- ables for all word vectors and sense vectors, within which each word’s vector ˆui is connected to all of its sense vectors vij, and the variables for sense vec- tors vij and vi′j′ are connected iff the corresponding senses are connected in the sense graph. For synonym selection, we follow Jauhar et al. in testing with ESL-50 (Turney, 2001), RD-300 (Jar- masz and Szpakowicz, 2004), and TOEFL-80 (Lan- dauer and Dumais, 1997), using maxSim for multi- Retrofitting then consists in optimizing the fol- lowing objective, where α is a sense-agnostic weight, and βr are relation-specific weights for 1379 Figure 2: Illustration of parallel-text-based sense graph. Figure 1: Illustration of WordNet-based sense graph. Figure 2: Illustration of parallel-text-based sense graph. Figure 1: Illustration of WordNet-based sense graph. sense models (Jauhar et al., 2015, eq. 9) to select the most similar word.1 For similarity rating, we again mirror Jauhar et al., testing with WS-353 (Finkel- stein et al., 2001), RG-65 (Rubenstein and Good- enough, 1965), MC-30 (Miller and Charles, 1991), and the designated test subset (1000 items) of MEN- 3k (Bruni et al., 2014), using avgSim (Jauhar et al., 2015, eq. 8) as the similarity rating, and evaluating model ratings against human similarity ratings via Spearman’s rank correlation coefficient (ρ).2 ∼5.8M lines of segmented Chinese-English paral- lel text from the DARPA BOLT project and the Broadcast Conversation subset of the segmented Chinese-English parallel data in the OntoNotes cor- pus (Weischedel et al., 2013).4 We perform word alignment with the Berkeley aligner (Liang et al., 2006). 4English was lemmatized post-alignment via lookup in the XTAG morphological database (XTAG Research Group, 2001). 3 Evaluation We filter out noisy alignments using the G- test statistic (Dunning, 1993), with a threshold se- lected during tuning on a development set. We set α (see Equation 1) to 1.0. Each sense- sense edge ⟨ei(cj), ei′(cj)⟩has individual weight 0 < βr ≤1, computed by obtaining the G-test statistic for the alignment of ei with cj and for the alignment of ei′ with cj, running these values through a logistic function, and averaging. Param- eters for these computations, as well as the G-test statistic threshold below which we filtered out noisy alignments, were selected during tuning on the de- velopment set. Initial word representations. We use the word2vec (Mikolov et al., 2013) skip-gram archi- tecture to train 80-dimensional word vectors (in keeping with Jauhar et al.), based on evidence that this model shows consistently strong performance on a wide array of tasks (Baroni et al., 2014; Levy et al., 2015). Training is on ukWaC (Ferraresi et al., 2008), a diverse 2B-word web corpus.3 Note that we have not currently incorporated spe- cial treatment for alignments of a single word to a multi-word phrase. This does create the possibil- ity of noisy or uninformative sense annotations (e.g., sense annotations corresponding to parts of aligned Chinese phrases) when such alignments are not fil- tered out by the G-test thresholding. 1Because it is not clear how multi-word phrases should best be treated (and this is not a question being investigated here), we filter out any questions containing multi-word phrases for any of the relevant items (probe or possible response), and any questions for which any of the relevant items is completely out of vocabulary (no vectors available) for any of the evaluated models. This leaves 48 items in ESL, 87 items in RD, and 77 items in TOEFL. 2The designated development set of MEN-3k (2000 items) was used for tuning. 3 1Because it is not clear how multi-word phrases should best be treated (and this is not a question being investigated here), we filter out any questions containing multi-word phrases for any of the relevant items (probe or possible response), and any questions for which any of the relevant items is completely out of vocabulary (no vectors available) for any of the evaluated models. This leaves 48 items in ESL, 87 items in RD, and 77 items in TOEFL. 2The designated development set of MEN-3k (2000 items) was used for tuning. 3To alleviate sparsity we lemmatized the ukWaC corpus. Runs without lemmatization produced weaker results. 3To alleviate sparsity we lemmatized the ukWaC corpus. Runs without lemmatization produced weaker results. Sense-graph construction from parallel text. To construct the sense graph per Section 2, we use Sense-graph construction from parallel text. To construct the sense graph per Section 2, we use 1Because it is not clear how multi-word phrases should best be treated (and this is not a question being investigated here), we filter out any questions containing multi-word phrases for any of the relevant items (probe or possible response), and any questions for which any of the relevant items is completely out of vocabulary (no vectors available) for any of the evaluated models. This leaves 48 items in ESL, 87 items in RD, and 77 items in TOEFL. Experimental conditions. We evaluate the fol- lowing experimental conditions: Skip-gram (SG) uses the un-retrofitted word2vec vectors, Word- 2The designated development set of MEN-3k (2000 items) was used for tuning. 3To alleviate sparsity we lemmatized the ukWaC corpus. Runs without lemmatization produced weaker results. 1380 Synonym Selection SYMM (%) ESL-48 RD-87 TOEFL-77 SG 58.3 58.6 71.4 WN 66.7 74.7 81.8 PD 68.8 62.1 80.5 PD-WN 70.8 79.3 84.4 Table 1: Synonym selection task results: accuracy Net (WN) retrofits using the WordNet-based sense graph, and Parallel Data (PD) retrofits using the sense graph built from parallel text. We also com- bine the two retrofitting approaches (PD-WN). For synonym selection, we compute maxSim over all sense pairs for WN and PD separately, and select the sense pair with the overall maximum cosine sim- ilarity across the two. For similarity rating, we explore two PD-WN combination approaches: for each word pair, we take the avgSim from each sep- arate model, and then we (a) take the average of the values given by the two models (avg), or (b) take the maximum value between the two models (max). Word similarity: avgSim SYMM (ρ) WS-353 RG-65 MC-30 MEN-1k SG .708 .729 .722 .763 WN .610 .725 .750 .739 PD .636 .777 .715 .769 PD-WN (avg) .666 .777 .742 .773 PD-WN (max) .630 .731 .758 .756 Table 2: Similarity rating task results Table 2: Similarity rating task results 4 Results raters were explicitly told to rate relatedness, rather than similarity, while the retrofitting process is in- tended to encourage similarity per se. If we exclude this set from consideration, we can observe that SG is outperformed by at least one sense-specific model in all cases.5 Table 1 shows that combining our new method with Jauhar et al.’s WN retrofitting performs best on synonym selection across all datasets, and both retrofitted models consistently outperform the no- retrofitting model (SG). Error analysis on RD-87, the only set on which WN substantially outperforms PD, suggests that PD’s errors are driven by the large number of lower frequency items that characterize this dataset. Given that WordNet is a hand-curated lexicon while the parallel data mirrors actual us- age, it is not surprising that the latter suffers when it comes to low frequency items. Note that as expected, the amount of training data has an impact on the quality of the alignments and of the sense graph. Retrofitting sense-specific embed- dings using only 300k sentence pairs, which repre- sent about 5% of the total training data, does not give clear benefit over word-form embeddings. Error analysis also indicates that PD performs particularly well on the synonym task precisely when one would expect: when the probe and the correct answer have an alignment to the same Chi- nese word form, so that the corresponding sense vec- tors are extremely close in vector space. Occasion- ally, PD yields “the wrong answer for the right rea- son”, choosing an option for which there is indeed a correct alignment that matches an alignment of the probe word. For instance, though the probe passage is intended to have the answer hallway, PD chooses ticket because both passage and ticket have a sense defined by alignment to the Chinese word 机票(jip- iao), meaning “air ticket”. Though this is a less fre- quent sense of passage, it is a reasonable one. 5We also explored using maxSim for similarity ratings, on the intuition that when human annotators give similarity judg- ments, they are likely to judge based on senses of the given words that are biased toward the words with which they are paired. However, top performance is similarly scattered when using maxSim for similarity scores and fails to improve over the SG baseline for two of the datasets. 6Sample sense-specific vectors and code for generat- ing a sense graph from parallel data can be accessed at http://ling.umd.edu/~aetting/retropd.html. 5We also explored using maxSim for similarity ratings, on the intuition that when human annotators give similarity judg- ments, they are likely to judge based on senses of the given words that are biased toward the words with which they are paired. However, top performance is similarly scattered when using maxSim for similarity scores and fails to improve over the SG baseline for two of the datasets. 6Sample sense-specific vectors and code for generat- ing a sense graph from parallel data can be accessed at http://ling.umd.edu/~aetting/retropd.html. Acknowledgments The authors would like to thank Sujay Kumar Jauhar for sharing software and data and for helpful dis- cussion. Thanks also to Manaal Faruqui and Pe- ter Turney for help in acquiring evaluation datasets, to Amittai Axelrod for his assistance with data, and to the anonymous reviewers for valuable comments and suggestions. This work was supported in part by an NSF Graduate Research Fellowship under Grant No. DGE 1322106. Any opinions, findings, and conclusions or recommendations expressed are those of the authors and do not necessarily reflect the views of the NSF. Eric H Huang, Richard Socher, Christopher D Manning, and Andrew Y Ng. 2012. Improving word representa- tions via global context and multiple word prototypes. In Proceedings of the 50th Annual Meeting of the Asso- ciation for Computational Linguistics: Long Papers- Volume 1, pages 873–882. Mario Jarmasz and Stan Szpakowicz. 2004. Roget’s thesaurus and semantic similarity. Recent Advances in Natural Language Processing III: Selected Papers from RANLP, 2003:111. Sujay Kumar Jauhar, Chris Dyer, and Eduard Hovy. 2015. Ontologically grounded multi-sense represen- tation learning for semantic vector space models. In Proceedings of NAACL, pages 683–693. Thomas K Landauer and Susan T Dumais. 1997. A so- lution to plato’s problem: The latent semantic analysis theory of acquisition, induction, and representation of knowledge. Psychological review, 104(2):211. 5 Conclusions and future work Building on Jauhar et al. (2015), we have presented an alternative means of deriving information about senses and sense relations to build sense-specific vector space representations of words, making use of parallel text rather than a manually constructed ontology. We show that this is a viable alterna- tive, producing representations that perform on par with those retrofitted to sense graphs based on Word- Net.6 Results on the similarity rating task (presented in Table 2) are less clearly interpretable, top perfor- mance being divided between the PD model and the combined models—with the exception of WS-353. We note that WS-353 is a test set for which human 1381 Based on these results, it would be interesting to evaluate further refinements of the sense graph: alignment-based senses could be clustered, or fur- ther filtered to reduce the impact of alignment noise; new edges could be added using other multilingual resources. Finally, it will be important to evaluate the effectiveness of the retrofitted word embeddings on extrinsic tasks that require disambiguating word meaning in context. Lev Finkelstein, Evgeniy Gabrilovich, Yossi Matias, Ehud Rivlin, Zach Solan, Gadi Wolfman, and Eytan Ruppin. 2001. Placing search in context: The con- cept revisited. In Proceedings of the 10th international conference on World Wide Web, pages 406–414. William A Gale, Kenneth W Church, and David Yarowsky. 1992. A method for disambiguating word senses in a large corpus. Computers and the Humani- ties, 26(5-6):415–439. Jiang Guo, Wanxiang Che, Haifeng Wang, and Ting Liu. 2014. Learning sense-specific word embeddings by exploiting bilingual resources. In Proceedings of COLING, pages 497–507. References Marco Baroni, Georgiana Dinu, and Germ´an Kruszewski. 2014. Don’t count, predict! A systematic compari- son of context-counting vs. context-predicting seman- tic vectors. In Proceedings of the 52nd Annual Meet- ing of the Association for Computational Linguistics, volume 1, pages 238–247. Els Lefever and Veronique Hoste. 2010. Semeval-2010 task 3: Cross-lingual word sense disambiguation. In Proceedings of the 5th International Workshop on Se- mantic Evaluation, pages 15–20. Elia Bruni, Nam-Khanh Tran, and Marco Baroni. 2014. Multimodal distributional semantics. Journal of Arti- ficial Intelligence Research, 49:1–47. Omer Levy, Yoav Goldberg, and Israel Ramat-Gan. 2014. Linguistic regularities in sparse and explicit word representations. In Proceedings of the Confer- ence on Natural Language Learning (CoNLLL), pages 171–180. Marine Carpuat and Dekai Wu. 2007. Improving statisti- cal machine translation using word sense disambigua- tion. In EMNLP-CoNLL, volume 7, pages 61–72. Omer Levy, Yoav Goldberg, and Ido Dagan. 2015. Im- proving distributional similarity with lessons learned from word embeddings. Transactions of the Associa- tion for Computational Linguistics, 3:211–225. Mona Diab and Philip Resnik. 2002. An unsupervised method for word sense tagging using parallel corpora. In Proceedings of the 40th Annual Meeting on Associ- ation for Computational Linguistics, pages 255–262. Percy Liang, Ben Taskar, and Dan Klein. 2006. Align- ment by agreement. In Proceedings of the main con- ference on Human Language Technology Conference of the North American Chapter of the Association of Computational Linguistics, pages 104–111. Ted Dunning. 1993. Accurate methods for the statistics of surprise and coincidence. Computational linguis- tics, 19(1):61–74. Adriano Ferraresi, Eros Zanchetta, Marco Baroni, and Silvia Bernardini. 2008. Introducing and evaluating ukWaC, a very large web-derived corpus of English. In Proceedings of the 4th Web as Corpus Workshop (WAC-4) Can we beat Google, pages 47–54. Tomas Mikolov, Kai Chen, Greg Corrado, and Jeffrey Dean. 2013. Efficient estimation of word representa- tions in vector space. arXiv preprint arXiv:1301.3781. 1382 George A Miller and Walter G Charles. 1991. Contex- tual correlates of semantic similarity. Language and Cognitive Processes, 6(1):1–28. George A Miller. 1995. Wordnet: a lexical database for English. Communications of the ACM, 38(11):39–41. Arvind Neelakantan, Jeevan Shankar, Alexandre Pas- sos, and Andrew McCallum. 2015. Effi- cient non-parametric estimation of multiple embed- dings per word in vector space. arXiv preprint arXiv:1504.06654. Hwee Tou Ng, Bin Wang, and Yee Seng Chan. 2003. Ex- ploiting parallel texts for word sense disambiguation: An empirical study. References In Proceedings of the 41st An- nual Meeting on Association for Computational Lin- guistics, pages 455–462. Joseph Reisinger and Raymond J Mooney. 2010. Multi- prototype vector-space models of word meaning. In Human Language Technologies: The 2010 Annual Conference of the North American Chapter of the As- sociation for Computational Linguistics, pages 109– 117. Herbert Rubenstein and John B Goodenough. 1965. Contextual correlates of synonymy. Communications of the ACM, 8(10):627–633. Fei Tian, Hanjun Dai, Jiang Bian, Bin Gao, Rui Zhang, Enhong Chen, and Tie-Yan Liu. 2014. A probabilistic model for learning multi-prototype word embeddings. In Proceedings of COLING, pages 151–160. Peter D Turney, Patrick Pantel, et al. 2010. From fre- quency to meaning: Vector space models of semantics. Journal of Artificial Intelligence Research, 37(1):141– 188. Peter Turney. 2001. Mining the web for synonyms: PMI- IR versus LSA on TOEFL. In Proceedings of the 12th European Conference on Machine Learning. Ralph Weischedel, Martha Palmer, Mitchell Marcus, Ed- uard Hovy, Sameer Pradhan, Lance Ramshaw, Nian- wen Xue, Ann Taylor, Jeff Kaufman, Michelle Fran- chini, et al. 2013. Ontonotes release 5.0 ldc2013t19. Linguistic Data Consortium, Philadelphia, PA. XTAG Research Group. 2001. A lexicalized tree adjoin- ing grammar for English. Technical Report IRCS-01- 03, IRCS, University of Pennsylvania. 1383
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Variant detection sensitivity and biases in whole genome and exome sequencing
BMC bioinformatics
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© 2014 Meynert et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE Open Access Abstract Background: Less than two percent of the human genome is protein coding, yet that small fraction harbours the majority of known disease causing mutations. Despite rapidly falling whole genome sequencing (WGS) costs, much research and increasingly the clinical use of sequence data is likely to remain focused on the protein coding exome. We set out to quantify and understand how WGS compares with the targeted capture and sequencing of the exome (exome-seq), for the specific purpose of identifying single nucleotide polymorphisms (SNPs) in exome targeted regions. Results: We have compared polymorphism detection sensitivity and systematic biases using a set of tissue samples that have been subject to both deep exome and whole genome sequencing. The scoring of detection sensitivity was based on sequence down sampling and reference to a set of gold-standard SNP calls for each sample. Despite evidence of incremental improvements in exome capture technology over time, whole genome sequencing has greater uniformity of sequence read coverage and reduced biases in the detection of non-reference alleles than exome-seq. Exome-seq achieves 95% SNP detection sensitivity at a mean on-target depth of 40 reads, whereas WGS only requires a mean of 14 reads. Known disease causing mutations are not biased towards easy or hard to sequence areas of the genome for either exome-seq or WGS. Conclusions: From an economic perspective, WGS is at parity with exome-seq for variant detection in the targeted coding regions. WGS offers benefits in uniformity of read coverage and more balanced allele ratio calls, both of which can in most cases be offset by deeper exome-seq, with the caveat that some exome-seq targets will never achieve sufficient mapped read depth for variant detection due to technical difficulties or probe failures. As WGS is intrinsically richer data that can provide insight into polymorphisms outside coding regions and reveal genomic rearrangements, it is likely to progressively replace exome-seq for many applications. Keywords: SNP, Sensitivity, Protein-coding genes, Next-generation sequencing, Whole genome sequencing, Exome sequencing Variant detection sensitivity and biases in whole genome and exome sequencing Alison M Meynert*, Morad Ansari, David R FitzPatrick and Martin S Taylor Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 *Correspondence: alison.meynert@igmm.ed.ac.uk MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, EH4 2XU Edinburgh, UK Background causal disease variants identified to date [2]. Addition- ally, experimental approaches to determine the function of candidate disease variants at protein coding or tran- script splice sites are well developed and accepted by the research community. The cost of sequencing DNA has decreased steeply since the introduction of next-generation short read technolo- gies [1]. It is now at the point where cohorts of whole human genomes are sequenced for study. However, inves- tigations of disease-causing variation continue to focus on the protein-coding exome, which is a small fraction of the whole genome. It contains fewer repetitive ele- ments than non-coding regions and contains most of the For these reasons, exome centric analysis will remain common in research and is increasingly used in clini- cal genetic settings [3]. The targeted capture followed by sequencing of specific regions, such as the 30 Mb human exome (exome-seq), has proven to be a cost-effective and productive strategy for the identification of single nucleotide polymorphisms (SNPs) and small insertions and deletions in this rich vein of the genome. However, *Correspondence: alison.meynert@igmm.ed.ac.uk MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Crewe Road, EH4 2XU Edinburgh, UK Page 2 of 11 Page 2 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 target capture kits estimated between 20X and 46X mean on-target depth was required to successfully genotype 95% of heterozygous SNPs, with the commercially avail- able kits at the higher end of that range. as sequencing technology rapidly improves and cost per sequenced nucleotide falls, there is likely to come a point where it is more economic to sequence a whole genome rather than target-capture and sequence, even if analysis is confined to just the exome. Where that point lies depends both on the costs of the technologies but also on the uni- formity of coverage and biases inherent in the data. In this work we set out to compare exome-seq with whole genome sequencing (WGS) in terms of their sensitivity to correctly detect known variants over the whole exome. g g We examine previously established measures of SNP detection sensitivity [9] in coding regions from exome- seq and WGS samples. Background SNP detection sensitivity can be measured both at a site level, considering the number of reads mapped over a given position in the reference genome, or as an overall estimate based on the mapped read depth across a region or regions (Figure 1). We com- puted the per-site measure for different sequencing tech- nologies and compared them directly when the per-site mapped depth is identical (point A, Figure 1). Because of the allele distribution bias in exome-seq, we expected that WGS would require fewer reads to successfully genotype heterozygous SNPs. The greater variability in coverage from exome-seq means that greater mean on-target depth should be required to identify the same proportion of SNPs in exome-seq as compared to WGS [10] (points B and C, Figure 1). We measured the estimated overall SNP detection sensitivity across a given set of target regions by using the per-site SNP detection sensitivity for the sequencing method combined with the coverage distri- bution for samples sequenced by the same method. This relates the overall sensitivity of a method to the mean on- target depth in the sample, which can be used to calculate the cost of sequencing to a given sensitivity. The process of exome-seq has known issues that impact negatively on SNP detection sensitivity. These include PCR amplification, which tends towards lower cover- age in GC-rich regions due to annealing during ampli- fication [4-6], and the preferential capture of reference sequence alleles, which biases the allele distribution away from alternate alleles at heterozygous SNP sites [7-9]. Exome-seq produces a relatively heterogeneous profile of read coverage over target regions when compared to the more homogeneous WGS [10]. Better uniformity of cov- erage yields improved SNP detection sensitivity across the regions of interest [9-11]. Previous estimates of the amount of sequencing required to accurately identify SNPs in WGS and exome- seq are variable. Bentley et al. estimated that 15X mapped read depth of WGS samples would be suffi- cient to detect almost all homozygous SNPs and 33X for almost all heterozygous SNPs [12]. 50X was esti- mated by Ajay et al. for all SNPs and small indels [13]. Depending on the capture kit, Clark et al. cal- culated that exome-seq required 80X mean on-target depth to reach the common threshold of 10X per-site depth in 90% or more of all targeted regions [10]. Our previous work on some of the original exome-seq Results and discussion Site level SNP detection sensitivity Results and discussion Site level SNP detection sensitivity Site level SNP detection sensitivity Site level SNP detection sensitivity Site level SNP detection sensitivity is the mapped read depth directly over a polymorphic site that is required to reliably call that polymorphism [9]. Figure 1 SNP detection sensitivity in exome and whole genome sequencing. Exome and whole genome sequencing mapped read depth across the exons of an example gene. The grey vertical lines denote exon boundaries. At point A, the depth is equal and we can compare the per-site SNP detection sensitivity. Points B and C are examples of unequal depth, where per-site sensitivity cannot be directly compared, but the overall estimated sensitivity of the region can be calculated to account for the variability in coverage. Figure 1 SNP detection sensitivity in exome and whole genome sequencing. Exome and whole genome sequencing mapped read depth across the exons of an example gene. The grey vertical lines denote exon boundaries. At point A, the depth is equal and we can compare the per-site SNP detection sensitivity. Points B and C are examples of unequal depth, where per-site sensitivity cannot be directly compared, but the overall estimated sensitivity of the region can be calculated to account for the variability in coverage. Page 3 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Ten human whole genome sequences (TCGA-WGS) and matched whole exome sequences from the same patients, plus ten additional exome samples (all TCGA- WXS), were obtained from The Cancer Genome Atlas (TCGA; only non-tumour samples were used). A fur- ther six human whole genome samples (1KG-WGS) were obtained from the 1000 Genomes Project [14], all aligned to the reference genome. An additional 13 exome samples were captured, sequenced, and aligned in house as part of two ongoing disease studies (HGU- WXS) (Methods, Additional file 1: Table S1, Additional file 2: Tables S2–S4, Additional file 1: Figures S1 and S2). We randomly downsampled all 49 alignments to simu- late shallower sequencing and called SNPs in the coding regions of the alignments as in our previous work [9]. gold standard (Additional file 1: Figure S4). We measured sensitivity as a function of the per-site depth for heterozy- gous (Figure 2) and homozygous (not shown) SNPs. We focused on heterozygous SNPs as the more challenging problem: only 2-3X per-site depth was required to accu- rately detect at least 95% of homozygous SNPs in all four data sets. Direct comparison of matched samples The matched WGS and exome-seq samples from TCGA were compared directly. Considering only SNPs in the regions targeted by the exome capture kit, 98.3 ± 0.007% of variant sites were called as polymorphic by both meth- ods with matched genotypes, and 0.3 ± 0.3% with mis- matched genotypes. A further 1.3 ± 0.4% were called as polymorphic by whole genome sequencing only, and 0.1 ± 0.1% by exome sequencing only. 93% of the sites called as polymorphic only by whole genome sequencing had greater mapped per-site depth in the WGS sam- ple than in the exome-seq sample (Additional file 1: Figure S7). Of these, 34% are at sites with no reads in the exome-seq sample, which could be due to probe failure or other technical problems. The majority of mismatched genotypes were cases where the whole genome sample was genotyped as het- erozygous and the exome sample was genotyped as homozygous (Additional file 1: Figure 8a and Table S7). Mismatches generally occurred at sites where the whole genome sample had higher per-site mapped depth than the exome sample (Additional file 1: Figure S8b). Some sites with very high mapped depth in the exome sample also had mismatched genotypes with the whole genome sample. This could be caused by random accumulation of the same sequencing error at a given position if that position is sequenced to very high mapped depth. Both of these results imply that improving uniformity of coverage will improve SNP detection sensitivity. The number of variants called from the TCGA-WGS data set did not improve greatly with grouped sample calling (mean of 37 heterozygous and 11 homozygous additional sites), though there were a large number of mismatched genotypes between the group calling and the single-sample calling. This was observed in only 6 of the samples; the other 4 all had ≤2 mismatched geno- types. The TCGA-WGS samples had both excellent mean on-target depth and uniformity of coverage, which made them easy to accurately genotype using single-sample variant calling. Grouped variant calling would therefore not provide the same boost as with the other data sets. We took the subset of coding SNPs where the alle- les and genotypes were identical in the full alignments between the TCGA-WGS and TCGA-WXS samples for the same individual, and compared the mapped depth of sequencing required to correctly identify the genotypes of both heterozygous and homozygous SNPs (Figure 3). Effect of grouped and single sample variant calling same generation of machine, this difference was likely caused by reference bias from the capture step. Low-depth exome sequencing projects may need to take this into consideration. Effect of grouped and single sample variant calling The results in this paper are derived from calling variants for one sample at a time; however, it is standard prac- tice to call variants in groups of samples (pooled calling) as this improves accuracy by allowing the use of reads across all samples at a position to determine the presence of a polymorphism. To investigate the relative benefits of pooled calling, we grouped our samples by data source, called variants on the full alignments for each group, and compared the results to the variants called on the full alignments by single sample calling (Additional file 2: Table S8a). All the data sets converged at 99% sensitivity for sites with between 15 and 20X mapped depth. The recall curves were virtually identical for variants drawn from the Ensembl 72 coding regions and each of the sets of targeted regions from the two exome sequencing data sets (Additional file 1: Figure S5, Additional file 1: Table S6). Specificity (1 - false positive rate) reached 99% at 9X for the 1KG-WGS data set and 8X for the other three (Additional file 1: Figure S6). For sites in HapMap 3.3, there were very few cases of mismatched genotypes between the two calling methods; the main difference was in additional sites called as poly- morphic when the samples were grouped. For all data sets, of the sites with mismatched genotypes or where only one method called the site as polymorphic, the mapped read depth was on average lower than for sites where genotypes were matched (Additional file 1: Figure S9). The two exome capture data sets benefited significantly from grouped sample calling, with a mean of 186 (594) heterozygous and 100 (326) homozygous additional sites genotyped for the HGU-WXS (TCGA-WXS) data set. These data sets had the most samples, which may have been the major cause of the improvement, or possibly the uneven coverage of the exomes was smoothed by the inclusion of multiple samples. Effect of grouped and single sample variant calling The 1KG-WGS data set also benefited to the same degree as the exome capture data sets for heterozygous sites (mean 317 additional), but not for homozygous sites (mean 52 additional), per- haps because the 1KG-WGS data set comprises two family trios, which would help to resolve heterozygous positions. Site level SNP detection sensitivity BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Page 4 of 11 Effect of grouped and single sample variant calling Site level SNP detection sensitivity The oldest data set (1KG-WGS) had the worst per- formance for heterozygous SNPs, requiring at least 13X to reach 95% sensitivity. This could be due to shorter read lengths or higher sequencing error rates on older technologies. The difference in per-site SNP detection sensitivity between this data set and the newer three data sets indicates that analysis of older data sets requires more stringent thresholds. We defined a gold-standard set of SNP calls for each sample, based on the full alignments (using all avail- able reads for the sample, i.e. not down sampled) and confined to known HapMap 3.3 variants (Additional file 2: Table S5, Additional file 1: Figure S3, Additional file 3). We validated the use of HapMap 3.3 variants as the gold standard in sample NA12878 by compar- ing results to those obtained using the Genome in a Bottle 2.18 highly confident variant call set [15] as the All of the newer three data sets, which are contempo- rary with each other, performed similarly. The TCGA- WXS samples reached 95% sensitivity at 10X, while the HGU-WXS and TCGA-WGS samples had a slight edge at 9X. There was a slight advantage in sensitivity for the newer TCGA-WGS data set as opposed to the two exome- seq data sets, though this equalized at 12-13X per-site depth. Given that read lengths are the same between these three data sets and most samples were sequenced on the Depth at SNV position Heterozygous SNP sensitivity 0.0 0.2 0.4 0.6 0.8 1.0 5 10 15 20 1KG−WGS HGU−WXS TCGA−WGS TCGA−WXS Figure 2 Site level heterozygous SNP detection sensitivity for exome and whole genome sequencing samples. Sensitivity is calculated from heterozygous HapMap 3.3 positions [16] located within coding sequence as determined by Ensembl 72 [17]. 95% sensitivity is reached at per-site mapped depths of 9X for the TCGA-WGS samples, 10X for the TCGA-WXS and HGU-WXS samples, and 13X for the 1KG-WGS samples. Figure 2 Site level heterozygous SNP detection sensitivity for exome and whole genome sequencing samples. Sensitivity is calculated from heterozygous HapMap 3.3 positions [16] located within coding sequence as determined by Ensembl 72 [17]. 95% sensitivity is reached at per-site mapped depths of 9X for the TCGA-WGS samples, 10X for the TCGA-WXS and HGU-WXS samples, and 13X for the 1KG-WGS samples. Page 4 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. Direct comparison of matched samples To accurately genotype 95% of heterozygous SNPs, the TCGA-WGS data set required a minimum per-site depth of 12X and the TCGA-WXS data set required 34X. For homozygous sites, the minimum per-site depths were 8X and 33X respectively. We also examined rare variants, as defined by absence from HapMap 3.3, presence in the Exome Variant Server ESP6500 (http://evs.gs.washington.edu/EVS) set at less than 0.01 minor allele frequency, and minimum geno- type quality at least 60 in each of the grouped and single sample call sets (Additional file 2: Table S8b). The 1KG- WGS, HGU-WXS, TCGA-WGS, and TCGA-WXS data sets gained a mean of an additional 0.28%, 0.25%, 1.23%, and 4.30% respectively of these rare variants by grouped Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Page 5 of 11 (a) Minimum depth for correct genotype call Cumulative density 0.0 0.2 0.4 0.6 0.8 1.0 0 10 20 30 40 Heterozygous 0.0 0.2 0.4 0.6 0.8 1.0 Homozygous WGS WXS (b) Difference (exome − genome) in minimum depth for correct genotype call Density 0.00 0.02 0.04 0.06 0 20 40 Heterozygous Homozygous re 3 Minimum per-site mapped depth required to correctly genotype a site in matched TCGA exome and genome samples. Coding at HapMap 3.3 positions [16] with identical genotypes and alleles between matched TCGA exome and genome samples in the full alignments. e minimum per-site mapped depth required for a correct genotype call in TCGA-WXS and TCGA-WGS matchd samples. b) The number of extra required to correctly identify a SNP in the TCGA-WXS sample. (a) Minimum depth for correct genotype call Cumulative density 0.0 0.2 0.4 0.6 0.8 1.0 0 10 20 30 40 Heterozygous 0.0 0.2 0.4 0.6 0.8 1.0 Homozygous WGS WXS (a) Cumulative density (b) Difference (exome genome) in minimum depth for correct genotype call Density 0.00 0.02 0.04 0.06 0 20 40 Heterozygous Homozygous (b) Density Difference (exome − genome) in minimum depth for correct genotype call Figure 3 Minimum per-site mapped depth required to correctly genotype a site in matched TCGA exome and genome samples. Coding SNPs at HapMap 3.3 positions [16] with identical genotypes and alleles between matched TCGA exome and genome samples in the full alignments. a) The minimum per-site mapped depth required for a correct genotype call in TCGA-WXS and TCGA-WGS matchd samples. b) The number of extra reads required to correctly identify a SNP in the TCGA-WXS sample. Characteristics of difficult target regions As has previously been noted for both whole genome and exome sequencing, regions of high G+C content and regions containing repetitive elements are gener- ally harder to sequence to high depth [19]. We define difficult regions based on poor coverage (see Methods) in at least half the samples from a given data set, and easy regions based on excellent coverage (see Methods) in all the samples from that set. Our samples show the expected characteristics, with the bulk of difficult regions occurring at G+C content above 60% (Additional file 1: Figure S11), and with a significantly higher proportion of difficult regions overlapping repetitive elements com- pared to relatively easy target regions (Additional file 1: Figure S12). The HGU-WXS data set also had a large number of difficult target regions that were of low G+C content. Because the classification of a region as difficult is based on at least half the samples in a data set, this was not caused by capture failure of one or a few samples; however, a larger scale failure could be implicated. Very few target regions were classed as difficult for the TCGA-WGS data set for either of the two exome capture target region sets. However, approximately one third of all regions identified as difficult in any of the four data sets were classed that way for both the TCGA-WXS and 1KG-WGS data set, and 15% for both the HGU-WXS and the 1KG-WGS data set (Additional file 1: Figure S13). Our estimates for WGS required mapped depth are lower than those from Bentley et al. (33X) [12] and Ajay et al. (50X) [13], though both were attempting to quan- tify detection of all or almost all SNPs rather than to a given percentage as here. It is unsurprising that the harder to sequence variants will require proportionally greater additional numbers of reads to accurately genotype. Addi- tionally, we are analysing only coding sequence variants, which are in the least repetitive portion of the human genome. The higher figures reported by the other WGS analyses will be influenced by the different qualities of non-coding sequence, especially repetitive regions. The TCGA-WXS and HGU-WXS exome-seq data sets used in this analysis can update the figures provided by Clark et al. Characteristics of difficult target regions of 80X mean on-target depth required for 10X mapped read depth in 90% of targeted regions [10]: a median of 59X mean on-target depth is needed for the same coverage in both of our more recent exome-seq data sets. The equivalent figure was 18X for the TCGA-WGS data set and 20X for the 1KG-WGS data set. To quantify the contributions of repetitive sequence and nucleotide composition to target difficulty, we iden- tified targets meeting our criteria for difficult (see Methods) in any of the samples for a data set. The num- ber of samples in which that target was defined as difficult was multiply regressed against target G+C content, pres- ence of annotated repeats and alignability [20]. All factors were significant to p < 0.001; however, their predic- tive power was slight (Additional file 1: Table S8). The adjusted R-squareds were 0.265, 0.086, 0.150, and 0.171 for 1KG-WGS, HGU-WXS, TCGA-WGS, and TCGA- WXS respectively. As the analysis was run on the intersec- tion of the target capture regions for the two exome-seq Direct comparison of matched samples Figure 3 Minimum per-site mapped depth required to correctly genotype a site in matched TCGA exome and genome samples. Coding SNPs at HapMap 3.3 positions [16] with identical genotypes and alleles between matched TCGA exome and genome samples in the full alignments. a) The minimum per-site mapped depth required for a correct genotype call in TCGA-WXS and TCGA-WGS matchd samples. b) The number of extra reads required to correctly identify a SNP in the TCGA-WXS sample. Page 6 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 calling, while losing only 0.31%, 0.04%, 0.19%, and 0.35% that were only called in the single sample method. The TCGA-WXS data set gained by far the most rare variants by use of the grouped calling, similar to the results for known common HapMap 3.3 sites. given a particular sequencing strategy. To demonstrate and at the same time discover if known disease causing mutations are preferentially located in easy or difficult to sequence regions of the genome: we obtained the loca- tions of such coding and splice variants from HGMD [18]. From these we generated the coverage distributions for disease-causing and disease-associated SNVs separately to compare their overall estimated sensitivity with cod- ing regions in general for both whole genome and exome sequencing. For 87,663 disease-causing and 2,241 disease- associated sites, we found no difference in the measure across all four of our sample sets (Additional file 1: Figure S10). Overall estimated sensitivity Using the depth of coverage distributions for every down- sampled and full alignment on the regions targeted by each of the two exome capture kits, and the per-site SNP detection sensitivity for each data set, we calculated the overall estimated sensitivity for each of the four data sets. We compared this measure to the mean on-target read depth across the targeted regions and found that the two whole genome data sets performed considerably better than each of the exome data sets (Figure 4). In order to reach an overall estimated 95% sensitivity for heterozy- gous SNPs in the targeted regions, the 1KG-WGS samples required at least 18X and the TCGA-WGS 14X mean on- target depth. The HGU-WXS samples required 41X mean on-target depth, and the TCGA-WXS samples 39X. This effect is almost entirely due to the lack of uniformity in coverage for the exome samples: The difference in per-site sensitivity is relatively slight between the two exome data sets and the TCGA-WGS data set (Figure 2), and both of them perform better than the 1KG-WGS data set on that measure. Cost of sequencing to a given level of sensitivity We compared exome and whole genome sequencing costs on current standard technology (Illumina HiSeq) with an exome capture kit of the same size as the Nimblegen SeqCap EZ Exome v3 (65Mbp) used for the HGU-WXS samples, assuming 60% of exome reads on target (Table 1) and holding the per sample cost of the exome capture kit constant. To achieve 93–94% overall estimated het- erozygous sensitivity in the coding regions of the genome, exome sequencing is 4.2X cheaper than whole genome sequencing (12 exome samples/lane vs. 1 lane/sample whole genome). Likewise, for 98–99% sensitivity, exome sequencing is 5.4X cheaper (4 exome samples/lane vs. 2 lanes/sample whole genome). The mean on-target depth needed for 95% SNP detec- tion sensitivity shown by our analysis of WGS data from multiple sources is also lower than previous estimates [12,13]. The earlier of these two estimates describes reads from the first next-generation sequencing experiments, which were shorter than the reads used for our WGS samples, and additionally contained no paired-end reads. The second estimate is more comparable in terms of data, and we conclude that improvement in variant calling algorithms is likely to be a factor in the difference here. Uniformity of coverage is clearly still a major issue for exome sequencing in terms of capturing a reason- able number of reads across all of the targeted regions. PCR amplification-free library preparation can mitigate the issue somewhat for WGS samples [4,5] but it is still required to provide a sufficiently large library for exome- seq samples. Allele distribution biases introduced by the reference bias of exome-seq target probes could be min- imised by the use of alternate probes containing common haplotypes, but the problem will remain for rare variants. The additional allele distribution bias introduced by treat- ing the reference genome as truth during computational analysis affects both WGS and again exome-seq and is not easily fixed for rare variants. We estimate that the cost per lane of sequence would have to be 15–20% of the current cost for the two meth- ods to reach cost parity, holding the cost of exome capture constant (Additional file 1: Figure S14). The projected $1000 genome at 30X depth enabled by the Illumina HiSeq X Ten (X10) system reaches this cost point for 93– 94% overall estimated heterozygous sensitivity in the cod- ing regions of the genome. Sensitivity at sites in Human Gene Mutation Database (HGMD) Overall estimated sensitivity is a useful measure that can be applied to more specific subsets of target regions. For instance, estimating how many known disease caus- ing or disease associated SNV sites can be recovered Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Page 7 of 11 Figure 4 Overall estimated sensitivity for targeted regions. Calculated from the per-site sensitivity for each data set combined with the depth of coverage distributions for samples across the regions targeted by each of the two exome capture kits. a) HGU-WXS (Nimblegen SeqCap EZ Exome v3). b) TCGA-WXS (Whole exome Agilent 1.1 plus boosters). Figure 4 Overall estimated sensitivity for targeted regions Calculated from the per-site sensitivity for each data set combin Figure 4 Overall estimated sensitivity for targeted regions. Calculated from the per-site sensitivity for each data set combined with the depth of coverage distributions for samples across the regions targeted by each of the two exome capture kits. a) HGU-WXS (Nimblegen SeqCap EZ Exome v3). b) TCGA-WXS (Whole exome Agilent 1.1 plus boosters). Page 8 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 for heterozygous SNPs [9]. The two data sets in this analysis from more recent capture kits (HGU-WXS and TCGA-WXS) show 40X is required for the same level of sensitivity. This progressive improvement in technology could partially explain the difference between our results and the higher mean on-target depth of 80X suggested by other previous analyses such as Clark et al. [10]. data sets; the particularly low R-squared for the HGU- WXS data set may be due to differences in probe design but is not due to a different set of targets. Cost of sequencing to a given level of sensitivity Holding the per sample exome kit cost constant, the X10 system claims to sequence genomes to 12X depth at 77% the cost of sequencing exomes to 29X depth, with roughly equivalent sensitiv- ity. However, for higher sensitivity of 98–99%, we estimate that WGS on the X10 system will still be 31% more expen- sive than exome-seq, and decreases in exome capture kit costs will likely keep the two methods at close to cost parity. The amount of raw sequencing is the main cost driver for both WGS and exome-seq, and the drop in cost to the $1000 human genome at 30X depth has brought the two methods roughly into parity. However, smaller sequenc- ing centres relying on the previous generation of machines will continue to charge three to four times exome-seq costs for the same level of SNP detection sensitivity across coding regions using WGS. When taking into account the considerably higher data storage requirements of WGS All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. Alignments Reads for the 13 HGU-WXS exomes were aligned to the hg19/GRCh37 assembly of the human genome ref- erence sequence with BWA 0.5.9 [21]. Duplicate reads were removed using the MarkDuplicates function of Picard 1.79 (http://picard.sourceforge.net). Reads were re- aligned around indels and quality scores re-calibrated using the Genome Analysis Toolkit (GATK) 2.2-8- gec077cd [22]. Full parameters are given in the Additional file 1: Supplementary Information and Additional file 4. We randomly down-sampled reads from exome align- ments using Picard DownsampleSam, which maintains read pair information. The probability of sampling each read varied from 0.1 to 0.9 at intervals of 0.1. Ethical approval and consent The samples used for in-house exome sequencing were collected under approval by the UK Multiregional Ethics Committee (References: 06/MRE00/76 and 04/MRE00/ 19). Conclusions Exome-seq target capture technology is clearly improv- ing. Our previous results from a solution-based target capture kit suggested a mean on-target depth of 46X was needed to obtain 95% overall estimated sensitivity Table 1 Cost of sequencing to achieve a given level of heterozygous SNV detection sensitivity Method Lane usage Mean on-target depth Sensitivity Cost Whole genome 1 lane/sample 11X 94.0% 4.60 2 lanes/sample 22X 98.5% 8.79 Exome 16 samples/lane 22X 91.1% 1.00 12 samples/lane 29X 93.4% 1.09 8 samples/lane 44X 95.9% 1.28 6 samples/lane 58X 96.9% 1.46 4 samples/lane 88X 98.1% 1.63 All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. Table 1 Cost of sequencing to achieve a given level of heterozygous SNV detection sensitivity All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. All costs have been normalised against the cheapest exome sequencing (16 samples per lane). Estimated costs include library preparation, exome capture and multiplexing where applicable, and paired-end sequencing on Illumina HiSeq. Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Page 9 of 11 Page 9 of 11 Page 9 of 11 Page 9 of 11 Target regions and the extra compute time required to perform align- ment and subsequent bioinformatic analyses on WGS samples, the cost difference will be further amplified. We defined a common set of target regions using the cod- ing regions of exons from Ensembl 72 [17]. The coding regions were merged so that every position was repre- sented only once, 50bp of flanking sequence added to each resulting region, and the regions merged again. We also used the provided targets for the Nimblegen SeqCap EZ Exome v3 kit, a set of targets labelled ‘Whole exome Agi- lent 1.1 RefSeq plus 3 boosters’ obtained directly from TCGA, and a merged set of these two targets. WGS provides a much richer data set, capturing information on polymorphisms over whole genome and potentially capturing genomic rearrangements. The dra- matically improved uniformity of read coverage and reduced bias of allele ratios in WGS, both lend themselves to improved detection of copy number changes and mea- surement of sample heterogeneity. These are likely to be extremely useful measures in some settings, such as for the sequencing of primary tumours whose analysis, even when focused on the exome, is confounded by copy num- ber change, sample heterogeneity and a desire to detect de novo mutations. Mapped read depth across all the target regions was calculated using the DepthOfCoverage tool from GATK 2.6-5-gba531bd [22]. The target regions were split into maximally 100bp non-overlapping tiles for further anal- ysis, with small tiles at target region edges. We defined difficult target region tiles as those with fewer than 50% of their bases covered at least 15X in the full align- ments for at least half of the samples in a given data set, and easy target region tiles as those with all their bases covered at least 15X in the full alignments for all the samples in a given data set. G+C content for tar- get regions was obtained using the GCContentByInterval tool from GATK GenomeAnalysisTK-2.5-2-gf57256b, and repeat element occurrences were mapped from Ensembl 73. HGMD disease causing mutations were obtained from the HGMD Professional database (March 2013 release). Exome capture and sequencing The 13 HGU-WXS exomes were captured using a Nim- blegen SeqCap EZ Exome v3 kit. Paired-end reads of 98 bp were generated on the Illumina HiSeq platform. Six whole genome samples were downloaded from the 1000 Genomes Project [14] Pilot 2 (high coverage family trios). Individuals with both whole genome and exome samples from TCGA were filtered for cases with similar num- bers of reads to the 1KG-WGS and HGU-WXS exome samples. From these, 20 individuals with exome samples labelled with the same set of target capture regions were randomly selected. Exome alignments for all 20 individ- uals and whole genome alignments for a random subset of 10 individuals were downloaded from TCGA Data Por- tal. Additional file 1: Table S1 summarises the technology used to generate each of the four data sets. Individuals with both whole genome and exome samples from TCGA were filtered for cases with similar num- bers of reads to the 1KG-WGS and HGU-WXS exome samples. From these, 20 individuals with exome samples labelled with the same set of target capture regions were randomly selected. Exome alignments for all 20 individ- uals and whole genome alignments for a random subset of 10 individuals were downloaded from TCGA Data Por- tal. Additional file 1: Table S1 summarises the technology used to generate each of the four data sets. Multiple linear regression of number of samples where a target region had less than 50% of bases covered at least 15X in the full alignment was performed using R lm on factors G+C content, presence of repeats, and alignability [20] with no interactions, where at least one sample met the criteria for a given data set. Alignabil- ity tracks were downloaded from the UCSC Genome Browser [23]. 36mer alignability was used for 1KG-WGS samples, 75mer for TCGA-WXS, and 100mer for HGU- WXS and TCGA-WGS, to best match the read lengths for each data set (Additional file 1: Table S1). Variants Variants were called on the full and down-sampled align- ments using the GATK 2.6-5-gba531bd UnifiedGeno- typer tool [24], one sample at a time (full parameters in Additional file 1: Supplementary Information). We obtained HapMap Phase III sites and genotypes from the project FTP site [16]. Variants from this set were mapped by position and alleles to called variants in the full and down-sampled alignments. Additional variant calls were made by grouping samples by data source and running the UnifiedGenotyper tool on the full alignments for samples within each group. Sensitivity Sensitivity for per-site mapped read depth and estimated overall sensitivity were calculated as in [9], using sites Page 10 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 facilitates improved mapping and assembly of (G+C)-biased genomes. Nat Methods 2009, 6(4):291–295. 5. Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 2011, 12(2):18. 6. 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Nucleic Acids Res 2012, 40(6):2426–2431. 9 Meynert AM Bicknell LS Hurles ME Jackson AP Taylor MS: Quantifying in HapMap 3.3 and the Ensembl 72 coding regions with minimum genotype quality of 60 in the full alignments. in HapMap 3.3 and the Ensembl 72 coding regions with minimum genotype quality of 60 in the full alignments. facilitates improved mapping and assembly of (G+C)-biased genomes. Nat Methods 2009, 6(4):291–295. 5. Aird D, Ross MG, Chen W-S, Danielsson M, Fennell T, Russ C, Jaffe DB, Nusbaum C, Gnirke A: Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biol 2011, 12(2):18. Acknowledgements We thank Edinburgh Genomics for providing information regarding the relative costs of whole genome sequencing and targeted exome capture followed by sequencing. This work was supported by the UK Medical Research Council and the Medical Research Foundation. 17. Flicek P, Ahmed I, Amode MR, Barrell D, Beal K, Brent S, Carvalho-Silva D, Clapham P, Coates G, Fairley S, Fitzgerald S, Gil L, García-Girón C, Gordon L, Hourlier T, Hunt S, Juettemann T, Kähäri AK, Keenan S, Komorowska M, Kulesha E, Longden I, Maurel T, McLaren WM, Muffato M, Nag R, Overduin B, Pignatelli M, Pritchard B, Pritchard E, et al: Ensembl 2013. Nucleic Acids Res 2013, 41(Database issue):48–55. Authors’ contributions AMM d MST d i d 15. Zook JM, Chapman B, Wang J, Mittelman D, Hofmann O, Hide W, Salit M: Integrating human sequence data sets provides a resource of benchmark SNP and indel genotype calls. Nat Biotechnol 2014, 32(3):246–251. AMM and MST designed the analysis and wrote the manuscript. AMM performed the analysis. MA and DRF provided and sequenced the HGU-WXS exomes. All authors read and approved the final manuscript. AMM and MST designed the analysis and wrote the manuscript. AMM performed the analysis. MA and DRF provided and sequenced the HGU-WXS exomes. All authors read and approved the final manuscript. 16. The International HapMap 3 Consortium: Integrating common and rare genetic variation in diverse human populations. Nature 2010, 467(7311):52–58. Availability of supporting data The TCGA-WXS and TCGA-WGS data are available through dbGaP and the Cancer Genome Hub (http:// cghub.ucsc.edu/) and 1KG-WGS data from the 1000 Genomes Project (http://www.1000genomes.org/data), see Additional file 1: Table S2 for accession identifiers. Reads for the HGU-WXS exome sequence data are avail- able upon request. VCF files containing the HapMap 3.3 SNP sites and associated genotype calls and read depths in the downsampled and full alignments used to generate the main results in this article are included as additional files. The command lines for producing the downsampled alignments and calling variants are included as a plain text additional file. 6. Veal CD, Freeman PJ, Jacobs K, Lancaster O, Jamain S, Leboyer M, Albanes D, Vaghela RR, Gut I, Chanock SJ, Brookes AJ: A mechanistic basis for amplification differences between samples and between genome regions. BMC Genomics 2012, 13:455. 7. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H, Turner DJ: A large genome center’s improvements to the Illumina sequencing system. Nat Methods 2008, 5(12):1005–1010. 8. Heinrich V, Stange J, Dickhaus T, Imkeller P, Krüger U, Bauer S, Mundlos S, Robinson PN, Hecht J, Krawitz PM: The allele distribution in next-generation sequencing data sets is accurately described as the result of a stochastic branching process. Nucleic Acids Res 2012, 40(6):2426–2431. 9. Meynert AM, Bicknell LS, Hurles ME, Jackson AP, Taylor MS: Quantifying single nucleotide variant detection sensitivity in exome sequencing. BMC Bioinformatics 2013, 14:195. 10. 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Additional file 4: Command lines. 12. Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, Boutell JM, Bryant J, Carter RJ, Keira Cheetham R, Cox AJ, Ellis DJ, Flatbush MR, Gormley NA, Humphray SJ, Irving LJ, Karbelashvili MS, Kirk SM, Li H, Liu X, Maisinger KS, Murray LJ, Obradovic B, Ost T, Parkinson ML, Pratt MR, et al: Accurate whole human genome sequencing using reversible terminator chemistry. Nature 2008, 456(7218):53–59. Abbreviations 1KG: 1000 Genomes Project; HGU: Human Genetics Unit; GATK: Genome Analysis ToolKit; TCGA: The Cancer Genome Atlas; TCGA-WXS: TCGA Whole eXome Sequence; TCGA-WGS: TCGA Whole genome sequence. 13. Ajay SS, Parker SC, Ozel Abaan H, Fuentes Fajardo KV, Margulies EH: Accurate and comprehensive sequencing of personal genomes. Genome Res 2011, 21(9):1498–1505. 14. 1000 Genomes Project Consortium, Durbin RM, Abecasis GR, Altshuler DL, Auton A, Brooks LD, Durbin RM, Gibbs RA, Hurles ME, McVean GA: A map of human genome variation from population-scale sequencing. Nature 2010, 467(7319):1061–1073. References 1. Wetterstrand KA: DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program. 2013. [http://www.genome.gov/ sequencingcosts/] 1. Wetterstrand KA: DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program. 2013. [http://www.genome.gov/ sequencingcosts/] 19. Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP: Sequencing depth and coverage: key considerations in genomic analyses. Nat Rev Genet 2014, 15(2):121–132. coverage: key considerations in genomic analyses. Nat Rev Genet 2014, 15(2):121–132. 20. Derrien T, Estellé J, Marco Sola S, Knowles DG, Raineri E, Guigó R, Ribeca P: Fast computation and applications of genome mappability. PloS One 2012, 7(1):30377. 21. Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25(14):1754–1760. 22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010, 20(9):1297–1303. 2. Cooper DN, Krawczak M, Antonarakis S. In The Metabolic and Molecular Bases of Inherited Disease. 7th edn. Edited by Scriver C, Beaudet A, Sly W, Valle D, Childs B, Kinzler K, Vogelstein B. New York: McGraw Hill; 1995:259–291. 2. Cooper DN, Krawczak M, Antonarakis S. In The Metabolic and Molecular Bases of Inherited Disease. 7th edn. Edited by Scriver C, Beaudet A, Sly W, Valle D, Childs B, Kinzler K, Vogelstein B. New York: McGraw Hill; 1995:259–291. 20. Derrien T, Estellé J, Marco Sola S, Knowles DG, Raineri E, Guigó R, Ribeca P: Fast computation and applications of genome mappability. PloS One 2012, 7(1):30377. 21. Li H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009, 25(14):1754–1760. 3. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A, Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS, Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL, Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis 3. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A, Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS, Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL, Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis f d li di d N E l J M d 2013 369(16) 1502 1511 3. Additional file 1: Supplementary Information. Supplementary methods, figures, and small tables. Additional file 2: Supplementary Tables. Large supplementary tables. Additional file 3: VCF files of HapMap 3.3 coding sites and genotypes. Additional file 4: Command lines. References Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A, Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS, Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL, Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ: Amplification-free Illumina sequencing-library preparation 3. Yang Y, Muzny DM, Reid JG, Bainbridge MN, Willis A, Ward PA, Braxton A, Beuten J, Xia F, Niu Z, Hardison M, Person R, Bekheirnia MR, Leduc MS, Kirby A, Pham P, Scull J, Wang M, Ding Y, Plon SE, Lupski JR, Beaudet AL, Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ: Amplification-free Illumina sequencing-library preparation 22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA: The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010, 20(9):1297–1303. Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ: Amplification-free Illumina sequencing-library preparation Gibbs RA, Eng CM: Clinical whole-exome sequencing for the diagnosis of mendelian disorders. N Engl J Med 2013, 369(16):1502–1511. 4. Kozarewa I, Ning Z, Quail MA, Sanders MJ, Berriman M, Turner DJ: Amplification-free Illumina sequencing-library preparation Page 11 of 11 Page 11 of 11 Meynert et al. BMC Bioinformatics 2014, 15:247 http://www.biomedcentral.com/1471-2105/15/247 23. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D: The human genome browser at UCSC. Genome Res 2002, 12(6):996–1006. 24. Depristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, Del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ: A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011, 43(5):491–498. doi:10.1186/1471-2105-15-247 Cite this article as: Meynert et al.: Variant detection sensitivity and biases in whole genome and exome sequencing. BMC Bioinformatics 2014 15:247. 23. References Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D: The human genome browser at UCSC. Genome Res 2002, 12(6):996–1006. 24. Depristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, Philippakis AA, Del Angel G, Rivas MA, Hanna M, McKenna A, Fennell TJ, Kernytsky AM, Sivachenko AY, Cibulskis K, Gabriel SB, Altshuler D, Daly MJ: A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011, 43(5):491–498. doi:10.1186/1471-2105-15-247 Cite this article as: Meynert et al.: Variant detection sensitivity and biases in whole genome and exome sequencing. BMC Bioinformatics 2014 15:247. doi:10.1186/1471-2105-15-247 Cite this article as: Meynert et al.: Variant detection sensitivity and biases in whole genome and exome sequencing. BMC Bioinformatics 2014 15:247. 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Im Not Sure We Had A Choice?: Decision Quality and The Use of Cardiac Implantable Electronic Devices In Older Adults With Cognitive Impairment
Cardiology and cardiovascular medicine
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Research Article Research Article Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Implantable Electronic Devices In Older Adults With Cognitive Impairment Nicole R. Fowler1,2,3*, C. Elizabeth Shaaban4, Alexia M. Torke1,2,3, Kathleen A. Lane5, Samir Saba6, and Amber E. Barnato7 1Indiana University Center for Aging Research, Indianapolis, IN, USA 2Regenstrief Institute, Inc., Indianapolis, IN, USA 3Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA 4Department of Epidemiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA 5Department of Biostatistics, Indiana University School of Medicine, Indianapolis, IN, USA 6Division of Cardiology, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA 7The Dartmouth Institute of Health Policy and Clinical Practice, Geisel School of Medicine at Dartmouth, Lebanon, NH, USA *Corresponding Author: Nicole R. Fowler, Indiana University School of Medicine, Indiana University Center for Aging Research, 1101 West 10th Street, Indianapolis, IN 46202, USA, Telephone: (317) 274-9021; E-mail: fowlern@iupui.edu Received: 30 January 2018; Accepted: 08 February 2018; Published: 12 February 2018 Keywords: Alzheimer’s Disease; Cardiac Implantable Electronic Device; Decision Making; Dementia Keywords: Alzheimer’s Disease; Cardiac Implantable Electronic Device; Decision Making; Dementia Abstract Background: The decision to implant a cardiac device in a person with Alzheimer’s disease or related dementia requires considering the possible trade-offs of quality of life (QOL) and quantity of life. This study measured the decision-making experience of patients with and without cognitive impairment (CI) who received a cardiac device and their family members who were involved in the decision. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 10 Methods and Results: Semi-structured interviews and questionnaires were administered with 15 patient-family member dyads. Interviews revealed few conversations between physicians, patients and family members about the patient’s cognitive status or about the benefits, risks, and long-term implications of the device for someone with CI. Participants largely stated that the decision to get the device was based on the patient’s functional status at the time of the implant, and not on expectations about future functioning. Patients with CI had more regret, measured with Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 10 10 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 the Decision Regret Scale (DRS), (p=0.037) and family members of patients without CI reported more decisional conflict, measured with the Decisional Conflict Scale (p=0.057). the Decision Regret Scale (DRS), (p=0.037) and family members of patients without CI reported more decisional conflict, measured with the Decisional Conflict Scale (p=0.057). Conclusions: Although CI impacts life expectancy and QOL, cognitive status was largely not discussed prior to device implant. Few differences were found between the experiences of dyads that included patients with or without CI. ogy and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-929 1. Introduction In the U.S., there are 5.5 million people with Alzheimer’s disease or a related dementia (AD) [1] and 6 million people with mild cognitive impairment (MCI) [2, 3]. The presence of AD and MCI are independent predictors of a shortened life expectancy [4-6]. Recent meta-analyses and large cohort studies indicate that people with AD have an average life expectancy of 4.5 years from diagnosis [4-8]. That is impacted by dementia subtype, patient age, and severity of impairment at diagnosis [4-8]. Family caregivers are frequently involved in medial decision-making for patients with AD regarding their medical care and treatment [9-12]. Most adults 65 and over have multimorbidity—the presence of two or more medical conditions, and this prevalence increases with age. Cardiac co-morbidities, such as coronary heart disease are common in older adults, including those with AD [13-15]. Moreover, some studies have found an association between atrial fibrillation and incident dementia, beyond its effect on stroke [16]. Among Medicare beneficiaries with AD, 29.7% have coronary heart disease, 27.8% have congestive heart failure (CHF), and 24.7% have cardiac dysrhythmias [17]. A potential treatment option for patients with these cardiac co-morbidities is the implantation of a cardiac implantable electronic device, such as a pacemaker or implantable cardioverter-defibrillator (ICD). Age and functional status at the time of device implant impact patient’s mortality post-device [18-21]. Despite having shorter life expectancies and other co-morbidities, many older adults with cognitive impairment (CI) receive cardiac devices, some beyond the time that it is clinically beneficial [12]. This raises questions about the risks of device placement in patients with CI as they are twice as likely to be admitted to a nursing home immediately and require long-term use of nursing homes after receiving a cardiac device [22]. The decision to implant a cardiac device in a person with CI involves unique considerations about the possible trade-offs of quality of life (QOL) and quantity of life [23]. Following device implantation, death from AD, which has been characterized by some as a state worse than death, may be prolonged [24-26]. We are unaware of any prior studies addressing decision-making by patients with CI, or their family members, regarding cardiac devices. The goals of this study are to compare the decision-making experience of a cohort of patients with and without CI who received a cardiac device as well as a family member who was involved in the decision to get a cardiac device. 2. Methods Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 We recruited participants from the University of Pittsburgh Alzheimer Disease Research Center (ADRC). All study procedures were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 We recruited participants from the University of Pittsburgh Alzheimer Disease Research Center (ADRC). All study procedures were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. We recruited participants from the University of Pittsburgh Alzheimer Disease Research Center (ADRC). All study procedures were approved by the Institutional Review Board (IRB) at the University of Pittsburgh. Patients in this study were active ADRC participants with or without CI, ≥50 years of age, able to read and speak English, and had received a single or dual chamber pacemaker and/or ICD after their baseline ADRC visit between 2002 and 2013. Inclusion criteria for family members included those who were named as the primary caregiver for an ADRC patient with MCI or AD or named by the non-cognitively impaired patient as the person who helped them make a decision about the device implantation. Patients were excluded from the study if they were too cognitively impaired to participate in an interview. Family members were excluded if they reported not being involved in the decision about the device implantation. We used a maximum variation sampling strategy for ADRC participants without CI and with varying levels of severity of cognitive impairment at the time of device implant [27]. Sampling for family members included someone for each living patient participant and for every deceased patient who met study criteria. Interviews were conducted with all family members and the subset of patients who were still alive and cognitively able to participate to obtain socio-demographic information, questionnaire, and semi-structured interview data. Additional research and clinical data were collected from the ADRC research database and from the patients’ medical records. 2.2 Measures The range of scores for the DRS is 0 to 100, where higher scores indicate more regret (and low satisfaction with the decision) [32-34]. Scores >25 are considered to reflect significant ambivalence about a decision and scores between 10-25 are defined as mild ambivalence [33]. Semi-structured interview data was collected from all enrolled family members and the seven patients who were still alive and cognitively able to participate, including five patients without CI and two patients with CI. Interviews were conducted in-person and over the phone and separately from the other dyad member. Interview questions were developed by the research team and piloted with the first two interviews. Two study team members (NRF and CES) conducted all in-depth interviews with questions that were broad and open-ended. We invited participants to relate their personal narratives regarding their experiences with decision-making about cardiac device implantation. The audio recordings were examined and transcribed, and no systematic differences were discerned between interviewers. Participants responded in an equally open and forthcoming way to both interviewers. To maximize the trustworthiness of our data collection, we continued recruiting participants until no new major themes emerged [35, 36]. 2.2 Measures Socio-demographic data collected from patient participants included age, sex, race, and ethnicity. For patients who had died, date of death was obtained from the ADRC research database and confirmed with the family member. For family members, we collected sex, ethnicity, race, and their relationship to the patient. Cognitive, functional, and clinical data collected included the Mini-Mental State Examination (MMSE) score [28] and the Functional Activities Questionnaire (FAQ), [29] a measure of activities of daily living closest to the date of device implant. Additionally, we reviewed all of the patient’s medical records at any point during the encounter for when the device was implanted to verify the date of device implant, type of device, device vendor, indication for the device, ACC/AHA Classification noted by the implanting physician, and to determine if any diagnosis of CI (AD, MCI or other) was noted. Questionnaire data focused on measuring known constructs of medical decision-making quality. To measure role in decision making, we used the Control Preferences Scale (CPS), which consists of one item with a five-point scale.[30] Patient and family participants were categorized as having an active, shared, or passive role in the decision regarding receipt of the cardiac device. We used the Decisional Conflict Scale (DCS) [31] to measure patients’ and family members’ level of uncertainty about getting a cardiac device. The DCS contains three sub-scales: (1) uncertainty, (2) factors contributing to the 12 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 uncertainty, and (3) perceptions of effective decision-making. The DCS includes 16 questions regarding a medical decision the participant has made or is about to make using a five-point Likert scale [31]. The range of scores for the DCS is 0 to100, where higher scores indicate more conflict. uncertainty, and (3) perceptions of effective decision-making. The DCS includes 16 questions regarding a medical decision the participant has made or is about to make using a five-point Likert scale [31]. The range of scores for the DCS is 0 to100, where higher scores indicate more conflict. To measure regret regarding the decision to get a cardiac device, we used the Decision Regret Scale (DRS), [32] which measures “distress or remorse after a health care decision”. 2.3 Analysis We conducted qualitative analyses of the interview data guided by the methods of grounded theory [36]. The steps included open coding, assigning topical codes to the text of the interviews to form a codebook, organizing themes, comparing the content of each new interview to the existing codebook categories and modifying the codebook accordingly and developing categories and defining the relationships among them and their possible implications [37]. To ensure reliability between the coders in identifying and assigning codes, each coder reviewed a random 10% sample of the transcripts coded by the other research team member. The crosschecking coder could either endorse or contest the existing coding. Following a consensus meeting of the coders, the contested codes were deleted, replaced, modified, or added to the codebook. Statistical analyses for the questionnaire data included descriptive analysis of the population, including means, standard deviations for continuous data and counts and percentages for categorical data. T-tests were used to compare the DRS and DCS scores of both patients and family members by the type of device and patient cognitive status at the time of implant. 3. Results 13 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 13 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Twenty-five patients received a pacemaker or ICD during the observation period; 10 patients or family members declined to participate. Of the15 patient-family member dyads who participated, all family members participated in an interview and completed questionnaires. Seven patients from the dyads were alive and cognitively able to participate in an interview and completed questionnaires. More patients with CI at the time of device implant were deceased or too impaired to participate at study enrollment than patients who were not cognitively impaired. The mean age (SD) of the patients at the time of device implant was 80.9 (SD 5.1). Sixty percent were male, and 100% were white. At the time of device implant, eight patients had a diagnosis of AD, two had MCI, and five had no CI. Nine patients received a pacemaker and six received an ICD. Twelve patients received a device for an AHA/ACA Class I indication defined as evidence and/or general agreement that the device would be beneficial, useful, and effective for the patient [12, 38, 39]. 2.3 Analysis Six had sinus node dysfunction, four received a device for the primary prevention of sudden cardiac death (SCD), three for atrioventricular block, and two for secondary prevention of SCD. Family members who participated in this study were mostly female (80%); eight were the spouse of the patient (53%), and seven (47%) were an adult child. Table 1 shows the descriptive data of the patient-family dyads. Family members who participated in this study were mostly female (80%); eight were the spouse of the patient (53%), and seven (47%) were an adult child. Table 1 shows the descriptive data of the patient-family dyads. Patient n=15 Caregiver n=15 Age*, years mean (SD) 80.9 (5.1) Sex, n (%) Male 9 (60) 3 (20) Female 6(40) 12 (80) Race, n (%) White 15 (100) 15 (100) Relationship to the patient, n(%) Spouse 8 (53.3) Adult child or child-in-law 7 (46.7) Cognitive status of patient,* n (%) AD 8 (53.3) Mild cognitive impairment 2 (13.3) Not impaired 5 (33.3) MMSE Score,* mean (SD) 24.7 (5.1) FAQ score,* mean (SD) 6.4 (7.9) Device Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 14 DOI: 10.26502/fccm.92920032 Pacemaker, n (%) 9 (60) Right ventricular only 2 Right atrial and right ventricular 7 ICD, n(%) 6 (40) Right ventricular only 2 Right atrial and right ventricular 3 Right atrial, right ventricular and left ventricular 1 Indications for the device Sinus node dysfunction 6 (40) Primary prevention of Sudden Cardiac Death 4 (26.7) Atrioventricular block 3 (20) Secondary prevention of Sudden Cardiac Death 2 (13.3) Indications for device therapy expressed in the standard ACC/AHA Classification format± I 12 (80) - II 0 - IIa 0 - IIb 3 (20) - III 0 - Role in the decision to get a cardiac device‡, n (%) Active or Shared 5 (71.4) 11 (73.3) Passive 2 (28.6) 4 (26.7) *Values at the time of device implant †Class I: Conditions for which there is evidence and/or general agreement that a given procedure or treatment is beneficial, useful, and effective. Class II: Conditions for which there is conflicting evidence and/or a divergence of opinion about the usefulness/efficacy of a procedure or treatment. Class IIa: Weight of evidence/opinion is in favor of usefulness/efficacy. Class IIb: Usefulness/efficacy is less well established by evidence/opinion. 2.3 Analysis Class III: Conditions for which there is evidence and/or general agreement that a procedure/treatment is not useful/effective and in some cases may be harmful. T bl 1 Ch t i ti f ti t d i 3.2 Qualitative Findings As shown in Table 2, three main themes emerged from the interviews: (1) the limited influence of the patient’s cognitive status in medical decision-making about cardiac co-morbidities and cardiac devices, (2) circumstances around the decision to get the device, and (3) discussions about the risks, benefits, and long-term implications of getting a cardiac device. Representative quotations for each of these themes from the patients and caregivers are shown in Table 2. Theme Quotation number Respondent information Representative Quotation Limited influence of the patient’s cognitive status in medical decision- making 1 Spouse of a patient with AD who received ICD “Oh yeah, [telling the doctor she has AD] that’s one of the first things I mention to all the doctors. In fact, they had a sitter several days with her [while in the hospital]. That’s one of the first things I mention to medical people. No one mentioned her AD when we discussed the pacemaker.” 2 Family member of a patient who had MCI at the time of their implant that progressed to AD at the time of the interview who received ICD “Once the [device] is in there, if you have other kinds of medical problems, it could keep you going kind of artificially rather than letting your life end in a natural kind of way. Just think about that. Is that what you want?” 3 Patient without CI who received pacemaker “Unless I am so debilitated, so sick, I don’t want to continue, but I can’t envision that at the moment.” Circumstances around the decision to get the device 4 Daughter of a patient with AD who received a pacemaker “I believed it was urgent. It was a priority according to the doctors. It wasn’t something that you could debate.” 5 Spouse of a patient with MCI who received an ICD for primary prevention of sudden cardiac death “Well, the doctor strongly advised it because he was [in] atrial fib, and so he advised us then to put it in. He just presented to us what difference it would make in him and, you know, that he’d have to feel better afterwards because it [the atrial fib] was weakening his heart. He really highly recommended that we do it.” 6 Family member of a patient without CI who received a pacemaker “My role was to see what he [the patient] decides and to discuss with it him. Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Table 1: Characteristics of patients and caregivers 15 Cardiol Cardiovascmed 2018; 2 (1): 010-026 3.2 Qualitative Findings Discuss the options, and let him make the decision.” Representative Quotation “Oh yeah, [telling the doctor she has AD] that’s one of the first things I mention to all the doctors. In fact, they had a sitter several days with her [while in the hospital]. That’s one of the first things I mention to medical people. No one mentioned her AD when we discussed the pacemaker.” “Well, the doctor strongly advised it because he was [in] atrial fib, and so he advised us then to put it in. He just presented to us what difference it would make in him and, you know, that he’d have to feel better afterwards because it [the atrial fib] was weakening his heart. He really highly recommended that we do it.” “My role was to see what he [the patient] decides and to discuss with it him. Discuss the options, and let him make the decision.” 16 16 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Ca diol Ca diovascmed 0 8; ( ): 0 0 0 6 O : 0. 650 /fccm.9 9 003 7 Patient without CI at the time of ICD implant but who had progressed to MCI at the time of the interview “I didn’t want it then, and I don’t want it now. I think the whole thing was ridiculous.” 8 Spouse of a patient without CI who received an ICD “On that [ICD] decision that was made – I don’t worry about it anymore. I think I made the right one. Even if it wasn’t the right one, it seems to be working out.” 9 Patient without CI and an ICD “I’m not sure I had a choice. My thing is, now that I look back, I felt that, if this is necessary, let’s do it.” 10 Patient without CI who received a pacemaker “They [the doctors] just suggested. It was almost like that is the way they thought it should be done. I mean, that I should have the pacemaker, and I went along with it, because I thought that is probably what I needed.” 11 Adult child of a patient with AD who received pacemaker “Obviously if it would prolong her life and preserve her QOL by doing it, then I would go for it. Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 3.3 CI was Not a Factor in Decision Making Few patients with CI or their family members reported having a discussion about the patient’s cognitive status with the physician. In all cases but one, it was unclear from the interviews and medical record reviews if the physician was even aware of the patient’s MCI or AD or if they believed that it was an important factor to be discussed. Only one out of ten patients with CI had a diagnosis or notation in the medical record. Because the patients and family members largely did not bring up the patient’s CI with the physician, there was no discussion about the impact of the device on prolonging life, improving QOL, or risks post-implantation in the setting of also having MCI or AD. Even in the one case where it was made explicit to the medical team about the patient’s AD, it was not discussed in the context of the cardiac device (Quote 1). None of the patients with cognitive impairment or the family members of patients with cognitive impairment, reported discussing their cognitive status with the physician implanting the device or its role in the decision to get a device. As previously mentioned, the medical records largely lacked information on the patients’ cognitive status; it was noted in only one case. Although family members acknowledged the patient’s MCI or AD during the interviews, opinions were mixed as to whether the CI should influence the decision-making process for getting a cardiac device. Most had a difficult time articulating the impact of AD on life expectancy and its role in medical decision-making about cardiac co-morbidities, and many were unaware of the trajectory and prognosis of AD. Patients and family members largely stated that the decision to get the device was based on the patient’s functional status at the time of the implant; however, a majority of them could imagine a time when the patient’s CI would be too severe to get a device and acknowledged that they would focus on QOL only (Quote 2). Regarding the future health status and the need for re-implantation or a new battery or device, all seven patients interviewed stated that they would make the same decision. However, half of them stated that they might reconsider based on their functional status at the time (Quote 3). 3.4.1 Urgent Need Both patients and family members perceived the decision to get the device as urgent. All patients received a device during an inpatient stay; no devices were placed electively, although many had a history of abnormal heart rhythm or cardiac disease (Quote 4). 3.2 Qualitative Findings But if all the surgery would do is maybe prolong her life, but if she no longer had any QOL, then I would be reluctant to do it.” Risks, benefits and long-term implications of getting a cardiac device 12 Adult child of a patient with MCI that received an ICD “When something like this happens, I think you don’t know what to ask. You’re just not informed enough as a layperson. We have no experience with this kind of thing, so you don’t even know what kind of questions you should ask, and if you should do something differently… Little things like how is that going to affect him, or how much does it cost, or can it wait... I don’t think we knew enough about what to ask.” 13 Family member of a patient with AD who received ICD “I would want to know how does it work; what are some of the side effects; what are the long-range aspects; can it keep someone alive; if they happened to go into a vegetative state, is it something that would cause someone to live longer than what they would normally want to live in a bad situation?” Table 2: Themes and Quotations “When something like this happens, I think you don’t know what to ask. You’re just not informed enough as a layperson. We have no experience with this kind of thing, so you don’t even know what kind of questions you should ask, and if you should do something differently… Little things like how is that going to affect him, or how much does it cost, or can it wait... I don’t think we knew enough about what to ask.” 17 3.4.6 Risks, benefits and long-term implications of getting a cardiac device Few family members and patients reported having a conversation about the benefits, risks, and long-term implications of getting the device. Many reported that they were unsure what types of questions to ask because they did not have any experience with cardiac devices (Quote 12). Many family members reported wishing they would have discussed more about the risks and alternatives to getting a cardiac device (Quote 13). Family members also expressed interest in learning more information regarding the benefits of the device and if it would increase the patient’s life expectancy and what specific symptoms should improve after the device is implanted. 3.4.4 Not a Decision An overwhelming majority of patients and family members did not acknowledge that getting the device was a decision and were strongly influenced by the presentation of information by the physician, especially regarding how getting it would impact symptom relief (Quote 9). In some cases, patients and family members reported that the physician described the implant of a cardiac device as “necessary” and would help the patient “feel better”. In other cases, patients and family members did not perceive getting the cardiac device as a choice, rather a decision that was made by their doctor and simply presented to them (Quote 10). 3.4.5 QOL Discussions with the patient’s physician tended to focus on the “necessity” of the device rather than the benefits and risks. Topics focused more on the benefits of getting the device, including enhanced QOL and symptom relief. Some family members thought or were told that that receipt of a device might have a positive impact on the patients’ cognitive function. Family members largely stated that the biggest factor was to improve the QOL for the patient (Quote 11). 3.4.2 The Physician’s Recommendation Many family members and patients believed that if the physician says the patient needs it, they must need it and there are no other options. Family members reported that the physician strongly advised that the patient get the device (Quote 5). 6). Regarding the role of the family member in decision-making, five stated that they went along with what the physician recommended without discussion, and ten reported that they were involved in the decision-making (Quote 18 ogy and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-929 Cardiol Cardiovascmed 2018; 2 (1): 010-026 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 3.4.3 Decision Regret When asked about whether the decision to get a cardiac device was a “good” or “bad” decision, or if they had regret, some patients reflected that it was not a “good decision” (Quote 7). Some family members of patients with CI also reported reflecting on whether getting the device was a “good decision” (Quote 8). 4. Conclusion The present study was undertaken to describe and compare the decision-making experience of patients with and without CI and their family members regarding cardiac devices. While a variety of circumstances precipitated the decision to implant a cardiac device, most felt that there was no decision to make and were greatly influenced by their physician’s recommendations. All patients in this study met the indications for the device; yet, few of the indications were described as clinically urgent in the medical record, despite what some dyads reported. The state of uncertainty about future events related to a decision is a key element of all decision-making, and decision makers often experience regret when decision outcomes, particularly poor outcomes, are compared with alternative outcomes had another option been chosen [38]. Even the most informed patients may have regret and psychological consequences if the outcomes of their treatment are not as expected or if treatment-related side-effects compromise their QOL [40]. However, this belief has not been assessed in adults who receive a cardiac device while already having CI or among their family members. Cognitive function and progression of AD affect prognosis and QOL of the patient and, therefore, should be incorporated into medical decision making. Circumstances in which CI co-occurs with other medical conditions will be increasingly common in our age of multimorbidity. The results of the present study suggest that CI is rarely incorporated despite the fact that functional status of the patient at the time of implant was a strong influence on decision-making. Indeed, most participants exhibited decision myopia [22] and had a difficult time articulating the relationship between having MCI or AD and its role in medical decision making about cardiac co-morbidities. Thus, further studies are required to analyze particular decision needs among patients with CI and their family caregivers. Additionally, patients with no CI were more likely to report that they made the decision to get the device either alone or with their doctor. In many cases, the patient’s description of the process was congruent with that of the family member. Family members of patients with CI at the time of device implant were more likely to report their role in the decision as either active or shared, both with and without the patient’s involvement. Many patients and family members described the physician’s role as active. 3.4.7 Decision Quality Measures Most participants described having an active role in the decision and levels of decisional regret and decisional conflict were low to moderate. While statistical tests should be interpreted cautiously in this small sample, there were some statistically significant differences between patients and families by patient cognitive status. Overall, patients had an average DRS score of 16.4 (SD 24.4, range 0-65) while the average DRS score for family members was 14.9 (SD 19.1, range 0-50). The average DRS score for patients with CI was 45 (SD 28.3), and for patients without CI, the average DRS score was 5 (SD 11.2) (p=0.03). There was no difference in DRS scores for patients who received a pacemaker vs. an ICD (p=0.12) or their family members (p=0.28). Patients had an average DCS score of 38.5 (SD 36.1, range, 3.1-100) while family members reported slightly lower DCS, with a mean DCS score 19 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 19 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 of 38.2 (SD 24.9, range 1.6-100). Family members of patients without CI reported higher levels of decisional conflict at the time of the decision to implant a device compared to family members of patients with CI (DCS scores 57.8 vs. 30.3, p=0.06) although differences did not meet statistical significance in this small sample. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 20 4. Conclusion Although patients and family members did not overwhelmingly report feelings of regret about getting a device in the questionnaires, expressions from the sample were more conflicted in the interviews. Some patients and family members had regret or questioned whether the decision was “the right one.” However, few patients endorsed a sense of regret, which may be related to the fact that few patients and family members reported any adverse events as a result of getting the device, which has been correlated with regret [41, 42]. If faced with the decision to replace the battery, most patients and family members felt they would make the same decision again; however, many said that they might reconsider based on their functional status at that time. 20 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Regarding medical decision-making, there are two main sources of conflict. The first is the inherent difficulty that arises from making decisions that have both positive and negative effects. The second source is from aspects of the decision that are modifiable (e.g., lack of knowledge about options and outcomes, unrealistic expectations, unclear values, unclear perceptions of those presenting information, social pressures, lack of support or mismatch between the preferred role in decision-making and the actual role, lack of skills and self-confidence to make the decision, and lack of other resources) [38]. In the present study, patients and family members reported limited discussion regarding their decision to get a cardiac device. There was little evidence that the physicians who were implanting the cardiac devices had knowledge of or engaged in discussions about the patient’s cognitive status; an indication of dementia was identified in only one medical record. This is consistent with previous research showing providers’ lack of awareness of patient’s cognitive status and a reluctance to have discussions about both the risks and benefits of device implantation and deactivation [41, 42]. Many noted that their physicians concentrated on the “necessity” of the device, and few family members felt that there was a real choice, deferring to the physician’s recommendations. This is consistent with previous studies about other cardiac devices, including left ventricular assist devices (LVADs) [43]. Some family members thought or were told that that receipt of a device might have an impact on the patients’ cognitive function; however, evidence is mixed to support this claim [45, 46]. 4. Conclusion The family members’ lack of informed thinking about the implications of the device on longer-term quantity and QOL in their loved one with CI, as well as a lack of competing death risks is similar to that reported in other studies of cardiac devices [22, 44, 47]. However, many family members reflected that they would have liked to have more discussion regarding the risks and alternatives to getting a cardiac device. Further studies will evaluate the experiences of dyads that included patients with CI who were eligible for cardiac devices, but chose not to receive them. Patients with AD and MCI endorsed more regret on the DRS, although the sample size limits our confidence in statistical comparisons. DRS scores for family members in the present study are comparable to what has been published in the literature for family caregivers who are involved in a decision for a family member with AD and who do not receive any support in the decision [48]. Whereas no differences in decisional conflict were observed in the patients irrespective of cognitive status, differences were noted in the family members. Among the family members of patients without CI who reported higher decisional conflict, they also reported being less certain about the risks and benefits at the time of implant compared to those family members of patients with CI. These findings are likely driven by the differences among the role of the caregiver in their role in medical decision-making (i.e., active vs. shared/passive). Although these results are counter to what we found regarding decisional regret and to what we hypothesized, they are similar to what has been reported in the literature for surrogate decision makers who do not receive any support for decision-making [49]. In the present study, patients received a pacemaker or ICD for a variety of reasons, including sinus node dysfunction, primary or secondary prevention of sudden cardiac death, and atrioventricular block. It is, therefore, possible that the decisions surrounding receipt of these devices may differ by device or indication. For example, primary prevention ICD is used to prolong life without improving QOL whereas a pacemaker for sinus node dysfunction could not only prolong life, but also improve QOL if the patient was experiencing syncope, fatigue, or palpitations. Moreover, the decision to receive a pacemaker for symptomatic bradycardia may be relatively Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. 4. Conclusion [ISSN 2572-9292] 21 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 straightforward, particularly in a patient with MCI, as compared with the decision to implant a primary prevention ICD without cardiac resynchronization therapy (CRT) in an asymptomatic patient, which poses considerable trade- offs. Thus, further studies with larger cohorts are necessary to explore the decision-making experiences of patient and caregiver dyads by cardiac device type. In addition to the small sample size, the present study is limited by the single site data and lack of racial or ethnic diversity in the sample, which have been shown by others to impact receipt of device [50]. In addition, for those patients who died following the procedure, the cause of death was unknown. Furthermore, the time since receiving the cardiac implant was considerable for some dyads (up to 11 years); thus, there may have been recall bias as well as survival bias, particularly for those who received a dual chamber pacemaker or ICD. Finally, there was no medical record data regarding the incidences of cardioversion or adverse events. In conclusion, although the decision to receive a cardiac device in a person with AD requires unique considerations about the possible trade-offs of quality versus quantity of life, the patients’ cognitive status was largely not discussed with their family or physician. Most patients and family members described the procedure as “necessary” to improve the patient’s QOL, but some patients with CI had expressed regret or questioned whether the decision was “right.” Both patients and family members expressed a wish to have more information regarding the risks, benefits, and alternatives, and a majority of family members could envision a time when the patient’s cognitive and functional impairment would be too severe to get a device and acknowledge that they would focus on QOL. Multimorbidity is the new normal in our aging society, and thus medical decision-making will increasingly involve trade-offs between various lifespan and QOL-increasing interventions, which have implications for how we prefer to live and die. A better understanding of medical decision-making in these circumstances is critical to assist patients in making decisions that are right for them. Acknowledgements We would like to thank MaryAnn Oakley, MA of the University of Pittsburgh ADRC for assistance with recruitment and Jeff Lee, MD with his assistance reviewing the medical records of patient participants. Sources of Funding This study was supported by the National Institutes of Health, National Institute on Aging grant P50 AG005133. References References 1. Association. As. 2017 Alzheimer's disease facts and figures. Alzheimers Dement 13 (2017): 325-373. 1. Association. As. 2017 Alzheimer's disease facts and figures. Alzheimers Dement 13 (2017): 325-373. 2. De Bruijn RF, Akoudad S, Cremers LG, Hofman A, Niessen WJ, et al. Determinants, MRI correlates, and prognosis of mild cognitive impairment: the Rotterdam Study. J Alzheimers Dis 42 (2014): S239-S249. 2. De Bruijn RF, Akoudad S, Cremers LG, Hofman A, Niessen WJ, et al. Determinants, MRI correlates, and prognosis of mild cognitive impairment: the Rotterdam Study. J Alzheimers Dis 42 (2014): S239-S249. 3. 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Conflicts of Interest Conflicts of Interest The authors Fowler, Shaaban, Torke, Lane, and Barnato have no conflict of interest regarding the publication of this article. The authors Fowler, Shaaban, Torke, Lane, and Barnato have no conflict of interest regarding the publication of this article. Saba has received research funding from the National Heart, Lung, and Blood Institute and significant research grant support from Boston Scientific. Saba has received research funding from the National Heart, Lung, and Blood Institute and significant research grant support from Boston Scientific. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 22 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. 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J Cardiovasc Electrophysiol 13 (2002): 1183-1199. 39. Mitty E and Post LF. Healthcare Decision Making. In: E. C. M. Boltz, T. Fulmer, & D. Zwicker ed. Evidence-based geriatric nursing protocols for best practice 4th ed. New York: Springer Publishing Company 2012: 562-578. 40. Davison BJ and Goldenberg SL. Decisional regret and quality of life after participating in medical decision-making for early-stage prostate cancer. BJU Int 91 (2003): 14-17. 41. Goldstein NE, Lampert R, Bradley E, Lynn J and Krumholz HM. Management of implantable cardioverter defibrillators in end-of-life care. Ann Intern Med 141 (2004): 835-838. 42. Russo JE. Original research: deactivation of ICDs at the end of life: a systematic review of clinical practices and provider and patient attitudes. Am J Nurs 111 (2011): 26-35. 43. Blumenthal-Barby JS, Kostick KM, Delgado ED, Volk RJ, Kaplan HM, et al. 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Strauss A and Corbin J. Grounded Theory Methodology. In: N. D. Y. Lincoln, ed. Handbook of Qualitative Research Thousand Oaks: Sage Publications 1994: 217-285. 37. Holton JA. The coding process and its challenges. In: K. C. A. Bryant, ed. The Sage handbook of grounded theory Thousand Oaks, CA: Sage Publications; 2007. 38. Gregoratos G, Abrams J, Epstein AE, Freedman RA, Hayes DL, et al. American College of Cardiology/American Heart Association Task Force on Practice Guidelines American College of Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 24 Cardiol Cardiovascmed 2018; 2 (1): 010-026 DOI: 10.26502/fccm.92920032 Cardiology/American Heart Association/North American Society for P and Electrophysiology C. ACC/AHA/NASPE 2002 guideline update for implantation of cardiac pacemakers and antiarrhythmia devices: summary article. Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 25 Citation: Nicole R. Fowler, C. Elizabeth Shaaban, Alexia M. Torke, Kathleen A. Lane, Samir Saba, and Am ber E. Barnat. “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Im plantable Electronic Devices In Older Adults With Cognitive Impairment. Cardiology and Cardiovascular Medicine 2 (2018): 010-026. References ACC/AHA/HRS 2008 Guidelines for Device-Based Therapy of Cardiac Rhythm Abnormalities: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the ACC/AHA/NASPE 2002 Guideline Update for Implantation of Cardiac Pacemakers and Antiarrhythmia Devices) developed in collaboration with the American Association for Thoracic Surgery and Society of Thoracic Surgeons. Heart Rhythm 5 (2008): e1-e62. 49. Hickman RL, Jr., Daly BJ and Lee E. Decisional conflict and regret: consequences of surrogate decision making for the chronically critically ill. Appl Nurs Res 25 (2012): 271-275. 50. Matlock DD, Peterson PN, Heidenreich PA, Lucas FL, Malenka DJ, et al. Regional variation in the use of implantable cardioverter-defibrillators for primary prevention: results from the National Cardiovascular Data Registry. Circ Cardiovasc Qual Outcomes 4 (2011): 114-121. 25 DOI: 10.26502/fccm.92920032 DOI: 10.26502/fccm.92920032 Cardiol Cardiovascmed 2018; 2 (1): 010-026 Citation: Nicole R. Fowler, C. Elizabeth Shaaban, Alexia M. Torke, Kathleen A. Lane, Samir Saba, and Am ber E. Barnat. “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Im plantable Electronic Devices In Older Adults With Cognitive Impairment. Cardiology and Cardiovascular Medicine 2 (2018): 010-026. Citation: Nicole R. Fowler, C. Elizabeth Shaaban, Alexia M. Torke, Kathleen A. Lane, Samir Saba, and Am ber E. Barnat. “I’m Not Sure We Had A Choice”: Decision Quality and The Use of Cardiac Im plantable Electronic Devices In Older Adults With Cognitive Impairment. Cardiology and Cardiovascular Medicine 2 (2018): 010-026. This article is an open access article distributed under the terms and conditions of the This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license 4.0 Cardiology and Cardiovascular Medicine - http://www.cardiolcardiovascmed.com/ - Vol. 2 No. 1 - Feb 2018. [ISSN 2572-9292] 26 26
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Linkage, Evaluation and Analysis of National Electronic Healthcare Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care
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Linkage, Evaluation and Analysis of National Electronic Healthcare Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care Katie Harron1*, Harvey Goldstein1,2, Angie Wade1, Berit Muller-Pebody3, Roger Parslow4, Ruth Gilbert1 Katie Harron1*, Harvey Goldstein1,2, Angie Wade1, Berit Muller-Pebody3, Roger P 1 Institute of Child Health, University College London, London, United Kingdom, 2 Graduate School of Education, University of Bristol, Bristol, United Kingdom, 3 Healthcare Associated Infection and Antimicrobial Resistance Department, Public Health England, London, United Kingdom, 4 Division of Epidemiology, University of Leeds, Leeds, United Kingdom Abstract Background: Linkage of risk-factor data for blood-stream infection (BSI) in paediatric intensive care (PICU) with bacteraemia surveillance data to monitor risk-adjusted infection rates in PICU is complicated by a lack of unique identifiers and under-ascertainment in the national surveillance system. We linked, evaluated and performed preliminary analyses on these data to provide a practical guide on the steps required to handle linkage of such complex data sources. Methods: Data on PICU admissions in England and Wales for 2003-2010 were extracted from the Paediatric Intensive Care Audit Network. Records of all positive isolates from blood cultures taken for children <16 years and captured by the national voluntary laboratory surveillance system for 2003-2010 were extracted from the Public Health England database, LabBase2. “Gold-standard” datasets with unique identifiers were obtained directly from three laboratories, containing microbiology reports that were eligible for submission to LabBase2 (defined as “clinically significant” by laboratory microbiologists). Reports in the gold-standard datasets were compared to those in LabBase2 to estimate ascertainment in LabBase2. Linkage evaluated by comparing results from two classification methods (highest-weight classification of match weights and prior-informed imputation using match probabilities) with linked records in the gold-standard data. BSI rate was estimated as the proportion of admissions associated with at least one BSI. Results: Reporting gaps were identified in 548/2596 lab-months of LabBase2. Ascertainment of clinically significant BSI in the remaining months was approximately 80-95%. Prior-informed imputation provided the least biased estimate of BSI rate (5.8% of admissions). Adjusting for ascertainment, the estimated BSI rate was 6.1-7.3%. Conclusion: Linkage of PICU admission data with national BSI surveillance provides the opportunity for enhanced surveillance but analyses based on these data need to take account of biases due to ascertainment and linkage error. This study provides a generalisable guide for linkage, evaluation and analysis of complex electronic healthcare data. Citation: Harron K, Goldstein H, Wade A, Muller-Pebody B, Parslow R, et al. (2013) Linkage, Evaluation and Analysis of National Electronic Healthcare Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care. PLoS ONE 8(12): e85278. doi:10.1371/ journal.pone.0085278 Editor: Caroline L Trotter, University of Cambridge, United Kingdom Copyright: © 2013 Harron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Harron K, Goldstein H, Wade A, Muller-Pebody B, Parslow R, et al. (2013) Linkage, Evaluation and Analysis of National Electronic Healthcare Data: Application to Providing Enhanced Blood-Stream Infection Surveillance in Paediatric Intensive Care. PLoS ONE 8(12): e85278. doi:10.1371/ journal.pone.0085278 Copyright: © 2013 Harron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received July 9, 2013; Accepted November 26, 2013; Published December 20, 2013 Copyright: © 2013 Harron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from the National Institute for Health Research Health Technology Assessment (NIHR HTA) programme [project number 08/13/47]. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS or the Department of Health. PICANet is funded by the National Clinical Audit and Patient Outcomes Programme via Healthcare Quality Improvement Partnership (HQIP), Health Commission Wales Specialised Services, NHS Lothian / National Service Division NHS Scotland, the Royal Belfast Hospital for Sick Children, Our Lady’s Children’s Hospital, Crumlin, Children’s University Hospital, Temple Street and The Harley Street Clinic, London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. * E-mail: Katie.harron.10@ucl.ac.uk Introduction processing, calculation of match weights or probabilities and errors due to mis-classification in the linkage process can have substantial effects on outcome measures[13–16]. We aim to describe the steps involved in preparing and linking routine data for enhanced BSI surveillance in PICU, which are generalisable to other administrative data of this type. Blood-stream infection (BSI) is an important cause of mortality, morbidity and substantial extra cost for paediatric patients, and paediatric intensive care units (PICU) have one of the highest rates of BSI of all specialties[1–4]. The national laboratory surveillance system coordinated by Public Health England (PHE, formerly the Health Protection Agency) collects data on microorganisms submitted by hospital laboratories in England and Wales[5]. Patient-level data on all children admitted to paediatric intensive care units (PICU) in England and Wales have been collected by the Paediatric Intensive Care Audit Network (PICANet) since 2003[6]. To date, no evaluation of the potential of linking these administrative data sources for national monitoring of risk-adjusted BSI trends in PICU has been performed[7,8]. Paediatric Intensive Care Infection Surveillance Paediatric Intensive Care Infection Surveillance Ethics Statement For PICANet, collection of personally identifiable data has been approved by the National Information Governance Board (Formerly the Patient Information Advisory Group) http:// www.nigb.nhs.uk/s251/registerapp and ethical approval granted by the Trent Medical Research Ethics Committee, ref. 05/MRE04/17. PICANet also has specific permission from the National Research Ethics Service for linkage with the PHE laboratory data on bloodstream infections using personal identifiers and to share PICANet data with PHE. An exemption under Section 251 of the NHS Act 2006 (previously Section 60 of the Health and Social Care Act 2001) allows PHE to receive patient-identifiable data from other organisations without patient consent in order to monitor infectious disease. Specific permission for the PICANet-PHE linkage has been granted by NIGB. Consent for the use of the data identifying individual PICUs in this study was obtained by the relevant PICANet unit leads. Data in PICANet and LabBase2 cannot be publicly deposited as it is personally-identifiable. Access to an anonymised form of the linked data may be requested from http://www.picanet.org.uk/. There are two main obstacles to linkage for enhanced BSI surveillance. Firstly, as a voluntary system, PHE’s surveillance database (LabBase2) does not capture complete BSI data from all laboratories[5]. Hospital laboratories are requested to report any clinically significant bacterial infections and clinically significant isolates from sterile sites such as blood, although there are no specific guidelines for judgement of clinical significance and non-clinically significant isolates or contaminants may also be present in the data. Data are not always captured consistently, with staffing issues and IT compatibility problems causing incomplete and variable reporting over time. Ascertainment of MRSA and MSSA within LabBase2 in 2008 was estimated at around 70% (based on mandatory reports for methicillin-resistant and methicillin- susceptible Staphylococcus aureus) although ascertainment for all-cause bacteraemia in children is unknown[9]. Secondly, linkage between data sources is complicated due to a lack of well-completed unique identifiers in LabBase2. For data such as these, the method of choice for linkage is often to calculate probabilistic match weights (or match probabilities) that measure the similarity between records from different sources, taking into account possible identifier errors or missing values[10,11]. These weights or probabilities are then used to classify record pairs as links or non-links. Linkage process Figure 1 displays the steps required for obtaining data for enhanced BSI surveillance through linkage between PICANet and LabBase2. Abstract Funding: This work was supported by funding from the National Institute for Health Research Health Technology Assessment (NIHR HTA) programme [project number 08/13/47]. The views and opinions expressed therein are those of the authors and do not necessarily reflect those of the HTA programme, NIHR, NHS or the Department of Health. PICANet is funded by the National Clinical Audit and Patient Outcomes Programme via Healthcare Quality Improvement Partnership (HQIP), Health Commission Wales Specialised Services, NHS Lothian / National Service Division NHS Scotland, the Royal Belfast Hospital for Sick Children, Our Lady’s Children’s Hospital, Crumlin, Children’s University Hospital, Temple Street and The Harley Street Clinic, London. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist 1 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 1 Data extraction and de-duplication Data on all PICU admissions for children <16 years for 2003-2010 were extracted from PICANet (n=109,654 records). Each PICANet record corresponded to an individual PICU admission within one of twenty-two PICUs admitting more than 200 children per year in England and Wales. Records of all positive isolates from blood culture captured by the national surveillance system were extracted for children <16 years between 2003-2010 extracted from LabBase2 (n=80,009). Classification is typically based on highest-weighted (HW) pairs, where the candidate record with the highest weight is accepted as a link, given it exceeds a pre-specified threshold. However, errors can be introduced if the highest-weighted record is not the correct match (false-matches), or if no candidate record exceeds the threshold (missed-matches). An alternative classification method is prior-informed imputation, which aims to avoid bias associated with these linkage errors. Prior-informed imputation works by accepting values for variables of interest within a multiple imputation framework, rather than by linking a complete record[12]. Values are selected according to Information from a prior distribution (based on match probabilities in candidate linking records) combined with a likelihood derived from unequivocally-linked records[12]. PICANet admission records could link to none, one or more LabBase2 specimen records. If an admission record linked to multiple specimens of the same organism (within 14 days), only the first specimen was retained. LabBase2 specimen records could link to more than one admission if a specimen fell within the timeframe for two admissions at once (if the admissions were consecutive). In this case, the specimen was linked to the earlier of the two admissions, so that each LabBase2 record linked to at most one admission record. There is a lack of practical guidance on the complex process required to link and analyse national administrative data such as PICANet and LabBase2. Methods used for data pre- December 2013 | Volume 8 | Issue 12 | e85278 PLOS ONE | www.plosone.org 2 Paediatric Intensive Care Infection Surveillance Figure 1. Steps involved in linkage of PICANet and LabBase2 for enhanced BSI surveillance in PICU. doi: 10.1371/journal.pone.0085278.g001 Figure 1. Steps involved in linkage of PICANet and LabBase2 for enhanced BSI surveillance in PICU. doi: 10 1371/journal pone 0085278 g001 Figure 1. Steps involved in linkage of PICANet and LabBase2 for enhanced BSI surveillance in PICU. Data extraction and de-duplication doi: 10.1371/journal.pone.0085278.g001 The representativeness of the gold-standard data was assessed by comparing characteristics of laboratories and PICUs providing gold-standard data with those that did not. Gold-standard data “Gold-standard” microbiology datasets were obtained directly from three hospital laboratories: Birmingham Children’s Hospital (BCH), Oxford University Hospital (OUH) and Royal London Hospital (RLH). These laboratories were chosen as they were able to provide microbiology data that were eligible for reporting to LabBase2 (i.e. defined as clinically significant my laboratory microbiologists) and included unique identifiers. The gold-standard datasets were used for two purposes: December 2013 | Volume 8 | Issue 12 | e85278 Ascertainment evaluation Incomplete reporting in LabBase2 was identified through manual inspection of plots of the total number of reports (all ages) of bacteraemia for individual laboratories by specimen month. Data were inspected for all ages, as numbers for children only were low, and reporting gaps were expected to relate to the laboratory as a whole rather than to an individual ward. Within individual laboratories, months during which no reports were submitted were defined as a reporting gap. In addition, months during which an unrealistically small number of reports were present were defined as having incomplete reporting. Unrealistic numbers of reports were identified through careful manual inspection of reports over time: due to the fluctuation of reports from month to month and variation in size of laboratories, a consistent definition of incomplete reporting could not be applied across all laboratories and so a conservative judgement on incomplete reporting was made. 1. To estimate ascertainment in LabBase2 by comparing records that were eligible for reporting to LabBase2 with records that actually appeared in LabBase2 (BCH and RLH) 2. To evaluate linkage error by comparing records linked using incomplete identifiers within LabBase2 with records linked using well-completed unique identifiers within the gold-standard data (BCH and OUH). Any uncertain links in the gold-standard data were verified with additional information from the hospital. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 3 Paediatric Intensive Care Infection Surveillance Lab-months classified as incomplete reporting were excluded from analysis. beginning with S, and the frequency-based weight represented this difference. For the remaining months, ascertainment of clinically significant BSI for children <16 years was estimated as the proportion of eligible records in the gold-standard data from BCH (March 2003-December 2010) and RLH (July 2006- December 2009) captured within LabBase2. BCH and RLH contained information on whether a specimen was deemed to be clinically significant (and therefore eligible for submission to LabBase2); this information was not available for OUH. The distribution of match weights for links and non-links was plotted to assess the performance of the match weights at separating links and non-links. Record pairs were then ordered by match weight and manually inspected to identify obvious non-links that had high weights, and probable links with low weights. Subsequent training datasets were obtained by retaining probable links identified through this review. Blocking The total number of pairwise comparisons between PICANet and LabBase2 would produce of 80,009 x 109,654 = 8,773,306,886 comparison pairs. Comparison pairs were therefore restricted to those where the specimen date fell within 3 days of a PICU admission, as errors in date variables were assumed to be trivial (unpublished analysis showed <1% of specimen dates were inconsistent with data upload dates). To further reduce the number of comparison pairs, several blocking variables were chosen, so that records were only compared if they agreed on at least one of Soundex, initial, postcode prefix, NHS number, hospital number or day of birth. This blocking scheme assumed that records not agreeing on any of these blocking variables did not belong to the same individual. For this project, manual review for uncertain links was not possible. This was because no additional external data was available, and uncertain links often contained only Soundex and date of birth, which did not provide enough information to positively determine link status by eye. Records were therefore classified as links or non-links based on a single cut-off weight, based on capturing probable links identified in the training datasets. A sensitivity analysis was performed by repeating this process with two different cut-offs. The first cut-off (relaxed threshold) aimed to capture as many of the probable links as possible. The second cut-off (conservative threshold) aimed to exclude as many non-links as possible. Any records with a match weight above the threshold was classified as a link, and all others were classified as non-links. Match probability calculation Completeness of common identifiers for linking varied between datasets and by time (identifiers were more complete in recent years). For LabBase2, completeness of identifiers varied by unit (Figure 2). For PICANet, date of birth and hospital number were 100% complete and the majority of other identifiers were >98% complete, with the exception of NHS number (85% complete). For both datasets, cleaning and data preparation were undertaken: NHS or hospital numbers such as “Unknown” or “9999999999” were set to null; generic names (e.g. “Baby”, “Twin 1”, “Infant Of”) were set to null; multiple variables were created for multiple surname and first names; postcodes beginning “ZZ” (indicating no UK postcode) were set to null. Match probabilities P(M|agreement pattern) were calculated to estimate the probability of a match given agreement on a joint set of identifiers. This avoided the assumption of independence between identifiers. Probabilities were derived as the number of links divided by the total number of pairs for each agreement pattern (based on probable links identified in the training datasets). For example, if 378 comparison pairs agreed on date of birth and Soundex but disagreed on sex, and 312 of these were probable links, the match probability for the agreement pattern [ 1,1,0 ] was 312/378=0.825. Classification of links 1: Highest-weight (HW) classification. Traditionally, candidate linking records are ordered by match weight, and only the comparison pair with the highest probabilistic weight is classified as a link. All remaining candidate records are discarded (highest-weight classification). Comparison pairs are classified into non-links, links and uncertain links, based on the value of the match weight. Uncertain links are then classified through manually inspecting the identifiers on each record, to determine whether or not they belong to the same individual. Manual inspection makes use of the fact that the human eye can recognise matches that a computer would discard (e.g. Liz and Elizabeth) and can involve the use of additional identifiers if available. Ascertainment evaluation This process was iterated a number of times, until match weights from consecutive training datasets were stable (Figure 3). Match weight calculation Match weight calculations were based on the Fellegi-Sunter method[11,17]. ‘Training’ datasets of record pairs assumed to be matches were used to estimate m- and u-probabilities P(agreement|match) and P(agreement|non-match) for individual identifiers. The first training dataset took records agreeing on NHS number or hospital number as assumed matches. The same training dataset was used to create a list of non-matches by cross-joining all record pairs and removing those agreeing on NHS number or hospital number. Frequency-based weights were calculated for surname, first name, sex and Soundex so that m- and u-probabilities were allowed to vary according to how rare or common a value was[18]. These were calculated by estimating m- and u- probabilities within groups for each identifier. For example, surnames beginning with Z were less common than surnames 2: Prior-informed imputation). Prior-informed imputation was performed as proposed by Goldstein et al, using Stat-JR software developed by the University of Bristol[12,19]. Linkage between PICANet and LabBase2 was ‘incomplete’, as PICANet records that did not have a BSI genuinely had no matching record in LabBase2. This is a special case for prior-informed December 2013 | Volume 8 | Issue 12 | e85278 PLOS ONE | www.plosone.org 4 Paediatric Intensive Care Infection Surveillance Figure 2. Completeness of identifiers in LabBase2 by PICU. doi: 10 1371/journal pone 0085278 g002 Figure 2. Completeness of identifiers in LabBase2 by PICU. doi: 10.1371/journal.pone.0085278.g002 derived using the unequivocally linked records and a set of PICANet predictor variables identified in previous analyses[7]. imputation, as all candidate records had the same outcome (link = BSI). For the remaining (equivocal) PICANet records, a prior distribution for BSI was created based on the maximum probability of a BSI in the candidate records (Figure 4). If the maximum candidate probability for BSI=1 if p, the probability that BSI=0 is 1-p. Prior-informed imputation uses match probabilities, rather than match weights, to avoid assuming independence between identifiers. If a PICANet record had a match probability>0.9, it was classed as “unequivocal” and the variable BSI was set equal to 1 (Figure 4). If a PICANet record had no candidate linking records (or the maximum candidate probability was <0.1), it was also classed as “unequivocal”, but BSI was set equal to 0. These cut-offs were based on previous simulation work for prior-informed imputation. Match weight calculation A likelihood for BSI was A modified (posterior) probability distribution was created by multiplying the above prior distribution by the likelihood and scaling to 1. For each equivocal PICANet record, BSI was set equal to 0 or 1 according to the highest modified probability for that record. If no probability exceeded 0.1, BSI was treated as PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 5 Paediatric Intensive Care Infection Surveillance Figure 3. Match weight calculation process. doi: 10.1371/journal.pone.0085278.g003 Figure 3. Match weight calculation process. doi: 10.1371/journal.pone.0085278.g003 doi: 10.1371/journal.pone.0085278.g003 subjective – for example, manual review or choice of thresholds. In our study, we present results based on two different probabilistic thresholds. missing and standard multiple imputation was used to impute a value based on the likelihood only. Five imputed datasets were produced and analysed separately, with results combined using Rubin’s rules[20]. Thirdly, comparisons of linked and unlinked data can be made, in order to identify potential sources of bias. This process can help to identify groups of subjects who may be missed from the linkage due to poor data quality. In our study, differences in the characteristics of linked and unlinked records were related to BSI: linked records corresponded to admissions with BSI and therefore represented children who had risk- factors for BSI. BSI rate: Evaluation of bias due to linkage error BSI rate was calculated as the percentage of admissions linked to >=1 BSI (within three days either side of admission). This crude rate reflects the proportion of admissions associated with an infection acquired either leading up to or during an admission. Finally, statistical techniques can be used to handle uncertainty in linkage within the analysis itself. In our study, we used prior-informed imputation to account for linkage error within analysis. There are a number of ways in which bias due to linkage error can be evaluated[21]. Firstly, comparisons with gold- standard data can be performed, based on the true match status of any record pair (known in the gold-standard data). Such data could be in the form of an external dataset including well-completed, unique identifiers, or a sample of records that have been subjected to extensive manual review. In our study, bias was estimated by comparing the BSI rate in gold-standard data obtained directly from two laboratories, with that estimated in the linked data, for each classification method. Match weight and probability calculation incomplete reporting for the entire study period (PICUs 9 and 10, Figure 5). Removing admissions during periods of incomplete reporting reduced the total number of admissions available for analysis from 109,654 to 78,525. Comparing the number of BSI records within BCH and RLH gold-standard datasets (defined as clinically significant by microbiologists) with BSI records captured by LabBase2 gave an estimated LabBase2 ascertainment (for clinically significant BSI in children <16 years) of 81.5% (95% confidence interval (CI) 79.9-83.1%; 1872/2298) and 79.5% (95% CI 75.1-83.9%; 260/327) for BCH and RLH respectively. As non-clinically significant specimens were also available in the gold-standard data, we identified that 181/2054 (8.8%) of LabBase2 reports for BCH were not clinically significant. incomplete reporting for the entire study period (PICUs 9 and 10, Figure 5). Removing admissions during periods of incomplete reporting reduced the total number of admissions available for analysis from 109,654 to 78,525. Comparing the number of BSI records within BCH and RLH gold-standard datasets (defined as clinically significant by microbiologists) with BSI records captured by LabBase2 gave an estimated LabBase2 ascertainment (for clinically significant BSI in children <16 years) of 81.5% (95% confidence interval (CI) 79.9-83.1%; 1872/2298) and 79.5% (95% CI 75.1-83.9%; 260/327) for BCH and RLH respectively. As non-clinically significant specimens were also available in the gold-standard data, we identified that 181/2054 (8.8%) of LabBase2 reports for BCH were not clinically significant. An initial three iterations of probabilistic weight calculation were performed. However, match weights did not stabilise. This was down to the failure of a number of assumptions underpinning probabilistic weight calculation. Firstly, all records should be equally likely to link. A small subset of LabBase2 records that did have well-completed data caused this assumption to fail, and weight calculations were dominated by agreement on NHS number, hospital number or name, making it difficult to distinguish between records containing only Soundex, date of birth and sex (Table 1). Secondly, agreement between identifiers should be independent. However, records with missing NHS number were also more likely to have missing surname, meaning that records failing to agree on NHS number were also likely to fail to agree on surname, meaning the independence assumption failed. The same was the case for Soundex and surname, and for separate elements of date of birth (records that disagreed on date of birth were disproportionately penalized). Finally, some weights produced for missing values were counter-intuitive (e.g. Ascertainment Figure 5 shows the variation and fluctuation in the total number of reports (all ages) submitted to LabBase2 per month for laboratories serving individual PICUs between 2003-2010. Manual inspection of data identified a total of 548/2596 lab- months with incomplete reporting. Two laboratories had Secondly, sensitivity analyses based on varying linkage criteria can be used to provide a range of plausible results. This is particularly useful when aspects of the linkage process are PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 6 Paediatric Intensive Care Infection Surveillance Figure 4. Prior-informed imputation for ‘incomplete’ linkage between PICANet and LabBase2. Predictor variables: Length of stay, age, admission type, admission source, renal status, quarter-year at admission. doi: 10.1371/journal.pone.0085278.g004 Figure 4. Prior-informed imputation for ‘incomplete’ linkage between PICANet and LabBase2. Predictor variables: Length of stay, age, admission type, admission source, renal status, quarter-year at admission. doi: 10.1371/journal.pone.0085278.g004 Match weight and probability calculation a higher weight for missing than agreement on day of birth). This was due to small m- and u-probabilities (<0.01) for missing values combined with the log-likelihood scaling convention. December 2013 | Volume 8 | Issue 12 | e85278 Blocking After removing record pairs that were not within the correct timeframe (more than 3 days outside admission), there were 3,081,719 record pairs to be compared. Blocking on NHS number, hospital number, day of birth, Soundex, initial and postcode prefix, provided a total of 1,803,808 comparison pairs. To deal with these problems, record pairs that included completed NHS number, hospital number, first name, surname, postcode and date of birth were extracted and a set of PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 7 Paediatric Intensive Care Infection Surveillance Figure 5. Total number of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. doi: 10.1371/journal.pone.0085278.g005 Figure 5. Total number of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. doi: 10.1371/journal.pone.0085278.g005 Figure 5. Total number of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. doi: 10 1371/journal pone 0085278 g005 er of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010. mber of reports (all ages) submitted to LabBase2 for laboratories serving PICUs between 2003-2010 85278.g005 deterministic rules applied (Table 2). Pairs agreeing on a deterministic rule were manually reviewed to remove false- matches based on disagreement between other identifiers, which was possible due to the high-level of identifier completeness. This deterministic process identified 6001 links. Table 1. Initial weight estimates based on first training dataset (records agreeing on NHS number or hospital number). Match weight Agreement between identifiers Disagreement between identifiers One or both identifiers missing NHS number 12.58 -7.94 -0.17 Hospital number 12.80 -2.23 0.10 Surname 6.20 -3.88 0.26 Soundex 5.26 -3.78 -0.46 First name 5.19 -3.22 0.25 Day of birth 1.28 -6.08 1.66 Month of birth 1.18 -6.81 1.66 Year of birth 0.91 -6.68 1.66 Sex 0.92 -5.63 -0.39 doi: 10.1371/journal.pone.0085278.t001 Final match weights were then calculated for the remaining records pairs, based on agreement or disagreement on date of birth (combined variable), Soundex, sex and location (Table 3). The relaxed threshold was set at 5, which was chosen to include the majority of probable and possible links whilst not including many non-links (Figure 6). The conservative threshold was set at 10, which was chosen to exclude the majority of non-links, whilst not excluding many probable or possible links. Calculation of joint match probabilities confirmed that the independence assumption did not hold (Table 4). BSI rate: Evaluation of bias due to linkage error A total of 6001 (deterministic), 6787 (highest-weighted conservative) and 8490 (highest-weighted relaxed) links were identified. Retaining only the first episode per admission and removing admissions within reporting gaps resulted in 3626, 4651 and 4043 admissions with BSI using each method. Prior- informed imputation identified 4549 admissions with BSI. Compared with gold-standard data, prior-informed imputation provided the least biased estimate of BSI rate (Table 5). After adjusting for -0.5% linkage bias and 80-95% estimated ascertainment, the crude BSI rate increased from 5.79% (initial PII estimate) to 6.13-7.28% (adjusted estimate). Representativeness of gold-standard data BCH and OUH had lower than average BSI rate (2.69% and 4.64% for respective PICUs compared with 5.79% overall). The PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 8 Paediatric Intensive Care Infection Surveillance Figure 6. Four iterations for match weight calculation. Lines=thresholds. doi: 10.1371/journal.pone.0085278.g006 Figure 6. Four iterations for match weight calculation. Lines=thresholds. doi: 10.1371/journal.pone.0085278.g006 Table 2. Identification of links through deterministic linkage. Deterministic rule Number of agreeing pairs Number of links Number of non-links NHS number or hospital number 4595 4586 9 First name, surname and date of birth 832 832 0 Postcode prefix and postcode suffix 538 416 122 Postcode prefix or postcode suffix and date of birth 94 52 42 At least 2 elements of date of birth and either first name or surname 1559 115 1444 Total reviewed 7618 6001 1617 doi: 10.1371/journal.pone.0085278.t002 Table 3. Final probabilistic match weights. Match weight Agreement Disagreement Soundex 5.18 -4.05 Date of birth 4.66 -6.89 Sex 0.91 -4.70 Location (PICU / lab) 5.53 -1.06 doi: 10.1371/journal.pone.0085278.t003 Table 2. Identification of links through deterministic linkage. Table 2. Identification of links through deterministic linkage. Table 2. Identification of links through deterministic linkage. Table 4. Match probabilities under independence and dependence assumptions. Table 2. Identification of links through deterministic linkage. Representativeness of gold-standard data Deterministic rule Number of agreeing pairs Number of links Number of non-links NHS number or hospital number 4595 4586 9 First name, surname and date of birth 832 832 0 Postcode prefix and postcode suffix 538 416 122 Postcode prefix or postcode suffix and date of birth 94 52 42 At least 2 elements of date of birth and either first name or surname 1559 115 1444 Total reviewed 7618 6001 1617 doi: 10.1371/journal.pone.0085278.t002 Agreement pattern Assuming dependence Assuming independence Sex Soundex Dob Location P(NM|g) P(M|g) P(NM|g) P(M|g) 0 0 0 0 1.000 0.000 1.000 0.000 0 0 0 1 1.000 0.000 1.000 0.000 0 0 1 0 1.000 0.000 1.000 0.000 0 0 1 1 1.000 0.000 0.998 0.002 0 1 0 0 1.000 0.000 1.000 0.000 0 1 0 1 0.993 0.007 0.999 0.001 0 1 1 0 0.883 0.117 0.957 0.043 0 1 1 1 0.600 0.400 0.530 0.470 1 0 0 0 1.000 0.000 0.999 0.001 1 0 0 1 1.000 0.000 1.000 0.000 1 0 1 0 0.998 0.002 0.999 0.001 1 0 1 1 0.949 0.051 0.883 0.117 1 1 0 0 1.000 0.000 1.000 0.000 1 1 0 1 0.985 0.015 0.980 0.020 1 1 1 0 0.175 0.825 <0 >1 1 1 1 1 0.009 0.991 <0 >1 doi: 10.1371/journal.pone.0085278.t004 Table 3. Final probabilistic match weights. Match weight Agreement Disagreement Soundex 5.18 -4.05 Date of birth 4.66 -6.89 Sex 0.91 -4.70 Location (PICU / lab) 5.53 -1.06 doi: 10.1371/journal.pone.0085278.t003 Table 3. Final probabilistic match weights. distribution of identifiers within LabBase2 was also different for these two PICUs. LabBase2 records from BCH were more distribution of identifiers within LabBase2 was also different for these two PICUs. LabBase2 records from BCH were more December 2013 | Volume 8 | Issue 12 | e85278 December 2013 | Volume 8 | Issue 12 | e85278 December 2013 | Volume 8 | Issue 12 | e85278 9 9 Paediatric Intensive Care Infection Surveillance distributed non-randomly. Although non-random error can introduce bias into results, prior-informed imputation has been shown to be particularly effective at handling this type of error[12]. Table 5. Estimated bias based on gold-standard data (BCH and OUH). Table 5. Estimated bias based on gold-standard data (BCH and OUH). Representativeness of gold-standard data Classification Total links BSI rate % Bias Gold-standard 426 3.87% Deterministic 125 1.14% -70.5% Highest-weighted: Relaxed threshold 492 4.47% 15.5 Highest weighted: Conservative threshold 418 3.80% -1.9 Prior-informed imputation 424 3.85% -0.5 doi: 10.1371/journal.pone.0085278.t005 Classification Total links BSI rate % Bias Gold-standard 426 3.87% Deterministic 125 1.14% -70.5% Highest-weighted: Relaxed threshold 492 4.47% 15.5 Highest weighted: Conservative threshold 418 3.80% -1.9 Prior-informed imputation 424 3.85% -0.5 doi: 10.1371/journal.pone.0085278.t005 This study demonstrates that linkage between routine datasets is complex and requires a number of steps. Firstly, calculation of appropriate match weights requires an iterative process and time-consuming manual review. Calculation of joint match probabilities avoids relying on independence assumptions that often fail, but the most effective ways of estimating such probabilities are still being debated. Current work is investigating this issue. likely to have completed Soundex, date of birth and sex, but less likely to have completed NHS number, name or postcode. LabBase2 records from OUH were more likely to have completed NHS number and name, but less likely to have completed postcode and date of birth. likely to have completed Soundex, date of birth and sex, but less likely to have completed NHS number, name or postcode. LabBase2 records from OUH were more likely to have completed NHS number and name, but less likely to have completed postcode and date of birth. Secondly, evaluation of data and linkage quality is required so that potential sources of bias can be identified. Bias due to linkage error can have dramatic effects on analyses based on linked data[13,14,16]. In particular, comparisons of units based on linked data may be biased by differing data quality, and such potential bias needs to be evaluated when using linked data for this purpose. Acknowledgements The authors would like to thank Tom Fleming (PICANet), Jim Gray, Ollie Bagshaw, Adrian Bigland (Birmingham Children’s Hospital), John Finney, Sarah Walker, Tim Peto, David Wyllie, Tariq Ali and Tim Whittington (Oxford University Hospital), Michael Millar and David Ball (Royal London Hospital) for helpful advice, contribution of data and facilitation of data retrieval for this paper. We would like to thank all the staff in participating hospitals who have collected data for PICANet. We are grateful to the UK Paediatric Intensive Care Society for continued support and to the members of the PICANet Steering Group and Clinical Advisory Group who are listed on our website http:// www.picanet.org.uk/participants.html. The authors would also like to thank the microbiology laboratories across England and Wales for their efforts in reporting to LabBase2. Gold-standard data provide a convenient means for evaluating both ascertainment and bias due to linkage error. This requires the assumption that the gold-standard datasets are representative of the larger dataset of interest. Firstly, we assumed that data capture from RLH and BCH reflected ascertainment in LabBase2 more generally, and that ascertainment was relatively constant over time. However, ascertainment based on these laboratories is likely to be overestimated, as RLH and BCH consistently submit data. Therefore final estimates of BSI rate may be under-estimated. Secondly, we assumed that bias due to linkage error in BCH and OUH was representative. Completeness of identifiers differed between laboratories, and linkage error was therefore Discussion Gold-standard data is one way to measure linkage bias, and this was practically possible in our study since linkage and analysis could be performed within the same department (PHE have permission to access patient-identifiable data for the purposes of surveillance). However, this is a special case, as clinical and identifiable data are often separated to protect patient privacy[22]. Appropriate evaluation of linkage success should be presented in reporting analysis based on linked data, to allow meaningful interpretation of results. Careful coordination between linkage and analysis is required so that research based on linked data can be reliable and transparent, whilst data confidentiality is preserved. Our study demonstrates that linkage of PICU admission data with national BSI surveillance is possible but that results based on these data vary according to the degree of under- ascertainment and bias introduced through linkage of incomplete or imperfect identifiers. We show that reporting gaps and under-ascertainment in national surveillance data lead to under-estimated rates of BSI, but that this can be measured through the use of gold-standard data. We also show that errors occurring during linkage can cause under- or over-estimated rates based on data linked using deterministic linkage only or highest-weight classification, but that prior- informed imputation can provide less biased results. Reasons for the under-ascertainment in LabBase2 cited by laboratories are lack of staffing provision, IT system compatibility issues and upload failures. We took a conservative approach to removing data points within periods of incomplete reporting, but this required a certain amount of subjectivity due to the fluctuating nature of BSI reports. PHE is currently developing a new surveillance system that aims to improve data capture. However, this type of data quality issue is relevant to many routinely collected datasets used for health research, and analyses based on these data need to carefully assess how poor data quality might affect results. References 11. Clark DE (2004) Practical introduction to record linkage for injury research. Injury Prev 10: 186-191. doi:10.1136/ip.2003.004580. PubMed: 15178677. 1. Nosocomial Infection National Surveillance Service (2007) Surveillance of Hospital-acquired Bacteraemia in English Hospitals. pp. 1997-2002. Public Health Laboratory Service. 1. Nosocomial Infection National Surveillance Service (2007) Surveillance of Hospital-acquired Bacteraemia in English Hospitals. pp. 1997-2002. Public Health Laboratory Service. 12. Goldstein H, Harron K, Wade A (2012) The analysis of record-linked data using multiple imputation with data value priors. Stat Med 31: 3481-3493. doi:10.1002/sim.5508. PubMed: 22807145. 2. Yogaraj JS, Elward AM, Fraser VJ (2002) Rate, risk factors, and outcomes of nosocomial primary bloodstream infection in pediatric intensive care unit patients. Pediatrics 110: 481-485. doi:10.1542/peds. 110.3.481. PubMed: 12205248. 13. Bohensky MA, Jolley D, Sundararajan V, Evans S, Pilcher DV et al. (2010) Data Linkage: A powerful research tool with potential problems. BMC Health Serv Res 10: 346-352. doi:10.1186/1472-6963-10-346. PubMed: 21176171. 3. Urrea M, Pons M, Serra M, Latorre C, Palomeque A (2003) Prospective incidence study of nosocomial infections in a pediatric intensive care unit. Pediatr Infect Dis J 22: 490-494. doi:10.1097/01.inf. 0000069758.00079.d3. PubMed: 12799503. 14. Schmidlin K, Clough-Gorr KM, Spoerri A, Egger M, Zwahlen M (2013) Impact of unlinked deaths and coding changes on mortality trends in the Swiss National Cohort. BMC Med Inform Decis Mak 13: 1-11. doi: 10.1186/1472-6947-13-S2-S1. PubMed: 23289362. 4. Elward AM, Hollenbeak CS, Warren DK, Fraser VJ (2005) Attributable cost of nosocomial primary bloodstream infection in pediatric intensive care unit patients. Pediatrics 115: 868-872. doi:10.1542/peds. 2004-0256. PubMed: 15805357. 15. Ford JB, Roberts CL, Taylor LK (2006) Characteristics of unmatched maternal and baby records in linked birth records and hospital discharge data. Paediatr Perinat EP 20: 329-337. doi:10.1111/j. 1365-3016.2006.00715.x. PubMed: 16879505. 5. Wilson J, Elgohari S, Livermore DM, Cookson B, Johnson A et al. (2011) Trends among pathogens reported as causing bacteraemia in England, 2004–2008. Clin Microbiol Infect 17: 451–458. doi:10.1111/j. 1469-0691.2010.03262.x. PubMed: 20491834. 16. Lariscy JT (2011) Differential Record Linkage by Hispanic Ethnicity and Age in Linked Mortality Studies. J Aging Health 23: 1263-1284. doi: 10.1177/0898264311421369. PubMed: 21934120. 6. Universities of Leeds and Leicester (2012) Paediatric Intensive Care Audit Network National Report 2009 - 2011 ISBN 9780853163121. Available:http://www.picanetorg.uk/Documents/General/ Annual_Report_2012/ Ninth_PICANet_Annual_Report_2009_2011_Summary_Report.pdf. Accessed 26 April 2013 17. Fellegi IP, Sunter AB (1969) A theory for record linkage. J Am Stat Assoc 64: 1183-1210. doi:10.1080/01621459.1969.10501049. 18. Author Contributions Conceived and designed the experiments: KH HG AW BMP RP RG. Performed the experiments: KH. Analyzed the data: KH. Contributed reagents/materials/analysis tools: HG RP BMP. Wrote the manuscript: KH HG AW BMP RP RG. December 2013 | Volume 8 | Issue 12 | e85278 PLOS ONE | www.plosone.org 10 Paediatric Intensive Care Infection Surveillance 10. Jaro MA (1995) Probabilistic linkage of large public health data files. Stat Med 14: 491-498. doi:10.1002/sim.4780140510. PubMed: 7792443. 22. Kelman CW, Bass AJ, Holman CDJ (2002) Research use of linked health data—a best practice protocol. Aust Nz. J Publ Heal 26: 251-255. Paediatric Intensive Care Infection Surveillance References DuVall SL, Kerber RA, Thomas A (2010) Extending the Fellegi-Sunter probabilistic record linkage method for approximate field comparators. J Biomed Inform 43: 24-30. doi:10.1016/j.jbi.2009.08.004. PubMed: 19683070. 7. Harron K, Wade A, Muller-Pebody B, Goldstein H, Parslow R et al. (2013) Risk-adjusted monitoring of blood-stream infection in paediatric intensive care: a data linkage study. Intensive Care Med 39: 1080-1087. doi:10.1007/s00134-013-2841-z. PubMed: 23404472. 19. Charlton CMJ, Michaelides DT, Cameron B, Szmaragd C, Parker RMA et al. (2012) Stat-JR software. Center for Multilevel Modelling, University of Bristol and Electronics and Computer Science. University of Southampton. 8. García Álvarez L, Aylin P, Tian J, King C, Catchpole M et al. (2011) Data linkage between existing healthcare databases to support hospital epidemiology. J Hosp Infect 79: 231–235. doi:10.1016/j.jhin. 2011.06.016. PubMed: 21868128. 20. Rubin D (1987) Multiple imputation for nonresponse in surveys: Wiley. 21. Harron K, Wade A, Muller-Pebody B, Goldstein H, Gilbert R. (2012) Opening the Black Box of Record Linkage. J Epidemiol Commun H Online First 9. Pearson A, Chronias A, Murray M (2009) Voluntary and mandatory surveillance for methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) bacteraemia in England. J Antimicrob Chemother 64: i11-i17. doi:10.1093/jac/dkp260. PubMed: 19675013. 22. Kelman CW, Bass AJ, Holman CDJ (2002) Research use of linked health data—a best practice protocol. Aust Nz. J Publ Heal 26: 251-255. 10. Jaro MA (1995) Probabilistic linkage of large public health data files. Stat Med 14: 491-498. doi:10.1002/sim.4780140510. PubMed: 7792443. 10. Jaro MA (1995) Probabilistic linkage of large public health data files. Stat Med 14: 491-498. doi:10.1002/sim.4780140510. PubMed: 7792443. PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e85278 11
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Native myeloperoxidase is required to make the experimental vasculitis model
Arthritis research & therapy
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LETTER Open Access Funding This work was supported by a grant from Ono Pharmaceutical (Osaka, Japan). Flow cytometry (FCM) using human neutrophils dem- onstrated the presence of ANCA in sera of group 2 but not group 1 (Fig. 1a). Correspondingly, sera of group 2 but not group 1 induced neutrophil extracellular traps (NETs) from tumor necrosis factor (TNF)-primed neu- trophils (Fig. 1b). Immunoblot of neutrophil lysates demonstrated that antibody reactive with the MPO light chain (14 kDa) was produced in group 1, whereas anti- bodies reactive with the MPO heavy chain (59 kDa) and light chain (14 kDa) were produced in group 2 (Fig. 1c). The collective findings indicated that the anti-MPO light chain antibody produced in group 1 did not bind to native MPO. Renal tissue damage represented by Native myeloperoxidase is required to make the experimental vasculitis model Mayu Nonokawa1, Ku Suzuki1, Hideyuki Hayashi1, Yuka Nishibata1, Sakiko Masuda1, Daigo Nakazawa2, Satoshi Tanaka3, Utano Tomaru4 and Akihiro Ishizu1* Availability of data and materials The data sheets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors’ contributions AI d i d h d M AI designed the study. MN, KS, HH, YN, SM, and AI acquired the data. All authors were involved in the interpretation of data and approval of the manuscript. UT and AI wrote the manuscript. Dear Editor, hematuria and erythrocyte casts in renal tubules was evident in group 2 but not group 1 regardless of the disease boost by LPS (Fig. 1d, e). The degree of pulmonary hemorrhage that represents capillaritis in the lungs tended to be severe in group 2 compared to group 1 (Fig. 1f). Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA) is a pathogenic autoantibody [1]. Wistar- Kyoto (WKY) rats immunized with human native MPO produce anti-human MPO antibody cross-reactive with rat MPO, resulting in the development of MPO-ANCA- associated vasculitis (MPO-AAV) [2]. MPO is a hetero- tetramer composed of two light chains (14 kDa) and two heavy chains (59 kDa) [3]. In this study, we examined if immunization of WKY rats with the recombinant light chain of human MPO could induce MPO-AAV. The majority of MPO-AAV patients produced MPO- ANCA that recognizes an epitope in the heavy chain of MPO, whereas a few number of patients produced MPO-ANCA against an epitope in the light chain of MPO [4, 5]. The collective findings suggested that the recombinant light chain of human MPO has a low potential to induce MPO-AAV in rats compared to na- tive human MPO. WKY rats (4–5 weeks old) were immunized with the recombinant light chain of human MPO (1600 μg/kg; Cloud-Clone, Katy, TX, USA; group 1) or human native MPO (1600 μg/kg; RayBiotech, Peachtree Corners, GA, USA; group 2) on day 0. These rats were given an intra- peritoneal injection of pertussis toxin (800 ng; Sigma- Aldrich, St. Louis, MO, USA) on days 0 and 2. A sub- group of group 1 was given an intraperitoneal injection of lipopolysaccharide (LPS; 100 μM/week; Sigma- Aldrich) through days 7 to 35. Urine samples were col- lected using a metabolic cage on day 40. All rats were euthanized on day 42. Consent for publication Not applicable Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. * Correspondence: aishizu@med.hokudai.ac.jp 1Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 0600812, Japan Full list of author information is available at the end of the article Nonokawa et al. Arthritis Research & Therapy (2019) 21:296 https://doi.org/10.1186/s13075-019-2084-7 Nonokawa et al. Arthritis Research & Therapy (2019) 21:296 https://doi.org/10.1186/s13075-019-2084-7 Author details 1 1Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 0600812, Japan. 2Department of Rheumatology, Endocrinology and Nephrology, Faculty of Ethics approval and consent to participate Experiments using rats were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals in Hokkaido University (Permission No. 15-0034). Experiments using human materials were permit- ted by the Ethics Committee of the Faculty of Health Sciences, Hokkaido Uni- versity (Permission No. 18-34). © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Nonokawa et al. Arthritis Research & Therapy (2019) 21:296 Page 2 of 3 Fig. 1 (See legend on next page.) Fig. 1 (See legend on next page.) Page 3 of 3 Nonokawa et al. Arthritis Research & Therapy (2019) 21:296 (See figure on previous page.) (See figure on previous page.) Fig. 1 Development of MPO-AAV. a ANCA detected by FCM. Human peripheral blood neutrophils were fixed with 4% paraformaldehyde, and then the plasma membrane of neutrophils was penetrated using permeabilization wash buffer (BioLegend, San Diego, CA, USA). Cells (1 × 106/ml) were allowed to react with 1:500 diluted rat sera for 30 min at room temperature (RT) followed by reaction with fluorescence-labeled secondary antibody. Concerning day 42 sera of group 1, the reactivity of 1:100 and 1:20 dilutions was also examined. To show the reactivity of anti-MPO heavy chain antibody to native MPO, a similar FCM was performed using the anti-MPO heavy chain monoclonal antibody (5 μg/ml; 4A4; Bio-Rad, Tokyo, Japan) as primary antibody and mouse IgG2b (5 μg/ml; BioLegend) as isotype control. b NET-forming neutrophils detected by FCM. Human peripheral blood neutrophils (1 × 106/ml) were treated with 5 ng/ml TNF-α for 15 min at 37 °C and then exposed to 10% rat sera. After incubation for 3 h at 37 °C, cells were next made to react with a plasma membrane-impermeable DNA-binding dye, SYTOX Green (Life Technologies, Carlsbad, CA, USA). After filtering out the debris with a mesh, the percolated cells were subjected for FCM. Histograms highlighted in green represent NET-forming neutrophils. The percentage of NET-forming neutrophils induced by group 2 sera was significantly higher than that induced by group 1 sera. c ANCA detected by immunoblotting. Lysates of human neutrophils boiled under reducing condition were electrophoresed (5 × 105 cells/lane) and then transferred to polyvinylidene difluoride membrane. References 1. Nakazawa D, Masuda S, Tomaru U, Ishizu A. Pathogenesis and therapeutic interventions for ANCA-associated vasculitis. Nat Rev Rheumatol. 2019;15(2): 91–101. 1. Nakazawa D, Masuda S, Tomaru U, Ishizu A. Pathogenesis and therapeutic interventions for ANCA-associated vasculitis. Nat Rev Rheumatol. 2019;15(2): 91–101. 2. Little MA, Smyth L, Salama AD, Mukherjee S, Smith J, Haskard D, et al. Experimental autoimmune vasculitis: an animal model of anti-neutrophil cytoplasmic autoantibody-associated systemic vasculitis. Am J Pathol. 2009; 174(4):1212–20. 3. Fiedler TJ, Davey CA, Fenna RE. X-ray crystal structure and characterization of halide-binding sites of human myeloperoxidase at 1.8 A resolution. J Biol Chem. 2000;275(16):11964–71. 3. Fiedler TJ, Davey CA, Fenna RE. X-ray crystal structure and characterization of halide-binding sites of human myeloperoxidase at 1.8 A resolution. J Biol Chem. 2000;275(16):11964–71. 4. Suzuki K, Kobayashi S, Yamazaki K, Gondo M, Tomizawa K, Arimura Y, et al. Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population. Microbiol Immunol. 2007;51(12):1215–20. 5. Gou SJ, Xu PC, Chen M, Zhao MH. Epitope analysis of anti-myeloperoxidase antibodies in patients with ANCA-associated vasculitis. PLoS One. 2013;8(4): e60530. After blocking the non-specific binding of antibodies, the membrane was incubated in diluted rat sera (day 42; group 1, 1:200 dilution; group 2, 1:1000 dilution) overnight at 4 °C. After rinsing with phosphate-buffered saline (PBS) with Tween 20 (PBS-T), the membrane was next incubated in the solution of horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT. After rinsing with PBS-T, the HRP activity on the membrane was detected by chemiluminescence using ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK). Blue arrowhead, MPO heavy chain (59 kDa); red arrowheads, MPO light chain (14 kDa). d Degree of hematuria assessed at urine sampling immediately by a dipstick (Siemens Healthineers, Erlangen, Germany). e Degree of renal tissue damage. Erythrocyte casts (yellow arrowheads) were counted in the maximum longitudinal section of the kidney. f Degree of pulmonary hemorrhage. The foci of pulmonary hemorrhage were counted in the maximum longitudinal section of the lung. Mann- Whitney U test was applied for statistical analyses between two non-parametric groups Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. 3Center for Cause of Death Investigation, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. 4Department of Pathology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. 3Center for Cause of Death Investigation, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. 4Department of Pathology, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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ABSTRACT characters performing a range of motions, which can serve as a valuable resource for future research. characters performing a range of motions, which can serve as a valuable resource for future research. Transforming neutral, characterless input motions to embody the distinct style of a notable character in real time is highly compelling for character animation. This paper introduces MOCHA, a novel online motion characterization framework that transfers both mo- tion styles and body proportions from a target character to an input source motion. MOCHA begins by encoding the input motion into a motion feature that structures the body part topology and cap- tures motion dependencies for effective characterization. Central to our framework is the Neural Context Matcher, which generates a motion feature for the target character with the most similar context to the input motion feature. The conditioned autoregres- sive model of the Neural Context Matcher can produce temporally coherent character features in each time frame. To generate the final characterized pose, our Characterizer network incorporates the characteristic aspects of the target motion feature into the in- put motion feature while preserving its context. This is achieved through a transformer model that introduces the adaptive instance normalization and context mapping-based cross-attention, effec- tively injecting the character feature into the source feature. We validate the performance of our framework through comparisons with prior work and an ablation study. Our framework can easily accommodate various applications, including characterization with only sparse input and real-time characterization. Additionally, we contribute a high-quality motion dataset comprising six different • Computing methodologies →Animation; Motion process- ing; Neural networks. • Computing methodologies →Animation; Motion process- ing; Neural networks. ACM Reference Format: ACM Reference Format: Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee. 2023. MOCHA: Real-Time Motion Characterization via Context Matching. In SIGGRAPH Asia 2023 Conference Papers (SA Conference Papers ’23), December 12–15, 2023, Sydney, NSW, Australia. ACM, New York, NY, USA, 11 pages. https://doi.org/10.1145/3610548.3618252 MOCHA: Real-Time Motion Characterization via Context Matching Sung-Hee Lee∗ sunghee.lee@kaist.ac.kr KAIST South Korea Sung-Hee Lee∗ sunghee.lee@kaist.ac.kr KAIST South Korea Jungdam Won jungdam@imo.snu.ac.kr Seoul National University South Korea Yuting Ye yuting.ye@meta.com Reality Labs, Meta USA Jungdam Won jungdam@imo.snu.ac.kr Seoul National University South Korea Deok-Kyeong Jang shadofex@kaist.ac.kr KAIST South Korea MOVIN Inc. South Korea Deok-Kyeong Jang shadofex@kaist.ac.kr KAIST South Korea MOVIN Inc. South Korea Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT h f i f i hi h Figure 1: Our characterization framework transforms neutral motions to express distinct style rization framework transforms neutral motions to express distinct style of characters in real-time. Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT T f i t l h t l i t ti t b d th characters performing a range of motions, which can serve as a valuable resource for future research s to express distinct style of characters in real-tim g p y ABSTRACT Transforming neutral, characterless input motions to embody the characters performing a range of motions, which can serve as a valuable resource for future research. MOCHA: Real-Time Motion Characterization via Context Matching Deok-Kyeong Jang shadofex@kaist.ac.kr KAIST South Korea MOVIN Inc. South Korea Yuting Ye yuting.ye@meta.com Reality Labs, Meta USA Jungdam Won jungdam@imo.snu.ac.kr Seoul National University South Korea Sung-Hee Lee∗ sunghee.lee@kaist.ac.kr KAIST South Korea Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT T f i t l h t l i t ti t b d th characters performing a range of motions, which can serve as a valuable resource for future research MOCHA: Real-Time Motion Characterization via Context Matching Deok-Kyeong Jang shadofex@kaist.ac.kr KAIST South Korea MOVIN Inc. South Korea Yuting Ye yuting.ye@meta.com Reality Labs, Meta USA Jungdam Won jungdam@imo.snu.ac.kr Seoul National University South Korea Sung-Hee Lee∗ sunghee.lee@kaist.ac.kr KAIST South Korea Figure 1: Our characterization framework transforms neutral motions to express distinct style of characters in real-time. ABSTRACT T f i t l h t l i t ti t b d th characters performing a range of motions, which can serve as a valuable resource for future research MOCHA: Real-Time Motion Characterization via Context Matching KEYWORDS Motion style transfer, Motion synthesis, Character animation, Deep learning 1 INTRODUCTION Transforming styleless motions to embody a particular character is invaluable for animating characters in feature films or in VR. As the popularity of interactive and immersive VR and AR applications grows, so does the need for real-time user motion characterization. In this paper, we propose a novel technique for transforming a va- riety of user motions into motions that express a specific character (e.g., Princess, Clown, etc.) in real-time. ∗Corresponding author This work is licensed under a Creative Commons Attribution International 4.0 License. SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia © 2023 Copyright held by the owner/author(s). ACM ISBN 979-8-4007-0315-7/23/12. https://doi.org/10.1145/3610548.3618252 We adopt an example-based approach, presuming the availability of a stylistic motion database for the target character. However, the character’s body proportions will not be an exact match to the user’s, and the character may not even possess a human-like physique. In this work, we posit that a character’s distinctive movement style is intricately linked with their unique body shape, so we need to solve both motion stylization and motion retargeting problems jointly. ∗Corresponding author SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia © 2023 Copyright held by the owner/author(s). ACM ISBN 979-8-4007-0315-7/23/12. https://doi.org/10.1145/3610548.3618252 Current data-driven motion stylization techniques often perceive style as a consistent feature within data. One approach is to extract SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee • We release a high-quality character motion dataset that con- tains a total of 6 characters performing various actions, with each action conducted with 5 emotions. • We release a high-quality character motion dataset that con- tains a total of 6 characters performing various actions, with each action conducted with 5 emotions. • We release a high-quality character motion dataset that con- tains a total of 6 characters performing various actions, with each action conducted with 5 emotions. • We release a high-quality character motion dataset that con- tains a total of 6 characters performing various actions, with each action conducted with 5 emotions. style elements from the target examples and apply them to the source motion [Aberman et al. 2020b; Jang et al. 2022]. Another approach treats style as a conditioning variable for motion gen- eration [Park et al. 2021; Tao et al. 2022a]. 2.1 Motion style transfer Research in motion style transfer centers around two fundamental questions: the definition and representation of style. Convention- ally, style is regarded as time-invariant variations within the same motion content, structure, or context, which can vary based on individual characteristics [Ma et al. 2010] or emotions [Amaya et al. 1996]. A lot of research investigate decomposition algorithms to factorize and parameterize style from motion content [Brand and Hertzmann 2000; Mason et al. 2018; Min et al. 2009; Rose et al. 1998; Shapiro et al. 2006; Unuma et al. 1995; Yumer and Mitra 2016]. Style can also be represented non-parametrically or as a label. For in- stance, Gaussian Process has been used to learn a latent space of pose styles [Grochow et al. 2004] or motion styles [Ikemoto et al. 2009; Wang et al. 2007]. More recently, explicit style labels are used to condition generative models to stylize the output motion [Smith et al. 2019; Tao et al. 2022b]. The style labels can also be latent learned from data [Park et al. 2021]. g To this end, we propose a novel framework that can characterize a user’s diverse motions in real-time. The basic idea is that when user performs a motion, we search for a target character’s motion with the most similar context from a motion database and then transfer the style elements of the found motion to the user’s motion. However, this approach has the disadvantages of storing character motion database, searching a suitable motion in real-time, as well as limiting target motions to the ones in the database. To improve upon this, inspired by learned motion matching technique [Holden et al. 2020], we train a neural network, dubbed Neural Context Matcher (NCM), to generate target motion features suitable for characterizing a source motion instead of searching a database. Modeled as a conditional VAE running autoregessively, the NCM can generate temporally coherent motion features for the target character suitable for the input motion, which is key to obtaining high-quality motion characterization for diverse motions. Treating style as a static feature limits its representative power. These approaches can often capture stereotypical pose features, but fall short at delivering complex and nuanced characteristics. Xia et al. [2015] addressed this issue by adapting style parameters in real-time based on local nearest-neighbors. They also contributed a dataset that supported many follow up research. Style may also be spatially varying. Motion Puzzle [Jang et al. 1 INTRODUCTION While these methods perform well on stereotypical locomotion datasets, we challenge their assumption by introducing a high quality and diverse dataset of professional performances. Our data clearly illustrates style as context-dependent. For example, the expression of "happiness" in a jumping motion manifests differently than in a crawling motion and their characteristics are not interchangeable. 2.1 Motion style transfer 2022] integrates style elements based on body parts into a single character. In our work, we consider style as non-parametric and context-dependent, and therefore choose a different class of approach more akin to motion matching [Clavet 2016].l In addition, the motions generated by our framework not only reflect the motion style aspects but also the target character’s body proportions, making additional motion retargeting to the target character unnecessary. Hence we call our technique motion char- acterization. This is possible because our framework encodes both motion styles and body proportions into motion feature and ef- fectively transfers them to the source motion. To the best of our knowledge, our work is the first that performs motion stylization and retagetting concurrently. Besides, we propose several crucial ideas, such as introducing contrastive loss and incorporating adap- tive instance normalization (AdaIN) [Huang and Belongie 2017] into transformer decoder, that significantly enhance the motion stylization quality. Recent research in motion style transfer is heavily influenced by the vast body of work in image style transfer. In this context, motion styles are analogous to image textures. The pioneering work of Neu- ral Style Transfer [Gatys et al. 2015] introduced the use of the Gram matrix of latent features for style representation, an approach later adopted in the motion domain [Du et al. 2019; Holden et al. 2016]. Its successor, the Adaptive Instance Norm (AdaIN) layer [Huang and Belongie 2017], has later become the predominant technique with widespread adoption (e.g., [Aristidou et al. 2022]). Addition- ally, Generative Adversarial Networks (GANs), in combination with contrastive and cycle-consistency losses [Zhou et al. 2016] have proven effective in self-supervised style learning [Aberman et al. 2020b; Dong et al. 2017], which we also apply in our work. Figure 1 shows snapshots of our characterization results. Various motions, such as walking, sitting and jumping (white character), can be characterized to match Zombie, Princess, and Clown. We demon- strate the effectiveness of our framework through comparisons with previous work and ablation study. Additionally, we showcase its capability for real-time live characterization from streamed motion data. We also show that our framework can accommodate sparse inputs, enabling its application in VR tracker-based motion capture systems. 4.1 Bodypart Encoder The bodypart encoder in Fig. 3 (a) consists of two components: body patch embedding, which reduces the spatial and temporal resolutions of the input motion while preserving bodypart structure, and transformer encoder, which uses a transformer-based structure to learn sequential motion dependencies. 4.1.1 Body patch embedding. Following the approach of flattening 2D patches input for standard vision transformer [Dosovitskiy et al. 2020], we employ body patch embedding, which maintains the graph structure of the human skeleton as much as possible. Specifically, we use spatial-temporal graph convolutional blocks (STGCN) [Yan et al. 2018] to reduce the spatial (joint) and temporal (frame) resolutions. STGCN blocks project an input motion into a sequential feature embedding E. We define embedding process as follows: 2.3 Puppeteering and Motion Retargeting Puppeteering and motion retargeting can be regarded as a special case of motion style transfer, where the control or source motion must be mapped and adapted to the target character’s design and style. Automatically mapping between two arbitrary characters remains a notable challenge with no unique answers. To address this, heuristic-based solution have been developed to analyze either the structure [Kry et al. 2009] or motion space between characters [Dontcheva et al. 2003; Seol et al. 2013]. For the less complex task of mapping between two bipedal skeletons - considered homeomor- phic graphs - graph convolutional networks have been successful [Aberman et al. 2020a; Park et al. 2021]. We therefore employ this strategy to extra motion features, facilitating retargeting across a diverse range of characters and styles. E = STGCN(X𝑖).𝑓𝑙𝑎𝑡𝑡𝑒𝑛(0, 1) + P ∈R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶, (2) E = STGCN(X𝑖).𝑓𝑙𝑎𝑡𝑡𝑒𝑛(0, 1) + P ∈R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶, (2) (2) where P denotes positional encoding with learnable parameters. E comprises a total of 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦patches with 𝐶channels, where 𝑛𝑏𝑜𝑑𝑦(= 6) is the number of bodyparts (head, spine, arms, and legs). X𝑖= [x]𝑖 𝑖−(𝑇−1), x = [𝑥𝑡 𝑗𝑥𝑟 𝑗¤𝑥𝑡 𝑗¤𝑥𝑟 𝑗], Y𝑖= [y]𝑖 𝑖−(𝑇−1), y = [𝑦𝑡 𝑗𝑦𝑟 𝑗¤𝑦𝑡 𝑗¤𝑦𝑟 𝑗], (1) (1) 4.1.2 Transformer encoder. We capture spatial-temporal dependen- cies of body patches by using a transformer structure. We feed the embedding sequence E into the transformer encoder 𝐸𝑏to gener- ate a source feature 𝑧= 𝐸𝑏(E). Each layer of the encoder consists of a multi-head self-attention module (MSA) and a feed-forward network (FFN). where 𝑗denotes all joints (including the pelvis), 𝑥𝑡∈R3 and 𝑥𝑟∈ R6 (6 for two orthogonal axes) are joint translations and rotations, ¤𝑥𝑡 𝑗∈R3 and ¤𝑥𝑟 𝑗∈R3 are joint linear and angular velocities local to root𝑖. Likewise, 𝑦𝑡, 𝑦𝑟, ¤𝑦𝑡 𝑗and ¤𝑦𝑟 𝑗denote joint translation, rotation, and linear and angular velocities local to the parent, with respect to root𝑖. As a result, the total dimensions of our human motion feature with 𝑇frames are X ∈R𝑇×𝑛𝑗𝑜𝑖𝑛𝑡×15 and Y ∈R𝑇×𝑛𝑗𝑜𝑖𝑛𝑡×15, where 𝑛𝑗𝑜𝑖𝑛𝑡is the number of joints. 𝑧′ = MSA(E) + E, 𝑧𝑖= FNN(𝑧′) + 𝑧′ ∈Z ⊂R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶. (3) 𝑧′ = MSA(E) + E, 𝑧= MSA(E) + E, 𝑧𝑖= FNN(𝑧′) + 𝑧′ ∈Z ⊂R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶. (3) (3) 𝑧𝑖= FNN(𝑧′) + 𝑧′ ∈Z ⊂R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶. 4.2 Neural Context Matcher After bodypart encoding, we search a character feature that shares a similar context with the source feature. This character feature will later be used to imbue its style aspects into the source feature. Inspired by learned motion matching (LMM) approach [Holden et al. 2020], we train the neural context matcher (NCM) to generate the best matching character feature. Unlike LMM, which performs matching every few frames, NCM runs at every frame to maximize responsiveness to the input motion, which is crucial for producing temporally continuous character features. To this end, we model the NCM using an autoregressive conditional variational autoencoder. 2.2 Motion Matching The major contributions of our work can be summarized as follows: Motion matching [Clavet 2016] provides continuous and control- lable animation in real-time for interactive gaming via nearest- neighbor search of motion features. To improve its scalability in memory and speed, learned motion matching [Holden et al. 2020] approximates this process using a neural network. Our Neural Con- tact Matcher (NCM) applies learned motion matching on learned context features to find the closest matching target motion, ensur- ing real-time and high quality output. • We present the first online motion characterization frame- work that can transfer both the motion style aspects and body proportions of characters to a variety of user motions. • Our C-VAE-based Neural Context Matcher model can effec- tively generate the target character’s motion feature with temporal coherency. SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia MOCHA: Real-Time Motion Characterization via Context Matching frame, the bodypart encoder transforms the input source motion into a feature vector (dubbed source feature) that structures human motion into six parts and captures sequential motion dependen- cies. Subsequently, given a target character, the NCM generates a corresponding feature of the target character (dubbed character feature) that shares the most similar context with the source motion. Lastly, the characterizer uses the source feature and the context- matched character feature to synthesize a characterized pose while preserving the source motion’s context. Figure 2: Illustration of the motion representation at frame 𝑖. We define the character forward facing direc- tion of current frame 𝑖as reference frame, which is denoted as root𝑖. Figure 2: Illustration of the motion representation at frame 𝑖. We define the character forward facing direc- tion of current frame 𝑖as reference frame, which is denoted as root𝑖. The bodypart encoder and characterizer are character-agnostic, allowing a single trained network to work for all characters. In contrast, the NCM is trained separately for each target character, so the number of NCMs increases linearly with the number of characters. 3 MOTION DATA REPRESENTATION AND PROCESSING Figure 2 illustrates the motion representation of our method. The reference frame of a motion, denoted as root𝑖, is located at the ground projection of the pelvis joint at current frame 𝑖and aligned to the ground normal direction and the pelvis’ forward facing direc- tion. A motion sequence at frame 𝑖is represented in two ways: X𝑖 and Y𝑖express each element with respect to root𝑖and with respect to the parent, respectively. E = STGCN(X𝑖).𝑓𝑙𝑎𝑡𝑡𝑒𝑛(0, 1) + P ∈R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶, (2) 4.3 Characterizer The NCM implicitly models a distribution of possible next character features that match the current source context feature given pre- vious character feature. Samples are drawn from this distribution and passed through the NCM decoder to create a character feature for next frame, one at a time in an autoregressive fashion (yellow part in Fig. 3 (a)). The characterizer transfers the style aspects (e.g., skeleton pro- portions, characteristic movements, etc.) of the character feature to the source feature. For this, the character transformer decoder with adaptive instance normalization (AdaIN) [Huang and Belongie 2017] and multi-head cross-attention generates characterized de- coded feature 𝑧𝑑(dubbed translated feature), which is then up- sampled with De-STGCN blocks to obtain the final characterized motion. 4.2.1 Context mapping. The context feature 𝑓(𝑧𝑖𝑠𝑟𝑐) ∈R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶 is extracted from encoded feature 𝑧𝑖via context mapping net- work. The context space made by the context mapping is character- agnostic, capturing shared information on context across character domains. It enables context matching between different characters, which is a crucial step in characterizing. The context mapping is learned from unlabeled motion data in an supervised manner with a set of loss terms as will be discussed in Sec. 6.5. 4.3.1 Character transformer decoder. After context matching, we feed the source feature and character feature into the transformer decoder. The character transformer decoder generates translated feature 𝑧𝑑= 𝐷𝑐(𝑧𝑖𝑠𝑟𝑐, 𝑧𝑖 𝑐ℎ𝑎) which merges the context of source motion and the character (style) aspects of target character motion. 4.3.1 Character transformer decoder. After context matching, we feed the source feature and character feature into the transformer decoder. The character transformer decoder generates translated feature 𝑧𝑑= 𝐷𝑐(𝑧𝑖𝑠𝑟𝑐, 𝑧𝑖 𝑐ℎ𝑎) which merges the context of source motion and the character (style) aspects of target character motion. As shown in Fig. 3 (b), instead of employing traditional trans- former decoder block, we model our decoder with the AdaIN, multi- head cross-attention module (MCA) with context mapping function, and FNN layer. The AdaIN module transfers the global statistics of character feature, as in [Jang et al. 2022], by taking 𝑧𝑖𝑠𝑟𝑐as input and injecting character feature 𝑧𝑖 𝑒,𝑐ℎ𝑎as: 4.2.2 Prior Net. The distribution over possible latent variable 𝑠𝑖∈ R𝐶for character feature 𝑧𝑖 𝑐ℎ𝑎is described by a learned prior [Rempe et al. 4 MOCHA FRAMEWORK Figure 3 (a) illustrates our motion characterization framework at runtime. Input to our framework is a motion sequence of length 𝑇 (1 second, 60 frames) from past to current frame 𝑖, represented with X𝑖. For output, we generate a characterized motion sequence of the same length, represented as Y𝑖, but only use the final characterized pose y𝑖. Our framework comprises a bodypart encoder, a neural con- text matcher (NCM), and a characterizer networks. At each time SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee Figure 3: Network configuration. (a) Overall architecture for motion characterization in run-time. Our framework consists of bodypart encoder, neural context matcher, and characterizer networks. (b) Detail of the characterizer transformer decoder block (𝐷𝑐). Figure 3: Network configuration. (a) Overall architecture for motion characterization in run-time. Our framework consists of bodypart encoder, neural context matcher, and characterizer networks. (b) Detail of the characterizer transformer decoder block (𝐷𝑐). 4.3 Characterizer 2021] conditioned on previous character feature 𝑧𝑖−1 𝑐ℎ𝑎and current source context feature 𝑓(𝑧𝑖𝑠𝑟𝑐): 𝑝(𝑠𝑖|𝑧𝑖−1 𝑐ℎ𝑎, 𝑓(𝑧𝑖 𝑠𝑟𝑐)) = N (𝑠𝑖; 𝜇(𝑧𝑖−1 𝑐ℎ𝑎, 𝑓(𝑧𝑖 𝑠𝑟𝑐)), 𝜎(𝑧𝑖−1 𝑐ℎ𝑎, 𝑓(𝑧𝑖 𝑠𝑟𝑐))), (4) 𝑧𝑑′′ = 𝛾(𝑧𝑖 𝑐ℎ𝑎) 𝑧𝑖𝑠𝑟𝑐−𝜇(𝑧𝑖𝑠𝑟𝑐) 𝜎(𝑧𝑖𝑠𝑟𝑐)  + 𝛽(𝑧𝑖 𝑐ℎ𝑎), (6) (4) (6) where 𝜎and 𝜇are the channel-wise mean and variance, respectively. AdaIN scales the normalized 𝑧𝑖𝑠𝑟𝑐with a learned affine transforma- tion with scales 𝛾and biases 𝛽generated by 𝑧𝑖 𝑐ℎ𝑎. In the second step, we feed the globally-stylized feature 𝑧𝑑′′ and character feature 𝑧𝑖 𝑐ℎ𝑎to MCA. We use the context feature 𝑓(𝑧𝑑′′) to generate the query 𝑄, the character context feature 𝑓(𝑧𝑖 𝑐ℎ𝑎) to generate the key 𝐾, and the character feature 𝑧𝑖 𝑐ℎ𝑎to generate the value 𝑉: which parameterizes a Gaussian distribution with diagonal covari- ance via a neural network. 4.2.3 NCM decoder. The character feature 𝑧𝑖 𝑐ℎ𝑎is predicted by the NCM decoder, which takes as input the latent variables 𝑠𝑖while being conditioned on previous character feature 𝑧𝑖−1 𝑐ℎ𝑎and current source context feature 𝑓(𝑧𝑖𝑠𝑟𝑐): 𝑄= 𝑓(𝑧𝑑′′)𝑊𝑞, 𝐾= 𝑓(𝑧𝑖 𝑐ℎ𝑎)𝑊𝑘, 𝑉= 𝑧𝑖 𝑐ℎ𝑎𝑊𝑣, (7) (7) 𝑧𝑖 𝑐ℎ𝑎= 𝐷𝑣(𝑠𝑖, 𝑧𝑖−1 𝑐ℎ𝑎, 𝑓(𝑧𝑖 𝑠𝑟𝑐)). (5) (5) where𝑊𝑞,𝑊𝑘,𝑊𝑣∈R𝐶×𝑑ℎ𝑒𝑎𝑑. Then, the output sequence 𝑧𝑑of the transformer decoder is obtained by We use transformer based C-VAE model for the NCM. In training phase, both NCM encoder and decoder are trained as detailed in Section 5.2 while only the decoder is used for inference. 𝑧′ 𝑑= MCA(𝑄, 𝐾,𝑉) + 𝑧𝑑′′, 𝑧𝑑= FNN(𝑧′ 𝑑) + 𝑧′ 𝑑. (8) (8) MOCHA: Real-Time Motion Characterization via Context Matching SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Figure 4: Stage-1 training process. A source motion X𝑖𝑠𝑟𝑐and a target motion X𝑖 𝑐ℎ𝑎are randomly selected from different char- acters in the motion dataset, and the networks are trained to make the characterized motion Y𝑖 𝑡𝑟𝑎preserve the context of X𝑖𝑠𝑟𝑐while reflecting the characteristic aspects of X𝑖 𝑐ℎ𝑎. 5.1.1 Identity and cycle loss. To design identity and cycle consis- tency losses, we first define a reconstruction loss that computes the difference of two motions both in terms of X and Y [Holden et al. 2020] as well as their velocities. L𝑟𝑒𝑐(eX, X) = 𝜆𝑙𝑜𝑐EX[∥eX −X∥1] + 𝜆𝑟𝑡EY[∥eY −Y∥1] + 𝜆𝑙𝑣𝑒𝑙EX[∥𝑉(eX) −𝑉(X)∥1] + 𝜆𝑟𝑣𝑒𝑙EY[∥𝑉(eY) −𝑉(Y)∥1], L𝑟𝑒𝑐(eX, X) = 𝜆𝑙𝑜𝑐EX[∥eX −X∥1] + 𝜆𝑟𝑡EY[∥eY −Y∥1] + 𝜆𝑙𝑣𝑒𝑙EX[∥𝑉(eX) −𝑉(X)∥1] + 𝜆𝑟𝑣𝑒𝑙EY[∥𝑉(eY) where X = 𝐹𝐾(Y), 𝑉(X) = X0−X1 ℎ , 𝑉(Y) = Y0−Y1 ℎ , ℎis time step, and 𝜆𝑙𝑜𝑐, 𝜆𝑟𝑡, 𝜆𝑙𝑣𝑒𝑙, 𝜆𝑟𝑣𝑒𝑙are the relative weights. 5 TRAINING Our training pipeline consists of two-stages; the bodypart encoder and characterizer are trained first, followed by training the NCM. Thus, in the first stage, our framework learns to extract encoded features from a motion and transfer style aspects of one motion (e.g., motion style and skeleton proportions) to the other motion to synthesize a characterized motion. At this stage, the context mapping network is also trained as a part of transformer decoder. The first stage training is conducted in an unsupervised way. The second stage trains the NCM in a supervised manner to generate target character features corresponding to the input context features from the source. To define the body patch-level context loss between 𝑓(𝑧𝑠𝑟𝑐) and 𝑓(𝑧𝑡𝑟𝑎), we use infoNCE loss [Oord et al. 2018]: 𝑙(ˆ𝑣, 𝑣+, 𝑣-) = −log " exp(ˆ𝑣· 𝑣+/𝜏) exp(ˆ𝑣· 𝑣+/𝜏) + Í𝑁 𝑛=1 exp(ˆ𝑣· 𝑣-𝑛/𝜏) # , (12) (12) where 𝜏is the temperature parameter, and 𝑣+ and 𝑣- denote positive and negative for ˆ𝑣. We set pseudo positive samples between body patch-level con- text of source 𝑓(𝑧𝑠𝑟𝑐) and characterized motion 𝑓(𝑧𝑡𝑟𝑎); for a body patch 𝑓(𝑧𝑡𝑟𝑎)𝑏∈𝑓(𝑧𝑡𝑟𝑎), we set its positive patch 𝑓(𝑧𝑠𝑟𝑐)𝑏as the patch in the same location in 𝑓(𝑧𝑠𝑟𝑐), and negative patches 𝑓(𝑧𝑠𝑟𝑐)𝐵\𝑏as all other patches.︁ 4.3 Characterizer To ensure that the characterized output motion not only maintains the overall context 𝑓(𝑧𝑠𝑟𝑐) ∈ R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶of source motion X𝑠𝑟𝑐, but also preserves context of body patches at a specific location between source and output, we introduce a body patch contrastive loss. For example, in Figure 5, the context of a princess leg at frame 𝑖−𝑡should be closer to that of input zombie leg at frame 𝑖−𝑡than the other patches of the same input.i 4.3 Characterizer 𝑉(X) represents the rate of change of X. Part of 𝑉(X) corresponds to the joint accelerations. Figure 4: Stage-1 training process. A source motion X𝑖𝑠𝑟𝑐and a target motion X𝑖 𝑐ℎ𝑎are randomly selected from different char- acters in the motion dataset, and the networks are trained to make the characterized motion Y𝑖 𝑡𝑟𝑎preserve the context of X𝑖𝑠𝑟𝑐while reflecting the characteristic aspects of X𝑖 𝑐ℎ𝑎. The identity loss ensures that the input motion remains un- changed when it is used for both the source and character motions: L𝑖𝑑= L𝑟𝑒𝑐(X𝑖𝑑 𝑠𝑟𝑐, X𝑠𝑟𝑐) + L𝑟𝑒𝑐(X𝑖𝑑 𝑐ℎ𝑎, X𝑐ℎ𝑎), (10) (10) where X𝑖𝑑 𝑠𝑟𝑐= 𝐹𝐾(𝐶𝐻(𝑧𝑠𝑟𝑐, 𝑧𝑠𝑟𝑐)) and 𝑧𝑠𝑟𝑐= 𝐵𝐸(X𝑠𝑟𝑐). X𝑖𝑑 𝑠𝑟𝑐is obtained by feeding X𝑠𝑟𝑐to both X𝑠𝑟𝑐and X𝑐ℎ𝑎in Fig. 4. Likewise, X𝑖𝑑 𝑐ℎ𝑎= 𝐹𝐾(𝐶𝐻(𝑧𝑐ℎ𝑎, 𝑧𝑐ℎ𝑎)) with 𝑧𝑐ℎ𝑎= 𝐵𝐸(X𝑐ℎ𝑎). 4.3.2 Body patch expanding and output. The translated feature 𝑧𝑑∈R( 𝑇 4 ∗𝑛𝑏𝑜𝑑𝑦)×𝐶is upsampled with additional De-STGCN blocks, which has symmetric architectures to that of STGCN, to yield out- put translated motion Y𝑖 𝑡𝑟𝑎as follows: 𝑐ℎ𝑎 To guarantee that the resulting motion Y𝑡𝑟𝑎(hence X𝑡𝑟𝑎) pre- serves the context of the source motion X𝑠𝑟𝑐and the characteristics of the character motion X𝑐ℎ𝑎, we employ cycle consistency loss [Choi et al. 2020]. Y𝑖 𝑡𝑟𝑎= De-STGCN(𝑅𝑒𝑠ℎ𝑎𝑝𝑒(𝑧𝑑)). (9) (9) L𝑐𝑦𝑐= L𝑟𝑒𝑐(X𝑐𝑦𝑐 𝑠𝑟𝑐, X𝑠𝑟𝑐) + L𝑟𝑒𝑐(X𝑐𝑦𝑐 𝑐ℎ𝑎, X𝑐ℎ𝑎), (11) where X𝑐𝑦𝑐 𝑠𝑟𝑐= 𝐹𝐾(𝐶𝐻(𝑧𝑡𝑟𝑎, 𝑧𝑠𝑟𝑐)), X𝑐𝑦𝑐 𝑐ℎ𝑎= 𝐹𝐾(𝐶𝐻(𝑧𝑐ℎ𝑎, 𝑧𝑡𝑟𝑎)), 𝑧𝑡𝑟𝑎= 𝐵𝐸(𝐹𝐾(Y𝑡𝑟𝑎)) and Y𝑡𝑟𝑎= 𝐶𝐻(𝑧𝑠𝑟𝑐, 𝑧𝑐ℎ𝑎). X𝑐𝑦𝑐 𝑠𝑟𝑐is obtained by feeding X𝑠𝑟𝑐and X𝑐ℎ𝑎to top and bottom inputs in Fig 4 to get Y𝑡𝑟𝑎, followed by feeding X𝑡𝑟𝑎(= 𝐹𝐾(Y𝑡𝑟𝑎)) and X𝑠𝑟𝑐to top and bottom inputs in Fig. 4 to get Y𝑐𝑦𝑐 𝑠𝑟𝑐. L𝑐𝑦𝑐= L𝑟𝑒𝑐(X𝑐𝑦𝑐 𝑠𝑟𝑐, X𝑠𝑟𝑐) + L𝑟𝑒𝑐(X𝑐𝑦𝑐 𝑐ℎ𝑎, X𝑐ℎ𝑎), (11) where X𝑐𝑦𝑐 𝑠𝑟𝑐= 𝐹𝐾(𝐶𝐻(𝑧𝑡𝑟𝑎, 𝑧𝑠𝑟𝑐)), X𝑐𝑦𝑐 𝑐ℎ𝑎= 𝐹𝐾(𝐶𝐻(𝑧𝑐ℎ𝑎, 𝑧𝑡𝑟𝑎)), 𝑧𝑡𝑟𝑎= 𝐵𝐸(𝐹𝐾(Y𝑡𝑟𝑎)) and Y𝑡𝑟𝑎= 𝐶𝐻(𝑧𝑠𝑟𝑐, 𝑧𝑐ℎ𝑎). X𝑐𝑦𝑐 𝑠𝑟𝑐is obtained by feeding X𝑠𝑟𝑐and X𝑐ℎ𝑎to top and bottom inputs in Fig 4 to get Y𝑡𝑟𝑎, followed by feeding X𝑡𝑟𝑎(= 𝐹𝐾(Y𝑡𝑟𝑎)) and X𝑠𝑟𝑐to top and bottom inputs in Fig. 4 to get Y𝑐𝑦𝑐 𝑠𝑟𝑐. (11) Finally, we pick the last frame pose y𝑖 𝑡𝑟𝑎from Y𝑡𝑟𝑎as final output. 4.3.3 Root motion. We maintain the root angular velocity of the source motion while scaling the linear velocity according to the ratio of the average hip velocities of the source motion X𝑖and the output motion Y𝑖 𝑡𝑟𝑎. This strategy allows for maintaining the overall shape of the root trajectory of the source motion, while varying the global linear velocity to match the target character. 5.1.2 Body patch contrastive loss. 6.1 Datasets We constructed a high-quality character motion dataset with five professional actors. The dataset comprises a total of 6 characters (Clown, Ogre, Princess, Robot, Zombie, and AverageJoe) performing various actions (Dance, Fight, Jump, Crawling, Run, Walk, and Sit) with 5 emotion variations for each action (Angry, Happy, Neutral, Sad, and Scared). Every character preserves the same body propor- tions as the actors, except we scale the forearm of the Ogre character to be twice as long to convey its style. The dataset contains a to- tal of 573k frames, captured at 60 fps, resulting in approximately 159 minutes of data. Since different emotions are manifested with unique styles, we consider a character-emotion pair as an individual character, such as “Neutral Zombie” and “Happy Ogre”. Figure 6: Training architecture of Neural Context Matcher. Once trained, only the decoder is used for inference. We additionally tested our algorithm on the Adult2child dataset [Dong et al. 2020] for quantitative evaluation.It consists of 17 sub- jects including nine adults (older than 18 years) and eight children (5-10 years old) performing actions such as Jump as high as you can in place, Punch, Kick, Walk, and Hop Scotch. We treat each subject as a character due to their personalized styles. Adult2child dataset is suitable for our evaluation since the skeleton proportions and motion styles vary depending on the subject. context loss work, with procedure to define positive and negative samples. context loss work, with procedure to define positive and negative samples. The total loss function of stage-1 is thus: (14) L𝑠𝑡𝑎𝑔𝑒1 = 𝜆𝑖𝑑L𝑖𝑑+ 𝜆𝑐𝑦𝑐L𝑐𝑦𝑐+ 𝜆𝑐𝑡𝑟L𝑐𝑡𝑟, (14) where 𝜆𝑖𝑑, 𝜆𝑐𝑦𝑐, and 𝜆𝑐𝑡𝑟are weights. where 𝜆𝑖𝑑, 𝜆𝑐𝑦𝑐, and 𝜆𝑐𝑡𝑟are weights. 6 EVALUATION AND EXPERIMENTS We conduct comparisons with other methods, perform an ablation study, and conduct additional experiments to demonstrate the utility of our framework. For visual animation results, please refer to the supplementary video. Figure 5: Illustration of body patch-level context contrastive loss. Blue box indicates a positive sample, while yellow boxes denote negative samples. Figure 6: Training architecture of Neural Context Matcher. Once trained, only the decoder is used for inference. 5.2 Stage-2 First, we qualitatively compare our method with two baselines: Motion Puzzle [Jang et al. 2022] and replacing NCM in MOCHA with nearest-neighbor search (Nearest Neighbor). Since Motion Puzzle only works for a single body proportion, we have to re- place its reconstruction loss with ours to accommodate skeleton variations at training. Note that Motion Puzzle conducts an offline, full sequence-to-sequence translation, while MOCHA is an online translator. In stage-2, the NCM is trained to infer a target character feature 𝑧𝑐ℎ𝑎that shares the same context with a source feature 𝑧𝑠𝑟𝑐. Briefly, this is achieved by providing the ground truth character feature 𝑧𝑐ℎ𝑎obtained from searching a target feature database D𝑡𝑎𝑟= [Z𝑡𝑎𝑟, 𝑓(Z𝑡𝑎𝑟)] with a context metric ||𝑓(𝑧𝑡𝑎𝑟)−𝑓(𝑧𝑠𝑟𝑐)||. To begin with, we roughly annotate the motion data with action labels (e.g., walk, jump, and crawl) to facilitate the nearest neighbor search; limiting the search only on the relevant subset of the same action label saves time and enhances accuracy of the results. Figure 7 compares the characterization result of Neutral Aver- ageJoe as the source (1st row) among MOCHA, Nearest Neighbor, and Motion Puzzle. Unlike MOCHA, both Motion Puzzle and the Nearest Neighbor method require a specific reference motion as input. Therefore, we manually selected a reference from each target character performing the most similar action (2nd row) for them. Compared to Motion Puzzle, MOCHA better preserves the unique style in each target character. Our results accurately capture the limping legs of the Neutral Zombie, the energetic waves of the Angry Clown, the elegantly crossed legs of the Princess, and the Figure 7 compares the characterization result of Neutral Aver- ageJoe as the source (1st row) among MOCHA, Nearest Neighbor, and Motion Puzzle. Unlike MOCHA, both Motion Puzzle and the Nearest Neighbor method require a specific reference motion as input. Therefore, we manually selected a reference from each target character performing the most similar action (2nd row) for them. As the NCM operates autoregressively, training the NCM en- coder and decoder is conducted with a sequence of source features 𝑧𝑖𝑠𝑟𝑐|𝑠 𝑖=0 and its corresponding sequence of target features 𝑧𝑖 𝑐ℎ𝑎|𝑠 𝑖=0 obtained with nearest neighbor search. These feature sequences represent continuous motions in motion database. In the training, the NCM transformer takes the previous character feature and cur- rent source context feature as condition to reconstruct the context- matched target character feature. 5.1 Stage-1 Figure 4 shows the training architecture in stage-1. Without the NCM, we randomly choose a source motion X𝑖𝑠𝑟𝑐and a character motion X𝑖 𝑐ℎ𝑎to train the bodypart encoder and the characterizer jointly. In addition to using common loss terms for learning style transfer, such as identity and cyclic consistency losses, we newly introduce a contrastive loss to enhance local context preservation both spatially and temporally. To simplify the notation, we use 𝐵𝐸(·) and 𝐶𝐻(𝑧𝑠𝑟𝑐,𝑧𝑐ℎ𝑎) to denote body part encoder and characterizer, and omit the superscript 𝑖. L𝑐𝑡𝑟= EX 𝐵 ∑︁ 𝑏 𝑙(𝑓(𝑧𝑡𝑟𝑎)𝑏, 𝑓(𝑧𝑠𝑟𝑐)𝑏, 𝑓(𝑧𝑠𝑟𝑐)𝐵\𝑏), (13) (13) ︁ where 𝑏∈{1, 2, . . . 𝐵} and 𝐵(= 𝑇/4 ∗𝑛𝑏𝑜𝑑𝑦) is the number of body patches. Figure 5 illustrates how body patch-level contrastive Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Figure 5: Illustration of body patch-level context contrastive loss. Blue box indicates a positive sample, while yellow boxes denote negative samples. Figure 6: Training architecture of Neural Context Matcher. Once trained, only the decoder is used for inference. Figure 5: Illustration of body patch-level context contrastive loss. Blue box indicates a positive sample, while yellow boxes denote negative samples. shape the latent variable 𝑠as a standard normal distribution (𝜇𝑝𝑟, 𝜎𝑝𝑟) using 𝑃𝑟𝑖𝑜𝑟𝑁𝑒𝑡. This process is illustrated in Fig. 6. For details on the training procedure, please see Algorithm 1 in supplementary material. At run-time, the encoder is discarded and the decoder is used to predict matched character features. At each time frame, we pass 𝑓(𝑧𝑖𝑠𝑟𝑐) through decoder to predict a matched character feature 𝑧𝑖 𝑐ℎ𝑎(Sec. 4.2). 5.2 Stage-2 At the same time, it attempts to Compared to Motion Puzzle, MOCHA better preserves the unique style in each target character. Our results accurately capture the limping legs of the Neutral Zombie, the energetic waves of the Angry Clown, the elegantly crossed legs of the Princess, and the MOCHA: Real-Time Motion Characterization via Context Matching SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Table 1: Frechet Motion Distance (FMD), context recognition accuracy (CRA), and style recognition accuracy (SRA) on sam- ples generated by each method as quantitative comparison. attention layer in the characterizing transformer decoder block (Fig- ure 3 (b)). To assess its impact, we trained a Characterizer without the AdaIN module. Results in Figure 8 shows that the ablated model cannot capture the Ogre’s raising arms style without using AdaIN. Methods FMD↓ SRA↑(%) CRA↑(%) Top1 Top5 Ground truth 43.61 84.76 - Ours 34.99 36.51 73.68 - Nearest Neighbor 32.80 23.62 69.06 - Motion Puzzle 39.51 31.78 70.7 - (a) MOCHA dataset. Methods FMD↓ SRA↑(%) CRA↑(%) Top1 Top5 Top1 Top5 Ground truth 96.46 99.77 58.97 85.60 Ours 25.98 68.57 95.21 38.18 74.45 Nearest Neighbor 29.30 63.32 93.20 45.96 79.55 (b) Adult2child dataset. 6.4.2 Effect of contrastive loss. To examine the effect of patch-level contrastive loss, we tested stage-1 training without the contrastive loss. Since the advantage of our contrastive loss is to preserve spatial-temporal context features across all characters, we randomly picked a source and a character motion to generate output. Figure 9 shows that the left and right legs of the output Ogre character are swapped (red circle) without patch-wise contrastive loss because attention module alone may mismatch the left leg of the source with the right leg of the character. In contrast, our model can distinguish the left and right side of the body. Additionally, despite significant differences of two motions, our method reasonably preserves the arm swinging motion of the source (red arrow), which cannot be achieved with the ablation model. 6.6 Applications 6.6.1 Input from unseen subjects. We test the effectiveness of our framework for inputs from unseen subjects with varying heights. Figure 11 (a) shows that our framework works successfully even when motions from unseen subjects with different body proportions are given as input. 6.6.2 Sparse input. We supposed that our framework could work with sparse inputs from the hip and end-effectors as they may contain essential information about context and style. To verify this, we reduced the input joints to only six (hip, head, hands, and feet) and retrained the entire framework. As shown in Figure 11 (b), our framework successfully characterizes motion as Neutral Princess from sparse input. This experiment suggests that our framework can accommodate 6-point tracker input data for real-time applications. 6.5 Context space analysisf wildly flailing arms of the Happy Zombie. In contrast, Motion Puzzle dampens the target style in favor of the source motion. Successful context matching across different characters is possible thanks to the shared character-agnostic context space constructed by the context mapping module. To visualize its result, we randomly sampled a character motion and computed its context feature as a query, then searched another character’s context database for the closest match. Figure 10 (a) shows semantically similar yet diverse postures across four different characters that match the query. In another experiment, given a source motion (X𝑠𝑟𝑐), we searched for the nearest context feature (𝑧𝑐ℎ𝑎) of a target character and generated a characterized motion (Y𝑡𝑟𝑎). Subsequently, both source and characterized motions were mapped to the context space as shown in Figure 10 (b). One can see that all three context feature points (𝑓(𝐵𝐸(X𝑠𝑟𝑐)), 𝑓(𝑧𝑐ℎ𝑎), 𝑓(𝐵𝐸(Y𝑡𝑟𝑎))) are located very close together, showing that the learned motion context indeed shares the same space across characters. Nearest Neighbor exhibits noticeable discontinuity in the output, due to the relatively sparse context space and insufficient temporal features used in search. It cannot respect the source motion context when similar context is not available for the target character. Thanks to the autoregressive CVAE, our method using NCM can mitigate these issues to produce smooth and consistent output. 6.3 Quantitative evaluation We quantitatively compare the degree of motion quality, context preservation, and style reflection with Motion Puzzle and the Near- est Neighbor method. Following prior work [Jang et al. 2022; Park et al. 2021], we use Frechet Motion Distance (FMD), context recog- nition accuracy (CRA), and style recognition accuracy (SRA) as metrics. We train the CRA classifier and the SRA classifier using vi- sion transformer [Dosovitskiy et al. 2020] for its superior accuracy. The two datasets, MOCHA and Adult2child, are each split into a 90% training set and a 10% test set. Results are detailed in Table 1. We didn’t apply CRA on the MOCHA dataset because it does not contain action labels. We also have to remove Motion Puzzle from the Adult2Child dataset comparison as it fails to train. When com- pared to Motion Puzzle, our method yields superior FMD and SRA scores, demonstrating higher motion quality and a more precise style reflection. A comparison with the Nearest Neighbor method underscores the significance of our NCM module, as we consistently achieve higher SRA scores due to superior temporal coherence. The better CRA score for Nearest Neighbor can be attributed to its direct usage of features from the database. 7 DISCUSSION AND CONCLUSION Yuzhu Dong, Sachin Paryani, Neha Rana, Aishat Aloba, Lisa Anthony, and Eakta Jain. 2017. Adult2Child: Dynamic Scaling Laws to Create Child-Like Motion. In Proceedings of the 10th International Conference on Motion in Games. 13:1–13:10. https://doi.org/10.1145/3136457.3136460 We introduced MOCHA, a motion characterization framework that can transform user motions to embody distinct style of characters in real-time, and demonstrated its effectiveness through a number of experiments and analysis. p g Mira Dontcheva, Gary Yngve, and Zoran Popović. 2003. Layered Acting for Character Animation. ACM Trans. Graph. 22, 3 (jul 2003), 409–416. https://doi.org/10.1145/ 882262.882285 Alexey Dosovitskiy, Lucas Beyer, Alexander Kolesnikov, Dirk Weissenborn, Xiaohua Zhai, Thomas Unterthiner, Mostafa Dehghani, Matthias Minderer, Georg Heigold, Sylvain Gelly, et al. 2020. An image is worth 16x16 words: Transformers for image recognition at scale. arXiv preprint arXiv:2010.11929 (2020). Our method has several limitations that necessitates further exploration. First, our method is most effective when a character motion dataset contains a single characteristic style per context. If multiple motions with different styles share the same context for a target character, our NCM may encounter difficulties in generating temporally consistent character features, leading to discontinuous motions. We observed such phenomena when consolidating all emotion sets of a character into one, thereby allowing emotional style variations within the same motion. 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This work was conducted during Deok-Kyeong Jang’s internship at Meta. Deok-Kyeong Jang would like to thank Michelle Hill for her help in capturing the dataset and the Meta team for their mentor- ship and support during his internship. Jungdam Won was partially supported by the New Faculty Startup Fund from Seoul National University, ICT (Institute of Computer Technology) at Seoul Na- tional University. Sung-Hee Lee was partially supported by the Technology Innovation Program (20011076) of KEIT. Wanli Ma, Shihong Xia, Jessica K Hodgins, Xiao Yang, Chunpeng Li, and Zhaoqi Wang. 2010. Modeling style and variation in human motion. In Proceedings of the 2010 ACM SIGGRAPH/Eurographics Symposium on Computer Animation. 21–30. https://doi.org/10.1109/IUCS.2010.5666642 Ian Mason, Sebastian Starke, He Zhang, Hakan Bilen, and Taku Komura. 2018. Few- shot Learning of Homogeneous Human Locomotion Styles. In Computer Graphics Forum, Vol. 37. Wiley Online Library, 143–153. https://doi.org/10.1111/cgf.13555 Jianyuan Min, Yen-Lin Chen, and Jinxiang Chai. 2009. Interactive Generation of Human Animation with Deformable Motion Models. ACM Trans. Graph. 29, 1, Article 9 (dec 2009), 12 pages. https://doi.org/10.1145/1640443.1640452 y g Animation with Deformable Motion Models. ACM Trans. Graph. 29, 1, Article 9 (dec 2009), 12 pages. https://doi.org/10.1145/1640443.1640452 Aaron van den Oord, Yazhe Li, and Oriol Vinyals. 2018. Representation learning with contrastive predictive coding. arXiv preprint arXiv:1807.03748 (2018). 7 DISCUSSION AND CONCLUSION More advanced methods for context learning is desirable to improve the characterization quality. Daniel Holden, Jun Saito, and Taku Komura. 2016. A deep learning framework for character motion synthesis and editing. ACM Transactions on Graphics (TOG) 35, 4 (2016), 1–11. https://doi.org/10.1145/2897824.2925975 Xun Huang and Serge Belongie. 2017. Arbitrary style transfer in real-time with adaptive instance normalization. In Proceedings of the IEEE International Conference on Computer Vision. 1501–1510. Leslie Ikemoto, Okan Arikan, and David Forsyth. 2009. Generalizing Motion Edits with Gaussian Processes. ACM Trans. Graph. 28, 1, Article 1 (feb 2009), 12 pages. https://doi.org/10.1145/1477926.1477927 Deok-Kyeong Jang, Soomin Park, and Sung-Hee Lee. 2022. Motion Puzzle: Arbitrary Motion Style Transfer by Body Part. ACM Trans. Graph. 41, 3, Article 33 (jun 2022), 16 pages. https://doi.org/10.1145/3516429 6.4 Ablation study 6.6.3 Live characterization from streamed motion data. We demon- strate the ability of our framework to characterize streamed motion data in real-time. Figure 11 (c) shows a snapshot of live characteri- zation of a streamed motion captured with Xsens Awinda sensor. The supplementary video shows that our method can produce suc- cessfully characterized motion even with network delays and noisy We conducted ablation studies to demonstrate the effectiveness of the AdaIN layer and the contrastive loss. For additional ablation studies, please refer to the supplementary material. 6.4.1 Effect of AdaIN.. The AdaIN module facilitates successful characterization by incorporating a global style before the cross Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia input. The overall inference time is under 16ms, achieving 60Hz or higher frame rate with a 2080 Ti GPU. Yuzhu Dong, Andreas Aristidou, Ariel Shamir, Moshe Mahler, and Eakta Jain. 2020. Adult2child: Motion Style Transfer Using CycleGANs. In Proceedings of the 13th ACM SIGGRAPH Conference on Motion, Interaction and Games. Article 13, 11 pages. https://doi.org/10.1145/3424636.3426909 T. Tao, X. Zhan, Z. Chen, and M. van de Panne. 2022a. Style-ERD: Responsive and Coherent Online Motion Style Transfer. In 2022 IEEE/CVF Conference on Computer SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia REFERENCESi Zhan, Z. Chen, and M. van de Panne. 2022a. Style-ERD: Responsive and Coherent Online Motion Style Transfer. In 2022 IEEE/CVF Conference on Computer MOCHA: Real-Time Motion Characterization via Context Matching Vision and Pattern Recognition (CVPR). IEEE Computer Society, Los Alamitos, CA, USA, 6583–6593. https://doi.org/10.1109/CVPR52688.2022.00648 Vision and Pattern Recognition (CVPR). IEEE Computer Society, Los Alamitos, CA, USA, 6583–6593. https://doi.org/10.1109/CVPR52688.2022.00648 p g Tianxin Tao, Xiaohang Zhan, Zhongquan Chen, and Michiel van de Panne. 2022b. Style-ERD: responsive and coherent online motion style transfer. In Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition. 6593–6603. Munetoshi Unuma, Ken Anjyo, and Ryozo Takeuchi. 1995. Fourier principles for emotion-based human figure animation. In Proceedings of the 22nd annual conference on Computer graphics and interactive techniques. 91–96. Jack M. Wang, David J. Fleet, and Aaron Hertzmann. 2007. Multifactor Gaussian Process Models for Style-Content Separation. In Proceedings of the 24th International Conference on Machine Learning (ICML ’07). 975–982. https://doi.org/10.1145/ 1273496.1273619 Shihong Xia, Congyi Wang, Jinxiang Chai, and Jessica Hodgins. 2015. Realtime style transfer for unlabeled heterogeneous human motion. ACM Transactions on Graphics (TOG) 34, 4 (2015), 1–10. https://doi.org/10.1145/2766999 p g Sijie Yan, Yuanjun Xiong, and Dahua Lin. 2018. Spatial temporal graph convolu- tional networks for skeleton-based action recognition. In Proceedings of the AAAI Conference on Artificial Intelligence, Vol. 32. i M Ersin Yumer and Niloy J Mitra. 2016. Spectral style transfer for human motion between independent actions. ACM Transactions on Graphics (TOG) 35, 4 (2016), 1–8. https://doi.org/10.1145/2897824.2925955 p g T. Zhou, P. Krahenbuhl, M. Aubry, Q. Huang, and A. A. Efros. 2016. Learning Dense Correspondence via 3D-Guided Cycle Consistency. In 2016 IEEE Conference on Com- puter Vision and Pattern Recognition (CVPR). IEEE Computer Society, Los Alamitos, CA, USA, 117–126. https://doi.org/10.1109/CVPR.2016.20 SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia Deok-Kyeong Jang, Yuting Ye, Jungdam Won, and Sung-Hee Lee Figure 7: Qualitative evaluation. Source motions of Neutral AverageJoe (top) are characterized with each method (3-5 rows). Reference motion is used for the Nearest Neighbor and Motion Puzzle. Figure 7: Qualitative evaluation. Source motions of Neutral AverageJoe (top) are characterized with each method (3-5 rows). Reference motion is used for the Nearest Neighbor and Motion Puzzle. MOCHA: Real-Time Motion Characterization via Context Matching SA Conference Papers ’23, December 12–15, 2023, Sydney, NSW, Australia g p y y Figure 8: Ablation study on AdaIN. Result obtained without AdaIN fails to capture the jumping style of Ogre. REFERENCESi ation study on AdaIN. Result obtained without AdaIN fails to capture the jumping style of Ogre. Figure 8: Ablation study on AdaIN. Result obtained without AdaIN fails to capture the jumpi Figure 9: Contrastive loss test. Ogre’s left and right leg are mistakenly swapped (red circle) without the contrastive loss. Furthermore, the contrastive loss helps retain the left arm’s swing phase of the source motion to the characterized motion (red arrows). Figure 9: Contrastive loss test. Ogre’s left and right leg are mistakenly swapped (red circle) without the contrastive loss. Furthermore, the contrastive loss helps retain the left arm’s swing phase of the source motion to the characterized motion (red arrows). Figure 10: (a) Four best matching motions in the entire datasets given the white character’s motion (top: running, bottom: walking). (b) A t-SNE visualization of the entire context features in the test dataset with colors differentiating characters. Three context feature points corresponding to a source, matched target, and output motion are located very closely to each other. Figure 10: (a) Four best matching motions in the entire datasets given the white character’s motion (top: running, bottom: walking). (b) A t-SNE visualization of the entire context features in the test dataset with colors differentiating characters. Three context feature points corresponding to a source, matched target, and output motion are located very closely to each other. Figure 11: Applications of our framework. Characterization results from an unseen subject (left), with sparse input from 6 joints (middle), and from streamed motion data (right). Figure 11: Applications of our framework. Characterization results from an unseen subject (left), with sparse input from 6 joints (middle), and from streamed motion data (right).
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Business decision analysis as a component of enterprise activity: theoretical aspect
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̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² ̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² ²ÂÀÍÞÒÀ Ò.Ì. Àíàë³òèêà á³çíåñ–ð³øåíü ÿê ñêëàäîâà ä³ÿëüíîñò³ ï³äïðèºìñòâà: òåîðåòè÷íèé àñïåêò Ïðåäìåò äîñë³äæåííÿ.  ñòàòò³ äîñë³äæåíî òåîðåòè÷íèé àñïåêò àíàë³òèêè á³çíåñ–ð³øåíü ÿê ñêëàäîâî¿ ä³ÿëüíîñò³ ï³äïðèºìñòâà â ñó÷àñíèõ óìîâàõ ãîñïîäàðþâàííÿ ñóá’ºêò³â ï³äïðèºìíèöòâà. Ìåòîþ íàïèñàííÿ ñòàòò³. Äîñë³äæåíî îñíîâíó ìåòó á³çíåñ–àíàë³òèêè òà âñòàíîâëåíî, ùî àíà- ë³òèêà á³çíåñ–ð³øåíü â êîðîòêîìó ñåíñ³ ñëîâà – öå àíàë³òè÷íà ñêëàäîâà, ùî ñóïðîâîäæóºòüñÿ âè- êîðèñòàííÿì ñó÷àñíèõ ï³äõîä³â ï³ä ÷àñ àíàë³çó ³íôîðìàö³¿ â îáëàñò³ ï³äãîòîâêè á³çíåñ–ð³øåíü. Ìåòîäîëîã³ÿ ïðîâåäåííÿ ðîáîòè.  øèðøîìó àñïåêò³, àíàë³òèêà á³çíåñ–ð³øåíü – öå çàãàëüíà ñóêóïí³ñòü ïðèíöèï³â ìåòîäîëîã³÷íî–îðãàí³çàö³éíîãî, à òàêîæ òåõíîëîã³÷íîãî ð³çíîãî ðîäó ðîçóìîâî¿ ä³ÿëüíîñò³, ùî äîçâîëÿº åôåêòèâíî ïðîâîäèòè îáðîáêó ³íôîðìàö³¿ ç ö³ëëþ ï³äâèùåííÿ ÿêîñò³ íàÿâ- íèõ ³ îòðèìàííÿ íîâèõ çíàíü, à òàêîæ ï³äãîòîâêè ³íôîðìàö³éíî–³íòåëåêòóàëüíî¿ áàçè äëÿ ïðèéíÿòòÿ ðàö³îíàëüíèõ óïðàâë³íñüêèõ á³çíåñ–ð³øåíü â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³ ñóá’ºêò³â ãîñïîäàðþâàííÿ. Ðåçóëüòàòè ðîáîòè. Ðîçãëÿíóòî îñíîâí³ ïðîöåñè, ÿê³ âèçíà÷àþòü àíàë³òèêó á³çíåñ–ð³øåíü ÿê ñïåöèô³÷íèé âèä ä³ÿëüíîñò³ íà ï³äïðèºìñòâ³. Âñòàíîâëåíî, ùî âèá³ð àëüòåðíàòèâè â õîä³ îáðàííÿ á³çíåñ–ð³øåííÿ çàëåæèòü â³ä: ìîæëèâîñò³ îö³íèòè åôåêòèâí³ñòü àëüòåðíàòèâíîãî ð³øåííÿ ³ íåîáõ³äí³ âèòðàòè äëÿ éîãî ðåàë³çàö³¿; ìåòîä³â, ÿê³ âèêîðèñòîâóâàëèñÿ â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³, à òàêîæ ðîçðîáëåí³ äî öüîãî ÷àñó ìîäåë³ äîñë³- äæåííÿ ï³äïðèºìíèöüêèõ îïåðàö³é; ìåòîä³â, ÿê³ äîçâîëèëè á àíàë³çóâàòè ñõîæ³ ïðîáëåìè.  ñòàòò³ ïðåäñòàâëåí³ ï³äõîäè äî âèíà÷åííÿ ïîíÿòòÿ «àíàë³òèêà á³çíåñ–ð³øåíü» òà òðè íàéâàæ- ëèâ³ø³ ñêëàäîâ³, ÿê³ îá’ºäíóº àíàë³òèêà á³çíåñ ð³øåíü. Äîñë³äæåíî îñíîâí³ ïðèíöèïè òà ñêëàäîâ³ àíàë³çó äàíèõ â á³çíåñ–àíàë³òèö³. Âèñíîâîê. Îðãàí³çàö³ÿ ïðîöåñó ïðèéíÿòòÿ á³çíåñ–ð³øåííÿ çàëåæèòü â³ä ïðîåêòóâàííÿ ³íôîðìà- ö³éíèõ òåõíîëîã³é äëÿ á³çíåñ–àíàë³òèêè â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³ òà ìຠáàçóâàòèñÿ íà ³íòåãðàö³¿ ¿õ ó ñèñòåìó óïðàâë³ííÿ á³çíåñ–ïðîöåñàìè ï³äïðèºìñòâà òà â³äïîâ³äàòè á³çíåñ–ìîäåë³ êîæíîãî ï³ä- ïðèºìñòâà ç óðàõóâàííÿì ìîæëèâîñò³ ¿õ ³íòåãðóâàííÿ ó íàÿâí³ ³íôîðìàö³éí³ ñèñòåìè ï³äïðèºìñòâà. Êëþ÷îâ³ ñëîâà: ï³äïðèºìñòâî, àíàë³ç, àíàë³òèêà, á³çíåñ–ð³øåííÿ, ñóá’ºêò ãîñïîäàðþâàííÿ, àíàë³òèê. ÅÊÎÍÎ̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßË Î̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ Óñòàíîâëåíî, ÷òî âûáîð àëüòåðíàòèâû â õîäå èçáðàíèÿ áèçíåñ–ðåøåíèÿ çàâèñèò îò: âîçìîæ- íîñòè îöåíèòü ýôôåêòèâíîñòü àëüòåðíàòèâíîãî ðåøåíèÿ è íåîáõîäèìûå ðàñõîäû äëÿ åãî ðåàëè- çàöèè; ìåòîäîâ, êîòîðûå èñïîëüçîâàëèñü â ïðåäïðèíèìàòåëüñêîé äåÿòåëüíîñòè, à òàêæå ðàçðà- áîòàííûå äî ñèõ ïîð ìîäåëè èññëåäîâàíèÿ ïðåäïðèíèìàòåëüñêèõ îïåðàöèé; ìåòîäîâ, êîòîðûå ïîçâîëèëè áû àíàëèçèðîâàòü ñõîæèå ïðîáëåìû.  ñòàòüå ïðåäñòàâëåíû ïîäõîäû ê îïðåäåëåíèþ ïîíÿòèÿ «àíàëèòèêà áèçíåñ–ðåøåíèé» è òðè âàæíåéøèõ ñêëàäîâûõ, êîòîðûå îáúåäèíÿåò àíàëèòèêà áèçíåñ ðåøåíèé. Èññëåäîâàíî îñíîâíûå ïðèíöèïû è ñîñòàâëÿþùèå àíàëèçà äàííûõ â áèçíåñ–àíàëèòèêå. Âûâîä. Îðãàíèçàöèÿ ïðîöåññà ïðèíÿòèÿ áèçíåñ–ðåøåíèÿ çàâèñèò îò ïðîåêòèðîâàíèÿ èí- ôîðìàöèîííûõ òåõíîëîãèé äëÿ áèçíåñ–àíàëèòèêè â ïðåäïðèíèìàòåëüñêîé äåÿòåëüíîñòè è äî- ëæíà áàçèðîâàòüñÿ íà èíòåãðàöèè èõ â ñèñòåìó óïðàâëåíèÿ áèçíåñ–ïðîöåññàìè ïðåäïðèÿòèÿ è ñîîòâåòñòâîâàòü áèçíåñ–ìîäåëè êàæäîãî ïðåäïðèÿòèÿ ñ ó÷åòîì âîçìîæíîñòè èõ èíòåãðàöèè â èìåþùèåñÿ èíôîðìàöèîííûå ñèñòåìû ïðåäïðèÿòèÿ. Êëþ÷åâûå ñëîâà: ïðåäïðèÿòèå, àíàëèç, àíàëèòèêà, áèçíåñ–ðåøåíèÿ, ñóáúåêò õîçÿéñòâîâàíèÿ, àíàëèòèê. Êëþ÷åâûå ñëîâà: ïðåäïðèÿòèå, àíàëèç, àíàëèòèêà, áèçíåñ–ðåøåíèÿ, ñóáúåêò õîçÿéñòâîâàíèÿ, àíàëèòèê ñëîâà: ïðåäïðèÿòèå, àíàëèç, àíàëèòèêà, áèçíåñ–ðåøåíèÿ, ñóáúåêò õîçÿéñòâîâàíèÿ, IVANYUTA T.M. 62 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   Àíàëèòèêà áèçíåñ–ðåøåíèé êàê ñîñòàâëÿþùàÿ ïðåäïðèÿòèÿ: òåîðåòè÷åñêèé àñïåêò Ïðåäìåò èññëåäîâàíèÿ.  ñòàòüå èññëåäîâàí òåîðåòè÷åñêèé àñïåêò àíàëèòèêè áèçíåñ–ðå- øåíèé êàê äåÿòåëüíîñòè ïðåäïðèÿòèÿ â ñîâðåìåííûõ óñëîâèÿõ õîçÿéñòâîâàíèÿ ñóáúåêòîâ ïðåä- ïðèíèìàòåëüñòâà. Öåëüþ íàïèñàíèÿ ñòàòüè. Èññëåäîâàíî îñíîâíóþ öåëü áèçíåñ–àíàëèòèêè è óñòàíîâëåíî, ÷òî àíàëèòèêà áèçíåñ–ðåøåíèé â êðàòêîì ñìûñëå ñëîâà – ýòî àíàëèòè÷åñêàÿ ñîñòàâëÿþùàÿ, êîòî- ðàÿ ñîïðîâîæäàåòñÿ èñïîëüçîâàíèåì ñîâðåìåííûõ ïîäõîäîâ ïðè àíàëèçå èíôîðìàöèè â îáëàñòè ïîäãîòîâêè áèçíåñ–ðåøåíèé. Ìåòîäîëîãèÿ ïðîâåäåíèÿ ðàáîòû.  áîëåå øèðîêîì àñïåêòå, àíàëèòèêà áèçíåñ–ðåøåíèé – ýòî îáùàÿ ñîâîêóïíîñòü ïðèíöèïîâ ìåòîäîëîãè÷åñêè–îðãàíèçàöèîííîãî, à òàêæå òåõíîëîãè- ÷åñêîãî ðàçíîãî ðîäà óìñòâåííîé äåÿòåëüíîñòè, ÷òî ïîçâîëÿåò ýôôåêòèâíî ïðîâîäèòü îáðàáîòêó èíôîðìàöèè ñ öåëüþ ïîâûøåíèÿ êà÷åñòâà ñóùåñòâóþùèõ è ïîëó÷åíèÿ íîâûõ çíàíèé, à òàêæå ïî- äãîòîâêè èíôîðìàöèîííî–èíòåëëåêòóàëüíîé áàçû äëÿ ïðèíÿòèÿ ðàöèîíàëüíûõ óïðàâëåí÷åñêèõ áèçíåñ–ðåøåíèé â ïðåäïðèíèìàòåëüñêîé äåÿòåëüíîñòè ñóáúåêòîâ õîçÿéñòâîâàíèÿ. Ðåçóëüòàòû ðàáîòû. Ðàññìîòðåíû îñíîâíûå ïðîöåññû, êîòîðûå îïðåäåëÿþò àíàëèòèêó áèç- íåñ–ðåøåíèé êàê ñïåöèôè÷åñêèé âèä äåÿòåëüíîñòè íà ïðåäïðèÿòèè. © ²ÂÀÍÞÒÀ Ò.Ì., 2021 61 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   Business decision analysis as a component of enterprise activity: theoretical aspect The subject of research. The article examines the theoretical aspect of business decision analysis as a component of the enterprise in modern business conditions of business entities. The purpose of writing. The main purpose of business analytics is studied and it is established that business decision analytics in the short sense of the word is an analytical component, which is accompanied by the use of modern approaches in the analysis of information in the field of business decision preparation. Methodology of work In a broader sense, business decision analytics is a general set of principles of methodological and organizational, as well as technological various kinds of mental activity, which allows you to effectively process information to improve the quality of existing and new knowledge, as well as preparing information and intellectual base for adoption rational managerial business decisions in business activities of business entities. The results of the work. The main processes that define the analysis of business decisions as a specific type of activity in the enterprise are considered. It is established that the choice of alternatives during the selection of a business solution depends on: the ability to assess the effectiveness of the alternative solution and the necessary costs for its implementation; methods used in business activities, as well as models developed so far to study business operations; methods that would analyze similar problems. The article presents approaches to the introduction of the concept of «business decision analyst» and the three most important components that unite business decision analytics. Researched the basic principles and components of data analysis in business intelligence. Conclusions. We concluded that the organization of the business decision–making process depends on the design of information technology for business intelligence in business and should be based on their integration into the business process management system of the enterprise and meet the business model of each enterprise taking into account their integration into existing information enterprise systems. Keywords: enterprise, analysis, analytics, business decisions, business entity, analyst. ³íôîðìàö³éíèõ ïîòîê³â, ÿê³ äîçâîëÿþòü, â çà- ëåæíîñò³ â³ä ¿õ ïîâíîòè, ïðèéìàòè åêîíîì³÷í³ òà óïðàâë³íñüê³ á³çíåñ–ð³øåííÿ. Ïîñòàíîâêà ïðîáëåìè. ijÿëüí³ñòü ï³äïðè- ºìñòâ â ðèíêîâèõ êîíêóðåíòíèõ óìîâàõ ãîñïî- äàðþâàííÿ çàëåæèòü â³ä âíóòð³øí³õ òà çîâí³øí³õ 62 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   Î̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ Ïðèéíÿòòÿ á³çíåñ–ð³øåííÿ áóäå çàëåæàòè â³ä ÿêîñò³, ñâîº÷àñíîñò³ íàäõîäæåííÿ ïîòîêó ³íôîð- ìàö³¿ é ñïåö³àë³ñòà, ÿêèé ïðîâîäèòü á³çíåñ–àíà- ë³ç, ç ìåòîþ çàäîâîëåííÿ ïîòðåá ï³äïðèºìñòâà (åêîíîì³÷íîãî ñóá’ºêòà). Business decision analysis as a component of enterprise activity: theoretical aspect øåíü ñïðè÷èíþþòü íåìîæëèâ³ñòü åôåêòèâíîãî óïðàâë³ííÿ ï³äïðèºìñòâîì áåç âèêîðèñòàííÿ ñó- ÷àñíèõ ³íôîðìàö³éíèõ òåõíîëîã³é, çîêðåìà, ìî- äåëåé, ìåòîä³â ³ çàñîá³â á³çíåñ–àíàë³òèêè. Íà ñüîãîäí³, åêîíîì³÷í³é ä³ÿëüíîñò³ ï³äïðè- ºìñòâ ïðèòàìàíí³ òàê³ îçíàêè, ÿê: êðèòè÷íèé ð³- âåíü êîíêóðåíö³¿ íà ðèíêó ³ ¿õ ÷óòëèâ³ñòü íà âïëèâ åêîíîì³÷íèõ ôàêòîð³â ³ ÷èííèê³â. Â÷åíèìè âñòàíîâëåíî, ùî á³çíåñ–àíàë³ç º íà- áîðîì çàâäàíü òà ìåòîäèê, ùî âèêîðèñòîâóºòü- ñÿ ç ö³ëëþ çàáåçïå÷èòè ðîçóì³ííÿ çàö³êàâëåíèìè ñòîðîíàìè ñòðóêòóðè, ïðàâèë òà ðîáîòè îðãàí³- çàö³¿ é çàïðîïîíóâàòè ð³øåííÿ, ÿê³ äîçâîëÿòü îð- ãàí³çàö³¿ äîñÿãòè ïîñòàâëåíèõ ö³ëåé.  ê³íöåâîìó ðåçóëüòàò³ – íåñòàá³ëüí³ñòü ñòàíó íà ðèíêó, ÿêà âèìàãຠîïåðàòèâíèõ ³ âèñîêîÿê³ñ- íèõ ð³øåíü ìåíåäæåð³â íà âñ³õ ð³âíÿõ óïðàâë³ííÿ ï³äïðèºìñòâîì. Òîìó ïèòàííÿ àíàë³òèêè á³çíåñ–ð³øåíü ÿê ñêëàäîâî¿ ä³ÿëüíîñò³ ï³äïðèºìñòâà º àêòóàëüíè- ìè ³ ïîòðåáóþòü óâàãè ç áîêó â÷åíèõ. Îñíîâîþ äëÿ ïðèéíÿòòÿ óïðàâë³íñüêèõ á³ç- íåñ– ð³øåíü º ïîâíà ³íôîðìàö³ÿ ïðî ñòàí á³çíå- ñó ³ òåíäåíö³é ðîçâèòêó ðèíêó, à êåðóâàííÿ òàêîþ ³íôîðìàö³ºþ – ñêëàäíèé ïðîöåñ, ùî âèìàãຠêîìïëåêñíîãî ï³äõîäó. Äîñë³äæóâàíà òåìà íàóêîâî¿ ñòàòò³ âàðòà óâà- ãè á³çíåñ–àíàë³òèê³â, íàóêîâö³â òà ôàõîâèõ ñïå- ö³àë³ñò³â. Îñíîâíà ìåòà àíàë³òèêè á³çíåñ–ð³øåíü – öå îð³ºíòàö³ÿ óïðàâë³íñüêîãî ïðîöåñó íà äîñÿãíåí- íÿ ö³ëåé, ùî ñòîÿòü ïåðåä ñóá’ºêòîì ãîñïîäà- ðþâàííÿ (ï³äïðèºìñòâîì, îðãàí³çàö³ºþ, ô³ðìîþ òà ³í.), çà ðàõóíîê çì³ùåííÿ àêöåíòó ç ä³àãíîñ- òèêè ìèíóëèõ ðåòðîñïåêòèâ íà ïðîãíîç ìàéáóò- í³õ ïåðñïåêòèâ, â³äñòåæåííÿ 䳿 âïëèâó âíóòð³øí³õ ³ çîâí³øí³õ ôàêòîð³â íà ä³ÿëüí³ñòü ï³äïðèºìñòâà ç ìåòîþ ïðèéíÿòòÿ ðàö³îíàëüíèõ á³çíåñ–ð³øåíü, ³íôîðìàö³éíî¿ òà êîíñóëüòàö³éíî¿ ï³äòðèìêè ïðè- éíÿòòÿ óïðàâë³íñüêèõ á³çíåñ–ð³øåíü [2]. Àíàë³ç îñòàíí³õ äîñë³äæåíü òà ïóáë³êàö³é. Îñíîâíîþ ì³ñ³ºþ äëÿ åêîíîì³êè êîæíî¿ êðà¿íè º çà- áåçïå÷åííÿ âèïåðåäæàþ÷èõ òåìï³â åêîíîì³÷íîãî çðîñòàííÿ ï³äïðèºìñòâ, ùî äîñÿãàºòüñÿ â õîä³ ïðè- éíÿòòÿ åôåêòèâíèõ á³çíåñ–ð³øåíü, ÿê³ ìîæóòü áóòè ïðèéíÿò³ â õîä³ ïðàâèëüíîãî ï³äáîðó ³íôîðìàö³¿ òà îáðàííÿ àëüòåðíàòèâíîãî ð³øåííÿ, ùî çàëåæèòü â ï³äïðèºìíèöòâ³ é â³ä òåõíîëîã³÷íèõ ³ííîâàö³é. Ïî÷èíàþ÷è ç 1980 ðîêó â çàõ³äí³é á³çíåñ– ïðàêòèö³ ç’ÿâèëîñÿ ïîíÿòòÿ á³çíåñ àíàë³òèêè (Business Analytics), ÿêå é íèí³ ïîâí³ñòþ îòîòîæ- íþºòüñÿ äåÿêèìè íàóêîâöÿìè ç á³çíåñ àíàë³çîì. Ïî÷èíàþ÷è ç 1980 ðîêó â çàõ³äí³é á³çíåñ– ïðàêòèö³ ç’ÿâèëîñÿ ïîíÿòòÿ á³çíåñ àíàë³òèêè (Business Analytics), ÿêå é íèí³ ïîâí³ñòþ îòîòîæ- íþºòüñÿ äåÿêèìè íàóêîâöÿìè ç á³çíåñ àíàë³çîì. Äî òåïåð³øíüîãî ÷àñó öå ïîíÿòòÿ îòðèìàëî äå- ÿêå òåîðåòè÷íå îá´ðóíòóâàííÿ, ùî çàñâ³ä÷óº âå- ëèêà ê³ëüê³ñòü íàóêîâèõ ïðàöü ³íîçåìíèõ â÷åíèõ ³ ïðàêòèê³â ç ö³º¿ ïðîáëåìàòèêè [1–5]. Àíàë³ç (ãðåö. ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   Business decision analysis as a component of enterprise activity: theoretical aspect – ðîçïàä, ðîç÷ëåíóâàííÿ, ðîçá³ð) – öå ëîã³÷íèé ïðèéîì, ìåòîä äîñë³äæåííÿ, ÿêèé ïîëÿãຠâ òîìó, ùî äîñë³äæóâàíèé ïðåäìåò ïî- äóìêè ðîçä³ëÿºòüñÿ íà ñêëàäîâ³ åëåìåíòè, ÿê³ ïî- ò³ì äîñë³äæóþòüñÿ îêðåìî ÿê ÷àñòèíà ðîçïîä³ëå- íîãî ö³ëîãî, äëÿ òîãî, ùîá âèä³ëåí³ â õîä³ àíàë³çó åëåìåíòè ç’ºäíàëèñÿ ç äîïîìîãîþ ³íøîãî ëîã³÷- íîãî ïðèéîìó – ñèíòåçó – â ö³ëå, çáàãà÷åíå íî- âèìè çíàííÿìè [3]. Äî òåïåð³øíüîãî ÷àñó öå ïîíÿòòÿ îòðèìàëî äå- ÿêå òåîðåòè÷íå îá´ðóíòóâàííÿ, ùî çàñâ³ä÷óº âå- ëèêà ê³ëüê³ñòü íàóêîâèõ ïðàöü ³íîçåìíèõ â÷åíèõ ³ ïðàêòèê³â ç ö³º¿ ïðîáëåìàòèêè [1–5]. Äî â³äîìèõ ïðåäñòàâíèê³â åêîíîì³÷íî¿ äóìêè, ÿê³ â ö³ëîìó äîñë³äæóâàëè òåìó ïðèñâÿ÷åíó àíà- ë³òèö³ á³çíåñ–ð³øåíü â åêîíîì³ö³ ìîæíà â³äíåñòè Áàðèëåíêî Â.²., Áîíäàðåíêî Â.Ã., Êîíðàä Êàðë- áåðãà, Ëÿøåíêî Î., Áàá³ÿ Ñ. Â., Øòåôàíà Á [1–5]. Ìåòà ñòàòò³. Äîñë³äèòè òåîðåòè÷íèé àñïåêò àíàë³òèêè á³çíåñ–ð³øåíü ÿê ñêëàäîâî¿ ä³ÿëüíîñò³ ï³äïðèºìñòâà â ñó÷àñíèõ óìîâàõ ãîñïîäàðþâàí- íÿ ñóá’ºêò³â ï³äïðèºìíèöòâà. Äî â³äîìèõ ïðåäñòàâíèê³â åêîíîì³÷íî¿ äóìêè, ÿê³ â ö³ëîìó äîñë³äæóâàëè òåìó ïðèñâÿ÷åíó àíà- ë³òèö³ á³çíåñ–ð³øåíü â åêîíîì³ö³ ìîæíà â³äíåñòè Áàðèëåíêî Â.²., Áîíäàðåíêî Â.Ã., Êîíðàä Êàðë- áåðãà, Ëÿøåíêî Î., Áàá³ÿ Ñ. Â., Øòåôàíà Á [1–5]. Òîìó àíàë³òèêà – öå êîìïëåêñíà íàóêîâà ñêëà- äîâà, â ÿê³é ìîæíà âèêîðèñòàòè ïðèíöèïîâî íîâ³, óð³çíîìàí³òíåíí³ ï³äõîäè ïðè àíàë³ç³ ³íôîðìàö³¿ â êîíòåêñò³ ï³äãîòîâêè óïðàâë³íñüêèõ ð³øåíü. Ìåòà ñòàòò³. Äîñë³äèòè òåîðåòè÷íèé àñïåêò àíàë³òèêè á³çíåñ–ð³øåíü ÿê ñêëàäîâî¿ ä³ÿëüíîñò³ ï³äïðèºìñòâà â ñó÷àñíèõ óìîâàõ ãîñïîäàðþâàí- íÿ ñóá’ºêò³â ï³äïðèºìíèöòâà. Àíàë³òèêà á³çíåñ–ð³øåíü – öå àíàë³òè÷íà ñêëà- äîâà, ùî ñóïðîâîäæóºòüñÿ âèêîðèñòàííÿì ñó- ÷àñíèõ ï³äõîä³â ï³ä ÷àñ àíàë³çó ³íôîðìàö³¿ â îá- ëàñò³ ï³äãîòîâêè á³çíåñ–ð³øåíü. Âèêëàä îñíîâíîãî ìàòåð³àëó. Ôóíêö³îíóâàí- íÿ ñó÷àñíèõ ï³äïðèºìñòâ ð³çíèõ ãàëóçåé åêîíîì³êè ó ðåàëüíèõ óìîâàõ õàðàêòåðíà ïîñò³éíîþ áîðîòü- áîþ ç íàðîñòàþ÷îþ êîíêóðåíö³ºþ, ÿêà ïîñò³éíî âèìàãຠâ³ä óïðàâë³íö³â ï³äâèùåííÿ îïåðàòèâíî- ñò³ ³ ÿêîñò³ ïðèéíÿòòÿ óïðàâë³íñüêèõ ð³øåíü. Ìîæíà çàçíà÷èòè, ùî ³ñíóþòü îñíîâí³ ïðîöåñè, ÿê³ âèçíà÷àþòü àíàë³òèêó á³çíåñ–ð³øåíü ÿê ñïåöè- ô³÷íèé âèä ä³ÿëüíîñò³ íà ï³äïðèºìñòâ³, ñåðåä ÿêèõ: Âåëèê³ îáñÿãè ðèíêîâî¿ ³íôîðìàö³¿, ÿêó íåîáõ³ä- íî ìîí³òîðèòè â õîä³ ïðèéíÿòòÿ óïðàâë³íñüêèõ ð³- 1. Àíàë³ç ö³ëåé óïðàâë³ííÿ ³ îá´ðóíòóâàííÿ çà- âäàíü ³íôîðìàö³éí–àíàë³òè÷íî¿ ðîáîòè. 63 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   ²×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² 2. Óïðàâë³ííÿ çáîðîì ³íôîðìàö³¿ ³ âèçíà÷åííÿ çíàíü äëÿ âèð³øåííÿ óïðàâë³íñüêèõ á³çíåñ–çà- äà÷ â óìîâàõ ì³íëèâî¿ åêîíîì³÷íî¿ ñèòóàö³¿. åôåêòèâíî ïðîâîäèòè îáðîáêó ³íôîðìàö³¿ ç ö³ë- ëþ ï³äâèùåííÿ ÿêîñò³ íàÿâíèõ ³ îòðèìàííÿ íî- âèõ çíàíü, à òàêîæ ï³äãîòîâêè ³íôîðìàö³éíî–³í- òåëåêòóàëüíî¿ áàçè äëÿ ïðèéíÿòòÿ ðàö³îíàëüíèõ óïðàâë³íñüêèõ á³çíåñ–ð³øåíü â ï³äïðèºìíèöüê³é ä³ÿëüíîñò³ ñóá’ºêò³â ãîñïîäàðþâàííÿ. 3. Àíàë³ç ³ îö³íêà îòðèìàíî¿ ³íôîðìàö³¿ ³ çíàíü â êîíòåêñò³ çàâäàíü óïðàâë³ííÿ, âèÿâëåííÿ ñóò³ ñïîñòåðåæóâàíèõ ïðîöåñ³â ³ ÿâèù. 4. Business decision analysis as a component of enterprise activity: theoretical aspect Ïîáóäîâà ìîäåëåé ïðåäìåòíî¿ ³ ïðîáëåìíî¿ îáëàñòåé, îá’ºêòà ñåðåäîâèùà éîãî ôóíêö³îíóâàí- íÿ, ïåðåâ³ðêà àäåêâàòíîñò³ ìîäåë³ ³ ¿¿ êîîðäèíàö³ÿ. 4. Ïîáóäîâà ìîäåëåé ïðåäìåòíî¿ ³ ïðîáëåìíî¿ îáëàñòåé, îá’ºêòà ñåðåäîâèùà éîãî ôóíêö³îíóâàí- íÿ, ïåðåâ³ðêà àäåêâàòíîñò³ ìîäåë³ ³ ¿¿ êîîðäèíàö³ÿ. Íàéãîëîâí³øèì ïðè âèð³øåíí³ çàâäàíü àíàë³- òèêè á³çíåñ–ð³øåíü, íåçàëåæíî â³ä ñôåðè ¿¿ çà- ñòîñóâàííÿ, çì³ñòó ³ õàðàêòåðó, º âèðîáëåííÿ íàéêðàùîãî óïðàâë³íñüêîãî á³çíåñ–ð³øåííÿ. ßê íàñë³äîê, âèá³ð àëüòåðíàòèâè â õîä³ îáðàííÿ á³ç- íåñ–ð³øåííÿ çàëåæèòü â³ä: 5. Ïëàíóâàííÿ ³ ïðîâåäåííÿ ìîäåëüíèõ åêñïå- ðèìåíò³â. 6. Ñèíòåç íîâèõ çíàíü, íåîáõ³äíèõ äëÿ âèð³øåíü óïðàâë³íñüêèõ á³çíåñ–çàäà÷. • ìîæëèâîñò³ îö³íèòè åôåêòèâí³ñòü àëüòåðíàòèâ- íîãî ð³øåííÿ ³ íåîáõ³äí³ âèòðàòè äëÿ éîãî ðåàë³çàö³¿; • ìåòîä³â, ÿê³ âèêîðèñòîâóâàëèñÿ â ï³äïðèºì- íèöüê³é ä³ÿëüíîñò³, à òàêîæ ðîçðîáëåí³ äî öüîãî ÷à- ñó ìîäåë³ äîñë³äæåííÿ ï³äïðèºìíèöüêèõ îïåðàö³é; • ìåòîä³â, ÿê³ äîçâîëèëè á àíàë³çóâàòè ñõî- æ³ ïðîáëåìè, ÿê îäíå ö³ëå, ùî çàáåçïå÷óâàëè á ïåðåãëÿä âåëèêî¿ ê³ëüêîñò³ àëüòåðíàòèâ, êîæíà ç ÿêèé áóëà á îïèñàíà âåëèêîþ ê³ëüê³ñòþ çì³ííèõ, Àíàë³òèêà á³çíåñ–ð³øåíü ïîâ’ÿçàíà ç ³íòåëåê- òóàëüíî–òåõíîëîã³÷íîþ ä³ÿëüí³ñòþ, ÿêà ñïðÿìî- âàíà íà âèð³øåííÿ çàâäàíü óïðàâë³ííÿ ³ ñèíòåçó íîâèõ çíàíü. Business decision analysis as a component of enterprise activity: theoretical aspect  øèðøîìó àñïåêò³, àíàë³òèêà á³çíåñ–ð³øåíü – öå çàãàëüíà ñóêóïí³ñòü ïðèíöèï³â ìåòîäîëî- ã³÷íî–îðãàí³çàö³éíîãî, à òàêîæ òåõíîëîã³÷íîãî ð³çíîãî ðîäó ðîçóìîâî¿ ä³ÿëüíîñò³, ùî äîçâîëÿº 64 ´ÎÐÌÓÂÀÍÍß ÐÈÍÊÎÂÈÕ Â“ÄÍÎÑÈÍ Â ³ÊÐÀŸÍ“ ™   ¹ Ñóòí³ñòü ï³äõîäó 1 Ìåòîäîëîã³÷íà îñíîâà ïðîöåñó îáðîáêè ³íôîðìàö³¿ 2 Ìåòîäîëîã³ÿ ï³çíàííÿ, ùî âèêîðèñòîâóº äëÿ îòðèìàííÿ íîâîãî çíàííÿ ÿê ñòðîãî íàóêîâ³, òàê ³ ³íòó¿òèâí³ ìåòîäè 3 Ôîðìà ìèñëåííÿ ³ ñâ³òîâ³ä÷óòòÿ, ùî ñïèðàºòüñÿ íà íàóêîâèé ï³äõ³ä 4 Ñóòí³ñíå çíàííÿ ïðî ïðîöåñè ðåàëüíîãî ñâ³òó 5 Çàñ³á ïåðåòâîðåííÿ ³íòó¿òèâíèõ óÿâëåíü ó ëîã³÷íèé, ðàö³îíàëüíèé ïëàí ìèñëåííÿ 6 Ôîðìà íàóêîâîãî çíàííÿ, ùî çàñòîñîâóºòüñÿ â ïðîöåñàõ óïðàâë³ííÿ, íàñàìïåðåä – äëÿ âèðîáëåííÿ óïðàâë³íñüêèõ ð³øåíü 7 Ñóêóïí³ñòü ìåòîä³â, çà äîïîìîãîþ ÿêèõ ìîæíà âèÿâëÿòè ïðèõîâàí³ ñìèñëè ó òåêñòàõ ³ ðåàëüíèõ ñîö³àëüíî–ïîë³òè÷íèõ òà åêîíîì³÷íèõ ïðîöåñàõ 8 ßäðî íàóêîâî–äîñë³äíî¿ ðîáîòè 9 Ïîòóæíèé ïëàñò ³íòåëåêòóàëüíî¿ êóëüòóðè, ùî âèêîðèñòîâóºòüñÿ åë³òîþ áóäü–ÿêîãî ñóñï³ëüñòâà äëÿ óïðàâë³ííÿ ñîö³óìîì 10 Êîðîòêà íàçâà ³íôîðìàö³éíî–àíàë³òè÷íî¿ ðîáîòè 11 Ñèíîí³ì ä³àëåêòè÷íî¿ ëîã³êè 12 Ñèíîí³ì ñèñòåìíîãî àíàë³çó 13 Ïðîöåñ óçàãàëüíåííÿ òà àíàë³çó ðîçð³çíåíèõ, íåïîâíèõ ³ ÷àñòî ñóïåðå÷ëèâèõ äàíèõ ïðî îá’ºêò äîñë³äæåííÿ 14 Ïðîöåñ âèÿâëåííÿ ïðè÷èííî–íàñë³äêîâèõ çàëåæíîñòåé ³ ïðîñòîðîâî÷àñîâèõ çâ’ÿçê³â ó áóäü–ÿêèõ îá’ºêòàõ 15 Ïðîöåñ ñèñòåìàòèçàö³¿ çì³ñòó çà äîïîìîãîþ ñõåìàòèçàö³¿, êîíñòðóþâàííÿ òà ìîäåëþâàííÿ ñóòí³ñíèõ åëåìåíò³â ³ çâ’ÿçê³â 16 Ïðîöåñ ïîä³ëó îá’ºêòà íà ñêëàäîâ³ ÷àñòèíè ³ ïîäàëüøîãî ¿õíüîãî îá’ºäíàííÿ ó ïåâíó ñèñòåìó 17 Ïðîöåñ âèÿâëåííÿ ïðîòèð³÷ â îá’ºêò³ ï³çíàííÿ, çâåäåííÿ ñêëàäíîãî äî ïðîñòîãî 18 Ïðèíöèï êîíñòðóêòèâíîãî ñïðîùåííÿ äëÿ âèÿâëåííÿ ôîðì âçàºìî䳿 åëåìåíò³â ö³ëîãî ³ ðîçêðèòòÿ âíóòð³øíüî¿ ñòðóêòóðè áóäü–ÿêîãî îá’ºêòà äîñë³äæåííÿ 19 Ñâîºð³äí³ çàñîáè äëÿ âèâ÷åííÿ ñóò³ ÿâèù, ïðåäìåò³â ³ ïðîöåñ³â ðåàëüíîñò³, ÿäðî áóäü–ÿêî¿ äîñë³äíî¿ ïðîãðàìè Äæåðåëî: ñêëàäåíî àâòîðîì íà îñíîâ³ äæåðåë 1–5]. Business decision analysis as a component of enterprise activity: theoretical aspect ϳäõîäè äî âèçíà÷åííÿ ïîíÿòòÿ «àíàë³òèêà á³çíåñ–ð³øåíü» 64 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   ¹ Ñóòí³ñòü ï³äõîäó 1 Ìåòîäîëîã³÷íà îñíîâà ïðîöåñó îáðîáêè ³íôîðìàö³¿ 2 Ìåòîäîëîã³ÿ ï³çíàííÿ, ùî âèêîðèñòîâóº äëÿ îòðèìàííÿ íîâîãî çíàííÿ ÿê ñòðîãî íàóêîâ³, òàê ³ ³íòó¿òèâí³ ìåòîäè 3 Ôîðìà ìèñëåííÿ ³ ñâ³òîâ³ä÷óòòÿ, ùî ñïèðàºòüñÿ íà íàóêîâèé ï³äõ³ä 4 Ñóòí³ñíå çíàííÿ ïðî ïðîöåñè ðåàëüíîãî ñâ³òó 5 Çàñ³á ïåðåòâîðåííÿ ³íòó¿òèâíèõ óÿâëåíü ó ëîã³÷íèé, ðàö³îíàëüíèé ïëàí ìèñëåííÿ 6 Ôîðìà íàóêîâîãî çíàííÿ, ùî çàñòîñîâóºòüñÿ â ïðîöåñàõ óïðàâë³ííÿ, íàñàìïåðåä – äëÿ âèðîáëåííÿ óïðàâë³íñüêèõ ð³øåíü 7 Ñóêóïí³ñòü ìåòîä³â, çà äîïîìîãîþ ÿêèõ ìîæíà âèÿâëÿòè ïðèõîâàí³ ñìèñëè ó òåêñòàõ ³ ðåàëüíèõ ñîö³àëüíî–ïîë³òè÷íèõ òà åêîíîì³÷íèõ ïðîöåñàõ 8 ßäðî íàóêîâî–äîñë³äíî¿ ðîáîòè 9 Ïîòóæíèé ïëàñò ³íòåëåêòóàëüíî¿ êóëüòóðè, ùî âèêîðèñòîâóºòüñÿ åë³òîþ áóäü–ÿêîãî ñóñï³ëüñòâà äëÿ óïðàâë³ííÿ ñîö³óìîì 10 Êîðîòêà íàçâà ³íôîðìàö³éíî–àíàë³òè÷íî¿ ðîáîòè 11 Ñèíîí³ì ä³àëåêòè÷íî¿ ëîã³êè 12 Ñèíîí³ì ñèñòåìíîãî àíàë³çó 13 Ïðîöåñ óçàãàëüíåííÿ òà àíàë³çó ðîçð³çíåíèõ, íåïîâíèõ ³ ÷àñòî ñóïåðå÷ëèâèõ äàíèõ ïðî îá’ºêò äîñë³äæåííÿ 14 Ïðîöåñ âèÿâëåííÿ ïðè÷èííî–íàñë³äêîâèõ çàëåæíîñòåé ³ ïðîñòîðîâî÷àñîâèõ çâ’ÿçê³â ó áóäü–ÿêèõ îá’ºêòàõ 15 Ïðîöåñ ñèñòåìàòèçàö³¿ çì³ñòó çà äîïîìîãîþ ñõåìàòèçàö³¿, êîíñòðóþâàííÿ òà ìîäåëþâàííÿ ñóòí³ñíèõ åëåìåíò³â ³ çâ’ÿçê³â 16 Ïðîöåñ ïîä³ëó îá’ºêòà íà ñêëàäîâ³ ÷àñòèíè ³ ïîäàëüøîãî ¿õíüîãî îá’ºäíàííÿ ó ïåâíó ñèñòåìó 17 Ïðîöåñ âèÿâëåííÿ ïðîòèð³÷ â îá’ºêò³ ï³çíàííÿ, çâåäåííÿ ñêëàäíîãî äî ïðîñòîãî 18 Ïðèíöèï êîíñòðóêòèâíîãî ñïðîùåííÿ äëÿ âèÿâëåííÿ ôîðì âçàºìî䳿 åëåìåíò³â ö³ëîãî ³ ðîçêðèòòÿ âíóòð³øíüî¿ ñòðóêòóðè áóäü–ÿêîãî îá’ºêòà äîñë³äæåííÿ 19 Ñâîºð³äí³ çàñîáè äëÿ âèâ÷åííÿ ñóò³ ÿâèù, ïðåäìåò³â ³ ïðîöåñ³â ðåàëüíîñò³, ÿäðî áóäü–ÿêî¿ äîñë³äíî¿ ïðîãðàìè Äæåðåëî: ñêëàäåíî àâòîðîì íà îñíîâ³ äæåðåë 1–5]. ϳäõîäè äî âèçíà÷åííÿ ïîíÿòòÿ «àíàë³òèêà á³çíåñ–ð³øåíü» 64 Î̲×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ Äîñë³äèìî îñíîâí³ ïðèíöèïè òà ñêëàäîâ³ àíàë³- çó äàíèõ â á³çíåñ–àíàë³òèö³: ÿê³ äàâàëè á ïîâíîòó êîæíî¿ àëüòåðíàòèâè òà áóëè âèì³ðþâàíèìè, à òàêîæ äàâàëè ìîæëèâ³ñòü ÿê³ñ- íî â³äîáðàæàòè íåâèçíà÷åí³ñòü ³ ðèçèêîâàí³ñòü îáðàíî¿ àëüòåðíàòèâè. Ïðîöåñ àíàë³çó (â ³íôîðìàö³éíîìó ï³äõîä³ äî àíàë³çó äàíèõ íåîáõ³äíèì º íàÿâí³ñòü òðüîõ êëþ- ÷îâèõ ñêëàäîâèõ: åêñïåðò, ã³ïîòåçà ³ àíàë³òèê). Äîñë³äæåííÿ òà çãðóïóâàííÿ ð³çíèõ òî÷îê çîðó, ùîäî àíàë³çó ³íôîðìàö³¿, ÿêà ïðèçâîäèòü äî áåç- ë³÷³ ï³äõîä³â äî âèçíà÷åííÿ ïîíÿòòÿ «àíàë³òèêè á³çíåñ–ð³øåíü», ïðåäñòàâëåíî â òàáëèö³. Âèá³ð ³ â³çóàë³çàö³ÿ äàíèõ (÷àñòî, ùîá îòðèìà- òè íîâ³ çíàííÿ ïðî ÿêèéñü îá’ºêò ÷è ÿâèùå, çîâ- ñ³ì íå îáîâ’ÿçêîâà ïîáóäîâà ñêëàäíèõ ìîäåëåé). Ìîäåëþâàííÿ (ïðîöåñ ïîáóäîâè ìîäåëåé â á³ç- íåñ–àíàë³òèö³ ñêëàäàºòüñÿ ç ïåâíèõ ïîñë³äîâíèõ åòàï³â). Çàëåæíî â³ä ñïåöèô³êè ñâî¿õ äîñë³äæåíü, ð³çí³ çàðóá³æí³ òà â³÷òèçíÿí³ â÷åí³ ³ ïðàêòèêè âêëàäàëè â ñâîº ðîçóì³ííÿ àíàë³òè÷íî¿ ä³ÿëüíîñò³ ð³çíèé çì³ñò. Òàê, ÷àñòèíà öèõ âèçíà÷åíü â³äíîñèòüñÿ íå äî ñóò³, à äî ôóíêö³é àíàë³òèêè á³çíåñ–ð³øåíü òà ôîðì ¿¿ ïðîÿâó. Âèñíîâîê Âèõîäÿ÷è ç âèùå âèêëàäåíîãî, ìîæíà ñïî- ñòåð³ãàòè, ùî ³íòåãðàö³ÿ ³íôîðìàö³éíèõ çàñîá³â â çàãàëüíó ³íôîðìàö³éíó ñèñòåìó ï³äïðèºìñòâà òà ³íôîðìàö³éíèõ òåõíîëîã³é, äàñòü ìîæëèâ³ñòü ïðîâîäèòè àíàë³òèêó åêîíîì³÷î¿ ³íôîðìàö³¿ òà äîçâîëèòü ïðèéìàòè åôåêòèâí³ á³çíåñ–ð³øåííÿ é øâèäêî âèçíà÷èòèñÿ ³ç àëüòåðíàòèâîþ. Á³çíåñ–àíàë³òèêà «íàãîëîøóº» íà òîìó, ùî ïðîöåñ îðãàí³çàö³¿ ³ àíàë³çó âæå ³ñíóþ÷èõ äàíèõ äëÿ ïðèéíÿò- òÿ åôåêòèâíèõ á³çíåñ–ð³øåíü º îäíèì ç íàéâàæ÷èõ ³ îñíîâíèõ çàâäàíü ìåíåäæìåíòó (óïðàâë³ííÿ). Á³çíåñ–àíàë³òèêà «íàãîëîøóº» íà òîìó, ùî ïðîöåñ îðãàí³çàö³¿ ³ àíàë³çó âæå ³ñíóþ÷èõ äàíèõ äëÿ ïðèéíÿò- òÿ åôåêòèâíèõ á³çíåñ–ð³øåíü º îäíèì ç íàéâàæ÷èõ ³ îñíîâíèõ çàâäàíü ìåíåäæìåíòó (óïðàâë³ííÿ). Ïîñò³éíå óäîñêîíàëåííÿ ³íòåëåêòóàëüíîãî àïà- ðàòó ñó÷àñíîãî óïðàâë³ííÿ ñòຠïåðøî÷åðãîâîþ ïðîáëåìîþ òåî𳿠³ ïðàêòèêè óïðàâë³ííÿ ï³äïðè- ºìñòâîì. Îðãàí³çàö³ÿ ïðîöåñó ïðèéíÿòòÿ á³çíåñ–ð³øåííÿ çíà÷íî çàëåæèòü â³ä çàñòîñóâàííÿ ³íôîðìàö³éíèõ òåõíîëîã³é äëÿ á³çíåñ–àíàë³òèêè â ï³äïðèºìíèöü- ê³é ä³ÿëüíîñò³ òà ìຠáàçóâàòèñÿ íà ³íòåãðàö³¿ ¿õ ó ñèñòåìó óïðàâë³ííÿ á³çíåñ–ïðîöåñàìè ï³äïðèºì- ñòâà òà â³äïîâ³äàòè á³çíåñ–ìîäåë³ êîæíîãî ï³äïðè- ºìñòâà ç óðàõóâàííÿì ìîæëèâîñò³ ¿õ ³íòåãðóâàííÿ ó íàÿâí³ ³íôîðìàö³éí³ ñèñòåìè ï³äïðèºìñòâà. Êåð³âíèêè, ÿê³ óñâ³äîìëþþòü, ùî ñòðàòåã³÷í³ àñïåêòè óïðàâë³ííÿ ñó÷àñíèì àâòîìàòèçîâàíèì ï³äïðèºìñòâîì, ³íòåãðîâàíèì ç çîâí³øí³ì ñå- ðåäîâèùåì, ïîòðåáóþòü ³íøî¿ òåîðåòè÷íî¿ áàçè â ïîð³âíÿíí³ ç, íàïðèêëàä, ïðèíöèïàìè óïðàâë³í- ñüêîãî ðàö³îíàë³çìó, òà çìîæóòü çíàéòè äîäàòêî- â³ ìîæëèâîñò³ âäîñêîíàëåííÿ âëàñíî¿ ä³ÿëüíîñò³ â êîíöåïö³¿ âñåçàãàëüíî¿ åêîíîì³÷íî¿ íàóêè [4]. Âàãîìîþ º ³íòåãðàö³ÿ á³çíåñ–àíàë³òèêè ç ñòðà- òåã³÷íèì àíàë³çîì, îñíîâíå ïðèçíà÷åííÿ ÿêîãî – öå ï³äãîòîâêà îñíîâè äëÿ ïðèéíÿòòÿ åêîíîì³÷íèõ òà óïðàâë³íñüêèõ á³çíåñ–ð³øåíü ç âèðîáëåííÿ ì³- ñ³¿, ö³ëåé, ñòðàòå㳿 ðîçâèòêó ï³äïðèºìñòâà ÷è ô³ðìè.  ïðîöåñ³ ñòðàòåã³÷íîãî àíàë³çó íåîáõ³äíî âè- ÿâèòè êîíêóðåíòí³ ïåðåâàãè îá’ºêòà äîñë³äæåííÿ, ùî çàáåçïå÷óþòü éîãî ê³íöåâ³ äîñÿãíåííÿ. Ó çàðóá³æí³é ïðàêòèö³ º äåÿêèé àðñåíàë ìåòî- ä³â ðèíêîâîãî àíàë³çó ÿêèé ìîæíà çàïîçè÷èòè ³ Êåð³âíèêè, ÿê³ óñâ³äîìëþþòü, ùî ñòðàòåã³÷í³ àñïåêòè óïðàâë³ííÿ ñó÷àñíèì àâòîìàòèçîâàíèì ï³äïðèºìñòâîì, ³íòåãðîâàíèì ç çîâí³øí³ì ñå- ðåäîâèùåì, ïîòðåáóþòü ³íøî¿ òåîðåòè÷íî¿ áàçè â ïîð³âíÿíí³ ç, íàïðèêëàä, ïðèíöèïàìè óïðàâë³í- ñüêîãî ðàö³îíàë³çìó, òà çìîæóòü çíàéòè äîäàòêî- â³ ìîæëèâîñò³ âäîñêîíàëåííÿ âëàñíî¿ ä³ÿëüíîñò³ â êîíöåïö³¿ âñåçàãàëüíî¿ åêîíîì³÷íî¿ íàóêè [4]. Business decision analysis as a component of enterprise activity: theoretical aspect Íà îñíîâ³ âèùåñêàçàíîãî ïîòð³áíî çàçíà÷è- òè ³ óçàãàëüíèòè, ùî îáîâ’ÿçêîâîþ ³íôîðìàö³é- íîþ ñêëàäîâîþ â õîä³ àíàë³òèêè á³çíåñ–ð³øåíü íà ï³äïðèºìñòâ³ º çàñòîñóâàííÿ ³íôîðìàö³éíèõ òåõíîëîã³é, ÿê³ âèð³øóâàòèìóòü ð³çíîìàí³òí³ çà- äà÷³ á³çíåñ–àíàë³òèêè. Õîò³ëîñÿ á ñêàçàòè, ùî àíàë³òèêà ïîñòຠÿê ³ åêîíîì³÷íà äèñöèïë³íà, ÿêà âêëþ÷ຠòðè íàé- âàæëèâ³øèõ ñêëàäîâ³: 1. Ìåòîäîëîã³þ ³íôîðìàö³éíî–àíàë³òè÷íî¿ ðîáîòè. Ñó÷àñí³ ³íôîðìàö³éí³ òåõíîëî㳿 äàþòü ìîæëè- â³ñòü çíà÷íî ñïðîùóâàòè ïðîöåñ àíàë³çó, à òàêîæ ï³äâèùóâàòè éîãî ïðîäóêòèâí³ñòü ³ ÿê³ñü. 2. Îðãàí³çàö³éíå çàáåçïå÷åííÿ ïðîöåñó àíàë³- òè÷íî¿ ðîáîòè. 3. Òåõíîëîãî–ìåòîäîëîã³÷íå çàáåçïå÷åííÿ ðîçðîáêè ³ ñòâîðåííÿ ³íñòðóìåíòàëüíèõ çàñîá³â äëÿ âåäåííÿ àíàë³òè÷íî¿ ðîáîòè íà ï³äïðèºìñòâ³. ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   Data about author Tetyana Ivanyuta, 4. Lyashenko O., Babij S. V. Novi informacijno– anality’chni texnologiyi doslidzhennya sy’stem uprav- 4. Lyashenko O., Babij S. V. Novi informacijno– anality’chni texnologiyi doslidzhennya sy’stem uprav- Äàííûå îá àâòîðå 3. Konrad Karlberg. By’znes–analy’z s y’spol’zovany’em Excel. Resheny’e by’znes–zadach, 4–e y’zdany’e = Busi- ness Analysis: Microsoft Excel. Moskva : «Vy’l’yams», 2013. – 576 s. 3. Konrad Karlberg. By’znes–analy’z s y’spol’zovany’em Excel. Resheny’e by’znes–zadach, 4–e y’zdany’e = Busi- ness Analysis: Microsoft Excel. Moskva : «Vy’l’yams», 2013. – 576 s. Ñïèñîê âèêîðèñòàíèõ äæåðåë Âàãîìîþ º ³íòåãðàö³ÿ á³çíåñ–àíàë³òèêè ç ñòðà- òåã³÷íèì àíàë³çîì, îñíîâíå ïðèçíà÷åííÿ ÿêîãî – öå ï³äãîòîâêà îñíîâè äëÿ ïðèéíÿòòÿ åêîíîì³÷íèõ òà óïðàâë³íñüêèõ á³çíåñ–ð³øåíü ç âèðîáëåííÿ ì³- ñ³¿, ö³ëåé, ñòðàòå㳿 ðîçâèòêó ï³äïðèºìñòâà ÷è ô³ðìè. 1. Áàðèëåíêî Â.È. Áèçíåñ–àíàëèç êàê âàæíûé âèä êîíñàëòèíãîâûõ óñëóã. ÐÈÑÊ: Ðåñóðñû, Èíôîðìàöèÿ, Ñíàáæåíèå, Êîíêóðåíöèÿ. ¹ 4. 2012 – Ñ. 202–207. 2. Áîíäàðåíêî Â.Ã. Ñó÷àñí³ ³ ìàéáóòí³ ³íôîêîìóí³êàö³éí³ òåõíîëî㳿 Óêðà¿íè: íàâ÷. ïîñ³á. [äëÿ ñòóäåíò³â âèùèõ íàâ÷. çàêë. çà íàïðÿìîì Òåëåêîìóí³êàö³¿ll] / Â.Ã. Áîíäàðåíêî, Â.Î. Ãðåáåíí³êîâ. – Ê.: Ðàä³îàìàòîð, 2013. – 160 ñ.  ïðîöåñ³ ñòðàòåã³÷íîãî àíàë³çó íåîáõ³äíî âè- ÿâèòè êîíêóðåíòí³ ïåðåâàãè îá’ºêòà äîñë³äæåííÿ, ùî çàáåçïå÷óþòü éîãî ê³íöåâ³ äîñÿãíåííÿ. 3. Êîíðàä Êàðëáåðã. Áèçíåñ–àíàëèç ñ èñïîëüçî- âàíèåì Excel. Ðåøåíèå áèçíåñ–çàäà÷, 4–å èçäàíèå = Business Analysis: Microsoft Excel. Ìîñêâà : «Âè- ëüÿìñ», 2013. – 576 ñ. Ó çàðóá³æí³é ïðàêòèö³ º äåÿêèé àðñåíàë ìåòî- ä³â ðèíêîâîãî àíàë³çó, ÿêèé ìîæíà çàïîçè÷èòè ³ ââåñòè â ïðàêòèêó ñòðàòåã³÷íîãî àíàë³çó. 65 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   ÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² ²×Ͳ ÏÐÎÁËÅÌÈ ÐÎÇÂÈÒÊÓ ÃÀËÓÇÅÉ ÒÀ ÂÈIJ ÅÊÎÍÎ̲×Íί IJßËÜÍÎÑÒ² linnya pidpry’yemstvamy’ //Innovacijna ekonomika. – 2012. – #. 3. – S. 20–25. linnya pidpry’yemstvamy’ //Innovacijna ekonomika. – 2012. – #. 3. – S. 20–25. 5. Shtefan B. Model’ informacijnoyi texnologiyi anality’chnogo zabezpechennya procesu vy’robny’cztva novoyi produkciyi / B.M. Shtefan // Ekonomichny’j analiz: zb. nauk. pr. 2015. – # 2. – T. 21. – S. 229–236. linnya pidpry’yemstvamy’ //Innovacijna ekonomika. – 2012. – #. 3. – S. 20–25. linnya pidpry’yemstvamy’ //Innovacijna ekonomika. – 2012. – #. 3. – S. 20–25. 4. Ëÿøåíêî Î., Áàá³é Ñ. Â. Íîâ³ ³íôîðìàö³éíî–àíà- ë³òè÷í³ òåõíîëî㳿 äîñë³äæåííÿ ñèñòåì óïðàâë³ííÿ ï³ä- ïðèºìñòâàìè //²ííîâàö³éíà åêîíîì³êà. – 2012. – ¹. 3. – Ñ. 20–25. 4. Ëÿøåíêî Î., Áàá³é Ñ. Â. Íîâ³ ³íôîðìàö³éíî–àíà- ë³òè÷í³ òåõíîëî㳿 äîñë³äæåííÿ ñèñòåì óïðàâë³ííÿ ï³ä- ïðèºìñòâàìè //²ííîâàö³éíà åêîíîì³êà. – 2012. – ¹. 3. – Ñ. 20–25. 5. Shtefan B. Model’ informacijnoyi texnologiyi anality’chnogo zabezpechennya procesu vy’robny’cztva novoyi produkciyi / B.M. Shtefan // Ekonomichny’j analiz: zb. nauk. pr. 2015. – # 2. – T. 21. – S. 229–236. 5. Shtefan B. Model’ informacijnoyi texnologiyi anality’chnogo zabezpechennya procesu vy’robny’cztva novoyi produkciyi / B.M. Shtefan // Ekonomichny’j analiz: zb. nauk. pr. 2015. – # 2. – T. 21. – S. 229–236. 5. Øòåôàí Á. Ìîäåëü ³íôîðìàö³éíî¿ òåõíîëî㳿 àíà- ë³òè÷íîãî çàáåçïå÷åííÿ ïðîöåñó âèðîáíèöòâà íîâî¿ ïðîäóêö³¿ / Á.Ì. Øòåôàí // Åêîíîì³÷íèé àíàë³ç: çá. íàóê. ïð. 2015. – ¹ 2. – Ò. 21. – Ñ. 229–236. ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™   References ê.å.í., äîöåíò, Íàö³îíàëüíîãî óí³âåðñèòåòó õàð÷îâèõ òåõíîëîã³é ê.å.í., äîöåíò, Íàö³îíàëüíîãî óí³âåðñèòåòó õàð÷îâèõ òåõíîëîã³é 1. Bary’lenko V.Y’. By’znes–analy’z kak vazhnûj vy’d konsalty’ngovûx uslug. RY’SK: Resursû, Y’nformacy’ya, Snabzheny’e, Konkurency’ya. # 4. 2012 – S. 202–207. 1. Bary’lenko V.Y’. By’znes–analy’z kak vazhnûj vy’d konsalty’ngovûx uslug. RY’SK: Resursû, Y’nformacy’ya, Snabzheny’e, Konkurency’ya. # 4. 2012 – S. 202–207. e–mail: te_ta_na@ukr.net e–mail: te_ta_na@ukr.net 2. Bondarenko V.G. Suchasni i majbutni infokomuni- kacijni texnologiyi Ukrayiny’: navch. posib. [dlya studentiv vy’shhy’x navch. zakl. za napryamom Telekomunikaciyill] / V.G. Bondarenko, V.O. Grebennikov. – K.: Radioama- tor, 2013. – 160 s. Äàííûå îá àâòîðå Èâàíþòà Òàòüÿíà Íèêîëàåâíà, ê.ý.í., äîöåíò, Íàöèîíàëüíîãî óíèâåðñèòåòà ïè- ùåâûõ òåõíîëîãèé e–mail: te_ta_na@ukr.net Data about author Tetyana Ivanyuta, Ph.D., Associate Professor, National University of Ïðåäìåò äîñë³äæåííÿ – ìåõàí³çìè ñòðàòåã³÷íîãî âòðó÷àííÿ áàãàòîð³âíåâîãî ñóá’ºêòèâíîãî ôàêòîðà â ñîö³àëüíî–åêîíîì³÷í³ ïðîöåñè. Ïðåäìåò äîñë³äæåííÿ – ìåõàí³çìè ñòðàòåã³÷íîãî âòðó÷àííÿ áàãàòîð³âíåâîãî ñóá’ºêòèâíîãî ôàêòîðà â ñîö³àëüíî–åêîíîì³÷í³ ïðîöåñè. Ïðåäìåò äîñë³äæåííÿ – çì³íà ïåðåäóìîâ îñâ³òíüîãî ïðîöåñó â ïîë³òèêî–åêîíîì³÷íîìó â³ä- òâîðåíí³. Ìåòîþ íàïèñàííÿ ñòàòò³ º âèÿâëåííÿ ìîæëèâîñòåé ³ âèìîã äî íàâ÷àííÿ äëÿ çðîñòàííÿ 䳺âîñò³ íàóêîâî–îñâ³òíüî–âèðîáíè÷èõ êëàñòåð³â ³ ñåðåäîâèùà çì³í. Ìåòîäîëîã³ÿ ïðîâåäåííÿ ðîáîòè áàçóºòüñÿ íà ïîºäíàíí³ ëîã³÷íîãî òà ³ñòîðè÷íîãî ï³äõîä³â â àíà- ë³ç³ ñîö³àëüíî–åêîíîì³÷íèõ ïðîöåñ³â ÿê îñíîâè ðåãóëÿòèâíèõ âïëèâ³â. Êîíöåíòðàö³ÿ çì³í, ùî äîç- âîëÿº õàðàêòåðèçóâàòè ñóñï³ëüñòâî ³ éîãî åêîíîì³êó, ÿê «çíàííÿ», «ï³çíàííÿ», «³íôîðìàö³¿» ³ òàê äàë³, çì³ùóº íå ò³ëüêè òðèâàë³ñòü, àëå ³ ôóíêö³¿, à òàêîæ çàâäàííÿ íàâ÷àííÿ. ßêùî ðàí³øå ³ñòîð³ÿ ïðèâ÷èëà äî ñèòóàö³¿, äå àêòèâíó ó÷àñòü â ïðîöåñàõ íàâ÷àííÿ áóëî äîñèòü êîðîòêèì ïåð³îäîì ³í- òåíñèâíî¿ ï³äãîòîâêè äî æèòòÿ â ñóñï³ëüñòâ³, à ðîëü ïåäàãîãà ÷àñîì êîíöåíòðóâàëàñÿ íàâêîëî ïå- ðåäà÷³ êèìîñü óæå âèðîáëåíèõ çíàíü, òî íîâà ðåàëüí³ñòü âèìàãຠâ³ä ëþäèíè ïîñò³éíîãî íàâ÷àííÿ (â òîìó ÷èñë³ ñàìîíàâ÷àííÿ), à ÿê³ñòü ä³ÿëüíîñò³ ïåäàãîãà íåâ³ä’ºìíî â³ä éîãî îñîáèñòî¿ (çîêðåìà, äóõîâíî–³íòåëåêòóàëüíî¿) òâîð÷îñò³. Äîñòàòîê ³íôîðìàö³¿ ï³äâèùóº ö³íí³ñòü ìåòîäîëîã³÷íîãî, êîí- öåïòóàëüíîãî çíàííÿ, ðîçóì³ííÿ ñèòóàö³éíèõ ³ ïðîöåñóàëüíèõ õàðàêòåðèñòèê, à òàêîæ âì³ííÿ êîí- âåðòóâàòè ¿õ â ñâ³äîìó àêòèâí³ñòü, ïåðø çà âñå, – ó âèðîáíè÷îìó æèòò³ òà åêîíîì³ö³ îñâ³òè. Ðåçóëüòàòè ðîáîòè – çä³éñíåíî àíàë³ç ìîæëèâîñòåé ï³äâèùåííÿ ðîë³ íàâ÷àííÿ ó ôîðìóâàíí³ íàóêîâî–îñâ³òíüî–âèðîáíè÷èõ êëàñòåð³â ³ ðîçâèòêó ñåðåäîâèùà çì³í. © ØÅÄßÊΠÂ.ª., 2021 66 ´ÎÐÌÓÂÀÍÍßÐÈÍÊÎÂÈÕ“ÄÍÎÑÈͳÊÐÀŸÍ“™  
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https://www.e3s-conferences.org/10.1051/e3sconf/201911301008/pdf
English
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Integration of heat pump and gas turbine combined cycle: market and climatic conditions for power plant flexibility enhancement
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ERROR: type should be string, got "https://doi.org/10.1051/e3sconf/201911301008 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons \nAttribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Integration of heat pump and gas turbine \ncombined cycle: market and climatic conditions \nfor power plant flexibility enhancement \nAndrea Giugno1, Alessandro Sorce1, Alessandra Cuneo2* and Stefano Barberis2 \n1 Thermochemical Power Group, University of Genoa, Via Montallegro 1, 16145 Genoa, Italy \n2 RINA Consulting, Via San Nazaro 19, 16145, Genoa, Italy Abstract. The increasing share of electricity produced from renewable \nenergy sources (RES), with the consequent strong penetration in the current \nenergy network, is causing a growing need of balancing power to \ncompensate power supply from such fluctuating sources. For these reasons, \nnowadays the power plants are requested to improve their operational \nflexibility, together with their global efficiency in part-load operation, for \nancillary services and to sustain the grid operability. A possible solution for \nflexibility enhancement is characterized by a highly efficient heat pump \nintegrated in a conventional natural gas combined cycle (CC). Such concept \ncan be applied both to power oriented combined cycle (POCC), to modify \nthe compressor intake temperature with a consequent increase or decrease \nof the power production, and both to cogeneration CC in association with \nDistrict Heating Network (DHN). In this work, a statistical analysis of \nclimatic data and their correlations with energy market condition will be \nperformed considering Italian context, to understand which the more \nsuitable conditions for such integrated system are. The analysis will be \nperformed on seasonal and daily basis. The final aim of this work is to \nidentify how such integrated system can be operated at its best in the \ndifferent Italian markets and climatic frames. E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 An innovative concept based on the coupling of a fast-cycling highly efficient Heat Pump \n(HP) with the CCGTs is here proposed [4], within the framework of an H2020 EU project \ncalled PUMP-HEAT [5]. This possible solution for flexibility enhancement is characterized \nby a highly efficient heat pump integrated in a CCGT, featuring cold/warm thermal storage \nand advanced control. Such concept can be applied both to power oriented combined cycle \n(POCC), to modify the compressor intake temperature with a consequent increase or decrease \nof the power production, and both to cogeneration CCGT in association with District Heating \nNetwork (DHN). In this work, a statistical analysis of climatic data and their correlations \nwith energy market condition will be performed considering the Italian market scenario, to \nunderstand which is the more suitable conditions for such integrated systems. The final aim is to identify how PUMP HEAT concept can be operated and the influences \nthat different Italian price zones and climatic conditions can have on its profitability. The \nPower Oriented (PO) combined cycle configuration is taken into consideration during this \nanalysis, with a particular attention on the continuous cooling mode (Figure 1). Such \noperational mode allows to modify the compressor intake temperature thanks to the cold \nenergy produced by the heat pump. The cooling of the inlet temperature increases the \nmaximum power output of the CC (about 10%) during peak hours, with also an increase of \nthe yearly efficiency and production. Figure 1. Power oriented HP-CC concept scheme working in continuous cooling Figure 1. Power oriented HP-CC concept scheme working in continuous cooling The heat pump produces cold energy using ambient air through a dedicate heat exchanger \n(ambient HX in Figure 1) as energy source. The performances of the heat pump in terms of \nCOP and the temperature that can be reached at the compressor inlet are dependent on the \nambient temperature. However, to consistently evaluate the HP performance, both ambient \ntemperature and humidity were considered. In addition, in this work, it was always \nconsidered to work at the maximum load of the heat pump, not considering off-design \nconditions. In fact, from the optimization analysis performed during the PUMP-HEAT \nproject, it resulted that this would be the condition that maximizes the economic benefit. However, a minimum threshold value of 5°C was set at the compressor inlet to avoid the risk \nof ice formation. E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 The CCGT model was validated in previous work with respect to the effect \nof ambient temperature. the effect of GT intake humidity on the CCGT overall performance \nwas neglected as conservative assumption as explained in [4]. 1 Introduction In the last years, a consistent evolution of combined cycle performance was shown, mainly \ncommanded by the market. The intermittent renewable capacity has increased dramatically \nin the last 10 years, due also to the EU target [1], having a major impact on markets. The \ngrowth of renewable energy sources is, of course, highly beneficial to the environment but \nintroduces intermittency into grid management. This rapid growth in variable generation is \ndriving the need for a more flexible power system and for a research and development \nstrategy to help achieve that. In such new energy scenario, the combined cycle gas turbines (CCGT) have to change \nthe way they operate: from a baseload to backup [2]. CCGT must now respond rapidly to \nvariations due to renewable supply and demand, requiring a more flexible cyclic pattern of \nuse [3]. Making conventional power plants more flexible can be a key strategy to integrate \nlarge shares of renewables more effectively in power systems [1,2]. 2 Italian electricity scenario The volatility of minimum and maximum prices \nof this region is much higher than in the rest of Italy and the other macro regions [8] compared to 2016), for 12.8% by hydroelectric production and for the remaining 16.3% by \ngeothermal, wind and photovoltaic sources (with an increase of 10.3% respect to the previous \nyear) [7]. The Italian electricity market operates in three different periods: (i) daily market \n(MGP), which trades most of the electricity purchase and sale transactions (ii) intra-day \nmarket (MI) consisting of seven sessions which allow market participants to modify the \nprograms defined in the daily market by sending additional sales or purchase offers and (iii) \nthe market for continuous trading of daily products (MPEG). The electricity system is divided \ninto zones classified into geographical zones, national virtual zones and foreign virtual zones. The geographical zones represent a part of the national network; there are currently six active \nzones: Northern Italy (NORD), Central-Northern Italy (CNOR), Central-Southern Italy \n(CSUD), Southern Italy (SUD), Sicily (SICI) and Sardinia (SARD). The MGP market is a \nmarginal market in which the price and volume of each hour are established from the point \nof equilibrium between supply and demand. Matched purchase offers referring to units of \nconsumption belonging to Italian geographical areas are valued at the single national price \n(PUN). The PUN price is equal to the average of the prices of the zones, by zonal \nconsumption and represents the purchase price for end customers. In the last years, due to \nthe increase of RES and to a reduction of the natural gas price, the PUN in all the zones has \ndecreased consistently (Figure 2). The Sicily zone, however, seems to be the most interesting \none where to study continuous cooling concept, with an average zonal price always higher \nrespect to the national PUN (+13% in 2017). The volatility of minimum and maximum prices \nof this region is much higher than in the rest of Italy and the other macro regions [8] Figure 2. Annual average zonal price on MGP (left) and monthly average zonal price in 2017 (right) \n[9] Figure 2. Annual average zonal price on MGP (left) and monthly average zonal price in 2017 (right) \n[9] Figure 2. 2 Italian electricity scenario Annual average zonal price on MGP (left) and monthly average zonal price in 2017 (rig\n[9] In this market scenario, the use of a heat pump (HP) to decrease the temperature at the \ncompressor inlet do not depend only on the electricity price but it is also related to the ambient \ntemperature. In the following sections, a statistical analysis to correlate the price and the \nambient temperature is performed, as well as a first order economic analysis of the proposed \nintegrated system (CCGT+HP). 2 Italian electricity scenario As part of the new National Energy Strategy, Italy set itself the goal of the phase out of coal \nby 2025, aiming to expand the share of renewable energy in final energy consumption to 28% \nby 2030 [6]. The Italian wholesale electricity market started to operate as an Exchange in \n2005 with the liberalization of the demand side bidding. In 2017 gross national production \namounting to 295.8 TWh was satisfied for 70.8% by thermoelectric production (+5.0% 2 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 compared to 2016), for 12.8% by hydroelectric production and for the remaining 16.3% by \ngeothermal, wind and photovoltaic sources (with an increase of 10.3% respect to the previous \nyear) [7]. The Italian electricity market operates in three different periods: (i) daily market \n(MGP), which trades most of the electricity purchase and sale transactions (ii) intra-day \nmarket (MI) consisting of seven sessions which allow market participants to modify the \nprograms defined in the daily market by sending additional sales or purchase offers and (iii) \nthe market for continuous trading of daily products (MPEG). The electricity system is divided \ninto zones classified into geographical zones, national virtual zones and foreign virtual zones. The geographical zones represent a part of the national network; there are currently six active \nzones: Northern Italy (NORD), Central-Northern Italy (CNOR), Central-Southern Italy \n(CSUD), Southern Italy (SUD), Sicily (SICI) and Sardinia (SARD). The MGP market is a \nmarginal market in which the price and volume of each hour are established from the point \nof equilibrium between supply and demand. Matched purchase offers referring to units of \nconsumption belonging to Italian geographical areas are valued at the single national price \n(PUN). The PUN price is equal to the average of the prices of the zones, by zonal \nconsumption and represents the purchase price for end customers. In the last years, due to \nthe increase of RES and to a reduction of the natural gas price, the PUN in all the zones has \ndecreased consistently (Figure 2). The Sicily zone, however, seems to be the most interesting \none where to study continuous cooling concept, with an average zonal price always higher \nrespect to the national PUN (+13% in 2017). 3 Statistical Analysis The distribution of the electricity price plays a major role over the profitability of CCGT \npower plants, presenting an average production cost, i.e. the Cost of Electricity, COE, aligned \nwith the average value of the PUN. The overall profitability of the proposed power \naugmentation application, continuous cooling by a HP, can be related to the electricity peak \nabsolute price and to the temperature and price peak coupling. In a broader approach, \nconsidering the use of a Thermal Energy Storage (TES) to accumulate the cold energy during \nthe off-peak periods, also the respective temperature and price minima must be considered. The daily temperature and price variation can increase the benefit related to TES installation, \nin particular when electrical price and temperatures maxima or minima occurs at the same 3 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 time. Also the width of the positive and negative peaks, i.e how many hours can be accounted \nas “high range” and “low range” can be related to the system profitability. Such extreme \nconditions were defined with respect to the daily variation ∆dailyx = max(𝑥) −min⁡(𝑥), as: time. Also the width of the positive and negative peaks, i.e how many hours can be accounted \nas “high range” and “low range” can be related to the system profitability. Such extreme \nconditions were defined with respect to the daily variation ∆dailyx = max(𝑥) −min⁡(𝑥), as: 𝐻𝑖𝑔ℎ⁡𝑟𝑎𝑛𝑔𝑒𝑥= [max(𝑥) −∆dailyx\n20\n, max(𝑥)] \n⁡⁡⁡⁡𝐿𝑜𝑤⁡𝑟𝑎𝑛𝑔𝑒𝑥= [min(𝑥) , min(𝑥) + ∆dailyx\n20\n] \n(1) (1) This definition allows preserving at least 90% of the ∆dailyx between high and low range \nof the variable, as depicted in Figure 3 for the temperature for a representative day. The points \nwith the x markers and ▼ are respectively for high and low range and are counted to \nevaluated the peak extension, in this case respectively 4 and 5 hours. Figure 3. Example of the peak analysis: red line, ambient temperature [°C; x high range value; ▼ low \nrange value \n∆dailyT \nHigh⁡rangeT \nLow⁡rangeT \n90%⁡∆dailyT Low⁡rangeT Figure 3. Example of the peak analysis: red line, ambient temperature [°C; x high range value; ▼ low \nrange value The statistical analysis considers six price zones and six geographical location, as \nreported in Table 1, while price data were acquired by the Italian Gestore del Mercato \nElettrico [9]. Table 1. Meteorological data measurement point [10] Table 1. 3 Statistical Analysis Meteorological data measurement point [10] \nZones \nLocation \nPosition [N lat., E long.] \nAltitude [m] \nNorth \nTurin / Bric della Croce (airport) \n45° 02', 07° 44' \n709 \nCNorth \nAncona / Falconara (airport) \n43° 37', 13° 22' \n12 \nCSouth \nRome / Fiumicino (airport) \n41° 48', 12° 14' \n2 \nSouth \nBari \n41° 8.17', 16° 45.78' \n42 \nSard \nCapo Caccia \n40° 34', 08° 10' \n200 \nSici \nTrapani-Birgi (airport) \n37° 54.7’, 12° 29.6' \n7 Figure 4 represents the average maximum and the minimum value of the electrical price \nwithin the six Italian zones. The size of the marker is proportional to the number of hours in \nthe high and low range (i.e. the peak extension). The distance between the two marker \ncentres, in the same month, is equal to the average ∆dailyx. The maximum price values are \nregistered in the North and in Center North during the first two months of the year, while the \nSouth presents always lower prices. This could be related to the high number of CCGT 4 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 production sites and wind farm installed in a region with a relative low power demand. It can \nbe noticed that the prices in all the zones experience a reduction until April (loosing up to 54 \n€/MWh) and then a recovery. The maximum price of Sicily shows a less pronounced \nreduction in price until April (Pmax=85.6 €/MWh) and then remains above 90 €/MWh. In \nthis zone, also the width of the peak price is different respect to the other zones in Italy. In \nSicily, the average of the high range is up to 6h/day during the hot season and the yearly \naverage is around 4 hours versus the average of 1.66 h/day of the other zones. Looking at the \ndaily price variation, for the first five zones the changes appear to be related to a seasonal \ntrend with a strong reduction during the hot months. production sites and wind farm installed in a region with a relative low power demand. It can \nbe noticed that the prices in all the zones experience a reduction until April (loosing up to 54 \n€/MWh) and then a recovery. The maximum price of Sicily shows a less pronounced \nreduction in price until April (Pmax=85.6 €/MWh) and then remains above 90 €/MWh. 3.1 Peak coupling of the ambient temperature and the electrical price 3.1 Peak coupling of the ambient temperature and the electrical price Under the proposed application perspective, another important effect that increases the \nbenefit of the inlet cooling system, is the location of the price and temperature peak within \nthe daytime. Figure 5. Monthly averaged daily peak positions: blue circle Zonal Electricity Price peak (max) value \n(size proportional to price), blue lines price peak extension; red circle ambient temperature peak (max) \nvalue (size proportional to temperature), red lines temperature peak extension Figure 5. Monthly averaged daily peak positions: blue circle Zonal Electricity Price peak (max) value \n(size proportional to price), blue lines price peak extension; red circle ambient temperature peak (max) \nvalue (size proportional to temperature), red lines temperature peak extension It can be noticed that the maximum daily temperature is registered around the first two \nhours after noon, apart from the northern locations in which the peak is delayed up to 5 pm. The duration of the peak is intense in Sicily (a year average of 3.1 h/day) and in Center-North \nlocations (2.89 h/day), with a slightly higher value in the summer months. The price peaks occur later in the day moving from North to South, from 5pm in the North \nzone and 5.30pm in the Center-North, to 6pm in the South. The Sicily zone differs also for \nthis parameter, with an average price peak that occurs at 7.25pm. However, the price enters \nthe high range at about 6pm, with a longer persistence in the summer months up to 11pm. The temperature and price peaks appear to be separated in time of 1-2 hours, except for the \nnorthern regions in which there is a partial overlap. 3 Statistical Analysis In \nthis zone, also the width of the peak price is different respect to the other zones in Italy. In \nSicily, the average of the high range is up to 6h/day during the hot season and the yearly \naverage is around 4 hours versus the average of 1.66 h/day of the other zones. Looking at the \ndaily price variation, for the first five zones the changes appear to be related to a seasonal \ntrend with a strong reduction during the hot months. Figure 4. Monthly averaged max daily peak and min off-peak zonal price, the size of the marker is \nproportional to the peak extension hours. Figure 4. Monthly averaged max daily peak and min off-peak zonal price, the size of the marker is \nproportional to the peak extension hours. The daily ambient temperature variation, which is evaluated as the difference between \nthe maximum and the minimum temperature value in the same month, presented in Table 2, \nshows a trend that increases during summer due to the increase of the solar radiance, except \nfor the South location. This is beneficial to the TES application, as they are usually employed \nduring the hot months. The monthly average maximum and minimum temperatures, not \nreported here, as expected, increase during the summer period. Table 2. Monthly average daily variation of ambient Temperature [K] Table 2. Monthly average daily variation of ambient Temperature [K] \nZones \nJan \nFeb \nMar \nApr \nMay \nJun \nJul \nAug \nSep \nOct \nNov \nDec \nNorth \n4.0 \n3.5 \n5.5 \n6.8 \n7.0 \n7.0 \n6.7 \n6.3 \n5.8 \n4.8 \n3.3 \n3.5 \nCNorth \n6.7 \n7.7 \n10.2 \n10.1 \n9.6 \n10.3 \n10.3 \n10.3 \n8.5 \n9.8 \n8.0 \n8.5 \nCSouth \n9.0 \n8.4 \n10.2 \n10.7 \n10.8 \n9.7 \n10.5 \n11.1 \n8.9 \n10.7 \n8.9 \n9.4 \nSouth \n5.0 \n6.1 \n6.0 \n6.3 \n5.6 \n5.3 \n5.3 \n6.0 \n6.9 \n6.4 \n6.5 \n5.7 \nSard \n4.5 \n4.5 \n4.6 \n4.9 \n5.5 \n5.4 \n6.0 \n6.5 \n4.4 \n4.3 \n3.5 \n3.1 \nSici \n7.1 \n7.7 \n9.0 \n9.2 \n9.0 \n10.5 \n9.2 \n10.0 \n7.4 \n7.6 \n6.8 \n6.7 5 5 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 SUPEHR19 Volume 1 4 Economic analysis The continuous inlet cooling system performance were calculated taking into account five \ndifferent heat-pump (HP) sizes [1, 3, 4, 5 and 7.5 MWe] and the six geographical zones \ndescribed in the previous section. Such locations were used as possible sites for the 6 6 6 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 installation of the CC+HP layout to perform the continuous cooling of the turbine intake to \nenhance the performance of the system and so evaluate the earnings that such system would \nguarantee. The HP are considered to operate to reach a target inlet temperature of the GT \nintake of 5°C, which has been identified as the minimum reachable temperature without the \nice formation risk. installation of the CC+HP layout to perform the continuous cooling of the turbine intake to \nenhance the performance of the system and so evaluate the earnings that such system would \nguarantee. The HP are considered to operate to reach a target inlet temperature of the GT \nintake of 5°C, which has been identified as the minimum reachable temperature without the \nice formation risk. The CC net power and efficiency dependence on the inlet GT temperature was obtained \nfrom the Gate Cycle calculation presented in [4] and so the earnings that such system would \nproduce over the year, taking 2017 as reference, were computed as: 𝐸𝑎𝑟𝑛𝑖𝑛𝑔𝑠⁡ =⁡ ∑𝐸𝑝𝑟𝑜𝑑,𝑖\n8760\n𝑖=1\n⁡∙⁡(𝑃𝑍,𝑖⁡−⁡(𝐶𝑓𝑖𝑥⁡+⁡𝐶𝑔𝑎𝑠\n𝜂𝐶𝐶\n))⁡; \n⁡⁡𝑖𝑓⁡𝑃𝑧> 𝐶𝑓𝑖𝑥⁡+⁡𝐶𝑔𝑎𝑠\n𝜂𝐶𝐶\n⁡⁡[€] \n(2) (2) Where 𝐸𝑝𝑟𝑜𝑑,𝑖 is the energy produced by the CC at the i-th hour as function of the \ntemperature, Pz is the zonal price, Cfix and Cgas represent the fixed costs of the plant and \nthe gas cost based on [9,11], and 𝜂𝐶𝐶 is the CC efficiency as function of the temperature. To \nestimate the cooling potential of a given climatic profile, the concept of Continuous \nEquivalent Cooling Degree Hours (ECDH) is introduced, based on [12], and evaluated as: 𝐸𝐶𝐷𝐻⁡ =⁡ ∑(𝑇𝑎𝑚𝑏,𝑖\n8760\n𝑖=1\n−⁡𝑇𝑟𝑒𝑓)⁡⁡⁡⁡⁡⁡𝑖𝑓⁡⁡𝑇𝑎𝑚𝑏,𝑖>⁡𝑇𝑟𝑒𝑓 \n(3) (3) (3) Where 𝑇𝑟𝑒𝑓 for this study is set to 5 °C for the reasons explained before. From Figure 6b \nit can be observed that the ΔEarnings, calculated as percentage variation from the earnings \nthat the same CC would guarantee in ambient conditions, tend to increase while moving from \nNorth to South of Italy, due to the temperature increase over the year which would allow a \nhigher exploitation of the continuous cooling system proposed. 5 Conclusions The statistical analysis of climatic data and their correlations with energy market condition \nwas performed considering Italian context for six different electricity market zones. The \nanalysis was also carried out on monthly and daily basis introducing a technique to identify \nthe range of extreme values of the daily distribution on the base of the daily price variation. It was highlighted how the daily price variation reduces during the hot months, while the \ntemperature daily variation increases, as expected, due to the increase in solar radiant energy \nduring the summer time. Finally, Sicily market zone, shows a strongly different behaviour, \ncharacterized by high and persistent maximum prices, with a yearly average of 20 €/MWh \nover the other zonal prices. Focusing on the Italian market, the profitability of a continuous inlet cooling system \nbecomes more attractive moving from north to south due to the increase of the average \ntemperatures. On the other side, the South electricity price, the lowest in the peninsula, has a \nlow profitability compared to the meteorological potential. The gain in power of the proposed \nsystem is proportional to the climatic condition summarized by the Continuous Equivalent \nCooling Degree Hours, plus the effect of the zonal price. Under the actual condition the Sicily \nmarket has the largest potential, maximizing both the temperature and electrical price effect. This project has received funding from the European Union’s Horizon 2020 research \nand innovation programme under Grant Agreement No 764706, PUMP-HEAT. (http://www.pumpheat.eu). 4 Economic analysis However, it can also be \nhighlighted that the South area (SUD) presents high ECDH, but low percentage earnings \ncompared to the other areas considered, due to the lower Pz. (a) \n(b) \nFigure 6 : (a) percentage power production and (b) percentage earnings over ambient conditions \nfor five different HP sizes and six different areas of Italy (a) (b) (b) (a) Figure 6 : (a) percentage power production and (b) percentage earnings over ambient conditions \nfor five different HP sizes and six different areas of Italy This trend becomes more evident increasing the HP size over 1 MW, as ΔEarnings \nbecomes lower than all the other areas apart from the North (NORD) one. This effect is 7 7 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 https://doi.org/10.1051/e3sconf/201911301008 mostly related to the PUN of the South being lower than the national mean and other areas \nconsidered, which does not allow a good exploitation of the power produced (Figure 6a). mostly related to the PUN of the South being lower than the national mean and other areas \nconsidered, which does not allow a good exploitation of the power produced (Figure 6a). The high PUN of Sicily allows to benefit more from this layout not only thanks to the \nhigher average temperatures, resulting in a maximum ΔEarnings of about 4% using a 7.5MW \nHP. On the other hand, from Figure 6a it can be observed that the percentage power variation \nconsidering Sicily and a 1MW HP is lower than SUD and SARD. This is due to the fact that \ntemperatures in Sicily are particularly high and then a 1MW HP would not be able to reach \nthe target temperature of 5°C, resulting in a power augmentation lower than the actual \npossibility. Indeed, also the worst result, obtained from using a 1MW HP in Northern Italy, \nwould guarantee an increase of earnings of about 0.8% for the year considered. Moreover, it can be highlighted that the lines corresponding to the HPs of 5 and 7.5MW \nbecome very close in terms of earnings, suggesting that when the investment cost comes in \nplace within the calculations, it would probably be more profitable to buy a 5MW HP which \nwould result in lower capital and installation costs. [ ]\ngo\ne g ewe de,\n7,\ny\n.\n[3] \nPrina, M. G., Garegnani, G., Moser, D., Oberegger, U. F., Vaccaro, R., Sparber, \nW., Gazzani, M., and Manzolini, G., 2015, “Economic and Environmental Impact of \nPhotovoltaic and Wind Energy High Penetration towards the Achievement of the Italian 20-\n20-20 Targets,” 2015 10th International Conference on Ecological Vehicles and Renewable \nEnergies, EVER 2015. This project has received funding from the European Union’s Horizon 2020 research \nand innovation programme under Grant Agreement No 764706, PUMP-HEAT. \n(http://www.pumpheat.eu). [4] \nSorce, A., Giugno, A., Marino, D., Piola, S., and Guedez, R., 2019, “Analysis of a [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. \n[2] \nAgora Energiewende, 2017, Flexibility in Thermal Power Plants. \n[3] \nPrina, M. G., Garegnani, G., Moser, D., Oberegger, U. F., Vaccaro, R., Sparber, \nW., Gazzani, M., and Manzolini, G., 2015, “Economic and Environmental Impact of \nPhotovoltaic and Wind Energy High Penetration towards the Achievement of the Italian 20-\n20-20 Targets,” 2015 10th International Conference on Ecological Vehicles and Renewable \nEnergies, EVER 2015. \n[4] \nSorce, A., Giugno, A., Marino, D., Piola, S., and Guedez, R., 2019, “Analysis of a References [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. [1] \nEuropean Parliament, 2018, Directive (EU) 2018/2001 of the European \nParliament and of the Council of 11 December 2018 on the Promotion of the Use of Energy \nfrom Renewable Sources. [2] \nAgora Energiewende, 2017, Flexibility in Thermal Power Plants. [3] \nPrina, M. G., Garegnani, G., Moser, D., Oberegger, U. F., Vaccaro, R., Sparber, \nW., Gazzani, M., and Manzolini, G., 2015, “Economic and Environmental Impact of \nPhotovoltaic and Wind Energy High Penetration towards the Achievement of the Italian 20-\n20-20 Targets,” 2015 10th International Conference on Ecological Vehicles and Renewable \nEnergies, EVER 2015. [4] \nSorce, A., Giugno, A., Marino, D., Piola, S., and Guedez, R., 2019, “Analysis of a 8 https://doi.org/10.1051/e3sconf/201911301008 E3S Web of Conferences 113, 01008 (2019) \nSUPEHR19 Volume 1 Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” \nProceedings of ASME Turbo Expo 2019: Turbomachinery Technical Conference and Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” \nProceedings of ASME Turbo Expo 2019: Turbomachinery Technical Conference and \nExposition GT2019. Combined Cycle Exploiting Inlet Conditioning Technologies for Power Modulation,” \nProceedings of ASME Turbo Expo 2019: Turbomachinery Technical Conference and \nExposition GT2019. [5] \n“Pump-Heat” [Online]. Available: www.pumpheat.eu. [Accessed: 30-Apr-2019] [5] \n“Pump-Heat” [Online]. Available: www.pumpheat.eu. [Accessed: 30-Apr-2019]\n[6]\nMinistero dello Sviluppo Economico, Proposta Di Piano Nazionale Integrato pe [5] \n“Pump-Heat” [Online]. Available: www.pumpheat.eu. [Accessed: 30-Apr-2019]. [6] \nMinistero dello Sviluppo Economico, Proposta Di Piano Nazionale Integrato per \nl’Energia Ed Il Clima. [7]\nTERNA 2018 D ti St ti ti i S ll’\ni El tt i\ni It li [ ]\np\n[\n]\np\np\n[\np\n]\n[6] \nMinistero dello Sviluppo Economico, Proposta Di Piano Nazionale Integrato per\nl’Energia Ed Il Clima. [7] \nTERNA, 2018, Dati Statistici Sull’energia Elettrica in Italia. [8] \nAntonelli, M., Desideri, U., and Franco, A., 2018, “Effects of Large Scale \nPenetration of Renewables: The Italian Case in the Years 2008–2015,” Renew. Sustain. Energy Rev. [9] \nGestore Mercati Energetici, “Dati Storici Mercati Gas” [Online]. Available: \nhttp://www.mercatoelettrico.org/It/download/DatiStoriciGas.aspx. [Accessed: 07-Apr-\n2019]. [10] \n“Weather in the World” [Online]. Available: https://rp5.ru/Weather_in_the_world. [Accessed: 30-Apr-2019]. [11] \nRicerca sul Sistema Energetico - RSE Spa, 2017, Energia Elettrica, Anatomia Dei \nCosti. [12] \nAncona, M. References A., Bianchi, M., Melino, F., and Peretto, A., 2015, “Power \nAugmentation Technologies: Part II — Thermo-Economic Analysis.” 9 9"
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Algumas Considerações Sobre Ensino e Aprendizagem na Disciplina Laboratório de Eletromagnetismo
Revista Brasileira de Ensino de Física
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cc-by
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Revista Brasileira de Ensino de F sica, vol. 24, no. 4, Dezembro, 2002 471 Algumas Consideraco~es Sobre Ensino e Aprendizagem na Disciplina Laborat orio de Eletromagnetismo Considerations About Learning and Teaching in the Electromagnetism Laboratory J. Humberto Dias da Silva Faculdade de Ci^ encias, Departamento de F sica Universidade Estadual Paulista, CEP 17033-360, Bauru, SP Recebido em 8 de maio, 2002. Aceito em 26 de setembro, 2002. Baseados por um lado em avancos conceituais recentes das pesquisas em ensino de ci^encias, e por outro em nossa experi^encia docente, propomos algumas re ex~oes sobre as praticas de ensino e aprendizagem na disciplina Laboratorio de Eletromagnetismo. Apresentamos e interpretamos resultados e comentarios dos estudantes, os quais forneceram elementos para a analise da efetividade de propostas de novo enfoque para esta disciplina. Based on recent research in science education, and on teaching experience, we analyze the learning and teaching process in the Electromagnetism laboratory for undergraduate physics students. Data and reasoning obtained from the answers of some groups of students are used to help the analysis of the e ectiveness of the changes proposed in this physics laboratory. I Introduc~ao Professores e pesquisadores da area de ensino de ci^encias geralmente imputam grande import^ancia ao espaco de aprendizado de fsica ocorrido nas disciplinas experimentais [1,2,3]. Aparentemente tambem existe um consenso entre docentes e estudantes de que deve haver mudancas na maneira tradicional de se enfocar estas disciplinas [1,4-7]. Apesar disto ha relatos de que esta import^ancia declarada algumas vezes e mais quest~ao de postura que de pratica efetiva [5,8], e tambem ha observac~oes crticas a respeito do real benefcio que estas disciplinas possam trazer ao processo de aprendizado dos estudantes [9]. A disciplina Laboratorio de Eletromagnetismo e a primeira disciplina experimental da parte espec ca que os estudantes dos cursos de Fsica realizam. Nela, em princpio, os estudantes t^em espaco propcio para realizar experimentos um pouco mais detalhados sobre o Eletromagnetismo Classico. Os experimentos geralmente apresentam um maior grau de complexidade em relac~ao aos experimentos das disciplinas basicas. Esta escolha de maior complexidade neste ponto do curso baseia-se na hipotese, nem sempre correta, de que o grau de maturidade e entendimento do estudante podem alcancar esta complexidade [6,10]. Esta e uma quest~ao que merece cuidado, pois corremos o risco de colocar o estudante em um emaranhado experimental, contribuindo para afastar dele o entendimento mais profundo sobre os conceitos que queremos trabalhar. Isto e especialmente valido quando tratamos das turmas dos cursos de Licenciatura em Fsica, pois alem de se defrontarem com quest~oes relativas ao conteudo espec co, estes estudantes tambem est~ao vivenciando procedimentos praticos de ensino em disciplinas experimentais. A tend^encia de reproduzir estes procedimentos praticos posteriormente com seus futuros alunos e grande. Baseados por um lado em avancos conceituais recentes das pesquisas em ensino de ci^encias, e por outro em nossa experi^encia docente junto aos estudantes do Curso de Licenciatura em Fsica da Unesp - Bauru, convidamos docentes e estudantes de Licenciatura em Fsica, para re etirmos sobre alguns aspectos relacionados aos processos de ensino e aprendizagem na disciplina Laboratorio de Eletromagnetismo, tentando analisar o que poderia ser mudado nestas praticas para que haja melhor aproximac~ao entre seus objetivos de aprendizagem e os resultados praticos observados. II Repensando alguns aspectos Nas pesquisas realizadas durante as tr^es ultimas decadas sobre o aprendizado de ci^encias pode-se notar consenso sobre pontos importantes pertinentes ao processo. Um destes pontos de consenso e a import^ancia de entender o aprendizado em termos de desenvolvi- 472 mento conceitual, em vez de simples acrescimo de novas informac~oes [11], ou de simples substituic~oes de concepc~oes existentes por novas [12]. Desenvolvimentos mais recentes [2,5] consistem em resgatar algumas propostas pioneiras sobre o tema [3] dando um enfoque investigativo as praticas de laboratorio. Estas pesquisas apresentam fortes implicaco~es para as praticas em sala de aula por mudarem a concepc~ao, adotada muitas vezes, de que as praticas de laboratorio devam ser meras ilustrac~oes da teoria. Quando abordados por este prisma os experimentos realizados adquirem uma orientac~ao rgida e dogmatica [2], na qual o criterio de verdade e por excel^encia o da autoridade da teoria, do docente, e do livro-texto. Segundo este ponto de vista os experimentos s~ao usados para veri car se existe ou n~ao uma concord^ancia dos resultados com a teoria, e, n~ao raro, quando existe a negativa, a rma-se que o experimento deve ser repetido ate que se consiga o resultado \adequado". Em contraposic~ao a algumas propostas de que os experimentos neste nvel devam necessariamente implicar em um alto grau de complexidade, observamos que, neste caso, a probabilidade de termos um enfoque tecnicista do problema apresentado e grande, conforme apontado por Salinas [2], Sebastia [4] e White [9]. Os objetivos desta disciplina n~ao s~ao prioritariamente tecnicos. Preferimos a proposta de Nedelsky [3] de que a func~ao central dos laboratorios de fsica e dar ao estudante a oportunidade de explorar os varios aspectos da relac~ao entre a descric~ao fsica da realidade e a propria realidade, acrescida dos aspectos motivacionais a nvel de experi^encia pessoal e de trabalho em grupos [8,9,10]. Quando os experimentos s~ao demasiadamente complexos n~ao ha tempo para o estudante re etir e analisar em detalhe o problema proposto. Nestes casos os grupos de estudantes acabam passando diretamente a execuc~ao do experimento, a qual e geralmente descrita em etapas bem de nidas pelo professor, sem antes ter uma noca~o clara do seu proposito. Os aspectos negativos deste tipo de pratica tambem s~ao observados por outros pesquisadores [4,6,9]. Neste caso jarg~oes do tipo \o experimento mostrou-se de acordo com a teoria", sem que nenhum espaco para levantar hipoteses tenha sido propiciado, muito pouco acrescentam ao entendimento dos estudantes sobre os aspectos fsicos e tambem sobre o aspecto da natureza da ci^encia que esta sendo \testada". Desta maneira, podemos considerar que os principais quesitos para escolher os problemas experimentais a serem abordados s~ao aqueles de proporcionar o adequado tema e o adequado nvel de hipoteses a serem testadas, de acordo com o estagio de desenvolvimento cognitivo, e com o interesse dos estudantes. J. Humberto Dias da Silva III Uma proposta baseada no processo investigativo A partir das considerac~oes sobre os problemas levantados acima, foi possvel elaborar uma proposta para a disciplina Laboratorio de Eletromagnetismo do curso de Licenciatura em Fsica da Unesp-Bauru. Nesta proposta cada experimento foi dividido em tr^es etapas. Na primeira etapa um problema aberto, tema do experimento, e apresentado aos estudantes a m de promover sua re ex~ao sobre o tema. O resultado das ideias que surgem durante esta re ex~ao e analisado, em classe e em re ex~ao do professor apos a aula. No segundo momento uma proposta de experimento, elaborada pelo professor sobre o mesmo tema, e apresentada aos estudantes. Nesta proposta levamos em considerac~ao, e tentamos propor como hipoteses, algumas ideias levantadas pelos estudantes no momento anterior. A analise dos resultados, a exposic~ao das discuss~oes, e o fechamento de algumas conclus~oes sobre o experimento proposto pelo professor, e realizada em sala, na ultima etapa. Nesta proposta, os aspectos de investigac~ao do trabalho de laboratorio no sentido proposto por Gil Perez [5] s~ao enfatizados. Resumimos os principais pontos da proposta em: i) Propor situac~oes-problema em vez de experimentos fechados. ii) Analisar, aproveitar, e valorizar as propostas dos estudantes, colocando-as quando possvel como hipoteses a serem testadas. iii) N~ao menosprezar nesta disciplina o potencial didatico de experimentos semi-quantitativos que envolvam a aplicac~ao pratica direta de conceitos, propiciando terreno adequado para o desenvolvimento conceitual e das habilidades dos estudantes. iv) Permitir que os estudantes tenham propostas sobre a maneira de encarar o problema fsico e de executar os experimentos. v) Propiciar cooperac~ao e integrac~ao dos grupos na soluc~ao dos problemas apresentados e na analise coletiva dos resultados obtidos. IV Exemplos de aplicac~ao Para ilustrar, vamos apresentar aqui dois experimentos realizados na disciplina durante o primeiro semestre de 2002. Apresentamos os experimentos com pequeno grau de orientac~ao propositalmente, para n~ao incorrer no erro de propor experimentos totalmente direcionados. Sob este ponto de vista vamos analisar que benefcios experimentos quantitativos ou semiquantitativos podem apresentar aos estudantes. No primeiro exemplo propusemos um experimento para medir o momento magnetico de uma barra imantada. Alguns pontos sobre a aplicabilidade que o conhecimento do momento magnetico possa ter no cotidiano dos estudantes foram salientados. Como ilustrac~ao citamos a quest~ao do dimensionamento dos alto falantes Revista Brasileira de Ensino de F sica, vol. 24, no. 4, Dezembro, 2002 de equipamentos sonoros, e as cabecas de gravac~ao de tas e cart~oes magneticos. Nenhum equipamento e trazido ao laboratorio, apenas a pequena barra cilndrica, objeto da proposta. Assim os estudantes s~ao confrontados com o problema, e apos um intervalo geralmente pequeno de maturac~ao, algumas propostas comecam a surgir, e desencadeiam um interessante processo de propostas onde os estudantes acabam mostrando muitas de suas concepc~oes a respeito do tema. Estas concepc~oes podem ser trabalhadas no momento em que s~ao expressas e tambem na aula seguinte. Nesta etapa a interac~ao que ocorre nos grupos e fundamental para o processo. A partir das quest~oes levantadas pelos grupos, o professor atento pode expor em sala ricos aspectos associados a historia da ci^encia, ao conhecimento formal, e a aplicac~oes praticas da disciplina. Algumas vezes isto e difcil de realizar no instante em que as quest~oes  importante que os pontos levantados peaparecem. E los grupos tambem sejam considerados pelo docente durante a semana que precede a realizac~ao do experimento, para serem retomados em melhores condic~oes na aula seguinte. As propostas dos estudantes para realizac~ao de experimentos para medir o momento magnetico de uma barra foram interessantes. Por exemplo, podemos citar a proposta de uma equipe que sugeriu a colocac~ao da barrinha em ^angulo com o campo de um grande im~a em forma de U, em um eixo perpendicular ao campo, para realizar movimento oscilatorio. O momento magnetico seria determinado a partir da frequ^encia de oscilac~ao. No esquema, reservamos o segundo par de aulas para tentar promover coletivamente uma evoluc~ao das representac~oes anteriormente apresentadas. Isto foi feito partindo dos conceitos apresentados pelos estudantes, enfatizando na proposta de experimento os pontos que apresentaram conceitos a serem trabalhados. Uma das possibilidades e introduzirmos alguns dos conceitos como hipoteses de veri cac~ao na proposta dos experimentos [5], de maneira que a mudanca conceitual possa ser trabalhada de forma mais natural, sem necessariamente enfocarmos o con ito entre a representac~ao previa do estudante e o conceito aceito dentro da teoria estudada. Isto propicia um carater de investigac~ao ao processo de aprendizagem, no sentido mais diretamente proposto por Gil Perez [5], mas que tambem e apontado como altamente bene co por outros autores [2-4,6,9]. Propusemos aos estudantes o experimento quantitativo classico da medida do momento magnetico de uma barra usando uma balanca de torc~ao e um solenoide. Neste experimento um campo magnetico causado por corrente contnua que percorre o solenoide provoca um pequeno torque na barra, o qual e equilibrado pelo torque mec^anico de uma balanca de torc~ao. Com a devida calibrac~ao e a considerac~ao de fatores como o campo magnetico terrestre, o comprimento ( nito) do solenoide, e a correta triangulac~ao para obtenc~ao do ^angulo de equilbrio, o experimento pode ser realizado com um erro em torno de 2%. Neste experimento ocorreram di culdades de visu- 473 alizac~ao por parte dos estudantes. A principal di culdade foi identi car corretamente os varios ^angulos que devem ser usados na execuc~ao do experimento. Ha ^angulos envolvidos na calibrac~ao da balanca, entre o eixo do solenoide e o eixo do im~a, o ^angulo de deex~ao da balanca ao aplicar o campo magnetico, e o ^angulo interno do solenoide, quando consideramos a aproximac~ao de solenoide nito. Houve di culdades tambem na determinac~ao do a^ngulo de de ex~ao a partir de triangulac~ao do feixe de laser. Com esta complexidade o tempo de duas horas foi escasso para a realizac~ao do experimento e algumas equipes optaram por retornar em horarios diversos para complementarem ou tomarem novos dados. Um detalhe importante, quando o tempo permite, e numerar algumas barras e pedir para que os varios grupos mecam cada uma delas, identi cando-as com as medidas. Isto e importante por possibilitar a comparac~ao entre os dados dos varios grupos, e permitir que o teste das hipoteses seja feito com troca de informac~oes e debates intermediados entre grupos. Assim, os estudantes encontram espaco, inexistente na maioria das propostas, para explicar suas hipoteses e debater suas ideias coletivamente. Este processo e rico e contribui de maneira signi cativa para o sucesso da proposta. Apesar das propostas e discuss~oes interessantes ocorridas, o resultado da analise neste experimento muitas vezes se perdeu na complexidade tecnica dos dados. Por este motivo optamos aqui por analisar em maior detalhe os resultados obtidos no experimento semi-quantitativo, apresentado a seguir. N~ao propomos com isto o abandono de propostas quantitativas. Porem e importante n~ao enfatizar demasiadamente os aspectos quantitativos dos experimentos em detrimento de outros aspectos igualmente importantes relativos ao aprendizado. Para citar um experimento semi-quantitativo utilizado na disciplina, recorremos a determinac~ao de superfcies equipotenciais e campos eletricos em geometrias diversas. O problema colocado foi: Como os campos eletricos podem ser medidos na pratica? Que experimento voc^e faria para medir os campos eletricos e mapear as supercies equipotenciais? Vejamos inicialmente algumas propostas feitas pelos estudantes: Grupo A - \colocamos um dipolo eletrico conhe- cido num campo eletrico cujo modulo desejamos saber. Atraves do torque provocado no dipolo, podemos calcular o modulo de E . Isto pode ser feito usando uma balanca de torc~ao, de maneira analoga ao experimento anterior". Comentario: esta proposta indica que o experimento sobre momento magnetico fez parte da bagagem dos estudantes no experimento seguinte. Uma interessante associac~ao entre o momento magnetico da barra e o momento de dipolo eletrico foi realizada, sem que nenhuma menc~ao formal tivesse sido feita pelo professor. Embora a proposta seja correta do ponto de vista 474 conceitual, os estudantes n~ao perceberam que ela seria de difcil realizac~ao pratica, pois n~ao se pode manter facilmente dipolos eletricos para servirem de prova, contrariamente ao que ocorre com os dipolos magneticos. Alem disso a proposta aparentemente n~ao leva em considerac~ao a variac~ao espacial do campo, e o consequente esforco de tomada de dados que teria que ser empregado para um mapeamento adequado do campo. Estes aspectos praticos negativos n~ao diminuem entretanto o interesse da proposta. Grupo B - \criar um campo eletrico uniforme em uma determinada regi~ao. Isso pode ser feito atraves de placas paralelas" (apresentam gura de geometria de placas paralelas, e duas cargas negativas colocadas em plano paralelo as placas, com linhas de campo corretamente desenhadas). \Se colocarmos duas cargas pun- tiformes iguais a uma dist^ancia l da placa carregada positivamente, havera uma forca exercida pelo campo eletrico E nas duas cargas e estas receber~ao uma certa acelerac~ao e velocidade como mostra a gura". Ilustra- ram com vetores F e v colocados em uma das cargas negativas e dirigidos para a placa positiva. Comentario: notamos que duas cargas foram utilizadas, provavelmente para denotar a const^ancia do campo. Observamos que aqui tambem a const^ancia do campo foi assumida. N~ao explicitaram como as grandezas mencionadas seriam medidas. Grupo C - Apresentam uma gura onde a parte externa da esfera de um gerador Van de Graa e ligada a uma placa plana que se encontra numa cuba com oleo e gr~aos de farinha. Existe outra placa paralela na cuba, porem nada esta conectado a esta. As placas est~ao a uma dist^ancia maior que seu comprimento. Linhas de campo (n~ao uniforme) foram desenhadas corretamente, porem as equipotenciais foram desenhadas como linhas paralelas as placas. \liga-se o positivo do Van de Gra- a em um dos eletrodos. Basicamente com isto pode-se ver as linhas de campo, e assim, ja e possvel imaginar as superfcies equipotenciais sabendo-se que as mesmas s~ao perpendiculares as linhas do campo eletrico. Podemos perceber que o campo eletrico e mais intenso na regi~ao onde ha maior concentrac~ao dos gr~aos de farinha, ou seja, proximo aos eletrodos. Nisso tudo considerando que os gr~aos de farinha n~ao adquirem carga no contato com o lquido (oleo). Uma possvel medida para o campo eletrico seria tirar uma foto da formac~ao das linhas de campo e contar os gr~aos de farinha numa determinada regi~ao, e com isso calcular a densidade por centmetro cubico. A mesma devera diminuir a medida que nos afastamos das placas". Comentario: os estudantes utilizaram um experimento demonstrativo apresentado a eles no laboratorio basico (Laboratorio de Fsica Geral IV), e acrescentaram o interessante processo de fotografar e medir a densidade de gr~aos para estimar o valor do campo. A proposta e interessante o su ciente para possvel utilizac~ao como hipotese a ser investigada no experimento proposto pelo professor. Infelizmente n~ao dispomos dos equipamentos fotogra cos necessarios para promover a J. Humberto Dias da Silva comparaca~o entre os valores de campo determinados desta maneira e os valores medidos no experimento. Nota-se a contradic~ao entre a a rmac~ao de que as equipotenciais deveriam ser perpendiculares ao campo com o desenho das equipotenciais paralelas as placas. Esta foi uma das raras propostas que levaram em conta variac~oes espaciais do campo, enquanto nos outros grupos foi observada uma tend^encia clara de n~ao se levar em conta estas possveis variac~oes espaciais. Na aula seguinte, correspondente a segunda etapa do experimento, propusemos um experimento para o mapeamento das superfcies equipotenciais utilizando uma cuba com agua, dois eletrodos de formas a escolher (pelo menos tr^es pares), uma fonte para polarizar, e um voltmetro digital para medir as diferencas de potencial. Algumas instruc~oes foram dadas para garantir um mapeamento adequado das equipotenciais. O intuito era propiciar condic~oes para que os estudantes \vissem" o campo a partir das equipotenciais, tendo a possibilidade de testar varios aspectos presentes nas suas concepco~es. Na sequ^encia apresentamos alguns resultados das interpretac~oes dos grupos: Grupo A - Relataram que o campo deveria ser paralelo ao eixo que une uma placa a uma carga circular pequena (identi cada por eles como puntiforme), apesar de terem calculado o campo utilizando as dist^ancias perpendiculares as equipotenciais. Grupo B - Esperavam que as superfcies equipotenciais para a simulac~ao de um dipolo fossem esferas conc^entricas com os centros dos polos. Grupo C - Utilizaram na determinac~ao dos campos apenas as linhas centrais de simetria nas tr^es geometrias escolhidas (duas placas, placa e ponto, dois pontos). Em nossa opini~ao o principal ponto relacionado ao conhecimento espec co deste experimento foi tratar com os estudantes o conceito de gradiente, e a consequente ligac~ao entre potencial e campo eletrico. Analogias com situac~oes de mapeamento topogra co de relevos, levou-os a um melhor entendimento das curvas equipotenciais, permitindo que calculassem o modulo e indicassem corretamente a direc~ao e o sentido do campo usando as equipotenciais tracadas. Antes desta fase, os estudantes citam a relac~ao E= grad (V) no corpo do relatorio, porem pouqussimas vezes conseguiram indicar, a partir das equipotenciais obtidas na pratica, uma maneira de calcular E usando esta relaca~o. Isto quer dizer que o docente que proponha que o campo eletrico seja calculado usando esta relac~ao em um roteiro de experimento, por melhor intencionado que seja, esta perdendo uma grande oportunidade de deixar que o estudante aprofunde seus conhecimentos, pensando ele proprio esta maneira de encarar o problema. Este ultimo experimento n~ao apresenta as vantagens quantitativas do primeiro, e talvez alguns professores possam acha-lo simples demais para um Laboratorio de Eletromagnetismo do terceiro ano. Porem em troca do aspecto quantitativo podemos citar algumas qualidades. Em primeiro lugar o unico equipamento necessario Revista Brasileira de Ensino de F sica, vol. 24, no. 4, Dezembro, 2002 a sua realizac~ao e um voltmetro, que atualmente pode ser adquirido a baixo custo por uma escola ou que talvez possa ser encontrado entre as ferramentas de trabalho de um estudante com pai eletricista. O experimento pode ser realizado utilizando-se como fonte de tens~ao uma bateria de 9V, ou um conjunto de pilhas de lanterna. Atraves das linhas equipotenciais bidimensionais tracadas o estudante pode descobrir a relac~ao entre potencial e campo eletrico, exempli cando este ultimo com uma maneira concreta, ate ent~ao provavelmente desconhecida. Colocados em quest~ao antes do experimento se o campo eletrico tinha uma exist^encia real ou se era mera abstrac~ao, os estudantes, mesmo neste grau, caram em sua maioria indecisos, e optaram muitas vezes por considerar que o campo era apenas uma entidade \teorica" que n~ao conseguiramos expressa-lo de uma maneira pratica. Entre os que responderam que o campo possua uma exist^encia real, muitos descreveram o experimento com cargas para testa-lo \soltase uma carga com massa conhecida e mede-se a acelerac~ao". Porem quando indagados a respeito de como seria possvel saber se o campo seria constante ou n~ao naquela regi~ao do espaco, nenhum fez menc~ao a alguma maneira de mapear o campo. Segundo nosso entendimento este experimento, embora semi quantitativo, apresenta um real valor no contexto da disciplina por ilustrar de maneira metodologicamente simples uma grandeza fsica muito importante para a formac~ao do estudante. V Comentarios dos estudantes sobre a proposta Durante o desenvolvimento da disciplina os estudantes zeram varios comentarios sobre as modi cac~oes propostas para a disciplina. Com o intuito de entender como a proposta foi recebida pelos estudantes, apresentamos aqui alguns pontos que consideramos importantes em seus comentarios. Ao conhecer a proposta da disciplina e nos primeiros experimentos houve duvidas quanto ao que se pretendia que fosse relatado, e quanto ao criterio que seria usado nas correc~oes dos trabalhos, em alus~ao clara a preocupac~ao com relac~ao aos criterios de avaliac~ao a serem adotados. Durante a realizac~ao dos primeiros experimentos houve um desconforto inicial em relac~ao aos procedimentos a serem utilizados nos experimentos propostos pelo professor, pois apesar de que os objetivos, e ate certo ponto os mecanismos principais a serem considerados foram estabelecidos, outros pontos foram deixados em aberto no experimento, entre eles a metodologia de aquisic~ao e interpretac~ao dos dados. Estas di culdades iniciais foram superadas durante o decorrer do semestre, e em nosso entender contriburam para desenvolver uma maior autonomia dos estudantes frente a situac~oes experimentais. Conforme apontado por Barolli [8], a superac~ao das di culdades 475 iniciais e os avancos da autonomia observados, podem ser atribudos em parte a melhor estruturac~ao dos grupos com o decorrer do tempo de interac~ao. Esta melhor estruturac~ao pode auxiliar no estabelecimento de ac~oes cooperativas que contribuem para a superaca~o das diculdades praticas. A possibilidade de aproveitar melhor suas proprias ideias foram pontos importantes sobre os quais os estudantes zeram comentarios em varias oportunidades manifestando clara aprovac~ao. Em nossa avaliac~ao este e um dos principais pontos favoraveis da proposta: propicia motivac~ao e envolvimento dos estudantes no sentido de utilizar os conhecimentos disponveis para abordar situac~oes praticas. Trocas de informac~oes entre os membros de cada grupo e entre os grupos da turma, e a intermediac~ao do professor foram favorecidas de maneira natural nesta din^amica. Uma di culdade n~ao claramente percebida pelos estudantes, mas externada em seus comentarios em varias ocasi~oes, foi a de considerar a proposta do professor como sendo a unica \correta" e de tentar avaliar a validade ou n~ao de sua proposta pela semelhanca com a proposta do professor. Tal postura dos estudantes e um problema real deste tipo de proposta e necessita uma atenc~ao especial do professor que se prop~oe a implementa-las. Esta e uma consequ^encia negativa de metodologias tradicionalmente colocadas para o estudante, e que merece intervenc~oes cuidadosas do professor. Apesar da grande import^ancia imputada por nos as propostas dos estudantes, o envolvimento posterior dos grupos com suas proprias propostas n~ao foi grande. Atribumos isto em parte a di culdade mencionada no paragrafo anterior e tambem a facilidade dos estudantes em modi carem suas propostas a partir do conhecimento de novos dados, ou de objec~oes consistentes que possam ser levantadas sobre seus argumentos. VI Considerac~oes nais Nesta abordagem do Laboratorio de Eletromagnetismo os estudantes tiveram a oportunidade de fazer uma proposta de experimento, de analisar os resultados de experimento proposto pelo professor, e de fazer uma analise comparativa entre sua proposta de experimento e aquele proposto pelo professor. Entretanto, o experimento realizado ainda n~ao e verdadeiramente uma investigac~ao proposta pelo grupo de estudantes, e sim uma investigac~ao dirigida. Apesar disto existe espaco para que alguns aspectos propostos diretamente pelos estudantes sejam enfocados: se o experimento proposto pelo professor contiver hipoteses de veri caca~o relacionadas as propostas as representac~oes previas dos estudantes, muitas das quais manifestaram-se na etapa inicial de proposic~ao, um dos importantes objetivos da disciplina sera atingido [3,5]. Nesta disciplina e importante termos em conta que, em muitas situac~oes, a inadequada complexidade dos experimentos propostos associada ao pequeno 476 tempo dedicado a re ex~ao sobre os conceitos e interpretac~ao dos resultados, pode infelizmente levar a poucos avancos na direc~ao desejada, ou seja ao efetivo aprofundamento do entendimento dos conceitos por parte dos estudantes. Este e um aspecto negativo, pois alem do entendimento espec co da disciplina os estudantes de licenciatura est~ao vivenciando exemplos dos processo de ensino e aprendizagem e dos relacionamentos em grupo [8,9,10] aplicados a soluc~ao de situac~oes praticas O papel dos grupos, o qual ja tem sua import^ancia em abordagens tradicionais de disciplinas de laboratorio, e fundamental para o sucesso deste tipo de proposta. A interac~ao entre os membros de cada grupo e entre grupos foi especialmente produtiva durante as proposic~oes dos estudantes. Neste momento houve uma forte interac~ao entre os membros de cada grupo, e uma abertura muito maior que a normalmente observada para a mediac~ao buscada junto ao professor. A mediac~ao do professor ocorreu principalmente quando algum membro propunha um experimento ao grupo e tentava explicar baseado em seu entendimento dos conceitos fsicos. As propostas e justi cativas geralmente causavam questionamentos, troca e confronto de ideias com os colegas. Isto tambem promoveu de maneira natural o papel do professor como mediador das discuss~oes. A di culdade rotineira de contarmos com menos tempo do que gostaramos foi potencializada nesta proposta. Para tratar da profus~ao de ideias, que ocorre quando deixamos um problema aberto para considerac~ao dos grupos, e necessario um intervalo de tempo geralmente n~ao disponvel nas propostas tradicionais. Para tentar superar esta di culdade foi adotado o procedimento de deixar uma semana para cada uma das tr^es etapas: propostas, realizac~ao, e discuss~oes (a disciplina e oferecida em duas horas semanais). Isto implica que um menor numero de experimentos pode ser tratado (em geral 4 a 5 por semestre!). Porem o fato de termos realizado menos experimentos n~ao signi ca que tenhamos abordado um numero menor de situac~oes praticas e tratado menos temas experimentais, alias, tem-se a impress~ao clara de que ocorre justamente o oposto. Pelas proprias respostas e atitudes dos estudantes, nos parece que com esta abordagem estamos mais proximos dos objetivo do laboratorio didatico: promover a oportunidade ao estudante de confrontar seus conceitos sobre a fsica com o resultado dos experimentos, e de repartir suas concepc~oes com os outros estudantes e com o professor [5,8,9,10]. O fato de podermos abranger os itens principais da proposta em maior ou menor grau em cada experimento, justi ca a diminuic~ao do numero de experimentos que podem ser trabalhados na disciplina pelo aumento da efetividade em que cada topico e trabalhado. Alem disso, pela riqueza de troca de experi^encia J. Humberto Dias da Silva que proporcionam, podemos dizer que ao inves de reduzir o numero de temas abordados (com a reduc~ao do numero de experimentos) o numero de temas abordados na disciplina e ampliado, pois ha espaco para se discutir aspectos historicos, aplicaco~es e abrang^encia dos conceitos. Longe de representar uma proposta fechada, este conjunto de ideias e um convite a docentes e estudantes de licenciatura a re etirem sobre o aprendizado no Laboratorio de Eletromagnetismo. Agradecimentos Agradeco aos estudantes do curso de Licenciatura em Fsica da Unesp - Bauru, e aos Professores Elizabeth Barolli e Fernando Bastos, pelas interessantes discuss~oes, ao Mestre Julio Cesar Ribeiro, cujo trabalho de orientac~ao me levou a repensar minhas proprias praticas de ensino, e a dois estimados professores de fsica experimental Jorge Ivan Cisneros e Curt Egon Hennies, do IFGW - Unicamp, que me propiciaram, enquanto estudante, o gosto pelas disciplinas experimentais. Refer^encias [1] D. Gil Perez e M. G. Ozamis, Ensen~anza de Las Ciencias y la Matematica, Editorial Popular S.A / Ministerio de Educacion y Ciencia, Espanha, 1992. [2] J. S. Salinas, L.C. de Cudmani, Revista de Ense~nanza de la Fsica, 5, 10 (1992). [3] L. Nedelsky, Am. J. Phys. 26, 51 (1958). [4] J.M. Sebastia, Ense~nanza de Las Ciencias, 5, 196 (1987). [5] D. Gil Perez, P. Valdes Castro. Ense~nanza de las Ci^encias, 14, 155 (1996). [6] M. E. Gonzales, Ense~nanza de las Ci^encias, 10, 206 (1992). [7] J.C. Ribeiro, O Papel do Professor no Laboratorio de Fsica Basica: Um Exemplo Ilustrativo, Dissertac~ao de Mestrado, Unesp-Bauru (2000). [8] E. Barolli, Re ex~oes sobre o Trabalho dos Estudantes no Laboratorio Didatico, Tese de Doutoramento, USPSP, (1998). [9] R.T. White, Int. J. Sci. Educ., 18, 76 (1996). [10] E. Barolli e A. Villani, Ci^encia e Educac~ao, 6, 1 (2000). [11] A Villani, Revista Brasileira de Ensino de Fsica, 6, 76 (1984). [12] E.F. Mortimer, Investigac~oes em Ensino de Ci^encias, 1, 22 (1996).
https://openalex.org/W2047871446
https://molecularcytogenetics.biomedcentral.com/counter/pdf/10.1186/1755-8166-7-S1-I57
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Vitiligo: a complex disease and a complex approach
Molecular cytogenetics
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Vitiligo: a complex disease and a complex approach Rasheedunnisa Begum1*, Yogesh S. Marfatia2, Naresh C Laddha1, Mitesh Dwivedi1, Mohmmad Shoab Mansuri1, Mala Singh1 From International Conference on Human Genetics and 39th Annual Meeting of the Indian Society of Human Genetics (ISHG) Ahmadabad, India. 23-25 January 2013 IL4, CTLA4, SOD2, SOD3, NALP1, MYG1, TNFA, TNFB, IFNG, IL10 and ICAM1 genes suggest that these genes are strongly associated with vitiligo susceptibility. We are also addressing the role of immune-regulatory genes with respect to their expression in skin along with the effect of selected cytokines on in vitro cultured melanocytes derived from healthy and vitiliginous human skin to have an insight towards vitiligo pathogenesis. We are also exploring the potential microRNAs involved in pathogen- esis of vitiligo. This integrated study will provide a better understanding of the role played by oxidative stress and autoimmunity in the pathogenesis of vitiligo in Gujarat population and also to develop selective therapy and the genetic marker/s for vitiligo. Vitiligo is an acquired, circumscribed hypomelanotic skin disorder, characterized by milky white patches due to loss of functional melanocytes from the epidermis. Prevalence of vitiligo is found to be very high in Gujarat i.e., ~8.8%. Vitiligo is a multifactorial polygenic disorder with a complex pathogenesis, linked with both genetic and non-genetic factors. Several theories have been proposed to explain the etiopathogenesis of vitiligo, but none of the hypotheses explains the entire spectrum of this disorder. We are addressing this complex disease in our Gujarat population with various approaches. Our study mainly deals with the evaluation of oxidative stress, autoimmune, genetic and neurochemical hypoth- eses in Gujarat vitiligo patients. We have shown that our vitiligo patients exhibit significant oxidative stress and thus, systemic oxidative stress could play a patho- physiological role in precipitation of vitiligo in Gujarat population. Our studies revealed that presence of increased antimelanocyte antibodies and the imbalance of T-cell (CD4+/CD8+ and Tregs) subsets along with their functional defects might result in melanocyte destruction in vitiligo patients. Our results on selected candidate genes in conferring oxidative stress and autoimmunity suggest that HLA-A*33:01, HLA-A*02:01,HLA-B*44:03, HLA- DRB1*07:01 and a few studied polymorphisms in IL4, CTLA4, SOD2, SOD3, GPX1, NALP1, MYG1, TNFA, TNFB, IFNG and IL10 genes are strongly associated with vitiligo susceptibility, whereas a few studied polymorph- isms in PTPN22, MBL2, ACE, CAT, G6PD and SOD1 genes are not found to be significantly associated with Gujarat vitiligo patients. * Correspondence: rasheedunnisab@yahoo.co.in 1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India Full list of author information is available at the end of the article Begum et al. Molecular Cytogenetics 2014, 7(Suppl 1):I57 http://www.molecularcytogenetics.org/content/7/S1/I57 SPEAKER PRESENTATION Open Access Vitiligo: a complex disease and a complex approach Gene expression studies of the Various factors such as the antioxidant status, LPO (oxidative stress) levels and antimelanocyte antibody titer decide the selective therapy for our vitiligo patients. The pathogenesis of vitiligo though partially understood still remains complex and enigmatic to a greater extent. Though the condition may be precipitated by multiple etiologies, the interaction of oxidative stress and immune system clearly appears to be the key convergent pathway that initiates and/or amplifies the enigmatic loss of melanocytes in vitiligo. 1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India. 2Department of Skin & V.D., Faculty of Medicine, The Maharaja Sayajirao University of Baroda, Vadodara, India. Authors’ details 1D f B 1Department of Biochemistry, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, India. 2Department of Skin & V.D., Faculty of Medicine, The Maharaja Sayajirao University of Baroda, Vadodara, India. Published: 21 January 2014 Published: 21 January 2014 doi:10.1186/1755-8166-7-S1-I57 Cite this article as: Begum et al.: Vitiligo: a complex disease and a complex approach. Molecular Cytogenetics 2014 7(Suppl 1):I57. doi:10.1186/1755-8166-7-S1-I57 Cite this article as: Begum et al.: Vitiligo: a complex disease and a complex approach. Molecular Cytogenetics 2014 7(Suppl 1):I57. © 2014 Begum et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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https://www.researchsquare.com/article/rs-1152091/latest.pdf
English
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Novel Type of References for BMI Aligned for Onset of Puberty - Using the QEPS Growth Model
Research Square (Research Square)
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Research Article Posted Date: February 11th, 2022 DOI: https://doi.org/10.21203/rs.3.rs-1152091/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Novel type of references for BMI aligned for onset of puberty – using the QEPS growth model Kerstin Albertsson-Wikland1*, Aimon Niklasson2, Lars Gelander1, Anton Holmgren2,3, Andreas F.M. Nierop1,4 p 1Department of Physiology/Endocrinology, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden 2Göteborg Pediatric Growth Research Center, Department of Pediatrics, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Sweden 3Department of Pediatrics, Halmstad Hospital, Halmstad, Sweden 4Muvara bv, Multivariate Analysis of Research Data, Leiderdorp, The Netherland, Short title: Pubertal BMI reference Short title: Pubertal BMI reference Keywords: pubertal growth, personalized growth, biological age, SDS, reference population 1 1 Abstract Objectives: Despite inter-individual variations in pubertal timing, growth references are conventionally constructed relative to chronological age (C-age). Thus, they are based on reference populations containing a mix of prepubertal and pubertal individuals, making them of limited use for detecting abnormal growth during adolescence. Recently we developed new types of height and weight references, with growth aligned to age at onset of the pubertal growth spurt (P-age). Here, we aim to develop a corresponding reference for pubertal BMI. Methods: The QEPS-height and weight models were used to define a corresponding QEPS- BMI model. QEPS-BMI was modified by the same individual, constitutional weight–height- factor (WHF) as computed for QEPS-weight. QEPS-BMI functions were computed with QEPS weight and height functions fitted on longitudinal measurements from 1418 individuals (698 girls) from GrowUp1990Gothenburg cohort. These individual BMI functions were used to develop BMI references aligned for height at AgeP5; when 5% of specific puberty-related (P-function) height had been attained. Pubertal timing, stature at pubertal onset, and childhood BMI, were investigated in subgroups of children from the cohort GrowUp1974Gothenburg using the new references. Methods: The QEPS-height and weight models were used to define a corresponding QEPS- BMI model. QEPS-BMI was modified by the same individual, constitutional weight–height- factor (WHF) as computed for QEPS-weight. QEPS-BMI functions were computed with QEPS weight and height functions fitted on longitudinal measurements from 1418 individuals (698 girls) from GrowUp1990Gothenburg cohort. These individual BMI functions were used to develop BMI references aligned for height at AgeP5; when 5% of specific puberty-related (P-function) height had been attained. Pubertal timing, stature at pubertal onset, and childhood BMI, were investigated in subgroups of children from the cohort GrowUp1974Gothenburg using the new references. Results: References (median, standard deviation score (SDS)) were generated for total BMI (QEPS-functions), for ongoing prepubertal growth (QE-function) vs C-age, and for total BMI and separated into BMI specific to puberty (P-function) and BMI gain from ongoing basic growth (QES-functions), allowing individual growth to be aligned based on P-age. Growth in basic BMI was greater than average for children categorized as tall and/or with high-BMI at puberty-start. In children categorized as short at puberty-start, P-function-related-BMI was greater than average. 2 2 Conclusions: Use of these new pubertal BMI references will make it possible for the first time to consider individual variations owing to pubertal timing when evaluating BMI. Abstract This will improve the detection of abnormal changes in body composition when used in combination with pubertal height and weight references also abnormal growth. Other benefits in the clinic will include improved growth monitoring during treatment for children who are overweight/obese or underweight. Furthermore, in research settings these new references represent a novel tool for exploring human growth. Conclusions: Use of these new pubertal BMI references will make it possible for the first time to consider individual variations owing to pubertal timing when evaluating BMI. This will improve the detection of abnormal changes in body composition when used in combination with pubertal height and weight references also abnormal growth. Other benefits in the clinic will include improved growth monitoring during treatment for children who are overweight/obese or underweight. Furthermore, in research settings these new references represent a novel tool for exploring human growth. 3 3 Background Unlike the previous reference developed around 2000 in Sweden that have used estimates of ongoing, childhood growth from the Infancy–Childhood– Puberty (ICP) growth model (11-14), we constructed prepubertal growth using the QE- functions of the QEPS-model (15, 16). Individual growth is described by the QEPS-model using four mathematical functions: a Q (Quadratic) and E (Exponential) function arising before birth and resulting in the prepubertal growth, to which a specific pubertal growth function, P, is added; the QE-growth is during puberty transformed and ended by a S (stop) function; resulting in basic, QES-function-growth during puberty (15). Thereafter, we also developed a QEPS-model for weight and constructed a reference for prepubertal weight gain during adolescence (17). of adolescence which is characterized by great inter-individual variations in growth because of differences between individuals in the C-age at which they enter puberty (8, 9). When in 2020 developing the most recent Swedish height references, to account for this we included a ‘prepubertal’ reference showing the growth that during adolescence is continuing independently of puberty (10). Unlike the previous reference developed around 2000 in Sweden that have used estimates of ongoing, childhood growth from the Infancy–Childhood– Puberty (ICP) growth model (11-14), we constructed prepubertal growth using the QE- functions of the QEPS-model (15, 16). Individual growth is described by the QEPS-model using four mathematical functions: a Q (Quadratic) and E (Exponential) function arising before birth and resulting in the prepubertal growth, to which a specific pubertal growth function, P, is added; the QE-growth is during puberty transformed and ended by a S (stop) function; resulting in basic, QES-function-growth during puberty (15). Thereafter, we also developed a QEPS-model for weight and constructed a reference for prepubertal weight gain during adolescence (17). of adolescence which is characterized by great inter-individual variations in growth because of differences between individuals in the C-age at which they enter puberty (8, 9). When in 2020 developing the most recent Swedish height references, to account for this we included a ‘prepubertal’ reference showing the growth that during adolescence is continuing of adolescence which is characterized by great inter-individual variations in growth because of differences between individuals in the C-age at which they enter puberty (8, 9). Background For many decades, body mass index (BMI) has been the most commonly used variable to define and evaluate body composition and weight status in children. In both research and clinical practice, BMI, which is expressed as kg/m2, has been widely used to define whether a child is of normal weight, underweight, overweight, or obese (1). While both height and weight increase with chronological age (C-age), this is not the case for BMI. BMI rises in infancy, then falls during childhood before rising again (2-4). The second rise in BMI is usually seen some years before puberty and continues during the adolescent years, with BMI only reaching adult levels sometime after adult height has been attained. In 2012, the international obesity task force (IOTF) used merged BMI data from different countries to create a worldwide-accepted reference with iso-lines defining overweight, obesity, and thinness (5), which has been especially valuable for international comparison of research studies. Beside this, there are national BMI references, such as those in Sweden which include risk estimation curves for obesity (3). The ongoing obesity epidemic complicates the construction of new weight and BMI references. Although the trend over time toward higher BMI seems partly to have plateaued in some high-income countries, BMI is still rising in many parts of the world (6). Recently in Sweden, for example, an increasing prevalence of overweight and obesity with age in both girls and boys has been reported (7). Such changes in BMI must not be incorporated in references reflecting optimal body composition. We addressed this issue in the recently updated Swedish references for weight and BMI by omitting individuals with obesity from the longitudinal BMI dataset used to develop the references (4). 4 It has been convention for growth references for height, weight, and BMI to be presented in relation to C-age, making them suboptimal for detecting abnormal growth during the period of adolescence which is characterized by great inter-individual variations in growth because of differences between individuals in the C-age at which they enter puberty (8, 9). When in 2020 developing the most recent Swedish height references, to account for this we included a ‘prepubertal’ reference showing the growth that during adolescence is continuing independently of puberty (10). Ethical approval Ethical approval was obtained from the Regional Ethics Review Board in Gothenburg (Ad 91-92/131 -93 and Ad 444-08 T062-09). Informed consent was given by participants and parents of individuals <18 years of age. Studies were conducted in accordance with the principles of the Declaration of Helsinki. Background When in 2020 developing the most recent Swedish height references, to account for this we included a ‘prepubertal’ reference showing the growth that during adolescence is continuing Recently, we have developed a new type of pubertal height reference describing growth aligned for onset of the specific P-function in the QEPS-model responsible for pubertal height gain; this reference provides information on total growth, as well as separating out the specific growth related to puberty from basic growth continuing during the pubertal years (10, 18). Subsequently, we developed a QEPS model for weight to construct corresponding references for pubertal weight gain (17). It now remains to develop a puberty-adjusted growth model for BMI and to use it to develop references that enable the separation of prepubertal and pubertal changes in BMI. 5 The aim of this study was to use the QEPS-models for height and weight to develop a growth model for BMI, and to develop new BMI references that take biological maturation of the 5 5 individual during adolescence into account. Like the previous references, data used to construct the new BMI reference will be obtained from longitudinally followed, healthy children born at term to non-smoking mothers and Nordic parents, selected from the GrowUp1990Gothenburg cohort born in Sweden (4). Separate references will be constructed for (a) prepubertal BMI (Q- and E-functions), (b) total BMI (modeled by the Q, E, P and S- functions), (c) BMI gain specific to puberty (by the P-function), and (d) BMI gain that is not specific to puberty (basic BMI, by the Q, E, and S-functions). Subgroups of children from the GrowUp1974Gothenburg cohort categorized based on the timing of puberty (early, average, late), height at start of puberty (tall, short) or body composition during childhood (high BMI, low BMI) will be used to explore the utility of the new references for monitoring BMI. reference Evaluation of the utility to research of the new pubertal BMI reference was made using data from healthy children from the GrowUp1974Gothenburg cohort (2177 subjects: 1081 girls). The impact of grouping children according to P-age, for age at onset of puberty, for height, and for childhood BMI was explored (see Supplemental Table S3 of Albertsson-Wikland et al (10)). 1. Reference population from GrowUp1990Gothenburg cohort 6 In total, 1418 individuals (698 girls) from the GrowUp1990Gothenburg cohort were included in the cohort used to construct the QEPS-BMI reference. This was the same population as used to develop earlier C-age references for total weight and BMI, and those for weight-for- height (see Supplemental Table S1 in Albertsson-Wikland et al (4)). The cohort included only healthy children (see Table 1 in Albertsson-Wikland et al (4)) who had Nordic parents and 6 6 were born in Sweden around 1990 at full term (gestational age (GA) 37–43 weeks) to non- smoking mothers. Information was available on longitudinal growth until adult height for all participants. For more information see Albertsson-Wikland et al. (4, 18). QEPS-BMI method The QEPS-height (15) and QEPS-weight models (17), were used to define a corresponding QEPS-BMI model. The QEPS-BMI model is expressed in kg0.5/m to be consistent with the QEPS-weight model, which was expressed in kg0.5 to enable the additive property of the QEPS weight functions. Where appropriate, we show BMI in kg/m2 in figures on the left axis and BMI in kg0.5/m on the right axis. For detailed information about the QEPS-BMI model see Supplement, including Supplemental Figures S1, S2, and S3. Like the QEPS-weight model, QEPS-BMI was modified by an individual ‘constitutional factor’, a weight–height factor (WHF): WHF=0, a ‘normal body constitution’, WHF>0, a heavy, and WHF<0, a lean body constitution. Traditional references according to C-age for total and prepubertal BMI were computed in two steps comparable to the ‘QEPS method used for the references’ section in Supplement of Albertsson-Wikland et al. (18), but with QEPS height functions replaced by corresponding QEPS-BMI functions. QEPS-BMI functions were computed with QEPS- 7 7 height functions fitted on height measurements and QEPS-weight functions fitted on weight measurements while including information from fitted QEPS-height functions. height functions fitted on height measurements and QEPS-weight functions fitted on weight measurements while including information from fitted QEPS-height functions. Comparison was made between the total BMI reference obtained using the QEPS-BMI method and the previously published BMI reference obtained by applying the LMS method (4). Both references had been created using the same data from the same population, see Supplemental Figure S4. height functions fitted on height measurements and QEPS-weight functions fitted on weight measurements while including information from fitted QEPS-height functions. Comparison was made between the total BMI reference obtained using the QEPS-BMI method and the previously published BMI reference obtained by applying the LMS method (4). Both references had been created using the same data from the same population, see Supplemental Figure S4. Median curves for the QEPS-derived and LMS-derived references were similar; however, as expected, variance was smaller for the QEPS-derived reference owing to computation with fitted functions that excluded residual variation. A new reference for pubertal BMI was then generated. The onset of puberty was identified based on height-specific P-function growth; the point at which 5% of height Pmax had been obtained (AgeP5) was used to define the onset of puberty (Supplemental Figure S2 in Albertsson-Wikland et al. (10))(16). Height for each individual was then aligned according to age at the onset of puberty. QEPS-BMI method To achieve this for BMI, all individual longitudinal QEPS-BMI-functions were aligned according to height at AgeP5. For more information see Supplement. Statistical evaluations Longitudinal growth data for each individual were exported to Matlab® (version 9.3 R2017b, The MathWorks). Estimation of individual QEPS height and weight parameters by nonlinear fitting was conducted using the Matlab Curve Fitting Toolbox. The fitting procedure for height is described in (15), and for weight in the Supplement (17). QEPS-BMI functions were computed with resulting QEPS height and weight functions. To simplify general mathematical formulae, age was assumed to be equal to age corrected for GA, here 37–43 weeks. All figures were prepared in Matlab; Figure 7 was finalized in Photoshop. 8 8 2. BMI references aligned for onset of puberty (P-age) The reference shown in Figure 2 is for total (QEPS) and basic (QES-function) BMI in kg/m2 (left axis) and kg0.5/m (right axis) for girls and boys aligned according to the onset of puberty, estimated as AgeP5 for height, P-age. Specific P-function growth has been included in the lower panel. BMI references in both figures are depicted from 4 years before to 10 years after the onset of the pubertal growth spurt to capture changes in BMI relative to the acceleration in height and weight that occurs during puberty. General differences in the timing of pubertal height and weight functions resulted in undulations of the aligned BMI functions (Supplemental Figure S3). 3. Rational for a puberty-aligned BMI reference Total BMI relative to C-age for girls and boys with an early, average, and late onset of puberty is shown in Figure 3. As expected, BMI gain occurred sooner than average in children with an early and later than average in children with a late onset of puberty. This highlights the inadequacy for many adolescents of using a C-age-based BMI reference. 1. Total and prepubertal BMI references vs chronological age (C-age) The novel BMI references in kg/m2 (left axis) and in kg0.5/m (right axis) according to C-age 4–20 years for girls and boys, Figure 1. Total BMI is shown in color (red/blue), and BMI gained independently of puberty, the prepubertal, (QE-function) BMI reference, in black. 4. Exploring pubertal BMI in subgroups from the GrowUp1974Gothenburg cohort Timing of puberty: Total (QEPS), basic (QES), and pubertal (P-function) BMI relative to the onset of puberty for subgroups of girls and boys with an early, average, and late onset of 9 9 puberty is shown in Figure 4. For girls in the early and late puberty onset groups, the total BMI gain and the gain in the basic component of BMI were somewhat higher than average; for boys in the early and late puberty onset groups, gains in total and basic BMI were similar to average. Puberty-specific BMI gain was greater than average in the early-puberty-onset group and lower than average in the late-puberty-onset group. Stature: The total (QEPS-functions), basic (QES-functions), and pubertal (P-function) BMI for girls and boys according to height at P-age, the age at the onset of puberty is shown in Figure 5. For girls and boys who were tall at the onset of puberty, the basic component of BMI was higher than average; for girls and boys who were short at the onset of puberty, this component was lower than average. In contrast, puberty-specific BMI gain was lower than average for the tall group and higher than average for the short group. Degree of childhood BMI: Total (QEPS-functions), basic (QES-functions), and pubertal (P- function) BMI for girls and boys according to the highest BMI during childhood (girls:3.5– 7yrs; boys: 3.5–8yrs), Figure 6. Gains in the basic component of BMI were higher than average in children in the high BMI group at the onset of puberty and lower than average in the low BMI group. In the low BMI groups, puberty-specific BMI gain was lower than average for girls and somewhat lower than average for boys. In the high BMI groups, puberty-specific BMI gain was somewhat higher than average for girls and similar to the average for boys. 5. Using the new BMI pubertal growth charts prospectively for an individual child In order to monitor pubertal BMI changes prospectively for an individual child using the new pubertal-age-BMI charts a manual procedure can be undertaken. Early identification of the ‘take-off’ in growth at the start of puberty can be achieved using the growth charts for total 10 and prepubertal height references (18). Figure 7 describes the prospective use of the pubertal- age-adjusted reference to assess total BMI. For corresponding changes in weight, see Figure 7 in Albertsson-Wikland et al. (17). and prepubertal height references (18). Figure 7 describes the prospective use of the pubertal- age-adjusted reference to assess total BMI. For corresponding changes in weight, see Figure 7 in Albertsson-Wikland et al. (17). New prepubertal and pubertal BMI references Here, we present the first pubertal BMI references adjusted for biological maturation based on age at onset of the pubertal growth spurt in the individual child. Thereby, the growth of any individual will be related only to peers with similar biological age and maturation. Here, we present the first pubertal BMI references adjusted for biological maturation based on age at onset of the pubertal growth spurt in the individual child. Thereby, the growth of any individual will be related only to peers with similar biological age and maturation. Another novel benefit of these BMI references is that they allow the separation of prepubertal and pubertal components of BMI gain. During adolescence this makes it possible to look in detail at the ongoing BMI gain that is unrelated to puberty in relation to the growth specific to puberty. As such, we expect these references to be of utility in the clinic and in research- based investigations of both puberty-independent and puberty-dependent changes in BMI. Moreover, a prepubertal BMI reference was also created, vs C-age, which will be particularly important when considering body composition as BMI changes in children in whom puberty is early or delayed. These new references complete a set of three growth references – for pubertal and prepubertal height, weight, and BMI – developed using growth functions based on QEPS growth models for height, weight, and BMI. In all cases, these references make it possible to align growth in the individual relative to the onset of pubertal growth in the individual (10, 17) and as a set, they will be valuable in pubertal and prepubertal individuals for evaluating growth in terms of height, weight, and BMI, as well as for evaluating the relationship between weight and height. To use growth references such as these effectively in healthcare and clinical settings, 11 11 estimation of the stage of pubertal maturation will be required alongside construction of the growth chart for the individual; this includes investigation of the genitalia and breast maturation in girls or testicular size in boys (14, 19). Using these three references alongside estimates of pubertal maturation will help to decide which reference for each variable to use, and will improve the precision with which we can evaluate changes in BMI and growth during adolescence (10). BMI changes preceding height changes The occurrence of characteristic sex-specific changes in weight and body fat mass before changes in pubertal growth suggest a relationship between energy storage in adipose tissue and pubertal maturation (20). By aligning BMI in relation to the onset of puberty, the new BMI references will allow us to explore in detail the changes in body composition (BMI and weight) that occur some years before the increase in height associated with puberty (Supplemental Figure S3). It is interesting to note that the BMI reference curves were observed to be undulating in places. This appears to be due to differences in the timing of puberty-related changes in height and weight, and to the type of tissue growth responsible for weight gain (e.g. fat vs muscle). Thus, residual variation around individual predicted BMI for normal body constitution might be related, not only to measuring errors, but also to individual variations in the relationship of fat and muscle distributions as estimated by body compartment analyses (dual-energy X-ray absorptiometry (DEXA)) or other comparable measurements (21). 12 Previously, we found sex differences in both the timing and amount of two types of weight: earlier gain of type A weight in girls and greater gain of type B weight in boys (Supplemental Figure S3). At present, we speculate that type A weight represents fat tissue because it correlates with the sex difference in the timing of early pubertal fat gain, as described in the 12 12 study by Vizmanos & Marti-Henneberg, and that type B weight represents muscle tissue; however, this needs to be confirmed in future studies (20, 22). Puberty-aligned BMI represents a relative balance between weight and height, making interpretation of these BMI results quite complex. Interestingly, the total BMI typical mean function for individual boys and girls became quite similar over time, whereas the P-function-related curve showed sex differences in both the timing and proportion of pubertal type A BMI gain and pubertal type B BMI gain (Supplemental Figure S3). DEXA studies will be needed to explore this observation in greater detail. Usefulness of a reference separating BMI growth functions during puberty The benefits of using a growth model to explore underlying growth regulating mechanisms have been discussed previously (17, 23, 24). A novel finding from our analysis of subgroups of children with different characteristics prior to entering puberty was that gain in basic BMI was greater in tall boys and girls, when compared to either the reference, or to boys and girls of average pubertal timing or stature. In contrast, lower-than-average basic BMI gain was observed for children who were short at the start of puberty, although their P-function-related BMI gain was greater than average. This is consistent with our previous investigations in children with different BMIs in which we found a positive relationship between BMI and greater than average gains in basic height and lower than average gains in puberty-specific height (25). We also found that a high BMI was associated with greater than average gains in basic BMI and that low BMI was associated with lower-than-average increases in basic BMI. 13 From a research perspective, the new BMI reference will be useful in the exploration of correlates for predicting future health. It is already well known that childhood obesity is an important factor associated with increased risk for reduced life expectancy. For example, increased BMI during adolescence is associated with elevated cardiovascular morbidity, the 13 development of type 2 diabetes and different types of cancer during adult life (26-28). It is also known that both prenatal and early postnatal nutrition have epigenetic effects on developmental programming resulting in cardiovascular diseases, diabetes, overweight, and obesity (29-32). As such, the ability to look at the different components of BMI during childhood will be useful for exploring which variables help us to predict the development of obesity. National growth screening and improved BMI monitoring during treatment Longitudinal follow up of children’s growth provides an important way of evaluating present and future population health and should be conducted at a national level (33). National monitoring of BMI-related child health will be more precise with the incorporation of insights from the BMI references described here. We anticipate that investigations using the new references will lead to re-evaluation of currently used BMI iso-line cutoffs especially during the pubertal years. With the exception of surgical treatment during late adolescence (34, 35), interventions to treat obesity during childhood are still generally unsuccessful (34). There is, therefore, a clear need for more clarity on the underlying causes of childhood obesity so that appropriate treatments can be developed. Gaining a greater understanding of the relationship of BMI to puberty will be an important step in this journey. Another condition where more in-depth understanding of BMI will be useful, is anorexia nervosa. Anorexia nervosa often occurs during the adolescent period, and monitoring severity of the disease and its treatment will benefit from using the new puberty onset aligned references for height, weight, and BMI (36- 38). The tool that we present here for investigating BMI changes in new ways and with higher precision than previously possible will be of great utility in these situations. 14 14 Limitations with using BMI One of the limitations associated with using BMI as a measure of body composition in growing individuals is that the correlation between BMI (w/h2) and height has been shown to change with C-age, especially during adolescence. Similarly, other relationships between weight and height vary with age, see Figure 2 in Karlberg & Albertsson-Wikland, where a beta-factor was introduced (2). When developing the current QEPS-BMI model we therefore implicitly applied the nonlinear regression coefficients within the QEPS weight model to allow a similar correlation to be maintained across different age ranges. This indicates that the standard formula for calculating BMI is not optimal for describing weight status during childhood and puberty (2). Human weight represents the sum of fat, muscle, body water, visceral organs, and bones in an individual. Despite the common use of BMI as a measure of adiposity or thinness, BMI cannot differentiate between these different tissues. A further limitation of using BMI as a measure of adiposity is that, while BMI correlates with fat mass in children on a group level, there is not always a correlation at the individual level (39, 40). Conclusion We present new BMI references that can be used to evaluate prepubertal BMI in relation to chronological age, as well as assessing pubertal and prepubertal changes in BMI relative to the onset of the pubertal growth spurt in the individual. When used together with comparable references developed using the QEPS-models for height and weight (10, 18), these novel BMI references will facilitate improved detection and monitoring of growth abnormalities during adolescence within the health care system. Furthermore, in research settings these new references represent a novel tool for exploring human growth. The availability of this type of 15 reference is an important step towards more meaningful and informative clinical monitoring of BMI development of the individual during the adolescent years. Declarations: Ethical approval: Ethical approval was obtained from the Regional Ethics Review Board in Gothenburg (Ad 91-92/131 -93 and Ad 444-08 T062-09). Informed consent was given by participants and parents of individuals <18 years of age. Studies were conducted in accordance with the principles of the Declaration of Helsinki. Acknowledgements: The authors are grateful for the contributions of the students of the 12th grade 1998/9 from the Gothenburg area schools, and their school nurses; the study team led by C Pfeiffer-Mosesson and A Olsson; for Data support given by L Ljungberg and B Bengtsson and for careful editing and language revision by H Crofts. Research funding: The authors acknowledge financial support from the Governmental grants under the ALF agreement, ALFGBG-719041, ALFGBG-812951, ALFGBG-965451, the Region Halland Grants, and the Foundation Växthuset for children. Competing interest: AFMN works for Muvara, Multivariate Analysis of Research Data, Statistical Consultation, the Netherlands. KAW, AN, AH, and LG declare that they have no competing interests. 16 Availability of data and materials: the data generated and analyzed during the current study are not publicly available due to that these data are part of several ongoing studies and will be publicly available at a later point in time but are available from the corresponding author on reasonable request. Author contributions: KAW is the principal investigator of the study populations used. AFMN performed the modeling work for the QEPS model described here and made the QEPS function estimations, reference values, and Figures 1–6 and Supplemental Figures S1– S4. AN made Figure 7. KAW, AN, AH, LG, and AFMN have all made substantial contributions to the conception, design, analysis, and interpretation of these data. KAW wrote the first draft and AFMN the methods part. All authors revised the manuscript critically for intellectual content and approved the final version to be submitted for publication. Consent of publication: Not applicable. REFERENCES Holmgren A, Niklasson A, Gelander L, Aronson AS, Nierop AFM, Albertsson-Wikland K. Insight into human pubertal growth by applying the QEPS growth model. BMC pediatrics. 2017; 17:107 17. Albertsson-Wikland K, Niklasson A, Gelander L, Holmgren A, Nierop AFM. Novel type of references for weight aligned for onset of puberty - using the QEPS growth model. BMC pediatrics. 2021; 21:507 18. Albertsson-Wikland K, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new Swedish reference for total and prepubertal height. Acta Paediatr. 2020; 109:754-63 19. Rollof L, Elfving M. 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Worldwide trends in body-mass index, underweight, overweight, an obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement studies in 128.9 million children, adolescents, and adults. Lancet. 2017; 390:2627-42 7. Bygdell M, Celind J, Lilja L, Martikainen J, Simonson L, Sjogren L, et al. Prevalence of overweight and obesity from 5 to 19 years of age in Gothenburg, Sweden. Acta Paediatr. 2021; 110:3349-55 8. Cole TJ. The development of growth references and growth charts. Annals of human biolog 2012; 39:382-94 9. Tanner JM, Whitehouse RH, Takaishi M. Standards from birth to maturity for height, weigh height velocity, and weight velocity: British children, 1965. I. Archives of disease in childhood. 1966; 41:454-71 10. Albertsson-Wikland KG, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new type of pubertal height reference based on growth aligned for onset of pubertal growth. J Pediatr Endocrinol Metab. 2020; 33:1173-82 11. Karlberg J. On the modelling of human growth. Statistics in medicine. 1987; 6:185-92 12. Karlberg J, Fryer JG, Engstrom I, Karlberg P. Analysis of linear growth using a mathematical model. II. From 3 to 21 years of age. Acta paediatrica Scandinavica. 1987; 337:12-29 13. Albertsson-Wikland K, Luo ZC, Niklasson A, Karlberg J. Swedish population-based longitudinal reference values from birth to 18 years of age for height, weight and head circumference. Acta Paediatr. 2002; 91:739-54 14. Gelander L, Hagenäs L, Albertsson-Wikland K. Tillväxtkurvan i skolhälsovården. Tillväxt och kroppslig mognad 6-18 år. Manual och Lilla tillväxtskolan. Stockholm: Kommentus; 2003 15. Nierop AF, Niklasson A, Holmgren A, Gelander L, Rosberg S, Albertsson-Wikland K. Modelli individual longitudinal human growth from fetal to adult life - QEPS I. J Theor Biol. 2016; 406:143-65 16. REFERENCES Statistics in medicine. 1987; 6:185-92 12. Karlberg J, Fryer JG, Engstrom I, Karlberg P. Analysis of linear growth using a mathematical model. II. From 3 to 21 years of age. Acta paediatrica Scandinavica. 1987; 337:12-29 13. Albertsson-Wikland K, Luo ZC, Niklasson A, Karlberg J. Swedish population-based longitudinal reference values from birth to 18 years of age for height, weight and head circumference. Acta Paediatr. 2002; 91:739-54 14. Gelander L, Hagenäs L, Albertsson-Wikland K. Tillväxtkurvan i skolhälsovården. Tillväxt och kroppslig mognad 6-18 år. Manual och Lilla tillväxtskolan. Stockholm: Kommentus; 2003 15. Nierop AF, Niklasson A, Holmgren A, Gelander L, Rosberg S, Albertsson-Wikland K. Modelling individual longitudinal human growth from fetal to adult life - QEPS I. J Theor Biol. 2016; 406:143-65 16. Holmgren A, Niklasson A, Gelander L, Aronson AS, Nierop AFM, Albertsson-Wikland K. Insight into human pubertal growth by applying the QEPS growth model. BMC pediatrics. 2017; 17:107 17. Albertsson-Wikland K, Niklasson A, Gelander L, Holmgren A, Nierop AFM. Novel type of references for weight aligned for onset of puberty - using the QEPS growth model. BMC pediatrics. 2021; 21:507 18. Albertsson-Wikland K, Niklasson A, Holmgren A, Gelander L, Nierop AFM. A new Swedish reference for total and prepubertal height. Acta Paediatr. 2020; 109:754-63 19. Rollof L, Elfving M. Evaluation of self-assessment of pubertal maturation in boys and girls using drawings and orchidometer. J Pediatr Endocrinol Metab. 2012; 25:125-9 20. Vizmanos B, Marti-Henneberg C. Puberty begins with a characteristic subcutaneous body fat mass in each sex. European journal of clinical nutrition. 2000; 54:203-8 18 21. Karlsson AK, Kullberg J, Stokland E, Allvin K, Gronowitz E, Svensson PA, et al. Measurements of total and regional body composition in preschool children: A comparison of MRI, DXA, and anthropometric data. Obesity (Silver Spring). 2013; 21:1018-24 22. Backman G. Gewichtswachstum des Mannes. Wilhelm Roux' Archiv für Entwicklungsmechanik der Organismen 1940; 140:285–314 23. Hochberg Z, Albertsson-Wikland K. Evo-Devo of Infantile and Childhood Growth. Pediatric research. 2008; 64:2-7 24. Hochberg Z. Evo-Devo of Child Growth: Treatise on Child Growth and Human Evolution, First Edition.: Wiley-Blackwell, Publishers; 2012 25. Holmgren A, Niklasson A, Nierop AF, Gelander L, Aronson AS, Sjoberg A, et al. Pubertal height gain is inversely related to peak BMI in childhood. Pediatric research. 2017; 26. Kindblom JM, Bygdell M, Sonden A, Celind J, Rosengren A, Ohlsson C. BMI change during puberty and the risk of heart failure. Journal of internal medicine. 2018; 283:558-67 27. REFERENCES Celind J, Ohlsson C, Bygdell M, Nethander M, Kindblom JM. Childhood Body Mass Index Is Associated with Risk of Adult Colon Cancer in Men: An Association Modulated by Pubertal Change in Body Mass Index. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2019; 28:974-9 28. Ohlsson C, Bygdell M, Nethander M, Rosengren A, Kindblom JM. BMI Change During Puberty Is an Important Determinant of Adult Type 2 Diabetes Risk in Men. J Clin Endocrinol Metab. 2019; 104:1823-32 29. Barker DJ, Osmond C, Kajantie E, Eriksson JG. Growth and chronic disease: findings in the Helsinki Birth Cohort. Annals of human biology. 2009; 36:445-58 30. Eriksson JG, Forsen TJ, Kajantie E, Osmond C, Barker DJ. Childhood growth and hypertension in later life. Hypertension. 2007; 49:1415-21 31. Forsdahl A. Living conditions in childhood and subsequent development of risk factors for arteriosclerotic heart disease. The cardiovascular survey in Finnmark 1974-75. Journal of epidemiology and community health. 1978; 32:34-7 32. Roseboom T, de Rooij S, Painter R. The Dutch famine and its long-term consequences for adult health. Early human development. 2006; 82:485-91 h h f ' h ld d 33. Janson A. The growth of nations' children. Acta Paediatr. 2021; 110:3151-2 34. Hagman E, Danielsson P, Lindberg L, Marcus C, Committee BS. Paediatric obesity treatment during 14 years in Sweden: Lessons from the Swedish Childhood Obesity Treatment Register- BORIS. Pediatric obesity. 2020; 15:e12626 35. Olbers T, Beamish AJ, Gronowitz E, Flodmark CE, Dahlgren J, Bruze G, et al. Laparoscopic Roux-en-Y gastric bypass in adolescents with severe obesity (AMOS): a prospective, 5-year, Swedish nationwide study. Lancet Diabetes Endocrinol. 2017; 5:174-83 36. Modan-Moses D, Yaroslavsky A, Pinhas-Hamiel O, Levy-Shraga Y, Kochavi B, Iron-Segev S, et al. Prospective Longitudinal Assessment of Linear Growth and Adult Height in Female Adolescents With Anorexia Nervosa. J Clin Endocrinol Metab. 2021; 106:e1-e10 37. Leger J, Fjellestad-Paulsen A, Bargiacchi A, Pages J, Chevenne D, Alison M, et al. One Year of GH Treatment for Growth Failure in Children With Anorexia Nervosa: A Randomized 38. Berkowitz SA, Witt AA, Gillberg C, Rastam M, Wentz E, Lowe MR. Childhood body mass index in adolescent-onset anorexia nervosa. Int J Eat Disord. 2016; 49:1002-9 39. Mei Z, Grummer-Strawn LM, Pietrobelli A, Goulding A, Goran MI, Dietz WH. 40. Freedman DS, Wang J, Maynard LM, Thornton JC, Mei Z, Pierson RN, et al. Relation of BMI to fat and fat-free mass among children and adolescents. Int J Obes (Lond). 2005; 29:1-8 Freedman DS, Wang J, Maynard LM, Thornton JC, Mei Z, Pierson RN, et al. Relation of BMI t fat and fat-free mass among children and adolescents. Int J Obes (Lond). 2005; 29:1-8 REFERENCES Validity of body mass index compared with other body-composition screening indexes for the assessment of body fatness in children and adolescents. The American journal of clinical nutrition. 2002; 75:978-85 19 40. 20 Figure 1: Chronological age reference for total BMI and prepubertal BMI (QE-function)) for girls (left) and boys (right) aged 4–20 years. Median total BMI (red/blue solid line, ±1SDS (red/blue dotted line)), and ±2SDS (red/blue dashed line) and median prepubertal BMI (black). Figure 1: Chronological age reference for total BMI and prepubertal BMI (QE-function)) for girls (left) and boys (right) aged 4–20 years. Median total BMI (red/blue solid line, ±1SDS (red/blue dotted line)), and ±2SDS (red/blue dashed line) and median prepubertal BMI (black). 21 21 Figure 2 upper panel: References in kg/m2 (left axis) and in kg0.5/m (right axis) for total BMI and the basic BMI (BMI gained independently of puberty (QES-function) for girls (red, left) and boys (blue, right). Curves are aligned for age at onset of pubertal height spurt (AgeP5). Median total BMI (red/blue solid line, ±1SDS (red/blue dotted line), and ±2SDS (red/blue dashed line)) and median basic BMI (black). General differences in timing between pubertal height and weight functions result in an undulation of aligned BMI functions. Figure 2 upper panel: References in kg/m2 (left axis) and in kg0.5/m (right axis) for total BMI and the basic BMI (BMI gained independently of puberty (QES-function) for girls (red, left) and boys (blue, right). Curves are aligned for age at onset of pubertal height spurt (AgeP5). Median total BMI (red/blue solid line, ±1SDS (red/blue dotted line), and ±2SDS (red/blue dashed line)) and median basic BMI (black). General differences in timing between pubertal height and weight functions result in an undulation of aligned BMI functions. Figure 2 upper panel: References in kg/m2 (left axis) and in kg0.5/m (right axis) for total BMI and the basic BMI (BMI gained independently of puberty (QES-function) for girls (red, left) and boys (blue, right). Curves are aligned for age at onset of pubertal height spurt (AgeP5). Median total BMI (red/blue solid line, ±1SDS (red/blue dotted line), and ±2SDS (red/blue dashed line)) and median basic BMI (black). General differences in timing between pubertal height and weight functions result in an undulation of aligned BMI functions. 22 Figure 2 lower panel: BMI gain resulting from the P-function of the QEPS (P-BMI) provides a puberty-specific BMI reference. REFERENCES Specific P-function-derived BMI as median, ±1SDS (dotted line), and ±2SDS (dashed line). The individual onset of puberty was identified and aligned based on the age at which 5% (AgeP5) of the total specific P-function growth (Pmax) for height had occurred. General differences in timing between pubertal height and weight functions result in an undulation of aligned BMI functions. Figure 2 lower panel: BMI gain resulting from the P-function of the QEPS (P-BMI) provides a puberty-specific BMI reference. Specific P-function-derived BMI as median, ±1SDS (dotted line), and ±2SDS (dashed line). The individual onset of puberty was identified and aligned based on the age at which 5% (AgeP5) of the total specific P-function growth (Pmax) for height had occurred. General differences in timing between pubertal height and weight functions result in an undulation of aligned BMI functions. 23 23 23 Figure 3: Median total BMI (in kg/m2, top; in SDS with 95% CI, bottom) according to chronological age for girls (left) and boys (right) from the GrowUp1974 Gothenburg cohort grouped according to onset of puberty: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― -). Data are visualized alongside the reference for median total BMI (thick solid lines) and ±2SDS (dotted red (left) and blue (right) lines). Figure 3: Median total BMI (in kg/m2, top; in SDS with 95% CI, bottom) according to chronological age for girls (left) and boys (right) from the GrowUp1974 Gothenburg cohort grouped according to onset of puberty: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― -). Data are visualized alongside the reference for median total BMI (thick solid lines) and ±2SDS (dotted red (left) and blue (right) lines). 24 igure 4. Median total, basic, and puberty-specific BMI gain (in kg/m2, top; in SDS with 95% CI bottom) relative to onset of puberty for girls (red at the top) and boys (blue at the bottom) grouped according to pubertal onset: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― - ). Median total BMI (QEPS-functions, left panels), basic BMI growth (QES-functions, middle panels), and specific pubertal BMI growth (P-function, right panels) from the GrowUp1974Gothenburg cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the new reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). REFERENCES bottom) relative to onset of puberty for girls (red at the top) and boys (blue at the bottom) grouped according to pubertal onset: early, <−1.5yrs (- - -), average, ±0.25yrs (• • •), and late, >+1.5yrs (- ― - ). Median total BMI (QEPS-functions, left panels), basic BMI growth (QES-functions, middle panels), and specific pubertal BMI growth (P-function, right panels) from the GrowUp1974Gothenburg cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the new reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). 25 Figure 5: Median total, basic, and puberty-specific BMI gain (in kg/m2, top; in SDS with 95% CI, bottom) relative to the onset of puberty for girls (red at the top) and boys (blue at the bottom) according to height at the onset of puberty: tall, >+1.5 SDS (- ― -) and short, <−1.5 SDS (- - -). Median total BMI (QEPS-functions, left panels), basic BMI growth (QES-functions, middle panels), and specific pubertal BMI growth (P-function, right panels) from the GrowUp1974Gothenburg cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the new reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). 26 Figure 6: Total, basic, and puberty-specific BMI gain (in kg/m2, top; in SDS with 95%CI, bottom) relative to the onset of puberty for girls (red at the top) and boys (blue at the bottom) according to BMI in childhood: high BMI, >+1.5SDS (- ― -) and low BMI, <−1.5SDS (- - -). Median total BMI (QEPS-functions, left panels), basic growth (QES-functions, middle panels), and specific pubertal growth (P-function, right panels) in girls and boys from the GrowUp1974Gothenburg cohort. Curves are aligned for age at onset of pubertal growth spurt. Data are visualized alongside the new reference for median total BMI (thick red or blue solid lines) and ±2SDS (red/blue dotted lines). 27 Figure 7: Guide for prospective use of the pubertal-age-adjusted reference for total BMI (kg/m2 and kg0.5/m) and total height (cm) for girls (♀) and boys (♂). Figure 7: Guide for prospective use of the pubertal-age-adjusted reference for total BMI (kg/m2 and kg0.5/m) and total height (cm) for girls (♀) and boys (♂). REFERENCES Figure 7: Guide for prospective use of the pubertal-age-adjusted reference for total BMI (kg/m2 and kg0.5/m) and total height (cm) for girls (♀) and boys (♂). Individual age adjustment is made using only the height measurement at the chronological age (C- age) at which puberty started (see upper left traditional C-age-reference for girls and boys) (14), by using the height increase from the individual prepubertal growth curve (as drawn in upper left panel) through the individual measuring points. This measurement is usually corresponding to the time when secondary sex characteristics develop; early breast development stage, B2 in girls (red, upper left panel), or testicular volume increase to 4-6 ml in boys (blue, upper left panel). Individual growth curves are shown for a girl aged 8 years and a boy aged 13-years at the start of puberty (15). Height and BMI according to C-age and P-age, ie the age adjusted to reflect the start of puberty, are shown for an 8-year-old girl and a 13-year-old boy (15). Height (cm) and BMI (in kg/m2 or kg0.5/m) according to C-age at the onset of puberty, and to P-age, after adjustment for age at onset of the pubertal growth spurt, are depicted as red dots (all panels), based only on age in the height references for each sex, respectively. Corresponding heights and BMIs are then moved to puberty- adjusted age = zero, in the P-age-references shown in the lower panels (left panels for height and right for BMI). Thereafter, all measured heights and BMIs are depicted at ages/times recalculated in relation to the specific onset of puberty in that individual, labelled on the x-axis as ‘Time from onset of growth spurt (years)’. Thus, changes in BMI and height in the years preceding the pubertal growth spurt can be evaluated using the novel references. Note, the BMI increase precedes the height increase in relation to puberty. For corresponding values for weight, see Figure 7 in Albertsson-Wikland et al. (17). 28 Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. 20220107aBMIpubSuppl.pdf
https://openalex.org/W4375921168
https://zenodo.org/record/3365363/files/cva-solution-2.pdf
English
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An Accurate Solution for Credit Valuation Adjustment (CVA) and Wrong Way Risk
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1 Email: tim_yxiao@yahoo.com Url: https://finpricing.com/ Tim Xiao1 Tim Xiao1 Journal of Fixed Income, 25(1) 84-95 Summer 2015 ABSTRACT This paper presents a Least Square Monte Carlo approach for accurately calculating credit value adjustment (CVA). In contrast to previous studies, the model relies on the probability distribution of a default time/jump rather than the default time itself, as the default time is usually inaccessible. As such, the model can achieve a high order of accuracy with a relatively easy implementation. We find that the valuation of a defaultable derivative is normally determined via backward induction when their payoffs could be positive or negative. Moreover, the model can naturally capture wrong or right way risk. Key Words: credit value adjustment (CVA), wrong way risk, right way risk, credit risk modeling, least square Monte Carlo, default time approach (DTA), default probability approach (DPA), collateralization, margin and netting. 1 1 For years, a widespread practice in the industry has been to mark derivative portfolios to market without taking counterparty risk into account. All cash flows are discounted using the LIBOR curve. But the real parties, in many cases, happen to be of lower credit quality than the hypothetical LIBOR party and have a chance of default. As a consequence, the International Accounting Standard (IAS) 39 requires banks to provide a fair-value adjustment due to counterparty risk. Although credit value adjustment (CVA) became mandatory in 2000, it received a little attention until the recent financial crises in which the profit and loss (P&L) swings due to CVA changes were measured in billons of dollars. Interest in CVA began to grow. Now CVA has become the first line of defense and the central part of counterparty risk management. CVA not only allows institutions to move beyond the traditional control mindset of credit risk limits and to quantify counterparty risk as a single measurable P&L number, but also offers an opportunity for banks to dynamically manage, price and hedge counterparty risk. The benefits of CVA are widely acknowledged. Many banks have set up internal credit risk trading desks to manage counterparty risk on derivatives. The earlier works on CVA are mainly focused on unilateral CVA that assumes that only one counterparty is defaultable and the other one is default-free. The unilateral treatment neglects the fact that both counterparties may default, i.e., counterparty risk can be bilateral. A trend that has become increasingly relevant and popular has been to consider the bilateral nature of counterparty credit risk. ABSTRACT Although most institutions view bilateral considerations as important in order to agree on new transactions, Hull and White (2013) argue that bilateral CVA is more controversial than unilateral CVA as the possibility that a dealer might default is in theory a benefit to the dealer. 2 CVA, by definition, is the difference between the risk-free portfolio value and the true (or risky or defaultable) portfolio value that takes into account the possibility of a counterparty’s default. The risk-free portfolio value is what brokers quote or what trading systems or models 2 2 normally report. The risky portfolio value, however, is a relatively less explored and less transparent area, which is the main challenge and core theme for CVA. In other words, central to CVA is risky valuation. In general, risky valuation can be classified into two categories: the default time approach (DTA) and the default probability approach (DPA). The DTA involves the default time explicitly. Most CVA models in the literature (Brigo and Capponi (2008), Lipton and Sepp (2009), Pykhtin and Zhu (2006) and Gregory (2009), etc.) are based on this approach. Although the DTA is very intuitive, it has the disadvantage that it explicitly involves the default time. We are very unlikely to have complete information about a firm’s default point, which is often inaccessible (see Duffie and Huang (1996), Jarrow and Protter (2004), etc.). Usually, valuation under the DTA is performed via Monte Carlo simulation. On the other hand, however, the DPA relies on the probability distribution of the default time rather than the default time itself. Sometimes the DPA yields simple closed form solutions. The current popular CVA methodology (Pykhtin and Zhu (2006) and Gregory (2009), etc.) is first derived using DTA and then discretized over a time grid in order to yield a feasible solution. The discretization, however, is inaccurate. In fact, this model has never been rigorously proved. Since CVA is used for financial accounting and pricing, its accuracy is essential. Moreover, this current model is based on a well-known assumption, in which credit exposure and counterparty’s credit quality are independent. Obviously, it can not capture wrong/right way risk properly. In this paper, we present a framework for risky valuation and CVA. In contrast to previous studies, the model relies on the DPA rather than the DTA. ABSTRACT Our study shows that the pricing process of a defaultable contract normally has a backward recursive nature if its payoff could be positive or negative. 3 An intuitive way of understanding these backward recursive behaviours is that we can think of that any contingent claim embeds two default options. In other words, when entering an 3 3 OTC derivatives transaction, one party grants the other party an option to default and, at the same time, also receives an option to default itself. In theory, default may occur at any time. Therefore, the default options are American style options that normally require a backward induction valuation. Wrong way risk occurs when exposure to a counterparty is adversely correlated with the credit quality of that counterparty, while right way risk occurs when exposure to a counterparty is positively correlated with the credit quality of that counterparty. For example, in wrong way risk exposure tends to increase when counterparty credit quality worsens, while in right way risk exposure tends to decrease when counterparty credit quality declines. Wrong/right way risk, as an additional source of risk, is rightly of concern to banks and regulators. Since this new model allows us to incorporate correlated and potentially simultaneous defaults into risky valuation, it can naturally capture wrong/right way risk. The rest of this paper is organized as follows: Section 2 discusses unilateral risky valuation and unilateral CVA. Section 2 elaborates bilateral risky valuation and bilateral CVA. Section 3 presents numerical results. The conclusions are given in Section 4. . All proofs and a practical framework that embraces netting agreements, margining agreements and wrong/right way risk are contained in the appendices. 1. Unilateral Risky Valuation and Unilateral CVA We consider a filtered probability space ( , F ,  0  t t F , P ) satisfying the usual conditions, where  denotes a sample space; F denotes a -algebra; P denotes a probability measure;  0  t t F denotes a filtration. The default model is based on the reduced-form approach proposed by Duffie and Singleton (1999) and Jarrow and Turnbell (1994), which does not explain the event of default endogenously, but characterizes it exogenously by a jump process. The stopping (or default) time 4 4  of a firm is modeled as a Cox arrival process (also known as a doubly stochastic Poisson process) whose first jump occurs at default and is defined as,  of a firm is modeled as a Cox arrival process (also known as a doubly stochastic Poisson process) whose first jump occurs at default and is defined as,        t s ds s h t 0 ) , ( : inf  (1) (1) where ) (t h or ) , ( t t h  denotes the stochastic hazard rate or arrival intensity dependent on an exogenous common state t, and  is a unit exponential random variable independent of t . where ) (t h or ) , ( t t h  denotes the stochastic hazard rate or arrival intensity dependent on an exogenous common state t, and  is a unit exponential random variable independent of t . It is well-known that the survival probability from time t to s in this framework is defined It is well-known that the survival probability from time t to s in this framework is defined by by          s t du u h Z t s P s t p ) ( exp ) , | ( :) , (   (2a) (2a) The default probability for the period (t, s) in this framework is defined by             s t du u h s t p Z t s P s t q ) ( exp 1 ) , ( 1 ) , | ( :) , (   (2b) (2b) Two counterparties are denoted as A and B. Let valuation date be t. 2 Here we use the recovery of market value (RMV) assumption. 1. Unilateral Risky Valuation and Unilateral CVA Under a risk-neutral measure, the value of this defaultable contract is the discounted expectation of all the payoffs and is given by     t T T T V t D X T t D E t V F | 1) ( ) , ( 1 ) , ( ) (         (4)     t T T T V t D X T t D E t V F | 1) ( ) , ( 1 ) , ( ) (         (4) (4) where Y is an indicator function that is equal to one if Y is true and zero otherwise. n indicator function that is equal to one if Y is true and zero otherwise. where Y is an indicator function that is equal to one if Y is true and zero otherwise. Although the DTA is very intuitive, it has the disadvantage that it explicitly involves the default time/jump. We are very unlikely to have complete information about a firm’s default point, which is often inaccessible. Usually, valuation under the DTA is performed via Monte Carlo simulation. The DPA relies on the probability distribution of the default time rather than the default time itself. We divide the time period (t, T) into n very small time intervals ( t ) and assume that a default may occur only at the end of each very small period. In our derivation, we use the approximation  y y  1 exp for very small y. The survival and the default probabilities for the period ( t , t t   ) are given by   t t h t t h t t t p t p          ) ( 1 ) ( exp ) , ( :) (ˆ (5a)   t t h t t h t t t q t q          ) ( ) ( exp 1 ) , ( :) (ˆ (5b) (5a) (5b) The binomial default rule considers only two possible states: default or survival. 1. Unilateral Risky Valuation and Unilateral CVA Consider a financial contract that promises to pay a 0  T X from party B to party A at maturity date T, and nothing before date T. All calculations in the paper are from the perspective of party A. The risk free value of the financial contract is given by   t F T F X T t D E t V ) , ( ) (  (3a)   t F T F X T t D E t V ) , ( ) (  (3a) where       du u r T t D T t ) ( exp ) , ( (3b) (3a) where       du u r T t D T t ) ( exp ) , ( (3b)       du u r T t D T t ) ( exp ) , ( (3b) (3b) where   t E F  denotes the expectation conditional on the t F , ) , ( T t D denotes the risk-free discount factor at time t for the maturity T and ) (u r denotes the risk-free short rate at time u ( T u t   ). Next, we turn to risky valuation. In a unilateral credit risk case, we assume that party A is default-free and party B is defaultable. Risky valuation can be generally classified into two 5 5 categories: the default time approach (DTA) and the default probability (intensity) approach (DPA). The DTA involves the default time explicitly. If there has been no default before time T (i.e., T   ), the value of the contract at T is the payoff T X . If a default happens before T (i.e., T t   ), a recovery payoff is made at the default time  as a fraction of the market value2 given by ) ( V where  is the default recovery rate and ) ( V is the market value at default. 1. Unilateral Risky Valuation and Unilateral CVA          t t t t V t t y E t t V t q t t p t t r E t V F F ) ( ) ( exp ) ( ) (ˆ ) ( ) (ˆ ) ( exp ) (             (6) where   ) ( ) ( ) ( 1 ) ( ) ( ) ( t c t r t t h t r t y       denotes the risky rate and  ) ( 1 ) ( ) ( t t h t c    is called the (short) credit spread. (6) Similarly, we have Similarly, we have     t t t t V t t t y E t t V            F ) 2 ( ) ( exp ) ( (7) (7) Note that  t t y   ) ( exp is t t F   -measurable. By definition, an t t F   -measurable random variable is a random variable whose value is known at time t t   . 1. Unilateral Risky Valuation and Unilateral CVA For the one-period ) , ( t t t   economy, at time t t   the asset either defaults with the default 2 Here we use the recovery of market value (RMV) assumption. 6 probability ) , ( t t t q   or survives with the survival probability ) , ( t t t p   . The survival payoff is equal to the market value ) ( t t V   and the default payoff is a fraction of the market value: ) ( ) ( t t V t t      . Under a risk-neutral measure, the value of the asset at t is the expectation of all the payoffs discounted at the risk-free rate and is given by          t t t t V t t y E t t V t q t t p t t r E t V F F ) ( ) ( exp ) ( ) (ˆ ) ( ) (ˆ ) ( exp ) (             (6) where   ) ( ) ( ) ( 1 ) ( ) ( ) ( t c t r t t h t r t y       denotes the risky rate and  ) ( 1 ) ( ) ( t t h t c    is called the (short) credit spread.          t t t t V t t y E t t V t q t t p t t r E t V F F ) ( ) ( exp ) ( ) (ˆ ) ( ) (ˆ ) ( exp ) (             (6) where   ) ( ) ( ) ( 1 ) ( ) ( ) ( t c t r t t h t r t y       denotes the risky rate and  ) ( 1 ) ( ) ( t t h t c    is called the (short) credit spread. 1. Unilateral Risky Valuation and Unilateral CVA Based on the taking out what is known and tower properties of conditional expectation, we have                 t i t t t t t t V t t i t y E t t V t t t y E t t y E t t V t t y E t V F F F F ) 2 ( ) ) ( exp ) 2 ( ) ( exp ) ( exp ) ( ) ( exp ) ( 1 0                         (8) (8) By recursively deriving from t forward over T and taking the limit as t  approaches zero, the risky value of the asset can be expressed as         t T t T V du u y E t V F ) ( ) ( exp ) ( (9) (9) We may think of ) (u y as the risk-adjusted short rate. Equation (9) is the same as Equation (10) in Duffie and Singleton [1999], which is the market model for pricing risky bonds. Using the DPA, we obtain a closed-form solution for pricing an asset subject to credit risk. Other good examples of the DPA are the CDS model proposed by J.P. Morgan (1999) and a more generic risky model presented by Xiao (2013a). 7 7 In theory, a default may happen at any time, i.e., a risky contract is continuously defaultable. This Continuous Time Risky Valuation Model is accurate but sometimes complex and expensive. For simplicity, people sometimes prefer the Discrete Time Risky Valuation Model that assumes that a default may only happen at some discrete times. A natural selection is to assume that a default may occur only on the payment dates. Fortunately, the level of accuracy for this discrete approximation is well inside the typical bid-ask spread for most applications (see O’Kane and Turnbull (2003)). From now on, we will focus on the discrete setting only, but many of the points we make are equally applicable to the continuous setting. For a derivative contract, usually its payoff may be either an asset or a liability to each party. 1. Unilateral Risky Valuation and Unilateral CVA Proposition 2: The unilateral risky value of the multiple-payment contract is given by Proposition 2: The unilateral risky value of the multiple-payment contract is given by Proposition 2: The unilateral risky value of the multiple-payment contract is given by            m i t i i j j j X T T F E t V 1 1 0 1) , ( ) ( F (11a) (11a) where 0T t  and     ) ( 1 ) , ( 1 1 ) , ( ) , ( 1 1 0 )) ( ( 1 1 1 1            j j j T V X j j j j T T T q T T D T T F j j  (11b)     ) ( 1 ) , ( 1 1 ) , ( ) , ( 1 1 0 )) ( ( 1 1 1 1            j j j T V X j j j j T T T q T T D T T F j j  (11b) e appendix (11b) Proof: See the appendix. Proof: See the appendix. The risky valuation in Proposition 2 has a backward nature. The intermediate values are vital to determine the final price. For a discrete time interval, the current risky value has a dependence on the future risky value. Only on the final payment date m T , the value of the contract and the maximum amount of information needed to determine the risk-adjusted discount factor are revealed. The coupled valuation behavior allows us to capture wrong/right way risk properly where counterparty credit quality and market prices may be correlated. This type of problem can be best solved by working backwards in time, with the later risky value feeding into the earlier ones, so that the process builds on itself in a recursive fashion, which is referred to as backward induction. The most popular backward induction valuation algorithms are lattice/tree and least square Monte Carlo. For an intuitive explanation, we can posit that a defaultable contract under the unilateral credit risk assumption has an embedded default option (see Sorensen and Bollier (1994)). 1. Unilateral Risky Valuation and Unilateral CVA Thus, we further relax the assumption and suppose that T X may be positive or negative. In the case of 0  T X , the survival value is equal to the payoff T X and the default payoff is a fraction of the payoff T X  . Whereas in the case of 0  T X , the contract value is the payoff itself, because the default risk of party B is irrelevant for unilateral risky valuation in this case. Therefore, we have Proposition 1: The unilateral risky value of the single-payment contract in a discrete-time setting is given by Proposition 1: The unilateral risky value of the single-payment contract in a discrete-time setting is given by   t F T X T t F E t V ) , ( ) (  (10a) (10a) where     ) ( 1 ) , ( 1 1 ) , ( ) , ( 0 T T t q T t D T t F T X      (10b) (10b) Proof: See the appendix. Here ) , ( T t F can be regarded as a risk-adjusted discount factor. Proposition 1 says that the unilateral risky valuation of the single payoff contract has a dependence on the sign of the payoff. If the payoff is positive, the risky value is equal to the risk-free value minus the discounted potential loss. Otherwise, the risky value is equal to the risk-free value. 8 8 Proposition 1 can be easily extended from one-period to multiple-periods. Suppose that a defaultable contract has m cash flows. Let the m cash flows be represented as 1 X ,…, m X with payment dates 1T ,…, m T . Each cash flow may be positive or negative. We have the following proposition. 1. Unilateral Risky Valuation and Unilateral CVA In other words, one party entering a defaultable financial transaction actually grants the other party an option to default. If we assume that a default may occur at any time, the default option is an 9 American style option. American options normally have backward recursive natures and require backward induction valuations. The similarity between American style financial options and American style default options is that both require a backward recursive valuation procedure. The difference between them is in the optimal strategy. The American financial option seeks an optimal value by comparing the exercise value with the continuation value, whereas the American default option seeks an optimal discount factor based on the option value in time. The unilateral CVA, by definition, can be expressed as The unilateral CVA, by definition, can be expressed as               m i t i i j j j i F X T T F T t D E t V t V t CVA 1 1 0 1) , ( ) , ( ) ( ) ( ) ( F (12) (12) Proposition 2 provides a general form for pricing a unilateral defaultable contract. Applying it to a particular situation in which we assume that all the payoffs are nonnegative, we derive the following corollary: Corollary 1: If all the payoffs are nonnegative, the risky value of the multiple-payments contract is given by            m i t i i j j j X T T F E t V 1 1 0 1) , ( ) ( F (13a) (13a) where 0T t  and where 0T t  and     ) ( 1 ) , ( 1 ) , ( ) , ( 1 1 1 1        j j j j j j j T T T q T T D T T F  (13b) (13b) The proof of this corollary is easily obtained according to Proposition 2 by setting  0 ) ( 1 1     j j T V X , since the value of the contract at any time is also nonnegative. 1. Unilateral Risky Valuation and Unilateral CVA e CVA in this case is given by The CVA in this case is given by The CVA in this case is given by                    m i t i i j j j j i F X T T T q T t D E t V t V t CVA 1 1 0 1 1 )) ( 1 )( , ( 1 1 ) , ( ) ( ) ( ) ( F  (14) (14) The current popular CVA model (e.g., equation (17) in Pykhtin and Zhu (2007) and equation (3) in Gregory (2009)) is quite different from above either equation (12) or equation (14). As a matter of fact, the current CVA model has never been rigorously proved. In order to reflect 10 the economic value of counterparty credit risk, to measure the profit and loss of a bank and to provide proper incentives to traders, a good CVA model must be not only rigorous and accurate but also feasible to implement. 2. Bilateral Risky Valuation and Bilateral CVA There is ample evidence that corporate defaults are correlated. The default of a firm’s counterparty might affect its own default probability. Thus, default correlation and dependence arise due to the counterparty relations. Default correlation can be positive or negative. The effect of positive correlation is usually called contagion, whereas the latter is referred to as competition effect. Two counterparties are denoted as A and B. The binomial default rule considers only two possible states: default or survival. Therefore, the default indicator j Y for party j (j=A, B) follows a Bernoulli distribution, which takes value 1 with default probability j q and value 0 with survival probability j p , i.e., j j p Y P  } 0 { and j j q Y P  } 1 { . The marginal default distributions can be determined by the reduced-form models. The joint distributions of a bivariate Bernoulli variable can be easily obtained via the marginal distributions by introducing extra correlations. Consider a pair of random variables ( A Y , B Y ) that has a bivariate Bernoulli distribution. The joint probability representations are given by Consider a pair of random variables ( A Y , B Y ) that has a bivariate Bernoulli distribution. 3 There are two default settlement rules in the market. The one-way payment rule was specified by the early ISDA master agreement. The non-defaulting party is not obligated to compensate the defaulting party if the remaining market value of the instrument is positive for the defaulting party. The two-way payment rule is based on current ISDA documentation. The non-defaulting party will pay the full market value of the instrument to the defaulting party if the contract has positive value to the defaulting party. 2. Bilateral Risky Valuation and Bilateral CVA The joint probability representations are given by (15a) AB B A B A p p Y Y P p       ) 0 ,0 ( : 00 (15a) AB B A B A q p Y Y P p       )1 ,0 ( : 01 (15b) AB B A B A p q Y Y P p       ) 0 ,1 ( : 10 (15c) AB B A B A q q Y Y P p       )1 ,1 ( : 11 (15d) 2   Y Y E ) )( ( h (15b) (15d) e j j q Y E  ) ( , j j j q p  2  ,   B B A A AB B A AB B B A A AB p q p q q Y q Y E           ) )( ( : where  where j j q Y E  ) ( , j j j q p  2  ,   B B A A AB B A AB B B A A AB p q p q q Y q Y E           ) )( ( : where AB  denotes the default correlation coefficient and AB  denotes the default covariance. 11 Table 1. Payoffs of a bilaterally defaultable contract This table displays all possible payoffs at time T. In the case of 0  T X , there are a total of four possible states at time T: i) Both A and B survive with probability 00 p . The contract value is equal to the payoff T X . ii) A defaults but B survives with probability 10 p . The contract value is T BX  , where B  represents the non-default recovery rate 3. B  =0 represents the one-way settlement rule, while B =1 represents the two-way settlement rule. iii) A survives but B defaults with probability 01 p . The contract value is T BX  , where B  represents the default recovery rate. iv) Both A and B default with probability 11 p . The contract value is T ABX  , where AB  denotes the joint recovery rate when both parties A and B default simultaneously. A similar logic applies to the case of 0  T X . State 0 ,0   B A Y Y 0 ,1   B A Y Y 1 ,0   B A Y Y 1 ,1   B A Y Y Comments A & B survive A defaults, B survives A survives, B defaults A & B default Probability 00 p 10 p 01 p 11 p Payoff 0  T X T X T BX  T BX  T ABX  0  T X T X T AX  T AX  T ABX  12 12 Suppose that a financial contract that promises to pay a T X from party B to party A at maturity date T, and nothing before date T where t T . The payoff T X may be positive or negative, i.e. the contract may be either an asset or a liability to each party. All calculations are from the perspective of party A. At time T, there are a total of four ( 4 22  ) possible states shown in Table 1. The risky value of the contract is the discounted expectation of the payoffs and is given by the following proposition. Table 1. Payoffs of a bilaterally defaultable contract oposition 3: The bilateral risky value of the single-payment contract is given by oposition 3: The bilateral risky value of the single-payment contract is given by       t t F F T A X B X T X T t k T t k T t D E X T t K E t V T T ) , ( 1 ) , ( 1 ) , ( ) , ( ) ( 0 0      (16a)       t t F F T A X B X T X T t k T t k T t D E X T t K E t V T T ) , ( 1 ) , ( 1 ) , ( ) , ( ) ( 0 0      (16a) where       t t F F T A X B X T X T t k T t k T t D E X T t K E t V T T ) , ( 1 ) , ( 1 ) , ( ) , ( ) ( 0 0      (16a) where (16a) where  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB B B AB A B AB A B B A B B A B B                (16b)  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB A A AB A B AB B A A B A A A B A                (16c)  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB B B AB A B AB A B B A B B A B B                (16b) (16b)  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB A A AB A B AB B A A B A A A B A                (16c)  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB A A AB A B AB B A A B A A A B A                (16c) (16c) Proof: See the appendix. Table 1. Payoffs of a bilaterally defaultable contract The bilateral CVA of the multiple-payment contract can be expressed as                   m i t i i j j j t i i F X T T K E X T t D E t V t V t CVA 1 1 0 1) , ( ) , ( ) ( ) ( ) ( F F (18)                   m i t i i j j j t i i F X T T K E X T t D E t V t V t CVA 1 1 0 1) , ( ) , ( ) ( ) ( ) ( F F (18)                   m i t i i j j j t i i F X T T K E X T t D E t V t V t CVA 1 1 0 1) , ( ) , ( ) ( ) ( ) ( F F (18) (18) 3. Numerical Results Table 1. Payoffs of a bilaterally defaultable contract  ) , ( 1 ) , ( 1 ) , ( ) , ( 1 0 )) ( ( 1 0 )) ( ( 1 1 1 1 1 1               j j A T V X j j B T V X j j j j T T k T T k T T D T T K j j j j (17b) (17b) where ) , ( 1  j j A T T k and ) , ( 1  j j B T T k are defined in Proposition 3. Proof: The proof is similar to Proposition 2 by replacing ) , ( 1  j j T T F with ) , ( 1  j j T T K . Proposition 4 says that the pricing process of a multiple-payment contract has a backward nature since there is no way of knowing which risk-adjusted discounting rate should be used without knowledge of the future value. Only on the maturity date, the value of the contract and the decision strategy are clear. Therefore, the evaluation must be done in a backward fashion, working from the final payment date towards the present. This type of valuation process is referred to as backward induction. There is a common misconception in the market. Many people believe that the cash flows of a defaultable financial contract can be priced independently and then be summed up to give the final risky price of the contract. We emphasize here that this conclusion is only true of the financial contracts whose payoffs are always positive. In the cases where the promised payoffs could be positive or negative, the valuation requires not only a backward recursive induction procedure, but also a strategic selection of different discount factors according to the market value in time. This coupled valuation process allows us to capture correlation between counterparties and market factors. Table 1. Payoffs of a bilaterally defaultable contract Proof: See the appendix. Proof: See the appendix. We may think of ) , ( T t K as the risk-adjusted discount factor. Proposition 3 tells us that the bilateral risky price of a single-payment contract can be expressed as the present value of the payoff discounted by a risk-adjusted discount factor that has a switching-type dependence on the sign of the payoff. Using a similar derivation as in Proposition 2, we can easily extend Proposition 3 from one-period to multiple-periods. Suppose that a defaultable contract has m cash flows. Let the m cash flows be represented as i X with payment dates iT , where i = 1,…,m. Each cash flow may be positive or negative. The bilateral risky value of the multiple-payment contract is given by Proposition 4: The bilateral risky value of the multiple-payment contract is given by            m i t i i j j j X T T K E t V 1 1 0 1) , ( ) ( F (17a) 13 13 where 0T t  and where 0T t  and  ) , ( 1 ) , ( 1 ) , ( ) , ( 1 0 )) ( ( 1 0 )) ( ( 1 1 1 1 1 1               j j A T V X j j B T V X j j j j T T k T T k T T D T T K j j j j (17b) where ) , ( 1  j j A T T k and ) , ( 1  j j B T T k are defined in Proposition 3. 3. Numerical Results In this section, we present some numerical results for CVA calculation based on the theory described above. First, we study the impact of margin agreements on CVA. The testing 14 portfolio consists of a number of interest rate and equity derivatives. The number of simulation scenarios (or paths) is 20,000. The time buckets are set weekly. If the computational requirements exceed the system limit, one can reduce both the number of scenarios and the number of time buckets. The time buckets can be designed fine-granularity at the short end (e.g., daily and then weekly) and coarse-granularity at the far end (e.g. monthly and then yearly). The rationale is that the calculation becomes less accurate due to the accumulated error from simulation discretization, and inherited errors from calibration of the underlying models, such as those due to the change of macro-economic climate. The collateral margin period of risk is assumed to be 14 days (2 weeks). For risk-neutral simulation, we use a Hull-White model for interest rate and a CIR (Cox- Ingersoll-Ross) model for hazard rate scenario generations a modified GBM (Geometric Brownian Motion) model for equity and collateral evolution. The results are presented in the following tables. Table 2 illustrates that if party A has an infinite collateral threshold   A H i.e., no collateral requirement on A, the CVA value increases while the threshold B H increases. Table 3 shows that if party B has an infinite collateral threshold   B H , the CVA value actually decreases while the threshold A H increases. This reflects the bilateral impact of the collaterals on the CVA. The impact is mixed in Table 4 when both parties have finite collateral thresholds. Table 2. The impact of collateral threshold B H on the CVA This table shows that given an infinite A H , the CVA increases while B H increases, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. Collateral Threshold B H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 19,550.91 20,528.65 21,368.44 22,059.30 Table 3. The impact of collateral threshold A H on the CVA This table shows that given an infinite A H , the CVA increases while B H increases, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. Collateral Threshold B H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 19,550.91 20,528.65 21,368.44 22,059.30 This table shows that given an infinite A H , the CVA increases while B H increases, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. Collateral Threshold B H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 19,550.91 20,528.65 21,368.44 22,059.30 Table 3. The impact of collateral threshold A H on the CVA Table 3. The impact of collateral threshold A H on the CVA 15 This table shows that given an infinite B H , the CVA decreases while A H increases, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. Collateral Threshold A H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 28,283.64 25,608.92 23,979.11 22,059.30 This table shows that given an infinite B H , the CVA decreases while A H increases, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. Collateral Threshold A H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 28,283.64 25,608.92 23,979.11 22,059.30 Table 4. The impact of the both collateral thresholds on the CVA The CVA may increase or decrease while both collateral thresholds change, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. This reflects the fact that the collaterals have bilateral impacts on the CVA. Collateral Threshold B H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) Collateral Threshold A H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 25,752.98 22,448.45 23,288.24 22,059.30 Table 5. The impact of wrong way risk on the CVA This table shows that the CVA increases while the negative correlation increases in the absolute value. We use an equity swap as an example and assume that there is a negative correlation between the equity price and the credit quality of party B. Correlation  0 -50% -100% CVA 165.15 205.95 236.99 Table 4. The impact of the both collateral thresholds on the CVA The CVA may increase or decrease while both collateral thresholds change, where B H denotes the collateral threshold of party B and A H denotes the collateral threshold of party A. This reflects the fact that the collaterals have bilateral impacts on the CVA. reflects the fact that the collaterals have bilateral impacts on the CVA. Collateral Threshold B H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) Collateral Threshold A H 10.1 Mil 15.1 Mil 20.1 Mil Infinite ( ) CVA 25,752.98 22,448.45 23,288.24 22,059.30 Next, we examine the impact of wrong way risk. Wrong way risk occurs when exposure to a counterparty is adversely correlated with the credit quality of that counterparty, while right way risk occurs when exposure to a counterparty is positively correlated with the credit quality of that counterparty. Wrong/right way risk, as an additional source of risk, is rightly of concern to banks and regulators. Some financial markets are closely interlinked, while others are not. For example, CDS price movements have a feedback effect on the equity market, as a trading strategy commonly employed by banks and other market participants consists of selling a CDS on a reference entity and hedging the resulting credit exposure by shorting the stock. On the other hand, Moody’s Investor’s Service (2000) presents statistics that suggest that the correlations between interest rates and CDS spreads are very small. 16 16 To capture wrong/right way risk, we need to determine the dependency between counterparties and to correlate the credit spreads or hazard rates with the other market risk factors, e.g. equities, commodities, etc., in the scenario generation. We use an equity swap as an example. Assume the correlation between the underlying equity price and the credit quality (hazard rate) of party B is . The impact of the correlation on the CVA is show in Table 5. The results say that the CVA increases when the absolute value of the negative correlation increases. 4. Conclusion This article presents a framework for pricing risky contracts and their CVAs. The model relies on the probability distribution of the default jump rather than the default jump itself, because the default jump is normally inaccessible. We find that the valuation of risky assets and their CVAs, in most situations, has a backward recursive nature and requires a backward induction valuation. An intuitive explanation is that two counterparties implicitly sell each other an option to default when entering into an OTC derivative transaction. If we assume that a default may occur at any time, the default options are American style options. If we assume that a default may only happen on the payment dates, the default options are Bermudan style options. Both Bermudan and American options require backward induction valuations. 17 Based on our theory, we propose a novel cash-flow-based framework (see appendix) for calculating bilateral CVA at the counterparty portfolio level. This framework can easily incorporate various credit mitigation techniques, such as netting agreements and margin agreements, and can capture wrong/right way risk. Numerical results show that these credit mitigation techniques and wrong/right way risk have significant impacts on CVA. A. Proofs Proof of Proposition 1: Under the unilateral credit risk assumption, we only consider the default risk when the asset is in the money. Assume that a default may only occur on the payment date. Therefore, the risky value of the asset at t is the discounted expectation of all possible payoffs and is given by               t t t F F F T T X T X X X T t F E X T t q T T t D E X T t q T T t p T t D E t V T T T ) , ( ) , ( ) ( 1 1 1 ) , ( 1 ) , ( ) ( ) , ( 1 ) , ( ) ( 0 0 0             (A1a) (A1a) where     ) ( 1 ) , ( 1 1 ) , ( ) , ( 0 T T t q T t D T t F T X      (A1b) (A1b) Proof of Proposition 2: Let 0 T t  . On the first payment day, let ) ( 1T V denote the risky Proof of Proposition 2: Let 0 T t  . On the first payment day, let ) ( 1T V denote the risky value of the asset excluding the current cash flow 1 X . According to Proposition 1, the risky value of the asset at t is given by the asset excluding the current cash flow 1 X . According to Proposition 1, the risky value i i b value of the asset excluding the current cash flow 1 X . A. Proofs According to Proposition 1, the risky value of the asset at t is given by     t F ) ( ) , ( ) ( 1 1 1 0 T V X T T F E t V   (A2a) (A2a) where       ) ( 1 ) , ( 1 1 ) , ( ) , ( 0 ) ( 1 0 1 0 1 1 T T t q T T D T T F X T V       (A2b) Similarly, we have     1 ) ( ) , ( ) ( 2 2 2 1 1 T T V X T T F E T V F   (A3)       ) ( 1 ) , ( 1 1 ) , ( ) , ( 0 ) ( 1 0 1 0 1 1 T T t q T T D T T F X T V       (A2b)       ) ( 1 ) , ( 1 1 ) , ( ) , ( 0 ) ( 1 0 1 0 1 1 T T t q T T D T T F X T V       (A2b) (A2b)     1 ) ( ) , ( ) ( 2 2 2 1 1 T T V X T T F E T V F   (A3) (A3) 18 Note that ) , ( 1 0 T T F is 1 T F -measurable. According to the taking out what is known and Note that ) , ( 1 0 T T F is 1 T F -measurable. A. Proofs Depending on whether the payoff is in the money or out of the money at T, we have          t T A X B X t T t T AB A A X t T AB B B X X T t k T t k T t D E X T t K E X T t p T T t p T T t p T T t p X T t p T T t p T T t p T T t p T t D E t V T T T T F F F F ) , ( 1 ) , ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) ( ) , ( ) ( ) , ( 1 ) , ( ) ( ) , ( ) ( ) , ( ) ( ) , ( 1 ) , ( ) ( 0 0 11 10 01 00 0 11 10 01 00 0                      (A6a) h          t T A X B X t T t T AB A A X t T AB B B X X T t k T t k T t D E X T t K E X T t p T T t p T T t p T T t p X T t p T T t p T T t p T T t p T t D E t V T T T T F F F F ) , ( 1 ) , ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) ( ) , ( ) ( ) , ( 1 ) , ( ) ( ) , ( ) ( ) , ( ) ( ) , ( 1 ) , ( ) ( 0 0 11 10 01 00 0 11 10 01 00 0                      (A6a) (A6a) where  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB B B AB A B AB A B B A B B A B B                (A6b) (A6b)  ) ( ) ( ) ( 1 ) , ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( T T T T t T t q T t q T T t q T t p T T t p T t q T T t p T t p T t k AB A A AB A B AB B A A B A A A B A                (A6c) A. Proofs According to the taking out what is known and tower properties of conditional expectation, we have tower properties of conditional expectation, we have                       t t t t t F F F F F F F ) ( ) , ( ) , ( ) ( ) , ( ) , ( ) , ( ) , ( ) ( ) , ( ) ( 2 1 0 1 2 1 1 0 1 2 2 1 2 2 1 1 0 1 1 0 1 1 1 0 1 1 T V T T F E X T T F E T V T T F E X T T F E T T F E X T T F E T V X T T F E t V j j j i i i j j j T T                 (A4) (A4) iving from 2 T forward over m T , where m m X T V  ) ( , we have By recursively deriving from 2 T forward over m T , where m m X T V  ) ( , we have By recursively deriving from 2 T forward over m T , where m m X T V  ) ( , we have            m i t i i j j j X T T F E t V 1 1 0 1) , ( ) ( F (A5) (A5) Proof of Proposition 3: We assume that a default may only occur on the payment date. Proof of Proposition 3: We assume that a default may only occur on the payment date. Proof of Proposition 3: We assume that a default may only occur on the payment date. p y y p y At time T, there are four possible states: 1) both A and B survive, 2) A defaults but B survives, 3) A survives but B defaults, and 4) both A and B default. The joint distributions of A and B are given by (15). B. A practical framework for calculating bilateral CVA We develop a practical framework for calculating bilateral CVA at counterparty portfolio level based on the theory described above. The framework incorporates netting and margin agreements, and captures right/wrong way risk. 19 Two parties are denoted as A and B. All calculations are from the perspective of party A. Let the valuation date be t. The CVA computation procedure consists of the following steps. B.1. One core element of the trading credit risk modeling is the Monte Carlo scenario generation (market evolution). This must be able to run a large number of scenarios for each risk factor with flexibility over parameterization of processes and treatment of correlation between underlying factors. Credit exposure may be calculated under real probability measure, while CVA or pricing counterparty credit risk should be conducted under risk-neutral probability measure. Due to the extensive computational intensity of pricing counterparty risk, there will inevitably be some compromise of limiting the number of market scenarios (paths) and the number of simulation dates (also called “time buckets” or “time nodes”). The time buckets are normally designed fine-granularity at the short end and coarse-granularity at the far end. The details of scenario generation are beyond the scope of this paper. B.2. For ease of illustration, we choose a vanilla interest rate swap, as interest rate swaps collectively account for around two-thirds of both the notional and market value of all outstanding derivatives (FinPricing (2015)) Assume that party A pays a fixed rate, while party B pays a floating-rate. Assume that there are M time buckets ( M T T T ,..., , 1 0 ) in each scenario and N cash flows in the sample swap. Let consider scenario j first. For swaplet i, there are four important dates: the fixing date f it , , the starting date s it , , the ending date e it , and the payment date p it , . In general, these dates are not coincidently at the simulation time buckets. The time relationship between swaplet i and the simulation time buckets is illustrated in Figure B1. 20 Figure B1: An interest rate swaplet j T 1  jT k T 1  kT f it , s it , e it , p it , Terms Rates Interest rate curve simulated at f it , Interest rate curve simulated at f it , Interest rate curve simulated at f it , Rates Figure B1: An interest rate swaplet This figure illustrates the time relationship between an interest rate swaplet and the simulation time buckets. The floating leg of the swaplet is reset at the fixing date f it , with the starting date s it , , the ending date e it , , and the payment date p it , . The simulation time buckets are 1 1,..., ,   k i i T T T . The simulated interest rate curve is starting at f it , . Both fixed rate payments and floating-rate payments occur on the same payment dates. The cash flow of swaplet i is determined at the fixing date f it , that is assumed to be between the simulation time buckets jT and 1  j T . First, we need to create an interest rate curve observed at f it , by interpolating the interest rate curves simulated at jT and 1  j T via either Brownian Bridge or linear interpolation. The linear interpolation is the expectation of the Brownian Bridge. B.2. Then we can calculate the payoff of swaplet i at scenario j as   ) , ( ) , ; ( , , , , , , e i s i e i s i f i i j t t R t t t F N     (B1) (B1) where N denotes the notional; ) , ; ( , , , e i s i f i t t t F denotes the simply compounded forward rate reset at f it , for the forward period ( s it , , e it , ); ) , ( , , e i s i t t  denotes the accrual factor or day count where N denotes the notional; ) , ; ( , , , e i s i f i t t t F denotes the simply compounded forward rate reset at f it , for the forward period ( s it , , e it , ); ) , ( , , e i s i t t  denotes the accrual factor or day count fraction for the period ( s it , , e it , ) and R denotes the fixed rate. fraction for the period ( s it , , e it , ) and R denotes the fixed rate. 21 The cash flow amount calculated by (B1) is paid on the payment date p it , . This value should be allocated into the nearest previous time bucket k T as: The cash flow amount calculated by (B1) is paid on the payment date p it , . This value should be allocated into the nearest previous time bucket k T as: e allocated into the nearest previous time bucket k T as: ) , ( ~ , , , , p i k i j i k j t T D    (B2) (B2) where ) , ( ,p i k t T D denotes the risk-free discount factor based on the interest rate curve simulated at k T . where ) , ( ,p i k t T D denotes the risk-free discount factor based on the interest rate curve simulated at k T . at k T . Cash flow generation for products without early-exercise provision is quite straightforward. B.2. For early-exercise products, one can use the approach proposed by Longstaff and Schwartz (2001) to obtain the optimal exercise boundaries and then the payoffs. B3. After generating cash flows for each deal, we need to aggregate them at counterparty portfolio level at each scenario and each time bucket. The cash flows are aggregated by either netting or nonnetting based on the netting agreements. A netting agreement is a provision that allows the offset of settlement payments and receipts on all contracts between two counterparties. Another important use of netting is the close-out netting that allows the offset of close-out values. For netting, we add all cash flows together at the same scenario and the same time bucket to recognize offsetting. The aggregated cash flow under netting at scenario j and time bucket k is given by   i i k j k j , , , ~ ~   (B3) (B3) For nonnetting, we divided cash flows into positive and negative groups and add them separately. In other words, the offsetting is not recognized. The aggregated cash flows under nonnetting at scenario j and time bucket k are given by            0 ~ 0 ~ ~ , , , , , , , , , m k j m m k j l k j l l k j k j if if      (B4) (B4) 22 Margin (or collateral) agreements For a more detailed discussion on pricing collateralized contract/portfolio, see Xiao (2013b). B5. After aggregating all cash flows via netting, one can price a portfolio in the same manner as pricing a single deal. We assume that the reader is familiar with the least square Monte Carlo valuation model proposed by Longstaff and Schwartz (2001) and thus do not repeat some well- known procedures for brevity. If the counterparty portfolio is collateralized, we can calculate the risky value based on equation (21) of Xiao (2013b). If there is no collateral agreement, we can price the portfolio according to Proposition 4 in this paper. CVA is by definition the difference between the risk-free portfolio value and the true (or risky or defaultable) portfolio value. FinPricing, 2015, Financial Market Solution, https://finpricing.com/product.html Pykhtin, Michael, and Steven Zhu, 2007, A guide to modeling counterparty credit risk, GARP Risk Review, July/August, 16-22. Reference Brigo, D., and Capponi, A., 2008, Bilateral counterparty risk valuation with stochastic dynamical models and application to Credit Default Swaps, Working paper. Duffie, Darrell, and Ming Huang, 1996, Swap rates and credit quality, Journal of Finance, 51, 921-949. Duffie, Darrell, and Kenneth J. Singleton, 1999, Modeling term structure of defaultable bonds, Review of Financial Studies, 12, 687-720. Duffie, Darrell, and Kenneth J. Singleton, 1999, Modeling term structure of defaultable bonds, Review of Financial Studies, 12, 687-720. Duffie, Darrell, and Kenneth J. Singleton, 1999, Modeling term structure of defaultable bonds, Review of Financial Studies, 12, 687-720. 23 23 Gregory, Jon, 2009, Being two-faced over counterparty credit risk, RISK, 22, 86-90. Hull, J. and White, A., 2013, CVA and wrong way risk, forthcoming, Financial Analysts Journal. Jarrow, R. A., and Protter, P., 2004, Structural versus reduced form models: a new information based perspective, Journal of Investment Management, 2, 34-43. Jarrow, Robert A., and Stuart M. Turnbull, 1995, Pricing derivatives on financial securities subject to credit risk, Journal of Finance, 50, 53-85. Lipton, A., and Sepp, A., 2009, Credit value adjustment for credit default swaps via the structural default model, Journal of Credit Risk, 5(2), 123-146. Longstaff, Francis A., and Eduardo S. Schwartz, 2001, Valuing American options by simulation: a simple least-squares approach, The Review of Financial Studies, 14 (1), 113-147. Moody’s Investor’s Service, 2000, Historical default rates of corporate bond issuers, 1920-99. J. P. Morgan, 1999, The J. P. Morgan guide to credit derivatives, Risk Publications. O’Kane, D. and S. Turnbull, 2003, Valuation of credit default swaps, Fixed Income Quantitative Credit Research, Lehman Brothers, QCR Quarterly, 2003 Q1/Q2, 1-19. Pykhtin, Michael, and Steven Zhu, 2007, A guide to modeling counterparty credit risk, GARP Risk Review, July/August, 16-22. 24 24 Sorensen, E. and T. Bollier, 1994, Pricing swap default risk, Financial Analysts Journal, 50, 23- 33. 33. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and credit risk modeling, Working paper. Xiao, T., 2013b, An economic examination of collateralization in different financial market, Working paper. 33. 33. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and credit risk modeling, Working paper. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and credit risk modeling, Working paper. Xiao, T., 2013a, The impact of default dependency and collateralization on asset pricing and credit risk modeling, Working paper. Reference Xiao, T., 2013b, An economic examination of collateralization in different financial market, Working paper. 25
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Evoluţia jurisprudenţei Curţii Europene a Drepturilor Omului cu privire la cererile interstatale
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1 Convenţia Europeană a Drepturilor Omului din 04.11.1950 https://www.echr.coe.int/Docu­ ments/Convention_RON.pdf CZU: 341.231.14 DOI: 10.5281/zenodo.5639377 CZU: 341.231.14 DOI: 10.5281/zenodo.5639377 Olga BENEŞ, master în drept, lector universitar (ORCID: 0000-0001-7853-9557) Olga BENEŞ, master în drept, lector universitar (ORCID: 0000-0001-7853-9557) Evolution of the Jurisprudence of the European Court of Human Rights on Interstate Requests Article 33 of the European Convention on Human Rights, entitled Interstate Cases, provides: „Any High Contracting Party may refer to the Court any violation of the provisions of the Convention and its Protocols by another High Contracting Party.“ To that end, the requesting State is acting rather to denounce one or more infringements alleged to have been suffered by private individuals who have been replaced in a certain way. Keywords: interstate demand, European public order, collective guarantee of rights and freedoms. Articolul 33 al Convenţiei Europene a Drepturilor Omului întitulat Cauze interstatale dispune: „Orice Înaltă Parte contractantă poate sesiza Curtea asupra oricărei pretinse încălcări a preve­ derilor Convenţiei şi ale Protocoalelor sale de către o altă Înaltă Parte Contractantă„. În acest sens, statul reclamant acţionează mai degrabă pentru a denunţa una sau mai multe încălcări pretinse a fi suferite de către persoane particulare cărora li se substituie într-un fel anume. Cuvinte-cheie: cerere interstatală, ordine publică europeană, garanţie colectivă a drepturilor şi libertăţilor. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 La acest capitol Convenţia Europeană a Drepturilor Omului a suferit o evoluţie foarte diferită de cea concepută de autorii săi, care la origini au decis instituirea unui caracter constrângător pentru statele care aveau deficienţe în materia protecţiei drepturilor omului, impunând prin aceasta obligaţia de a se supune Curţii Europene. „Recursul interstatal, prevăzut ca un instrument cheie de către autorii Convenţiei, în scopul de a împiedica un stat membru să admită dictatura, trebuia să constituie me­ canismul principal. Protecţia drepturilor fundamentale, apărea într-un fel oarecare pe planul doi. Dar n-a fost cazul, recursul interstatal care la origini trebuia să constituie pivotul sistemului a fost de fapt abandonat pe parcurs de zece ani“2. Majoritatea plângerilor care au apărut pe parcurs se referă la situaţiile de criză sau de conflict, printre care metodele de interogare folosite de autorităţile britanice în anii 1971—1975 pe parcursul tulburărilor din Irlanda de Nord, operaţiunile militare desfăşurate de Turcia în Ciprul de Nord în anul1974, conflictul armat dintre Georgia şi Rusia din 2008 şi mai recent, evenimentele din Crimeea şi Estul Ucrainei din 2014. Încă în cauza Austria c. Italiei din 1963, Comisia a dispus în raportul său că „Orice Parte contractantă poate sesiza Comisia cu privire la orice încălcare a dispozi­ ţiilor Convenţiei care crede că poate fi imputată unei alte Părţi contractante, că prin acest articol, Înaltele Părţi Contractante au autorizat, în consecinţă, oricare dintre ele să aducă în faţa Comisiei orice presupusă încălcare a dispoziţiilor Convenţiei, indife­ rent dacă victimele acestei încălcări sunt sau nu resortisanţi ai statului solicitant, fie că presupusa violare prejudiciază sau nu în special interesele acestui stat, că un stat contractant, atunci când sesizează Comisia în temeiul articolului 24 (33), nu trebuie, prin urmare, să fie considerat ca acţionând pentru a-şi asigura propriile drepturi, ci mai degrabă ca adresând Comisiei o întrebare care aduce atingere ordinii publice a europene“3. În acest caz, statul reclamant acţionează mai puţin în apărarea drepturilor sale proprii, aşa cum este cazul în faţa Curţii Internaţionale de Justiţie, dar are scopul de „a denunţa una sau mai multe încălcări pretinse a fi suferite de către persoane particulare cărora li se substituie într-un fel anume,“4 încălcări care aduc atingere ordinii publice europene. 2 Petitti L.E., Decaux E., Imbert P.H. La Convention Européenne des Droits de l’Homme. Com­ mentaire article par article. Réflexions sur les principes et les mécanismes de la Convention. Paris : Economica, 1999. p. 27. 3 Rapport de la Com.EDO, Autriche c. Italie, 788/60, 30.03.1963. http://hudoc.echr.coe.int/app/ conversion/pdf/?library=ECHR&id=001-27972&filename=001-27972.pdf 4 CEDO, Georgia c. Rusiei (II), n° 38263/08, dec. din 13.12. 2011. https://hudoc.echr.coe.int/ eng#{%22itemid%22:[%22001-108097%22]} 4 CEDO, Georgia c. Rusiei (II), n° 38263/08, dec. din 13.12. 2011. https://hudoc.echr.coe.int/ eng#{%22itemid%22:[%22001-108097%22]} Evolution of the Jurisprudence of the European Court of Human Rights on Interstate Requests Article 33 of the European Convention on Human Rights, entitled Interstate Cases, provides: „Any High Contracting Party may refer to the Court any violation of the provisions of the Convention and its Protocols by another High Contracting Party.“ To that end, the requesting State is acting rather to denounce one or more infringements alleged to have been suffered by private individuals who have been replaced in a certain way. words: interstate demand, European public order, collective guarantee of rights and freedoms. Articolul 33 al Convenţiei Europene a Drepturilor Omului întitulat Cauze interstatale dispune: „Orice Înaltă Parte contractantă poate sesiza Curtea asupra oricărei pretinse încălcări a preve­ derilor Convenţiei şi ale Protocoalelor sale de către o altă Înaltă Parte Contractantă„. În acest sens, statul reclamant acţionează mai degrabă pentru a denunţa una sau mai multe încălcări pretinse a fi suferite de către persoane particulare cărora li se substituie într-un fel anume. Cuvinte-cheie: cerere interstatală, ordine publică europeană, garanţie colectivă a drepturilor şi libertăţilor. Curtea Europeană a Drepturilor Omului nu poate să se sesizeze din oficiu, ea fiind competentă de a statua asupra plângerilor de încălcare a Convenţiei Europene a Drepturilor Omului şi în acest scop trebuie să fie sesizată cu cereri individuale sau interstatale. Convenţia distinge două tipuri de cereri: cererile individuale depuse conform articolului 34 al Convenţiei şi cererile interstatale conform articolului 33 al Convenţiei. Articolul 33 al Convenţiei Europene a Drepturilor Omului întitulat Cauze inter­ statale dispune: „Orice Înaltă Parte contractantă poate sesiza Curtea asupra oricărei pretinse încălcări a prevederilor Convenţiei şi ale Protocoalelor sale de către o altă Înaltă Parte Contractantă“1. Astfel, prin această dispoziţie, Convenţia Europeană a Drepturilor Omului îi acordă Curţii Europene o competenţă în materie de diferende între state, competenţă exercitată pe parcursul timpului foarte rar. 32 2 Petitti L.E., Decaux E., Imbert P.H. La Convention Européenne des Droits de l’Homme. Com­ mentaire article par article. Réflexions sur les principes et les mécanismes de la Convention. Paris : Economica, 1999. p. 27. 5 Annuaire de la Convention Europeenne des Droits de l’Homme. Commission et Cour Eu­ ropeenne des Droits de l’Homme 1958-1959. Springer Science+Business Media, B.V. 1960. PP.179 https://books.google.md/books?id=7HrwCAAAQBAJ&pg=PA174&lpg=PA174&dq= Requete+No.+176/56+Par+cette+premiere+requ%C3%8ATe,+introduite+le+7+mai+I956,+l e+Gouvernement+hellenique&source=bl&ots=GrHVB1gQ9j&sig=ACfU3U0QmZDxdLI8u X58AE8lL6gT_1oHjg&hl=en&sa=X&ved=2ahUKEwjYjf2l9PDmAhVMCewKHUHRBQ0Q 6AEwAHoECAkQAQ#v=onepage&q=Requete%20No.%20176%2F56%20Par%20cette%20 premiere%20requ%C3%8ATe%2C%20introduite%20le%207%20mai%20I956%2C%20le%20 Gouverne-ment%20hellenique&f=false b d s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Pentru prima dată Comisia a fost sesizată cu două cereri interstatale ale guver­ nului elen împotriva Regatului Unit. Aceste două cereri făceau referinţă la aplicarea Convenţiei în insula Cipru. Prin prima cerere nr. 176/56, depusă la 7 mai 1956, Guvernul elen susţinea că derogarea de la dispoziţiile Convenţiei notificate de către 2 Petitti L.E., Decaux E., Imbert P.H. La Convention Européenne des Droits de l’Homme. Com­ mentaire article par article. Réflexions sur les principes et les mécanismes de la Convention. Paris : Economica, 1999. p. 27. 33 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Guvernul britanic cu privire la Cipru în temeiul articolului 15 al Convenţiei, nu era conformă şi că condiţiile de fond cerute de acest articol nu erau îndeplinite în speţă. Prin cererea 299/57 din 17 iulie 1957, guvernul elen sesiza Comisia de 49 de cazuri de „tortură sau servicii asimilabile torturii“ produse în Cipru şi de care guvernul britanic se făcea responsabil. De asemenea, el a contestat compatibilitatea unei serii de legi şi regulamente de urgenţă în vigoare în Cipru cu dispoziţiile Convenţiei. În special, el a susţinut că legislaţia care prevedea impunerea pedepsei cu biciuirea şi anumite pedepse colective încălca articolul 3 din Convenţie, de la care articolul 15 interzice Părţilor contractante derogarea, chiar şi în caz de război sau alt „pericol public care ameninţă viaţa naţiunii“. La rândul său, Guvernul britanic a negat încălcarea Convenţiei bazându-se fie pe însăşi definiţia drepturilor şi libertăţilor recunoscute de Convenţie, alteori pe existenţa în Cipru a unui „pericol public care ameninţă viaţa naţiunii“ în sensul articolului 15 citat mai sus5. Guvernul britanic cu privire la Cipru în temeiul articolului 15 al Convenţiei, nu era conformă şi că condiţiile de fond cerute de acest articol nu erau îndeplinite în speţă. Prin cererea 299/57 din 17 iulie 1957, guvernul elen sesiza Comisia de 49 de cazuri de „tortură sau servicii asimilabile torturii“ produse în Cipru şi de care guvernul britanic se făcea responsabil. De asemenea, el a contestat compatibilitatea unei serii de legi şi regulamente de urgenţă în vigoare în Cipru cu dispoziţiile Convenţiei. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 În special, el a susţinut că legislaţia care prevedea impunerea pedepsei cu biciuirea şi anumite pedepse colective încălca articolul 3 din Convenţie, de la care articolul 15 interzice Părţilor contractante derogarea, chiar şi în caz de război sau alt „pericol public care ameninţă viaţa naţiunii“. La rândul său, Guvernul britanic a negat încălcarea Convenţiei bazându-se fie pe însăşi definiţia drepturilor şi libertăţilor recunoscute de Convenţie, alteori pe existenţa în Cipru a unui „pericol public care ameninţă viaţa naţiunii“ în sensul articolului 15 citat mai sus5. În procedura din perioada respectivă s-a pus întrebarea de a şti care erau prero­ gativele Comisiei atunci când un stat invocând articolul 15, deroga de la obligaţiile prevăzute de Convenţie. În această situaţie, Comisia a considerat că era „competentă să se pronunţe cu privire la existenţa unui pericol public, care conform articolului 15 îi acordă statului dreptul de a deroga de la obligaţiile prevăzute de Convenţie“. De asemenea, Comisia a considerat că era „competentă să se pronunţe asupra fap­ tului dacă măsurile luate de către o Parte conform articolului 15 al Convenţiei, au fost în strictă conformitate cu exigenţele situaţiei“. În acest sens, ea a precizat că „guvernul trebuie să păstreze o anumită marjă de apreciere cu privire la stricteţea măsurii cerută de situaţie“6. Deoarece în perioada dată nici Grecia, nici Regatul Unit nu acceptase competenţa obligatorie a Curţii Europene a Drepturilor Omului şi odată cu reglementarea politică a problemei cipriote, Comisia a decis să pună capăt procedurii fără a se pronunţa pe fond în cauza dată. În următoarea cauză Austria c. Italiei nr.788/60 din 11 iulie 1960, Austria a invocat încălcarea de către guvernul italian a dispoziţiilor articolului 6 al Convenţiei contestând procedurile care au adus la pedepsirea persoanelor în speţă, acestea fiind considerate ilegale şi contrare Convenţiei Europene a Drepturilor Omului, şi anume dispoziţiilor care enunţă regulile cu privire la o bună administrare a justiţiei şi a articolului 14 care interzice orice discriminare în prevalarea drepturilor garantate 34 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 de Convenţie7. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Fiind chemată să se expună asupra obligaţiilor statelor părţi ce re­ zultă din textul Convenţiei, Comisia a enunţat că: „…rezultă că obligaţiile asumate de statele contractante prin Convenţie au, în esenţă un caracter obiectiv deoarece vizează protejarea drepturilor fundamentale ale indivizilor împotriva abuzurilor statelor contractante mai degrabă decât crearea unor drepturi subiective şi reci­ proce între acestea, că natura obiectivă a angajamentelor menţionate apare şi în mecanismul instituit de Convenţie, că acest mecanism se bazează pe conceptul de garanţie colectivă, de către statele contractante, a drepturilor şi libertăţilor definite în Convenţie“8. Prin decizia Comisiei în această cauză s-a instituit dreptul fiecărei Părţi contractante de a pune în aplicare mecanismul instituit prin articolul 24 al Convenţiei chiar şi pentru fapte anterioare ratificării sale de către statul reclamant, rolul acestui mecanism fiind protecţia „patrimoniului comun, de tradiţii politice, de idealuri, de libertate şi de preeminenţă a dreptului, astfel asigurând garanţia colectivă a unor drepturi enunţate în Declaraţia Universală9“. Deci, după cum observăm, în această perioadă se enunţă scopul recursului interstatal, şi anume, garantarea colectivă a drepturilor şi libertăţilor fundamentale, acesta fiind consolidat printr-o serie de cauze care au urmat depuse de Danemarca, Franţa, Germania, Suedia şi Ţările de Jos împotriva Turciei la 1 iulie 1982. Chiar dacă cetăţenii acestor state nu erau direct ameninţaţi de situaţia stabilită, aceste state totuşi au contestat în faţa Curţii Europene atingerile aduse drepturilor şi libertăţilor comise de către autorităţile turce în urma unei lovituri de stat militare intervenite în septembrie 1980. Se consideră că sub acest aspect cauza Irlanda c. Regatului Unit10 din 10 martie 1976, poate fi considerată un punct de încercare, anunţând un moment de cotitură deoarece a reprezentat o importanţă şi o semni­ ficaţie particulară în măsura în care punea problema recurgerii la anumite metode de represiune, şi aceasta nu într-un regim dictatorial dar în cadrul unei democraţii. Această cauză este de o importanţă deosebită în definirea anumitor termeni cu privire la articolul 3 CEDO şi determinarea marjei pentru stabilirea diferenţei între calificativele tratamentului uman sau inuman, umilitor sau degradant, care trebuie să fie apreciate în condiţiile şi contextul cauzei. Astfel, regula de principiu care a fost stabilită enunţă că: „Aplicarea sistematică a metodelor în scopul de a instiga o persoană să dea informaţii prezintă similitudini clare cu metodele de tortură sistematică cunoscută pe parcursul secolelor. 7 Annuaire de la Convention Europeenne des Droits de l’Homme. Commission et Cour Eu­ ropeenne des Droits de l’Homme 1962. Springer Science+Business Media, B.V. 1963, p.47; Commission et Cour%20 Europeenne des Droits de l’Homme 1958-1959. 8 Rapport de la Com.EDO, Autriche c. Italie, 788/60, 30.03.1963, p. 9. 9 Ibidem, p.8. 10 CtEDO, cauza Irlanda c. Regatului Unit, hot. din 18.01.1978. https://hudoc.echr.coe.int/eng #{%22itemid%22:[%22001-62064%22]} 10 CtEDO, cauza Irlanda c. Regatului Unit, hot. din 18.01.1978. https://hudoc.echr.coe.int/eng #{%22itemid%22:[%22001-62064%22]} 11 Annuaire de la Convention Europeenne des Droits de l’Homme. 1976. La Haye: Martinus Nijhoff, 1977. p.795 12 CtEDO, cauza Cipru c. Turciei nr. 25781/94, hot. din 12 .05.2014. https://hudoc.echr.coe.int/ fre#{%22itemid%22:[%22001-144153%22]} 13 CtEDO, cauza Cipru c. Turciei nr. 25781/94, hot. din 12 .05.2014. Opinia comună a judecă­ torului PINTO DE ALBUQUERQUE şi VUČINIĆ https://hudoc.echr.coe.int/fre#{%22item id%22:[%22001-144153%22]} s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Chiar dacă cele cinci metode cunoscute drept metode de dezorientare sau de privare senzorială nu lasă în mod necesar urme grave, Comisia vede în aceasta un sistem modern de tortură care se 35 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 află în aceiaşi categorie cu sistemele aplicate anterior pentru a obţine informaţii sau mărturii“11. O altă hotărâre care a fost de o importanţă particulară în evoluţia jurispruden­ ţei este Cipru c. Turciei12, pronunţată de Marea Cameră la 12 mai 2014 şi vizează noţiunea de satisfacţie echitabilă în cadrul cererilor interstatale. Pentru prima dată Curtea impune statului pârât obligaţia să plătească despăgubiri altui stat, şi anume 90 milioane de Euro cu titlu de satisfacţie echitabilă în cadrul unei cauze intersta­ tale, statul destinatar fiind şi el obligat să distribuie această sumă victimelor sub supravegherea Comitetului de Miniştri în termenul fixat de Curte. Referindu-se la caracterul satisfacţiei echitabile, Curtea reaminteşte că logica articolului 41 cu privire la satisfacţia echitabilă decurge direct din regulile şi principiile dreptului internaţional public cu privire la răspunderea statului pentru încălcarea unei obligaţii ce decurge dintr-un tratat şi trebuie interpretată în acest context. Având în vedere natura Convenţiei Europene a Drepturilor Omului, anume individul şi nu statul este direct afectat prin încălcarea unuia sau mai multor drepturi garantate de Convenţie şi deci, o satisfacţie echitabilă într-o cauză interstatală trebuie să fie întotdeauna în beneficiul victimelor individuale. i În urma acestui precedent şi alte state membre ar putea invoca sume importante în baza încălcărilor repetate, continue sau multiple a drepturilor fundamentale mai ales având în vedere faptul că cererile interstatale sunt adesea izvorâte dintr-un context belicos între statele părţi. Astfel, exemplul acestei hotărâri poate aduce la creşterea numărului cererilor interstatale în cadrul cărora s-ar obţine o indemnizaţie globală pentru o multitudine de victime. Exprimând opinia lor comună referitor la acest precedent, unii judecători au atras atenţia că: „Hotărârea Cipru c. Turciei (satisfacţia echitabilă) este cea mai importantă contribuţie la pacea în Europa în istoria Curţii Europene a Dreptu­ rilor Omului. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Mesajul adresat satelor membre ale Consiliului Europei este clar: statele membre care fac război, invadează alte state membre sau susţin interven­ ţiile militare în alte state membre trebuie să plătească pentru actele lor ilegale şi consecinţele acestor acte, în timp ce victimele şi familiile lor cât şi statele ale căror sunt resortisanţi au un drept dobândit şi executoriu de a fi compensate în mod corespunzător şi pe deplin de către statul beligerant responsabil. Războiul şi consecinţele sale tragice nu mai pot fi tolerate în Europa, iar statele membre care nu respectă acest principiu trebuie să răspundă pentru actele lor în faţa justiţiei, fără alte consecinţe în plan politic“13. 36 14 CtEDO, cauza Cipru c. Turciei nr. 25781/94, hot. din 12 .05.2014. Opinia comună a judecă­ torilor ZUPANČIČ, GYULUMYAN, DAVÍD THÓR BJÖRGVINSSON, NICOLAOU, SAJÓ, LAZAROVA TRAJKOVSKA, POWER-FORDE, VUČINIĆ ET PINTO DE ALBUQUERQUE. https://hudoc.echr.coe.int/fre#{%22itemid%22:[%22001-144153%22]} p p j torilor ZUPANČIČ, GYULUMYAN, DAVÍD THÓR BJÖRGVINSSON, NICOLAOU, SAJÓ, LAZAROVA TRAJKOVSKA, POWER-FORDE, VUČINIĆ ET PINTO DE ALBUQUERQUE. https://hudoc.echr.coe.int/fre#{%22itemid%22:[%22001-144153%22]} 15 CtEDO, cauza Georgia c. Rusiei (I) nr.13255/07, hot. din 03.07.2014. https://hudoc.echr.coe. int/fre#{%22itemid%22:[%22001-145552%22]} 16 CtEDO, cauza Georgia c. Rusiei (I) nr.13255/07, hot. sat. echit. din 31.01.2019. https://www. doctrine.fr/d/CEDH/HFJUD/GRANDCHAMBER/2019/CEDH001-189852 17 CtEDO, cauza Slovenia c. Croatia nr. 5155/16, cerere din 15.09.2016. https://www.google. com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwiPk5KDhYXnAhUlsa QKHRnJDicQFjABegQIBBAC&url=http%3A%2F%2Fhudoc.echr.coe.int%2Fapp%2Fconve rsion%2Fpdf%2F%3Flibrary%3DECHR%26id%3D003-6427775 8450864%26filename%3D Grand%2520Chamber%2520hearing%2520Slovenia%2520v.%2520Croatia.pdf&usg=AOvV aw15y2jR8ia5B23O6XFBC5v8 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Atenţionând asupra importanţei acestei hotărâri, 10 din cei 17 judecători ai Marii Camere au menţionat în opinia lor că: „Această hotărâre anunţă începutul unei noi ere în implementarea drepturilor omului protejate de Curte şi marchează o etapă importantă în ceea ce priveşte respectarea statului de drept în Europa. Este pentru prima dată în istoria Curţii când aceasta face o declaraţie specifică cu privire la scopul şi efectul uneia dintre hotărârile sale în contextul executării.“14 Altă cauză care consolidează jurisprudenţa Curţii în materie de satisfacţie echita­ bilă în cazurile interstatale este Georgia c. Rusiei (I)15, cu privire la expulzarea colectivă a resortisanţilor georgieni de către autorităţile ruse începând cu luna octombrie 2006 şi până în ianuarie 2007. În hotărârea sa asupra fondului cauzei din 3 iulie 2014, Curtea a stabilit aplicarea de către Federaţia Rusă a unei a politici coordonate de arestare, detenţie şi expulzare a resortisanţilor georgieni care a constituit o practică administrativă, în sensul jurisprudenţei Curţii, astfel încălcându-se articolul 4 din Protocolul nr.4, articolele 3 şi 13 combinat cu articolele 5§1 şi 3. În această hotărâre Curtea a enumerat cele trei condiţii necesare de a fi reunite pentru ca să stabilească dacă se justifică acordarea unei satisfacţii echitabile în cadrul cauzelor interstatale: capătul de acuzare formulat de guvernul reclamant care trebuie să se refere la încăl­ carea drepturilor fundamentale a resortisanţilor săi sau a altor persoane, posibilitatea de identificare a victimelor şi obiectivul principal al procedurii. Astfel, Curtea a hotărât în hotărârea cu privire la satisfacţie echitabilă, că Rusia trebuie să plătească Georgiei 10 milioane de Euro pentru prejudiciul moral, care să fie repartizat unui grup de victime de circa 1500 de resortisanţi georgieni16. În prezent pe rolul Curţii sunt opt cereri pendinte dintre care o cerere Slovenia c.Croatiei, două cereri Georgia c. Rusiei şi 5 cereri depuse de Ucraina c. Rusiei. Cauza Slovenia c. Croaţiei17 nr. 54155/16 se referă la lipsa de echitate, la parţialitatea şi discriminarea din partea instanţelor croate în cadrul procedurilor iniţiate de o bancă slovenă Ljubljanska banka d.d., pentru încasarea datoriilor de la companiile croate. La rândul său Georgia a depus două plângeri împotriva Rusiei, în anul 2008 cu privire la conflictul armat dintre Georgia şi Rusia din 2008 şi consecinţele sale, 14 CtEDO, cauza Cipru c. 18 CtEDO, cauza Ucraina c. Federaţiei Ruse nr. 20958/14, cerere din 13.03.2014. https://www. google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwjBuuuy5ILnA hUOkxQKHfuJB24QFjABegQIARAC&url=https%3A%2F%2Fhudoc.echr.coe.int%2Fapp% 2Fconversion%2Fpdf%3Flibrary%3DECHR%26id%3D003-6498862-8572168%26filename% 3DAudience%2520de%2520Grande%2520Chambre%2520Ukraine%2520c.%2520Russie%2 520%2528Crim%25E9e%2529.pdf&usg=AOvVaw3cuONJfE0Z8mCUmHUr-Nyi s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 Turciei nr. 25781/94, hot. din 12 .05.2014. Opinia comună a judecă­ torilor ZUPANČIČ, GYULUMYAN, DAVÍD THÓR BJÖRGVINSSON, NICOLAOU, SAJÓ, LAZAROVA TRAJKOVSKA, POWER-FORDE, VUČINIĆ ET PINTO DE ALBUQUERQUE. https://hudoc.echr.coe.int/fre#{%22itemid%22:[%22001-144153%22]} 17 CtEDO, cauza Slovenia c. Croatia nr. 5155/16, cerere din 15.09.2016. https://www.google. com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=2ahUKEwiPk5KDhYXnAhUlsa QKHRnJDicQFjABegQIBBAC&url=http%3A%2F%2Fhudoc.echr.coe.int%2Fapp%2Fconve rsion%2Fpdf%2F%3Flibrary%3DECHR%26id%3D003-6427775 8450864%26filename%3D Grand%2520Chamber%2520hearing%2520Slovenia%2520v.%2520Croatia.pdf&usg=AOvV aw15y2jR8ia5B23O6XFBC5v8 37 s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 care se află pe rolul Marii Camere, şi în 2018 cu privire la pretinsele deteriorări ale situaţiei drepturilor omului de-a lungul frontierei administrative dintre teritoriul controlat de Georgia, Abhazia şi Osetia de Sud. Începând cu anul 2014, Ucraina depune mai multe cereri împotriva Federaţiei Ruse şi în prezent pe rolul Curţii regăsim cinci cereri pendinte: două în faţa Marii Camere, una cu privire la situaţia din Crimeea şi alta la cea din partea de Est a Ucrai­ nei. Alte trei cereri sunt pendinte în faţa unei camere: una cu privire la presupusa răpire de copii din estul Ucrainei şi transferul lor temporar în Rusia în 2014, alta referitor la detenţia şi acuzarea resortisanţilor ucraineni pentru diferite infracţiuni şi una privind incidentul din strâmtoarea Kerch din noiembrie 2018, care a dus la capturarea a trei nave de război ucrainene şi a echipajului lor. Este interesantă abordarea Curţii în cererea Ucraina c. Federaţiei Ruse din 13 martie 2014, cu privire la măsurile provizorii cerute de guvernul ucrainean referitor la obligarea imediată a Federaţiei Ruse să se abţină de la orice intervenţie care ar constitui ameninţări la viaţa şi sănătatea populaţiilor civile de pe teritoriul lor, în special în Crimeea. Curtea Europeană a acceptat să ia măsuri provizorii conform articolului 39 al Regulamentului său, dar în acelaşi timp ea s-a adresat ambelor părţi contractante, atât Ucrainei cât şi Rusiei „să se abţină de a lua orice măsură şi, în special de natură militară, care ar putea implica pentru populaţia civilă, încălcări ale drepturilor garantate de Convenţie, inclusiv cele care ar putea pune în pericol viaţa şi sănătatea populaţiei civile“18. Pentru o gestionare mai eficientă a cazurilor interstatale rezultate din cererile formulate de Ucraina împotriva Federaţiei Ruse, Curtea a decis anul trecut să soluţioneze toate plângerile referitoare la evenimentele din Crimeea în contextul cauzei nr. 20958/14 şi toate reclamaţiile referitoare la evenimentele din estul Ucrainei şi la Donbass în contextul cauzei Ucraina v. Rusia (V), nr. s t u di i j u r i dic e u n i v e r si ta r e • Nr. 1 / 2021 8019/16.l Aflându-ne deci în câmpul recursului interstatal, putem spune că respectarea drepturilor omului este o bază esenţială a existenţei societăţilor democratice care sunt mai bine plasate decât alte regimuri pentru a evita conflictele în cadrul statelor Consiliului Europei, acestea având legături strânse între ele prin acorduri de ordin politic, economic şi social astfel încât să se asigure o veritabilă ordine europeană. 38
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Regulatory and Mechanistic Actions of Glucocorticoids on T and Inflammatory Cells
Frontiers in endocrinology
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Regulatory and Mechanistic Actions of Glucocorticoids on T and Inflammatory Cells Ana C. Liberman1†, Maia L. Budziñski1†, Clara Sokn1, Romina Paula Gobbini1, Anja Steininger1 and Eduardo Arzt1,2* Ana C. Liberman1†, Maia L. Budziñski1†, Clara Sokn1, Romina Paula Gobbini1, Anja Steininger1 and Eduardo Arzt1,2* 1 Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA) – CONICET – Partner Institute of the Max Planck Society, Buenos Aires, Argentina, 2 Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina Glucocorticoids (GCs) play an important role in regulating the inflammatory and immune response and have been used since decades to treat various inflammatory and autoim- mune disorders. Fine-tuning the glucocorticoid receptor (GR) activity is instrumental in the search for novel therapeutic strategies aimed to reduce pathological signaling and restoring homeostasis. Despite the primary anti-inflammatory actions of GCs, there are studies suggesting that under certain conditions GCs may also exert pro-inflammatory responses. For these reasons the understanding of the GR basic mechanisms of action on different immune cells in the periphery (e.g., macrophages, dendritic cells, neutro- phils, and T cells) and in the brain (microglia) contexts, that we review in this chapter, is a continuous matter of interest and may reveal novel therapeutic targets for the treatment of immune and inflammatory response. INTRODUCTION Living organisms must sustain a dynamic equilibrium in order to maintain homeostasis and survival which is constantly challenged by internal or external stressors. In order to appropriately cope with stressful stimuli, they have developed a highly conserved regulatory system. This neuroendocrine system consists mainly of the hypothalamic–pituitary–adrenal (HPA) axis and the autonomic nerv- ous system. Glucocorticoids (GCs), are the end-product of the HPA axis, and play an important role in the maintenance of both resting and stress-related responses. If the stress response is dysregu- lated, homeostasis is altered and probably a wide range of adverse effects may appear on many vital physiological functions, such as growth, development, metabolism, reproduction, immune response, cognition, and behavior. †These authors have contributed equally to this work. Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Specialty section: This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology Received: 12 January 2018 Accepted: 25 April 2018 Published: 16 May 2018 GCs act on almost all types of cells and in particular in the immune cells they have been shown to have powerful immunosuppressive and anti-inflammatory activities (1–5). As a result of their anti-inflammatory properties, GCs are widely used to help treat many different conditions, such as allergic, autoimmune, inflammatory, and hematological alterations. Interestingly, an accumulating body of evidence now strongly suggests that GCs can have both pro- and anti-inflammatory roles under specific conditions. The pro-inflammatory activity of GCs is most apparent in the central nervous system (CNS). These opposite effects work together in order to resolve cellular responses to inflammatory stimuli and also as a protective mechanism “priming” the immune cells to efficiently respond to the noxa or stressor and then restore homeostasis (6). Keywords: glucocorticoids, inflammation, FKBP51, transactivation, transrepression Edited by: Ana Rosa Pérez, CONICET, Argentina Reviewed by: Tullio Florio, Università di Genova, Italy Luciano David D’Attilio, CONICET Rosario, Argentina *Correspondence: Eduardo Arzt earzt@iboba-mpsp-conicet.gov.ar †These authors have contributed equally to this work. Edited by: Ana Rosa Pérez, CONICET, Argentina Reviewed by: Tullio Florio, Università di Genova, Italy Luciano David D’Attilio, CONICET Rosario, Argentina Keywords: glucocorticoids, inflammation, FKBP51, transactivation, transrepression Review published: 16 May 2018 doi: 10.3389/fendo.2018.00235 Citation: Liberman AC, Budziñski ML, Sokn C, Gobbini RP, Steininger A and Arzt E (2018) Regulatory and Mechanistic Actions of Glucocorticoids on T and Inflammatory Cells. Front. Endocrinol. 9:235. doi: 10.3389/fendo.2018.00235 May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org 1 GC-Mediated Immune Regulation Liberman et al. nuclear localization signals, which are important for GR nuclear translocation. The DBD also contains the nuclear export signal sequence (NES) which targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex. Upon peripheral or cerebral immune stimulation, the HPA axis is activated. When the immunogenic stress occurs in the brain, local inflammatory components activate the HPA axis. However, if the challenge takes place outside the brain, multiple pathways bring together stimulatory signals from the periphery to the HPA axis. Mounting evidence suggests that cytokine signals access to the brain through different pathways. These pathways mainly include: cytokines passing across the blood–brain barrier; by specific saturable transport molecules on the brain endothelium; activation of endothelial cells of brain capillaries that release second messengers within the brain parenchyma; transmission of cytokine signals via afferent nerve fibers and finally by periph- erally activated monocytes that can enter into the brain (7–11). The induction of these different mechanisms modulates cytokine activity in the brain (12–14).h Some degree in the heterogeneity in GR proteins may result from alternative splicing (30) (Figure 1B). The specificity and sensitivity of different target tissues to GCs has been reported to be related to GR isoforms (30). The GRα is the predominant isoform, and it is the one that transduces GCs signaling in the cell (31). There are other four additional splice variants identified: GRβ, GRγ, GR-A, and GR-P. GRβ differs from GRα in the carboxy terminal sequence, rendering GRβ non-responsive to GCs (32, 33), with no transcription of target genes. Therefore, GRβ can be described as a dominant negative inhibitor of GRα activity. GRβ does not bind GC agonists, however, it does bind to the GR antag- onist RU-486 (34). GRβ can inhibit GRα transcriptional activity by different molecular mechanisms including competition for glucocorticoid response elements (GRE), interference with the activity of coregulators, and formation of inactive dimers (35, 36). In most tissues, GRβ is expressed at very low levels. Citation: However, abundant GRβ expression has been described especially in some inflammatory cells, such as lymphocytes and macrophages, and have been related to GCs resistance in diseases such as asthma (37), rheumatoid arthritis (38), ulcerative colitis (39), systemic lupus erythematosus (40), and acute lymphoblastic leukemia and chronic lymphocytic leukemia (41, 42). Considering that GRβ can inhibit GRα activity, the modulation of GRα/GRβ expression ratios may be an interesting approach to regulate GC sensitiv- ity (42, 43). In addition, eight alternative translation initiation sites increase the repertory of GR proteins to almost 40 distinct isoforms of GR protein (44) (Figure 1B). The accurate regulation of the HPA axis activity is critical, since GC imbalances can result in many different pathological conditions (13, 15). Long-term treatment with GCs may result in a plethora of harmful undesired side effects, such as diabetes, hypertension, growth retardation, dyslipidemia, osteoporosis, glaucoma, muscle atrophy, and is also related to many important behavioral alterations, among others (16, 17). Chronic exposure to GCs can also be associated with GC insensitivity, reducing the efficacy of the therapy (18). Also, alterations or deficits in the HPA axis response are tightly associated with a wide range of autoim- mune and inflammatory diseases (19–24). l In this review, we will discuss the role of GCs on the immune and inflammatory cells in the periphery and also the physiologi- cal importance and mechanisms implicated in the apparent para- doxical functions of GCs in the brain in order to appropriately maintain a coordinated homeostatic response. At the cellular level, GC availability is also modulated by enzymes of the 11β-hydroxysteroid dehydrogenase (11β-HSD) family, mainly 11β-HSD1 and 11β-HSD2 which regulate the conversion of active cortisol into inactive cortisone. 11β-HSD1 favors the conversion of cortisol from cortisone, increasing local GC activity (45). In contrast, 11β-HSD2 catalyzes cortisol to cortisone, thereby reducing GC availability. Thus, the balance in the expression of these two enzymes in a given tissue or cell, regulates GC-mediated responses. In addition, some studies show that inflammatory cytokine signaling modulates the relative expression of 11β-HSD genes, favoring 11β-HSD1 and inhibiting 11β-HSD2 (46, 47), adding another level of regulation of GC activity.i Frontiers in Endocrinology  |  www.frontiersin.org THE GLUCOCORTICOID RECEPTOR (GR) As a small lipophilic hormone, GCs can rapidly diffuse into cells and exert their main actions. These actions are elicited by the binding of GCs to their intracellular receptor, the GR. The GR is a hormone-activated transcription factor (TF) that belongs to the superfamily of nuclear hormone receptors (25). GR is a modular protein composed of three distinct regions with different func- tions (Figure  1A). The N-terminal domain (NTD) contains a transactivation domain called activation function 1 (AF1) that is responsible for the transcriptional activation and is implicated in the association with coregulators and the basal transcription machinery. The DNA-binding domain (DBD) is composed of two zinc fingers that have been shown to be important for GR homodimerization and DNA-binding specificity. The hinge region, which separates the DBD from the ligand binding domain (LBD), is a flexible linker structure which is implicated in allow- ing proper DNA binding, dimerization, and nuclear translocation of the receptor (26). The C-terminal LBD, contains the ligand binding site and a second transactivation domain (AF2) regulated by hormone binding (27). The AF2 transactivation domain is important for the interaction with co-chaperones, coregulators, and other TFs (28). The LBD also encompasses a dimer interface which is critical for GR function and the binding of the heat shock protein (Hsp) 90 (29). The DBD and LBD both contain y Another important level for fine-tuning the cellular response to GCs in different environmental situations is the modulation of GR activity by posttranslational modifications (PTMs). These PTMs include phosphorylation, acetylation, ubiquitination, and sumoylation, which may accurately regulate GR activity in response to diverse external stimuli (48) (Figure  1A). In particular, SUMO conjugation has been extensively described to modulate GR transcriptional activity (49–52). GR contains three consensus sumoylation sites. Two sumoylation sites located at the NTD have been demonstrated to be part of the synergy control (SC) motif sequence (50). The SC motifs consist of short regulatory sequences which are important for inhibiting the synergistic transactivation. SUMO conjugation to the two May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org 2 GC-Mediated Immune Regulation Liberman et al. Figure 1 | The glucocorticoid receptor (GR) structure, isoforms, and mechanisms of transcriptional regulation. (A) Full human GRα protein has an N-terminal domain (NTD), a DNA-binding domain (DBD), a ligand binding domain (LBD) and a hinge region (H) between DBD and LBD. THE GLUCOCORTICOID RECEPTOR (GR) They have different associated functions, e.g., transactivation, dimerization, nuclear localization, DNA binding, and heat-shock protein 90 binding. The receptor can be post-translationally modified by phosphorylation (P), ubiquitination (U), acetylation (A) and sumoylation (S). Regions associated with transactivation (activation function 1 and 2: AF1 and AF2) are shown. (B) The GR has various isoforms which result from alternative splicing and multiple transcriptional start sites at exon 2. The colors indicate NTD (red, exon 2 DBD (green, exons 3–4), H (light blue, exon 5) and LBD (dark blue, exon 5–9). The 5′ and 3′-untranslated regions are colored in gray. There are five patterns of alternative splicing that result in GR isoforms α, β, P, γ, A. Each of them has eight translational variants (A, B, C1, C2, C3, D1, D2, D3) depending on the transcriptional start site (“*” denotes an alternative splice donor site in the intron between exons 3 and 4). (C) The GR, carrying GC ligand, translocates to the nucleus and regulates gene expression. GR can directly activate/inactivate gene expression by interacting with GREs/nGREs, it can bind to GREs and modulate gene transcription by interacting with neighboring DNA-bound transcription factors (TFs) (composite mechanism) and it can act by attaching itself to DNA-bound TFs (tethering mechanism). Abbreviations: TF, transcription factor; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; TFRE, transcription factor response element. Liberman et al. GC-Mediated Immune Regulat Figure 1 | The glucocorticoid receptor (GR) structure, isoforms, and mechanisms of transcriptional regulation. (A) Full human GRα protein has an N-terminal domain (NTD), a DNA-binding domain (DBD), a ligand binding domain (LBD) and a hinge region (H) between DBD and LBD. They have different associated functions, e.g., transactivation, dimerization, nuclear localization, DNA binding, and heat-shock protein 90 binding. The receptor can be post-translationally modified by phosphorylation (P), ubiquitination (U), acetylation (A) and sumoylation (S). Regions associated with transactivation (activation function 1 and 2: AF1 and AF2) are shown. (B) The GR has various isoforms which result from alternative splicing and multiple transcriptional start sites at exon 2. The colors indicate NTD (red, exon 2), DBD (green, exons 3–4), H (light blue, exon 5) and LBD (dark blue, exon 5–9). The 5′ and 3′-untranslated regions are colored in gray. There are five patterns of alternative splicing that result in GR isoforms α, β, P, γ, A. GCs ANTI-INFLAMMATORY ACTIONS The GR forms complex with chaperone molecules, such as Hsp90 and 70, and immunophilins, such as FKBP51, FKBP52, Cyp44, and PP5 (63). FKBP51 binds to the unbound GR and reduces GR activity mainly by reducing GR hormone binding and its nuclear translocation. Therefore, FKBP51 is considered as an inhibitor of GR transcriptional activity. Upon ligand binding, the GR exchanges FKBP51 for FKBP52, which is able to interact with the dynein motor protein, facilitating GR translocation to the nucleus (64). Interestingly, FKBP51 overexpression has been associated with GC resistance in autoimmune diseases. FKBP51 expression was found to be enhanced in sputum samples from patients with chronic obstructive pulmonary disease (65). Moreover, in a genome-wide profiling focused on the iden- tification of epithelial gene markers of asthmatic patients and response to corticosteroids, GC treatment was found to induce FKBP51 expression, which in turn was associated with a poor response to corticosteroids, suggesting a role of FKBP51 in GC resistance (66, 67). Also, enhanced expression of FKBP51 has been found in bone marrow cells in patients with rheumatoid arthritis (68). Evidence also suggests that FKBP51 modulates NFĸB-dependent gene expression, with possible implications for various inflammatory and immune pathways (69–73). Considering that GR is a key modulator of immune and inflam- matory responses, FKBP51 dysregulation may provide the basis for a role of FKBP51 in these processes (66). Moreover, FKBP51 has recently been shown to be a target of SUMO conjugation and that sumoylation of FKBP51 is necessary for its association to Hsp90 and modulates FKBP51-mediated inhibition of GR activ- ity in neuronal cells (58). In the brain, FKBP51 has been shown to be important for the development of psychiatric diseases and the response to antidepressant treatment, suggesting that regulation of FKBP51 activity might be an interesting approach for modulating GR outcome in the stress response and also in the inflammatory context (74–76). l It has been shown that several of the undesirable metabolic side effects associated with chronic GC treatment are mediated via transactivation. However the anti-inflammatory effects of GCs are mainly mediated via the transrepression elicited by a mono- meric GR with the activity of TFs, such as NFĸB and AP1 (1–3, 85). These TFs are involved in the activation of pro-inflammatory and immunoregulatory genes, such as inflammatory cytokines, cytokine receptors, adhesion molecules, and chemotactic pro- teins that play a key role for the coordination of the inflamma- tory response (1, 86–88). THE GLUCOCORTICOID RECEPTOR (GR) A genome-wide analysis of GR sumoylation impact on gene expression, showed that genes differentially regulated by this PTM are mostly related to proliferation and apoptosis pathways and also strongly suggests that sumoylation can regulate genome-wide chromatin occupancy of the GR (56). Also, GR SUMO conjugation is influenced by other PTMs such as phosphorylation in order to fine-tune GR transcriptional activity in a target gene-specific manner (57). Important coregulators of the GR are also modified by SUMO conjugation, such as Hsp90, GRIP1, and also FKBP51, further regulating GR activity (58–62). Therefore, PTMs that impact on the GR but also on key molecules that fine-tune its activity, helps to understand the complexity of GR-mediated regulation of its target gene expression (2, 48). Once in the nucleus, the activated GR can regulate gene expression by different mechanisms known as genomic effects (Figure  1C) (27). The genomic mechanism involves changes in the levels of specific genes: binding of GR to GREs in the promoters of its target genes and activation of transcription (transactivation); DNA binding of the GR with other TFs to “composite” elements which contain a GRE and an overlapping response element of another TF (binding can lead to gene activa- tion or repression); or binding of the GR to a TF (e.g., NFĸB; or AP1) by means of a “tethering” mechanism without contacting DNA, to influence the activity of the TF (this mechanism is considered to be the prevailing mechanism for transrepression) (2, 77, 78). Furthermore, GR-mediated transcriptional repression can be exerted via GR binding to a negative GRE (nGRE) (79). Binding to these nGRE prevent receptor dimerization through a strong negative cooperativity and alters the conformation of GR residues that are critical for transcriptional activation so that negative regulation is accomplished (80). A growing body of evidence shows that GC can also mediate non-genomic actions that do not require protein synthesis and are implicated in rapid cellular responses. For example, in the cytoplasm the activated GR can acutely interact with signaling pathways, such as PI3K, JNK, 14-3-3 proteins, and components of the T  cell receptor signaling complex (81), modulating pro-inflammatory gene expression. In thymocytes, the activated GR can translocate to mitochondria and induce a rapid apoptotic response (82). In addition, membrane-bound GR on monocytes was reported to mediate non-genomic effects (82). THE GLUCOCORTICOID RECEPTOR (GR) On the other hand, binding of GCs to GR can modify the recruitment of different factors such as the multiprotein chaperone complex that participate in many signaling pathways, modifying secondary signaling cascades and, therefore, may further regulate the immune response (78, 83). GCs may also exert anti-inflammatory responses by direct nega- tive interaction with components of the MAPK pathway, such as ERK, c-Jun NH2-terminal kinases (JNK), and p38 isoforms (p38) regulating their activity (84). Further studies are required to clarify the implications of non-genomic GC-mediated activity in the immune and inflammatory context. THE GLUCOCORTICOID RECEPTOR (GR) Each of them has eight translational variants (A, B, C1, C2, C3, D1, D2, D3) depending on the transcriptional start site (“*” denotes an alternative splice donor site in the intron between exons 3 and 4). (C) The GR, carrying GC ligand, translocates to the nucleus and regulates gene expression. GR can directly activate/inactivate gene expression by interacting with GREs/nGREs, it can bind to GREs and modulate gene transcription by interacting with neighboring DNA-bound transcription factors (TFs) (composite mechanism) and it can act by attaching itself to DNA-bound TFs (tethering mechanism). Abbreviations: TF, transcription factor; GRE, glucocorticoid response element; nGRE, negative glucocorticoid response element; TFRE, transcription factor response element. May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org Liberman et al. GC-Mediated Immune Regulation NTD sumoylation sites is responsible for the functional effect of the SC motifs and thereby they inhibit GR activity (50, 53) (Figure 1A). It has also been demonstrated that in the presence of the sumoylation enhancer, RSUME (54), a SUMO peptide is conjugated to the third sumoylation site located in the LBD of the GR. Sumoylation in the LBD may be important for inducing GR-mediated transcriptional regulation during stress adaptation (55) (Figure 1A). A genome-wide analysis of GR sumoylation impact on gene expression, showed that genes differentially regulated by this PTM are mostly related to proliferation and apoptosis pathways and also strongly suggests that sumoylation can regulate genome-wide chromatin occupancy of the GR (56). Also, GR SUMO conjugation is influenced by other PTMs such as phosphorylation in order to fine-tune GR transcriptional activity in a target gene-specific manner (57). Important coregulators of the GR are also modified by SUMO conjugation, such as Hsp90, GRIP1, and also FKBP51, further regulating GR activity (58–62). Therefore, PTMs that impact on the GR but also on key molecules that fine-tune its activity, helps to understand the complexity of GR-mediated regulation of its target gene expression (2, 48). NTD sumoylation sites is responsible for the functional effect of the SC motifs and thereby they inhibit GR activity (50, 53) (Figure 1A). It has also been demonstrated that in the presence of the sumoylation enhancer, RSUME (54), a SUMO peptide is conjugated to the third sumoylation site located in the LBD of the GR. Sumoylation in the LBD may be important for inducing GR-mediated transcriptional regulation during stress adaptation (55) (Figure 1A). Frontiers in Endocrinology  |  www.frontiersin.org GCs ANTI-INFLAMMATORY ACTIONS By all these different mechanisms, GCs regulate important functions, not only in the periphery but also in the brain.t GCs can regulate the maturation, survival, and migration toward the lymph nodes and motility of dendritic cells (DCs), and also inhibit their immunogenic functions (Figure 2). GCs were shown to reduce the ability of DCs to stimulate T cells by inhibiting the upregulation of co-stimulatory molecules and cytokines, such as IL6, IL12, and TNFα and by inducing the tolerance-inducing transcription factor GILZ (119–125). The distinct actions exerted by GCs in immature and mature DCs are due to differential expression of GR translational isoforms (126). Synthetic analogs of GC are often employed in the clinic in the therapy of allergic, inflammatory, and autoimmune disorders (97–99). It is generally accepted that GR-mediated transrepres- sion holds the beneficial anti-inflammatory action, whereas their side effects are due mainly to the direct binding of GR to GREs as depicted before (98–100). However, transactivation is also necessary for the induction of several anti-inflammatory genes, such as MAP kinase phosphatase 1 (101), glucocorticoid-induced leucine zipper (102), and inhibitor kappa B-alpha (IĸBα) (85). Therefore, the ideal GC analogs should be those that have high repressive activity against inflammatory mediators, but low transactivation activity, causing minimal side effects. Several steroidal and nonsteroidal ligands have been reported to have this dissociated function between transactivation and transrepressive mechanisms (97–99, 103). These compounds were shown to repress the activity of key inflammatory and immune TFs in vivo (104–107). However, GCs can induce gene expression not only by binding to GRE, but also in combination with other TFs and also by binding to promoter regions in a mechanism that does not involve GR dimerization or DNA interaction; therefore, unexpected secondary side effects might appear (78).lf GCs are important modulators of neutrophilia (Figure  2). Leukocyte extravasation is the movement of leukocytes out of the circulation and toward the site of tissue damage or infection. Rolling, adhesion, activation, and transmigration are necessary to arrive to the damaged tissue. GCs can modulate each of these steps. Rolling and adhesion is mediated by the interaction of the leukocyte integrins with the endothelial counterparts, which are inhibited by GCs (127–129). Also, GCs increase the number of circulating neutrophils in the blood stream by favoring their egress from the bone marrow and also inhibiting their migration to inflammatory sites by hindering the expression of adhesion molecules (32, 129, 130). GCs ANTI-INFLAMMATORY ACTIONS The first example of the transrepressive mechanism was the inhibitory interaction described between GR and AP1 (89), which results in the inhibition of IL2 expression (90). NFĸB is present in almost all immune cells and regulates the expression of inflammatory cytokines. Thus, inhibition of NFĸB activity is an important feature for GR-mediated anti- inflammatory activity (85, 91). It also inhibits NFAT-dependent IL2 transcription (92). The main mechanism of the GR action over these TFs is via transrepression: the activated GR acts by binding proximal to the NFĸB or AP1-binding site and interacts Frontiers in Endocrinology  |  www.frontiersin.org May 2018  |  Volume 9  |  Article 235 4 GC-Mediated Immune Regulation Liberman et al. with these TFs inhibiting gene expression (93). The transrepres- sion mechanism is not restricted to these TFs, but has expanded including among others, CREB, STAT, and T-bet (1–3, 94). with these TFs inhibiting gene expression (93). The transrepres- sion mechanism is not restricted to these TFs, but has expanded including among others, CREB, STAT, and T-bet (1–3, 94). anti-inflammatory macrophages (111). The intrinsic molecular mechanism involves a prolonged induction of the extracellular signal-regulated kinase/MAPK (ERK/MAPK) pathway resulting in inhibition of caspase activities and expression of anti-apoptotic genes (111). GCs can also improve the phagocytic activity of these cells and stimulate the clearance of harmful elements, such as neutrophil clearance (112–114). GCs also suppress immunostim- ulatory functions of these cells and inhibit the release of various pro-inflammatory mediators, such as cytokines, chemokines, and reactive oxygen through different mechanisms (115, 116). Functional clustering of GC-regulated genes by human anti- inflammatory macrophages by microarray technology indicated induction of phagocytosis and motility as well as repression of adhesion, apoptosis, and oxidative burst (117, 118). Alterations in chromatin structure have been reported to be important for regulating GC actions. The GR can differentially interact with proteins that have histone acetyltransferase (HAT) activity, but also with histone deacetylases and kinases that can influence the chromatin environment modifying chromatin accessibility and further regulating immune and inflammatory gene expression (3). In addition, chromatin accessibility has been reported to pre-determine GR binding patterns and, therefore, is critical for cell-specific outcome, providing new molecular basis for the tissue selectivity (95, 96). GCs ANTI-INFLAMMATORY ACTIONS GCs exert distinct immunomodulatory actions on T  cells (Figure 2). GCs decrease the number of circulating T cells by favoring their migration back to the bone marrow and second- ary lymphoid tissues or through the induction of chemokine receptors, adhesion molecules, and matrix metalloproteinases (131, 132). The steroid hormone also favors T  cells apoptosis. GC-induced apoptosis of T cells requires the dimerization of the GR (133) and is mediated via the induction of Puma and Bim expression (134–137). The relative expression of distinct GR isoforms increases the susceptibility of T cells to GC-induced cell death (138). Helper T (Th) cells are important players of the adap- tive immunity (1). Upon antigen stimulation, naive Th cells can differentiate into different subsets: Th1, Th2, Th17, or regulatory T (Tregs) cells among others, each with specific effector functions. Th1 cells express the lineage-specific TF T-bet and STAT4 and release pro-inflammatory cytokines, such as IFNγ and IL2 (139). Th1 cells help in the activation of effector T cells, natural killer (NK)  cells, and macrophages at the site of infection, promote effective immune responses against intracellular pathogens and are also implicated in autoimmune pathologies. Th2 lymphocytes selectively express the TF GATA3 and are characterized by the expression of IL5, IL4, IL10, and IL13 and are important for the GCs may exert acute anti-inflammatory effects through the release of annexin-A1 (ANXA1) (108). Originally, this protein was suggested to have anti-inflammatory actions because it was described to inhibit phospholipase A2 (109). However, ANXA1 has been reported to regulate different cellular processes, such as migration, growth, differentiation, apoptosis, membrane fusion during exocytosis, lipid metabolism, and cytokine expression. Importantly, in the HPA axis, ANXA1 has been reported to play a critical role in the negative feedback exerted by GCs, therefore, affecting hypothalamic-releasing hormones secretion possibly via non-genomic mechanisms (110). GCs ACTIVITY ON PERIPHERAL IMMUNE CELLS They also suppress GATA3 activity in Th2 cells inhibiting the expression of IL-4 and IL-5. The action of GCs toward Th17 and regulatory T cells is not yet well understood. Figure 2 | Glucocorticoid (GC) activity on periphery immune cells. GCs act upon almost every immune cell type. GCs promote an anti-inflammatory state on both monocytes and macrophages. GCs prevent monocytes into entering apoptosis and inhibit the liberation of pro-inflammatory mediators by both types of cells. Particularly in macrophages, GCs promote phagocytosis and motility, while they inhibit adhesion, apoptosis and oxidative burst. They also act upon neutrophils function by inhibiting rolling, adhesion and activation. GCs act toward dendritic cells by promoting their maturation, survival, migration and motility, and at the same time GCs inhibit their ability to activate T cells by suppressing the production of pro-inflammatory molecules. A naïve helper T (Th) cell can differentiate into different Th lineages and GCs exert different actions. They act upon Th1 by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory molecules such as IL-2 and IFNγ. They also suppress GATA3 activity in Th2 cells inhibiting the expression of IL-4 and IL-5. The action of GCs toward Th17 and regulatory T cells is not yet well understood. (94) (Figure 2). Also GCs where shown to reduce mRNA and protein levels of T-bet (94). The activity of the Th2-specific TF GATA3 is also suppressed by GCs via two main mechanisms: first by GR-mediated inhibition of GATA3 translocation into the nucleus and second by the inhibition of GATA3 phospho- rylation by GC-induced MKP1 expression (147, 148) (Figure 2). Furthermore, STAT6 activity also involved in Th2 differentiation is inhibited by GCs (149). How GCs modulate Th17-mediated responses has not been extensively studied, and the importance of Th17 modulation by GCs for the suppression of allergic or autoimmune diseases remains unclear (150). In rheumatoid arthritis, GC treatment diminished IL17 levels (151). In addition, in rat lymphocytes methylprednisolone inhibited IL17 expres- sion due to the inhibition of RORγt expression (152) (Figure 2). However, several studies strongly suggest that GC resistance is associated with a pathogenic inflammatory Th17 phenotype that is refractory to GCs (150, 153, 154). Recently, a gene-expression profiling to characterize the steroid-resistant phenotype showed proper eradication of extracellular pathogens (140). Also, Th2 cells activate B cells to produce antibodies and play a triggering role in the activation/recruitment of eosinophils and mast cells in allergic responses. GCs ACTIVITY ON PERIPHERAL IMMUNE CELLS GCs mediate immunosuppressive functions by acting on almost all types of immune cells (Figure 2). GCs can regulate the phe- notype, survival, and functions of monocytes and macrophages which have crucial roles in tissue homeostasis and innate immu- nity. GCs exhibit anti-apoptotic effects promoting the survival of May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org 5 GC-Mediated Immune Regulation Liberman et al. Figure 2 | Glucocorticoid (GC) activity on periphery immune cells. GCs act upon almost every immune cell type. GCs promote an anti-inflammatory state on both monocytes and macrophages. GCs prevent monocytes into entering apoptosis and inhibit the liberation of pro-inflammatory mediators by both types of cells. Particularly in macrophages, GCs promote phagocytosis and motility, while they inhibit adhesion, apoptosis and oxidative burst. They also act upon neutrophils function by inhibiting rolling, adhesion and activation. GCs act toward dendritic cells by promoting their maturation, survival, migration and motility, and at the same time GCs inhibit their ability to activate T cells by suppressing the production of pro-inflammatory molecules. A naïve helper T (Th) cell can differentiate into different Th lineages and GCs exert different actions. They act upon Th1 by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory molecules such as IL-2 and IFNγ. They also suppress GATA3 activity in Th2 cells inhibiting the expression of IL-4 and IL-5. The action of GCs toward Th17 and regulatory T cells is not yet well understood. Figure 2 | Glucocorticoid (GC) activity on periphery immune cells. GCs act upon almost every immune cell type. GCs promote an anti-inflammatory state on both monocytes and macrophages. GCs prevent monocytes into entering apoptosis and inhibit the liberation of pro-inflammatory mediators by both types of cells. Particularly in macrophages, GCs promote phagocytosis and motility, while they inhibit adhesion, apoptosis and oxidative burst. They also act upon neutrophils function by inhibiting rolling, adhesion and activation. GCs act toward dendritic cells by promoting their maturation, survival, migration and motility, and at the same time GCs inhibit their ability to activate T cells by suppressing the production of pro-inflammatory molecules. A naïve helper T (Th) cell can differentiate into different Th lineages and GCs exert different actions. They act upon Th1 by decreasing T-bet transcriptional activity and suppressing the production of pro-inflammatory molecules such as IL-2 and IFNγ. Frontiers in Endocrinology  |  www.frontiersin.org GCs ACTIVITY ON PERIPHERAL IMMUNE CELLS IL17-producing Th17 cells selectively express RORγt and also RORα (141, 142). Th17 cells play an important role in autoimmune diseases and in host defense against infec- tion. Treg cells mainly express the TF Foxp3 and inhibit effector T-cell differentiation and proliferation and suppress autoimmune and allergic responses (143). GCs inhibit the expression of many T cell cytokines (1) and can produce a shift from Th1-mediated cellular immunity to mediating humoral Th2 responses at physi- ological doses or chronic treatment (144). Upon acute treatment with GCs, they inhibit the synthesis of Th1 cytokines like IL2 and IFNγ and reduce STAT4 activity (145) and also reduce Th2 cytokines expression (146). The molecular mechanism by which GCs inhibit Th1 responses involves the reduction of T-bet transcriptional activity by the inhibitory interaction between GR and T-bet that results in diminished binding of T-bet to DNA May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org 6 GC-Mediated Immune Regulation Liberman et al. by intraparenchymal endotoxin injection (174). Mice treated with the GR antagonist RU486 before intracerebral LPS administration showed an increase in the pro-inflammatory response, which in turn induced neuronal death. These findings suggest that GCs are important for protecting the brain during innate immunity (175, 176). Interestingly, when mice lacking GR in microglia were chal- lenged with an intracerebral administration of LPS, the activation of the toll-like receptor 4 signaling pathway induced cellular lesion, and also neuronal and axonal damage (177). In addition, microglial cell cultures have reduced motility and increased amoeboid morphology in the absence of GR expression. This study strongly suggests that microglial GR is the principal media- tor preventing neuronal degeneration triggered by LPS and that it also contributes to the protection of other cell types (177), having an important role in promoting neuronal survival.hl that Th17 cells have restricted genome-wide responses to GCs and that they are refractory to GC inhibition at this level. In addi- tion, Th17 cells were sensitive to suppression with the calcineurin inhibitor, cyclosporine A, suggesting that the clinical efficacy of cyclosporine A in the treatment of steroid resistance may be due to its selective inhibition of Th17 cells (155). Another interesting study has shown that Th17 cells are insensitive to GC-induced apoptosis and had high levels of BCL-2, knockdown of which sen- sitized Th17 cells to GC-induced cell death (156). GCs ACTIVITY ON PERIPHERAL IMMUNE CELLS Also, lung Th17 development in the murine severe asthma model was enhanced by GCs, supporting a role of Th17 cells in GC-refractory inflam- matory conditions such as asthma (157).fl In contrast to the inhibitory effect of GCs on pro-inflamma- tory effector T  cells, it has been shown that Treg cells, which are key suppressors of T cell-dependent immune responses, are enhanced upon dexamethasone treatment by being more resist- ant to GC-induced cell death (158) (Figure 2). Also, GCs where shown to amplify IL2-dependent expansion of Treg cells and to enhance their capacity to reduce experimental autoimmune encephalomyelitis (EAE) in mice (159). In addition, GCs increase the percentage of Treg cells that express Foxp3 in patients with multiple sclerosis (160). In vivo, T cell-specific targeted GR dele- tion in pregnant animals undergoing EAE, resulted in a reduction of Treg population and a loss of pregnancy-induced protection, suggesting that steroid hormones can shift the immunological balance in favor of Tregs via differential engagement of the GR in T cells (161). However, others have found that GC treatment suppresses the expression of Foxp3 Tregs in an EAE model (162) and also in lungs of allergic mice (163).l The majority of GC pro-inflammatory activity has been described in animal models of acute or chronic stress which occurred previous to peripheral or cerebral immune challenges. For instance, acute stressors were reported to induce the expres- sion of pro-inflammatory cytokines in specific brain regions, such as the hippocampus, following LPS peripheral challenge (178–180). GCs were also found to upregulate microglial activa- tion markers including the toll-like receptor 2 pro-inflammatory pathway (178, 181) (Figure 3A). It was also shown that chronic unpredictable stress was able to potentiate LPS-mediated activa- tion of NFĸB activity in the frontal cortex and hippocampus via GC production (182). Also, chronically stressed animals that were Figure 3 | Glucocorticoids (GCs) actions in the brain. Acute stressors promote an inflammatory phenotype in the brain. (A) In the microglia, GCs bind to the glucocorticoid receptor (GR) which then promotes the translation of the toll-like receptor 2 (TLR2) by interacting with STAT5 and NFƙB response elements. TLR2 then exerts a pro-inflammatory response by promoting the production of inflammatory cytokines. (B) In macrophages, GCs promote the expression of the purinergic receptor P2Y2R which then produces IL-6 in response to ATP. Moreover, GCs enhance the expression of NLRP3 which in turn promotes the production of pro-inflammatory cytokines. GCs ACTIVITY ON PERIPHERAL IMMUNE CELLS In addition to their well-studied anti-inflammatory and immunosuppressive activity, an increasing body of evidence has revealed situations in which GCs have the opposite effect. This has been shown to depend on the dose, timing, duration of exposure, and cell population or tissue analyzed (164). The paradoxical pro- inflammatory role of GCs is mostly evident in the brain, where accumulating evidence show that GCs elicit different immune responses depending on the affected brain regions. Frontiers in Endocrinology  |  www.frontiersin.org CONCLUSION GCs are widely used in the clinic to control not only peripheral, but also CNS inflammatory response. However, the prolonged administration of this steroid hormone is often ineffective and can even worsen the outcome of the disease. Considering the known undesirable metabolic side effect, the induction of pro- inflammatory responses and the existence of GC resistance, GCs should be used carefully. Future research should be focused not only in understanding the molecular basis of GCs side effects and resistance, but also in dissecting how GCs induce pro- inflammatory responses in order to avoid serious detrimental consequences, particularly in the brain. In the future, a combi- nation of different therapeutic approaches may lead to a more effective treatment and may help to lower the doses or duration of GC treatment thus minimizing the risk of toxicity and drug resistance (196). Finally, taking into account inter-individual differences in patient responsiveness to GC treatment, where different molecular mechanisms might be implicated, future directions should be in support of a customized and personal- ized treatment to meet individual patient needs. 3. Ratman D, Vanden Berghe W, Dejager L, Libert C, Tavernier J, Beck IM, et al. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering. Mol Cell Endocrinol (2013) 380:41–54. doi:10.1016/j.mce.2012.12.014 FUNDING This work was supported by grants from the Max Planck Society, Germany; the University of Buenos Aires; CONICET; the Agencia Nacional de Promoción Científica y Tecnológica, Argentina; and FOCEM-Mercosur (COF 03/11). diseases. Microbiol Mol Biol Rev (2016) 80:495–522. doi:10.1128/MMBR. 00064-15 GCs ACTIONS IN THE BRAIN There is a significant body of evidence indicating that GCs can suppress the innate immunity in the brain after a peripheral or cerebral challenge (23). In this way, in adrenalectomized mice, there is an induction in the levels of pro-inflammatory cytokines in the brain following LPS injection (165–168). Studies also dem- onstrated that GCs inhibit the release of pro-inflammatory media- tors in microglial cells treated with LPS (169, 170). Experiments performed in vivo support these findings by revealing that dexa- methasone causes a strong reduction in LPS induction of NFĸB expression in the brain (171). In addition, COX inhibitors were demonstrated to increase the expression of pro-inflammatory genes in the brain during systemic inflammation by reducing the activation of the HPA axis and the release of GCs (172, 173). This same effect took place when the GR antagonist RU486 was administrated (172, 173). Also, systemic inflammation, through the increase in circulating GCs, has been reported to have the ability to prevent the cerebral innate immune response induced Figure 3 | Glucocorticoids (GCs) actions in the brain. Acute stressors promote an inflammatory phenotype in the brain. (A) In the microglia, GCs bind to the glucocorticoid receptor (GR) which then promotes the translation of the toll-like receptor 2 (TLR2) by interacting with STAT5 and NFƙB response elements. TLR2 then exerts a pro-inflammatory response by promoting the production of inflammatory cytokines. (B) In macrophages, GCs promote the expression of the purinergic receptor P2Y2R which then produces IL-6 in response to ATP. Moreover, GCs enhance the expression of NLRP3 which in turn promotes the production of pro-inflammatory cytokines. May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org 7 GC-Mediated Immune Regulation Liberman et al. injected with LPS in the prefrontal cortex or the hippocampus, exhibited microglia activation, an increase in pro-inflammatory mediators and loss of astroglia and neurons. These effects were reduced with RU486 administration (183, 184). The prefrontal cortex is important in many brain functions and is a target for neurodegenerative diseases. It has been reported that in this brain region, TNFα expression and activation of MAPK signal- ing pathway is upregulated by chronic stress after intracortical LPS injection in a GR-dependent manner suggesting a syner- gistic effect between inflammation and stress. This fact could ultimately explain the relationship described between stress and some neurodegenerative pathologies (183, 184). GCs ACTIONS IN THE BRAIN For example, GR activation during chronic stress increases LPS-induced NFκB activation and TNFα, IL1β, and iNOS expression in the hippocampus and frontal cortex, AUTHOR CONTRIBUTIONS AL: wrote, discussed, and corrected the manuscript. MB: discussed and corrected the manuscript. CS: discussed and cor- rected the manuscript, performed the figures. RG: discussed and corrected the manuscript, performed the figures. AS: corrected the manuscript. EA: discussed and corrected the manuscript. 4. Refojo D, Liberman AC, Holsboer F, Arzt E. Transcription factor-mediated molecular mechanisms involved in the functional cross-talk between GCs ACTIONS IN THE BRAIN In order to investigate if stress-induced GCs is responsible for the response of brain immune cells to pro-inflammatory stimuli, animals were acutely stressed and 24 h later hippocampal microglia were challenged with LPS ex vivo. Treatment in vivo with RU486 and adrenalectomized inhibited the microglial pro-inflammatory response, indicating that stress-induced GCs are able to sensitize the microglial pro-inflammatory function (185, 186). Therefore, stress may act “priming” central innate immunity to a subse- quent immune challenge by making the neuroimmune context more responsive to inflammation, also favoring GC insensitivity or reducing the HPA response (187). In addition, acute restraint stress, inescapable tail shock and other stressors induce many inflammatory mediators, reduce immunoregulatory proteins and trigger microglia activation and proliferation (188–193). In addition, GCs have been reported to increase the expression of the purinergic receptor P2Y2R (Figure 3B) which promotes the secretion of inflammatory mediators in response to ATP (194). Recent data also indicate that GCs induce the expression of NLRP3 (NLRP3: nucleotide-binding domain, leucine-rich- containing family, pyrin domain-containing 3) in macrophages, which is a critical component of the inflammasome (Figure 3B). The GC-dependent induction of NLRP3 sensitizes the cells to extracellular ATP and significantly enhances the ATP-mediated release of pro-inflammatory molecules. This effect was specific for GCs and dependent on the GR and suggests that GCs sen- sitize the initial inflammatory response in the context of acute cellular damage or death (32). In addition, GCs and TNFα were shown to coregulate immune gene expression when combined (195). These results suggest that the final outcome of GCs pro- or anti-inflammatory activity depends on the activation state and signaling context. GCs are also able to modulate the inflamma- tory response to LPS in different ways according to the brain region (180, 182). For example, GR activation during chronic stress increases LPS-induced NFκB activation and TNFα, IL1β, d iNOS i i h hi d f l but exhibits contrary effects in the hypothalamus (182). It is important to keep in mind that a pro-inflammatory context does not necessarily mean that damage will take place. Timing is a key parameter that will determine the final outcome of the inflammatory response. While exaggerated inflammation can favor neuronal dysfunction and cell death, pro-inflammatory mediators may at first induce the removal of the pathogen, the recruitment of immune cells and initiate tissue remodeling in order to appropriately cope with the pathogen and therefore, restoring homeostasis. GCs ACTIONS IN THE BRAIN injected with LPS in the prefrontal cortex or the hippocampus, exhibited microglia activation, an increase in pro-inflammatory mediators and loss of astroglia and neurons. These effects were reduced with RU486 administration (183, 184). The prefrontal cortex is important in many brain functions and is a target for neurodegenerative diseases. It has been reported that in this brain region, TNFα expression and activation of MAPK signal- ing pathway is upregulated by chronic stress after intracortical LPS injection in a GR-dependent manner suggesting a syner- gistic effect between inflammation and stress. This fact could ultimately explain the relationship described between stress and some neurodegenerative pathologies (183, 184). In order to investigate if stress-induced GCs is responsible for the response of brain immune cells to pro-inflammatory stimuli, animals were acutely stressed and 24 h later hippocampal microglia were challenged with LPS ex vivo. Treatment in vivo with RU486 and adrenalectomized inhibited the microglial pro-inflammatory response, indicating that stress-induced GCs are able to sensitize the microglial pro-inflammatory function (185, 186). Therefore, stress may act “priming” central innate immunity to a subse- quent immune challenge by making the neuroimmune context more responsive to inflammation, also favoring GC insensitivity or reducing the HPA response (187). In addition, acute restraint stress, inescapable tail shock and other stressors induce many inflammatory mediators, reduce immunoregulatory proteins and trigger microglia activation and proliferation (188–193). In addition, GCs have been reported to increase the expression of the purinergic receptor P2Y2R (Figure 3B) which promotes the secretion of inflammatory mediators in response to ATP (194). Recent data also indicate that GCs induce the expression of NLRP3 (NLRP3: nucleotide-binding domain, leucine-rich- containing family, pyrin domain-containing 3) in macrophages, which is a critical component of the inflammasome (Figure 3B). The GC-dependent induction of NLRP3 sensitizes the cells to extracellular ATP and significantly enhances the ATP-mediated release of pro-inflammatory molecules. This effect was specific for GCs and dependent on the GR and suggests that GCs sen- sitize the initial inflammatory response in the context of acute cellular damage or death (32). In addition, GCs and TNFα were shown to coregulate immune gene expression when combined (195). These results suggest that the final outcome of GCs pro- or anti-inflammatory activity depends on the activation state and signaling context. GCs are also able to modulate the inflamma- tory response to LPS in different ways according to the brain region (180, 182). Frontiers in Endocrinology  |  www.frontiersin.org diseases. Microbiol Mol Biol Rev (2016) 80:495–522. doi:10.1128/MMBR. 00064-15 3. Ratman D, Vanden Berghe W, Dejager L, Libert C, Tavernier J, Beck IM, et al. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering. Mol Cell Endocrinol (2013) 380:41–54. doi:10.1016/j.mce.2012.12.014 4. Refojo D, Liberman AC, Holsboer F, Arzt E. Transcription factor-mediated molecular mechanisms involved in the functional cross-talk between 1. Liberman AC, Druker J, Perone MJ, Arzt E. 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No use, distribution or reproduction is permitted which does not comply with these terms. May 2018  |  Volume 9  |  Article 235 Frontiers in Endocrinology  |  www.frontiersin.org 14
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Parametric Decay Instabilities during Electron Cyclotron Resonance Heating of Fusion Plasmas, Problems and Possibilities
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1 Introduction fusion research, decays to two daughter waves once the pump wave amplitude exceeds a threshold determined by the nonlinear interaction strength between the three waves. Energy conservation in the three-wave process requires the frequency, f, and wave vector, k, of the pump wave to equal the sum of those of the daughter waves, i.e., Parametric decay instabilities (PDIs) of millimeter wave radiation from gyrotron sources used for electron cy- clotron resonance heating (ECRH) of magnetically con- fined fusion plasmas have been a topic of significant inter- est in recent years. The main reason for this is that PDIs can generate strong microwave signals capable of dam- aging microwave diagnostics [1], as well as transfering a significant fraction of the ECRH power from electromag- netic waves to quasi-electrostatic plasma waves, altering the heating and current drive characteristics from those expected based on linear ECRH theory [2, 3]. However, the microwave signals caused by PDIs can also serve as plasma diagnostics, e.g., allowing the occurrence of O- X-B heating to be confirmed [4, 5] and the properties of edge-localized modes to be explored [6]. Additionally, the low-frequency waves produced by some PDIs also al- low generation of a moderate number of fast ions in pure ECRH plasmas [7–11]. In the present Paper, we provide an overview of the ECRH PDIs studied in recent years and assess their potential impact on the O-mode ECRH scenar- ios planned for ITER [12]. f0 = f1 + f2, k0 = k1 + k2, (1) (1) where subscript 0 refers to the pump wave, while sub- scripts 1 and 2 refer to the daughter waves. Equation (1) requires the daughter waves excited by PDIs to be shifted in frequency relative to the pump wave, meaning that their frequencies will often be outside the protective diagnostic filters around the ECRH frequency in the case of ECRH PDIs. This is the root cause of the microwave diagnos- tics damage associated with such PDIs [1]. Traditionally, magnetic confinement fusion ECRH PDIs were only ex- pected in connection with extremely high power ECRH provided by pulsed free-electron maser sources [13] or in scenarios with lower ECRH power where a significant fraction of the ECRH power would reach the upper hy- brid resonance (UHR) with X-mode polarization [4, 5, 9– 11, 14–30]. PDIs are expected in the above cases due to the occurrence of electric fields with large amplitudes. Parametric Decay Instabilities during Electron Cyclotron Resonance Heating of Fusion Plasmas, Problems and Possibilities S.K. Hansen1,∗, S.K. Nielsen2, J. Stober3, J. Rasmussen2, M. Salewski2, M. Willensdorfer3, M. Hoelzl3, the ASDEX Upgrade Team4 S.K. Nielsen2, J. Stober3, J. Rasmussen2, M. Salewski2, M. Willensdorfer3, M. Hoelzl3, M. Stejner2, and 4 1Plasma Science and Fusion Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2Department of Physics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark 3Max-Planck-Institut für Plasmaphysik, D-85748 Garching b. München, Germany 4See Author List in H. Meyer for the ASDEX Upgrade Team, Nucl. Fusion 59, 112014 (2019) 1Plasma Science and Fusion Center, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 2Department of Physics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark 3Max-Planck-Institut für Plasmaphysik, D-85748 Garching b. München, Germany 4See Author List in H. Meyer for the ASDEX Upgrade Team, Nucl. Fusion 59, 112014 (2019) Abstract. We review parametric decay instabilities (PDIs) expected in connection with electron cyclotron resonance heating (ECRH) of magnetically confined fusion plasmas, with a specific focus on conditions relevant for the ITER tokamak. PDIs involving upper hybrid (UH) waves are likely to occur in O-mode ECRH scenarios at ITER if electron density profiles allowing trapping of UH waves near the ECRH frequency are present. Such PDIs may occur near the plasma center in ITER full-field scenarios heated by 170 GHz O-mode ECRH and on the high-field side of half-field ITER plasmas heated by 110 GHz or 104 GHz O-mode ECRH. Additionally, 110 GHz O-mode ECRH of half-field ITER scenarios may have low ECRH absorption, due to the electron cyclotron resonance being located on the high-field side of the main plasma. This potentially allows PDIs driven by a significant amount of ECRH radiation reaching the UH resonance in X-mode to occur, as X-mode radiation can be generated by reflection of unabsorbed O-mode radiation from the high-field side wall. The occurrence of PDIs during ECRH may damage microwave diagnostics, such as the electron cyclotron emission and low-field side reflectometer systems at ITER, as well as complicate the calculation of heating and current drive characteristics. However, if PDIs are induced in a controlled manner, they may provide novel diagnostic tools and allow the generation of a moderate fast ion population in plasmas heated only by ECRH. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). EPJ Web of Conferences 277, 01002 (2023) EC21 EPJ Web of Conferences 277, 01002 (2023) EC21 EPJ Web of Conferences 277, 01002 (2023) https://doi.org/10.1051/epjconf/202327701002 ∗e-mail: soerenkh@psfc.mit.edu 2 Theoretical Background ITER is a large tokamak with minor radius a = 2 m and major radius R0 = 6.2 m [12]. The magnetic field strength, B, is approximately proportional to 1/R, with R being the distance from the symmetry axis. Thus, B ≈B0 R0 R , (2) (2) where B0 is the field at the magnetic axis; in full-field sce- narios, B0 = 5.3 T, while B0 = 2.65 T in half-field scenar- ios [12]. For quasi-perpendicular ECRH injection in the non-relativistic limit, absorption occurs when f0 is equal to multiples of the electron cyclotron frequency [61], [ , , , ], p [ , ] In spite of previous expectations, strong PDI-like mi- crowave signals were observed in connection with ECRH of plasma scenarios with tearing modes at the TEXTOR tokamak that did not fulfill any of the above criteria [37, 38]. This was explained in terms of ECRH PDIs in- volving trapped daughter waves [39], for which the con- vective losses normally limiting the occurrence of PDIs in inhomogeneous plasmas [31–33] are essentially removed. Specifically, the TEXTOR results at higher densities were explained in terms of decay of X-mode ECRH waves into two trapped UH waves near the O-point of the magnetic is- land [40–47]. PDIs of this kind have also been found at the ASDEX Upgrade tokamak [1, 6, 34], in low-temperature plasma filament experiments [2], and in particle-in-cell simulations [48–50]. The PDIs leading to microwave di- agnostics damage at ASDEX Upgrade [1] and significant anomalous X-mode ECRH absorption in low-temperature plasma filaments [2] were both of the above type. More recently, an explanation of strong microwave signals ob- served at the Wendelstein 7-X stellarator in terms of decay of an X-mode ECRH wave into a trapped UH wave and a propagating X-mode wave around the O-point of a divertor island [51, 52], has also provided a potential explanation of the strong microwave signals observed at lower densi- ties in TEXTOR [37, 38]. Apart from the PDIs driven by X-mode ECRH, PDIs driven by O-mode ECRH, relevant for the full-field ITER scenarios [12], have also been in- vestigated. Particularly, decay of an O-mode ECRH wave to a trapped UH wave and a low-frequency lower hybrid wave has been investigated theoretically [53–56], in a low- temperature plasma filament [3], and at the FTU tokamak [57–59]. 1 Introduction This is due to the fact that X-mode waves cannot reach the UHR directly from the usual low- field side ECRH launcher position in tokamaks, owing to the R-cutoffalways occurring on the low-field side of the UHR. The analysis of PDIs at the UHR is distinct for strongly overdense plasmas [18, 19, 22, 29], commonly found in spherical tokamaks, and underdense/weakly over- dense plasmas [17, 24–28, 30], commonly found in con- ventional tokamaks and during plasma start-up, owing to the different dispersion properties of the electron Bernstein waves (EBWs) involved in the PDI in these cases. Specific scenarios in which PDIs at the UHR are of interest in con- nection with magnetic confinement fusion include funda- mental X-mode [9–11, 17] and O-mode [9, 11, 21, 24–28] ECRH with optically thin/gray resonances, O-X-B heating [4, 5, 18–20, 29], and EBW start-up [35, 36]. pedestals could lead to PDIs near the plasma edge during O-mode ECRH [60]. In this Paper, we review the basic conditions under which the above PDIs may occur and as- sess their relevance for the O-mode ECRH scenarios pro- posed for ITER [12], in a manner similar to the investiga- tions in Section 4 of [1], but covering different scenarios. The remainder of the Paper is organized as follows. Section 2 discusses the theoretical background of the em- ployed criteria. In Section 3, the criteria are applied to the O-mode ECRH scenarios planned at ITER. Finally, Sec- tion 4 presents our conclusions. 1 Introduction These large amplitude electric fields allow the convective losses normally suppressing PDIs in inhomogeneous plas- In a PDI, a strong quasi-coherent pump wave, e.g., the millimeter waves used for ECRH in magnetic confinement https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023) EC21 EPJ Web of Conferences 277, 01002 (2023) mas [31–33] to be overcome. In the free-electron maser case, the electric field amplitude is large enough to over- come the losses in the bulk plasma [13], while the UHR case relies on enhancement of the electric field ampli- tude associated with X-mode waves near the UHR, where their group velocity becomes very small [34]. Although PDIs involving X-mode waves reaching the UHR can oc- cur at relatively low ECRH power levels without strict re- quirements on the plasma conditions [25, 26], they have generally only been considered to play a role in certain ECRH scenarios. This is due to the fact that X-mode waves cannot reach the UHR directly from the usual low- field side ECRH launcher position in tokamaks, owing to the R-cutoffalways occurring on the low-field side of the UHR. The analysis of PDIs at the UHR is distinct for strongly overdense plasmas [18, 19, 22, 29], commonly found in spherical tokamaks, and underdense/weakly over- dense plasmas [17, 24–28, 30], commonly found in con- ventional tokamaks and during plasma start-up, owing to the different dispersion properties of the electron Bernstein waves (EBWs) involved in the PDI in these cases. Specific scenarios in which PDIs at the UHR are of interest in con- nection with magnetic confinement fusion include funda- mental X-mode [9–11, 17] and O-mode [9, 11, 21, 24–28] ECRH with optically thin/gray resonances, O-X-B heating [4, 5, 18–20, 29], and EBW start-up [35, 36]. mas [31–33] to be overcome. In the free-electron maser case, the electric field amplitude is large enough to over- come the losses in the bulk plasma [13], while the UHR case relies on enhancement of the electric field ampli- tude associated with X-mode waves near the UHR, where their group velocity becomes very small [34]. Although PDIs involving X-mode waves reaching the UHR can oc- cur at relatively low ECRH power levels without strict re- quirements on the plasma conditions [25, 26], they have generally only been considered to play a role in certain ECRH scenarios. 2 Theoretical Background Additionally, it has recently been suggested that trapped low-frequency waves existing as a consequence of the strong gradients in the ITER and ASDEX Upgrade | fce| = eB 2πme ≈eB0 2πme R0 R , (3) (3) where e is the elementary charge and me is the electron mass. Additionally in the cold plasma limit, O-mode radi- ation has a cutoffat the electron plasma frequency, fpe = s e2ne 4π2ϵ0me , (4) (4) where ne is the electron density and ϵ0 is the permittivity of vacuum. The UHR occurs for X-mode radiation at the UH frequency, fUH = q f 2pe + f 2ce, (5) (5) in cold plasmas, although we note that its properties are substantially influenced by warm plasma effects at elec- tron temperatures Te ≳3 keV [1, 62]. ECRH radia- tion with a frequency of f0 will encounter the cold UHR ( f0 = fUH) at an ne value of nUH e ≈ϵ0me e2 4π2 f 2 0 −e2B2 0 m2e R2 0 R2 . (6) (6) We investigate nUH e for O-mode ECRH radiation at ITER, as such radiation may drive a PDI involving a trapped UH wave and a low-frequency lower hybrid wave in the pres- ence of an inhomogeneous ne profile with a local maxi- mum slightly above nUH e [53–56]. Additionally, in connec- tion with O-mode ECRH at ITER, we investigate whether scenarios are likely to occur in which a significant amount of X-mode radiation reaches the UHR upon reflection offthe high-field side wall, as PDIs have been observed 2 EPJ Web of Conferences 277, 01002 (2023) EPJ Web of Conferences 277, 01002 (2023) EC21 https://doi.org/10.1051/epjconf/202327701002 Figure 1. nUH e , nG e , ¯nmin e , f0 = |fce|, and f0 = 2|fce| of the O- mode ECRH scenarios planned for ITER [12]. The top panel shows the situation for the full-field scenario (B0 = 5.3 T, Ip = 15 MA) heated by the main ECRH system, f0 = 170 GHz. The middle and bottom panels show the situations for O-mode ECRH of the half-field scenario (B0 = 2.65 T, Ip = 7.5 MA) with f0 = 110 GHz and f0 = 104 GHz, respectively. in such scenarios at ASDEX Upgrade, even without UH wave trapping [23–26]. We note that the trapping-ne of the UH wave is slightly different from the value in Eq. 3 Potential O-Mode ECRH PDI Scenarios at ITER Figure 1. nUH e , nG e , ¯nmin e , f0 = |fce|, and f0 = 2|fce| of the O- mode ECRH scenarios planned for ITER [12]. The top panel shows the situation for the full-field scenario (B0 = 5.3 T, Ip = 15 MA) heated by the main ECRH system, f0 = 170 GHz. The middle and bottom panels show the situations for O-mode ECRH of the half-field scenario (B0 = 2.65 T, Ip = 7.5 MA) with f0 = 110 GHz and f0 = 104 GHz, respectively. We plot nUH e , nG e , ¯nmin e , along with the (cold) fundamen- tal (f0 = | fce|) and second-harmonic (f0 = 2| fce|) electron cyclotron resonances (ECRs) for the O-mode ECRH sce- narios planned at ITER [12] in Fig. 1. The plots cover R ∈[4.11 m, 8.48 m], corresponding to the R-range of the ITER vacuum vessel in the midplane, while the R-ticks at R0 −a = 4.2 m, R0 = 6.2 m, and R0 + a = 8.2 m represent the extent of the main plasma in the midplane [12]. The top panel of Fig. 1 shows the situation for fun- damental O-mode ECRH of the full-field ITER scenario (B0 = 5.3 T, Ip = 15 MA) using the main ITER ECRH system operating at f0 = 170 GHz [12]. In this case, nUH e attains values from 0 to nG e in the central part of the plasma. Since the ECRH waves are injected from the low-field side (large R) [12] and thus encounter the re- gion with nUH e ∈]0, nG e [ before they are absorbed around the fundamental ECR, PDIs involving decay of an O-mode ECRH wave to a trapped UH wave and a low-frequency lower hybrid wave are likely occur if a region allowing UH wave trapping is present near the plasma center in full-field ITER scenarios. It is also possible to operate plasmas with core ne-values approaching 2nG e when the plasma is fueled by pellets [64], in which case trapped UH wave PDIs may be possible over a wider range of the full- field ITER plasmas; the requirement that ne < nG e at the plasma edge means that there will always be some point with R < R0 + a at which f0 = fUH in stable plasmas. 2 Theoretical Background (6), since it is down-shifted relative to f0 by the frequency of the low-frequency lower hybrid wave in accordance with Eq. (1). However, this shift can be ignored, as Eq. (6) al- ready ignores finite-Te effects, which will modify the UHR condition in the core of ITER plasmas [1, 12, 62]. Equa- tion (6) should thus be considered a rough estimate of the ne-value at which UH wave trapping and field enhance- ment of X-mode ECRH waves may occur. In addition to nUH e , we consider the ne-limits and typi- cal ne-values at ITER. To avoid disruptions, ne should not exceed the Greenwald density, nG e [1020 m−3] = Ip [MA] π(a [m])2 , (7) (7) (7) at the plasma edge [63, 64]; Ip is the plasma current, which is 15 MA in standard full-field ITER scenarios and 7.5 MA in half-field ITER scenarios [12]. We note that the core- ne can exceed nG e in the case of peaked plasma profiles obtained by pellet fueling [64] and that the empirical nG e given by Eq. (7) may underestimate the ne-limit in ITER [65], meaning that nG e does not represent a hard limit on ne in ITER, but still serves as a useful upper estimate of the edge-ne. Further, the line-averaged ne values leading to the minimum L−H threshold, ¯nmin e , which are representative of the lower bound of ne in the core of typical H-mode plas- mas, are given by 5×1019 m−3 in full-field ITER scenarios and 2.5 × 1019 m−3 in half-field ITER scenarios according to [12, 66]. 4 Conclusion We have investigated the potential occurrence of PDIs in O-mode ECRH scenarios at ITER [12], using an approach similar to that employed for the ITER X-mode ECRH sce- narios in Section 4 of [1]. We found that PDIs are likely to occur near the UHR in connection with phenomena leading to non-monotonic ne-profiles allowing UH wave trapping in a wide variety of O-mode ECRH scenarios at ITER, as well as in situations with X-mode radiation re- flected offthe high-field side wall reaching the UHR with- out restrictions on the ne-profiles in scenarios with limited ECRH absorption. Specifically, trapped UH wave PDIs may occur near the plasma center during 170 GHz ECRH in full-field scenarios (B0 = 5.3 T, Ip = 15 MA), while PDIs requiring limited ECRH absorption are unlikely in these scenarios. Trapped UH wave PDIs may also occur on the high-field side during 110 GHz or 104 GHz ECRH of half-field scenarios (B0 = 2.65 T, Ip = 7.5 MA). Such scenarios may further allow PDIs requiring limited ECRH absorption at sufficiently low B0. For 110 GHz ECRH, a B0 > 2.66 T was found to make such PDIs unlikely for ECRH beams launched from the outboard midplane, while a less strict requirement of B0 > 2.52 T was found to apply for 104 GHz ECRH. It is possible to mitigate the poten- tial damage to microwave diagnostics due to ECRH PDIs by installing additional filters around the frequencies ex- pected to be affected and by minimizing the abilitiy of the radiometers to pick up strong signals generated inside the ECRH beams [1]. While the f0 = 110 GHz and f0 = 104 GHz cases are similar in terms of their behavior with respect to PDIs in- volving a trapped UH wave, the conclusions regarding the possibility of having a significant amount of X-mode radi- ation reaching the UHR due to reflections from the high- field side wall and driving PDIs differ. For f0 = 110 GHz, the middle panel of Fig. 1 shows that the fundamental ECR is located on the far high-field side, slightly outside the main plasma, meaning that its absorption is highly de- pendent on the precise value of B0 and the angle at which the O-mode ECRH waves are launched [24–26, 61]. 3 Potential O-Mode ECRH PDI Scenarios at ITER Opera- tion with pellet fueling above nG e would allow the trapped UH wave PDIs to occur near the plasma center and on the low-field side in addition to the high-field side; once again, the requirement of ne < nG e at the plasma edge means that there will always be some point with R < R0 + a at which f0 = fUH in stable plasmas. of PDIs relying on a significant amount of X-mode ECRH power reaching the UHR. In the case of f0 = 104 GHz, shown in the bottom panel of Fig. 1, the fundamental ECR occurs well inside main plasma around the midplane for B0 = 2.65 T, so no PDIs relying on X-mode ECRH waves reaching the UHR are expected in this case. Additionally, Bmin 0 ≈2.52 T for f0 = 104 GHz, meaning that operation at B0 values somewhat below 2.65 T is also possible without the occurrence of PDIs relying on X-mode ECRH power around the UHR. Finally, we note that 104 GHz radiation might also be preferable for avoiding strong trapped UH wave PDIs involving X-mode ECRH pump waves [62]. 3 Potential O-Mode ECRH PDI Scenarios at ITER Trapped UH wave PDIs in the ITER full-field scenarios may create strong microwave signals with frequency shifts ∼10 GHz from f0 = 170 GHz [1]. This makes them a po- tential risk to the ITER electron cyclotron emission (ECE) and low-field side reflectometer systems, operating in the frequency ranges 70−1000 GHz [67, 68] and 30−165 GHz [69], respectively. PDIs involving a significant amount of X-mode ECRH power reflected from the high-field side wall reaching the UHR are unlikely to occur in the full- field ITER scenario, as the fundamental ECR is located close to the plasma center and therefore expected to be op- tically thick for the O-mode ECRH waves [61]. The middle and bottom panels of Fig. 1 show the sit- uation for fundamental O-mode ECRH of the half-field 3 https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023) ITER scenario (B0 = 2.65 T, Ip = 7.5 MA) using the two lower-frequency ECRH options considered for ITER to enable ECRH start-up in the one-third-field scenarios [12], f0 = 110 GHz (middle panel) and f0 = 104 GHz (bottom panel). In these cases, nUH e covers the range from 0 to nG e on the high-field side of the plasma. Since the ECRH waves are still injected from the low-field side, they will encounter the region with nUH e ∈]0, nG e [ before potentially being absorbed at the fundamental ECR [61]; the second- harmonic ECR for f0 = 110 GHz which occurs far on the low-field side, outside the main plasma, is optically thin due to the low ne and Te in this region [61]. This means that 110 GHz and 104 GHz ECRH waves are also likely to drive PDIs involving decay of the O-mode ECRH waves into trapped UH waves and low-frequency lower hybrid waves in regions allowing UH wave trapping on the high- field side in ITER half-field scenarios. Such trapped UH wave PDIs would again pose a risk to the ECE [67, 68] and low-field side reflectometer [69] systems at ITER. References [1] S.K. Hansen, A.S. Jacobsen, M. Willensdorfer, S.K. Nielsen, J. Stober, K. Höfler, M. Maraschek, R. Fis- cher, M. Dunne, the EUROfusion MST1 team et al., Plasma Phys. Control. Fusion 63, 095002 (2021) [21] S. Kubo, M. Nishiura, K. 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This mechanism has been demonstrated experimentally in ASDEX Upgrade [24–26] and can be as- sessed for given ITER plasma conditions and ECRH beam geometries using the theory of [24, 30]. We note that by slightly increasing B0, such that the fundamental ECR oc- curs inside the last closed flux surface at the midplane, the absorption of 110 GHz ECRH radiation from the midplane launchers can be made more certain, which should also limit the occurrence of PDIs relying on X-mode radiation reaching the UHR. The minimum value of B0 at which ECR absorption around the midplane is guaranteed, Bmin 0 , is given by the condition that f0 = | fce| at R = R0 −a, from which Eq. (3) yields While trapped UH wave PDIs have been found to lead to diagnostics damage [1] and significant anomalous ECRH absorption [2, 3], which would generally be detri- mental to the operation of the ITER ECRH system, we note that PDIs may also allow novel diagnostics and heat- ing schemes to be implemented. For instance, PDIs in- volving X-mode radiation reaching the UHR are being considered for generating fast ions with ECRH in spherical tokamaks [7, 8], although we note that the efficacy of this scheme has yet to be demonstrated, and trapped UH wave PDIs occurring near the plasma edge have already been used to investigate edge-localized mode and L-mode blob characteristics at ASDEX Upgrade [6]. Since PDIs involv- ing low-frequency trapped waves in the density pedestal are expected in connection with O-mode ECRH at ITER [60], it would be of particular interest to assess their value as an edge diagnostic. Bmin 0 ≈2π f0 me e R0 −a R0 . (8) (8) For f0 = 110 GHz, we find Bmin 0 ≈2.66 T, so operating at B0 slightly above 2.65 T should prevent the occurrence 4 https://doi.org/10.1051/epjconf/202327701002 EPJ Web of Conferences 277, 01002 (2023) References Tartari, E. Alessi, O. D’Arcangelo, J. Instrum. 15, C01046 (2020) [40] A.Yu. Popov, E.Z. Gusakov, Plasma Phys. Control. 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Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate
Brazilian journal of food technology/Brazilian Journal of Food Technology
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Aline Cássia Figueira1* , Valdecir Luccas1 1Instituto de Tecnologia de Alimentos, Cereal Chocotec, Campinas/SP - Brasil. 1Instituto de Tecnologia de Alimentos, Cereal Chocotec, Campinas/SP - Brasil. *Corresponding Author: Aline Cássia Figueira, Instituto de tecnologia de Alimentos, Cereal Chocotec, Avenida Brasil, 2880, Jardim Brasil, CEP: 13070-178, Campinas/SP – Brasil, e-mail: aline.cassia.figueira@gmail.com Cite as: Figueira, A. C., & Luccas, V. (2022). Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate. Brazilian Journal of Food Technology, 25, e2022033. https://doi.org/10.1590/1981-6723.03322 ORIGINAL ARTICLE ORIGINAL ARTICLE Abstract Cocoa butter is one of the most important ingredients in chocolate production as it is responsible for important characteristics of chocolates such as hardness, snap, mold shrinkage, fat bloom stability and melting. It appears that factors such as the geographic area and climate where cocoa is grown directly affect the chemical composition of cocoa butter, so fruits grown in Africa and Asia have greater thermal stability. In this sense, two samples of cocoa butter, a Brazilian one from the south of Bahia and a commercial one (consisting of a mixture of butters from different origins) were studied and compared. The results obtained in this study showed that Brazilian cocoa butter had a higher content of Saturated Fatty Acids (SFA) and a higher content of unsaturated triacylglycerols compared to commercial butter. It also showed a faster polymorphic transition and a higher maximum solids content compared to commercial cocoa butter, indicated by isothermal crystallization analysis. Based on the analyzes carried out, it was found that this cocoa butter studied, from the Forasteiro amelonado species and coming from several farms in the south of Bahia, presented greater thermal stability in relation to commercial cocoa butter, i.e., different from that presented in other studies in the literature. Keywords: Thermal behavior; Fatty acids; Triacylglycerols; Isothermal crystallization; Cooling curve; Solid curve. Cite as: Figueira, A. C., & Luccas, V. (2022). Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate. Brazilian Journal of Food Technology, 25, e2022033. https://doi.org/10.1590/1981-6723.03322 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Caracterização físico-química de manteigas de cacau nacional e comercial utilizadas no Brasil para fabricação de chocolate Caracterização físico-química de manteigas de cacau nacional e comercial utilizadas no Brasil para fabricação de chocolate Aline Cássia Figueira1* , Valdecir Luccas1 Aline Cássia Figueira1* , Valdecir Luccas1 Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 1 Introduction Cocoa butter is one of the most important raw materials used in chocolate production, being one of the ingredients with the highest value-added product, corresponding to 25 and 36%, or more, of the value of the final product. It is also one of the most important ingredients, where it represents the continuous phase of chocolate and acts as a dispersing matrix for cocoa, milk, and sugar solids. The importance of this ingredient is also related to the quality present in the final product, since it is responsible for the characteristics of hardness, snap (breakdown at room temperature), complete melting in the mouth, contraction during demolding and chocolate shine, in addition to the product stability to fat bloom (Suri & Basu, 2022). Among the most predominant fatty acids in the structure of cocoa butter, oleic acid (C18:1), stearic acid (C18:0) and palmitic acid (C16:0) stood out (Jin et al., 2021). The literature has shown that factors such as geographic area and climatic conditions where cocoa is grown directly influence the composition of triacylglycerols (TAGs) and the fatty acid profile of cocoa butter, thus modifying its melting point and solids profile. According to Beckett (2008), the closer to the equator the cocoa was grown, the harder the butter would be. In addition, cocoa butter from fruits grown in South America contains a higher content of oleic acid than those from fruits grown in Africa and Asia (Beckett, 2008). Studies have been shown that Brazilian cocoa butter had a lower content of monounsaturated triacylglycerols and a higher content of di and polyunsaturated triacylglycerols (Ribeiro et al., 2012). In this way, it has a softer texture, which interferes with the thermal stability of chocolates produced with this raw material and, consequently, contributes to a reduction in its commercial value. According to Beckett (2008), while the cocoa butter from Malaysia had a Solid Fat Content (SFC) of 81%, the Brazilian one had only approximately 65% of this content. For this reason, most of the cocoa butter used by Brazilian industries in chocolate making is imported, as they are more resistant to heat, but more expensive. As an alternative to this issue, many cocoa processors in Brazil sell a blend of cocoa butters from different origins, but this blend has a percentage of only 20 to 30% of national butter in the mixture, which is considered a low value. HIGHLIGHTS: • Oleic, stearic and palmitic acids are the most predominant fatty acids of cocoa butter • Oleic, stearic and palmitic acids are the most predominant fatty acids of cocoa butter • The Brazilian sample presented characteristics close to the commercial sample • The Brazilian sample presented characteristics close to the commercial sample p p p • It is not recommended to use only the solids curve for selecting cocoa butters for application in chocolates • It is not recommended to use only the solids curve for selecting cocoa butters for application in chocolates Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 Resumo A manteiga de cacau é um dos ingredientes mais importantes na fabricação de chocolate, pois é responsável por características importantes desse produto como dureza, snap, contração na desmoldagem, estabilidade ao fat bloom e derretimento. Fatores como a área geográfica e o clima onde o cacaueiro é cultivado afetam diretamente a composição química da manteiga de cacau, de modo que frutos cultivados na África e Ásia apresentam maior estabilidade térmica. Nesse sentido, duas amostras de manteiga de cacau, uma brasileira proveniente do sul da Bahia e uma comercial (constituída por uma mistura de manteigas de diversas origens) foram estudadas e comparadas. Os resultados obtidos neste estudo mostraram que a manteiga de cacau brasileira apresentou maior teor de ácidos graxos saturados e maior teor de triacilgliceróis disaturados (S2U), em relação à manteiga comercial. This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distributio and reproduction in any medium, provided the original work is properly cited. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 1/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Também apresentou uma transição polimórfica mais rápida e um teor máximo de sólidos maior em relação à manteiga de cacau comercial, indicados pela análise de isoterma de cristalização. Com base nas análises realizadas, constatou-se que esta manteiga de cacau estudada, da espécie de cacaueiro forasteiro amelonado, apresentou uma maior estabilidade térmica em relação à manteiga de cacau comercial, resultado diferente dos apresentados em outros estudos da literatura. Palavras-chave: Comportamento térmico; Ácidos graxos; Triacilgliceróis; Isoterma de cristalização; Curva de resfriamento; Curva de sólidos. 1 Introduction In this way, this work aimed to evaluate the physicochemical characteristics of Brazilian cocoa butter sample, from the south of Bahia and commercial one (blend of cocoa butter from different origins). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 2/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. 2.2.2 Triacylglycerol composition The triacylglycerol composition was determined according to the AOCS Ce 5-86 methodology (American Oil Chemists’ Society, 2009). A CGC model Agilent 6850 Series Gc System and capillary column DB-17 HT Agilent Catalog: 122-1811 (50% phenyl – ethylpolysiloxane) with dimensions of 10 m, internal diameter of 0.25 mm and 0. 15 μm film were used. The chromatograph operating conditions were: column flow = 1.0 mL/min.; linear velocity = 40 cm/sec; detector temperature: 375 °C; injector temperature: 360 °C; Oven temperature: 250 – 350 °C (5 °C/min), 350 °C for 20 minutes; carrier gas: Helium; injected volume: 1.0 μL, split 1:100; and sample concentration: 10 mg/mL tetrahydrofuran (THF). TAGs were identified through the comparison of retention times, according to the procedures of Antoniosi Filho et al. (1995). The quantity was determined by peak area normalization. The analysis was carried out in triplicate. 2.1 Material The Brazilian cocoa butter used in this study was granted by the Cocoa Innovation Center (CIC), being extracted from cocoa beans of the Forasteiro amelonado species, from several farms located in the south of Bahia. Commercial cocoa butter was obtained from the company Olam Cocoa, Brazil, that it was a commercial cocoa butter, made up of a mixture of butters from different origins, the proportion of which was not known. 2.2.1 Fatty acids composition The fatty acid composition was determined according to the AOCS Ce 2-66 method (American Oil Chemists’ Society, 2009). A Capillary Gas Chromatography (CGC) model Agilent 6850 Series GC System and an Agilent DB-23 capillary column (50% cyanopropyl) - methylpolysiloxane, with dimensions of 60 m, internal diameter of 0.25 mm and 0.25 μm thickness of movie were used. The identification of fatty acids was performed by comparing the retention time obtained with the respective standards. The analysis was performed in triplicate. The conditions used for chromatograph operation were: column flow = 1.0 mL/min.; linear velocity = 24 cm/sec; detector temperature: 280°C; injector temperature: 250 °C; oven temperature: 110 °C for 5 min, 110 – 215 °C (5 °C/min), 215 °C for 24 min.; carrier gas: Helium; and injected volume: 1.0 μL. The identification of the fatty acids present in the sample was carried out by comparing the retention times of the peaks with those of the respective fatty acid standards. The quantification of fatty acids was determined through the normalization of the peak areas obtained by the chromatogram, expressed in mass percentage. The analysis was carried out in triplicate. 2.3 Statistical analysis The results obtained were submitted to the t test for two samples to determine significant differences in the means at a significance level of 5% (p ≤ 0.05), using the Minitab 19 software. 2.2.5 Isothermal crystallization The sample was melted and kept at 70 °C for 1 h in a TCON 2000 dry bath, with a temperature range of 0 to 70 °C (Duratech, USA), to erase its crystalline history. After this period, the sample was kept in a thermal bath at 17.5 °C ± 0.5 °C and readings were taken every 1 min in a Bruker pc120 Minispec NMR Spectroscopy (Rheinstetten, Germany), until reaching equilibrium (250 min). The crystallization kinetics was characterized according to the induction period data, which corresponds to the time required for the beginning of the formation of a stable nucleus and maximum SFC (SFCmax). The methodology used for the isothermal crystallization analysis was proposed by Ribeiro et al. (2009). The analysis was carried out in duplicate. 2.2.6 Cooling curve The cooling curves of the cocoa butter samples were obtained on a Multitherm TC temperature meter (Buhler, Switzerland). The sample was heated to 70 °C and packed in aluminum capsules, specific to the equipment. The equipment's cooling compartment was kept at a temperature of 17.6 °C for the analysis. The result was evaluated using the Buhler Crystallization Index (BCI) (Zeng et al., 2021). 2.2.4 Melting point The melting point of the samples was obtained through the solids curve, corresponding to the temperature at which the sample presented 4% of solids. 2.2.3 Solid Fat Content (%SFC) The SFC of cocoa butters was determined according to the AOCS Cd 16b-93 method, using a Bruker pc120 Minispec Nuclear Magnetic Resonance Spectroscopy (NMR Spectroscopy) (Silberstreifen, Rheinstetten, Germany) (American Oil Chemists’ Society, 2009). First, the sample was tempered using Duratech Tcon 2000 dry baths (Carmel, USA), where it was subjected to 0 °C for 90 min, 26 °C for 40 h and, again, 0 °C for 90 min. After this preparation, the sample was kept for 1 h at the temperatures analyzed (10, 20, 25, 30 and 35 °C) before taking the reading. The analysis was carried out in triplicate. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3.1 Fatty acids composition The results presented in Table 1 indicate that the fatty acids present in greater amounts in cocoa butter are palmitic (C16:0), stearic (C18:0) and oleic (C18:1) acids, which represent more than 90% of the fatty acids in the sample. The levels obtained for these fatty acids are close to the values found in the literature, which vary from 24 to 27% for palmitic acid, 33 to 36% for stearic acid and 32 to 35% for oleic acid (Ribeiro et al., 2012). According to Beckett (2008), the fact that cocoa butter is relatively simple in relation to the main fatty acids makes this fat have very interesting physical properties, such as the fact that it melts in a small temperature range, and melts quickly in the mouth, besides being responsible for the glow and snap of chocolates. It was also verified that the national cocoa butter had a higher content of Saturated Fatty Acids (SFA) and a lower content of Unsaturated Fatty Acids (UFAs) in relation to commercial cocoa butter. The literature has shown that factors such as geographic area and climatic conditions where cocoa is grown directly influence the composition of TAGs and the fatty acid profile of cocoa butter, modifying its melting point and solid profile. According to Beckett (2008), the closer to the equator the cocoa was grown, the harder the butter would be. In addition, cocoa butter from fruits grown in South America contains a higher content of oleic acid than those from fruits grown in Africa and Asia (Beckett, 2008). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 4/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Table 1. Fatty acids composition (%) present in commercial and national cocoa butter (CB). Table 1. Fatty acids composition (%) present in commercial and national cocoa butter (CB). 3.1 Fatty acids composition Fatty acids National CB Commercial CB C14:0 – myristic 0.1 ± 0.00 0.1 ± 0.02 C16:0 – palmitic 26.7 ± 0.02 25.1 ± 0.01 C16:1 – palmitoleic 0.3 ± 0.03 0.3 ± 0.01 C17:0 – margaric 0.3 ± 0.00 0.3 ± 0.00 C18:0 – stearic 33.6 ± 0.03 33.3 ± 0.24 C18:1 – oleic 34.3 ± 0.03 35.1 ± 0.10 C18:2 – linoleic 2.9 ± 0.02 4.1 ± 0.26 C18:3 – linolenic 0.2 ± 0.00 0.3 ± 0.02 C20:0 – arachidic 1.2 ± 0.00 1.1 ± 0.01 C20:1 – gadoleic 0.1 ± 0.01 0.1 ± 0.00 C22:0 – behenic 0.2 ± 0.00 0.2 ± 0.00 C24:0 – lignoceric 0.1 ± 0.00 0.0 ± 0.02 ∑ Saturated 62.2 ± 0.05 60.2 ± 0.21 ∑ Unsaturated 37.8 ± 0.09 39.8 ± 0.14 3.2 TAG composition The TAGs are the major components of oils and fats, and they are responsible for the properties of these lipids, such as spreadability, melting in the mouth, crystallization profile and type of polymorph. Table 2 present the TAG composition of South of Bahia and commercial cocoa butter. For both butters, it was possible to obtain 14 types of TAGs. The TAGs of the types Palmitic-Oleic-Palmitic (POP), Palmitic-Oleic- Stearic (POSt) and Stearic-Oleic-Stearic (StOSt) were the predominant ones, where they presented a total of 84.5% in national butter and 78.5% in commercial butter. These values agree with the range of these triglycerides reported in the literature, which varies from 72 to 93% (Jin et al., 2021). However, contrary to what was expected, commercial cocoa butter had a higher content (9.8%) of TAGs of the types Palmitic-Oleic-Oleic (POO) and Stearic-Oleic-Oleic (StOO) compared to national butter (6.6%). In a study carried out by Ribeiro et al. (2012), the authors found a higher percentage of POO and StOO in Bahia (8.5%) and Espírito Santo (9.3%) butters in relation to the commercial butters evaluated (around 5%). Furthermore, national cocoa butter presented lower content of TAGs trisaturated (0.9%) compared to commercial cocoa butter (1.3%). These TAGs have a higher melting point and can accelerate the fat crystallization. Table 2. Triacylglycerol composition present in commercial and national cocoa butter (CB). TAG (%) National CB Commercial CB PPP 0.2 ± 0.0 0.2 ± 0.1 PPSt 0.4 ± 0.2 0.5 ± 0.1 POP 20.6 ± 0.2 17.6 ± 0.2 PLP 1.9 ± 0.2 2.1 ± 0.0 POSt 40.6 ± 0.8 37.4 ± 0.9 POO 3.3 ± 0.3 4.5 ± 0.0 PLSt 2.7 ± 0.2 3.5 ± 0.4 PLO 0.4 ± 0.1 0.7 ± 0.4 PStSt 0.3 ± 0.5 0.6 ± 0.3 StOSt 23.3 ± 0.2 23.5 ± 0.4 StOO 3.3 ± 0.4 5.3 ± 0.1 StLO 1.5 ± 0.0 2.0 ± 0.0 OOO 0.2 ± 0.3 0.9 ± 0.2 StOA 1.3 ± 0.0 1.3 ± 0.4 ΣS3 0.9 ± 0.7 1.3 ± 0.5 ΣS2U 90.4 ± 1.5 85.3 ± 2.3 ΣU2S 8.5 ± 0.8 12.5 ± 0.4 P P l iti ( t t d) St St i ( t t d) O Ol i ( t t d) A A hidi ( t t d) L Li l i (di t t d) Table 2. Triacylglycerol composition present in commercial and national cocoa butter (CB). Braz. J. P: Palmitic (saturated); St: Stearic (saturated); O: Oleic (monounsaturated); A: Arachidic (saturated); L: Linoleic (diunsaturated Triacylglycerols: S3 = trisaturated, S2U = disaturated–monounsaturated e U2S = diunsaturated–monosaturated. urated); St: Stearic (saturated); O: Oleic (monounsaturated); A: Arachidic (saturated); L: Linoleic (diunsaturated). 3.3 Solid Fat Content (%SFC) One of the indications of the fat melting profile is through the solids curve, which relates the solid fraction of fat in relation to its total mass (SFC) when the sample is subjected to different temperatures. The solids curves obtained for the national CB and commercial CB are shown in Figure 1. At all temperatures evaluated, Bahia cocoa butter presented a lower solid fat content in relation to commercial cocoa butter, presenting a similar behavior to that obtained by Ribeiro et al. (2012). The authors evaluated the SFC of different commercial cocoa butters, where, at 10 °C, this content ranged from 83.9 to 85.4%, while for national butter, the value obtained by the authors was 77.4%. In the present work, at the same temperature, it was possible to obtain a value of 80% for national butter and 86.4% for commercial butter. In a study carried out by Shukla (2006), the authors obtained a variation in SFC from 74.8 to 83.7%, at a temperature of 25 °C, for butters from Ghana, India, Nigeria, Malaysia, and Sri Lanka. In the present work, at 25 °C, a value of 57.5% was obtained for national butter and 65.4% for commercial butter, lower values compared to imported butters. Ribeiro et al. (2012) also obtained values like those obtained in this study for Bahia and commercial butter. The authors stated that national cocoa butters, especially those obtained in Espírito Santo (Brazil), have greater softness in relation to imported cocoa butters and, therefore, to be used in tropical regions, they need to be mixed with butters from foreign origins to obtain higher levels of SFC at various temperatures. Importantly, other analyses, such as isothermal crystallization and thermal behavior, must also be considered before determining whether one cocoa butter is harder than the other. Figure 1. Solid Fat Content (SFC) of national cocoa butter and commercial cocoa butter (CB). Figure 1. Solid Fat Content (SFC) of national cocoa butter and commercial cocoa butter (CB). Through the solids curve, it was possible to quantify several physical parameters of fats. A very important parameter that can be extracted from the solids curve is the variation of the solid fat content from 25 to 35 °C, called ∆S. This parameter is widely used in the food industry to assess the quality of cocoa butter (Luccas, 2001). 3.2 TAG composition Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 5/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3.4 Melting point From the solids curve, it was also possible to obtain the value of the melting temperature of the samples, which corresponded to the temperature at which the solid fat content of the sample was equivalent to 4%. It is considered that at this value the fat is completely in the liquid state, with no residual fraction of solids (Ribeiro et al., 2012). The melting temperature obtained for each of the butters evaluated is shown in Table 4. According to Shukla (2006), the melting temperature of cocoa butter could vary in a range of 32 to 35 °C. This variation in temperature is not relevant because cocoa butter has a relatively uniform chemical composition, since the predominant TAGs in cocoa butter (POP, POS and SOS) have a uniform chemical structure, where oleic acid occupies the second position (Suri & Basu, 2022). The values determined for the national CB and commercial CB are within this temperature range, and both values were very close. Table 4. Melting point of national and commercial cocoa butter (CB). Samples Melting point (°C) National CB 34.40 ± 0.04 Commercial CB 34.66 ± 0.02 Table 4. Melting point of national and commercial cocoa butter (CB). Table 4. Melting point of national and commercial cocoa butter (CB). 3.3 Solid Fat Content (%SFC) It is desirable that fats used for chocolate production have high values of ∆S, thus indicating that the fat has good melting properties in the mouth, associated with the release of flavor and sensation of freshness. The solid fat content at 25 °C represents the hardness of the fat. Fats with higher values of S25 °C and lower values of ∆S25 °C – 35 °C are characterized by being harder and with greater resistance to heat, respectively. The presence of solid fat at temperatures above 35 °C is undesirable, since it causes a sensory perception of waxy fat, and is easily perceptible during tasting (Luccas, 2001). For cocoa butter to be used in the manufacture of chocolates, it must have a solid fat content above 50% at 25 °C and must not have high solids content at 35 °C (Luccas, 2001). The values obtained for S25 °C, ∆S25 °C – 35 °C, and S35 °C are shown in Table 3. The cocoa butters showed significant differences (p < 0.05) concerning the fat content solid at 25 °C and 35 °C, where commercial butter showed higher SFC values than Bahia cocoa butter. Regarding the ΔS values, it was found that commercial cocoa butter presented a higher value compared to Bahia cocoa butter, with a significant difference between the samples at a significance level of 5%. Both butters had low solids content at 35 °C, as expected for cocoa butter samples. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 6/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Table 3. Comparison between the physical characteristics of national cocoa butter (Bahia) and commercial cocoa butter (CB). Samples S25 °C ∆S25 °C - 35 °C S35 °C National CB (Bahia) 57.51 ± 0.04b 57.30 ± 0.16b 0.21 ± 0.17b Commercial CB 65.38 ± 0.19a 63.96 ± 0.34a 1.42 ± 0.15a Values with different letters in the same column are significantly different (p ≤ 0.05). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 3.5 Isothermal crystallization The graph of Figure 2 shows the curves obtained from the isothermal crystallization of the analyzed cocoa butters. The isothermal crystallization analysis was performed at 17.5 °C, according to Marangoni & McGauley (2003). It is possible to verify that, in both cocoa butters, it was possible to obtain a crystallization with an induction period called “𝜏𝜏1” and in a second step, a second induction period called “𝜏𝜏2” was obtained. In addition, the crystallization curve showed a sigmoidal shape. Ribeiro et al. (2012) also observed this behavior of cocoa butter. It follows that the first induction time (𝜏𝜏1) is related to the time required for the formation of a crystal nucleus. The presence of this plateau in the cocoa butter isothermal crystallization is related to its polymorphic transition, where the transition from a less stable to a more stable form occurs. Figure 2. Isothermal crystallization at 17.5 °C of national and commercial cocoa butters (CB). Figure 2. Isothermal crystallization at 17.5 °C of national and commercial cocoa butters (CB). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 7/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Table 5 presents the results of the two induction periods (𝜏𝜏1 and 𝜏𝜏2) and the maximum solids content obtained for both butters. It is found that national cocoa butter had a higher 𝜏𝜏1 value than commercial one. This behavior may be related to the TAG composition of the fat. According to Suri & Basu (2022), different types of cocoa butter submitted to the same crystallization condition may have different crystallization rates. This fact can be attributed to the minority components present in cocoa butter, such as glycolipids, phospholipids and tri-saturated TAGs, which can act as “seeds” or crystallization nuclei and accelerate the crystallization of fat (Suri & Basu, 2022). Based on the data of the TAG composition of the cocoa butters in this study, it was possible to note that commercial cocoa butter had a TAG content (S3) of approximately 40% more than national cocoa butter (Bahia). Such TAGs had a higher melting point, thus being the first to crystallize, therefore the commercial butter started to crystallize first. 3.5 Isothermal crystallization 𝜏 Regarding the values of 𝜏𝜏2, it appears that the national cocoa butter showed a faster polymorphic transition than the commercial one. This fact can be attributed to the TAG composition of cocoa butters, since national cocoa butter had a higher content of symmetrical TAGs (90.4%) in relation to commercial cocoa butter (85.3%), and these TAGs are responsible for accelerating the polymorphic transition of these fats. Regarding the maximum solids content, it appears that, although the national cocoa butter has presented itself as softer by the solids curve analysis, when evaluated at a temperature of 17.5 °C, it could reach equilibrium with a higher solids content when compared to commercial cocoa butter. Table 5. Induction periods and SFCmax obtained for national and commercial cocoa butters (CB). Samples τ1 (min) τ2 (min) SFCmax (%) National CB 22.00 ± 0.71a 55.00 ± 1.41a 73.77 ± 0.42a Commercial CB 14.75 ± 0.35b 62.00 ± 5.60a 59.00 ± 0.71b Values with different letters in the same column are significantly different (p ≤ 0.05). Table 5. Induction periods and SFCmax obtained for national and commercial cocoa butters (CB). Table 6. Results obtained by analyzing the cooling curve of national and commercial cocoa butters (CB). Samples Tn (°C) tn (min) Q (°C/min) BCI National CB 18.8 16.7 0.22 5.4 Commercial CB 20.2 9.2 0.12 3.5 Tn corresponds to the temperature at the beginning of crystallization; tn corresponds to the time of start of crystallization; Q corresponds to the crystallization rate. 3.6 Cooling curve The cooling curve provides information on the crystallization of cocoa butter, making it possible to assume whether it has the necessary or desired hardness for making chocolates. The analysis is based on a calorimetric principle, where heat is released from cocoa butter during cooling. The analysis is performed at a temperature of 17.6 °C to maximize the expansion of the cocoa butter exothermic reaction. The BCI index was created by Buhler (manufacturer of the equipment used) and evaluates the cooling curve of cocoa butter as a whole, considering the nucleation and crystals growth. Through this value it is possible to know the crystallization of the raw material used is suitable for chocolate production. According to the manufacturer, fats with indexes above 4.0 are recommended for chocolate production, as they have high hardness and rapid crystallization. However, some companies use cocoa butter to make chocolates with BCI values up to 3.5. Table 6 presents the values determined for the cocoa butters in this study. It is verified that national cocoa butter (Bahia) presented a BCI value of 5.4, which is characterized as a very hard fat, with rapid crystallization. The commercial cocoa butter evaluated had an index of 3.5, thus characterizing a butter with moderate crystallization capacity (Zeng et al., 2021). Furthermore, the data obtained for crystallization start temperatures also showed that domestic cocoa butter started to crystallize before commercial cocoa butter, suggesting it is a harder cocoa butter. Table 6. Results obtained by analyzing the cooling curve of national and commercial cocoa butters (CB). Samples Tn (°C) tn (min) Q (°C/min) BCI National CB 18.8 16.7 0.22 5.4 Commercial CB 20.2 9.2 0.12 3.5 Tn corresponds to the temperature at the beginning of crystallization; tn corresponds to the time of start of crystallization; Q corresponds to the crystallization rate. Table 6. Results obtained by analyzing the cooling curve of national and commercial cocoa butters (CB). Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 8/9 Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. Physicochemical characterization of national and commercial cocoa butter used in Brazil to make chocolate Figueira, A. C., & Luccas, V. 4 Conclusion The results obtained in this study showed that Brazilian cocoa butter had a higher content of SFA and a higher content of unsaturated TAGs compared to commercial cocoa butter. In addition, national cocoa butter also showed a faster polymorphic transition and a higher SFCmax compared to commercial cocoa butter. Although Brazilian cocoa butters are characterized by being softer when compared to butters originated from countries such as Ghana and Malaysia, it could be noted in the sample studied, Forasteiro amelonado species, which came from several farms located in the South of Bahia, that this species presented physicochemical characteristics close to the commercial sample, used by national chocolate industries, unlike the results presented in other studies. This indicated that, probably, the South region of Bahia has some species consisting of significant thermal resistance. References American Oil Chemists’ Society – AOCS. (2009). Official methods and recommended practices of the American Oil Chemists’ Society (6th ed). Champaign: AOCS. Beckett, S. T. (2008). The science of chocolate. Cambridge: RSC Paperbacks. Jin, J., Jin, Q., Akoh, C., & Wang, X. (2021). StOSt-rich fats in the manufacture of heat-stable chocolates and their potential impacts on fat bloom behaviors. Trends in Food Science & Technology, 118, 418-430. http://dx.doi.org/10.1016/j.tifs.2021.10.005 Jin, J., Jin, Q., Akoh, C., & Wang, X. (2021). StOSt-rich fats in the manufacture of heat-stable chocolates and their potential impacts on fat bloom behaviors. Trends in Food Science & Technology, 118, 418-430. http://dx.doi.org/10.1016/j.tifs.2021.10.005 Luccas, V. (2001). Fracionamento térmico e obtenção de gorduras de cupuaçu alternativas à manteiga de cacau para uso na fabricação de chocolate (Tese de doutorado). Universidade Estadual de Campinas, Faculdade de Engenharia Química, Campinas. Luccas, V. (2001). Fracionamento térmico e obtenção de gorduras de cupuaçu alternativas à manteiga de cacau para uso na fabricação de chocolate (Tese de doutorado). Universidade Estadual de Campinas, Faculdade de Engenharia Química, Campinas. Marangoni, A. G., & McGauley, S. E. (2003). Relationship between crystallization behavior and structure in cocoa butte Growth & Design, 3(1), 95-108. http://dx.doi.org/10.1021/cg025580l Marangoni, A. G., & McGauley, S. E. (2003). Relationship between crystallization behavior and structure in cocoa butter. Crystal Growth & Design, 3(1), 95-108. http://dx.doi.org/10.1021/cg025580l Ribeiro, A. P. B., Grimaldi, R., Gioielli, L. A., Santos, A. O., Cardoso, L. P., & Gonçalves, L. A. G. (2009). Thermal behavior, microstructure, polymorphism, and crystallization properties of zero trans fats from soybean oil and fully hydrogenated soybean oil. Food Biophysics, 4(2), 106-118. http://dx.doi.org/10.1007/s11483-009-9106-y Ribeiro, A. P. B., Silva, R. C., Gioielli, L. A., Almeida Gonçalves, M. I., Grimaldi, R., Gonçalves, L. A. G., & Guenter Kieckbusch, T. (2012). Physico-chemical properties of Brazilian cocoa butter and industrial blends. Part I - Chemical composition, solid fat content and consistency. Grasas y Aceites, 63(1), 79-88. http://dx.doi.org/10.3989/gya.069011 Shukla, V. (2006). Cocoa butter, cocoa butter equivalents, and cocoa butter substitutes. In C. Akoh (Ed.), Handbook of functional lipids (pp. 279 -307). Boca Raton: CRC Press. Suri, T., & Basu, S. (2022). Heat resistant chocolate development for subtropica and tropical climates: A review. Food Science & Nutrition, 62(20), 5603-5622. PMid:33635177. Suri, T., & Basu, S. (2022). Heat resistant chocolate development for subtropica and tropical climates: A review. Food Science & Nutrition, 62(20), 5603-5622. PMid:33635177. Zeng, Y., Braun, P., Keller, J. Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 References P., Paggios, K., Jacob, J., & Pirhalla, S. (2021). Exothermal analysis of cocoa butter, cocoa liquor, and chocolate mass with MultiTherm TC™. The Green Machine, 32(2), 24-29. http://dx.doi.org/10.21748/inform.02.2021.24 Zeng, Y., Braun, P., Keller, J. P., Paggios, K., Jacob, J., & Pirhalla, S. (2021). Exothermal analysis of cocoa butter, cocoa liquor, and chocolate mass with MultiTherm TC™. The Green Machine, 32(2), 24-29. http://dx.doi.org/10.21748/inform.02.2021.24 Funding: Acknowledgement to FAPESP (The São Paulo Research Foundation) for the financial support: projects numbers 2019/09376-0 and 2020/07015-7. Received: Apr. 04, 2022; Accepted: July 16, 2022 Received: Apr. 04, 2022; Accepted: July 16, 2022 Received: Apr. 04, 2022; Accepted: July 16, 2022 Associate Editor: Maria Gabriela Bello Koblitz Associate Editor: Maria Gabriela Bello Koblitz Braz. J. Food Technol., Campinas, v. 25, e2022033, 2022 | https://doi.org/10.1590/1981-6723.03322 9/9
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Spread of Amphibian Chytrid Fungus across Lowland Populations of Túngara Frogs in Panamá
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Spread of Amphibian Chytrid Fungus across Lowland Populations of Túngara Frogs in Panamá Sofía Rodríguez-Brenes1*, David Rodriguez2, Roberto Ibáñez3,4, Michael J. Ryan1,3 Sofía Rodríguez-Brenes1*, David Rodriguez2, Roberto Ibáñez3,4, Michael J. Ryan1,3 1 Department of Integrative Biology, University of Texas, Austin, Texas, United States of America, 2 Department of Biology, Texas State University, San Marcos, Texas, United States of America, 3 Smithsonian Tropical Research Institute, Balboa, Panamá, 4 Departamento de Zoología, Universidad de Panamá, Panamá, Panamá Sofía Rodríguez-Brenes1*, David Rodriguez2, Roberto Ibáñez3,4, Michael J. Ryan1,3 1 Department of Integrative Biology, University of Texas, Austin, Texas, United States of America, 2 Department of Biology, Texas State University, San Marcos, Texas, United States of America, 3 Smithsonian Tropical Research Institute, Balboa, Panamá, 4 Departamento de Zoología, Universidad de Panamá, Panamá, Panamá 1 Department of Integrative Biology, University of Texas, Austin, Texas, United States of America, 2 Department of Biology, Texas State University, San Marcos, Texas, United States of America, 3 Smithsonian Tropical Research Institute, Balboa, Panamá, 4 Departamento de Zoología, Universidad d Panamá, Panamá, Panamá a1111 * sofiarb@utexas.edu * sofiarb@utexas.edu * sofiarb@utexas.edu Abstract Chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is an emergent infectious disease partially responsible for worldwide amphibian population declines. The spread of Bd along highland habitats (> 500 meters above sea level, m a.s.l.) of Costa Rica and Panamá is well documented and has been linked to amphibian popula- tion collapses. In contrast, data are scarce on the prevalence and dispersal of Bd in lowland habitats where amphibians may be infected but asymptomatic. Here we describe the spread (2009 to 2014) of Bd across lowland habitats east of the Panamá Canal (< 500 m a.s.l.) with a focus on the Túngara frog (Physalaemus [Engystomops] pustulosus), one of the most common and abundant frog species in this region. Highland populations in western Panamá were already infected with Bd at the start of the study, which was consistent with previous studies indicating that Bd is enzootic in this region. In central Panamá, we col- lected the first positive samples in 2010, and by 2014, we detected Bd from remote sites in eastern Panamá (Darién National Park). We discuss the importance of studying Bd in low- land species, which may serve as potential reservoirs and agents of dispersal of Bd to high- land species that are more susceptible to chytridiomycosis. OPEN ACCESS Citation: Rodríguez-Brenes S, Rodriguez D, Ibáñez R, Ryan MJ (2016) Spread of Amphibian Chytrid Fungus across Lowland Populations of Túngara Frogs in Panamá. PLoS ONE 11(5): e0155745. doi:10.1371/journal.pone.0155745 Editor: Louise A. Rollins-Smith, Vanderbilt University School of Medicine, UNITED STATES Received: September 29, 2015 Accepted: May 3, 2016 Published: May 13, 2016 Editor: Louise A. Rollins-Smith, Vanderbilt University School of Medicine, UNITED STATES Received: September 29, 2015 Accepted: May 3, 2016 Published: May 13, 2016 Copyright: © 2016 Rodríguez-Brenes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Louise A. Rollins-Smith, Vanderbilt University School of Medicine, UNITED STATES Received: September 29, 2015 Accepted: May 3, 2016 Published: May 13, 2016 Editor: Louise A. Rollins-Smith, Vanderbilt University School of Medicine, UNITED STATES Received: September 29, 2015 Accepted: May 3, 2016 Published: May 13, 2016 Copyright: © 2016 Rodríguez-Brenes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Published: May 13, 2016 Copyright: © 2016 Rodríguez-Brenes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Data Availability Statement: All data are available from the DRYAD Digital Repository (doi:10.5061/ dryad.6bp92). Data Availability Statement: All data are available from the DRYAD Digital Repository (doi:10.5061/ dryad.6bp92). Wildlife extinctions are not typically attributed to infectious diseases, yet there are a few exam- ples showing pathogens as the direct cause of local extinctions [1–3]. Some of them have been linked to chytridiomycosis, an emerging infectious disease [4,5]. In amphibians, chytridiomy- cosis results from a skin infection caused by the chytrid fungus Batrachochytrium dendrobati- dis (Bd). In the tropics, the most severe declines have been documented in comparatively cooler and humid areas above 500 m [6], most likely because lower temperatures (17–25°C) are optimal for Bd growth and water facilitates the propagation and dispersal of the aquatic, flagellated Bd zoospores [7]. Although the rapid spread of Bd into apparently Bd-free areas Funding: This work was funded by National Science Foundation (http://www.nsf.gov/), IBN 0517328 to MJR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 1 / 8 Amphibian Chytrid Spreads across Tropical Lowlands throughout the world is well-documented [8–12], the process by which Bd spreads is still not clearly understood [13]. Bd has rapidly spread throughout the highlands of Central America. In the late 1980s, the dis- appearance of golden toads (Incilius periglenes) and declines of other anuran populations in the protected cloud forest of Monteverde, Costa Rica (Fig 1), were the first alerts to what later became a predictable pattern of declines spreading towards Panamá [14,15]. Shortly thereafter, between 1993 and 1997 additional cases of amphibian population declines or extinctions were reported in the highlands close to the border with Panamá at Las Tablas, Costa Rica, and Fortuna, Panamá (Fig 1)[16,17]. The sites experiencing population declines were located at altitudes above 500 m, and the fastest declining species were stream dwellers with aquatic tadpoles [6]. Bd was spreading west to east in a wave like pattern [8]. High elevation sites in western and central Panamá were then intensively studied prior to and during the arrival of Bd, these studies documented amphib- ian population declines after the arrival of Bd in this region (Fig 1)[16–19]. Only two studies have investigated Bd infection spread in eastern Panamá. Rebollar et al. [20] sampled populations along the lowlands of east Panamá, and Woodhams et al. Methods We sampled Túngara frog populations in Panamá from 2009 to 2014, each year during the rainy season (June to November), which falls within their reproductive season. In most cases, populations were sampled during two reproductive seasons. Lowland sites in east and central Panamá ranged from 12 to 67 m in altitude (Fig 1). We also sampled highland populations at El Valle (elevation 600 m; Fig 1) in central Panamá, and at Chiriquí near the town of Cuesta de Piedra (elevation 460–967 m; Fig 1) in western Panamá. This altitude is the highest known Túngara population in Panamá. Chiriquí was also our western most site and was located 30 km east of sites where other amphibian species experienced declines in 1993 [17]. In central Panamá, we sampled two lowland sites east of the Panamá Canal, Gamboa and Summit, ranging from 46 to 98 m in elevation (Fig 1). These two lowland sites are located on the eastern side of the Panamá Canal but are separated by the Chagres River. Gamboa is a small town north of the river that is surrounded by rainforest. Here we included Pipeline Road, which intersects a protected rainforest in the Soberanía National Park. Summit is located south of the river and includes portions of the Soberanía National Park. We sampled along the main road, on trails and dirt roads within the National Park. In east Panamá, we sampled four sites. Metetí and Yaviza are located along and at the very end of the Inter-American Highway, respectively (Fig 1). Here, we sampled disturbed, defor- ested habitats, in puddles on dirt roads and cattle ranches. Further east, along the Tuira River, we sampled at El Real (Fig 1), a small town surrounded by less disturbed habitat and in Darién National Park. In Darién National Park, we sampled around the Rancho Frío field station (Fig 1) at an average of 50 m of elevation. This site is predominantly primary rainforest with a tall canopy, thick lianas and an understory dominated by palms. Data Availability Statement: All data are available from the DRYAD Digital Repository (doi:10.5061/ dryad.6bp92). [19] sam- pled sites in central Panamá. These studies did not detect a clear pattern of wave-like spread from west to east as observed in the highlands of Panamá. It is also possible that another wave of Bd from South America crossed into Panamá [8]. Currently, the dynamics of Bd spread in the lowlands of Panamá, where environmental conditions are not ideal for Bd, are still unclear and require additional investigation. Evaluating the spread of a pathogen in a single, abundant, and well-characterized host can help to predict spread dynamics while controlling for host phylogenetic diversity. The Túngara frog (Physalaemus [Engystomops] pustulosus) is a common species occupying lowland habitats ranging from Mexico to Colombia, Venezuela, and a small portion of the Guyana Shield. Its Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops] pustulosus) populations (open triangles). Year in parenthesis corresponds to year of decline or the year that Bd was first detected. doi:10.1371/journal.pone.0155745.g001 Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops] pustulosus) populations (open triangles). Year in parenthesis corresponds to year of decline or the year that Bd was first detected. Fig 1. Sites where Bd has been detected in the past (filled circles), and sites where we sampled Túngara frog (Physalaemus [Engystomops] pustulosus) populations (open triangles). Year in parenthesis corresponds to year of decline or the year that Bd was first detected. doi:10.1371/journal.pone.0155745.g001 doi:10.1371/journal.pone.0155745.g001 PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 2 / 8 Amphibian Chytrid Spreads across Tropical Lowlands range parallels many highland regions where Bd and amphibian declines have been extensively documented [8–13]. The adults and tadpoles of this species use both permanent and ephemeral bodies of water in habitats ranging from urban areas to pristine forests. Thus, the Túngara frog is an ideal species in which to characterize the spread of Bd through populations of a lowland host. Here, we (i) report the Bd infection of túngara frogs at two sites west of the Panamá Canal where Bd is enzootic, and (ii) present data documenting the spread of Bd in the lowlands of central and east Panamá from 2009 to 2014. Time scale of surveys We first sampled El Valle in 2009 and then Chiriquí in 2010, where Bd is thought to have arrived in the mid-1990s according to previous estimations [13]. In 2010, we also sampled Gamboa and Summit in central Panamá. Woodhams et al. [19] reported Bd in Summit in 2007. We then sampled Metetí and Yaviza in eastern Panamá during 2011, where we expected the front of the Bd wave to be just arriving. In 2013, we sampled all the populations previously surveyed. In 2014 we sampled in Gamboa, Yaviza and finally Darién National Park, which was the eastern most site and where we expected P. pustulosus populations to be Bd naive. Results In total, we sampled 1695 P. pustulosus adults across Panamá from 2009 to 2014 (Fig 1). Sam- ple size, prevalence, and 95% binomial confidence intervals are shown in Table 1. We consid- ered sites with sample sizes greater than 60 individuals and no positive samples as Bd-negative [25]. The site Chiriquí, in western Panamá, was positive for Bd in 2010 and 2013. In central Panamá, El Valle was positive for Bd in 2009. In 2010, Gamboa samples were negative for Bd, but Summit, which is only 8 km to the south, was positive. Bd reached Gamboa in 2011, and in following years we detected Bd positive samples in both Summit and Gamboa. By 2014, preva- lence had reached 26% in Gamboa (Table 1). Populations in Metetí were Bd naive in 2011 but positive in 2013. Farther east, Yaviza was naive for Bd in 2013, but by 2014 prevalence reached approximately 6%. By 2014, Bd was also present in P. pustulosus populations from El Real and Rancho Frío, Darién National Park (Table 1). Bd sampling We toe-clipped individuals to avoid recapture. To avoid cross contamination, we captured adults by hand using a new pair of nitrile gloves for each individual and kept them isolated in a plastic bag until processing. We swabbed the ventral area using a sterile cotton tip dry swab (Medical Wire & Equipment, model MW113 and MW110) following established procedures [21]. Swabs were stored in 90% ethanol or they were kept frozen until extraction, thus we expect no effects of sample storage on estimates of prevalence or infection intensity [22]. To 3 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands avoid potential cross-contamination between sites, we bleached our rubber boots and vehicle tires and then rinsed them with tap water before leaving the collection site. Real time quantitative PCR To determine the prevalence of Bd in each population and the intensity of infection for individ- ual frogs, we processed the swabs using quantitative PCR (qPCR) following the protocol devel- oped by Boyle et al. [23] and modified by Kriger et al. [24]. For samples collected from 2009 to 2012, we used a Roche LightCycler 480 system with a high confidence setting to detect positive samples. For samples collected during 2009–2010, we did not quantify the number of zoo- spores due to lack of Bd standards. For samples collected during 2013 and 2014, we used Taq- Man1 Fast Advanced Master Mix (Applied Biosystems) and a StepOnePlus™system. We used a dilution series of genomic DNA from strain JEL423 as our standard reference for the estima- tions of infection intensity in samples collected from 2011 to 2014. We calculated prevalence by using the ratio of positive samples to total samples per population and calculated 95% bino- mial confidence intervals. For each population, we calculated average infection intensity using the average number of zoospore equivalents (z.e.) inferred from qPCR among positive individuals. To calculate the rate of Bd spread across sites east of the Canal, we used the distance between sites and the year we first detected infected frogs. We sampled frogs from June to November. If Bd arrived after sampling was completed for a given year, then we would have detected it the following year. We could not calculate the rate of spread in western Panamá because Bd was already present at the beginning of our sampling period. Thus, we approximated when Tún- gara frogs were first infected based on historical reports for sites near Chiriquí [8,13,19]. Ethical approval All applicable institutional and/or national guidelines for the care and use of animals were fol- lowed. The protocol was approved by the Institutional Animal Care and Use Committee of the Smithsonian Tropical Research Institute (protocol number: 2011-0825-2014-02), and by the Autoridad Nacional del Ambiente (permit numbers: SE/A-81-09, SE/A-73-10, SE/A-48-10, SC/A-28-11, SE/A-83-11, SE/A-42-11, SE/A-30-12, SE/A-47-13, SC/A-9-14) Amphibian Chytrid Spreads across Tropical Lowlands Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi- dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by l it d Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi- dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by l it d Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi- dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by longit de Table 1. Bd prevalence and infection intensity per site and year in Túngara frog (Physalaemus [Engystomops] pustulosus) populations sampled in this study (n = number of individuals sampled; Positive = number of individuals detected positive for Bd; 95% CI = 95% binomial distribution confi- dence intervals; Average intensity = average of number of zoospore equivalents in infected frogs per population). Sites are arranged west to east by longitude Site Year N Positive Prevalence % (95% CI) Average Intensity (± St.Dev) Chiriquí 2010 41 11 27 (14–43) data not available 2013 38 16 42 (26–60) 3570 (± 12454) El Valle 2009 5 3 60 (15–95) data not available Gamboa 2010 321 0 0 (0–0.5) 0 2011 111 7 6 (3–13) 86 (± 117) 2012 205 26 13 (8–18) 1617 (± 5485) 2013 166 35 21 (15–29) 536 (± 2061) 2014 84 22 26 (17–37) 209 (± 833) Summit 2010 12 2 17 (2–48) data not available 2011 108 2 2 (0–6) 10 (± 7) 2013 120 17 14 (9–22) 89 (± 193) Metetí 2011 91 0 0 (0–4) 0 2013 94 2 2 (0–8) 7 (± 3) Yaviza 2013 68 0 0 (0–0.5) 0 2014 41 3 7 (2–20) 6 (± 1) El Real 2014 40 2 5 (1–17) 6 (± 1) Rancho Frío 2014 150 11 7 (4–13) 48 (± 101) doi:10.1371/journal.pone.0155745.t001 The rates of spread, however, vary substantially. Specifically, our data suggest that it took approximately one year for Bd to move the 8 km distance between Summit and Gamboa, which are separated by the Chagres River. In contrast, the rate of Bd spread from Summit to Metetí was 65 km/year, and 42 km/year from Metetí to Yaviza. All data are available from the DRYAD Digital Repository (doi:10.5061/dryad.6bp92). The rates of spread, however, vary substantially. Specifically, our data suggest that it took approximately one year for Bd to move the 8 km distance between Summit and Gamboa, which are separated by the Chagres River. In contrast, the rate of Bd spread from Summit to Metetí was 65 km/year, and 42 km/year from Metetí to Yaviza. All data are available from the DRYAD Digital Repository (doi:10.5061/dryad.6bp92). All data are available from the DRYAD Digital Repository (doi:10.5061/dryad.6bp92). Rates of spread of Bd in Túngara frogs If Bd spread eastward from Summit in central Panamá to Darién in eastern Panamá, then the front moved at an average rate of 54 km/year among these lowland Túngara frog populations. PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 4 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands We cannot discern whether the source of infection of the Gamboa populations was from Summit Túngara frogs or from other species of frogs in Gamboa. Regardless, there is a rela- tively large time lag between when Bd was detected in Túngara frogs from Gamboa compared to when Bd was first detected in Túngara frogs from Summit. The mechanism by which Bd spreads is unknown, but it has been suggested that Bd could survive and could be carried in mud, and thus easily dispersed by humans [26]. In Túngara frogs, Bd did not spread as fast as expected from Summit to Gamboa, which are only 8 km apart and connected by a well-traveled road and bridge over the Chagres River. Certain geographic features, like rivers, could impede the spread of Bd. The Chagres River, which is about 100 m wide and has water all year long, separates Gamboa and Summit. Genetic studies demonstrate that the Chagres River is a geographical barrier for gene flow between Túngara frog populations [27], thus it is possible that limited migration between these populations could slow the spread of Bd whether Túngara frogs contracted Bd from conspecifics or heterospecifics. Bd does seem to have spread rapidly in the lowlands towards eastern Panamá. An alternative explanation for the spread of Bd from east to west through- out all of Panamá is that populations in Darién, and elsewhere in far eastern Panamá, were infected by a wave coming from the south [8]. These two scenarios should be tested through a phylogenetic analysis of Bd throughout the Túngara frog's range in Central and South America. As such data are currently unavailable, it is difficult to determine the recent origin of Bd in eastern Panamá; therefore, our estimates of rate of spread should be viewed with this potential caveat in mind. Woodhams et al. [19] conservatively estimated that Bd spread east and would have reached Tortí in September of 2012, but [28] reported two positives out of 93 samples of other species of frogs at this site in 2010. There are no other published reports on the pres- ence of Bd among amphibian species from this specific area. In 2011, we sampled in Metetí, approximately 70 km southeast of Tortí, and found this site to be Bd naive. Rebollar et al. PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 Discussion Data for the spread of Bd in the lowlands of Middle America are scarce. Here we estimated the rate of spread of Bd in Túngara frogs based on the first detection of Bd, and we assumed that Bd spread in a wave-like fashion [8]. In the lowlands of Panamá, Bd spread at a similar rate among Túngara frogs (54 km/year) when compared to other amphibian species tested in this area (30–174 km/year) [19]. There was one exception. The rate of spread from Summit across the Chagres River to Gamboa was slower than the average (8 km/year). In western Panamá, we sampled in Chiriquí, at elevations where amphibian population declines have been severe in the past [16]. Since Bd is now enzootic in this area [19] and at El Valle in central Panamá, it is not surprising that the Túngara frog populations from Chiriquí and El Valle were positive in 2010, and 2009, respectively. We also found the highest average loads of Bd in Chiriquí (3570 z.e., Table 1). We could not estimate the rate of Bd dispersal among Túngara frog populations between Chiriquí and El Valle as they were already infected at the time we sampled. In 2007, Woodhams et al. [19] reported Bd positive individuals (30% prevalence) among three species at Soberanía National Park, including the Summit area where we sampled in 2010, and they suggested that Bd was already enzootic during their study. In the same year, Gamboa, was still naive and we most likely sampled before Bd arrived. In 2011, we detected Bd at Gamboa for the first time, one year earlier than previously reported for this area [20]. 5 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 References 1. MacPhee RDE, Greenwood AD. Infectious disease, endangerment, and extinction. Int J Evol Biol. 2013 Jan 16; 2013:e571939. 1. MacPhee RDE, Greenwood AD. Infectious disease, endangerment, and extinction. Int J Evol Biol. 2013 Jan 16; 2013:e571939. 2. McCallum H. Disease and the dynamics of extinction. Philos Trans R Soc B Biol Sci. 2012 Oct 19; 367 (1604):2828–39. 3. Smith KF, Sax DF, Lafferty KD. Evidence for the role of infectious disease in species extinction and endangerment. Conserv Biol. 2006 Oct 1; 20(5):1349–57. PMID: 17002752 3. Smith KF, Sax DF, Lafferty KD. Evidence for the role of infectious disease in species extinction and endangerment. Conserv Biol. 2006 Oct 1; 20(5):1349–57. PMID: 17002752 4. Collins JP, Crump ML. Extinction in Our Times: Global Amphibian Decline. Oxford University Press; 2009. 477 p. 4. Collins JP, Crump ML. Extinction in Our Times: Global Amphibian Decline. Oxford University Press; 2009. 477 p. 5. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012 Apr 12; 484(7393):186–94. doi: 10.1038/ nature10947 PMID: 22498624 5. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, et al. Emerging fungal threats to animal, plant and ecosystem health. Nature. 2012 Apr 12; 484(7393):186–94. doi: 10.1038/ nature10947 PMID: 22498624 6. Brem FMR, Lips KR. Batrachochytrium dendrobatidis infection patterns among Panamanian amphib- ian species, habitats and elevations during epizootic and enzootic stages. Dis Aquat Organ. 2008 Sep 24; 81(3):189–202. doi: 10.3354/dao01960 PMID: 18998584 6. Brem FMR, Lips KR. Batrachochytrium dendrobatidis infection patterns among Panamanian amphib- ian species, habitats and elevations during epizootic and enzootic stages. Dis Aquat Organ. 2008 Sep 24; 81(3):189–202. doi: 10.3354/dao01960 PMID: 18998584 7. Piotrowski JS, Annis SL, Longcore JE. Physiology of Batrachochytrium dendrobatidis, a chytrid patho- gen of amphibians. Mycologia. 2004 Jan 1; 96(1):9–15. PMID: 21148822 8. Lips KR, Diffendorfer J, Mendelson JR, Sears MW. Riding the wave: reconciling the roles of disease and climate change in amphibian declines. PLoS Biol. 2008 Mar; 6(3):e72. doi: 10.1371/journal.pbio. 0060072 PMID: 18366257 9. Farrer RA, Weinert LA, Bielby J, Garner TWJ, Balloux F, Clare F, et al. Multiple emergences of geneti- cally diverse amphibian-infecting chytrids include a globalized hypervirulent recombinant lineage. Proc Natl Acad Sci. 2011 Nov 15; 108(46):18732–6. doi: 10.1073/pnas.1111915108 PMID: 22065772 10. Fisher MC, Garner TWJ, Walker SF. Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host. [20] recorded Bd positive samples from Nuevo Vigía in 2012, just 26 km east of Metetí, where we detected low Bd prevalence in 2013, thus supporting a wave-like Bd spread from west to east in Túngara frogs. Species that carry Bd asymptomatically and share the habitat with more vulnerable species can potentially function as Bd spreaders. Túngara frog population declines have not been reported and were not evident during our study. If the prevalence and dispersal of Bd is den- sity dependent, then the spread of Bd along the lowlands might be enhanced by abundant and apparently resistant species serving as reservoirs. Túngara frogs are known to disperse between breeding sites at distances up to 200 m [29]. They also share the habitat with a wide variety of species; thus, they could contribute to the rapid dispersal of Bd if they are effective carriers. Moreover, if most species in the lowlands are less susceptible to Bd or Bd is less viru- lent [7], then the high diversity and abundance of hosts could further facilitate the dispersal of Bd. Chytridiomycosis is linked to some of the most severe population declines and extinctions of wildlife yet recorded. While substantial efforts have been aimed at understanding the spread and pathogenicity in the more vulnerable highland frog species, we know relatively little about the dynamics of Bd in tropical lowland regions of the world. Lowland species, however, could be reservoirs and dispersal agents between areas where amphibian species are more vulnerable to Bd infection. As highlighted by our results, even though lowland regions are typically char- acterized by less favorable climatic conditions for Bd [7], by harboring asymptomatic Bd infec- tions, lowland amphibian populations could potentially play an important role in the spread of Bd across tropical regions. 6 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands Author Contributions Conceived and designed the experiments: SRB MJR. Performed the experiments: SRB MJR RI DR. Analyzed the data: SRB DR RI. Contributed reagents/materials/analysis tools: SRB MJR DR RI. Wrote the paper: SRB MJR DR RI. Acknowledgments For help in the field we thank Emma and Lucy Ryan, Tony Alexander, Alex Jordan, Meghan Still, Ty Hoskin, Mahudy Díaz, Samuel Sucre, and for logistical support we thank SENA- FRONT, ANAM, and STRI. We thank Teofil Nakov and Robert Puschendorf for comments on the manuscript. We also thank Michael R.J. Forstner at Texas State University for instrumenta- tion support, and Mahudy Díaz at STRI for laboratory assistance. PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 References Annu Rev Microbiol. 2009; 63(1):291–310. 11. James TY, Litvintseva AP, Vilgalys R, Morgan JAT, Taylor JW, Fisher MC, et al. Rapid global expan- sion of the fungal disease chytridiomycosis into declining and healthy amphibian populations. PLoS Pathog. 2009 May 29; 5(5):e1000458. doi: 10.1371/journal.ppat.1000458 PMID: 19478871 12. Velo-Antón G, Rodríguez D, Savage AE, Parra-Olea G, Lips KR, Zamudio KR. Amphibian-killing fun- gus loses genetic diversity as it spreads across the New World. Biol Conserv. 2012 Feb; 146(1):213–8. 13. Phillips BL, Puschendorf R. Do pathogens become more virulent as they spread? Evidence from the amphibian declines in Central America. Proc R Soc B Biol Sci [Internet]. 2013 Sep 7 [cited 2013 Jul 29]; 280(1766). Available: http://rspb.royalsocietypublishing.org/content/280/1766/20131290 14. Pounds JA, Crump ML. Amphibian declines and climate disturbance: the case of the Golden Toad and the Harlequin Frog. Conserv Biol. 1994 Mar 1; 8(1):72–85. 15. Pounds JA, Fogden MPL, Savage JM, Gorman GC. Tests of null models for amphibian declines on a tropical mountain. Conserv Biol. 1997; 11(6):1307–22. 16. Lips KR. Mass mortality and population declines of anurans at an upland site in western Panama. Con- serv Biol. 1999; 13(1):117–25. 17. Lips KR. Decline of a tropical montane amphibian fauna. Conserv Biol. 1998; 12(1):106–17. 7 / 8 PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 Amphibian Chytrid Spreads across Tropical Lowlands 18. Lips KR, Brem F, Brenes R, Reeve JD, Alford RA, Voyles J, et al. Emerging infectious disease and the loss of biodiversity in a neotropical amphibian community. Proc Natl Acad Sci U S A. 2006 Feb 28; 103 (9):3165–70. PMID: 16481617 19. Woodhams DC, Kilburn VL, Reinert LK, Voyles J, Medina D, Ibáñez R, et al. Chytridiomycosis and amphibian population declines continue to spread eastward in Panama. EcoHealth. 2008 Sep 1; 5 (3):268–74. doi: 10.1007/s10393-008-0190-0 PMID: 18807089 20. Rebollar EA, Hughey MC, Harris RN, Domangue RJ, Medina D, Ibáñez R, et al. The lethal fungus Batrachochytrium dendrobatidis is present in lowland tropical forests of far eastern Panamá. PLoS ONE. 2014 Apr 16; 9(4):e95484. doi: 10.1371/journal.pone.0095484 PMID: 24740162 21. Hyatt A, Boyle D, Olsen V, Boyle D, Berger L, Obendorf D, et al. Diagnostic assays and sampling proto- cols for the detection of Batrachochytrium dendrobatidis. Dis Aquat Organ. 2007; 73:175–92. PMID: 17330737 22. Sluys MV, Kriger KM, Phillott AD, Campbell R, Skerratt LF, Hero JM. PLOS ONE | DOI:10.1371/journal.pone.0155745 May 13, 2016 References Storage of samples at high tem- peratures reduces the amount of amphibian chytrid fungus Batrachochytrium dendrobatidis DNA detectable by PCR assay. Dis Aquat Organ. 2008 Aug 27; 81(2):93–7. doi: 10.3354/dao01953 PMID: 18924373 23. Boyle D, Boyle D, Olsen V, Morgan J, Hyatt A. Rapid quantitative detection of chytridiomycosis (Batra- chochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay. Dis Aquat Organ. 2004; 60:141–8. PMID: 15460858 24. Kriger KM, Hero J, Ashton KJ. Cost efficiency in the detection of chytridiomycosis using PCR assay. Dis Aquat Organ. 2006 Jul 25; 71(2):149–54. PMID: 16956062 25. Skerratt LF, Berger L, Hines HB, McDonald KR, Mendez D, Speare R. Survey protocol for detecting chytridiomycosis in all Australian frog populations. Dis Aquat Organ. 2008 Jul 7; 80(2):85–94. doi: 10. 3354/dao01923 PMID: 18717061 26. Johnson ML, Speare R. Possible modes of dissemination of the amphibian chytrid Batrachochytrium dendrobatidis in the environment. Dis Aquat Organ. 2005 Jul 18; 65(3):181–6. PMID: 16119886 27. Lampert KP, Rand AS, Mueller UG, Ryan MJ. Fine-scale genetic pattern and evidence for sex-biased dispersal in the túngara frog, Physalaemus pustulosus. Mol Ecol. 2003 Dec 1; 12(12):3325–34. PMID: 14629349 28. Küng D, Bigler L, Davis LR, Gratwicke B, Griffith E, Woodhams DC. Stability of microbiota facilitated by host immune regulation: informing probiotic strategies to manage amphibian disease. PLoS ONE. 2014 Jan 29; 9(1):e87101. doi: 10.1371/journal.pone.0087101 PMID: 24489847 29. Marsh DM, Fegraus EH, Harrison S. Effects of breeding pond isolation on the spatial and temporal dynamics of pond use by the Tungara frog, Physalaemus pustulosus. J Anim Ecol. 1999 Jul 1; 68 (4):804–14. 8 / 8
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https://zenodo.org/records/5825210/files/Improvement%20of%20test%20methods%20and%20criteria%20for%20evaluation%20of%20resistance%20to%20flame%20propagation%20of%20long%20elements%20of%20the%20wiring%20system.pdf
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Improvement of test methods and criteria for evaluation of resistance to flame propagation of long elements of the wiring system
Eastern-European journal of enterprise technologies
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IMPROVEMENT OF TEST METHODS AND CRITERIA FOR EVALUATION OF RESISTANCE TO FLAME PROPAGATION OF LONG ELEMENTS OF THE WIRING SYSTEM Using a substrate of a wooden board covered with a layer of tissue paper with an areal density of (21±9) g/m2, adopted for testing other elements of the electrical wiring system, a cable was identified that is not resistant to flame propagation. It is proposed to use this substrate for testing the flame propagation of cables instead of a substrate made of a double layer of filter paper with a surface density of (80±15) g/m2. In one of three experiments, a cable that was not resistant to flame propagation was found based on the criterion of the presence of ignition of the substrate located under it. To reduce the risk of making an incorrect decision on compliance, it is proposed that the assessment of long elements of the wiring system be carried out according to the rules established for cable ducts, trays and ladders in EN 50085-1 and IEC 61537. R o s t y s l a v K r a v c h e n k o Head of Department Department of Technical Regulation Center of Research in Fire Protection** P a v l o I l l i u c h e n k o Corresponding author Head of Department* E-mail: illuchenko@ukr.net A n d r i i O n y s h c h u k Junior Researcher* O l e k s a n d r Z a z y m k o Researcher* *Department of Electrical Products Research and Testing Center** **Institute of Public Administration and Research in Civil Protection Vyshhorodska str., 21, Kyiv, Ukraine, 04074 For the AVVG cable with an outer diameter of 10 mm to 60 mm, when it touches the blue flame cone of 1 kW, the correlation coefficient of the dependence of the length of the charred part on the diameter was 0.969. For a distance of 100 mm between the sample and the burner along its axis, a correlation coefficient of 0.985 was obtained. It is proposed to test cables under the second condition recommended in IEC 60695-11-2. For two conduits, flame propagation was revealed when exposed to a 1 kW flame for 120 s and 240 s. However, for these conduits, flame propagation did not occur under standard conditions of exposure to such a flame for 20 s and 25 s. IMPROVEMENT OF TEST METHODS AND CRITERIA FOR EVALUATION OF RESISTANCE TO FLAME PROPAGATION OF LONG ELEMENTS OF THE WIRING SYSTEM To identify long elements of the wiring system that are not resistant to flame propagation, it is proposed to test them at a duration of exposure to a flame of 1 kW, established for cables in IEC 60332-1-2 Keywords: electrical product, electrical and optical cable, conduit, fire safety, flame propagation, wiring system Received date 26.10.2021 Accepted date 01.12.2021 Published date 29.12.2021 How to Cite: Kravchenko, R., Illiuchenko, P., Onyshchuk, A., Zazymko, O. (2021). Improvement of test methods and criteria for evaluation of resistance to flame spread of long elements of the wiring system. Eastern-European Journal of Enterprise Technolo­ gies, 6 (10 (114)), 57–68. doi: https://doi.org/10.15587/1729-4061.2021.249105 How to Cite: Kravchenko, R., Illiuchenko, P., Onyshchuk, A., Zazymko, O. (2021). Improvement of test methods and criteria for evaluation of resistance to flame spread of long elements of the wiring system. Eastern-European Journal of Enterprise Technolo­ gies, 6 (10 (114)), 57–68. doi: https://doi.org/10.15587/1729-4061.2021.249105 Ecology Ecology UDC 614.841.41 The test methods for flame propagation of long elements of the electrical wiring system, in particular, cables, cable conduits and ducts, are analyzed, and differences in them are found in the test conditions and criteria for evaluating the resistance to flame propagation. DOI: 10.15587/1729-4061.2021.249105 2. Literature review and problem statement In [3], a new model was developed for the rate of heat re­ lease during combustion of a horizontally located cable tray located at the side wall. The resulting model makes it possi­ ble to reduce the error in determining the peak rate of heat release during the combustion of the cable tray. However, it is advisable to identify discrepancies in the results obtained by the developed model and standardized test methods, for example, using the Steiner tunnel. The European Committee for Electrotechnical Stan­ dardization (CENELEC) for the method [13] has defined an additional criterion for assessing the length of the carbon part of the cable [17]. However, with the introduction of an additional classification criterion [8], other important cri­ teria for assessing the length of the carbon part of the cable were excluded. Also CENELEC for method [13] has improved the re­ quirements for test equipment to ensure constant exposure to a flame source for a specified duration of its applica­ tion [18]. At the same time, such an improvement has not been proposed for the method [14]. An extended model for estimating the speed of flame propagation during combustion of a vertically located ca­ ble tray, depending on the distance between the cables, is proposed in [4]. However, it is desirable to investigate the change in flame propagation speed for cables placed on a cable ladder. Testing of cables on cable ladders is accepted by international and European standards. The research results presented in [19] established a sig­ nificant variance in the test results according to the meth­ od [13] of samples of identical cables obtained in different laboratories. The author notes that if the obtained length of the charred part of the sample is more than 152 mm, the test­ ing laboratory runs the risk of making an incorrect decision on the conformity of the cables. However, the factors that influenced the reproducibility of the comparative test results have not been identified. The work [5] presents the results of studies of the effect of the distance between cables and their layers on the height and width of the flame when burning cables laid in the tun­ nel. The paper also presents data on the speed of horizontal flame propagation along the cables. At the same time, it is ad­ visable to determine the dependence of the horizontal flame propagation on the transverse dimensions of the cables. 2. Literature review and problem statement When using a 1 kW flame source, the International Elec­ trotechnical Commission has developed methods for testing the flame propagation of other long elements of the wiring system [20–24]. At the same time, these methods differ in terms of test conditions and criteria for evaluating the results, which can influence the decision on the conformity of such elements. This applies to standardized methods for testing cables with a 1 kW flame. The results of studies of the influence of the angle of inclination of the cable (0–20°) laid in the tunnel and the speed of longitudinal air flows (0.35–0.65 m/s) on the heat­ ing temperature of the tunnel ceiling from a burning cable are presented in [6]. But the work does not provide import­ ant data on the influence of these parameters on the speed of propagation of the flame of the cables. In order to reduce the risk of fire in buildings associated with cables, methods for testing cables laid in bundles for flame propagation have been improved [7]. These methods make it possible to additionally determine the indicators of heat release and smoke production. On the basis of these and other methods, a new classification of cables for reaction to fire has been developed. This classification is implemented in EN 13501-6 [8] to fulfill the requirements of Regula­ tion (EU) No 305/2011 [9]. At the same time, the disadvan­ tage of this classification is not taking into account all the criteria for assessing the resistance to flame propagation of single-laid cables. The lack of an estimate for the duration of self-combustion and the flammable ability of particles sepa­ rated from single laid cables can affect their classification. It should be noted that in practice, standardized test meth­ ods for products compared to non-standardized ones are those, based on the results of which, a decision is made on its compli­ ance and use at facilities. The perfection of these methods is an important influential factor in ensuring the fire safety of both the product itself and the facilities on which it is used. At the same time, for economic reasons, improvements in such methods, especially in terms of significant changes in the design of test equipment, are slowly being introduced into practice. The complex of factors influencing the assessment of the fire hazard of electrical products is defined in [2]. 2. Literature review and problem statement Their influence on the assessment of resistance to flame propaga­ tion of long elements of the wiring system has not yet been adequately studied. To reduce the cost of testing cables of the same brand with different transverse dimensions, a procedure for ex­ tended application of the results for fiber-optic cables has been developed [10]. Such a procedure has been proposed for the classification of electrical cables according to their reaction to fire. All this leads to research on the resistance to flame propagation of long elements of the electrical wiring system under conditions of exposure to a 1 kW flame source. At the same time, work [11] did not find a clear relation­ ship between fire hazard indicators and the main parameter χ of the extended application procedure for electrical cables. Therefore, further research is advisable to improve the pro­ cedure for the extended application of cable test results. 1. Introduction At the same time, the distance between the torch and the sample was not given in accordance with the distance recommended by the basic IEC standard. 1. Introduction ance of these systems with fire safety requirements ensures compliance with such requirements of each of their elements. Electrical wiring systems are elements of power supply, power supply and control systems for various equipment and communication systems in buildings and vehicles. Fires as­ sociated with electrical wiring systems can spread the flame over a considerable distance from the source of the fire. In the process of their combustion, smoke, toxic and corrosive volatile combustion products can be released in a dangerous amount for people and property. Under certain conditions, the development of a fire from electrical wiring systems led to catastrophic consequences. In electrical wiring systems, potentially fire hazardous elements are electrical and optical cables and wires (hereinaf­ ter referred to as cables), conduits, trunkings, ductings, trays, ladders, liquid tight sheathings and powertracks. For long elements of the electrical wiring system, one of the measures to reduce the emission of hazardous volatile combustion products is to limit the spread of the flame. Considering that fire hazard tests of long elements of the wiring system are performed separately, their test con­ ditions, criteria for evaluating the results, as well as the rules for making a decision on compliance should not differ significantly. The main preventive measure to reduce the risk of fire from electrical products is the fire hazard test [1]. The prior­ ity is the testing of finished products [2]. At the same time, in standard methods, the specified characteristics, on which the results of assessing the resis­ tance to flame propagation of long elements of the wiring In practice, fire hazard testing of all combinations of wiring systems is problematic. In this regard, the compli­ 57 s is an open access article under the Creative Commons CC BY license 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 system depend, are different, therefore, studies to determine the characteristics that significantly affect such an assess­ ment are relevant. part of samples [13] and the incendiary ability of particles separated from them [14]. For these methods, the procedure for measuring the outer diameter of the samples was deter­ mined and the requirements for the duration of the applica­ tion of a flame source of 1 kW to samples of flat cables were improved [15, 16]. 3. The aim and objectives of research The aim of research is to ensure the fire safety of facilities by improving test methods and criteria for assessing the re­ sistance to flame propagation of long elements of the wiring system. This will help to reduce the number of people killed and injured and the amount of damage from fires. To meet the requirements of technical regulations [9, 12], The International Electrotechnical Commission (IEC) has developed methods for determining the length of the charred 58 Ecology To achieve the aim, the following objectives were set: flame propagation of the cable, a test method was applied ac­ cording to IEC 60332-1-3 [14, 16]. During the experiments, let’s additionally use a base made of a white pinewood board 0 25 700−  mm long, 0 25 300− mm wide and 10 mm thick, covered with tissue paper with an areal density of (21±9) g/m2. – to analyze the standardized test methods for flame propagation of long elements of the wiring system; – to determine the influence of the characteristics of the bases, placed under the sample of long elements of the wiring system, on their flammability; The distance between the base and the point of applica­ tion of the 1 kW flame was 200 mm. – to determine the influence of the characteristics of the base, placed under the sample of long elements of the wiring system, on the assessment of resistance to the propagation of flame of the cable; Samples for each experiment were six lengths of 4×240 AVVG cable, 54.6 mm in diameter each. The duration of the application of the test flame to the cable samples was 240 s. – to determine the influence of the duration of appli­ cation of the flame source to the sample and the distance between the burner and the sample on the assessment of the resistance to flame propagation of cables; In the course of the experiments, the length of the charred part and the duration of self-combustion of the sam­ ples were additionally determined. – to determine the effect of the duration of application of the flame source to the sample on the assessment of resistance to flame propagation of cable conduits trunkings and ductings. 4. 4. Methodology for studying the influence of flame application conditions on the assessment of resistance to flame propagation of cables To study the influence of the duration of application of the flame source to the sample and the distance between the burner and the sample on the assessment of resistance to flame propagation of cables, test methods and criteria for evaluating the results were used according to stan­ dards [13–17]. 4. 1. Experimental conditions h h d f Research methods of testing and criteria for assessing the resistance to flame propagation of long elements of the wiring system [8, 13–17, 20–24] were carried out using ana­ lytical and experimental methods. Additional experiments were carried out on the distance between the burner and the sample, which is measured along the axis of the burner, equal to 100 mm. To eliminate the influence on the results of experimental studies of the conditions of air conditioning and the environ­ ment and the size of the samples: In all experiments, the distance between the base and the point of application of the 1 kW flame was 200 mm. The samples for the experiments were six cable sections AVVG 4×2.5, AVVG 4×16, AVVG 4×35, AVVG 4×120 and AVVG 4×240 with diameters of 10 mm, 19 mm, 26 mm, 42 mm, and 61 mm. 1) samples and bases for assessing the inflammatory capacity of particles separated from them were kept for at least 48 hours at a temperature of (23±2) °С and a relative humidity of (50±10) %; In the course of the experiments, the duration of self-com­ bustion of the samples was additionally determined. 2) the experiments were carried out at a temperature of (23±2) °С, a relative air humidity of no more than 75 % and an air flow rate of no more than 0.1 m/s; 2) the experiments were carried out at a temperature of (23±2) °С, a relative air humidity of no more than 75 % and an air flow rate of no more than 0.1 m/s; 3) samples of length 25 0 600+  mm were subjected to tests. 4. 5. Methods for studying the effect of the duration of the application of the flame on the assessment of resistance to flame propagation of cable conduitstrunk­ ings and ductings 3) samples of length 25 0 600+  mm were subjected to tests. 4. 2. Methodology for studying the effect of the char­ acteristics of bases on their flammability To study the influence of the characteristics of the bases placed under the sample of the long elements of the electrical wiring system on their flammability, two bases were used. To study the effect of the duration of the application of the flame source to the sample on the assessment of resis­ tance to flame propagation of cable conduits trunkings and ductings, test methods and criteria for evaluating the results were used in accordance with the standards [21, 23]. The first base consisted of a white pinewood board 0 25 700− mm long, 0 25 300−  mm wide and 10 mm thick, covered with tissue paper with an areal density of (21±9) g/m2. Additionally, experiments were carried out on the du­ ration of the application of the flame source, established for cables [13]. The second base was a double layer of filter paper with an areal density of (80±15) g/m and a square shape with a side of (300±10) mm. The samples for the experiments were: The samples for the experiments were: The source of the formation of burning drops was a tele­ phone wire with polyethylene insulation TRP 2×0.5. a) six segments: a) six segments: 1) corrugated cable conduit made of polyamide with an outer diameter of 16 mm and a wall thickness of 0.5 mm; The bases were placed under a horizontally located wire at distances of 150 mm, 200 mm and 600 mm. 2) corrugated polypropylene cable conduit with an outer diameter of 50 mm and a wall thickness of 0.8 mm; Five experiments were performed at each distance. h h b f d h h 3) rigid PVC cable conduit with an outer diameter of 52 mm and a wall thickness of 4.1 mm; During the experiments, the number of drops at which the base ignited was recorded, and the time from the beginning of the fall of the first burning drop until the base ignited. 4) corrugated cable conduits made of polyethylene with an outer diameter of 62.5 mm and a wall thickness of 1.4 mm; b) three segments: 5. Results of experimental studies on resistance to flame propagation of long elements of the wiring system ditioning of samples are specified compared to other long elements of the electrical wiring system. The temperature and relative humidity of the air of such elements is consis­ tent with the conditions for the conditioning of electrical products [25, 26]. 5. 1. The results of the analysis of standardized test methods for flame propagation of long elements of the wiring system The duration of conditioning samples of cable conduits, liquid tight sheathings and cable trunkings, ductings, trays, ladders (Table 1), respectively, is 5 and 3.5 times longer than the duration of conditioning samples of electrical products established in the fundamental standards [25, 26]. For ca­ bles, this duration is 2 times less. Tables 1, 2 give the characteristics of test methods and criteria for assessing the resistance to flame propagation of long elements of the wiring system. 5. Results of experimental studies on resistance to flame propagation of long elements of the wiring system According to the Table 1, for cable conduits and liquid tight sheathings, more stringent conditions for the con­ Table 1 Characteristics of test methods and criteria for evaluating the resistance to flame propagation of long elements of the wiring system Characteristics Value or description Cables [13–17] Powertracks [20] Cable trunk­ ings, ductings, trays, ladders [21, 22] Cable conduits [23] liquid tight sheath­ ings [24] Sample length (E*), mm 600±25 675±10 Conditioning conditions for samples (23±5) °С; (50±20) %; ≥16 h not defined ≥168 h after manufactur­ ing (23±2) °С; (50±10) %; ≥240 h Conditioning conditions of the base to determine the flammability of particles separated from the sample (23±2) °С; (50±10) %; ≥4 h not defined Temperature during testing, °С 23±10 20±5 Chamber volume for protection against air flows, m3 ≥1 not used Dimensions of the metal fence, mm×mm×mm (1200±25)× ×(300±25)×(450±25) ( ) +25 +25 0 0 1300 25 700  450 × × ± × ( ) +25 +25 0 0 1300 25 300 450 ± × × × Clamp width (support) (A*), mm not defined (≈25 mm accord�­ ing to IEC 332-1:1979) 25 Distance between clamps (resistors) (D*), mm 550±5 550±10 Distance between the lower clamp and the point of application of the flame source to the sample (C*), mm 75±10 100±5 Distance between the upper edge of the lower clamp and the bottom of the guard (F*), mm ≈75 500±10; 550±10 (for trays and ladders) Width of white pine board covered with tissue paper, mm not used 0 25 700 0 25 300 Distance between burner and sample (B*), mm 62±16 (blue cone height) 100±10 Duration of application of the flame source to the sample, s t±2 (Table 2) 60±2 +1 0t (Table 2) Distance between the point of application of the flame source to the sample and the upper limit of the rounded zone of the sample (L1), mm <425±5 1) <400±15 Distance between the point of application of the flame source to the sample and the lower boundary of the carbon zone of the sample (L2), mm ≤65±5 1) not defined <50±5 Length of the carbon part of the sample (L3), mm ≤425 2) not defined Standard base condition double layer of filter paper 3) does not ignite 1) tissue paper 4) does not ignite and the wood board is not heated the tissue paper does not ignite Duration of self-combustion of the sample (taf), s not defined ≤30 The number of samples that must withstand the test, pcs. 4. 3. Methodology for studying the influence of the characteristics of the bases on the assessment of resis­ tance to the flame propagation of the cable 4. 3. Methodology for studying the influence of the characteristics of the bases on the assessment of resis­ tance to the flame propagation of the cable 1) blind cable ducting with transverse dimensions 50×20 mm, complying with EN 50085-1 [21]; 2) cable trunking with transverse dimensions 60×40 mm, which complies with EN 50085-1 [21]. To study the influence of the characteristics of the foun­ dations under the sample on the assessment of resistance to 59 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 5. Results of experimental studies on resistance to flame propagation of long elements of the wiring system 5. Results of experimental studies on resistance to flame propagation of long elements of the wiring system 1 or 2 additional 1 or 12 additional 1 or 3 or 2 additional 3 *See Fig. 1: 1) The criterion does not apply for cables covered by EN 13501-6 [8]. 2) The criterion applies only to cables that are subject to EN 13501-6 [8]. 3) Filter paper with a basis weight of (80±15) g/m2 and a square shape with a side of (300±10) mm. 4) Packing with surface density (21±9) g/m2. Characteristics of test methods and criteria for evaluating the resistance to flame propagation of long elements of the wiring system 60 Ecology Table 2 Table 2 Table 2 Duration of application of a flame source of 1 kW to samples of long elements of the electrical wiring system Cables [12, 13] Cable conduits, liquid tight sheathings [22, 23] Outer diameter, mm t, s Wall thick­ ness, mm t, s more no more than more no more than – 25 60 – 0.5 15 0.5 1.0 20 1.0 1.5 25 1.5 2.0 35 2.0 2.5 45 2.5 3.0 55 3.0 3.5 65 3.5 4.0 75 4.0 4.5 85 25 50 120 4.5 5.0 130 50 75 240 5.0 5.5 200 5.5 6.5 300 75 – 480 6.5 – 500 * For cables with a non-circular cross-section in which the ratio between the length of the longer and shorter axis does not exceed 3, the nominal value of the length of the shorter axis is chosen as the outer diameter. For cables with a non-circular cross-section, in which the ratio between the length of the longer and shorter axes is in the range from 3 to 16, the sum of the lengths of the longer and shorter axes, divided by 3.14 (π), is chosen as the outer diameter. For cables in which the ratio between the longer and shorter lengths exceeds 16, the test criteria are defined in the product standards, and if they are not available, in the agreement between the manufacturer and the buyer. – Tissue paper Flame Back wall Steel bar (for conduits and liquid tight sheathings) Clamp Sample located in the center of the horizontal plane White pinewood board 10 45° ± 2° 100 ± 10 F A A D E 0 25 450 Fig. 1. Table 3 For all long elements of the wiring system, the distance between the clamps is 550 mm (Table 1). Taking into ac­ count the width of the clamps, a specimen length of 600 mm is sufficient. Experimental data on the inflammatory capacity of standard bases Basis character­ istics Distance between cable and base, m The number of burning parti­ cles at which the base ignited * Duration from the beginning of the fall of the burning particles to the base ignition*, s Double layer of filter paper 150 6 3.7 200 7 5.5 600 9 7.9 White pinewood board covered with a layer of tissue paper 150 2 1.1 200 2 1.5 600 3 2.6 *Average value over five experiments. Experimental data on the inflammatory capacity of standard bases According to the Table 1, the distance between the torch and the sample, which is not recommended in IEC 60695-11-2 [27], is set for cables to ensure reproducibil­ ity of results. The results of the study on the influence of this factor on the assessment of resistance to flame propagation of cables are presented in clause 5. 4. According to the Tables 1, 2 for long elements of the wiring system, different durations of application of the flame source are determined. The results of the study on the influ­ ence of this factor on the assessment of resistance to flame propagation of cables, cable conduits trunkings and ductings are presented in clauses 5. 4 and 5. 5. For cables other than those intended for use in buildings, the permissible length of the charred part of the sample can be in the range from 425 to 490 mm (Table 1). At the same time, for cables intended for use in buildings, the spread of the flame downward from the point of application of the flame source is not limited. The research results presented in Table 3 prove that: 1) standard white pinewood board base covered with a layer of tissue paper is more flammable than a double layer of filter paper; For other long elements of the wiring system, a stricter permissible flame spread of 400 mm upward from the point of application of the flame source is set than for cables. Table 3 2) ability of standard bases to ignite does not signifi­ cantly decrease with an increase in the distance between them and the source of the formation of burning particles according to the distance between the source of formation of burning particles and the base in the range from 150 mm to 600 mm. A stricter permissible level of flame spread of 50 mm downward from the point of application of the flame source is established for liquid tight sheathings. When this level is selected for all elements of the electrical wiring system, with the permissible length of the carbon part of 425 mm, there is an admissible level of flame prop­ agation upward from the point of application of the flame source of 375 mm. Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Regarding the conditioning of the bases for assessing the inflammatory capacity of particles separated from the samples, such conditions are determined only for cables (Ta­ ble 1). Moreover, such conditions for the base are not consis­ tent with the conditions of sample conditioning. According to the Table 1 rules for deciding on the com­ pliance of all three samples for cable conduits and liquid tight sheathings with more stringent ones. Less stringent rules are established for powertracks when a decision on the test data of one or an additional sample is sufficient. At the same time, more samples have been identified for cable trunkings, ductings, trays and ladders for testing, which contribute to obtaining more reliable results. To prevent the influence of extraneous air currents on the combustion process, the cables are tested in a protective chamber (Table 1). Also for these purposes, such a chamber is used to test other electrical products in accordance with fundamental standards [25, 26]. No such chamber is provid­ ed for testing powertracks, cable trunkings, ductings, trays, ladders and liquid tight sheathings. 5. 2. Influence of the characteristics of the bases placed under the sample of long elements of the electrical wiring system on their flammability 5. 2. Influence of the characteristics of the bases placed under the sample of long elements of the electrical wiring system on their flammability When testing all elements of the wiring system, the total ambient temperature is (23±2) °С. At this temperature, it is customary to test electrical products in accordance with standards [25, 26]. According to these standards, the rela­ tive humidity should not exceed 75 %. The data of experimental studies on the ability to ignite standard bases for determining the igniting ability of parti­ cles (drops) separated from a burning cable TRP 2×0.5 are presented in Table 3. 5. Results of experimental studies on resistance to flame propagation of long elements of the wiring system Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) Duration of application of a flame source of 1 kW to samples of long elements of the electrical wiring system Duration of application of a flame source of 1 kW to samples of long elements of the electrical wiring system * For cables with a non-circular cross-section in which the ratio between the length of the longer and shorter axis does not exceed 3, the nominal value of the length of the shorter axis is chosen as the outer diameter. For cables with a non-circular cross-section, in which the ratio between the length of the longer and shorter axes is in the range from 3 to 16, the sum of the lengths of the longer and shorter axes, divided by 3.14 (π), is chosen as the outer diameter. For cables in which the ratio between the longer and shorter lengths exceeds 16, the test criteria are defined in the product standards, and if they are not available, in the agreement between the manufacturer and the buyer. Tissue paper Flame Back wall Steel bar (for conduits and liquid tight sheathings) Clamp Sample located in the center of the horizontal plane White pinewood board 10 45° ± 2° 100 ± 10 F A A D E 0 25 450 Fig. 1. Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) Tissue paper Flame Back wall Steel bar (for conduits and liquid tight sheathings) Clamp Sample located in the center of the horizontal plane White pinewood board 10 45° ± 2° 100 ± 10 F A A D E 0 25 450 Fig. 1. Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) Sample located in the center of the horizontal plane 100 ± 10 Flame Back wall White pinewood board Tissue paper . 1. Scheme of testing long elements of the wiring system for flame propagation (legend – according to Table 1) 61 61 6/10 ( 114 ) 2021 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 Table 4 5. 4. Influence of the conditions of use of a flame source on the assessment of resistance to flame propaga­ tion of cables The data of experimental studies on the influence of the duration of the application of the flame source to the sample and the distance between the burner and the sample to assess the resistance to flame propagation of cables are presented in Table 5. The duration of the application of the flame source to the sample increases with an increase in the outer diameter of the cable (Table 2). But it is constant for certain ranges of the outer diameter of the cable. Therefore, to analyze the results, it is better to consider the curves of the dependence of the resis­ tance to flame propagation on the outer diameter of the cable. According to the Table 5, linear dependences of the aver­ age values of the length of the charred part and the duration of self-combustion of cables on their outer diameter (d) are plotted, which are shown in Fig. 2, 3. 5. 3. Influence of the characteristics of the bases un­ der the sample on the assessment of resistance to flame propagation of the cable The data of experimental studies on the influence of the characteristics of the bases under the sample on the assess­ ment of resistance to flame propagation of the AVVG 4×240 cable are presented in Table 4. For cables and other elements of the electrical wiring system, various bases have been determined for assess­ ing the flammability of particles separated from samples and (Table 1). Also, different distances are set between the point of application of the ignition source and the base. The results of the study on the influence of these factors on the flammability of the bases and the assessment of the resistance to flame propagation of cables are presented in clauses 5. 2 and 5. 3. The research results presented in Table 4 prove that l f dd l f 1) application of an additional criterion for assessing re­ sistance to flame propagation – the flammability of particles separated from the sample increases the efficiency of detect­ ing unsuitable cables; 2) standard white pinewood board backing covered with a layer of tissue paper is more effective in locating unsuitable cables than a double layer of filter paper; 2) standard white pinewood board backing covered with a layer of tissue paper is more effective in locating unsuitable cables than a double layer of filter paper; For long elements of the wiring system, in addition to cables, such a criterion as the duration of self-combustion of samples, not exceeding 30 s is used. To obtain additional data on the fire hazardous properties of cables, such a crite­ rion was applied during experimental studies. 3) assessment of the resistance of cables to flame propa­ gation based on test data from only one sample may lead to an incorrect decision on the conformity of the cables. 3) assessment of the resistance of cables to flame propa­ gation based on test data from only one sample may lead to an incorrect decision on the conformity of the cables. 5. 3. Influence of the characteristics of the bases un­ der the sample on the assessment of resistance to flame propagation of the cable 62 Ecology Table 4 Data of experimental studies on resistance to flame propagation of AVVG 4×240 cable samples using various standard bases Sample number L1/L2/L3, mm taf, s Combustion of the standard base placed under the sample Double layer of filter paper 1 166/7/173 15 no 2 175/8/183 19 no 3 176/11/187 27 yes White pinewood board covered with a layer of tissue paper 4 158/9/167 10 yes 5 170/7/177 26 yes 6 172/10/182 20 yes Note: Legend – according to Table 1 Table 4 The convergence of the results of the duration of self-combustion of the samples was influenced by the prop­ erties of the insulating materials placed under the shell, and the size of the zones of oxygen access to the place of combustion of such materials formed as a result of damage 1 2 3 4 5 AVVG 4×240 cable Blue cone of flame touches the sample surface 1 240 131/10/141 20.0 no 2 240 145/15/160 25.4 no 3 240 135/15/150 21.2 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 240 151/11/162 25.8 no 5 240 155/15/170 24.2 no 6 240 148/12/160 21.4 no Note: Legend – according to Table 1 Continuation of Table 5 2 1 L3 = 1,339d + 74 r = 0,969 L3 = 1,113d + 96,5 r = 0,985 0 20 40 60 80 100 120 140 160 180 0 10 20 30 40 50 60 70 L3, mm d, mm Fig. 2. Linear dependences of the length of the charred part of the cables on their outer diameter (d): 1 – distance between the torch and the sample, measured along the torch axis – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient 2 1 taf = 0,352d - 4,36 r = 0,801 taf = 0,426d - 1,985 r = 0,983 -5 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 taf, s d, mm Fig. 3. Linear dependences of the duration of self-combustion cables on their outer diameter (d): 1 – distance between the burner and the sample, measured along the axis of the burner – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient Also, at a distance of 100 mm, a better correlation was obtained between the results of testing different diameters of the AVVG cable than when the blue cone touched the sample surface. To assess the effect of the distance be­ tween the burner and the sample on the convergence of the results according to the Table 5, the rms values of the length of the charred part and the duration of self-com­ bustion of the investigated AVVG cables are calculated, presented in Table 6. Table 4 Table 6 The value of the standard deviation of the length of the charred part and the duration of self-combustion of cable samples of the AVVG brand 1 2 3 4 5 AVVG 4×240 cable Blue cone of flame touches the sample surface 1 240 131/10/141 20.0 no 2 240 145/15/160 25.4 no 3 240 135/15/150 21.2 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 240 151/11/162 25.8 no 5 240 155/15/170 24.2 no 6 240 148/12/160 21.4 no Note: Legend – according to Table 1 Continuation of Table 5 2 1 L3 = 1,339d + 74 r = 0,969 L3 = 1,113d + 96,5 r = 0,985 0 20 40 60 80 100 120 140 160 180 0 10 20 30 40 50 60 70 L3, mm d, mm Fig. 2. Linear dependences of the length of the charred part of the cables on their outer diameter (d): 1 – distance between the torch and the sample, measured along the torch axis – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient 1 2 3 4 5 AVVG 4×240 cable Blue cone of flame touches the sample surface 1 240 131/10/141 20.0 no 2 240 145/15/160 25.4 no 3 240 135/15/150 21.2 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 240 151/11/162 25.8 no 5 240 155/15/170 24.2 no 6 240 148/12/160 21.4 no Note: Legend – according to Table 1 Continuation of Table 5 1 2 3 4 5 AVVG 4×240 cable Blue cone of flame touches the sample surface 1 240 131/10/141 20.0 no 2 240 145/15/160 25.4 no 3 240 135/15/150 21.2 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 240 151/11/162 25.8 no 5 240 155/15/170 24.2 no 6 240 148/12/160 21.4 no Note: Legend – according to Table 1 Continuation of Table 5 2 1 L3 = 1,339d + 74 r = 0,969 L3 = 1,113d + 96,5 r = 0,985 0 20 40 60 80 100 120 140 160 180 0 10 20 30 40 50 60 70 L3, mm d, mm Fig. 2. Table 6 Fig. 2. Linear dependences of the length of the charred part of the cables on their outer diameter (d): 1 – distance between the torch and the sample, measured along the torch axis – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient The value of the standard deviation of the length of the charred part and the duration of self-combustion of cable samples of the AVVG brand The value of the standard deviation of the length of the charred part and the duration of self-combustion of cable samples of the AVVG brand Cable mark size RMS В=62 mm В=100 mm L3, mm taf, s L3, mm taf, s AVVG 4×2,5 5.68 0.306 4.16 1.206 AVVG 4×16 3.46 0.231 2.52 0.529 AVVG 4×35 3.06 1.528 2.52 5.69 AVVG 4×120 1.629 3.64 3.89 2.5 AVVG 4×240 9.5 2.84 5.29 2.23 Note: Legend according to Table 1 2 1 taf = 0,352d - 4,36 r = 0,801 taf = 0,426d - 1,985 r = 0,983 -5 0 5 10 15 20 25 30 0 10 20 30 40 50 60 70 taf, s d, mm Fig. 3. Linear dependences of the duration of self-combustion cables on their outer diameter (d): 1 – distance between the burner and the sample, measured along the axis of the burner – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient From Table 6, there is in four out of five cases better convergence of the results at a distance of 100 mm than when the blue flame cone touches the sample surface. As for the standard deviation of the duration of self-combustion of the samples, the best con­ vergence at a distance of 100 mm between the burner and the sample was obtained only in two out of five cases. Fig. 3. Linear dependences of the duration of self-combustion cables on their outer diameter (d): 1 – distance between the burner and the sample, measured along the axis of the burner – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient In Fig. Table 4 Linear dependences of the length of the charred part of the cables on their outer diameter (d): 1 – distance between the torch and the sample, measured along the torch axis – 100 mm; 2 – blue cone of flame touches the sample surface; r – the correlation coefficient Also, at a distance of 100 mm, a better correlation was obtained between the results of testing different diameters of the AVVG cable than when the blue cone touched the sample surface. To assess the effect of the distance be­ tween the burner and the sample on the convergence of the results according to the Table 5, the rms values of the length of the charred part and the duration of self-com­ bustion of the investigated AVVG cables are calculated, presented in Table 6. Table 6 Table 4 Table 5 Table 5 Experimental data on the influence of the duration of application of the flame source to the sample and the distance between the burner and the sample on the assessment of the resistance to flame propagation of cables Sample number Duration of the ignition source type, s L1/L2/L3, mm taf, s Combustion of the standard base placed under the sample 1 2 3 4 5 AVVG 4×2,5 cable Blue cone of flame touches the sample surface 1 60 60/12/72 3.6 no 2 60 65/15/80 4.2 no 3 60 70/13/83 4.0 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 60 93/13/106 3.0 no 5 60 90/14/104 4.0 no 6 60 100/12/112 5.4 no AVVG 4×16 cable Blue cone of flame touches the sample surface 1 60 90/11/101 1.0 no 2 60 88/13/101 1.0 no 3 60 95/12/107 1.4 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 60 110/11/121 3.0 no 5 60 109/14/123 4.0 no 6 60 105/13/118 3.8 no AVVG 4×35 cable Blue cone of flame touches the sample surface 1 60 99/15/114 1.0 no 2 60 100/18/118 4.0 no 3 60 103/17/120 3.6 no The distance between the torch and the sample, measured along the torch axis – 100 mm мм 4 60 115/11/126 13.6 no 5 60 110/18/128 6.0 no 6 60 108/15/123 8.4 no AVVG 4×120 cable Blue cone of flame touches the sample surface 1 120 123/10/133 7.4 no 2 120 113/18/131 1.2 no 3 120 115/15/130 1.0 no The distance between the torch and the sample, measured along the torch axis – 100 mm 4 120 125/14/139 14.4 no 5 120 115/16/131 15.0 no 6 120 129/13/142 19.0 no Experimental data on the influence of the duration of application of the flame source to the sample and the distance between the burner and the sample on the assessment of the resistance to flame propagation of cables Experimental data on the influence of the duration of application of the flame source to the sample and the di the burner and the sample on the assessment of the resistance to flame propagation of cables he influence of the duration of application of the flame source to the sample and the distance between ner and the sample on the assessment of the resistance to flame propagation of cables 63 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 6/10 ( 114 ) 2021 In Fig. Table 4 2, 3 it is possible to see that with an increase in the duration of application of the ignition source, the length of the charred part and the duration of self-combustion of the AVVG cable with an outer diameter from 10 mm to 60 mm increase At the same time the values of these in­ Also, at a distance of 100 mm, a better correlation was obtained between the results of testing different diameters of the AVVG cable than when the blue cone touched the sample surface. To assess the effect of the distance be­ tween the burner and the sample on the convergence of the results according to the Table 5, the rms values of the length of the charred part and the duration of self-com­ bustion of the investigated AVVG cables are calculated, presented in Table 6. Table 6 The value of the standard deviation of the length of the charred part and the duration of self-combustion of cable samples of the AVVG brand Cable mark size RMS В=62 mm В=100 mm L3, mm taf, s L3, mm taf, s AVVG 4×2,5 5.68 0.306 4.16 1.206 AVVG 4×16 3.46 0.231 2.52 0.529 AVVG 4×35 3.06 1.528 2.52 5.69 AVVG 4×120 1.629 3.64 3.89 2.5 AVVG 4×240 9.5 2.84 5.29 2.23 Note: Legend according to Table 1 From Table 6, there is in four out of five cases better convergence of the results at a distance of 100 mm than when the blue flame cone touches the sample surface. As for the standard deviation of the duration of self-combustion of the samples, the best con­ vergence at a distance of 100 mm between the burner and the sample was obtained only in two out of five cases. Table 7 Experimental data on resistance to flame propagation of cable conduits trunkings and ductings Sample number Duration of the ignition source type, s L1/L2/L3, mm taf, s Combustion of the standard base placed un­ der the sample Corrugated polyamide cable conduit with 16 mm outer diameter and a 0.5 mm wall thickness 1 15 115/15/130 5 no 2 15 125/14/139 7 no 3 15 122/18/140 9 no 4 60 190/23/213 108 yes 5 60 154/21/175 84 yes 6 60 170/24/194 97 yes Corrugated polypropylene cable conduit with 50 mm outer diameter and 0.8 mm wall thickness 1 120 burned out (Fig. 4) ≥30 yes 2 120 burned out ≥30 yes 3 120 burned out ≥30 yes 4 20 62/18/80 16 no 5 20 65/19/84 21 no 6 20 74/23/97 26 no Rigid PVC cable conduit with 52 mm outer diameter and 4.1 mm wall thickness 1 85 99/18/117 0 no 2 85 105/13/118 0 no 3 85 102/13/115 0 no 4 240 118/11/129 0 no 5 240 123/18/141 0 no 6 240 126/17/143 0 no Corrugated polyethylene cable conduit with 62.5 mm outer diameter and 1.4 mm wall thickness 1 25 136/28/164 22 no 2 25 129/27/156 18 no 3 25 138/29/167 24 no 4 240 burned out (Fig. 5) ≥30 yes 5 240 burned out ≥30 yes 6 240 burned out ≥30 yes Blind cable ducting with cross dimensions 50 mm×20 mm, which complies with EN 50085-1 [19] 1 60 185/37/222 0 no 2 60 148/27/175 0 no 3 60 131/31/162 0 no Cable trunking with cross dimensions 60mm×40mm, which com­ plies with EN 50085-1 [19] 1 120 125/29/154 0 no 2 120 127/36/163 0 no 3 120 149/35/184 0 no Note: Legend according to Table 1 Experimental data on resistance to flame propagation of cable conduits trunkings and ductings Sample number Duration of the ignition source type, s L1/L2/L3, mm taf, s Combustion of the standard base placed un­ der the sample Corrugated polyamide cable conduit with 16 mm outer diameter and a 0.5 mm wall thickness Fig. 4. Corrugated polypropylene conduits that spreads the flame Fig. 5. Corrugated polyethylene conduit that spreads the flame Rigid PVC cable conduit with 52 mm Fig. 5. Table 6 2, 3 it is possible to see that with an increase in the duration of application of the ignition source, the length of the charred part and the duration of self-combustion of the AVVG cable with an outer diameter from 10 mm to 60 mm increase. At the same time, the values of these in­ dicators do not exceed the threshold values of 425 mm and 30 s, respectively. The convergence of the results of the duration of self-combustion of the samples was influenced by the prop­ erties of the insulating materials placed under the shell, and the size of the zones of oxygen access to the place of combustion of such materials, formed as a result of damage to the shell. As a result, it can be considered that the use of the rec­ ommended distance of 100 mm [2] will improve the conver­ gence and reproducibility of the results of testing cables for flame propagation. At a distance of 100 mm (curve 1), larger values of the indicators were obtained than when the blue cone touched the surface of the sample (curve 2). 64 Fig. 4. Corrugated polypropylene conduits that spreads the flame 5. 5. Influence of the duration of the application of the flame on the resistance to flame propagation of conduits trunkings and ductings The data of experimental studies on the influence of the duration of the application of the flame source to the sample on the assessment of resistance to flame propagation of cable conduits and ducts are presented in Table 7. 6. Discussion of research results on resistance to flame propagation of long elements of electrical wiring systems duration of the application of the flame source to the samples set for cables (Table 2). If flame-resistant materials are used, damage to such elements occurs directly in the zone of influence of the ig­ nition source. In this case, an increase in the duration of the application of the flame source will only lead to the burnout of materials in the zone of influence of this source. At the same time, the duration of self-combustion can reach a peak and further decrease. According to the principles set forth in [2], the condi­ tions for the impact of a 1 kW flame source on long elements of the electrical wiring system and the criteria for evaluating the results, as far as possible, should be the same. According to the results presented in Tables 4, 5, it is advisable to evaluate the resistance to flame propagation of cables according to such criteria as the presence of particles capable of ignition and the duration of self-combustion no more than 30 s. Assessment by such indicators is necessary for making a decision on the introduction of additional fire protection for cables at facilities. Taking this into account, it is promising to evaluate the long elements of the wiring system by the speed of flame propagation. Also, according to the test results provided in Tables 5, 7 and Fig. 2, 3, it is advisable to choose for the cables the dis­ tance between the torch and the sample recommended in [27]. This is due to the need to ensure the convergence and repro­ ducibility of the results, since at a distance of about 100 mm, the parameters of the 1 kW flame source are checked. The results are presented in Tables 3, 4, found that the generally accepted basis for evaluating the flammability of particles separated during the combustion of electrical prod­ ucts is more effective than that selected for cables. Therefore, the use of a base consisting of a white pine board covered with tissue paper will help reduce the risk of making the wrong decision about cable compliance. The permissible distance between the clamps for the sample (Table 1) allows for all elements of the electrical wiring system to use for testing samples with the same and smaller length mm, adopted for cables. 6. Discussion of research results on resistance to flame propagation of long elements of electrical wiring systems This makes it possi­ ble to eliminate the influence of the effects associated with the longitudinal heating of the samples under the influence of the ignition source. The specified dimensional tolerance will avoid the problems of fixing specimens due to the short length. Also, the results presented in Table 3, enable all el­ ements of the wiring system to establish the same dis­ tance (200±5) mm between the point of application of the flame source and the base. Since the thermal energy of burn­ ing particles becomes less when falling from a greater height, determining such a distance will help to reduce the risk of making an incorrect decision in assessing the resistance to flame propagation of these elements. It is provided that the length of the samples is the same, eliminating the influence on the results of their transverse dimensions allows to choose the optimal duration of appli­ cation of the ignition source to the samples. Therefore, it is promising to conduct relevant studies. It is not possible to determine the specified distance less than the overall dimensions of the burner [27] and the estab­ lished distance between it and the sample (Table 1). Also, reducing the specified distance allows to test all the long elements of the wiring system in a metal fence with the same height of 1200 mm (Table 1). This will help to min­ imize the number of pieces of test equipment. The content of excess moisture in the samples and the base, which is located under them, in excess of that which may be contained in electrical products during use, can neg­ atively affect the process of their combustion. Evaluation of the resistance of cables to flame propaga­ tion only according to the test data of one sample can lead to an incorrect decision about their compliance (Table 4). This also applies to powertracks. For the elements of the electrical wiring system, the conditions of sample conditioning are different (Table 1). To eliminate the effect of this factor, for all elements of the wiring system, equal conditioning conditions for the samples and the base can be determined, established in the fundamental stan­ dards [25, 26]. Such conditions are holding the samples and the base for at least 48 hours at a temperature of (23±2) °С and a relative humidity of (50±10) %. Table 7 Corrugated polyethylene conduit that spreads the flame The results of experimental studies presented in Table 6 prove that the duration of the ignition source seems to be a significant factor that affects the compliance of long ele­ ments of the wiring system with the requirements for resis­ tance to flame propagation. The duration of the operation of a flame source of the type specified in IEC 61386-1 [23] may not ensure that the conduits reach thermal energy sufficient to maintain stable combustion and flame propagation. This was revealed using the duration of application of the ignition source of the type set for cables in IEC 60332-1-2 [13]. This also applies to the test method for flame propaga­ tion of liquid tight sheathings [24]. For cable trunkings and ductings, an increase in the du­ ration of the ignition source like this revealed their inconsis­ tency. At the same time, additional testing of these products, cable trays, ladders and powertracks with a flame source dura­ tion of more than 60 s will provide confidence in the decision on their proper resistance to flame propagation. 65 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 duration of the application of the flame source to the samples set for cables (Table 2). 6. Discussion of research results on resistance to flame propagation of long elements of electrical wiring systems At the same time, for safe and economic reasons, it is advisable to conduct exper­ imental studies to determine the optimal duration of condi­ tioning samples of long elements of the wiring system. To obtain reliable results, the decision rules established for cable trunkings and ductings, trays and ladders can be considered optimal (Table 1). At the same time, it is ad­ visable to conduct experimental studies on the number of samples exceeding six. With an increase in the duration of the application of the flame source to the samples of long elements of the electrical wiring system, an increase in the length of the charred part and the duration of self-combustion of the samples was ob­ tained (Tables 5, 7 and Fig. 2–5). This is due to the transfer of more thermal energy from the ignition source to the sam­ ples, which helps to maintain their combustion. The combustion processes of electrical products can be affected by ambient temperature, relative humidity and at­ mospheric pressure. In this regard, it is advisable to test long elements of the wiring system under conditions that give the worst result, but are limited by actual operating conditions. Therefore, it is promising to carry out studies to identify the influence of such factors on the resistance to flame propa­ gation of long elements of the wiring system. At the same time, such studies are difficult in terms of maintaining stable environmental parameters. At the same time, the levels of these indicators, as it is known, depend on the properties of the used insulating ma­ terials, their dimensions, the energy of the ignition source, oxygen availability and environmental conditions. If materials that are not resistant to flame propagation are used, the short duration of the application of the flame source to them is not enough to initiate stable combustion. An increase in the duration of the application of the flame source to the samples made it possible to identify combusti­ ble conduits (Table 7). The combustion processes of samples of electrical prod­ ucts can be influenced by air currents. To limit their impact, protective chambers are used [25, 26]. Among the long ele­ ments of the wiring system, such chambers are intended for testing only cables (Table 1). 7. Conclusions 1. It was proposed to assess the resistance to flame propaga­ tion of long elements of the wiring system to meet the require­ ments of IEC 60695-1-30 [2], according to the same criteria: 1. It was proposed to assess the resistance to flame propaga­ tion of long elements of the wiring system to meet the require­ ments of IEC 60695-1-30 [2], according to the same criteria: [ ] g – the length of the carbon part up and down from the point of application, the value of which does not exceed 375 mm and 50 mm, respectively; – the length of the carbon part up and down from the point of application, the value of which does not exceed 375 mm and 50 mm, respectively; – the presence and absence of ignition of the base located under the sample at a distance of (200±5) mm; – the duration of self-combustion, the value of which does not exceed 30 s. 2. It was found that at a distance of 200 mm below the source of the formation of burning particles, the base of a wood­ en board covered with a layer of tissue paper with a surface den­ sity of (21±9) g/m2 was occupied by 2 burning particles, and the base was made of a double layer of filter paper with a surface density (80±15) g/m2 – from 7 burning particles. At the speci­ fied distance between the point of application of the 1 kW flame source to the cable and the first base, its ignition was detected, and when the second base was used, it did not occur. The use of the first base, which is accepted for testing other elements of the wiring system, increases the efficiency of detecting cables that are not resistant to flame propagation. 2. It was found that at a distance of 200 mm below the source of the formation of burning particles, the base of a wood­ en board covered with a layer of tissue paper with a surface den­ sity of (21±9) g/m2 was occupied by 2 burning particles, and the base was made of a double layer of filter paper with a surface density (80±15) g/m2 – from 7 burning particles. 6. Discussion of research results on resistance to flame propagation of long elements of electrical wiring systems In this regard, for all elements of the wiring system, it is advisable to take the most stringent requirements for the The disadvantage of using protective chambers is the de­ pletion of oxygen during combustion of samples in a confined 66 Ecology space. This affects the course of the normal combustion pro­ cess. Therefore, for the use of protective chambers for testing elements of the wiring system, their internal dimensions should be justified. criterion of the presence of a base fire located under the cable. To reduce the risk of making an incorrect decision on compliance, it is proposed to evaluate long elements of the wiring system according to the rules established for cable trunkings, ductings, trays and ladders in the stan­ dards [21, 22]. The results obtained were discussed at the International Scientific and Practical Conference [28]. 4. It was found for the AVVG cable in four out of five cases a better convergence of the results along the length of the charred part of the samples at a distance between the burner and the sample, which is 100 mm along the burner axis, than when the 1kW thief flame cone touches the sample surface. But in three out of five cases, the best convergence of the results of the duration of self-combustion was obtained under the latter condition. Under the first condition, the maximum values of the length of the carbon part of 170 mm and the duration of self-combustion of 25.8 s were obtained, and under the second condition, respectively, 160 mm and 25.4 s. Under the first condition, for the linear dependence of these indicators on the diameter of the AVVG cable within the outer diameter from 10 mm to 60 mm, the correlation co­ efficients were 0.985 and 0.983, respectively, and under the second condition, 0.969 and 0.801, respectively. Based on the generalization of these results and the recommendations of IEC 60695-11-2 [27], it was proposed to test cables under the first condition. 7. Conclusions At the speci­ fied distance between the point of application of the 1 kW flame source to the cable and the first base, its ignition was detected, and when the second base was used, it did not occur. The use of the first base, which is accepted for testing other elements of the wiring system, increases the efficiency of detecting cables that are not resistant to flame propagation. 5. For two cable conduits that meet the requirements of IEC 61386-1 [22], for the duration of the application of a flame of 1 kW for 20 s and 25 s, it is found that they spread the flame for the duration of the application of such a flame source for 120 s and 240 s. To increase the efficiency of de­ tecting long elements of the wiring system that are unstable to flame propagation, it was proposed to test such elements with a flame application duration of 1 kW, set for cables in IEC 60332-1-2 [13]. 3. In one of three experiments, a cable was found that was not resistant to flame propagation, based on the References 1. IEC 60695-1-10:2016 Fire hazard testing – Part 1-10: Guidance for assessing the fire hazard of electrotechnical products – General guidelines (2016). Geneva: International Electrotechnical Commission, 50. 1. IEC 60695-1-10:2016 Fire hazard testing – Part 1-10: Guidance for assessing the fire hazard of electrotechnical products – General guidelines (2016). 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Brussels: European Committee for Standardization, 30. doi: http:// doi.org/10.3403/30348257 g/ / 9. Regulation (EU) No. 305/2011 of the European Parliament and of the Council of 9 March 2011 laying down harmonized conditions for the marketing of construction products and repealing Council Directive 89/106/EEC (2011). OJ L 88, 4.4.2011, 5–43. 9. Regulation (EU) No. 305/2011 of the European Parliament and of the Council of 9 March 2011 laying down harmonized conditions for the marketing of construction products and repealing Council Directive 89/106/EEC (2011). OJ L 88, 4.4.2011, 5–43. 10. Johansson, R., Post, J., Försth, M. (2015). Extended field of application (EXAP) for reaction-to-fire Euro-classification of optical fibre cables. SP Technical Research Institute of Sweden. SP Report, 49. 10. Johansson, R., Post, J., Försth, M. (2015). Extended field of application (EXAP) for reaction-to-fire Euro-classification of optical fibre cables. SP Technical Research Institute of Sweden. SP Report, 49. 7. Conclusions 67 67 6/10 ( 114 ) 2021 Eastern-European Journal of Enterprise Technologies ISSN 1729-3774 11. Kaczorek-Chrobak, K., Fangrat, J. (2019). Influence of Constructional-Material Parameters on the Fire Properties of Electric Cables. Energies, 12 (23), 4569. doi: http://doi.org/10.3390/en12234569 12. Directive 2014/35/EU of the European Parliament and of the Council of 26 February 2014 on the harmonisation of the laws of the Member States relating to the making available on the market of electrical equipment designed for use within certain voltage limits. OJ L 96, 29.3.2014. Р. 357–374. 13. IEC 60332-1-2:2004 Tests on electric and optical fibre cables under fire conditions – Part 1-2: Test for vertical flame propagation for a single insulated wire or cable – Procedure for 1 kW pre-mixed flame (2004). Geneva: International Electrotechnical Commission, 21. doi: http://doi.org/10.3403/03171042 14. IEC 60332-1-3:2004 Tests on electric and optical fibre cables under fire conditions – Part 1-3: Test for vertical flame propagation for a single insulated wire or cable – Procedure for determination of flaming droplets/particles (2004). Geneva: International Electrotechnical Commission, 21. 15. IEC 60332-1-2:2004/AMD1:2015 Tests on electric and optical fibre cables under fire conditions – Part 1-2: Test for vertical flame propagation for a single insulated wire or cable – Procedure for 1 kW pre-mixed flame (2015). Geneva, International Electrotechnical Commission, 9. 16. IEC 60332-1-3:2004/AMD1:2015 Tests on electric and optical fibre cables under fire conditions – Part 1-3: Test for vertical flame propagation for a single insulated wire or cable – Procedure for determination of flaming droplets/particles (2015). Geneva, International Electrotechnical Commission, 9. doi: http://doi.org/10.3403/03172788 17. 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Geneva: International Electrotechnical Commission, 285. 21. 7. Conclusions EN 50085-1:2005 Cable trunking systems and cable ducting systems for electrical installations – Part 1: General requirements (2005). Brussels: European Committee for Electrotechnical Standardization, 43. 22. IEC 61537:2006 Cable management – Cable tray systems and cable ladder systems (2006). Geneva: International Electrotechnical Commission, 161. 23. IEC 61386-1:2008+AMD1:2017 Conduit systems for cable management – Part 1: General requirements (2017). Geneva: International Electrotechnical Commission, 159. 24. EN 50369:2005 Liquid tight sheathing systems for cable management (2 Standardization, 25. doi: http://doi.org/10.3403/30102733u 24. EN 50369:2005 Liquid tight sheathing systems for cable management (2005). Brussels: European Committee for Electrotechnical Standardization, 25. doi: http://doi.org/10.3403/30102733u 25. IEC 60695-11-10:2013 Fire hazard testing – Part 11-10: Test flames – 50 W horizontal and vertical flame test meth doi: http://doi.org/10.3403/01860485u 26. IEC 60695-11-20:2015 Fire hazard testing – Part 11-20: Test flames – 500 W flame test method. Brussels. doi: http://doi.org/ 10.3403/01859086u 27. IEC 60695-11-2:2017 Fire hazard testing – Part 11-20: Test flames – 1 kW pre-mixed flame – Apparatus, confirmatory test arrangement and guidance (2017). Geneva: International Electrotechnical Commission, 40. doi: http://doi.org/10.3403/30338608 27. IEC 60695 11 2:2017 Fire hazard testing Part 11 20: Test flames 1 kW pre mixed flame Apparatus, confirmatory test arrangement and guidance (2017). Geneva: International Electrotechnical Commission, 40. doi: http://doi.org/10.3403/30338608 28. Kravchenko, R. I., Illiuchenko, P. O., Onyshchuk, A. Ye. (2021). Udoskonalennia metodiv vyprobuvannia ta kryteriivotsinky stiikosti do poshyrennia polumia dovhykh elementiv systemy elektroprovodky. Problemy nadzvychainykh sytuatsii (PES-2021). Kharkiv: Natsionalnyi universytet tsyvilnoho zakhystu Ukrainy, 49–50. 28. Kravchenko, R. I., Illiuchenko, P. O., Onyshchuk, A. Ye. (2021). Udoskonalennia metodiv vyprobuvannia ta kryteriivotsinky stiikosti do poshyrennia polumia dovhykh elementiv systemy elektroprovodky. Problemy nadzvychainykh sytuatsii (PES-2021). Kharkiv: Natsionalnyi universytet tsyvilnoho zakhystu Ukrainy, 49–50. 68 68
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Reference Intervals (RIs) in veterinary medicine
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ABSTRACT ABSTRACT Reference Intervals (RIs) are necessary in veterinary clinical pathology to provide a data base in order to compare results obtained from healthy versus diseased animals. Data are obtained from laboratory tests and, depending on whether they have a Gaussian or non- Gaussian distribution, are processed through statistical tests to define the RIs. This process begins with a healthy reference population made up of individuals who have been initially chosen based on inclusion or exclusion criteria. It is frequently challenging to have a large number of healthy individuals on which to establish de novo RIs, especially in wild and exotic animals. However, the use of reference intervals in daily clinical practice remains a fundamental instrument for therapeutic and diagnostic decisions, but it must always be accompanied by clinical findings that can confirm the hypothesis. Received August 7, 2023; In final form August 7, 2023; Published March 2024 Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 License, which per distribution, and reproduction in any medium, provided the original author and source are credited. Corresponding author: Martina Quagliardi, e-mail: martina.quagliardi@unicam.it ACTA IMEKO ISSN: 2221-870X March 2024, Volume 13, Number 1, 1 - 5 March 2024 | Volume 13 | Number 1 | 1 Keywords: reference intervals; clinical pathology; veterinary medicine; laboratory data; statistical test Keywords: reference intervals; clinical pathology; veterinary medicine; laboratory data; statistical test Citation: M. Quagliardi, L. Galosi, G. Rossi, A. Roncarati, A. Gavazza, Reference Intervals (RIs) in veterinary medicine, Acta IMEKO 5. DOI: 10.21014/actaimeko.v13i1.1615 L. Galosi, G. Rossi, A. Roncarati, A. Gavazza, Reference Intervals (RIs) in veterinary medicine, Acta IMEKO, vol. 13 (2024) no. 1, pp. 1 eko.v13i1.1615 Section Editor: Leopoldo Angrisani, Università degli Studi di Napoli Federico II, Naples, Italy Received August 7, 2023; In final form August 7, 2023; Published March 2024 4. EXAMPLES Breed is another crucial parameter to consider within the same species; numerous studies have been conducted to identify the main hematobiochemical differences between different breeds and standard RIs, with the majority of these studies focusing on dogs. Greyhounds have been extensively studied in both veterinary transfusion medicine and clinical pathology. Their haematological and biochemical values are found to differ from those of the general canine population, indicating distinct physiological characteristics [15]. Numerous deviations have been observed in this breed, including higher HCT, MCV, MCHC, RBC counts and HGB concentrations. However, PLT count and total white blood cell (WBC) counts tend to be lower [15], [16]. Significant differences in values were also detected in newborn Martina Franca donkeys in the first three weeks of life. Compared to horse foals, Martina Franca donkey foals exhibited lower red blood cells (RBC) count, hematocrit (HCT) and hemoglobin (HGB) levels in their first few days of life. Additionally, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) also resulted lower. These findings underline that RIs should be made to particular breeds species during the newborn stage [8]. There are hematological differences even among sighthounds of the same category, such as Whippet, Greyhound, Italian Greyhound, Sloughi, Saluki, Borzoi, Pharaoh Hound and Azawakh. In a study of 2013, it was documented that Whippets had the laboratory profile that matched Greyhounds the closest and, with the exception of Pharaoh Hounds, Italian Greyhounds demonstrated significantly higher alanine aminotransferase (ALT) activity than the other sighthound breeds [17]. Calves are another species in which hematological values have been observed to deviate from adult RIs. Both neonatal Holstein and Norwegian Red Breed calves present higher RBC and PLT counts during the first period of life. However, at the same time, MCV and MCHC values decrease [9], [10]. Recent studies on sighthounds have revealed that most RIs available in veterinary textbooks cannot be confirmed for Italian Greyhounds (Piccolo Levriero Italiano-PLI). Only a handful of these variants can be interpreted as peculiarities unique to this breed, perhaps as a result of its predisposition for racing. 3. METHODS OF DETERMINATION Generally, a RI of an analyte for a given population could be determined in three ways: calculated de novo, transferred from a previous RI when a method or instrument is changed or validated from a previously established or transferred RI [3]. In contrast to calves, during the early stages of life, dogs tend to have lower RBC count, HCT, total proteins and creatinine levels; conversely, alkaline phosphatase (ALP), creatine kinase (CK) and MCV increase [11], [12]. The ASVCP (American Society of Veterinary Clinical Pathology) published the reference interval guidelines in order to determine de novo RI in veterinary species, based on the CLSI (Clinical Laboratory and Standards Institutes) recommendations of 2008 [5], [6]. In addition to the influence of age, it is important that the animals are non-treated with drugs that can affect test results, such as corticosteroids that increase neutrophil count and liver enzymes; because of their action, their use is an exclusion criterion for reference individuals in order to determine de novo RIs [5]. De novo determination of RIs is the most used and frequent procedure in veterinary medicine; an "a priori" method is advised for determining inclusion or exclusion criteria for the starting population, whereas a "a posteriori" approach is utilised when pre-existing data have not yet been mined to generate reference values. The reference individuals chosen form the reference population, are described as “an undefined number of individuals that represent the demographic for which the reference intervals will be used” [5]. Moreover, if the animals are females, it is essential that they are not late pregnant or in early lactations; these physiologic states are uncontrollable pre-analytical variables, such as age. It is known from published studies that this state could affect clinical pathology tests. For example, in Standardbred mares during peripartum (last month of gestation and first week after parturition), a reduction of HGB, HCT and WBC has been found, as well as in many other serum analytes; it is important to place the attention in these temporary haematological changes, which greatly differentiate the mares in perinatal from those not in lactation and not pregnant [13]. The samples must be carried out from animals of a particular species, using a mixture of breeds, ages and gender. The animals of the population must be adults, clinically and serological testing healthy, since the age is a physiological pre-analytical variable that could induce different results in clinical pathology tests. 1. INTRODUCTION Firstly, these intervals are used to help identify abnormalities in a sick patient, compared to known values from healthy patients. It is important for a RI to represent a large, heterogeneous and appropriate demographic population in order to undertake a precise comparison [2]. Reference Intervals (RIs) in veterinary medicine have a fundamental role in the everyday medical practices and application. This topic could be strictly associated to the field of clinical pathology, but, at the same time they are implicitly used by everyone that processes and interprets laboratory data. RIs of laboratory tests are calculated from a group or population of healthy adult animals (hence they are called population-based reference intervals). In fact, without a RI it would be very difficult to determine if the results of a laboratory test are altered or normal; other terms that could be used for RI include normal, normal values and reference range [1]. In fact, reference values are used to describe the dispersion of variables in healthy individuals, comprising 95 % of the healthy population. In addition, they describe fluctuations observed in healthy populations or individuals that make the definition of health and health status. For this, it remains a fundamental tool in order to make clinical decisions for the patients [3]. The terms "range" and "interval" have rather different meanings and should not be used interchangeably. A "range" refers to the difference between two values, the highest and lowest observations, and is a single whole number, whereas a "interval" only includes the values between two reference limits and the reference limits themselves. On the other hand, the term “normal value” is also discouraged, since it is difficult to define the concept of “normality”, often many variations may appear “abnormal” due to age, physiology, diet, environment or other non-pathological factors [1], [2]. This emphasises the first significant drawback of the reference intervals, which is that they differ when the composition of the group of healthy animals used to construct them varies. The most significant influencing factors are race, age, sex, environment, diet, and lifestyle choices; as a result, each laboratory should establish its own reference ranges based on the population of animals that visit the facility. However, this is frequently impractical, with the exception of large facilities that receive a large volume of samples [4]. 1. INTRODUCTION March 2024 | Volume 13 | Number 1 | 1 ACTA IMEKO | www.imeko.org puppies has identified three different RIs, corresponding to 3 phases of 15 days, starting from 16th day after birth [12]. 3. METHODS OF DETERMINATION For instance, hematocrit and protein concentration are lower and lymphocyte count is higher in younger animals compared to adults. The impact of age has been observed in a variety of species, including dogs, cats, horses, calves, and donkeys, thus this selection criterion needs to be handled with extreme caution. A similar study was carried out in Holstein dairy cows, covering the first month after calving; actually 39 analytes out of 52 tested were found significantly different due to lactation period. These differences due to the lactation phase can be statistically validated and biologically demonstrated, but they can also be affected by pre-analytic factors, such as herds, parity groups and management, which must be considered [14]. The majority of age-related changes are primarily associated to the growth and differentiation that are unique to young animals, such as higher metabolism and grater tissue maturation. A study conducted on Dutch warmblood foals that were raised on pasture with free movement demonstrated that age has significant effects on several hematological values, with the exception of leucocytes (band-shaped, eosinophilic, basophilic and monocytes), platelets (PLT), creatinine, creatine phosphokinase (CPK), pCO2 and sodium, potassium and calcium. Moreover, it appeared that raising conditions may impact other characteristics, although the clinical significance of these effects may be minimal [7]. ACTA IMEKO | www.imeko.org 4. EXAMPLES In fact, they present higher MCH, MCHC, cell haemoglobin concentration mean (CHCM), albumin, calcium and iron, while In clinical practice of small animals, it is important for clinicians to be aware of the temporal variations in biochemical and hematologic values for dog puppies during the first two months of life to prevent misinterpretation of findings [11]. During this period, haematological parameters change significantly, and a study conducted on 101 clinically healthy dog March 2024 | Volume 13 | Number 1 | 2 ACTA IMEKO | www.imeko.org large unstained cells (LUC) is lower. At the same time RIs for total proteins and cholesterol resulted wider and the morphology of RBC and reticulocytes different [18]. reference intervals. In most cases, the breed-specific data overlapped with the standard reference intervals, suggesting minimal impact on clinical interpretation [31]. However, certain breeds exhibited unique characteristics, such as the Birman, which has been found to have physiologically elevated levels of creatinine and serum symmetric dimethylarginine (SDMA) concentrations [32]. Standard RIs were also compared with populations of other dog breeds, underlining the presence of breed-specific variation in blood values. For example, Bernese Mountain dogs were found to require new RIs for 7 analytes (eosinophils, MCHC, ALP, γ-glutamyltransferase (GGT), total bilirubin, amylase, and cholesterol) [19], while in Shetland sheepdogs, cholesterol and glucose resulted to need specific RIs because of differences depending on breed peculiarities [20]. As mentioned above, in veterinary medicine it is often challenging to obtain the numbers suggested by guidelines, particularly in wild species or exotic animals. As a result, practitioners often have to make do with limited data sets where one has to make do with small numbers of samples that have been managed to collect. Concerning the French mastiff Dogue de Bordeaux (DDB), age was found to significantly affect RIs for HGB, HCT, MCHC, WBC, neutrophil, lymphocyte, and monocyte counts. Additionally, DDBs have higher RIs for HGB, HCT, MCV, MCHC and mean platelet volume (MPV), while lower RIs for reticulocytes counts, platelets by impedance (PLT-I) and optical count (PLT-O) and plateletcrit. For this reason, new RIs for RBC and PLT have been recommended to avoid pathological suspicions of polycythaemia and thrombocytopenia in this breed [21]. 5. STATISTICAL METHODS The hematologic profile of Miniature Dachshunds has been found to be like Greyhounds, with higher RBC count, packed cell volume (PCV) and hemoglobin concentration [26]. On the other hand, a 1995 Australian study reported that bile acids measurement may not be very useful in Maltese dogs, as they may have "artifactually" elevated serum bile acids due to unknown reacting substances [27]. According to the guidelines, the nonparametric ranking approach with 90 % confidence intervals should be used to determine new Reference Intervals from a minimum of 120 Reference Individuals (CI) [6]. Based on the number of individuals, it is necessary to test if data follow a Gaussian distribution. Then, in order to determine reference limits and intervals, methods are used to remove outliers and select the central 95 % of reference values [1], [37]. The Japanese breeds, such the Akita or Shiba Inu and Hokkaido, are perhaps the ones with the most well-known hematological changes related to the breed in daily clinical practice. It has been observed in clinically healthy dogs that they have physiological microcytosis (red blood cells smaller than normal), resulting in lower MCV, MCH, MCHC, higher red cell distribution width (RDW) and significant anisocytosis (variation in red cell size) on smear exam [28]. When data do not follow a Gaussian distribution, a possible solution is applying a simple nonparametric method like percentile rank. This is called rank-percentile method, and it is used when there are more than 40 individuals not normally distributed [2]. Then, parametric (mean ± 2sd) or non-parametric (percentiles) methods can be used in order to establish reference limits [1]. By the way, veterinary transfusion centers and related databases can often be an excellent source for creating de novo RIs and studying breed differences for hematological and biochemical values. This is due to donor dogs being par excellence a healthy animal and must respect well-defined characteristics [29] and to the large number of samples processed. A recent study carried out through the blood donor database of the EMOVET-UNIPG blood bank and transfusion unit found significant differences in 5 hunting dog breeds (Ariégeois, Bleu de Gascogne, Bracco Italiano, Segugio Italiano, Briquet Griffon Vandeen) for 12 significant differences in hematologic and serum biochemical analytes; new RIs for HCT, MCH, MCHC, RDW, PLT, monocytes, eosinophils, albumin, urea, creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were supplied for at least one breed [30]. 4. EXAMPLES For example, for the 50th EAAM (European Association for Aquatic Mammals) Annual Symposium of 2023 that took place in Valencia in March, two posters were presented regarding the RIs of hematological and biochemical parameters in a particular species of dolphin, Risso’s dolphin (Grampus griseus) [33], [34]. As for other exotic species, also in this case the bibliography available is very little numerous and the number of individuals involved in the studies is very small [35], [36]. Despite the small number of individuals (9 prepuberal females), the study on the RIs of hematological and biochemical parameters in Risso’s dolphins presented at the 50th EAAM Annual Symposium in Valencia in March 2023 is noteworthy for the high number of samples under analysis (818 blood samples). This represents the first investigation on a large number of samples in this species and may serve as a preliminary study for future research on age and sex differences. There are also breed-specific hematologic peculiarities observed in small dog breeds. For instance, hereditary poodle macrocytosis, a clinically silent condition, has been reported in some Miniature and Toy Poodles [22]. In Cairn and Norfolk Terriers, a condition known as hereditary macrothrombocytopenia has been identified, which is caused by mutations in the beta1-tubulin gene [23]. This condition is prevalent in the Cavalier King Charles Spaniel (CKCS) [24], that have been demonstrated to have a significantly lower whose platelet count than the general RI [25]. 6. CONCLUSIONS DOI: 10.1016/j.theriogenology.2014.04.004 Laboratories should report the type of RIs used and how it was calculated, since incorrect interpretation of laboratory data can lead to clinical errors and is often underestimated. This is due to the fact that the reference intervals are "statistical artifacts" and the percentage of probability that a healthy animal taken at random from the population has an anomalous value is 5 % [4]. [9] H. C. Brun-Hansen, A. H. Kampen, A. Lund, Hematologic values in calves during the first 6 months of life, Vet. Clin. Pathol. 35(2) (2006), pp. 182-187. DOI: 10.1111/j.1939-165X.2006.tb00111.x DOI: 10.1111/j.1939-165X.2006.tb00111.x [10] N. Panousis, N. Siachos, G. Kitkas, E. Kalaitzakis, M. Kritsepi- Konstantinou, G. E. Valergakis, Hematology reference intervals for neonatal Holstein calves, Res Vet Sci. 118 (2018), pp. 1-10. DOI: 10.1016/j.rvsc.2018.01.002 As a matter of fact, when multiple analytes are measured, the more analytes measured, the greater the risk of interpreting values as pathological when they are actually due to chance. On the other hand, just as a value falling within the reference limits does not mean that the presence of underlying pathologies can be excluded, meanwhile the mere fact that a value falls within the reference limits does not imply that the patient is healthy [4]. [11] E. Rosset, B. Rannou, G. Casseleux, K. Chalvet-Monfray, S. Buff, Age-related changes in biochemical and hematologic variables in Borzoi and Beagle puppies from birth to 8 weeks, Vet. Clin. Pathol. 41(2) (2012), pp. 272-282. DOI: 10.1111/j.1939-165X.2012.00415.x DOI: 10.1111/j.1939-165X.2012.00415.x [12] R. Rørtveit, B. K. Saevik, A. V. Eggertsdóttir, E. Skancke, F. Lingaas, S. I. Thoresen, J. H. Jansen, Age-related changes in hematologic and serum biochemical variables in dogs aged 16-60 days, Vet. Clin. Pathol. 44(1) (2015), pp. 47-57. DOI: 10.1111/vcp.12220 However, the importance of reference intervals remains fundamental in clinical practice and must always be accompanied by concrete clinical findings on the patient. In conclusion, this paper highlights the importance of reference intervals (RIs) in clinical pathology. While RIs are used in other areas of veterinary medicine that involve measurements such as diagnostic imaging, with other units of measurements and values, the focus of this discussion has been on the importance of RIs in interpreting laboratory test results. [13] J. Mariella, A. Pirrone, F. Gentilini, C. Castagnetti, Hematologic and biochemical profiles in Standardbred mares during peripartum, Theriogenology 81(4) (2014), pp. 526-534. DOI: 10.1016/j.theriogenology.2013.11.001 [14] P. Moretti, S. Paltrinieri, E. Trevisi, M. Probo, A. Ferrari, A. 5. STATISTICAL METHODS In the reference interval guidelines of ASVCP there is a table that recommends procedures for establishing RIs based on reference sample size and distribution; for example, when reference samples are ≥ 20 and < 40, if the distribution is Gaussian, it could be used a parametric method, while, if non- Gaussian, a robust method [5]. This large number of required data are often impossible to achieve in veterinary clinical pathology, but at the same time it is stated that “the smaller the sample is, the higher is the degree of uncertainty in the estimation of reference limits” [5]. Consequently, for small samples, it is better to report them graphically in histograms or dot plot and subsequently use different methods in order to see which one could fit better [38]. This large number of required data are often impossible to achieve in veterinary clinical pathology, but at the same time it is stated that “the smaller the sample is, the higher is the degree of uncertainty in the estimation of reference limits” [5]. Consequently, for small samples, it is better to report them graphically in histograms or dot plot and subsequently use different methods in order to see which one could fit better [38]. This was tested in a study that randomly selected a small sample from a large sample group, showing that the RI estimated from small samples was highly variable and dissimilar from the This was tested in a study that randomly selected a small sample from a large sample group, showing that the RI estimated from small samples was highly variable and dissimilar from the Several feline breeds have been evaluated for hematological and biochemical measurements in comparison to standard March 2024 | Volume 13 | Number 1 | 3 ACTA IMEKO | www.imeko.org conditions from birth to 5 months of age, Vet Q. 23(2) (2001), pp. 92-95. / conditions from birth to 5 months of age, Vet Q. 23(2) (2001), pp. 92-95. RI of the large whole sample. In conclusion, when there are small samples, the bias and not normality of distribution increase [38]. This is why reporting all values and forgoing calculations when there are less than 20 samples is preferable [39]. pp. 92-95. DOI: 10.1080/01652176.2001.9695090 DOI: 10.1080/01652176.2001.9695090 [8] [8] M. C. Veronesi, A. Gloria, S. Panzani, M. P. Sfirro, A. Carluccio, A. REFERENCES [1] S. L. Stockham, M. A. Scott, Introductory concepts, in: Fundamentals of Veterinary Clinical Pathology 2nd ed., Ames Iowa, Blackwell Pub, 2008, pp 16-20, ISBN 9780813800769 [15] S. Zaldívar-López, L. M. Marín, M. C. Iazbik, N. Westendorf- Stingle, S. Hensley, C. G. Couto, Clinical pathology of Greyhounds and other sighthounds, Vet. Clin. Pathol. 40(4) (2011), pp. 414-425. DOI: 10.1111/j.1939-165X.2011.00360.x [2] K. S. Latimer, J. R. Duncan, Generating and interpreting test results: test validity, quality control, reference values, and basic epidemiology, in: Duncan & Prasse's Veterinary Laboratory Medicine: Clinical Pathology, 5th ed. Chichester West Sussex UK, Wiley-Blackwell, 2011, pp. 369-373, ISBN 9780813820149. [16] C. Campora, K. P. Freeman, F. I. Lewis, G. Gibson, F. Sacchini, M. J. Sanchez-Vazquez, Determination of haematological reference intervals in healthy adult greyhounds, J. Small Anim. Pract. 52(6) (2011), pp. 301-309. DOI: 10.1111/j.1748-5827.2011.01070.x y pp [3] A. Geffré, K. Friedrichs, K. Harr, D. Concordet, C. Trumel, J.P. Braun, Reference values: a review, Vet. Clin. Pathol. 38(3) (2009), pp. 288-298. DOI: 10.1111/j.1939-165X.2009.00179.x DOI: 10.1111/j.1748-5827.2011.01070.x [17] I. Uhríková, A. Lačňáková, K. Tandlerová, V. Kuchařová, K. Řeháková, E. Jánová, J. Doubek, Haematological and biochemical variations among eight sighthound breeds, Aust. Vet. J. 91(11) (2013), pp. 452-459. DOI: 10.1111/avj.12117 [4] S. Paltrinieri, W. Bertazzolo, A. Giordano, Come fare e intepretare correttamente un esame: errori preanalitici e interpretativi - How to do and interpret correctly examination: analytical and interpretative errors, in: Patologia clinica del cane e del gatto: approccio pratico alla diagnostica di laboratorio - Clinical pathology of the dog and cat: practical approach to laboratory diagnostics, Italia, ed. Elsevier Health Sciences Italy, 2015, pp. 18.20, ISBN 8821431592 [In Italian] [18] P. Scarpa, B. Ruggerone, S. Gironi, T. Vitiello, S. Paltrinieri, Haematological and biochemical reference intervals in healthy racing and retired Italian Greyhounds, Acta Vet. Hung. 68(1) (2020), pp. 71-78. DOI: 10.1556/004.2020.00006 [5] K. R. Friedrichs, K. E. Harr, K. P. Freeman, B. Szladovits, R. M. Walton, K. F Barnhart, J. Blanco-Chavez; American Society for Veterinary Clinical Pathology, ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics, Vet. Clin. Pathol. 41(4) (2012), pp. 441-453. DOI: 10.1111/vcp.12006 [19] L. Nielsen, M. Kjelgaard-Hansen, A. L. Jensen, A. T. Kristensen, Breed-specific variation of hematologic and biochemical analytes in healthy adult Bernese Mountain dogs, Vet. Clin. Pathol. 39(1) (2010), pp. 20-28. DOI: 10.1111/j.1939-165X.2009.00186.x [20] B. Ruggerone, M. Giraldi, S. Paltrinieri, P. 5. STATISTICAL METHODS Contri, Blood analysis in newborn donkeys: hematology, biochemistry, and blood gases analysis, Theriogenology 82(2) (2014), pp. 294-303. 6. CONCLUSIONS Minuti, A. Giordano, Reference intervals for hematological and biochemical parameters, acute phase proteins and markers of oxidation in Holstein dairy cows around 3 and 30 days after calving, Res. Vet. Sci. 114 (2017), pp. 322-331. DOI: 10 1016/j rvsc 2017 06 012 DOI: 10.3390/ani10071212 [31] S. Paltrinieri, F. Ibba, G. Rossi, Haematological and biochemical reference intervals of four feline breeds, J. Feline Med. Surg. 16(2) (2014), pp. 125-136. DOI: 10.1177/1098612X13499337 [23] M. E. Gelain, W. Bertazzolo, G. Tutino, E. Pogliani, F. Cian, M. K. Boudreaux, A novel point mutation in the β1-tubulin gene in asymptomatic macrothrombocytopenic Norfolk and Cairn Terriers, Vet. Clin. Pathol. 43(3) (2014), pp. 317-321. DOI: 10.1111/vcp.12168 [32] S. Paltrinieri, M. Giraldi, A. Prolo, P. Scarpa, E. Piseddu, M. Beccati, B. Graziani, S. Bo, Serum symmetric dimethylarginine and creatinine in Birman cats compared with cats of other breeds, J. Feline Med. Surg. 20(10) (2018), pp. 905-912. DOI: 10.1177/1098612X17734066 [24] H. D. Pedersen, J. Häggstrom, L. H. Olsen, K. Christensen, A. Selin, M. L. Burmeister, H. Larsen, Idiopathic asymptomatic thrombocytopenia in Cavalier King Charles Spaniels is an autosomal recessive trait, J. Vet. Intern. Med. 16(2) (2002), pp. 169-173. DOI: 10.1111/j.1939-1676.2002.tb02350.x [33] A. Gavazza, M. Quagliardi, G. Rossi, I. Jen, K. Mu, A. Roncarati, L. Galosi, B. Biancani. Hematological parameters in subadult female Risso’s dolphins (Grampus griseus), Proc. of the EAAM 51st annual symposium, Oceanografic-Valencia, Spain, 7-11 March 2023, pp. 43. [25] J. Kelley, L. C. Sharkey, P. W. Christopherson, A. Rendahl, Platelet count and plateletcrit in Cavalier King Charles Spaniels and Greyhounds using the Advia 120 and 2120, Vet. Clin. Pathol. 43(1) (2014), pp. 43-49. pp [34] B. Biancani, M. Quagliardi, G. Rossi, I. Jen, K. Mu, A. Roncarati, L. Galosi, A. Gavazza. Biochemical parameters in subadult female Risso’s dolphins (Grampus griseus), Proc. of the EAAM 51st annual symposium, Oceanografic-Valencia, Spain, 7-11 March 2023, pp. 41. [26] A. R. Torres, S. E. Cassle, M. Haymore, R. C. Hill, Hematologic differences between Dachshunds and mixed breed dogs, Vet. Clin. Pathol. 43(4) (2014), pp. 519-524. DOI: 10.1111/vcp.12178 [35] P. Natchtigall, J. L. Pawloski, J. P. Schroeder, S. Sinclair, Successful maintenance and research with a formerly stranded Risso’s dolphin (Grampus griseus), Aquatic Mammals 16(1) (1990), pp. 8- 13. p [27] P. L. Tisdall, G. B. Hunt, G. Tsoukalas, R. Malik, Post-prandial serum bile acid concentrations and ammonia tolerance in Maltese dogs with and without hepatic vascular anomalies, Aust. Vet. J. 72(4) (1995), pp. 121-126. DOI: 10.1111/j.1751-0813.1995.tb15029.x [36] K. Shirai, T. Sataki, Haematological findings in captive dolphins and whales, Aust. Vet. J. 75(7) (1997), pp. 512-514. DOI: 10.1111/j.1751-0813.1997.tb14384.x [28] O. Aniołek, A. Barc, A. Jarosińska, G. March 2024 | Volume 13 | Number 1 | 5 REFERENCES Scarpa, Hematologic and biochemical reference intervals in Shetland Sheepdogs, Vet. Clin. Pathol. 47(4) (2018), pp. 617-624. DOI: 10.1111/vcp.12665 [6] Clinical and Laboratory Standards Institute (CLSI) document EP28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline-Third Edition. Wayne, PA, 2008, ISBN 978-1-56238-682-5. [21] R. Lavoué, A. Geffré, J. P. Braun, D. Peeters, F. Granat, N. Bourgès-Abella, C. Trumel, Breed-specific hematologic reference intervals in healthy adult Dogues de Bordeaux, Vet. Clin. Pathol. 43(3) (2014), pp. 352-361. DOI: 10.1111/vcp.12151 y [7] H. Brommer, M. M. Sloet van Oldruitenborgh-Oosterbaan, B. Kessels, Haematological and blood biochemical characteristics of Dutch warmblood foals managed under three different rearing March 2024 | Volume 13 | Number 1 | 4 ACTA IMEKO | www.imeko.org [22] O. W. Schalm, Erythrocyte macrocytosis in miniature and toy poodles, Canine Practice 3 (1976), pp. 55-57. DOI: 10.3390/ani10071212 DOI: 10.3390/ani10071212 Zdzislaw, Evaluation of frequency and intensity of asymptomatic anisocytosis in the Japanese dog breeds Shiba, Akita, and Hokkaido, Acta Veterinaria Brno 86 (2017), pp. 385-391. DOI: 10 2754/avb201786040385 [37] P. S. Horn, L. Feng, Y. Li, A. J. Pesce, Effect of outliers and nonhealthy individuals on reference interval estimation, Clin. Chem. 47(12) (2001), pp. 2137-2145. DOI: 10.1093/clinchem/47.12.2137 [38] A. Geffré, J. P. Braun, C. Trumel, D. Concordet, Estimation of reference intervals from small samples: an example using canine plasma creatinine, Vet Clin Pathol. 38(4) (2009), pp. 477-484. DOI: 10.1111/j.1939-165X.2009.00155.x [29] Gazzetta Ufficiale della Repubblica Italiana, Linea guida relativa all’esercizio delle attività sanitarie riguardanti la medicina trasfusionale in campo veterinario - Guideline on the practice of transfusion medical health activities in the veterinary field, n.25, 1 February 2016. Online [Accessed 15 February 2024] https://www.fnovi.it/sites/default/files/LineeGuidaMedicinaTr asfus_0.pdf [In Italian] [39] J. P. Braun, D. Concordet, A. Geffré, N. Bourges Abella, C. Trumel, Confidence intervals of reference limits in small reference sample groups, Vet. Clin. Pathol. 42(3) (2013), pp. 395- 398. / DOI: 10.1111/vcp.12065 [30] A. Miglio, A. Gavazza, D. Siepi, F. Bagaglia, A. Misia, M. T. Antognoni, Hematological and Biochemical Reference Intervals for 5 Adult Hunting Dog Breeds Using a Blood Donor Database, Animals 10(7) (2020), art. No. 1212. March 2024 | Volume 13 | Number 1 | 5 ACTA IMEKO | www.imeko.org
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Which factors determine adaptation to drought amongst farmers in Northern Thailand? Investigating farmers’ appraisals of risk and adaptation and their exposure to drought information communications as determinants of their adaptive responses
Mitigation and adaptation strategies for global change
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Mitig Adapt Strateg Glob Change (2024) 29:6 https://doi.org/10.1007/s11027-023-10099-w Mitig Adapt Strateg Glob Change (2024) 29:6 https://doi.org/10.1007/s11027-023-10099-w ORIGINAL ARTICLE Abstract Drought communications constitute an important source of learning about climate risks and responses that can assist adaptation decision-making amongst those whose livelihoods are threatened by drought. This paper applies Protection Motivation Theory to explore associations between drought communications and attitudes towards drought risk and adap- tation amongst farmers in Northern Thailand. The analysis reveals links between drought communications, farmers’ adaptation appraisal, and their adaptation decisions, whilst links with risk appraisal are minimal. The results highlight positive feedbacks between adaptation experience and appraisal and reveal a weak negative relationship between risk appraisal and adaptation appraisal. The findings imply benefits to framing drought com- munications in terms of the efficacy and attainability of suitable adaptations, rather than simply highlighting drought risks or providing drought warnings, to best enable farmers to build drought resilience. Keywords  Protection Motivation Theory · Drought risk perceptions · Climate change resilience · Thailand Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Received: 3 February 2022 / Accepted: 19 December 2023 / Published online: 11 January 2024 © The Author(s) 2024 Extended author information available on the last page of the article Which factors determine adaptation to drought amongst farmers in Northern Thailand? Investigating farmers’ appraisals of risk and adaptation and their exposure to drought information communications as determinants of their adaptive responses Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 1  Introduction Agriculture is sensitive to water availability, with climatic extremes leading to substantial yield losses (Lesk et al. 2016). Droughts constrain crop productivity and pose an ongoing threat to global food production which is likely to increase due to climate change, popula- tion growth and pressure on water resources (Daryanto et al. 2017; Leng and Hall 2019). Building agricultural drought resilience is of crucial importance to global food security and human well-being (Carrão et al. 2016; Challinor et al. 2014). However, many factors limit effective adaptation, including informational, attitudinal and behavioural barriers (Howden et  al. 2007). Understanding how to overcome these barriers and increase the adaptive 23451 56789) 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 2 of 21 6 capacity of farmers must be prioritised to support agricultural development, crisis preven- tion and vulnerability reduction (Lipper et al. 2014). As such, research has increasingly focused on how farmers’ understandings and perceptions of climate change determine their adaptation decisions (Deressa et al. 2011; Mertz et al. 2009; Sutcliffe et al. 2016), with studies addressing the cognitive processes that link climate perceptions with decisions to implement action (Grothmann and Patt 2005; Truelove et al. 2015). The attitudinal factors that determine decisions to employ a protective measure in response to a threat (i.e. adaptation) have been modelled using the Protection Motivation Theory (PMT) conceptual framework. The PMT framework identifies two cognitive path- ways which are believed to determine whether individuals decide to employ protective behaviour. These pathways are risk appraisal and adaptation appraisal. Risk appraisal com- bines an assessment of the potential severity of a negative impact with the likelihood that such an impact will occur. Adaptation appraisal combines an assessment of how effectively a proposed response would curtail the threat of the negative impact (response efficacy), with an assessment of the capacity of the individual to employ that particular response (self-effi- cacy), along with an assessment of the potential costs of doing so (Milne et al. 2000).i PMT was first employed in the 1970s within social psychology studies of health behaviours (Rogers 1975) and has since been highly influential across a range of disci- plinary areas including business studies, computing and environmental science. It has been applied in relation to a wide array of different threats, including information secu- rity (Haag et al. 2021; Herath and Rao 2009), pollution risks (Wang et al. Mitig Adapt Strateg Glob Change (2024) 29:6 Page 3 of 21 6 Key questions: 1. To what extent do PMT and drought communication variables influence adaptation outcome variables?f 1. To what extent do PMT and drought communication variables influence adaptation outcome variables?f 2. Which determinants affect adaption and risk appraisal? f 3. What role do socioeconomic factors (age, education and wealth) play in determining access to drought communications? f 3. What role do socioeconomic factors (age, education and wealth) play in determining access to drought communications? Based on these questions, we discuss the extent to which PMT variables predict adapta- tion outcome variables and how institutional drought communications influence farmers’ motivations and actions to protect their production activities from the risk of drought. By using the PMT framework to understand the relative importance of farmers’ appraisals of risk and of adaptive responses within their agricultural decision-making and illustrating the influence of drought communications, the paper provides communication design rec- ommendations that should increase farmers’ implementation of adaptations to reduce the negative impacts of drought on agricultural livelihoods. 1  Introduction 2019) and the threat of extreme weather and climate change (Bubeck et al. 2012; Kuruppu and Liv- erman 2011; Grothmann and Patt 2005; Grothmann and Reusswig 2006). Most studies related to extreme weather have concentrated on flood protection, often in a European context (e.g. Babcicky and Seebauer 2017; Bamberg et al. 2017; Bradford et al. 2012; Poussin et al. 2014). Few studies have used PMT to explore drought adaptation in the Global South, although a body of research is building in Asian (e.g. Keshavarz and Kar- ami 2016; Truelove et al. 2015) and African (e.g. Gebrehiwot and van der Veen 2015; Tabe-Ojong et al. 2020; Wens et al. 2021) contexts. Knowledge and understanding of climate change are positively associated with farm- ers’ adaptation intentions (Ngo et al. 2020). Studies have noted the influence of different sources of information for risk and adaptation appraisal (Milne et  al. 2000), including social networks (Babcicky and Seebauer 2017; Haer et al. 2016) and public institutions (Grothmann and Reusswig 2006), and the role of institutions in communicating climate information to influence local level responses (Dorward et al. 2020; Haer et al. 2016; Stey- nor et al. 2021). The provision of drought warnings, forecasts and advice can effectively build resilience to drought by mitigating impacts and leading to faster recoveries within farming communities in developing countries (Ewbank et al. 2019). Optimising the provi- sion of this information is an increasingly key necessity for policy makers, climate services and agricultural development practitioners. This paper aims to build understanding of the processes that determine adaptation to drought amongst farmers in Northern Thailand. In it, we explore and compare the roles that risk appraisal and adaptation appraisal play as determinants of past adaptations and their perceived success and as determinants of responsiveness to official adaptation recom- mendations and the desire to implement further adaptations in future. We also investigate the extent to which exposure to and perceptions of both formal and informal drought com- munications are associated with these adaptation outcomes. 1 3 1 Mitig Adapt Strateg Glob Change (2024) 29:6 2.3.1  Adaptation outcome variables The first set of questions within the questionnaire collected data from respondents on their adaptation actions and perceptions. In particular, respondents provided informa- tion about the number of adaptations they had undertaken in the past (Ai), whether or not they considered these adaptations to have successfully reduced their longer term vulnerability to drought (Aii), whether they had changed anything about their agricul- tural production activities in response to having a received a drought warning (Aiii) and whether or not they had a desire to implement any further adaptations that they had not yet been able to (Aiv) (see Table 1 for further details). 2.2  Sampling and data collection The research team purposively selected a sample of villages exposed to different lev- els of drought over the last decade. Villages both within and outside of formal irriga- tion zones were selected to represent a range of typical agricultural production profiles (such as rice, longan and livestock production). According to the customary context, the village headperson (a village elder who acts as the community leader) in each vil- lage was contacted initially, and the research team requested their permission to con- duct the research. The village headperson then invited farm household heads to partici- pate. As opposed to random sampling of households, this method may have resulted in a degree of sampling bias, meaning that the sample should not be interpreted as fully representative. In particular, readers should bear in mind the possibility that due to the selection of participants by the village headperson, older, wealthier farmers may be over-represented, whilst poorer, younger farmers and female-headed farms may be under-represented. However, this sampling approach was considered the most appropri- ate given the local socio-cultural context, and the validity of the analysis reported herein is not contingent on a perfectly representative sample. Trained enumerators undertook data collection after obtaining informed consent. In total, 176 questionnaires were com- pleted in January 2020 with a close to even distribution of respondents coming from the provinces of Chiang Mai (n = 41), Lamphun (n = 45), Kamphaeng Phet (n = 45) and Tak (n = 45). The structured questionnaire collected socioeconomic, agricultural, risk appraisal, adaptation appraisal, adaptation behaviour and drought communications data from farmers. The questionnaire was originally written in English, translated into Thai and administered before translation back to English for analysis. Questionnaires were anonymised, and the questionnaire and dataset can be found at https://​doi.​org/​10.​17862/​ cranf​ield.​rd.​16553​136. 2.1  Case study As Southeast Asia’s second largest economy, Thailand’s development significantly affects its neighbours, and the country plays an important role in policy-making within the wider region (Kiguchi et al. 2021). Agricultural production in Thailand, like other countries in Southeast Asia, is important for global food supply, with the region as a whole contribut- ing 40% of global rice exports (Yuan et al 2022). However, rice yields have stagnated in Thailand in recent decades, as they have in several neighbouring countries, with drought having played a major role in crop losses in Thailand, Cambodia and Myanmar since 2015 (Venkatappa et al. 2021). Across Southeast Asia, there is an increasing risk of agricultural drought in the near term (Amnuaylojaroen and Chanvichit 2019), with projections sug- gesting countries across the region will experience longer, more intense and more severe droughts prior to 2050 due to the impacts of climate change under a range of probable emission scenarios (Supharatid and Nafung 2021). Identifying strategies to support farmers to stabilise and increase yields whilst reducing their vulnerability to drought is therefore a key concern for policy makers across the region that may look to Thailand as an example. Drought has already had major impacts on the availability of water for irrigation in Thailand in recent years (Kiguchi et al. 2021), with this problem set to grow in the near future (Chang et al. 2021; Singhrattna and Babel 2011). Due to the recent incidence and severity of agricultural drought impacts, the Ping River catchment (a major tributary of Thailand’s main interior river, the Chao Phraya) was chosen for data collection. Signifi- cant regional droughts took place in 2015–2016 and 2019–2020 (Khan and Gilani 2021; Pak-uthai 2019), with reports that the timing of government drought warnings and advice hampered effective farm responses (Khan and Gilani 2021). Despite the major agricultural impacts and increasing risk of drought in Thailand, few studies have focused on climate risk communications (Arunrat et al. 2017; Lebel et al. 2018). As such, Northern Thailand provides a suitable context for investigating how institutional drought communications 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 4 of 21 6 6 currently support farmers to build resilience to drought and for identifying strategies to optimise such support in future. 2.3.2  PMT variables Respondents were then asked to indicate agreement (measured on a five-point scale from strongly agree to strongly disagree) with statements constructed to investigate 1 3 Page 5 of 21  6 Mitig Adapt Strateg Glob Change (2024) 29:6 6 1 3 Table 1   Description and questionnaire numbers for key adaptation, PMT and drought communication variables Variable name Variable type Variable composition and explanation Total adaptation count (Ai) Continuous Total count of number of past adaptations indicated by the respondent Proportion adaptations successful (Aii) Continuous Total count of adaptations perceived to reduce vulnerability to drought impacts in future divided by grand total of adaptations Adapted in response to drought warning (Aiii) Dichotomous (0, 1) Yes/no response to questions, “During previous droughts was your household informed that a drought was coming? Did your household farm change anything as a result of receiving this information?” Desires to adapt further (Aiv) Dichotomous (0, 1) Yes/no response to question, “To reduce the impacts of droughts in the long-term, are there any adapta- tions you would like to undertake but did not yet?” Adaptation appraisal Continuous Sum of adaptation cost scores subtracted from sum of adaptation and self-efficacy statement scores. 2.3.2  PMT variables (“Appropriate adaptation strategies exist for protecting farms like mine from drought” + “I feel confi- dent implementing adaptation strategies for protecting my household farm from drought” + “I consider myself well informed about drought”) minus (“I work hard every day, so I feel too tired to implement the necessary adaptation strategies” + “Farm work is too busy, so there is no extra time to implement the necessary adaptation strategies” + “I have not got enough money to implement the necessary adaptation strategies”) Risk appraisal Continuous Combined scale (sum) consisting of three statements “My household farm is likely to be affected by drought in the next five years” + “The impacts of drought on my farm are likely to be severe” + “The impacts of drought on my household farm are worse than before” Drought information types count Continuous Sum of drought information types received by respondent Drought information channels count Continuous Sum of information channels through which drought information was received Drought information efficacy scores Continuous Agreement (using a five-point scale) that drought information had: helped them prepare for drought (“helped prepare”); arrived early enough in the season to be of use (“early enough”); was relevant to the household’s farming activities (“relevant”); was accurate (“accurate”); was in the correct format (“best format”); and whether the organisation providing the information was receptive to receiving farmer drought and adaptation information back (“receptive”). These measures were combined to form a single variable Number of other farmers drought communica- tions were received from Ordinal Number of farmers (ranked groups) that had informed household a drought was coming and what to do Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 6 of 21 6 their levels of adaptation appraisal and risk appraisal (Milne et al. 2000) (see Table 1). An overall adaptation appraisal score was calculated, based on the sum of perceived self-efficacy and perceived response efficacy scores, less the sum of perceived response costs scores. Similarly, an overall risk appraisal score was based on the sum of per- ceived vulnerability and perceived severity scores. 2.3.4  Land and asset scores In contexts where agricultural households use some of their produce for subsistence, mon- etary income may not accurately reflect household wealth. This necessitates assessing farm household wealth in terms of asset and land ownership. Scores for land and asset owner- ship were calculated following Cordova (2009) and Filmer and Pritchett (2001), whereby standardised scores for a range of culturally prevalent assets are derived from the frequency of their ownership (represented using a dummy variable as either 0 or 1) amongst respond- ent households in the dataset. In the case of land ownership, since this was a continuous variable, farm sizes were split into five bands (at cut points of 95%, 75%, 50%, 25% and 5% of the sample) represented by dummy variables. Standardised scores were then calculated based on frequencies for each band, and scores for household land ownership were made up by adding these scores cumulatively, so that those with land in the highest band accu- mulated the scores for the band they were in as well as all the lower bands. 2.3.3  Drought communications The questionnaire then collected information on whether respondents had received drought warnings, which types of drought monitoring information they had received (for example, weather forecasts, river discharge information and irrigation availability forecasts) and which channels drought information was received through, in addition to the perceived effi- cacy of that information. These were converted into counts of drought information types and channels and a perceived drought information efficacy score (Table 1). 3  Results In the “3” section, RQ1 presents the factors influencing the four adaptation outcome variables using individual regression models. In each case, the influence of the PMT and drought communication variables on the adaptation outcome variables is described, and other influential variables are identified. RQ2 explores factors affecting adaptation and risk appraisal, and RQ3 identifies relationships between adaptation, drought communications and socioeconomic characteristics (age, education and wealth). 3.1.1  Total adaptation count (regression model Ai) Model Ai was significant (F(8) = 12.232, p = .000) and accounted for 37% of the variance in the total adaptation count scores (with an adjusted R2 of .37). Of the PMT variables, only adaptation appraisal significantly predicted total adaptation count, risk appraisal did not. Of the adaptation appraisal components, the statement “Appropriate adaptation strat- egies exist for protecting my household farm from drought” was a stronger determinant than the aggregate “adaptation appraisal” variable; hence, only the “Appropriate…” state- ment variable was selected for inclusion in the model. Of the socioeconomic factors, only age was found to contribute significantly, with a negative relationship evident between age and the number of adaptations employed. In terms of drought communication variables, the count of types of drought information received significantly predicted total adapta- tion counts, as did agreement with the information efficacy statements, “early enough”, “accurate”, “relevant” and “best format”, and the number of farmers that information had been received from. Whilst the relationship between total adaptation count and the state- ments “relevant” and “accurate” were positive, the relationships with the statements “early enough” and “best format” were negative, suggesting that as the number of adaptations undertaken increased, agreement with these particular statements declined (see Table 2). 2.3.5  Statistical analysis Relationships between the four adaptation outcome variables (Ai–Aiv), PMT variables and drought communications variables, in addition to socioeconomic (age, education and wealth), and agricultural and policy factors (formal irrigation support and drought compen- sation provision) were investigated using multiple regression analyses and, where relevant, t-tests, ANOVA, chi-square, Fisher’s exact test and Pearson’s and Spearman’s correlation coefficients. Linear regression was used to identify significant determinants of the two con- tinuous adaptation outcome variables Ai and Aii (“total adaptation count” and “propor- tion adaptations successful”, respectively). Binary logistic regression was used to construct models for the two dichotomous adaptation outcome variables Aiii and Aiv (“adapted in response to drought warning” and “desires further adaptation”, respectively), with interac- tion terms investigated for potential effects (Field 2018). f The regression analysis sought to investigate relationships between the PMT variables (adaptation appraisal and risk appraisal), the drought information variables and the four adap- tation outcome variables (Ai–Aiv). Alternative regression analyses were run in turn incorpo- rating either the compound adaptation and risk appraisal scores or their disaggregated com- ponent variables (such as, for example, agreement scores with the statements representing Mitig Adapt Strateg Glob Change (2024) 29:6 Page 7 of 21 6 response efficacy and self-efficacy). This was done in order to compare results and determine whether the compound scores or the disaggregated scores were more powerful predictors of the independent adaptation variable (Babazadeh et al. 2017). Where the separate component factors proved to have stronger predictive power than the compound variables, these were included preferentially within the regression models presented in the results.i response efficacy and self-efficacy). This was done in order to compare results and determine whether the compound scores or the disaggregated scores were more powerful predictors of the independent adaptation variable (Babazadeh et al. 2017). Where the separate component factors proved to have stronger predictive power than the compound variables, these were included preferentially within the regression models presented in the results.i Following a principle of parsimony (Field 2018), variables found not to significantly predict the outcome variable were excluded (Braun and Oswald 2011). Variables were entered into the models hierarchically, with those expected to have the largest influence entered first. 3.1.2  Proportion of adaptations perceived as successful for longer term drought vulnerability reduction (regression model Aii) R2 .21 (Cox & Snell) and .285 (Nagelkerke). Model χ2 (5) = 36.573, p = .000)i Table 4   Binary logistic regression results showing predictors of whether the household changed anything in response to receiving a drought warning (model Aiii). R2 .21 (Cox & Snell) and .285 (Nagelkerke). Model χ2 (5) = 36.573, p = .000) 95% confidence interval B Standard error Wald statistic Sig. Odds ratio Lower Upper (Constant) −0.562 0.366 2.36 0.124 0.57 Received financial drought compensation × fruit production −1.182 0.433 7.454 0.006 0.307 0.131 0.716 Instances of drought in last 10 years 0.346 0.149 5.411 0.02 1.413 1.056 1.891 Adaptation appraisal 0.44 0.196 5.055 0.025 1.552 1.058 2.277 Received weather forecast information 1.148 0.52 4.874 0.027 3.153 1.138 8.74 adaptation appraisal, animal husbandry and land within a supported irrigation zone, was significant (F(5) = 5.132, p = .000) and accounted for 16% of the variance in the propor- tion of adaptations perceived as successful. All included variables significantly determined the proportion of adaptations perceived as successful, with negative relationships pertain- ing with wealth and animal husbandry, indicating that wealthier farmers and those that raised livestock tended to perceive a lower proportion of their past adaptations as success- ful than other farmers (see Table 3). 3.1.2  Proportion of adaptations perceived as successful for longer term drought vulnerability reduction (regression model Aii) Analysis showed that the drought communications variables did not improve model Aii, and this group of variables was excluded. The final model, which incorporated age, wealth, 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 8 of 21 Table 2   Linear model of predictors of total adaptation count (model Ai, adjusted R2 = .37, F(8) = 12.232, p =.000) Unstandardised coefficients Standard- ised coef- ficients t Sig. 95.0% confidence interval for B B Std. error Beta Lower bound Upper bound (Constant) 1.140 1.394 0.818 0.415 −1.615 3.896 “Appropriate adaptation strategies exist for protecting my household farm from drought” 0.839 0.145 0.386 5.774 0.000 0.552 1.126 Age group −0.712 0.230 −0.206 −3.089 0.002 −1.167 −0.256 Count of different drought monitoring information types received 0.478 0.138 0.254 3.453 0.001 0.204 0.751 “The information provided arrives early enough in the season to be useful to my household farm” −0.527 0.168 −0.233 −3.140 0.002 −0.859 −0.195 “The information provided is usually relevant to the farming in the household farm activi- ties” 0.605 0.253 0.207 2.393 0.018 0.105 1.105 “I am confident that the information provided is accurate” 0.722 0.224 0.243 3.217 0.002 0.278 1.165 “The information is provided in the best format for farmers to access and understand it easily” −0.845 0.261 −0.273 −3.240 0.001 −1.360 −0.329 Information was received from how many farmers 0.171 0.066 0.173 2.572 0.011 0.040 0.302 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 9 of 21 Page 9 of 21 6 Table 3   Linear model of predictors of proportion of adaptations perceived to successfully reduce vulner- ability to future droughts (model Aii, adjusted R2 = .16, F(5) = 5.132, p = .000) Unstandardised coef- ficients Standardised coefficients Sig. 95.0% confidence interval for B B Std. error Beta Lower bound Upper bound (Constant) 0.231 0.174 0.186 −0.112 0.575 Age 0.136 0.047 0.221 0.004 0.044 0.228 Wealth −0.013 0.005 −0.182 0.017 −0.023 −0.002 Adaptation appraisal 0.075 0.026 0.222 0.004 0.025 0.126 Household raises animals −0.135 0.054 −0.184 0.014 −0.242 −0.028 Household has land in official irrigation zone 0.14 0.057 0.179 0.016 0.027 0.252 le 3   Linear model of predictors of proportion of adaptations perceived to successfully reduce vulner- ity to future droughts (model Aii, adjusted R2 = .16, F(5) = 5.132, p = .000) Table 4   Binary logistic regression results showing predictors of whether the household changed anything in response to receiving a drought warning (model Aiii). 3.1.4   Respondent indicated a desire to implement further adaptations (regression model Aiv) Model Aiv was significant (χ2 (4) = 26.164, p = .000) and accounted for 23% of the vari- ance. Within the model, the odds that a farmer would indicate wanting to make further drought adaptations were significantly increased (by 1.44) for each additional drought adaptation that they had previously implemented (Table  5). The odds also increased if they indicated lacking money for implementing adaptations, feeling well informed about drought risks or perceiving drought information to be in the right format for farmers. The odds were lowered amongst farmers that indicated stronger agreement that the drought information received had been relevant (Table 5). Whilst this last finding was unexpected, a potential explanation could be that a perceived lack of relevant drought information caused farmers to delay implementing their desired adaptations.i Whilst risk appraisal was not a significant predictor of a desire to implement further adaptations in this model, a t-test revealed that farmers that indicated a desire to adapt fur- ther had significantly higher risk appraisal scores than farmers that indicated that they did not wish to implement any further changes (x ̅  = 11.9 vs x ̅  = 11.2, t = 1.985, p = 0.049). 3.1.3  Respondent adapted their agricultural practice in response to receiving a drought warning (regression model Aiii) Model Aiii, which used binary logistic regression to investigate decisions to adapt in response to official advice, was significant (χ2 (5) = 36.573, p = .000) and accounted for 29% of the variance. Both fruit production and rice production were found to interact sig- nificantly with having received financial drought compensation, although when both fruit and rice were included in the same model, the significance of the rice × compensation 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 10 of 21 6 interaction was lost (Table 4). Nevertheless, this result points to a potentially important relationship between past receipt of drought compensation and behavioural responses to drought information amongst producers of these crops, which reduced the likelihood that households would have changed something about their agricultural or livelihood activities in response to receiving drought information. However, given the cross-sectional nature of our data, we cannot discern whether failing to adapt in response to advice came before or after receiving compensation. The other significant determinants (all of which increased the likelihood that households would indicate having changed something in response to the information they received) were the number of droughts that the household had experi- enced over the last 10 years, the respondent’s adaptation appraisal score and if the respond- ent had received weather forecast information (Table 4). Amongst these variables, having received a weather forecast had an odds ratio of 3.15, meaning that the odds of households adapting in response to official advice were 3.15 times greater amongst households that had received weather forecast information than amongst those that had not. For every addi- tional instance of drought the household reported experiencing in the last decade, the odds that they had adapted in response to a drought warning were increased by 1.41, and for every unit increase in adaptation appraisal, the odds increased by 1.56.i Although not found to contribute significantly to the logistic regression, growers that indicated having received information via a social network messaging group were statisti- cally more likely to have changed something in response to official advice (χ2 = 3.991, p = .046), as were growers that had been educated to high school level or above (χ2 = 7.780, p = .005). 3.2.1  Adaptation appraisal Multiple significant associations were found using Pearson’s correlation coefficient between adaptation appraisal and the drought information communications variables. In 1 3 Page 11 of 21  6 Mitig Adapt Strateg Glob Change (2024) 29:6 Table 5   Binary logistic regression results showing predictors of “Are there any further changes you would like to implement on the farm, but that you did not yet?” (model Aiv). R2 .17 (Cox & Snell) and .229 (Nagelkerke). Model χ2 (4) = 26.164, p = .000) 95% C.I. for EXP (B) B Standard error Wald statistic Sig. Odds ratio Lower Upper Constant −5.852 2.027 8.334 0.004 0.003 “I have not got enough money to implement the necessary adaptation strategies” 0.626 0.212 8.689 0.003 1.869 1.233 2.833 Total number of adaptations 0.365 0.111 10.869 0.001 1.44 1.159 1.789 “I consider myself to be well informed about drought risks” 0.633 0.264 5.746 0.017 1.883 1.122 3.158 “The information provided is usually relevant to the farming in the household farm activities” −0.959 0.409 5.512 0.019 0.383 0.172 0.853 “The information is provided in the best format for farmers to access and understand it easily” 1.037 0.394 6.935 0.008 2.82 1.304 6.102 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 12 of 21 6 particular, confidence in implementing adaptation strategies correlated with the number of types of climate information that had been received (r = .23, p = .04), the perception that climate information had helped the farmer prepare in the past (r = .40, p = .001) and that the information was provided in the best format for farmers (r = .36, p =.001).i Using multiple regression, adaptation appraisal scores were significantly determined by education (Beta .271, p = .001), the total number of adaptations that had been undertaken in the past (Beta .220, p = .005), the proportion of past adaptations perceived as successful (Beta .213, p = .007) and the drought information efficacy score (Beta .199, p = .013). The model was significant (F, 10.478; p = .000), and these four variables accounted for a fifth of the variance in adaptation appraisal (adjusted R2 = .21).i Wealth correlated significantly with adaptation appraisal (r = .207, p = .006), but did not contribute significantly to the regression model due to high semi-partial correlation with education. 3.2.1  Adaptation appraisal Older farmers (aged over 60) were also found to have significantly lower adaptation appraisal scores (mean = 0.27 vs 0.63 for younger farmers, t = −2.161, p = .032). Adaptation appraisal was lower if a respondent perceived there to be higher costs asso- ciated with undertaking adaptations. Respondents’ perceptions of adaptation costs were measured via their scores for the availability of time, energy and finance for implementing adaptations. Whilst correlation between the availability of time and energy for implement- ing adaptations was high (.61, p = .000), there were no significant associations between financial capacity to adapt and these other factors, suggesting that financial constraints operate independently for a large proportion of the respondents.ii Spearman’s rank tests identified significant weak positive correlations between age and agreement that the respondent could not implement adaptations due to insufficient energy and time (ρ (rho) =.20, p = .009 and ρ (rho) = .16, p = .032, respectively). Conversely, edu- cation correlated negatively with all three types of perceived adaptation cost: energy (ρ (rho) = −.25, p =.001), time (ρ (rho) = −.32, p = .000) and money (ρ (rho) = −.24, p = .002). 1 3 3.3  RQ3: What role do socioeconomic factors (age, education and wealth) play in determining access to drought communications? Statistical tests demonstrated the role of socioeconomic factors in determining access to drought warnings. Older farmers reported receiving drought communications through significantly fewer channels (mean = 1.0 vs 1.3 for farmers aged under 60, t = −2.883, p = .004) and tended to report receiving drought communications from a smaller num- ber of other farmers (although p = .059). Respondents in older age groups were less likely to have received drought warnings that were accompanied by advice on what to do (Fisher’s exact test = 14.998, p = .003), as were respondents with lower education levels (Fisher’s exact test = 33.219, p = .000). Wealth was also a factor, with the highest mean household asset and land wealth scores found within households that received both a drought warning and advice on what to do (10.0), compared to lower scores amongst those that only received warnings (8.9), and those that received neither (5.4), F(2172) = 5.416, p = .005. Adaptation appraisal scores followed the same pattern, with the lowest mean scores in households that received no warning or advice (−0.85), mean scores of 0.36 in households that only received the drought warning and mean scores of 0.73 in households that received both the warning and advice, F(2169) = 15.419, p = .000. Respondents that received drought warnings accompanied by adaptation advice were significantly more likely to have changed something as a result, than those that received the drought warning alone (67.5% compared to only 25.0%, χ2 = 24.696, p = .000). 3.2.2  Risk appraisal Farmers’ risk appraisal was not statistically associated with any of the climate information variables, apart from a weak positive association with how many other farmers the infor- mation was received from (r = .183, p = .016). Additionally, those that received informa- tion at a village meeting had significantly stronger mean agreement that their household was likely to be negatively affected by drought in the next 5 years, than those who received the information by other means (mean = 3.98 vs 3.59, t = 2.655, p = .021). Growers that experienced drought in the most recent 3 years before the survey (in Janu- ary 2020) displayed significantly higher agreement with the risk statements “The impacts of drought on my household farm are likely to be severe” (mean = 4.02, t = 2.951, p = .004) and “The impacts of drought on my farm are worse than before” (mean = 4.25, t = 5.316, p = .000) than those that did not have such recent experience of drought (means = 3.44 and 3.29, respectively). A positive correlation was observed with the total number of economic and crop drought impacts indicated by the farmer (r = .262, p = .000), and a weak negative correlation was observed between risk appraisal and adaptation appraisal (r = −.169, p = .026). These findings suggest that direct experience of drought influences risk appraisal, with more recent experiences having a more pronounced effect. f The three variables, years since last drought experience (Beta −.216, p = .011), number of farmers climate information was received from (Beta .185, p = .027) and household 1 3 Page 13 of 21 Mitig Adapt Strateg Glob Change (2024) 29:6 6 engages in animal husbandry (Beta .139, p = .096), resulted in a significant model (F = 5.721, p = .001) with an R2 of .096. 4.1  Conceptual diagram Figure 1 provides a conceptual overview of the main relationships reported in the results above, with the routes connecting the four adaptation outcome variables with risk appraisal and adaptation appraisal illustrated. Also shown are other sociodemographic and policy factors found to have significant effects on either adaptation outcomes or the cognitive pathways associated with adaptation appraisal and/or risk appraisal. Being in a formal irri- gation zone is indicated as a policy intervention since farmers based in these areas benefit from irrigation zone policies which provide additional government-funded support. The results synthesised in Fig. 1 show that adaptation appraisal interacted directly with all four of the adaptation outcome variables whilst only a weak association was found between risk appraisal and one of the adaptation outcome variables. This lends weight to the idea (reported in the context of flooding by Babcicky and Seebauer 2019) that, although there may be two separate PMT pathways, only the one operating through adaptation or cop- ing appraisal leads to the implementation of protective measures. As such, the results sup- port Babcicky and Seebauer (2019), suggesting that the traditional interpretation of PMT, where high risk appraisal and high adaptation appraisal combined lead to the implementa- tion of protective measures, may not be empirically accurate in this kind of context. 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 14 of 21 6 Fig. 1   The relationships represented are those uncovered using regression analysis, but the diagram addi- tionally includes two relationships identified using other statistical tests: a negative correlation between adaptation and risk appraisal and a difference in mean risk appraisal relating to desire to implement further adaptations (both represented by dashed lines). These additions are considered relevant to the discussion. Arrows represent the direction of influence that was modelled, with line thickness representing the strength of the relationship and colour representing whether the relationship was positive (green) or negative (red). For simplicity, symbols are used to represent groups of variables as described in the key. Where the model- ling identified relationships with sub-components of these variable groups, the individual variable is identi- fied using a label at the middle of the relationship line 1 3 4.3  To what extent do institutional drought communications influence farmers’ motivations and actions to protect their production activities from the risk of drought? Whilst direct personal experience of climate impacts strongly shapes adaptation motiva- tions (Niles et al. 2015), climate and adaptation information received through personal and institutional networks also affects adaptation decision-making (Babcicky and Seebauer 2017). Further, contrary to the suggestion that top-down campaigns may have limited capacity to affect the uptake of protective measures against extreme climate events (Haer et al. 2016), our results indicate that institutional drought communications (via commu- nity, private or government institutions) interact significantly with adaptation attitudes and actions amongst agricultural producers. The diamonds in Fig. 1 illustrate where drought communications or direct policy interventions (the provision of drought compensation or official irrigation support) significantly predicted adaptation outcome variables. It is evi- dent that both the size of a farmer’s local drought information network and the nature of the drought information they receive via institutional channels interact significantly with adap- tation intention and behaviour. The study has shown that receiving more different types of drought information as well as the perceived efficacy of the information received are both positively associated with higher confidence in implementing adaptations and with the number of adaptations farmers have employed in the past. Additionally, our findings sup- port the importance that drought information is accompanied by practical advice on what to do to limit negative impacts (Ewbank et al. 2019; Haer et al. 2016; Koerth et al. 2013).i Although the institutional drought communication measures significantly contributed to adaptation outcome variables (namely, adaptation appraisal scores, past adaptation counts and whether respondents reported having adapted in response to official advice), they appeared not to contribute to respondents’ drought risk appraisal. Instead, risk appraisal was influenced by local communication factors, such as the number of farmers drought informa- tion was received from, and whether information was received at a village meeting. This supports findings that local and informal risk communications are more likely to alter risk perceptions (Binh et al. 2020). Additionally, risk appraisal was influenced by direct personal experience of drought, supporting the suggestion that factors which are more psychologi- cally close (either in time, space or socially) determine risk perceptions more strongly than official communications (Binh et al. 2020; Niles et al. 2015; Steynor et al. 4.2  To what extent do PMT variables predict adaptation outcomes? Looking at the relationship between PMT variables and the adaptation outcome variables analysed, this study reveals a much more pronounced connection between adaptation and adaptation appraisal than between adaptation and risk appraisal, which reflects findings from many previous PMT studies exploring a range of different risks and contexts (Bubeck et al. 2012; Milne et al. 2000; Poussin et al. 2014; Sheeran et al. 2014; Truelove et al. 2015). It has been argued that risk appraisal could be higher prior to an adaptation being undertaken, but then decline in light of adaptation success (Bamberg et al. 2017). Our finding of significantly higher mean risk appraisal scores amongst respondents that indicated wanting to implement further adaptations supports this possibility. However, since no significant associations were found between adaptation counts and other more stable factors that correlate with risk appraisal (for example, the number of droughts and drought impacts respondents reported having experienced), the authors argue that the findings suggest adaptation appraisal is the more robust behavioural predictor of the two PMT constructs (Milne et al. 2000). The finding of a weak negative correlation between adaptation appraisal and risk appraisal illustrates that, rather than being purely additive, it is possible that high levels of risk perception may lower perceived capacity to adapt, or vice versa. The conceptual diagram in Fig. 1 depicts positive feedbacks between adaptation and adaptation appraisal, showing that direct experience of implementing adaptation and perceived adaptation success may increase cognitive readiness to adapt in future. These findings suggest that direct experience, observation and learning about adaptation and drought risk continually reshape perceptions, illustrating processes of adaptation as continuous rather than finite 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 15 of 21 6 and suggesting that adaptation experience and a pro-adaptive mindset are mutually rein- forcing. This identification of positive adaptation feedbacks mirrors those identified by Noll et al. (2022) in the context of flood adaptation. On the basis that adaptation actions may trigger further adaptation intentions, drought communications and adaptation cam- paigns should not be regarded as standalone responses, but rather as elements within long- term support programmes geared towards developing cultures of resilience and capacity- building (Ewbank et al. 2019). This finding is of particular relevance for countries where agricultural impacts from drought are projected to increase in coming decades. 4.3  To what extent do institutional drought communications influence farmers’ motivations and actions to protect their production activities from the risk of drought? 2021).iil fi The finding of a significant influence of local farmer communications on risk appraisal and past adaptation points to the role of social connectedness in facilitating access to infor- mation and as an important determinant of adaptation responses. Moreover, the appar- ent role of smartphone messaging group membership in enhancing responsiveness to official advice should not be overlooked. Overall, these findings highlight the benefits of strengthening opportunities for all farmers to participate in local and institutional drought 1 3 3 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 16 of 21 6 communications networks via locally appropriate and accessible means, particularly in agricultural development contexts similar to the case study presented here. communications networks via locally appropriate and accessible means, particularly in agricultural development contexts similar to the case study presented here. 4.4  Which types of farmers are less likely to implement successful drought adaptations and why? Demographic variables such as age and education are important determinants of disaster warning response, with evidence that different segments of at risk populations may com- prehend information differently (Mayhorn and McLaughlin 2014). Our findings reflect socio-economic patterns well-established by diffusion of innovations scholars (E. M. Rogers 2003), namely that education, age and wealth determine capacity to implement new behav- iours. The study found that older farmers were more likely to indicate adaptation constraints, such as a lack of time or energy. Older farmers were also likely to receive drought informa- tion from fewer local farmers and via fewer communication channels. As such, this study highlights that older farmers may be less able to adapt to drought and supports findings that older individuals may have different communication needs and support should be tai- lored accordingly (Walkling and Haworth 2020). As drought information communications move online in future, the need may grow to implement additional communication strategies which ensure that older and poorer population segments do not end up missing out.i The study findings also suggest that livestock producers experience drought risks and impacts differently, resulting in measurably higher risk appraisal scores and lower per- ceived success rates for past adaptations. This indicates that households which focus on livestock production may likewise benefit from specific targeted drought support, reflecting findings from different regional contexts that drought responses within the livestock sector are different to other agricultural sectors (Salmoral et al. 2020). 4.5  How might drought communications and policy be improved to support adaptation? The absence of a strong link between risk appraisal and adaptation behaviour (aligning with many other PMT studies) suggests that seeking to heighten risk appraisal by incor- porating fear appeals within official drought communications is unlikely to enhance adaptation behaviour amongst farmers and signals the possibility that fear appeals could be counterproductive. Whilst this study supports the communication of targeted drought information to farmers, emphasis should be placed upon accompanying drought informa- tion with positive messages about household coping capacity and practical tailored advice about adaptation strategies which enhance farmers’ perceptions of both self- and adapta- tion efficacy (Binh et al. 2020; Ong and Araral 2021).i fi Although the findings do not support risk appraisal directly determining adaptation actions, higher risk appraisal levels may promote adaptation desire and/or intention (Milne et  al. 2000). The findings indicated that risk appraisal levels may be higher following a direct experience of drought, but then decline thereafter. Drought communications should be targeted to take advantage of potential synergies between aspects of risk and adaptation appraisal (Sheeran et al. 2014), by ensuring that drought adaptation information and physical/ financial assistance are made available soon after farming households have directly experi- enced drought, since this may lead to greater uptake than if only provided at a later stage.i The findings indicate that the constraint of not having enough money to implement the nec- essary adaptations may be delaying farmers from implementing desired further adaptations. 1 3 Mitig Adapt Strateg Glob Change (2024) 29:6 Page 17 of 21 6 Given the lack of a correlation between financial and the other adaptation constraints meas- ured (time and energy), providing financial support to farmers who indicate an inclination to adapt may boost the uptake of drought adaptation measures. In some instances, pursuing this proactive approach to building drought resilience may be preferable to providing drought com- pensation after losses have already been incurred, particularly given the negative relationship between compensation for major crops such as fruit and rice and responsiveness to official drought warnings and adaptation advice. This finding reflects Tabe-Ojong et al. (2020), who show that relief assistance may negatively influence household resilience strategies for coping with extreme weather events. 4.5  How might drought communications and policy be improved to support adaptation? However, given that our study is cross-sectional, it is not pos- sible to establish whether compensation arrived before or after farmers failed to take action in response to official drought advice; hence, longitudinal research on this issue is warranted. 5  Conclusion The study supports protective behavioural changes (in this case, implementation of drought adaptations) being more closely linked to adaptation appraisal than risk appraisal. Further, it has indicated that an inverse relationship can pertain between these PMT constructs. Both institutional and local drought communications contribute in important ways to adaptation actions and perceptions, but institutional communications appear to operate more through pathways leading to adaptation via adaptation appraisal. Conversely, local communications are of greater significance for risk appraisal, although the study has not found evidence to support a direct pathway from risk appraisal to adaptation. Institutional drought com- munications should include adaptation advice to promote adaptation behaviour amongst agricultural producers, with drought communications framed in positive terms, emphasis- ing the efficacy of adaptation recommendations and the feasibility of implementing them for the producers themselves. Policy makers should employ measures that support farmer participation in both local and institutional drought communication networks. The study shows that farmers who are older, less educated or poorer are less likely to adapt to drought due to perceived costs and reduced exposure to and assimilation of drought information. More research is needed on how to shape and supply institutional drought and adaptation communications to meet the needs of these segments of agricultural populations more effectively. Whilst financial support may sufficiently address adaptation constraints for some, evidence of time and energy constraints for, particularly, older farmers indicate a need for different targeting of adaptation support for these farmers, including the provision of social safety nets in cases where adaptation is improbable. Evidence that those practicing ani- mal husbandry perceive greater levels of drought risk and lower levels of adaptation success than other agricultural producers also suggests a need for specific targeted support.i i Understanding adaptation as a continuous (rather than finite) process, characterised by feedbacks between experience and appraisal, means that efforts to build drought resilience must go beyond providing assistance only when drought is forecast or causing measur- able impacts. Efforts to reduce drought vulnerability should provide long-duration, con- tinuous institutional support for resilience building. Such support needs to engender pro- adaptive mindsets across all sections of agricultural communities by improving social and institutional drought communication networks, ensuring that organisations are receptive to climate and adaptation information fed back by farmers and developing habits across the board of engaging in ongoing climate, drought and adaptation dialogue. Declarations Ethics approval  The study gained ethical approval through the Cranfield University Research Ethics System (CURES) CURES/9419/2019. Competing interests  The authors declare no competing interests. Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. 5  Conclusion 3 1 Mitig Adapt Strateg Glob Change (2024) 29:6 6  Page 18 of 21 6 Acknowledgements  We wish to thank the wider members of the STAR project team, the CMU students who performed the interviews and the participant farmers for their time. Author contribution  DR, CS and IH acquired the funding. CS, DR, IH and GS developed the survey instru- ment and conceived the original scope of the article. LP, SV and CE coordinated the fieldwork and transla- tions. CS developed the statistical analysis. CS drafted the manuscript. All authors reviewed, edited and approved the manuscript. Funding  This project is funded through a NERC (Natural Environment Research Council, UK) grant for the STAR project (Strengthening Thailand’s Agricultural drought Resilience) grant number NE/S003223/1 and Thailand Science Research and Innovation (TSRI) grant number is RDG6130017. Data availability  The dataset and questionnaire for this study can be found in the Cranfield Online Resea Data (CORD) 10.17862/cranfield.rd.16553136. References https://​doi.​org/​10.​1016/j.​accre.​2021.​03.​001 62–171. https://​doi.​org/​10.​1016/j.​accre.​2021.​03.​001 p g j Cordova A (2009) Methodological note: measuring relative wealth using household asset indicators. In: AmericasBarometer Insights Series, vol 6. https://​www.​vande​rbilt.​edu/​lapop/​insig​hts/​I0806​en_​v2.​ pdf. 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J Environ Manag 237:15–23. https://​ doi.​org/​10.​1016/j.​jenvm​an.​2019.​02.​070 Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 Chloe Sutcliffe1,2 · Ian Holman1   · Daniel Goodwin1,3 · Gloria Salmoral1 · Liwa Pardthaisong4 · Supattra Visessri5,6 · Chaiwat Ekkawatpanit7 · Dolores Rey1 * Ian Holman i.holman@cranfield.ac.uk Chloe Sutcliffe chloesutcliffe@rhs.org.uk Daniel Goodwin daniel.goodwin@utas.edu.au Gloria Salmoral gsalmoral@icatalist.eu Liwa Pardthaisong liwa.p@cmu.ac.th Supattra Visessri supattra.vi@chula.ac.th Chaiwat Ekkawatpanit chaiwat.ekk@kmutt.ac.th Dolores Rey dolores.rey-vicario@ec.europa.eu * Ian Holman i.holman@cranfield.ac.uk Chloe Sutcliffe chloesutcliffe@rhs.org.uk Daniel Goodwin daniel.goodwin@utas.edu.au Gloria Salmoral gsalmoral@icatalist.eu Liwa Pardthaisong liwa.p@cmu.ac.th Supattra Visessri supattra.vi@chula.ac.th Chaiwat Ekkawatpanit chaiwat.ekk@kmutt.ac.th Dolores Rey dolores.rey-vicario@ec.europa.eu 1 Centre for Water, Environment and D Bedfordshire MK43 0AL, UK 2 Environmental Horticulture Departm 3 School of Social Sciences, Universit Chaiwat Ekkawatpanit chaiwat.ekk@kmutt.ac.th Dolores Rey dolores.rey-vicario@ec.europa.eu 1 Centre for Water, Environment and Development, Cranfield University, College Road, Cranfield, Bedfordshire MK43 0AL, UK 2 Environmental Horticulture Department, Royal Horticultural Society, Wisley GU23 6BQ, UK 3 School of Social Sciences, University of Tasmania, Hobart, Australia 4 Department of Geography, Faculty of Social Sciences, Chiang Mai University, Chiang Mai, Thailand 5 Department of Water Resources Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand 6 Disaster and Risk Management Information Systems Research Unit, Chulalongkorn University, Bangkok, Thailand 7 Department of Civil Engineering, King Mongkut’s University of Technology Thonburi, Bangkok, Thailand 1 3
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Rapid Adsorption Enthalpy Surface Sampling (RAESS) to Characterize Nanoporous Materials
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Rapid adsorption enthalpy surface sampling (RAESS) to characterize nanoporous materials Emmanuel Ren François-Xavier Coudert Emmanuel Ren, François-Xavier Coudert To cite this version: Emmanuel Ren, François-Xavier Coudert. Rapid adsorption enthalpy surface sampling (RAESS) to characterize nanoporous materials. Chemical Science, 2023, 14 (7), pp.1797-1807. ￿10.1039/D2SC05810C￿. ￿hal-03955453v2￿ Emmanuel Ren, François-Xavier Coudert. Rapid adsorption enthalpy surface sampling (RAESS) to characterize nanoporous materials. Chemical Science, 2023, 14 (7), pp.1797-1807. ￿10.1039/D2SC05810C￿. ￿hal-03955453v2￿ Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction associated physical and chemical properties. These databases can be computationally screened to retrieve key structure– property relationships, potential theoretical limitations and promising new structures.7,8 This is particularly true in the case of nanoporous materials, which have been extensively studied in high-throughput screening methodologies9 for the identi- cation of top-performing materials for applications such as electrocatalysis,10 photocatalysis,11 heterogeneous catalysis,12 membrane separation,13 adsorptive separation,14 adsorptive storage,15 mechanical behaviors,16 etc. 1 Molecular adsorption has many large-scale industrial applica- tions in our society, including uid storage, molecular separa- tion, and purication, and is therefore a very active area of research in both chemistry and materials science. Improvement in the performance of adsorption-based processes could reduce the environmental impact of separation and purication applications by replacing energy-intensive processes such as cryogenic distillation.1,2 In the energy industry, the use of nanoporous solids is a promising low-energy alternative to pressurized gas tanks for the storage of fuel such as H2 or CH4.3 Adsorption is also foreseen as a crucial component in the next generation of carbon capture and storage systems.4 To further unfold the potential of this technology, the design of materials for targeted applications needs to be rened. Because high-throughput screening is performed on the scale of hundreds of thousands or even millions of structures, there is a need for faster computational methods to predict the materials' properties, to be able to analyze larger and larger databases. In the eld of adsorption, several measures have been proposed in order to study the performance of a material for the adsorption of a specic guest molecule. The most accurate, but also the most computationally expensive one, is the grand canonical Monte Carlo (GCMC) simulation. On the other end of the scale, the use of simple geometric descriptors (pore size, pore volume, and surface area) has also been proposed as proxies for various adsorption- related metrics. As the eld of computational chemistry is turning more and more towards machine learning (ML), the development of a wide range of rapidly calculable descriptors17 is an exciting research focus especially to speed up computational screenings. Although the nal steps of this design process can only be achieved by experimentation and engineering, computational simulations can play a key role in guiding the design process and speeding up the discovery of materials for targeted appli- cations. aCEA, DES, ISEC, DMRC, Univ. Montpellier, Marcoule, France bChimie ParisTech, PSL University, CNRS, Institut de Recherche de Chimie Paris, 75005 Paris, France. E-mail: fx.coudert@chimieparistech.psl.eu † Electronic supplementary information (ESI) available: Additional information is available in the supporting information le, raw data are available online at https://github.com/fxcoudert/citable-data and the RAESS code is available at https://github.com/coudertlab/RAESS. See DOI: https://doi.org/10.1039/d2sc05810c HAL Id: hal-03955453 https://hal.science/hal-03955453v2 Submitted on 15 Feb 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License This article is licensed under a Creative Commons Attribution 3.0 Received 19th October 2022 Accepted 2nd January 2023 DOI: 10.1039/d2sc05810c rsc.li/chemical-science Received 19th October 2022 Accepted 2nd January 2023 Accepted 2nd January 2023 DOI: 10.1039/d2sc05810c rsc.li/chemical-science DOI: 10.1039/d2sc05810c rsc.li/chemical-science Chemical Science View Article Online View Journal | View Issue Rapid adsorption enthalpy surface sampling (RAESS) to characterize nanoporous materials† Cite this: Chem. Sci., 2023, 14, 1797 All publication charges for this article have been paid for by the Royal Society of Chemistry Cite this: Chem. Sci., 2023, 14, 1797 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. rticle is licensed under a Creative Commons Attribution 3.0 Unported Licence. nder a Creative Commons Attribution 3.0 Unported Licence. Emmanuel Ren ab and François-Xavier Coudert *b All publication charges for this article have been paid for by the Royal Society of Chemistry Molecular adsorption in nanoporous materials has many large-scale industrial applications ranging from separation to storage. To design the best materials, computational simulations are key to guiding the experimentation and engineering processes. Because nanoporous materials exist in a plethora of forms, we need to speed up the existing simulation tools to be able to screen databases of hundreds of thousands of structures. Here, we describe a new algorithm that quickly calculates adsorption enthalpies by sampling the surface of the material instead of the whole porous space. This surface sampling has been tested on the CoRE MOF 2019 database and has been proven to be more than 2 orders of magnitude faster than the gold standard method (Widom insertion), with an acceptable level of error on an enthalpy value of 0.34 kJ mol−1, and is therefore proposed as a valuable addition to the high- throughput screening toolbox. This article is licensed under a Creative Commons Attribution 3 All simulations are performed for xenon adsorption on struc- tures of the CoRE MOF 2019 database at innite dilution and at 298 K. Krypton adsorption has also been considered in order to see the viability of the method in prediction of Xe/Kr selective mate- rials (see full details in ESI† Section S4). Adsorption at another temperature (600 K) was tested using the nal algorithm, and the results are presented in ESI† Section S2. Other databases have also been briey explored to test the robustness of the proposed algo- rithm. A subset of a hypothetical MOF database, the ToBaCCo database,24 has been screened (Section S6.1†), because it contains very different MOFs according to the diversity analysis of Moosavi et al.25 (for instance, the pores are larger). An amorphous material database (containing 205 structures) has also been screened and the algorithm identied some top materials for xenon adsorption (Section S6.2†). One such biased sampling methodology was recently proposed and used in a computational screening study of adsorptive separation of xenon from krypton. Simon et al. used a machine learning model to screen over 670 000 structures based on geometrical descriptors and one energy descriptor that mainly explains the accuracy of the nal model. To calcu- late this descriptor, the authors used the average of the inter- action energy over the Voronoi network to account for the exponential contribution of the energy in the Henry constant. According to the authors, this approach can sample the most favorable sites of the structure without wasting computational time on unfavorable ones. However, because the sampled points are at the center of the cages, they may not always be the most attractive sites especially for large pores. This approximate approach makes this sampling interesting for quickly producing an energy-based ML descriptor, but it could not replace a Widom insertion for assessing the adsorption performance. Other biased sampling methods that calculate the integrals of the adsorption enthalpy and the Henry constant could be applied, instead. In this work we propose one way of exploiting prior chemical knowledge of adsorption—the fact that it occurs near the surface of the pores—to efficiently sample the nanoporous space. Widom insertion19 is implemented in RASPA2. It consists in randomly inserting a single molecule inside an existing molecular system to measure an interaction energy. 1 Introduction Thanks to initiatives such as the Materials Genome project,5,6 we now have databases of hundreds of thousands of structures at our disposal, including both experimentally known and theoretically predicted structures, along with their For low-pressure adsorption, a widely used characterization parameter is the adsorption enthalpy DHads, which describes the affinity of an adsorbate molecule with the internal surface of a porous framework. In theory,18 this value is derived from the continuous Boltzmann average of the interaction energies Eint of the adsorbate with the framework over the entire porous space, Chem. Sci., 2023, 14, 1797–1807 | 1797 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article View Article Online Edge Article View Article Online Chemical Science Chemical Science and this integral is calculated using a discrete summation over a set of well-chosen points i (note that the −RT term comes from the ideal gas approximation): and this integral is calculated using a discrete summation over a set of well-chosen points i (note that the −RT term comes from the ideal gas approximation): © 2023 The Author(s). Published by the Royal Society of Chemistry 2.1 Benchmark Before describing the core components of our surface sampling algorithm, we briey present the other simulation tools used in the article, for comparison and benchmarking purposes. We used the RASPA2 soware for calculations such as Widom insertion and surface area calculations.20 For the Voronoi sampling technique, we used the Zeo++ soware to determine the positions of the Voronoi nodes.21 All the calculations were performed by considering the CoRE MOF 2019 (ref. 22) struc- tures as rigid. In order to speed up the surface sampling, our algorithm exploits the symmetry of the material, looping over all symmetry-inequivalent atoms. Since the space groups (and symmetry operations) of the nanoporous structures in the CoRE MOF database were not specied, we used a Python script based on the Spglib library to determine them.23 The unique atoms are dened according to the symmetry determined by the afore- mentioned Python script. DHads ¼ Ð EintðrÞeEintðrÞ=RTdr Ð eEintðrÞ=RTdr  RTx P i Eiexpð Ei=RTÞ P i expð Ei=RTÞ  RT (1) (1) This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. However, in molecular simulations, a complete sampling of the free volume can be extremely cumbersome. Therefore, random samplings are used to reduce simulation time, for example in the Widom insertion method.19 Still, convergence of the random sampling of space requires a large number of samples, and most of the points may not contribute signi- cantly to the Boltzmann average (i.e., they might have high energy). It is possible to reduce the computational cost further by reducing the number of sampled points, to try and capture only those with the highest contribution to the actual value of enthalpy. But, how can we choose these points a priori, without rst calculating their energy? 3.1 Beyond Widom insertion Widom insertion is a standard calculation method that consists in randomly inserting a single molecule inside an existing molecular system by randomly choosing its center and its rotation angle.19 By measuring the interaction energy of the molecule inserted, one can obtain the excess free energy DFexc difference associated with its insertion into the framework, i.e., the species chemical potential mi. In the context of adsorption, this method has been used to randomly insert a molecule in the empty porous framework: aer many cycles, the simulation has generated a diverse enough sample of points with different interaction energies Eint, yielding the adsorption free energy DFads = −RT ln(hexp(−Eint/RT)i), the Henry constant KH, and the adsorption enthalpy, DHads (eqn (1)), which has the opposite sign of the zero-loading isosteric heat of adsorption Q0 st. The Henry constant18,41,42 KH (in mol kg−1 Pa−1) associated with the adsorption inside a crystalline framework of mass density rf at temperature T can be derived using the following eqn (2): This article is licensed under a Creative Commons Attribution 3.0 Unpo To model the van der Waals interactions, we used Lennard-Jones (LJ) truncated and shied potentials with a 12 Å cut-offwithout tail corrections. The atoms of the structures were modeled using the LJ parameters from the universal force eld UFF.28 For xenon we used the following LJ parameters:29 3Xe = 221.0 K and sXe = 4.100 Å.30 To determine cross interaction parameters between xenon and the host atoms, we used the Lorentz–Berthelot combi- nation rules.31 Throughout the article, all interaction energies are calculated with these same parameters, and while the exact results in terms of adsorption enthalpies depend on the force eld chosen, the goal of this article is the comparison of different methodologies. We want to stress that the sampling method proposed herein could be used with any other force eld. Other analytical forms could be used, or other LJ parameters could be chosen; for example, it is standard to mix Dreiding32 for the organic part and UFF for the inorganic part of the MOF structures. This article is licensed under a Creative Commons Attribution 3 In adsorption simula- tions, these interaction energies of the randomly inserted adsor- bate are typically used to determine values of the Henry constant KH and of adsorption enthalpy DHads at the zero-loading limit. The surface area calculation implemented in RASPA2 follows a very standard “rolling ball” algorithm26 based on hard spheres with sizes determined by the sigma value of the Lennard-Jones potential. First, a probe-molecule samples the spherical surface of the atoms of the framework. A portion of the sphere is excluded, because the probe overlaps with another atom. Each framework atom has an area of adsorbable surface, and the sum of all the areas gives the total adsorbable surface area. The values given by these simulations are then compared to the values given by our algorithm to check the consistency of our implementation. We propose a novel algorithm for rapid adsorption enthalpy characterization, based on the reduction of the sampling space from 3D to 2D. This method is more accurate than the previous Voronoi sampling strategy. Moreover, the dimension reduction coupled with symmetric properties makes it faster than the stan- dard Widom insertion method. This algorithm has been tested on xenon and is directly applicable to any spherical adsorbate model; and it can then be adapted to polyatomic adsorbate molecules as well. This algorithm can be used in the future to speed up the calculation of adsorption properties in regular or ML-assisted high-throughput computational screenings of nanoporous materials. In mathematics, a tessellation of a given space corresponds to a partition into non overlapping sub-spaces. In the Voronoi tessellation, named aer Georgy Feodosevich Voronoy , a set of points (seeds) are associated with a tessellation of regions (Voronoi cells) so that each seed has a cell that is closer to it than any other seeds.27 © 2023 The Author(s). Published by the Royal Society of Chemistry 1798 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science View Article Online Chemical Science View Article Online 2.3 Simulation box To design a versatile simulation tool, we use periodic boundary conditions to create a rectangular simulation box for structures with non-rectangular unit cells. An extended neighbor list is created from the atoms of the translated rectangular boxes within the chosen cutoff. When looping over the unique atoms of the framework in the surface sampling, this neighbor list is restrained to a shorter neighbor list to be used in the interaction energy calculation, like in most molecular simulation algo- rithms.33 To evaluate the effect of the neighbor list, we tested the nal implementation without the implementation of the neighbor list, and the simulation ran for 12.6 s instead of 0.34 s (37 times slower) without altering the accuracy. This shows that the implementation of an efficient neighbor list is a key point in our algorithm. 3 2.2 Force eld p y p g The method we found to be the most efficient (slightly faster) for the typical values of n that we consider (between 100 and 300 000) consists in wrapping a string of points around the sphere in a spiral manner. This technique is closer to laying a uniform grid over the surface than a “random sampling” of the surface, which avoids redundant sampling points. The height h = r cos f (where (r, q, f) are the spherical coordinates) of the sphere is uniformly divided into n points; for each of these heights we chose an angle q in the orthogonal plane space so that the difference between two consecutive angles is the golden number.40 This method is referred to as the spherical Fibonacci mapping. While it is not the optimal solution to the Thomson problem, its uniformity is rather good for our purposes (we have checked that its inuence on the calculated properties is negligible) and the computational cost is lower. It also gives a convergence for values of n smaller than the random distribution methods described above, allowing us to use smaller sampling sizes, which is why we used this method for the rest of the surface sampling simulations presented in this article. © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article Applied in materials science, the Voronoi cells associated with each atom of the framework can be used to determine key geometrical descriptors (void volume, accessible surface area, and pore sizes). At the vertices of each cell, there are Voronoi nodes that were used in the Voronoi energy calculation pre- sented by Simon et al.14 To compare our algorithm to a Voronoi sampling, we used the interaction energy values at the Voronoi nodes to calculate a Boltzmann average. These proxies for the adsorption enthalpy are then indirectly compared to the adsorption enthalpies calculated by our surface sampling. n. To generate random 3D unit vectors, one approach is to draw random vectors in the corresponding cube, rejecting the points that are not inside the sphere. A simple normalization of the remaining vectors gives a random sampling of the sphere surface.36 The same result can also be achieved by generating three normally distributed random values and normalizing the vector obtained by these numbers.36,37 This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Another technique to obtain a uniform distribution is to imagine using a simulation of n charged points on the surface of a sphere and minimize their electrostatic repulsion. This method, based on the Thomson problem,38 relies on numerical optimization and can become very expensive if n is high.39 2.4 Sphere sampling algorithm In our proposed method as in the surface area calculation algorithm,26,34 we rely heavily on the use of a uniform sampling of n points on the surface of a sphere, but this problem can be quite challenging in and of itself. In fact, except for very specic values of n, there is not a general analytical solution to the problem, only numerical approximations. During the develop- ment of our algorithm, we tested several existing methodolo- gies35 to achieve this sampling. KH ¼ hexpð Eint=RTÞi rfRT ¼ 1 rfRT 1 Nsample X i˛sample expð Ei=RTÞ The rst technique is to rely on random sampling, with no guarantee of uniformity, but which should converge for a large (2) Chem. Sci., 2023, 14, 1797–1807 | 1799 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article View Article Online Edge Article View Article Online Chemical Science where i represents the point of a sample used in practice to calculate the integral. approximation of the adsorption enthalpy. However, this sampling assumes that the nodes are close to the real, most favorable, adsorption sites. Or to put it differently, the adsorption sites need to be at the center of the pores, which is only true for structures with pore sizes close to the adsorbate size. If the free volume has been thoroughly explored, the Boltzmann average of the host/guest interaction energies converges to the adsorption enthalpy at innite dilution. The Widom insertion method is very accurate, meaning that it converges to the “perfect” value of the adsorption enthalpy—for a given choice of interaction parameters—in the limit of innite sampling. However, it is computationally expensive, and a lot of computational resources are wasted during this sampling in the calculation of interaction energies that have a negligible contribution to the overall Boltz- mann average (points of high energy). Therefore, we can improve this method if we manage to sample preferentially points with the most negative interaction energies. To achieve this improvement we need to identify the characteristics of the adsorption sites that will have the highest weight (“count the most”) in the nal average, while avoiding sampling parts of space where points will have marginal contribution. To check the accuracy of this sampling technique, we compared it to our reference sampling, the Widom insertion with 100 000 cycles. Fig. This article is licensed under a Creative Commons Attribution 3. The Voronoi sampling and the surface sampling presented in this article are examples of biased sampling methods, that follow this idea. The change in the sampling technique can dramatically improve the computation time required, and even a slight improvement in computational efficiency can have a non- negligible impact when dealing with datasets containing thou- sands of structures. This article focuses on biased sampling tech- niques to speed up adsorption enthalpy calculations. As a proof of concept, we only consider monoatomic adsorbates (in our tests, we used xenon) or adsorbates that can be modeled as a sphere (which is common in molecular simulations of species such as CH4). However, the methodology can be adapted to rigid polyatomic adsorbates, where the algorithm would need to be adapted by sampling the rotational degree of freedom of the adsorbed molecule. The root mean square error (RMSE) and the mean absolute error (MAE) for Voronoi sampling are respectively 6.78 kJ mol−1 and 2.01 kJ mol−1, if we consider all structures in our set, which seem too high to be useful for screening purposes. However, non- porous materials would be screened out a priori in any high- Fig. 1 Scatter-plot of the enthalpies calculated by a Voronoi sampling compared to the enthalpies calculated by a 100k-step Widom inser- tion simulation of xenon in the structures of CoRE MOF 2019. The points are labeled according to the largest cavity diameter (LCD) belonging to one of the intervals. In all the comparisons in this paper, we chose to take a Widom insertion simulation with 100 000 cycles (a very large number) as a ground truth or reference for the adsorption enthalpy values of every structure of the CoRE MOF 2019 database.22 2.4 Sphere sampling algorithm 1 compares the enthalpy computed in the Voronoi sampling with the reference adsorption enthalpy (ground truth)— showing at the same time the largest cavity diameter for each porous framework. The correlation between the values of enthalpy is very good only for a restricted number of structures with enthalpy of around −50 kJ mol−1. For structures with higher enthalpy, the correlation starts to degrade, and becomes very poor for small-pore structures. For the points in purple, the largest cavity diameter is lower than the kinetic diameter of a xenon, where the sampling of the Voronoi nodes is clearly insufficient. In addition, the accuracy loss at the other points (larger pores) can be explained by the fact that the pores are slightly bigger and the center of the pore is not a good approximation of adsorption site position anymore: the adsorption sites are actually closer to the pore surface than to the center of the pore. This conclusion is what prompted us to propose a new sampling scheme based on the molecular surface of the pore space, which we will detail in the next sections. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This is reinforced by the very low computational cost of the method. The Voronoi tessellation performed using the Zeo++ soware is extremely quick and can output the positions of the Voronoi nodes in 0.28 s (measured as an average over all the structures of the CoRE MOF 2019 database), on a typical workstation (a single Intel Xeon Platinum 8168 core at 2.7 GHz). While a simple Python code for the energy calculation took around 27 s per structure, we benchmarked that a C++ opti- mized implementation can perform the Voronoi sampling in around 0.4 s. We only need to remember that this method takes a few hundred milliseconds per structure, while a Widom insertion needs approximately hundreds of seconds per struc- ture. A Voronoi sampling is therefore 2 to 3 orders of magnitude quicker than a full sampling of the pore space. 3.3.1 Initial implementation. We present here our initial implementation of the surface sampling algorithm, and its basic principles. This rst implementation is a relatively basic one and already performs well compared to the other methods. In the next sections, we rene it with two additional features that will improve its accuracy and its speed. This initial implementation speeds up the calculation of adsorption enthalpy in nanoporous materials by sampling interaction energies only near the surface. It is illustrated in Fig. 2. For this purpose, a loop over all unique atoms (as dened by crystalline symmetry) is performed. And for each atom, a sphere around its position is sampled using a uniform distri- bution around it; these points will be called sampling points and we can change the number of sampling points. The default radius chosen for the sampling spheres is the distance rmin = 21/ 6sij to the minimum of the LJ potential between atoms of type i (belonging to the framework) and j (the guest), corresponding to the strongest possible pair interaction (although the neighboring atoms will of course have an inuence). Aer calculating the interaction energy at each of the sampled points, a Boltzmann average of these energies corresponds to a biased adsorption enthalpy, as described by eqn (1). This preliminary study identied a fast method for adsorption enthalpy calculations that can be widely used in screening proce- dures, but has limited accuracy for quantitative prediction. 3.2 Voronoi sampling The use of the Voronoi decomposition of the pore space of mate- rials for their geometric characterization has been widely employed in computational studies in the last decade,21 in particular since it was made easily available as part of the Zeo++ soware package.43 Its use was extended recently to implement a novel sampling scheme, in a study proposing the ML-assisted screening of nanoporous materials for xenon/krypton separation. In this article, Simon et al.14 relied on a Voronoi tessellation of the nanoporous materials and assigned the potential adsorption sites (i.e., the sampling points) at the nodes of this decomposition. The Voronoi tessellation identies the vertices of polygons that corre- spond to the closest regions of each atom of the structure. These vertices (or Voronoi nodes) are the points equidistant to at least four atoms of the structure, and they can be associated with adsorption sites since they are positioned near the center of the pores. It is possible to calculate the host/guest interaction energies at every Voronoi node, and average them to obtain an Fig. 1 Scatter-plot of the enthalpies calculated by a Voronoi sampling compared to the enthalpies calculated by a 100k-step Widom inser- tion simulation of xenon in the structures of CoRE MOF 2019. The points are labeled according to the largest cavity diameter (LCD) belonging to one of the intervals. © 2023 The Author(s). Published by the Royal Society of Chemistry 1800 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science View Article Online Chemical Science View Article Online This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. It raised important questions on the importance of selecting sampling points within the pore space of materials, and we wanted to develop an intermediate technique that is both fast and accurate for the prediction of adsorption enthalpy. For this purpose, we developed and optimized a new sampling technique that focuses the sampling on the surface of the material, which is expected to make up for the main aws of the Voronoi sampling. Edge Article Voronoi sampling and faster than Widom insertion. Our initial idea is based on a series of theoretical considerations: (1) the strong adsorption sites are near the surface of the material; (2) by changing the problem from 3D to 2D sampling we can reduce the complexity; and (3) the algorithm can scale with the number of unique atoms in the structure (and not with the size of the unit cell), which is efficient because many porous frameworks have high symmetry. The rst consideration ensures that this method will be more accurate than a Voronoi sampling, and the last two made us think that a well-optimized code would be fast. To conrm these hypotheses, we will analyze both the accuracy and the speed of this new algorithm and compare them to those of existing methods. throughput workow, as they would not be of interest. We can only consider the structures with large enough cavities, larger than 3.7 Å (a bit lower than 3.96 Å Xe kinetic diameter). Thereby, the RMSE and MAE drop respectively to 2.11 kJ mol−1 and 1.55 kJ mol−1, which can be considered acceptable for a quick estimation of the guest–host affinity, but not for an accurate adsorption enthalpy calculation. Voronoi sampling and faster than Widom insertion. Our initial idea is based on a series of theoretical considerations: (1) the strong adsorption sites are near the surface of the material; (2) by changing the problem from 3D to 2D sampling we can reduce the complexity; and (3) the algorithm can scale with the number of unique atoms in the structure (and not with the size of the unit cell), which is efficient because many porous frameworks have high symmetry. The rst consideration ensures that this method will be more accurate than a Voronoi sampling, and the last two made us think that a well-optimized code would be fast. To conrm these hypotheses, we will analyze both the accuracy and the speed of this new algorithm and compare them to those of existing methods. This article is licensed under a Creative Commons Attribution 3.0 Fig. 3 RMSE convergence of our algorithm (left) compared to a 100k- step Widom insertion simulation (right) for xenon adsorption in all the structures of the CoRE MOF 2019 database. In order to validate the accuracy of the approximation made using this sampling, we applied this algorithm with 300 000 sampling points per unique atom. The results are illustrated in Fig. S1 and S2 and Table S1 of the ESI.† There is a good numerical agreement with the reference calculations; the RMSE and MAE are only around 0.90 kJ mol−1 and 0.66 kJ mol−1 considering all the structures from the database. Moreover, there is no noticeable difference in RMSE when considering the structures with a pore size above 3.7 Å (as determined by the largest cavity diameter, or LCD). Unlike Voronoi sampling, this method gives a consistent accuracy across all the structures of the database with a lower error. The fact that the RMSE error is below 1 kJ mol−1 is quite promising, and validates our intuition that this new sampling technique can be an intermediate between to the two previous methods (Voronoi and Widom). Because we have no physical model that would predict the optimal value of the sampling sphere, we followed a statistical approach. We studied the inuence of the l parameter on both the accuracy and the computation time, and the results are presented in Fig. 4. The RMSE turns out to be relatively high at around 0.90 kJ mol−1 for a radius of the sphere lower than the rmin, and it then decreases for larger values of radius to reach a plateau at around 0.35 kJ mol−1. We conrm that by increasing the sampling sphere radius we can improve the accuracy of our algorithm, and nd that for values of l higher than 1.6, the accuracy is stabilized. We also nd that increasing the sphere radius negatively impacts the computational effi- ciency, since it increases the number of neighbors considered in the energy calculation. Aer proving the good accuracy of the method, we are now exploring the computation time required. We see in Fig. 3 that the method reaches an RMSE below 1.0 kJ mol−1 very quickly for an average CPU time of 1.2 s (Table S1†), corresponding to 2000 sampling points per atom. © 2023 The Author(s). Published by the Royal Society of Chemistry 3.3 Construction of a surface sampling algorithm In this section we describe the development of our surface sampling algorithm, with the goal of being more accurate than Fig. 2 Schematic description of our surface sampling based on the three main steps of the algorithm: the loop over the unique atoms, the spiral sampling around each atom, and the energy averaging. The adsorbate is represented by the point i and is moved across all the points around the unique atoms of the structure. Fig. 2 Schematic description of our surface sampling based on the three main steps of the algorithm: the loop over the unique atoms, the spiral sampling around each atom, and the energy averaging. The adsorbate is represented by the point i and is moved across all the points around the unique atoms of the structure. Chem. Sci., 2023, 14, 1797–1807 | 1801 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article View Article Online Edge Article View Article Online Fig. 3 RMSE convergence of our algorithm (left) compared to a 100k- step Widom insertion simulation (right) for xenon adsorption in all the structures of the CoRE MOF 2019 database. Chemical Science Chemical Science Chemical Science and adopting a fast rejection criterion. The rejection of high- energy points with little contribution to the nal enthalpy value can reduce the simulation time, whereas the size of the sampling sphere can improve the accuracy. The initially chosen sphere size only takes into account the interaction with the closest atom; we therefore chose to set it at the minimum of Lennard-Jones potential. However, the interaction with the neighboring atoms can further stabilize the adsorbate, so sampling further from this minimum could in consequence increase the accuracy of our surface sampling method. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. 3.3.2 Size of the sampling sphere. The validity of the initial algorithm is based on the assumption that the adsorption site is at the minimum of the Lennard-Jones potential. It will only perform well if the closest atom contributes to almost all the interactions, but in real frameworks other neighboring atoms contribute to the host/guest interaction as well. We have found that in the vast majority of materials, the adsorption sites are located farther apart compared to the LJ potential minimum, in order to maximize the contribution of all atoms—and because of the dissymmetry of the interaction potential well. In order to see if this could be introduced in our algorithm, we imple- mented a parameter l, and the sampling sphere radius is now dened using Rl = ls, where s is the distance at which the LJ potential is zero. If l = 21/6, we fall back to our initial denition of the sampling sphere, and the adsorbent is at the minimum of the LJ potential of the atom. If l = 1, the sampling sphere is at the zero of the LJ potential, and by increasing this parameter, we can check if our intuition was right. This article is licensed under a Creative Commons Attribution 3.0 can optimize the parameter on a small diverse sample of the unseen data. strong and points with non negligible contribution would end up being rejected. This rejection condition is schematically represented in Fig. 5. 3.3.3 Rejection conditions. As shown above, our algorithm has better accuracy than Voronoi sampling, but its initial implementation was several times slower, which could make it unsuitable for screening applications in high-throughput workows, where the number of structures to be screened can reach one million or more. To reduce the computational expense, we thought of rejecting the points with little contri- bution to the nal enthalpy, i.e., the largely positive interaction energies that would vanish in the exponential of the Boltzmann average. This rejection condition is expected to speed up the calcula- tions, since the energy calculation is avoided for the rejected sampling points. The energy calculation accounts for the largest portion of the CPU time spent on the surface sampling. For the structure KAXQIL,44 the Lennard-Jones potential calculation represents up to 90% of the calculation time for 100 000 sampling points per sphere (with the initial algorithm). The higher the factor m, the more rejections there would be. But, if too many points are rejected, the accuracy will drop. Here again, we used a statistical analysis to determine the optimal value of m, making our sampling faster without compromising the accuracy of the enthalpy calcu- lation. The results are displayed in Fig. 6. Inspired by typical methods for accessible surface calcula- tions, we implemented a hard sphere rejection condition based on the distance to neighbors. If the adsorbate is too close to another atom of the structure, the sampling point is rejected, i.e., its energy is not calculated (or considered to be innite). We based this distance threshold on the sij parameter of the Lennard-Jones potential. To determine the optimal threshold, we introduced a factor m with real values between 0 and 1 that changes the size of the hard sphere rejection condition. If the guest–host distance is lower than dm = m × s, then the point is rejected. If m = 0, then there is no rejection condition. And if m = 1, we reject all points with a positive energy interaction with at least one atom of the structure. This condition could be a bit The values of RMSE and time in Fig. This article is licensed under a Creative Commons Attribution 3.0 This is far less than the 150 s (Table S2†) required for a Widom insertion to reach its plateau value, for an RMSE of 0.10 kJ mol−1 with 12 000 cycles. Moreover, the Widom insertion needs around 14 s to reach a similar RMSE of 1.0 kJ mol−1, which is still slower than the surface sampling. We can conclude that this initial implementation of the surface sampling is faster than a standard Widom insertion, with a good accuracy. By choosing an optimal sampling sphere, we can more than halve the error, while increasing the computation time by around 20 percent, when comparing the case l = 1.6 with l = 1.1 (close to rmin). In most cases, it will be an acceptable trade- off. However, in a case where the computation time is crucial, like in a rapid screening, the optimal choice might not be to increase the sampling sphere at l = 1.6 but to have it lower at l = 1.4 or l = 1.2, and have an RMSE at around 0.5 kJ mol−1—still quite acceptable. The new scale parameter introduced in this section can therefore be tweaked to serve the users' purpose, whether it is to focus on the accuracy or to optimize the computation speed. If one wants to use it on a completely different database under very different conditions, then one can either choose a default value that works ne (e.g. l = 1.4) or one However, this initial implementation of the method is slower than a Voronoi sampling that only needs to sample around 1600 points on average, instead of 13 000 sampled points on average (if we multiply by the average number of unique atoms). The sampling part would take approximately 0.15 s, and the Voronoi node generation 0.28 s, so our surface sampling algorithm remains 2 to 3 times slower (implemented in an identically compiled language, in this case C++). In order to improve the accuracy and performance, we have further tweaked the surface sampling method, adjusting the size of the sampling sphere © 2023 The Author(s). Published by the Royal Society of Chemistry 1802 | Chem. Sci., 2023, 14, 1797–1807 Fig. 6 Influence of the rejection coefficient m on the average CPU time required for a simulation of 100k sampling points and the RMSE compared to the reference adsorption enthalpy. © 2023 The Author(s). Published by the Royal Society of Chemistry This article is licensed under a Creative Commons Attribution 3.0 The averaging is performed only on the structures with a largest cavity diameter (LCD) greater than 3.7 Å. Chemical Science View Article Online View Article Online Fig. 4 Influence of the sampling sphere radius Rl on the average CPU time required for a simulation of 100k sampling points and the RMSE, compared to the reference adsorption enthalpy. The averaging is performed only on the structures with a largest cavity diameter (LCD) higher than 3.7 Å. Fig. 6 Influence of the rejection coefficient m on the average CPU time required for a simulation of 100k sampling points and the RMSE compared to the reference adsorption enthalpy. The averaging is performed only on the structures with a largest cavity diameter (LCD) greater than 3.7 Å. Edge Article Chemical Science Edge Article This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Fig. 4 Influence of the sampling sphere radius Rl on the average CPU time required for a simulation of 100k sampling points and the RMSE, compared to the reference adsorption enthalpy. The averaging is performed only on the structures with a largest cavity diameter (LCD) higher than 3.7 Å. Fig. 6 Influence of the rejection coefficient m on the average CPU time required for a simulation of 100k sampling points and the RMSE compared to the reference adsorption enthalpy. The averaging is performed only on the structures with a largest cavity diameter (LCD) greater than 3.7 Å. This article is licensed under a Creative Commons Attribution 3.0 On very large databases, one could consider that this increase in the required computational time is not worth the accuracy improvement, and one could decide to keep a smaller sampling sphere. If this method is transposed to different molecular systems, its parameters should be tested on the specic database and adsorbate of interest. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. This article is licensed under a Creative Commons Attribution 3.0 Unported Fig. 7 Comparison of the RMSE to the reference Widom insertion (100k cycles) and the average computation time for different types of enthalpy calculation methods. The surface sampling calculations were all performed with 2k sampling points on each sphere and the Widom simulations were performed using 12k cycles. These values corre- spond to the value at the convergence identified using Fig. 3. 3.4.2 Calculation of the Henry constant and surface area. The main goal of our sampling algorithm is to calculate adsorption enthalpy at the zero-loading limit. But the method can also calculate the Henry constant and surface area of the materials at the same time, without signicant additional computational cost. The Henry constant is a key metric for assessing the affinity of an adsorbate to a nanoporous structure. The A/B gas selectivity at low pressure is dened as a ratio of the Henry constants of components A and B. This important property can be calculated using eqn (2) in a Widom insertion calculation. Instead of using the interaction energies at the Widom inserted points, we can now use the surface sampled points to get an approximate value for the Henry constant. This article is licensed under a Creat higher values of m that trade a bit more accuracy in exchange of further speed gains. For the simulations considered in Fig. 6, the use of a rejec- tion condition m = 0.85 makes the simulation four times faster than the standard algorithm. As we will see in the next section, the combination of optimal values for the l and m parameters generates an algorithm with very interesting performance compared to Voronoi sampling or Widom insertion. Using the optimized set of parameters for surface sampling, we assessed the performance of our algorithm on the values of the Henry constant by comparing them to ground truth ob- tained by 100 000 cycles of Widom insertion. This article is licensed under a Creative Commons Attribution 3.0 6 are averaged only on the most interesting structures for xenon adsorption (LCD $ 3.7 Å). For m # 0.85, increasing the value of m improves the speed of the calculation without changing the RMSE.‡ For high values of m, the rejection condition is too strong and we reject points with non-negligible contribution to the overall enthalpy. The RMSE increases as a consequence. If we want to keep the accuracy unchanged, the optimal value is therefore m x 0.85, because it gives the lowest computation time with a similar RMSE. We note that it would be possible, in specic cases, to explore Fig. 5 Simplified 2D representation of the principle of rejection conditions and the concept of sampling spheres inside the one-dimensional channels of a nanoporous material. Fig. 5 Simplified 2D representation of the principle of rejection conditions and the concept of sampling spheres inside the one-dimensional channels of a nanoporous material. Chem. Sci., 2023, 14, 1797–1807 | 1803 © 2023 The Author(s). Published by the Royal Society of Chemistry Edge Article View Article Online Edge Article View Article Online Chemical Science Fig. 7 Comparison of the RMSE to the reference Widom insertion (100k cycles) and the average computation time for different types of enthalpy calculation methods. The surface sampling calculations were all performed with 2k sampling points on each sphere and the Widom simulations were performed using 12k cycles. These values corre- spond to the value at the convergence identified using Fig. 3. accurate and 26% faster than Voronoi sampling, and it is also about 430 times faster than a Widom insertion with 12k cycles. Finally, we suggest that the values of the parameters optimized in this work might need adjustment when applied to other adsorption systems. The optimal m parameter depends on the size of the adsorbent, and it should be tweaked differently when considering another adsorbent. For instance, the set of structures used for the optimization of m depends on the size of their cavities, and the 3.7 Å threshold chosen here would need to be changed according to the kinetic diameter of the adsorbate. Furthermore, as aforementioned in the section on rejection conditions, it is possible to trade-offa bit of accuracy for faster simulations espe- cially in high-throughput screenings where speed is extremely important. Similarly, in the case of xenon, the cost of increasing the sphere size is around 10 to 20%. This article is licensed under a Creative Commons Attribution 3.0 Since the Henry constant corresponds to the exponential of an adsorption free energy and we are more interested in the precision of the free energy, we are using a log-scale evaluation metric. For surface sampling, the log-RMSE of KH is equal to 0.2, which means that the order of magnitude of the values is well predicted (Table S4†). If we consider the derived free energy DFads = −RT log(rfRTKH), the RMSE is of the order of 1.1 kJ mol−1 reached in about 1 s (Table S6†), whereas for Widom insertion, this level of error is also reached in a similar amount of time and 0.1 kJ mol−1 of RMSE is reached in about 86 s (Table S7†). For free energy calculations, surface sampling is still 86 times faster to converge. If we consider that the main target is the adsorption enthalpy, the Henry constant can be calculated with little additional computational cost and with reasonable accuracy: we get two thermodynamic properties of interest for the price of one. © 2023 The Author(s). Published by the Royal Society of Chemistry 1804 | Chem. Sci., 2023, 14, 1797–1807 This article is licensed under a Creative Commons Attribution 3.0 Unport p y Finally, although the algorithm in its present form can already be applied in a wide range of applications, additional develop- ment work could allow us to generalize it to polyatomic adsor- bates. For instance, we would need to work on a denition of the molecular radius for nonspherical adsorbates as well as all the orientation conformations of the adsorbent. We could imagine making the distance to the surface depend on the orientation of the adsorbate or sample a band volume on the surface. Although the best implementation of surface sampling for polyatomic adsorbates remains an open question, in theory it should be possible to apply it to more complex adsorbates than spherical noble gas. This would add more complexity to the algorithm but would not change the fundamental speed up due to surface sampling, since these orientation moves are also performed in other standard methodologies. To improve the accuracy even more, we could test hybrid samplings with multiple sampling spheres, or a combination of Voronoi nodes and sampling spheres. Another idea could be to have fractions of spheres that are oriented toward the center of pores given by the Voronoi node. In theory, having a wider variety of sampling points can only improve the sampling. There are therefore multiple possible sampling techniques that could be built around the method introduced herein. The code is made freely available on the group's GitHub, where further development will be released. 4 Conclusions and perspectives In the present article, we described a novel algorithm for the high-speed calculation of adsorption enthalpy in nanoporous materials that takes a unique approach to reduce the sampling necessary. This new algorithm is based on the core principle of dimensional reduction, from a volume problem to a surface one. The algorithm is proven to be signicantly faster than the reference Widom insertion (random sampling of porous space). Moreover, the error associated is found to be in the order of 0.4 kJ mol−1, tested throughout the entire CoRE MOF 2019 data- base, for xenon adsorption. Even when compared to existing very fast sampling techniques such as Voronoi sampling, this surface sampling technique requires similar CPU time, combined with a better accuracy. Based on these results, this algorithm has important potential for applications in the current computational analysis workows of material databases, such as high-throughput screening studies. For instance, this algorithm can be used to get a fast approximation of the low-loading adsorption enthalpy of a molecule inside nanoporous materials. This cheap evalu- ation of enthalpy can be used to screen out the structures with little affinity with the targeted adsorbate molecule. It can also be used as a thermodynamic descriptor for selectivity prediction in a machine learning model, as performed by Simon et al.14 The computational speed-up brought about by this novel method- ology can also enable the screening of materials databases at a larger scale in the future. Data availability Data and code related to this study are available from our group repository at https://github.com/fxcoudert/citable-data. The RAESS code is available at https://github.com/coudertlab/RAESS. 3.4 Final surface sampling algorithm 3.4.1 Performance comparison. For the calculation of adsorption enthalpy, our proposed surface sampling method is a good compromise between the accuracy of Widom insertion (full sampling of the porous space) and the speed of a less accurate method such as Voronoi sampling. The performance of our algo- rithm, including the two new features (sampling sphere scaling and rejection criterion) is illustrated in Fig. 7, where we can see the improvement brought about by each feature and how it compares to reference simulations. All CPU times are calculated using the smallest possible number of sampling points so that the respective algorithms reach convergence. With the implementation of a rejection condition, we nd that surface sampling is even quicker than Voronoi sampling. Moreover, the increase in the size of the sampling sphere makes the surface sampling much more accu- rate, reaching an RMSE of 0.33 kJ mol−1 and an MAE of 0.21 kJ mol−1. The ideal set of parameters, determined for porous mate- rials from the CoRE MOF 2019 database, is (l = 1.6, m = 0.85) in order to combine the lowest error and smallest computational cost. By combining both of these new features into the algorithm, we have a nal surface sampling method with an RMSE of 0.33 kJ mol−1 and an average computation time of 0.34 s per structure. According to the data in Table S3,† it is about 6 times more The same goes for the determination of the surface area. We can adapt our algorithm to count the number of points of the sampling spheres that have a negative energy. These represent the points where a guest molecule can favorably interact; © 2023 The Author(s). Published by the Royal Society of Chemistry 1804 | Chem. Sci., 2023, 14, 1797–1807 Chemical Science View Article Online Chemical Science View Article Online Chemical Science View Article Online This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. where V is the volume of the cell; a is the number of atoms of the cell; Naccessible(a) is the number of accessible points around the atom a; Ntotal is the total number of sampling points; r(a) is the radius of the sampling sphere around the atom a. When we set l = 1, we are sampling spheres that have a radius of s and it is equivalent to considering hard spheres all dened using s (convention used by RASPA2 to calculate surface areas). If we compare simulation with l = 1, we obtain surface areas that are very close to the one obtained by RASPA2 (see Fig. S11 in the ESI†). However, when we consider l = 1.6, we lose the accor- dance previously obtained and the points are weakly correlated at the log-scale (see Fig. S10 in the ESI†). The difference can be explained by the fact that the sphere size is larger, but the proportion of adsorbable points also changes. The relationship between these two adsorption surface areas is not trivial at all. Since the calculation of surface areas is quite cheap, this implementation would not be very useful, except for having a rough idea of the surface area. where V is the volume of the cell; a is the number of atoms of the cell; Naccessible(a) is the number of accessible points around the atom a; Ntotal is the total number of sampling points; r(a) is the radius of the sampling sphere around the atom a. When we set l = 1, we are sampling spheres that have a radius of s and it is equivalent to considering hard spheres all dened using s (convention used by RASPA2 to calculate surface areas). If we compare simulation with l = 1, we obtain surface areas that are very close to the one obtained by RASPA2 (see Fig. S11 in the ESI†). However, when we consider l = 1.6, we lose the accor- dance previously obtained and the points are weakly correlated at the log-scale (see Fig. S10 in the ESI†). The difference can be explained by the fact that the sphere size is larger, but the proportion of adsorbable points also changes. The relationship between these two adsorption surface areas is not trivial at all. © 2023 The Author(s). Published by the Royal Society of Chemistry This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Since the calculation of surface areas is quite cheap, this implementation would not be very useful, except for having a rough idea of the surface area. Edge Article therefore when dividing it by the number of sampled points, we obtain a proportion of the adsorbable area of the sphere. Summing this over all atoms, we obtain the total surface area. This implementation is summed up in eqn (3): a simple Lennard-Jones interaction potential, this sampling technique could equally be used to speed up samplings of space based on more expensive modeling strategies, including polar- izable force elds or density functional theory (DFT) calculations. In the literature, the need for cheap ab initio grade thermody- namic properties is usually fullled by using an importance sampling method based on a classical force eld.45 In our method, the description of surface sampling is independent of any force eld, and the sampling spheres can be dened according to kinetic radius, van der Waals radius or any other physically relevant distance. Consequently, given a denition of atomic radii, it is possible to dene a surface on which to carry out other types of simulations such as neural network potential, DFT or any other force elds. Although the accuracy or relevance of such a sampling remains an open question, the approach will undeniably speed up the simulations. This could even be applied to calculate adsorption enthalpies while considering intrinsic structure exibility,46 a task whose main drawback is the high computation time required. Since surface sampling is hundreds of time faster than standard methodologies, we could use hundreds of snapshots in a exibility-aware calculation. SA ¼ 1 V X a˛cell NaccessibleðaÞ Ntotal 4prðaÞ2 (3) (3) Notes and references 18 S. Kar and C. Chakravarty, Computational evaluation of Henry's constants and isosteric heats of sorption for Lennard-Jones sorbates in Na-Y zeolite, Mol. Phys., 2001, 99, 1517–1521. ‡ In fact, what we observe is a deterioration of the accuracy for structures with small pores because the probability of rejection in a conned space is really high and all sampled points end up rejected. But these points are not considered, if we apply the condition on the cavity size (LCD $ 3.7 Å). This article is licensed under a Creative Commons Attribution 3.0 19 B. Widom, Some Topics in the Theory of Fluids, J. Chem. Phys., 1963, 39, 2808–2812. 1 D. S. Sholl and R. P. Lively, Seven chemical separations to change the world, Nature, 2016, 532, 435–437. 20 D. Dubbeldam, S. Calero, D. E. Ellis and R. Q. Snurr, RASPA: molecular simulation soware for adsorption and diffusion in exible nanoporous materials, Mol. Simul., 2016, 42, 81–101. 2 National Academies of Sciences, Engineering, and Medicine, A Research Agenda for Transforming Separation Science, National Academies Press, 2019. 21 T. F. Willems, C. H. Rycro, M. Kazi, J. C. Meza and M. Haranczyk, Algorithms and tools for high-throughput geometry-based analysis of crystalline porous materials, Microporous Mesoporous Mater., 2012, 149, 134–141. 3 R. Morris and P. Wheatley, Gas Storage in Nanoporous Materials, Angew. Chem., Int. 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Author contributions Both authors designed the study, analysed the results, wrote and revised the article. E. R. wrote the RAESS soware and ran the molecular simulations. We note, moreover, that the speed of our method resides in the sampling technique itself, rather than in the actual energy calculation. While we have benchmarked it in this work for © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci., 2023, 14, 1797–1807 | 1805 Edge Article View Article Online View Article Online Chemical Science a Xenon/Krypton Mixture?, Chem. Mater., 2015, 27, 4459– 4475. a Xenon/Krypton Mixture?, Chem. Mater., 2015, 27, 4459– 4475. Notes and references Snurr, Topologically Guided, Automated Construction of Metal– Organic Frameworks and Their Evaluation for Energy- Related Applications, Cryst. Growth Des., 2017, 17, 5801– 5810. 8 E. Ren and F.-X. 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English
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Challenges to the Israeli healthcare system: attracting medical students to primary care and to the periphery
Israel journal of health policy research
2,018
cc-by
11,047
Abstract Background: The greatest challenges facing healthcare systems include ensuring a sufficient supply of primary care physicians and physicians willing to work in rural or peripheral areas. Especially challenging is enticing young physicians to practice primary care in rural/peripheral areas. Identifying medical students interested in primary care and in residencies in Israel’s periphery should aid the healthcare leadership. It may be particularly important to do so during the clinical years, as this is the stage at which many future physicians begin to crystallize their specialty and location preferences. Methods: Questionnaires, distributed to 6 consecutive 5th-year classes of the Hebrew University – Hadassah School of Medicine, from 2010 to 2016, elicited information on criteria for choosing a career specialty, criteria for choosing a residency program and whether one-time monetary grants authorized in the 2011 physicians’ union contract would attract students to residencies in the periphery. Results: Completed questionnaires were returned by 511 of 740 (69%) students. Ninety-eight (19%) were interested in a primary care residency, 184 (36%) were unsure and 229 (45%) were not interested. Students interested in primary care were significantly less interested in specialties that perform procedures/surgeries and in joining a medical school faculty, while being more inclined towards specialties dealing with social problems, controllable lifestyles and working limited hours. The percentage of students interested in primary care was stable during the study period. Forty-eight of the students indicated interest in residencies in the country’s periphery, and 42% of them were also interested in primary care residencies. Overall, only 3.7% of students were interested in both a primary care residency and a residency in the periphery. Thirty percent of the students indicated that the monetary incentives tempted them to consider a residency in the periphery. Fifty-three percent of these students reported that they did not yet know the geographic area where they wished to do their residency, as compared to only 22% among those not interested in incentives. (Continued on next page) * Correspondence: charles@hadassah.org.il Correspondence: charles@hadassah.org.il 1Department of Anesthesiology and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: charles@hadassah.org.il 1Department of Anesthesiology and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel Full list of author information is available at the end of the article Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 https://doi.org/10.1186/s13584-018-0218-z Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 https://doi.org/10.1186/s13584-018-0218-z Open Access Challenges to the Israeli healthcare system: attracting medical students to primary care and to the periphery es Weissman1*, Rachel Yaffa Zisk-Rony2, Alexander Avidan1, Uriel Elchalal3 and Howard Tandete Background specialists and pediatricians, especially in the county’s peripheral areas. This shortage is predicted to increase as the population grows, ages, and life spans lengthen. Furthermore, the primary care physician population is aging as many physicians who emigrated from the former Soviet Union in the 1990’s are reach- ing retirement age [8]. Therefore, it is necessary to at- tract more medical students to primary care residencies. To increase recruitment it is important that the medical education and healthcare system leadership better understand the characteristics of stu- dents interested in pursuing primary care and how they differ from those without such interests. The leadership needs similar information on students in- terested in residency positions in the county’s periphery. The greatest challenges facing healthcare systems in- clude providing sufficient numbers of primary care and rural physicians [1]. Especially challenging is en- ticing young physicians to establish primary care practices in rural areas [1, 2]. Among the counties where such challenges exist are the United States, Australia and Canada [1, 3, 4]. Israel suffers from a similar problem with shortages of family medicine specialists in peripheral areas of the country. The lat- ter include the northern and southern regions of the country which encompass the majority of the county’s rural districts. These areas chronically suffer from physician shortages, greater infant mortality and lower life expectancy than the rest of the country [5]. In an attempt to remedy this maldistribution, the physicians’ union contract of 2011 included both pay increases for practicing in the periphery and one- time monetary incentives for moving and committing to work in the periphery [6, 7]. This programs also provided incentives for medical students interested in residencies in specialties suffering workforce short- ages. The original program excluded family medicine residents from both the incentives for work in the periphery and the incentives to work in distressed specialties. However, in 2015 the Israel Ministry of Health began to provide financial incentives to family medicine residents willing to train in peripheral areas. The present study compared the characteristics of Israeli 5th-year (out of 6 years) medical students interested in primary care residencies and residencies in peripheral areas with those without such interests. The dataset used was collected over a 6-year period from a single Israeli medical school and includes over 500 students. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 (Continued from previous page) Conclusions: This study provides the healthcare leadership with information on the characteristics of the students at a centrally-located medical school who tend to be more interested in primary care and in working in the periphery. Specifically, the study found that students interested in primary care desire a positive life/work balance, something that Israeli non-hospital primary care practice provides. Students considering residencies in the periphery were similarly inclined. Moreover, about a third of students had positive thoughts about monetary incentives for residencies in peripheral hospitals. These students should be identified early during their clinical experience so that attempts to recruit them to the periphery can commence before their specialty and location preferences have fully crystallized. Parallel studies should be performed at additional Israeli medical schools. Keywords: Medical students, Medical education, Residency, Medical specialty selection, Career choice Data analysis Data were entered into Microsoft Excel (Redmond, WA) spreadsheets and analyses were performed with Systat 12 (San Jose CA). Background The two hypotheses tested were that the student interest in primary care would be greater among women medical students and that interest in residencies in peripheral areas would be greater among students who attended high school in the northern and southern regions. We also explored whether the one-time monetary grants approved in the 2011 physicians’ union contract would encourage students to consider a residency in a peripheral hospital. The ultimate goal was to provide the medical education and healthcare leaderships with the attributes that typify students interested in primary care and/or rural practice. Fifth-year students were studied since our previous study revealed that most had already begun the process of deciding on a specialty [9]. In order for the healthcare leadership to influence specialty decisions, it is important to be cognizant of the thought patterns of the students early in their decision process. The Israeli healthcare system is dependent on primary care physicians to provide comprehensive out-patient care and to serve along with community spe- cialists and internal Health Maintenance Organization regulations and pre-authorization systems as gate- keepers for secondary and tertiary care. These primary care physicians, family medicine specialists, pediatricians, internists and general practitioners, are based in health maintenance organization clinics. Despite family medicine and pediatrics being popular specialties among Israeli medical students, there is still a nationwide shortage of family medicine Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 3 of 17 Page 3 of 17 Incentives The data set was divided into three groups as per the responses to the multiple- choice query “As the result of the union contract of 2011, residents in peripheral hospitals receive a one-time monetary incentive and higher salaries: (1) These incentives attract me to a residency in the periphery (2) I already plan to do a residency in the periphery (3) The incentives do not attract me to a residency in the periphery”. The differences between the characteristics of the three groups were determined. After two small (15 students) preliminary studies designed to identify problems and test the question- naire’s user-friendliness, the questionnaires were distributed to the 5th year classes of the Hebrew University – Hadassah School of Medicine in Jerusalem during the 2010–2011, 2011–2012, 2012– 2013, 2013–2014, 2014–2015 and 2015–2016 school- years. A parallel article examining medical student subgroups also utilized this dataset [10]. Based on prior research demonstrating significant gender differences associated with specialty selection, an a priori decision was made to separately analyze and compare the male and female data [11]. Responses to multiple choice questions are pre- sented as frequency distributions. When the Likert Scale results were considered continuous variables, statistical analyses were performed using all 5 points. When presented as categorical variables the Likert Scale results were compressed into three categories, (the two points representing negative tendencies and the two points representing positive tendencies were each combined). The percentages of total responses for each of the three categories (positive tendency, middle point and negative tendency) were then computed. Rural (periphery) workforce p p y Initial data analysis showed that a significant number of students interested in primary care were inter- ested in a residency in the county’s periphery. There- fore, a post-hoc examination was made of the characteristics of students interested in a residency in the country’s periphery. The dataset was divided into two groups based on the answer to the 5-point Likert scale question: “Are you interested in a resi- dency in the country’s periphery?” Group A included the two points representing positive tendencies while Group B included the two points representing nega- tive tendencies. Methods included the two points representing positive tendencies; Group 2 included the neutral point; and Group 3 the two points representing negative tendencies. This permitted us to compare students interested and not interested in a primary care resi- dency, while also examining those who were unsure. The results from each of the 6 school-years were compared to determine whether there were differ- ences between years. This study included students from 6 consecutive 5th-year classes of the Hebrew University – Hadassah School of Medicine in Jerusalem (2010–2016). It utilized a questionnaire to examine various aspects of the medical specialty selection process. The ques- tionnaire was based on the results of factor analysis from a questionnaire used previously [9]. This per- mitted us to reduce repeat Likert scale questions thus providing space for new ones that investigated additional issues. Among the new topics examined were the influence of family and colleagues on specialty and residency program decisions as well as the interests of the students in academic pursuits. The questionnaire included multiple choice questions, free-text queries and 5-point Likert scales. In addition to demographic information, the questionnaire elicited information about: (1) Whether the student had already consid- ered a specialty for their residency, which specialty or specialties they were considering (free-text), when they had first considered a specialty and whether and when (prior to beginning medical school or when during the first 5 years of medical school) they had changed their mind; (2) The criteria for choosing a career specialty {20 items, 10 new, 5-point Likert scale}; (3) The criteria for choosing a residency pro- gram {20 items, 9 new, 5-point Likert scale}; and (4) The importance of interest in a specific specialty when choosing a residency {3 new items, multiple choice}. (5) Whether the one-time monetary grants authorized in the 2011 physicians’ union contract would attract them to a residency in a peripheral hospital (1 new item, final 4 classes). Rural (periphery) workforce y Differences between students expressing and not interested in a residency in the country’s periphery are found in Table 4 (Appendix B). Results of multivariate and logistic regression analyses are in Table 3. Criteria for specialty and residency program selection were subjected to factor analysis (principal components analysis) using varimax rotation with set eigenvalues of ≥1.0. The data were also analyzed with hierarchal cluster analysis. Interactions between the replies to the questions “Are you interested in a residency in the country’s periphery?” and “Are you interested in a primary care residency?” revealed that 4% of all the students were interested in both a primary care residency and a residency in the periphery (Appendix C). The Institutional Review Board of the Hadassah Medical Organization approved this study. Completion of the questionnaire by the student was considered tacit consent. Discussion The present study identified several medical student characteristics associated with interest in a primary care residency and those interested in a residency in the per- iphery among 5th year students at the Hadassah-Hebrew University Medical School. Primary care h Ninety-eight (19%) students were interested in pursuing a primary care residency, 184 (36%) were unsure and 229 (45%) were not interested. Demo- graphic information is found in Table 1. Interest in primary care among 5th-year students was stable over the 6-year study period ranging from 17%–21% annually. Compared to students not interested in primary care, those interested in primary care were signifi- cantly less interested in a specialty with procedures/ surgeries and becoming “members of a medical school faculty”, while being more inclined towards a specialty dealing with social problems (Tables 2 and 3). When choosing a residency program, students attracted to primary care were more interested than their colleagues in a residency in the country’s periphery. Alternately, 42% of the 48 students who indicated interest in a residency program in the country’s periphery were also interested in a primary care residency (Table 2). When asked about specialties they were considering, students interested in primary care expressed significantly more interest in family medicine and pediatrics than those not interested in Incentives Completed questionnaires were returned by 511 of 740 (69%) 5th-year medical students. Responses to the query about monetary incentives for a residency in peripheral hospitals are in Tables 5 (Appendix D). Thirty percent of the students reported that the incentives interested them, while another 6% had already decided to do a residency in the periphery. The relationships between the responses to this question and those to the query “are you interested in a residency in the country’s periphery?” revealed that 82% of the students who replied they were not attracted by the incentives indicated that they were not interested in a residency in the periphery while those that reported that the incentives interested them showed less aversion (20% negative tendency and 54% positive tendency) to a residency in the periphery. Primary care The dataset was divided into three groups based on the answer to the 5-point Likert scale question: “Are you interested in a primary care residency?” Group 1 Page 4 of 17 Page 4 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 primary care. Among the latter, none were consider- ing family medicine (Table 1). For continuous data, differences between the groups were analyzed using analysis of variance with Tukey post-hoc tests. Categorical data were analyzed using χ2 or Fisher exact tests, as appropriate. A p value < 0. 05 was assumed to represent statistical significance. Univariate linear regression analysis was used to examine the association between the answers to two queries. Backward multivariate and logistic regression analyses were performed with the dependent variable being either interest in a residency in primary care or a peripheral hospital. The independent variables were the demographic parameters and specialty and resident selection criteria. As can be seen in Tables 1 and 2 (Appendix A), there was similar interest in a primary care residency between female (17%) and male (21%) students. Com- parisons between men and women students interested in primary care showed that women rated lifestyle is- sues, such as family time, more highly than men and were more interested in pediatrics (Tables 1 and 2, Appendix A). Primary care There were many differences between 5th-year Israeli medical students interested and not interested in a Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 5 of 17 Table 1 Primary care - demographic and other information Primary care Undecided No primary care Primary care vs no primary care Primary care Females vs males Females Males N 98 184 229 41 57 Female 42% 51% 49% Male 58% 50% 51% NS Age (years) 18–20 0% 0% 0.4% 0% 0% 21–23 25% 14% 17% 27% 23% 24–26 19% 31% 29% 34% 9% 27–29 37% 42% 39% 27% 44% 30–32 14% 10% 12% 10% 18% + 32 5% 4% 3% NS 2% 7% p < 0.01 Marital status Single 70% 63% 73% 66% 72% Married 30% 36% 26. % 34% 26% Divorced 1% 1% 0.4% NS 0% 2% NS Thought of a specialty when started Yes 85% 79% 82% 85% 84% No 15% 21% 18% NS 15% 16% NS When did you start thinking of a specialty? Pre - med school 25% 29% 26% 27% 23% Year 1 4% 4% 4% 6% 2% Year 2 4% 4% 2% 3% 4% Year 3 4% 3% 5% 0% 6% Year 4 56% 42% 48% 62% 51% Year 5 9% 19% 16% NS 3% 13% NS Have you changed your mind? Yes 61% 59% 63% 59% 62% No 40% 41% 38% NS 41% 38% NS When did you change your mind? Future residency location Primary care Year 1 0% 0% 2% 0% 0% Year 2 0% 3% 1% 0% 0% Year 3 2% 1% 2% 0% 4% Year 4 41% 44% 42% 35% 46% Year 5 57% 52% 54% NS 65% 50% NS Specialties under consideration Family medicine 16% 2% 0% 14% 17% Pediatrics 41% 42% 24% 54% 30% Internal med 26% 30% 32% 26% 26% Ob/Gyn 19% 20% 22% 34% 7% Emergency med 4% 0% 2% 0% 7% Surgical* 20% 31% 31% 9% 30% Other 22% 44% 39% p < 0.01 20% 30% p < 0.01 High school location Israel 97% 97% 96% 95% 98% Other 3% 3% 4% NS 5% 2% NS High school location in israel Jerusalem 32% 25% 23% 26% 36% Central 29% 37% 44% 31% 29% North 26% 28% 23% 33% 21% South 13% 9% 10% 10% 14% Other 0% 1% 1% NS 0% 0% NS Future residency location Israel 98% 98% 99% 97% 98% Other 2% 2% 1% NS 3% 2% NS Table 1 Primary care - demographic and other information Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 6 of 17 Table 1 Primary care - demographic and other information (Continued) Table 1 Primary care - demographic and other information (Continued) Primary care Undecided No primary care Primary care vs no primary care Primary care Females vs males Females Males Perferred residency location in Israel Jerusalem 28% 18% 23% 23% 31% Central 22% 32% 34% 13% 29% North 17% 11% 9% 23% 12% South 1% 1% 2% 3% 0% Don’t know 33% 39% 32% p < 0.05 39% 29% p < 0.01 *Surgical specialties male students. Students inclined toward primary care were less interested in academic pursuits, such as being academic faculty members. Reduced interest in academic activities was also observed among Japanese medical students with preferences for primary care residency. Students inclined toward primary care were more interested in lifestyle: spend- ing time with their families, working limited hours and working only during the daytime. Percent of "agree" and "agree much" responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis aclusters per cluster analysis nalysis he higher value in a statistically significant pair p y Bold result indicates the higher value in a statistically significant pair d "agree much" responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis nalysis Primary care This import- ance of lifestyle was more pronounced in female than Table 2 Primary care - selection criteria Primary care Undecided No primary care Primary care vs no primary Primary care Females vs males Females Males N 98 184 229 41 57 98 Criteria for choosing a specialty Time with family (1) 76% 73% 65% p < 0.03 85% 68% p < 0.05 Specialty with team work 57% 54% 45% p < 0.04 56% 58% NS Influence of spouse 43% 43% 31% p < 0.007 49% 39% p < 0.05 Specialty that deals with social issues (3) 42% 35% 22% p < 0.001 51% 35% p < 0.009 Daytime work only (1) 26% 29% 16% p < 0.03 34% 19% p < 0.03 Work only in the community 9% 3% 4% p < 0.001 12% 7% NS Procedures/surgery 43% 41% 57% p < 0.01 34% 49% p < 0.02 High salary 36% 48% 51% p < 0.002 34% 38% NS Opportunity for research (2) 34% 37% 44% p < 0.05 27% 39% NS Academic faculty member 19% 26% 30% p < 0.05 24% 16% NS Criteria for choosing a residency Much supervision by senior physicians 53% 45% 37% p < 0.001 68% 42% p < 0.04 Limited work hours 29% 30% 17% p < 0.001 33% 27% NS Short residency (<4.5. years) 28% 15% 12% p < 0.001 29% 26% NS Much clinic time (2) 23% 16% 5% p < 0.001 20% 25% NS Hospital in the periphery (3) 19% 7% 6% p < 0.001 20% 19% NS Leading department (1)a 67% 77% 79% p < 0.03 71% 65% NS Large hospitial 52% 55% 62% p < 0.04 59% 47% NS Family living location 78% 75% 64% NS 88% 70% p < 0.05 Making clinical decisions on your own 66% 50% 55% NS 56% 74% p < 0.05 Pre-determined work hours (2) 47% 48% 35% NS 59% 39% p < 0.05 Influence of family 42% 37% 31% NS 54% 33% p < 0.01 Many on-call shifts 10% 11% 12% NS 5% 14% p < 0.03 Percent of "agree" and "agree much" responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis aclusters per cluster analysis Table 2 Primary care - selection criteria Weissman et al. Primary care Israel Journal of Health Policy Research (2018) 7:28 Page 8 of 17 Table 3 Regression analysis (Continued) Demographics/criteria Multivariable Backward regression coefficient Backward logistic regression p Odds ratio 95% confidence limits P Monetary incentives for residency in the periphery r - 0.58 Opportunity for research −0.126 0.047 0.580 0.360–0.933 0.025 Specialty advancing rapidly −0.143 0.022 0.620 0.393–0.90 0.041 Specific location −0.204 0.001 0.428 0.275–0.667 0.001 Primary care 0.115 0.023 Specialty that deals with social issues 0.126 0.008 1.530 1.044–2.242 0.029 Peripheral hospital 0.258 0.001 2.835 1.782–4.511 0.001 entering general internal medicine and pediatric practices [17]. The major reasons cited for the dearth of students entering primary care in the United States are relatively low incomes in the face of high student debt burdens, many administrative tasks and time pressures [16]. Many medical schools have in- stituted programs to attract more students to pri- mary care, with a multi-year exposure to primary care being more successful than adding a single pri- mary care course to a conventional curriculum [18]. Other countries face similar problems. In Vietnam less than a third of commune (collective farming communities) health stations are staffed by a phys- ician even though the number of medical school graduates almost tripled between 2004 and 2011 [19]. The reasons include poor working conditions, low income and lack of opportunities for career de- velopment [19]. In a survey of 9499 South Korean medical students only 2.2% expressed interest in family medicine [20]. family medicine [12]. Lack of interest in academic endeavors is problematic since it reduces the number of family medicine faculty members able to serve as medical student mentors. This lack of mentors might decrease the ability to attract students to the specialty. p y It is important to note that the query on the questionnaire was about the broader area of primary care and not specifically about family medicine. Unlike a previous study where we found a female- predominance among 6th year Israeli medical students interested in family medicine, in the present study we did not find such predominance [13]. Furthermore, the proportion of women medical students interested and not interested in primary care was comparable. Similarly, upon multiple regression analysis, interest in primary care was not associated with being female. We thus failed to prove our hypothesis that interest in primary care would be greater among women than men medical students. Primary care Israel Journal of Health Policy Research _#####################_ Page 7 of 17 Table 3 Regression analysis Demographics/criteria Multivariable Backward regression coefficient Backward logistic regression p Odds ratio 95% confidence limits P Interest in a primary care residency r = 0.43 Male gender 0.264 0.015 2.251 1.195–4.241 0.012 Criteria for choosing a specialty Opportunity for research −0.096 0.033 0.755 0.578–0.987 0.04 High salary −0.207 0.002 0.580 0.376–0.893 0.013 Influency of spouse 0.122 0.018 Specialty that deals with social issues 0.146 0.004 1.442 1.079–1.927 0.013 Work only in the community 0.278 0.001 Wide range of medical problems 1.512 1.004–2.277 0.048 Criteria for choosing a residency program r = 0.45 Leading department −0.125 0.053 0.581 0.395–0.854 0.006 Influence of family 0.094 0.026 1.414 1.083–1.847 0.011 Short residency (< 4.5 years) 0.159 0.001 1.524 1.122–2.069 0.007 Peripheral hospital 0.188 0.001 Much supervision by senior physicians 0.202 0.001 1.836 1.340–2.515 0.001 Much clinic time 0.207 0.001 1.495 1.110–2.013 0.008 Interest in a residency in a peripheral hospital r = 0.43 Older age 0.157 0.004 1.732 1.144–2.621 0.009 Have considered a specialty −0.234 0.048 High school location (periphery) 0.138 0.002 1.546 1.089–2.193 0.015 Criteria for choosing a specialty Wide range of medical problems 0.144 0.008 Time with family 0.145 0.009 1.686 1.001–2.839 0.049 Work only in the community 0.178 0.002 1.701 1.147–2.524 0.008 Influence of spouse 1.488 1.019–2.173 0.039 Private practice −0.109 0.012 Specialty that deals with social issues 0.123 0.003 Narrow range of medical problems 0.298 0.001 2.156 1.377–3.376 0.001 Criteria for choosing a residency program r = 0.52 Specific location in Israel −0.106 0.031 Leading department −0.280 0.001 0.480 0.091–0.791 0.004 Limited work hours 0.108 0.031 Family living location 0.115 0.046 Many on-call shifts 0.121 0.011 Teaching students 0.144 0.001 1.563 1.061–2.304 0.024 Physical challenge 0.157 0.001 Primary care 0.168 0.002 Much clinic time 0.198 0.003 1.893 1.288–2.783 0.001 Much supervision by senior physicians 0.045 1.010–2.455 0.045 Influence of family 1.430 1.030–1.986 0.022 Weissman et al. Primary care Therefore, in many countries with large rur Table 4 Residency in a peripheral hospital Periphery No periphery Periphery vs no Periphery N 48 382 Gender Female 45% 48% Male 55% 52% NS Age (years) 18–20 0% 0.3% 21–23 17% 18% 24–26 19% 30% 27–29 42% 39% 30–32 17% 10% + 32 6% 3% NS Marital status Single 63% 71% Married 38% 29% Divorced 0% 1% NS High school location Israel 100% 97% Other 0% 3% NS High school location in Israel Jerusalem 17% 27% Central 31% 42% North 38% 22% South 15% 8% Other 0% 1% p < 0.05 Residency location Israel 100% 98% Other 0% 2% NS Future residency location in Israel Jerusalem 13% 26% Central 16% 36% North 40% 7% South 13% 1% Don’t know 18% 31% p < 0.01 Specialties under consideration Family medicne 0% 2% Pediatrics 46% 30% Internal medicine 24% 32% Ob/Gyn 15% 21% Emergency medicine 5% 1% Surgical specialties 32% 30% Other specialties 32% 35% p < 0.05 Criteria for choosing a specialty Time with family 85% 66% p < 0.001 Controllable lifestyle 75% 65% p < 0.04 Influency of spouse 56% 37% p < 0.01 Specialty that deals with social issues 46% 27% p < 0.004 Table 4 Residency in a peripheral hospital (Continued) Periphery No periphery Periphe vs no Periphe Work only in the community 19% 3% p < 0.00 Narrow range of medical problems 10% 2% p < 0.00 Advancing rapidly 48% 62% p < 0.05 Opportunity for research 29% 41% p < 0.03 Criteria for choosing a residency program Family living location 81% 69% p < 0.04 Teaching students 57% 40% p < 0.05 Pre-determined work hours 56% 41% p < 0.05 Influence of family 52% 33% p < 0.0 Much supervision bysenior physicians 50% 42% p < 0.03 Primary care 42% 15% p < 0.00 Limited work hours 42% 21% p < 0.03 Much clinic time 33% 8% p < 0.00 Leading department 58% 79% p < 0.00 Large hosptial 47% 60% p < 0.00 Percent of “agree” and “agree much” responses on 5-point Likert Scale Table 4 Residency in a peripheral hospital (Continued) Periphery No periphery Periphery vs no Periphery Work only in the community 19% 3% p < 0.001 Narrow range of medical problems 10% 2% p < 0.001 Advancing rapidly 48% 62% p < 0.05 Opportunity for research 29% 41% p < 0.03 Criteria for choosing a residency program Family living location 81% 69% p < 0.04 Teaching students 57% 40% p < 0.05 Pre-determined work hours 56% 41% p < 0.05 Influence of family 52% 33% p < 0.02 Much supervision bysenior physicians 50% 42% p < 0.03 Primary care 42% 15% p < 0.0004 Limited work hours 42% 21% p < 0.03 Much clinic time 33% 8% p < 0.001 Leading department 58% 79% p < 0.0001 Large hosptial 47% 60% p < 0.003 Percent of “agree” and “agree much” responses on 5-point Likert Scale Table 4 Residency in a peripheral hospital (Continued) greater interest in short (in years) residency programs with limited hours and with much time spent in clinics. Primary care This variance with our previous studies is attributable to primary care incorporating general internal medicine, general pediatrics and some aspects of ob- stetrics/gynecology, in addition to family medicine. We previously found that In Israel, internal medicine and obstetrics/gynecology residencies attract many male students [13]. When asked which specialties they were considering, students interested in primary care listed pediatrics and internal medicine more fre- quently than family medicine. Shortages of primary care physicians are generally attributed to low salaries, lack of prestige and glamor; long hours with frequent on-call responsibil- ities; and lack of a controllable lifestyle [21, 22]. The situation in Israel differs from other countries in that primary care physicians mainly work in health main- tenance clinics, receive salaries comparable to other physicians, have few on-call obligations and have set hours [8]. This was reflected in our previous study where Israeli 6th-year students rated family medicine and pediatrics as specialties with controllable life- styles and positive relationships between controllable lifestyle and remuneration [13]. This was also found in the present study, where compared to 5th-year students not interested in primary care, those inter- ested in primary care wanted a specialty with time for family involving only daytime work and practice in the community (i.e. outside the hospital). This interest profile was similarly demonstrated by their In many countries, attracting medical students to primary care careers is a daunting task [14]. The reasons for this difficulty differ between countries [15]. In the United States, the proportion of medical students selecting primary care specialties dropped from 73% in 1996 to 44% in 2008, although subse- quently there has been some stabilization [16]. More- over, more internal medicine and pediatric residents are choosing to subspecialize, reducing the numbers Weissman et al. Primary care Israel Journal of Health Policy Research (2018) 7:28 Page 9 of 17 Table 4 Residency in a peripheral hospital Periphery No periphery Periphery vs no Periphery N 48 382 Gender Female 45% 48% Male 55% 52% NS Age (years) 18–20 0% 0.3% 21–23 17% 18% 24–26 19% 30% 27–29 42% 39% 30–32 17% 10% + 32 6% 3% NS Marital status Single 63% 71% Married 38% 29% Divorced 0% 1% NS High school location Israel 100% 97% Other 0% 3% NS High school location in Israel Jerusalem 17% 27% Central 31% 42% North 38% 22% South 15% 8% Other 0% 1% p < 0.05 Residency location Israel 100% 98% Other 0% 2% NS Future residency location in Israel Jerusalem 13% 26% Central 16% 36% North 40% 7% South 13% 1% Don’t know 18% 31% p < 0.01 Specialties under consideration Family medicne 0% 2% Pediatrics 46% 30% Internal medicine 24% 32% Ob/Gyn 15% 21% Emergency medicine 5% 1% Surgical specialties 32% 30% Other specialties 32% 35% p < 0.05 Criteria for choosing a specialty Time with family 85% 66% p < 0.001 Controllable lifestyle 75% 65% p < 0.04 Influency of spouse 56% 37% p < 0.01 Specialty that deals with social issues 46% 27% p < 0.004 greater interest in short (in years) residency program with limited hours and with much time spent clinics. This grouping of interests indicates a desi for positive life/work balance, something that Isra non-hospital primary care practice provides. A rece study of Israeli family medicine residents report similar findings. Specifically, more than 85% of res dents reported that factors that positively influen their choice included the ability to combine wor family, and free time; direct, meaningful contact wi patients; the diversity of patients and medical cond tions; and attractive working conditions [23]. Th interest profile is similar to those reported from oth countries among students interested in primary ca and family medicine [12, 24]. However, primary ca in isolated Israeli rural village has been reported lead to unclear boundaries between private life an physician roles leading to problems with life/wo balance [25]. This may be among the reasons for th shortage of primary care practitioners in the country periphery. Rural (periphery) workforce Worldwide, rural areas often suffer physician shor ages. Primary care This grouping of interests indicates a desire for positive life/work balance, something that Israeli non-hospital primary care practice provides. A recent study of Israeli family medicine residents reported similar findings. Specifically, more than 85% of resi- dents reported that factors that positively influence their choice included the ability to combine work, family, and free time; direct, meaningful contact with patients; the diversity of patients and medical condi- tions; and attractive working conditions [23]. This interest profile is similar to those reported from other countries among students interested in primary care and family medicine [12, 24]. However, primary care in isolated Israeli rural village has been reported to lead to unclear boundaries between private life and physician roles leading to problems with life/work balance [25]. This may be among the reasons for the shortage of primary care practitioners in the country’s periphery. Rural (periphery) workforce Rural (periphery) workforce Worldwide, rural areas often suffer physician short- ages. Therefore, in many countries with large rural Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 10 of 17 Table 5 Incentives - residency in periphery 1. Incentive interests me 2. Plan peripheral residency 3. Rural (periphery) workforce Incentives don’t interest me 1 vs 3 1 vs 2 2 vs 3 Leading department (1)a 69% 45% 85% p < 0.0002 p < 0.004 p < 0.0009 Specific location 51% 68% 70% p < 0.0001 NS NS Large hospitial 50% 26% 68% p < 0.005 p < 0.04 p < 0.0006 Opportunity for research 17% 5% 29% p < 0.02 p < 0.01 p < 0.0005 Much supervision by senior physicians 44% 20% 46% NS NS p < 0.04 Percent of “agree” and “agree much” responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis aClusters per cluster analysis Bold result indicares the higher value in a statistically significant pair Table 5 Incentives - residency in periphery (Continued) areas, such as the United States, Canada and Australia, emphasis has been placed on encouraging more medical students to become rural primary care physicians [26, 27]. To attract students to rural areas, medical schools have programs that expose students to rural practice and have increased the recruitment of students from rural areas [28]. The current study showed that of the more than 500 5th-year Israeli students studied, 8.9% were considering residency in the country’s periphery. This percentage is greater than that reported in our previous study of 5th year students (4.6%) and might be attributable to the re- cent introduction of monetary incentives (one-time grants and salary increases) for physicians choosing to train and practice in the periphery. Shortages of rural physicians frequently includes a lack of primary care physicians; a situation also present in Israel. Among students interested in primary care, 19% would choose a residency in the periphery. Alternately, among students considering a residency in the periph- ery, 42% were interested in primary care. This attrac- tion to primary care among students interested in living in rural areas was also observed among Japanese medical students [29]. However, when we examined our overall student sample, only 3.7% of the 5th-year students reported interest in both primary care and peripheral residencies. the periphery”, 63% had gone to high school in the northern or southern areas. Therefore, we confirmed the hypothesis that interest in residencies in periph- eral areas is greater among students who attended high school in peripheral regions. Similar observa- tions were made in Kenya, United States, Japan and Australia where students of rural origin were more interested in rural practice [2, 11, 28, 29]. Rural (periphery) workforce Incentives don’t interest me 1 vs 3 1 vs 2 2 vs 3 N 106 20 223 Percent 30% 6% 64% Gender Female 48% 47% 50% Male 52% 53% 50% NS NS NS Age (years) 18–20 0% 0% 0% 21–23 22% 20% 19% 24–26 27% 15% 31% 27–29 36% 50% 35% 30–32 10% 10% 11% + 32 5% 5% 3% NS NS p < 0.02 Marital status Single 68% 60% 72% Married 30% 40% 27% Divorced 2% 0% 1% NS p< 0.05 p < 0.02 High school location in Israel Jerusalem 27% 5% 25% Central 36% 32% 40% South 10% 26% 10% Other 1% 0% 1% NS p < 0.01 NS Future residency location in Israel Jerusalem 14% 10% 29% Central 20% 0% 41% North 14% 55% 8% South 0% 20% 1% Don’t know 53% 15% 22% p< 0.03 p < 0.01 p< 0.01 Specialties under consideration Pediatrics 39% 31% 31% Internal medicine 21% 6% 35% Ob/Gyn 29% 19% 15% Emergency medicine 0% 13% 1% Surgical specialties 32% 31% 29% Other specialties 31% 31% 37% p < 0.04 p < 0.01 p < 0.02 Criteria for choosing a specialty Time with family (1) 78% 60% 63% p < 0.004 p < 0.04 NS Specialty that deals with social issues (3) 35% 35% 25% p < 0.02 NS NS Advancing rapidly (2) 51% 45% 69% p < 0.0007 NS p < 0.04 Opportunity for research (2) 29% 5% 47% p < 0.0001 p < 0.04 p < 0.0001 Controllable lifestyle (1) 74% 50% 66% NS NS NS Independent practice 54% 40% 51% NS p < 0.03 p < 0.03 High salary 51% 20% 45% NS p < 0.006 p < 0.01 Procedures/surgery 45% 32% 51% NS NS p < 0.04 Private practice 37% 10% 404% NS p < 0.008 p < 0.009 Criteria for choosing a residency Controllable lifestyle 73% 45% 58% p < 0.04 p < 0.03 NS Primary care 27% 32% 15% p < 0.001 NS p < 0.002 Hospital in the periphery (3) 8% 55% 4% p < 0.001 p < 0.0007 p < 0.001 Intellectual challenge (1)a 8% 60% 87% p < 0.004 NS p < 0.007 Table 5 Incentives - residency in periphery Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 11 of 17 Table 5 Incentives - residency in periphery (Continued) 1. Incentive interests me 2. Plan peripheral residency 3. Rural (periphery) workforce These results have potential healthcare policy implications. Firstly, they can contribute to decisions about whether to admit more medical school applicants residing in the periphery in an attempt to reduce the shortage of physicians practicing there. Secondly, the results can also contribute to decisions about whether to include rural medicine rotations during the clinical years to provide all students with expos- ure to such medical practice [30]. The characteristics of students considering a residency in the periphery were similar to those interested in a primary care residency. Although this may be partially attributable to the many students interested in a primary care residency, it also likely reflects that those interested in residencies in the per- iphery are more interested in life-work balance and are less interested in a residency in a large hospital and in a leading department. Regression analysis showed that attending high school in Israel’s south and north was associated with interest in residency in the same regions. Notably, significantly more students interested in residencies in the periphery reported that the locale of their family was an important criterion for choosing a residency program. Furthermore, among students who responded to the question concerning the effects of one-time monetary incentives to do a residency in the periphery, “I already plan to do a residency in Strengths and limitations The strength of this study is that the large number of students studied allowed us to examine subgroups, such as those interested in primary care. A further strength was its multi-year design showing that the proportion of students interested in primary care and peripheral hospitals remained steady throughout the study. The major limitation is that the study was performed in a single institution that is located in the center of the country and is focused on academic medicine. Hence the findings are not automatically generalizable to the entire Israeli medical student population, and parallel studies should be carried out at additional Israeli medical schools.1 Conclusions h h The characteristics of students showing interest in primary care and practice in peripheral areas, that were delineated by this study, should aid department heads and residency program directors in identifying potential residents. Moreover, the present study re- vealed that for Israeli medical students the 5th-year is an important juncture in their choice of a medical specialty. Eighty percent had already considered vari- ous specialties. Although, about a quarter had begun their considerations before beginning medical school, the majority had begun during their 4th and 5th- years. Furthermore, 60% of those who had begun the thought process had already changed their minds. Therefore, the 4th and 5th years of medical school appear to be an opportune time to market the various specialties to medical students and might also be the time to begin informing them about residency pro- grams. Since the students’ decisions as to specialty and residency program decisions have major influ- ences on the composition and geographic distribution of the future physician workforce, it is for the health- care leadership to take the initiative and provide the students with direction, counseling and information to help them with their choices. Incentives Alternately, they were less interested in re- search opportunities, a rapidly advancing specialty and a residency in a leading department in a large hospital. For medical educators and healthcare leaders these results point to a group with distinct character- istics who might be encouraged to join residency pro- grams in the periphery. The challenge is to better characterize this student group, identify them early during their specialty/residency program decision process and provide positive information and counsel- ing about residency programs and lifestyle in the per- iphery. Thirty percent of the residents working in Israel’s periphery reported that the incentives had in- fluenced them greatly, even though initially they had intended to work there [23]. Previous studies have shown that Israeli primary care practitioners in the periphery are more satisfied and had a broader scope of practice than urban practitioners [29, 31, 32]. While the aim is to market residency programs in the periphery to the students, the results of this study show that a third of the students reported that the influence of family was an important part of their residency selection process. Therefore, consideration should be given to including spouses in recruitment efforts. from such schools [23]. Yet, unlike the foreign graduates who only enter the Israeli healthcare sys- tem as interns, having had exposure to the various specialties in foreign healthcare systems during med- ical school, the Israeli medical students are part of the Israeli healthcare system while they are medical students providing the Israeli healthcare leadership the opportunity to directly expose them to Israeli primary and rural practices early in their clinical experience. A further limitation is that there might be selection criteria that were not included in the questionnaire. However, both the factor and cluster analyses showed few factors and clusters indicating that a wide-variety of topics were queried. Incentives The last four years of the study provided an oppor- tunity to explore the student’s thoughts about the monetary incentives for rural residency included in the 2011 union contract [7]. Although only 6% were already planning to do a residency in the periphery, 30% indicated that the incentives induced them to consider a residency there. Fifty-three percent of the latter students reported that they did not yet know Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Page 12 of 17 Page 12 of 17 where they wished to do their residency as opposed to 22% among those not interested in the incentives. Moreover, there were more differences between the two groups. Students interested in the incentives were more interested in primary care residencies, special- ties dealing with social issues, specialties providing family time and residencies affording controllable life- styles. Alternately, they were less interested in re- search opportunities, a rapidly advancing specialty and a residency in a leading department in a large hospital. For medical educators and healthcare leaders these results point to a group with distinct character- istics who might be encouraged to join residency pro- grams in the periphery. The challenge is to better characterize this student group, identify them early during their specialty/residency program decision process and provide positive information and counsel- ing about residency programs and lifestyle in the per- iphery. Thirty percent of the residents working in Israel’s periphery reported that the incentives had in- fluenced them greatly, even though initially they had intended to work there [23]. Previous studies have shown that Israeli primary care practitioners in the periphery are more satisfied and had a broader scope of practice than urban practitioners [29, 31, 32]. While the aim is to market residency programs in the periphery to the students, the results of this study show that a third of the students reported that the influence of family was an important part of their residency selection process. Therefore, consideration should be given to including spouses in recruitment efforts. where they wished to do their residency as opposed to 22% among those not interested in the incentives. Moreover, there were more differences between the two groups. Students interested in the incentives were more interested in primary care residencies, special- ties dealing with social issues, specialties providing family time and residencies affording controllable life- styles. Endnote 1Our hypothesis is that the findings for the other medical schools will be similar to what we found for Hebrew University-Hadassah. This is because a) Des- pite its location in the center of the country, the Hebrew University- Hadassah Medical School draws students from across the country; b) Earlier studies of family medicine specialty preferences found similar- ities between this medical school and the total for all Israeli medical schools [23]. Another limitation is that the study only included Israeli medical students when half the medical in- ternship workforce is comprised of Israeli who grad- uated from foreign medical schools and immigrants and 58% of the family medicine residents graduated Weissman et al. Endnote Israel Journal of Health Policy Research (2018) 7:28 Page 13 of 17 Table 6 Primary care - selection criteria Primary care Undecided No primary care Primary care vs no primary Primary care Females vs males Females Males N 98 184 229 41 Criteria for choosing a specialty Time with family (1) 75.51% 72.68% 65.07% p < 0.03 85.37% 68.42% p < 0.05 Specialty with team work 57.14% 53.80% 44.98% p < 0.04 56.10% 57.89% NS Influence of spouse 42.55% 42.62% 31.14% p < 0.007 48.65% 38.60% p < 0.05 Specialty that deals with social issues (3) 41.84% 34.62% 21.93% p < 0.001 51.22% 35.09% p < 0.009 Daytime work only (1) 25.51% 28.73% 16.23% p < 0.03 34.15% 19.30% p < 0.03 Work only in the community 9.18% 2.73% 3.51% p < 0.001 12.20% 7.02% NS Procedures/surgery 42.86% 40.98% 56.58% p < 0.01 34.15% 49.12% p < 0.02 High salary 36.08% 48.09% 51.09% p < 0.002 34.15% 37.50% NS Opportunity for research (2) 33.67% 37.16% 43.67% p < 0.05 26.83% 38.60% NS Academic faculty member 19.39% 25.68% 30.26% p < 0.05 24.39% 15.79% NS Bedside specialty 93.88% 92.93% 92.07% NS 92.68% 94.74% NS Wide range of medical problems 79.38% 71.04% 68.12% NS 75.61% 82.14% NS Controllable lifestyle (1) 69.39% 73.91% 64.19% NS 78.05% 63.16% NS Advancing rapidly (2) 56.12% 61.41% 58.77% NS 51.22% 59.65% NS Independent practice 51.02% 60.11% 52.40% NS 56.10% 47.37% NS Private practice 39.80% 36.96% 42.48% NS 41.46% 38.60% NS Influence of family 12.37% 9.78% 8.81% NS 15.00% 10.53% NS Specialty that my coleagues choosea 2.06% 1.64% 0.00% NS 0.00% 3.57% NS Influence of classmatesa 2.06% 1.63% 0.44% NS 0.00% 3.57% NS Narrow range of medical problemsa 2.04% 3.26% 3.49% NS 0.00% 3.51% NS Criteria for choosing a residency Much supervision by senior physicians 53.06% 45.11% 36.68% p < 0.001 68.29% 42.11% p < 0.04 Limited work hours 29.17% 29.89% 16.67% p < 0.001 32.50% 26.79% NS Short residency (< 4.5. Endnote Years) 27.55% 15.30% 11.79% p < 0.001 29.27% 26.32% NS Much clinic time (2) 22.45% 16.30% 4.82% p < 0.001 19.51% 24.56% NS Hospital in the periphery (3) 19.39% 6.52% 6.11% p < 0.001 19.51% 19.30% NS Leading department (1)a 67.35% 77.17% 79.04% p < 0.03 70.73% 64.91% NS Large hospitial 52.04% 54.64% 61.84% p < 0.04 58.54% 47.37% NS Intellectual challenge (1)a 83.67% 82.07% 83.41% NS 87.80% 80.70% NS Family living location 77.55% 74.86% 63.76% NS 87.80% 70.18% p < 0.05 Controllable lifestyle 69.39% 70.49% 60.70% NS 73.17% 66.67% NS Making clinical decisions on your own 66.33% 49.46% 55.02% NS 56.10% 73.68% p < 0.05 Specific location in Israel 65.31% 64.48% 63.88% NS 68.29% 63.16% NS Much “action” 52.04% 33.15% 46.49% NS 51.22% 52.63% NS Pre-determined work hours (2) 46.94% 48.37% 35.37% NS 58.54% 38.60% p < 0.05 Physical challenge 47.96% 36.96% 46.93% NS 41.46% 52.63% NS Teaching students 43.88% 43.48% 43.42% NS 39.02% 47.37% NS Influence of family 41.84% 37.16% 31.00% NS 53.66% 33.33% p < 0.01 Opportunity for research 20.41% 25.00% 27.95% NS 19.51% 21.05% NS Many on-call shifts 10.20% 11.41% 11.79% NS 4.88% 14.04% p < 0.03 Percent of “agree” and “agree much” responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis Weissman et al. Endnote Israel Journal of Health Policy Research (2018) 7:28 Page 14 of 17 Appendix B Table 7 Residency in a peripheral hospital Periphery No periphery Periphery vs no periph N 48 382 Criteria for choosing a specialty Time with family 85.42% 66.23% p < 0.001 Controllable lifestyle 75.00% 65.01% p < 0.04 Influency of spouse 56.25% 37.40% p < 0.01 Specialty that deals with social issues 45.83% 27.03% p < 0.004 Work only in the community 18.75% 2.62% p < 0.001 Narrow range of medical problems 10.42% 1.83% p < 0.001 Advancing rapidly 47.92% 62.30% p < 0.05 Opportunity for research 29.17% 40.94% p < 0.03 Bedside specialty 91.67% 93.10% NS Wide rangeof medical problems 62.50% 72.44% NS Independent practice 60.42% 55.24% NS Specialty with teamwork 54.17% 47.52% NS Procedures/surgery 50.00% 47.24% NS High salary 50.00% 47.64% NS Private practice 47.92% 41.05% NS Daytime work only 27.08% 21.78% NS Influence of family 14.89% 10.76% NS Academic faculty member 18.75% 28.61% NS Influence of classmate 4.17% 1.31% NS Specialty that my coleagues choose 2.08% 0.79% NS Criteria for choosing a residency Family living location 81.25% 68.59% p < 0.04 Teaching students 56.25% 40.05% p < 0.05 Pre-determined work hours 56.25% 40.73% p < 0.05 Influence of family 52.08% 32.98% p < 0.02 Much supervision by senior physicians 50.00% 42.15% p < 0.03 Primary care 42.22% 14.72% p < 0.0004 Limited work hours 41.67% 21.26% p < 0.03 Much clinic time 33.33% 8.38% p < 0.001 Leading department 58.33% 78.59% p < 0.0001 Large hosptial 46.81% 60.05% p < 0.003 Intellectual challenge 77.08% 82.77% NS Controllable lifestyle 77.08% 61.56% NS Specific location 75.00% 65.79% NS Making clinical decisions on your own 62.50% 53.26% NS Much “action” 43.75% 40.31% NS Physical challenge 41.67% 41.10% NS Short residency 22.92% 15.18% NS Opportunity for research 20.83% 25.85% NS Many on-call shifts 16 67% 9 14% NS Page 15 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 9. Weissman C, Zisk-Rony RY, Schroeder JE, Weiss YG, Avidan A, Elchalal U, Tandeter H. Medical specialty considerations by medical students early in their clinical experience. Isr J Health Policy Res. 2012;1:13. References The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. 1. Allen SM, Ballweg RA, Cosgrove EM, Engle KA, Robinson LR, Rosenblatt RA, Skillman SM, Wenrich MD. Challenges and opportunities in building a sustainable rural primary care workforce in alignment with the Affordable Care Act: the WWAMI program as a case study. Acad Med. 2013;88:1862–9. 1. Allen SM, Ballweg RA, Cosgrove EM, Engle KA, Robinson LR, Rosenblatt RA, Skillman SM, Wenrich MD. Challenges and opportunities in building a sustainable rural primary care workforce in alignment with the Affordable Care Act: the WWAMI program as a case study. Acad Med. 2013;88:1862–9. 1. Allen SM, Ballweg RA, Cosgrove EM, Engle KA, Robinson LR, Rosenblatt RA, Skillman SM, Wenrich MD. Challenges and opportunities in building a sustainable rural primary care workforce in alignment with the Affordable Care Act: the WWAMI program as a case study. Acad Med. 2013;88:1862–9. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. 8. OECD Reviews of Health Care Quality: Israel 2012: Raising Standards OCED Library http://www.oecd-ilibrary.org/social-issues-migration-health/oecd- reviews-of-health-care-quality-israel-2012_9789264029941-en Accessed 26 Feb 2017. 8. OECD Reviews of Health Care Quality: Israel 2012: Raising Standards OCED Library http://www.oecd-ilibrary.org/social-issues-migration-health/oecd- reviews-of-health-care-quality-israel-2012_9789264029941-en Accessed 26 Feb 2017. Authors’ contributions CW – Designed study, collected and analyzed data, wrote and edited manuscript. RYZ – Equal contribution with first author – designed study, edited questionnaire, analyzed data, edited, commented and revised manuscript. AA – Reviewed and edited questionnaire, reviewed data, edited and commented on manuscript. UA – Reviewed and edited questionnaire, edited and commented on manuscript. HT – Designed study, revised questionnaire, reviewed manuscript. All the authors read and approved the final manuscript. 2. Duffrin C, Diaz S, Cashion M, Watson R, Cummings D, Jackson N. Factors associated with placement of rural primary care physicians in North Carolina. South Med J. 2014;107:728–33. 3. Sempowski IP. Effectiveness of financial incentives in exchange for rural and underserviced area return-of-service commitments: systematic review of the literature. Can J Rural Med. 2004;9:82–8. 4. Del Mar C. New investments in primary care in Australia. BMC Health Serv Res. 2011;11:39. Appendix C Appendix C Table 8 In “residency in the periphery” and “primary care residency” Residency in the periphery Not intersted Neutral Interested Total Primary Not interested 37.4% 4.7% 2.7% 44.8% Care Neutral 22.7% 11.0% 2.3% 36.0% Residency Interested 10.4% 5.1% 3.7% 19.2% Total 70.5% 20.7% 8.8% Appendix D Table 9 Primary care - selection criteria 1 Incentive interests me 2 Plan peripheral residency 3 Incentives don’t interest me 1 vs 3 1 vs 2 2 vs 3 N 106 20 223 Criteria for choosing a specialty Time with family (1) 78.30% 60.00% 63.39% p < 0.004 p < 0.04 NS Specialty that deals with social issues (3) 35.24% 35.00% 25.11% p < 0.02 NS NS Advancing rapidly (2) 51.43% 45.00% 68.75% p < 0.0007 NS p < 0.04 Opportunity for research (2) 29.25% 5.00% 46.64% p < 0.0001 p < 0.04 p < 0.0001 Bedside specialty 95.28% 95.00% 92.79% NS NS NS Wide range of medical problems 73.58% 63.16% 73.66% NS NS NS Controllable lifestyle (1) 73.58% 50.00% 66.07% NS NS NS Independent practice practice 53.77% 40.00% 51.28% NS p < 0.03 p < 0.03 High salary 50.48% 20.00% 45.09% NS p < 0.006 p < 0.01 Specialty with team work 48.11% 55.00% 50.89% NS NS NS Procedures/surgery 45.28% 31.58% 50.67% NS NS p < 0.04 Influence of spouse 41.35% 45.00% 34.84% NS NS NS Private practice 36.79% 10.00% 39.64% NS p < 0.008 p < 0.009 Daytime work only (1) 23.30% 21.05% 19.20% NS NS NS Academic faculty member 22.86% 10.53% 31.25% NS NS NS Influence of family 9.43% 10.00% 11.21% NS NS NS Work only in the community 4.72% 10.00% 3.14% NS NS NS Narrow range of medical problemsa 4.72% 5.00% 2.68% NS NS NS Specialty that my coleagues choosea 1.90% 5.00% 0.90% NS NS NS Influence of classmatesa 0.95% 0.00% 1.34% NS NS NS Criteria for choosing a residency Controllable lifestyle 73.33% 45.00% 58.12% p < 0.04 p < 0.03 NS Primary care 27.18% 31.58% 15.32% p < 0.001 NS p < 0.002 Hospital in the periphery (3) 7.55% 55.00% 3.85% p < 0.001 p < 0.0007 p < 0.001 Intellectual challenge (1)a 80.19% 60.00% 87.05% p < 0.004 NS p < 0.007 Leading department (1)a 68.87% 45.00% 84.82% p < 0.0002 p < 0.004 p < 0.0009 Specific location 50.94% 68.42% 69.82% p < 0.0001 NS NS Large hospitial 50.00% 26.32% 67.71% p < 0.005 p < 0.04 p < 0.0006 Opportunity for research 16.98% 5.00% 29.02% p < 0.02 p < 0.01 p < 0.0005 Family living location 68.87% 70.00% 69.06% NS NS NS Making clinical decisions on your own 49.06% 50.00% 58.04% NS NS NS Weissman et al. Ethics approval and consent to participate 5. Israel Ministry of Health: Disparity in health services and ways of coping with it. 2015, Jerusalem; www.health.gov.il/publicationsfiles/inequality-2015. pdf (Accessed 12 Mar 2017). The Institutional Review Board of the Hadassah Medical Organization approved this study. Completion of the questionnaire by the student was considered tacit consent. The Institutional Review Board of the Hadassah Medical Organization approved this study. Completion of the questionnaire by the student was considered tacit consent. 6. Weil LG, Bin Nun G, Mckee M. Recent physician strike in Israel: a health system under stress? Israel J Health Policy Res. 2013;2:33. 7. Simonstein F. Priorities in the Israeli health care system. Med Health Care Philos. 2013;16:341–7. Acknowledgements The authors thank the many students who participated in this study. The authors thank the many students who participated in this study. Received: 15 November 2017 Accepted: 25 April 2018 / Appendix C Israel Journal of Health Policy Research (2018) 7:28 Page 16 of 17 Table 9 Primary care - selection criteria (Continued) 1 Incentive interests me 2 Plan peripheral residency 3 Incentives don’t interest me 1 vs 3 1 vs 2 2 vs 3 Much “action” 45.28% 40.00% 43.75% NS NS NS Much supervision by senior physicians 43.81% 20.00% 46.43% NS NS p < 0.04 Limited work hours 43.40% 30.00% 40.18% NS NS NS Physical challenge 42.45% 30.00% 41.70% NS NS NS Teaching students 38.10% 50.00% 48.21% NS NS NS Influence of family 32.38% 30.00% 34.38% NS NS NSb Pre-determined work hours (2) 25.71% 25.00% 20.72% NS NS NS Short residency 15.09% 10.00% 16.52% NS NS NS Much clinic time (2) 13.33% 20.00% 9.38% NS NS NS Many on-call shifts 12.26% 10.00% 10.71% NS NS NS Percent of “agree” and “agree much” responses on 5-point Likert Scale Numbers in parenthesis are the results of factor analysis aClusters per cluster analysis Bold results represent the higher value of a statistically significant pair Table 9 Primary care - selection criteria (Continued) Jerusalem, Israel. 4Department of Family Medicine, Ben Gurion University Joyce and Irving Goldman School of Medicine, Be’er Sheva, Israel. Acknowledgements Received: 15 November 2017 Accepted: 25 April 2018 / Received: 15 November 2017 Accepted: 25 April 2018 / Funding Israel National Institute for Health Policy Research. Funding Israel National Institute for Health Policy Research. Author details 1 10. Avidan A, Weissman C, Elchalal U, Tandeter H, Zisk-Rony RY. Medical specialty selection criteria of Israeli medical students early in their clinical experience: subgroups. Isr J Health Policy Res. 2018;7:20. 1Department of Anesthesiology and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel. 2Hadassah Henrietta Szold School of Nursing, Hebrew University, Jerusalem, Israel. 3Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, Hebrew University Hadassah School of Medicine, 1Department of Anesthesiology and Critical Care Medicine, Hadassah-Hebrew University Medical Center, Hebrew University – Hadassah School of Medicine, Kiryat Hadassah, POB 12000, 91120 Jerusalem, Israel. 2Hadassah Henrietta Szold School of Nursing, Hebrew University, Jerusalem, Israel. 3Department of Obstetrics and Gynecology, Hadassah-Hebrew University Medical Center, Hebrew University Hadassah School of Medicine, 11. Dossajee H, Obonyo N, Ahmed SM. Career preferences of final year medical students as a medical school in Kenya – a cross sectional study. BMC Medical Education. 2016;16:5. 11. Dossajee H, Obonyo N, Ahmed SM. Career preferences of final year medical students as a medical school in Kenya – a cross sectional study. BMC Medical Education. 2016;16:5. Page 17 of 17 Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 12. Kawamoto R, Ninomiya D, Kasai Y, Kusunoki T, Ohtsuka N, Kumagi T, Abe M. Factors associated with the choice of general medicine as a career among Japanese medical students. Med Educ Online. 2016;21:29448. 13. Weissman C, Tandeter H, Zisk-Rony RY, Weiss YG, Elchalal U, Avidan A. Schroeder JE Israeli medical student's perceptions of six key medical specialties. Israel J Health Policy Res. 2013;2:19. 14. Petterson SM, Liaw WR, Tran C, Bazemore AW. Estimating the residency expansion required to avoid projected primary care physician shortages by 2035. Ann Fam Med. 2015;13:107–14. 15. Kringos D, Boerma W, Bourgueil Y, Cartier T, Dedeu T, Hasvold T, Hutchinson A, Lember M, Oleszczyk M, Rotar Pavlic D, Svab I, Tedeschi P, Wilm S, Wilson A, Windak A, Van der Zee J, Groenewegen P. The strength of primary care in Europe: an international comparative study. Br J Gen Pract. 2013;63:e742–50. 16. Teng VC, Lin SY. Renewing US medical students’ interest in primary care: bridging the role model gap. Postgrad Med. 2014;90:1–2. 17. Messinger CJ, Hafler J, Khan AM, Long T. Recent trends in primary care interest and career choices among medical students at an academic medical institution. Teach Learn Med. 2017;29:42–51. Author details 1 18. Pfarrwaller E, Sommer J, Chung C, Maisooeuve H, Nendaz M, Perron NJ, Haller DM. Impact of interventions to increase the proportion of medical students choosing a primary career: a systemic review. J Gen Intern Med. 2015;30:1349–58. 19. Giang KB, Minh HV, Hien NV, Ngoc NM, Hinh ND. Knowledge of primary health care and career choice at primary health care settings among final year medical students – challenges to human resources for health in Vietnam. Global Pub Heal. 2017;10(sup 1):S120–30. 20. Kim YY, Kim UN, Kim YS, Lee JS. Factors associated with the specialty choice of Korean medical students: a cross-sectional survey. Hum Resource Health. 2016;14:45. 21. Gold JA, Barg FK, Margo K. Undergraduate students’ perspectives on primary care. J Prim Care Comm Health. 2014;5:279–83. 22. Clinite KL, DeZee KJ, Durning SJ, Kogan JR, Blevins T, Chou CL, Diemer G, Dunne DW, Fagan MJ, Hartung PJ, Kazantsev SM, Mechaber HF, Paauw DS, Wong JG, Reddy ST. Lifestyle factors and primary care specialty selection: comparing 2012-2013 graduating and matriculating medical students’ thoughts on specialty lifestyle. Acad Med. 2014;89:1483–9. 23. Ashkenazi Y, Gordon M, Yankellevich A, Rosen B. Attracting medical residents to the periphery and to medical specialties in crisis following the 2011 collective agreement. Jerusalem: Myers-JDC-Brookdale Institute Smokler Center for Health Policy Research; 2017. 24. Kiobassa K, Miksch A, Hermann K, Loh A, Szecsenyi J, Joos S, et al. Becoming a general practitioner – which factors have more impact on career choice of medical students? BMC Fam Pract. 2011;12:25. 25. Monnickendam SM, Borkan JM, Matalon A, Zalewski S. Trials and tribulations of country doctors: a qualitative study of doctor-patient relationships in rural Israel. Isr J Med Sci. 1996;32:239–47. 26. Royston PJ, Mathieson K, Leafman J, Ojan-Sheehan O. Medical student characteristics predictive of intent for rural practice. Rural Remote Health. 2012;12:2107. 27. Russell DJ, Wakerman J, Humphreys JS. What is a reasonable length of employment for health workers in Australian rural and remote primary healthcare services? Aust Health Rev. 2013;37:256–61. 28. Puddey IB, Mercer A, Playford DE, Riley GJ. Medical student selection criteria and socio-demographic factors as predictors of ultimately working rurally after graduation. BMC Med Educ. 2017;17:1. 29. Kawamoto R, Uemoto A, Ninomiya D, Hasegawa Y, Ohtsuka N, Kusunoki T, Kumagi T, Abe M. Characteristics of Japanese medical students associated with their intention for rural practice. Rural Remote Health. 2015;15:3112. 30. Williamson MI, Wilson R, McKechnie R, Ross J. Weissman et al. Israel Journal of Health Policy Research (2018) 7:28 Author details 1 Does the positive influence of an undergraduate rural placement persist into postgraduate years? Rural Remote Health. 2012;12:2011. 31. Fennig S, Yuval D, Greenstein M, Rabin S, Weingarten M. Job satisfaction among certified and non-certified general practitioners. Isr Med Assoc J. 2000;2:823–7. 32. Biderman A, Shvartzman P, Anson O, Almagor G, Grol R. 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Single-Dose Microparticle Delivery of a Malaria Transmission- Blocking Vaccine Elicits a Long-Lasting Functional Antibody Response
Current molecular medicine
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R.R. Dinglasan*,1, J.S. Armistead1,#, J.F. Nyland2,#, X. Jiang3,4 and H.Q. Mao3,4 1W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205, USA 2Department of Pathology, Microbiology & Immunology, University of South Carolina School of Medicine, 6439 Garner's Ferry Road, Columbia, SC 29209, USA 3Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA 3Department of Materials Science and Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA 4 4Translational Tissue Engineering Center, Whitaker Biomedical Engineering Institute, Johns Hopkins School of Medicine, 400 North Broadway, Baltimore, MD 21287, USA 4Translational Tissue Engineering Center, Whitaker Biomedical Engineering Institute, Johns Hopkins School of Medicine, 400 North Broadway, Baltimore, MD 21287, USA Abstract: Malaria sexual stage and mosquito transmission-blocking vaccines (SSM-TBV) have recently gained prominence as a necessary tool for malaria eradication. SSM-TBVs are unique in that, with the exception of parasite gametocyte antigens, they primarily target parasite or mosquito midgut surface antigens expressed only inside the mosquito. As such, the primary perceived limitation of SSM-TBVs is that the absence of natural boosting following immunization will limit its efficacy, since the antigens are never presented to the human immune system. An ideal, safe SSM-TBV formulation must overcome this limitation. We provide a focused evaluation of relevant nano-/microparticle technologies that can be applied toward the development of leading SSM-TBV candidates, and data from a proof-of-concept study demonstrating that a single inoculation and controlled release of antigen in mice, can elicit long-lasting protective antibody titers. We conclude by identifying the remaining critical gaps in knowledge and opportunities for moving SSM-TBVs to the field. Keywords: Antigen, controlled release, immunity, malaria, midgut, mosquito, nanotechnology, natural boosting, sexual stages, transmission-blocking vaccine. Send Orders of Reprints at reprints@benthamscience.net Send Orders of Reprints at reprints@benthamscience.net Current Molecular Medicine 2013, 13, 479-487 479 © 2013 Bentham Science Publishers *Address correspondence to this author at the W. Harry Feinstone Department of Molecular Microbiology & Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Rm. E5646, Baltimore, MD 21205, USA; Tel: +1-410-614-4839; Fax: +1-410-955-0105; E-mail: rdinglas@jhsph.edu #These authors contributed equally to this manuscript. Single-Dose Microparticle Delivery of a Malaria Transmission- Blocking Vaccine Elicits a Long-Lasting Functional Antibody Response R. Dinglasan*,1, J.S. Armistead1,#, J.F. Nyland2,#, X. Jiang3,4 and H.Q. Mao3 INTRODUCTION vivax Efficacy >85% efficacy and reduction of the Case Reproductive Rate (R0) below 1 Duration Effective for at least 2 years Safety Vaccine has a safety and efficacy profile comparable to Hepatitis B vaccine Target Population All age groups Administration Route Administered orally, or by intramuscular or subcutaneous injection or other innovative device Immunization Schedule A single dosage schedule that can be administered by mass administration or clinic-based programs. Booster dose after 2 years may be required Minimal schedule is three doses, administered over 6 months Stability & Storage Minimum shelf life of 36 months and can be stored at ambient temperature and withstand freeze thawing Minimum is stability at 37°C for 30 minutes and 2 years at 2-8°C Co-administration No interference or interactions with other vaccines expected to be concurrently administered Target Product Profile of a SSM-TBV “natural boosting” issue (Table 1). Furthermore, we have also used APN1 as a model antigen to directly address the above issue using nano- and microparticle technologies. [14, 15], thus it is possible that gametocyte exposure in the NHPs following challenge was responsible for boosting. This study further supported the notion that boosting would increase the efficacy and utility of SSM- TBVs but raised the question of the need for highly potent adjuvants such as FCA, which is considered a serious obstacle in human vaccine development. An ideal SSM-TBV formulation with a highly immunogenic antigen must therefore have the following characteristics: (i) it should be safe; (ii) it should not require a cold-chain; (iii) it should easily be administered; and (iv) a single immunization should confer long-lasting protection. A biodegradable microparticle (BMP) system, which provides sustained release of antigen and adjuvant properties, is capable of meeting these challenges. Several recent studies have demonstrated the utility of this general vaccine approach in vertebrate models [36-40]. Microparticle size is an important determinant for cell uptake [41, 42] and may also influence the antigen release rate [43]. In line with this, recent studies have shown that smaller particle delivery systems are effective in eliciting a robust immune response to the target immunogen [44- 47]. The bioabsorption rate of BMPs and antigen release rate can be engineered to provide boosting from weeks to several months. INTRODUCTION (gamete, zygote, or ookinete) or Anopheles mosquito midgut surface antigens that are only expressed in the mosquito. As such, one of the potential limitations of the TBV approach is that since the antigens are never naturally presented to the human immune system, the absence of natural boosting following immunization will limit their efficacy [8-13]. A complete P. knowlesi model in non-human primates (NHP) has been used to test the “natural boosting” hypothesis for Plasmodium gamete antigens [13]. It was found that following a two- dose immunization regimen using 105-107 P. knowlesi microgametes and macrogametes in a Freund’s complete adjuvant (FCA), the majority of the monkeys maintained a high level of functional transmission- blocking antibody titer for more than 1 year. Furthermore, annual challenge infections over a six year period were found to be sufficient for boosting and transmission-blocking immunity persisted in the majority of splenectomized NHPs. Importantly, as expected, they observed that transmission-blocking activity waned over time in the absence of boosting and that the challenge infection resulted in an increase in gamete-specific antibody levels. Although the likely gamete antigens had not yet been fully characterized at the time of this study, it was already known that gametocytes and gametes shared surface antigens The malaria eradication research agenda has re- emphasized the need for effective sexual stage and mosquito transmission-blocking vaccines (SSM-TBV) [1], which prevents malaria parasite development in its mosquito vector and the subsequent cascade of secondary infections [2-5]. SSM-TBVs, in general, work through the action of inhibitory antibodies [5-7]. Thus, the minimum objective of immunization is to induce high titer antibodies sustainable for at least one transmission season (~3-6 months), but preferably for 2 years. Achieving this minimum goal would theoretically drive the case reproductive rate, (R0) <1. A summary of the target product profile (TPP) for SSM- TBVs is shown in Table 1. With the exception of Plasmodium falciparum or P. vivax gametocyte surface antigens that are expressed in the human, SSM-TBVs are considered unique in that they target parasite #These authors contributed equally to this manuscript. /13 $58.00+.00 © 2013 Bentham Science Publishers 0 Current Molecular Medicine, 2013, Vol. 13, No. 4 480 Dinglasan et al. Table 1. The Proposed Target Product Profile (TPP) for a Malaria Sexual Stage and Mosquito Transmission-Blocking Vaccine (SSM-TBV) [61] Target Product Profile of a SSM-TBV Indication Prevention of transmission of P. falciparum or P. INTRODUCTION Particles carrying single or multiple antigens can arguably mimic viral antigen presentation thus rapidly inducing a potent and long- lasting cellular and humoral response either by direct immune stimulation of antigen presenting cells (APCs) or/and by delivering antigen to the lymph node [30, 37, 48]. In fact, virosomes follow this approach and have shown to be effective carriers for proteins and subunit vaccines against a variety of pathogens, including malaria [49], but to date, this approach has not been used to deliver SSM-TBV antigens. With these goals in mind, we conducted proof-of-concept studies to test the hypothesis that safe biodegradable microparticles can mimic natural boosting through sustained release of antigen and, in doing so, elicit significant transmission- blocking antibodies against Plasmodium. The four leading SSM-TBVs (Table 2) include two gametocyte surface antigens, Pfs230 [16-20] and Pfs48/45 [21], the ookinete surface protein Pfs25 [22] and the Anopheles gambiae alanyl aminopeptidase N (APN1), which is an abundant, midgut-specific apical microvilli surface glycoprotein that has been shown to mediate ookinete invasion and oocyst development [7, 23]. Of these, only Pfs25 and APN1 are expressed explicitly inside the mosquito midgut. Note that the goal of this report is not to evaluate the complete repertoire of proven and possible SSM-TBV candidates, and the reader is directed to several excellent reviews for additional information [3, 4, 24-29]. Among the four leading candidates, only Pfs25 has completed Phase I clinical trials, albeit with equivocal results [29]. Efforts are underway to produce the full-length Pfs/Pvs230 [30-32] and Pfs48/45 antigens [33-35], which have proven to be a difficult undertaking using different expression platforms due to their size and/or conformation, as well as the high A+T content of plasmodial genes; and these issues have a direct impact on vaccine process development. The APN1 antigen, on the other hand, does not require the full- length antigen, is highly immunogenic [7] and is entering process development, with an optimistic initiation of Phase I clinical trials within the next 3-4 years. Since Pfs25 and APN1-based vaccines are the least likely to benefit from boosting following natural infection, we focused on these two antigens in this article to examine their current state of development, as well as similarities and differences in the context of several identified target product profiles and the Current Molecular Medicine, 2013, Vol. 13, No. 4 481 Current Molecular Medicine, 2013, Vol. 13, No. 4 481 Single-Dose Microparticle Delivery Table 2. Preparation of Biodegradable Microparticles (BMPs) with Different Size Range and Different Antigen Loading Levels Recombinant APN1 was produced in E. coli as previously described [23]. Polylactofate (PLE) was used to prepare BMPs. PLE is a poly(lactide-co- glycolide) derivative with good biocompatibility and better control of biodegradation rate and physical properties [50, 51] (Fig. 1A). BMPs were prepared by a modified double emulsion method [50], and characterized by scanning electron microscopy. The release kinetics of APN1 from BMPs was characterized by monitoring the concentration of APN1 using ELISA. To modulate APN1 release, we used bovine serum albumin (BSA) as a filler protein. MATERIALS AND METHODS MATERIALS AND METHODS Immunizations BALB/c female mice were immunized with either (A) recombinant APN1 in PBS in suspension with alum, or (B) recombinant APN1 in PBS emulsified with incomplete Freund’s adjuvant (IFA), or (C) BMP- encapsulated recombinant APN1 delivered with alum, or (D) BMP encapsulated APN1 with IFA or (E) empty BMP with alum or (F) empty BMP with IFA. For all treatment groups, mice received 2 g antigen/mouse/ dose. At day 0, mice received a subcutaneous (s.c.) injection of the appropriate inoculum in a volume of 100 l per mouse. At 2, 4 and 6 weeks post priming, mice in the Control cohorts (treatments A and B, above) were boosted intraperitoneally (i.p.) with the same dose of the inoculum per mouse, whereas the BMP cohorts were boosted only with PBS. At these time points, each mouse was bled to collect sera for anti-APN1 antibody titer determination via ELISA (Fig. 1C). INTRODUCTION Update of the Current Status and Characteristics of the Leading SSM-TBV Candidates Target Antigen Current Status Attributes P230 Recombinant antigen expression through a variety of systems including plant, cell free wheat germ systems. Present in the gametocyte and can confer natural boosting [10, 19] Immunogenicity is poor and requires a strong adjuvant [19, 62, 63] Molecule is large, resulting in difficulty in expression and maintenance of conformational epitopes [63] P48/45 Recombinant antigen expression using E. coli (codon harmonized) Conformational epitopes necessitates an appropriate expression system [33] Immunogenic protein in animals (alum) and is further enhanced by using a strong adjuvant [33] P25 Phase I clinical trials + Conjugated to recombinant Pseudomonas aeruginosa ExoProtein A [62] Phase I Clinical trial of ExoProtein A product is ongoing Immunogenic varies depending on route [63] but is generally considered poorly immunogenic by itself and may require a strong adjuvant [29, 64-66] or conjugation to a molecular adjuvant or protein carrier [67] Reactogenic formulations prevented continuation of the first Phase I clinical trial [65] Successfully produced the small immunogen in yeast and plants [68] APN1 Entering Process Development Immunogenic in mice [7] and non-human primates (Dinglasan, unpublished) using alum as adjuvant Does not require an adjuvant for complete seroconversion in mice [7] Update of the Current Status and Characteristics of the Leading SSM-TBV Candidates ELISA and Cytokine Assay ELISAs were performed as previously described, using recombinant APN1 as coating antigen [7]. For cytokine assays, the spleen was removed and homogenized at 10% wt/vol in 2% fetal bovine serum/minimal essential medium, and supernatants stored at -80°C until used. Cytokines were measured in tissue homogenates using bead-based multiplex cytokine kits (Bio-Plex, Bio-Rad), according to manufacturer’s instructions. The limits of detection were as follows: interleukin (IL)-1, 1.32 pg/ml, IL-1, 1.70 pg/ml; IL-2, 1.98 pg/ml; IL-3, 1.32 pg/ml; IL-4, 2.43 pg/ml; IL-5, 1.69 pg/ml; IL-5, 1.69 pg/ml; IL-6, 1.02 pg/ml; IL-9, 1.36 pg/ml; IL-10, 1.04 pg/ml; IL-12/23 p40, 1.15 pg/ml; IL-12 p70, 1.20 pg/ml; IL-13, 1.57 pg/ml; IL-17a, 1.44 pg/ml; interferon (IFN)-, 1.30 pg/ml; eotaxin, 1.70 pg/ml; granulocyte-colony stimulating factor, 1.69 pg/ml; granulocyte-macrophage-colony stimulating factor, 1.58 pg/ml; monocyte chemo-attractant protein, 1.71 pg/ml; macrophage inflammatory protein (MIP)-1, 1.57 pg/ml; MIP-1, 1.20 pg/ml; RANTES, 0.95 pg/ml; tumor necrosis factor (TNF)-, 1.73 pg/ml. Cytokine measurements below the limit of detection as determined by the standard curve for each individual cytokine were assigned a value of the limit of detection/2 for statistical analysis and plotting. Statistical significance was determined by One- way ANOVA with Bonferroni Post Test,  = 0.05. Preparation of Biodegradable Microparticles (BMPs) with Different Size Range and Different Antigen Loading Levels Preparation of Biodegradable Microparticles (BMPs) with Different Size Range and Different Antigen Loading Levels Transmission-Blocking Assays * . /*   +#, #       )        +#! % %! * . /* Fig. (1). Polylactofate biodegradable microparticles for single inoculation delivery of a malaria transmission-blocking vaccine antigen. (A) Structure of polylactofate (PLE). (B) Scanning electron micrographs of three batches of BMPs with 0.52%, 3.53% and 6.77% protein loading, respectively (Scale bars = 10 m). (C) Effect of protein loading level on the cumulative release profile of encapsulated proteins from BMPs. (D) Immunization dosing regimens for BMP and control groups, and functional analysis by direct feeding assay (DFA). Transmission-Blocking Assays The Direct Feeding Assays (DFA) were conducted as previously described [7] at 2 months and at 6 months post-priming immunization (Fig. 1D). Since Plasmodium oocyst numbers are generally overdispersed in our system, statistical significance was assessed using the non-parametric Mann Whitney U Test,  = 0.05. 482 Current Molecular Medicine, 2013, Vol. 13, No. 4 Dinglasan et al. Fig. (1). Polylactofate biodegradable microparticles for single inoculation delivery of a malaria transmission-blocking vaccine antigen. (A) Structure of polylactofate (PLE). (B) Scanning electron micrographs of three batches of BMPs with 0.52%, 3.53% and 6.77% protein loading, respectively (Scale bars = 10 m). (C) Effect of protein loading level on the cumulative release profile of encapsulated proteins from BMPs. (D) Immunization dosing regimens for BMP and control groups, and functional analysis by direct feeding assay (DFA).                                              !" # ! $ !% "&'$( )'* ( +#, #       )        +#! % %! *    - - - . /* # 0  1 482 Current Molecular Medicine, 2013, Vol. 13, No. 4 Dinglasan et al.                      # 0 1 Fig. (1). Polylactofate biodegradable microparticles for single inoculation delivery of a malaria transmission-blocking vaccine antigen. (A) Structure of polylactofate (PLE). (B) Scanning electron micrographs of three batches of BMPs with 0.52%, 3.53% and 6.77% protein loading, respectively (Scale bars = 10 m). (C) Effect of protein loading level on the cumulative release profile of encapsulated proteins from BMPs. (D) Immunization dosing regimens for BMP and control groups, and functional analysis by direct feeding assay (DFA).                         !" # ! $ !% "&'$( )'* ( +#, #       )        +#! % %! RESULTS The two significantly different cytokines (E) IL-2 and (F) IL-5 and o  2 3            # 0%" %  #             # 0%" %  # *4  *4  *4  *4+5 *4+5 *4+5      #  .4  .4  .4+5 .4+5 .4+5 *4  *4  *4  *4+5 *4+5 *4+5     6  ! % 7*$ 6  ! % 7*$  #  .4  .4  .4+5 .4   #  .4+5 .4+5 8 8 8 8  .  #  6 . +9'"$$"#( 4 +9'"$$"#( 4 .  #  6 .      .  #  6 . 5 8 8 8 8  )65'"$$"#(  4            # 0%" %  #  * * * * * *      #  .4  .4  .4+5 .4+5 .4+5 6  ! % 7*$ 0%" %  # 0%" %  #             # 0%" %  # *4 *4 *4 * * *     6  ! % 7*$  #  .4  .4  .4+5 .4   #  .4+5 .4+5  0%" %  # +9'"$$"#( 4 .  #  6 .      5 2 8 8 8 8  .  #  6 . +9'"$$"#( 4 2 5 +9'"$$"#( 4 3 .  #  6 . 8 8 8 8  )65'"$$"#(  4 3 Fig. (2). Characterization of the immune response and activity of antibodies elicited following immunization with APN1. (A) APN1-specific antibody titers (at bleeds 1-3) for mice that received only a single inoculation of BMP encapsulated APN1 with alum or IFA. RESULTS gambiae (Keele) mosquitoes for groups in (C) at two months nization. For A-D: Median oocyst numbers are represented by the horizontal line; control infections were f hed, unimmunized mouse; and the P-value was determined by Mann Whitney U Test and asterisks (*) indica cance at  = 0.05. (E-G) APN1-BMP induces pro-T-cell and B-cell cytokines. Twenty-three cytokines m genized spleen samples from mice that received either BMP (empty) or APN1-encapsulated BMPs. Data e of tissue basis (corrected for spleen weight). RESULTS single inoculation of APN1-containing BMPs plus IFA or Alum alone (Fig. 2A) mounted a relatively poor antibody response in comparison to a prime and 3- boost regimen of APN1 plus IFA/Alum (Fig. 2C). Surprisingly, the immunoglobulin subtypes (IgG1, IgG2a, and IgG2b) generated in the group that received a single immunization of APN1-BMPs/alum were similar to that elicited by the APN1-alum (data not shown). We generated PLE BMPs (Fig. 1B) and optimized the protocol for controlling the protein antigen loading levels. We then used loading level as a parameter to adjust the release rate of the antigen. Using bovine serum albumin (BSA) as a model antigen, we have shown that the amount of antigen released from the BMPs can be controlled by loading level as shown in Fig. (1C). For example, BMPs with 3.53% protein loading level released protein antigen at a rate of ~ 104 ng/day per mg of BMPs, after an initial burst release of 9.3% of the total protein loaded. These release rates amounted to a release of approximately 15% of total protein within the first 22 days. For this pilot study, we used BMPs with 3.53% of protein loading level. We compared the humoral response of mice using the schedule outlined in Fig. (1D). Mice immunized with a To determine the short-term and long-term efficacy of transmission-blocking serum antibodies against P. berghei we performed direct feeding assays (DFAs) two weeks following the final boost in the control group at 2 months (60 days) and at 6 months (180 days) (Figs. 1D, 2B, D). We compared parasite development in mosquitoes that were fed on four groups: (i) control cohort (primed with APN1/alum followed by three Current Molecular Medicine, 2013, Vol. 13, No. 4 483 Single-Dose Microparticle Delivery 2). Characterization of the immune response and activity of antibodies elicited following immunization PN1-specific antibody titers (at bleeds 1-3) for mice that received only a single inoculation of BMP encapsulate or IFA. (B) Direct Feeding Assay to assess short-term transmission-blocking potential of mouse APN1 anti modium berghei (ANKA 2.34) in Anopheles gambiae (KEELE) mosquitoes for groups in (A) at two months nization (see Fig. 1D). (C) APN1-specific antibody titers (at bleeds 1-3, at two week intervals) for mice that rec either alum or IFA as adjuvant. (D) Direct Feeding Assay to assess short-term transmission-blocking potent antisera against P. berghei (ANKA 2.34) in An. RESULTS (B) Direct Feeding Assay to assess short-term transmission-blocking potential of mouse APN1 antisera against Plasmodium berghei (ANKA 2.34) in Anopheles gambiae (KEELE) mosquitoes for groups in (A) at two months post-priming immunization (see Fig. 1D). (C) APN1-specific antibody titers (at bleeds 1-3, at two week intervals) for mice that received APN1 with either alum or IFA as adjuvant. (D) Direct Feeding Assay to assess short-term transmission-blocking potential of mouse APN1 antisera against P. berghei (ANKA 2.34) in An. gambiae (Keele) mosquitoes for groups in (C) at two months post-priming immunization. For A-D: Median oocyst numbers are represented by the horizontal line; control infections were from an age- matched, unimmunized mouse; and the P-value was determined by Mann Whitney U Test and asterisks (*) indicate statistical significance at  = 0.05. (E-G) APN1-BMP induces pro-T-cell and B-cell cytokines. Twenty-three cytokines measured in homogenized spleen samples from mice that received either BMP (empty) or APN1-encapsulated BMPs. Data expressed on pg/g of tissue basis (corrected for spleen weight). The two significantly different cytokines (E) IL-2 and (F) IL-5 and one cytokine, TNF-, which was not significantly different (G), are shown. Data presented as box and whiskers plots with outliers identified as dots. Median is the horizontal line within the box. Statistical significance was determined by one way ANOVA with Bonferroni Post Test,  = 0.05. 484 Current Molecular Medicine, 2013, Vol. 13, No. 4 Dinglasan et al. Table 3 Direct Feeding Assays (DFA) to Assess Long-Term Transmission-Blocking Potential of Mouse APN1 Antisera Against Plasmodium berghei (ANKA 2.34) in Anopheles gambiae (Keele) Mosquitoes. DFAs were Performed at 6 Months Post-Priming Immunization (see Fig. 1D) Table 3 Direct Feeding Assays (DFA) to Assess Long-Term Transmission-Blocking Potential of Mouse APN1 Antisera Against Plasmodium berghei (ANKA 2.34) in Anopheles gambiae (Keele) Mosquitoes. DFAs were Performed at 6 Months Post-Priming Immunization (see Fig. 1D) Group (Mouse #) N Median Oocyst # (Range) % Inhibition Prevalence P-Value Long-Term APN1-Alum Control (M3) 23 82 (1-181)  100%  APN1-BMP-Alum (M4) 22 8.5 (0-84) 90 82% <0.0001 APN1-BMP-Alum (M5) n.d.     APN1-BMP-IFA (M4) 32 16 (0-124) 81 59% <0.0001 APN1-BMP-IFA (M5) 22 0 (0-1) 100 9% <0.0001 Groups: APN1-Alum Control = recombinant APN1 + alum, using a prime + 3 boost immunization regimen (age-matched with BMP groups); APN1-BMP =APN1-BMP + alum (single inoculation); APN1-BMPIFA=APN1-BMP +Incomplete Freund's adjuvant (single inoculation). RESULTS n.d., not determined since the mouse did not survive the mosquito feeding. P-value determined by Mann Whitney U Test,  = 0.05. Groups: APN1-Alum Control = recombinant APN1 + alum, using a prime + 3 boost immunization regimen (age-matched with BMP groups); APN1-BMP =APN1-BMP + alum (single inoculation); APN1-BMPIFA=APN1-BMP +Incomplete Freund's adjuvant (single inoculation). n.d., not determined since the mouse did not survive the mosquito feeding. P-value determined by Mann Whitney U Test,  = 0.05. boosts); (ii) treatment group receiving a single inoculation of APN1-BMPplus alum, (iii) treatment group receiving a single inoculation of APN1-BMP plus IFA, and (iv) control (naïve/unimmunized or empty BMP immunized) infected mice. At 60 days, both the APN1-alum and APN1-IFA immunized controls elicited functional transmission-blocking antibodies against P. berghei (Fig. 2B). Despite the lower antibody titer observed previously, APN1-BMP-immunized mice generated a significant level of functional antibody titers that can effectively inhibit oocyst development in An. gambiae (Fig. 2C, D). We observed that at 6 months post-priming immunization, serum from mice immunized with APN1/alum, following a standard immunization regimen, no longer contained any transmission-blocking antibodies [refer to median oocyst number/prevalence for APN1-Alum Control (M3)]. In contrast, individual mice that received either APN1-BMPs/alum or APN1-BMPs/IFA still retained functional transmission-blocking antibody (Table 3). Cytokine levels analyzed by multiplex assay also demonstrated that APN1-BMPs significantly induced splenic pro-T-cell and B-cell cytokines such as IL-2 and IL-5. These data suggest a cell-specific immune effect rather than a general inflammatory process in response to BMP dosing, thereby validating the specificity of the immune response to the vaccine formulation (Fig. 2D- F). gambiae mosquitoes; and (3) that with a potent adjuvant such as incomplete Freund’s adjuvant, immunization with BMPs elicits and maintains transmission-blocking titers in mice for 6 months. Furthermore, the protracted release kinetics of model antigen over 16 days in vitro by our PLE BMP demonstrates a more controlled profile as compared to gel core liposome or conventional liposome particles which have been shown to exhibit a 50% cumulative percentage release of antigen at 10-15 days and 5 days, respectively [44]. These data suggest that larger microparticles allow for enhanced control over the release profile. Recently, it was shown that incorporation of TLR9 agonists in 1-m gel core liposomes can significantly enhance the immune response to the poorly immunogenic Pfs25 SSM-TBV antigen [44]. Thus, the use of molecular adjuvants as filler molecules may also be considered in future formulations. RESULTS Taken together with our proof-of-concept data, we anticipate that co-encapsulation of adjuvant and administration of different BMPs with different release profiles (e.g. burst and fast release serve as priming and sustained/delayed release as boosting dose) will significantly enhance the overall immune responses. DISCUSSION Although nano- and microparticle technology has been already shown to potentiate the immune response to pathogen-derived antigens [52, 53], including malaria [44, 45, 49, 54], its use in TBV delivery while previously postulated [9], remained relatively untested [44]. Our small scale study adds to the growing body of data, and moreover, successfully demonstrates that (1) APN1-BMPs with alum adjuvant elicit antigen-specific antibody titers after single dose immunization and induce the production of cell- activation rather than broad-spectrum pro-inflammatory cytokines; (2) the functional transmission-blocking activity of APN1 antisera against P. berghei from mice immunized with a single dose of APN1-BMP in An. CONCLUSION AND FUTURE PERSPECTIVES Vaccines are traditionally developed with the prospect of eventual parenteral administration, and the TPP for SSM-TBVs suggests that this is the primary consideration for the development of the leading candidates (Table 1). Given the uniqueness of the SSM-TBV approach it is argued that non-classical concepts for vaccine delivery may be more suitable. In this section we highlight some concerns surrounding the use of NPs and BMPs when considering vaccine delivery not only through parenteral, oral or mucosal routes, but specifically via cutaneous immunization. REFERENCES [1] Alonso PL, Brown G, Arevalo-Herrera M, et al. A research agenda to underpin malaria eradication. PLoS Med 2011; 8(1): e1000406. ( ) [2] Carter R. Transmission blocking malaria vaccines. Vaccine 2001; 19(17-19): 2309-2314. ( ) [3] Dinglasan RR, Jacobs-Lorena M. Flipping the paradigm on malaria transmission-blocking vaccines. Trends Parasitol 2008; 24(8): 364-370. [4] Lavazec C, Bourgouin C. Mosquito-based transmission blocking vaccines for interrupting Plasmodium development. Microbes Infect 2008; 10(8): 845-849. ( ) Sinden RE. A biologist's perspective on malaria vaccine development. Hum Vaccin 2010; 6(1): 3-11. While there are clear opportunities for the utility of BNPs and BMPs in the development of the next generation of SSM-TBVs, the current working model by many vaccine developers remains generally conservative. This is rightly so, since malaria vaccines must be low cost to allow for general distribution. The huge number of vaccine doses to cover the more than one third of the world’s population is likely to be borne by public-private partnerships and other novel funding models. However, there is hope for this approach since the prevailing strategy has been more recently revisited by the PATH Malaria Vaccine Initiative [59]. One of the biggest benefits of the BNP/BMP approach, namely the potential to mimic natural boosting, is quite attractive, especially in light of the prediction that titers of antibody (produced either naturally or following vaccination) against sexual stage and mosquito antigens will likely wane over time [60]. Furthermore, there is optimism Ranawaka GR, Fleck SL, Blanco AR, Sinden RE. Characterization of the modes of action of anti-Pbs21 malaria transmission-blocking immunity: ookinete to oocyst differentiation in vivo. Parasitology 1994; 109 (Pt 4): 403-411. Ranawaka GR, Fleck SL, Blanco AR, Sinden RE. Characterization of the modes of action of anti-Pbs21 malaria transmission-blocking immunity: ookinete to oocyst differentiation in vivo. Parasitology 1994; 109 (Pt 4): 403-411. Mathias DK, Plieskatt JL, Armistead JS, et al. Expression, immunogenicity, histopathology, and potency of a mosquito- based malaria transmission-blocking recombinant vaccine. Infect Immun 2012; 80(4): 1606-1614. Mathias DK, Plieskatt JL, Armistead JS, et al. Expression, immunogenicity, histopathology, and potency of a mosquito- based malaria transmission-blocking recombinant vaccine. Infect Immun 2012; 80(4): 1606-1614. ( ) Kaslow DC. Immunogenicity of Plasmodium falciparum sexual stage antigens: implications for the design of a transmission blocking vaccine. Immunol Lett 1990; 25(1-3): 83-86. [9] Kaslow DC. Transmission-blocking immunity against malaria and other vector-borne diseases. Curr Opin Immunol 1993; 5(4): 557-565. Kaslow DC. Does Route of Delivery Matter? It has been shown that size also has a direct influence on the effectiveness of delivery when the route of administration is considered. Intradermal or subcutaneous inoculation of BNPs and BMPs bypasses the issue of tissue barriers and proteolytic environments, in the case of oral administration. However, the clear potential of this technology lies in the idea of needle-free vaccination. The use of BNP and BMPs as carriers for transcutaneous or cutaneous immunization has been extensively studied [57, 58] and it is well recognized that the main barrier for trans- or percutaneous delivery of antigen payload to the rich population of APCs in the epidermis and dermis is the stratum corneum lipid bilayer overlaying the epidermis [57]. Passive diffusion of antigen carried via nanocarriers through intercellular or follicular routes to access to the APCs in the epidermis and preferably the dermis has been demonstrated, strongly implying that presentation is size dependent [58]. Does Size Matter? In the context of SSM-TBVs, it remains to be seen if different carrier modes can further potentiate the humoral response to confer long-term protection. that by leveraging the potential advantages conferred by particle-based approaches, the community will ultimately see the incorporation of vaccine antigens targeting different life stages of the parasite in a single particle formulation. ABBREVIATIONS ABBREVIATIONS BMP = Biodegradable microparticle NP = Nanoparticle TBV = Transmission-blocking vaccine SSM-TBV = Sexual stage and mosquito TBV APN1 = Alanyl aminopeptidase N 1 PLGA = Poly-lactic-co-glycolic acid FCA = Freund’s complete adjuvant IFA = Incomplete Freund’s adjuvant. ACKNOWLEDGEMENTS This work was supported by the Bloomberg Family Foundation and the Johns Hopkins Malaria Research Institute Pilot Grant Award Program. The authors thank Hilary Hurd and Paul Eggleston for the Anopheles gambiae KEELE strain. CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest. Does Size Matter? It has been shown that 40 nm polystyrene nanoparticles (NP) that are surface-coated with antigen can be targeted to the lymph nodes to generate a robust immune response [46-48, 55]. NPs have also Current Molecular Medicine, 2013, Vol. 13, No. 4 Single-Dose Microparticle Delivery 485 been shown to increase the breadth and avidity of the humoral response to a Plasmodium vivax blood stage antigen [37, 45] arising in part through a synergistic effect of surface displayed and encapsulated antigen in a single formulation. However, it is likely that the nature of the particle, the characteristics of the antigen, including intrinsic immunogenicity and molecular size, presence of conformational antibody epitopes, as well as the type of immune response that should be engendered will have a direct influence on the selection of biodegradable nanoparticles (BNP) vs BMP as carrier (reviewed in [56, 57]). It was found that larger particles engender a Type 2 response while smaller, virus-sized particles induced a largely cell-mediated Type 1 response [46]. An interesting approach would be to use different BNP and BMP carriers, leveraging the advantages of antigen targeting and antigen depot effect endowed by each type of particles to reach a specific immune response endpoint [36]. In the context of SSM-TBVs, it remains to be seen if different carrier modes can further potentiate the humoral response to confer long-term protection. been shown to increase the breadth and avidity of the humoral response to a Plasmodium vivax blood stage antigen [37, 45] arising in part through a synergistic effect of surface displayed and encapsulated antigen in a single formulation. However, it is likely that the nature of the particle, the characteristics of the antigen, including intrinsic immunogenicity and molecular size, presence of conformational antibody epitopes, as well as the type of immune response that should be engendered will have a direct influence on the selection of biodegradable nanoparticles (BNP) vs BMP as carrier (reviewed in [56, 57]). It was found that larger particles engender a Type 2 response while smaller, virus-sized particles induced a largely cell-mediated Type 1 response [46]. 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Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse
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Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,* 1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science and Technology, Kowloon, Hong Kong, China *Correspondence: linqiang@scsio.ac.cn (Q.L.); boqianpy@ust.hk (P.-y.Q.) Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052 ª 2020 The Author(s). Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,* 1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science and Technology, Kowloon, Hong Kong, China *Correspondence: linqiang@scsio.ac.cn (Q.L.); boqianpy@ust.hk (P.-y.Q.) Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052 ª 2020 The Author(s). Report Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,* 1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science and Technology, Kowloon, Hong Kong, China *Correspondence: linqiang@scsio.ac.cn (Q.L.); boqianpy@ust.hk (P.-y.Q.) Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052 ª 2020 The Author(s). Report The Innovation Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,* 1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Chunyan Li,1,2 Yongxin Li,2,3 Geng Qin,1 Zelin Chen,1 Meng Qu,1 Bo Zhang,1 Xue Han,1 Xin Wang,1 Pei-yuan Qian,2,4,* and Qiang Lin1,2,* 1CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China Bio-Resources and Ecology, South China Sea Institute of Oceanology, Innovation of South China Sea Ecology and Environmenta ces, 510301 Guangzhou, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong L 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 2Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), 511458 Guangzhou, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science and Technology, Kowloon, Hong Kong, China 3Department of Chemistry, The University of Hong Kong, Pokfulam, Hong Kong, Hong Kong SAR, China 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Labo and Technology, Kowloon, Hong Kong, China y y g g g g g g 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science and Technology, Kowloon, Hong Kong, China g Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Hong Kong University of Science Chi 4Department of Ocean Science and Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laborator and Technology, Kowloon, Hong Kong, China Received: June 23, 2020; Accepted: October 12, 2020; Published: November 25, 2020; https://doi.org/10.1016/j.xinn.2020.100052 ª 2020 The Author(s). PUBLIC SUMMARY - Male seahorses epitomize the unique characteristic of male pregnancy - Retinoic acid (RA) plays key regulatory roles in brood pouch formation and pregnancy - RA could influence the brood pouch formation by regulating steroid hormone metabolism - RA may contribute to the successful gestation by regulating antioxidant defense ll ll www.cell.com/the-innovation The Innovation Changes in Genes and Metabolites during Brood Pouch Formation and Pregnancy The results of two comparisons, that is, for pouch formation (unformed [UF] versus newly formed [NF]) and pregnancy (pregnant [PG] versus [NF]), of the lined seahorses (H. erectus) at UF, NF, and PG stages are shown in Fig- ure 1A. In total, we quantified and annotated 21,232 transcripts and 210 me- tabolites (Tables S1–S4 and S13). Principal-component analysis (PCA) of both transcripts and metabolites showed large variations among seahorses at different stages of brood pouch development (Figures 1B and 1C). A total of 141 differentially expressed genes (DEGs) and 73 significantly different metabolites (SDMs) were identified in pouch formation analysis (Figure 1D; Tables S5 and S6), while 2,533 DEGs and 121 SDMs were identified in preg- nancy comparison (Figure 1E; Tables S14 and S15). INTRODUCTION Syngnathid fish possess the unique reproductive strategy of male preg- nancy, in which the male possesses an embryo-incubating structure called a brood pouch.1,2 Both the structure and position of the brood pouch vary substantially among syngnathids and pouch ultrastructure and functions differ even among closely related species.3 Among syngnathid fish, sea- horses (Hippocampus) not only have the most complex pouch structure but also experience the most significant physiological changes during em- bryo incubation.4 A brood pouch is not present in juvenile seahorses but rather appears with growth in male seahorses.5 Male seahorses first form a baggy structure from the primordium, followed by differentiation and estab- lishment of brood pouch-specific tissues, which ultimately form a pouch with well-developed blood vessels capable of incubating embryos.2 Seahorses have placenta-like tissues that facilitate pregnancy, which are similar to the placental structure in mammals.4 Moreover, the significant morphological and functional changes of the brood pouch that occur during gestation are equivalent to those in the uterus of the mammals.6 For example, it has been suggested that heavily vascularized tissues in the brood pouch are related to gas exchange between the embryos and the father,7 while abun- dant C-type lectins present during early incubation mayhave an important im- mune protective role for developing embryos.8 Although a few studies have sought to identify specific genes and pathways associated with pouch func- y ( ) The expression levels of most DEGs were higher in seahorses in the NF stage and lower in the PG stage (Figure 2A). Two pathways (retinol meta- bolism and ABC transporters) were significantly enriched for both pouch for- mation and pregnancy (p < 0.01, Figure 2B; Tables S7–S10). Meanwhile, SDMs exhibited diverse changes (Figure 2C). Specifically, aminoacyl-tRNA biosynthesis, nitrogen metabolism, as well as phenylalanine, tyrosine, and tryptophan biosynthesis were the three significantly enriched SDM pathways identified for both pouch formation and pregnancy (p < 0.05, Figure 2D; Ta- bles S16 and S17). Of the 32 co-enriched pathways for transcriptome and metabolome analyses, 6 pathways, including aminoacyl-tRNA biosynthesis, were co-enriched for both pouch formation and pregnancy (Figure 2E; Tables S18 and S19). In particular, five SDMs and ten DEGs were mapped in the ste- roid hormone metabolism pathway (Figure 2F; Table S20). Regulatory Role of Retinoic Acid in Male Pregnancy of the Seahorse This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). tion,9,10 a comprehensive analysis of the regulatory gene and metabolite net- works responsible for control of brood pouch formation and pregnancy has not yet been conducted. Seahorses epitomize the exuberance of evolution. They have the unique characteristic of male pregnancy, which includes the carrying of many embryos in a brood pouch that incubates and nourishes the embryos, similar to the mammalian placenta. However, the regulatory networks underlying brood pouch formation and pregnancy remain largely un- known. In this study, comparative transcriptomic and metabolomic profiling on the lined seahorse Hippocampus erectus, with unformed, newly formed, and pregnant brood pouches identified a total of 141 and 2,533 differentially expressed genes together with 73 and 121 significantly differential metabolites related to brood pouch formation and pregnancy, respectively. Specifically, integrative omics analysis re- vealed that retinoic acid (RA) synthesis and signaling pathway played essential roles in the formation of the brood pouch and pregnancy. RA might function upstream of testosterone and progesterone, thereby directly influencing brood pouch formation by regulating the expression of fshr and cyp7a1. Our results also revealed that RA regulates antioxi- dant defenses, particularly during male pregnancy. Alternatively, preg- nancy caused a consistent decrease in RA, canthaxanthin, astaxanthin, and glutathione synthetase, and an increase in susceptibility to oxidative stress, which may balance brood pouch development and reproduction in seahorses and pave the way to successful gestation. Retinoic acid (RA) has multiple functions in a wide range of biological pro- cesses, particularly in early organogenesis induction and patterning,11–14 as well as in innate and adaptive immunity.15 RA receptors are involved in the regulation of androgen biosynthesis,16,17 which is necessary for the produc- tion of seahorse pouches.4 Due to the pleiotropic roles of RA in the immune system,15 any deviation from the required concentration of RA can result in oxidative stress, while impacting normal growth and differentiation.18 Howev- er, the effects of RA signaling in syngnathids remain unclear despite the diverse brooding types and structures present in this family. In this study, we aimed to investigate the molecular mechanism underlying brood pouch development by combining both transcriptomic and metabolomic analyses of the lined seahorse (Hippocampus erectus) at different pouch develop- mental stages with specific focus on the regulatory role of RA in brood pouch formation and pregnancy. Figure 1. Transcriptome and Metabolome Mapping of Transcripts and Metabolites over Different Brood Pouch Developmental Stages in Seahorses (A) Illustration of un- formed (UF), newly formed (NF), and pregnant (PG) brood pouches of seahorses. Two comparisons for the brood pouch formation and pregnancy are labeled. Principal- component analysis (PCA) for RNA-seq (B) and metab- olome (C) data. Each point represents one biological repli- cate and points with different colors represent seahorses at different pouch developmental stages. Volcano plots showing the relative abundance of transcripts (D) and me- tabolites (E) in comparison for pouch formation and preg- nancy. Important transcripts and metabolites are marked. Potential Multiple Functions of RA in the Development of the Retinol metabolism was significantly enriched in DEGs identified during both pouch formation and pregnancy. In fact, it was also the most signifi- cantly enriched pathway during pouch formation (Figure 2B; Tables S9 and S10). Expression level of six genes related to RA synthesis and signaling consistently decreased during pregnancy (Figure 3A; Table S11). In addition, 54 RA target genes were differentially expressed over different stages of pouch formation and pregnancy (Table S11). Expression of most RA target genes consistently decreased during pregnancy, while expression of several genes were increased in UF seahorses (e.g., cholesterol 7-alpha-monooxyge- nase [cyp7a1]) and PG (e.g., cytochrome b [558] subunit beta [cybb]) brood pouches (Table S11). These RA target genes participate in diverse intercon- nected functions, including steroid hormone metabolism (e.g., cyp7a1 and follicle-stimulating hormone receptor [fshr]19), tissue remodeling (e.g., collagen alpha-1 (IV) chain [col4a1],20 collagen alpha-2 (IV) chain [col4a2],21 and keratin 18 [krt18]22), organ development (e.g., fshr23 and paired-like ho- meodomain 2 [pitx2]24), and immune defense (e.g., cybb25 and transient re- INTRODUCTION Upstream metab- olites of the pathway (prolactin [PRL] and progesterone [PR]) were abundant in seahorses in the UF stage, while downstream metabolites of the pathways ll The Innovation 1, 100052, November 25, 2020 1 The Innovation Report A A A B D E C Figure 1. Transcriptome and Metabolome Mapping of Transcripts and Metabolites over Different Brood Pouch Developmental Stages in Seahorses (A) Illustration of un- formed (UF), newly formed (NF), and pregnant (PG) brood pouches of seahorses. Two comparisons for the brood pouch formation and pregnancy are labeled. Principal- component analysis (PCA) for RNA-seq (B) and metab- olome (C) data. Each point represents one biological repli- cate and points with different colors represent seahorses at different pouch developmental stages. Volcano plots showing the relative abundance of transcripts (D) and me- tabolites (E) in comparison for pouch formation and preg- nancy. Important transcripts and metabolites are marked. (testosterone [TE], 5-beta-androstane-3,17-dione and 5-alpha-pregnane-3,20- dione) were abundant in seahorses in the PG stage (Figure 2E). Unlike meta- bolic regulation, most DEGs participating in steroid hormone metabolism showed the highest expression levels in seahorses in the NF stage. ceptor potential c Intriguingly, five g RA target genes, pregnancy (Figu B D E C B C B C D E D E D D (testosterone [TE], 5-beta-androstane-3,17-dione and 5-alpha-pregnane-3,20- dione) were abundant in seahorses in the PG stage (Figure 2E). Unlike meta- bolic regulation, most DEGs participating in steroid hormone metabolism showed the highest expression levels in seahorses in the NF stage. (testosterone [TE], 5-beta-androstane-3,17-dione and 5-alpha-pregnane-3,20- dione) were abundant in seahorses in the PG stage (Figure 2E). Unlike meta- bolic regulation, most DEGs participating in steroid hormone metabolism showed the highest expression levels in seahorses in the NF stage. ceptor potential cation channel subfamily M member 2 [trpm2]26) (Figure 3B). Intriguingly, five genes involved in RA synthesis and signaling, as well as 18 RA target genes, were also differentially expressed in H. abdominalis during pregnancy (Figure 3A; Table S12). Four RA target genes (fshr, col4a2, col4a1, and krt18) were differentially expressed during pregnancy in both H. erectus and H. abdominalis (Tables S11 and S12). In addition, data from a previous publication suggest that genes involved in RA signaling and syn- thesis tended to have higher expression levels in the pituitary and gonad, whereas RA target genes tended to have higher expression in the brood pouch (Figure S2).11 Antioxidant Defenses Regulated by RA during Brood Pouch Formation and Pregnancy 27 Canthaxanthin, acting as an antioxidant,27 showed the highest fold change (7-fold) in seahorses in the NF stage compared with the UF stage, whereas ophthalmic acid, an oxidant,28 showed the greatest (5.9-fold) change in sea- horses in the UF stage compared with the NF stage (Figure 4A). Compared with seahorses in the NF stage, pregnant seahorses had lower levels of an- tioxidants (canthaxanthin, 19.1-fold; astaxanthin, 50.3-fold) (Figure 4A). Expression of glutathione S-transferase (GST) also significantly decreased in pregnant seahorses (Table S6). Ophthalmic acid and canthaxanthin showed reverse correlations with the same DEGs identified during pouch formation (Figure 4B; Table S21). During The Innovation 1, 100052, November 25, 2020 The Innovation 1, 100052, November 25, 2020 www.cell.com/the-innovation 2 The Innovation Report Report A B C D E F re 2. Expression Pattern and Enrichment Analysis of DEGs and SDMs over Different Pouch Developmental Stages (A–D) Heatmaps of DEGs (A) and S ficantly enriched pathways for DEGs (p < 0.01) (B) and SDMs (p < 0.05) (D). Pink and green bar charts in (B) represent pathways enriched for brood pouch pregnancy processes, respectively; while the blue and orange bar charts in (D) indicate pathways enriched for SDMs identified for pouch formation and p esses, respectively. (E) Co-enriched pathways for both DEGs and SDMs. Co-enriched pathways for both brood pouch formation and pregnancy are la nging patterns of DEGs and SDMs involved in steroid hormone metabolism. The left graph shows a simplified pathway of steroid hormone metabolism and h h h i DEG d SDM ti i ti i t id h t b li B A C D D C E F E Figure 2. Expression Pattern and Enrichment Analysis of DEGs and SDMs over Different Pouch Developmental Stages (A–D) Heatmaps of DEGs (A) and SDMs (C). Significantly enriched pathways for DEGs (p < 0.01) (B) and SDMs (p < 0.05) (D). Pink and green bar charts in (B) represent pathways enriched for brood pouch formation and pregnancy processes, respectively; while the blue and orange bar charts in (D) indicate pathways enriched for SDMs identified for pouch formation and pregnancy processes, respectively. (E) Co-enriched pathways for both DEGs and SDMs. Co-enriched pathways for both brood pouch formation and pregnancy are labeled. (F) Changing patterns of DEGs and SDMs involved in steroid hormone metabolism. Antioxidant Defenses Regulated by RA during Brood Pouch Formation and Pregnancy 27 The left graph shows a simplified pathway of steroid hormone metabolism and the right graph shows changes in DEGs and SDMs participating in steroid hormone metabolism. ll The Innovation 1, 100052, November 25, 2020 3 A B Report The Innovation A B A B Figure 3. Role of RA in Seahorse Brood Pouch Formation and Pregnancy Detailed gene expression data are shown in Tables S11 and S12. (A) Role of RA synthesis and signaling during pregnancy of two seahorse species, H. erectus and H. abdominalis. RA synthesis and signaling pathway is simplified from Duester.11 Upward and downward arrows indicate upregu- lation and downregulation of genes and metabolites during pouch pregnancy, respectively. Each arrow represents one gene. (B) Summarized functions of RA target genes in seahorse brood pouch formation and pregnancy. RA might function upstream of testosterone and progesterone by regulating the expression of fshr and cyp7a1. Oval circles indicate RA target genes that were significantly differentially expressed during pouch formation or pregnancy and three rectangles indicate diverse functions that may be regulated by RA. A Figure 3. Role of RA in Seahorse Brood Pouch Formation and Pregnancy Detailed gene expression data are shown in Tables S11 and S12. A B (A) Role of RA synthesis and signaling during pregnancy of two seahorse species, H. erectus and H. abdominalis. RA synthesis and signaling pathway is simplified from Duester.11 Upward and downward arrows indicate upregu- lation and downregulation of genes and metabolites during pouch pregnancy, respectively. Each arrow represents one gene. g (B) Summarized functions of RA target genes in seahorse brood pouch formation and pregnancy. RA might function upstream of testosterone and progesterone by regulating the expression of fshr and cyp7a1. Oval circles indicate RA target genes that were significantly differentially expressed during pouch formation or pregnancy and three rectangles indicate diverse functions that may be regulated by RA. Antioxidant Defenses Regulated by RA during Brood Pouch Formation and Pregnancy 27 B B B reported,13,30,31 and patterned expression of RA-related genes may play spe- cific roles (e.g., tissue remodeling, organ development, and immune defense) in seahorse brood pouch development.9 For example, PITX2, a transcription factor known for its role in left-right symmetry,24,32 may control seahorse brood pouch symmetry by regulating the Wnt signaling and collagen-related genes.33 Meanwhile, CYBB, a super-oxide-generating enzyme conserved in most eukaryotic groups, is involved in antioxidant immune defense as it is activated in response to pathogens.25 Diverse and pleiotropic functions of genes in the brood pouch may be responsible for their diverse expression pattern (Table S11). pregnancy, both canthaxanthin and astaxanthin were core metabolites of the regulatory network and showed a positive correlation with many genes, particularly those participating in transportation, including ATP-binding cassette superfamily G member 2 (abcg2) (Figure4C; Table S22). Meanwhile, canthaxanthin showed a positive correlation with RA receptor responder pro- tein 3 (rarres3), and astaxanthin showed a positive correlation with UDP glu- curonosyltransferase 2 family, polypeptide A2 (ugt2a2) and beta,beta-caro- tene 15,150-dioxygenase (bco1) during pregnancy (Figure 4C). Rarres3, ugt2a2, and bco1 were all involved in RA metabolism and signaling (Tables S10 and S11). Contradictory results have been reported regarding the relationship be- tween the RA signaling pathway and steroid hormone metabolism. On the one hand, conditional deletion of aldehyde dehydrogenase 1 family member A2 (aldh1a2), the enzyme that metabolizes the vitamin A-intermediate retinal- dehyde into RA, results in embryonic pituitary dysmorphology and alters hor- mone expression.34 On the other hand, TE has been reported as the key up- stream signal that controls RA biogenesis by promoting the expression of genes encoding RA synthesizing enzymes.35 Our results indicate that RA may function upstream of steroid hormone metabolism in seahorses as demonstrated by the consistent decrease in levels of upstream molecules of steroid hormone metabolism with RA during pregnancy (Figures 2F and 3A). Therefore, PRL, PR, and TE play important roles in the maintenance of brood pouch structure and function of brood pouch.4,36 Whether RAregulates pouch development by steroid hormones requires further investigation. DISCUSSION (B and C) Significant correlations between significantly different antioxidants and DEGs (correlation coefficient >0.9 and p < 0.01). Fonts of antioxidants and genes involved in transporting or related to RA are in bold. Metabolites and genes are indicated with blue and green background colors, respectively. Blue and green lines indicate significant positive and negative correlations, respectively. (B) Correlations between canthaxanthin and ophthalmic acid with other DEGs identified for brood pouch formation. (C) Correlations between canthaxanthin (left) and astaxanthin (right) with other DEGs identified during pregnancy. ca4 grik4 Figure 4. Interactional Network of Antioxidant Defense in Seahorse Brood Pouch Development (A) Boxplots showing the changing tendency of one oxidant and two antioxidants over different pouch developmental stages in seahorses. Each point represents one replicate (n = 12). Center line, median; box limits, upper and lower quartiles; whiskers, 1.53 the interquartile range. (B and C) Significant correlations between significantly different antioxidants and DEGs (correlation coefficient >0.9 and p < 0.01). Fonts of antioxidants and genes involved in transporting or related to RA are in bold. Metabolites and genes are indicated with blue and green background colors, respectively. Blue and green lines indicate significant positive and negative correlations, respectively. (B) Correlations between canthaxanthin and ophthalmic acid with other DEGs identified for brood pouch formation. (C) Correlations between canthaxanthin (left) and astaxanthin (right) with other DEGs identified during pregnancy. ity, and the metabolic rate of pregnant seahorses increases from 10% to 52% over pre-gravid levels.42 The increasing metabolic rate could induce oxidative stress during pregnancy by consuming antioxidants and enzymes (Fig- ure 4A), which may weaken the immune defense43 and facilitate successful gestation. The trade-off between immunological tolerance and embryo rejec- tion accompanies the evolution of unique male pregnancy.44 Accordingly, the increased susceptibility to oxidative stress in seahorses during pregnancy might result from the necessity to strike a balance between reproduction and survival that contributes to seahorse reproduction.45,46 intermediate filament family of cytoskeletal proteins;22 all three of which may be involved in brood pouch tissue remodeling. Our results may indicate the various roles of RA in seahorse brood pouch formation and pregnancy by regulating downstream genes with diverse functions. DISCUSSION In this study, we quantified transcripts and metabolites over different sea- horse brood pouch developmental stages and suggested multiple roles of RA in pouch formation and pregnancy by regulating steroid hormone meta- bolism and antioxidant defense. In seahorses, interruption of PRL synthesis leads to disruption of brooding tissues and spontaneous abortions during pregnancy as PRL production is essential for PR and TE secretion.4 However, no study has examined changes in sex steroid hormones over different stages of brood pouch development or how they affect pouch development in seahorses. Here, we detected high levels of PRL and PR in seahorses before pregnancy and high levels of TE during pregnancy (Figure 2F). Further- more, we indicated the different roles of upstream and downstream metab- olites of steroid hormone metabolism in seahorse brood pouch formation and pregnancy (Figure 2F). In addition, we observed nonsynchronous regula- tion at the transcriptional and metabolic levels (Figure 2F). Owing to their diverse roles, the concentrations of sex steroids fluctuate during the breeding cycle of the lined seahorses.29 Future research on specific sex hormones may help to better illustrate the complex roles of steroid hormone meta- bolism in brood pouch development. pouch development by steroid hormones requires further investigation. DEGs that participate in RA signaling and pathway, or that are regulated by RA, were also identified in H. abdominalis, suggesting a conserved role of RA in Hippocampus brood pouch development (Figure 3A; Tables S11 and S12). Of the four RA target genes identified in comparison of the pregnancy pro- cess in both H. erectus and H. abdominalis, fshr was reportedly expressed during the luteal phase in the secretory endometrium of the mammalian uterus23 and may be involved in the formation of placenta-like tissue during pregnancy.37 FSHR may also be involved in pouch formation by affecting TE production.19 Meanwhile, COL4A1 and COL4A2 are the major structural com- ponents of basement membranes20,21 and KRT18 is a member of the In addition to the roles of steroid hormones, our results may indicate that RA regulates 54 downstream target genes with diverse functions, indicating its potential roles in seahorse brood pouch development (Figure 3). DISCUSSION In fact, involvement of RA in teleost development and regeneration has been widely The Innovation 1, 100052, November 25, 2020 www.cell.com/the-innovation www.cell.com/the-innovation 4 The Innovation Report nme2 kirrel3 hgf plxna1 igfbp1 dlgap4 wnt9a clock zmynd8 pdp1 epn3 il17re nalcn gpm6a pex11b sv2a dnm1 map7d2 six1b syt1 methocarbamol ndrg1 methionine sulfoxide nmes1 cacna1i adenosine 5'-monophosphate astaxanthin shank1 cdo1 cecr1a bco2 hykk cep170b grik2 pdcd4 aatk gdi1 12-hydroperoxy-9z,13e,15-octadecatrienoic acid sema6b scg5 camsap2 cngb1 adprhl1 ppfia2 dhcr24 fosl1 ugt2a2 cdh24 adgrb2 pcsk2 syp ncan dnaaf5 tmem260 epb41l3 tnip1 mapt vdac2 oprm1 abcg2 cebpb gabrb2 pcdhgc5 col25a1 grik4 sbk2 rin2 osbpl7 kbp acot2 olfr139 erich3 scn8a pcdhac2 grm3 gria3 cpne2 ampd2 rap1gap mdga2 nav1 gmeb1 col28a1 pcbp4 pfkm uridine diphosphate-n-acetylglucosamine efna2 43347 kcnj11 tnmd taurocholic acid maltotetraose fry slc27a2 cacna2d1 shank3 srl tnrc6c fam184b grin2a qpct sgcb hsd17b12a chrng wfikkn2 egfl6 ciart tubb2a pcdhga11 kcnab1 kcnma1 myo16 rab3c cga ptprn ank3 sepiapterin d-norvaline spon1 5-deoxy-5'-methylthioadenosine cxxc4 adora1 ldlr chd4 slc22a23 ttbk1 zfhx3 sh2d3c rtn1 slc5a3 hapln1 elp6 gfi1b gyc88e acadl grin2b trpm3 rfx1 pomcb guca2a gpt2l zearalenone camkv arhgef28 igsf9 argininosuccinic acid map2 sgip1 cdk5r1 gss lmtk3 gnao1 nrxn2 tnk2 lrp1b kiaa1549 slc4a3 astn1 kcnj12 napb slc1a2 l-cysteine-glutathione gisulfide celsr3 unc80 atp1a1 sez6 ank1 1-methylguanosine gad2 map1a sphkap tubb4b tenm4 csrp3 pcdh15 prickle2 caskin1 grin1 trpc5 akap6 map6 nup210 syn2 cpne5 srcin1 stox2 erc2 zfhx4 dlgap2 pg(16:0/0:0)[u] serpine1 lect1 scn2a ano10 gltscr1l prex2 lta4h tkt cacna2d2 rarres3 chd3 ptch1 abcc4 ppm1b mgat5b klhl41b wdr31 klhl33 acsf2 slc2a9 adnp2 il22ra2 canthaxanthine 8-hydroxy-2-deoxyguanosine slc22a3 amacr kcng2 siah1 ca4 hs3st3b1 thap4 mansc1 arhgef10 0e+00 2e−04 4e−04 6e−04 Stage opthalmic_acid 0e+00 5e−05 1e−04 Stage canthaxanthine 0e+00 2e−05 4e−05 Stage astaxanthin lect1 scn2a ano10 gltscr1l prex2 lta4h tkt cacna2d2 rarres3 chd3 ptch1 abcc4 ppm1b mgat5b klhl41b wdr31 klhl33 acsf2 slc2a9 adnp2 il22ra2 canthaxanthine 8-hydroxy-2-deoxyguanosine slc22a3 amacr kcng2 siah1 ca4 hs3st3b1 thap4 mansc1 arhgef10 astaxanthin shank1 cdo1 cecr1a bco2 hykk cep170b grik2 pdcd4 aatk gdi1 12-hydroperoxy-9z,13e,15-octadecatrienoic acid sema6b scg5 camsap2 cngb1 adprhl1 ppfia2 dhcr24 fosl1 ugt2a2 abcg2 cebpb gabrb2 pcdhgc5 col25a1 grik4 sbk2 rin2 osbpl7 kbp B A C Figure 4. Interactional Network of Antioxidant Defense in Seahorse Brood Pouch Development (A) Boxplots showing the changing tendency of one oxidant and two antioxidants over different pouch developmental stages in seahorses. Each point represents one replicate (n = 12). Center line, median; box limits, upper and lower quartiles; whiskers, 1.53 the interquartile range. DISCUSSION In addition, different tis- sue-specific expression pattern of genes participating in RA signaling and synthesis (Figure 3A) and RA target genes (Figure 3B) may indicate that changes in RA level was resulted from the differential expression of genes in the pituitary and gonad, which further regulated pouch development by regulating the expression of RA target genes in the brood pouch (Figure S2). Meanwhile, in mammals, RA is required to promote differentiation of the mesenchyme into the future uterus and vagina by acting on the Mullerian duct, and plays a vital role in morphological and functional differentiation of female reproductive organs.38,39 Therefore, future studies comparing the specific morphological and functional changes in the reproductive organs of female mammals and male seahorses during pregnancy post-RA chal- lenge may better illustrate the role of RA in male and female pregnancy. In addition, the protective role of transporters against oxidative stress may be responsible for their positive correlation with antioxidants (Figure 4C). For example, ABCG2, an ABC transporter that discharges a variety of toxic sub- stances, protects cells from reactive oxygen species-mediated cell dam- age.47 We also detected a positive correlation between astaxanthin and genes participating in RA signaling (Figure 4C). The consistent decreasing trend in RA expression and antioxidants, as well as their correlation during pregnancy, may suggest that antioxidant defense during brood pouch devel- opment is partly under the control of RA. Low RA levels in the cell milieu are relevant to the expression of several antioxidants and antioxidant enzymes, including GST;18 in mammals, a higher oxidative state caused by RA treat- ment promotes secretion of vascular endothelial growth factor, which may be necessary for successful pregnancy.48 Therefore, decreasing RA and an- tioxidants in the seahorse during pregnancy indicates that an RA-related anti- oxidant defense mechanism plays a key role in successful reproduction dur- ing seahorse pregnancy, which is similar to that in mammals. Correlations This study serves as the first application of metabolome in seahorse for identifying important pathways and metabolites possibly related to brood pouch formation and pregnancy. As the co-enriched pathways of DEGs and SDMs identified in both pouch formation and pregnancy, aminoacyl- tRNA biosynthesis may participate in the formation of placenta-like tissues during male pregnancy by facilitating the growth of collagenous fibers.40 Furthermore, antioxidant defense regulation, which is necessary to maintain a stable low concentration of reactive oxygen species,41 plays a key role in male pregnancy (Figure 4). DISCUSSION (B and C) Significant correlations between significantly different antioxidants and DEGs (correlation coefficient >0.9 and p < 0.01). Fonts of antioxidants and genes involved in transporting or related to RA are in bold. Metabolites and genes are indicated with blue and green background colors, respectively. Blue and green lines indicate significant positive and negative correlations, respectively. (B) Correlations between canthaxanthin and ophthalmic acid with other DEGs identified for brood pouch formation. (C) Correlations between canthaxanthin (left) and astaxanthin (right) with other DEGs identified during pregnancy. DISCUSSION 0e+00 2e−04 4e−04 6e−04 Stage opthalmic_acid 0e+00 5e−05 1e−04 Stage canthaxanthine A C 0e+00 2e−05 4e−05 Stage astaxanthin B 0e+00 2e−04 4e−04 6e−04 Stage opthalmic_acid 0e+00 5e−05 1e−04 Stage canthaxanthine 0e+00 2e−05 4e−05 Stage astaxanthin B A C B A A C nme2 kirrel3 hgf plxna1 igfbp1 dlgap4 wnt9a clock zmynd8 pdp1 epn3 il17re nalcn gpm6a pex11b sv2a dnm1 map7d2 six1b syt1 methocarbamol ndrg1 methionine sulfoxide nmes1 cacna1i adenosine 5'-monophosphate astaxanthin shank1 cdo1 cecr1a bco2 hykk cep170b grik2 pdcd4 aatk gdi1 12-hydroperoxy-9z,13e,15-octadecatrienoic acid sema6b scg5 camsap2 cngb1 adprhl1 ppfia2 dhcr24 fosl1 ugt2a2 cdh24 adgrb2 pcsk2 syp ncan dnaaf5 tmem260 epb41l3 tnip1 mapt vdac2 oprm1 abcg2 cebpb gabrb2 pcdhgc5 col25a1 grik4 sbk2 rin2 osbpl7 kbp acot2 olfr139 erich3 scn8a pcdhac2 grm3 gria3 cpne2 ampd2 rap1gap mdga2 nav1 gmeb1 col28a1 pcbp4 pfkm uridine diphosphate-n-acetylglucosamine efna2 43347 kcnj11 tnmd taurocholic acid maltotetraose fry slc27a2 cacna2d1 shank3 srl tnrc6c fam184b grin2a qpct sgcb hsd17b12a chrng wfikkn2 egfl6 ciart tubb2a pcdhga11 kcnab1 kcnma1 myo16 rab3c cga ptprn ank3 sepiapterin d-norvaline spon1 5-deoxy-5'-methylthioadenosine cxxc4 adora1 ldlr chd4 slc22a23 ttbk1 zfhx3 sh2d3c rtn1 slc5a3 hapln1 elp6 gfi1b gyc88e acadl grin2b trpm3 rfx1 pomcb guca2a gpt2l zearalenone camkv arhgef28 igsf9 argininosuccinic acid map2 sgip1 cdk5r1 gss lmtk3 gnao1 nrxn2 tnk2 lrp1b kiaa1549 slc4a3 astn1 kcnj12 napb slc1a2 l-cysteine-glutathione gisulfide celsr3 unc80 atp1a1 sez6 ank1 1-methylguanosine gad2 map1a sphkap tubb4b tenm4 csrp3 pcdh15 prickle2 caskin1 grin1 trpc5 akap6 map6 nup210 syn2 cpne5 srcin1 stox2 erc2 zfhx4 dlgap2 pg(16:0/0:0)[u] serpine1 lect1 scn2a ano10 gltscr1l prex2 lta4h tkt cacna2d2 rarres3 chd3 ptch1 abcc4 ppm1b mgat5b klhl41b wdr31 klhl33 acsf2 slc2a9 adnp2 il22ra2 canthaxanthine 8-hydroxy-2-deoxyguanosine slc22a3 amacr kcng2 siah1 ca4 hs3st3b1 thap4 mansc1 arhgef10 Stage Stage Stage lect1 scn2a ano10 gltscr1l prex2 lta4h tkt cacna2d2 rarres3 chd3 ptch1 abcc4 ppm1b mgat5b klhl41b wdr31 klhl33 acsf2 slc2a9 adnp2 il22ra2 canthaxanthine 8-hydroxy-2-deoxyguanosine slc22a3 amacr kcng2 siah1 ca4 hs3st3b1 thap4 mansc1 arhgef10 astaxanthin shank1 cdo1 cecr1a bco2 hykk cep170b grik2 pdcd4 aatk gdi1 12-hydroperoxy-9z,13e,15-octadecatrienoic acid sema6b scg5 camsap2 cngb1 adprhl1 ppfia2 dhcr24 fosl1 ugt2a2 abcg2 cebpb gabrb2 pcdhgc5 col25a1 grik4 sbk2 rin2 osbpl7 kbp C Figure 4. Interactional Network of Antioxidant Defense in Seahorse Brood Pouch Development (A) Boxplots showing the changing tendency of one oxidant and two antioxidants over different pouch developmental stages in seahorses. Each point represents one replicate (n = 12). Center line, median; box limits, upper and lower quartiles; whiskers, 1.53 the interquartile range. Detection of DEGs Related to RA in Seahorse We first recorded RA-related genes identified in previous studies, including genes participating in the RA synthesis and signaling pathway,11 as well as those with expres- sion regulated by RA (RA target genes).56 We then searched for RA-related genes from the DEGs identified by comparing both the pouch formation and pregnancy process of H. erectus. To better illustrate the role of RA in Hippocampus, we also searched RA- related genes from among the DEGs identified in the pregnancy process of the pot- bellied seahorse Hippocampus abdominalis.9 We then used in-house R scripts to identify RA-related DEGs associated with the pregnancy process of both species and investigated their expression tendency. To reduce errors caused by inconsistency in gene names from different studies, only gene symbols generated by the Ensembl dataset were used when gene lists identified from different studies were compared. Ethical Approval All the experiments were carried out with an approval from the Experimental Animal Ethics Committee of the South China Sea Institute of Oceanology, Chinese Academy of Sciences, China. DISCUSSION Reproduction isan energetically demanding activ- ll The Innovation 1, 100052, November 25, 2020 5 The Innovation The Innovation Report olome analysis and 3 out of the 12 samples were randomly selected and used for tran- scriptome analysis9,49 (Figure S1). between antioxidants and genes with different functions could indicate the synergistic network of molecules involved in brood pouch development and require further research. Certain limitations were noted in this study. First, is the potential bias asso- ciated with the sampling design for transcriptome analysis. This may result in allometric changes in organ properties due to the samples being collected from animals in different stages. However, quantifying and extracting RNA from half a testis or the pituitary gland from one seahorse is challenging due to their light weight. Although this bias likely had minimal effects on com- parison of the pouch pregnancy process, since both NF and PG seahorses contain mature tissues, it is important to consider these issues when inter- preting transcriptome data for the pouch formation process. In addition, similar to what has been reported in previous seahorse studies,9,49 we used three replicates per group for transcriptomic analysis and chose edgeR to identify DEGs due to its power in dealing with a low number of replicates.50 Despite the reasonable DEG dataset we obtained, Schurch et al.50 stated that only a tiny fraction of DEGs can be detected with three replicates per group. Hence, future transcriptome studies with a higher number of replicates will enrich the gene dataset responsible for pouch development. Furthermore, although we carefully interpreted the correlation networks, we did not focus on the specific correlations between a single transcript and metabolite due to the low sampling size (n = 6) used for Pearson correlation analysis. Hence, although an increased sample size may serve to generate more convincing correlation results, we believe that the reasonable positive correlations pre- sented here between antioxidants and RA-related genes and transporters during pregnancy will not be significantly impacted. Experimental Materials A total of 100 lined seahorses were obtained from the Shenzhen Seahorse Breeding Center (Shenzhen, China) in August 2017. These seahorses were categorized into three developmental stages according to brood pouch morphology: (1) unformed (UF) stage comprising 50 seahorses (5.0–5.7 cm in length and 0.39–0.54 g in weight) with unformed brood pouches; (2) newly formed (NF) stage, which included 25 male seahorses (6.2–7.4 cm in length and 0.78–1.43 g in weight) with newly formed brood pouches; and (3) pregnant (PG) stage, including 25 male seahorses (9.0–9.3 cm in length and 2.42–3.55 g in weight) during pregnancy. Detection of RA, PRL, PR, and TE As a complement to the non-target metabolome, using enzyme-linked immu- nosorbent assay (ELISA) we detected levels of PRL, PR, and TE due to their re- ported roles in seahorse brood pouch development.4 RA was also detected using the same method. There were six replicates per developmental stage. Muscle tis- sues were used for RA, PRL, PR, and TE detection. Muscles in each sample were maintained at 2C–8C after melting and were homogenized by grinding in PBS solution (pH 7.4). These samples were centrifuged at 3,000 rpm for 20 min and the supernatant was collected and reserved for detection. RA (cat. no. RJ- 27871), PRL (cat. no. RJ-21563), TE (cat. no. RJ-21574), and PR (cat. no. RJ- 21719) were all detected according to the ELISA manufacturer's instructions (Re- njie, Shanghai, China). In brief, the purified antibody was used to coat microtiter plate wells and produce a solid-phase antibody. The muscle supernatant was then added to the wells and combined with a horseradish peroxidase (HRP)- labeled antibody to form an antibody-antigen-enzyme-antibody complex. 3,30,5,50-Tetramethylbenzidine (TMB) was added for coloration after washing thoroughly; TMB turned blue after HRP catalysis and yellow after the addition of sulfuric acid solution. Absorbance (optical density) was measured at 450 nm using a microplate reader (Synergy H1, BioTek, USA), and the concentra- tion of the target protein in the sample was determined using the standard curve method. RNA Sequencing and Detection and Analysis of DEGs q g y RNA (three replicates per stage) was extracted using TRIzol (Invitrogen, USA) and RNA quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Each replicate was used to generate an independent library. The RNA sequencing paired-end (PE) libraries were prepared using a NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA), following the manufacturer's protocols. Each library was sequenced on an Illumina HiSeq platform (Illumina, San Diego, CA, USA) with 150 bp PE reads. A total of approximately 489 million clean PE reads were generated. After reads filtering and removal of rRNA-mapped reads,51 high-quality reads were mapped to the reference genome of the lined seahorse (NCBI accession number: PRJNA347499)52 and gene abundance was quantified using the FPKM (fragments per million mapped reads) method.53 Based on the expression pattern of all genes, PCA was performed using R package gmodels (http://www. r-project.org/) for sample relationship analysis. DEGs were detected with the edgeR package using the following criteria: >2-fold change >2 and false discovery rate <0.05.50,54 Using the annotations of all identified transcripts as a reference, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted using DAVID v.6.8 (https://david.ncifcrf.gov/) and KOBAS software,55 respectively. GO terms or KEGG pathways with a calculated p value < 0.01 were considered significantly enriched. Conclusion We showed the general trends in transcript and metabolite changes over different stages of seahorse brood pouch development and highlighted the potential roles of RA, and its target genes, in brood pouch formation and pregnancy. This first application of metabolome analysis in the lined sea- horse uncovered an increased susceptibility to oxidative stress in seahorses during pregnancy, which may have resulted from decreased RA expres- sions. We also identified certain molecules with important functions in sea- horse brood pouch formation and pregnancy, which will promote future specific functional brood pouch studies post-RA and stress challenge in syngnathids. Metabolite Extraction, Detection, and Analysis ( All remaining tissues from the 36 seahorses (12 replicates per stage) were freeze- dried separately (Boyikang, Beijing, China) and made into powder using a tissue crush- ing apparatus (Guangzhou Good, Guangzhou, China). After mixing evenly, approxi- mately 20 mg of tissue per sample was collected for UHPLC-QE Orbitrap/MS analysis according to the standard protocol (Biotree, Shanghai, China). The resulting data ma- trix, including the peak number, sample name, and normalized peak area was imported to the SIMCA 14.1 software package (v.14.1, Umea, Sweden) for PCA. Metabolites with variable importance for the projection values > 1 and p value < 0.05 were identi- fied as SDMs. Commercial databases, including KEGG (http://www.genome.jp/kegg/) and MetaboAnalyst (http://www.metaboanalyst.ca/) were utilized to search for metabolite pathways. Pathways with a calculated p value < 0.05 were considered significantly enriched. REFERENCES (2005). The male seahorse synthesizes and se- cretes a novel C-type lectin into the brood pouch during early pregnancy. FEBS J. 272, 1221–1235. 36. Scobell, S.K., and MacKenzie, D.S. (2011). Reproductive endocrinology of syngnathi- dae. J. Fish Biol. 78, 1662–1680. 9. 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A cost of reproduction: oxidative stress susceptibility is associated with increased egg production in Drosophila mela- nogaster. Exp. Gerontol. 36, 1349–1359. 20. Raija, S., Haka-Risku, T., Prockop, D.J., et al. (1987). Complete primary structure of the a1-chain of human basement membrane (type IV) collagen. FEBS Lett. 225, 188–194. 47. Nie, S., Huang, Y., Shi, M., et al. (2018). Protective role of ABCG2 against oxidative stress in colorectal cancer and its potential underlying mechanism. Oncol. Rep. 40, 2137–2146. 21. Killen, P.D., Francomano, C.A., Yamada, Y., et al. (1987). Partial structure of the human alpha 2(IV) collagen chain and chromosomal localization of the gene (COL4A2). Hum. Genet. 77, 318–324. 48. Wu, J., Hansen, J.M., Hao, L., et al. (2011). Retinoic acid stimulation of VEGF secretion from human endometrial stromal cells is mediated by production of reactive oxygen species. J. Physiol. (Lond.) 589 (Pt 4), 863–875. 22. Lebherzeichinger, D., Krenn, C.G., and Roth, G.A. (2013). Integrative Analysis of Metabolome and Transcriptome 23. La Marca, A., Carducci Artenisio, A., Stabile, G., et al. (2005). Evidence for cycle-depen- dent expression of follicle-stimulating hormone receptor in human endometrium. Gynecol. Endocrinol. 21, 303–306. For each of the two processes (pouch formation and pregnancy), two separate KEGG analyses were conducted for identified DEGs and SDMs. We then compared the enriched KEGG pathways of the DEGs and SDMs identified for the same process to identify the co-enriched pathways of transcriptome and metabolome analysis. Pear- son correlation coefficients between DEGs and SDMs identified for comparison of the same pouch process were further calculated. DEGs and SDMs with a correlation coef- ficient >0.9 and p value < 0.01 were imported to Cytoscape (v.3.3.0),57 which visual- ized and clarified the metabolomic and transcriptome data and built networks be- tween genes and metabolites. Here, we focused on the entire correlation network between the transcriptome and metabolome, rather than one specific correlation be- tween a single transcript and metabolite. Moreover, according to the results published previously,58,59 we did not further correct the p values. 24. Torlopp, A., Khan, M.A., Oliveira, N.M., et al. (2014). The transcription factor Pitx2 po- sitions the embryonic axis and regulates twinning. Elife 3, e03743. 25. Aguirre, J., and Lambeth, J.D. (2010). Nox enzymes from fungus to fly to fish and what they tell us about Nox function inmammals.Free Radic. Biol. Med.49, 1342–1353. l d d ( ) h h l d 26. Knowles, H., Li, Y., and Perraud, A.L. (2013). The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation. Immunol. Res. 55, 241–248. 27. Surai, A.P., Surai, P.F., Steinberg, W., et al. (2003). Effect of canthaxanthin content of the maternal diet on the antioxidant system of the developing chick. Br. Poult. Sci. 44, 612–619. 28. Soga, T., Baran, R., Suematsu, M., et al. (2006). Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione con- sumption. J. Biol. Chem. 281, 16768–16776. Tissue Sampling The seahorse dissection and sampling processes were performed on ice. After dissection, UF-stage seahorse gonads were observed under a microscope, and only male seahorses with testes were retained. Embryos were gently removed from preg- nant seahorses before sampling. A total of 18 individual seahorses in each of the three stages were used. Muscles from six seahorses from each stage were used to detect RA, PRL, PR, and TE; another 12 seahorses in each stage were used for transcriptome and metabolome analyses. From each animal, half of the pituitary gland, testis, and brood pouch tissue were dissected, pooled, and quickly frozen in liquid nitrogen until transcriptome analysis. For the UF stage, skin tissue was collected from the primordial brood pouch area and designated as the pouch sample. After transcriptome sampling, all remaining tissues from each animal were then frozen separately in liquid nitrogen for metabolome analysis. Finally, 12 samples from each stage were used for metab- www.cell.com/the-innovation The Innovation 1, 100052, November 25, 2020 6 The Innovation Report SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j.xinn.2020. 100052. 57. Kohl, M., Wiese, S., and Warscheid, B. (2011). Cytoscape: software for visualization and analysis of biological networks. Methods Mol. Biol. 696, 291–303. 58. Hao, R., Du, X., Yang, C., et al. (2019). Integrated application of transcriptomics and metabolomics provides insights into unsynchronized growth in pearl oyster Pinctada fucata martensii. Sci. Total Environ. 666, 46–56. REFERENCES Keratin 18 and heat-shock pro- tein in chronic kidney disease. Adv. Clin. Chem. 62, 123–149. ll The Innovation 1, 100052, November 25, 2020 7 Innovation Report 49. Qin, G., Zhang, Y., Zhang, B., et al. (2020). Environmental estrogens and progestins disturb testis and brood pouch development with modifying transcriptomes in male-pregnancy lined seahorse Hippocampus erectus. Sci. Total Environ. 715, 136840. 59. Li, B., Song, K., Meng, J., et al. (2017). Integrated application of transcriptomics and metabolomics provides insights into glycogen content regulation in the Pacific oyster Crassostrea gigas. BMC Genomics 18, 713. ACKNOWLEDGMENTS 50. Schurch, N.J., Schofield, P., Gierlinski, M., et al. (2016). How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? RNA 22, 839–851. This research was supported by the National Natural Science Foundation of China (41825013, 41806189), China; the K.C. Wong Education Foundation, China; the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (GML2019ZD0407), China; the Guangdong Special Support Program of Youth Scientific and Technological Innovation (2017TQ04Z269), China; and the ChinaPost- doctoral Science Foundation grant (2019M663151), China. 51. Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359. 52. Lin, Q., Qiu, Y., Gu, R., et al. (2017). Draft genome of the lined seahorse, Hippocampus erectus. Gigascience 6, 1–6. 53. Kim, D., Pertea, G., Trapnell, C., et al. (2013). TopHat2: accurate alignment of tran- scriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36. WEB RESOURCES 55. Wu, J., Mao, X., Cai, T., et al. (2006). KOBAS server: a web-based platform for auto- mated annotation and pathway identification. Nucleic Acids Res. 34, W720–W724. Sequencing raw data of transcriptome were deposited at NCBI under the accession num- ber PRJNA628893. 56. Balmer, J.E., and Blomhoff, R. (2002). Gene expression regulation by retinoic acid. J. Lipid Res. 43, 1773–1808. The authors declare no conflicts of interest. 54. Wang, L., Feng, Z., Wang, X., et al. (2010). Degseq: an R package for identi- fying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136–138. DECLARATION OF INTERESTS The authors declare no conflicts of interest. LEAD CONTACT WEBSITE http://sourcedb.scsio.cas.cn/cn/rck/jcqn/202004/t20200410_5536905.html. The Innovation 1, 100052, November 25, 2020 www.cell.com/the-innovation 8
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Applying robust control theory to solve problems in bio-medical sciences: study of an apoptotic model
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Article Creative Commons: Attribution-No Derivative Works 3.0 Creative Commons: Attribution-No Derivative Works 3.0 Open Access Open Access Trujillo, M., Becerra, V. M. and Hadjiloucas, S. ORCID: https://orcid.org/0000-0003-2380-6114 (2014) Applying robust control theory to solve problems in bio-medical sciences: study of an apoptotic model. Journal of Physics Conference Series, 490 (1). 012140. ISSN 1742-6588 doi: https://doi.org/10.1088/1742-6596/490/1/012140 Available at https://centaur.reading.ac.uk/38003/ It is advisable to refer to the publisher’s version if you intend to cite from the work. See Guidance on citing . nk to this article DOI: http://dx.doi.org/10.1088/1742-6596/490/1/012140 Publisher: Institute of Physics All outputs in CentAUR are protected by Intellectual Property Rights law, including copyright law. Copyright and IPR is retained by the creators or other copyright holders. Terms and conditions for use of this material are defined in the End User Agreement . www.reading.ac.uk/centaur www.reading.ac.uk/centaur Home Search Collections Journals About Contact us My IOPscience Applying robust control theory to solve problems in bio-medical sciences: study of an apoptotic model This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 J. Phys.: Conf. Ser. 490 012140 (http://iopscience.iop.org/1742-6596/490/1/012140) This content has been downloaded from IOPscience. Please scroll down to see the full text. 2014 J. Phys.: Conf. Ser. 490 012140 (http://iopscience.iop.org/1742-6596/490/1/012140) View the table of contents for this issue, or go to the journal homepage for more Download details: Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Published under licence by IOP Publishing Ltd 1 E-mail: s.hadjiloucas@reading.ac.uk Abstract. Biological models of an apoptotic process are studied using models describing a system of differential equations derived from reaction kinetics information. The mathematical model is re-formulated in a state-space robust control theory framework where parametric and dynamic uncertainty can be modelled to account for variations naturally occurring in biological processes. We propose to handle the nonlinearities using neural networks. 1 To whom any correspondence should be addressed. 1. Introduction Robust control theory has been considered as a tool in the biomedical community for model validation, optimal experiment design for hypothesis testing, systems oriented drug design, target identification, or multi-target drug dosage optimization. Unfortunately practical application of this method is limited to models that can be linearized without substantial loss of quantitative or qualitative features. Since most problems arising in biomedical applications do not fall in this category, we present a different perspective where traditional parameter sensitivity analysis is translated into sensitivity of biological features that can be measured experimentally. In this study, biological models of an apoptotic process are studied using models describing a system of differential equations derived from reaction kinetics information. The mathematical model is re-formulated in a state-space robust control theory framework where parametric and dynamic uncertainty can be modelled to account for variations naturally occurring in biological processes. Since the standard robust control theory framework is valid for linear systems only, we re-formulate the problem as piecewise linear allowing for specific non-linearities. We propose to handle the nonlinearities using neural networks. The approach overcomes the limitations imposed by forced linearization and extends the applicability of robust control theory to problems encountered within the Bio-medical community. IP Address: 134.225.215.136 This content was downloaded on 14/03/2014 at 14:10 Please note that terms and conditions apply. 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 M Trujillo 1, V M Becerra and S Hadjiloucas School of Systems Engineering, University of Reading, RG6 6AY, UK M Trujillo 1, V M Becerra and S Hadjiloucas School of Systems Engineering, University of Reading, RG6 6AY, UK 2. Apoptosis model p p Apoptosis is the programed death that regulates the disposal of cells that have been damaged or are no longer required by an organism. Mis-regulation of this process can lead to pathological conditions such as cancer when cells do not die or Alzheimer’s condition where cells die more than needed. Many models of apoptosis are available in the literature and choosing an adequate model can be a difficult process. The current study focuses on the EARM (Extrinsic Apoptosis Reaction Model) described in [1] because in the process of creating, evaluating, calibrating and evolving this model, as 1 To whom any correspondence should be addressed. 1 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 nces 2013 IOP Publishing doi:10.1088/1742-6596/490/1/012140 doi:10.1088/1742-6596/490/1/012140 this is now broadly accepted in the literature as a comprehensive one, where the associated experimental parameters are well calibrated. Furthermore, the model incorporates the latest hypotheses associated to the different reaction mechanisms postulated in the literature. this is now broadly accepted in the literature as a comprehensive one, where the associated experimental parameters are well calibrated. Furthermore, the model incorporates the latest hypotheses associated to the different reaction mechanisms postulated in the literature. p The model of the extrinsic apoptotic pathway published in [1] is formed by four sub-circuits that interact. These describe the entire pathway leading to the apoptotic process. The first circuit starts by the addition of tumor necrosis factor (TNF). The TNF-related apoptosis-inducing ligand (TRAIL), binds to its receptor forming the death-inducing signalling complexes (DISC) and activating pro caspase 8 (pro-C8). The second circuit represents the activation of pro-C8 produces caspase 8 (C8). C8 directly activates caspase-3 (C3) which is responsible for the cleavage of Poly (ADP-ribose) polymerase (PARP), an essential cell substrate for cell survival. The third circuit is a feed-forward loop where C8 promotes the cleavage of Bid to form tBid which activates Bax. Activated Bax promotes the formation of pores in the mitochondria to translocate Smac and cytochrome c (CyC) to the cytosol. Smac binds to X-linked IAP (XIAP) preventing it from inhibiting C3. CyC binds to Apaf and then attracts caspase 9 (C9) to form the apoptosome which in turn produces more C3. 2. Apoptosis model The fourth circuit is a feedback loop where the production of C3 activates caspase 6 (C6) to produce more C8. Figure 1. Simplified block diagram of the apoptosis model divided in four sub-circuits. The input is the death inducing ligand TRAIL and the output is the amount of cleaved PARP. The four blocks are the receptor complex, mitochondria, caspace cascade and feedback. Figure 1. Simplified block diagram of the apoptosis model divided in four sub-circuits. The input is the death inducing ligand TRAIL and the output is the amount of cleaved PARP. The four blocks are the receptor complex, mitochondria, caspace cascade and feedback. The model has 18 species with non-zero initial conditions and 40 additional species that represent modified forms of the initial 18 species (cleaved and short-lived intermediate species). When these species have moved across membranes, they are annotated differently. All species interact via 28 biochemical reactions that use 70 rate reactions including forward, reverse and catalytic rates for each reaction. The biochemical reactions were then translated into 58 ordinary differential equations. y q It has been proposed and experimentally tested by the authors of the model that the apoptotic network works as a variable delay snap-action switch described by the following function: ( ) [ ( ) ] (1) (1) (1) where c is the amount of substrate cleaved at time t, f is the fraction cleaved at the end of the reaction, Td is the delay between TRAIL addition and half the total substrate cleaved (cPARP) and Ts is the switching time between initial and total effector caspase cleavage. This concept is illustrated with the response of cPARP for an initial concentration of TRAIL (Figure 2a). where c is the amount of substrate cleaved at time t, f is the fraction cleaved at the end of the reaction, Td is the delay between TRAIL addition and half the total substrate cleaved (cPARP) and Ts is the switching time between initial and total effector caspase cleavage. This concept is illustrated with the response of cPARP for an initial concentration of TRAIL (Figure 2a). The authors of the model found that Td depends on TRAIL dose, but Ts and f are independent of TRAIL dose. The model parameters were calibrated to follow experimental values of Td, Ts and f. 3. Nonlinear robust control approach The most obvious application of robust control for the apoptosis network is to specify desirable behaviour, translate it as performance functions, define boundaries for uncertain parameters and dynamics and design a controller that achieves the required performance for all the possible uncertain scenarios. However, the apoptosis pathway is not yet fully understood, many contradicting models describe different possible hypotheses and experimental data cannot be yet generated to test them. The model we selected has gone through various iterations of modelling, experimental data fitting and calibration but since the experimental measurements of variables in individual cells destroy the cell, it is not possible yet to monitor the same cell through all its stages in the process from reception of the apoptosis-inducing ligand to cell death. Hence we focus in the use of robust control to provide further understanding of the process, before we could design controllers to intervene at the cellular level. 2. Apoptosis model The cell is destined to die when 10% of the total amount of PARP is cleaved therefore, the value of cPARP = 0.1 PARP is the threshold necessary for cell death (Figure 2b). Experiments with TRIAL doses from 2 to 1,000 ng/ml corresponded to a variation in Td from 140 to 660 minutes. We assume that reducing the TRIAL dose enough to prevent reaching the cPARP threshold will create a cell survival steady 2 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 doi:10.1088/1742-6596/490/1/012140 state for long enough to compare the set of parameter values key for differentiating death and survival states. In figure 2b four species are shown as representative of the different stages of the apoptotic process. The initiator caspase C8* is the only concentration that has a slow increment during the delay period Td followed by a sudden increment after reaching the switching time Ts. Figure 2. a) cPARP trajectory in response to ligand treatment as described in (1), b) main responses of apoptotic system species; initiator caspaces, mitochondria pores, effector caspaces and cleaved PARP. 0 2 4 6 8 10 x 10 5 cPARP time (hrs) cPARP response 0 1 2 3 4 5 6 0 2 4 6 8 10 x 10 4 time (hrs) Main responses of apoptotic system C8*(x5) Mito pores C3(x10) cPARP(x0.1) Cell survival Cell death cPARP threshold Td Ts f (a) (b) 0 2 4 6 8 10 x 10 5 cPARP time (hrs) cPARP response Td Ts f (a) 0 1 2 3 4 5 6 0 2 4 6 8 10 x 10 4 time (hrs) Main responses of apoptotic system C8*(x5) Mito pores C3(x10) cPARP(x0.1) Cell survival Cell death cPARP threshold (b) Figure 2. a) cPARP trajectory in response to ligand treatment as described in (1), b) main responses of apoptotic system species; initiator caspaces, mitochondria pores, effector caspaces and cleaved PARP. ( ) 3.1. General robust control problem Almost any dynamical system can be put in the general form described in figure 3a. In this general form P is a plant consisting of the nominal dynamics of our system augmented with performance functions defined by the user to force the system to satisfy design requirements, K is the controller to be designed to satisfy performance and stability requirements for all the systems resulting from the combination of the plant P with the uncertain block ∆ which contains the allowed variations in parameter space and un-modelled dynamics that may affect the system. w is a vector of the external inputs to the system, z are the outputs to be controlled, u are the inputs to the controller, y are the measurements available from the system, wp and zp are the output and input to the uncertainty block. Figure 3. a) General plant P and controller K, b) Robust control problem for uncertainty ∆, c) Plant and controller integrated in M for the analysis of robust performance P K w z y u M ∆ w z wp zp a) b) P K ∆ w z y u wp zp c) P K ∆ w z y u wp zp P K w z y u ) M ∆ w z wp zp c) c) b) a) Figure 3. a) General plant P and controller K, b) Robust control problem for uncertainty ∆, c) Plant and controller integrated in M for the analysis of robust performance 3 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 The mathematical representation of the perturbed plant P is given by The mathematical representation of the perturbed plant P is given by The mathematical representation of the perturbed plant P is given by { ̇ ( ) ( ) ( ) ( ) ( ) ( ) (2) (2) where x is the state vector of the nominal system, ( ) are the dynamics of the system as described in the nominal model, and are functions of x. 3.1. General robust control problem The nonlinear H∞ problem is to find a controller action u that results in an L2-gain of the system P from w to z less or equal to a given while maintaining system stability. This is an optimisation problem where the allowed disturbance has to be maximized while the control energy has to be minimized. Solutions to this problem come from theories such as differential games, disipativity, viscosity among others. The final result is a Hamilton- Jacobi-Isaacs equation (HJIE) of the form: ( ) ( ) ( ) [ ( ) ( ) ( ) ( )] ( ) ( ) ( ) ( ) (3) (3) Finding a storage function ( ) that satisfies the HJI in most cases is not straightforward. Once a function ( ) satisfying equation 3 is found, the minimum energy control and the maximum disturbance allowed for the system in figure 3 a) are given by: ( ) ( ) ( ) ( ), ( ) ( ) ( ) (4) (4) system given in figure 3 b) the perturbed plant becomes For the system given in figure 3 b) the perturbed plant becomes { ̇ ( ) ( ) ( ) [ ( ) ( )] ( ) [ ( ) ( )] ( ) ( ) ( ) (5) { ̇ ( ) ( ) ( ) [ ( ) ( )] ( ) [ ( ) ( )] ( ) ( ) ( ) (5) (5) where and are unknown functions that belong to the set of admissible uncertainties and are the system parameters that may vary over time in a defined range [2]. For the purpose of this paper, we are not concerned with the design of a controller since we consider the apoptosis network as a whole system with an integrated controller. Figure 3b shows the general framework used in robust control theory for analysis rather than synthesis where P and K form a single block M. This framework allows us to ask how big can the perturbation be and still permit the system to achieve the desired performance. Then, by manipulating the weighting functions (performance criteria) and the allowed uncertainties we can perform sensitivity analysis to measure the impact of various parameters in the system, test different hypothesis for model validation/invalidation and identify underlying mechanisms and interactions between species of the system. 3.2. References [1] Albeck J G, Burke J M, Spencer S L, Lauffenburger D A and Sorger P K 2008 PLoS Biology, 6(12): e299. [2] Abu-Khalaf M, Huang J and Lewis F L 2006 Nonlinear H2/H∞ Constrained Feedback Control (Springer-Verlag London) [3] Gaudet S, Spencer S L, Chen W W, Sorger P K, 2012, PLoS Computational Biology, 1-15 [4] Aliyu M D S 2011 Nonlinear H∞-control, Hamiltonian systems and Hamilton-Jacobi equation (CRC Press, Taylor &Francis, Boca Raton FL, USA) 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 2nd International Conference on Mathematical Modeling in Physical Sciences 2013 IOP Publishing Journal of Physics: Conference Series 490 (2014) 012140 doi:10.1088/1742-6596/490/1/012140 g doi:10.1088/1742-6596/490/1/012140 can identify which once affect the most each feature. To study the effects that variations in the system have on the defined features of interest we have to model such variations as uncertainties ∆ and incorporate them in the system M. Parametric uncertainty is the variation in parameter space that we want to explore and dynamic uncertainty accounts for the unknown dynamics taking place in the system. When a mixture of both uncertainty types has to be analyzed then ∆ becomes a structured uncertainty block that can be separated from the plant as in figure 3b. A solution for the HJIE from equation 3 can be found indirectly by using iterative processes. The HJI is broken in a sequence of linear partial differential equations and the aim becomes finding a possible storage function ( ). In each iteration step, the performance of an initial stabilizing controller is improved until a satisfactory solution to the HJI is found through several iterations. Even though finding ( ) for the linear partial differential equations at each iteration is less complicated than finding it for the HJIE, nevertheless, it is still a difficult task that needs to be solved. One possible way is by approximating ( ) with a neural network at each iteration. A more complete description of the process can be found in [4]. 3.1. General robust control problem Performance functions, modeling uncertainty and solving the HJIE with Neural networks The performance functions are used to modify the nominal system to include desired trajectories. In the case of the apoptosis network, it was suggested in [3] that four features of the time trajectories from cytosolic Smac and cPARP could define the difference between a normal and a pathological response. These are: 1) tPARP which is the time it takes 50% of PARP to be cleaved, 2) tMOMP which is the time it takes for half the Smac to translocate from the mitochondria, 3) tswitch which is the time between the start and finish of PARP cleavage and 4) fPARP which is the amount of PARP cleaved after the experiment or simulation. It is possible then to identify values of interest for these features and define performance functions accordingly. By defining structural perturbations on various species we 4 4. Conclusion So far, because of its implementation complexity, robust control theory has failed to gain popularity as a tool in the bio-medical field. Non-linear robust control theory is still under development and soft computing alternatives are constantly introduced to overcome the complexity of solving problems that cannot be tackled by linearization. The HJIE approach should be seen as an extension to linear - control theory, providing a unifying approach in treating disturbances using a nonlinear counterpart of the Riccati equations which are conventionally used to solve linear problems. Different soft computing methodologies such as neural networks and fuzzy systems theory are worthwhile exploring to overcome the difficulties of solving nonlinear HJI equations. A particular advantage in the general formulation developed for the apoptotic process described in section 3.1 is that it clarifies the parameters that need to be estimated (for example the different uncertainties introduced in the model and their inter-relationship to other biochemical pathways). Furthermore, because of the nice stability and stabilizability properties of passive systems, such as (global) asymptotic stabilizability by pure-gain output-feedback it is considerably desirable in pharmacological or radiological intervention to render the associated biochemical non-linear systems passive. In non-linear control theory, it is well understood that if the state space of a system is reachable from the origin x=0, by an appropriate choice of an input u(t) so that its output is finite, then it can be rendered dissipative. A further goal of the proposed formulation is, therefore, to identify the control points in biochemical reactions that would render interconnected pathways passive through the control of the supply rate (in our case the cPARP threshold in Fig. 2). Since most biochemical pathways can be seen as interconnected dissipative sub-systems of processes described by storage functions, the approach should have a wide domain of applications across Systems Biology. The present work, therefore, serves also as an intellectual scaffolding upon which other Systems-Biology problems can be formulated where seemingly non-linear processes can be controlled though the control of the supply rate to an identified storage function of the system. References 5
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Supplementary Table 1 from Notch3 Pathway Alterations in Ovarian Cancer
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ENTARY TABLE S1. Notch Pathway Alterations in Patients with HGS-OvCa ENTARY TABLE S1. Notch Pathway Alterations in Patients with HGS-OvCa SUPPLEMENTARY TABLE S1. Notch Pathway Alterations in Patients with HGS-OvCa Supplementary Table 1a. Clinical Characteristics of Patients with Notch Pathway Alterations ementary Table 1a. Clinical Characteristics of Patients with Notch Pathway ti Supplementary Table 1b. Mutations of Notch2, Notch3 and Notch 4 in Patients with HGS- OvCa Strand NOTCH2 4853 1 120292618 120292618 + Silent SNP T T C NOTCH2 4853 1 120262689 120262689 + Missense_MutatiSNP C C T NOTCH2 4853 1 120269944 120269944 + Missense_MutatiSNP C C A NOTCH2 4853 1 120294980 120294980 + Missense_MutatiSNP T T C NOTCH3 4854 19 15137882 15137882 + Missense_MutatiSNP G G T NOTCH3 4854 19 15139142 15139142 + Missense_MutatiSNP G G C NOTCH3 4854 19 15142228 15142228 + Silent SNP G G A NOTCH3 4854 19 15142296 15142296 + Silent SNP G G A NOTCH3 4854 19 15159004 15159004 + Silent SNP G G A NOTCH3 4854 19 15139118 15139118 + Silent SNP C C A NOTCH3 4854 19 15142145 15142145 + Missense_MutatiSNP A A C NOTCH3 4854 19 15161209 15161209 + Missense_MutatiSNP A A G NOTCH4 4855 6 32280085 32280085 + Missense_MutatiSNP T T A NOTCH4 4855 6 32296287 32296287 + Silent SNP C C G NOTCH4 4855 6 32296776 32296776 + Missense_MutatiSNP C C T NOTCH4 4855 6 32298323 32298323 + Missense_MutatiSNP G G A NOTCH4 4855 6 32271779 32271779 + Missense_MutatiSNP G G T Variant- Type Reference- Allele Tumor- Seq_Allele1 Tumor- Seq_Allele2 Symbol Gene Id Chromosome Start- position End- position Variant- Classification Supplementary Table 1b. Mutations of Notch2, Notch3 and Notch 4 in Patients with HGS- OvCa ** Notch3 downregulation, and other alterations. *Notch 3 alterations ( amplification, upregulation, and mutation) nregulation, and other alterations. OvCa Supplementary Table 1c. Clinical Characteristics of Patients with NOTCH3 Alterations mentary Table 1c. Clinical Characteristics of Patients with NOTCH3 Alteration *Notch 3 alterations ( amplification, upregulation, and mutation ** Notch3 downregulation, and other alterations. ** Notch3 downregulation, and other alterations.
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Biostratigraphic partition of the Upper Jurassic and Lower Cretaceous (Oxfordian - Lower Berriasian) deposits of the Crimean Mountains for foraminiphers
Vìsnik Harkìvsʹkogo nacìonalʹnogo unìversitetu ìmenì V.N. Karazìna. Serìâ Geologìâ. Geografìâ. Ekologìâ
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БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ За результатами форамініферового аналізу проведено детальне біостратиграфічне розчленування верхньої юри та нижньої крейди (оксфорд-нижній беріас) Гірського Криму. В розрізах виділено п’ять форамініферових комплексів, які хара- ктеризують верхньоюрський (нижньо- верхньооксфордський, нижньокімериджський, верхньотитонський) та нижньокрей- довий (нижньоберіаський) вік порід. Аналіз форамініферових комплексів дозволив в кожному розрізі виявити біостратигра- фічні підрозділи - зони: в нижньому оксфорді - Lenticulina quenstedti-Globuligerina oxfordiana; у верхньому окс- форді - Lenticulina russiensis-Epistomina uhligi; у верхньому титоні - Anchispirocyclina lusitanica-Melathrokerion spirialis, у нижньому беріасі (нижню зону) Protopeneroplis ultragranulatus-Siphoninella antiqua та верстви у нижньому кімериджі – з Epistomina praetatariensis-Globuligerina parva. Виявлені форамініферові зони прослідковуються нами в різних структурно- фаціальних зонах (зона Lenticulina quenstedti-Globuligerina oxfordiana – в Судацько-Феодосійській; зона - Lenticulina russiensis-Epistomina uhligi – в Ай-Петрі-Бабуганській та Судацько-Феодосійській; верстви з Epistomina praetatariensis- Globuligerina parva - в Судацько-Феодосійській; зона Anchispirocyclina lusitanica-Melathrokerion spirialis – в Сухоріченсько- Байдарській, Демерджі-Карабійській та Судацько-Феодосійській; зона Protopeneroplis ultragranulatus-Siphoninella antiqua – в Демерджі-Карабійській та Судацько-Феодосійській). Для кожної форамініферової зони наведена характеристика, яка включає комплекс характерних видів, види, які зникають, з’являються та види, які продовжують своє існування, а також окремі особливості. За літологічними особливостями та палеонтологічною характеристикою у відкладах досліджуваних розрізів виявлені світи та підсвіти. В результаті дослідження уточнені біостратиграфічні характеристики розрізів та доповнено палеонтологічну характеристику верхньоюрських та нижньокрейдових відкладів Гірського Криму. Ключові слова: оксфорд, кімеридж, титон, беріас, форамініферовий комплекс, форамініферова зона, структурно-фа- ціальна зона, Гірський Крим. р р Ю. Б. Доротяк. БИОСТРАТИГРАФИЧЕСКОЕ РАСЧЛЕНЕНИЕ ВЕРХНЕЮРСКИХ И НИЖНЕМЕЛОВЫХ (ОКС- ФОРД - НИЖНИЙ БЕРРИАС) ОТЛОЖЕНИЙ ГОРНОГО КРЫМА ЗА ФОРАМИНИФЕРАМИ. По результатам форами- ниферового анализа проведено детальное биостратиграфическое расчленение верхней юры и нижнего мела (оксфорд-ниж- ний берриас) Горного Крыма. В разрезах установлено пять фораминиферовых комплексов, которые характерезуют верхне- юрский (нижне- верхнеоксфордский, нижнекимериджский, верхнетитонский) и нижнемеловой (нижнеберриасский) возраст пород. Анализ фораминиферовых комплексов позволил в каждом разрезе выявить биостратиграфические подразделе- ния - зоны: в нижнем оксфорде - Lenticulina quenstedti-Globuligerina oxfordiana; в верхнем оксфорде - Lenticulina russiensis- Epistomina uhligi; в верхнем титоне - Anchispirocyclina lusitanica-Melathrokerion spirialis, в нижнем берриасе (нижнюю зону) Protopeneroplis ultragranulatus-Siphoninella antiqua и слои в нижнем кимеридже – с Epistomina praetatariensis-Globuligerina parva. Виявленные фораминиферовые зоны прослеживаются нами в разных структурно-фациальных зонах (зона Lenticulina quenstedti-Globuligerina oxfordiana – в Судакско-Феодосийской; зона - Lenticulina russiensis-Epistomina uhligi – в Ай-Петри- Бабуганской и Судакско-Феодосийской; слои с Epistomina praetatariensis-Globuligerina parva - в Судакско-Феодосийской; зона Anchispirocyclina lusitanica-Melathrokerion spirialis – в Сухореченско-Байдарской, Демерджи-Карабийской и Судакско-Фео- досийской; зона Protopeneroplis ultragranulatus-Siphoninella antiqua – в Демерджи-Карабийской и Судакско-Феодосийской). Вісник Харківського національного університету імені В.Н. Каразіна Вісник Харківського національного університету імені В.Н. Каразіна ( ) Юлія Богданівна Доротяк, мол. наук. співроб., Інститут геологічних наук НАН України, вул. О. Гончара, 55-б, м. Київ, 01601, Україна, е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 © Доротяк Ю. Б. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Чайковський публікують уточнену схему стратиграфії юрських відкладів Гірського Криму з пояснювальною запискою (наведений ярусний, зональний поділ, 31 світа і 8 горизонтів) [25]. В 1993 р. складена В.В. Пермяковим, М.М. Пермя- ковою, Б.П. Чайковським уточнена стратиграфі- чна схема юрських відкладів Гірського Криму (структурно-фаціальне районування, доповни- лась зональна шкала, встановлені і виділені гори- зонти, які є основними одиницями регіональних стратиграфічних підрозділів) [26]. Формування мети статті. Метою статті є стратиграфічне розчленування і кореляція верх- ньоюрських та нижньоберіаських відкладів Гірсь- кого Криму на підставі вивчення форамініфер, та використання отриманих результатів для біостра- тиграфічних розчленувань. Виклад основного матеріалу. Відклади окс- фордського ярусу поширені на території Гірсь- кого Криму досить широко і тісно пов’язані з під- стилаючими верхньокеловейськими породами. За амонітами для нижнього оксфорду Криму виді- лено зону Cardioceras cordatum, якій видповідає форамініферова зона Lenticulina quenstedti - Globuligerina oxfordiana [30]. Вивчення беріаських відкладів Гірського Криму пов′язують з вивченням фауни з «пачки феодосійських мергелів» характерного маркую- чого горизонту розрізу беріаса Східного Криму. В 1974 р. І.Г. Сазоновою і Н.Т. Сазоновим пропону- ється перша схема зонального розчленування примежових відкладів титону і беріасу за амоні- тами [5; 29]. У 1984 р. В.В. Пермяковим [17] за комплексом форамініфер, коралів, брахіопод, двостулкових, брюхоногих та головоногих молю- сків виділено двоякірну світу із стратотипу в Дво- якірній долині, але сам стратотип не був описа- ний. Починаючи з 1984 р. і до сьогодні прово- дяться детальні дослідження примежових відкла- дів юри та крейди в розрізах Гірського Криму, а саме літологічні, магнітостратиграфічні [2], мак- рофауністичні (амоніти, двостулкові молюски, брахіоподи, белемніти, корали, криноідеі, аптихи) [2-5], мікрофауністичні (форамініфери, остра- коди) [4; 7, 9, 10, 12; 13, 14], палінологічні (спори та пилок, диноцисти) [10] та дослідження наноп- ланктону [18-23]. Відклади верхнього оксфорду пов’язані пос- туповим переходом, як з підстиляючими нижньо- оксфордськими, так і перекриваючими нижньокі- мериджськими відкладами. В межах Гірського Криму верхній оксфорд підрозділяється на дві амонітові зони: Gregoryceras transversarium і Epipeltoceras bimammatum. За форамініферами виділено зону Lenticulina russiensis-Epistomina uhligi, яка простежується впродовж всього пізд- нього оксфорду [30]. Кімериджські відклади поширені на території Гірського Криму і утворюють єдиний комплекс порід з оксфордськими відкладами. Виділений лише нижній під’ярус, в якому за амонітами виді- лена лона Streblites tenuilobatus, а за форамініфе- рами встановлені верстви з Epistomina praetata- riensis - Globuligerina parva [30]. Відклади верх- нього кімериджу впевнено не встановлені, це по- яснюється розмивом або переривом в осадконако- пиченні. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Для каждой фораминиферовой зоны приведена характеристика, которая включает комплекс характерных видов, исчезаю- щие виды, появляющиеся, и виды, продолжающие свое существование, а также отдельные особенности. По литологичным особенностям и палеонтологической характеристики в отложениях изучаемых разрезов виявлены свиты и подсвиты. В ре- зультате исследования уточнена биостратиграфическая характеристика разрезов и дополнена палеонтологическая харак- теристика верхнеюрских и нижнемеловых отложений Горного Крыма. Ключевые слова: оксфорд, кимеридж, титон, берриас, фораминиферовый компле турно-фациальная зона, Горный Крым. Вступ. Відклади верхньої юри і крейди в Гір- ському Криму поширені досить широко і предста- влені – оксфордським, кімериджським, титонсь- ким і беріаським ярусами. Ступінь вивченості цих відкладів неоднакова. Найбільш детально вивчені відклади оксфордського, титонського та беріась- кого ярусів. За основу біостратиграфічного по- ділу верхньоюрських та нижньокрейдових відкла- дів Гірського Криму нами взята «Стратиграфічна схема верхньоюрських та нижньокрейдових відк- ладів Гірського Криму» 2013 р. [20]. дових відкладів Гірського Криму висвітлюється в численних роботах, та особливо детально в узага- льнюючих працях, а саме «Стратиграфія УРСР. Том 7. Юра» [27], «Геологія СРСР. Т. 8. Крим» [6], «Стратиграфія та форамініфери верхньої юри та нижньої крейди Криму» [15], «Нижня крейда заходу і півдня України» [16], «Юрські відклади півдня України» [17], «Беріас Гірського Криму» [5], «Стратиграфія верхнього протерозою та фа- нерозою України» [20] та в статтях і тезах [1,3,14]. В 1969 р Є.А. Успенською розроблена стра- тиграфічна схема верхньоюрських відкладів Криму за амонітами [27]. В 1970 р. Є.А. Успен- https://doi.org/10.26565/2410-7360-2018-49-04 В 1969 р Є.А. Успенською розроблена стра- тиграфічна схема верхньоюрських відкладів Криму за амонітами [27]. В 1970 р. Є.А. Успен- https://doi.org/10.26565/2410-7360-2018-49-04 Аналіз останніх досліджень і публікацій. Історія вивчення верхньоюрських та нижньокрей- © Доротяк Ю. Б. - 49 - - 49 - Серія «Геологія. Географія. Екологія», випуск 49 У зв’язку із складною геологічною будовою досліджуваної території та тектонічним району- ванням до цього часу залишається багато невирі- шених питань, в тому числі і остаточно не встано- влена межа примежових відкладів титону та бері- асу. Базою для остаточних висновків може слугу- вати детальний біостратиграфічних аналіз відкла- дів межі юри та крейди Гірського Криму. ською і В.В. Пермяковим удосконалюється стра- тиграфічна схема (наведено районування Гірсь- кого Криму зональний поділ, літологічна і фауні- стична характеристика, виділені світи - ескі-орди- нська, бітацька, уточнено положення перерв) [28]. В 1984 р. В.В. Пермяковим і М.В. Ваніною публі- кується стратиграфічна схема юрських відкладів Гірського Криму (затверджена УРМСК в 1982 р.), яка складається з загальної, регіональної шкал і кореляції місцевих стратиграфічних підрозділів [17]. В 1991 р. В.В. Пермяков, М.М. Пермякова, Б.П. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ та продовжують своє існування поо- динокі нижньооксфордські види Epistomina nemunensis G r i g . , Saracenaria cornucopiae (S c h w a g e r ). Відклади зони Lenticulina russiensis-Epistomina uhligi простежені у нижній частині манджильської світи. (р , ) В Ай-Петрі-Бабуганській СФЗ відклади вер- хнього окфорду представлені мергелем сірим гли- нистим щільним нешаруватим. Форамініферові угрупування багаточисельні, представлені бенто- сними формами. За кількістю видів форми з сек- реційною черепашкою (68%) переважають над аг- лютинуючими (32%). Характерними видами для данної зони є Texstularia jurassica G ü m b e l ., Epistomina stelligeraformis M j a t l . Виявлені ску- пчення Marsonella doneziana D a і n , Lagena apiculata (R e u s s ), Epistomina limbata K a p t ., E. stelligeraformis M j a t l . В угрупуваннях прису- тні бореальні форми - Lenticulina polovinkaensis Put., L. sibirensis (Kosyr.), L. nordformis Put. et Rom. та продовжують своє існування поо- динокі нижньооксфордські види Epistomina nemunensis G r i g . , Saracenaria cornucopiae (S c h w a g e r ). Відклади зони Lenticulina russiensis-Epistomina uhligi простежені у нижній частині манджильської світи. (р ) В Ай-Петрі-Бабуганській СФЗ відклади вер- хнього окфорду представлені мергелем сірим гли- нистим щільним нешаруватим. Форамініферові угрупування багаточисельні, представлені бенто- сними формами. За кількістю видів форми з сек- реційною черепашкою (68%) переважають над аг- лютинуючими (32%). Характерними видами для данної зони є Texstularia jurassica G ü m b e l ., Epistomina stelligeraformis M j a t l . Виявлені ску- пчення Marsonella doneziana D a і n , Lagena apiculata (R e u s s ), Epistomina limbata K a p t ., E. stelligeraformis M j a t l . В угрупуваннях прису- тні бореальні форми - Lenticulina polovinkaensis Put., L. sibirensis (Kosyr.), L. nordformis Put. et Rom. та продовжують своє існування поо- динокі нижньооксфордські види Epistomina nemunensis G r i g . , Saracenaria cornucopiae (S c h w a g e r ). Відклади зони Lenticulina russiensis-Epistomina uhligi простежені у нижній частині манджильської світи. В нижньому беріасі за амонітами виділяють зону Berriasella jacobi в межах, якої встановлено за планктонними форамініферами шари з Globuligerina gulekhensis, в нижній частині яких виділені зони за бентосними форамініферами: ни- жня зона Protopeneroplis ultragranula- tus - Siphoninella antiqua і верхня зона Quadratina tunassica - Siphoninella antiqua. Сукупність цих зон відповідає об'єму амонітовій зоні Berriasella jacobi [5; 30]. [5; 30]. ОКСФОРДСЬКИЙ ЯРУС. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Але, в останні роки отримані деякі до- кази присутності верхнього кімериджу в розрізах Ялтинського амфітеатру [1; 14], та у південно-схі- дному Криму [3], проте стратиграфія верхнього кімериджу на теперішній час недостатньо обґрун- тована і в стратиграфічній схемі 2013 року не ви- діляється. На сьогодні стратиграфічні схеми верхньоюр- ських та нижньокрейдових відкладів Гірського Криму удосконалені, зкорельовані з МСШ 2016 р., відповідно змінено назву деяких амоніто- вих зон, уточнено вік та обсяг окремих світ, роз- ширена палеонтологічна характеристика, прове- дена кореляція з суміжними регіонами. У схему введена шкала за форамініферами, яка розроблена по верхній юрі К.І. Кузнецовою, а по нижній крейді Т.М. Горбачик. Макрофауна (амоніти, дво- стулкові молюски, гастроподи і корали) за даними В.В. Пермякова, Р.Й. Лещуха, В.В. Аркадьєва, Є.Ю. Барабошкіна [30]. Титонські відклади широко поширені на тери- торії Гірського Криму та залягають без чітко ви- раженого кутового неузгодження на кімериджсь- ких відкладах і, можливо, раннього титону, при- падає перерва в осадконакопиченні. Стратиграфі- чно відклади нижнього титону розділені на дві амонітові лони: Kossmatia richteri, яка співпадає з верствами з Epistomina ventriosa - Texstularia densa, і лона Semiformiceras semiforme – - 50 - Вісник Харківського національного університету імені В.Н. Каразіна Бабуганській СФЗ - відслонення 49 км дороги Ялта-Севастополь та в Судацько-Феодосійській СФЗ, а саме у розрізах відслонень поблизу с. Бо- гатівка на півострові Меганом, св. № 3 на г. Кара- даг (рис. 1, 2) [8; 11]. Бабуганській СФЗ - відслонення 49 км дороги Ялта-Севастополь та в Судацько-Феодосійській СФЗ, а саме у розрізах відслонень поблизу с. Бо- гатівка на півострові Меганом, св. № 3 на г. Кара- даг (рис. 1, 2) [8; 11]. відповідає зоні Astacolus laudatus - Epistomina omninoreticulata [30]. У верхньому титонському під’ярусі за форамініферами виділено зону Anchispirocyclina lusitanica - Melathrokerion spirialis, яка співпадає з амонітовою лоною Paraulacosphinctes transitorius [30]. (р , ) В Ай-Петрі-Бабуганській СФЗ відклади вер- хнього окфорду представлені мергелем сірим гли- нистим щільним нешаруватим. Форамініферові угрупування багаточисельні, представлені бенто- сними формами. За кількістю видів форми з сек- реційною черепашкою (68%) переважають над аг- лютинуючими (32%). Характерними видами для данної зони є Texstularia jurassica G ü m b e l ., Epistomina stelligeraformis M j a t l . Виявлені ску- пчення Marsonella doneziana D a і n , Lagena apiculata (R e u s s ), Epistomina limbata K a p t ., E. stelligeraformis M j a t l . В угрупуваннях прису- тні бореальні форми - Lenticulina polovinkaensis Put., L. sibirensis (Kosyr.), L. nordformis Put. et Rom. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Нижній окс- фордський під’ярус. Зона Lenticulina quenstedti- Globuligerina oxfordiana. Нижньооксфордські від- клади досліджувалися в Судацько-Феодосійській структурно-фаціальній зоні (далі скорочення СФЗ) в розрізах відслонень поблизу с. Богатівка на півострові Меганом, Янишарській бухті та в св. № 2 на г. Карадаг (рис. 1, 2) [11]. Відклади представлені зеленувато-сірими темно-сірими алевритистими, піскуватими глинами. Угрупо- вання форамініфер виділені з глинистих фацій. Дані угруповання представлені бентосними (93%) і планктонними (7%) формами. За кількістю видів форми з секреційною черепашкою (60%) перева- жають над аглютинуючими (33%), але за кількі- стю екземплярів їх співвідношення зворотнє, в де- яких розрізах, а саме св. 2 на г. Карадаг, відсло- нення поблизу с. Богатівка. Одним з важливих компонентів даного фауністичного угрупування є присутність у відкладах нижнього оксфорду пла- нктонних форм. Globuligerina oxfordiana (Grig.) – це вид вузького стратиграфічного і широкого гео- графічного поширення. Він зустрічається у розрі- зах нижнього оксфорду в обмеженій кількості (10-30 екз, на 200 г сухої породи). Угрупування малочисельні, рідше багаточисельні. Характер- ними видами для данної зони є Lenticulina attenuatа (Kub. et Zw.), Epistomina nemunensis Grig., Spirillina kübleri M j a t l Globuligerina oxfordiana (G r i g .). Виявлені скупчення Ammodiscus cf. uglicus ehremeevae D a i n , Psammatodendron cf. dichotomicum Neagu, Epistomina nemunensis Grig., Ophthalmidium dilatatum P a a l z ., O. tenuisimum , O. cf. stuifense (Paalz.), Sigmoilina milioliniformis (P a a l z .). Від- клади зони Lenticulina quenstedti-Globuligerina oxfordiana простежені у верхній частині судацької світи. [ ; ] ОКСФОРДСЬКИЙ ЯРУС. Нижній окс- фордський під’ярус. Зона Lenticulina quenstedti- Globuligerina oxfordiana. Нижньооксфордські від- клади досліджувалися в Судацько-Феодосійській структурно-фаціальній зоні (далі скорочення СФЗ) в розрізах відслонень поблизу с. Богатівка на півострові Меганом, Янишарській бухті та в св. № 2 на г. Карадаг (рис. 1, 2) [11]. Відклади представлені зеленувато-сірими темно-сірими алевритистими, піскуватими глинами. Угрупо- вання форамініфер виділені з глинистих фацій. Дані угруповання представлені бентосними (93%) і планктонними (7%) формами. За кількістю видів форми з секреційною черепашкою (60%) перева- жають над аглютинуючими (33%), але за кількі- стю екземплярів їх співвідношення зворотнє, в де- яких розрізах, а саме св. 2 на г. Карадаг, відсло- нення поблизу с. Богатівка. Одним з важливих компонентів даного фауністичного угрупування є присутність у відкладах нижнього оксфорду пла- нктонних форм. Globuligerina oxfordiana (Grig.) – це вид вузького стратиграфічного і широкого гео- графічного поширення. Він зустрічається у розрі- зах нижнього оксфорду в обмеженій кількості (10-30 екз, на 200 г сухої породи). Угрупування малочисельні, рідше багаточисельні. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Янишарська бухта, 17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Рис. 1. Карта-схема фактичного матеріалу. Рис. 1. Карта-схема фактичного матеріалу. Умовні позначення: Умовні позначення: Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська, ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина, 5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла, 10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47), 13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. Янишарська бухта, 17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Умовні позначення: Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська, ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина, 5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла, 10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47), 13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. Янишарська бухта, 17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська, ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина, 5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла, 10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47), 13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. Янишарська бухта, 17. м. Св. Іллі, 18. Двоякірна бухта, 19. с. Богатівка (п-в Меганом). – відслонення з фауною; – відслонення пусті; – свердловини Zw.), Spirillina kübleri Mjatl. Разом з тим вияв- лені скупчення Rhizammina indivisa Brady, Psammatodendron cf. dichotomicum Neagu. Відк- лади верств з Epistomina praetatariensis- Globuligerina parva простежені у верхній частині манджильської світи. переважають над аглютинуючими (2%). Характе- рними видами є Texstularia densa Gofman, Ophthalmidium sigmoiliniformis (A n t . ), Lenticulina nodosa (R e u s s ), L. uspenskajae K . K u z ., L. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ Характер- ними видами для данної зони є Lenticulina attenuatа (Kub. et Zw.), Epistomina nemunensis Grig., Spirillina kübleri M j a t l Globuligerina oxfordiana (G r i g .). Виявлені скупчення Ammodiscus cf. uglicus ehremeevae D a i n , Psammatodendron cf. dichotomicum Neagu, Epistomina nemunensis Grig., Ophthalmidium dilatatum P a a l z ., O. tenuisimum , O. cf. stuifense (Paalz.), Sigmoilina milioliniformis (P a a l z .). Від- клади зони Lenticulina quenstedti-Globuligerina oxfordiana простежені у верхній частині судацької світи. В Судацько-Феодосійській СФЗ верхньоо- ксфордські відклади представлені сланцевими глинами, глинистими алевролітами та мергелями зеленувато-сірими піщанистими, з яких виділені форамініферові угрупування. Угрупування мало- чисельні, представлені бентосними формами. За кількістю видів форми з секреційною черепаш- кою (98%) переважають над аглютинуючими (2%). Характерними видами є Epistomina volgensis Mjatl., E. uhligi Mjatl. Виявлені скупчення Epistomina limbata K a p t . В верхньооксфордсь- ких угрупуваннях продовжують існувати пооди- нокі верхньокеловейські Lenticulina tumida (M j a t l . ) , L. parmula G o f . , Epistomina mosquensis U h l i g . та нижньооксфордські Epistomina nemunensis G r i g . , Saracenaria cornucopiae (S c h w a g e r ). Відклади зони Lenticulina russiensis - Epistomina uhligi просте- жені у нижній частині манджильської світи. КІМЕРИДЖСЬКИЙ ЯРУС. Нижній кіме- риджський під’ярус. Верстви Epistomina praetatariensis-Globuligerina parva. Відклади ниж- нього кімериджу досліджувалися в Судацько-Фе- одосійській СФЗ в розрізі відслонення поблизу с. Богатівка на півострові Меганом (Рис. 1, 2) [11]. Відклади представлені товщею глин з сидери- тами, з яких виділені форамініферові угрупу- вання. Дані угрупування малочисельні, представ- лені бентосними формами, серед яких аглютину- ючі форми представлені більшістю екземплярів (74%) в порівнянні з секреційними (26%). Харак- терними видами є Lenticulina simplex (Kubl. et Верхній оксфордський під’ярус. Зона Lenticulina russiensis-Epistomina uhligi. Верхньоо- ксфордські відклади досліджувалися в Ай-Петрі- Верхній оксфордський під’ярус. Зона Lenticulina russiensis-Epistomina uhligi. Верхньоо- ксфордські відклади досліджувалися в Ай-Петрі- - 51 - Серія «Геологія. Географія. Екологія», випуск 49 Рис. 1. Карта-схема фактичного матеріалу. Умовні позначення: Структурно-фаціальні зони: І – Сухоріченсько-Байдарська, ІІ – Ай-Петрі-Бабуганська, ІІІ – Демерджі-Карабійська, ІV – Судацько-Феодосійська. Розрізи: 1. с. Тилове, 2 с. Орлине, 3. 49 км дороги Ялта-Севастополь, 4. Богата Ущелина, 5. Ай-Петринська Яйла, 6. м. Іограф, 7. г. Ай-Петрі, 8. Урочище Панагії, 9. г. Карабі-Яйла, 10. с. Красноселівка (р. Тонасу), 11. с. Красноселівка (с. Кучук-Узень), 12. хр. Суук-Су (св. 47), 13. смт Богатоє (св. 54), 14. г. Карадаг (св. 2), 15. г. Карадаг (св. 3), 16. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ immensa K. K u z ., Astacolus laudatus G o f m a n , Epistomina ventriosa Esp. et Sigal, Pseudonodosaria tutkovskii (M j a t l . ), Globospirillina caucasica G o f . , Trocholina alpina (Leupold), T. solecensis Biel. et Poz., Ramulina spinata A n t . , Discorbis agalarovae Ant. D. crimicus S c h k h i n a . Виявлені скупчення Epistomina ventriosa E s p . e t S i g a l , Globospirillina caucasica Go f . , Spirillina kübleri M j a t l . В угрупуваннях ще продовжують існу- вання деякі кімериджські види Lenticulina cf. essica Putrja, L. cf. acutata P u t r j a , L. cf. pertiosa P u t r j a , а також з'являються поодинокі нижньо- беріаські, а саме Ammobaculites cf. incostans gracielis (B r a t . e t B r a n d ), Stomatostoecha cf. enisalensis Gorb., Dentalina cf. nana R e u s s , Siphoninella antique G o r b . , Trocholina cf. burlini G o r b . Відклади зони Anchispirocyclina lusitanica-Melathrokerion spirialis простежені у верхній частині деймен-деринської світи. ТИТОНСЬКИЙ ЯРУС. Верхній титонсь- кий під’ярус. Зона Anchispirocyclina lusitanica- Melathrokerion spirialis. У вивчених автором роз- різах мікропалеонтологічно охарактеризовані тільки верхньотитонські відклади. Дані відклади досліджувалися в Сухоріченсько-Байдарській СФЗ (відслонення с. Орлине, с. Тилове), в Демер- джі-Карабійській СФЗ (відслонення на околицях с. Боксан північний схил г. Карабі-Яйла) і в Суда- цько-Феодосійській СФЗ (відслонення с. Красно- селівка, Двоякірна бухта та свердловина № 54 район смт Богате) (Рис. 1, 2) [7; 9; 10; 12]. ТИТОНСЬКИЙ ЯРУС. Верхній титонсь- кий під’ярус. Зона Anchispirocyclina lusitanica- Melathrokerion spirialis. У вивчених автором роз- різах мікропалеонтологічно охарактеризовані тільки верхньотитонські відклади. Дані відклади досліджувалися в Сухоріченсько-Байдарській СФЗ (відслонення с. Орлине, с. Тилове), в Демер- джі-Карабійській СФЗ (відслонення на околицях с. Боксан північний схил г. Карабі-Яйла) і в Суда- цько-Феодосійській СФЗ (відслонення с. Красно- селівка, Двоякірна бухта та свердловина № 54 район смт Богате) (Рис. 1, 2) [7; 9; 10; 12]. ТИТОНСЬКИЙ ЯРУС. Верхній титонсь- кий під’ярус. Зона Anchispirocyclina lusitanica- Melathrokerion spirialis. У вивчених автором роз- різах мікропалеонтологічно охарактеризовані тільки верхньотитонські відклади. Дані відклади досліджувалися в Сухоріченсько-Байдарській СФЗ (відслонення с. Орлине, с. Тилове), в Демер- джі-Карабійській СФЗ (відслонення на околицях с. Боксан північний схил г. Карабі-Яйла) і в Суда- цько-Феодосійській СФЗ (відслонення с. Красно- селівка, Двоякірна бухта та свердловина № 54 район смт Богате) (Рис. БІОСТРАТИГРАФІЧНЕ РОЗЧЛЕНУВАННЯ ВЕРХНЬОЮРСЬКИХ ТА НИЖНЬОКРЕЙДОВИХ (ОКСФОРД - НИЖНІЙ БЕРІАС) ВІДКЛАДІВ ГІРСЬКОГО КРИМУ ЗА ФОРАМІНІФЕРАМИ 1, 2) [7; 9; 10; 12]. В Сухоріченсько-Байдарській СФЗ відклади представлені перешаруванням алевритистих вап- няків, сидеритів і глин. Форамініферові угрупу- вання виділені з глин. Угрупування малочисельні, представлені бентосними формами. За кількістю видів форми з секреційною черепашкою (98%) - 52 - Вісник Харківського національного університету імені В.Н. Каразіна В Демерджі-Карабійській СФЗ відклади пред- ставлені перешаруванням глинистих і піскуватих вапняків, карбонатних глин. Форамініферові угрупування виділені з глин. Угрупування мало- чисельні, представлені бентосними формами. За кількістю видів форми з секреційною черепаш- кою (92%) переважають над аглютинуючими (8%). Характерними видами є Stomatostoecha compressa Gorb., Texstularia densa Gof., Melathrokerion spirialis Gorb. , Belorussiella taurica G o r b . , Lenticulina. vistulae B i e l . E t P o z . , L. immensa K . K u z . , Epistomina ventriosa Esp. et Sigal, Globospirillina caucasica Gof. , Trocholina alpina (Leup.). В угрупуваннях з′являються поодинокі нижньоберіаські види: Lenticulina cf. neocomiana (R o m . ), Trocholina molesta G o r b . Разом з тим виявлені скупчення Epistomina ventriosa Esp. et Sigal, Trocholina molesta G o r b . Відклади зони Anchispirocyclina lusitanica-Melathrokerion spirialis простежені у ни- жній частині беденекирської світи. molesta G o r b . Відклади зони Anchispirocyclina lusitanica-Melathrokerion spirialis простежені у ни- жній частині беденекирської світи. В Судацько-Феодосійській СФЗ відклади представлені вапняками глинистими, сірими, пе- літоморфними, які поступово заміщуються вапня- ками оолітовими, глинами алевритистими вапни- стими, строкатими, дуже міцними, темно-сірими, зеленувато-сірими глинами з частими прошар- ками світло-сірих і рожевих вапняків, глинами алевролітовими темно-зеленувато-сірими плитча- стими з прошарками вапняків брунатно-сірих мі- цних детритових. Форамініферові угрупування виділені з глинистих фацій. Угрупування багато- чисельні, рідше малочисельні, представлені бен- тосними (99%) і планктонними (1%) формами. За кількістю видів форми з секреційною черепаш- кою (84%) переважають над аглютинуючими Рис. 2.1. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів (оксфорд-нижній беріас) Гірського Криму Рис. 2.1. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів (оксфорд-нижній беріас) Гірського Криму Рис. 2.1. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів (оксфорд-нижній беріас) Гірського Криму - 53 - Серія «Геологія. Географія. Екологія», випуск 49 Рис. 2.2. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів (оксфорд-нижній беріас) Гірського Криму (15%) В данному угрупованні виявлені планк- (Leup ) Discorbis crimicus Schok D agalarovae Рис. 2.2. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів (оксфорд-нижній беріас) Гірського Криму Рис. 2.2. Біостратиграфічна схема верхньоюрських-нижньокрейдових відкладів (оксфорд-нижній беріас) Гірського Криму (Leup.), Discorbis crimicus Schok., D. agalarovae Ant., D. infracretaceous Schok. В угрупуваннях з′являються поодинокі нижньоберіаські види: Gaudryina cf. alternans Gorb., Paleotexstularia crimica Gorb., Dorothia cf. praeoxycona Moul., Verneuilina angularis Gorb., Lenticulina cf. neocomiana (R o m . ), L. macra Gorb., Trocholina molesta G o r b . , T. burlini Gorb., T. elongata (Leup.), Siphoninella antique Gorb. Разом з тим ви- явлені скупчення Anchispirocyclina lusitanica (Egger), Lenticulina macra Gorb., Marginulina cf. mollis K. Kuz., Epistomina ventriosa Epit. Вісник Харківського національного університету імені В.Н. Каразіна et Sig., Globospirillina caucasica (Hoff.), Spirillina kübleri (Leup.), Discorbis crimicus Schok., D. agalarovae Ant., D. infracretaceous Schok. В угрупуваннях з′являються поодинокі нижньоберіаські види: Gaudryina cf. alternans Gorb., Paleotexstularia crimica Gorb., Dorothia cf. praeoxycona Moul., Verneuilina angularis Gorb., Lenticulina cf. neocomiana (R o m . ), L. macra Gorb., Trocholina molesta G o r b . , T. burlini Gorb., T. elongata (Leup.), Siphoninella antique Gorb. Разом з тим ви- явлені скупчення Anchispirocyclina lusitanica (Egger), Lenticulina macra Gorb., Marginulina cf. mollis K. Kuz., Epistomina ventriosa Epit. et Sig., Globospirillina caucasica (Hoff.), Spirillina kübleri (15%). В данному угрупованні виявлені планк- тонні форми (Globuligerina gulekhensis Gorb.), які представлені поодинокими видами. Характер- ними видами є Glomospira multivoluta Rom., Anchispirocyclina lusitanica (Egger), Stomatostoecha compressa Gorb., Melathrokerion spirialis Gorb., M. eospirialis Gorb., Texstularia densa Gof., Belorussiella taurica Gorb., Lenticulina immensa K. Кuz., Astacolus laudatus (Reuss), Saracenaria tsaramandrosoensis Epit. et Sig., Pseudonodosaria tutkovskii (Mjatl.), Ophthalmidium sigmoiliniformis (Ant.), Ramulina spinata Ant., Globospirillina caucasica (Goff.), Trocholina alpinа - 54 - Вісник Харківського національного університету імені В.Н. Каразіна існик Харківського національного університе spirialis Gorb., Belorussiella taurica Gorb., Verneuilina angularis Gorb., V. subminuta Gorb., Lenticulina cf. ambanjabensis Espit. et Sig., Discorbis praelongus Gorb., D. crimicus Schok., D. infracretaceous Schok., Pseudolamarckina reussi (A n t . ), Globospirillina neocomiana (Moul.), Siphoninella antique Gorb., Protopeneroplis ultragranulatus Gorb., Trocholina alрina (Leup.), T. molesta Gorb. T. burlini Gorb., T. elongatа (Leup.). В угрупуваннях з′являються поодинокі крейдові види: Triplasia emslandensis acuta Bart. et Br., Gaudryina neocomica Chal., G. gradata Bert., Gaudryiadhella onachensis (Sig.), Quinqueloculina infravalanginiana Bart., Lenticulina neocomiana Gorb., L. macra Gorb., L. cf. onachensis onachensis, L. cf. guttata guttata (ten Dam), L. cf. circumcidanea (Bert.), Saracenaria cretacea Gorb., S cf. navicula (Orb.), Vaginulina recta Reuss, V. truncata Reuss, V. cf. hagenovi (Reuss), Vaginulinopsis cf. grata (Reuss), Nodosaria grassulariformis Bass., N. cf. concinna Reuss, N. paupercula Reuss та ін. Також продовжують іс- нувати поодинокі титонські види - Ammobaculites hagni Bhal. et Ab., Ammodiscus cf. veteranus Kosyr., Gaudryina vadaszi Cush. et Glaz., Ophthalmidium sigmoiliniformis (Ant.), Astacolus praesibirensis Kos., A. aquilonicus (Mjatl.), A. laudatus (Reuss), Lenticulina uspenskajae K. Kuz., L. gregaria Putrja, L. cf. tumidiuscula Pjat., L. cf. tanatcheva Putrja, Epistomina ventriosa Espit. et Sig., Discorbis agalarovae Ant., Trocholina solecensis Biel. et Poz. Разом з тим виявлені скупчення Glomospira gordialis (Jon. et Par.), Epistomina caracаlla anterion Bart. et Br., Discorbis crimicus Schok., Globospirillina caucasica (Hoff.), Lenticulina neocomiana Gorb., L. macra Gorb., Trocholina molesta Gorb. T. elongatа (Leup.). Вісник Харківського національного університету імені В.Н. Каразіна Відклади зони Protopeneroplis ultragranulatus-Siphoninella antiqua простежені у верхній частині двоякірної світи. (Mjatl.), Trocholina elongata (Leup.). Відклади зони Anchispirocyclina lusitanica-Melathrokerion spirialis простежені у нижній частині двоякірної світи. БЕРІАСЬКИЙ ЯРУС. Нижній беріаський під’ярус. Зона Protopeneroplis ultragranulatus - Siphoninella antiqua. Нижньобері- аські відклади досліджувались в Демерджі-Кара- бійській СФЗ (відслонення на околицях с. Боксан північний схил г.Карабі-Яйла) і в Судацько-Фео- досійській СФЗ (відслонення с. Красноселівка, Двоякірна бухта, мис Св. Іллі та свердловинах № 54 район смт Багате, № 47 хр. Суук-Су) (Рис. 1, 2) [7; 9; 10; 12]. В Демерджі-Карабійській СФЗ відклади пред- ставлені глинами карбонатними, з яких виділені форамініферові угрупування. Угрупування мало- чисельні, представлені бентосними формами. За кількістю видів форми з секреційною черепаш- кою (72%) переважають над аглютинуючими (28%). Характерними видами є Triplasia cf. emslandensis acuta B a r t . e t B r a n d , Lenticulina macra G o r b . , Discorbis crimicus S c h o h . , Trocholina molesta G o r b . , T. durlini G o r b . , T. alpina (L e u p . ). В угрупуваннях ще продовжують існувати поодинокі титонські види: Stomatostoecha compressa Gorb., Ammobaculites hagni B h a l l a e t A b b a s , A. infravolgensis M j a t l . , A. subaequalis M j a t l . , Melathrokerion spirialis G o r b . , Texstularia densa G o f ., Quinqueloculina podlubiensis T e r e s . , Lenticulina uspenskajae K. Kuz., Epistomina cf. ventriosa E s p . e t S i g . Відклади зони Protopeneroplis ultragranulatus-Siphoninella antiqua простежені у верхній частині беденекірської світи. р р В Судацько-Феодосійській СФЗ відклади представлені глинами алевритистими, строка- тими, щільними, вапнистими, пісковиками сі- рими дрібнозернистими глинистими, вапняками глинистими, сірими, алевролітами піскуватими та пісковиками глинистими дрібнозернистими та глинами темно-сірими, перешаруванням зелену- вато-сірих тонкоплитчастих глин, алевролітів, ко- ричневато-сірих масивних вапняків і мергелів те- мно-зеленувато-сірих міцних плитчастих, вапня- ком темно-брунатним дрібнозернистим з тонкими лінзами чорної і сірої глини. Форамініферові угрупування виділені з алевролітів, мергелів, глин, вапняків. Угрупування багаточисельні, рі- дше малочисельні, представлені бентосними (99%) і планктонними (1%) формами. За кількі- стю видів форми з секреційною черепашкою (82%) переважають над аглютинуючими (17%). Планктоні форми представлені поодинокі видами Globuligerina gulekhensis (Gorb. et Por.)., G. cf. caucasica Gorb. et Por.. Характерними видами є Stomatostoecha enisalensis Gorb., Melathrokerion Висновки. Література р ур 1. Анікеєва О.В., Жабіна Н.М. Умови седиментації верхньоюрських відкладів Гірського Криму (Ялтинський ам- фітеатр) [Текст] / О.В. Анікеєва, Н.М. Жабіна // Викопна фауна і флора України: Палеоекологічний та стра- тиграфічний аспекти. – Київ, 2009. – С. 99-103. 2. Аркадьев В.В. Новые данные по биостратиграфии берриасских отложений бассейна р. Тонасу (Горный Крым) [Текст] / В.В. Аркадьев, Т.Н. Богданова, С.В. Лобачева // Второе всерос. совещ. «Меловая система России: проблемы стратиграфии палеогеографии». – СПб., 2005. – С. 111-135. 3. Аркадьев В.В., Рогов М.А. Новые данные по биостратиграфии и аммонитам верхнего кимериджа и титона Восточного Крыма [Текст] / В.В. Аркадьев, М.А. Рогов // Стратиграфия. Геол. корреляция. – 2006 (а). – Т. 14, № 2. – С. 90-104. 4. Аркадьев В.В. Биостратиграфия пограничних отложений юры и мела Восточного Крыма [Текст] / В.В. Ар- кадьев, А.А. Федорова, Ю.Н. Савельева, Е.М. Тесакова // Стратиграфия. Геологическая корреляция. 2006. (б) – Т. 14, № 3. – С. 84-112. , 5. Аркадьев В.В. Берриас Горного Крыма [Текст] / В.В. Аркадьев, Т.Н. Богданова, А.Ю. Гужиков, С.В. Лобачева Н.В. Мышкина, Е.С. Платонов, Ю.Н. Савельева, О.В. Шурекова, Б.Т. Янин. – Санкт-Петербург: Лема, 2012. – 473 с. 6. Геология СССР. Т. 8. Крым. Ч. 1. Геологическое описание [Текст] / под. ред. А.В. Сидоренко. – М.: Недра, 1969. – 575 с. 7. Доротяк Юлія Стратифікація титон-беріаських відкладів південно-східної частини Гірського Криму за фо- рамініферами [Текст] / Юлія Доротяк // Палеонтол. зб. – Львів, 2007. – № 39. – C. 125-130. 8. Доротяк Ю.Б. Розподіл форамініфер в оксфордських відкладах Гірського Криму [Текст] / Ю.Б. Доротяк // Палеонтологічні дослідження в Україні: історія, сучасний стан та перспективи: Зб. наук. праць ІГН НАН України. – Київ, 2007. – С. 134-139. 9. Доротяк Ю.Б. Фораминиферовые комплексы пограничных титон-берриасских отложений Горного Крыма [Текст] / Ю.Б. Доротяк // Біостратиграфічні основи побудови стратиграфічних схем фанерозою України: Зб. наук. праць ІГН НАН України. – Київ, 2008. – С. 78-82. 10. Доротяк Ю.Б. Xарактеристика пограничных отложений юры и мела в Горном Крыму (фораминиферы, изве- стковый н аннопланктон, диноцисты, палинокомплексы) [Текст] / Ю.Б. Доротяк, А.В. Матвеев, Е.А. Шевчук // Викопна фауна і флора України: Палеоекологічний та стратиграфічний аспекти: Зб. наук. праць ІГН НАН України. – Київ, 2009. – С. 108-117. 11. Доротяк Ю.Б. Палеогеографія пізньокеловейсько – ранньокімериджського басейну Гірського Криму [Текст] / Ю.Б. Доротяк // Проблеми обґрунтування регіональних стратонів фанерозою України: Матеріали XXXVII сесії Палеонтол. т-ва НАН України. – Київ, 2016. – С. 47-49. 12. Доротяк Ю.Б., Клименко Ю.В. Вісник Харківського національного університету імені В.Н. Каразіна За результатами досліджень авто- ром в розрізах Гірського Криму за форамініфе- рами встановлені біостратиграфічні підрозділи зони: в нижньому оксфорді - Lenticulina quenstedti-Globuligerina oxfordiana; у верхньому оксфорді - Lenticulina russiensis-Epistomina uhligi; у верхньому титоні - Anchispirocyclina lusitanica- Melathrokerion spirialis та у нижньому беріасі, ни- жню зону - Protopeneroplis ultragranulatus- Siphoninella antiqua; і верстви в нижньому кімери- джі з Epistomina praetatariensis-Globuligerina parva. Зона нижнього оксфорду Lenticulina quenstedti-Globuligerina oxfordiana є в верхній ча- стині судацької світи Судацько-Феодосійській СФЗ; зона верхнього оксфорду Lenticulina russiensis-Epistomina uhligi простежується в ниж- ній частині яйлинської світи в Ай-Петрі-Бабуган- ській та в нижній частині манджильської світи в Висновки. За результатами досліджень авто- ром в розрізах Гірського Криму за форамініфе- рами встановлені біостратиграфічні підрозділи зони: в нижньому оксфорді - Lenticulina quenstedti-Globuligerina oxfordiana; у верхньому оксфорді - Lenticulina russiensis-Epistomina uhligi; у верхньому титоні - Anchispirocyclina lusitanica- Melathrokerion spirialis та у нижньому беріасі, ни- жню зону - Protopeneroplis ultragranulatus- Siphoninella antiqua; і верстви в нижньому кімери- джі з Epistomina praetatariensis-Globuligerina parva. Зона нижнього оксфорду Lenticulina quenstedti-Globuligerina oxfordiana є в верхній ча- стині судацької світи Судацько-Феодосійській СФЗ; зона верхнього оксфорду Lenticulina russiensis-Epistomina uhligi простежується в ниж- ній частині яйлинської світи в Ай-Петрі-Бабуган- ській та в нижній частині манджильської світи в - 55 - Серія «Геологія. Географія. Екологія», випуск 49 Судацько-Феодосійській СФЗ; зона верхнього ти- тону Anchispirocyclina lusitanica-Melathrokerion spirialis простежується у верхній частині деймен- деринської світи в Сухоріченсько-Байдарській, у нижній частині беденекирської світи в Демерджі- Карабійській та у нижній частині двоякірної світи Судацько-Феодосійській СФЗ; зона нижнього бе- ріасу Protopeneroplis ultragranulatus-Siphoninella antiqua – простежується у верхній частині бедене- кірської світи Демерджі-Карабійській та у верх- ній частині двоякірної світи Судацько-Феодо- сійській СФЗ; верстви нижнього кімериджу з Epistomina praetatariensis-Globuligerina parva про- стежуються у верхній частині манджильської світи в Судацько-Феодосійській СФЗ. В резуль- таті проведених мікрофауністичних досліджень верхньоюрських та нижньокрейдових (оксфорд- нижній беріас) відкладів у досліджених автором розрізах встановлені біостратиграфічні підроз- діли за форамініферами та доповнено палеонтоло- гічну характеристику даних відкладів Гірського Криму. UDC 56.076:551.762(477) ( ) Yuliya Dorotyak, Junior Researcher, Institute of Geological Sciences of NAS of Ukraine, 55-b O. Honchara St., Kyiv, 01601, Ukraine, е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 ( ) Yuliya Dorotyak, Junior Researcher, Institute of Geological Sciences of NAS of Ukraine, 55-b O. Honchara St., Kyiv, 01601, Ukraine, е-mail: dorotyak78@gmail.com, http://orcid.org/0000-0002-0498-1892 ( ) Yuliya Dorotyak, Junior Researcher, Institute of Geological Sciences of NAS of Ukraine, 55-b O. 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Стратиграфія верхнього протерозою, палеозою та мезозою України [Текст] / Гол. ред. П.Ф. Гожик, ІГН НАН України. – Київ: Логос, 2013. – 638 с. UDC 56.076:551.762(477) BIOSTRATIGRAPHIC PARTITION OF THE UPPER JURASSIC AND LOWER CRETACEOUS (OXFORDIAN - LOWER BERRIASIAN) DEPOSITS OF THE CRIMEAN MOUNTAINS FOR FORAMINIPHERS Introduction. Upper Jurassic and Lower Cretaceous deposits in the Crimean Mountains are widespread and are represented by all stages - Oxfordian, Kimmeridgian, Tithonian, and Berriasian. These deposits have been unevenly studied. The most studied deposits are the Oxfordian, Tithonianian and Berriasian stages. The biostratigraphic division of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea is based on "Stratigraphic scheme of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea" 2013. been unevenly studied. The most studied deposits are the Oxfordian, Tithonianian and Berriasian stages. The biostratigraphic division of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea is based on "Stratigraphic scheme of the Upper Jurassic and Lower Cretaceous deposits of the Mountain Crimea" 2013. Purpose. Detailed biostratigraphic devision of the Upper Jurassic and Lower Cretaceous (Oxfor- dian - Lower Berriasian) of the Mountain Crimea as results of foraminiferal analysis. g p pp p Purpose. Detailed biostratigraphic devision of the Upper Jurassic and Lower Cretaceous (Oxfor- dian - Lower Berriasian) of the Mountain Crimea as results of foraminiferal analysis. Results of the study. There are five foraminiferal complexes in sections that characterize Upper Jurassic (Lower Upper Oxfordian, Lower Kimmeridgian, Upper Tithonian) and Lower Cretaceous (Lower Berriasian) age. Analysis of foraminiferal complexes allowed us to establish biostratigraphical zones in each section: ox- fordiana in the Lower Oxfordian Lenticulina quenstedti-Globuligerina; in the Upper Oxfordian Lenticulina russiensis-Epistomina uhligi; in the Upper Tithonian Anchispirocyclina lusitanica-Melathrokerion spirialis; in the Lower Berriasian (lower zone) Protopeneroplis ultragranulatus-Siphoninella antiqua and layers in the Lower Kimmeridgian with Epistomina praetatariensis-Globuligerina parva. Specified foraminiferal zones were traced by us in different structural-facial zones (zone Lenticulina quenstedti-Globuligerina oxfordiana - in Sudak-Feodosia; zone Lenticulina russiensis-Epistomina uhligi - in Ai-Petri-Babugansk and Sudak-Feodosia; strata layers with Epistomina praetatariensis-Globuligerina parva - in Sudak-Feodosia; zone Anchispirocyclina lusitanica-Melathrokerion spirialis - in Sukhorychensk-Baidar, Demerdzhi-Karabijskaya and Sudak-Feodo- siya; the zone of Protopeneroplis ultragranulatus-Siphoninella antiqua in Demerdzhi-Karabijskaya and Sudak- - 57 - рія «Геологія. Географія. Екологія», випуск 4 Feodosiya). Characteristic features of each foraminiferа zone include a complex of species, disappearing spe- cies, and species that continue to exist, as well as particular features of the complexes. According to lithological peculiarities and paleontological characteristics, suite and sub suite are defined. Feodosiya). Characteristic features of each foraminiferа zone include a complex of species, disappearing spe- cies, and species that continue to exist, as well as particular features of the complexes. References f 1. Anikeyeva O.V., Zhabina N.M. (2009). Sedimentation conditions of the Upper Jurassic deposits in the Crimean Moun- tains, Yalta Amphitheater. Fossil flora and fauna of Ukraine: paleoecological and stratigraphic aspects: Proceedings of the Institute of Gological Sciences of the NAS of Ukraine. Kyiv, 99-103. f f g f f y 2. Arkadiev V.V., Bogdanova T.N., Guzhikov A.Yu., Lobacheva S.V., Myshkina N.V., Platonov E.S., Savelyeva Yu.N., Shurekova O.V., Yanin B.T. (2012). Berriasian stage of the Mountainous Crimea. V.V. Arkadiev, T.N. Bogdanova (Editors). St.-Petersburg: Lema, 473. 3. Arkadyev V.V., Rogov M.A. (2006). New data on biostratigraphy and ammonites of the Upper Kimmeridgian and Tithonian of the Eastern Crimea. Stratigraphy. Geol. correlation.T. 14, No. 2, 90-104. 4. Geology of the USSR. (1969) T. 8. Crimea. Part 1. Geological description: Sidorenko A.V. (Ch. ed.). Moscow: Nedra, 575. lia (2007). The stratification of the Tithonian-Berriassian sediments of the foraminifers in the South-East rimean Mountain Paleontologichnyy zbirnyk №39 125 130 5. Dorotyak Yulia (2007). The stratification of the Tithonian-Berriassian sediments of the foraminifers in part of the Crimean Mountain. Paleontologichnyy zbirnyk, № 39, 125-130. 6. Dorotyak Yu.B. (2007). The distribution of foraminifers in Oxford sediments of the Mountainous Crimea. Paleonto- logical studies in Ukraine: history, present-day state and prospects: Proceedings of the Institute of Geological Sci- ences of the NAS of Ukraine. Kyiv, 134-139. 7. Dorotyak Yu.B. (2008). Phoraminiferۥs complex of the frontier Tithonian-Berriassian sediments of the Crimean Moun- tains. Biostratigraphic fundamentals of creating the stratigraphic schemes of the Phanerozoic of Ukraine: Proceed- ings of the Institute of Geological Sciences of the NAS of Ukraine. Kyiv, 78-82. 8. Dorotyak Yu.B., Matveev A.V., Shevchuk Е.A. (2009). Characteristics of the conterminal scurfs drainage area of Ju- rassic and Cretaceous in the Crimean Mountains (foraminifers, cacareous nanoplankton, dynocysts, palinocom- plexes). Fossil flora and fauna of Ukraine: Paleoecological and stratigraphic aspects: Proceedings of the Institute of Geological Sciences of the NAS of Ukraine. Kyiv, 108-117. 9. Dorotyak Yu.B. (2016). Paleogeography of the Late Callovian - Earley Kimmeridgian Basin of the Crimean Moun- tains. Problems of justification of regional stratigraphic units of Phanerozoic of Ukraine: Proceedings of XXXVII session of Paleontological society of NAS of Ukraine. Kyiv, 47-49. f g y f f y 10. Dorotyak Yu.B., Klimenko Yu.V. (2017). Foraminifers and sponges from the boundary sediments of the Jurassic and Cretaceous of the eastern part of the Crimean Mountains. BIOSTRATIGRAPHIC PARTITION OF THE UPPER JURASSIC AND LOWER CRETACEOUS (OXFORDIAN - LOWER BERRIASIAN) DEPOSITS OF THE CRIMEAN MOUNTAINS FOR FORAMINIPHERS According to lithological peculiarities and paleontological characteristics, suite and sub suite are defined. Conclusions. As a result of the study, the biostratigraphic characteristics of the sections have been spec- ified and the paleontological characteristics of the Upper Jurassic and Lower Cretaceous deposits of the Moun- tain Crimea have been supplemented. Keywords: Oxfordian, Kimmeridgian, Tithonian, Berriasian, foraminiferal complexes, foraminiferal zone, structural-facial zone, Crimean Mountains Keywords: Oxfordian, Kimmeridgian, Tithonian, Berriasian, foraminiferal complexes, foraminiferal zone, structural-facial zone, Crimean Mountains References Paleontological society of Ukraine is 40 years: Proceedings of XXXIX session of Paleontological society of NAS of Ukraine. Kyiv, 89-91. f f g y f f y 11. Zhabina N.M., Anikeyeva O.V. (2012). New data on the age of the biaxial world (Southeast Crimea). Proceedings of XXXIV session of Paleontological society of NAS of Ukraine. Kyiv, 41-43. 12. Kuznetsova K.I., Gorbachik T.H. (1985). Upper Jurassic and Lower Cretaceous stratigraphy and foraminifers of the Crimea. Vachrameev (responsible). Moscow: Nayka, 132. 12. Kuznetsova K.I., Gorbachik T.H. (1985). Upper Jurassic and Lower Cre Crimea. Vachrameev (responsible). Moscow: Nayka, 132. p y . Leschukh R.Y. (1992). Lower Cretaceous of the West and the South of Ukraine. Kyiv: Nauk.Dumka. 208. 14. Leschukh R.Y., Permyakov V.V., Polukhtovich B.M. (1999). Jurassic deposits of southern Ukraine. Lviv: Eurovision, 336. 15. Permyakov V.V, Permyakova M.N., Tchaikovsky P.P. (1991). New scheme of stratigraphy of the Jurassic deposits of the Crimean Mountains. Pres. AU Ukr. Institute of Geological Sciences; 91-12. Kyiv, 38. V.V., Permyakova M.N., Sedenko O.G., Tchaikovsky P.P. (1993). New local stratigraphic subdivisions in Mountains. Geol journ. № 2, 79-81. 16. Permyakov V.V., Permyakova M.N., Sedenko O.G., Tchaikovsky P.P. (1993). New local stratigraphic the Crimean Mountains. Geol journ. № 2, 79-81. the Crimean Mountains. Geol journ. № 2, 79 81. 17. Stratigraphy of the Jurassic deposits in the Ukrainian SSR. Vol. VII. Jurassic. (1969). Kyiv: Naukova Dumka, 101-124. 17. Stratigraphy of the Jurassic deposits in the Ukrainian SSR. Vol. VII. Jurassic. (1969). Kyiv: N 101-124. of the Jurassic deposits in the Ukrainian SSR. Vol. VII. Jurassic. (1969). Kyiv: Naukova Dumka, 18. Stratigraphic scheme of the Jurassic sediments of Ukraine. (1970). Kyiv: Naukova Dumka, 28. g p f f ( y 19. Stratigraphic scheme of Cretaceous deposits of Ukraine and explanatory note. (1971). Kyiv: Na 20. Hozhyk P.F. ed. (2013). Stratigraphy of Upper Proterozoic and Phanerozoic of Ukraine. Vol.1. Stratigraphy of Upper Proterozoic, Paleozoic and Mesozoic of Ukraine. Kyiv Institute of Geological Sciences of the NAS of Ukraine. Logos, 637. - 58 -
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A therapeutic antibody targeting osteoprotegerin attenuates severe experimental pulmonary arterial hypertension
Nature communications
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Corresponding author(s): Last updated by author(s): Lawrie, Allan Sep 27, 2019 Corresponding author(s): Last updated by author(s): Lawrie, Allan Sep 27, 2019 Corresponding author(s): Last updated by author(s): Lawrie, Allan Sep 27, 2019 research | reporting summary Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist . Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure fo in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Ch Policy information about availability of data The data that support the findings of this study are available from the corresponding author upon reasonable request. 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Statistics For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section. n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. 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For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section. n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. nature research | reporting summary nature research | reporting summary Statistics F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable. For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above. Life sciences study design arch | reporting summary he therapeutic efficacy of the Ky3 antibody was tested using antibody derived from 2 separate manufacturing batches in greater tha istinct studies. nvestigators were blinded to treatment groups. All animals were assigned a unique ID and phenotype recorded in a bespoke researc atabase. Sheffield investigators were blinded to antibody labels until after all the phenotype data were collected. Field-specific reporting hat is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections nature research | reporting summary Animals and other organisms Animals and other organisms Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals Wild animals Field-collected samples Ethics oversight N h f ll i f i h l f h d l l b id d i h i Reported in the methods section of the manuscript N/A N/A UK Home office License and Institution Protocol approval as stated in manuscript methods Note that full information on the approval of the study protocol must also be provided in the manuscript. Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. 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Life sciences study design All studies must disclose on these points even when the disclosure is negative. Sample size Data exclusions Replication Randomization Blinding Sample size was calculated using in house data obtained from previous studies. A sample size of at least 4 animals was used to provide greater than 95% power to detect a difference in RVSP of 10 mmHg with a SD of 3 mmHg with 95% confidence. Additional animals were studied in large group comparisons and to obtain sufficient tissue for analysis and allow for multiple comparison testing. No data were excluded from the study. The therapeutic efficacy of the Ky3 antibody was tested using antibody derived from 2 separate manufacturing batches in greater than 3 distinct studies. Animals were randomised to treatment regime and investigators blinded to treatment labels Investigators were blinded to treatment groups. All animals were assigned a unique ID and phenotype recorded in a bespoke research database. Sheffield investigators were blinded to antibody labels until after all the phenotype data were collected. Sample size Data exclusions Replication Randomization Blinding Sample size was calculated using in house data obtained from previous studies. A sample size of at least 4 animals was used to provide greater than 95% power to detect a difference in RVSP of 10 mmHg with a SD of 3 mmHg with 95% confidence. Additional animals were studied in large group comparisons and to obtain sufficient tissue for analysis and allow for multiple comparison testing. No data were excluded from the study. The therapeutic efficacy of the Ky3 antibody was tested using antibody derived from 2 separate manufacturing batches in greater than 3 distinct studies. Animals were randomised to treatment regime and investigators blinded to treatment labels Investigators were blinded to treatment groups. All animals were assigned a unique ID and phenotype recorded in a bespoke research database. Sheffield investigators were blinded to antibody labels until after all the phenotype data were collected. Sample size Sample size was calculated using in house data obtained from previous studies. A sample size of at least 4 animals was used to provide greater than 95% power to detect a difference in RVSP of 10 mmHg with a SD of 3 mmHg with 95% confidence. Additional animals were studied in large group comparisons and to obtain sufficient tissue for analysis and allow for multiple comparison testing. Policy information about studies involving human research participants Policy information about studies involving human research participants Patients were recruited from those undergoing lung transplant at Royal Papworth Hospital NHS Foundation Trust. Informed consent was obtained from all patients who contributed samples to the study. Patients were recruited from those undergoing lung transplant at Royal Papworth Hospital NHS Foundation Trust. Informed consent was obtained from all patients who contributed samples to the study.
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Intraoperative blood transfusion volume is an independent risk factor for postoperative acute kidney injury in type A acute aortic dissection
BMC cardiovascular disorders
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© The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​ mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Liu et al. BMC Cardiovasc Disord (2020) 20:446 https://doi.org/10.1186/s12872-020-01727-3 Liu et al. BMC Cardiovasc Disord (2020) 20:446 https://doi.org/10.1186/s12872-020-01727-3 Open Access Abstract Background:  Type A acute aortic dissection is a life-threatening disease associated with adverse clinical outcomes. Acute kidney injury (AKI) is common after surgery. However, the relationship between intraoperative blood transfu- sion and postoperative AKI remains unclear. Methods:  The records of 130 patients who underwent type A acute aortic dissection surgery from January 2015 to December 2018 were retrospectively analyzed. According to the Kidney Disease Improving Global Outcomes criteria, postoperative AKI was defined based on serum creatinine concentration. Multivariable logistic regression analysis was applied to estimate the independent association between intraoperative blood transfusion volume and the risk of postoperative AKI. Results:  Postoperative AKI was observed in 82 patients (63.08%). The in-hospital mortality was 16.15% (n = 21). Multi- variate logistic regression showed that the amount of intraoperative blood transfusion was independently associated with the risk of postoperative AKI in a dose-dependent manner. Every 200 ml increment of blood transfusion volume was associated with a 31% increase in AKI risk (odds ratio 1.31 and 95% confidence interval 1.01–1.71). Conclusions:  Intraoperative transfusion volume may increase the incidence of postoperative AKI. The mechanism and influence of transfusion thresholds on AKI need to be explored in the future. Keywords:  Blood transfusion, Acute kidney injury, Type A acute aortic dissection dissection, patients often suffer from unstable hemody- namics, coagulopathy, and organ malperfusion. Acute kidney injury (AKI) is a common complication and has been reported as an important risk factor for mortality in patients undergoing cardiac surgery [1, 2]. More than 30% of patients undergoing cardiac surgery with cardio- pulmonary bypass (CPB) are likely to develop AKI and approximately 3% of patients need renal replacement therapy (RRT) for severe AKI. Various risk factors for AKI, including volume depletion, hypotension, anemia, and blood transfusion, were identified in reports [3, 4]. Freeland and colleagues reported that blood transfusion Intraoperative blood transfusion volume is an independent risk factor for postoperative acute kidney injury in type A acute aortic dissection Yanli Liu1†, Yuqiang Shang2†, Ding Long1 and Li Yu1* Background Acute aortic dissection remains a clinical emergency characterized by anterior chest or back pain and has high morbidity and mortality, especially for type A acute aortic dissection. Unlike other elective cardiac sur- gery, in emergency aortic repair for type A acute aortic Full list of author information is available at the end of the article *Correspondence: yuliwhzxyy@163.com †Yanli Liu and Yuqiang Shang have contributed equally to this work 1 Intensive Care Unit, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China Full list of author information is available at the end of the article Outcome variables A AKI was categorized in accordance with the Kidney Dis- ease: Improving Global Outcomes criteria. Postopera- tive AKI was defined by the changes in serum creatinine within 48 h after surgery and classified as stage 1, stage 2, and stage 3 on the basis of postoperative serum cre- atinine. Stage 1 AKI showed more than 50% increase or 0.3 mg/dl increase in serum creatinine. Stage 2 AKI showed 100% increase in serum creatinine. Stage 3 AKI showed 200% or higher or 4  mg/dl increase in serum creatinine or initiation of renal replacement therapy [13]. The last serum creatinine value before surgery was defined as the baseline serum creatinine value. The indi- cations for RRT included acidosis, electrolyte distur- bance and fluid overload. The initiation timing of RRT was determined by the clinician. Our primary endpoint was the occurrence of AKI within 48 h after surgery. Sec- ondary endpoints were stages 2–3 AKI within 48 h after Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 2 of 8 was an independent predictor of postoperative AKI in patients undergoing coronary artery bypass grafting, aor- tic or mitral valve surgery, or combined coronary artery bypass grafting and valve surgery [5]. Previous studies have also probed into the relationship between blood transfusion and AKI, suggesting a 10–20% increase in the risk of AKI in patients with cardiac surgery after being given each unit of perioperative blood transfusion [6–10]. Reasonable perioperative management might curb the development of kidney injury [11, 12]. However, the rela- tionship between intraoperative blood transfusion and postoperative AKI in patients undergoing type A acute aortic dissection surgery remains unclear. For this reason, we conducted a retrospective cohort study on patients with type A acute aortic dissection surgery performed with CPB, and further explored the dose–response asso- ciation between intraoperative blood transfusion volume and postoperative AKI. (Ethics approval NO: 2020.163). Data were anonymous, and the requirement for informed consent was waived. The following patients were not eligible for participation: death within 24  h after surgery; renal artery dissection or occlusion; preoperative shock (a systolic blood pres- sure < 90 mmHg); dialysis-dependent before surgery; no serum creatinine measures after surgery; missing blood transfusion volume. Finally, a total of 130 patients among 158 participants were eligible for the study (Fig. 1). Surgical technique All participants underwent right axillary artery can- nulation for CPB. CPB was initiated after systemic hep- arinization maintaining an activated clotting time longer than 480  s. Cooling was initiated after CPB was estab- lished. The ascending aorta was clamped and cold blood cardioplegia was infused through the coronary ostia to accomplish cardiac arrest. During CPB, the mean arte- rial pressure was maintained between 50 and 70 mmHg. An arterial blood gas monitoring was performed every 30  min and the required hemoglobin level was at least 80 g/L. Whether to replace an aortic valve depended on the condition of the aortic valve. If there was moderate or severe aortic regurgitation, Bentall procedure was per- formed. If the severity of aortic regurgitation was mild, only ascending aorta replacement was performed. For patients with aortic arch involvement in the dissection, total arch replacement combined with stented elephant trunk implantation or hybrid operation was performed. Moderate hypothermia circulatory arrest or deep hypo- thermia circulatory arrest and selective cerebral per- fusion were necessitated in this procedure. Moderate hypothermia was defined as nasopharyngeal tempera- ture between 22 and 25 °C, while deep hypothermia was defined as nasopharyngeal temperature between 18 and 21 °C. After selective cerebral perfusion was terminated, rewarming was initiated. Types of intraoperative blood transfusion included erythrocytes, fresh frozen plasma, platelets and cryoprecipitate. The blood transfusion vol- ume depended on intraoperative bleeding and hemo- globin concentration during CPB. Study population Th d The study was a retrospective cohort design, includ- ing patients who underwent type A acute aortic dis- section surgery in the Center Hospital of Wuhan from January 2015 to December 2018. The diagnosis of type A acute aortic dissection was in accordance with con- trast-enhanced computed tomography. This study was approved by the ethics committees of this hospital Fig. 1  Flowchart of patients underwent type A acute aortic dissection surgery Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 3 of 8 Page 3 of 8 regression was applied to estimate the risk of postop- erative AKI. The variables used in multivariable analysis included clinical factors that are known to increase post- operative AKI to control confounding bias. Nonlinear relationship was explored between intraoperative blood transfusion volume and AKI risk following emergent tho- racic aortic surgery via the smoothing plot generated by generalized additive model with an adjustment for poten- tial confounders. All of the analyses were performed using R (https​://www.R-proje​ct.org, version 3.5.2) and EmpowerStats (https​://www.empow​ersta​ts.com, X&Y Solutions, Inc., Boston, MA) with a two-sided signifi- cance threshold of P < 0.05. surgery, prolonged ventilation, length of stay in the ICU, incidence of renal replacement therapy, adult respira- tory distress syndrome (ARDS), perioperative myocardial infarction, stroke, redo surgery, and in-hospital death. Baseline characteristics A total of 130 patients with type A acute aortic dissection were included in this study (Table  1) with average age 54.74 ± 11.84 years old. 101 patients (77.69%) were males. The major comorbidities included hypertension (62.31%), diabetes (6.15%), coronary artery disease (5.38%), chronic obstructive pulmonary disease (4.62%), and stroke (6.15%).CPB time was 193 (154–233) min, aortic cross clamp time was 101 (81–124) min, and circulatory arrest time was 16 (0–24) min. 82 patients (63.08%) developed AKI, 31 patients (23.85%) on stage 1 AKI, and 51 patients (39.23%) on stage 2 or 3 AKI. Demographic and perioperative characteristics strati- fied according to Improving Global Outcomes criteria [13] were shown in Table 2. The type of procedure, pump time, and the incidence of blood transfusion were associ- ated with AKI development after surgery. No significant difference was found between patients with AKI and those without AKI in terms of age, sex, diabetes, and Cir- culatory arrest time. Clinical datah Worse in-hospital outcomes were observed in patients with AKI, which contributed to approximately 14.62% of in-hospital death. Patients with AKI had long intuba- tion time and ICU stay, and patients with stage 3 AKI were susceptible to renal replacement therapy. The major adverse events, including perioperative ARDS, periopera- tive myocardial infarction, stroke, and redo surgery, were more frequent in patients with AKI than those without. The demographic characteristics were collected dur- ing patient hospitalization from the electronic medical record system. Information of preoperative hemoglobin, eGFR, preoperative and postoperative serum creatinine levels, type of surgical procedure, time on bypass, aortic cross clamp time, circulatory arrest time, the amount and type of intraoperative blood transfusion, reoperation and other events after surgery were also collected. Statistical analysish BMC Cardiovasc Disord (2020) 20:446 Page 4 of 8 Table 1  Demographic characteristics of patients at baseline AKI, acute kidney injury; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure Characteristics All patients (n = 130) Age (years), mean (SD) 54.74 (11.84) Gender, n (%)  Male 101 (77.69)  Female 29 (22.31) Hypertension, n (%) 81 (62.31) Diabetes mellitus, n (%) 8 (6.15) COPD, n (%) 6 (4.62) Stroke, n (%) 8 (6.15) Coronary artery disease, n (%) 7 (5.38) SBP (mmHg), mean (SD) 133.63 (28.41) DBP (mmHg), mean (SD) 72.92 (19.31) eGFR [ml/(min.1.73m2)], mean (SD) 91.86 (40.27) Preoperative hemoglobin (g/l), mean (SD) 122.96 (21.58) Emergency operation, n (%) 88 (67.69) CPB time (min), median (IQR) 193 (154–233) Aortic cross clamp time (min), median (IQR) 101 (81–124) Circulatory arrest time (min), median (IQR) 16 (0–24) Total arch replacement, n (%) 76 (58.46) Semi-arch replacement, n (%) 3 (2.31) Stented elephant trunk, n (%) 72 (55.38) Bentall procedure, n (%) 55 (42.31) Ascending aorta replacement, n (%) 73 (56.15) David procedure, n (%) 2 (1.54) Intraoperative erythrocytes use (ml), median (IQR) 2300 (1300–3675) Intraoperative fresh frozen plasma use (ml), median (IQR) 900 (550–1775) Intraoperative platelets use (ml), median (IQR) 1200 (600–1200) Intraoperative cryoprecipitate use (ml), median (IQR) 500 (213–519) Intraoperative blood transfusion (ml), median (IQR) 4975 (2756–6819) Postoperative AKI, n (%) 82 (63.08) Length of ICU (day) 5.0 (3.0–10.0) Length of mechanical ventilation (day) 2.5 (1.0–6.0) In-hospital mortality, n (%) 21 (16.15) Table 1  Demographic characteristics of patients at baseline AKI, acute kidney injury; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ICU intensive care unit; SD standard deviation; SBP systolic blood pressure Characteristics All patients (n = 130) Age (years), mean (SD) 54.74 (11.84) Gender, n (%)  Male 101 (77.69)  Female 29 (22.31) Hypertension, n (%) 81 (62.31) Diabetes mellitus, n (%) 8 (6.15) COPD, n (%) 6 (4.62) Stroke, n (%) 8 (6.15) Coronary artery disease, n (%) 7 (5.38) SBP (mmHg), mean (SD) 133.63 (28.41) DBP (mmHg), mean (SD) 72.92 (19.31) eGFR [ml/(min.1.73m2)], mean (SD) 91.86 (40.27) Preoperative hemoglobin (g/l), mean (SD) 122.96 (21.58) Emergency operation, n (%) 88 (67.69) CPB time (min), median (IQR) 193 (154–233) Aortic cross clamp time (min), median (IQR) 101 (81–124) Circulatory arrest time (min), median (IQR) 16 (0–24) Total arch replacement, n (%) 76 (58.46) Semi-arch replacement, n (%) 3 (2.31) Stented elephant trunk, n (%) 72 (55.38) Bentall procedure, n (%) 55 (42.31) Ascending aorta replacement, n (%) 73 (56.15) David procedure, n (%) 2 (1.54) Intraoperative erythrocytes use (ml), median (IQR) 2300 (1300–3675) Intraoperative fresh frozen plasma use (ml), median (IQR) 900 (550–1775) Intraoperative platelets use (ml), median (IQR) 1200 (600–1200) Intraoperative cryoprecipitate use (ml), median (IQR) 500 (213–519) Intraoperative blood transfusion (ml), median (IQR) 4975 (2756–6819) Postoperative AKI, n (%) 82 (63.08) Length of ICU (day) 5.0 (3.0–10.0) Length of mechanical ventilation (day) 2.5 (1.0–6.0) In-hospital mortality, n (%) 21 (16.15) Table 1  Demographic characteristics of patients at baseline Table 1  Demographic characteristics of patients at baseline AKI, acute kidney injury; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure complications, including bleeding, ARDS, acute myo- cardial infarction, stroke, AKI, and infection. Statistical analysish Postoper- ative AKI is a common complication of cardiac surgery with CBP. Volume depletion, hypotension, anemia, and blood transfusion increase the incidence of AKI [3, 4]. In this study, We explored the relationship between intraoperative blood transfusion volume and postop- erative AKI for patients with type A acute aortic dis- section surgery and found that intraoperative blood transfusion volume was an independent risk factor for postoperative AKI. Over the last decade, the risk of blood transfusion for AKI has been the focus of many observational studies. Freeland found that patients with postoperative AKI seemed to have a high incidence of increased with an increment of blood transfusion volume, and the curve tends to go down when the amount of blood transfusion is above 4000  ml (Fig. 2). In dose–response analyses, every 200 ml increment of blood transfusion vol- ume could lead to a 31% (odds ratio 1.31 and 95% confi- dence interval 1.01–1.71) increase in AKI risk when the amount of blood transfusion was below 4000 ml. However, null volume association was observed when the amount of blood transfusion was more than 4000 ml (Table 3). Statistical analysish No difference was observed in the pre-transfusion Hgb level between patients with and without AKI. Patients with AKI received much more amount of blood transfu- sion than those without AKI. A nonlinear relationship between intraoperative blood transfusion volume and AKI was explored. We observed the risk of postoperative AKI The parameters were summarized as mean ± standard deviation (SD) for normally distributed continuous vari- ables, median with interquartile range for continuous variables with skewed distribution, and frequency (per- centage) for categorical variables. Multivariable logistic Liu et al. Discussion Type A acute aortic dissection is a severe disease related to increased morbidity and mortality. Patients undergoing cardiac surgery suffer from a series of Liu et al. Discussion BMC Cardiovasc Disord (2020) 20:446 Page 5 of 8 Table 2  Comparison of demographic, preoperative, intraoperative, and postoperative characteristics between patients with AKI and those without AKI AKI, acute kidney injury; ARDS, adult respiratory distress syndrome; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure No AKI Stage 1 AKI Stage 2 or 3 AKI P value N 48 31 51 – Age (years), mean (SD) 53.15 (11.61) 57.13 (11.22) 54.78 (12.39) 0.35 Male, n (%) 33 (68.75%) 27 (87.10%) 41 (80.39%) 0.16 Hypertension, n (%) 22 (45.83%) 25 (80.65%) 34 (66.67%) 0.006 Diabetes mellitus, n (%) 1 (2.08%) 3 (9.68%) 4 (7.84%) 0.37 COPD, n (%) 2 (4.17%) 1 (3.23%) 3 (5.88%) 1.00 Stroke, n (%) 3 (6.25%) 2 (6.45%) 3 (5.88%) 1.00 Coronary artery disease, n (%) 1 (2.08%) 4 (12.90%) 2 (3.92%) 0.12 SBP (mmHg), mean (SD) 131.90 (22.70) 125.39 (27.54) 140.27 (32.43) 0.06 DBP (mmHg), mean (SD) 71.02 (16.53) 66.71 (21.57) 78.49 (19.15) 0.018 eGFR [ml/(min.1.73m2)], mean (SD) 100.57 (30.65) 71.53 (22.21) 94.84 (51.22) 0.007 Preoperative hemoglobin (g/l), mean (SD) 125.87 (18.39) 120.77 (22.74) 124.53 (22.44) 0.61 Emergency operation, n (%) 21 (43.75%) 25 (80.65%) 42 (82.35%) < 0.001 CPB time (min), median (IQR) 176 (120–221) 173 (132–212) 210 (185–244) 0.001 Aortic cross clamp time (min), median (IQR) 96 (76–118) 96 (74–107) 120 (95–127) 0.004 Circulatory arrest time (min), median (IQR) 12 (0–20) 15 (0–24) 18 (8–24) 0.26 Nasopharyngeal temperature (°C), mean (SD) 20.9 (2.1) 20.8 (2.1) 20.4 (2.5) > 0.05 Total arch replacement, n (%) 20 (41.67%) 18 (58.06%) 38 (74.51%) 0.004 Semi-arch replacement, n (%) 2 (4.17%) 1 (3.23%) 0 (0.00%) 0.35 Stented elephant trunk, n (%) 19 (39.58%) 17 (54.84%) 36 (70.59%) 0.008 Bentall procedure, n (%) 27 (56.25%) 14 (45.16%) 14 (27.45%) 0.014 Ascending aorta replacement, n (%) 21 (43.75%) 17 (54.84%) 35 (68.63%) 0.044 Intraoperative erythrocytes use (ml), median (IQR) 1750 (1050–3375) 2200 (1400–2775) 2950 (1950–4350) 0.007 Intraoperative fresh frozen plasma use (ml), median (IQR) 850 (550–1200) 1000 (575–1550) 1000 (550–1925) 0.55 Intraoperative platelets use (ml), median (IQR) 600 (300–1200) 1200 (900–1200) 1200 (750–1200) 0.006 Intraoperative cryoprecipitate use (ml), median (IQR) 350 (0–500) 475 (250–525) 500 (250–560) 0.11 Intraoperative blood transfusion(ml), median (IQR) 2825 (1988–5538) 4400 (2750–5800) 4850 (3650–6875) 0.007 ARDS, n (%) 8 (16.67%) 13 (41.94%) 31 (60.78%) < 0.001 Perioperative myocardial infarction, n (%) 0 (0.00%) 0 (0.00%) 2 (3.92%) 0.342 Redo surgery, n (%) 2 (4.17%) 3 (9.68%) 13 (25.49%) 0.007 Length of ICU (day), median (IQR) 4.00 (2.75–5.25) 6.00 (3.50–6.50) 9.00 (3.00–14.50) 0.001 Length of mechanical ventilation (day), median (IQR) 2.00 (1.00–3.00) 2.00 (1.00–4.00) 5.00 (2.00–7.50) < 0.001 In-hospital mortality, n (%) 2 (4.17%) 5 (16.13%) 14 (27.45%) 0.005 Table 2  Comparison of demographic, preoperative, intraoperative, and postoperative characteristics between patients with AKI and those without AKI f demographic, preoperative, intraoperative, and postoperative characteristics between patients h AKI AKI, acute kidney injury; ARDS, adult respiratory distress syndrome; COPD, chronic obstructive pulmonary; CPB, cardiopulmonary bypass; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; ICU, intensive care unit; SD, standard deviation; SBP, systolic blood pressure blood transfusion [5]. Discussion Koch et al. reported that periop- erative red blood cell transfusion was the independent predictor of postoperative AKI in patients undergoing isolated coronary artery bypass grafting [7]. Mean- while, blood transfusion was strongly associated with AKI in a retrospective cohort study of patients with acute coronary syndrome undergoing PCI, which was consistent with our study to some extent [3]. However, some studies reported null association between blood transfusion and postoperative AKI, presumably due to the limited sample size and heterogeneous patients’ characteristics [14, 15]. To the best of our knowledge, the current study is the first to investigate the association of intraopera- tive blood transfusion volume with postoperative AKI in patients undergoing type A acute aortic dissection surgery. We observed a nonlinear relationship between intraoperative blood transfusion volume and postop- erative AKI. A 31% increment of the postoperative AKI risk was observed for every 200  ml of blood transfu- sion received when the total volume was lower than 4000  ml. However, no difference was observed when the blood transfusion volume was higher than 4000 ml. Different from the nonlinear association reported in Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 6 of 8 Fig. 2  The nonlinear relationship between intraoperative blood transfusion volume and postoperative AKI. The results were generated utilizing generalized additive model and adjusted for age, gender, hypertension, diabetes mellitus, eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery. The red line indicates the risk of AKI and the blue dot line indicates 95% confidence intervals was correlated with worse in-hospital outcomes. These associations were confirmed in prior studies [16, 17].h i The mechanisms through which blood transfusion volume increased the risk of postoperative AKI are not illuminated, but several potential explanations are con- sidered. The pathogenesis of AKI is mostly related to inflammation, renal hypoxia, impairment of tissue oxy- gen delivery, and oxidative stress. Patients who undergo cardiac surgery with CPB are subjected to the initiation phase of ischemia–reperfusion kidney injury and the extension phase of kidney injury. The initiation phase is characterized by renal artery vasoconstriction and increased oxygen consumption. Discussion Patients in the initia- tion phase have an increased risk of aggravation to the extension phase, which may lead to AKI development if they cannot recover or if they suffer from other exter- nal risk factors, such as blood transfusion, ischemia, and anemia [10, 18, 19]. Red blood cell transfusion is com- mon during cardiac surgery with CPB. Red blood cells during storage undergo a series of changes, including decreased deformability, increased fragility, progressive hemolysis, and accumulation of free hemoglobin and iron that may accelerate organ tissue dysfunction [20– 22]. Macrophages under normal circumstances dispose of red blood corpuscles and release iron to the circula- tion bound to transferrin. However, a certain amount of free hemoglobin and iron released by macrophages fails to bind to the iron binding sites on the iron-carrier pro- tein transferrin in circulation after blood transfusion and is toxic to the kidneys [10, 23]. Also, dysregulation of renal haemodynamics is typical of AKI. Platelets play a crucial part in renal haemodynamic processes by regu- lating the endothelial vascular permeability. Activation of platelets could release granules and microvesicles, which are associated with the pathophysiology of AKI [24, 25]. Other evidence further links blood transfusion and AKI. Patients with persistent positive fluid balance are likely inclined to suffer from kidney injury. Increased kidney interstitial pressure and kidney venous pressure due to Fig. 2  The nonlinear relationship between intraoperative blood transfusion volume and postoperative AKI. The results were generated utilizing generalized additive model and adjusted for age, gender, hypertension, diabetes mellitus, eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery. The red line indicates the risk of AKI and the blue dot line indicates 95% confidence intervals Fig. 2  The nonlinear relationship between intraoperative blood transfusion volume and postoperative AKI. The results were generated utilizing generalized additive model and adjusted for age, gender, hypertension, diabetes mellitus, eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery. The red line indicates the risk of AKI and the blue dot line indicates 95% confidence intervals our study, Karkouti et  al. Discussion Fluid overload causes visceral and peripheral edema, which leads to difficult organic functioning and delays the AKI diagnosis due to the dilution of serum creatinine [26, 27]. A randomized trial concluded that goal-directed therapy decreased blood transfusion and the incidence of AKI attributing to this effect to improved fluid management [28]. Thus, the important risk factors may be interrelated to AKI development. the excess fluid that overwhelms the limited capacity of the kidneys lead to the decrease in glomerular filtra- tion rate. Fluid accumulation in patients may be asso- ciated with increased mortality. Fluid overload causes visceral and peripheral edema, which leads to difficult organic functioning and delays the AKI diagnosis due to the dilution of serum creatinine [26, 27]. A randomized trial concluded that goal-directed therapy decreased blood transfusion and the incidence of AKI attributing to this effect to improved fluid management [28]. Thus, the important risk factors may be interrelated to AKI development. Abbreviations AKI A k d 7. Koch CG, Li L, Duncan AI, Mihaljevic T, Cosgrove DM, Loop FD, et al. Morbidity and mortality risk associated with red blood cell and blood- component transfusion in isolated coronary artery bypass grafting. CRIT Care Med. 2006;34(6):1608–16. Abbreviations AKI: Acute kidney injury; ARDS: Adult respiratory distress syndrome; COPD: Chronic obstructive pulmonary; CPB: Cardiopulmonary bypass; DBP: Diastolic blood pressure; eGFR: Estimated glomerular filtration rate; ICU: Intensive care unit; RRT​: Renal replacement therapy; SD: Standard deviation; SBP: Systolic blood pressure. 8. De Santo L, Romano G, Della CA, de Simone V, Grimaldi F, Cotrufo M, et al. Preoperative anemia in patients undergoing coronary artery bypass grafting predicts acute kidney injury. J Thorac Cardiovasc Surg. 2009;138(4):965–70. References 1. Li Y, Yang N, Duan W, Liu S, Yu S, Yi D. Acute aortic dissection in China. Am J Cardiol. 2012;110(7):1056–61. 1. Li Y, Yang N, Duan W, Liu S, Yu S, Yi D. Acute aortic dissection in China. Am J Cardiol. 2012;110(7):1056–61. 2. Ko T, Higashitani M, Sato A, Uemura Y, Norimatsu T, Mahara K, et al. Impact of acute kidney injury on early to long-term outcomes in patients who underwent surgery for type a acute aortic dissection. Am J Cardiol. 2015;116(3):463–8. 3. Karrowni W, Vora AN, Dai D, Wojdyla D, Dakik H, Rao SV. Blood transfusion and the risk of acute kidney injury among patients with acute coronary syndrome undergoing percutaneous coronary intervention. Circ Cardio- vasc Interv. 2016;9(9):e003279. Competing interests Competing interests The authors declare that they have no competing interests. Discussion found a linear relationship between blood transfusion volume and risk of AKI in a randomized goal-directed fluid resuscitation study [10]. In their study, non-cardiac surgery patients were included and the amount of blood transfusion was rela- tively small, which might account for the difference. f AKI is a common adverse event for type A acute aor- tic dissection. Our study also evaluated the relation- ship between postoperative AKI and in-hospital major adverse events. An association was found between the incidence of AKI and major adverse events. Patients with AKI were associated with long ventilation time and prolonged ICU stay. Moreover, the severity of AKI Table 3  Multivariable logistic regression analysis to estimate the independent association between blood transfusion volume and the risk of postoperative AKI Model 1: Crude model Model 2: Adjusted for age, gender, hypertension, and diabetes mellitus Model 3: Additional adjust for eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery Variable Model 1 P value Model 2 P value Model 3 P value Blood transfusion volume ≤ 4000 ml  Increase per 200 ml 1.29 (1.08, 1.53) 0.004 1.30 (1.07, 1.57) 0.008 1.31 (1.01, 1.71) 0.044 Blood transfusion volume > 4000 ml  Increase per 200 ml 1.02 (0.96, 1.07) 0.57 1.01 (0.96, 1.07) 0.70 1.02 (0.96, 1.08) 0.61 Table 3  Multivariable logistic regression analysis to estimate the independent association betwe volume and the risk of postoperative AKI j g , g , yp , Model 3: Additional adjust for eGFR, emergency operation, CPB time, aortic cross clamp time, nasopharyngeal temperature, total arch replacement, semi-arch replacement, stented elephant trunk, Bentall procedure, ascending aorta replacement, intraoperative erythrocytes use, intraoperative platelets use and redo surgery Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 7 of 8 University of Science and Technology for his contribution to the statistical analysis. University of Science and Technology for his contribution to the statistical analysis. the excess fluid that overwhelms the limited capacity of the kidneys lead to the decrease in glomerular filtra- tion rate. Fluid accumulation in patients may be asso- ciated with increased mortality. Ethics approval and consent to participate t cs app o a a d co se t to pa t c pate This study was approved by the ethics committees of the central hospital of Wuhan (Ethics approval NO: 2020.163). The requirement for informed consent was waived. Author details 1 Author details 1 Intensive Care Unit, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. 2 Department of Cardiothoracic Surgery, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China. Received: 20 February 2020 Accepted: 6 October 2020 Received: 20 February 2020 Accepted: 6 October 2020 Authors’ contributions YL and LYL conceived and supervised the study; LYL and SYQ designed experi- ments; LYL, SYQ and LD performed information acquisition and examination analysis; LYL wrote the manuscript; LD and YL made manuscript revisions. All authors read and approved the final manuscript. Conclusions Our study showed that the amount of intraoperative blood transfusion was an independent risk factor for postoperative AKI in patients with type A acute aor- tic dissection. Intraoperative transfusion volume might increase the incidence of postoperative AKI, and the mechanism underlying transfusion thresholds on AKI should be further investigated. 4. Mehta RL, Burdmann EA, Cerda J, Feehally J, Finkelstein F, Garcia-Garcia G, et al. Recognition and management of acute kidney injury in the Inter- national Society of Nephrology 0by25 Global Snapshot: a multinational cross-sectional study. Lancet. 2016;387(10032):2017–25. 5. Freeland K, Hamidian JA, Duvall LM, Mancini MC. Postoperative blood transfusion is an independent predictor of acute kidney injury in cardiac surgery patients. J Nephropathol. 2015;4(4):121–6. 6. Brown JR, Kramer RS, MacKenzie TA, Coca SG, Sint K, Parikh CR. Determi- nants of acute kidney injury duration after cardiac surgery: an externally validated tool. Ann Thorac Surg. 2012;93(2):570–6. Availability of data and materials y The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Study limitationsh This study is subjected to several limitations. Firstly, the causality could not be established because of the nature of the observational study. Thus, further prospective intervention study is needed. Secondly, type A acute aor- tic dissection surgery is a complicated procedure. We are often forced into the situation where multiple transfu- sions are required.The appropriate timing of blood trans- fusion could not be determined, which is an interesting topic to study in the future. Thirdly, because the sample size for this study is relative small, perhaps the influ- ence of blood transfusion volume becomes less impor- tant. Fourthly, multiple factors may cause postoperative AKI after aortic surgery, including preoperative shock or hypotension, renal artery dissection or occlusion, anemia, surgical trauma, prolonged use of CPB, hypo- thermia, and extensive blood product, during the study period, it is very difficult to take all factors into consider- ation. Fortunately, most important variables are included in our study to discuss the incidence of AKI. Fifthly, the residual confounding and bias could not be totally addressed in the observational study. We gratefully thank Dr. Liangkai Chen from the Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety Huazhong Acknowledgements Boyle JM, Moualla S, Arrigain S, Worley S, Bakri MH, Starling RC, et al. Risks and outcomes of acute kidney injury requiring dialysis after cardiac transplantation. Am J Kidney dis. 2006;48(5):787–96. 14. Boyle JM, Moualla S, Arrigain S, Worley S, Bakri MH, Starling RC, et al. Risks and outcomes of acute kidney injury requiring dialysis after cardiac transplantation. Am J Kidney dis. 2006;48(5):787–96. 24. Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F, et al. Intravital videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol Renal Physiol. 2002;282(6):F1150–5. p y 15. Ho J, Reslerova M, Gali B, Nickerson PW, Rush DN, Sood MM, et al. Serum creatinine measurement immediately after cardiac surgery and predic- tion of acute kidney injury. Am J Kidney Dis. 2012;59(2):196–201. 25. Jansen M, Florquin S, Roelofs J. The role of platelets in acute kidney injury. Nat Rev Nephrol. 2018;14(7):457–71. 26. Grams ME, Estrella MM, Coresh J, Brower RG, Liu KD. Fluid balance, diuretic use, and mortality in acute kidney injury. Clin J Am Soc Nephrol. 2011;6(5):966–73. 26. Grams ME, Estrella MM, Coresh J, Brower RG, Liu KD. Fluid balance, diuretic use, and mortality in acute kidney injury. Clin J Am Soc Nephrol. 2011;6(5):966–73. 16. Zhou H, Wang G, Yang L, Shi S, Li J, Wang M, et al. Acute kidney injury after total arch replacement combined with frozen elephant trunk implantation: incidence, risk factors, and outcome. J Cardiothor Vasc Anesthesia. 2018;32(5):2210–7. 27. Avila MO, Rocha PN, Zanetta DM, Yu L, Burdmann EA. Water balance, acute kidney injury and mortality of intensive care unit patients. J Bras Nefrol. 2014;36(3):379–88. 27. Avila MO, Rocha PN, Zanetta DM, Yu L, Burdmann EA. Water balance, acute kidney injury and mortality of intensive care unit patients. J Bras Nefrol. 2014;36(3):379–88. 17. Hoste EA, Clermont G, Kersten A, Venkataraman R, Angus DC, De Bacquer D, et al. RIFLE criteria for acute kidney injury are associated with hospital mortality in critically ill patients: a cohort analysis. Crit Care. 2006;10(3):R73. 28. Weber CF, Gorlinger K, Meininger D, Herrmann E, Bingold T, Moritz A, et al. Point-of-care testing: a prospective, randomized clinical trial of efficacy in coagulopathic cardiac surgery patients. Anesthesiology. 2012;117(3):531–47. 28. Weber CF, Gorlinger K, Meininger D, Herrmann E, Bingold T, Moritz A, et al. Point-of-care testing: a prospective, randomized clinical trial of efficacy in coagulopathic cardiac surgery patients. Anesthesiology. 2012;117(3):531–47. 18. Acknowledgements Redfors B, Bragadottir G, Sellgren J, Sward K, Ricksten SE. Acute renal failure is NOT an “acute renal success”—a clinical study on the renal oxygen supply/demand relationship in acute kidney injury. Crit Care Med. 2010;38(8):1695–701. Acknowledgements We gratefully thank Dr. Liangkai Chen from the Department of Nutrition and Food Hygiene, Hubei Key Laboratory of Food Nutrition and Safety Huazhong Liu et al. BMC Cardiovasc Disord (2020) 20:446 Liu et al. BMC Cardiovasc Disord (2020) 20:446 Page 8 of 8 9. Bove T, Calabro MG, Landoni G, Aletti G, Marino G, Crescenzi G, et al. The incidence and risk of acute renal failure after cardiac surgery. J Cardio- thorac Vasc Anesth. 2004;18(4):442–5. 20. Tinmouth A, Fergusson D, Yee IC, Hebert PC. Clinical consequences of red cell storage in the critically ill. Transfusion. 2006;46(11):2014–27. 21. Donadee C, Raat NJ, Kanias T, Tejero J, Lee JS, Kelley EE, et al. Nitric oxide scavenging by red blood cell microparticles and cell-free hemoglobin as a mechanism for the red cell storage lesion. Circulation. 2011;124(4):465–76. 10. Karkouti K. Transfusion and risk of acute kidney injury in cardiac surgery. Br J Anaesth. 2012;109(Suppl 1):i29–38. 10. Karkouti K. Transfusion and risk of acute kidney injury in cardiac surgery. Br J Anaesth. 2012;109(Suppl 1):i29–38. pp 11. Rosner MH, Okusa MD. Acute kidney injury associated with cardiac surgery. Clin J Am Soc Nephrol. 2006;1(1):19–32. 11. Rosner MH, Okusa MD. Acute kidney injury associated with cardiac surgery. Clin J Am Soc Nephrol. 2006;1(1):19–32. 22. Almac E, Ince C. The impact of storage on red cell function in blood transfusion. Best Pract Res Clin Anaesthesiol. 2007;21(2):195–208. 12. Vanmassenhove J, Kielstein J, Jorres A, Biesen WV. Management of patients at risk of acute kidney injury. Lancet. 2017;389(10084):2139–51. 12. Vanmassenhove J, Kielstein J, Jorres A, Biesen WV. Management of patients at risk of acute kidney injury. Lancet. 2017;389(10084):2139–51. 12. Vanmassenhove J, Kielstein J, Jorres A, Biesen WV. Management of patients at risk of acute kidney injury. Lancet. 2017;389(10084):2139–51. 13. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120(4):c179–84. 23. Lasocki S, Longrois D, Montravers P, Beaumont C. Hepcidin and anemia of the critically ill patient: bench to bedside. Anesthesiology. 2011;114(3):688–94. p y j y 13. Khwaja A. KDIGO clinical practice guidelines for acute kidney injury. Nephron Clin Pract. 2012;120(4):c179–84. y y 13. Khwaja A. KDIGO clinical practice guideline Nephron Clin Pract. 2012;120(4):c179–84. 13. Khwaja A. KDIGO clinical practice guidelin Nephron Clin Pract. 2012;120(4):c179–84. 24. Yamamoto T, Tada T, Brodsky SV, Tanaka H, Noiri E, Kajiya F, et al. Intravital videomicroscopy of peritubular capillaries in renal ischemia. Am J Physiol Renal Physiol. 2002;282(6):F1150–5. 14. Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 19. Ho J, Lucy M, Krokhin O, Hayglass K, Pascoe E, Darroch G, et al. Mass spectrometry-based proteomic analysis of urine in acute kidney injury following cardiopulmonary bypass: a nested case-control study. 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A common polymorphism in <i>SNCA</i> is associated with accelerated motor decline in <i>GBA</i>-Parkinson’s disease
Journal of neurology, neurosurgery and psychiatry
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A common polymorphism in SNCA is associated with accelerated motor decline in GBA-­Parkinson’s disease A growing number of genetic susceptibility factors have been identified for Parkinson’s disease (PD). The combination of inherited risk variants is likely to affect not only risk of developing PD but also its clinical course. Variants in the GBA gene are particularly common, being found in approximately 5% to 10% of patients, and they lead to more rapid disease progression.1 However, the effect of concomitant genetic risk factors on disease course in GBA-­PD is not known. Sequencing of the GBA gene was carried out in 114 patients in the CamPaIGN cohort, as described here.7 A further 16 patients underwent targeted genetic screening for common GBA variants using the Illumina Multi-­Ethnic Genotyping Array (MEGA) chip. Genetic analysis of the SNCA rs356219 polymorphism had also been previously performed in 124 patients from the cohort.2 GBA variants were identified in 20 patients, which were included in survival analyses. Of these, 7 carried the high-­risk (G/G) SNCA geno- type, with 13 carrying low-­risk (G/A or A/A) genotypes. There were no differences in age at diagnosis nor in baseline assess- ments including total and part III Unified Parkinson’s Disease Rating Scale scores, Hoehn and Yahr scores, MMSE scores or levodopa equivalent doses between these SNCA genotype groups. All carriers of the G/G genotype were male, compared with 53.8% in the low-­risk group (p=0.03) (online supplementary table 1). The aggregation of α-synuclein, encoded by the SNCA gene, is central to the patho- genesis of PD. The SNCA rs356219 A/G polymorphism alters the risk of developing PD, with homozygotes for guanine (G/G) having an increased risk compared with carriers of an adenine (G/A or A/A) at this locus.2 The relationship between glucoce- rebrosidase (the enzyme encoded by the GBA gene) and α-synuclein is complex. These proteins have been shown to interact directly in vitro, as well as to influence the intracellular levels and processing of each other, potentially in a bidirectional feedback loop.3 4 Interestingly, a recent genome-­wide association study found that the presence of this SNCA polymorphism was associated with an increased likelihood of developing PD in GBA mutation carriers.5 We therefore hypothesised that the presence of the SNCA rs356219 polymorphism would accelerate the clinical course of GBA variant-­associated PD. Here, we report on the effect of this SNCA polymorphism on clinical outcomes within the GBA-­PD population. This cohort contained four patients with the non-­coding c. 762–18 T>A variant (online supplementary table 2). Letter G/G carriers reached HY3 within 3 years of diagnosis. informed consent was obtained from all subjects. Newly diagnosed patients were followed up with assessments every 2 years for up to 18 years. Time to development of dementia (defined as Mini-­Mental State Examination (MMSE) score of 24 or less, with fulfilment of Diagnostic and Statistical Manual of Mental Disorders IV criteria), progression to postural instability (Hoehn and Yahr stage three (HY3)) and death were determined. To account for potential confounders, time to HY3 was also compared using a Cox regression model controlling for sex and age at diagnosis. The G/G SNCA genotype was associated with a HR for progression to HY3 of 3.8 (95% CI 0.9 to 16.1), p=0.07, adjusted p=0.21) when controlling for these confounders. Because all the SNCA G/G carriers were male, we also performed a survival analysis in which only male subjects were included, to further control for sex as a potential confounder. An increased risk of progression to HY3 was again observed in G/G carriers in comparison to A carriers (p=0.036, adjusted p=0.11; online supple- mentary figure 1). SNCA genotype had no effect on progression to HY3 in non-­carriers of GBA variants (n=85) in the CamPaIGN cohort (p=0.88, adjusted p=2.65; online supplementary figure 2). A common polymorphism in SNCA is associated with accelerated motor decline in GBA-­Parkinson’s disease While this has been reported to be a potential risk factor for PD,8 its importance is not clear. We therefore also performed analysis after exclusion of these patients and found that progression to HY3 was greater in the G/G carrier group, with a HR of 5.3 (95% CI 1.1 to 26.2; p=0.041, adjusted p=0.12). To our knowledge, no previous studies have investigated the influence of concom- itant genetic risk factors on the progression of GBA-­PD. Here we report that the SNCA rs356219 polymorphism significantly affects motor progression in GBA-­PD, with the G/G genotype associated with a particularly aggressive disease course. This effect was not observed in patients with PD without GBA variants, suggesting that it was relatively specific to GBA-­PD. Survival analyses for the aforementioned outcome measures were performed, with statistical significance determined using log-­rank tests. P-­values were adjusted for multiple comparisons using the Bonfer- roni method. Among carriers of GBA vari- ants, there were no differences between the high-­risk and low-­risk SNCA groups for time to dementia (p=0.29, adjusted p=0.86) or death (p=0.43, adjusted p=1.28) (figure  1A,B). Progression to HY3, however, was significantly faster in the G/G SNCA group (p=0.02, adjusted p=0.07), with mean time to development of postural instability 2.0 years (95% CI 1.3 to 2.7) compared with 4.9 years (95% CI 2.5 to 7.3) in A carriers (figure 1C). All This new study therefore raises the inter- esting possibility that GBA variants and the G/G SNCA rs356219 polymorphism act synergistically to accelerate pathology and clinical progression in PD. GBA muta- tions are thought to increase the risk of PD predominantly through perturbations in the lysosome-­autophagy system—a system important in α-synuclein clearance. Longitudinal data from GBA-­variant carriers were analysed from the community-­ based ‘Cambridgeshire Incidence of Parkinson’s disease from General Prac- tice to Neurologist’ (CamPaIGN) cohort (n=142).6 This study was approved by the local ethics committee and written Figure 1  Survival analyses in GBA-­PD patients comparing those with high-­risk and low-­risk variants in the SNCA rs356219 polymorphism. Kaplan-­Meier curves for time to (A) dementia, (B) death and (C) postural instability. A, adenine, G, guanine; H&Y3, Hoehn and Yahr stage three; PD, Parkinson’s disease. Figure 1  Survival analyses in GBA-­PD patients comparing those with high-­risk and low-­risk variants in the SNCA rs356219 polymorphism. Kaplan-­Meier curves for time to (A) dementia, (B) death and (C) postural instability. A, adenine, G, guanine; H&Y3, Hoehn and Yahr stage three; PD, Parkinson’s disease. PostScript PostScript References 1 Cilia R, Tunesi S, Marotta G, et al. Survival and dementia in GBA-­associated Parkinson’s disease: the mutation matters. Ann Neurol 2016;80:662–73. Twitter Thomas Foltynie @foltynie Twitter Thomas Foltynie @foltynie Twitter Thomas Foltynie @foltynie 2 Goris A, Williams-­Gray CH, Clark GR, et al. Tau and alpha-­synuclein in susceptibility to, and dementia in, Parkinson’s disease. Ann Neurol 2007;62:145–53. 2 Goris A, Williams-­Gray CH, Clark GR, et al. Tau and alpha-­synuclein in susceptibility to, and dementia in, Parkinson’s disease. Ann Neurol 2007;62:145–53. Contributors  TBS wrote the manuscript. TBS, MC, SW-­R, GL and TF performed the research. TF, CRS, RAB and CHW-­G developed the original concepts and provided critical review of the manuscript. Contributors  TBS wrote the manuscript. TBS, MC, SW-­R, GL and TF performed the research. TF, CRS, Contributors  TBS wrote the manuscript. TBS, MC, SW-­R, GL and TF performed the research. TF, CRS, RAB and CHW-­G developed the original concepts and provided critical review of the manuscript. RAB and CHW-­G developed the original concepts and provided critical review of the manuscript. RAB and CHW-­G developed the original concepts and provided critical review of the manuscript. 3 Mazzulli JR, Xu Y-­H, Sun Y, et al. Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell 2011;146:37–52. 3 Mazzulli JR, Xu Y-­H, Sun Y, et al. Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell 2011;146:37–52. Funding  The CamPaIGN study has received financial support from the Wellcome Trust, the Medical Research Council, Parkinson’s UK and the Patrick Berthoud Trust. CHW-­G is supported by a Research Councils UK/UK Research and Innovation Fellowship awarded by the Medical Research Council (MR/R007446/1). RAB is supported by the Wellcome Trust Stem Cell Institute (Cambridge 203151/Z/16/Z). RAB is a National Institute for Health Research (NIHR) Senior Investigator (NF-­ SI-0616-10011). TBS received financial support from the Cure Parkinson’s Trust. The study is also supported by the NIHR Cambridge Biomedical Research Centre Dementia and Neurodegeneration Theme (reference number 146281). CRS’ work is supported in part by National Institutes of Health grants R01AG057331, U01NS100603, R01AG057331, and the American Parkinson Disease Association. Illumina MEGA Chip genotyping was made possible by a philanthropic investment from Dooley LLC (to Brigham & Women’s Hospital and CRS). 4 Yap TL, Velayati A, Sidransky E, et al. Membrane-­ bound α-synuclein interacts with glucocerebrosidase and inhibits enzyme activity. Mol Genet Metab 2013;108:56–64. 5 Blauwendraat C, Reed X, Krohn L, et al. PostScript Thomas B Stoker ‍ ‍ ,1,2 Marta Camacho,1 Sophie Winder-­Rhodes,1 Ganqiang Liu,3,4,5 4 5 6 Clemens R Scherzer,4,5 Thomas Foltynie ‍ ‍ ,6 Roger A Barker,1,2 Caroline H Williams-­Gray1 1John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK 1John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK To cite Stoker TB, Camacho M, Winder-­Rhodes S, et al. J Neurol Neurosurg Psychiatry 2020;91:673–674. Received 9 October 2019 Revised 18 February 2020 Accepted 9 March 2020 Published Online First 2 April 2020 2Wellcome Trust ‑ Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK 3School of Medicine, Sun Yat-­Sen University, 2Wellcome Trust ‑ Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge, UK 3School of Medicine, Sun Yat-­Sen University, Guangzhou, Guangdong, China 4Advanced Center for Parkinson’s Disease Research, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts, USA 5Precision Neurology Program, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts USA J Neurol Neurosurg Psychiatry 2020;91:673–674. doi:10.1136/jnnp-2019-322210 Hospital, Boston, Massachusetts, USA 5 ORCID iDs 5Precision Neurology Program, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts, USA 5Precision Neurology Program, Harvard Medical School, Brigham and Women’s Hospital, Boston, Massachusetts USA Thomas B Stoker http://​orcid.​org/​0000-​0001-​5186-​ 7630 Thomas B Stoker http://​orcid.​org/​0000-​0001-​5186-​ 7630 6Department of Clinical and Movement Neurosciences, UCL Institute of Neurology, London, UK 6Department of Clinical and Movement Neurosciences, UCL Institute of Neurology, London, UK Thomas Foltynie http://​orcid.​org/​0000-​0003-​0752-​ 1813 Thomas Foltynie http://​orcid.​org/​0000-​0003-​0752-​ 1813 We acknowledge that our sample size is small, but these preliminary observations raise the possibility that GBA variants and the G/G SNCA rs356219 polymorphism synergistically alter motor progression in PD. In this study we have considered the time from diagnosis to the development of important clinical milestones in PD. The time to diagnosis may vary between patients, so it should be recognised that this does not necessarily reflect disease dura- tion. Time from disease onset to the devel- opment of clinical milestones may be more representative of disease duration, but onset is very difficult to ascertain accurately, given that many patients experience prodromal symptoms that are not initially attributed to PD. Correspondence to Dr Thomas B Stoker, John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, Cambridgeshire CB2 0PY, UK; ​tbs26@​cam.​ac.​uk Correspondence to Dr Thomas B Stoker, John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, Cambridgeshire CB2 0PY, UK; ​tbs26@​cam.​ac.​uk PostScript Furthermore, glucocerebrosidase and α-sy- nuclein have been shown to interact directly in vitro and to coexist in Lewy bodies of patients with PD, with a greater propor- tion of Lewy bodies containing glucocere- brosidase in patients with GBA mutations compared with those in patients with PD not carrying a GBA variant.4 9 It has been speculated that mutant glucocerebrosidase potentiates the aggregation of α-synuclein, and it is therefore feasible that SNCA vari- ants such as the rs356219 polymorphism alters the degree to which GBA variants predispose to PD pathology and disease progression. It has also been suggested that α-synuclein impedes the transit of glucoce- rebrosidase from the endoplasmic reticulum to the lysosome, further supporting the idea that the relationship between these two proteins is directly important in the patho- genesis of GBA-­PD.3 The proposed syner- gistic interaction between variants in the GBA gene and SNCA gene therefore could also potentially be explained by differences in the degree to which α-synuclein impairs normal glucocerebrosidase processing asso- ciated with specific SNCA variants. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://​creativecommons.​org/​licenses/​by/​ 4.​0/. of our study is that all subjects in the G/G group were male, and though this was accounted for in the Cox regression model and by performing the additional male-­ only analysis, it would be important to investigate the relationship between these two genetic risk factors in a large cohort, to reduce the effect of such confounders and to allow for the stratification of G/G carriers into those with severe and those with mild GBA variants. © Author(s) (or their employer(s)) 2020. Re-­use permitted under CC BY. Published by BMJ. © Author(s) (or their employer(s)) 2020. Re-­use permitted under CC BY. Published by BMJ. ► ►Additional material is published online only. To view please visit the journal online (http://​dx.​doi.​org/​ 10.​1136/​jnnp-​2019-​322210). A common polymorphism in SNCA is associated with accelerated motor decline in GBA-­Parkinson’s disease J Neurol Neurosurg Psychiatry June 2020 Vol 91 No 6 673 PostScript References Genetic modifiers of risk and age at onset in GBA associated Parkinson’s disease and Lewy body dementia. Brain 2020;143:234–48. There is a degree of genotype–phenotype correlation in GBA-­PD, with severe muta- tions accelerating disease course to a greater extent than less severe variants.10 However, because our sample size was small, it was not possible to stratify the patients with GBA-­PD into those with non-­severe and severe GBA variants, so our GBA-­PD population was genetically heterogeneous (online supplementary table 2). Mean age at diagnosis was approximately 5 years later in G/G carriers which may potentially have contributed to their accelerated disease course. However, there were no differences in time to dementia or death, and when age at diagnosis was accounted for in the Cox regression model, the accelerated progres- sion to HY3 persisted. Another limitation 6 Williams-­Gray CH, Mason SL, Evans JR, et al. The campaign study of Parkinson’s disease: 10-­year outlook in an incident population-­based cohort. J Neurol Neurosurg Psychiatry 2013;84:1258–64. 7 Winder-­Rhodes SE, Evans JR, Ban M, et al. Glucocerebrosidase mutations influence the natural history of Parkinson’s disease in a community-­based incident cohort. Brain 2013;136:392–9. 7 Winder-­Rhodes SE, Evans JR, Ban M, et al. Glucocerebrosidase mutations influence the natural history of Parkinson’s disease in a community-­based incident cohort. Brain 2013;136:392–9. 8 Moors TE, Paciotti S, Ingrassia A, et al. Characterization of brain lysosomal activities in GBA-­Related and sporadic Parkinson’s disease and dementia with Lewy bodies. Mol Neurobiol 2019;56:1344–55. Competing interests  None declared. Competing interests  None declared. Patient consent for publication  Not required. Provenance and peer review  Not commissioned; externally peer reviewed. Provenance and peer review  Not commissioned; externally peer reviewed. 9 Goker-­Alpan O, Stubblefield BK, Giasson BI, et al. Glucocerebrosidase is present in α-synuclein inclusions in Lewy body disorders. Acta Neuropathol 2010;120:641–9. 9 Goker-­Alpan O, Stubblefield BK, Giasson BI, et al. Glucocerebrosidase is present in α-synuclein inclusions in Lewy body disorders. Acta Neuropathol 2010;120:641–9. 10 Liu G, Boot B, Locascio JJ, et al. Specifically neuropathic Gaucher’s mutations accelerate cognitive decline in Parkinson’s. Ann Neurol 2016;80:674–85. 674 J Neurol Neurosurg Psychiatry June 2020 Vol 91 No 6
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Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new areas
Biological invasions
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Biol Invasions (2024) 26:1969–1983 https://doi.org/10.1007/s10530-024-03290-w Biol Invasions (2024) 26:1969–1983 https://doi.org/10.1007/s10530-024-03290-w ORIGINAL PAPER Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new areas Marta Kolanowska   · Agnieszka Rewicz · James D. Ackerman Received: 29 November 2023 / Accepted: 5 March 2024 / Published online: 2 April 2024 © The Author(s) 2024 Received: 29 November 2023 / Accepted: 5 March 2024 / Published online: 2 April 2024 © The Author(s) 2024 Abstract  Climate change plays an increasing role in the global biodiversity crisis. Alteration in local cli- matic conditions not only can negatively affect native biodiversity but also can accelerate the introduction and spread of invasive species. In this study the eco- logical niche modelling approach was used to evalu- ate possible changes in the distribution of suitable niches of invasive orchid Eulophia graminea within its native (Asia) and non-native geographical range (America, Australia). We mapped the current poten- tial range of this species and analysed three various projections of future climate (for 2100) each with four different climate change scenarios (SSPs). Cal- culated niche overlap indexes indicated low similarity of niches occupied by native and invasive populations of E. graminea and Australian populations seem to be the most unique, while American and Asian groups share partially similar niches. The occurrence of the American population of E. graminea was correlated especially with the temperature seasonality, while the Asian and Australian populations with annual pre- cipitation and precipitation of the wettest quarter. As indicated in our analyses within Asia and America, E. graminea does not occupy all climatically suit- able niches. On the other hand, in Australia the spe- cies studied already occupies all appropriate niche space. Climate change will likely be favorable for species studied to expand its range if the biotic com- ponents of its niche space (e.g., mycorrhizal fungi) will respond similarly. The most significant range expansion is predicted to occur in Australia which is interesting considering the marginally suitable habi- tats that E. graminea currently occupies. Keywords  Climate projection · Intercontinental invasion · Niche modelling · Shared socioeconomic pathways Introduction Orchidaceae is the second largest family of flow- ering plants with more than 31,000 species (POWO 2023). Representatives of this group are found around the world, except polar regions and very dry deserts (Dressler 1981). Nonetheless, the highest orchid diversity is in the humid tropics (Vitt et  al. 2023). Despite the great variation in morphology and physi- ology observed within Orchidaceae (Zhang et  al. 2018), relatively few species are considered to be invasive or weedy (Ackerman 2007; Daehler 1998; Randall 2017). In all biological invasions, there are barriers to overcome at every stage of an invasion (Blackburn et  al. 2011). Major constraints to orchid establish- ment and spread involve symbioses. The first barrier is seed production. Fruit set in orchids is pollination limited, which is exacerbated by generally having one or very few pollinators (Ackerman et  al. 2023; Tremblay et  al. 2005). Although fruits can contain hundreds to millions of tiny, wind-dispersed seeds which to a certain extent compensates for low fruit set (Arditti and Ghani 2000; Sonkoly et al. 2016), disper- sal is strongly leptokurtic and establishment is seed limited (Ackerman et al. 1996; Brzosko et al. 2017). Like most plants, orchids that have become invasive usually arrived at novel locations through the horti- cultural trade (Rojas-Sandoval and Ackerman 2021; van Kleunen et al. 2018), but subsequent stages of an invasion, establishment and spread, generally require seed production. If a novel location lacks a fauna that can provide pollinator services, then self-pollination and vegetative propagation are the only means for population growth and dispersal. Less than a quarter of orchid species have the capacity to autonomously self-pollinate (Ackerman et  al. 2023), further con- straining which orchids would be able to become established and spread. Biological invasions are complex ecological pro- cesses which depend on propagule pressure, species invasive potential, and the susceptibility of commu- nities to invasion (Chaffin et al. 2016; Chown et al. 2015; Lowry et  al. 2012; Simberloff et  al. 2013). Moreover, this process is currently accelerated by globalization which circumvents natural biogeo- graphic barriers and increases propagule pressure, and accidental or intentional releases of non-native species (Hänfling and Kollmann 2002; Meyerson and Mooney 2007). Global warming can accelerate the spread of invasive species (Demertzis and Ili- adis 2018) which can exacerbate climate change by increasing wetland methane and terrestrial nitrous oxide emissions (Bezabih Beyene et al. 2022). Introduction Supplementary Information  The online version contains supplementary material available at https://​doi.​ org/​10.​1007/​s10530-​024-​03290-w. Human activities are responsible for the accumula- tion of alien plant species across most regions of the world, a process that continues to this day (Seebens et al. 2017). Most of these species have been inten- tionally introduced for agriculture, forage, forestry, but most commonly as ornamentals (Beaury et  al. 2021; Dodd et al. 2015; Rojas-Sandoval and Acker- man 2021). The latter pathway is particularly egre- gious as domestic gardens select species with traits M. Kolanowska (*) · A. Rewicz  Department of Geobotany and Plant Ecology, University of Lodz, Ul. Banacha 12/16, 90‑237 Lodz, Poland e-mail: marta.a.kolanowska@gmail.com M. Kolanowska (*) · A. Rewicz  Department of Geobotany and Plant Ecology, University of Lodz, Ul. Banacha 12/16, 90‑237 Lodz, Poland e-mail: marta.a.kolanowska@gmail.com J. D. Ackerman  Department of Biology, University of Puerto Rico, 17 Avenue Universidad Suite 1701, San Juan, PR 00925‑2537, USA ol.: (01 123456789) 3 1970 M. Kolanowska et al. fundamental niche shift (Müller-Schärer et al. 2004), release from dispersal barriers (Smith et  al. 2020) or biotic constraints (realized niche shifts) (Mitchell et al. 2006). Also the spread of exotics is expected to be influenced by the physiological response of alien and native species to environmental changes and the subsequent changes in ecological interactions (Robin- son et al. 2020).l that are associated with invasion success (Guo et al. 2019). Unsurprisingly, many plants escape cultiva- tion, spread into novel areas thereby becoming inva- sive with potential negative impacts on the envi- ronment, local biota, and human well-being (van Kleunen et al. 2018). Non-indigenous plants, once introduced may become invasive without continued intervention by humans if environmental and biotic conditions are conducive for establishment and spread, even into undisturbed native vegetation (Mashhadi and Rado- sevich 2004) where they can be a major threat to native biodiversity and alter ecosystem functions (Lowry et al. 2012; Simberloff et al. 2013; Vitousek and Walker 1989). On the other hand, invasions may also create novel communities under human-impacted conditions that may provide ecosystem services such as soil conservation, nutrient cycling, wildlife habi- tat, carbon storage, watershed protection, and miti- gate species extinctions (Lugo 2009). Whether or not the net outcome of biological invasions is negative, innocuous, or positive is likely context dependent, either environmentally, biologically, sociologically, or combinations of them all (Daehler 2003; Osborne and Gioria 2022). List of localities List of localities Localities of E. graminea were compiled from the Global Biodiversity Information Facility (GBIF 2022), herbarium specimens and field excursions. Only records which could be georeferenced with the precision of 1 km were used in ENM analyses and the duplicate presence records (records within the same grid cell) were removed using MaxEnt. The complete list of localities used in our study is available as Sup- plementary Table  S1. Due to the lack of sufficient location data, the samples from South Africa were not included in further analyses. Introduction Forecasting biological invasions is crucial for managing non-native species but any predictions of potential spread of exotics, should consider potential species niche shifts resulting from evolved environ- mental tolerances (fundamental niche) or the pres- ence of novel conditions in the invaded range (real- ized niche) (Tingley et al. 2014). The niche shift in newly occupied regions (Rodder and Lotters 2009; Stiels et  al. 2015; Zhu et  al. 2017) can result from The second hurdle to establishment success upon arriving at a novel location is the need to form an association with orchid mycorrhizal fungi (OMF). Orchid seeds are extremely small, lack endosperm 1 Vol:. 3 (1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1971 The reasons for the geographical differences in inva- siveness are unknown.f and are dependent on OMF to provide resources for successful germination (McCormick et  al. 2018). Often this symbiosis is maintained into adulthood, although the OMF that provide for successful germi- nation are not necessarily the same that associate with adult plants (Bayman et al. 2016; Phillips et al. 2020; Zhang et  al. 2018). While some OMF have a very broad distribution, on a local scale they are not ubiq- uitous so that local orchid populations may associate with different sets of fungi that may involve niche and/or spatial segregation (Fernández et  al. 2023; McCormick et al. 2018; Swift et al. 2019), although this is not always the case (Suarez et al. 2016). The spatial distribution of the fungi may be dependent on biophysical factors which, in turn, would affect the distribution of their orchid symbionts (Izuddin et al. 2019; Jacquemyn et al. 2016). The aim of this study is to evaluate differences in environmental niche preferences of E. graminea in various geographical regions, to visualize current non-native potential range of this orchid and to esti- mate the invasive potential of this species under vari- ous climate change scenarios using ecological niche modelling (ENM). Machine learning-based models are commonly used to estimate distribution of suita- ble niches of invasive species and predict their further spread in non-native areas (Cunze et al. 2020; Fand et al. 2020; Paclibar and Tadiosa 2019; Stiels et al. 2011). Methods q y ) A rapidly spreading orchid species which is already present on several continents (Ackerman and González-Orellana 2021; Juárez Gutiérrez et al. 2023) is Eulophia graminea Lindl. (chinese crown orchid, grass leaved Eulophia). This terrestrial spe- cies is native to southern and central Asia. It produces rounded pseudobulbs and long, thin, linear leaves. The long inflorescence of E. graminea is composed of numerous flowers with greenish tepals and 3-lobed, white-pinkish lip. As summarized by Chang et  al. (2010), there are several characters of E. graminea that likely facilitate its invasiveness. The quick ger- mination and fast rhizome production enhance seed- ling survival and the short juvenile stage increases the expansive potential of this orchid. While pollinator availability is an important factor affecting long-term survival of most orchids, flowers of E. graminea are autogamous (Chang et al. 2010). Its dispersibility is exemplified by being one of the few orchid species to colonize remnants of Krakatau 25 years after the 1883 explosion (Partomihardjo 2003).i Climatic niche similarity We used a principal components analysis (PCA) to assess niche variability within the native and non- native distribution of E. graminea populations (from America, Asia and Australia). Each population was characterized by a set of 21 traits of which 19 were related to the climate data (Fick and Hijmans 2017), one described land cover (European Commission 2003), and one soil type (Hengl et  al. 2017). To reduce bias, samples were spatially filtered at 5 km. Calculations were made with the software pack- ages PAST ver. 4.03 (PAST PAleontological STatis- tics Version 3.20). The data matrix (Supplementary Table S2) was transformed (square root) before per- forming the ordination analysis. The first report of the chinese crown orchid outside its native range came from Australian Northern Ter- ritories (Macrae 2002; Pemberton 2013). Later it was recorded in South Africa (O’Conner et al. 2006) and USA (Pemberton et al. 2008). While the populations in Australia and Africa have not significantly spread since initial observations, its rapid range expansion in Florida has continued and has reached into the West Indies (Ackerman and González-Orellana 2021; Juárez Gutiérrez et al. 2023; Singhurst et al. 2020). 1 Vol.: (01 3 123456789) 1972 M. Kolanowska et al. for four Shared Socio-economic Pathways (SSPs): 1–2.6, 2–4.5, 3–7.0 and 5–8.5 (Li et al. 2021; McGee et al. 2000; Meinshausen et al. 2020). SSPs are tra- jectories adopted by the Intergovernmental Panel on Climate Change (IPCC), comprising narrative descriptions of future world development (Riahi et al. 2017). SSP storylines describe contrasting visions of future society and the assumed climate change chal- lenges, with global warming in 2100 ranging from a low of 3.1 °C to a high of 5.1 °C above pre-industrial levels. O’Neill et al. (2017). Three different simula- tions of future climate developed by Coupled Model Intercomparison Project Phase 6 (CNRM), Goddard Institute for Space Studies (GISS), and Institute for Numerical Mathematics (INM) were used. We chose these projections because they present the most differ- ent simulations of maximum temperature and precipi- tation within study areas (Supplementary Figure S4). Climatic niche modelling The modelling of the current and future distribu- tion of the species studied was done using the maximum entropy method implemented in Max- Ent version 3.3.2 (Elith et  al. 2011; Phillips et  al. 2006; Phillips and Dudik 2008), which is based on presence-only observations. Bioclimatic variables in 30 arc-seconds of interpolated climate surface downloaded from WorldClim v. 2.1 were used for the modelling (Fick and Hijmans 2017). The study area was divided into three geographical regions— America (0.15°S–44.16°N, 127.64–58.20°W), Asia (10.99°S–41.80°N, 67.54–159.78°E), and Australia (10.79–17.39°S, 125.98–138.09°E). Pearsons’ correlation coefficient was computed using SDMtoolbox  2.3 for ArcGIS (Brown 2014; Brown et  al. 2017) (Supplementary Table  S3) and highly correlated (> 0.8) variables were removed from ENM analyses to prevent problems associated with auto-correlation. The final list of bioclimatic variables used in the analyses is provided in Table 1. In all analyses the maximum number of itera- tions was set to 10,000 and convergence threshold to 0.00001. The neutral (= 1) regularization multiplier value and auto features were used. The “random seed" option provided a random test partition and back- ground subset for each run and 20% of the samples We predicted the future extent of E. graminea cli- matic niches for 2080–2100 by using four projections Table 1   Climatic variables used in ENM analyses (marked with +) Variable code Variable description Geographical region America Australia Asia bio1 Annual mean temperature + + + bio2 Mean diurnal range [mean of monthly (max temp–min temp)] + + + bio3 Isothermality (bio2/bio7) (× 100) + + + bio4 Temperature seasonality (standard deviation × 100) bio5 Max temperature of warmest month + bio6 Min temperature of coldest month bio7 Temperature annual range (bio5-bio6) bio8 Mean temperature of wettest quarter + + bio9 Mean temperature of driest quarter + bio10 Mean temperature of warmest quarter bio11 Mean temperature of coldest quarter bio12 Annual precipitation + + + bio13 Precipitation of wettest month bio14 Precipitation of driest month + + + bio15 Precipitation seasonality (coefficient of variation) + + + bio16 Precipitation of wettest quarter bio17 Precipitation of driest quarter + bio18 Precipitation of warmest quarter + + + bio19 Precipitation of coldest quarter Table 1   Climatic variables used in ENM analyses (marked with +) Table 1   Climatic variables used in ENM analyses (marked with +) Variable code Variable description 1 Vol:. Climatic niche modelling ( Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1973 were used as test points. The run was performed as a bootstrap with 100 replicates. The output was set to logistic. We used the “fade by clamping” function in MaxEnt was used to prevent extrapolations outside the environmental range of the training data (Owens et  al. 2013). All analyses of GIS data were carried out using ArcGis 10.6 (Esri, Redlands, CA, USA). The evaluation of the created models was made using the area under the curve (AUC) (Mason and Graham 2002) and True Skill Statistic (TSS) (Čengić et  al. 2020; Shabani et al. 2016). Results Differences in occupied niches between geographical groups Differences in occupied niches between geographical groups The PCA analyses revealed the highest popula- tion density of E. graminea from America clustered in the center of the PCA plot, with distinct separate populations originating from Australia. Populations of the orchid from Asia were the most dispersed and occupied the right part of the PCA plot (Fig. 1). The analysis indicated that the first two principal compo- nents explained 73.8% of the total variance. The first component represented 41.1% of the total variance and the second component accounted for 32.7%. PC1 is correlated with bio16, bio12 and bio4. PC2 is cor- related with bio14, bio16 and bio19. The first com- ponent demonstrates positive correlations with bio16 and bio12, and negative correlations with bio4. The second component demonstrates positive correlations with bio14 and bio19 (Supplementary Figure  S5). The occurrence of the American population of E. graminea was correlated especially with bio4, while the Asian and Australian populations with bio16 and bio12. To visualize changes in the distribution of suit- able niches of the orchid, we used SDMtoolbox 2.3 for ArcGIS (Brown 2014; Brown et  al. 2017). To compare the distribution model created for current climatic conditions with future predictions all SDMs were converted into binary rasters and projected using the Goode homolosine as a projection. The presence thresholds used in the analyses equaled the calculated minimum training presence threshold (Liu et al. 2005). Based on obtained models the overlap of the cli- matic niches between the native and the introduced ranges was assessed using Schoener’s D and I statis- tics (Warren et al. 2008). Vol : (0123456789) Fig. 1   PCA ordination diagram of studied localities of Eulo- phia graminea according to 21 environmental predictors (19 were related to the climate data, one described land cover and one soil type). Legend: black dots—North American popula- tions, red dots–Asian populations, green dots–Australian popu- lations) Fig. 1   PCA ordination diagram of studied localities of Eulo- phia graminea according to 21 environmental predictors (19 were related to the climate data, one described land cover and one soil type). Legend: black dots—North American popula- tions, red dots–Asian populations, green dots–Australian popu- lations) 1 ol.: (0 3 ) 1974 M. Kolanowska et al. The sites occupied among geographical groups did not differ significantly in annual temperature (bio1) (Supplementary Figure  S6); however, non- native populations seemed to be adapted to slightly higher temperatures (Asia 21.0–28.0  °C, America 23.0–35.0  °C, Australia 26.5–30.0  °C). Models evaluation and limiting factors Models evaluation and limiting factors All created models received high scores of AUC (0.958–0.999) and mostly high scores of TSS (0.748–0.991) tests. Both sensitivity and specificity were also generally high indicating good reliability of presented modelling results (Table 3). According to the jackknife test of variable impor- tance, for both Asian and Australian populations bio12 (annual precipitation) was the variable with the highest gain when used in isolation, and the same variable decreased the most when it was omit- ted. Thus, it not only is the most informative, but also contains information not represented by the other variables. Models of American populations received different scores. The variable with highest gain when used in isolation was bio8 (mean tem- perature of the wettest quarter) while bio1 (annual mean temperature) decreased the gain the most when it was omitted (Supplementary Figure S5). Differences in occupied niches between geographical groups The mean diurnal range of temperature (bio2) was narrower in American group (Asia 3–15 °C, America 3–8 °C, Australia 4–17.5  °C). Australian populations have narrower tolerance for isothermality (bio3; Asia— 18–58, America 12–85, Australia 56–66). Consid- ering annual precipitation (bio12) Australian popu- lations are characterized by narrower tolerance for rainfall (Asia – 800 to 1800 mm, America − 1000 to 2000 mm, Australia 1700–2000 mm) and precipi- tation of the driest month (bio14; Asia 20–550 mm, America 50–550 mm, Australia − 0.5 to 6.5 mm). The precipitation seasonality (bio15) did not dif- fer significantly among geographical groups (Asia 5–110, America 10–100, Australia 108–137), but precipitation of the warmest quarter was highly variable among regions (bio18; Asia 800–5800 mm, America 200–2000 mm, Australia 90–930 mm). Current potential orchid range Calculated niche overlap indexes (Table 2) indi- cated low similarity of niches occupied by native and invasive populations of E. graminea. Austral- ian populations seem to be the most unique, while American and Asian groups share partially similar niches. Generally, current potential range of E. graminea is consistent with the known distribution of the spe- cies populations; nonetheless, Asian and Ameri- can models indicated presence of suitable niches in some areas not occupied by this orchid (Fig. 2). Within Asian study area, suitable niches of E. graminea are located in New Guinea which is out- side species known native range. In America, our model indicated Lesser Antilles, Jamaica, Domi- nica and Haiti as additional areas suitable for E. graminea occurrence. The Australian model is con- sistent with the distribution of known populations of the orchid. 1 Vol:. (1234567890) model indicated Lesser Antilles, Jamaica, Domi nica and Haiti as additional areas suitable for E. graminea occurrence. The Australian model is con- sistent with the distribution of known populations of the orchid. Table 2   Niche overlap calculated using Schoener’s D and I statistics D\I America Asia Australia America x 0.5919 0.0018 Asia 0.4670 x 0.0221 Australia 0.0002 0.0103 x Table 3   Scores of model reliability tests and value of minimum training presence Modelled area AUC​ TSS Sensitivity Specificity Minimum training presence logistic threshold America 0.986 0.959 0.977 0.982 0.0215 Asia 0.958 0.748 0.978 0.769 0.0295 Australia 0.999 0.9912 1 0.991 0.2174 Table 2   Niche overlap calculated using Schoener’s D and I statistics Table 3   Scores of model reliability tests and value of minimum training presence Modelled area AUC​ TSS Sensitivity Specificity Minimum training presence logistic threshold America 0.986 0.959 0.977 0.982 0.0215 Asia 0.958 0.748 0.978 0.769 0.0295 Australia 0.999 0.9912 1 0.991 0.2174 1 Vol:. ( 3 (1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1975 Fig. 2   Current distribution of suitable niches of E. graminea in Asia (A), North America (B), and northern Australia (C) t distribution of suitable niches of E. graminea in Asia (A), North America (B), and northern Australia (C) Fig. 2   Current distribution of suitable niches of E. graminea in Asia (A), North America (B), and northe Changes in the distribution of suitable niches of E. graminea two predictions the coverage of suitable niches of E. graminea will be several times larger than currently recorded (Table 4, Fig. 5). In these scenarios species will expand its range in Melville and Bathusts islands, and south from Darwin to West Daly region. In some scenarios additional niches will also become avail- able for E. graminea around the South Alligator River estuary. All analysed projections indicate that E. graminea will expand its native range in Asia (Table 4, Fig. 3); however, niches located currently in foothills of New Guinean Highlands, southern foothills of Mül- ler mountains in Borneo, Indian Eastern Ghats will become unsuitable for the orchid. CNRM projec- tions also indicate south-eastern Himalayan foothills, steppes and savannas of Myanmar as areas of poten- tial range contraction for E. graminea. Expansion will mostly occur in south-western and south-central foot- hills of Himalayas, areas around Indian Chota Nagpur Plateau and Vindhya Range, Chinese Sichuan Plain, southern South Korea, and southern Honshu in Japan.i Potential versus observed geographical range Potential versus observed geographical range Every species has a fundamental niche of which only a portion is occupied (realized niche). The elements constituting both types of niche cannot be completely described but can be estimated as a hypervolume of various biophysical parameters (Blonder et al. 2014). During the expansion into non-native geographical regions invasive species generally occupy the same hypervolume constituted by the fundamental niche of the species as defined by its native range (Aravind et al. 2022). North American invasive populations will benefit from global warming and coverage of suitable niches of the orchid will expand for 36–193% (Table  4, Fig. 4), generally into the Great Plains, and north- eastern directions within US Coastal Plain. The range contraction is expected to occur (not in all scenarios) in Guatemala and Belize around the Belize River; Puerto Rico along the foothills of the Sierra de Cayey and Cordillera Central, and the Mogotes; Dominican Republic in lowlands north of Cordillera Central, south of the Cordillera Oriental, and the Cordillera Septentrional; Haiti along Massif du Nord and foot- hills of Massif de la Hotte; Jamaica primarily around Westmoreland and Saint Catherine parishes; Cuba along western foothills of the Sierra Cristal, western and northern foothills of Sierra Maestra (Supplemen- tary Figure S7). As indicated in our analyses even within Asia, E. graminea does not occupy all climatically suit- able niches. That may be caused either by geographi- cal barriers preventing spread, unmeasured natural or human-induced abiotic conditions (Wraith et  al. 2020), or by constraining biotic factors in unoccu- pied areas, e.g. lack of symbiotic mycorrhizal fungi (Downing et  al. 2020), presence of herbivores and pathogens (Meena et  al. 2018; Meena and Mani 2022). However, it should be noted that the actual sensibility of natural orchid populations to patho- gens is still little recognized (Melendez and Ack- erman 1993). A similar situation of uninhabited Australian populations of E. graminea will likely become extinct in GISS projection, but their potential invasive range will be larger than currently observed in both CNRM and INM simulations. In the latter 1 ol.: (01 3 123456789) 1976 M. Kolanowska et al. Table 4   Changes in the coverage ­(km2) of the suitable niches of E. Potential versus observed geographical range 3 (1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1977 Fig. 3   Changes in the dis- tribution of suitable niches of E. graminea in Asia in various climate change scenarios - s (Recart et  al. 2013). The invasive orchid was the preferred host of a native, orchid specialist weevil, Stethobaris polita, previously regarded as a relatively rare beetle. Flower and fruit damage to S. plicata was high with significant demographic consequences, but population growth rates remained positive (Fal- cón et al. 2017). Elevated beetle populations resulted in increased weevil attack on the native orchid, sig- nificantly reducing fruit production. Two other com- mon, non-indigenous orchids on the island also serve as hosts, Arundina graminifolia and Dendrobium crumenatum, and may elevate weevil populations as well, with possible impacts on native species (Fos- ter and Ackerman 2021). The other orchid which is considered by local authorities to constitute a threat to native plants is Disa bracteata which invaded Australia in 1944 (Wapstra et al. 2020). However, in this case there is no evidence of negative effects on the Australian flora. We are unaware of any studies designed to detect the consequences of E. graminea invasions. comparison, American and Asian groups are more similar to each other, but still the overlap in occupied niches between these regions is low. Impact of global warming. Climate change can accelerate non-native plant invasions by altering environmental conditions, which may shift the geographical distribution of suit- able niche components and affect normal regimes of habitat disturbance (e.g., the frequency, duration, and severity of climatic events). Further change may occur through human responses to these changes (Adhikari et al. 2019; Blumenthal et al. 2013; Dukes and Mooney 1999; Turbelin and Catford 2021). Climate change will likely be favorable for E. graminea to expand its range if the unaccounted biotic components of its niche space (e.g., mycorrhi- zal fungi) will respond similarly. The most significant range expansion is predicted to occur in Australia (in 2 of 3 projections) which is interesting consider- ing the marginally suitable habitats that it currently occupies. The two most obvious ecological constraints for any orchid species persistence are (1) availability of pollen vectors and (2) presence of microbial sym- bionts in the environment. The former is not likely a problem for E. Potential versus observed geographical range graminea  Geographical region Projection SSP scenario Range expansion No range change Range contraction Change (%) North America CNRM 1–2.6 503,775.4 576,167.5 27,525.28 + 79 2–4.5 770,379.3 582,260.7 21,432.05 + 124 3–7.0 882,609.6 476,440.1 127,252.6 + 125 5–8.5 1,295,440 470,612.6 133,080.1 + 193 GISS 1–2.6 444,069.4 544,644.5 59,048.22 + 64 2–4.5 456,440.7 543,423.5 60,269.27 + 66 3–7.0 580,439.4 528,220.5 75,472.24 + 84 5–8.5 526,820.6 484,518 119,174.7 + 68 INM 1–2.6 264,659.7 556,143.1 47,549.68 + 36 2–4.5 366,845.5 546,330.8 57,361.96 + 51 3–7.0 722,937.2 561,093.5 42,599.26 + 113 5–8.5 929,173.8 569,635.7 34,057.05 + 148 Asia CNRM 1–2.6 1,591,586 5,189,896 328,331.2 + 23 2–4.5 2,059,589 5,150,549 367,678.3 + 31 3–7.0 2,415,786 4,936,159 582,068.7 + 33 5–8.5 2,645,666 4,403,998 1,114,230 + 28 GISS 1–2.6 1,286,665 5,206,398 311,829.6 + 18 2–4.5 2,058,696 5,307,062 211,165.9 + 33 3–7.0 2,463,861 5,351,687 166,540.4 + 42 5–8.5 2,688,171 5,150,799 367,428.1 + 42 INM 1–2.6 988,160.4 5,203,643 314,584.9 + 12 2–4.5 1,479,287 5,274,574 243,653.5 + 22 3–7.0 1,943,816 5,056,149 462,078.8 + 27 5–8.5 2,182,075 5,137,480 380,747.9 + 33 Australia CNRM 1–2.6 1975.163 503.5561 564.6961 + 132 2–4.5 13,809.15 1068.252 0 + 1293 3–7.0 7569.475 977.3913 90.86087 + 700 5–8.5 7622.123 701.412 366.8402 + 679 GISS 1–2.6 0 0 1068.252 − 100 2–4.5 0 0 1068.252 − 100 3–7.0 0 0.849167 1067.403 − 100 5–8.5 0 71.33003 996.9221 − 93 INM 1–2.6 8646.219 838.977 229.2751 + 788 2–4.5 7750.348 1068.252 0 + 726 3–7.0 2418.428 990.1288 78.12337 + 219 5–8.5 855.1112 723.4903 344.7618 + 48 Table 4   Changes in the coverage ­(km2) of the suitable niches of E. graminea potential range is observed in North America; how- ever, because unfilled niches are located on islands, time and geographical barriers are most probable reasons for unoccupied but potentially suitable areas in this region. Time is a factor as this invasion is relatively recent having been first noted in 2007 and new populations are discovered every year. We have anecdotal evidence that stratified dispersal is likely involved: long-distance dispersal to islands from Florida (USA) via the landscape trade in topsoil and potted plants; and local wind-dispersal of the dust- like seeds. In Australia the species is not expected to spread under current conditions since it already occupies all appropriate niche space which is only marginally suitable based on our analyses of its native range. In 1 Vol:. Fig. 3   Changes in the dis- tribution of suitable niches of E. graminea in Asia in various climate change scenarios Fig. 5   Changes in the dis- tribution of suitable niches of E. graminea in Australia in various climate change scenarios Fig. 4   Changes in the dis- tribution of suitable niches of E. graminea in North America in various climate change scenarios Potential versus observed geographical range While broad-scale analyses across the geographical regions are needed to uncover the importance of fungal partner(s) for orchid long- term survival, current results suggest that availabil- ity of OMF may not be a constraint to any of these invasive orchids now or in the future under climate change scenarios. Nonetheless, the orchid and its OMF symbionts will not necessarily respond to cli- mate change similarly (Kolanowska 2023). Although OMF have garnered most of the attention because of their importance in seed germination, other microbial endophytes can have fitness consequences by affect- ing plant growth, resistance to pathogens, and toler- ance capacity to biotic and abiotic stresses. We know very little about these interactions in an ecological or biogeographical context (Bayman et  al. 2002; Sar- saiya et al. 2019). The other factor which was not included in our analyses was geographical distribution of mating system. We only know that flowers of native Taiwan populations are autogamous (Chang et  al. 2010), but in other regions, native or non-native, reproduc- tion in E. graminea may be pollinator-dependent. This may be important because orchid species and their pollinators will not necessarily respond to cli- mate change in the same manner so potential range shifts of both should assessed (Kolanowska 2021; Kolanowska et al. 2021). Some orchids are characterized by very special- ized relationships with mycorrhizal partners and for these species predictions of future distribu- tion should be accompanied with the analyses of changes in the potential ranges of their symbionts (Kolanowska 2023). Unfortunately, little is known of the endophytic biota of E. graminea (Downing et al. 2020) and even less of their geographical dis- tribution so ENM of the ecological relationships of this species was not possible. Obviously, considering human impact on E. graminea, the most damaging is direct habitat destruction which cannot be predicted and included in simulations. However, the ecological ampli- tude of this orchid is remarkable. It grows in sandy beaches, coastal grasslands, lowland shrubs and open forests. It also does very well in lawns, road- sides, parks, home and hotel gardens (Ackerman and González-Orellana 2021; Chang et  al. 2010). This broad tolerance and ability to adapt to vari- ous environments constitutes an advantage of E. graminea in human-altered ecosystems. Potential versus observed geographical range graminea since the flowers appear Orchids are generally not considered to be harm- ful where they have invaded. However, a study con- ducted in Puerto Rico demonstrated apparent com- petition between mixed populations of introduced Spathoglottis plicata on a native orchid, Bletia patula 1 ol.: (01 3 123456789) 1978 M. Kolanowska et al. 1 3 Vol:. (1234567890) Fig. 4   Changes in the dis- tribution of suitable niches of E. graminea in North America in various climate change scenarios Fig. 5   Changes in the dis- tribution of suitable niches of E. graminea in Australia in various climate change scenarios 1 3 Vol:. (1234567890) Fig. 5   Changes in the dis- tribution of suitable niches of E. graminea in Australia in various climate change scenarios Fig. 5   Changes in the dis- tribution of suitable niches of E. graminea in Australia in various climate change scenarios 1 Vol:. ( 1 Vol:. ( 3 (1234567890) Climate change will likely facilitate invasion of Asian orchid Eulophia graminea into new… 1979 to be self-pollinating, perhaps by the same mecha- nism as E. maculata (Chang et al. 2010; González- Díaz and Ackerman 1988). The flowers contain small quantities of nectar (0.5  μl; (Ackerman and González-Orellana 2021) so the potential for a mutu- alistic plant-pollinator interaction exists, but thus far no pollinators have been reported (Ackerman et  al. 2023). On the other hand, E. graminea cannot escape the need for OMF. Based on expectations of bipartite networks, invasive or widespread native orchids likely specialize on a widespread OMF, or be a generalist in the number of OMF that they can exploit (Acker- man 2007; Bascompte et al. 2003; Vázquez and Aizen 2004). Downing et  al. (Downing et  al. 2020) com- pared the OMF of E. graminea from its native range in southwest China and the OMF of the species in its invasive range (Florida, USA) and concluded that E. graminea is a generalist with respect to its OMF as 10 of 18 fungal strains tested had successfully germi- nated seeds ex situ. The OMF are known for only two other invasive orchids, Eulophia (as Oeceoclades) maculata and Disa bracteata and both orchids asso- ciate with widespread OMF (Bayman et  al. 2016; Bonnardeaux et al. 2007). Potential versus observed geographical range While broad-scale analyses across the geographical regions are needed to uncover the importance of fungal partner(s) for orchid long- term survival, current results suggest that availabil- ity of OMF may not be a constraint to any of these invasive orchids now or in the future under climate change scenarios. Nonetheless, the orchid and its OMF symbionts will not necessarily respond to cli- mate change similarly (Kolanowska 2023). Although OMF have garnered most of the attention because of their importance in seed germination, other microbial endophytes can have fitness consequences by affect- ing plant growth, resistance to pathogens, and toler- ance capacity to biotic and abiotic stresses. We know very little about these interactions in an ecological or biogeographical context (Bayman et  al. 2002; Sar- saiya et al. 2019). However, currently it is not possible to evaluate the possible alterations in the soil physical, chemical or biotic properties resulting from global warming. According to our data (Supplementary Table  S2) E. graminea can grow in eleven different soil types (acrisols, arenosols, cambisols, ferralsols, fluvisols, gleysols, histosols, leptosols, luvisols, phaeozems, vertisols) and apparently has rather broad tolerance for various substrates. to be self-pollinating, perhaps by the same mecha- nism as E. maculata (Chang et al. 2010; González- Díaz and Ackerman 1988). The flowers contain small quantities of nectar (0.5  μl; (Ackerman and González-Orellana 2021) so the potential for a mutu- alistic plant-pollinator interaction exists, but thus far no pollinators have been reported (Ackerman et  al. 2023). On the other hand, E. graminea cannot escape the need for OMF. Based on expectations of bipartite networks, invasive or widespread native orchids likely specialize on a widespread OMF, or be a generalist in the number of OMF that they can exploit (Acker- man 2007; Bascompte et al. 2003; Vázquez and Aizen 2004). Downing et  al. (Downing et  al. 2020) com- pared the OMF of E. graminea from its native range in southwest China and the OMF of the species in its invasive range (Florida, USA) and concluded that E. graminea is a generalist with respect to its OMF as 10 of 18 fungal strains tested had successfully germi- nated seeds ex situ. The OMF are known for only two other invasive orchids, Eulophia (as Oeceoclades) maculata and Disa bracteata and both orchids asso- ciate with widespread OMF (Bayman et  al. 2016; Bonnardeaux et al. 2007). Declarations Bayman P, Gonzalez E, Fumero J et al (2002) Are fungi nec- essary? How fungicides affect growth and survival of the orchid Lepanthes rupestris in the field. J Ecol 90:1002–1008 Conflict of interest  The authors declare that they have no fi- nancial or non-financial conflicts of interest. Consent for publication  All authors provide their consent for publication. Bayman P, Mosquera-Espinosa A, Saladini-Aponte C et  al (2016) Age-dependent mycorrhizal specifi city in an invasive orchid, Oeceoclades maculata. Am J Bot 103:1880–1889 Beaury EM, Patrick M, Bradley BA (2021) Invaders for sale: the ongoing spread of invasive species by the plant trade industry. Front Ecol Environ 19:550–556 Bezabih Beyene B, Li J, Yuan J et al (2022) Non-native plant invasion can accelerate global climate change by increas- ing wetland methane and terrestrial nitrous oxide emis- sions. 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Trends Ecol Evol 26:333–339 Blonder B, Lamanna C, Violle C et  al (2014) The n-dimen- sional hypervolume. Glob Ecol Biogeogr 23:595–609 Blumenthal DM, Resco V, Morgan JA et  al (2013) Invasive forb benefits from water savings by native plants and car- bon fertilization under elevated ­CO2 and warming. New Phytol 200:1156–1165 Bonnardeaux Y, Brundrett M, Batty A et al (2007) Diversity of mycorrhizal fungi of terrestrial orchids: compatibility webs, brief encounters, lasting relationships and alien invasions. Mycol Res 111:51–61 Brown J (2014) SDMtoolbox: a python-based GIS toolkit for landscape genetic, biogeographic and species distribution model analyses. Declarations Methods Ecol Evol 5:694–700 Brown JL, Bennett JR, French CM (2017) SDMtoolbox  2.0: the next generation Python-based GIS toolkit for land- scape genetic, biogeographic and species distribution model analyses. PeerJ 5:e4095 Model deficiencies Author contributions  All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Marta Kolanowska, James Ackerman and Agnieszka Rewicz. The first draft of the manu- script was written by Marta Kolanowska and all authors com- mented on previous versions of the manuscript. All authors read and approved the final manuscript. This study, as any other simulation, has its limita- tions. The models of future distribution of suitable niches of E. graminea are based exclusively on the climatic data. The grass leaved Eulophia is a terrestrial species and depends on the soil proper- ties which may also be altered by climate changes. 1 ol.: (01 3 123456789) 1980 M. Kolanowska et al. home” or finding a “new home”? Biodivers Conserv 31:2625–2638 Funding  The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. 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Evaluation of Volumetric Change of Intracerebral Hemorrhage in Patients Treated with Thrombolysis for Intraventricular Hemorrhage
Neurocritical care
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Abstract Background:  Intraventricular hemorrhage (IVH) is often caused by irruption of intracerebral hemorrhage (ICH) of basal ganglia or thalamus into the ventricular system. Instillation of recombinant tissue plasminogen activator (rtPA) via an external ventricular drainage (EVD) has been shown to effectively decrease IVH volumes while the impact of rtPA instillation on ICH volumes remains unclear. In this series, we analyzed volumetric changes of ICH in patients with and without intrathecal lysis therapy. Methods:  Between 01/2013 and 01/2019, 36 patients with IVH caused by hemorrhage of basal ganglia, thalamus or brain stem were treated with rtPA via an EVD (Group A). Initial volumes were determined in the first available com‑ puted tomography (CT) scan, final volumes in the last CT scan before discharge. During the same period, 41 patients with ICH without relevant IVH were treated without intrathecal lysis therapy at our neurocritical care unit (Group B). Serial CT scans were evaluated separately for changes in ICH volumes for both cohorts using OsiriX DICOM viewer. The Wilcoxon signed-rank test was performed for statistical analysis in not normally distributed variables. Results:  Median initial volume of ICH for treatment Group A was 6.5 ml and was reduced to 5.0 ml after first instilla‑ tion of rtPA (p < 0.01). Twenty-six patients received a second treatment with rtPA (ICH volume reduction 4.5 to 3.3 ml, p < 0.01) and of this cohort further 16 patients underwent a third treatment (ICH volume reduction 3.0 ml to 1.5 ml, p < 0.01). Comparison of first and last CT scan in Group A confirmed an overall median percentage reduction of 91.7% (n = 36, p < 0.01) of ICH volumes and hematoma resolution in Group A was significantly more effective compared to non-rtPA group, Group B (percentage reduction = 68%) independent of initial hematoma volume in the regression analysis (p = 0.07, mean 11.1, 95%CI 7.7–14.5). There were no adverse events in Group A related to rtPA instillation. Conclusion:  Intrathecal lysis therapy leads to a significant reduction in the intraparenchymal hematoma volume with faster clot resolution compared to the spontaneous hematoma resorption. Furthermore, intrathecal rtPA applica‑ tion had no adverse effect on ICH volume. Keywords:  Intraventricular hemorrhage, Intracerebral hematoma, Intrathecal lysis, Volumetric change Keywords:  Intraventricular hemorrhage, Intracerebral hematoma, Intrathecal lysis, Volumetric chang Introduction *Correspondence: Franziska.staub‑bartelt@med.uni‑duesseldorf.de 1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article *Correspondence: Franziska.staub‑bartelt@med.uni‑duesseldorf.de 1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article Spontaneous intracerebral hemorrhage (ICH) is a life- threatening event leading to high mortality rates and permanent disability in surviving patients [1]. Intraven- tricular hemorrhage (IVH)—an extension of the ICH Evaluation of Volumetric Change of Intracerebral Hemorrhage in Patients Treated with Thrombolysis for Intraventricular Hemorrhage Franziska Staub‑Bartelt1*, Jasper Hans van Lieshout1, Thomas Beez1, Rainer Kram2, Daniel Hänggi1 and Kerim Beseoglu1 © 2020 The Author(s), corrected publication 2020 © 2020 The Author(s), corrected publication 2020 Neurocrit Care (2021) 34:529–536 https://doi.org/10.1007/s12028-020-01054-7 Neurocrit Care (2021) 34:529–536 https://doi.org/10.1007/s12028-020-01054-7 *Correspondence: Franziska.staub‑bartelt@med.uni‑duesseldorf.de 1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article Methods due to irruption of the bleeding into the ventricular system—is reported in more than 50% of patients with ICH and its volume is among the main predictors for poor outcome [2–5]. Due to its high impact on mor- tality and morbidity, diverse treatment strategies for IVH have been evaluated in the past. However, a clini- cal benefit from reduction in IVH due to irrigation of fibrinolytic substances through an external ventricular drainage (EVD) has been difficult to demonstrate in humans. Studies provide evidence for a reduction in all-cause mortality but shows no improvement in func- tional outcome [6–9]. We performed a retrospective analysis of a single-center cohort to investigate the effect of intrathecal recombinant tissue plasminogen activator (rtPA) on the volumetric changes of ICH associated with intrathecal lysis therapy. The study was approved by the local ethical committee (Study number: 2018-295). Reporting of this study was according to the strengthening the reporting of observa- tional studies in epidemiology (STROBE) guidelines for observational studies (Supplementary Material) [11]. 530 Patients Intraparenchymal hematoma expansion following intrathecal lysis therapy of IVH could explain these dis- appointing results, since neurological impairment due to ICH is directly associated with the clot size [10]. The aim of our study was to determine the effect of intrath- ecal lysis therapy on the volume of ICH. We here report our findings in 36 patients with ICH and concomitant IVH treated with intrathecal fibrinolysis and compared ICH volumes and occurrence of adverse events with a group of patients with ICH either with or without IVH treated with EVD alone. We identified all consecutive patients admitted to our neurocritical care unit at the University Hospital Düssel- dorf with an IVH due to ICH and treated with rtPA via an EVD, between January 2013 and January 2019. Inclu- sion criteria were: (1) age 18 years and older, (2) origin of hemorrhage located either at the basal ganglia, thala- mus or brain stem with relevant extension into any ven- tricle, (3) availability of initial and follow-up computed tomography (CT) scans and (4) at least one documented treatment with intrathecal lysis (Fig. 1). A relevant IVH was defined as a blood volume impending to cause a Fig. 1  Flowchart illustrating patient screening and selection. Patients were screened regarding general criteria (age, localization of ICH, EVD, no other surgical intervention) and then divided into two groups. Group A patients receiving intrathecal lysis, Group B patients without intrathecal lysis. Afterwards, patients with missing data (e.g., no follow-UP CT scans) in both cohorts were excluded Fig. 1  Flowchart illustrating patient screening and selection. Patients were screened regarding general criteria (age, localization of ICH, EVD, no other surgical intervention) and then divided into two groups. Group A patients receiving intrathecal lysis, Group B patients without intrathecal lysis. Afterwards, patients with missing data (e.g., no follow-UP CT scans) in both cohorts were excluded 531 hydrocephalus as evaluated by the treating physician with obstruction of the Foramina Monroi and/or the aqueduct. of 2 ml saline solution in order to guarantee intrathecal application. The EVD was then closed for 30–45  min, depending on individual patient tolerance. Upon reopen- ing of the EVD, the drainage system was kept at a reduced pressure threshold (10–12  mmHg) to achieve sufficient clearance of blood. This procedure was repeated every 12 h until a sufficient reduction in intraventricular blood in the third and/or fourth ventricle was achieved. Patients We performed repeat CT imaging every 24  h to determine the treatment effect as reported in previous publications [12, 13]. For definition of a control group (Group B), we fur- thermore screened the patient database for patients with (1) age 18 years and older, (2) ICH located at either basal ganglia, thalamus or brain stem, (3) receiving an EVD without an indication for intrathecal lysis (e.g., no or insignificant amounts of IVH) treated during the same period of time. Patients who underwent surgery for evacuation of the intracerebral hematoma, patients with lobar ICH and ICH related to aneurysm rupture, arteriovenous malfor- mation rupture or neoplasm as well as traumatic hemor- rhages were excluded. Outcome Primary outcome measure was the median volume (ml) of ICH on repeat CT imaging. Hematoma volume for every patient at every defined point of time was meas- ured by one of the authors (FSB) using Region of Interest (ROI) volumetry in OsirixLite (Pixmeo SARL, Switzer- land). The hematoma outline, defined as hyperdensity compared to brain parenchyma, was outlined manually on axial CT slices with 2–5  mm thickness. Hematoma volume was then calculated automatically using ROI vol- umetry function of the software (Fig. 2).hi Indication for EVD insertion in patients with ICH with or without IVH (Group A and Group B) were uncon- sciousness with indication for intubation, intubation due to other reasons (e.g., pulmonary insufficiency) and was established to monitor ICPs during the sedation/acute unconscious phase where patients could not undergo adequate neurological assessments. Additionally, for patients with ICH and IVH (Group A) indication for intrathecal lysis required EVD insertion. Indication for intrathecal lysis were: hydrocephalus/neurological dete- rioration due to tri-or tetra ventricular blood collection, particularly with localization in the III and IV ventricle. All patients included in Group A were treated with rtPA ­(Actilyse®, Boehringer-Ingelheim, Germany) within 12 h after initial CT scan. For each application, 2.5 mg rtPA was dissolved in 2 ml of saline solution. Actilyse was instilled directly into the ventricle followed by instillation Indication for EVD insertion in patients with ICH with or without IVH (Group A and Group B) were uncon- sciousness with indication for intubation, intubation due to other reasons (e.g., pulmonary insufficiency) and was established to monitor ICPs during the sedation/acute unconscious phase where patients could not undergo adequate neurological assessments. Additionally, for patients with ICH and IVH (Group A) indication for intrathecal lysis required EVD insertion. Indication for intrathecal lysis were: hydrocephalus/neurological dete- rioration due to tri-or tetra ventricular blood collection, particularly with localization in the III and IV ventricle. The first available CT scan showing ICH/IVH was defined as “initial” scan irrespective whether further CT scans without any intervention existed or not. Outcome First fol- low-up CT after first instillation of rtPA was defined as “first,” second available CT scan after repeated instilla- tion was defined as “second,“ third CT scan after further instillation of rtPA was defined as “third.“ Accordingly last available CT scan before discharge of the patients was defined as “final.“ CT scans were generally done All patients included in Group A were treated with rtPA ­(Actilyse®, Boehringer-Ingelheim, Germany) within 12 h after initial CT scan. For each application, 2.5 mg rtPA was dissolved in 2 ml of saline solution. Actilyse was instilled directly into the ventricle followed by instillation Fig. 2  Example for the region of interest (ROI) volumetry using OsirixLite (Pixmeo SARL, Switzerland). Axial CT scans were checked for slides show‑ ing hyperdensity relating to ICH, and hematoma outlines were marked on corresponding slides (left). Hematoma volume was then calculated automatically using ROI volumetry function of the software (right) Fig. 2  Example for the region of interest (ROI) volumetry using OsirixLite (Pixmeo SARL, Switzerland). Axial CT scans were checked for slides show‑ ing hyperdensity relating to ICH, and hematoma outlines were marked on corresponding slides (left). Hematoma volume was then calculated automatically using ROI volumetry function of the software (right) 532 Patients from Group A showed a significantly higher clot resolution rate as compared to Group B (p < 0.02, Fig. 4). In a regression analysis with hematoma volume reduction in percent (mean 72.1%; 95%CI 65.3–78.8%) as the dependent variable and initial hematoma volume and intraventricular rtPA application as independent variables only rtPA application (proportion 0.46; 95%CI 0.35–0.57) contributed significantly (p < 0.01) to the model. Initial hematoma volume (mean 11.1 ml, 95%CI 7.7–14.5 ml) was excluded (p = 0.07).i within 24  h after rtPA application. The number of CT scans were not representative for the number of intrathe- cal lysis administration. For the 41 patients from Group B only analyses of volume for the time periods “initial” and “final” were conducted. Statistical Analysis Categorical data are presented as counts and percentages and continuous variables as means with standard error of the mean (SEM) or medians with interquartile ranges (IQRs), depending on the normality of the data. Median time between initial CT and first administra- tion of rtPA was 10.0 h (IQR 6.8–14.0 h). For the primary outcome measure, we calculated median and interquartile ranges (IQR) instead of mean values and standard deviation in order to avoid distor- tion by outliers and extremities due to the small sample size. The Shapiro–Wilk test was used to test for normal distribution. Hereafter, non-parametric testing was used for related samples to determine changes in hematoma volumes over the treatment period (Wilcoxon signed- rank test). Group differences between Group A and B were evaluated using Mann–Whitney U test. Addition- ally, we performed a regression analysis with hematoma volume reduction (in %) as dependent variable and rtPA application and initial hematoma volume as contributing independent variables. The Type I error was set at 0.05 and the tests were 2-tailed. Statistical analyses were per- formed using IBM SPSS Statistics Version 26 (IBM Cor- poration, USA). 34 patients received repeated CT scans every 24 h after instillation of either one or two doses of rtPA per day. In 2 patients (subject 1 and 2), the interval between initial CT scan, start of lysis and first control CT was doubled to 48 h. Both patients’ treatment were finalized after the first CT scan. The median time period between the ini- tial and final CT scan for Group A was 10.0 days (IQR 7.0–14.0). The median period between initial and final CT scan for Group B was 11 days (IQR 9.0–17.0). No patient experienced an increase in hematoma volume after application of rtPA. However, 6 patients showed a hematoma progression between initial CT scan and start of intrathecal lysis (Table 2). All of these patients were treated with rtPA after hematoma progres- sion and showed a significant hematoma reduction by the time of discharge (p = 0.03). Furthermore, 3 patients of Group B showed higher hematoma volume at final CT scan compared to initial CT scan (subject 10, 21, 34, for detailed volumes please refer to Table  2 of supplement material). As these patients received no intervention, hematoma growth was due to natural progression and not related to any intervention. Results We included 36 patients with IVH due to ICH treated with rtPA via an EVD (Group A, Supplement Material Table 1) and 41 patients with ICH with or without IVH without intrathecal rtPA application (Group B, Supple- ment Material Table 2). All epidemiological data includ- ing main risk factors for ICH are summarized in Table 1. Median initial ICH volume in cohort A (n = 36) was 6.5 ml (IQR 5.3–12.6 ml) and was reduced to 5.0 ml (IQR 3.0–9.9  ml) after first treatment with intrathecal rtPA (p < 0.01). Overall, we did not see any infection related to intrath- ecal lysis therapy. Permanent shunting was performed in 55% of patients in Group A and 34% of patients in Group B during hospitalization. Median time from initial CT to permanent shunting for Group A was 9.5 days (IQR 7–12) and for Group B 11 days (IQR 9–12) (Table 1). 26 out of 36 patients received a second instillation of rtPA. Follow-up CT scan showed a decrease in median hematoma volume for this subgroup [4.5  ml (IQR 3.1– 9.5 ml) to 3.3 ml (IQR 1.6–8.4 ml); p < 0.01]. A further 16 out of 26 patients received a third intrathecal application of rtPA, which further decreased the mean hematoma volume from 3.0 ml (IQR 1.4–8.5 ml) to 1.5 ml (IQR 0.7– 6.5 ml); (p < 0.01, Fig. 3).i Discussion Our analysis reveals three relevant aspects. First, intra- ventricular application of rtPA in patients with ICH and concomitant IVH significantly decreases parenchymal hematoma volume without direct application into the hematoma. Second, rtPA application accelerates intra- parenchymatous hematoma reduction compared to rtPA-untreated patients. Third, the intrathecal applica- tion of rtPA does not increase the risk of parenchymal hematoma expansion or hemorrhagic complications.hfi Comparison of initial and final hematoma volume in Group A showed an absolute median decrease from 6.5 ml (IQR 5.3–12.6 ml) to 0.5 ml (IQR 0.0–3.9 ml). In Group B, median hematoma volume decreased from ini- tially 8.9 ml (IQR4.3–13.1 ml) to 3.1 ml (IQR 0.6–7.1 ml). The efficacy of intraventricular clot resolution by appli- cation of rtPA via an EVD has been demonstrated before 533 Table 1  Details and comparison of patient Group A and B Bold values represent significant values (p < 0.05) Details of all patients from Group A (intrathecal lysis) and Group B (no intrathecal lysis). Location thalamus and brainstem have been grouped due to the small group size. Discussion IQR interquartile range, CT scan computed tomography scan Intrathecal lysis Group A No intrathecal lysis Group B p N 36 41 Female  Absolute (%) 23 (64%) 14 (34%) < 0.01 Age  Median (IQR) 67 (59–74) 63 (52–70) 0.32 Location absolute (%)  Basal Ganglia 26 (72%) 30 (73%) 0.93  Thalamus or Brainstem 10 (28%) 11 (27%) Clot volume on CT scan (ml) Median (IQR)  Initial 6.5 (5.3–12.6) 8.9 (4.3–13.1) 0.74  After first lysis 5.0 (3.0–9.9) N/A  After second lysis 3.3 (1.6–8.4) N/A  After third lysis 1.5 (0.7–6.5) N/A  Final 0.5 (0–3.9) 3.1 (0.6–7.1) 0.02 Reduction in clot volume in percent initial to final CT scan  Mean (95% confidence interval) 81.8 (73.5–90.2) 63.4 (53.4–73.4) < 0.01 Time initial CT scan—1st lysis (h)  Median (IQR) 10.0 (6.8–14.0) N/A Days (initial–final CT scan)  Median (IQR) 10 (7–14) 11 (9–17) 0.20 Risk factors for IVH Absolute (%)  Arterial hypertension 33 (92%) 32 (78%) 0.10  Diabetes 4 (11%) 6 (15%) 0.65  Coronary artery disease 7 (19%) 3 (7%) 0.12  Alcohol 5 (14%) 3 (7%) 0.35  Nicotine 11 (31%) 5 (12%) 0.05  Antiplatelet 7 (19%) 4 (10%) 0.23  Anticoagulation 6 (17%) 4 (10%) 0.37 EVD-related infections (%) 0 0 – CSF shunt rate (%) 20 (55%) 14 (34%) 0.24 Timing of permanent shunting in days  Median (IQR) 9.5 (7–12) 11 (9–12) 0.26 Table 1  Details and comparison of patient Group A and B Details and comparison of patient Group A and B [13]; however, the focus was placed on IVH and to our knowledge data on the effect on the intraparenchymatous hematoma has not been published before.i hydrocephalus and allow for ICP monitoring. This simultaneously provides clinicians with a route for application of rtPA without the need for an additional intervention. Our analysis demonstrates that the intra- ventricular rtPa application leads to significant reduc- tion in ICH volume. It appears likely that rtPA diffuses into the parenchyma via the breach in the ventricle wall and the local effects are comparable to direct appli- cation of fibrinolytic agents into the intraparenchy- mal hematoma without the necessity of an additional Direct application of fibrinolytic agents into an intracerebral hematoma via image-guided catheter placement significantly reduces clot volume but inves- tigated patients had no IVH and required surgery to place the catheter [10]. In patients with deep-seated hematomas extending into the ventricles, insertion of an EVD is necessary in most cases to treat concomitant 534 Fig. Discussion 3  Visualization of hematoma volumes in Group A. Hematoma volume (ml) in the initial CT scan and after first, second and third application of recombinant tissue plasminogen activator (rtPA) as well as in the final CT scan before discharge. Circles signify patients receiving a repeated rtPA dose after CT scan, dots signify patients who did not receive further rtPA application. Patient #14 from Group A was omitted in this figure for illustra‑ tive reasons due to a very high hematoma volume; however, the value was included when calculating the medians Fig. 3  Visualization of hematoma volumes in Group A. Hematoma volume (ml) in the initial CT scan and after first, second and third application of recombinant tissue plasminogen activator (rtPA) as well as in the final CT scan before discharge. Circles signify patients receiving a repeated rtPA dose after CT scan, dots signify patients who did not receive further rtPA application. Patient #14 from Group A was omitted in this figure for illustra‑ tive reasons due to a very high hematoma volume; however, the value was included when calculating the medians catheter [10]. However, the underlying biophysiological processes remain obscure in detail. suffered from symptomatic hemorrhages, only three of them (1.2%) during the dosing phase [14]. Hematoma expansion in 6 patients from the treated cohort occurred before intrathecal therapy and was thus unrelated to rtPA application and in line with previously reported hema- toma progression during the first 24 h after hemorrhage [15]. Compared to the spontaneous hematoma resorp- tion rate, treated patients showed a significantly greater reduction in ICH hematoma volume over time. Comparable to other reports, we did not experience rebleeding complications attributable to rtPA applica- tion. In the MISTIE III trial, no significant difference in the prevalence of adverse events like symptomatic bleeding between the standard medical care and inter- ventional group was observed, even if rtPA was instilled directly into the residual hematoma after surgical clot removal [10]. Similarly, the CLEAR III trial showed a very low risk for new bleeding events under rtPA instillation therapy. It was reported that 2.4% (n = 6) of the patients Study limitations include the retrospective design and particularly the small number of patients in both cohorts. Regarding the retrospective character of the study, we would like to underline the following limitation. Fig. 4  Comparison of initial and final hematoma volume in Group A and B. Initial and final hematoma volume for patients receiving intrathecal lysis therapy (Group A, circles) compared to patients without intrathecal lysis therapy (Group B, dots). Comparison of hematoma volume in the final CT scan demonstrated a significant difference between both groups (p < 0.02). Patient #14 from Group A was omitted in this figure for illustrative reasons due to a very high hematoma volume; however, the value was included when calculat‑ ing the medians for any reliable assessment. Still, the procedure described was safe with regard to hematoma progression and seems to have no negative consequences on neurological out- come, but we cannot provide evidence for the treatment protocol with regard to improved functional outcome. for any reliable assessment. Still, the procedure described was safe with regard to hematoma progression and seems to have no negative consequences on neurological out- come, but we cannot provide evidence for the treatment protocol with regard to improved functional outcome. In addition to the small sample size, the median ICH size was considerably smaller compared to larger cohort studies as the MISTIE III trial. In our analysis, we focused on the effect of intrathecal lysis on ICH volumes; therefore, patients with larger ICH clot sizes with indica- tion for any surgical intervention (e.g., minimal invasive treatments, evacuation of ICH) addressing the ICH were excluded as hematoma volumes would not have been comparable regarding the specific aim of the present study. According to its aim and study design, this study con- firmed feasibility, safety and efficacy in clot resolution and our findings provide a basis for further prospective data collections. A controlled prospective study would be required to further elucidate, if the effect demonstrated is transferable to larger patient cohorts. om cohort A with a spontaneous hematoma expansion before initiation of intrathecal rtPA (n = 6) ble 2  Patients from cohort A with a spontaneous hematoma expansion before initiation of intrathec Table 2  Patients from cohort A with a spontaneous hematoma expansion before initiation of intrathecal rtPA (n = 6) A subgroup of patients in Group A showing details of hematoma progression between initial CT scan and last CT before intrathecal lysis. As not all patients received repeated CT scans before treatment, CTs on admission were used for calculation of initial hematoma volume. Discussion The rel- evance of faster ICH clot resolution for neurological out- come was not evaluated in the present data analysis. This was due to missing neurological outcome parameters in a large number of patients which excluded possibilities 535 Fig. 4  Comparison of initial and final hematoma volume in Group A and B. Initial and final hematoma volume for patients receiving intrathecal lysis therapy (Group A, circles) compared to patients without intrathecal lysis therapy (Group B, dots). Comparison of hematoma volume in the final CT scan demonstrated a significant difference between both groups (p < 0.02). Patient #14 from Group A was omitted in this figure for illustrative reasons due to a very high hematoma volume; however, the value was included when calculat‑ ing the medians At last, limitations of CT evaluation need to be dis- cussed. First, hematoma evaluation was not blinded to the groups. Secondly, the software that was used (Osi- rix Lite) is a free software version of Osirix MD, a widely used medical image viewer enabling various tools for processing of MRI/CT scans and other medical imag- ing. The full version is FDA cleared and CE IIa labelled and might be used for radiological diagnosis. We decided to use Osirix Lite as this is a freely available high-qual- ity software version even though some tools are missing compared to Osirix MD. For CT evaluation, ROI volume- try was needed and still available through Osirix Lite. In order to verify the results, we randomly evaluated hema- toma volumes by ABC/2 score and could not find any major discrepancy. A last potential confounder might be the decrease in hematoma density over time due to deg- radation of hemoglobin. Approximately 3–20 days after acute onset of ICH, the appearance of intraparenchymal hematoma in CT scans becomes less intense [16, 17]. The median time between initial CT and initiation of therapy in the intervention Group A was 10 h followed by repeti- tive CT scans every 24 h (except in 2 subjects). Thus, the treatment group received the majority of CT scans in the hyperacute and acute ICH stages, where the clot appears to be clearly hyperdense allowing a clear identification of parenchyma clot borders in the ROI volumetry. For comparison of initial and last CT in both cohorts, the decrease in hematoma density might have had an influ- ence on evaluation. Group values are stated as median values with interquartile ranges Patient Location CT scan admission CT scan pre-lysis 1st CT scan post-lysis CT scan discharge Volume (ml) Volume (ml) Volume (ml) Volume (ml) 8 Basal ganglia 22.9 33.1 24.4 3.4 11 Basal ganglia 5.9 23.3 15.3 9.1 13 Basal ganglia 30.7 34.0 32.1 18.5 18 Basal ganglia 0.1 3.8 2.7 0.0 28 Basal ganglia 25.1 33.0 32.8 32.6 29 Basal ganglia 5.3 5.9 5.7 3.7 14.4 [5.3–25.1] 28.1 [10.3–33.1] 19.8 [5.7–32.1] 6.4 [3.4–18.5] A subgroup of patients in Group A showing details of hematoma progression between initial CT scan and last CT before intrathecal lysis. As not all patients received repeated CT scans before treatment, CTs on admission were used for calculation of initial hematoma volume. Group values are stated as median values with interquartile ranges 536 by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​mmons​.org/licen​ses/by/4.0/. Conclusion We could demonstrate that intraventricular rtPA applica- tion significantly reduces ICH volumes in patients with IVH secondary to ICH in a small patient cohort. Fur- thermore, intrathecal thrombolysis leads to accelerated reduction in ICH volume compared to patients treated with EVD alone. The risk of procedure-related complica- tions was not increased, especially with regard to expan- sion of intraparenchymatous hematoma. Nevertheless, a prospective randomized trial will be mandatory to define new treatment strategies. Conflict of interest 10. Hanley DF, et al. Efficacy and safety of minimally invasive surgery with thrombolysis in intracerebral haemorrhage evacuation (MISTIE III): a ran‑ domised, controlled, open-label, blinded endpoint phase 3 trial. Lancet. 2019;393(10175):1021–32. The authors declare that they have no competing interests and all authors have nothing to disclose. The authors declare that they have no competing interests and all authors have nothing to disclose. Ethics Approval and Consent to Participate 11. von Elm E, et al. The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observa‑ tional studies. Lancet. 2007;370(9596):1453–7. The present retrospective study only reports data that were collected at our local neurosurgical intensive care ward. According to local legislation research, associates are allowed to use data that is directly accessible for research without previous informed consent of the patient when used in scientific context according. Due to anonymization, disclosure of identity of individual persons is impossible. The study was approved by the local ethical committee, Heinrich-Heine University, Faculty of Medicine, Düsseldorf, Germany (Study Number: 2018-295-RetroDeuA). 12. Webb AJ, et al. Resolution of intraventricular hemorrhage varies by ventricular region and dose of intraventricular thrombolytic: the clot lysis: evaluating accelerated resolution of IVH (CLEAR IVH) program. Stroke. 2012;43(6):1666–8. 12. Webb AJ, et al. Resolution of intraventricular hemorrhage varies by ventricular region and dose of intraventricular thrombolytic: the clot lysis: evaluating accelerated resolution of IVH (CLEAR IVH) program. Stroke. 2012;43(6):1666–8. 13. Naff N, et al. Low-dose recombinant tissue-type plasminogen activa‑ tor enhances clot resolution in brain hemorrhage: the intraventricular hemorrhage thrombolysis trial. Stroke. 2011;42(11):3009–16. 13. Naff N, et al. Low-dose recombinant tissue-type plasminogen activa‑ tor enhances clot resolution in brain hemorrhage: the intraventricular hemorrhage thrombolysis trial. Stroke. 2011;42(11):3009–16. Source of Support No funding was received. Source of Support No funding was received. References 1. Delcourt C, et al. Intracerebral hemorrhage location and outcome among INTERACT2 participants. Neurology. 2017;88(15):1408–14. 1. Delcourt C, et al. Intracerebral hemorrhage location and outcome among INTERACT2 participants. Neurology. 2017;88(15):1408–14. Abbreviations IVH: Intraventricular hemorrhage; ICH: Intracerebral hemorrhage; rtPA: Recombinant tissue plasminogen activator; EVD: External ventricular drainage; STROBE: Strengthening the Reporting of Observational Studies in Epidemiol‑ ogy; CT: Computed tomography. 4. Young WB, et al. Prognostic significance of ventricular blood in supraten‑ torial hemorrhage: a volumetric study. Neurology. 1990;40(4):616–9. 5. Nieuwkamp DJ, et al. Treatment and outcome of severe intraventricular extension in patients with subarachnoid or intracerebral hemorrhage: a systematic review of the literature. J Neurol. 2000;247(2):117–21. Availability of Data and Materials 14. Dey M, et al. Bleeding and infection with external ventricular drainage: a systematic review in comparison with adjudicated adverse events in the ongoing clot lysis evaluating accelerated resolution of intraventricular hemorrhage phase III (CLEAR-III IHV) trial. Neurosurgery. 2015;76(3):291– 300 di i 301 14. Dey M, et al. Bleeding and infection with external ventricular drainage: a systematic review in comparison with adjudicated adverse events in the ongoing clot lysis evaluating accelerated resolution of intraventricular hemorrhage phase III (CLEAR-III IHV) trial. Neurosurgery. 2015;76(3):291– 300 di i 301 All data generated or analyzed during this study are included in this published article and its supplementary information files. Electronic supplementary material The online version of this article (https​://doi.org/10.1007/s1202​8-020-01054​-7) contains supplementary material, which is available to authorized users. 2. Trifan G, Arshi B, Testai FD. Intraventricular hemorrhage severity as a predictor of outcome in intracerebral hemorrhage. Front Neurol. 2019;10:217. 3. Tuhrim S, et al. Volume of ventricular blood is an important determinant of outcome in supratentorial intracerebral hemorrhage. Crit Care Med. 1999;27(3):617–21. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Received: 18 January 2020 Accepted: 9 July 2020 Published online: 31 July 2020 Author details 1 f 6. Dey M, et al. External ventricular drainage for intraventricular hemor‑ rhage. Curr Neurol Neurosci Rep. 2012;12(1):24–33. 1 Department of Neurosurgery, Medical Faculty, Heinrich-Heine University Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. 2 Department of Anesthesiology, Medical Faculty, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany. 7. Wang D, et al. Local fibrinolytic therapy for intraventricular hemor‑ rhage: a meta-analysis of randomized controlled trials. World Neurosurg. 2017;107:1016–1024 e1. 8. Gaberel T, et al. Intraventricular fibrinolysis versus external ventricular drainage alone in intraventricular hemorrhage: a meta-analysis. Stroke. 2011;42(10):2776–81. Source of Support Source of Support No funding was received. Acknowledgements Acknowledgements Open Access funding provided by Projekt DEAL. Open Access funding provided by Projekt DEAL. 9. Hanley DF, et al. Thrombolytic removal of intraventricular haemor‑ rhage in treatment of severe stroke: results of the randomised, multicentre, multiregion, placebo-controlled CLEAR III trial. Lancet. 2017;389(10069):603–11. g p 300 ; discussion 301. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted 15. Brott T, et al. Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke. 1997;28(1):1–5. 15. Brott T, et al. Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke. 1997;28(1):1–5. 16. Macellari F, et al. Neuroimaging in intracerebral hemorrhage. Stroke. 2014;45(3):903–8. 17. Osborn AG. Osborn’s brain imaging, pathology, and anatomy. Salt Lake City: Amirsys Publication; 2018.
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Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease, Exercise and Altitude Exposure
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Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI) Interested in publishing with us? Contact book.department@intechopen.com Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com Open access books available Countries delivered to Contributors from top 500 universities International authors and editors Our authors are among the most cited scientists Downloads We are IntechOpen, the world’s leading publisher of Open Access books Built by scientists, for scientists 14% 191,000 210M TOP 1% 154 7,200 Chapter 7 Additional information is available at the end of the chapter http://dx.doi.org/10.5772/60698 © 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease, Exercise and Altitude Exposure Anthony M.J. Sanchez, Robin Candau, Audrey Raibon and Henri Bernardi Anthony M.J. Sanchez, Robin Candau, Audrey Raibon and Henri Bernardi Additional information is available at the end of the chapter http://dx.doi.org/10.5772/60698 Additional information is available at the end of the chapter 1. Introduction Skeletal muscle exhibits remarkable adaptive capabilities in response to various stimuli such as loading conditions (resistance training, microgravity), contractile activity (electrical stimulations, endurance exercise), environmental factors (altitude exposure), or nutritional interventions. To access this great capacity, a plethora of quantitative and functional adaptations are involved. Changes in the size of adult muscle, in response to these external stimuli, are mainly due to the growth of individual muscle fibers rather than an increase in fiber number [1]. The control of muscle mass is dependent upon a balance between anabolic and catabolic processes. Hypertrophy is associated with increased protein synthesis, while atrophy is characterized by increased degradation of muscle proteins and/or a decrease in protein translation. The initiation of protein synthesis is mainly mediated by a signaling pathway in which the mammalian/mechanistic target of rapamycin complex 1 (MTORC1), a multiprotein complex composed of MTOR (mammalian/mechanistic target of rapamycin), RPTOR (regu‐ latory-associated protein of MTOR), mLST8/GβL (MTOR-associated protein LST8 homolog), DEPTOR (DEP domain containing MTOR-interacting protein), and PRAS40 (proline-rich Akt substrate of 40 kDa) [2,3]. MTORC1 by phosphorylating its substrates S6K1 (ribosomal protein p70S6 kinase 1) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) controls skeletal muscle protein translation and hypertrophy [4-7]. The Insulin signaling pathway leads to the activation of MTORC1 through the activation of the kinases PI3K (phosphatidylinositol 3-kinase), PDK1 (phosphoinositide-dependent kinase-1), and Akt. Akt, also known as protein kinase B (PKB), inactivates tuberous sclerosis complex 1/2 (TSC1/2), promoting MTOR activation by Rheb-GTP [8-10]. Akt also phosphorylates and inactivates the glycogen synthase kinase 3β (GSK3β), resulting in the activation of the eukaryotic translation initiation factor 2B (eIF2B) and increased protein synthesis [5,11]. Muscle atrophy leads to a state of weakness and emaciation of the body, which is encountered, for example, in the terminal phase of certain diseases or chronic infections such as cancer, AIDS, diabetes, bacterial infections, and nerve degeneration [12]. Muscle atrophy is also observed during aging, immobilization, and stress or trauma to the muscle and is associated with increased proteolysis. Protein degradation is essentially modulated by two conserved path‐ ways: the ATP-dependent ubiquitin-proteasome system and the autophagy pathways. The first one has been particularly involved in the degradation process after the discovery of two E3 ubiquitin ligases (E3 ligases), MAFbx/atrogin-1 (muscle atrophy F-box) and MuRF1 (muscle RING finger-1), which are both overexpressed in various models of atrophy (fasting, cancer, diabetes, immobilization, and other stresses) [13,14]. Abstract Autophagy is an evolutionarily conserved intracellular system that selectively eliminates protein aggregates, damaged organelles, and other cellular debris. It is a self-cleaning process critical for cell homeostasis in conditions of energy stress. Autophagy has been until now relatively overlooked in skeletal muscle, but recent data highlight its vital role in this tissue in response to several stress conditions. The most recognized sensors for autophagy modulation are the adenosine monophos‐ phate (AMP)-activated protein kinase (AMPK) and the mechanistic target of rapamycin (MTOR). AMPK acts as a sensor of cellular energy status by regulating several intracellular systems including glucose and lipid metabolisms and mitochon‐ drial biogenesis. Recently, AMPK has been involved in the control of protein synthesis by decreasing MTOR activity and in the control of protein breakdown programs. Concerning proteolysis, AMPK notably regulates autophagy through FoxO transcrip‐ tion factors and Ulk1 complex. In this chapter, we describe the functioning of the different autophagy pathways (macroautophagy, microautophagy, and chaperone- mediated autophagy) in skeletal muscle and define the role of macroautophagy in response to physical exercise, a stress that is well assumed to be a key strategy to counteract metabolic and muscle diseases. The effects of dietary factors and altitude exposure are also discussed in the context of exercise. Keywords: Cachexia, Endurance exercise, Hypoxia, Proteolysis, Sarcopenia 172 Muscle Cell and Tissue 1. Introduction The invalidation of these proteins confers a resistance to certain types of induced atrophy, suggesting a critical role in the catabolism for the ubiquitin-proteasome pathway [13]. The function of E3 ligases is to ubiq‐ uitinate specific proteins to target them for recognition by the 26S proteasome where they are eliminated. Other E3 ligases like zinc-finger protein 216 (ZNF216), the mitochondrial E3 ubiquitin protein ligase 1 (Mul1), and the tripartite motif-containing protein 32 (Trim32) have been shown to play an important role in skeletal muscle atrophy [15-17]. Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 173 The autophagy signaling, which constitutes the second pathway, is important for maintain‐ ing cell metabolism and organelle turnover. It involves the degradation of substrates by hydrolases into a vesicle called lysosome [18]. Recent evidence demonstrates cross talk and cooperation between the ubiquitin-proteasome system and autophagy [19,20]. The impor‐ tance of this pathway in skeletal muscle was long much neglected, and autophagy was thought to be a nonselective degradation system. However, it is now well recognized that autophagy machinery is critical for muscle homeostasis and organelle turnover in response to cellular stress like physical exercise or hypoxia [21]. Importantly, the energy sensor AMPK (5'-adenosine monophosphate-activated protein kinase) has been involved in the regulation of both protein translation pathway and protein degradation systems, with a particular interest in the regula‐ tion of autophagy program for the last few years. The present chapter focuses on the role of autophagy in skeletal muscle homeostasis. We will describe the functioning of the autophagy signaling pathways (i.e., macroautophagy, microautophagy, and chaperone-mediated autophagy) and detail the regulation of macroautophagy by both AMPK and MTOR, with the final goal to discuss the potential applications of recent discoveries concerning autophagy- related pathologies. The involvement of autophagy in response to physical exercise (acute and chronic exercise) and altitude exposure is thereafter discussed. 2.1. The autophagy pathways 2.1.1. Description of the system 2.1.1. Description of the system Autophagy has been discovered during nutrient privation and can be referred to as “self- eating” as cells degrade their own constituents to maintain cellular homeostasis in response to various injuries like starvation and hypoxia and in pathological conditions, including cancer, muscular dystrophy, and neurodegenerative diseases. One purpose of the starvation-in‐ duced autophagy is to degrade materials to provide amino acids and free fatty acids in order to preserve metabolism and ATP levels when extracellular nutrients reach hazardous low levels [22]. Moreover, autophagy also eliminates protein aggregates as well as unwanted and dysfunctional organelles. The term autophagy embraces macroautophagy, microautophagy, and chaperone-mediated autophagy that we will describe hereafter. Macroautophagy and microautophagy are conserved from yeast to humans, and these processes were originally described as bulk degradation mechanisms. However, these two processes can be selective for targeting different organelles, and we distinguish mitophagy selective for degradation of mitochondria; pexophagy, selective for degradation of peroxisomes; xenopha‐ gy, selective for degradation of intracellular bacteria and virus; reticulophagy, selective for endoplasmic reticulum; heterophagy, selective for substances taken in by phagocytosis; golgiphagy, selective for Golgi apparatus; ribophagy, selective for ribosomes; crinophagy, specific for the contents (proteins, peptides) of secretory granules; glycophagy, selective for glycogen; and lipophagy, selective for lipid droplets [23-29]. Among these different varieties of Muscle Cell and Tissue 174 autophagy, mitophagy has been the most studied in the last decade, and this process involves notably two Parkinson’s disease factors, the RING-between-RING E3 ligase Parkin and the mitochondrial kinase PINK1 (PTEN-induced putative kinase protein 1), PTEN being “phospha‐ tase and tensin homolog” [30,31]. After mitochondrial potential depolarization, PINK1 promotes Parkin activation through phosphorylation of its ubiquitin-like domain [32]. In addition to these factors, the mitochondrial E3 ligase Mul1 can also be involved in mitophagy by the degradation of the mitochondrial fusion protein Mfn2 (mitofusin-2) and the stabiliza‐ tion of the dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation [33]. Autophagy by providing a turnover of the cellular components plays a central role in the homeostasis of the cell. It is a key mechanism by which a starving cell reallocates nutrients from unnecessary to more-essential processes. Macroautophagy, microautophagy, and chaperone- mediated autophagy lead cytoplasmic substrates inside lysosomes in which their contents are digested by a battery of acidic hydrolases [34]. Four essential ubiquitous proteases have been identified: the cathepsins B, D, H, and L [35,36]. 2.1.2. Microautophagy Microautophagy is localized directly at the level of the lysosome which directly engulfs cytosol components by invagination, protrusion, and/or elimination of the lysosomal limiting mem‐ brane. It is implicated in the degradation of long half-life proteins in numerous cell types and does not respond to classical stimuli inducing chaperone-mediated autophagy and macroau‐ tophagy [51]. In contrast to macroautophagy, microautophagy has not been extensively studied in skeletal muscle, and its functions in muscle proteolysis have to be more characterized [35]. 2.1.1. Description of the system High levels of these cathepsins are expressed in tissues exhibiting high rates of protein turnover like the kidney, spleen, liver, or placenta, while low concentrations of cathepsins are found in tissues with lower protein turnover as skeletal muscles [37-40]. Similar enzymatic properties were reported for different muscles, independent of their metabolic and contractile type. However, their concentrations differ according to the fiber type. Indeed, slow-twitch oxidative muscles exhibit higher levels of cathepsins than the fast-twitch glycolytic muscles [35], suggesting a more important activity of this system in oxidative muscle. This data is in agreement with the fact that oxidative muscles present a more important protein turnover and a greater translational activity than glycolytic muscles. Although skeletal muscle expresses cathepsins B, D, H, and L, they play distinctive roles. During fusion of myoblasts into myotubes, several groups have reported an increase in the expres‐ sion and activity of lysosomal cathepsins, in particular cathepsin B [41-46]. Several studies showed that the expression of cathepsin L is induced during various forms of skeletal muscle atrophy including starvation [12,36,47-49]. Increase in cathepsin D activity has been reported in muscles of dystrophic mice and chicken and in muscles of patients with Duchenne muscu‐ lar dystrophy [35,50]. 2.1.4. Macroautophagy Macroautophagy, often referred to as autophagy or autophagosome-lysosome system, is an evolutionarily conserved intracellular system that coordinates and oversees the degradation of damaged organelles as mitochondria, peroxisomes, or ribosomes, intracellular pathogens, and unused long-lived proteins [61]. More than 30 autophagy-specific genes (Atgs) have been identified and are known to facilitate the sequestration of cytoplasmic substrates into a double- membrane vesicle called autophagosome or autophagic vacuole. Atgs are essential mediators of autophagy, by controlling the formation of the autophagosome. Autophagosome fuses with lysosome to form an autolysosome (also called autophagolysosome). 2.1.3. Chaperone-mediated autophagy Chaperone-mediated autophagy (CMA) is a selective form of autophagy that has only been described in mammalian cells to date [52,53]. In this form of autophagy, only cytosolic proteins that possess the consensus pentapeptide Lys-Phe-Glu-Arg-Gln (KFERQ) are recognized. The Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 1 175 KFERQ-like sequence is recognized by the heat-shock cognate protein of 73 kDa (hsc73) then targeted to lysosomes for degradation. This targeting needs the binding of the complex protein substrates hsc73 to the lysosome-associated membrane protein type 2A (LAMP-2A) and to a multi-molecular chaperone complex including hsp40, hsp70, and hsp90 at the cytosolic side of the lysosomal membrane [54]. LAMP-2A is a glycoprotein present at the lysosomal mem‐ brane which acts as a CMA receptor. The substrates are then unfolded and translocated across the lysosomal membrane with the hsc73 protein. Unlike other forms of autophagy, CMA is very selective in substrate degradation and cannot eliminate organelles [55-57]. The use of an antibody directed to the KFERQ amino acid sequence substrates showed that proteins containing this sequence are conserved in skeletal muscle during starvation, while they are degraded in the liver and heart [58]. Moreover, the absence of consensus sequence in most myofibrillar proteins indicates that this degradation pathway is not implicated in their degradation. However, Nishino and colleagues showed that LAMP-2 deficiency is the primary defect in human Danon disease, a pathology characterized by myopathy and cardiomyopathy with massive accumulation of autophagic vacuoles [59,60].Thus, as microautophagy, CMA has to be more characterized in the context of muscle atrophy. 2.2.1. Initiation Macroautophagy (autophagy) is initiated in response to a multitude of factors including nutrient deprivation and oxidative stress. The activation of autophagy during muscle wasting was shown by the accumulation of autophagosomes in muscles of fasted transgenic GFP-LC3 mice [62]. Studies showed that during starvation-induced atrophy, FoxO3a (forkhead box class O3a) regulates the transcription of several Atgs, including Atg4B, LC3B (microtubule- associated protein 1A/1B-light chain 3B), Beclin-1, Vps34 (vacuolar protein sorting 34)/PI3K class III, Gabarapl1 (GABAA receptor-associated protein-like 1), Atg12, and Ulk2 (unc-51-like kinase 2) [63,64]. Initiation of the autophagy processes involves the activation of the unc-51- like kinase 1 (Ulk1, also called Atg1 in yeast)/Atg13/FIP200/Atg101 and the Beclin-1/Vps34 complexes. These proteins operate in conjunction with several Atgs to mediate the assembly of the autophagosomal membrane [65-68]. Ulk1 also phosphorylates Beclin-1 at Ser-14 following amino acid withdrawal, and this stage is necessary for the Vps34 lipid kinase Muscle Cell and Tissue 176 activation and full autophagy induction [69]. In yeast and mammalian cells, Atg1 or Ulk1 (respectively) activity is suppressed under nutrient-rich conditions by MTORC1 (phosphory‐ lation of Ulk1 at Ser-757) [65,70-73]. In addition, MTOR inhibition and its subsequent dissoci‐ ation from Ulk1 are critical for Beclin-1/Vps34 complex activation by Ulk1 [69]. 2.3. Implication of autophagy in cell homeostasis and disease Autophagy plays a prominent role in the maintaining of cell homeostasis by selectively eliminating protein aggregates, damaged organelles, and other nonactive cellular debris [79,80]. This process is required for normal cellular function, but it is increasingly apparent that it can have both beneficial and detrimental effects on cells and tissues, depending on the origin of its activation [81]. Physiological function of basal autophagy in maintaining tissue homeostasis has been demonstrated in several tissues as the brain, liver, heart, striated muscle, intestine, pancreas, and adipose tissue [82-89]. Furthermore, exciting reports suggest that autophagy may contribute to counteract the deleterious effects of aging by limiting the deposition of aggregated proteins and damaged mitochondria [90-92]. By blocking apoptosis, autophagy preserves cell survival by providing endogenous metabolites when exogenous substrates are lacking [93]. Thus, at regular levels of activation, autophagy may represent the first step to restore homeostasis. However, when the autophagic capacity is submerged, apoptosis takes over [94]. Nevertheless, the relationship between autophagy and apoptosis appears to be extremely complex, and additional data are necessary to clarify the situation, especially in the context of disease. In numerous pathologies as diabetes, obesity, cancer, heart failure, and neurodegenerative, infectious, and inflammatory disease, autophagy activity is affected [34,95,96]. For instance, it has been reported that the lack in Beclin-1 expression decreases autophagy flux and leads to increased risk of breast and prostate cancers [97]. However, the systematic beneficial effect of autophagy should be tempered. Thus, the role of autophagy in cancer is ambivalent, and this process can be involved in both the promotion and the prevention of this disease. In the first stage of the malignancy (i.e., tumor initiation), inhibition of autophagy may allow the growth of initial cancerous cells, and thus autophagy can act as a suppressor of cancer [98,99]. When cancer is established, transformed cells may need autophagy to survive, especially in nutrient- limiting condition [100]. In addition, for patient undergoing treatment such as chemotherapy, cancer cells could use autophagy to protect themselves from the stress induced by the therapy. Other reports indicate also that glycogen storage disease type II (also called Pompe disease) – an autosomal recessive metabolic disorder – is a pathology attributable in part to mutations of Atgs. In this disease, muscle and nerve cells are damaged by an accumulation of glycogen into the lysosomes caused by a deficiency of the lysosomal acid alpha-glucosidase enzyme [101,102]. 2.2.2. Maturation of autophagosomes The maturation and completion of the autophagic vacuole is facilitated by a ubiquitin-like conjugation-signaling cascade that culminates with the binding of phosphatidylethanolamine (PE) to the cytosolic form of LC3 (LC3-I) to form LC3-II. LC3, a mammalian homolog of yeast Atg8, is a protein with a molecular mass of 17 kDa that is distributed ubiquitously in mam‐ malian tissues. Two other LC3 homologs are GABAA receptor-associated protein (Gabarap) and Golgi-associated ATPase enhancer (GATE-16). LC3-phosphatidylethanolamine conjugate (LC3-II) is recruited to the autophagosomes [74,75]. LC3-II plays here a structural role that allows the elongation and the formation of the mature autophagosome. The mature autopha‐ gosome fuses with the lysosome to form an autolysosome (Fig.1). Concomitantly, LC3-II in autolysosomal lumen is degraded by lysosomal hydrolases; thus, turnover of the autophago‐ somal protein LC3-II reflects autophagic activity [76]. LC3 also interacts with the autophagy adapter p62/SQSTM1 (sequestosome 1), which contains multiple apparent protein-protein interaction domains. p62 binding to LC3 permits the entry of ubiquitinated cargo into the autophagosome [77]. In addition, the Atg12-Atg5-Atg16 complex plays an important role in the maximal promotion of LC3 lipidation and autophagy induction [78]. Figure 1. Processing of the macroautophagy system Figure 1. Processing of the macroautophagy system Figure 1. Processing of the macroautophagy system Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 1 Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 177 2.4. The roles of autophagy in skeletal muscle Compared to other tissues like the liver or pancreas, autophagy in skeletal muscle exhibits a low protein turnover and a small size of autophagosomes. These peculiar characteristics have probably constituted a brake for the detection of autophagy in this tissue for a long time. Associated to the use of transgenic mice expressing LC3 fused with GFP, autophagy process can be now easily visualized [62]. Conversely to liver or pancreas in which autophagy is activated transiently for a few hours, in skeletal muscle, autophagy can be activated for several days [62]. As it was shown in other tissues, muscular autophagy is activated by nutrient deprivation or by the absence of growth factors [108]. Although it was reported that the mRNAs coding for Atgs are present in abundance in skeletal muscle [109], the role and the regulation of basal autophagy have been poorly characterized in this tissue until recently. In order to assess the function of autophagy in skeletal muscle, Masiero and colleagues performed experiments on mice deprived of the Atg7 gene, a gene necessary to the unfolding of the autophagy program [86,110]. Importantly, mice showed obvious signs of muscular weakness and atrophy exacerbated during ageing. Mice presented an accumulation of degraded proteins and free radicals, a deterioration of the internal cellular structures, and an activation of the apoptotic program. The authors clearly defined that inhibition of basal autophagy does not protect from skeletal muscle atrophy induced by denervation or starvation, but on the contrary, contributes to its degeneration. Similar muscle alterations have been obtained in muscle-specific Atg5-/- mice [87], confirming the necessity to have regular autophagic flux in the cells, even during atrophy. In many conditions varying from fasting, denervation, inactivity, microgravity, various pathologies as cancer, AIDS, sepsis, diabetes, cardiac failure, and myopathies, autophagy is overactive and pathologic, thus leading altered metabolism and muscle loss [81,108,111]. Contribution of autophagy to muscle loss begins to be clarified with the use of different animal models and innovative techniques. Inactivation of autophagic flux by LC3 silencing partially prevents FoxO3-mediated muscle atrophy [63] and atrophy caused by the expression of mutant SOD1 G93A in skeletal muscle [112]. In another model, atrophy induced by L-type calcium channel (DHPR) inactivation is linked to the expression of autophagic genes including LC3, Vps34, BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3), and cathepsin L (for the lysosome) [113]. 2.3. Implication of autophagy in cell homeostasis and disease In many neurodegenerative disorders such as Parkinson’s, Huntington’s, and Alzheimer’s diseases, accumulation of autophagic vesicles has been also observed [103-105]. Regarding Alzheimer’s disease, it has been reported that Atg7 influences the accumulation of amyloid β (Aβ) peptides, resulting in aggregation into plaques in the brain [106]. In this model, autophagy seems to participate to the disease progression since it is involved in the generation of Aβ peptides. In summary, by removing misfolded proteins and abnormal organelles, autophagy can be considered as a critical mechanism for cell protection. On the contrary, by destroying excessive fraction of cytosol and organelles, too high levels of autophagy represent a side mechanism responsible of the initiation of pathologies [94,107]. Muscle Cell and Tissue 178 2.4. The roles of autophagy in skeletal muscle During sepsis, an upregulation of autophagy was found in parallel to mitochondrial injury and decreased biogenesis, especially in locomotor muscles [114]. Reactive oxygen species overproduction by altered mitochondria is now considered as a critical signal for the promotion of skeletal muscle autophagy, thus finding an opening to practical prospects for treatment of disease [115]. Accordingly, antioxidant supplement can lead to an inhibition of skeletal muscle autophagy through a reduction of oxidative stress and an increase in antioxidant capacity [116]. Taken together, these recent results confirm that excessive activation of autophagy is a critical factor for muscle wasting determinism, and strategies attempted to control autophagy level will be promising. Data concerning sarcopenia are also specific. Sarcopenia is an age-related loss of muscle mass and strength, which is associated with increased autophagy, apoptosis, and exacerbated proteolysis [117]. Elevated peroxisome proliferator-activated receptor-coactivator α (PGC-1α) expression in muscle during aging contributes to reduce the proteolytic activity associated Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 179 with atrophy. In sarcopenia, attenuation of the degradative processes and the maintenance of mitochondrial function contribute to the preservation of muscle integrity [118]. In summary, as already described for cancer cells, autophagy in muscle is a complex process that can be, according to its activation level, beneficial or deleterious. During disease, the systematic question is to determine whether autophagy plays a protective function, has a causative role, or is a result of the disease process itself. In summary, as already described for cancer cells, autophagy in muscle is a complex process that can be, according to its activation level, beneficial or deleterious. During disease, the systematic question is to determine whether autophagy plays a protective function, has a causative role, or is a result of the disease process itself. 3.1. The AMP-activated protein kinase AMPK is a serine/threonine protein kinase highly conserved through evolution. AMPK is a heterotrimeric complex composed of a catalytic subunit (AMPK-α) and two regulatory subunits (AMPK-β and AMPK-γ). Humans have seven genes encoding AMPK subunits (α1, α2, β1, β2, γ1, γ2, γ3) that can form at least 12 αβγ heterotrimers, increasing the diversity of its functions [119]. The catalytic α subunit contains the threonine phosphorylation site that upon phosphorylation leads to AMPK activation [120]. AMPK acts as a sensor of cellular energy status by regulating several intracellular systems including glucose and lipid metab‐ olisms and mitochondrial biogenesis [121]. Thus, AMPK activation leads to increased glycol‐ ysis flux [122] and fatty acid oxidation [123-126] and on the contrary, to an inhibition of glycogenogenesis [127,128] and cholesterol and fatty acid biosynthesis [129-131]. The enzyme also increases the expression of PPARα (peroxisome proliferator-activated receptor α) target genes and PGC-1 leading to mitochondrial biogenesis and enhanced oxidative metabolism of muscle cells [132,133]. AMPK is activated by a large variety of cellular signals that decrease cellular ATP levels and increase AMP in response to different kinds of stress like electrical- stimulated muscle contraction, exercise, hypoxia, and heat shock or under conditions of nutrient deprivation [124,134,135]. The recognized enzymes in the regulation of AMPK under energy stress conditions are LKB1 (liver kinase B1), CaMKK (calmodulin-dependent protein kinase kinase), and TAK-1 (transforming growth factor beta-activated kinase 1). 3.2. Regulation of skeletal muscle autophagy by AMPK The role of AMPK in protein turnover has been clearly defined in recent years. AMPK has been involved in the control of protein synthesis and the repression of skeletal muscle mass by inhibiting MTOR activity [136,137] in two ways: AMPK phosphorylates TSC2 (tuberous sclerosis complex 2) at Thr-1227 and Ser-1345 and RPTOR at Ser-722 and Ser-792, leading to a reduction of MTORC1 activity. Several studies also showed that AMPK increases protein degradation through the modulation of the ubiquitin-proteasome and the autophagosome- lysosome pathways [138,139]. AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribonucleo‐ side, an AMPK activator) treatment increases the expression of the E3 ligases MAFbx/ Atrogin-1 and MuRF1 in muscles cells. In addition, increase of autophagic flux by AMPK has been reported in several muscle models as C2C12 myoblasts, C2C12 myotubes, and primary myotubes [138,139]. Two major signaling pathways were characterized in AMPK-induced muscular autophagy (Fig.2). Muscle Cell and Tissue 180 Figure 2. Role of AMPK in skeletal muscle protein turnover The first one concerns the activation of the forkhead box class O proteins (FoxO), notably involved in the regulation of protein breakdown, energy metabolism, and mitochondrial turnover [140]. FoxO factors also play an important role in exercise-induced angiogenesis by limiting it during the first days of training program [141,142]. Activation of FoxO3a by AMPK leads to an increase in several Atgs expression, including LC3-II and Gabarapl1 that act as promoters of autophagosome fabrication [139]. AMPK directly interacts with FoxO3a and phosphorylates it on Ser-588, a residue known to lead to FoxO3a activation [139,143]. The upregulation of several Atgs by FoxO factors have been described in Drosophila larval fat body [144], mammalian cardiomyocytes [145], hepatocytes [146], and colorectal cancer cells [147]. Regarding the regulation of FoxO3a subcellular localization in muscle cells, while long treatments (i.e., 24 h) with AMPK activators do not change FoxO3a nuclear content, an increase in the total protein level is notable after 30 min. With a short time course (30 min–6 h), the activation of AMPK by AICAR leads to a relocalization of FoxO3a into the nucleus [139]. Tong and colleagues reported that AICAR treatment causes FoxO3a nuclear relocation through a decrease in FoxO3a phosphorylation at Thr-318/321 [148]. However, Greer and colleagues have reported an increase of FoxO3a transcriptional activity without any change in the nuclear content of the factor after AMPK activation by 2-deoxyglucose (2DG) in HEK293T cells [143]. 3.2. Regulation of skeletal muscle autophagy by AMPK These data strongly suggest that FoxO3a relocalization into the nucleus is not necessarily required to increase its transcriptional activity. A possibility is that AMPK may also control FoxO3a protein stability. The second pathway involves modulation of the Ulk1 complex A multiprotein complex Figure 2. Role of AMPK in skeletal muscle protein turnover Figure 2. Role of AMPK in skeletal muscle protein turnover The first one concerns the activation of the forkhead box class O proteins (FoxO), notably involved in the regulation of protein breakdown, energy metabolism, and mitochondrial turnover [140]. FoxO factors also play an important role in exercise-induced angiogenesis by limiting it during the first days of training program [141,142]. Activation of FoxO3a by AMPK leads to an increase in several Atgs expression, including LC3-II and Gabarapl1 that act as promoters of autophagosome fabrication [139]. AMPK directly interacts with FoxO3a and phosphorylates it on Ser-588, a residue known to lead to FoxO3a activation [139,143]. The upregulation of several Atgs by FoxO factors have been described in Drosophila larval fat body [144], mammalian cardiomyocytes [145], hepatocytes [146], and colorectal cancer cells [147]. Regarding the regulation of FoxO3a subcellular localization in muscle cells, while long treatments (i.e., 24 h) with AMPK activators do not change FoxO3a nuclear content, an increase in the total protein level is notable after 30 min. With a short time course (30 min–6 h), the activation of AMPK by AICAR leads to a relocalization of FoxO3a into the nucleus [139]. Tong and colleagues reported that AICAR treatment causes FoxO3a nuclear relocation through a decrease in FoxO3a phosphorylation at Thr-318/321 [148]. However, Greer and colleagues have reported an increase of FoxO3a transcriptional activity without any change in the nuclear content of the factor after AMPK activation by 2-deoxyglucose (2DG) in HEK293T cells [143]. These data strongly suggest that FoxO3a relocalization into the nucleus is not necessarily required to increase its transcriptional activity. A possibility is that AMPK may also control FoxO3a protein stability. The second pathway involves modulation of the Ulk1 complex. A multiprotein complex composed of AMPK, MTORC1, Ulk1, FIP200, and Atg13 has been identified in muscle cells (Fig.3) [139]. These data fit with the model found in other cell types showing that, under basal Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 1 181 conditions, MTORC1 prevents autophagy by interacting with Ulk1 [72]. 3.2. Regulation of skeletal muscle autophagy by AMPK Under nutrient-rich conditions, phosphorylation of Ulk1 by MTORC1 represses Ulk1 kinase activity and its ability to interact with Atg13 or FIP200; thereby, it coordinates the autophagy response [65,149]. In muscle cells, activation of AMPK (by AICAR treatment) or inhibition of MTORC1 (by Torin1 treatment or amino acid privation) removes AMPK, MTOR, and RPTOR from Ulk1 [139]. These events are known to induce the Ulk1-dependent phosphorylation of Atg13 and FIP200, leading to the initiation of autophagy [70]. Proteomics screens of autophagy [150] and a co-immunoprecipitation study performed in HEK293T cells [151] showed that AMPK interacts with both Ulk1 and Ulk2. In muscle cells, Ulk1 also acts as an interacting partner of AMPK, and Ser-467 site identified by Egan and colleagues is also phosphorylated by AMPK [139]. Ulk1 phosphorylation by AMPK may participate to conformational changes and thus disrupts the interaction between Ulk1 and MTORC1, in agreement with the suppression of MTORC1 anti-autophagy activity in the Ulk1 complex [72]. Moreover, Ulk1 phosphorylation by AMPK may directly activate Ulk1 kinase activity. Indeed, in vitro studies showed that Ulk1 is highly phosphorylated and that purified Ulk1 can phosphorylate itself and requires autophosphorylation for stability [152]. In mam‐ mals, Ulk1 phosphorylation by AMPK is critical for mitochondrial homeostasis and cell survival during starvation [153]. In summary, AMPK regulates Ulk1 activity by decreasing MTORC1 activity and by phosphorylating Ulk1 [121]. Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Time-course studies have been performed in muscle cells in order to better understand the dynamics of UlK1 complex following autophagy induction [139]. Interestingly, AMPK dissociates from Ulk1 3 h after AICAR treatment [139]. In agreement with these observations, in HeLa cells, AMPK is associated with Ulk1 only under nutrient-rich condition, and it dissociates from Ulk1 5 min after starvation [154]. Thus, in normal condition, Ulk1 is associated Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Figure 3. The Ulk1/Atg13/FIP200/MTORC1/AMPK complex Time-course studies have been performed in muscle cells in order to better understand the dynamics of UlK1 complex following autophagy induction [139]. Interestingly, AMPK dissociates from Ulk1 3 h after AICAR treatment [139]. In agreement with these observations, in HeLa cells, AMPK is associated with Ulk1 only under nutrient-rich condition, and it dissociates from Ulk1 5 min after starvation [154]. Thus, in normal condition, Ulk1 is associated Muscle Cell and Tissue 182 with AMPK; upon AICAR treatment, the complex remains stable for 3 h and then dissociates. As suggested by another group, Ulk1 dissociation from AMPK could permit to Ulk1 complex to be more active [154]. Conversely, this dissociation can constitute a negative regulatory feedback as proposed by Loffler et al. [155]. The authors showed that Ulk1 could mediate phosphorylation of AMPK on the regulatory subunits, constituting an inhibitory feedback control. Further works have to define the molecular mechanisms for these events, especially in skeletal muscle. 4. Autophagy, exercise and altitude exposure However, Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 183 reticulophagy has not been studied yet in skeletal muscle, especially in response to exercise. Further works are needed to clarify the possible clearance of important organelles, such as ribosomes or endoplasmic reticulum, during physical exercise. Exercise promotes better “cell health”; it would be not surprising to discover that exercise increases the turnover of such organelles like it is strongly suggested for mitochondria [21]. The rise of autophagy is essential to prevent mitochondrial damage during endurance exercise. Although acute inhibition of autophagy prior to exercise seems to not significantly affect performance, it leads to accumulation of dysfunctional mitochondria and augmentation of oxidative stress especially during eccentric contraction [164]. Thus, autophagy has a critical role in mitochondria quality control during acute exercise. In addition, autophagy is highly involved in exercise training-induced adaptations. Autophagy-deficient mice present attenu‐ ated improvement of endurance capacity in response to endurance training. In parallel to lower increases of basal autophagy flux, these mice show attenuated raises of mitochondrial content and angiogenesis [165], explaining the poor response to the training program. The dietary factors have to be considered in autophagy response to acute exercise since essential amino acids (EAA) or carbohydrate (CHO) intake modulate protein turnover. Jamart et al. (2014) found that exercise performed in the fasted state permits a higher raise in auto‐ phagic flux indexes compared with the fed state. Concerning resistance exercise, few works showed a depression of autophagy markers after such an exercise like the study by Fry et al. conducted in humans [166]. In addition, autophagic flux markers can be depressed following EAA and carbohydrate (CHO) ingestion after resistance exercise [167]. To date, autophagy seems non-critical for muscle adaptations to resistance training [161]. However, an exception occurs during aging in which both endurance and resistance training are able to reverse the drop of autophagy regulatory proteins that occurs [168,169] (Fig.4). Little is known regarding protein turnover pathways, especially autophagy, in response to exercise performed during altitude exposure in humans. Such an environment can induce a state of hypoxia that is exacerbated according to the level of altitude considered. Hypoxia results in decreased oxygen availability and leads to several hormonal, cardiorespiratory, and muscular adjustments in order to preserve cell homeostasis. Long-lasting hypoxia can cause a diminution of skeletal muscle mass and a reduction of muscle oxidative capacity. 4. Autophagy, exercise and altitude exposure Attractive data concerning the role of autophagy during exercise are emerging. Autophagic vacuole formation during physical exercise was observed for the first time by Salminen and coworkers in 1984 with electron microscopy [156]. Nevertheless, there were no further studies on the topic until recently. In the last decade, data supporting the importance of autophagy in muscle homeostasis in response to exercise have been numerous, starting with a study from Bonaldo’s team that showed that mice presenting impaired autophagy develop severe muscle weakness (i.e., accumulation of defective mitochondria, exacerbated apoptosis, muscle degeneration, and atrophy) [157]. Thereafter, other studies highlight that chronic inactivation of autophagy leads to a loss of metabolic effects related to exercise and drastic decreases in endurance performance [158]. Concerning autophagy modulation in response to acute exercise in humans, studies by Jamart and colleagues [159] were the first to demonstrate a raise of autophagy-regulatory genes and autophagic flux markers after ultraendurance exercise. By showing that AMPK and FoxO3a regulate in a coordinated way autophagy and ubiquitin-proteasome pathways during ultraendurance exercise, the authors gave an important picture of the cross-regulation of both degradation pathways in response to long-lasting endurance exercise [160]. Regarding more common endurance exercises, the modulation of muscle protein turnover, autophagy, and mitochondrial dynamics markers has been investigated thereafter in mice in response to different exercises conducted or not until exhaustion. Endurance exercise quickly initiates the autophagy pathway through Ulk1 activation resulting in an increase of autophagic flux, especially near exhaustion [161]. A rise in the phosphorylation of DRP1, a GTPase essential for mitochondrial fission, quickly occurred during exercise without any change in the expression of fusion markers (OPA1 and Mfn2). These data are consistent with an increase in mitophagy (i.e., the degradation of mitochondria by autophagy) since exacerbated fission can lead to mitochondrial fragmentation. Noteworthy, exercise decreases the activity of the main protein synthesis pathway (i.e., Akt/MTOR signaling pathway), from 90 min of moderate exercise (40– 50 % of VO2max), concomitantly to an increase in the phosphorylation of a marker of endoplas‐ mic reticulum stress (eiF2α Ser-51) [161]. Others studies reported an increase of endoplasmic reticulum stress by the evaluation of the content of the double-stranded RNA-activated protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), the ER stress-induced transcription factor C/EBP homologous protein (CHOP), and the X-box binding protein 1 (XBP1s), in response to both ultraendurance [162] and moderate-intensity exercise [163]. 4. Autophagy, exercise and altitude exposure In agree‐ ment with an alteration of protein synthesis flux, hypoxia impairs the overload-induced increase of the PI3K/Akt/MTOR signaling pathway in rats [170]. Regarding degradation pathways in humans, while ubiquitin-proteasome system seems not positively modulated by environmental hypoxia, an upregulation of skeletal muscle autophagic flux markers has been found during acute normobaric hypoxia (10.7 % O2) and after exercise conducted in such an environment [171,172]. Another study investigated the effects of acute high-altitude exposure (at 5,300 m altitude) in the course of the Caudwell Research Expedition to Mt. Everest [173]. The authors notably found an upregulation of heat shock cognate 71 kDa protein involved in chaperone-mediated autophagy and a reduction of protein translation markers. Taken together, these studies seem to highlight a preventive role of autophagy for energy expenditure and an activation of chaperone-mediated autophagy during acute high-altitude exposure. However, the effects of chronic altitude exposure on autophagy and its combination with training remain to be characterized to date. Chronic hypoxia leads to a change in oxidative Muscle Cell and Tissue 184 Figure 4. Exercise and autophagy in skeletal muscle. Adapted from Sanchez AMJ et al. 2014. Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise. American Journal of Physiology – Regulatory, In‐ tegrative and Comparative Physiology 307(8): R956-R969 [21]. Essential amino acids (EAA); carbohydrate (CHO) Figure 4. Exercise and autophagy in skeletal muscle. Adapted from Sanchez AMJ et al. 2014. Autophagy is essential to support skeletal muscle plasticity in response to endurance exercise. American Journal of Physiology – Regulatory, In‐ tegrative and Comparative Physiology 307(8): R956-R969 [21]. Essential amino acids (EAA); carbohydrate (CHO) metabolism [174,175]; it is likely that autophagy, especially mitochondrial autophagy, be amended during such an exposure. Consistent with this, experiments performed in cells showed that mitochondrial autophagy is induced by chronic hypoxia through HIF-1 (hypoxia- dependent factor-1) and BNIP3, and this regulation constitutes a preventive response that is necessary to avoid accumulation of reactive oxygen species and cell death [176]. The effects of exposition to moderate altitude (i.e., 1,500–3,000 m) have also to be definite since it concerns a larger population (athletes, general tourist population, and highlanders) compared to high altitude. Research of this type is leading to a better understanding of the autophagy-mediated turnover of organelles like mitochondria in response to exercise and altitude exposure. 4. Autophagy, exercise and altitude exposure Finding optimal training strategies represents an important objective to enhance exercise adaptations in both athletes and patients with metabolic or muscle diseases, including COPD (chronic obstructive pulmonary disease) that can cause systemic hypoxia and loss in muscle capability. 5. Conclusions In summary, autophagy represents a crucial mechanism for cell homeostasis and cell survival. Autophagy constitutes a recycling process that degrades used or flawed internal structures Autophagy, a Highly Regulated Intracellular System Essential to Skeletal Muscle Homeostasis — Role in Disease… http://dx.doi.org/10.5772/60698 1 185 into amino acid, enabling cells to survive in difficult circumstances. The identification of Ulk1 as a direct target of AMPK represents a significant step toward understanding how cellular energy stress regulates autophagy machinery in muscle cells. Based on the data presented here, it is clearly conceivable that the autophagy pathway must be considered in muscle pathologies such as sarcopenia and myopathies. Fine molecular studies on AMPK/FoxO3a and AMPK/ Ulk1 axes will offer new and promising strategies in the treatment of muscular loss associated not only to several pathologies like cancer, AIDS, and neuromuscular diseases but also to age- related disorders. Further investigations must be conducted in order to better understand whether exercise-induced autophagy can also prevent skeletal muscle diseases. In addition, autophagy is involved in skeletal muscle adaptation to endurance exercise in a healthy population. While autophagy is important to prevent mitochondria alteration and exacerbated oxidative stress in response to severe acute exercise, its role in the improvement of endurance capacity has also been demonstrated in response to endurance training. In addition, the feeding pattern and the environment, especially altitude exposure, have to be considered since they represent additional modulators of protein turnover and autophagy pathway. Regular exercise is associated with an enhanced quality of life and represents the most profitable way to limit metabolic disorders and the loss of muscle mass. As a result, these research directions are of major interest in the battle against a wide range of diseases and have to be sustained. Acknowledgements Dr AMJ Sanchez thanks the “Cité de l’excellence sportive Sud de France” of Font-Romeu (France). This work was supported by the department PHASE from INRA and by the Uni‐ versité de Montpellier 1, Faculté des Sciences du Sport. Miss A. Raibon holds a graduate fellowship from INRA, from the region Languedoc-Roussillon, and from INCA. The authors also thank Pr Raviola for the helpful discussions. Author details Anthony M.J. Sanchez1*, Robin Candau2, Audrey Raibon2 and Henri Bernardi2 *Address all correspondence to: anthony.sanchez@univ-perp.fr 1 Laboratoire Européen Performance Santé Altitude, EA4604, Université de Perpignan Via Domitia, Département STAPS Font-Romeu, France 1 Laboratoire Européen Performance Santé Altitude, EA4604, Université de Perpignan Via Domitia, Département STAPS Font-Romeu, France 2 INRA, UMR866, Dynamique Musculaire et Métabolisme, Université de Montpellier Montpellier, France 2 INRA, UMR866, Dynamique Musculaire et Métabolisme, Université de Montpellier 2 INRA, UMR866, Dynamique Musculaire et Métabolisme, Université de Montpellier, Montpellier, France Montpellier, France List of abbreviations 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; Aβ, amyloid β; AIDS, acquired immune deficiency syndrome; AMPK, 5'-adenosine monophosphate-activated protein kinase; Atg, autophagy-specific gene; BCL2, B-cell lymphoma 2; BNIP3, BCL2/ adenovirus E1B 19 kDa-interacting protein-3; CaMKK, Ca2+/calmodulin-dependent protein kinase; CHO, carbohydrate; CHOP, C/EBP homologous protein; CMA, chaperone-mediated autophagy; COPD, chronic obstructive pulmonary disease; DEPTOR, DEP domain-containing MTOR-interacting protein; EAA, essential amino acids; eIF2B, eukaryotic translation initiation factor 2B; eIF2α, eukaryotic initiation factor 2α; FIP200, focal adhesion kinase (FAK) family- interacting protein of 200 kDa; FoxO, forkhead box class “other” O; Gabarapl1, GABAA receptor-associated protein-like 1; GATE-16, Golgi-associated ATPase enhancer; GSK3β, glycogen synthase kinase 3β; hsc73, heat-shock cognate protein of 73 kDa; HIF-1, hypoxia- dependent factor-1; IGF-1, insulin-like growth factor-1; LAMP-2A, lysosome-associated membrane protein 2A; LC3, microtubule-associated protein 1A/ 1B light chain 3; LKB1, liver kinase B1; MAFbx/atrogin-1, muscle atrophy F-box; Mfn2, mitofusin-2; mLST8/GβL, MTOR- associated protein LST8 homolog; MTOR, mechanistic/mammalian target of rapamycin; MTORC1, mechanistic/mammalian target of rapamycin complex 1; Mul1, mitochondrial E3 ubiquitin protein ligase 1; MuRF1, muscle RING-finger protein-1; OPA1, optic atrophy 1; p62/ SQSTM1, sequestosome 1; PDK1, phosphoinositide-dependent kinase-1; PE, phosphatidyle‐ thanolamine; PERK, double-stranded RNA-activated protein kinase R (PKR)-like endoplasmic reticulum kinase; PGC-1, peroxisome proliferator activator receptor γ coactivator-1; PI3K, phosphatidylinositol 3-kinase; PINK1, PTEN-induced putative kinase protein 1; PKB/Akt, Muscle Cell and Tissue 186 protein kinase B; PPARα, peroxisome proliferator-activated receptor α; PRAS40, proline-rich Akt substrate of 40 kDa; PTEN, phosphatase and tensin homolog; Rheb, ras homologous enriched in brain; RPTOR, regulatory-associated protein of MTOR, complex 1; ROS, reactive oxygen species; S6K1, p70S6 kinase 1; TAK-1, transforming growth factor β-activated kinase 1; Trim32, tripartite motif-containing protein 32; TSC21/2, tuberous sclerosis complex 1/2; Ulk, unc-51-like kinase; Vps34, vacuolar protein sorting 34; XBP1s, X-box binding protein 1; ZNF216, zinc-finger protein 216 References [1] Glass DJ (2005) Skeletal muscle hypertrophy and atrophy signaling pathways. 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A System for Converting and Recovering Texts Managed as Structured Information
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A System for Converting and Recovering Texts Managed as Structured Information OPEN Edgardo Samuel Barraza Verdesoto 1,3,4*, Marlly Yaneth Rojas Ortiz 3 & Richard de Jesus Gil Herrera 2 Edgardo Samuel Barraza Verdesoto 1,3,4*, Marlly Yaneth Rojas Ortiz 3 & Richard de Jesus Gil Herrera 2 This paper introduces a system that incorporates several strategies based on scientific models of how the brain records and recovers memories. Methodologically, an incremental prototyping approach has been applied to develop a satisfactory architecture that can be adapted to any language. A special case is studied and tested regarding the Spanish language. The applications of this proposal are vast because, in general, information such as text way, reports, emails, and web content, among others, is considered unstructured and, hence, the repositories based on SQL databases usually do not handle this kind of data correctly and efficiently. The conversion of unstructured textual information to structured one can be useful in contexts such as Natural Language Generation, Data Mining, and dynamic generation of theories, among others. Written communication is a type of information that has a basic structure well defined in each language which is useful in information ­processing1. Additionally, some other types of communication can be totally or partially converted into text whereupon the final processing is carried ­out2,3. Written communication is a type of information that has a basic structure well defined in each language which is useful in information ­processing1. Additionally, some other types of communication can be totally or partially converted into text whereupon the final processing is carried ­out2,3. i Several applications have been developed in information processing following principles or attributes of the text, for instance, to build ontologies or micro-theories4–7 which are convenient for automatic decision-making ­tasks8. Furthermore, In process automation, the speed at which the information is produced reduces human performance and delays the decision-making processes, this has generated the urgent need to delegate some decisions to machines and create applications for resolving these ­problems9–11.h pp g p The information retrieval and the generation of natural language promote the generation of sentences/phrases with meaning from a large amount of data generated by interactivity, shared repositories, and homogeneous or heterogeneous data sources, one of these types of application is presented ­in12 where is exposed a framework able to generate three classes of question and answers from corpora: fill in the gaps, multiple choice, and shuffled sentences. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports 1Universidad Americana de Europa (UNADE), Cancún, México. 2Universidad Internacional de la Rioja, Logroño, Spain. 3Research Department, Tecnológica Autónoma de Bogotá (FABA), Bogotá, Colombia. 4Universidad de Santander (UDES), Bogotá, Colombia. *email: edgardo.barraza@correo.faba.edu.co Scientific Reports | (2022) 12:22249 Conceptual framework p Object‑action dissociation/integration. These studies and approaches suggest that the information held in the brain is a set of clusters (cores) that could be affected by the ambiguity and the context in both, the dissociation and integration. Likewise, according to some theories, the brain saved our memories in two ways: semantically and the episodic ­way13,14, this latter manner is very important to explain the development of the strategy followed in this paper. Historically, the dissociation of the information by the human brain was observed when comparing Broca’s aphasic agrammatical patients, whose speech involves the use of very few verbs in contrast with other anomic patients that had great difficulty finding concrete ­nouns15. Initially, the major difficulty with verbs for Broca’s patients was interpreted based on the highest syntactical complexity of verbs compared to ­nouns16–18. However, the idea that verbs are, in general, harder to produce has been undermined in other studies where it is indicated that patients with anomic difficulties produce verbs more easily than ­nouns19,20. From a neurophysiological point of view, there are different opinions and theoretical ­proposals21, of which three hypotheses have been put for- ward regarding verb-noun storage issues within neural networks: partial separation of verbs and nouns22,23, word separation based on morphosyntax24, and separation between actions and objects25,26. Psycholinguistics also agree that exist, in the brain, the distinction between various grammatical categories, particularly between verbs and nouns, and propose three possible starting points or context to access the information: availability of information related to the grammatical class, a required grammatical knowledge, and the independence between the definition of grammatical class and the semantic differences27–30. A considerable number of studies have dealt with aspects associated with the dissociation of the information within the human mind and the conclusions are ­similar31, there are a dissociation between verbs(actions) and nouns (objects).h j The counterpart of the dissociation process is the integration process. According ­to32, grammatical informa- tion is relevant to understanding and producing sentences, but a plausible conclusion suggests that the gram- matical class information is not a lexical property that can be retrieved automatically; instead, this property is likely to play an important role in the context of a sentence. Fundamentally, the role of the grammatical class in sentence processing is modulated by the linguistic differences regarding the way as words of certain grammati- cal classes are used within sentences. A System for Converting and Recovering Texts Managed as Structured Information OPEN The framework aims to create a pedagogical tool able to automatically generate tests in the context of a topic, the parser divides texts according to the processed language and prepares the type of question selected. These approaches that allow recording and recovering of fragments or whole texts from a repository, conceiv- ing and improving strategies applied in recovering unstructured information are very important in the current Computer Science.h This article introduces an architecture that allows building applications capable of dissociating texts/sentences in subsets of cores with properties and simple operations such as those that the algebraic groups incorporate. These operations are preferable because they have properties that promote a straightforward and reliable manner to retrieve a whole text or part of it by keeping the structure of the language. Additionally, relationships between subsets are incorporated because they play an essential role in maintaining the meaning of the recovered text/ sentences.h The main objective of this manuscript is to show the design of a system with the capability of processing sentences (part of the text), storing them in databases, and finally, recovering them while keeping the original text’s basic meaning. To reach that, it is reviewing some previous concepts and experiences about linguistic computational to support the architectural design; it is described and justified how the algebraic groups help in the organization of the components of sentences for storage and recovering them while keeping the meaning and structure; Also, it is treated how to design an architecture for a processing system the objects/data as structured information (into structured databases), and finally, it is shown the functionality of a system for some illustrative instances and test cases for the Spanish language. 1Universidad Americana de Europa (UNADE), Cancún, México. 2Universidad Internacional de la Rioja, Logroño, Spain. 3Research Department, Tecnológica Autónoma de Bogotá (FABA), Bogotá, Colombia. 4Universidad de Santander (UDES), Bogotá, Colombia. *email: edgardo.barraza@correo.faba.edu.co | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ This paper is organized as follows. Firstly, a theoretical framework under which the proposal is based will be explored. Secondly, a general architecture will be proposed that incorporates each one of the elements exposed in the theoretical framework. Thirdly, an approach, based on the architecture presented, applied to the Spanish Language will be analyzed. Finally, the findings and future works will be exhibited. Conceptual framework In all languages, verbs commonly require higher processing than nouns at various levels, firstly, because the processing of verbs is about events and could exist many elements that will have to be integrated. Secondly, the verb syntax also demands more processing because verbs should be connected to other words to convey their meaning. Lastly, nouns are linked to objects, but they might refer to events too, and it is necessary its disambiguation. In conclusion, the effects of the grammatical class in the retrieval and representation of simple words are more productive when the context is ­present33–35. In addition, Neuroscience states that there are two kind of memory for storing and remembering facts and events consciously; such events are stored in the episodic memory such as a storyteller whereas the semantic memory records the same event as part of our overall knowledge (dictionary). In specific, the episodic memory is intended as a repository in our brain where is recorded an event similar to a text well-written13,14. p y In summary, there are two processes well conceived in our brain, dissociation and integration of an event These tasks are the fundamentals of the proposal in this paper. From sentences to clusters of words. The word classification has been a normal practice in linguistics, computer sciences, and education, among others (see Fig. 1); this practice normally has different targets and results. Furthermore, as an instance, ConceptNet is a project based on the sense common concept that was conceived as a semantic network containing lots of things that the computers should know about the ­world36–38. Another example is the WordNet project which resembles a thesaurus in that words are grouped based on their meanings, the result is a network that can be browseable ­easily39–42.i g y A text is more complex than simple words, it is a texture that relates, firstly, words to create sentences, sec- ondly paragraphs, and ignoring other structures, finally several paragraphs directly or indirectly (e.g. using the anaphor) linked between one another result in a text. Each language has rules to build sentences and paragraphs. According ­to43, there are various ways to classify and describe the languages, but a very common is the order of each one of their main components (Subject, Object, and Verb) in the sentence: • Subject-Object-Verb (SOV). This is the most frequent type of word order in spoken languages. • Subject-Verb-Object (SVO). Conceptual framework It is a relevant type of word order because of its speakers worldwide. • Verb-Subject-Object (VSO): It represents a relatively small set of languages. • Verb-Object-Subject (VOS): Very few languages use this kind of order. • Subject-Object-Verb (SOV). This is the most frequent type of word order in spoken languages. j j ( )h q yp p g g Subject-Verb-Object (SVO). It is a relevant type of word order because of its speakers worldwide. j j p y g g • Verb-Object-Subject (VOS): Very few languages use this kind of order. Some approaches use such classifications to divide sentences, expressions, paragraphs, and texts, and, ultimately, to generate categories that are used in specific ­applications44,45. Additionally, other applications use these charac- teristics in a reverse way, for instance, to build sentences and paragraphs, or concatenate textual expressions from the same or different sources for generating new expressions; this is being applied in Human Machine Interfaces (HMI) ­development46. On the other hand, a text not only has nouns and verbs, else other types of words with different purposes, e.g., emphasizing words, which to join small sentences to produce effects like generalization or itemization, etc. These words play an important role to decide how the relations between words, sentences, https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Figure 1. Some word classifications techniques. Source: Extracted and adapted ­from37,42. Figure 1. Some word classifications techniques. Source: Extracted and adapted ­from37,42. and paragraphs are. They can be linked to verbs or nouns, e.g. the determinants which comply with the function of generalization or quantification of ­nouns47.h and paragraphs are. They can be linked to verbs or nouns, e.g. the determinants which comply with the function of generalization or quantification of ­nouns47.h i In48 was analyzed the preliminary results focused on the dissociation of sentences in clusters. The sentences studied were in the Spanish Language. Section 2 of this reference exposes why it is necessary to migrate from String-set dependence to another algebraic structure for modeling a sentence, and why this algebraic structure must be an Abelian group, it also supplied the proof. In summary, the dissociation between verbs and nouns, mainly, is a convenient strategy to generate new sentences, also, it is important to create an adequate environ- ment for it. Algebraic environment. Conceptual framework Modern Algebra is a discipline that deals with the properties of the sets and their elements, and the operations that can be executed within them. Modern algebra classifies the sets as semigroups, https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Figure 2. Dissociation of a sentence. Source: Own elaboration. Figure 2. Dissociation of a sentence. Source: Own elaboration. monoids, groups, rings, and fields; all of these are named algebraic structures. These classifications depend on the number and type of properties that the operation fulfills. i If the elements of sentences are treated like components of an algebraic set, then such components could be used to build phrases and new sentences easily by applying an operation that complies with certain properties. This section shows that converting the conventional algebraic structure of the set of strings (sentences) to a structure more adequate allows for reaching this purpose.i q g p p A class very important for this approach is the groups, specifically, the Abelian groups49 these last ones have significant properties that guarantee that by operating elements of a dissociated sentence, the original sentence can be rebuilt; A key property is to be commutative because it allows that the result of an operation among ele- ments will be the same, although the operands change their place in the operation. g p g p p A sentence could be treated as an ordered set of strings which implies an algebraic structure very simple, but this structure does not is adequate because each string in the sentence complies with a function depending on its position in it, if the sentence is dissociated and then it is reassembled, this last process must guarantee that the product is at least coherent with the structure of the language. p g g A sentence could be treated as an ordered set of strings which implies an algebraic structure very simple, it is ordered because each string in the sentence complies with a function depending on its position in it, if the sentence is dissociated into strings and later is required its reassembling, this last process must guarantee that the final sentence keeps the structure of the language and its meaning. These conditions comply if the set gener- ated in the dissociation has associated an operation with certain properties which will be shown in this section. Conceptual framework Components Description [v0 : v1 : · · · : vk], [n0 : n1 : · · · : nk] or X Components: Each component is a vector of vectors. First-internal-vector only contains verbal clusters (v-elements). Second-internal-vector only nominal clusters (n-elements). X generalizes the internal vectors. k  −element : Represents a null string. The subscript is the position in the internal vector. [0/∞], [0/∞ Ŵ −element A component with only  −elements in all internal vectors. Figure 3. Dissociation of a Spanish sentence. Mapping from Pvn to Ovn . Source: Own elaboration. Table 1. Set Ovn and description of its components. Source: Own elaboration. Components Description [v0 : v1 : · · · : vk], [n0 : n1 : · · · : nk] or X Components: Each component is a vector of vectors. First-internal-vector only contains verbal clusters (v-elements). Second-internal-vector only nominal clusters (n-elements). X generalizes the internal vectors. k  −element : Represents a null string. The subscript is the position in the internal vector. [0/∞], [0/∞ Ŵ −element A component with only  −elements in all internal vectors. n1 : · · · : nk] or X Components: Each component is a vector of vectors. First-internal-vector only contains verbal clusters (v-elements). Second-internal-vector only nominal clusters (n-elements). X generalizes the internal vectors. Table 1. Set Ovn and description of its components. Source: Own elaboration. able 1. Set Ovn and description of its components. Source: Own elaboration. Figure 3. Dissociation of a Spanish sentence. Mapping from Pvn to Ovn . Source: Own elaboration. Figure 3. Dissociation of a Spanish sentence. Mapping from Pvn to Ovn . Source: Own elaboration. E0 + E3 =null + “Fred′′ +′′ quiere ir a′′ + “Hong Kong′′ =“Fred quiere ir a Hong Kong′′(correct) E2 + E4 =null + “sitios turisticos′′ +′′ quiere ir′′ +′′ a Hong Kong′′ =“sitios turisticos quiere ir a Hong Kong′′(no meaning) So far, this strategy revolves around the closure property and other properties such as associative, and the neutral element; but this is not enough to guarantee structure and meaning, at least compared to the source text. Conceptual framework To improve this proposal is necessary to include more properties to the set along with the operation, this is only possible by exploring other possible set types that can build up an algebraic structure more useful, and thus, it is decisive to map Pvn to another set that will be named Ovn . Table 1 shows the new set and its components. Mapping F : P →O : Let us define F as: • Pairs < vi, nj > belong Pvn with different index ( i = j ) will be mapped to Ŵ in Ovn. • Pairs < vi, nj > belong Pvn with different index ( i = j ) will be mapped to Ŵ in Ovn. j gf j pp • All pairs mapped must contain at least a n-element, then, pairs such as < vi,  > will be mapped to Ŵ in j gf j pp • All pairs mapped must contain at least a n-element, then, pairs such as < vi,  > will be mapped to Ŵ in Ovn. • Additionally, if couples such as < vi, ni > exist in Pvn , then, elements such as < , ni > will be mapped to Ŵ in Ovn. • All pairs mapped must contain at least a n-element, then, pairs such as < vi,  > will be mapped to Ŵ in Ovn. • Additionally, if couples such as < vi, ni > exist in Pvn , then, elements such as < , ni > will be mapped to Ŵ in Ovn. • Additionally, if couples such as < vi, ni > exist in Pvn , then, elements such as < , ni > will be mapped to Ŵ in Ovn. Figure 3 show the mapping made from Pvn to Ovn for the example. In O the operation used also change and it is defined as follows Figure 3 show the mapping made from Pvn to Ovn for the example. In Ovn the operation used, also, change, and it is defined as follows: In Ovn the operation used, also, change, and it is defined as follows: • Dual. It is Dual because of whether two components are operated, then the operation takes place indepen- dently in each internal vector. This property allows to separate completely verbs and nouns. • Positional. It is Positional because the operation is carried out by two elements with the same subscript. Conceptual framework p p p Supposing the following sentence in the Spanish language: ”Fred quiere ir a Hong Kong y visitar sitios turísticos” (English meaning: ”Fred wants to go to Hong Kong and visit tourist places”), and it is dissociated in strings with a word each one. One scenario for creating Natural Language from this dissociation will be to use the conventional algebraic structure of strings which is composed of the set of strings, and an operator able to join the strings and generate others (closure property). In this algebraic structure the closure property functions as follows: String-set = {“Fred′′, “Hong Kong′′, “quiere′′, “ir′′, “a′′,′′ y′′,′′ visitar′′,′′ sitios′′, turisticos} A new string = “Fred′′ + “quiere′′ + “ir′′ + “a′′ + “Hong Kong′′ = “Fred quiere ir a Hong Kong′′ String-set = {“Fred′′, “Hong Kong′′, “quiere′′, “ir′′, “a′′,′′ y′′,′′ visitar′′,′′ sitios′′, turisticos} A new string = “Fred′′ + “quiere′′ + “ir′′ + “a′′ + “Hong Kong′′ ′′ ng = “Fred′′ + “quiere′′ + “ir′′ + “a′′ + “Hong Kong′′ = “Fred quiere ir a Hong Kong′′ But the closure property is not enough, because the generation of a new string in natural language must ensure structure and meaning, and this is not completely possible in this set with this operation, for example: A new string = “ir′′ + “quiere′′ + “a′′ + “Fred′′ + “Hong Kong′′ = “ir quiere a Fred Hong Kong′′(no meaning) = “ir quiere a Fred Hong Kong′′(no meaning) = “ir quiere a Fred Hong Kong′′(no meaning) A possible solution is to divide the sentence into adequate strings forming ordered sets of clusters. The sets generated by this process will be named Kn (noun-cores/noun-clusters) and Kv(verb-cores/verb-clusters)), but the set used to generate phrases would be the Cartesian product of these sets ( Kv × Kn = Pvn ). In this strategy, the verb-core must contain the null string because the SVO languages (Spanish and English, among others) allow generating phrases without verbs. Figure 2 shows a dissociation following the heuristics ­in48:hh The operation should destroy the operand pairs and apply the operation concatenation or plus (+). This method can generate several sentences, but any of them without meaning, or, at least in the context of the original sentence: Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ Table 1. Set Ovn and description of its components. Source: Own elaboration. Conceptual framework To create the sentence starting from this new core is applied a process that states that for each position in the vectors a verb is concatenated with the noun corresponding and the result will be added to the next result as follows: =′′(quiere ir′′ +′′ a Hong Kong)′′ =′′quiere ir′′ +′′ a Hong Kong′′ +′′ (y visitar′′ +′′ sitios turisticos)′′ =′′quiere ir a Hong Kong y visitar sitios tursticos′′ =′′quiere ir′′ +′′ a Hong Kong′′ +′′ (y visitar′′ +′′ sitios turisticos)′′ =′′quiere ir a Hong Kong y visitar sitios tursticos′′ It is too easy to deduct that this new operation in Ovn is commutative, i.e., the result is the same, although the operands will change their position. This commutative structure is known as an Abelian monoid structure and, ­in48, and by including the symmetrical element, is converted to an Abelian group.i An algebraic structure as has been defined is very useful because reduces the complexity in the reconstruc- tion of phrases because the operation is easy to implement and its behavior is similar to the add operation in the numbers by managing sentences as sets of discrete cores. In the section ”An approach” will be explained that a sentence can generate several Abelian groups, and each one can generate sentences separately. Conceptual framework This • Dual. It is Dual because of whether two components are operated, then the operation takes place indepen- dently in each internal vector. This property allows to separate completely verbs and nouns.h yh p p y p p y Positional. It is Positional because the operation is carried out by two elements with the same subscript. This property allows to implement commutativity. For example: This operation is the same that: [0 : v1 : 2][0 : n1 : 2] + [0 : 1 : v2][0 : 1 : n2] =[0 + 0 : v1 + 1 : 2 + v2][0 + 0 : n1 + 1 : 2 + n2] =[0 : v1 : v2][0 : n1 : n2] =v1 + n1 + v2 + n2 same that: [0 : v1 : 2][0 : n1 : 2] + [0 : 1 : v2][0 : 1 : n2] =[0 + 0 : v1 + 1 : 2 + v2][0 + 0 : n1 + 1 : 2 + n2] =[0 : v1 : v2][0 : n1 : n2] =v1 + n1 + v2 + n2 https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ Figure 4. Use case for the architecture of the system. Source: Own elaboration. igure 4. Use case for the architecture of the system. Source: Own elaboration. ? [0 :′′ quiere ir′′ : 2][0 :′′ a Hong Kong′′ : 2] + [0 : 1 :′′ y visitar′′][0 : 1 :′′ sitios turisticos′′]? =? [0 + 0 :′′ quiere ir′′ + 1 : 2 +′′ y visitar′′][0 + 0 :′′ a Hong Kong′′ + 1 : 2 +′′ sitios turisticos′′]? =? [0 :′′ quiere ir′′ :′′ y visitar′′][0 :′′ a Hong Kong′′ :′′ sitios turisticos′′]? Methodology Th This section proposes an architecture of a system, for dissociating and recovering texts and sentences, based on the concepts, theories, and regulations aforementioned. Figure 4 shows a scheme of the system based on use cases ­view50. The system would include three major sub-systems: dissociation, memory and recovery. The two first sub-systems will be activated serially and immediately after a reading takes place, and the latter process is executed when a query promotes the generation of sentences. Nevertheless, in terms of the information pro- cessing associated with each sub-system, they operate independently. The entire system is conceptualized as a framework that could be up-gradable and enriched with plug-in modules.h The class diagram is shown in Fig. 5. h g g And the activities diagram is shown in Fig. 6, this last diagram is only for dissociating, because the recovery depends on the implementation which is shown in Section ”An approach”. Dissociation subsystem. The function of the dissociation subsystem is to split a text/sentence into special units. As previously mentioned in section From sentences to clusters of words, all languages share a common char- acteristic which is the identification of three basic clusters within a sentence: Subject(S), Verb(V), and Object(O). They can occur within a sentence in a different order depending on the language. hf In this paper, the expression SOV-trio or simply SOV will be used to represent the trio that models a sentence or a text. Given that Subject and Object have similarities both will be treated as (S). Additionally, each of the components of a SOV will be named a core.h The cores may contain one or more words from the sentence. For example, it is possible to have a verb fol- lowed by another verb in the same core, as in the following sentence: ”Fred quiere ir a Hong Kong y visitar sitios turísticos” the two verbs (”quiere ir”) constitute a V core. Once a SOV is generated, this is dispatched to the memory subsystem. Strategies to generate SOVs l d b As explained in section Object-action dissociation/integration, there is a consensus about the dissociation between actions (verbs) and objects (nouns) inside the human mind. However,32 emphasizes the existence of problems by establishing the grammar category that can generate confusion between verbs and nouns, this also Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Class diagram of the framework. Source: Own elaboration. Figure 5. Class diagram of the framework. Source: Own elaboration. Figure 6. Activities diagram of the framework. Source: Own elaboration. Figure 6. Activities diagram of the framework. Source: Own elaboration. Figure 6. Activities diagram of the framework. Source: Own elaboration. https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. A generic UML sequence diagram for the dissociation process. Source: Own elaboration. Figure 7. A generic UML sequence diagram for the dissociation process. Source: Own elaboration. can happen in the process of dissociation in this subsystem. To dissociate the sentences correctly, the subsystem should implement modules such as: can happen in the process of dissociation in this subsystem. To dissociate the sentences correctly, the subsystem should implement modules such as: can happen in the process of dissociation in this subsystem. To dissociate the sentences correctly, the subsystem should implement modules such as: • Syntactic Analysis (Parsing). An ordinary parser generates a syntax tree from which the SOVs can be rapidly built. Although this strategy is good, it does not avoid that the syntax tree generated may require the involve- ment of some other heuristic processes to “refine” the creation of the cores, for instance, in cases of slang interpretation as is shown in Fig. 7, Parser module of, routine parserMethod(). d d d l fi ll • Dictionaries and conjugators. Sometimes, parsers can produce an incorrect word classification, especially when the parser has not well-trained in a particular language, in such case it is necessary to perform an analysis and debugging process over these words. For this purpose, software like dictionaries and conjuga- tors modules could be useful to validate the category as is shown in Fig. 7, Parser module, routine correct- • Grouping of elements. The dissociation in cores requires identifying elements like determinants, adverbs, prepositions, conjunctions, etc., in such a way that they will be inserted in the adequate core. This process should be customized for each language as is shown in Fig. 7, Groups module. To summarize, some procedures, syntactic-semantic strategies, and heuristics should be implemented to help in building the S/O/V cores correctly. Memory subsystem. An important function of the memory system is to store the information generated by the dissociation subsystem. Hence, it is mandatory to build a structure that guarantees order and efficiency. Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Therefore, the memory system should contain a repository to save the SOVs generated by each text read inter- related between them. This storage should maintain these cores in such a way that can be retrieved in the exact order as they were read. According to these principles, the implementation should comply with the following conditions: • SQL-database. The type of database towards has been addressed in this research is the SQL-database because it is the most used to store information. The main idea is to save the texts in such a way that their elements will be organized in groups or clusters representing sets that, joining them, can reproduce the source without losing their meaning. g g • Repository based on queries of cores. Firstly, a repository based on query means that uses SQL technol- ogy to save and recover information. Secondly, the queries can be attended by modules that recover cores, compare against the queries, determine similarity, and create sentences, paragraphs, and full texts as of the cores chosen. g g • Repository based on queries of cores. Firstly, a repository based on query means that uses SQL technol- ogy to save and recover information. Secondly, the queries can be attended by modules that recover cores, compare against the queries, determine similarity, and create sentences, paragraphs, and full texts as of the cores chosen. https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Figure 8. Relationship between groups generated by the sentence: ”Fred quiere ir a Hong Kong y visitar sitios turísticos”. Source: Own elaboration. Figure 8. Relationship between groups generated by the sentence: ”Fred quiere ir a Hong Kong y visitar sitios turísticos”. Source: Own elaboration. • Meta-engine. Each implementation should program a meta-engine that works over the database in a superior layer that the database engine, this should be equipped with the algebraic operation explained in subsection Algebraic environment and mappings between groups to integrate them and build a part or whole original text. • Meta-engine. Each implementation should program a meta-engine that works over the database in a superior layer that the database engine, this should be equipped with the algebraic operation explained in subsection Algebraic environment and mappings between groups to integrate them and build a part or whole original text. The Engineh The queries are expected in natural language and it would transform into a set of SOVs. The key is to com- pare SOVs for finding the closest results. The strategies to match the SOVs.can be wide. An example could be to establish matches of SOVs that contain elements that could respond contextually to the query as in Fig. 9. The degree of coincidence will be the measure.h g This strategy could recover sentences that do not answer the query completely, hence, it would be important to implement another stage. For instance, that compares sentences in a logical context. This can be carried out by converting the query and the text recovered into small text-theories that can be matched logically. Dynamic structureh The dissociation in SOVs and the mapping create sets distributed and connected in terms of their original semantic content. Figure 8 shows a scheme that illustrates the relationship between the sets of SOVs (Abelian group).h g p The nodes will be related with adequate functions (mapping) to guarantee that the recovery of the part, or the whole, of a sentence/text will be executed correctly as will be explained later. Recovery subsystem. The purpose of this subsystem is to generate, in a dynamic way, a sentence/text part or entirely. This subsystem is closely interrelated to the dynamic structure because this subsystem is composed of the functions that connect the nodes.h The Engineh An approachh This section has a summary of a prototype designed as a layered framework that could be used for any language characterized as S-V-O (Spanish, English, etc.) The most relevant layers of the dissociation processes are the following: 1. Identifying the language. This first layer has been designed to identify the language of the text and divide it into sentences, and finally, their results will send to the next layer one at a time.h 1. Identifying the language. This first layer has been designed to identify the language of the text and divide it into sentences, and finally, their results will send to the next layer one at a time.h i y y 2. Planning. This second layer chooses the modules required to dissociate the sentences based on the language recognized. This layer makes flexible the framework because it allows changing the rules of dissociation depending on the language to be processed.h i 2. Planning. This second layer chooses the modules required to dissociate the sentences based on the language recognized. This layer makes flexible the framework because it allows changing the rules of dissociation depending on the language to be processed.h p g g g p 3. Reaction layer. This layer is related to the strategies to generate SOVs which were described in the para- graph Strategies to generate SOVs. Figure 7 is shown the execution of two modules in a pipeline way, but new modules could be included to improve the results, this will depend on the implementation. The name of this layer is due to the modules chosen by the plan layer being triggered dynamically and executed like a chain reaction in a pipeline. p g g g p 3. Reaction layer. This layer is related to the strategies to generate SOVs which were described in the para- graph Strategies to generate SOVs. Figure 7 is shown the execution of two modules in a pipeline way, but new modules could be included to improve the results, this will depend on the implementation. The name of this layer is due to the modules chosen by the plan layer being triggered dynamically and executed like a chain reaction in a pipeline. An approachh In this implementation, the modules created in the reaction layer dealt with sentences in the Spanish language (S-V-O language) and were organized in three linear phases following the guidelines described in the paragraph In this implementation, the modules created in the reaction layer dealt with sentences in the Spanish language (S-V-O language) and were organized in three linear phases following the guidelines described in the paragraph https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Source: Extracted ­from52. In this approach, there are two types of determinants p-det and a J-det; both interrelate the sets with func- tions, but the Abelian group pointed by a J-det is considered optional in the rebuild of the sentence. All of these properties were established empirically.h The restoring process is not the reverse operation exactly, else it is a complex process that executes tasks from the repository trying to preserve syntax and the original semantics. This purpose is successful due to the properties of the Abelian groups ­(see52) and the hierarchy of sets created by the determinants in the dissociation process. The process is shown in Fig. 10h ph p g The sets are operated and mapped in a domino way from the core where the matching occurs up until the root of the hierarchy. For example, in Fig. 10, if the core matched is in G3 and corresponds to ” n2 = críticas” then the recovered sentence will be: “La marcha programada, para el próximo 26 de marzo, recibía críticas”. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 9. Creating a sentence from a query. Source: Own elaboration. Figure 9. Creating a sentence from a query. Source: Own elaboration. Figure 9. Creating a sentence from a query. Source: Own elaboration. Strategies to generate SOVs and shown in Fig. 7. In the first phase, each sentence is processed by a linguistic tool, commanded by the VISL ­parser51 reaction layer, in this stage, it, also, corrects possible inconsistencies generated by the parser as the wrong classification of the words, e.g., some words classified as nouns or vice versa. The information produced by the parser is significant, therefore, it is discriminated, and sent to the next module in the pipeline. The second phase receives the information and classification and applies heuristics for generating, initially, raw-clusters, then refined by another heuristic, and finally to produce the set Ov . Lastly, in the third phase, the set Ov is saved in a standard database (SQL-style). The heuristics applied in this approach are not extendable to other languages. However, currently, they are being tested in the English Language, also S-V-O language, to measure their effectiveness in it. Each sentence is organized in Abelian groups hierarchically organized with a binary operation capable of building phrases (see Fig. 3). The Abelian groups obey the specifications done in section Algebraic environment.ih g Table 2 shows the classification established heuristically for the Spanish language cores in this approach. This process involves a loop where neighboring words that comply with certain conditions are packed into a single class named: nominal core (S), determinant, and verbal core (V). A determinant is used to interrelate Abelian groups as in Fig. 8 in the paragraph Dynamic structure. It is important note that the punctuation signs are useful to create these categories, some are part of the determinants and other are par of the verbal cores or nominal cores, for example, in Fig. 8 the nominal core n6 in G2 include a comma: ”, la Paz y la justicia”, similarly, the verbal cores v5 in G3 : ”, recibía”. https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | www.nature.com/scientificreports/ Table 2. Final categorization scheme. Category Description p, J Determinants v, V Verbal core n Noun core Table 2. Final categorization scheme. Category Description p, J Determinants v, V Verbal core n Noun core Table 2. Final categorization scheme. Figure 10. Scheme to recover sentences. Source: Extracted ­from52. Figure 10. Scheme to recover sentences. Proposal comparison Currently, generating sentences and small texts is a task very significant in several fields of Computer Science. The approach named Rhetorical Structure Theory (RST)53 is one of the first proposals created to divide the discourse and has been the inspiration for Natural Language Generation (NLG) schemes. Th d d d h d h h h ll ( l d ll ) The strategy used in RST to divide the texts into cores and organize them hierarchically (nucleus and satellite) requires training of neural ­networks54,55. The relevance of the framework presented in this document is that it does not need training. g With respect to the generation of sentences, the implementations and approaches are very exigent. According ­to56, they should carry out several complicated tasks as below: . Determining the information relevant. This part is associated with the context and scope, basically related to the ­searching57–60. 2. Determining the order as the words should appear. Some approaches try to resolve this part from texts by collecting, recovering, and organizing sentences inside ­them61,62hfi 3. Determining: how should be the information aggregated? This stage is considered very difficult because the information can be provided by several sources or it is not the correct response to any query. Some works use the context to resolve discrepancies or the domain to explore the ­sources63,64. Scientific Reports | (2022) 12:22249 | https://doi.org/10.1038/s41598-022-26304-w www.nature.com/scientificreports/ 4. Determining the right words and phrases (verbs and nouns). This part contains two stages but they will be joined in one because can be carried out jointly. In this part the sentence is organized in one of the following structures: SVO, SOV, VSS, VOS, also, it is analyzed the verb ­times65.h 4. Determining the right words and phrases (verbs and nouns). This part contains two stages but they will be joined in one because can be carried out jointly. In this part the sentence is organized in one of the following structures: SVO, SOV, VSS, VOS, also, it is analyzed the verb ­times65.h y 5. Combining words and phrases to generate well-formed sentences. This phase builds the sentences, sometimes through templates, or grammar-based techniques, among ­others66,67. y 5. Combining words and phrases to generate well-formed sentences. This phase builds the sentences, someti through templates, or grammar-based techniques, among ­others66,67. Conclusions and future worksh The high demand for information has caused an increasingly important in the automation of processes such as decision-making, pattern recognition, and interaction human-machine, among others. Several of these processes require the use of the text, either to understand queries, generate reports, or answer in natural language, hence, building applications with these functions takes a greater relevance. This paper presents an architecture for dis- sociating the text/sentences, saving it in a SQL database, and recovering it without loss of meaning. This is highly productive in process automation because the textual information is converted from unstructured to structured format and the queries and other processes in natural language can be more efficient.hi g gfi The suggested system has been inspired and based on processes verified by scientists related to the dissociation of the information inside the human brain, memory models in the Neuroscience field, and the structure of the languages in Linguistic and Psycholinguistic disciplines. The proposed framework divides a sentence/text into clusters like the brain dissociates the speech into nominal and verbal categories. The scheme will divide the text/ sentence into sets of cores named nominal cores and verbal cores, and implement an algebraic operation that can be used to generate new sentences that keep the original meaning without loosing the structure of the language. This proposal was applied by the approach studied in the last section successfully.h h The explored implementation resolved a great part of the challenges described in the paper by implementing a framework with abstract modules that can be custom implemented, for instance, the processes described in the architecture, the generic abstract modules for different languages, and the recovery modules, among others. dd ll h l l f h h l b h f b h f Additionally, the implementation creates a solution for the Spanish language by using heuristics for both dissociation and recovery processes. The application suggests interrelating the algebraic sets by employing func- tions to recover the whole or part of the textual information by maintaining the meaning. The approach shows that for the Spanish language is possible to have an implementation. ­In68 is exposed several proposals of NLG. A system has been proposed for converting unstructured textual information to be computationally man- aged structured information. This proposal has been tested in an approach for the Spanish language successfully. Conclusions and future worksh Future works will be addressed to implement this framework for other languages and to generate applications for these approaches. Proposal comparison In the framework exposed in this document, the first three steps are part of the recovery system in this frame- work, specifically, corresponding to the engine searching. the last two steps can be resolved by responding to the queries and executing the algebra of the groups and mapping between them which are tasks easy and efficient. All of these show a framework simple to implement. Received: 1 February 2022; Accepted: 13 December 2022 Data availabilityh The current document has been focused on the discussion about a framework able to compose strategies to divide sentences and texts into cores to save them in a SQL-databases. Under this context the data used to verify the effectiveness belong to other work where the purpose was to study heuristics for dividing the sentences into these clusters, these data do not form part of the current research. Therefore, all data generated or analyzed dur- ing this study are included in this published article. 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Neural generative rhetorical structure parsing. arXiv preprint arXiv:​1909.​1 (2019). https://doi.org/10.1038/s41598-022-26304-w Scientific Reports | (2022) 12:22249 | Author contributions Edgardo Barraza is the main author, he generated the main idea of the paper and its proof previously published in another journal.Richard de Jesús is the doctoral tutor of Edgardo Barraza and he contributed to the direction of the research.Marlly Rojas wrote part of the paper and she was fundamental in final review © The Author(s) 2022 Additional information Correspondence and requests for materials should be addressed to E.S.B.V. Correspondence and requests for materials should be addressed to E.S.B.V. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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Historical contingency and spatial processes rather than ecological niche differentiation explain the distribution of invasive goldenrods (Solidago and Euthamia)
Plant ecology
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Plant Ecol (2016) 217:565–582 DOI 10.1007/s11258-016-0601-1 Historical contingency and spatial processes rather than ecological niche differentiation explain the distribution of invasive goldenrods (Solidago and Euthamia) Magdalena Szymura . Tomasz H. Szymura Received: 19 January 2016 / Accepted: 5 April 2016 / Published online: 13 April 2016  The Author(s) 2016. This article is published with open access at Springerlink.com the species that was first introduced occupied available habitats and prevented the establishment of other species. Solidago canadensis was distributed ran- domly throughout the entire region and did not differ from S. altissima in its ecology. Euthamia graminifo- lia differed from the other goldenrods in habitat preferences and environmental requirements. Its dis- tribution was strongly spatially structured, clumping around initial infestation sites. The extents of the differences resulting from ecological niche differen- tiation and the limitation of long-range dispersal are unclear. We argue that the under-representation of E. graminifolia in abandoned fields originates from the inhibition of establishment in habitats already occu- pied by other goldenrods. The results underline that the effect of historical contingency can be more prominent than ecological niche differentiation on invasive species distribution and invasion effectiveness. Abstract The effectiveness of invasion depends on the interacting environmental factors and biology of the invader. The effects of these interactions are contingent on their contexts and difficult to predict. Among the most successful plant invaders in Europe are the goldenrods (Solidago and Euthamia genera). We assessed the roles of ecological niche differenti- ation and historical contingency on their distribution in Silesia (Central Europe, studied area approximately 32,000 km2). The distributions of the two most common species (S. gigantea and S. altissima) were clumped, and the species dominated different areas. The distribution was unrelated to ecological niche differences but originates rather from the history of invasion and subsequent density-dependent spread; Communicated by Scott J Meiners. Electronic supplementary material The online version of this article (doi:10.1007/s11258-016-0601-1) contains supple- mentary material, which is available to authorized users. Keywords Biological invasions  Dispersal limitation  Ecological contingency  Habitat preferences  Invasion effectiveness  Local distribution  Priority effect  Range infilling Keywords Biological invasions  Dispersal limitation  Ecological contingency  Habitat preferences  Invasion effectiveness  Local distribution  Priority effect  Range infilling M. Szymura (&) Department of Agroecosystems and Green Areas Management, Wrocław University of Environmental and Life Sciences, Grunwaldzki Sq. 24A, 50-363 Wrocław, Poland M. Szymura (&) Department of Agroecosystems and Green Areas Management, Wrocław University of Environmental and Life Sciences, Grunwaldzki Sq. 24A, 50-363 Wrocław, Poland Introduction 2013; Vannette and Fukami 2014; Marchante et al. 2015; Young et al. 2015). Numerous studies of species’ interactions indicate that the order of arrival into an ecosystem, the so-called priority effect (Shulman et al. 1983), can influence local community assembly (Fukami 2010; Grman and Suding 2010; Kardol et al. 2013; Putten et al. 2013; Zefferman 2015; Fraser et al. 2015). As a result, ecological contingencies can govern the suc- cess of colonisation of dispersing individuals and limit the distribution of invasive species (Ricklefs 2010; Fraser et al. 2015). The priority effect should be particularly strong when interacting species are sim- ilar in resource use, or when the early-arriving species strongly affect the environment, and the late-arriving species have resource high requirements (Vannette and Fukami 2014). The native ranges of these species partially overlap but differ in extent: S. altissima has the largest range, the ranges of S. gigantea and E. graminifolia are similar but narrower than that of S. altissima, while the range of S. canadensis is relatively the smallest (Weber and Schmid 1998; Weber 2001; Semple and Cook 2006). They are considered as broad tolerant in terms of soil characteristics, but S. gigantea is usually associated with moist to wet habitats, while the S. altissima is sensitive to flooding (Weber 2000; Abra- hamson et al. 2005; Weber and Jakobs 2005). In the native range, several goldenrod species can co-occur, but the particular combination of the species present at one site can vary according to site conditions (Rhoads and Block 2000; Abrahamson et al. 2005). Observa- tion at the local scale in Pennsylvania, USA, showed that S. altissima and S. gigantea were associated with neutral soils, whereas E. graminifolia achieved high abundance on acidic soils. S. altissima occurred more often on well-drained soils, S. gigantea on moister soils with stable water levels over time, whereas E. graminifolia preferred clay-rich soils. In Europe, S. altissima and S. gigantea are also considered as broad tolerant in terms of soil and climate conditions. They primarily invade abandoned fields and meadows as well as ruderal and disturbed habitats (Weber 2000; Weber and Jakobs 2005; Szymura and Szymura 2013; Bartha et al. 2014). Nonetheless, it appears that S. altissima prefers drier sites occurring along roadsides, railway lines and disturbed places around settlements, while S. Introduction T. H. Szymura Department of Ecology, Biogeochemistry and Environmental Protection, University of Wrocław, Maksa Borna Sq. 9, 50-328 Wrocław, Poland Invasions of alien species alter biodiversity, landscape structure, ecosystem functions and services, and the 12 3 Plant Ecol (2016) 217:565–582 566 2003). Four alien goldenrod species (Solidago and Euthamia) invade Central Europe: S. gigantea Aiton, S. canadensis L., S. altissima L. (S. canadensis var. scabra (Muhl.) Torr. and Gray) and E. graminifolia (L.) Nutt. Alien Solidago species cause the decrease of plant species richness (Hejda et al. 2009; Del Fabbro et al. 2013; Fenesi et al. 2015a; Pal et al. 2015), alter spontaneous succession (Bornkamm 2007; Bartha et al. 2014) and negatively influence the diversity of birds (Sko´rka et al. 2010) and insects (Moron´ et al. 2009). Due to their locally high abundance and substantial environmental impact, their populations need to be controlled (Sheppard et al. 2006; Sko´rka et al. 2010; Fenesi et al. 2015a). local economy, as well as human health and well- being (Chapuis-Lardy et al. 2006; Pejchar and Mooney 2009; Hejda et al. 2009; Pysˇek and Richard- son 2010; Vila` et al. 2011; Hulme et al. 2014, 2015). The total number of invasive species is still increasing, and habitats may be invaded by multiple species (Kuebbing et al. 2013; Kuebbing and Nun˜ez 2015). Thus, knowledge about the interactions between non- native species becomes critical for understanding their distribution and abundance, particularly from the perspectives of nature conservation and management (Kuebbing et al. 2013; Kuebbing and Nun˜ez 2015). Anthropogenic species movement is the primary driver of plant invasion (Chytry´ et al. 2008; Pysˇek and Richardson 2010; Kuebbing and Nun˜ez 2015). How- ever, the subsequent effectiveness of invasion, con- sidered as the efficient distribution and habitat infilling within the new range and/or the abundance of individuals, is driven by the interaction of environ- mental factors (land-use system, climate, habitat disturbances and resident vegetation) and the biology of the invasive species (Lake and Leishman 2004; Pysˇek and Richardson 2008; Catford et al. 2009; van Kleunen et al. 2010; Essl et al. 2011; Johnson et al. 2012; Warren et al. 2013). The effects of these interactions are highly contingent on their context (Chamberlain et al. 2014), e.g. plant community composition of the new territory, specifically the presence of interacting species, availability of suit- able space and resources, and the time a population has been established (Putten et al. Introduction gigantea is more frequent on river banks and moist areas often connected with river networks (Guzikowa and May- cock 1986; Weber 2001, 2011; Weber and Jakobs 2005). The presence of E. graminifolia was related to One of the most successful worldwide plant invaders are the goldenrods (Pysˇek 1998; Weber 123 123 Plant Ecol (2016) 217:565–582 567 limitation and historical contingency in determining the distribution of invasive goldenrods in Silesia, Central Europe. We tested the differences among species with respect to the following aspects: (a) habi- tat preferences of studied species, (b) composition of co-occurring vascular plant species, (c) climatic and topographical variables and (d) soil properties. To assess the potential role of priority effects and the subsequent density-dependent processes that lead to infilling by one species in a particular area, we modelled the spatial structure of plot locations, effects of distance from initial infestation sites and the environmental heterogeneity on goldenrod distribu- tion. Finally, the quantitative effects of each of these factors on species distribution were separated. moist and wet habitats disturbed by humans (Guzi- kowa and Maycock 1986). There is a lack of systematic measurements of primary environmental gradients that can be used for testing the hypothesis concerning the distribution of invasive goldenrods or for modelling their distribu- tion. This is important because an increase of abun- dance and infilling available habitats within the species secondary range is occurring (Szymura and Szymura 2011; Bartha et al. 2014; Fenesi et al. 2015a, b). The relationship of a particular goldenrod species distribution with environmental conditions is unclear, particularly the recently observed tendency of S. gigantea to occupy drier habitats (Gu¨sewell et al. 2005; Weber and Jakobs 2005; La´nı´kova´ et al. 2009; Bartha et al. 2014). The establishment of goldenrods from seedlings on abandoned lands occurs soon after the abandonment, whereas germination of seeds in undisturbed, well- established native vegetation is rather infrequent (Hartnett and Bazzaz 1985; Bartha et al. 2014; Fenesi et al. 2015b). After establishing at a new site, the subsequent spread of goldenrods is almost exclusively clonal via horizontal rhizomes, and the death of an established genet is a rare event (Meyer and Schmid 1999a, b). The age of single genets of S. altissima and S. canadensis has been reported to be 20–100 years (Whitham 1983; Carson and Root 2000). Conse- quently, goldenrods last for a long time during mid- successional stages of vegetation (Cain 1990; Wise et al. Introduction 2006; Bornkamm 2007; Bartha et al. 2014; Fenesi et al. 2015a), and in such well-established stands, the recruitment of new individuals from seeds is rather unlikely (Hartnett and Bazzaz 1985; Meyer and Schmid 1999a). Therefore, it could be hypothe- sised that the pattern of goldenrod distribution is influenced by propagule pressure related to the invasion history; species that occur first may infill the available habitats and dominate the local vegeta- tion. Since the number of goldenrod populations is increasing, the constant presence of their propagules increases the probability of successful colonisation when a gap occurs. Because goldenrods are similar with respect to the use of environmental resources and strongly affect the environment, the historical contin- gency caused by priority effect should restrict their distribution. Studied species Studied species We studied four alien goldenrod species (Solidago and Euthamia): S. gigantea Aiton, S. canadensis L., S. altissima L. and E. graminifolia (L.) Nutt. E. gramini- folia is most commonly recognised in Europe as Solidago graminifolia (L.) Elliot; however, based on anatomical and DNA studies (Semple et al. 1981, 1984), the taxon should be classified to Euthamia Nutt. genus. The taxonomical problem concerned also considers S. canadensis and S. altissima as separate taxa. In several articles, these taxa are reported as two varieties of S. canadensis s.l.: var. canadensis and var. scabra (Guzikowa and Maycock 1986; Weber 1997; Weber and Schmid 1998). Among the primary traits that differ between S. altissima and S. canadensis, the length and arrangement of hair covering the stem and leaves and the shape and margins of the leaves (Weber 1997; Semple and Cook 2006), micro-morphological characteristics of leaf epidermis (Szymura and Wolski 2011) and rhizome systems (Schmid et al. 1988) have been described. In this study, the aforementioned species were separated into S. canadensis and S. altissima. Goldenrods were introduced from North America in the eighteenth century and distributed to gardens in different parts of Europe (Hitchmough et al. 2004). After a short time, the goldenrods escaped from cultivation. At present, S. canadensis s.l. and S. gigantea are aggressive invaders that are widespread In this study, we assessed the relative roles of ecological niche differentiation, long-range dispersal 12 3 3 Plant Ecol (2016) 217:565–582 568 et al. 2005). Each of the plots consisted of a buffer with a radius of 282 m (area 25 ha) within which the alien goldenrods were sampled. The centre of the buffer and the buffer extent were determined in the field using a GPS receiver. The land use/land cover (LULC) in the centre of the buffer was noted. If a population of alien goldenrods was found, the vegetation was sampled. The sampling plot was sized 10 9 10 m (area 100 m2), and the cover of all vascular plants found was assessed using the Braun–Blanquet abundance scale. The nomenclature of plant species fallows to Mirek et al. (2002). The Polish vegetation database housed all the vegetation data (Kacki and Sliwinski 2012). Plots were established wherever Solidago first occurred, regardless of its abundance. If a few separated populations of a goldenrod species were found within the buffer, the stand placed nearest the buffer centre was sampled. Studied species If another species of alien goldenrods, forming a separate stand, was found, an additional sampling plot was established. By this way, in a particular buffer, no sampling plot (absence of goldenrods), a plot with a single goldenrod taxon, a plot with two or more goldenrod species or separate plots with different goldenrod taxa in each could be established. For each sampling plot, the LULC was noted, and the cover of trees and shrub canopy was visually assessed (canopy). The climatic conditions in Europe (Weber and Schmid 1998; Weber 2001; Schlaepfer et al. 2008). In contrast, E. graminifolia is present only at a few localities in Europe but can form dense, monospecific stands (Weber and Schmid 1998; Kompała-Ba˛ba and Ba˛ba 2006; Dajdok and Nowak 2007; Szymura and Szymura 2013). In Poland, the occurrence of S. gigantea was noted in 1853, S. canadensis s.l. in 1872 and E. graminifolia in 1885 (Guzikowa and Maycock 1986; Tokarska-Guzik 2003). Studied area and sampling plot design Studied area and sampling plot design The fieldwork was performed in the Silesia region (Poland, Central Europe, sampled area approximately 32,000 km2, Fig. 1a). The studied area was mostly lowlands with a rather small portion of foothills and mountains (Fig. 1b); the land was mostly used for agriculture (approximately 64 %) and forestry (ap- proximately 28 %). Within the study region (Fig. 1a), we established 309 plots, placed at the nodes of 10 9 10 km regular grid. The altitudinal range of plots was from 50 to 1100 m above sea level; however, most of the plots were placed at an altitude below the 300 m a.s.l. The total annual precipitation varied between 533 and 858 mm, and the average annual temperature varied between 3.5 and 9.2 C (Hijmans Fig. 1 Studied region (panel a, dark grey) and pattern of historical (before 1939) infestation of particular goldenrod species (according to Guzikowa and Maycock 1986) in Silesia on the background of altitude and main rivers (panel b) Fig. 1 Studied region (panel a, dark grey) and pattern of historical (before 1939) infestation of particular goldenrod species (according to Guzikowa and Maycock 1986) in Silesia on the background of altitude and main rivers (panel b) 123 Fig. 1 Studied region (panel a, dark grey) and pattern of historical (before 1939) infestation of particular goldenrod species (according to Guzikowa and Maycock 1986) in Silesia on the background of altitude and main rivers (panel b) 12 12 3 Plant Ecol (2016) 217:565–582 569 occurred. Then, the difference between the proportion of goldenrods and those in the buffer centre with respect to particular species and habitat type was calculated. A negative value indicates that a particular goldenrod taxon was under-represented in a given habitat type, whereas a positive value indicated that it was over-represented. The significance of these differences compared to random was assessed with a v2 test. [average annual temperature (tempaver), the maximal temperature of the warmest month (tempmax), the minimal temperature of the coldest month (tempmin) and the annual sum of precipitation (precipitation)] were determined from the climatic model of Hijmans et al. (2005). Using the digital elevation model (Jarvis et al. 2008), the altitude was defined and the topographic wetness index (TWI) was calculated. Based on the LULC in the buffer centres and in the sampling plots, LULC was categorised. Studied area and sampling plot design A total of 11 types of habitats were distinguished: (1) urban, industrial and transport areas (urbanised areas); (2) unpaved roads as footpaths, skidder trail in forests and sporadically used agricultural roads (unpaved roads); (3) drainage ditches and strips of land along paved roads (road verges); (4) habitat edges, e.g. forest/grassland ecotones; (5) various anthropogenic vegetation types, other than agriculture—allotments, orchards, gardens, parks (green areas); (6) meadows and pastures (grasslands); (7) forests and scrublands; (8) rivers, ditches, embankments or escarpments; (9) arable fields; (10) unmanaged strips of lands along fencing and other types of borders (property bound- aries); and (11) abandoned lands. Sampling of the plots gave us knowledge about the frequency and distribution of each goldenrod species. For 78 sam- pling plots selected using a stratified-random method, topsoil samples were collected. As strata, species identity and geographical space were used. We tended to sample as much as possible of rare species (E. graminifolia and S. canadensis) and to obtain spatially diversified In additionsamples of common species (S. altissima and S. gigantea). The selected 78 plots were the ‘topsoil subset’. Soil samples from the top 20 cm were taken with a soil auger from four randomly chosen positions and mixed into one sample repre- senting the plot. Then the subsamples collected from upper soil layer (approximately 20 cm) were mixed into one sample representing the plot. In the labora- tory, the samples were assessed for soil texture, pH of H2O, carbon (C), total nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg) and calcium (Ca) content. The tendency of the studied species to cluster or occur with a regular or random distribution was tested using Ripley’s Kd function. To verify whether the patches of vegetation invaded by particular goldenrod taxa differed with respect to vascular plant species composition, we analysed the similarity (ANOSIM) and, additionally, performed an ordination with non-metric multidimensional scaling (NMDS) using Bray–Curtis distance. As a grouping factor in the ANOSIM, the occurrence of particular goldenrod taxa was used; in the case of plots invaded by multiple goldenrod species, we classified it as a ‘mixture’. Because the results of NMDS ordination would be under the strong influence of the presence of particular goldenrod taxa, they were excluded from the analysis. Studied area and sampling plot design The significance of differences in environmental traits across species was tested using permutation and Kruskal–Wallis analysis of variance on ranks with a Nemenyi–Damico–Wolfe–Dunn post hoc test (Hol- lander and Wolfe 1999; Hothorn et al. 2013). For all plots, the shortest distances to the initial invasion location in Silesia were calculated. Data regarding the initial invasion (distribution before WW II) were derived from Guzikowa and Maycock (1986; Fig. 1b). As the authors of that article did not distinguish S. altissima, we calculated the distances to the locations of initial infestations by E. gramini- folia (minEut), S. gigantea (minGig) and S. canaden- sis sensu lato (minCan). For modelling goldenrod spatial distributions, we used the principal coordinate analysis of neighbour matrices (PCNMs; Borcard and Legendre 2002; Dray et al. 2006). The geographical coordinates of the plots were used to compute PCNM, from which vectors that represented statistically significant positive spatial cor- relations were selected, using Moran’s I (Borcard and Legendre 2002; Borcard et al. 2004). The PCNM vectors were calculated independently for the entire dataset (N = 232) as well as for the topsoil subset (N = 78). Statistical methods The percentage of different habitat types found in the buffer centres was calculated, including the percent- age of habitats where particular goldenrod species 12 3 3 570 Plant Ecol (2016) 217:565–582 All environmental data, distances to initial invasion sites and the PCNM vectors were used as explanatory variables in a canonical correspondence analysis (CCA) of alien goldenrod distribution using pres- ence/absence data. Two independent analyses were performed. First, using the all predictor variables for the entire dataset. The second analysis used the canopy, topsoil properties, the shortest distances and PCNM vectors for the topsoil subset. Within the analysis, models exclusively for environmental data (environ- ment) were separately constructed, which were used as the explanatory variables solely for the shortest distances (distance) and for the spatial data alone (space) as explanatory variables. In these models, using forward selection procedures, the variables that signif- icantly shaped alien goldenrod distribution were selected. Finally, combined models, including signif- icant environmental, spatial and distance variables, were constructed. For these combined goldenrod distribution models, variance partitioning (Borcard et al. 1992; Peres-Neto et al. 2006) was used to quantify the amount of variation explained by the three different groups of factors (space, environment and distance). In addition, correlations between the site scores obtained in the CCA models with space as explanatory variables on one hand and species data, environment and the distance group of variables on the other hand were checked to determine which species and/or which variables were associated with the spatial structures of goldenrod population. The significance of the correla- tions was checked using the Monte Carlo permutation method (Hothorn et al. 2013). Solidago canadensis occurred most often as a few ramets in the 10 9 10 m plots (‘?’ according to the Braun–Blanquet scale), S. altissima and S. gigantea mostly covered up to 5 % of the plot (‘1’ in B–B scale), while E. graminifolia covers most often 5–25 % of the plot area (‘2’ in B–B scale). However, S. altissima and S. gigantea in some plots can cover more than 50 % of the plot (‘4’ and ‘5’ according to B– B scale, Online Resource A). The distributions of S. gigantea and S. altissima were clustered (values of Ripley’s Kd function above random ranges, Fig. 3), and their ranges did not overlap entirely within the studied region. S. canaden- sis was randomly distributed throughout the region. The range of E. Statistical methods graminifolia was restricted to one area, but within that range, its distribution was random. Goldenrods were most often found on abandoned lands and road verges. The number of plots for particular species and habitat types is listed in Online Resource B. All species avoided arable fields and urbanised areas but were over-represented on road verges (Fig. 4). We observed differences between the percentage of habitats invaded by E. graminifolia and other species. E. graminifolia was over-represented on unpaved roads and, in contrast to other species, under- represented on abandoned lands but did not signifi- cantly avoid forests and scrublands. The most frequent species co-occurring with the goldenrods were Cirsium arvense (57.3 %), Artemisia vulgaris (56.9 %), Urtica dioica (50.0 %), Achillea Fig. 2 Percentage of plots with particular goldenrod species growing in monocultures or mixtures with other goldenrod taxa. In total, 232 plots were sampled with one or more goldenrod species on each Statistical analyses were performed using ‘vegan’ and ‘coin’ packages in R environment (Oksanen et al. 2008; Hothorn et al. 2013) and PASSAGE (Rosenberg and Anderson 2011) software for spatial analysis. Fig. 3 Maps of the studied species distribution (grey dots) and values of Ripley’s Kd function of different spatial extents. The grey area on Ripley’s Kd graphs shows ranges for random distribution. The crosses on the maps denote the centres of the studied buffers Results Among the analysed 309 buffers, 232 sampling plots were established. Monospecific stands of S. gigantea (N = 92, 40 %) or S. altissima (N = 88, 38 %) were found in most of the plots. In contrast, plots with a mixture of these two species (N = 23, 10 %) rarely occurred. The two remaining species, S. canadensis (N = 21) and E. graminifolia (N = 9), were much rarer and mostly occurred in stands along with other goldenrod species (Fig. 2). Fig. 2 Percentage of plots with particular goldenrod species growing in monocultures or mixtures with other goldenrod taxa. In total, 232 plots were sampled with one or more goldenrod species on each 12 123 Plant Ecol (2016) 217:565–582 571 and Dactylis glomerata cies found in the sample plots Resource C. The results of there were no significant 06, p = 0.660) between the rent goldenrod species with nt species composition. The plots invaded by particular upper panel) was consistent with particular species composition. The exception was the tendency of E. graminifolia to concentrate on the right side of the graph with forest species. The ordination of species (Fig. 5, lower panel) suggests that the first axis is related to the gradient from frequently disturbed habitats (left wing of the graph, arable weeds and annual or biennial ruderal species such as Tripleurospermum inodorum, Conyza canadensis and Setaria viridis) through mead- Fig. 3 Maps of the studied species distribution (grey dots) and values of Ripley’s Kd function of different spatial extents. The grey area on Ripley’s Kd graphs shows ranges for random distribution. The crosses on the maps denote the centres of the studied buffers millefolium (49.1 %) and Dactylis glomerata (43.1 %). All of the species found in the sample plots are listed in Online Resource C. The results of ANOSIM showed that there were no significant differences (R = -0.006, p = 0.660) between the plots invaded by different goldenrod species with respect to vascular plant species composition. The NMDS ordination of plots invaded by particular goldenrod taxa (Fig. 5, upper panel) was consistent with the results of ANOSIM; that is, the goldenrod species did not have any tendency to form clusters with particular species composition. The exception was the tendency of E. graminifolia to concentrate on the right side of the graph with forest species. The ordination of species (Fig. Results 5, lower panel) suggests that the first axis is related to the gradient from frequently disturbed habitats (left wing of the graph, arable weeds and annual or biennial ruderal species such as Tripleurospermum inodorum, Conyza canadensis and Setaria viridis) through mead- ows/grassland vegetation (centre of the graph, Ar- rhenatherum elatius, D. glomerata and Lolium Kd function of different spatial extents. The grey area on Ripley’s Kd graphs shows ranges for random distribution. The crosses on the maps denote the centres of the studied buffers with particular species composition. The exception was the tendency of E. graminifolia to concentrate on the right side of the graph with forest species. The ordination of species (Fig. 5, lower panel) suggests that the first axis is related to the gradient from frequently disturbed habitats (left wing of the graph, arable weeds and annual or biennial ruderal species such as Tripleurospermum inodorum, Conyza canadensis and Setaria viridis) through mead- ows/grassland vegetation (centre of the graph, Ar- rhenatherum elatius, D. glomerata and Lolium millefolium (49.1 %) and Dactylis glomerata (43.1 %). All of the species found in the sample plots are listed in Online Resource C. The results of ANOSIM showed that there were no significant differences (R = -0.006, p = 0.660) between the plots invaded by different goldenrod species with respect to vascular plant species composition. The NMDS ordination of plots invaded by particular goldenrod taxa (Fig. 5, upper panel) was consistent with the results of ANOSIM; that is, the goldenrod species did not have any tendency to form clusters 12 3 3 Plant Ecol (2016) 217:565–582 572 Fig. 4 Differences between the percentages of habitat types calculated for the goldenrods and the buffer centres. The positive values reveal over-representation of goldenrod species in the particular type, while the negative values show under- representation. The asterisks show statistical significance Fig. 4 Differences between the percentages of habitat types calculated for the goldenrods and the buffer centres. The positive values reveal over-representation of goldenrod species in the particular type, while the negative values show under- representation. The asterisks show statistical significance Fig. 4 Differences between the percentages of habitat types calculated for the goldenrods and the buffer centres. The positive values reveal over-representation of goldenrod species Fig. 4 Differences between the percentages of habitat types calculated for the goldenrods and the buffer centres. 123 Results The positive values reveal over-representation of goldenrod species in the particular type, while the negative values show under- representation. The asterisks show statistical significance Fig. 5 Results of NMDS ordination. The upper panel shows the ordination of the plots. Because of the low number of plots with E. graminifolia (asterisks) and S. canadensis (grey trapeze) in these two groups, plots where these two species occur in combination with S. altissima and/or S. gigantea were also included. The group mixture (grey dots) consists of plots with a mixture of S. gigantea and S. altissima, exclusively. The lower panel shows the ordination of the species. For legibility, only the species, distribution of which was best explained by the ordination model, and/or the most frequent species were shown. The species labels consist of the first three letters of genus and species Latin names. The full names are given in Online Resource C perenne) up to relatively undisturbed forest vegetation (right wing, tree and shrub species such as Sorbus aucuparia, Corylus avellana and Quercus robur). The second axis can be interpreted as decreasing moisture gradient from moderate (upper part of the graph, Oxalis acetosella, Pinus sylvestris and Equisetum pratense) to wet habitats (lower part, Phragmites australis, Stellaria media and Filipendula ulmaria). Goldenrods occurred in a wide range of soils with respect to pH, nutrient content and texture. We found significant differences between taxa with respect to environmental variables (Table 1). E. graminifolia occurred in more over-shaded plots than S. altissima and S. canadensis; however, the latter species were also sporadically found under canopy cover. We also found differences between E. graminifolia and the remaining species with respect to the average precipitation (S. gigantea) and minimum temperature (S. altissima and S. canadensis); nonetheless, the ranges of these climatic variables overlapped among species. Analysis of the topsoil subset also revealed differences of E. Results graminifolia in lower K and Mg contents and a tendency to occur on more sandy soils 123 123 Plant Ecol (2016) 217:565–582 573 Table 1 The average ± standard deviation and range (in parenthesis) of environmental variables for each goldenrod species, the shortest distance to the initial infestation location (distance) and the results of statistical tests (v2, p) Table 1 The average ± standard deviation and range (in parenthesis) of environmental variables for each goldenrod species, the shortest distance to the initial infestation location (distance) and the results of statistical tests (v2, p) Table 1 The average ± standard deviation and range (in parenthesis) of environmental variables for each goldenrod species, the shortest distance to the initial infestation location (distance) and the results of statistical tests (v2, p) S. altissima S. canadensis S. gigantea E. Results The valu of PCNM vectors that significantly shaped goldenr distribution plotted against geographical coordinat the entire dataset and topsoil subset is not equal to the num Table 2 Results of canonical correspondence analysis (CC (R2, F and p) of the entire dataset and topsoil subset R2 F p Entire dataset Environment 0.031 6.531 0.0 Distance 0.081 10.053 0.0 Space 0.169 6.531 0.0 Topsoil subset Environment 0.161 4.152 0.0 Distance 0.121 3.402 0.0 Space 0.208 4.786 0.0 123 are presented in Online Resource D. It was found that not only the distribution of all the studied goldenrod species but also the canopy, precipitation and the distance (minCan, minEut) were correlated with the CCA axis calculated on the basis of PCNM vectors (Table 3). This implies that the environmental vari- ables were not randomly distributed throughout the studied region but spatially structured and that the PCNM variables also well reflected the distance. The combined model that joins environment, distance and space variables significantly explained approximately 19.6 % of goldenrod distribution variability (Fig. 6, lower panel). The combined model explained the distribution variabilities of 32.5 % of E. graminifolia, 23.5 % of S. altissima, 15.8 % of S. gigantea and 5.6 % of S. canadensis. The procedure of variation partitioning showed that space explains far more variation than the environment. The fraction of variation explained by pure distance was relatively less; however, the high amount of explained variation was shared with space. The fraction of explained variation shared by these three components concur- rently was relatively small (Fig. 6, lower panel). The first ordination axis, explaining most of the variation, well modelled the distribution of E. graminifolia. The second axis reflected the differences between S. altissima and S. gigantea (Fig. 6, lower panel). compared to S. altissima as well as on soils with a higher C/N ratio compared to S. canadensis (Table 1). The results of CCA of the entire dataset and the topsoil subset are presented in Table 2. The CCA model results, developed for the entire dataset, using the environmental variables showed that the distribu- tion of goldenrods was significantly shaped by precipitation and canopy. Their distribution was also significantly influenced by the shortest distance to initial infestation sites (distance) of E. graminifolia (minEut) and S. canadensis (minCan). It was found that the distance explained more variation than the environment (Table 2). Results graminifolia Entire dataset N observations 127 21 129 9 Canopy 11.7b ± 21.4 10.2b ± 20.3 17.2a,b ± 27.7 33.3a ± 28.2 v2 = 9.166 (0.0 to 100.0) (0.0 to 70.0) (0.0 to 100.0) (0.0 to 80.0) p = 0.024 Precipitation 590.8a,b ± 38.7 592.0a ± 32.3 580.7b ± 34.8 600.7a ± 24.5 v2 = 9.736 (535.0 to 737.0) (556.0 to 671.0) (533.0 to 675.0) (567.0 to 648.0) p = 0.017 TWI 19.9 ± 2.3 19.5 ± 2.6 19.8 ± 2.0 21.3 ± 0.8 v2 = 7.611 (9.0 to 22.7) (11.2 to 22.7) (12.2 to 22.9) (20.2 to 22.5) p = 0.051 Altitude 204.9 ± 106.3 218.3 ± 95.8 185.5 ± 83.3 181.0 ± 33.6 v2 = 4.723 (53.0 to 674.0) (98.0 to 424.0) (86.0 to 487.0) (147.0 to 246.0) p = 0.198 Tempaver 8.1 ± 0.7 8.0 ± 0.6 8.1 ± 0.5 8.3 ± 0.1 v2 = 2.358 (5.4 to 9.2) (6.7 to 8.9) (6.4 to 8.9) (8.1 to 8.4) p = 0.514 Tempmax 23.1 ± 0.9 23.0 ± 0.8 23.2 ± 0.6 23.3 ± 0.2 v2 = 1.197 (19.2 to 24.5) (21.3 to 23.8) (20.9 to 24.2) (23.1 to 23.5) p = 0.760 Tempmin -5.7a ± 1.0 -5.7a ± 0.8 -5.6a,b ± 0.7 -5.0b ± 0.2 v2 = 9.729 (-8.3 to -4.0) (-7.2 to -4.3) (-7.7 to -4.0) (-5.4 to -4.8) p = 0.019 Distance* 53.2a ± 37.2 20.8b ± 16.9 11.9b ± 6.5 v2 = 80.744 (0.2 to 152.2) (2.2 to 108.5) (2.2 to 21.2) p = 0.000 Topsoil subset N observations 46 18 43 8 Canopy 12.1 ± 21.1 10.8 ± 22.0 12.2 ± 21.4 31.9 ± 29.8 v2 = 7.145 (0.0 to 80.0) (0.0 to 70.0) (0.0 to 80.0) (0.0 to 80.0) p = 0.066 pH 5.9 ± 0.8 5.8 ± 0.9 5.8 ± 0.9 5.7 ± 1.1 v2 = 0.502 (4.3 to 7.9) (4.3 to 8.0) (4.3 to 8.0) (4.4 to 7.1) p = 0.922 C 3.3 ± 3.4 2.1 ± 1.0 3.7 ± 4.0 4.2 ± 3.9 v2 = 3.769 (0.3 to 20.0) (0.6 to 4.1) (0.6 to 20.3) (1.3 to 13.6) p = 0.296 N 1.6 ± 1.3 1.2 ± 0.4 1.5 ± 0.9 1.3 ± 0.7 v2 = 1.799 (0.3 to 9.1) (0.7 to 1.8) (0.6 to 4.7) (0.6 to 2.9) p = 0.622 C/N 2.0a,b ± 0.7 1.7b ± 0.5 2.1a,b ± 0.9 2.9a ± 1.1 v2 = 10.485 (0.9 to 4.7) (0.9 to 2.7) (0.9 to 5.3) (1.5 to 4.8) p = 0.014 P 47.7 ± 58.7 38.7 ± 26.6 47.9 ± 53.8 13.4 ± 8.4 v2 = 7.036 (3.8 to 279.5) (6.2 to 102.8) (3.6 to 211.2) (5.9 to 29.5) p = 0.069 K 155.2a ± 122.0 141.4a,b ± 96.7 120.2a,b ± 111.5 41.9b ± 26.2 v2 = 11.139 (20.0 to 545.0) (20.0 to 355.0) (5.0 to 520.0) (20.0 to 95.0) p = 0.011 Mg 98.2a ± 55.7 84.0a,b ± 47.4 80.9a,b ± 64.5 50.7b ± 32.2 v2 = 8.428 (22.2 to 234.8) (25.4 to 170.0) (17.1 to 380.6) (22.2 to 115.0) p = 0.032 Ca 710.1 ± 883.5 760.9 ± 1135.2 709.5 ± 837.1 1351.0 ± 1664.1 v2 = 0.356 (147.0 to 4206.0) (170.0 to 4206.0) (18.0 to 3372.0) (18.0 to 4206.0) p = 0.953 Sand 55.4c ± 21.2 55.2b,c ± 22.4 66.8a,b ± 18.7 81.3a ± 5.2 v2 = 14.434 (21.0 to 90.0) (27.0 to 90.0) (27.0 to 91.0) (73.0 to 88.0) p = 0.001 Silt 41.3a ± 19.4 41.8a,b ± 20.6 31.1b,c ± 17.0 17.6c ± 5.0 v2 = 14.252 (10.0 to 73.0) (10.0 to 68.0) (9.0 to 68.0) (11.0 to 25.0) p = 0.002 Clay 3.3a ± 2.3 2.9a,b ± 2.0 2.1b,c ± 1.8 1.1c ± 0.4 v2 = 13.386 (0.0 to 12.0) (0.0 to 6.0) (0.0 to 8.0) (1.0 to 2.0) p = 0.002 12 Plant Ecol (2016) 217:565–582 574 Table 1 continued S. Results altissima S. canadensis S. gigantea E. graminifolia Precipitation 595.7 ± 41.9 596.0 ± 33.1 582.7 ± 30.9 601.5 ± 26.0 v2 = 4.484 (540.0 to 720.0) (556.0 to 671.0) (536.0 to 671.0) (567.0 to 648.0) p = 0.215 Distance* 55.2a ± 39.6 20.4b ± 17.7 13.3b ± 8.9 v2 = 31.595 (7.4 to 152.2) (2.2 82.8) (2.3 to 25.9) p = 0.000 Asterisk indicates the Distance was calculated for S. canadensis s.I. Bold values indicate statistically significant results at p level 0.05 The different superscript letters denote significant differences between pairs of taxa. The sum of observations for particular taxa for the entire dataset and topsoil subset is not equal to the number of studied plots since two or more species can occur on some plots Bold values indicate statistically significant results at p level 0.05 y g p The different superscript letters denote significant differences between pairs of taxa. The sum of observations for particular taxa for the entire dataset and topsoil subset is not equal to the number of studied plots since two or more species can occur on some plots The different superscript letters denote significant differences between pairs of taxa. The sum of observations for particular taxa for the entire dataset and topsoil subset is not equal to the number of studied plots since two or more species can occur on some plots compared to S. altissima as well as on soils with higher C/N ratio compared to S. canadensis (Table The results of CCA of the entire dataset and t topsoil subset are presented in Table 2. The CC model results, developed for the entire dataset, usi the environmental variables showed that the distrib tion of goldenrods was significantly shaped precipitation and canopy. Their distribution was al significantly influenced by the shortest distance initial infestation sites (distance) of E. graminifo (minEut) and S. canadensis (minCan). It was fou that the distance explained more variation than t environment (Table 2). However, the space repr sented by PCNM vectors better described the va ability of goldenrod distribution (Table 2). particular, the first CCA axis adequately modell the distribution of E. graminifolia (Fig. 6, upp panel, high positive scores), while the second CC axis well represented the contrast between the plo invaded by S. gigantea (high positive scores) and altissima (moderate and negative scores). Results However, the space repre- sented by PCNM vectors better described the vari- ability of goldenrod distribution (Table 2). In particular, the first CCA axis adequately modelled the distribution of E. graminifolia (Fig. 6, upper panel, high positive scores), while the second CCA axis well represented the contrast between the plots invaded by S. gigantea (high positive scores) and S. altissima (moderate and negative scores). The values of PCNM vectors that significantly shaped goldenrod distribution plotted against geographical coordinates Table 2 Results of canonical correspondence analysis (CCA) (R2, F and p) of the entire dataset and topsoil subset R2 F p Entire dataset Environment 0.031 6.531 0.005 Distance 0.081 10.053 0.005 Space 0.169 6.531 0.005 Topsoil subset Environment 0.161 4.152 0.005 Distance 0.121 3.402 0.005 Space 0.208 4.786 0.005 123 Table 2 Results of canonical correspondence analysis (CCA) (R2, F and p) of the entire dataset and topsoil subset The ordination of the topsoil subset showed that goldenrod distributions were significantly shaped by C/N ratio and Mg and the percentage of silt in the soil among the environmental components. The shortest distances to initial infestation sites (distance) of E. graminifolia (minEut) and S. canadensis (minCan), as well as the four PCNM vectors, also significantly influenced goldenrod distribution, when the distance and space group of factors were analysed (Table 2). The distribution of S. gigantea, S. altissima and E. 12 575 Plant Ecol (2016) 217:565–582 graminifolia correlated with the CCA axis calculated The first ordination axis again reflected the contrast Fig. 6 Results of CCA ordination for the entire dataset. On the upper panel, the values of site scores of CCA with exclusively spatial variables as constraints plotted against geographical coordinates of plots are shown. The lower panel shows the results for the combined model (space, environment and distance) of alien goldenrod distribution: the variation partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location from S. canadensis s.l. plots; minEut the shortest distance to the initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V2–V30 PCNM vectors Fig. 6 Results of CCA ordination for the entire dataset. On the upper panel, the values of site scores of CCA with exclusively spatial variables as constraints plotted against geographical coordinates of plots are shown. Results The lower panel shows the results for the combined model (space, environment and distance) of alien goldenrod distribution: the variation partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location from S. canadensis s.l. plots; minEut the shortest distance to the initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V2–V30 PCNM vectors The first ordination axis again reflected the contrast between the sites invaded by E. graminifolia and the remaining species, while the second axis represented the differentiation between S. altissima and S. gigan- tea (Fig. 7, lower panel). The details of the topsoil subset analysis are shown in the Online Resource E. graminifolia correlated with the CCA axis calculated from the PCNM vectors (Table 3). We also found that silt and Mg, as well as minCan and minEut, were correlated with the site score of the first CCA axis calculated using the space components (Table 3). Finally, the combined model of goldenrod distribu- tions explained 28.3 % of the dataset variation (Fig. 7, lower panel). The pure space component explained more variation than solely environment fraction and distance, while the fraction of shared variation was relatively high (Fig. 7, lower panel). The combined model explained the distribution variabilities of 46.6 % of E. graminifolia, 33.3 % of S. altissima, 19.9 % of S. gigantea and 10.3 % of S. canadensis. graminifolia correlated with the CCA axis calculated from the PCNM vectors (Table 3). We also found that silt and Mg, as well as minCan and minEut, were correlated with the site score of the first CCA axis calculated using the space components (Table 3). Finally, the combined model of goldenrod distribu- tions explained 28.3 % of the dataset variation (Fig. 7, lower panel). The pure space component explained more variation than solely environment fraction and distance, while the fraction of shared variation was relatively high (Fig. 7, lower panel). The combined model explained the distribution variabilities of 46.6 % of E. graminifolia, 33.3 % of S. altissima, 19.9 % of S. gigantea and 10.3 % of S. canadensis. Solidago altissima and S. gigantea Solidago altissima and S. gigantea The frequently observed patchy distributions of inva- sive plant species can result from both dispersal 3 Plant Ecol (2016) 217:565–582 576 Table 3 Correlations between species data, environmental variables and the shortest distances significantly shaping species distribution, from one side, and canonical axes derived from CCA analysis with PCNM vectors as explanatory variables, on the other The correlations were tested separately for the entire dataset as well as topsoil subset Components Entire dataset Topsoil subset CCA1 CCA2 CCA1 CCA2 S. altissima Z = -13.332 Z = -11.001 Z = -6.335 Z = -5.739 p \ 0.001 p \ 0.001 p \ 0.001 p \ 0.001 S. canadensis n.s. Z = -5.353 Z = -1.263 Z = 0.4496 p \ 0.001 p = 0.206 p = 0.653 S. gigantea Z = 12.825 Z = 12.733 Z = 6.876 Z = -7.104 p \ 0.001 p \ 0.001 p \ 0.001 p \ 0.001 E. graminifolia Z = 5.413 Z = -5.251 Z = 4.725 Z = 2.498 p \ 0.001 p \ 0.001 p \ 0.001 p = 0.012 Precipitation n.s. Z = -3.2814 – – p = 0.001 Canopy Z = 2.182 n.s. – – p = 0.029 C/N – – Z = 1.8002 Z = 0.654 p = 0.072 p = 0.513 Mg – – Z = -2.688 Z = 0.968 p = 0.007 p = 0.333 Silt – – Z = -3.824 Z = 1.614 p = 0.001 p = 0.106 minEut Z = -6.337 Z = -2.508 Z = -4.633 Z = 1.672 p \ 0.001 p = 0.012 p \ 0.001 p = 0.094 minCan Z = 4.636 Z = 1.767 Z = 3.954 Z = -1.717 p \ 0.001 p = 0.077 p \ 0.001 p = 0.086 essential for successful invasion is competition with resident vegetation (Levine et al. 2003; Vila´ and Weiner 2004; van Kleunen et al. 2010; Gioria and Osborne 2014; Kuebbing and Nun˜ez 2015). Based on the results, there is no reason to assume that native vegetation in areas invaded by these two taxa differs. The results of ANOSIM and NMDS reveal that the two species did not differ with respect to the composition of co-occurring vascular plant species. On the other hand, the distributions of these species were well modelled using geographical space. Solidago altissima and S. gigantea Specif- ically, the location of initial infestation sites, prior to the rapid spread phase on invasion (Fig. 1b), was well correlated with the present-day distribution of spe- cies. This suggests that the observed pattern resulted from a limitation in the local distribution within the invaded ranges. However, these species are known to have efficient long-range dispersal (Weber 2000; Weber and Jakobs 2005). We suggest that priority effect limited goldenrod distributions (Shulman et al. 1983; Vannette and Fukami 2014); that is, the limitation and lack of suitable habitats (Warren et al. 2013). Local dispersal, which leads to infilling gaps within the invaded range, drives the landscape cover- age of the invader and, usually, its environmental impact (Wangen and Webster 2006; Miller and Matlack 2010; Warren et al. 2013; Bartha et al. 2014). Because of this, the observation of aggregated patterning without other analysis does not provide an explanation of the ecological processes and limita- tions, which determine the observed distribution (Warren et al. 2013). In contrast to previous analyses regarding the differentiation of ecological niches between S. altissima and S. gigantea, our study reveals little differentiation of climatic and soil conditions, and none in the invaded habitats. Despite the fact that in the native range, S. altissima is considered as preferring drier sites than S. gigantea (Weber and Jakobs 2005; Weber 2000), we did not find such environmental preferences in the studied region based both on TWI and plant species composition. A factor 123 123 Plant Ecol (2016) 217:565–582 577 Fig. 7 Results of CCA ordination for topsoil subset. On the upper panel, the values of site scores of CCA with exclusively spatial variables as constraints plotted against geographical coordinates of plots are shown. The lower panel shows the results for the combined model (space, environment and distance) of alien goldenrod distribution: the variation partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location from S. canadensis s.l. plots; minEut the shortest distance to the initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V1–V18 PCNM vectors Fig. 7 Results of CCA ordination for topsoil subset. On the upper panel, the values of site scores of CCA with exclusively spatial variables as constraints plotted against geographical coordinates of plots are shown. Euthamia graminifolia Euthamia graminifolia and Nowak 2007), suggesting that the species may overcome the dispersal barrier to invasion (Szymura and Szymura 2013). This species outstand from the other goldenrod species as it was under-represented on abandoned lands, key habitats of invasive Solidago in Central Europe and did not tend to avoid forests. This species was also more frequent within linear habitats such as road verges and unpaved roads. This suggests that the species is more shade tolerant than the others and is perhaps less efficient in colonisation and occupation of open habitats. In contrast, in the native range, it is a typical species of open prairie landscape (Parker et al. 1993; Lake et al. 2014). In garden experiments, this species is highly competitive and can significantly decrease the biomass of S. altissima and S. gigantea through underground competition (Szymura and Szy- mura 2016). Therefore, its under-representation in open areas is more likely attributed to the limited long- range distribution of this species and to low seed germination in the new range. The proportion of germinating E. graminifolia seeds in the native range of Canada reaches 78 % (White et al. 2009), but in Europe, very few mature seeds could germinate, compared to the much higher percentages of S. altissima and S. gigantea (Voser-Huber 1983; Szy- mura 2012). Moreover, many of the abandoned fields in the studied region were already occupied by S. gigantea, which would additionally limit the expan- sion of E. graminifolia. Low germination of E. graminifolia is compensated by efficient re-growth by rhizomes, which comprises a significant part of the clonal biomass (Szymura and Szymura 2015). Thus, its over-representation on linear habitats can be attributed to the vegetative spread along road verges and unpaved roads. Ineffective long-range dispersal also explained the limited range of its distribution, which did not expand considerably since the initial establishment in Europe (Guzikowa and Maycock 1986; Weber 2001). There were also likely fewer E. graminifolia introduction sites, compared to the other species as it is considered less ornamental than S. altissima and S. gigantea and has been less frequently used in horticulture (Weber and Schmid 1998; Weber 2001). In Silesia, its propagules were probably trans- ported accidentally along with ornamental trees into the local arboretum (Dajdok and Nowak 2007). E. Euthamia graminifolia graminifolia is sporadically reported from open aban- doned fields and new localities of this species have been found (Kompała-Baba and Baba 2006; Dajdok y ) The results suggest differences in ecological niche of E. graminifolia compared to the Solidago species. However, the fraction of variability shared by space, environment and distance in the topsoil model was high. Thus, the presence of E. graminifolia on more shaded habitats, on sandy soils with a high C/N ratio and low content of K and Mg, as well as higher minimal temperature, cannot unequivocally be con- sidered as its ecological optima. The observed pattern could also result from both limited distribution and priority effects. The potential European range of E. graminifolia, determined on the basis of its climatic preferences, is comparable to other goldenrods (We- ber 2001). In North America, this species occupies areas with slightly lower temperatures than the areas occupied by S. altissima and S. gigantea (Weber 2001). In Silesia, we observed an opposite temperature reaction. However, these differences appear to be caused mostly by its limited distribution, because E. graminifolia sites were exclusively within the lowland part of the region, where the minimal temperatures are higher than those in highland areas. Moreover, the differences in soil chemical composition can be attributed to typical properties of forest soils, on which E. graminifolia was most commonly found, while the remaining species often invaded more fertile post-agricultural lands. Solidago altissima and S. gigantea The lower panel shows the results for the combined model (space, environment and distance) of alien goldenrod distribution: the variation partitioning results on the left and the CCA biplot on the right. minCan the shortest distance to the initial infestation location from S. canadensis s.l. plots; minEut the shortest distance to the initial infestation location from E. graminifolia plots, E. gram E. graminifolia, S. alti S. altissima, S. cana S. canadensis, S. giga S. gigantea, V1–V18 PCNM vectors 1999a). In the case of S. altissima, the proportion of seeds germinated in patches of vegetation was 0.008 %; however, the emergence of seedlings in artificially created openings was high (Meyer and Schmid 1999a). The authors concluded that offspring coming from seeds did not influence the dynamics of established populations of S. altissima but, most probably, are important in colonisation of new, open habitats with disturbed soil. As a result, we observed regions dominated by either S. altissima or S. gigantea. The association of S. gigantea with river valleys seems to be the effect of early colonisation patterns, rather than strict preferences for wet habitats in Central Europe. goldenrod species that first occupied the particular area blocked the establishment of further species from seeds. In subsequent stages, the populations produce a large number of diaspores that increase the probabil- ity of invasion of the remaining suitable habitats within the neighbourhood (Meyer and Schmid 1999a). Results of garden experiments showed that the two species are similar with respect to their competitiveness (Szymura and Szymura 2016); thus, the competitive exclusion of one species by another is not likely during later stages of invasion. In addition, the probability that new species could establish from seeds in a dense, well-established stand is minimal (Hartnett and Bazzaz 1985; Meyer and Schmid 12 123 123 Plant Ecol (2016) 217:565–582 578 Solidago canadensis O´ va´ri M, Komoly C, Sutyinszki Z, Szabo´ G, Csatho´ AI, Juha´sz M, Penksza K, Molna´r Z (2014) Impact of mid- successional dominant species on the diversity and pro- gress of succession in regenerating temperate grasslands. Appl Veg Sci 17:201–213 O´ va´ri M, Komoly C, Sutyinszki Z, Szabo´ G, Csatho´ AI, Juha´sz M, Penksza K, Molna´r Z (2014) Impact of mid- successional dominant species on the diversity and pro- gress of succession in regenerating temperate grasslands. 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Ecology 89:1541–1553 Acknowledgments The study was supported by Grants: NCN (N N305 401438), the Wrocław University of Environmental and Life Sciences, and the University of the Wrocław. The authors thank the Editor and Anonymous Reviewers for their comments, which helped us to improve the manuscript. Dajdok Z, Nowak A (2007) Solidago graminifolia (L.) Elliott in Poland—spreading and habitat preferences. Solidago canadensis In: Tokarska- Guzik B et al (eds) Plant invasions: human perception, ecological impacts and management. Backhuys Publishers, Leiden, pp 101–116 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unre- stricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons license, and indicate if changes were made. Del Fabbro C, Gu¨sewell S, Prati D (2013) Allelopathic effects of three plant invaders on germination of native species: a field study. Biol Invasions 16:1035–1042 Dray S, Legendre P, Peres-Neto PR (2006) Spatial modelling: a comprehensive framework for principal coordinate analy- sis of neighbour matrices (PCNM). Ecol Model 196:483–493 Essl F, Dullinger S, Rabitsch W, Hulme PE, Hu¨lber K, Jarosˇı´k V, Kleinbauer I, Krausmann F, Ku¨hn I, Nentwig W, Vila` M, Genovesi P, Gherardi F, Desprez-Loustau ML, Roques A, Pysˇek P (2011) Socioeconomic legacy yields an inva- sion debt. Proc Natl Acad Sci USA 108(1):203–207 Solidago canadensis Solidago is known as one of the most complex genera of higher plants, and its variability has further increased due to hybridisation, introgression and ecological factors (Beaudry and Chabot 1957; Beau- dry 1963; Weber 1997). In the native range, S. canadensis consists of diploid (2n = 18) and tetra- ploid plants (2n = 36), while S. altissima consists of hexaploid (2n = 54), triploid (2n = 27) and tetraploid (2n = 36) cytotypes (Semple et al. 1981, 1984, 2015). Both taxa, S. canadensis var. canadensis and S. altissima, were introduced from North America to Europe (Wagenitz 1979; Weber 1997). However, in Europe, the taxonomical status of S. canadensis and S. altissima is unclear, because both are represented exclusively by diploids (Weber 2000). These species can form hybrids in the native range. Hybridisation has 12 123 Plant Ecol (2016) 217:565–582 579 also been observed in Europe between S. canadensis s.l. and native S. virgaurea (Pliszko 2013; Migdałek et al. 2014). Thus, it could be also hypothesised that the random pattern of S. canadensis distribution, overlapping extensively with the ranges of S. altissima and S. gigantea, could be related to the hybrid nature of this species. However, the populations examined here were checked for their chromosome number (Szymura et al. 2015), and the results showed that all S. canadensis and S. altissima were exclusively diploids, whereas S. gigantea was tetraploid. These findings allow us to reject the hypothesis about the hybrid nature of local S. canadensis. The traits used to distinguish S. altissima and S. canadensis primarily refer to the presence or absence of hairs on stems and leaves as well as leaf epidermis (Weber 1997; Semple and Cook 2006; Szymura and Wolski 2011). There- fore, they could not be considered as functional traits, which influence the fitness and competitiveness of individuals, and are likely not under natural selection. S. altissima and S. canadensis did not differ in functional traits, biomass production and allocation, as well as ploidy levels (Szymura and Szymura 2013, 2015; Szymura et al. 2015). 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Hierarchical Over-the-Air Federated Edge Learning
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24/10/2024 05:55 Hierarchical Over-the-Air Federated Edge Learning / Aygun, O.; Kazemi, M.; Gunduz, D.; Duman, T. 2022-:(2022), pp. 3376-3381. (Intervento presentato al convegno 2022 IEEE International Confere Communications, ICC 2022 tenutosi a COEX, kor nel 2022) [10.1109/ICC45855.2022.9839230]. Terms of use: The terms and conditions for the reuse of this version of the manuscript are specified in the publish policy. For all terms of use and more information see the publisher's website. Institute of Electrical and Electronics Engineers Inc. This is the peer reviewd version of the followng article: 24/10/2024 05:55 Hierarchical Over-the-Air Federated Edge Learning / Aygun, O.; Kazemi, M.; Gunduz, D.; Duman, T. 2022-:(2022), pp. 3376-3381. (Intervento presentato al convegno 2022 IEEE International Confere Communications, ICC 2022 tenutosi a COEX, kor nel 2022) [10.1109/ICC45855.2022.9839230]. Terms of use: The terms and conditions for the reuse of this version of the manuscript are specified in the publish policy. For all terms of use and more information see the publisher's website. Institute of Electrical and Electronics Engineers Inc. This is the peer reviewd version of the followng article: This is the peer reviewd version of the followng article: Hierarchical Over-the-Air Federated Edge Learning / Aygun, O.; Kazemi, M.; Gunduz, D.; Duman, T. M.. - 2022-:(2022), pp. 3376-3381. (Intervento presentato al convegno 2022 IEEE International Conference on Communications, ICC 2022 tenutosi a COEX, kor nel 2022) [10.1109/ICC45855.2022.9839230]. Terms of use: The terms and conditions for the reuse of this version of the manuscript are specified in the publishing policy. For all terms of use and more information see the publisher's website. Institute of Electrical and Electronics Engineers Inc. e s o use The terms and conditions for the reuse of this version of the manuscript are specified in the publishing policy. For all terms of use and more information see the publisher's website. 24/10/2024 05:55 I. INTRODUCTION Extensive amounts of collected data from various sources such as mobile phones and Internet-of-things (IoT) sensors have enabled the accelerating rise of machine learning (ML) algorithms, aiming to assemble all the data in a cloud server to obtain representative datasets for model training. This, however, brings out growing concerns regarding the privacy, cost, and latency of traditional ML algorithms. Firstly, data owners have become more sensitive about sharing their data; secondly, the increasing quality of data results in higher communication costs; and finally, solutions that work in real- time are faced with latency issues [1]. To overcome these problems, a decentralized approach called federated learning (FL) has been introduced, where the transmission of data is not required since models are trained locally instead of using a centralized server for training [2]. In order to make distant MUs more resilient to the chan- nel effects, we propose hierarchical over-the-air federated learning (HOTAFL), where MUs communicate with their corresponding ISs through wireless links. In this setup, each MU shares its local training result with its corresponding IS through OTA (cluster) aggregation. After several local itera- tions with the MUs in their clusters, the ISs send the results to the PS to complete the global aggregation, which constitutes one global iteration. We examine the performance of HOTAFL and compare the results with those of the conventional FL and error-free HFL both through analytical results and numerical experiments. The results show that the proposed framework outperforms conventional OTA FL and leads to a better model accuracy and faster convergence. In FL, several data owners called mobile users (MUs) are selected based on some criteria such as their computing capability, data quality, available power, and location [3]. Each MU in the federation trains a local model using its own data and computing power in every iteration. After each MU completes its local stochastic gradient descent (SGD) computation, only the weight updates are sent to a parameter server (PS) that performs model aggregation and sends back the updated global model to MUs for the next iteration. Despite its superiority over traditional ML, adverse channel effects in wireless setups and increased communication costs The paper is organized as follows: in Sections II and III, we introduce the HOTAFL framework as well as its com- munication model. In Section IV, we provide a convergence analysis of HOTAFL under convexity assumptions on the loss functions. Ozan Ayg¨un’s research in this study is supported by Turkcell A.S. within the framework of 5G and Beyond Joint Graduate Support Programme coordinated by Information and Communication Technologies Authority. Hierarchical Over-the-Air Federated Edge Learning Ozan Ayg¨un1, Mohammad Kazemi1, Deniz G¨und¨uz2 and Tolga M. Duman1 1Dept. of Electrical and Electronics Engineering, Bilkent University, Ankara, Turkey 2Dept. of Electrical and Electronic Engineering, Imperial College London, London, UK {ozan, kazemi, duman}@ee.bilkent.edu.tr, d.gunduz@imperial.ac.uk have arisen some concerns about the feasibility of conventional FL in practical scenarios. To address the communication cost concerns, over-the-air (OTA) aggregation [4] has become a popular method in wireless schemes thanks to its efficient strategy that allocates all the users to the same bandwidth, thereby handling the transmission and aggregation of the gradient updates simultaneously (over the air). For this frame- work, one approach to deal with the channel effects (particu- larly when there is no transmit side channel state information) is to increase the number of receive antennas at the PS [5]. Nevertheless, the disparity among the channel gains is still a critical factor when some MUs are far away from the PS. Abstract—Federated learning (FL) over wireless communica- tion channels, specifically, over-the-air (OTA) model aggregation framework is considered. In OTA wireless setups, the adverse channel effects can be alleviated by increasing the number of receive antennas at the parameter server (PS), which performs model aggregation. However, the performance of OTA FL is limited by the presence of mobile users (MUs) located far away from the PS. In this paper, to mitigate this limitation, we propose hierarchical over-the-air federated learning (HOTAFL), which utilizes intermediary servers (IS) to form clusters near MUs. We provide a convergence analysis for the proposed setup, and demonstrate through theoretical and experimental results that local aggregation in each cluster before global aggregation leads to a better performance and faster convergence than OTA FL. v:2112.11167v1 [cs.LG] 21 Dec 2021 arXiv:2112.11167v1 [cs.LG] 21 Dec Index Terms—machine learning, over-the-air communication, clustering, hierarchical federated learning. Recent developments on FL include device selection algo- rithms [6], efficient communication schemes [4], [7]–[11], het- erogeneity of data [12], and power and latency analysis [13], [14]. Although Federated Averaging [2] is the most common way to perform global aggregation in error-free setups, OTA communication has been preferred for wireless FL [5], [12], [15]. Furthermore, hierarchical federated learning (HFL) has been gaining increasing attention, where the objective is to utilize intermediate servers (IS) to form clusters to reduce communication costs. There exist studies on HFL on latency and power analysis [16], [17], resource allocation [18], [19], and performance analysis for non-independent and identically distributed (i.i.d.) data [20]. However, there is no work on HFL with OTA taking into account practical wireless channel models, which motivates this work. y g y D. Gunduz acknowledges support from UK EPSRC through CHIST-ERA project CONNECT (CHISTERA-18-SDCDN-001, EPSRC-EP/T023600/1). B. OTA Communication We now consider the scheme referred as OTA communica- tions, for which the links between the users and the ISs are wireless with OTA aggregation, however, the links between ISs and the PS is assumed to be error-free. Since a common wireless medium is used in local aggregations, noisy versions of the model updates ∆θIS,c(t) are received at the ISs. In our setup, the ISs are equipped with K antennas, and we assume perfect channel state information (CSI) at the receivers. For the k-th antenna, the received signal at the c-th IS can be written as1 We consider a hierarchical and iterative approach to min- imize (1) consisting of global, local, and user iterations. In every cluster iteration, the MUs carry out τ user iterations on their own, then send their model updates to their corresponding ISs for local iteration. I local iterations are performed at the IS in every cluster before all the local models are forwarded to the PS for global aggregation. At the j-th user iteration of the i-th local iteration, the weight update is performed employing stochastic gradient descent (SGD) for the m-th user in the c-th cluster as follows yi IS,c,k(t) = M X m=1 hi m,c,k(t) ◦xi m,c,k(t) + zi IS,c,k(t), (8) θi,j+1 m,c (t) = θi,j m,c(t) −ηi,j m,c(t)∇Fm,c(θi,j m,c(t), ξi,j m,c(t)), (2) (8) where ηi,j m,c(t) is the learning rate, ∇Fm,c(θi,j m,c(t), ξi,j m,c(t)) denotes the stochastic gradient estimate for the weight vector θi,j m,c(t) and a randomly sampled batch of data samples ξi,j m,c(t) from the dataset of the m-th user in the c-th cluster at the t-th global, i-th local and j-th user iteration. Initially, θ1,1 m,c(t) = θi IS,c(t), ∀i ∈[I] where [I] ≜{1, 2, . . . , I}, and θ1 IS,c(t) = θP S(t), where θP S(t) is the global model at the PS at the t-th global iteration and θi IS,c(t) denotes the local model of IS in the c-th cluster at the i-th local iteration. The purpose of employing ISs is to accumulate the local model differences within each cluster more frequently in smaller areas before obtaining the global model θP S(t) for the next iteration. where ◦denotes the element-wise product, xi m,c,k(t) ∈CN, zi IS,c,k(t) ∈CN with independent and identically distributed (i.i.d.) entries zi,n IS,c,k(t) ∼CN(0, σ2 z). 1Note that the setup here can be efficiently implemented in practice using orthogonal frequency-division multiplexing (OFDM). II. SYSTEM MODEL After completing I local iterations in each cluster, ISs send their model updates to the PS, which can be written as After completing I local iterations in each cluster, ISs send their model updates to the PS, which can be written as The objective of HOTAFL is to minimize a loss function F(θ) with respect to the model weight vector θ ∈R2N, where 2N is the model dimension. Our system consists of C clusters each containing an IS and M MUs as depicted in Fig. 1. The dataset of the m-th MU in the c-th cluster is denoted as Bm,c, and we define B ≜PC c=1 PM m=1 |Bm,c|. We have ∆θP S,c(t) = θI+1 IS,c(t) −θP S(t). (6) (6) The global update rule is ∆θP S(t) = 1 C PC c=1 ∆θP S,c(t). Using recursion, we can conclude that The global update rule is ∆θP S(t) = 1 C PC c=1 ∆θP S,c(t). Using recursion, we can conclude that ∆θP S(t) = 1 MC C X c=1 I X i=1 M X m=1 ∆θi m,c(t). (7) (7) F(θ) = C X c=1 M X m=1 |Bm,c| B Fm,c(θ), (1) (1) After the global aggregation, the model at the PS is updated as θP S(t + 1) = θP S(t) + ∆θP S(t). where Fm,c(θ) ≜ 1 |Bm,c| P u∈Bm,c f(θ, u), with f(θ, u) de- noting the corresponding loss of u-th data sample. A. Ideal Communication We refer to the case where all the communication among all the units is error-free as the ideal communication scenario. In this case, after performing SGD, each MU calculates its model difference to be sent to its corresponding IS as ∆θi m,c(t) = θi,τ+1 m,c (t) −θi IS,c(t). (3) (3) Then, the local aggregation at the c-th cluster is performed as Then, the local aggregation at the c-th cluster is performed as ∆θi IS,c(t) = 1 M M X m=1 ∆θi m,c(t), (4) θi+1 IS,c(t) = θi IS,c(t) + ∆θi IS,c(t). (5) (4) conclude the paper in Section VI. (5) I. INTRODUCTION We present our numerical results in Section V, and MU Local Aggregation Global Aggregation Cluster IS PS Fig. 1: HOTAFL system model. Also, note that ∇Fm,c(θi,j m,c(t), ξi,j m,c(t)) is an unbiased estima- tor of ∇Fm,c(θi,j m,c(t)), i.e., Eξ  ∇Fm,c(θi,j m,c(t), ξi,j m,c(t))  = ∇Fm,c(θi,j m,c(t)), where the expectation is over the random- ness due to the SGD. B. OTA Communication Knowing the CSI perfectly, the c-th IS combines the received signals as yi IS,c(t) = 1 K PK k=1 PM m=1 hi m,c,k(t) ∗ ◦ yi IS,c,k(t). For the n-th symbol, it can be written as θP S(t + 1) = θP S(t) + ∆ˆθP S(t), (17) where ∆ˆθP S(t) =  ∆ˆθ1 P S(t) ∆ˆθ2 P S(t) · · · ∆ˆθ2N P S(t) T . (17) n S,c(t)=Pt M X m=1  1 K K X k=1 |hi,n m,c,k(t)|2 ∆θi,n,cx m,c (t) | {z } yi,n,sig IS,c (t) (signal term) + Pt K M X m=1 M X m′=1 m′̸=m K X k=1 (hi,n m,c,k(t))∗hi,n m′,c,k(t)∆θi,n,cx m′,c (t) | {z } yi,n,itf IS,c (t) (interference term) + 1 K M X m=1 K X k=1 (hi,n m,c,k(t))∗zi,n c,k(t) | {z } yi,n,no IS,c (t) (noise term) . (11) yi,n IS,c(t)=Pt M X m=1  1 K K X k=1 |hi,n m,c,k(t)|2 ∆θi,n,cx m,c (t) | {z } yi,n,sig IS,c (t) (signal term) B. OTA Communication The channel coeffi- cients are modelled as hi m,c,k(t) = p βm,c gi m,c,k(t), where gm,c,k(t) ∈CN with entries gi,n m,c,k(t) ∼CN(0, σ2 h) (i.e., Rayleigh fading), βm,c is the large-scale fading coefficient modeled as βm,c = (dm,c)−p, where p represents the path loss exponent, and dm,c denotes the distance between the m- th user in the c-th cluster and the IS in that cluster. 1) Local Aggregation: In OTA communication, in order to increase the spectral efficiency, the model differences are grouped to form a complex vector ∆θi,cx m,c ∈CN with the following real and imaginary parts yn P S(t) = C X c=1 xn P S,c(t) (15) = C X c=1 I X i=1 Re  yi,n,sig IS,c (t) PtMσ2 h | {z } yn P S,1(t) + C X c=1 I X i=1 Re  yi,n,itf IS,c (t) PtMσ2 h | {z } yn P S,2(t) + C X c=1 I X i=1 Re  yi,n,no IS,c (t) PtMσ2 h | {z } yn P S,3(t) . (16) (15) ∆θi,re m,c(t) ≜  ∆θi,1 m,c(t), ∆θi,2 m,c(t), . . . , ∆θi,N m,c(t) T , (9a) ∆θi,im m,c (t)≜  ∆θi,N+1 m,c (t), ∆θi,N+2 m,c (t), . . . , ∆θi,2N m,c (t) T. (9b) Under the assumption that there is no inter-cluster interference, the received signal for the k-th antenna in the c-th cluster at the i-th local iteration can be represented as Under the assumption that there is no inter-cluster interference, the received signal for the k-th antenna in the c-th cluster at the i-th local iteration can be represented as + C X c=1 I X i=1 Re  yi,n,no IS,c (t) PtMσ2 h | {z } yn P S,3(t) . (16) (16) yi IS,c,k(t) = Pt M X m=1 hi m,c,k(t) ◦∆θi,cx m,c(t) + zi IS,c,k(t), (10) {z yn P S,3(t) The received signal at the PS is then recovered as ∆ˆθn P S(t) = 1 C Re{yn P S(t)}, ∆ˆθn+N P S (t) = 1 C Im{yn P S(t)}. Finally, the global aggregation is performed as The received signal at the PS is then recovered as ∆ˆθn P S(t) = 1 C Re{yn P S(t)}, ∆ˆθn+N P S (t) = 1 C Im{yn P S(t)}. Finally, the global aggregation is performed as where Pt is the transmit power constant at the t-th global iteration. IV. CONVERGENCE ANALYSIS In this section, we present a convergence analysis of the proposed HOTAFL algorithm. Define the optimal solution as θ∗≜arg minθ F(θ), the minimum values of the total and the local loss functions as F ∗= F(θ∗) and F ∗ m,c, respectively, and the bias in the dataset as Γ ≜F ∗−PC c=1 PM m=1 Bm,c B F ∗ m,c ≥ 0. In addition, assume that the learning rate of the overall system does not change in local iterations, i.e., ηi,j m,c(t) = η(t). Therefore, we can write the global update rule as yIS,c ( ) g + Pt K M X m=1 M X m′=1 m′̸=m K X k=1 (hi,n m,c,k(t))∗hi,n m′,c,k(t)∆θi,n,cx m′,c (t) | } + Pt K M X m=1 M X m′=1 m′̸=m K X k=1 (hi,n m,c,k(t))∗hi,n m′,c,k(t)∆θi,n,cx m′,c (t) | {z } yi,n,itf IS,c (t) (interference term) {z yi,n,itf IS,c (t) (interference term) + 1 K M X m=1 K X k=1 (hi,n m,c,k(t))∗zi,n c,k(t) | {z } yi,n,no IS,c (t) (noise term) . (11 θi,j+1 m,c (t) = θi,j m,c(t) −η(t)∇Fm,c(θi,j m,c(t), ξi,j m,c(t)), (18) (11) (11) which can also be written as which can also be written as yi,n,no IS,c (t) (noise term) θi,j+1 m,c (t)−θi,1 m,c(t) = −η(t) j X l=1 ∇Fm,c(θi,l m,c, ξi,l m,c(t)). (19) Aggregated model differences can be recovered by Assumption 1. All the loss functions are L-smooth and µ- strongly convex; i.e., ∀v, w ∈R2N, ∀m ∈[M], ∀c ∈[C], Assumption 1. All the loss functions are L-smooth and µ- strongly convex; i.e., ∀v, w ∈R2N, ∀m ∈[M], ∀c ∈[C], ∆ˆθi,n IS,c(t) = 1 PtMσ2 h ¯βc Re{yi,n IS,c(t)}, (12a) ∆ˆθi,n+N IS,c (t) = 1 PtMσ2 h ¯βc Im{yi,n IS,c(t)}, (12b) (12a) , (12b) model differ- Fm,c(v)−Fm,c(w)≤⟨v−w,∇Fm,c(w)⟩+ L 2 ∥v −w∥2 2, (20) Fm,c(v)−Fm,c(w)≥⟨v−w,∇Fm,c(w)⟩+ µ 2 ∥v −w∥2 2. (21) (12b) where ¯βc = PM m=1 βm,c. After estimating the model differ- ence values, the cluster model update is written as Assumption 2. The expected value of the squared l2 norm of the stochastic gradients are bounded; i.e., ∀j ∈[τ], i ∈[I], θi+1 IS,c(t) = θi IS,c(t) + ∆ˆθi IS,c(t), (13) (13) (13) Eξ h ∇Fm,c(θi,j m,c(t), ξi,j m,c(t)) 2 2 i ≤G2, (22) which translates to ∀n∈[2N], Eξ  ∇Fm,c(θi,j,n m,c , ξi,j,n m,c (t))  ≤G. Theorem 1. V. SIMULATION RESULTS We consider a hierarchical system with one PS and C = 4 non-overlapping clusters, each containing one IS with K = 5MC receive antennas and M = 5 MUs. Users are randomly placed in the clusters in such a way that their distance to the PS is between 0.5 and 3, while having a distance between 0.5 and 1 with their corresponding IS. We also define α = PM m=1 PC c=1 dm,c PD d=1 dd as a measure of relative closeness of the MUs to their corresponding IS compared to the PS, where dm,c is the distance between m-th user in c-th cluster to the c-th IS and dd is the distance between the d-th user and the PS. α is set to 0.4 in the simulations. We use MNIST [21] and CIFAR-10 [22] datasets with Adam optimizer [23], and considered both i.i.d. and non-i.i.d. data distributions. In the i.i.d. case, data samples are randomly distributed among MUs, while in the non-i.i.d. case, the training data is divided into 5MC groups each consisting of data with only one label. Then, 5 groups are assigned to each user randomly. For CIFAR10, we use the neural network given in [5] with 2N = 307498 whereas for MNIST, we employ a one-layer neural network with 784 input and 10 output neurons with 2N = 7850. We consider a hierarchical system with one PS and C = 4 non-overlapping clusters, each containing one IS with K = 5MC receive antennas and M = 5 MUs. Users are randomly placed in the clusters in such a way that their distance to the PS is between 0.5 and 3, while having a distance between 0.5 and 1 with their corresponding IS. We also define α = PM m=1 PC c=1 dm,c PD d=1 dd as a measure of relative closeness of the MUs Proof: Let us define auxiliary variable v(t + 1) = θP S(t) + ∆θP S(t). Then, we have ∥θP S(t+1)−θ∗∥2 2 =∥θP S(t+1)−v(t+1) + v(t+1)−θ∗∥2 2 = ∥θP S(t + 1) −v(t + 1)∥2 2 + ∥v(t + 1) −θ∗∥2 2 + 2⟨θP S(t + 1) −v(t + 1), v(t + 1) −θ∗⟩. (25) P d=1 dd to their corresponding IS compared to the PS, where dm,c is the distance between m-th user in c-th cluster to the c-th IS and dd is the distance between the d-th user and the PS. IV. CONVERGENCE ANALYSIS Since the third term in Y (a) is independent of η(a), even for lim t→∞η(t) = 0, we have lim t→∞E[F(θP S(t))] −F ∗̸= 0. This term is also inversely proportional to M, C, and K. + σ2 zIN P 2a M 2C2Kσ2 h M X m=1 C X c=1 βm,c ¯β2c Lemma 1. We have E h θP S(t + 1) −v(t + 1) 2 2 i ≤η2(t)τ 2G2I M 2C2 M X m1=1 C X c1=1 β2 m1,c1 K ¯ β2c1 +  M X m2=1 C X c2=1 A1I  + M X m=1 M X m′=1 m′̸=m C X c=1 η2(t)τ 2G2Iβm,cβm′,c M 2C2K ¯β2c + σ2 zIN P 2 t M 2C2Kσ2 h M X m=1 C X c=1 βm,c ¯β2c . (26) Proof: See Appendix A. E h θP S(t + 1) −v(t + 1) 2 2 i ≤η2(t)τ 2G2I M 2C2 M X m1=1 C X c1=1 β2 m1,c1 K ¯ β2c1 +  M X m2=1 C X c2=1 A1I  + M X m=1 M X m′=1 m′̸=m C X c=1 η2(t)τ 2G2Iβm,cβm′,c M 2C2K ¯β2c Three scenarios are considered: baseline with error-free transmissions, FL with OTA aggregation over a wireless medium, and HOTAFL. We set the total number of global iterations T to 200, the mini-batch size to |ξi m,c(t)| = 500, σ2 h = 1 and the path loss exponent p to 4. The noise variance is σ2 z = 10 for the MNIST, σ2 z = 1 for the CIFAR-10 training. Also, the power multiplier is set to Pt = 1 + 10−2t for HOTAFL, Pt = 1.5 + 10−2t for conventional FL, t ∈[T]. Three scenarios are considered: baseline with error-free transmissions, FL with OTA aggregation over a wireless medium, and HOTAFL. We set the total number of global iterations T to 200, the mini-batch size to |ξi m,c(t)| = 500, σ2 h = 1 and the path loss exponent p to 4. The noise variance is σ2 z = 10 for the MNIST, σ2 z = 1 for the CIFAR-10 training. Also, the power multiplier is set to Pt = 1 + 10−2t for HOTAFL, Pt = 1.5 + 10−2t for conventional FL, t ∈[T]. (26) Proof: See Appendix A. IV. CONVERGENCE ANALYSIS In HOTAFL, for 0 ≤η(t) ≤min{1, 1 µτI }, the Eξ h ∇Fm,c(θi,j m,c(t), ξi,j m,c(t)) 2 2 i ≤G2, (22) Eξ h ∇Fm,c(θi,j m,c(t), ξi,j m,c(t)) 2 2 i ≤G2, (22) translates to ∀n∈[2N], Eξ  ∇Fm,c(θi,j,n m,c , ξi,j,n m,c (t))  ≤G. (22) where ∆ˆθi IS,c(t) =  ∆ˆθi,1 IS,c(t) ∆ˆθi,2 IS,c(t) · · · ∆ˆθi,2N IS,c (t) T . where ∆ˆθi IS,c(t) =  ∆ˆθi,1 IS,c(t) ∆ˆθi,2 IS,c(t) · · · ∆ˆθi,2N IS,c (t) T . 2) Global Aggregation: This part is similar to the case of ideal communication. The only difference is that the aggre- gated signals are estimates of the actual model differences. Letting xP S,c(t) be the transmitted signal from the c-th IS, its n-th symbol can be written as Theorem 1. In HOTAFL, for 0 ≤η(t) ≤min{1, 1 µτI }, the global loss function can be upper bounded as E  ∥θP S(t) −θ∗∥2 2  ≤  t−1 Y a=1 X(a)  ∥θP S(0)−θ∗∥2 2+ t−1 X b=1 Y (b) t−1 Y a=b+1 X(a), (23) h X( ) (1 ( )I ( ( )( 1))) d xn P S,c(t) = ∆θn P S,c(t) + j∆θn+N P S,c (t). (14) (14) Then, using (6), (11), (14) and recursion, the received signal for 1≤n≤N (similarly for N+1≤n≤2N) can be written as where X(a) = (1 −µη(a)I (τ −η(a)(τ −1))) and where X(a) = (1 −µη(a)I (τ −η(a)(τ −1))) and Corollary 1. Assuming L-smoothness, after T global itera- tions, the loss function can be upper bounded as Y (a)=η2(a)τ 2G2I M 2C2 M X m1=1 C X c1=1 β2 m1,c1 K ¯ β2c1 +  M X m2=1 C X c2=1 A1I  + M X m=1 M X m′=1 m′̸=m C X c=1 η2(a)τ 2G2Iβm,cβm′,c M 2C2K ¯β2c + σ2 zIN P 2a M 2C2Kσ2 h M X m=1 C X c=1 βm,c ¯β2c + (1 + µ(1 −η(a)) η2(a)IG2 τ(τ −1)(2τ −1) 6 + η2(a)I(τ 2 + τ −1)G2 + 2η(a)I(τ −1)Γ, (24) with A1 = 1 −βm1,c1 ¯βc1 −βm2,c2 ¯βc2 + βm1,c1βm2,c2 ¯βc1 ¯βc2 . E [F(θP S(T)) −F ∗] ≤L 2 E h ∥θP S(T) −θ∗∥2 2 i ≤L 2  T −1 Y n=1 X(n)  ∥θP S(0)−θ∗∥2 2+ L 2 T −1 X p=1 Y (p) T −1 Y n=p+1 X(n). (28) Remark. Lemma 2. We have Lemma 2. We have E h v(t+1)−θ∗ 2 2 i ≤(1−µη(t)I(τ −η(t)(τ−1)))E h θP S(t)−θ∗ 2 2 i + (1 + µ(1 −η(t)) η2(t)IG2 τ(τ −1)(2τ −1) 6 + η2(t)I(τ 2 + τ −1)G2 + 2η(t)I(τ −1)Γ. (27) Accuracy plots are presented in Figs. 2-4, where ¯P is the average transmit power. The results show that bringing the servers closer to the users enhances learning accuracy significantly. One reason for the improved performance is that the cluster structure enables the MUs share their model differences with a local server closer than the PS, reducing the adverse effects of the large-scale wireless channel effects. Accuracy plots are presented in Figs. 2-4, where ¯P is the average transmit power. The results show that bringing the servers closer to the users enhances learning accuracy significantly. One reason for the improved performance is that the cluster structure enables the MUs share their model differences with a local server closer than the PS, reducing the adverse effects of the large-scale wireless channel effects. Another reason is that MUs receive updated models even without communicating with the PS due to local aggregations. We also observe that although more initial power is given to FL, the user updates do not reflect on the global model as much as HOTAFL does due to the effects of the wireless channel. More local iterations enables faster convergence but uses more transmit power due to the accumulating nature of IS. Even though the noise variance is high when compared to (27) Proof: The proof is similar to that of Lemma 2 in [5]. Proof: The proof is similar to that of Lemma 2 in [5]. Lemma 3. E [⟨θP S(t + 1) −v(t + 1), v(t + 1) −θ∗⟩] = 0. Proof: We have E[⟨θP S(t+1)−v(t+1), v(t+1)−θ∗⟩]= E h ⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩ i . Then, know- ing that channel realizations are independent of the user and cluster updates at the same global iteration t, we have E h ⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩ i =0. Lemma 3. E [⟨θP S(t + 1) −v(t + 1), v(t + 1) −θ∗⟩] = 0. Proof: We have E[⟨θP S(t+1)−v(t+1), v(t+1)−θ∗⟩]= E h ⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩ i . Then, know- ing that channel realizations are independent of the user and cluster updates at the same global iteration t, we have E h ⟨∆ˆθP S(t)−∆θP S(t),θP S(t)+∆θP S(t)−θ∗⟩ i =0. V. SIMULATION RESULTS α is set to 0.4 in the simulations. We use MNIST [21] and CIFAR-10 [22] datasets with Adam optimizer [23], and considered both i.i.d. and non-i.i.d. data distributions. In the i.i.d. case, data samples are randomly distributed among MUs, while in the non-i.i.d. case, the training data is divided into 5MC groups each consisting of data with only one label. Then, 5 groups are assigned to each user randomly. For CIFAR10, we use the neural network given in [5] with 2N = 307498 whereas for MNIST, we employ a one-layer neural network with 784 input and 10 output neurons with 2N = 7850. Next, we provide upper bounds on the three terms of (25). Next, we provide upper bounds on the three terms of (25). h i Recursively iterating through the results of Lemmas 1, 2, and 3 concludes the theorem. Lemma 2. We have MNIST data with τ = 1. 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Accuracy HOTAFL Ideal, I=4 HOTAFL Ideal, I=2 FL Ideal HOTAFL, I = 4, ̄P = 3.29 HOTAFL, I = 2, ̄P = 2.76 FL, ̄P = 4.80 g. 4: Test accuracy for i.i.d. CIFAR 10 data with τ 5. 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0 1 2 3 4 5 Upper bound, [F(θPS(t))] −F*(×10−3) FL FL Ideal HOTAFL, I = 2 HOTAFL Ideal, I = 2 HOTAFL, I = 4 HOTAFL Ideal, I = 4 0 75 100 125 1 Number of Global Iterations,t Fig. 3: Test accuracy for non-i.i.d. MNIST data with τ = 3. Fig. 5: Convergence rate for i.i.d. MNIST data with τ = 1. the transmit power, deploying 5MC = 100 receive antennas almost mitigates the noise and the interference terms [5]. Increasing τ compensates the accuracy under more complex data structure. In Fig. 5, we compare the convergence rates of conventional FL and HOTAFL using the upper bound in (28), with 2N = 7850, L = 10, µ = 1, G2 = 1, Γ = 1, η(t) = 5·10−2−2 · 10−5t, Pt = 1 + 10−2t, β = 3, ∥θP S(0) −θ∗∥2 2 = the transmit power, deploying 5MC = 100 receive antennas almost mitigates the noise and the interference terms [5]. Increasing τ compensates the accuracy under more complex data structure. In Fig. 5, we compare the convergence rates of conventional FL and HOTAFL using the upper bound in (28), with 2N = 7850, L = 10, µ = 1, G2 = 1, Γ = 1, η(t) = 5·10−2−2 · 10−5t, Pt = 1 + 10−2t, β = 3, ∥θP S(0) −θ∗∥2 2 = 103. It can be seen that the convergence rate of HOTAFL is very close to that of the ideal case, and it becomes almost the same when the number of local iterations is increased. VI. CONCLUSIONS where A1 = 1 −βm1,c1 ¯βc1 −βm2,c2 ¯βc2 + βm1,c1βm2,c2 ¯βc1 ¯βc2 In this work, we have proposed HOTAFL where ISs are employed to create clusters to bring the server-side closer to the areas where MUs are more densely located. Our framework includes OTA cluster aggregations, where the MUs send their model updates to the ISs through a wireless channel with path loss and fading. We have examined the performance and convergence rate of HOTAFL through theoretical limits as well as model training where MNIST and CIFAR-10 datasets are used with both i.i.d. and non-i.i.d. data distributions. The results show that employing a cluster-based hierarchical model outperforms the conventional FL. Proof: Using (7) and (12), we have E ∆ˆθn P S,1(t) −∆θn P S(t) 2 = E h 1 M 2C2 M X m1=1 M X m2=1 C X c1=1 C X c2=1 I X i1=1 I X i2=1 ∆θi1,n m1,c1(t) × ∆θi2,n m2,c2(t)  1 − 1 Kσ2 h ¯βc1 K X k1=1 |hi1,n m1,c1,k1(t)|2 E ∆ˆθn P S,1(t) −∆θn P S(t) 2 = E h 1 M 2C2 M X m1=1 M X m2=1 C X c1=1 C X c2=1 I X i1=1 I X i2=1 ∆θi1,n m1,c1(t) × ∆θi2,n m2,c2(t)  1 − 1 Kσ2 h ¯βc1 K X k1=1 |hi1,n m1,c1,k1(t)|2 − 1 Kσ2 h ¯βc2 K X k2=1 |hi2,n m2,c2,k2(t)|2 + 1 K2σ4 h ¯β2c1 K X k1=1 K X k2=1 |hi1,n m1,c1,k1(t)|2|hi2,n m2,c2,k2(t)|2i . (31) − 1 Kσ2 h ¯βc2 K X k2=1 |hi2,n m2,c2,k2(t)|2 + 1 K2σ4 h ¯β2c1 K X k1=1 K X k2=1 |hi1,n m1,c1,k1(t)|2|hi2,n m2,c2,k2(t)|2i . (31) Lemma 2. We have 2N X n=1 E ∆ˆθn P S,1(t) −∆θn P S(t) 2 = 1 M 2C2 M X m1=1 C X c1=1 I X i1=1 β2 m1,c1 K ¯ β2c1 E  ∆θi1 m1,c1(t) 2 2  +  M X m2=1 C X c2=1 I X i2=1 2N X n=1 A1E  ∆θi1,n m1,c1(t)∆θi2,n m2,c2(t)  , (30) where A1 = 1 −βm1,c1 ¯βc1 −βm2,c2 ¯βc2 + βm1,c1βm2,c2 ¯βc1 ¯βc2 Proof: Using (7) and (12), we have 2N 2 103. It can be seen that the convergence rate of HOTAFL is very close to that of the ideal case, and it becomes almost the same when the number of local iterations is increased. (30) Lemma 2. We have h i Recursively iterating through the results of Lemmas 1, 2, and 3 concludes the theorem. 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Accuracy HOTAFL Ideal, I=4 HOTAFL Ideal, I=2 FL Ideal HOTAFL, I = 4, ̄P = 29.40 HOTAFL, I = 2, ̄P = 27.35 FL, ̄P = 44.22 Fig. 4: Test accuracy for i.i.d. CIFAR-10 data with τ = 5. 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0 1 2 3 4 5 Upper bound, [F(θPS(t))] −F*(×10−3) FL FL Ideal HOTAFL, I = 2 HOTAFL Ideal, I = 2 HOTAFL, I = 4 HOTAFL Ideal, I = 4 Fig. 5: Convergence rate for i.i.d. MNIST data with τ = 1. Lemma 4. We have 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Accuracy HOTAFL Ideal, I=4 HOTAFL Ideal, I=2 FL Ideal HOTAFL, I = 4, ̄P = 0.10 HOTAFL, I = 2, ̄P = 0.07 FL, ̄P = 0.24 Fig. 2: Test accuracy for i.i.d. MNIST data with τ = 1. 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Accuracy HOTAFL Ideal, I=4 HOTAFL Ideal, I=2 FL Ideal HOTAFL, I = 4, ̄P = 3.29 HOTAFL, I = 2, ̄P = 2.76 FL, ̄P = 4.80 Fig. 3: Test accuracy for non-i.i.d. MNIST data with τ = 3. 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Accuracy HOTAFL Ideal, I=4 HOTAFL Ideal, I=2 FL Ideal HOTAFL, I = 4, ̄P = 0.10 HOTAFL, I = 2, ̄P = 0.07 FL, ̄P = 0.24 0 25 50 75 100 125 150 175 200 Number of Global Iterations,t 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 Accuracy HOTAFL Ideal, I=4 HOTAFL Ideal, I=2 FL Ideal HOTAFL, I = 4, ̄P = 29.40 HOTAFL, I = 2, ̄P = 27.35 FL, ̄P = 44.22 Fig. 4: Test accuracy for i.i.d. CIFAR-10 data with τ = 5. Fig. 2: Test accuracy for i.i.d. MNIST data with τ = 1. 1 0 Fig. 2: Test accuracy for i.i.d. In the following lemmas, we will bound each of these terms. Summing over all the symbols and using the independence of channel coefficients result in (30). Lemma 6. [14] D. Liu and O. Simeone, “Privacy for free: Wireless federated learning via uncoded transmission with adaptive power control,” IEEE J. Sel. Areas Commun., vol. 39, no. 1, pp. 170–185, 2021. 2N X n=1 E ∆ˆθn P S,3(t) 2 = σ2 zIN P 2 t M 2C2Kσ2 h M X m=1 C X c=1 βm,c ¯β2c . (34) pp [15] M. M. Amiri, D. Gunduz, S. R. Kulkarni, and H. Vincent Poor, “Convergence of federated learning over a noisy downlink,” IEEE Trans. Wireless Commun., pp. 1–16, 2021. Proof: Using the independence of channel coefficients, for 1 ≤n ≤N, we have [16] M. S. H. Abad, E. Ozfatura, D. Gunduz, and O. Ercetin, “Hierarchical federated learning across heterogeneous cellular networks,” in ICASSP 2020-2020 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP). IEEE, 2020, pp. 8866–8870. E ∆ˆθn P S,3(t) 2 = E h M X m=1 C X c=1 I X i=1 K X k=1 1 PtMCKσ2 h ¯βc × Re hi,n m,c,k(t) ∗zi,n c,k(t) 2i = E h M X m=1 C X c=1 I X i=1 K X k=1 1 P 2 t M 2C2K2σ4 h ¯β2c × Re hi,n m,c,k(t) ∗zi,n c,k(t) 2i = σ2 zI 2P 2 t M 2C2Kσ2 h M X m=1 C X c=1 βm,c ¯β2c . (35) E ∆ˆθn P S,3(t) 2 = E h M X m=1 C X c=1 I X i=1 K X k=1 1 PtMCKσ2 h ¯βc × Re hi,n m,c,k(t) ∗zi,n c,k(t) 2i pp [17] L. Liu, J. Zhang, S. Song, and K. B. Letaief, “Client-edge-cloud hierarchical federated learning,” in ICC 2020-2020 IEEE International Conference on Communications (ICC). IEEE, 2020, pp. 1–6. [18] S. Luo, X. Chen, Q. Wu, Z. Zhou, and S. Yu, “HFEL: Joint edge asso- ciation and resource allocation for cost-efficient hierarchical federated edge learning,” IEEE Trans. Wireless Commun., vol. 19, no. 10, pp. 6535–6548, 2020. × Re hi,n m,c,k(t)  zi,n c,k(t)  i = E h M X m=1 C X c=1 I X i=1 K X k=1 1 P 2 t M 2C2K2σ4 h ¯β2c × Re hi,n m,c,k(t) ∗zi,n c,k(t) 2i = σ2 zI 2P 2 t M 2C2Kσ2 h M X m=1 C X c=1 βm,c ¯β2c . (35) [19] J. Wang, S. Wang, R.-R. Chen, and M. Ji, “Local averaging helps: Hi- erarchical federated learning and convergence analysis,” arXiv preprint arXiv:2010.12998, 2020. [20] C. Briggs, Z. APPENDIX A For the n-th symbol, we have ∆ˆθn P S(t) = P3 l=1 ∆ˆθn P S,l(t), so using the independence of channel coefficients, we have E  ||θP S(t+1)−v(t+1)||2 2  = E  ∆ˆθP S(t)−∆θP S(t) 2 2  = 2N X n=1 (E ∆ˆθn P S,1(t)−∆θn P S(t) 2 + 3 X l=2 E ∆ˆθn P S,l(t) 2 . (29) Summing over all the symbols and using the independence of channel coefficients result in (30). In the following lemmas, we will bound each of these terms. Lemma 5. We have [2] B. McMahan, E. Moore, D. Ramage, S. Hampson, and B. A. y. Arcas, “Communication-Efficient Learning of Deep Networks from Decentralized Data,” Proceedings of the 20th International Conference on Artificial Intelligence and Statistics (AISTATS), pp. 1273–1282, 2017. 2N X n=1 E ∆ˆθn P S,2(t) 2 = M X m=1 M X m′=1 m′̸=m C X c=1 I X i=1 βm,cβm′,c M 2C2K ¯β2c E  ∆θi m′,c(t) 2 2  . (32) [3] D. Gunduz, D. B. Kurka, M. Jankowski, M. M. Amiri, E. Ozfatura, and S. Sreekumar, “Communicate to earn at the edge,” IEEE Commun. Mag., vol. 58, no. 12, pp. 14–19, 2020. pp [4] M. Mohammadi Amiri and D. Gunduz, “Machine learning at the wire- less edge: Distributed stochastic gradient descent over-the-air,” IEEE Trans. Signal Process., vol. 68, pp. 2155–2169, 2020. [5] M. M. Amiri, T. M. Duman, D. Gunduz, S. R. Kulkarni, and H. V. P. Poor, “Blind federated edge learning,” IEEE Trans. Wireless Commun., vol. 20, no. 8, pp. 5129–5143, 2021. Proof: For 1 ≤n ≤N, using the independence of channel coefficients, we have pp [6] M. M. Amiri, S. R. Kulkarni, and H. V. Poor, “Federated learning with downlink device selection,” arXiv preprint arXiv:2107.03510, 2021. E ∆ˆθn P S,2(t) 2 = E h M X m=1 M X m′=1 m′̸=m C X c=1 I X i=1 1 MCKσ2 h ¯βc E ∆ˆθn P S,2(t) 2 = E h M X m=1 M X m′=1 m′̸=m C X c=1 I X i=1 1 MCKσ2 h ¯βc × K X k=1 Re hi,n m,c,k(t) ∗hi,n m′,c,k(t)∆θi,n m′,c(t) 2i = E h M X m=1 M X m′=1 m′̸=m C X c=1 I X i=1 βm,cβm′,c 2M 2C2K ¯β2c × ∆θi,n m′,c(t) 2 + ∆θi,n+N m′,c (t) 2 + ∆θi,n m,c(t)∆θi,n m′,c(t) −∆θi,n+N m,c (t)∆θi,n+N m′,c (t)  i (33) [7] G. Zhu, Y. Wang, and K. Lemma 6. Fan, and P. Andras, “Federated learning with hierarchical clustering of local updates to improve training on non-iid data,” in 2020 International Joint Conference on Neural Networks (IJCNN). IEEE, 2020, pp. 1–9. (35) pp [21] Y. LeCun, “The MNIST database of handwritten digits,” http://yann. lecun. com/exdb/mnist/, 1998. The same result holds for N + 1 ≤n ≤2N. Combining the two results concludes the lemma. [22] A. Krizhevsky et al., “Learning multiple layers of features from tiny images,” 2009. Combining the results in Lemmas 4, 5, and 6 and applying Assumption 2 with (19) completes the proof of Lemma 1. [23] D. P. Kingma and J. Ba, “Adam: A method for stochastic optimization,” arXiv preprint arXiv:1412.6980, 2014. APPENDIX A Huang, “Broadband analog aggregation for low-latency federated edge learning,” IEEE Trans. Wireless Commun., vol. 19, no. 1, pp. 491–506, 2020. [8] G. Zhu, Y. Du, D. G¨und¨uz, and K. Huang, “One-bit over-the-air aggregation for communication-efficient federated edge learning: Design and convergence analysis,” IEEE Trans. Wireless Commun., vol. 20, no. 3, pp. 2120–2135, 2021. pp [9] M. Chen, N. Shlezinger, H. V. Poor, Y. C. Eldar, and S. Cui, “Communication-efficient federated learning,” Proceedings of the Na- tional Academy of Sciences, vol. 118, no. 17, 2021. [10] B. Tegin and T. M. Duman, “Blind federated learning at the wireless edge with low-resolution adc and dac,” IEEE Trans. on Wireless Com- mun., 2021. × ∆θi,n m′,c(t) 2 + ∆θi,n+N m′,c (t) 2 + ∆θi,n m,c(t)∆θi,n m′,c(t) −∆θi,n+N m,c (t)∆θi,n+N m′,c (t)  i (33) [11] ——, “Federated learning over time-varying channels,” in IEEE Global Communications Conference (GLOBECOM), Madrid, Spain, Dec. 2021. [12] T. Sery, N. Shlezinger, K. Cohen, and Y. Eldar, “Over-the-air federated learning from heterogeneous data,” IEEE Trans. Signal Process., vol. 69, pp. 3796–3811, 2021. Obtaining the expressions for N + 1 ≤n ≤2N in a similar manner and combining the two, results in (32). [13] C. T. Dinh, N. H. Tran, M. N. Nguyen, C. S. Hong, W. Bao, A. Y. Zomaya, and V. Gramoli, “Federated learning over wireless networks: Convergence analysis and resource allocation,” IEEE/ACM Transactions on Networking, vol. 29, no. 1, pp. 398–409, 2020. Lemma 6. REFERENCES [1] W. Y. B. Lim, N. C. Luong, D. T. Hoang, Y. Jiao, Y. C. Liang, Q. Yang, D. Niyato, and C. Miao, “Federated learning in mobile edge networks: A comprehensive survey,” IEEE Commun. Surveys Tuts., vol. 22, no. 3, pp. 2031–2063, 2020. [1] W. Y. B. Lim, N. C. Luong, D. T. Hoang, Y. Jiao, Y. C. Liang, Q. Yang, D. Niyato, and C. Miao, “Federated learning in mobile edge networks: A comprehensive survey,” IEEE Commun. Surveys Tuts., vol. 22, no. 3, pp. 2031–2063, 2020.
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Supplementary Figure 1 from Phosphoproteomics Identifies Driver Tyrosine Kinases in Sarcoma Cell Lines and Tumors
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B R R R+INSR R R+INSR C M t EGFR Ctr EGFR Met siRNA INSR Ctr IGF1R INSR IGF1R siRNA INSR Ctr IGF1R INSR IGF1R siRNA Met β-actin β-actin IGF1R β-actin IGF1R Ctr Src Ctr Fyn Fyn siRNA Ctr Lyn Lyn siRNA β-actin β-actin Lyn B R R R+INSR R R+INSR C M t EGFR Ctr EGFR Met siRNA INSR Ctr IGF1R INSR IGF1R siRNA INSR Ctr IGF1R INSR IGF1R siRNA Met β-actin β-actin IGF1R β-actin IGF1R Ctr Src Ctr Fyn Fyn siRNA Ctr Lyn Lyn siRNA β-actin β-actin Lyn R R+INSR R R+INSR C R Ctr IGF1R INSR IGF1R NA R Ctr IGF1R INSR IGF1R NA n R n R Ctr Lyn Lyn siRNA β-actin Lyn B R R R+INSR R R+INSR C M t EGFR Ctr EGFR Met siRNA INSR Ctr IGF1R INSR IGF1R siRNA INSR Ctr IGF1R INSR IGF1R siRNA Met β-actin β-actin IGF1R β-actin IGF1R Ctr Src Src RNA Ctr Fyn Fyn siRNA Ctr Lyn Lyn siRNA actin β-actin β-actin Lyn R EGFR Met INS siR INS siR β-ac IGF β-ac IGF Ctr Fyn Fyn siRNA β-actin
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Bayesian Semiparametric Density Deconvolution in the Presence of Conditionally Heteroscedastic Measurement Errors
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S.1 S.1 SUPPLEMENTARY MATERIALS Supplementary Materials to Bayesian Semiparametric Density Deconvolution in the Presence of Conditionally Heteroscedastic Measurement Errors Abhra Sarkar and Bani K. Mallick Department of Statistics, Texas A&M University, 3143 TAMU, College Station, TX 77843-3143 USA abhra@stat.tamu.edu and bmallick@stat.tamu.edu John Staudenmayer Department of Mathematics and Statistics, University of Massachusetts, Amherst, MA 01003-9305 USA jstauden@math.umass.edu Debdeep Pati Department of Statistics, Florida State University, Tallahassee, FL 32306-4330 USA debdeep@stat.fsu.edu Raymond J. Carroll Department of Statistics, Texas A&M University, 3143 TAMU, College Station, TX 77843-3143 USA carroll@stat.tamu.edu SUPPLEMENTARY MATERIALS S.2 Quadratic B−spline Bases t3 = A t4 t5 t6 t7 t8 t9 = B 0.0 0.5 1.0 Figure S.1: Plot of 9 quadratic (q = 2) B-splines on [A, B] defined using 11 knot points that divide [A, B] into K = 6 equal subintervals. Quadratic B−spline Bases Figure S.1: Plot of 9 quadratic (q = 2) B-splines on [A, B] defined using 11 knot points that divide [A, B] into K = 6 equal subintervals. −4 −2 0 2 4 0.0 0.1 0.2 0.3 0.4 0.5 Density Scaled error −4 −2 0 2 4 0.0 0.1 0.2 0.3 0.4 0.5 Density Scaled error Figure S.2: Skew-normal densities with mean=0, variance=1 and varying skewness parameter λ. The solid line is the density of SN(· | 0, 1, 0), the special case of standard normal distribution. The dashed line is the density of SN(· | 0, 1, 7). The dotted line is the density of SN(· | 0, 1, ∞) corresponding to the special case of a half-normal density. Figure S.2: Skew-normal densities with mean=0, variance=1 and varying skewness parameter λ. The solid line is the density of SN(· | 0, 1, 0), the special case of standard normal distribution. The dashed line is the density of SN(· | 0, 1, 7). The dotted line is the density of SN(· | 0, 1, ∞) corresponding to the special case of a half-normal density. Table S.1: Combined p-values for 4! = 24 nonparametric tests of association between Wj1 and Cj2j3j4 = {(Wj2 −Wj3)/(Wj2 −Wj4)} for various j1 ̸= j2 ̸= j3 ̸= j4 for 25 regularly consumed dietary components for which daily intakes were recorded in the EATS study. See Section 3 for additional details. S.1 Quadratic B-Splines Used to Model Variance Func- tions in Section 2 of the Main Paper Consider knot-points t1 = t2 = t3 = A < t4 < · · · < B = tK+3 = tK+4 = tK+5, where t3:(K+3) are equidistant with δ = (t4 −t3). For j = 3, 4, . . . , (K + 2), define b2,j(X) =        {(X −tj)/δ}2/2 if tj ≤X < tj+1, −{(X −tj+1)/δ}2 + (X −tj+1)/δ + 1/2 if tj+1 ≤X < tj+2, {1 −(X −tj+2)/δ}2 if tj+2 ≤X < tj+3, 0 otherwise. b2,j(X) =        {(X −tj)/δ}2/2 −{(X −tj+1)/δ}2 + (X −tj+1)/δ + 1/2 {1 −(X −tj+2)/δ}2 0 Also define b2,1(X) =  {1 −(X −t1)/δ}2/2 if t3 ≤X < t4, 0 otherwise. b2,2(X) =    −{(X −t3)/δ}2 + (X −t4)/δ + 1/2 if t3 ≤X < t4, {1 −(X −t4)/δ}2/2 if t4 ≤X < t5, 0 otherwise. b2,K+1(X) =    {(X −tK+1)/δ}2/2 if tK+1 ≤X < tK+2, −{(X −tK+2)/δ}2 + (X −tK+2)/δ + 1/2 if tK+2 ≤X < tK+3, 0 otherwise. b2,K+2(X) =  {(X −tK+2)/δ}2/2 if tK+2 ≤X < tK+3, 0 otherwise. if t3 ≤X < t4, otherwise. if t3 ≤X < t4, if t4 ≤X < t5, otherwise. Supplementary Materials to Bayesian Semiparametric Density Deconvolution in the Presence of Conditionally Heteroscedastic Measurement Errors SUPPLEMENTARY MATERIALS S.3 Dietary Component P-value combined from 4!=24 tests Truncation Limit ς = 0.05 Truncation Limit ς = 0.50 Kendall’s τ Test Spearman’s ρ Test Kendall’s τ Test Spearman’s ρ Test 1 Calcium 1 1 0.511 0.984 2 Carbohydrate 1 1 0.824 1 3 Carotene 1 1 0.816 0.993 4 Cholesterol 1 1 0.978 1 5 Copper 1 1 0.982 1 6 Monosaturated Fat 1 1 0.777 1 7 Polysatuared Fat 1 1 1 1 8 Saturated Fat 1 1 0.987 1 9 Fiber 1 1 0.627 0.995 10 Folate 1 1 1 1 11 Iron 1 1 0.996 1 12 Magnesium 1 1 1 1 13 Niacin 1 1 0.910 0.999 14 Phosphorus 0.986 1 0.769 0.986 15 Potassium 1 1 0.989 1 16 Protein 1 1 0.969 1 17 Riboflavin 1 1 1 1 18 Sodium 1 1 0.856 0.999 19 Thiamin 1 1 1 1 20 Vitamin A 1 1 0.999 1 21 Vitamin B6 1 1 0.985 1 22 Vitamin B12 1 1 0.999 1 23 Vitamin C 0.980 1 0.507 0.970 24 Vitamin E 1 1 1 1 25 Zinc 1 1 1 1 able S.1: Combined p-values for 4! = 24 nonparametric tests of association between Wj1 a j2j3j4 = {(Wj2 −Wj3)/(Wj2 −Wj4)} for various j1 ̸= j2 ̸= j3 ̸= j4 for 25 regularly consum ietary components for which daily intakes were recorded in the EATS study. See Section 3 f dditional details. SUPPLEMENTARY MATERIALS S.4 S.4 S.1 Quadratic B-Splines Used to Model Variance Func- tions in Section 2 of the Main Paper S.2 Additional Simulation Experiments In this section, we present the results of additional simulation experiments when the true density of interest is a normalized mixture of B-splines: f 3 X(X) ∝P7 k=1 b2,k(X)ck with c = (c1, . . . , c7)T = (0, 0, 2, 0.1, 1, 0, 0)T and equidistant knots on [−2, 6]. The normalizing constant was estimated by numerical integration on a grid of 500 equidistant points in [−2, 6]. The true values of X were generated from f 3 X using the inverse cumulative distribution function method. We recall that the SRB approach of Staudenmayer, et al. (2008) models fX by normalized mixture of B- splines and assumes normality of the scaled errors. The SRB approach and the three methods we proposed in the main paper are compared over a factorial combination of three sample sizes (n = 250, 500, 1000), nine different types of distributions for the scaled errors (Table 1 and Figure 1), and one variance function v(X) = (1 + X/4)2. For each subject, mi = 3 replicates were simulated. The estimated MISEs are presented in Table S.2. Results for error distribution (i) are summarized in Figure S.3. The results show that the deconvolution approaches proposed in Section 2 of the main paper outperform the SRB model in all 27 (3 × 9) cases, even in scenarios when the measurement errors were normally distributed and hence the truth actually conformed to the SRB model. This may SUPPLEMENTARY MATERIALS S.5 be attributed to the fact that Models I, II and III estimate fX by a flexible infinite mixture model, where the number of mixture components that are ‘active’ in the data is inferred semiautomatically from the data making it an adaptive data dependent approach. On the other hand, the SRB model estimates the density of interest by a mixture of normalized B-Splines with a fixed number of components. Model III, we recall, also relaxes parametric assumptions on the measurement errors, accommodating skewness, multimodality and heavy tails and resulting in huge reductions in MISE over other models when the measurement errors are heavy-tailed. Table S.2: Mean integrated squared error (MISE) performance of density deconvolution models described in Section 2 of this article (Models I, II and III) compared with the model of Stauden- mayer, et al. (2008) (Model SRB) for different scaled error distributions when the true density of interest is a mixture of splines. The true variance function was v(X) = (1 + X/4)2. See Section S.2 for additional details. The minimum value in each row is highlighted. True and estimated densities of interest True and estimated densities of interest −2 0 2 4 6 0.0 0.1 0.2 0.3 0.4 X Density True and estimated densities of interest −4 −2 0 2 4 0.0 0.4 0.8 Scaled error Density True, estimated and assumed densities of errors −2 −1 0 1 2 3 4 5 0 2 4 6 8 10 X Variance function True and estimated variance functions Figure S.3: Results for heavy-tailed error distribution (i) with sample size n=1000 corresponding to 25th percentile MISE. The true density fX is a normalized mixture of B-splines. See Section S.2 for additional details. The top panel shows the estimated densities under different models. The bottom left panel shows estimated densities of scaled errors under Model-II (dashed line) and Model-III (solid bold line) superimposed over a standard Normal density (solid line). The bottom right panel shows estimated variance functions under different models. For the top panel and the bottom right panel, the solid thin line is for Model-I; the dashed line is for Model-II; the solid bold line is for Model-III; and the dot-dashed line is for the Model of Staudenmayer, et al. (2008). In all three panels the bold gray lines represent the truth. −2 0 2 4 6 0.0 0.1 0.2 0.3 0.4 X Density −4 −2 0 2 4 0.0 0.4 0.8 Scaled error Density True, estimated and assumed densities of errors −2 −1 0 1 2 3 4 5 0 2 4 6 8 10 X Variance function True and estimated variance functions −2 −1 0 1 2 3 4 5 0 2 4 6 8 10 X Variance function True and estimated variance functions True, estimated and assumed densities of errors −4 −2 0 2 4 0.0 0.4 0.8 Scaled error Density True, estimated and assumed densities of errors True and estimated variance functions Density X Figure S.3: Results for heavy-tailed error distribution (i) with sample size n=1000 corresponding to 25th percentile MISE. The true density fX is a normalized mixture of B-splines. See Section S.2 for additional details. The top panel shows the estimated densities under different models. The bottom left panel shows estimated densities of scaled errors under Model-II (dashed line) and Model-III (solid bold line) superimposed over a standard Normal density (solid line). The bottom right panel shows estimated variance functions under different models. S.2 Additional Simulation Experiments True Error Distribution Sample Size MISE ×1000 SRB Model1 Model2 Model3 (a) 250 8.66 4.58 4.74 4.68 500 4.80 3.63 3.74 3.87 1000 4.03 2.57 2.75 2.68 (b) 250 9.13 5.77 4.38 4.48 500 5.12 3.76 3.53 3.56 1000 4.68 2.83 2.50 2.72 (c) 250 6.35 4.74 4.35 4.16 500 6.08 3.15 3.85 3.07 1000 3.93 2.54 2.96 1.93 (d) 250 6.31 5.17 5.95 3.61 500 3.70 3.91 6.36 2.70 1000 2.92 2.75 7.08 2.03 (e) 250 8.73 5.74 5.31 4.06 500 7.42 5.63 3.70 3.01 1000 7.99 3.37 2.35 1.90 (f) 250 8.86 5.32 5.39 5.19 500 4.64 3.87 3.83 3.12 1000 3.31 2.47 3.00 2.35 (g) 250 22.77 12.51 12.61 3.45 500 19.66 17.66 17.09 2.25 1000 40.55 22.66 16.36 1.50 (h) 250 11.15 6.61 6.38 3.96 500 8.34 9.38 7.18 3.22 1000 13.69 9.91 7.98 2.03 (i) 250 17.49 12.25 13.55 3.28 500 32.99 20.40 15.19 2.42 1000 40.67 19.47 12.18 1.17 Table S.2: Mean integrated squared error (MISE) performance of density deconv described in Section 2 of this article (Models I, II and III) compared with the mo mayer, et al. (2008) (Model SRB) for different scaled error distributions when the interest is a mixture of splines. The true variance function was v(X) = (1 + X/4 S.2 for additional details. The minimum value in each row is highlighted. Table S.2: Mean integrated squared error (MISE) performance of density deconvolution models described in Section 2 of this article (Models I, II and III) compared with the model of Stauden- mayer, et al. (2008) (Model SRB) for different scaled error distributions when the true density of interest is a mixture of splines. The true variance function was v(X) = (1 + X/4)2. See Section S.2 for additional details. The minimum value in each row is highlighted. Table S.2: Mean integrated squared error (MISE) performance of density deconvolution models described in Section 2 of this article (Models I, II and III) compared with the model of Stauden- mayer, et al. (2008) (Model SRB) for different scaled error distributions when the true density of interest is a mixture of splines. The true variance function was v(X) = (1 + X/4)2. See Section S.2 for additional details. The minimum value in each row is highlighted. SUPPLEMENTARY MATERIALS S.6 True and estimated densities of interest For the top panel and the bottom right panel, the solid thin line is for Model-I; the dashed line is for Model-II; the solid bold line is for Model-III; and the dot-dashed line is for the Model of Staudenmayer, et al. (2008). In all three panels the bold gray lines represent the truth.
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Design Patterns in Beeping Algorithms (extended abstract)
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Design Patterns in Beeping Algorithms (extended abstract) Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari To cite this version: Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari. Design Patterns in Beeping Algorithms (extended abstract). 20th International Conference on Principles of Distributed Systems (OPODIS), Dec 2016, Madrid, Spain. ￿hal-01472903￿ To cite this version: Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari. Design Patterns in Beeping Algorithms (extended abstract). 20th International Conference on Principles of Distributed Systems (OPODIS), Dec 2016, Madrid, Spain. ￿hal-01472903￿ To cite this version: Arnaud Casteigts, Yves Métivier, John Michael Robson, Akka Zemmari. Design Patterns in Beeping Algorithms (extended abstract). 20th International Conference on Principles of Distributed Systems (OPODIS), Dec 2016, Madrid, Spain. ￿hal-01472903￿ Abstract We consider networks of processes which interact with beeps. In the basic model defined by Cornejo and Kuhn [5], which we refer to as the BL variant, processes can choose in each round either to beep or to listen. Those who beep are unable to detect simultaneous beeps. Those who listen can only distinguish between silence and the presence of at least one beep. Stronger variants exist where the nodes can also detect collision while they are beeping (BcdL) or listening (BLcd), or both (BcdLcd). Beeping models are weak in essence and even simple tasks are difficult or unfeasible with them. This paper starts with a discussion on generic building blocks (design patterns) which seem to occur frequently in the design of beeping algorithms. They include multi-slot phases: the fact of dividing the main loop into a number of specialised slots; exclusive beeps: having a single node beep at a time in a neighbourhood (within one or two hops); adaptive probability: increasing or decreasing the probability of beeping to produce more exclusive beeps; internal (resp. peripheral) collision detection: for detecting collision while beeping (resp. listening); and emulation of collision detection: for enabling this feature when it is not available as a primitive. We then provide algorithms for a number of basic problems, including colouring, 2-hop colour- ing, degree computation, 2-hop MIS, and collision detection (in BL). Using the patterns, we formulate these algorithms in a rather concise and elegant way. Their analyses (in the full ver- sion) are more technical, e.g. one of them relies on a Martingale technique with non-independent variables; another improves that of the MIS algorithm in [8] by getting rid of a gigantic constant (the asymptotic order was already optimal). Finally, we study the relative power of several variants of beeping models. In particular, we explain how every Las Vegas algorithm with collision detection can be converted, through emulation, into a Monte Carlo algorithm without, at the cost of a logarithmic slowdown. We prove that this slowdown is optimal up to a constant factor by giving a matching lower bound. Digital Object Identifier 10.4230/LIPIcs... Keywords. Beeping models, Design patterns, Collision detection, Colouring, 2-hop colouring, Degree computation, Emulation. Design Patterns in Beeping Algorithms (extended abstract)∗ A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari LaBRI, University of Bordeaux {acasteig, metivier, robson, zemmari}@labri.fr LaBRI, University of Bordeaux {acasteig, metivier, robson, zemmari}@labri.fr HAL Id: hal-01472903 https://hal.science/hal-01472903v1 Submitted on 21 Feb 2017 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. ∗This research has been supported by ANR projects DESCARTES (ANR-16-CE40-0023) and ESTATE (ANR-16-CE25-0009-03). A full version is available on arXiv (http://arxiv.org/abs/1607.02951) licensed under Creative Commons License CC-BY Leibniz International Proceedings in Informatics Schloss Dagstuhl – Leibniz-Zentrum für Informatik, Dagstuhl Publishing, Germany 1.1 Contributions The contributions of this paper are manifold. As a warm-up, we start by identifying generic building blocks (design patterns) which seem to occur often in the design of beeping algo- rithms. Then we present a number of algorithms for various graph problems which improve upon previous solutions. Finally, we generalise existing emulation techniques for using col- lision detection if it is not available, and we prove them optimal w.h.p. up to a constant factor. Due to space limitations, this version of the paper omits (in its core) most complexity analyses and some proofs. However, both are available respectively in Appendix A and B of the present paper, as well as in the arXiv version whose reference is given in the first page. 1.1.1 Design patterns. We identify a number of common building blocks in beeping algorithms, including multi-slot phases: the fact of dividing the main loop into a (typically constant) number of slots having specific roles (e.g., contention among neighbours, collision detection, termination detection); exclusive beeps: the fact of having a single node beep at a time in a neighbourhood (within one or two hops, depending on the needs); adaptive probability: increasing or decreasing the probability of beeping in order to maximise the number of exclusive beeps; internal (resp. peripheral) collision detection: the fact of detecting collision while beeping (resp. listening); and emulation of collision detection: the fact of detecting collisions even when it is not available as a primitive. As we show in the paper, these patterns make it possible to formulate the algorithms in a rather concise and elegant way. (extended abstract) (extended abstract) In beeping models, the only communication capabilities offered to the nodes are to beep or to listen. Several variants exist. In [5], a node that beeps is unable to detect whether other nodes have beeped simultaneously. When listening, it can distinguish between silence or the presence of at least one beep, but it cannot distinguish between one and several beeps. In Section 6 of [1], beeping nodes can detect whether other nodes are beeping simultaneously. In [10] and Section 4 of [1], yet another variant is considered where listening nodes can tell the difference between silence, one beep, and several beeps. In this paper, we denote the ability to detect collision while beeping (internal collision) by Bcd and that of detecting collision while listening (peripheral collision) by Lcd. The absence of such ability is denoted by B and L, respectively. The existing models can be reformulated using the cartesian product of these capabilities. Hence, the basic model introduced by Cornejo and Kuhn in [5] is BL; the model considered by Afek et al. in [1] (Section 6) and Jeavons et al. in [8] is BcdL; and the model considered in [10] and in Section 4 of [1] is BLcd. To the best of our knowledge, BcdLcd was only used in a previous work of the authors [3]. Although some variants are stronger than others, all beeping models remain extremely weak in essence. Yet, they are relevant to account for real-world applications or phenomena. For instance, they reflect the features of a network at the lowest levels (physical and MAC layers), where a node can probe or emit signals, with or without collision detection. At a higher level of abstraction, beeping models also reflect some communication patterns in biology [4, 1, 9]. 1 Introduction Distributed computing is concerned with various assumptions, like the structure of the network (trees, rings, planar graphs, etc.) or knowledge available to the nodes (network size, identifiers, port numbering, etc.). Another important aspect is the size of messages, which may range from unbounded, to logarithmic size, to constant size. As a natural goal is to reduce assumptions as much as possible. Typically, when a problem is solved in some strong model, the community strives to solve it in weaker models. In a recent series of works [5, 10, 1, 7, 8, 6], new models were explored that are even weaker than constant size messages. They are called beeping models. XX:2 Design Patterns in Beeping Algorithms (extended abstract) 1.1.2 Algorithms and analyses for basic graph problems. We present, or analyse algorithms for a number of basic graph problems, including colouring, 2-hop colouring, degree computation, Maximal Independent Set (MIS) and 2-hop MIS. Quite A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari XX:3 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Model Time (# slots) Message size Knowledge # colours BcdL O(log n + ∆) expected and w.h.p. ≃1 bit (BcdL beeps) None O(log n + ∆) BcdL O K(log n + log2 K) w.h.p. ≃1 bit (BcdL beeps) Upper bound K on the max degree of G K Table 1 Randomised Las Vegas colouring algorithms on graphs with n vertices. Table 1 Randomised Las Vegas colouring algorithms on graphs with n vertices. often, the design of algorithms is easier and more natural if collision detection is assumed as a primitive, e.g., in BcdLcd or BcdL. Furthermore, emulation techniques such as those described later in this paper enable safe and automatic translations of algorithms into weaker models like BL. For this reason, our algorithms are expressed using whichever model is the most convenient. First, we present a Las Vegas (i.e. guaranteed result, uncertain time) colouring algorithm in the BcdL model, with time complexity of O(log n + ∆) slots w.h.p., where ∆is the maximum degree in G. Its analysis relies on a martingale technique with non-independent random variables, which makes use of a result by Azuma [2] (details in appendix). In fact, the phenomenon is quite ubiquitous in beeping models: the algorithm terminates in the first moment when every node has produced an exclusive beep at least once within its (1- hop) neighbourhood. This stopping time is made more complex by the use of the adaptive probability pattern mentioned above. Another algorithm for 2-hop colouring is given, this time in the BcdLcd model, with slot complexity O(log n+∆2) w.h.p. Both algorithms require no knowledge on G. However, both can result in arbitrarily many colours (in fact, one per slot). If the nodes know an upper bound K ≥∆, a different strategy is proposed that uses at most K + 1 colours. However, the slot complexity becomes O(K(log n + log2 K)) w.h.p. for colouring (trade K for K2 in the 2-hop variant). Note that this complexity is not thought to be tight. The results are summarised on Table 1. 1.1.3 Collision detection and emulation techniques. Classical considerations on symmetry breaking in anonymous beeping networks, see for example [1] (Lemma 4.1), imply that there is no Las Vegas internal collision detection algorithm in the beeping models BL and BLcd. Likewise, there is no Las Vegas peripheral collision detection algorithm in the beeping models BL and BcdL. Since collision detection is required to detect exclusive beeps with certainty, and this pattern is central in most beeping algorithms, this implies that a large range of algorithms cannot exist in a Las Vegas version in these models. We study the cost of detecting collision when it is not available, typically in BL, and present generic techniques to emulate collision detection probabilistically in order to trans- form Las Vegas algorithms with collision detection into Monte Carlo algorithms (uncertain result, guaranteed time) in BL. These techniques generalise that of Algorithm 3 in [1], where a similar strategy is encapsulated into the algorithm. We show how, given 0 < ϵ < 1, any collision in the neighbourhood of a given node can be detected in O(log( 1 ϵ )) slots with error at most ϵ, and similarly it can be detected in O(log n) slots w.h.p. Ensuring that this is true for any node requires more time. By union bound, it holds that O(log( n ϵ )) slots are sufficient with error ϵ and that O(log n) slots are sufficient w.h.p. We prove that this technique is essentially optimal (asymptotically and up to a constant factor) by giving a matching lower bound. Precisely, we prove that some topologies require Ω(log n) slots to break symmetries w.h.p. Finally, we provide two generic procedures that can be used in an algorithm to emulate collision detection when it is not available (e.g. in BL). These proce- dures are EmulateBcdinBL(), to detect collision while beeping, and EmulateLcdinBL(), to detect collision while listening. We illustrate their use in the case of the computation of a MIS given in BcdL, thus obtaining a Monte Carlo algorithm in BL. Design Patterns in Beeping Algorithms Design Patterns in Beeping Algorithms 1.2 Organisation of the paper In Section 2 we present the model and give further definitions. Section 3 introduces design patterns in a tutorial manner. These patterns are then used in Section 4 to describe the various algorithms. For the sake of readability, the corresponding analyses are put together in Section A. Finally, Section 5 presents our contribution on collision detection and emulation techniques. An extra bibliography is provided in Section ?? on related questions. 1.1.2 Algorithms and analyses for basic graph problems. Based on the observation that degree computation is strongly related to 2-hop colouring, we present an adaptation of the algorithm for this problem, with same slot complexity, that is, O(log n + ∆2) w.h.p. In fact, the random process induced by this algorithm is the same as that of colouring, except that it occurs in the square of the graph (whence the ∆2 term). Algorithmically, the main loop contains more specialised slots (e.g., one for peripheral collision reporting), but still a constant number of them, which keeps the asymptotics unchanged. We then turn our attention to the 2-hop MIS problem, which shares common traits and patterns with 2-hop colouring and degree computation and, regarding the high-level purpose of each phase, with the MIS algorithm from [8]. The running time is however shorter than that of 2-hop coulouring and degree computation (and the analysis quite different) due to the fact that exclusive beeps cause whole neighbourhoods to terminate at once. In fact, we prove that the slot complexity of this algorithm is O(log n) w.h.p. with a “reasonable” constant factor of 76. Noteworthily, the number of phases (i.e. iterations of the main loop) for the 2-hop MIS is exactly the same as what the analogue for classical MIS would produce in the square of the graph. As a consequence, our analysis also improves substantially that of the MIS algorithm presented in [8], where a gigantic constant factor (i.e. one larger than e25) is used. An earlier analysis in [11] yielded a better, yet huge constant of 2 × 1011. Although constant factors are less meaningful in general, the gap in this case is one between practical and unpractical running times. Furthermore, the contribution it not as much in the constant itself than in the analysis techniques that achieve it. XX:4 Distributed Randomised Algorithm. A randomised (or probabilistic) algorithm is an algorithm which makes choices based on given probability distributions. A distributed randomised algorithm is a collection of local randomised algorithms (in our case, all identical). A Las Vegas algorithm is a randomised algorithm whose running time is not determin- istic, but still finite with probability 1, and that always produces a correct result. A Monte Carlo algorithm is a randomised algorithm whose running time is deterministic, but whose result may be incorrect with a certain probability. Put differently, Las Vegas algorithms have uncertain execution time but certain result, and Monte Carlo algorithms have cer- tain execution time but uncertain result. Classical considerations on symmetry breaking in anonymous beeping networks (see for instance Lemma 4.1 in [1]), imply that: ▶Remark. There is no Las Vegas (and a fortiori no deterministic) algorithm in BL which allows a node to distinguish between an execution where it is isolated and one where it has exactly one neighbour. From this remark we deduce that there is no Las Vegas counting algorithm in BL, which advocates the use of stronger models. In what follows, we consider whichever model is the most convenient and provide Las Vegas algorithms in these models. We then present canonical emulation techniques to turn any such algorithm into a Monte Carlo one in BL. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari addition, processors can perform an unrestricted amount of local computation in-between two slots (in effect, our algorithms require little computation). ▶Remark. In general, nodes are active or passive. When they are active they beep or listen; in the description of algorithms we say explicitly when a node beeps meaning that a non beeping active node listens. The time complexity, also called slot complexity, is the maximum number of slots needed until every node has terminated. Our algorithms are typically structured into phases, each of which corresponds to a small (constant or logarithmic) number of slots. In the algorithm, we specify which one is the current slot by means of a switch instruction with as many case statements as there are slots in the phase. Phases repeat until some condition holds for termination. ▶Remark. An algorithm given in a beeping model induces an algorithm in the (synchronous) message passing model. Thus, given a problem, any lower bound on the round complexity in the message passing model also holds for slot complexity in the beeping model. 3 Design patterns for beeping algorithms As a warm-up, this section presents a number of design patterns which seem to occur frequently in the design of beeping algorithms. The concept of pattern refers here to reusable solutions to common problems. These patterns are then used to describe algorithms in the other sections. 2 Network Model and Definitions We consider a wireless network and we follow definitions given in [1] and [5]. The network is anonymous: unique identifiers are not available to distinguish the processes. Possible communications are encoded by a graph G = (V, E) where the nodes V represent processes and the edges E represent pairs of processes that can hear each other. We denote by ∆the maximum degree of G. The neighbourhood of a vertex v, denoted N(v), is the set of vertices adjacent to v (at distance 1 from v). We define N(v) by including v itself in N(v). We also use the set of vertices at distance at most 2 from v called the 2-neighbourhood of v and denoted N2(v) (or N2(v) if it includes v). Finally, we write log n for the binary logarithm of n. Time is divided into discrete synchronised time intervals (rounds) also called slots (follow- ing the usual terminology in wireless networks). All processes wake up and start computation in the same slot. In each slot, all processors act in parallel and either beep or listen. In XX:5 (extended abstract) goal is all the more difficult to achieve that the nodes cannot use identifiers nor even port numbers in their basic exchanges. If we assume that a node that is beeping can detect whether another node beeps simultaneously (Bcd), then this feature can be used to take exclusive decision if indeed it beeps alone. We call this an exclusive beep. Algorithm 1 illustrates an empty shell of algorithm that relies on repeated attempts to produce exclusive beeps. Most, if not all algorithms rely implicitly on this pattern as a basis. Algorithm 1: Exclusive beeps (using Bcd). repeat beep with some probability; if I beeped alone then do something exclusive; ... until finished; Algorithm 1: Exclusive beeps (using Bcd). Algorithm 1: Exclusive beeps (using Bcd). Algorithm 1: Exclusive beeps (using Bcd). repeat beep with some probability; if I beeped alone then do something exclusive; ... 2-hop exclusive beeps. For some problems like 2-hop colouring, 2-hop MIS, or computation of the degree (all dis- cussed in this paper), the level of mutual exclusion offered by exclusive beeps is not sufficient and the algorithm requires that a node be the only one to beep at distance 2. Assuming collision can also be detected upon listening (Lcd), one can design a 2-slots pattern whereby non-beeping neighbours report if they have heard more than one beep. Hence, if a node produced an exclusive beep in the first slot, and none of its neighbours reported a collision in the second, then it knows that it has produced a 2-hop exclusive beep (see Algorithm 2). Algorithm 2: Two-hops exclusive beeps (using BcdLcd). repeat switch slot do slot 1 // contending beep with some probability; slot 2 // detection of peripheral collision if several neighbours beeped in slot 1 then beep after slot 2 if I beeped alone in slot 1 and no neighbour beeped in slot 2 then do something 2-hop exclusive ... until finished; Algorithm 2: Two-hops exclusive beeps (using BcdLcd). repeat switch slot do slot 1 // contending beep with some probability; slot 2 // detection of peripheral collision if several neighbours beeped in slot 1 then beep after slot 2 if I beeped alone in slot 1 and no neighbour beeped in slot 2 then do something 2-hop exclusive ... until finished; Algorithm 2: Two-hops exclusive beeps (using BcdLcd). until finished; Exclusive beeps. Beeping algorithms operate in synchronous periods called slots, which are equivalent to the concept of rounds in message passing models. Most problems in distributed computing require some node v to take exclusive decisions at times (i.e., with respect to vertices of N(v) or N2(v)), which requires some type of symmetry breaking. In beeping networks, this XX:6 Adaptive probability. As far as feasibility and expressivity are concerned, the next design pattern is not crucial. However, it plays a central role in terms of performance. Adaptive probability consists in adapting the probability to beep in the next phase depending on the outcome of previous phases. Typically, if a collision occurs, the probability is reduced, and if no one beeps, it is increased. Since the nodes do not know how many neighbours are contending with them, this technique proves useful in optimizing the odds of producing exclusive beeps. The values Algorithm 3: Adaptive beeping probability (using BcdLcd). Float p ←1/2 // say repeat beep with probability p; if I beeped alone then do something exclusive; else if no one beeped then increase p; else decrease p; until finished; Algorithm 3: Adaptive beeping probability (using BcdLcd). given to the probabilities in Algorithm 3 are left unspecified. There are several options. In this paper, we use a doubling/halving pattern, that is, p is increased to 2p (up to 1/2), and it is decreased to p/2 (without limit). A similar doubling/halving pattern was used in [11]. One could also increment or decrement the denominator of p as done in [3]. The consequences of choosing one over the other are not discussed here. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari which have not yet performed some action beep. If the slot remain silent, then a form of local termination is detected: nodes are in a terminal state. which have not yet performed some action beep. If the slot remain silent, then a form of local termination is detected: nodes are in a terminal state. Multi-slot phases. The example in Algorithm 2 illustrates another common aspect of beeping algorithms, namely multi-slot phases. The expressivity of a single beep is rather poor, but several combined slots can achieve elaborate behavior. In Algorithm 2, one slot is devoted to con- tending and another to peripheral collision detection. The whole compound is then called a phase. Another common task is termination detection. In a termination slot, all nodes XX:7 4.1 Colouring The colouring problem consists of assigning a colour to every node in the network, such that no two neighbours have the same colour. We first consider the case that no extra information is available to the nodes. Then we consider that (an upper bound on) the maximum degree is known. 4 Algorithms for basic graph problems We now present algorithms for a number of problems, including colouring (with or without knowledge on the degree), 2-hop colouring, computation of the degree and 2-hop MIS. These algorithms are based on various combinations of the patterns presented in Section 3. All algorithms are Las Vegas, and they rely on medium to strong primitives (BcdL to BcdLcd models) depending on the needs. The adaptation of these algorithms in the weakest model (BL) is discussed in Section 5. We also recall Jeavons et al.’s Las Vegas algorithm for the MIS [8] problem and discuss its relations with our 2-hop MIS algorithm. Whenever using the adaptive probability pattern in algorithms, for generality, we stick to the terms increase and decrease (as opposed to our analyses, in which these actions are instantiated to doubling and halving the probability). Collision detection. Most algorithms in this paper use collision detection as a built-in primitive, referred to as Bcd for detection on beeping and Lcd for detection on listening. However, this feature is not always available as a primitive. An important question is the transformation of a (high- level) algorithm using Bcd or Lcd (or both) into one that works in the weakest BL model. This question is the topic of Section 5, in which we study generic mechanisms to achieve this goal. Essentially, each slot that requires collision detection can be replaced with a logarithmic number of slots (in the size of various quantities depending on the desired guarantees) where the ties are broken w.h.p. We provide dedicated procedures that generalise the technique used internally to one of the algorithms in [1]. Besides complexity, the price to pay is that the algorithm becomes Monte Carlo instead of Las Vegas, that is, the result is correct only probabilistically (though possibly w.h.p.). We present a matching lower bound showing that these procedures are essentially optimal. XX:8 Design Patterns in Beeping Algorithms A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari with time, thus it is at most the same (at most, because some phases may not produce exclusive beeps). Colouring without knowledge. Informally, the algorithm proceeds as follows (see Algorithm 4 for details). Initially, every node is uncoloured (nil). In every phase, each node increments a counter. Uncoloured nodes contend with each other to produce an exclusive beep, and when one succeeds, it takes the current value of the counter as its colour and retires. An adaptive probability is used to regulate the probability of beeping among uncoloured nodes. Local termination (a node and its neighbours are coloured) detection is not explicitly handled here, though we could add a termination slot where uncoloured nodes are the only ones to beep. Algorithm 4: A Las Vegas colouring algorithm in BcdL (without knowledge). Float p ←1/2; Integer colour ←nil; Integer counter ←0; repeat beep with probability p; if I beeped alone then colour ←counter else if no one beeped then increase p; else decrease p; counter ←counter + 1; until colour ̸= nil; The running time of this algorithm is of O(log n + ∆) phases w.h.p as well as on average (none of both imply the other trivially). Note that this is also the number of slots, since each phase consists of a constant number of slots. As for the number of colours, it is incremented XX:9 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari 4.2 2-hop colouring A 2-hop colouring of a graph G is a colouring such that any two nodes at distance ≤2 have different colours. In other words, it is a colouring of the square of G, the graph where an edge exists between nodes which are neighbours in G or share a common neighbour in G. Colouring with a bound K on the maximum degree ∆. If a bound K ≥∆is known, then one can obtain a better colouring using at most K + 1 colours. The algorithm follows the same lines as Algorithm 4, i.e. a colour counter is incremented in each phase, and its current value is chosen by those nodes who produced an exclusive beep. The main difference (see Algorithm 5 for details) is that only those colours within {0, . . . , K} are considered and thus the counter is incremented modulo K +1. Conflicts of colours are avoided by keeping a phase idle if the corresponding value was already taken in the past (locally). To do so, when a node takes a colour, it re-beeps in a new slot called confirmation slot to inform its neighbours that they must remove the current colour from their list of authorized colours. Accordingly, the uncoloured will contend in a phase only if the current colour is still available (otherwise, they wait). An adaptive probability is used similarly to Algorithm 4, except that idle phases are not considered as silent (the probability is not updated in these phases). Algorithm 5: A Las Vegas colouring algorithm in BcdL (knowing K ≥∆). Colours = {0, · · · , K}; Float p ←1/2; Integer colour ←nil; Integer counter ←0; repeat if counter ∈Colours then switch slot do slot 1 // contending beep with probability p slot 2 // confirmation if I beeped alone in slot 1 then colour ←counter; beep; else if no one beeped then increase p; else decrease p; if someone beeped in slot 2 then Colours ←Colours \ {counter} counter ←(counter + 1) mod (K + 1); until colour ̸= nil; Algorithm 5: A Las Vegas colouring algorithm in BcdL (knowing K ≥∆). Regarding performance, the only difference between this algorithm and Algorithm 4 is that a growing number of phases are idle in each neighbourhood, inflicting a slow down to the algorithm. Managing the dependencies here proved more difficult and we “only” managed to prove that the number of phases is O K(log n + log2 K)  w.h.p. However, the algorithm is believed to be faster. (extended abstract) 4.3 Degree computation Let us recall that 2-hop exclusive beeps allow a node v to perform an exclusive action within a radius of distance 2. This feature was used in Section 4.2 to assign unique colours. At it turns out, the pattern is very versatile and it can be used to count the degree of a node as well. The strategy consists in replacing the colour-related action in slot 2 (second if-then block) by an action aiming at having v counted in the degree of its neighbours (then v stops contending and keeps on reporting collisions, as before). Precisely, a new confirmation slot is inserted wherein v re-beeps if indeed it produced a 2-hop exclusive beep. Upon hearing the confirmation beep, all of v’s neighbours increment a local counter that eventually amounts to their degree. Termination proceeds in the same way as for the 2-hop-colouring algorithm (i.e. uncounted nodes beep in a termination slot). Up to a constant factor which accounts for the additional confirmation slot in each phase, the running time of this algorithm is again O(log n + ∆2) w.h.p. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Once we realize that the execution produced here is the same as what Algorithm 4 would produce in the square of G, analysis of this algorithm is straightforward. The only difference is that the maximal number of contenders of a node becomes ∆2 instead of ∆. Thus Algorithm 6 takes O(log n + ∆2) phases (and slots) w.h.p., and the number of colours cannot exceed the same value. 4.4 Jeavons et al.’s Las Vegas Algorithm for the MIS in BcdL We recall here Jeavons et al.’s Las Vegas Algorithm for the MIS [8]. This algorithm uses an adaptive probability to maximize the frequency of exclusive beeps (with a doubling/halving pattern for p, starting at 1/2). If a node v produces an exclusive beep, it enters the MIS (by the end of the first slot), then it uses a confirmation slot to inform its neighbours, all of which terminate together with v. Since the whole neighbourhood shuts down at once, the algorithm progresses faster than, for instance, the basic colouring algorithm discussed above. This algorithm was already proven by Jeavons et al. to terminate within O(log n) slots with a huge constant factor (larger than e25). With a bound K on the maximum degree ∆. The same idea can be applied as in the 1-hop variant, i.e., taking colours between 0 and K2 + 1 (instead of K + 1) and incrementing the counter accordingly (mod K2 + 1). As a result, at most K2 + 1 colours are used, with time complexity O(K2(log n + log2 K)) w.h.p. 2-hop colouring without knowledge. A similar strategy is used as in Algorithm 4 (colouring), except that exclusive beeps are replaced with 2-hop exclusive beeps. Whenever a node produces such a beep, it takes the current value of the counter as colour. Since no other node has beeped within distance 2, the colouring is legal. Contrary to the 1-hop colouring, the collaboration of a node remains crucial even after it becomes coloured. Indeed, this node must keep on reporting peripheral collisions to its neighbours. As a result, instead of retiring from computation, coloured nodes keep on listening until all of their neighbours are coloured, which is detected using an extra termination slot. Details are given in Algorithm 6. Four slots are used in total, the first two being devoted to the management of 2-hop exclusive beeps (see Section 3 for details). The third slot manages a (2-hop) adaptive probability based on beeps heard at distance one (slot 1) or at distance two (slot 3 itself). Finally, slot 4 is the termination slot. Algorithm 6: A Las Vegas 2-hop-colouring algorithm in BcdLcd (without knowledge). Float p ←1/2; Integer colour ←nil; Integer counter ←0; repeat switch slot do slot 1 // contending slot if colour = nil then beep with probability p; slot 2 // peripheral collision detection (and consequences) if several neighbours beeped in slot 1 then beep if I beeped alone in slot 1 and heard no beep in slot 2 then colour ←counter slot 3 // adaptive probability if someone beeped in slot 1 then beep if colour = nil then if no beep heard in slot 1 nor 3 then increase p else decrease p slot 4 // termination slot if colour = nil then beep counter ←counter + 1 until no beep heard in slot 4; Algorithm 6: A Las Vegas 2-hop-colouring algorithm in BcdLcd (without knowledge). XX:11 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari ▶Lemma 1. Let v be a node. If a collision occurs in the neighbourhood of v, then v detects it in O log( 1 ϵ )  sub-phases (slots) with probability at least 1 −ϵ, and in O (log n) sub-phases (slots) with probability 1 −o 1 n2  . 5 Collision detection and emulation techniques In Section 4, we have considered collision detection as a built-in primitive. Depending on the algorithms, we assumed that collision detection was possible while beeping (Bcd) or while listening (Lcd). This assumption is convenient because it allows one to design Las Vegas algorithms for all the considered problems. Unfortunately, we know since [1] that no Las Vegas algorithms can be designed for most problems without collision detection, that is, in the BL model. One has to turn to Monte Carlo instead, which means that the result is correct only with some probability (possibly w.h.p.). In this section, we investigate the cost of building a probabilistic collision detection primitive in the BL model, inspired by a technique from [1]. Then we adapt it into two generic emulation procedures, one for detecting collision while beeping, the other while listening. These procedures can then be used to translate any Las Vegas algorithml in BcdL, BLcd, or BcdLcd, into a Monte Carlo algorithm in BL. The cost is a logarithmic slowdown of the execution, which we prove is essentially optimal (for sufficiently large n). (extended abstract) within a (small) constant factor. Interestingly, our analysis of this algorithm improves much over that of [8], taking the huge constant down to 76 (i.e., making the algorithm practical). within a (small) constant factor. Interestingly, our analysis of this algorithm improves much over that of [8], taking the huge constant down to 76 (i.e., making the algorithm practical). 5.1 Collision detection The impossibility for a node in BL to distinguish between begin alone or having neighbours has strong implications. For instance, in the colouring problem, it means that two neighbours could possibly end up with the same colour. In the MIS problem, two neighbours could enter the MIS. In fact, there is no guarantee on the correctness of basic patterns like exclusive beeps or 2-hop exclusive beeps, which are at the basis of most (if not all) Las Vegas algorithms. We present a (Monte Carlo) algorithm for detecting collisions in BL. This procedure generalises the technique used in Algorithm 3 of [1], which consists of replacing each slot that requires collision detection in the original model, with several BL slots in which symmetries are probabilistically broken. Of course, the more slots, the more reliable the detection. 4.5 Computing a 2-hop MIS In this problem, we must select a set of nodes (the MIS) such that no pair of selected nodes are within distance 2 and no node can be added further to the set. This algorithm is a combination of those of other 2-hop algorithms seen above, and Jeavons et al’s MIS algorithm. That is, the same structure of algorithm is used as for 2-hop colouring or degree computation, except that whenever a node produces a 2-hop exclusive beep, it enters the MIS and informs its neighbours (using the confirmation slot) that they will not be in the MIS. This algorithm takes the same number of phases than what the (1-hop) MIS algorithm would produce in the square of the graph, that is, O(log n) w.h.p.. The number of slots is higher due to using additional slots for managing 2-hop exclusive beeps, but it remains The algorithm. The same argument, combined with the second claim of Lemma 1 proves the second claim. ◀ The algorithm. Assume a collision occurs between some nodes u1 and u2 in the neighbourhood of v (one of them being possibly v itself). It is detected if u1 and u2 choose a different slot in at least one of the k sub-phases. The probability that this does not happen is 1 2 k. This probability is less than ϵ (resp. o 1 n2  ) for any k ≥log( 1 ϵ ) (resp. 2 log(n)). Observe that if collisions occur between more than two nodes in the neighbourhood of v, this cannot decrease the odds of a successful detection (to the contrary, the odds can only increase). ◀ Proof. Assume a collision occurs between some nodes u1 and u2 in the neighbourhood of v (one of them being possibly v itself). It is detected if u1 and u2 choose a different slot in at least one of the k sub-phases. The probability that this does not happen is 1 2 k. This probability is less than ϵ (resp. o 1 n2  ) for any k ≥log( 1 ϵ ) (resp. 2 log(n)). Observe that if collisions occur between more than two nodes in the neighbourhood of v, this cannot decrease the odds of a successful detection (to the contrary, the odds can only increase). ◀ ▶Corollary 2. Let G be a graph. If collisions occur in the neighbourhood of an arbitrary number of nodes, then all of them detect collision after at most O log( n ϵ )  sub-phases (slots) with probability at least 1 −ϵ, and after at most O (log n) sub-phases (slots) w.h.p. Proof. Assume collisions occur in G and let T denote the number of sub-phases before all concerned nodes detect collision. Clearly T = max{Tv | v ∈V }, where Tv is the time it takes to any node v to decide collision. By the same argument as in the proof of Lemma 1, together with union bound, it holds that Pr  T > log n ϵ  ≤ n × Pr  Tv > log n ϵ  (1) = n × 1 2log( n ϵ ) = ϵ (2) (1) (2) which proves the first claim. The same argument, combined with the second claim of Lemma 1 proves the second claim. ◀ which proves the first claim. The algorithm. Each slot that requires collision detection (Bcd or Lcd) is replaced with a number of sub- phases, each consisting of two BL slots. For instance, if a node wishes to beep with collision detection in the original algorithm, it will choose one of the two slots (u.a.r.) in each of the sub-phases and will beep in that slot (listen in the other). If it hears a beep while listening in the other slot, then an internal collision is detected. Similarly, if a node wishes to listen with collision detection in the original algorithm, it will listen in both slots of each sub-phase. A peripheral collision is detected if a beep is heard in both slots of a same sub-phase. The procedure is detailed by Algorithm 7, where k is the number of sub-phases used. False positives never happen, but real collisions might still go unnoticed, with probability inversely related to k. We are interested in determining how large k should be to guarantee that a given node detects a collision in its neighbourhood with a given probability. The stronger question asks how many sub-phases are required to guarantee that none of the nodes fails to detect a collision. XX:13 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Algorithm 7: Collision detection algorithm in BL (with parameter k) Boolean collision ←false; Integer i ←0; while i < k do if v wishes to beep then Flip a coin; if heads then beep in slot 1; listen in slot 2; else listen in slot 1; beep in slot 2; if another beep was heard then collision ←true else listen in both slots; if beeps are heard in both slots then collision ←true; i ←i + 1; return collision; Algorithm 7: Collision detection algorithm in BL Boolean collision ←false; Integer i ←0; while i < k do if v wishes to beep then Flip a coin; if heads then beep in slot 1; listen in slot 2; else listen in slot 1; beep in slot 2; if another beep was heard then collision ←true else listen in both slots; if beeps are heard in both slots then collision ←true; i ←i + 1; return collision; Algorithm 7: Collision detection algorithm in BL (with parameter k) Proof. 5.2 Emulation procedures Based on this tie-breaking mechanism, we define two probabilistic emulation procedures whose purpose is to replace beep or listen instructions with collision detection in BL. XX:14 Design Patterns in Beeping Algorithms (extended abstract) (extended abstract) (extended abstract) Both are Monte Carlo in the sense that detection is only guaranteed with some proba- bility. The first procedure, EmulateBcdinBL(), is given by Algorithm 8 and the second, EmulateLcdinBL(), by Algorithm 9. Both procedures are parametrized by an integer k > 1, which accounts for the number of sub-phases that are used in each invocation of the proce- dure (k controls the error bound). They return true if a collision has been detected, false otherwise. Both are Monte Carlo in the sense that detection is only guaranteed with some proba- bility. The first procedure, EmulateBcdinBL(), is given by Algorithm 8 and the second, EmulateLcdinBL(), by Algorithm 9. Both procedures are parametrized by an integer k > 1, which accounts for the number of sub-phases that are used in each invocation of the proce- dure (k controls the error bound). They return true if a collision has been detected, false otherwise. Before the execution each vertex generates a sequence s of k random bits (u.a.r.) which will be the ones used in each sub-phase. The reason why this is made once at the beginning rather than in each invocation is a technicality that relates to preventing an additional union bound in the analysis (more k would be needed to guarantee that each invocation is successful if the numbers are drawn every time). Algorithm 8: A Procedure to emulate a Bcd in the BL model. Procedure EmulateLcdinBL(in : Integer k, Array s; out : Boolean collision) Boolean collision ←false; Integeri ←0; repeat if s[i] then beep in slot 1; listen in slot 2; else listen in slot 1; beep in slot 2; if another beep was heard then collision ←true; i ←i + 1 until i = k; End Procedure Algorithm 8: A Procedure to emulate a Bcd in the BL model. Procedure EmulateLcdinBL(in : Integer k, Array s; out : Boolean collision) Boolean collision ←false; if another beep was heard then collision ←true; End Procedure Algorithm 9: A Procedure to emulate a Lcd in the BL model. Using the procedures. In the listings of our algorithms (see Section 4), listen instructions are implicit. By default, a node listens if it does not beep. Emulation procedures should be used explicitly for both beep and listen primitives, in order for the nodes to remain synchronized (since each of them takes logarithmically many rounds to be carried out). Therefore, whenever a node calls EmulateBcdinBL or EmulateLcdinBL, the other nodes should call one of these or wait the corresponding amount of time. Likewise, the procedures should not be interrupted even after a collision with a given neighbor is detected, to preserve synchrony with other neighbours or farther nodes. (extended abstract) Procedure EmulateLcdinBL(in : Integer k; out : Boolean beep, Boolean collision) Boolean beep ←false; Boolean collision ←false; Integer i ←0; repeat switch slot do slots 1 and 2 listen end of phase: if a beep was heard in any slot then beep ←true if a beep was heard in both slots then collision ←true i ←i + 1 until i = k; End Procedure until i = k; End Procedure Hence, the value of k depends on the bound we require on the probability of error, a straightforward adaptation of the above analysis gives us the values of Lemma 3. ▶Lemma 3. For any ε > 0, and any n > 0: 1. if k = ⌈log 1 ε  ⌉, the procedures are correct for a given node with probability 1 −ε XX:15 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari 4ms 2ms 1s 2s 2ms + 2s 4s 2ms + 4s ... 2ms + 2is 2is ... ... 2ms + 1s ... Figure 1 The wheel gadget used in the proof of optimality for emulation. 4ms 2ms 1s 2s 2ms + 2s 4s 2ms + 4s ... 2ms + 2is 2is ... ... 2ms + 1s ... 2s Figure 1 The wheel gadget used in the proof of optimality for emulation. 2. if k = ⌈log n ε  ⌉, the procedures are correct for any node with probability 1 −ε 3. if k = ⌈2 log(n)⌉, the procedures are correct for any node w.h.p. Observe that in general, the size of the network n is not known to the nodes, which is an obstacle to achieving the second and third types of guarantees. However, it is reasonable in practice to assume that the nodes know an upper bound on n, e.g., when a network of wireless sensors is deployed. The upper bound may even be loose without much consequence: so long as it is polynomial in n, the slowdown factor remains of the same order. 5.3 Optimality of the emulation In this section we prove that the emulation procedures presented in Section 5.2 are essentially optimal (i.e. asymptotically and up to a constant factor), namely, we prove a Ω(log n) lower bound on the number of slots required to detect collision in some graphs called wheels. A (m, s)-wheel, illustrated in Figure 1, is a graph W = (V, E) such that V = u1, . . . u4ms, the edges E are all the (ui−1, ui) (arithmetic modulo 4ms) plus m spokes, that is edges (uis, u(i+2m)s) (1 ≤i ≤2m), where the wheel can be odd (all spokes with i odd) or even (all spokes with i even). The even and odd (m, s)-wheels are isomorphic. We consider only situations in which all vertices uis are in the same state, a state in which they wish to beep and all other vertices are in the same internal state, a state in which they do not wish to beep. Thus vertices at the ends of spokes and no others must conclude that there is a collision. The slot complexity of any algorithm which detects collision in such a graph with high probability is to be Ω(log n). Due to space limitations, the full proofs are relegated to Appendix B. We provide, however, a minimal sentence of insight for each. Considering a computation of a collision detecting algorithm on a wheel, we define, for any t > 0, bi t as the signal (beep or not) from ui to all its neighbours at time t, and, for any (extended abstract) t ≥0, Bi t the sequence bi 1 · · · bi t . Then, we define the event Et for a spoke uis, u(i+2m)s as follows: Et = n (Bis t = B(i+2m)s t ) ∧(Bis+1 t = B(i+2m)s+1 t ) ∧(Bis−1 t = B(i+2m)s−1 t ) o . ▶Lemma 4. For any t (0 ≤t < s), it holds that Pr (Et) ≥2−3t. The proof proceeds by induction on t, with base case t = 0. (Full proof in Appendix B.) The proof proceeds by induction on t, with base case t = 0. (Full proof in Appendix B.) If Et holds for the spoke (uis, u(i+2m)s), we say that the spoke fails to break symmetry within time t. This happens with probability at least 2−3t and, if it happens, the existence of the spoke has had no influence on the computation up to time t. In particular, whenever uis beeped, u(i+2m)s also beeped and so neither has ever heard the other beep. ▶Theorem 5. For any Monte Carlo algorithm A which detects collision in W, if A halts in less than log2 n/4 rounds with probability greater than 3/4 then for some situations in some wheels, A gives incorrect results for some vertices with probability greater than 1/4. The proof proceeds using the wheel gadget of Figure 1 and Lemma 4 to characterize the rate at which the symmetry induced by the spokes can be broken. (Full proof in Appendix B.) ▶Corollary 6. The complexity of a Monte Carlo algorithm which detects collision with high probability in the BL model is Ω(log n). ▶Corollary 6. The complexity of a Monte Carlo algorithm which detects collision with high probability in the BL model is Ω(log n). A Complexity analysis This appendix section provides the analysis and proofs of the running time complexity of the algorithms presented in Section 4. A.1 Colouring algorithm without knowledge Informally the execution progresses as follows. There is a first period of adjustment in which the probabilities will converge towards “good values”. Then the probability that an exclusive beep is produced in a given phase in a given neighbourhood remains bounded in some ways. Loosely speaking, the final bound is essentially obtained by a repetition of the corresponding periods ∆times. More precisely, we prove the following theorem: ▶Theorem 7. There are constants α, β and γ such that for any graph G = (V, E) of n vertices and maximum degree ∆, the number of phases of Algorithm 4 to colour all the nodes in G is: 1. less than α(∆+ log n) with probability 1 −o n−1 , 2. less than β(∆+ log n) on average, 3. less than γ(∆+log n) with probability 1−o (n−c), for any c > 1. (This result is stronger than 1, but the proof is more difficult, which is why we keep both.) 3. less than γ(∆+log n) with probability 1−o (n−c), for any c > 1. (This result is stronger than 1, but the proof is more difficult, which is why we keep both.) 3. less than γ(∆+log n) with probability 1−o (n−c), for any c > 1. (This result is stronger than 1, but the proof is more difficult, which is why we keep both.) References 1 Y. Afek, N. Alon, Z. Bar-Joseph, A. Cornejo, B. Haeupler, and F. Kuhn. Beeping a maximal independent set. Distributed Computing, 26(4):195–208, 2013. 2 K. Azuma. Weighted sums of certain dependent random variables. Tohoku Mathematical Journal, Second Series, 19(3):357–367, 1967. 3 A. Casteigts, Y. Métivier, J. Michael Robson, and A. Zemmari. Counting in one-hop beeping networks. CoRR, abs/1605.09516, 2016. 4 J. Collier, N. Monk, P. Maini, and J. Lewis. Pattern formation by lateral inhibition with feedback: a mathematical model of delta-notch intercellular signalling. Journal of Theo- retical Biology, 183(4):429–446, 1996. 5 A. Cornejo and F. Kuhn. Deploying wireless networks with beeps. In DISC, pages 148–162, 2010. 6 S. Gilbert and C. Newport. The computational power of beeps. In Proc. of 29th Interna- tional Symposium on Distributed Computing (DISC), 2015. 7 B. Huang and Th. Moscibroda. Conflict resolution and membership problem in beeping channels. In DISC, pages 314–328, 2013. 8 P. Jeavons, A. Scott, and L. Xu. Feedback from nature: simple randomised distributed algorithms for maximal independent set selection and greedy colouring. Distributed Com- puting, DOI 10.1007/s00446-016-0269-8, 2016. 9 S. Navlakha and Z. Bar-Joseph. Distributed information processing in biological and com- putational systems. Commun. ACM, 58(1):94–102, 2015. 10 J. Schneider and R. Wattenhofer. What is the use of collision detection (in wireless net- works)? In DISC, pages 133–147, 2010. 11 A. Scott, P. Jeavons, and L. Xu. Feedback from nature: an optimal distributed algorithm for maximal independent set selection. In PODC, pages 147–156, 2013. XX:17 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A.1.2 Available Neighbours. In a given round any ui has a well defined probability ai of being available that is able to take the current colour since no neighbour claims it. These probabilities are far from being independent. We will argue that, except for cases where pdec (the probability of a decrease in d) is at least 2/5, the average decrease in M ′ is always minimised when all ai = 0. Consider a situation where pdec < 2/5 and some ai > 0. We can decrease ai to 0 with no change to the other aj by adding an infinite number of vertices adjacent to ui but to no other vertex in N(v). This will change the average increase/decrease in q and d; qi will be halved instead of doubled with probability ai, decreasing q by 3qi/2 and so decreasing f(q) by at most 6aiqi on average. The probability of a decrease in d is decreased by ai times the probability that ui claims and no other uj takes the colour. This last probability is the product of qi and the conditional probability that no other uj takes the colour given that ui claims. But, since the probabilities of ui claiming and of some uj (j ̸= i) taking the colour are negatively correlated or independent, this conditional probability is at most the unconditional probability that no uj (j ̸= i) takes the colour and so greater than the probability that no uj takes the colour, namely 1 −pdec ≥3/5, giving an average decrease in d (respectively M ′) reduced by more than 3aiqi/5 (resp. 6aiqi). Thus the decrease in the measure is decreased more by the d component than it is increased by the change in q. Repeating this process at most d times we arrive at a situation with a smaller mean decrease in M ′ than the initial one and either pdec ≥2/5 or all ai = 0 so, to lower-bound the decrease in M ′, we need only consider such situations. (extended abstract) if the degree of v decreases by more than 1, the change in M ′ only includes −10 in total for the change in d rather than −10 for each neighbour removed; if v takes a colour, M ′ is decreased by just 10 whatever the values of p at v and its neighbours; if v takes a colour, M ′ is decreased by just 10 whatever the values of p at v and its neighbours; in a round where v is already coloured, M ′ is decreased by 1. This ensures that: if the algorithm has not terminated at v, M ′ ≥M, meaning that M ′ dominates M; if M ′ ≤0, the algorithm has terminated at v; M ′ decreases at each round by a value in [−3 . . . 11] (since, log p can only change by ±1, f(q) can only change by up to ±2, and if d decreases because a neighbour u takes the colour, u beeped in the round and so p has halved). M ′ decreases at each round by a value in [−3 . . . 11] (since, log p can only change by ±1, f(q) can only change by up to ±2, and if d decreases because a neighbour u takes the colour, u beeped in the round and so p has halved). A.1.1 Local Average Time Complexity. First, we give an overview. We define pv as the probability that vertex v claims the colour in a given round and qv as the sum of p over all neighbours of v which we will call ui (1 ≤i ≤d) where d is v’s degree in the residual graph (taken as 0 if v has been eliminated from the graph). We define a measure M of the distance from a given situation to the goal where p = 1/2, qv ≤1/2, d = 0 as follows: M = −log(p) + f(q) + 10d where f is the function defined as follows: f(x) = 4x if x ≤1, for x > 1, f is the piecewise linear approximation to 2 log2 4x wheref is interpolated linearly between f(2i) = 2i + 4 and f(2i+1) = 2i + 6. We note the following properties of f which will be used in what follows: We note the following properties of f which will be used in what follows: f(x) is continuous for x > 0, except at powers of 2, f is differentiable with derivative ≤4, except at powers of 2, f is differentiable with derivative ≤4, f(x) −f(x/2) = 2 for x ≥1, f(x) −f(x/2) = 2x for x ≤1. We show that in any round, the mean decrease in M is at least 1. Then after a number of rounds equal to the initial M (≤1 + 2 log(2d) + 10d), M is reduced on average to 0 unless the algorithm has already terminated at v and after O(log n) further rounds, the algorithm has terminated at v with probability o(n−2) and so it has terminated everywhere with probability o(n−1). Intuitively, we expect both p and q to decrease initially until q < 1/2, after which p will re-ascend until it is at least close to 1/2 and then d will start to descend. We actually analyse the variation in a random variable M ′ which dominates the r.v. M. The r.v. M ′ is initially equal to M but its changes may be slightly different from those of M in the following ways: q ≥1: q decreases to q/2, reducing f(q) M ′ is decreased by at least 2 −1 = 1. M ′ is decreased by at least 2 −1 = 1. q ≥1: q decreases to q/2, reducing f(q) by 2 and log p can decrease by at most 1 so that M ′ is decreased by at least 2 −1 = 1. A.1.4 Time Complexity w.h.p.. We define the sequence of r.v.’s (Mk)0≤k≤t as follows M ′ 0 = M0 and for any k ≥1, Mk is the value of M ′ after time k. We also define the sequence (Gk)0≤k≤t as the sequence of residual graphs, i.e., G0 = G and Gk+1 is the graph obtained from Gk after round k +1 (each vertex which succeeds in beeping alone is removed from the graph). Then for any k ≥1: E (Mk | G1, G2, · · · , Gk−1) ≤Mk−1 −1. (3) E (Mk | G1, G2, · · · , Gk−1) ≤Mk−1 −1. E (Mk | G1, G2, · · · , Gk−1) ≤Mk−1 −1. (3) Hence, (Mk)k≥0 is a super-martingale with respect to (Gk)k≥0. We define the r.v. Dk = Mk −Mk−1 for any k ≥1 and we denote µ = E (Dk). We also introduce the r.v.: 4 3 + 3 D′′ k = − 4 µ −3Dk + 3µ + 3 µ −3 . D′′ k = − 4 µ −3Dk + 3µ + 3 µ −3 . Then, it is easy to see that E (D′′ k) = −1 and Pr (−11 ≤D′′ k ≤3) = 1. N d fi th (M ′′) f ll M ′′ M d f k ≥ Then, it is easy to see that E (D′′ k) = −1 and Pr (−11 ≤D′′ k ≤3) = 1. Then, it is easy to see that E (D′′ k) = −1 and Pr (−11 ≤D′′ k ≤3) = 1. Now, define the r.v. (M ′′ k )k≥0 as follows: M ′′ 0 = M0 and for any k ≥1, M ′′ k = M ′′ k−1 + D′′ + 1 Then (M ′′)k≥0 is a martingale with respect to (Gk)k≥0 Now, define the r.v. (M ′′ k )k≥0 as follows: M ′′ 0 = M0 and for any k ≥1, M ′′ k = M ′′ k−1 + D′′ k + 1. Then (M ′′ k )k≥0 is a martingale with respect to (Gk)k≥0. Now, define the r.v. (Mk )k≥0 as follows: M0 = M0 and for any k ≥1, Mk = Mk−1 + D′′ k + 1. Then (M ′′ k )k≥0 is a martingale with respect to (Gk)k≥0. M ′′ k )k≥0 is a martingale with respect to (Gk)k≥0. We apply Theorem 18 of [2] to our martingale M ′′ t with expectation M0. A.1.4 Time Complexity w.h.p.. Since the increments (D′′ k +1)k≥0 are in [−10..4] and have mean 0, their variance is upper bounded by the case of a distribution with values −10 and 4 with probabilities 2/7 and 5/7 respectively, giving variance of 40 and maximum discrepancy from the mean of 10. Applying the theorem with t = 2M0 + 174 ln n and λ = t −M0, we see that the probability that Mt ≥0 is less than Pr (M ′′ t ≥t) which is at most: e(−λ2/2(40t+10λ/3)), and we claim that this is o(n−2). This is because λ2/2(40t + 10λ/3) >> 2 ln n, i.e. λ2 >> 4 ln n(40t + 10λ/3). and we claim that this is o(n−2). This is because λ2/2(40t + 10λ/3) >> 2 ln n, i.e. λ2 >> 4 ln n(40t + 10λ/3). (Proof of this claim: λ2/13 >> 4 ln n(10λ/3) because λ >> 520 ln n/3; 12λ2/13 > 12(t2 −2M0t)/13 = 12t(t −2M0)/13 = 12t(174 ln n)/13 >> 160t ln n. Adding these two gives the claim.) (Proof of this claim: λ2/13 >> 4 ln n(10λ/3) because λ >> 520 ln n/3; 12λ2/13 > 12(t2 −2M0t)/13 = 12t(t −2M0)/13 = 12t(174 ln n)/13 >> 160t ln n. Adding these two gives the claim.) Then taking α = 174, this proves the first claim of Theorem 7. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari q < 1: q decreases to q/2, decreasing f(q) by 2q while p doubles with probability at least 1 −q (and halves with probability at most q). This gives a mean decrease in M ′ of at least 2q + (1 −2q) = 1. A.1.5 Average Time Complexity. We first prove the following lemma: A.1.3 The mean decrease in M ′. We consider cases depending on the value q. First note that if pdec ≥2/5, the mean decrease in M ′ is at least 10(2/5) −2 −1 = 1. So in the other cases we suppose that all ai = 0 so that q is halved. In the case where q < 1, we need to consider what happens when no ui claims the colour. If p < 1/2 this is that p increases, decreasing M ′ by 1; if p = 1/2 it is that, with probability 1/2, v takes the colour so that d decreases by 1, decreasing M ′ by 10 so that on average M ′ decreases by 5. Accordingly we suppose that the former happens. q ≥1: q decreases to q/2, reducing f(q) by 2 and log p can decrease by at most 1 so that M ′ is decreased by at least 2 −1 = 1. XX:19 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari ▶Lemma 8. Let v be any vertex in G, and t > 0. We have: ▶Lemma 8. Let v be any vertex in G, and t > 0. We have: Pr (v is not coloured at time t) ≤e− 3 260 (t−2M0). Proof. Let Tv denote the time before v gets coloured. Then, by discussions above, taking λ = t −M0 in Theorem 18 of [2]: Pr (Tv −M0 > λ) ≤e − λ2 2(40t+ 10 3 λ) . XX:20 A.1.6 Time Complexity With Very High Probability. A.1.6 Time Complexity With Very High Probability. To prove the last claim, let c > 1 and take t = 260 3 (c + 1) ln n + 2M0 in Lemma 8, this gives: To prove the last claim, let c > 1 and take t = 260 3 (c + 1) ln n + 2M0 in Lemma 8, this gives: To prove the last claim, let c > 1 and take t = 260 3 (c + 1) ln n + 2M0 in Pr  Tv > 260 3 (c + 1) ln n + 2M0  ≤ 1 nc+1 = o  1 nc  . Thus, taking γ = 87 proves the last claim of Theorem 7. XX:20 Design Patterns in Beeping Algorithms On the other hand, λ ≥t −2M0 and hence, a simple computation yields: λ2 > 3 13(t −2M0)(4t + λ 3 ), λ2 > 3 13(t −2M0)(4t + λ 3 ), which proves the lemma. Back to Theorem 7. Let T denote the time before all the vertices in the graph are coloured. Then: E (T) = X t≥1 Pr (T ≥t) . Now, let t0 = 2M0 + 260 3 ln n then: E (T) = t0 X t=1 Pr (T ≥t) + X t>t0 Pr (T > t) ≤ 2M0 + 260 3 ln n + X t>t0 Pr (T > t) . On the other hand, for any t > 0: On the other hand, for any t > 0: Pr (T > t) ≤ X v∈V Pr (Tv > t) ≤ne− 3 260 (t−2M0). Yielding: E (T) ≤ 2M0 + 260 3 ln n + n X t>t0 e− 3 260 (t−2M0) = 2M0 + 260 3 ln n + n X t>0 e− 3 260 (t+t0−2M0) = 2M0 + 260 3 ln n + X t>0 e−3t 260 = 2M0 + 260 3 ln n + 1 1 −e− 3 260 . Taking β = 87, this proves the second claim of Theorem 7. E (T) ≤ 2M0 + 260 3 ln n + n X t>t0 e− 3 260 (t−2M0) = 2M0 + 260 3 ln n + n X t>0 e− 3 260 (t+t0−2M0) = 2M0 + 260 3 ln n + X t>0 e−3t 260 = 2M0 + 260 3 ln n + 1 1 −e− 3 260 . Taking β = 87, this proves the second claim of Theorem 7. Taking β = 87, this proves the second claim of Theorem 7. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari We have the following theorem: ▶Theorem 9. Let G be a graph of size n, let K be an upper bound on the maximum degree of G. Algorithm 5 computes a K + 1 colouring of G in at most O K(log n + log2 K)  slots w.h.p. Proof. We consider the Colouring algorithm in which every node has the same upper bound K on the maximum degree. We consider both the basic algorithm in which v uses the current value of |Colours| to decide its beeping probability and also the modified algorithm in which it uses the value at the start of the current cycle. We recall that by a cycle we mean K phases considering the |Colours| colours. We consider Pk the probability that vertex v survives uncoloured over k cycles. In what follows: In what follows: i ranges over 1..k, c ranges over the Ci colours possible for v at the start of cycle i, u ranges over the neighbours of v still uncoloured at the start of cycle i, pu(i, c) is the probability that u beeps at colour c in cycle i. First we consider the probability p that v survives uncoloured in a single phase using a colour c ∈colours(v). Then: p = Pr (v does not beep at colour c in cycle i) p = Pr (v does not beep at colour c in cycle i) + Pr (v does beep and some neighbour u also beeps) , + Pr (v does beep and some neighbour u also beeps) , but Pr (v does beep) ≥1/2Ci and the beeping probabilities of v and its neighbours are independent giving: p ≤ (1 −1/2Ci) + Pr (some neighbour beeps) /2Ci = (1 −1/2Ci) (1 + Pr (some neighbour beeps) /(2Ci −1)) ≤ (1 −1/2Ci) 1 + X u pu(i, c)/(2Ci −1) ! . ≤ (1 −1/2Ci) 1 + X u pu(i, c)/(2Ci −1) ! . After the first phase, pu(i, c) and Ci are random variables dependent on what has happened so far, and we consider the tree of all possible executions up to k cycles, where each tree node has its own value of p. It is easily shown by induction that Pk is upper bounded by the maximum over all paths in this tree of the product of the values of p along the path. A.2 Colouring algorithm knowing K ≥∆. ▶Remark. We can consider the modified colouring algorithm, deduced from Algorithm 5, defined in the following way. By a cycle we mean K rounds considering the K colours. Now, every vertex uses the value of |Colours| at the start of each cycle to decide the beeping probability it uses throughout this cycle. XX:21 A.3 2-hop colouring To calculate a 2-hop colouring of a graph G, we need to calculate a colouring of the “square” of G, that is the graph with the same vertices as G and an edge between any pair v and w of vertices which either are neighbours in G or have a common neighbour in G. In this context, Theorem 7 becomes: ▶Theorem 10. There are constants α, β and γ such that for any graph G = (V, E) of n vertices and maximum degree ∆, the number of phases of Algorithm 6 to calculate a 2-hop colouring in G is: 1. less than α(∆2 + log n) with probability 1 −o n−1 , 2. less than β(∆2 + log n) on average, 3. less than γ(∆2 + log n) with probability 1 −o (n−c), for any c > 1. 3. less than γ(∆2 + log n) with probability 1 −o (n−c), for any c > 1. ▶Remark. The same transformation can be done starting from Algorithm 5 when we know an upper bound of the maximum degree. ▶Remark. The same transformation can be done starting from Algorithm 5 when we know an upper bound of the maximum degree. (extended abstract) Firstly base: in cycle i, v has Ci colours available and so has less than Ci neighbours; each neighbour u has P c base ≤1/2, giving, for this cycle, P u P c base/(2Ci −1) ≤1/4 so that P i P u P c base/(2Ci −1) ≤k/4. i u c Secondly δ: For the modified algorithm δ = 0. In the basic algorithm, a node uj initially has K colours available and when (if) this number decreases from l to l −1, pu(i, c) increases from 1/2l to 1/2(l −1) and this increase of 1/2l(l −1) affects δ only for the, at most, l −1 colours still to be considered in this cycle so that P c δ for a cycle is at most P l 1/2l, the sum being taken over those l for which the number of colours is reduced from l. This gives an upper bound on P i P c δ/(2Ci −1) of log K/2(2j + 1) since Ci > j and so P u P i P c δ/(2Ci −1) < P j log K/2(2j + 1) < log2 K/4. j Hence, by standard arguments, after k = O(log n + log2 K) cycles for the basic algo- rithm or O(log n) cycles for the modified algorithm, v has probability o(1/n2) of remaining uncoloured and the graph has probability o(1/n) of having any uncoloured node. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari We fix a path which gives this maximum and bound the product for this path. We have the probability of surviving cycle i ≤(exp(−1/2) ∗Q c(1 + P u pu(i, c)/(2Ci −1))) ≤ exp(−1/2+P c P u pu(i, c)/(2Ci−1)) and so Pk ≤exp(−k/2+P i P c P u pu(i, c)/(2Ci−1)). We will give an upper bound on P i P c P u pu(i, c)/(2Ci −1). We number v’s neighbours in the initial graph from 1 to deg(v) in decreasing order of their lifetime, that is the number of phases in which they remain uncoloured. Thus as long as uj is not coloured the degree of v in the residual graph is at least j and so |colours(v)| > j. We write pu(i, c) as base+δ where base = 1/2Ci and δ is what has been added as a result of colours(u) being decreased before colour c and we will bound P i P u P c base/(2Ci −1) and P u P i P c δ/(2Ci −1) separately. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari We finally write l(q) for log(5 max{q, 1/5}), that is l(q) = max{log(5q), 0}. Then, we have the following theorem: We finally write l(q) for log(5 max{q, 1/5}), that is l(q) = max{log(5q), 0}. Then, we have the following theorem: ▶Theorem 11. For any t ≥0 and for any vertex v, its probability of remaining active after the next t phases is at most αt0−t for the constant α = 21/36 ≈1.01944. Proof. Note that α3 log q = q3 log α = q1/12. The proof will be by induction on t. We have t0 ≥2, so that if t = 0, αt0−t > 1 and the claim is trivially true. Let t > 0. After one phase which does not add v or a neighbour to the MIS we have by induction that the probability of remaining active for the following t −1 phases is at most αt′ 0−t+1 where t′ 0 is the new value of t0, namely 3l(q′) −2 log p′. So we conclude that the probability of survival is upper bounded by the mean of the random variable which is αt′ 0−t+1 if v survives the first phase and 0 otherwise. We refer to this mean as the bound and note that it is dependent on what happens outside the neighbourhood of v. ◀ We will come back to the proof of the Theorem, but we first prove the following lemma: ▶Lemma 12. The bound is maximised when what happens outside the neighbourhood of v is that every neighbour u of v is inhibited from joining the MIS by some external neighbour beeping and no neighbour of v becomes inactive through another vertex (outside N(v)) joining the MIS. Proof. Clearly a vertex ouside N(v) joining the MIS can only affect the bound by reducing q which reduces the bound. Consider any external behaviour E in which some u is not inhibited; we will show that the bound is increased or unchanged if the behaviour is changed to E′ in which u is inhibited and there is no change for any other neighbours of v. A.4 Analysis of Jeavons et al.’s Las Vegas Algorithm for the MIS in BcdL In [8], Jeavons et al. give and analyse a Las Vegas beeping algorithm to compute a MIS in the model BcdL. They prove that for any graph G with n vertices, their algorithm terminates in at most K0 log n phases, with probability at least 1 −o(n−1) and K0 ≥e25. The starting point of our work is the observation made by Scott et al. at the end of Section 4 in [11]: “Our simulations show that in practice the constants are rather lower”. We verify this observation by proving that the number of phases taken by the Jeavons et al. algorithm on any graph with n vertices is at most 76 log n w.h.p. We first introduce some notation that we will use in this section. If a neighbour of v beeps (in a slot), we say that v is “inhibited” (in that slot). For any vertex v, we define the following sum: qv = X u∈N(v) pu. We also note q∗ v = max{qv, 1/5} and finally t0 = 3 log(5q∗ v) −2 log pv. We omit the subscript v where there is no risk of ambiguity. XX:23 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari (In a given graph there may be no such E′ but we consider the maximum possible over any graph containing the neighbourhood N(v).) We consider fixed beeping decisions of all vertices in N(v) except u and show that with these decisions E′ gives a value of the bound greater than or equal to that of E. We consider three cases: Some neighbour of v which is neither u nor a neighbour of u enters the independent set: Note that this is determined by the fixed beeping decisions and the external behaviour other than as it affects u. Hence this happens for E iffit also happens for E′ and in each case the bound is 0. Some neighbour of u in N(v) beeps: pu will be halved whether or not u is inhibited by E′ and so both p′ and q′ and the probability of survival are the same for E and E′. The bound is identical in the two cases. Otherwise: Let the value of p′ be p0 if u does not beep and p1 if u does beep. p1 ≤p0. Let the value of q′ be q0 if u does not beep and is not inhibited, q1 if it beeps and is inhibited and q2 if it does not beep and is inhibited. Note that if u beeps and is not inhibited, u enters the independent set and v does not survive. We have q1 ≥q0/4 since, at most, u’s beeping can result in a vertex w halving qw when otherwise it would have doubled it. Similarly q2 ≥q0/4 and q2 ≥q0 −3pu/2 since the inhibition results in pu being halved rather than potentially doubled. The bounds are thus puα3l(q1)−2 log(p1)−t+1 + (1 −pu)α3l(q2)−2 log(p0)−t+1 in the inhibited case and (1 −pu)α3l(q0)−2 log(p0)−t+1 in the uninhibited case. We claim that the ratio of the inhibited bound to the uninhibited is at least 1. This ratio ≥puα3l(q1)+(1−pu)α3l(q2) (1−pu)α3l(q0) Let the value of q′ be q0 if u does not beep and is not inhibited, q1 if it beeps and is inhibited and q2 if it does not beep and is inhibited. Note that if u beeps and is not inhibited, u enters the independent set and v does not survive. We have q1 ≥q0/4 since, at most, u’s beeping can result in a vertex w halving qw when otherwise it would have doubled it. (extended abstract) (since p1 ≤p0) Remember that pu is a power of 1/2. We consider four subcases: (since p1 ≤p0) Remember that pu is a power of 1/2. We consider four subcases: ( ) Remember that pu is a power of 1/2. We consider four subcases: q0 ≤1/5: l(q1) = l(q2) = l(q0) = 0 and the ratio ≥(pu + 1 −pu)/(1 −pu) > 1. 1/5 < q0 and pu ≥1/8: We use the bounds q1 ≥q0/4 and q2 ≥q0/4 giving that the ratio is at least (pu + 1 −pu)α−6/(1 −pu) = α−6/(1 −pu) ≥α−6(8/7) ≥1. 1/5 < q0 ≤4/5 and pu ≤1/16: We use the bounds q1 ≥q0/4 and q2 ≥q0 −3pu/2 and the fact that for 0 < x ≤15/32, (1 −x)1/12 > 1 −4/3(x/12) so that the ratio is at least puα−6/(1 −pu) + (1 −3pu/2q0)3 log α ≥puα−6 + (1 −15pu/2)1/12 ≥puα−6 + (1 −(15pu/2)/12 × (4/3)) ≥1 + pu(α−6 −5/6) > 1. q0 > 4/5 and pu ≤1/16: Using the same bounds as in the previous subcase the ratio is greater than pu 1−pu α−6 +α3(l(q0−3pu/2)−l(q0)) > pu 1−pu α−6 +α3(l(4/5−3pu/2)−l(4/5)) and this is the bound already used for the case with q0 = 4/5 and the same value of pu and so is greater than or equal to 1. q0 > 4/5 and pu ≤1/16: Using the same bounds as in the previous subcase the ratio is greater than pu 1−pu α−6 +α3(l(q0−3pu/2)−l(q0)) > pu 1−pu α−6 +α3(l(4/5−3pu/2)−l(4/5)) and this is the bound already used for the case with q0 = 4/5 and the same value of pu and so is greater than or equal to 1. This ends the proof that E′ gives a value for the bound at least as great as that for E. The lemma is then proved by a simple induction on the number of uninhibited vertices. We return to the inductive proof. Using the lemma we will always take q′ = q/2 giving probability of survival ≤α3l(q/2)−2 log p′−t+1. We consider five cases. q ≥2/5: We have l(q/2) = l(q)−1 and p′ ≥p/2 giving P(survival) ≤α3(l(q)−1)−2(log p−1)−t = α3l(q)−2(log p)−t as claimed. q ≥2/5: We have l(q/2) = l(q)−1 and p′ ≥p/2 giving P(survival) ≤α3(l(q)−1)−2(log p−1)−t+ = α3l(q)−2(log p)−t as claimed. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari Similarly q2 ≥q0/4 and q2 ≥q0 −3pu/2 since the inhibition results in pu being halved rather than potentially doubled. XX:24 Design Patterns in Beeping Algorithms XX:24 (extended abstract) 1/5 ≤q < 2/5 and p < 1/2: The probability that a neighbour of v beeps is less than q so that pv is doubled with probability at least 1 −q and halved in the remaining cases. In all cases l(q/2) = 0. Hence P(survival) ≤α−2 log(p)−t+1((1 −q)α−2 + qα2) and our claim is that it is at most α3 log(5q)−2 log(p)−t. That is the claim is valid since (1 −q)α−1 + qα3 ≤α3 log(5q) in the range 1/5 ≤q < 2/5. (It is valid at q = 1/5 since 4α−1 +α3 < 5 and at q = 2/5 since 3α−1 +2α3 < 5α3; between these two limits, the left hand side is linear and the right hand side ((5q)3 log α) has a negative second derivative so the inequality holds there also.) 1/5 ≤q < 2/5 and p = 1/2: With probability greater than 1−q no neighbour of v beeps and then v has probability 1/2 of entering the independent set; otherwise pv remains 1/2. On the other hand, if a neighbour does beep, pv becomes 1/4. In all cases l(q/2) = 0. Thus the probability of survival ≤α2−t+1((1 −q)/2 + qα2) and the claim is that it is at most α3log2(5q)+2−t. That is the claim is valid if (1 −q)α/2 + qα3 ≤α3 log(5q) a weaker condition than in the previous case. q < 1/5 and p < 1/2: The probability that a neighbour of v beeps is less than 1/5 so that pv is doubled with probability at least 4/5 and halved in the remaining cases. In all cases l(q) decreases or is unchanged. Hence P(survival) ≤α3l(q)−2 log(p)−t+1((4/5)α−2+ (1/5)α2) and this is less than α3l(q)−2 log p−t as claimed, again since 4α−1 + α3 < 5. q < 1/5 and p = 1/2: With probability greater than 4/5 no neighbour of v beeps and then v has probability 1/2 of entering the independent set; otherwise pv remains 1/2. On the other hand, if a neighbour does beep, q decreases and pv becomes 1/4. Hence P(survival) ≤(2α3l(q/2)−2 log(1/2)−t+1+α3l(q/2)−2 log(1/4)−t+1)/5 ≤α3l(q)−2 log(1/2)−t+1(2+ α2)/5 which is at most α3l(q)−2 log(1/2)−t as claimed since 2 + α2 < 5α−1. α2)/5 which is at most α3l(q)−2 log(1/2)−t as claimed since 2 + α2 < 5α−1. B Missing proofs This appendix section provides the missing proofs of Section 5. Proof of Lemma 4. By induction on t. Clearly the claim is true for t = 0. We suppose that Et−1 is true and we consider probabilities conditional on the values of Bt−1 for is −1, is, is + 1, (i + 2m)s −1, (i + 2m)s and (i + 2m)s + 1. ( ) ( ) ( ) ( ) ( ) We will show that the probability that bis t = b(i+2m)s t and bis−1 t = b(i+2m)s−1 t and bis+1 t = b(i+2m)s+1 t is at least 2−3. The three events: (i+2 ) The three events: bis t = s(i+2m)s b bis−1 t = b(i+2m)s−1 t bis+1 t = b(i+2m)s+1 t are independent. bis t = s(i+2 b ( bt sb bis−1 t = b(i+2m)s−1 t bis+1 t = b(i+2m)s+1 t t b bis−1 t = b(i+2m)s−1 t t t bis+1 t = b(i+2m)s+1 t are independent. For the first, uim and u(i+2m)s started in the same state and have sent and heard identical signals. Thus they have the same probability of beeping at the next round and so have probability at least 1/2 of either both beeping or neither. For the second, the two chains (u(i−1)s · · · uis−1) and (u(i+2m−1)s · · · u(i+2m)s−1) started in the same states, have received the same signals from uis and u(i+2m)s, and have sent the same signals. Thus, again the two vertices uis−1 and u(i+2m)s−1 have the same conditional probability of beeping and so probability at least 1/2 of making the same choice. The argument for the third event is identical. This proves that the three events happen with probability at least 2−3 yielding that the probability of event Et is lower bounded by 2−3t. ◀ Proof of Theorem 5. For simplicity we consider wheels (m, s) where s is a power of 2 and m = 24s−2/s so that s = log2 n/4. We consider a computation on this wheel without specifying whether it is the odd or even wheel. By Lemma 4, the probability that a given spoke i breaks symmetry within time s −1 is at most 1 −23−3s < exp(−23−3s) and this is independent for all spokes so that the probability that every spoke breaks symmetry in the even case in time s −1, is at most exp(−23−3sm) = exp(−2s+1/s) < 1/4. A.5 The Case of the 2-hop MIS Las Vegas Algorithm in BcdLcd The 2-hop MIS algorithm simulates the MIS algorithm in the square of G; knowing that the complexity depends only on the number of vertices of the graph we deduce from the previous section: ▶Theorem 14. The number of phases taken by the 2-hop MIS algorithm on any graph with n nodes is less than 76 log n w.h.p. A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari ▶Corollary 13. The number of phases taken by Jeavons et al.’s algorithm on any graph with n nodes is, in fact, less than 76 log n w.h.p. Proof. Since initially pv = 1/2 and qv < n/2 where the graph has n vertices, we conclude that t0 < 3 log(5n/2) −2 log(1/2) < 3 log n + 6 so that after t = 76 log2 n phases, every vertex v has probability n−2 of still being active and therefore the algorithm has terminated with probability 1 −o(n−1). ◀ ▶Remark. The number of phases before the probability is 1 −o(n−1) compares well with the value of K0 ≥e25 proved in [8]. This completes the proof of the theorem. We end this section by the following corollary: We end this section by the following corollary: XX:25 A. Casteigts, Y. Métivier, J.M. Robson, and A. Zemmari XX:26 Design Patterns in Beeping Algorithms XX:26 B Missing proofs Hence the (extended abstract) probability that the algorithm halts and some spoke fails to break symmetry is greater than 1/2. If, in the even case, spoke i fails to break symmetry, vertex ui hears the same signals from its neighbours in the odd and even cases and, so, if it terminates the algorithm in this time, it has the same probability of deciding collision in the two cases. Hence it gives the wrong response in one case with probability at least 1/2. Hence there is a vertex which gives the wrong response in the odd or even case with probability greater than 1/4. probability that the algorithm halts and some spoke fails to break symmetry is greater than 1/2. If, in the even case, spoke i fails to break symmetry, vertex ui hears the same signals from its neighbours in the odd and even cases and, so, if it terminates the algorithm in this time, it has the same probability of deciding collision in the two cases. Hence it gives the wrong response in one case with probability at least 1/2. Hence there is a vertex which gives the wrong response in the odd or even case with probability greater than 1/4. Thus if an algorithm halts in time o(log n) with probability ≥3/4, for sufficiently large n it halts in time less than s and so its probability of giving an incorrect result is at least 1/4 for some initial conditions. It follows that the same is true for any algorithm halting in expected time o(log n). ◀
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Influence of posterior corneal astigmatism on postoperative refractive astigmatism in pseudophakic eyes after cataract surgery
BMC ophthalmology
2,016
cc-by
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© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: konkonhotaru@yahoo.co.jp 1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1 Amakubo, Tsukuba, Ibaraki 305-8576, Japan 2Hitachi General Hospital, 2-1-1 Jonancho, Hitachi, Ibaraki 317-0077, Japan Full list of author information is available at the end of the article Abstract Background: To examine the influence of posterior corneal astigmatism on postoperative refractive astigmatism in pseudophakic eyes after cataract surgery. Methods: The study enrolled 64 pseudophakic eyes of 50 patients (71.8 ± 9.9 years old, mean ± standard deviation) who had undergone phacoemulsification with non-toric IOL implantation. Refractive astigmatism was measured using an auto ref-keratometer with a 0.01- diopter (D) scale. Two types of corneal astigmatism were calculated using anterior segment optical coherence tomography; keratometric and total corneal astigmatism. Keratometric astigmatism was obtained based on anterior corneal curvature alone and total corneal astigmatism was calculated using both anterior and posterior corneal curvatures. The difference between refractive and corneal astigmatism was computed as the vector difference using 1) refractive and keratometric astigmatism and 2) refractive and total corneal astigmatism. Results: The mean refractive, keratometric, and total corneal astigmatism was 0.92 ± 0.48 D, 0.87 ± 0.44 D, and 0.94 ± 0.46 D, respectively. The difference between refractive and keratometric astigmatism (0.70 ± 0.40 D, mean vector of 0.30 D axis 164°) was significantly larger than the difference between refractive and total corneal astigmatism (0.63 ± 0.38 D, mean vector of 0.12 D axis 137°) (P = .019). Conclusions: The difference between refractive and total corneal astigmatism, calculated using both anterior and posterior corneal curvatures, was significantly smaller than the difference between refractive and keratometric astigmatism using anterior corneal astigmatism alone, implying that the latter overestimates the true postoperative refractive astigmatism and can cause cylindrical inaccuracy after cataract surgery. Keywords: Posterior astigmatism, Cataract surgery, Toric intraocular lens Keywords: Posterior astigmatism, Cataract surgery, Toric intraocular lens Influence of posterior corneal astigmatism on postoperative refractive astigmatism in pseudophakic eyes after cataract surgery aki Sano1,2,3*, Takahiro Hiraoka4, Yuta Ueno4, Hideo Itagaki2, Tomohiro Ogami3 and Tetsuro Osh Sano et al. BMC Ophthalmology (2016) 16:212 DOI 10.1186/s12886-016-0391-1 Sano et al. BMC Ophthalmology (2016) 16:212 DOI 10.1186/s12886-016-0391-1 Background accuracy of preoperative measurements of corneal astig- matism is often discussed [2, 3]. It has been reported that uncorrected astigmatism of greater than one diopter (D) in magnitude significantly deteriorates uncorrected visual acuity in pseudophakic eyes [1]. The introduction of toric intraocular lens (IOL) technology has made it possible to offer better and more stable uncorrected visual acuity to patients with astigma- tism. In clinical practice, however, some patients still present with postoperative refractive astigmatism of un- known origin even with toric IOL implantation, and the Both anterior and posterior corneal curvatures con- tribute to total corneal astigmatism [4], but less atten- tion has been directed to posterior corneal curvature [4–10]. This is because traditionally anterior and poster- ior corneal surfaces in normal eyes were thought to be almost parallel in shape. In addition, refractive power of the posterior surface is much smaller than the anterior surface due to the small difference in refractive index between the corneal stroma and the aqueous humor. Thus, in general, ophthalmologists tended to believe that detailed examination of posterior corneal shape and curve is not necessary [7]. Therefore, keratometric * Correspondence: konkonhotaru@yahoo.co.jp 1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1 Amakubo, Tsukuba, Ibaraki 305-8576, Japan 2Hitachi General Hospital, 2-1-1 Jonancho, Hitachi, Ibaraki 317-0077, Japan Full list of author information is available at the end of the article Sano et al. BMC Ophthalmology (2016) 16:212 Page 2 of 9 Page 2 of 9 corrected visual acuity of less than 0.8 (decimal) (20/25 snellen), history of toric IOL implantation, or cases with surgical complication. Informed consent was obtained from all patients. The research was approved by the Institutional Review Board of the University of Tsukuba Hospital and conducted according to the tenets of the Declaration of Helsinki. astigmatism which is calculated based on anterior cor- neal measurements only has been used clinically to rep- resent total corneal astigmatism, assuming a fixed posterior/anterior curvature ratio to estimate the contri- bution of posterior corneal power [4]. Newer technologies, such as slit-scanning videokera- toscope, Scheimpflug device, and anterior segment optical coherence tomography (AS-OCT), are now available for measuring anterior as well as posterior corneal shapes [4]. Results obtained with these de- vices demonstrated that keratometric astigmatism calculated based on the measurements of anterior corneal surface alone significantly differ from that based on both anterior and posterior corneal mea- surements [4, 5]. Background The posterior corneal surface tends toward against-the-rule astigmatism pattern in comparison with the anterior corneal surface [7]. Therefore, in eyes with with-the-rule astigmatism, keratometric astigmatism overestimates total corneal astigmatism, whereas in eyes with against-the-rule astigmatism, keratometric astigmatism underestimates total corneal astigmatism [4, 7, 10]. These discrepan- cies seem to be explained by the fact that corneal thickness profile is not uniform between horizontal and vertical directions, i.e., the cornea is thicker in the vertical than the horizontal directions [11]. Thus, it is not always true that the anterior and posterior corneal curvatures have a constant and linear rela- tionship [9, 11], and posterior corneal astigmatism should be at least partially responsible for postopera- tive refractive astigmatism of unknown origin [2, 3]. The preoperative evaluation included measurements of objective refractive power obtained using an auto ref- keratometer (RC-5000, Tomey Corporation, Nagoya, Japan) with a 0.01-D scale, axial length measured by contact applanation ultrasound (AL-1100, Tomey Corporation, Nagoya, Japan), and keratometric and total corneal powers measured by an AS-OCT (SS-1000, CASIA, Tomey Corporation, Nagoya, Japan). We took automatically-calculated values of total corneal power from the AS-OCT based on the actual measures of anterior and posterior corneal power. A standard phacoemulsification technique was per- formed through a 3-mm superior sclerocorneal one- plane incision under topical anesthesia. Aspheric and non-toric IOLs were implanted in all patients. One month after surgery, we measured postoperative refractive power using the RC-5000 with a 0.01-D scale and keratometric power and total corneal powers using the AS-OCT, and then assessed the correlation among postoperative refractive, keratometric, and total corneal astigmatism. In addition, the vector differences between postoperative refractive astigmatism and two types of corneal astigmatism (keratometric and total corneal astigmatism) were calculated and compared with each other. In this study, all measurements were based on the data from the annular ring with 3 mm in diameter around the corneal apex. The above findings highlight the need to clarify the effect of posterior corneal astigmatism on postoperative refractive astigmatism after cataract surgery. Several pre- vious reports have already demonstrated the relationship between posterior corneal astigmatism and total corneal astigmatism [4, 5, 12]. However, the relationship be- tween posterior corneal astigmatism and postoperative refractive astigmatism of unknown origin in patients undergoing cataract surgery has not been examined in detail. AS-OCT AS-OCT The AS-OCT is a non-contact, non-invasive three- dimensional imaging system based on the principle of “Swept Source” OCT. This system uses light of 1,310- nm wavelength and achieves resolutions of 10 μm (axial) and 30 μm (transverse) to obtain 30,000 axial-scans per second. The scan range diameter is 10.0 mm, and 16 radial cross-sectional images were obtained within 0.34 s per measurement, with each image containing 512 measurement points [13–15]. All measurements were taken by experienced examiners (MS and YU). Two im- ages were obtained for each eye, and the better image was selected for data analysis. Methods This study included consecutive eligible patients who had undergone phacoemulsification with non-toric IOL implantation at the University of Tsukuba Hospital from November 2012 to March 2013. Patients who had cor- neal or retinal disease and a history of ocular surgery other than cataract surgery or ocular injury were excluded. Patients were also excluded if they met any of the following criteria: postoperative decimal best- Background In this study, we investigated the influence of pos- terior corneal astigmatism on postoperative refractive astigmatism in pseudophakic eyes with non-toric IOL implantation. refractive index of the cornea ¼ 1:376 In this study, the AS-OCT was used to measure and calculate keratometric and total corneal astigmatism. Keratometric astigmatism was calculated as the differ- ence in keratometric power between the steepest and flattest meridians, whereas total corneal astigmatism was calculated based on total corneal power without regard to keratometric power. Corneal astigmatism calculation Keratometric power was calculated using the kerato- metric index (1.3375) and the radius of anterior corneal curvature, while total corneal power was calculated Sano et al. BMC Ophthalmology (2016) 16:212 Page 3 of 9 Page 3 of 9 based on the refractive power of the anterior and poster- ior corneal surface as well as corneal thickness. based on the refractive power of the anterior and poster- ior corneal surface as well as corneal thickness. Vector difference between refractive and corneal astigmatism In this study, we computed vector difference between postoperative refractive astigmatism and each of two types of corneal astigmatism (keratometric and total cor- neal astigmatism) using equations below [16], and these differences were compared with each other to simulate the influence of actual posterior corneal astigmatism on refractive astigmatism in pseudophakic eyes after non- toric IOL implantation, in which internal astigmatism induced by IOL itself is theoretically regarded as 0 D. Statistical analyses y Two types of corneal astigmatism (keratometric and total corneal astigmatism) were compared using the paired t-test. The mean magnitude of difference between refractive and keratometric astigmatism, and difference between refractive and total corneal astigmatism was also compared using the paired t-test. The mean differ- ences in magnitude between refractive and each of two types of corneal astigmatism according to the types of preoperative keratometric astigmatism, such as ATR, WTR or oblique astigmatism were compared using the Wilcoxon signed-rank test. Using Pearson’s correlation and Bland-Altman plots, the correlation between post- operative refractive and two types of corneal astigmatism were examined. In addition, after all patients were divided into two groups based on the magnitude of difference between postoperative refractive and total corneal astigmatism, various parameters such as age, spherical equivalent refraction, refractive astigmatism, keratometric astigmatism, total corneal astigmatism, axial length, and IOL power were compared between the two groups with greater than 0.5 D and less than 0.5 D difference using Student’s t-test. P values < 0.05 were considered statistically significant. Statistical analyses were carried out using StatView version 5.0 (SAS Inc., Cary, NC). keratometric power ¼ 1:3375 ‐ 1:0 ð Þ=r keratometric index ¼ 1:3375 keratometric power ¼ 1:3375 ‐ 1:0 ð Þ=r keratometric index ¼ 1:3375 r ¼ radius of anterior corneal curvature total corneal power ¼ Pa þ Pb  d  Pa  Pb=1:376 Pa ¼ refractive power of the anterior cornea Pb ¼ refractive power of the posterior cornea d ¼ corneal thickness Pa ¼ refractive power of the anterior cornea Pb ¼ refractive power of the posterior cornea d ¼ corneal thickness refractive index of the cornea ¼ 1:376 Results This study enrolled 64 eyes (33 right; 51.6%) of 50 patients (25 women; 50.0%) with a mean age of 71.8 ± 9.9 (SD: standard deviation) years (range 33 to 92 years). The age distribution of patients was shown in Fig. 1. The lenses implanted in the study group were SN60 WF (50 eyes) (Alcon, Fort Worth, TX), iSert Micro251 (10 eyes) (HOYA, Tokyo, Japan) and ZCB00V (4 eyes) (Abbott Medical Optics, Santa Ana, CA). xr ¼ refractive astigmatism  Cos 2  axis ð Þ yr ¼ refractive astigmatism  Sin 2  axis ð Þ xc ¼ corneal astigmatism  Cos 2  axis ð Þ yc ¼ corneal astigmatism  Sin 2  axis ð Þ xr ¼ refractive astigmatism  Cos 2  axis ð Þ yr ¼ refractive astigmatism  Sin 2  axis ð Þ xc ¼ corneal astigmatism  Cos 2  axis ð Þ yc ¼ corneal astigmatism  Sin 2  axis ð Þ Fig. 1 The age distribution of patients was shown In the formulas, the angle of the axis of astigmatism is doubled to give the correct x and y values. Cylinder ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi xr ‐ xc ð Þ2 þ yr ‐ yc ð Þ2 p Angle ¼ 1=2  Arc tan y=x ð Þ If x and y > 0 then Axis ¼ Angle If x < 0 then Axis ¼ Angle þ 90 ○ If x > 0 and y < 0 then Axis ¼ Angle þ 180 ○ Cylinder ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi xr ‐ xc ð Þ2 þ yr ‐ yc ð Þ2 p Angle ¼ 1=2  Arc tan y=x ð Þ If x and y > 0 then Axis ¼ Angle If x < 0 then Axis ¼ Angle þ 90 ○ If x > 0 and y < 0 then Axis ¼ Angle þ 180 ○ Refractive astigmatism was corrected to the corneal plane using the following equation: Fc = (1000 x Fs)/ {1000 - (Fs x d)} (Fc = refractive power (D) at the corneal plane, Fs = refractive power (D) at the spectacle plane, and d = vertex distance (12 mm)) [17]. Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. Results BMC Ophthalmology (2016) 16:212 Page 4 of 9 Table 2 Postoperative patients’ data Parameter Mean ± SD Range Postoperative days (days) 36.0 ± 12.0 20–77 Spherical equivalent refraction (D) −1.03 ± 1.42 −5.88–1.48 Refractive astigmatism (D) 0.92 ± 0.48 −2.28–−0.12 Keratometric astigmatism (D) 0.87 ± 0.44 0.18–2.46 Total corneal astigmatism (D) 0.94 ± 0.46 0.09–2.68 Difference between refractive and keratometric astigmatism (D) (Mean vector) 0.70 ± 0.40 (0.30 Axis 164°) 0.09–1.72 Difference between refractive and total corneal astigmatism (D) (Mean vector) 0.63 ± 0.38 (0.12 Axis 135°) 0.04–1.81 SD standard deviation, D diopter Table 1 shows preoperative patient data. The mean magnitude of keratometric astigmatism was 0.76 ± 0.46 D (range 0.03 to 2.76 D), and the mean axial length was 23.67 ± 1.69 mm (range 20.98 to 27.55 mm). Astigma- tism types were categorized as against-the-rule (ATR) (steepest meridian 0 to 29° or 150 to 180°), with-the-rule (WTR) (steepest meridian 60 to 119°), or oblique (stee- pest meridian 30 to 59° or 120 to 149°), and the number of eyes for each group was 23, 19, and 22, respectively. Table 2 Postoperative patients’ data Table 2 shows postoperative patient data. The mean magnitude of objective refractive, keratometric and total corneal astigmatism was 0.92 ± 0.48 D, 0.87 ± 0.44 D, and 0.94 ± 0.46 D, respectively. Total corneal astigma- tism was significantly larger than keratometric astigma- tism (P = .0015, paired t-test) (Fig. 2). The mean magnitude of difference was 0.70 ± 0.40 D between refractive and keratometric astigmatism, and 0.63 ± 0.38 D between refractive and total corneal astigmatism, with a sig- nificant difference between them (P = .019, paired t-test). correlation between refractive and total corneal astig- matism (r = 0.598, P < .0001, Fig. 4) seemed stronger than that between refractive and keratometric astig- matism (r = 0.515, P < .0001, Fig. 3). Figures 5 to 6 are the Bland-Altman plots showing the relation between postoperative refractive and two types of corneal astigmatism, with the mean values of individual mea- surements plotted on the horizontal axis and the dif- ferences of individual measurements plotted on the vertical axis. It was found that the correlation be- tween refractive and total corneal astigmatism (Fig. 6) is stronger than that between refractive and kerato- metric astigmatism (Fig. 5). Figures 7 to 9 show doubled-angle plots for each astigmatism. Discussion Using AS-OCT, we examined the anterior and posterior corneal curvatures in pseudophakic eyes after phacoe- mulsification. There are several tools available for the measurement of anterior and posterior corneal curva- tures. Among them, OCT-based topography boasts the shortest measurement time as well as high resolution. Tang et al. reported that the repeatability of corneal power measurements obtained using a Fourier-domain OCT system (RTVue, Optovue, Inc. Fremont, CA) was comparable to that of measurements obtained by Placido-ring topography [18]. Szalai et al. reported that AS-OCT had better reliability for measurements of pos- terior corneal power, anterior and posterior corneal astigmatism, and apical pachymetry in comparison to Scheimpflug imaging [13]. Because the repeatability and reliability of OCT-based topography have been estab- lished [13, 18], we used the AS-OCT to examine both anterior and posterior corneal curvatures. Figure 11 shows difference in vector between postop- erative refractive and total corneal astigmatism. The mean vector was 0.12 D axis 135°. When compared between Figs. 10 and 11, the mean difference in vec- tor between refractive and total corneal astigmatism was closer to 0 D. Table 3 shows the patient data for two groups separated by the magnitude of difference between postoperative refractive and total corneal astigmatism: greater than 0.5 D and less than 0.5 D, respectively. Statistical analysis showed no differences between the two groups in terms of patient age, post- operative spherical equivalent, postoperative refractive, keratometric, and total corneal astigmatism, axial length, or IOL power. Figure 11 shows difference in vector between postop- erative refractive and total corneal astigmatism. The mean vector was 0.12 D axis 135°. When compared between Figs. 10 and 11, the mean difference in vec- tor between refractive and total corneal astigmatism was closer to 0 D. Table 3 shows the patient data for two groups separated by the magnitude of difference between postoperative refractive and total corneal astigmatism: greater than 0.5 D and less than 0.5 D, respectively. Statistical analysis showed no differences between the two groups in terms of patient age, post- operative spherical equivalent, postoperative refractive, keratometric, and total corneal astigmatism, axial length, or IOL power. Fig. 4 A significant correlation was observed between the magnitude of postoperative refractive and total corneal astigmatism (Pearson’s correlation coefficient; r = 0.598, P < .0001) In clinical practice, we sometimes encounter unex- pected postoperative refractive astigmatism after cataract surgery even with toric IOLs. Results 5 Differences between the magnitude of postoperative refractive and keratometric astigmatism plotted against their average (Bland-Altman plots) Results The mean vector of postoperative refractive, keratometric, and total corneal astigmatism was 0.42 D axis 175° (Fig. 7), 0.16 D axis 15° (Fig. 8), and 0.39 D axis 5° (Fig. 9), respectively. Figure 10 shows difference in vector between postoperative refractive and keratometric astigmatism. The mean vector was 0.30 D axis 164°. We also calculated the mean difference in magnitude and vector between refractive and each of two types of corneal astigmatism according to the types of preopera- tive keratometric astigmatism, such as ATR, WTR or oblique astigmatism. The mean difference in magnitude and vector between refractive and keratometric astigma- tism were 0.58 ± 0.32 D (0.14 D Axis126°), 0.62 ± 0.31 D (0.44 D Axis2°), 0.89 ± 0.48 D (0.52 D Axis165°), respect- ively. The vector difference between refractive and total corneal astigmatism were 0.62 ± 0.34 D (0.29 D Axis103°), 0.52 ± 0.28 D (0.22 D Axis5°), 0.75 ± 0.48 D (0.32 D Axis153°), respectively. As for the mean difference in magnitude between postoperative refract- ive and total corneal astigmatism, there were signifi- cant differences in eyes with WTR or oblique astigmatism (P = .006, P = .007), but not in eyes with ATR astigmatism (P = .330). Fig. 2 The mean magnitude of postoperative keratometric and total corneal astigmatism was significantly different (P = .015, paired t-test) Fig. 2 The mean magnitude of postoperative keratometric and total corneal astigmatism was significantly different (P = .015, paired t-test) Figures 3 and 4 show correlations between postopera- tive refractive and two types of corneal astigmatism (keratometric and total corneal astigmatism). The Table 1 Preoperative patients’ data Parameter Mean ± SD Range Age (year) 71.8 ± 9.9 33–92 Sex (male: female) 25: 25 Right: Left 33: 31 Keratometric astigmatism (D) 0.76 ± 0.46 0.03–2.76 Type of keratometric astigmatism (eyes) ATR (0–29°, 150–180°) 23 WTR (60–119°) 19 oblique (30–59°, 120–149°) 22 Axial length (mm) 23.67 ± 1.69 20.98–27.55 SD standard deviation, D diopter, ATR against-the-rule, WTR with-the-rule Sano et al. BMC Ophthalmology (2016) 16:212 Page 5 of 9 Fig. 3 A significant correlation was observed between the magnitude of postoperative refractive and keratometric astigmatism (Pearson’s correlation coefficient; r = 0.515, P < .0001) Fig. 5 Differences between the magnitude of postoperative refractive and keratometric astigmatism plotted against their average (Bland-Altman plots) Fig. 5 Differences between the magnitude of postoperative refractive and keratometric astigmatism plotted against their average (Bland-Altman plots) Fig. Discussion When we use toric IOL in In clinical practice, we sometimes encounter unex- pected postoperative refractive astigmatism after cataract surgery even with toric IOLs. When we use toric IOL in Fig. 4 A significant correlation was observed between the magnitude of postoperative refractive and total corneal astigmatism (Pearson’s correlation coefficient; r = 0.598, P < .0001) Fig. 6 Differences between the magnitude of postoperative refractive and total corneal astigmatism plotted against their average (Bland-Altman plots) Fig. 6 Differences between the magnitude of postoperative refractive and total corneal astigmatism plotted against their average (Bland-Altman plots) Fig. 6 Differences between the magnitude of postoperative refractive and total corneal astigmatism plotted against their average (Bland-Altman plots) Fig. 6 Differences between the magnitude of postoperative refractive and total corneal astigmatism plotted against their average (Bland-Altman plots) Fig. 4 A significant correlation was observed between the magnitude of postoperative refractive and total corneal astigmatism (Pearson’s correlation coefficient; r = 0.598, P < .0001) Sano et al. BMC Ophthalmology (2016) 16:212 Page 6 of 9 Fig. 7 Double-angle plots of postoperative refractive astigmatism. The mean vector of the astigmatism (represented by the grey rhombus, larger than the other plots) was 0.41 D axis 175° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 9 Double-angle plots of postoperative total corneal astigmatism. The mean vector of the astigmatism (represented by the grey rhombus larger than the other plots) was 0.38 D axis 4° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 7 Double-angle plots of postoperative refractive astigmatism. The mean vector of the astigmatism (represented by the grey rhombus, larger than the other plots) was 0.41 D axis 175° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 9 Double-angle plots of postoperative total corneal astigmatism. The mean vector of the astigmatism (represented by the grey rhombus larger than the other plots) was 0.38 D axis 4° (each ring = 0.5 D, outer ring = 3.0 D) cataract surgery, a particular IOL model is selected by assuming that astigmatism is derived entirely from the cornea and crystalline lens. That is to say, if other factors producing astigmatism exist, they must cause unex- pected postoperative refractive astigmatism. Analyzing postoperative refractive astigmatism of unknown origin may help more accurate astigmatism correction. Discussion BMC Ophthalmology (2016) 16:212 Page 7 of 9 Page 7 of 9 Fig. 11 Double-angle plots of difference in vector between postoperative refractive and total corneal astigmatism. The mean vector of the difference (represented by the grey rhombus larger than the other plots) was 0.12 D axis 135° (each ring = 0.5 D, outer ring = 3.0 D) investigated internal astigmatism in eyes implanted with non-toric IOL, and reported that the mean vector of in- ternal astigmatism, corresponding to difference between refractive and keratometric astigmatism in our study, was 0.24 D and 0.38 D, respectively, which coincided approximately with our results (0.30 D). The mean difference in vector and magnitude between postoperative refractive and total corneal astigmatism was closer to 0 D as compared to that between postoper- ative refractive and keratometric astigmatism. This sug- gests that preoperative actual measurement of posterior corneal astigmatism may lead to more accurate postop- erative astigmatism correction. When we further exam- ined depending on the types of astigmatism such as ATR, WTR, oblique astigmatism, the mean differences in magnitude and vector between postoperative refract- ive and total corneal astigmatism was closer to 0 D in eyes with WTR or oblique astigmatism, but not in eyes with ATR astigmatism. We are unaware of the exact rea- son why eyes with ATR astigmatism had no similar ten- dency. This issue should be examined in a larger population because the number of eyes with ATR astig- matism was somewhat small in the current study. Although several previous reports have already men- tioned the discrepancy in posterior corneal astigmatism between actual and estimated values [4, 5], this is the first report to elucidate the more detailed influences of posterior corneal astigmatism on total refractive error in pseudophakic eyes. Based on the current findings, it can be concluded that incorporating the data of posterior corneal curvature into preoperative IOL power calcula- tion results in better refractive outcomes after cataract surgeries with toric IOLs. Fig. 11 Double-angle plots of difference in vector between postoperative refractive and total corneal astigmatism. The mean vector of the difference (represented by the grey rhombus larger than the other plots) was 0.12 D axis 135° (each ring = 0.5 D, outer ring = 3.0 D) than that between postoperative refractive and kerato- metric astigmatism (Figs. 3, 4, 5 and 6). Discussion Theoretically, considering that astigmatism is derived entirely from the cornea and crystalline lens, refractive astigmatism must be equal to corneal astigmatism, in cases of non-toric IOL insertion eyes. Therefore, the above findings imply that the relationship between refractive and total corneal astigmatism is more theoretical than that between re- fractive and keratometric astigmatism. We also calcu- lated the difference between postoperative refractive and two types of corneal astigmatism (keratometric and total corneal astigmatism) (Figs. 10 and 11). Difference between refractive and keratometric astigmatism was also referred to as “internal astigmatism” in some reports [2, 6, 19]. Teus et al. [6] and Tejedor et al. [2] g The examination of individual cases showed that 56.0 and 17.2% of eyes showed difference between postopera- tive refractive and total corneal astigmatism of greater than 0.5 D and 1.0 D, respectively. This means that ap- proximately half of eyes that are planned to undergo toric IOL implantation may exhibit astigmatism correc- tion errors greater than 0.5 D postoperatively even if actual measurement data of posterior corneal astigma- tism is incorporated into preoperative IOL power calcu- lation. We tried to find the associated factors which cause the greater difference, but no predictive factors could be identified (Table 3). The difference between postoperative refractive astigmatism and total corneal astigmatism may involve any unknown astigmatism other than corneal and lenticular astigmatism (e.g., ret- inal, vitreous) [18, 19]. Further studies should be con- ducted to clarify the causes other than posterior corneal astigmatism which induce postoperative refractive errors. Table 3 Patients’ data of two groups Parameter (postoperative data) greater than 0.5 D less than 0.5 D P value Total number (eyes) 36 (56%) 28 (44%) - Age (year) 73.6 ± 10.1 69.4 ± 9.4 .093 Spherical equivalent refraction (D) −0.80 ± 1.16 −1.33 ± 1.68 .143 Refractive astigmatism (D) 0.92 ± 0.55 0.91 ± 0.39 .868 Keratometric astigmatism (D) 0.93 ± 0.48 0.81 ± 0.38 .296 Total corneal astigmatism (D) 0.97 ± 0.50 0.90 ± 0.40 .553 Axial length (mm) 23.44 ± 1.45 23.97 ± 1.94 .209 IOL power (D) 21.38 ± 3.50 20.09 ± 3.67 .159 D diopter Table 3 Patients’ data of two groups Parameter (postoperative data) greater than 0.5 D less than 0.5 D P value Table 3 Patients’ data of two groups Table 3 Patients’ data of two groups There are some limitations in this study. Discussion In this study, we researched influence of posterior corneal astigmatism on postoperative refractive astigmatism by comparing keratometric and total corneal astigmatism in pseudophakic eyes after non-toric IOL implantation. We found that the mean magnitude of keratometric and total corneal astigmatism were significantly different (Fig. 2). Moreover, the correlation between postoperative refractive and total corneal astigmatism was stronger Fig. 10 Double-angle plots of difference in vector between postoperative refractive and keratometric astigmatism. The mean vector of the difference (represented by the grey rhombus larger than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer ring = 3.0 D) y, p Fig. 8 Double-angle plots of postoperative keratometric astigmatism. The mean vector of the astigmatism (represented by the grey rhombus larger than the other plots) was 0.16 D axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 10 Double-angle plots of difference in vector between postoperative refractive and keratometric astigmatism. The mean vector of the difference (represented by the grey rhombus larger than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 8 Double-angle plots of postoperative keratometric astigmatism. The mean vector of the astigmatism (represented by the grey rhombus larger than the other plots) was 0.16 D axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 10 Double-angle plots of difference in vector between postoperative refractive and keratometric astigmatism. The mean vector of the difference (represented by the grey rhombus larger than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 10 Double-angle plots of difference in vector between postoperative refractive and keratometric astigmatism. The mean vector of the difference (represented by the grey rhombus larger than the other plots) was 0.30 D axis 164° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 8 Double-angle plots of postoperative keratometric astigmatism. The mean vector of the astigmatism (represented by the grey rhombus larger than the other plots) was 0.16 D axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Fig. 8 Double-angle plots of postoperative keratometric astigmatism. The mean vector of the astigmatism (represented by the grey rhombus larger than the other plots) was 0.16 D axis 13° (each ring = 0.5 D, outer ring = 3.0 D) Sano et al. Author details 1 1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1 Amakubo, Tsukuba, Ibaraki 305-8576, Japan. 2Hitachi General Hospital, 2-1-1 Jonancho, Hitachi, Ibaraki 317-0077, Japan. 3Seinan Medical Center hospital, 4 1Department of Ophthalmology, University of Tsukuba Hospital, 2-1-1 Amakubo, Tsukuba, Ibaraki 305-8576, Japan. 2Hitachi General Hospital, 2-1-1 Jonancho, Hitachi, Ibaraki 317-0077, Japan. 3Seinan Medical Center hospital, 2190 Sakaimachi, Sashimagun, Ibaraki 306-0433, Japan. 4Department of Ophthalmology, Faculty of Medicine, University of Tsukuba, 2-1-1 Amakubo, Tsukuba, Ibaraki 305-8576, Japan. Discussion First, we only simulated the influence of posterior corneal astigmatism Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. BMC Ophthalmology (2016) 16:212 Page 8 of 9 on postoperative refractive astigmatism in pseudophakic eyes after non-toric IOL implantation. Prospective stud- ies should be conducted to compare the surgical out- comes between eyes in which preoperative IOL power calculation is done using total corneal power (including actual measurements of the posterior corneal curvature) and keratometric power (neglecting actual measurement of the posterior corneal curvature). Second, the degree of preoperative corneal astigmatism was relatively small among our study population. Similar research will be necessary also in eyes with larger corneal astigmatism, because candidates for toric IOL implantation generally have considerable corneal astigmatism. Third, we didn’t consider the influence of tilt and dislocation of im- planted IOLs on postoperative refraction. There is a possibility that these factors affect postoperative refract- ive astigmatism. Further studies should also be con- ducted to elucidate this point. Ethics approval and consent to participate This is a clinical study, and the research was approved by the Institutional Review Board of the University of Tsukuba Hospital and conducted according to the tenets of the Declaration of Helsinki. The trial registration (No. H 24–92) was requested on November 30, 2012. Written informed consent to participate was obtained for each subject prior to the study. Abbreviations 6. Teus MA, Arruabarrena C, Hernández-Verdejo JL, Sales-Sanz A, Sales- Sanz M. Correlation between keratometric and refractive astigmatism in pseudophakic eyes. J Cataract Refract Surg. 2010;36(10):1671–5. doi:10.1016/j.jcrs.2010.05.010 [PubMed]. AS-OCT: Anterior segment optical coherence tomography; ATR: Against- the-rule; D: Diopter; IOL: Intraocular lens; SD: Standard deviation; WTR: With- the-rule 7. Ueno Y, Hiraoka T, Beheregaray S, Miyazaki M, Ito M, Oshika T. Age-related changes in anterior, posterior, and total corneal astigmatism. J Refract Surg. 2014;30(3):192–7. doi:10.3928/1081597X-20140218-01 [PubMed]. 7. Ueno Y, Hiraoka T, Beheregaray S, Miyazaki M, Ito M, Oshika T. Age-related changes in anterior, posterior, and total corneal astigmatism. J Refract Surg. 2014;30(3):192–7. doi:10.3928/1081597X-20140218-01 [PubMed]. Author’s contributions MS was responsible for, collection of data, analysis and interpretation of results and wrote the first draft of the manuscript. YU participated in its design and data collection. TH and HI and TO (Tomohiro Ogami) revised the intellectual content of the draft. TO (Tetsuro Oshika) supervised the study. All authors reviewed and approved the final manuscript. Conclusions This study showed that the relationship between refract- ive and total corneal astigmatism is more intimate than that between refractive and keratometric astigmatism in pseudophakic eyes, and the vector difference is closer to 0 D in the former than in the latter. If we use actual measurements of the posterior corneal curvature for evaluations prior to cataract surgery, the amount of unexpected postoperative refractive astigmatism might be reduced, resulting in improved uncorrected visual acuity. However, even after eliminating the discrepancy between actual and estimated values of posterior corneal astigmatism, some amount of postoperative refractive astigmatism of unknown origin persisted. Our findings warrant further investigation to find causes of unex- pected astigmatism correction errors beyond posterior corneal astigmatism in order to improve visual function after toric IOL implantation. Received: 19 April 2016 Accepted: 24 November 2016 Received: 19 April 2016 Accepted: 24 November 2016 References 1. Hayashi K, Manabe S, Yoshida M, Hayashi H. Effect of astigmatism on visual acuity in eyes with a diffractive multifocal intraocular lens. J Cataract Refract Surg. 2010;36(8):1323–9. doi:10.1016/j.jcrs.2010.02.016 [PubMed]. 2. Tejedor J, Guirao A. Agreement between refractive and corneal astigmatism in pseudophakic eyes. Cornea. 2013;32(6):783–90. doi:10.1097/ICO. 0b013e31826dd44b [PubMed]. 3. Frings A, Katz T, Steinberg J, Druchkiv V, Richard G, Linke SJ. Ocular residual astigmatism: effects of demographic and ocular parameters in myopic laser in situ keratomileusis. J Cataract Refract Surg. 2014;40(2):232–8. doi:10.1016/j. jcrs.2013.11.015 [PubMed]. 4. Koch DD, Ali SF, Weikert MP, Shirayama M, Jenkins R, Wang L. Contribution of posterior corneal astigmatism to total corneal astigmatism. J Cataract Refract Surg. 2012;38(12):2080–7. doi:10.1016/j. jcrs.2012.08.036 [PubMed]. 5. Ho JD, Tsai CY, Liou SW. Accuracy of corneal astigmatism estimation by neglecting the posterior corneal surface measurement. Am J Ophthalmol. 2009;147(5):788–95. doi:10.1016/j.ajo.2008.12.020. 795.e1-2 [PubMed]. Competing interests p g The authors declare that they have no competing interest. Acknowledgements The authors would like to acknowledge all members of the Department of Ophthalmology, Faculty of Medicine, University of Tsukuba, Hitachi General Hospital and Seinan Medical Center hospital. The authors have no public or private financial support to disclose. None of the authors has a financial or proprietary interest in any material or method mentioned herein. 8. Dubbelman M, Sicam VA, Van der Heijde GL. The shape of the anterior and posterior surface of the aging human cornea. Vision Res. 2006;46(6–7): 993–1001 [PubMed]. 9. Ho JD, Tsai CY, Tsai RJ, Kuo LL, Tsai IL, Liou SW. Validity of the keratometric index: evaluation by the Pentacam rotating Scheimpflug camera. J Cataract Refract Surg. 2008;34(1):137–45. doi:10.1016/j.jcrs.2007. 09.033 [PubMed]. 10. Ho JD, Liou SW, Tsai RJ, Tsai CY. Effects of aging on anterior and posterior corneal astigmatism. Cornea. 2010;29(6):632–7. doi:10.1097/ICO. 0b013e3181c2965f [PubMed]. Funding None. 11. Ueno Y, Hiraoka T, Miyazaki M, Ito M, Oshika T. Corneal thickness profile and posterior corneal astigmatism in normal corneas. Ophthalmology. 2015; 122(6):1072–8. doi:10.1016/j.ophtha.2015.01.021 [PubMed]. 10. Ho JD, Liou SW, Tsai RJ, Tsai CY. Effects of aging on anterior and posterior corneal astigmatism. Cornea. 2010;29(6):632–7. doi:10.1097/ICO. 0b013e3181c2965f [PubMed]. 9. Ho JD, Tsai CY, Tsai RJ, Kuo LL, Tsai IL, Liou SW. Validity of the keratometric index: evaluation by the Pentacam rotating Scheimpflug camera. J Cataract Refract Surg. 2008;34(1):137–45. doi:10.1016/j.jcrs.2007. 09.033 [PubMed]. AS-OCT: Anterior segment optical coherence tomography; ATR: Against- the-rule; D: Diopter; IOL: Intraocular lens; SD: Standard deviation; WTR: With- the-rule g The authors would like to acknowledge all members of the Department of Ophthalmology, Faculty of Medicine, University of Tsukuba, Hitachi General Hospital and Seinan Medical Center hospital. The authors have no public or private financial support to disclose. None of the authors has a financial or proprietary interest in any material or method mentioned herein. Availability of data and materials Data can be shared upon request. Availability of data and materials Data can be shared upon request. Availability of data and materials Data can be shared upon request. Page 9 of 9 Sano et al. BMC Ophthalmology (2016) 16:212 Sano et al. BMC Ophthalmology (2016) 16:212 12. Koch DD, Jenkins RB, Weikert MP, Yeu E, Wang L. Correcting astigmatism with toric intraocular lenses: effect of posterior corneal astigmatism. J Cataract Refract Surg. 2013;39(12):1803–9. doi:10.1016/j.jcrs.2013.06.027 [PubMed]. 13. Szalai E, Berta A, Hassan Z, Módis Jr L. Reliability and repeatability of swept- source Fourier-domain optical coherence tomography and Scheimpflug imaging in keratoconus. J Cataract Refract Surg. 2012;38(3):485–94. doi:10. 1016/j.jcrs.2011.10.027 [PubMed]. 14. Nakagawa T, Maeda N, Higashiura R, Hori Y, Inoue T, Nishida K. Corneal topographic analysis in patients with keratoconus using 3-dimensional anterior segment optical coherence tomography. J Cataract Refract Surg. 2011;37(10):1871–8. doi:10.1016/j.jcrs.2011.05.027 [PubMed]. 15. Yamaguchi T, Ohnuma K, Tomida D, Konomi K, Satake Y, Negishi K, Tsubota K, Shimazaki J. The contribution of the posterior surface to the corneal aberrations in eyes after keratoplasty. Invest Ophthalmol Vis Sci. 2011;52(9): 6222–9. doi:10.1167/iovs.11-7647 [PubMed]. 16. Holladay JT, Moran JR, Kezirian GM. Analysis of aggregate surgically induced refractive change, prediction error, and intraocular astigmatism. J Cataract Refract Surg. 2001;27(1):61–79 [PubMed]. 17. Visser N, Berendschot TT, Bauer NJ, Nuijts RM. Vector analysis of corneal and refractive astigmatism changes following toric pseudophakic and toric phakic IOL implantation. Invest Ophthalmol Vis Sci. 2012;53(4):1865–73. doi:10.1167/iovs.11-8868 [PubMed]. 18. Tang M, Chen A, Li Y, Huang D. Corneal power measurement with Fourier- domain optical coherence tomography. J Cataract Refract Surg. 2010;36(12): 2115–22. doi:10.1016/j.jcrs.2010.07.018 [PubMed]. 19. Srivannaboon S. Internal astigmatism and its correlation to corneal and refractive astigmatism. J Med Assoc Thai. 2003;86(2):166–71 [PubMed]. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step:
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Statistical Considerations in Demonstrating CMC Analytical Similarity for a Biosimilar Product
Statistics in biopharmaceutical research
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WAGTTBAS What A Great Time To Be A Statistician! 2015 Duke Industry Stat Symposium 1 This presentation reflects the views of the authors and should not be construed to represent FDA’s views or policies. 2 2 OUTLINES 2015 Duke Industry Stat Symposium 3 y g gg • Clinicians or scientists are interested to measure the drug effect instead of p-values. • Significance of drug effect can be derived from confidence interval 2015 Duke Industry Stat Symposium 4 I. Introduction I. Introduction • The debate between reporting p-value of statistical test or confidence interval in clinical trial, a few years ago was meaningful when both CI and statistical test lead to the consistent decision to the hull hypothesis. • However, it was never clearly emphasized on the difference on hypothesis test and general confidence interval estimation. • However, it was never clearly emphasized on the difference on hypothesis test and general confidence interval estimation. • Furthermore, due to the recent development of statistical testing in drug development, the duality may lead to some of the difficulties in constructing test-based confidence interval to be consistent the with significance test. • We illustrate the problems with a few examples. • Furthermore, due to the recent development of statistical testing in drug development, the duality may lead to some of the difficulties in constructing test-based confidence interval to be consistent the with significance test. • We illustrate the problems with a few examples. • We illustrate the problems with a few examples. 2015 Duke Industry Stat Symposium 5 5 ng Mean Difference of Normal Outcomes ), , ( ~ 2 i i i N   i = T, R represent the outcome of test ducts. est of mean difference: study objective is to test the mean difference   versus       R T A H : 0. Assuming equal sample size n and variance 2 , the ul unbiased test we use is the t-test with the following biased estimate of      R T and 2s the estimate of ance . T is a monotone statistic that reaches its maximum te at 0       R T . 2015 Duke Industry Stat Symposium 6 II. Comparing Mean Difference of Normal Outcomes Assuming ), , ( ~ 2 i i i N X   i = T, R represent the outcome of test reference products. II. Comparing Mean Difference of Normal Ou Assuming ), , ( ~ 2 i i i N X   i = T, R represent the outcome of test reference products. Assuming ), , ( ~ 2 i i i N X   i = T, R represent the outcome of test reference products. Considering test of mean difference: Considering test of mean difference: When the study objective is to test the mean difference       R T H : 0 versus       R T A H : When the study objective is to test the mean difference When the study objective is to test the mean difference       R T H : 0 versus       R T A H : where is ≥ 0. Assuming equal sample size n and variance 2 , the most powerful unbiased test we use is the t-test with the following statistic     R T H : 0 versus       R T A H : where is ≥ 0. Assuming equal sample size n and variance 2 , the most powerful unbiased test we use is the t-test with the following statistic n s T 2  with ˆ the unbiased estimate of      R T and 2s the estimate of common variance . T is a monotone statistic that reaches its maximum type I error rate at 0       R T . with ˆ the unbiased estimate of      R T and 2s the estimate of common variance . T is a monotone statistic that reaches its maximum type I error rate at 0       R T . 2015 Duke Industry Stat Symposium 6 6 ng distribution of T at 0       R T is t with degrees of freedom 2(n-1). he null hypothesis if T > t(0.975, 2(n-1)). er hand,      R T can be estimated with a (1-α)% interval ))) 1 ( 2,2 / 1 ( 2 ˆ )), 1 ( 2,2 / 1 ( 2        n t s n t s   o reject H0 if the lower confidence level is greater than wer confidence limit can be derived from the test d its sampling distributions. decisions made with both significance test and interval are consistent although they may be derived ntly.  The sampling distribution of T at 0       R T is t with degrees of freedom of freedom 2(n-1).  The sampling distribution of T at 0       R T is t with degrees of freedom of freedom 2(n-1). freedom of freedom 2(n 1).  We reject the null hypothesis if T > t(0.975, 2(n-1)).  We reject the null hypothesis if T > t(0.975, 2(n-1)).  We reject the null hypothesis if T > t(0.975, 2(n-1)).  On the other hand,      R T can be estimated with a (1-α)% confidence interval ))) 1 ( 2,2 / 1 ( 2 ˆ )), 1 ( 2,2 / 1 ( 2 ˆ (          n t s n t s CI    We will also reject H0 if the lower confidence level is greater than 0.  That the lower confidence limit can be derived from the test statistic and its sampling distributions.  That the lower confidence limit can be derived from the test statistic and its sampling distributions.  In this case, decisions made with both significance test and confidence interval are consistent although they may be deriv independently.  In this case, decisions made with both significance test and confidence interval are consistent although they may be derived independently. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 7 III. Comparing Mean Difference of Binary Outcomes Assuming ), ( ~ i i P B X i = T, R represent the binary outcome of test and reference products. Let us consider significance test, non-inferiority and equivalence tests under the setting separately. Significance test: Significance test: When comparing two proportions, the significance testing hypotheses are, 0 : 0   R T P P H versus 0 :   R T A P P H Let R T P P    , the unbiased estimate of is R T P P ˆ ˆ ˆ    . The asymptotic test of the hypotheses is a score test in the form that ) ˆ ˆ ( ˆ ˆ R T R T P P e P P Z    (III.1) ) ( e is the standard error of estimation. 2015 Duke Industry Stat Symposium 8 Significance test: When comparing two proportions, the significance testing hypotheses are, 0 : 0   R T P P H versus 0 :   R T A P P H Let R T P P    , the unbiased estimate of is R T P P ˆ ˆ ˆ    . The asymptotic test of the hypotheses is a score test in the form that ) ˆ ˆ ( ˆ ˆ R T R T P P e P P Z    (III.1) ) ( e is the standard error of estimation. 2015 Duke Industry Stat Symposium 8 8 est is monotone, i.e. if 2 1    the Z value of 1 the Z , the type I error rate of Z reaches its maximum at = ng distribution of the statistic is derived from 0 |   Z . y, the standard error is derived with restriction to = 0. 0 |)   R P ] = n P P /) 1( 2  (III.2) 2 /) R P . When using this significance test, we reject the s if Z > Z(1 -α/2) asmptotically. stimate the standard error ) ˆ ˆ ( R T P P e  without to null hypothesis, we have ) ˆ ˆ ( R T P P e  = P P ) 1(   Since the test is monotone, i.e. if 2 1    the Z value of 1 the Z value of 2 , the type I error rate of Z reaches its maximum at = 0.  The sampling distribution of the statistic is derived from 0 |   Z . Accordingly, the standard error is derived with restriction to = 0. It leads to with 2 /) ( R T P P P   . When using this significance test, we reject the null hypothesis if Z > Z(1 -α/2) asmptotically.  When we estimate the standard error ) ˆ ˆ ( R T P P e  without restriction to null hypothesis, we have ) ˆ ˆ ( R T P P e  = P P P P R R T T ) 1( ) 1(    (III 3) n 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 9  It can be shown that n P P P P R R T T ) 1( ) 1(     n P P /) 1( 2  . That means restricted standard error is at least as large as the unrestricted standard error.  The conventional confidence interval of is ) 2 / 1( ˆ (     Z n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( ˆ     Z n P P P P R R T T ) 1( ) 1(    ).  Using conventional confidence interval, one may claim superiority if ) 2 / 1( ˆ     Z n P P P P R R T T ) 1( ) 1(    > 0. It is inconsistent to the significance test constructed under null hypothesis.  It can be shown that n P P P P R R T T ) 1( ) 1(     n P P /) 1( 2  . That means restricted standard error is at least as large as the unrestricted standard error. est is monotone, i.e. if 2 1    the Z value of 1 the Z , the type I error rate of Z reaches its maximum at = ng distribution of the statistic is derived from 0 |   Z . y, the standard error is derived with restriction to = 0. 0 |)   R P ] = n P P /) 1( 2  (III.2) 2 /) R P . When using this significance test, we reject the s if Z > Z(1 -α/2) asmptotically. stimate the standard error ) ˆ ˆ ( R T P P e  without to null hypothesis, we have ) ˆ ˆ ( R T P P e  = P P ) 1(  It is also pointed out the other two test statistics with small to moderate sample sizes, the type I error rate is not controlled.  With a continuity correction, the asymptotic test based on (III.1) becomes ) ˆ ˆ ( / 1 ˆ ˆ R T R T P P e n P P Z      With a continuity correction, the asymptotic test based on (III.1) becomes ) ˆ ˆ ( / 1 ˆ ˆ R T R T P P e n P P Z      The continuity correction adjusted confidence interval is then, ( ) 2 / 1( / 1 ˆ (      Z n n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( / 1 ˆ      Z n n P P P P R R T T ) 1( ) 1(    )  It was pointed out by Farrington and Manning (1990, SIM ) that all three statistics converge to the standard normal distribution. But they argued that test statistic using (II.2) is both theoretically correct and convergent to N(0,1) faster. It is also pointed out the other two test statistics with small to moderate sample sizes, the type I error rate is not controlled. ecomes ) ( R T P P e  e continuity correction adjusted confidence interval is then, ) 2 / 1( /   Z n n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( / 1 ˆ      Z n n P P P P R R T T ) 1( ) 1(    ) was pointed out by Farrington and Manning (1990, SIM ) that three statistics converge to the standard normal distribution. ut they argued that test statistic using (II.2) is both theoretically rrect and convergent to N(0,1) faster. It is also pointed out the her two test statistics with small to moderate sample sizes, the pe I error rate is not controlled. est is monotone, i.e. if 2 1    the Z value of 1 the Z , the type I error rate of Z reaches its maximum at = ng distribution of the statistic is derived from 0 |   Z . y, the standard error is derived with restriction to = 0. 0 |)   R P ] = n P P /) 1( 2  (III.2) 2 /) R P . When using this significance test, we reject the s if Z > Z(1 -α/2) asmptotically. stimate the standard error ) ˆ ˆ ( R T P P e  without to null hypothesis, we have ) ˆ ˆ ( R T P P e  = P P ) 1(   The conventional confidence interval of is ) 2 / 1( ˆ (     Z n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( ˆ     Z n P P P P R R T T ) 1( ) 1(    ).  Using conventional confidence interval, one may claim superiority if ) 2 / 1( ˆ     Z n P P P P R R T T ) 1( ) 1(    > 0. It is inconsistent to the significance test constructed under null hypothesis. standard error.  The conventional confidence interval of is ) 2 / 1( ˆ (     Z n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( ˆ     Z n P P P P R R T T ) 1( ) 1(    ).  Using conventional confidence interval, one may claim superiority if ) 2 / 1( ˆ     Z n P P P P R R T T ) 1( ) 1(    > 0. It is inconsistent to the significance test constructed under null hypothesis 2015 Duke Industry Stat Symposium 10  With a continuity correction, the asymptotic test based on (III.1) becomes ) ˆ ˆ ( / 1 ˆ ˆ R T R T P P e n P P Z      The continuity correction adjusted confidence interval is then, ( ) 2 / 1( / 1 ˆ (      Z n n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( / 1 ˆ      Z n n P P P P R R T T ) 1( ) 1(    )  It was pointed out by Farrington and Manning (1990, SIM ) that all three statistics converge to the standard normal distribution. But they argued that test statistic using (II.2) is both theoretically correct and convergent to N(0,1) faster. est is monotone, i.e. if 2 1    the Z value of 1 the Z , the type I error rate of Z reaches its maximum at = ng distribution of the statistic is derived from 0 |   Z . y, the standard error is derived with restriction to = 0. 0 |)   R P ] = n P P /) 1( 2  (III.2) 2 /) R P . When using this significance test, we reject the s if Z > Z(1 -α/2) asmptotically. stimate the standard error ) ˆ ˆ ( R T P P e  without to null hypothesis, we have ) ˆ ˆ ( R T P P e  = P P ) 1(   The continuity correction adjusted confidence interval is then, ( ) 2 / 1( / 1 ˆ (      Z n n P P P P R R T T ) 1( ) 1(    , ) 2 / 1( / 1 ˆ      Z n n P P P P R R T T ) 1( ) 1(    ) 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 11 Non-inferiority and equivalence tests inferiority and equivalence tests When comparing two proportions, the non-inferiority hypotheses     R T P P H : 0 versus     R T A P P H : re δ > 0 is a non-inferiority constant margin. asymptotic test of the hypotheses is a score test in the form that ) ˆ ˆ ( ˆ ˆ R T R T P P e P P Z      (III.4) ) ˆ ˆ ( 1 ˆ ˆ R T R T P P e n P P Z       with continuity correction, re ) ( e is the standard error of estimation. The sampling distribution e statistic is derived from     | Z . Accordingly, the standard error is ed as the maximum likelihood estimate restricted to = - δ. It can hown as (Farrington and Manning, 1990) n P P P P P P e R R T T R T / )] ~ 1( ~ ) ~ 1( ~ [ |) ˆ ˆ (        2015 Duke Industry Stat Symposium 12 y q hen comparing two proportions, the non-inferiority hypotheses When comparing two proportions, the non-inferiority hypotheses re, with a =2 b =-[2+ T p + R p + 3δ] c =δ2 +δ(2 T p +2) + T p + R p d = - T p δ(1+δ) where T p and R p are the sample proportions of test and reference respectively, T P~ = R P~ + δ. Again without restriction, we have ) ˆ ˆ ( R T P P e  = n P P P P R R T T ) 1 ( ) 1 (    are,     R T P P H : 0 versus     R T A P P H : where δ > 0 is a non-inferiority constant margin. The asymptotic test of the hypotheses is a score test     R T P P H : 0 versus     R T A P P H :   R T P P H : 0 versus   R T A P P H : where δ > 0 is a non-inferiority constant margin. The asymptotic test of the hypotheses is a score test in the fo where δ > 0 is a non-inferiority constant margin. y g ptotic test of the hypotheses is a score test in the form that ) ˆ ˆ ( ˆ R T P P P    (III.4) y g The asymptotic test of the hypotheses is a score test in the form that mptotic test of the hypotheses is a score test in the form that ˆ ˆ P P  ) ˆ ˆ ( ˆ ˆ R T R T P P e P P Z      ) ˆ ˆ ( 1 ˆ ˆ R T R T P P e n P P Z       w 12 where T P~ and R P~ are the maximum likelihood estimates of PT and PR restricted to H0. For testing against H0, T P~ and R P~ are shown to be the solutions in (δ, 1) of the following equation aX3 + bX2 + cX +d =0 with a =2 b =-[2+ T p + R p + 3δ] c =δ2 +δ(2 T p +2) + T p + R p d = - T p δ(1+δ) nfidence decision is not different from the one for superiority test we compare its lower limit with – δ. This confidence decision is not different from the one for superiority test except we compare its lower limit with – δ. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 13 test ence test consists of two one-sided hypotheses     R P versus     R T A P P H : 1    R P versus    R T A P P H : 2 ting both null hypotheses, one shows that    R P istic corresponds to testing the second one-sided s ) ˆ ˆ R R P P   (III.5) ) ˆ ˆ 1 ˆ R T R P P n P     with continuity correction. d error is derived as the maximum likelihood estimate = δ. It can be derived as n P P P P R R T T / )] ~ 1( ~ ) ~ 1( ~ [ |)      2015 Duke Industry Stat Symposium 14 The equivalence test consists of two one-sided hypotheses     R T P P H : 01 versus     R T A P P H : 1    R T P P H : 02 versus    R T A P P H : 2 When rejecting both null hypotheses, one shows that       R T P P The test statistic corresponds to testing the second one-sided hypotheses is hypotheses is ) ˆ ˆ ( ˆ ˆ R T R T P P e P P Z      (III.5) 14 where TP~ and R P~ are the maximum likelihood estimates of PT and PR restricted to H02. For testing against H0, TP~ and RP~ are shown to be the solutions in (δ, 1) of the following equation 3 2 with On the other hand, using the confidence interval, the decision of equivalence is derived with the unrestricted maximum likelihood estimate ) ˆ ˆ ( R T P P e  = n P P P P R R T T ) 1 ( ) 1 (    15 The inconsistency applies to any distribution(such as Bernoulli and Poisson) of which the variance is a function is linearly dependent to the mean 2015 Duke Industry Stat Symposium 16 lternative Comparisons of Normal Outcomes st for exchangeability hypotheses involving both mean and y of significance test and confidence interval decision rules istent. for a probability hypothesis of non-inferiority such as ) 1(5.0 ) Pr( : vs. ) 1(5.0 ) P L X X H P L X R T a R        ecified margin and P a pre-specified percentage. rmality assumption, ) 2, ( ~ 2    R T R T N X X   , Tsong and nd Dong and Tsong (2015) showed the one-sided tolerance ) of R T X X  with significance level 1-α/2 and coverage 5(1+P). One reject the null hypothesis if P L > L. It is an IV. Alternative Comparisons of Normal Outcomes VI.1 Considering test for exchangeability hypotheses involving both mean and variance, the duality of significance test and confidence interval decision rules may not be as consistent. For example, for a probability hypothesis of non-inferiority suc For example, for a probability hypothesis of non-inferiority such as ) 1(5.0 ) Pr( : vs. ) 1(5.0 ) Pr( : 0 P L X X H P L X X H R T a R T         where L is a pre-specified margin and P a pre-specified percentage. p p y yp y 5.0 ) Pr( : vs. ) 1(5.0 ) Pr( : 0 L X X H P L X X H R T a R T        where L is a pre-specified margin and P a pre-specified percentage. 0 ) Pr( : vs. ) 1(5.0 ) Pr( : 0 L X X H P L X X H R T a R T        where L is a pre-specified margin and P a pre-specified percentage.  Under the normality assumption, ) 2, ( ~ 2    R T R T N X X   , Tsong and Shen (2007) and Dong and Tsong (2015) showed the one-sided tolerance interval ) , (  P L of R T X X  with significance level 1-α/2 and coverage percentage 0.5(1+P). One reject the null hypothesis if P L > L. It is an exact test. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 17 However , for an equivalence hypotheses •Corresponding to the two one-sided tests, •Corresponding to the two one-sided tests, •Corresponding to the two one-sided tests, p g , 2015 Duke Industry Stat Symposium 18  Test based confidence interval is then ) , (  P L ∩ ) , ( P U  . On the other hand, if we use a regular two-sided tolerance interval with 1 – α confidence level and P coverage, we are considering a tolerance interval ) , ( kS X X kS X X R T R T     with k determined by the sample size n, α and P for two-sided tolerance interval.  In this case, the regular confidence interval provides a different decision rule that significance test.  Test based confidence interval is then ) , (  P L ∩ ) , ( P U  . On the other hand, if we use a regular two-sided tolerance interval with 1 – α confidence level and P coverage, we are considering a tolerance interval 18 2015 Duke Industry Stat Symposium n the other hand, if we use a regular two-sided tolerance terval with 1 – α confidence level and P coverage, we are onsidering a tolerance interval ) , ( kS X X kS X X R T R T     ith k determined by the sample size n, α and P. ne may reject the null hypothesis if L kS X X R T    . ther using approximation method or exact method, this terval provides no assurance that ) , ( kS X X R T    covers ss that < 0.5(1-P) at 1 - α/2 level. this case, the regular confidence interval provides a fferent decision rule from the significance test. On the other hand, if we use a regular two-sided tolerance interval with 1 – α confidence level and P coverage, we are considering a tolerance interval ) , ( kS X X kS X X R T R T     with k determined by the sample size n, α and P.  One may reject the null hypothesis if L kS X X R T    .  Either using approximation method or exact method, this interval provides no assurance that ) , ( kS X X R T    covers less that < 0.5(1-P) at 1 - α/2 level.  In this case, the regular confidence interval provides a different decision rule from the significance test. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 19 VI.2 Asymptotic Tests for Variance-Adjusted Equivalence w Normal Endpoints (Chen, Weng, Dong & Tsong, 2015) Test equivalence hypothesis •Two one-sided hypotheses VI.2 Asymptotic Tests for Variance-Adjusted Equivalence Normal Endpoints (Chen, Weng, Dong & Tsong, 2015) Test equivalence hypothesis Two one sided hypotheses VI.2 Asymptotic Tests for Variance-Adjusted Equivalence wit Normal Endpoints (Chen, Weng, Dong & Tsong, 2015) Test equivalence hypothesis VI.2 Asymptotic Tests for Variance-Adjusted Equivalence with Normal Endpoints (Chen, Weng, Dong & Tsong, 2015) Test equivalence hypothesis •Two one-sided hypotheses symptotic Tests for Variance-Adjusted Equivalence with al Endpoints (Chen, Weng, Dong & Tsong, 2015) equivalence hypothesis one-sided hypotheses 2015 Duke Industry Stat Symposium 20 – Unconstrained maximum likelihood estimates – Unconstrained uniformly minimum variance unbiased estimates (Ahn and Fessler, 2003) – Constrained maximum likelihood estimates (Farrington and Manning, 1990; Ng, Gu, and Tang, 2007; Stucke and Kieser, 2013) – Constrained maximum likelihood estimates (Farrington and Manning, 1990; Ng, Gu, and Tang, 2007; Stucke and Kieser, 2013) 2015 Duke Industry Stat Symposium 21 • Under , if , the log-likelihood function • Under , if , the log-likelihood function 22 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 22 Statistical inference for – Based on – Variance estimates of zL – estimates , including MLE, UMVUE, constrained MLE – Test statistic – P-value = – Reject if p-value < Similar test procedure for 23 2015 Duke Industry Stat Symposium • Statistical inference for – Based on – Variance estimates of zL – estimates , including MLE, UMVUE, constrained MLE – Test statistic – P-value = – Reject if p-value < Similar test procedure for 23 2015 Duke Industry Stat Symposium 23 Type I error rate comparison based on simulation Set cL=1.5, cU=1.5, effect size=1.5, Equal and unequal sample size Generate Repeat times for each parameter configuration Significance level for each one-sided test 24 2015 Duke Industry Stat Symposium Type I error rate comparison based on simulation 24 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 25 2015 Duke Industry Stat Symposium 26 27 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 28 Power comparisons based on simulation • Set cL=1.5 and cU=1.5 • Equal and unequal sample size • • Effect size = -2.0(0.1)2.0 • Generate • Repeat times for each parameter configuration • Significance level for each one-sided test 2015 Duke Industry Stat Symposium Power comparisons based on simulation • Set cL=1.5 and cU=1.5 • Equal and unequal sample size • • Effect size = -2.0(0.1)2.0 • Generate • Repeat times for each parameter configuration • Significance level for each one-sided test 2015 Duke Industry Stat Symposium Power comparisons based on simulation 29 30 Power comparisons based on simulation 2015 Duke Industry Stat Symposium mparisons based on simulation Power comparisons based on simulation 30 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 30 31 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 31 • Bioassays are experiment to measure biological activity (potency) of a drug as a function of concentration/dose; • Relative Potency : ratio of the conc. of the test product that produces the same biological response as one unit of the conc. of the reference product 32 conc. of the reference product ρ < 1: Test drug produces higher response (lower conc. can produce the same response as the ref.); 2015 Duke Industry Stat Symposium VI.3 Statistical Methods for Parallelism Test of Bioassays (Shao, Dong, Torigoe & Tsong, 2015) • Bioassays are experiment to measure biological activity (potency) of a drug as a function of concentration/dose; ρ < 1: Test drug produces higher response (lower conc. can produce the same response as the ref.); 2015 Duke Industry Stat Symposium 32 Parallel-Line Model 33 • Independent • Normality • Homogenous variances of residuals Relative potency 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 33 Equivalence Test based on Slope Ratio • Hypothesis: test if the ratio of slopes is close to 1. S L T H    : 0 S U T    S U T S L a H        : Linearized hypothesis or Use Wald test with restricted and unrestricted standard error 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 34 Pr (Conclude Parallel | Not Parallel) Pr (Conclude Parallel | Not Parallel) Equivalence Test based on Slope Ratio: Fieller’s Method 35 quivalence Test based on Slope Ratio: Fieller s Method 2 2 2 / , ~ (0, ) T S T S T S N           2015 Duke Industry Stat Symposium 3 2 2 2 / , ~ (0, ) T S T S T S N           2015 Duke Industry Stat Symposium 35 Simulation Studies • Output of simulation studies: • Questions to be answered: – Can those methods control the type I error rate (≤ 5%)? – What is the coverage of CI-based approaches (close to 90%)? – What are the impact of sample size and variance on the Type I error rate and coverage? – Is the decision rule of Test Stat consistent with CI? Type I Error Rate Pr (Conclude Parallel | Not Parallel) Coverage Pr (CI Covers the True Value of Par.) 2015 Duke Industry Stat Symposium • Output of simulation studies: • Questions to be answered: – Can those methods control the type I error rate (≤ 5%)? – What is the coverage of CI-based approaches (close to 90%)? – What are the impact of sample size and variance on the Type I error rate and coverage? – Is the decision rule of Test Stat consistent with CI? 36 Type I Error Rate Pr (Conclude Parallel | Not Parallel) Coverage Pr (CI Covers the True Value of Par.) 2015 Duke Industry Stat Symposium • Output of simulation studies: Type I Error Rate Pr (Conclude Parallel | Coverage Pr (CI Covers the True • Output of simulation studies: Type I Error Rate Pr ( Coverage Pr ( • Output of simulation studies: Pr (CI Covers the True Value of Par.) Questions to be answered: – Can those methods control the type I error rate (≤ 5%)? – What is the coverage of CI-based approaches (close to 90%)? – What are the impact of sample size and variance on the Type I error rate and coverage? – Can those methods control the type I e – What is the coverage of CI-based appro – What is the coverage of CI-based approaches (close to 90 – What are the impact of sample size and variance on the Typ error rate and coverage? – Is the decision rule of Test Stat consistent with CI? 2015 Duke Industry Stat Symposium 36 Ha : 0.80 < βT / βS < 1.25 2015 Duke Industry Stat Symposium 37 Simulation Studies: Type I Error Rate (5%) 38 ation Studies: Type I Error Rate (5%) 2015 Duke Industry Stat Symposium Simulation Studies: Type I Error Rate ( 2015 Duke Industry Stat Symposium 38 ation Studies: Type I Error Rate (5%) 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 38 Simulation Studies: Coverage (Target = 90%) 39 ulation Studies: Coverage (Target 90%) 2015 Duke Industry Stat Symposium Simulation Studies: Coverage (Target 9 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 39 Type I Error Rate vs. Coverage • When the coverage of confidence interval can reach (1-2α) 100%, say 90%, the type I error rate may not reach α (5%): – The definition of coverage is consistent with decision rule of the significant test, not the equivalence test; • When the coverage of confidence interval can reach (1-2α) 100%, say 90%, the type I error rate may not reach α (5%): – The definition of coverage is consistent with decision rule of the significant test, not the equivalence test; 40 Fieller βT/βS βT βS SD.β Coverage (%) RR_low (%) RR_Up (%) RR_Tost (%) N = 5 1.25 5 4 0.5 90.04 79.71 5 1.77 N = 50 1.25 5 4 0.5 90.09 87.4 4.97 0.12 N = 1000 1.25 5 4 0.5 89.93 87.84 4.97 0.02 H0: βT / βS ≠ λL H0: λL < βT / βS < λU 2015 Duke Industry Stat Symposium eller βT/βS βT βS SD.β Coverage (%) RR_low (%) RR_Up (%) RR_Tost (%) = 5 1.25 5 4 0.5 90.04 79.71 5 1.77 = 50 1.25 5 4 0.5 90.09 87.4 4.97 0.12 = 1000 1.25 5 4 0.5 89.93 87.84 4.97 0.02 H0: βT / βS ≠ λL H0: λL < βT / βS < λU H0: λL < βT / βS < λU 2015 Duke Industry Stat Symposium 40 • Fieller’s Method: • Fieller’s Method: Fieller s Method: – provides a reliable inference for the ratio of slopes – controls the type I error rate; – provides a reliable inference for the ratio of slopes – controls the type I error rate; – However, this method is solvable only when both slopes are significant. – However, this method is solvable only when both slopes are significant. • A confidence interval with a 90% two-sided coverage may not assure a type error rate of 5% for equivalence test. • A confidence interval with a 90% two-sided coverage ma not assure a type error rate of 5% for equivalence test. 2015 Duke Industry Stat Symposium 2015 Duke Industry Stat Symposium 41 Conclusion and recommendation In many situations the standard error estimated under null hypothesis is different from the one estimated without restriction. It leads to the failing of consistency in decision making using a significance test and a traditional confidence interval in various situations. Therefore, in various situations, estimation using regular confidence interval should be done after significance testing in order to maintain the consistency of decision making. 2015 Duke Industry Stat Symposium 42 May I answer any question? 2015 Duke Industry Stat Symposium 43
https://openalex.org/W2908766845
https://www.scielo.br/j/lajss/a/VTGCDZkmjH8kLqFxbD5kBtd/?lang=en&format=pdf
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Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete
Latin American Journal of Solids and Structures
2,019
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THEMATIC SECTION - CILAMCE 2018: IBERO-LATIN AMERICAN CONGRESS ON COMPUTATIONAL METHODS IN ENGINEERING Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavinaa,b*  Keywords bond slip da Keywords bond-slip, damage, plasticity, multi-scale, reinforced concrete. Abstract Here we present a multi-scale model to carry out the computation of brittle composite materials reinforced with fibers, and we show its application to standard reinforced concrete. The computation is carried out within an operator-split framework on the macro-scale, which allows for different failure mechanisms to develop in separate phases, as both the concrete and the bond-slip exhibit non-linear behavior. The computations on the micro-scale are performed for each constituent separately. The reinforcement is taken to be linear elastic, and the bond-slip is handled as a plastic deformation. The standard elastoplastic procedure is used to compute the bond stresses, combined with the X-FEM methodology to give the global representation of slip. The crack development in concrete, on the other hand, is described with a damage model with exponential softening, where ED-FEM is used to represent localized failure. A numerical example is shown to test the developed methodology. Ivica Kozarb  niversité de Technologie de Compiègne, Sorbonne Universités, Laboratoire Roberval de Mécanique, Centre de Rech 200 Compiègne, France. E-mail: tea.rukavina@utc.fr, adnan.ibrahimbegovic@utc.fr a Université de Technologie de Compiègne, Sorbonne Universités, Laboratoire Roberval de Mécanique, Centre de Recherche Royallieu, 60200 Compiègne, France. E-mail: tea.rukavina@utc.fr, adnan.ibrahimbegovic@utc.fr b University of Rijeka, Faculty of Civil Engineering, Radmile Matejcic 3, 51000 Rijeka, Croatia. E-mail: ivica.kozar@uniri.hr niversity of Rijeka, Faculty of Civil Engineering, Radmile Matejcic 3, 51000 Rijeka, Croatia. E-mail: ivica.kozar@uniri. *Corresponding author Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Received: October 25, 2018. In Revised Form: October 30, 2018. Accepted: October 31, 2018. Available online: November 01, 2018 http://dx.doi.org/10.1590/1679-78255341 1 INTRODUCTION (2010), where a Drucker-Prager non-associative plasticity model is used, but still the issue with introducing a special element persists. That is why we have decided to simplify the handling of bond-slip, but without sacrificing the model capabilities. Building on top of the work of Ibrahimbegovic et al. (2010), we are proposing a novel and efficient way to take into account the slip between the reinforcing bar and the surrounding concrete. Instead of having a special finite element for every constituent (i.e. a CST element for concrete, a truss bar for steel, and a Q4 element for bond-slip), we have encapsulated all three ingredients in a single finite element, without the need to worry about the nodal connections between the concrete, steel, and interface part. In the next chapter, the formulation of the model is presented, with its specific micro−macro treatment of the interaction of three different constituents, and after that we show the results of the numerical simulation of a reinforced concrete specimen. 1 INTRODUCTION To model fiber-reinforced composite materials, one has to take into account the behavior of each of the constituents, and their interaction. In the case of fiber-reinforced concrete (FRC), we are dealing with concrete, steel fibers, and the interface between them. We can consider standard reinforced concrete as a special case of such a material, with its specific features. First of all, there are not more than a few reinforcement bars embedded in concrete (compared to a large number of randomly oriented short fibres in FRC), that is usually straight, larger in diameter, and anchored at its ends. This facilitates the numerical implementation, as the steel reinforcement can lie along the finite element’s edge, and the anchorage prevents the pull-out of the bar. One of the most important choices to make when dealing with the interaction of two different materials (steel and concrete in this case), is the treatment of the interface between them. Many authors have opted for the insertion of a special interface element, i.e. Ožbolt et al. (2002), Kohnehpooshi and Jaafar (2017), while others have opted for a layered description of reinforced concrete, where each layer can represent a different material, i.e. Jukić et al. (2014) or Šćulac at al. (2014). An example of the utilisation of a special interface element can be found in the work of Dominguez et al. (2005), where a degenerated Q4 element is used to handle the relative displacements between the steel and concrete. Despite the fact that such a description of slip can provide many benefits, it can be gruesome to Received: October 25, 2018. In Revised Form: October 30, 2018. Accepted: October 31, 2018. Available online: November 01, 2018 http://dx.doi.org/10.1590/1679-78255341 1/11 1/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete handle and implement. A simplification regarding the constitutive law implementation in this kind of Q4 element can be found in Ibrahimbegovic et al. (2010), where a Drucker-Prager non-associative plasticity model is used, but still the issue with introducing a special element persists. handle and implement. A simplification regarding the constitutive law implementation in this kind of Q4 element can be found in Ibrahimbegovic et al. 2.1 Displacement field approximation We start by defining the bond-slip as the relative displacement between steel and concrete 𝜶𝑏𝑏𝒅𝑐𝒅𝑠 𝜶𝜶𝑏𝑏𝑏𝑏= 𝒅𝒅𝑐𝑐−𝒅𝒅𝑠𝑠 (1) 𝒅𝑠 where 𝜶𝜶𝑏𝑏𝑏𝑏 represents the slip on the interface along the reinforcement bar, 𝒅𝒅𝑐𝑐 is the concrete displacement, and 𝒅𝒅𝑠𝑠 is the steel displacement. This kind of description allows us to write the displacement field of a single finite element as sum of the standard and the enriched part, according to the X-FEM methodology (Fries and Belytschko, 2010) 𝒖𝒙𝑛𝑁𝒅𝑐𝑛𝑁𝜓𝜶𝑏𝑏 𝒖𝒖(𝒙𝒙)|𝛺𝛺𝑒𝑒= ∑ 𝑖𝑖=1 𝑛𝑛 𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖 𝑐𝑐+ ∑ 𝑖𝑖=1 𝑛𝑛 𝑁𝑁𝑖𝑖𝜓𝜓𝜶𝜶𝑖𝑖 𝑏𝑏𝑏𝑏 (2) 𝑁𝑖𝜓𝛺 𝒖𝒖(𝒙𝒙)|𝛺𝛺𝑒𝑒= ∑ 𝑖𝑖=1 𝑛𝑛 𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖 𝑐𝑐+ ∑ 𝑖𝑖=1 𝑛𝑛 𝑁𝑁𝑖𝑖𝜓𝜓𝜶𝜶𝑖𝑖 𝑏𝑏𝑏𝑏 (2) Equation (2) exploits the partition of unity property of standard shape functions 𝑁𝑁𝑖𝑖, that allows for any other function (in this case the enrichment function 𝜓𝜓) to be reproduced exactly on the element domain 𝛺𝛺𝑒𝑒, (Melenk and Babuška, 1996). The enrichment function 𝜓𝜓 is equal to the Heaviside function that takes the value one in the elements that contain the reinforcement bar (that is located at coordinate 𝑦𝑦), and zero in all the other elements 𝜓𝐻𝑦𝑦𝑦 𝜓𝜓= 𝐻𝐻(𝑦𝑦) = ൜ 1, 𝑦𝑦= 𝑦𝑦 0, 𝑦𝑦≠𝑦𝑦 (3) 𝜓𝜓= 𝐻𝐻(𝑦𝑦) = ൜ 1, 𝑦𝑦= 𝑦𝑦 0, 𝑦𝑦≠𝑦𝑦 (3) 𝜓𝜓= 𝐻𝐻(𝑦𝑦) = ൜ 1, 𝑦𝑦= 𝑦𝑦 0, 𝑦𝑦≠𝑦𝑦 (3) Since in our case the fiber coincides with the lower edge of the enriched element, as shown on Figure 1, we can simplify equation (2) by exploiting the properties of the enrichment function 𝒖𝒙𝑁𝒅𝑁𝛼 ∑ 𝑖𝑖=1 3 𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖 𝑐𝑐+ ∑ 𝑖𝑖=1 2 𝑁𝑁𝑖𝑖𝛼𝛼𝑖𝑖 𝑏𝑏𝑏𝑏 Figure 1: Finite element with enriched degrees of freedom (the reinforcement bar is shown in bold). 𝒖𝒖(𝒙𝒙)|𝛺𝛺𝑒𝑒= ∑ 𝑖𝑖=1 3 𝑁𝑁𝑖𝑖𝒅𝒅𝑖𝑖 𝑐𝑐+ ∑ 𝑖𝑖=1 2 𝑁𝑁𝑖𝑖𝛼𝛼𝑖𝑖 𝑏𝑏𝑏𝑏 (4) Figure 1: Finite element with enriched degrees of freedom (the reinforcement bar is shown in bold). 2/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete The multi-scale characteristics of our model can be observed in the treatment of the failure mechanisms on two levels, macro and micro. 2.1 Displacement field approximation The macro-scale computations are divided in two phases, according to the macro-scale operator-split procedure: the macro-global phase (where the concrete and the steel are taken into account), and the macro-local phase (where the redistribution of slip is computed, with the steel bar acting as a coupling term). But, that is not all, since every constituent is taken care of within a micro-scale computation, which can have its own operator-split solution procedure, as in the case of concrete and bond-slip, giving rise to micro-local and micro-global computations. All of this is important for ensuring the best possible description of the behavior of each constituent, with its own peculiar phases related to specific failure mechanisms. Also, it is important to carry out all the computations in a defined order, based on experimental observations on composite specimens. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 2.2 Micro-scale computations 3/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavina et Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Since the softening part of the response is controlled by an anisotropic multi-surface damage model, we have to consider each direction at the discontinuity surface separately. This kind of model can account for the crack opening in mode I (in the normal direction) and mode II (in the tangential direction). In the following equations the subscripts 1 and 2 denote the direction, and 𝒏𝒏 and 𝒎𝒎 stand for the normal and tangential vector on the discontinuity, respectively. The damage functions for the discontinuity are then 𝜙𝒕𝒏𝜎𝑞𝑐 𝜙𝜙ധ1 = 𝒕𝒕⋅𝒏𝒏−൫𝜎𝜎ധ𝑓𝑓−𝑞𝑞ധ𝑐𝑐൯ 𝜙𝜙ധ2 = |𝒕𝒕⋅𝒎𝒎| −൬𝜎𝜎ധ𝑠𝑠− 𝜎𝜎ന𝑠𝑠 𝜎𝜎ന𝑓𝑓𝑞𝑞ധ𝑐𝑐൰ (8) 𝒕𝜎𝑓𝜎𝑠𝑞 𝜙𝜙ധ1 = 𝒕𝒕⋅𝒏𝒏−൫𝜎𝜎ധ𝑓𝑓−𝑞𝑞ധ𝑐𝑐൯ 𝜙𝜙ധ2 = |𝒕𝒕⋅𝒎𝒎| −൬𝜎𝜎ധ𝑠𝑠− 𝜎𝜎ന𝑠𝑠 𝜎𝜎ന𝑓𝑓𝑞𝑞ധ𝑐𝑐൰ 𝒕 (8) where 𝒕𝒕 is the traction at the discontinuity, 𝜎𝜎ധ𝑓𝑓 is the ultimate stress in tension, and 𝜎𝜎ധ𝑠𝑠 is the ultimate stress in shear. The stress-like softening variable 𝑞𝑞ധ𝑐𝑐 is the same for both directions, and it depends on the chosen softening law, i.e. linear or exponential softening. 𝒕𝜶𝑐 where 𝒕𝒕 is the traction at the discontinuity, 𝜎𝜎ധ𝑓𝑓 is the ultimate stress in tension, and 𝜎𝜎ധ𝑠𝑠 is the ultimate stress in shear. The stress-like softening variable 𝑞𝑞ധ𝑐𝑐 is the same for both directions, and it depends on the chosen softening law, i.e. linear or exponential softening. 𝒕𝜶𝑐 The constitutive equation relating the traction at the discontinuity 𝒕𝒕 and the crack opening 𝜶𝜶𝑐𝑐 is based on a traction-separation cohesive law 𝒕𝐐𝜶𝑐 (9) 𝒕𝒕= 𝐐𝐐ന−1𝜶𝜶𝑐𝑐 (9) where 𝐐𝐐ന is the damage compliance tensor for the discontinuity. The handling of the displacement jump inside the element is done through the introduction of incompatible mode functions 𝑴𝑴. The total displacement field of a single element is then a sum of the standard and the incompatible part 𝒖𝑵𝒅𝑐𝑴𝜶𝑐 (10) 𝒖𝒖= 𝑵𝑵𝒅𝒅𝑐𝑐+ 𝑴𝑴𝜶𝜶𝑐𝑐 from which it follows that the strain can be written as 𝜺𝑩𝒅𝑐𝐆𝜶𝑐 (11) 𝜺𝜺= 𝑩𝑩𝒅𝒅𝑐𝑐+ 𝐆𝐆෩𝜶𝜶𝑐𝑐 where 𝑩𝑩 is the matrix of the shape functions’ derivatives, and 𝑮𝑮 consists of the regular part of the incompatible shape functions’ derivatives. where 𝑩𝑩 is the matrix of the shape functions’ derivatives, and 𝑮𝑮 consists of the regular part of the incompatible shape functions’ derivatives. 2.2 Micro-scale computations To be able to couple the three constituents, each one of them has to provide the stress value that is computed from its own constitutive law and kinematics (Figure 2). Steel computation. Since the steel reinforcement is taken to be linear elastic, its constitutive 𝜎𝑠𝐸𝑠𝜀𝑠 omputation. Since the steel reinforcement is taken to be linear elastic, its constitutive equation is 𝐸𝜀𝑠 (5) (5) 𝜎𝜎𝑠𝑠= 𝐸𝐸𝑠𝑠𝜀𝜀𝑠𝑠 n (5), 𝜎𝜎𝑠𝑠 is the stress, 𝐸𝐸𝑠𝑠 is the Young’s modulus for steel, and 𝜀𝜀𝑠𝑠 is the strain that is defined as 𝑑𝑠 In equation (5), 𝜎𝜎𝑠𝑠 is the stress, 𝐸𝐸𝑠𝑠 is the Young’s modulus for steel, and 𝜀𝜀𝑠𝑠 is the strain that is defined as 𝜀𝑠𝑁𝑑𝑠 In equation (5), 𝜎𝜎𝑠𝑠 is the stress, 𝐸𝐸𝑠𝑠 is the Young’s modulus for steel, and 𝜀𝜀𝑠𝑠 is the strain that is defined as 𝜀𝑠𝑁𝑑𝑠 (6) where the steel displacements are computed according to (1). Figure 2: Constitutive law for each material: (a) damage model for concrete; (b) linear elasticity for steel; (c) elastoplasticity for bond-slip. Figure 2: Constitutive law for each material: (a) damage model for concrete; (b) linear elasticity for steel; (c) elastoplasticity for bond-slip. Concrete computation. The chosen material model for concrete is the damage model with isotropic hardening, that can take into account the formation of micro-cracks in the bulk, combined with a localized failure part that describes the opening of the macro-crack at the discontinuity. We will give here just a short description of the model, since all the details regarding the formulation and implementation can be found in the work of Brancherie and Ibrahimbegovic (2009), or Do et al. (2017) for applications in dynamics. 𝜙 The damage functions 𝜙𝜙 are used to check the admissibility of the stress state, and for the hardening part, this function is defined as 𝜙𝑐𝝈𝑐𝜎𝑞𝑐 𝜙𝜙ത𝑐𝑐= ||𝝈𝝈𝑐𝑐||𝑫𝑫𝑒𝑒− 1 √𝐸𝐸𝑐𝑐(𝜎𝜎𝑓𝑓−𝑞𝑞ത𝑐𝑐) ≤0 𝑫𝑒𝑫𝑪 (7) 𝐸 (7) where 𝑫𝑫𝑒𝑒 is the undamaged elastic compliance tensor for the bulk material. It is computed as the inverse of the elastic constitutive matrix: 𝑫𝑫𝑒𝑒= (𝑪𝑪𝑒𝑒)−1. In the above equation, ||𝝈𝝈𝑐𝑐||𝑫𝑫𝑒𝑒 represents the norm of 𝝈𝝈𝑐𝑐in the stress space, 𝐸𝐸𝑐𝑐 is Young’s modulus for concrete, 𝜎𝜎𝑓𝑓 is the stress at the first cracking, and 𝑞𝑞ത𝑐𝑐 is the stress-like hardening variable that controls the damage threshold evolution. 2.2 Micro-scale computations The final value of stress in concrete can then be computed from 𝝈𝑐𝑪𝑒𝑒𝜺𝑐 (12) 𝝈𝝈𝑐𝑐= 𝑪𝑪𝑒𝑒𝑒𝑒𝜺𝜺𝑐𝑐 and the value of the elasto-damage modulus 𝑪𝑪𝑒𝑒𝑒𝑒 has to be chosen according to the current phase of the computation, which is determined from the damage functions described in (7-8). 𝜎𝑏𝑏 and the value of the elasto-damage modulus 𝑪𝑪𝑒𝑒𝑒𝑒 has to be chosen according to the current phase of the computation, which is determined from the damage functions described in (7-8). 𝜎𝑏𝑏 Bond-slip computation. The main novelty in this work is the description of bond-slip through a one-dimensional elasto-plastic computation. Since the bond-slip law is given as a relationship between the bond stress 𝜎𝜎𝑏𝑏𝑏𝑏 and the slip 𝛼𝛼𝑏𝑏𝑏𝑏, instead of the plastic strain 𝜀𝜀𝑝𝑝 that is usually employed in this kind of computations, we will have the plastic slip 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝. The computation is carried out within an operator-split solution procedure (see Ibrahimbegovic, 2009) where the evolution equations of internal variables are solved in the local phase, on the level of the Gauss numerical integration point of each element, and the equilibrium equations are solved globally, on the level of the whole structure. We start by an elastic trial step, in which the plastic multiplier 𝛾𝛾𝑏𝑏𝑏𝑏 is equal to zero, and the values of all internal variables are frozen (they take the value from the previous time step) 𝛼𝑏𝑏𝑝𝑡𝑡𝑡𝑡𝑡𝛼𝑏𝑏𝑝 𝛼𝛼𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑝𝑝,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝛼𝛼𝑛𝑛 𝑏𝑏𝑏𝑏,𝑝𝑝 𝜉𝜉𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝜉𝜉𝑛𝑛 𝑏𝑏𝑏𝑏 𝑞𝑞𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝑞𝑞𝑛𝑛 𝑏𝑏𝑏𝑏= −𝐾𝐾𝑏𝑏𝑏𝑏,ℎ𝜉𝜉𝑛𝑛 𝑏𝑏𝑏𝑏 (13) 𝑞𝑞𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝑞𝑞𝑛𝑛 𝑏𝑏𝑏𝑏= −𝐾𝐾𝑏𝑏𝑏𝑏,ℎ𝜉𝜉𝑛𝑛 𝑏𝑏𝑏𝑏 (13) 4/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete 𝜉𝑞 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete 𝜉𝑞 Tea Rukavina et al. In the above equations, 𝜉𝜉𝑏𝑏𝑏𝑏 represents the hardening variable, 𝑞𝑞𝑏𝑏𝑏𝑏 is the stress-like hardening variable for bond-slip, and 𝐾𝐾𝑏𝑏𝑏𝑏,ℎ is the hardening modulus. The trial value of bond stress is then computed as 𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝐾𝑏𝑏𝛼𝑏𝑏𝛼𝑏𝑏𝑝 𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= 𝐾𝐾𝑏𝑏𝑏𝑏(𝛼𝛼𝑛𝑛+1 𝑏𝑏𝑏𝑏 −𝛼𝛼𝑛𝑛 𝑏𝑏𝑏𝑏,𝑝𝑝) (14) where 𝐾𝐾𝑏𝑏𝑏𝑏 is the bond-slip tangent modulus. The term in the brackets represents the elastic slip 𝛼𝛼𝑏𝑏𝑏𝑏,𝑒𝑒𝑒𝑒, since the total slip can be represented as the sum of the elastic and the plastic part: 𝛼𝛼𝑏𝑏𝑏𝑏= 𝛼𝛼𝑏𝑏𝑏𝑏,𝑒𝑒𝑒𝑒+ 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝. The final value of stress is then 𝜎𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝐾𝑏𝑏𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏 = 𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡−𝐾𝐾𝑏𝑏𝑏𝑏𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏sign(𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) (20) 𝐾𝑏𝑏 𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏 = 𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡−𝐾𝐾𝑏𝑏𝑏𝑏𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏sign(𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) (20) The elastoplastic tangent modulus in the elastic phase is equal to 𝐾𝐾𝑏𝑏𝑏𝑏, and in the plastic phase, it has the value 𝐶𝑒𝑒𝑏𝑏𝐾𝑏𝑏𝐾𝑏𝑏 The elastoplastic tangent modulus in the elastic phase is equal to 𝐾𝐾𝑏𝑏𝑏𝑏, and in the plastic phase, it ha 𝐶𝐶𝑛𝑛+1 𝑒𝑒𝑒𝑒,𝑏𝑏𝑏𝑏= 𝐾𝐾𝑏𝑏𝑏𝑏𝐾𝐾𝑏𝑏𝑏𝑏,ℎ 𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (21) 2.3 Macro-scale computations𝑑 𝐶𝐶𝑛𝑛+1 𝑒𝑒𝑒𝑒,𝑏𝑏𝑏𝑏= 𝐾𝐾𝑏𝑏𝑏𝑏𝐾𝐾𝑏𝑏𝑏𝑏,ℎ 𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (21) 2.2 Micro-scale computations The total slip is computed from the nodal values of slip, 𝛼𝛼𝑖𝑖 𝑏𝑏𝑏𝑏, which are obtained from the local iteration of the macro-level computation 𝛼𝑏𝑏𝑁𝑏𝑏𝛼𝑏𝑏 𝛼𝛼𝑛𝑛+1 𝑏𝑏𝑏𝑏 = ∑ 𝑖𝑖=1 2 𝑁𝑁𝑖𝑖 𝑏𝑏𝑏𝑏𝛼𝛼𝑖𝑖 𝑏𝑏𝑏𝑏 (15) The bond-slip shape functions 𝑁𝑁𝑖𝑖 𝑏𝑏𝑏𝑏 are actually the product of the standard shape functions for concrete, and the X-FEM enrichment function 𝜓𝜓, as given in equation (2). For our case it follows 𝑁𝑏𝑏𝑥𝑁𝒙𝜓𝒙𝑁𝑥 𝑁𝑁𝑖𝑖 𝑏𝑏𝑏𝑏(𝑥𝑥) = 𝑁𝑁𝑖𝑖(𝒙𝒙)𝜓𝜓(𝒙𝒙) = 𝑁𝑁𝑖𝑖(𝑥𝑥) (16) since the enrichment function is a Heaviside function. Here, 𝑁𝑁𝑖𝑖(𝑥𝑥) are the linear shape functions for a truss bar. To check if the trial value of stress is admissible, we introduce the yield function 𝜙𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜏𝑞𝑏𝑏𝑡𝑡𝑡𝑡𝑡 since the enrichment function is a Heaviside function. Here, 𝑁𝑁𝑖𝑖(𝑥𝑥) are the linear shape functions for a truss bar. To check if the trial value of stress is admissible, we introduce the yield function 𝜙𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝜏𝑞𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝜙𝜙𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡= |𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡| −(𝜏𝜏𝑦𝑦−𝑞𝑞𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) 𝜏𝑦 (17) where 𝜏𝜏𝑦𝑦 is the limit value of the bond stress. We have two cases to consider: if the trial value of the yield function is negative or zero, the step is indeed elastic and the trial values are accepted as final. If not, we have to proceed to the plastic step to correct the value of bond stress due to the plastic slip activation. where 𝜏𝜏𝑦𝑦 is the limit value of the bond stress. We have two cases to consider: if the trial value of the yield function is negative or zero, the step is indeed elastic and the trial values are accepted as final. If not, we have to proceed to the plastic step to correct the value of bond stress due to the plastic slip activation. p, we have to compute a new value for the plastic multiplier In the plastic step, we have to compute a new value for the plastic multiplier 𝛾𝑏𝑏𝜙𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏 = 𝜙𝜙𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (18) which is used to update the internal variables according to𝛼𝛼𝛾𝜎 𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏 = 𝜙𝜙𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (18) 𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏 = 𝜙𝜙𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡 𝐾𝐾𝑏𝑏𝑏𝑏+𝐾𝐾𝑏𝑏𝑏𝑏,ℎ (18) which is used to update the internal variables according to 𝛼𝑏𝑏𝑝𝛼𝑏𝑏𝑝𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 which is used to update the internal variables according to 𝛼𝑏𝑏𝑝𝛼𝑏𝑏𝑝𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 𝛼𝛼𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑝𝑝= 𝛼𝛼𝑛𝑛 𝑏𝑏𝑏𝑏,𝑝𝑝+ 𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏sign(𝜎𝜎𝑛𝑛+1 𝑏𝑏𝑏𝑏,𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡) 𝜉𝑏𝑏𝜉𝑏𝑏𝛾𝑏𝑏 𝜉𝜉𝑛𝑛+1 𝑏𝑏𝑏𝑏 = 𝜉𝜉𝑛𝑛 𝑏𝑏𝑏𝑏+ 𝛾𝛾𝑛𝑛+1 𝑏𝑏𝑏𝑏 (19) The final value of stress is then 𝜎𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡𝐾𝑏𝑏𝛾𝑏𝑏𝜎𝑏𝑏𝑡𝑡𝑡𝑡𝑡 2.3 Macro-scale computations By inserting the approximation of the virtual displacement field into the weak form of the equilibrium equations, and after gathering the terms related to the concrete displacements 𝑑𝑑𝑐𝑐, and the ones related to the bond-slip displacements 𝛼𝛼𝑏𝑏𝑏𝑏, we are left with two systems of equations 𝒓𝑐𝑐𝔸𝑛𝒇𝑐𝑖𝑖𝑖𝑒𝔸𝑛𝑛𝑛𝒇𝑠𝑖𝑖𝑖𝑒𝒇𝑒𝑒𝑒𝟎 𝒓𝒓𝑐𝑐𝑐𝑐= 𝔸𝔸 𝑛𝑛 𝑒𝑒=1𝒇𝒇𝑐𝑐,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒+ 𝔸𝔸 𝑛𝑛𝑛𝑛𝑛𝑛 𝑒𝑒=1𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒−𝒇𝒇𝑒𝑒𝑒𝑒𝑒𝑒= 𝟎𝟎 5/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 materials: application to reinforced Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavina et al. 𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠= 𝔸𝔸 𝑛𝑛𝑛𝑛𝑛𝑛 𝑒𝑒=1 ቀ𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒+ 𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒ቁ= 𝟎𝟎 𝑛 (22) Here, 𝑛𝑛 denotes the number of elements, and 𝑛𝑛𝑛𝑛𝑛𝑛 the number of enriched elements (the ones containing the steel bar). The global equation (22a) is solved with a fixed slip, and in the local one (22b) the redistribution of slip is computed. The external force vector and the internal force vectors for concrete, steel, and bond-slip, are defined in the following manner 𝒇𝑐𝑖𝑖𝑖𝑒𝑩𝑇𝝈𝑐𝑑𝑑 𝒇𝒇𝑐𝑐,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑇𝑇𝝈𝝈𝑐𝑐𝑑𝑑𝑑𝑑 𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑠𝑠𝐴𝐴𝑠𝑠𝑑𝑑𝑑𝑑 𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑑𝑑𝑑𝑑 𝒇𝒇𝑒𝑒𝑒𝑒𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑵𝑵𝑵𝑵𝑑𝑑𝛺𝛺𝑒𝑒+ ∫𝛤𝛤𝜎𝜎𝑵𝑵𝑵𝑵𝑑𝑑𝛤𝛤𝜎𝜎 (23) 𝑩𝑠𝐴𝑠𝐴 𝒇𝒇𝑐𝑐,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑇𝑇𝝈𝝈𝑐𝑐𝑑𝑑𝑑𝑑 𝒇𝒇𝑠𝑠,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑠𝑠𝐴𝐴𝑠𝑠𝑑𝑑𝑑𝑑 𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑑𝑑𝑑𝑑 𝒇𝒇𝑒𝑒𝑒𝑒𝑒𝑒= ∫𝛺𝛺𝑒𝑒𝑵𝑵𝑵𝑵𝑑𝑑𝛺𝛺𝑒𝑒+ ∫𝛤𝛤𝜎𝜎𝑵𝑵𝑵𝑵𝑑𝑑𝛤𝛤𝜎𝜎 𝑩𝑠𝐴 𝒇𝒇𝑏𝑏𝑏𝑏,𝑖𝑖𝑖𝑖𝑖𝑖,𝑒𝑒= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝜎𝜎𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑑𝑑𝑑𝑑 𝒇𝑒𝑒𝑒𝑵𝑵𝑑𝛺𝑒𝑵𝑵𝑑𝛤 (23) In equation (23), 𝑩𝑩𝑠𝑠 is the matrix of derivatives of the shape functions for the steel bar, 𝐴𝐴𝑠𝑠 is the cross-sectional area of the bar, and 𝐴𝐴𝑏𝑏𝑏𝑏 is the bond-slip area that is computed as the circumference of the bar (it is the part of the steel bar in contact with concrete). In the external force vector, 𝒃𝒃 represents the volumetric forces, and 𝒕𝒕 is the imposed traction acting at the boundary 𝛤𝛤𝜎𝜎. 3 NUMERICAL RESULTS The proposed methodology was implemented in FEAP (Finite Element Analysis Program), developed at Berkeley by professor R. L. Taylor (2014). All the described constituents are implemented into a single finite element that allows for an efficient computation of their interaction. 𝜙 To test the performance of the developed formulation, we have performed a tension test on a concrete specimen of dimensions 400x100x100 mm3, reinforced by a steel bar of diameter 𝜙𝜙=16 mm. Since our model is 2D (as shown of Figure 3.), the area of the bar and the bond-slip area are divided by the thickness of the specimen. Figure 3: Geometry of the reinforced concrete specimen in 2D. Figure 3: Geometry of the reinforced concrete specimen in 2D. Figure 3: Geometry of the reinforced concrete specimen in 2D. We consider the steel bar to be fixed at both ends, as it is anchored in concrete. The material properties used in the model are listed here: 𝐸𝑐𝜈𝑐𝜎𝑓𝐾𝜎𝑓 • for concrete: 𝐸𝐸𝑐𝑐= 45700MPa, 𝜈𝜈𝑐𝑐= 0.2, 𝜎𝜎ത𝑓𝑓= 3.5MPa, 𝐾𝐾ℎ= 1000MPa, 𝜎𝜎ധ𝑓𝑓= 4MPa, 𝜎𝑓𝑤𝑤𝑤𝑤𝛽𝑐 • for concrete: 𝐸𝐸𝑐𝑐= 45700MPa, 𝜈𝜈𝑐𝑐= 0.2, 𝜎𝜎ത𝑓𝑓= 3.5MPa, 𝐾𝐾ℎ= 1000MPa, 𝜎𝜎ധ𝑓𝑓= 4MPa, 𝜎𝑓𝑤𝑤𝑤𝑤𝛽𝑐 • for concrete: 𝐸𝐸𝑐𝑐= 45700MPa, 𝜈𝜈𝑐𝑐= 0.2, 𝜎𝜎ത𝑓𝑓= 3.5MPa, 𝐾𝐾ℎ= 1000MPa, 𝜎𝜎ധ𝑓𝑓= 4MPa, 𝜎𝜎ധ𝑓𝑓,𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤= 3.8MPa, 𝛽𝛽𝑐𝑐= 20; 𝐸𝑠 • for the steel bar: 𝐸𝐸𝑠𝑠= 210000MPa, 𝐴𝐴𝑠𝑠= 2.01mm2; • for the bond: 𝐾𝐾𝑏𝑏𝑏𝑏= 30N/mm3, 𝜏𝜏𝑦𝑦= 6MPa, 𝐾𝐾𝑏𝑏𝑏𝑏,ℎ= 0.03N/mm3, 𝐴𝐴𝑏𝑏𝑏𝑏= 0.5mm. 𝜈𝑐𝛽𝑐𝜎 • for the steel bar: 𝐸𝐸𝑠𝑠= 210000MPa, 𝐴𝐴𝑠𝑠= 2.01mm2; • for the bond: 𝐾𝐾𝑏𝑏𝑏𝑏= 30N/mm3, 𝜏𝜏𝑦𝑦= 6MPa, 𝐾𝐾𝑏𝑏𝑏𝑏,ℎ= 0.03N/mm3, 𝐴𝐴𝑏𝑏𝑏𝑏= 0.5mm. 𝜈𝑐𝛽𝑐𝜎 Among the properties listed above, 𝜈𝜈𝑐𝑐 is the Poisson’s ratio for concrete, and 𝛽𝛽𝑐𝑐 is the parameter that controls the exponential softening. 𝜎𝜎ധ𝑓𝑓,𝑤𝑤𝑤𝑤𝑤𝑤𝑤𝑤 is the ultimate stress for a strip of weakened elements in the middle of the specimen where the crack will appear. Figure 4: Finite element mesh with enriched elements shown in grey. 𝑢 Figure 4: Finite element mesh with enriched elements shown in grey. 𝑢 The specimen is fixed at the left-hand side, and there is an imposed displacement 𝑢𝑢ത= 0.5 mm acting on the right-hand side. The finite element mesh shown on Figure 4 consists of 800 CST elements, where the triangle sides of each element have the dimension 10 mm. There are 40 enriched elements along the reinforcement bar. 𝑥 The specimen is fixed at the left-hand side, and there is an imposed displacement 𝑢𝑢ത= 0.5 mm acting on the right-hand side. 2.3 Macro-scale computations By linearizing (22), we obtain the following system of equations 𝑲𝑐𝑐𝑭𝑐𝑐𝛥𝒅𝑐𝒓𝑐𝑐 ቂ𝑲𝑲𝑐𝑐𝑐𝑐 𝑭𝑭𝑐𝑐𝑐𝑐 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐 𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐 𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ (26) ቂ𝑲𝑲𝑐𝑐𝑐𝑐 𝑭𝑭𝑐𝑐𝑐𝑐 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐 𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐 𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ ቂ𝑲𝑲𝑐𝑐𝑐𝑐 𝑭𝑭𝑐𝑐𝑐𝑐 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐 𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐 𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ (26) ቂ𝑲𝑲𝑐𝑐𝑐𝑐 𝑭𝑭𝑐𝑐𝑐𝑐 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠ቃቂ𝛥𝛥𝒅𝒅𝑐𝑐 𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏ቃ= −ቂ𝒓𝒓𝑐𝑐𝑐𝑐 𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠ቃ (26) where the tangent stiffness matrices are defined as 𝑲𝑐𝑐𝑩𝑐𝑇𝑪𝑒𝑒𝑩𝑐𝑑𝑑𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑 where the tangent stiffness matrices are defined as 𝑲𝑐𝑐𝑩𝑐𝑇𝑪𝑒𝑒𝑩𝑐𝑑𝑑𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑 𝑲𝑲𝑐𝑐𝑐𝑐= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑐𝑐,𝑇𝑇𝑪𝑪𝑒𝑒𝑒𝑒𝑩𝑩𝑐𝑐𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑭𝑭𝑐𝑐𝑐𝑐= 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠 𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠 𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐾𝐾𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 (25) 𝑲𝑲𝑐𝑐𝑐𝑐= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑐𝑐,𝑇𝑇𝑪𝑪𝑒𝑒𝑒𝑒𝑩𝑩𝑐𝑐𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑭𝑭𝑐𝑐𝑐𝑐= 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑠𝑠𝑠𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑𝑩𝑠𝑇𝐾𝑏𝑏𝐴𝑏𝑏𝑩𝑠𝑑𝑑 𝑲𝑲𝑐𝑐𝑐𝑐= ∫𝛺𝛺𝑒𝑒𝑩𝑩𝑐𝑐,𝑇𝑇𝑪𝑪𝑒𝑒𝑒𝑒𝑩𝑩𝑐𝑐𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑭𝑐𝑐𝑭𝑠𝑠𝑠𝛤𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑 𝑭𝑭𝑐𝑐𝑐𝑐= 𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑠𝑠𝑠𝑩𝑠𝑇𝐸𝑠𝐴𝑠𝑩𝑠𝑑𝑑𝑩𝑠𝑇𝐾 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠 𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠 𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐾𝐾𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠= ∫𝛤𝛤𝑠𝑠 𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐸𝐸𝑠𝑠𝐴𝐴𝑠𝑠𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑+ ∫𝛤𝛤𝑠𝑠 𝑒𝑒𝑩𝑩𝑠𝑠,𝑇𝑇𝐾𝐾𝑏𝑏𝑏𝑏𝐴𝐴𝑏𝑏𝑏𝑏𝑩𝑩𝑠𝑠𝑑𝑑𝑑𝑑 (25) (25) The system (24) is handled within the macro-scale operator-split solution procedure, that gives us two equations to solve sequentially 𝑲𝛥𝒅𝑐𝒓𝑐𝑐 𝑲𝑲෡ 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠 (26) 𝑲 𝑲𝑲෡ 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠 (26) 𝑲 𝑲𝑲෡ 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠 𝑲 𝑲𝑲෡ 𝛥𝛥𝒅𝒅𝑐𝑐= −𝒓𝒓𝑐𝑐𝑐𝑐 𝑯𝑠𝑠𝑠𝛥𝜶𝑏𝑏𝒓𝑠𝑠𝑠 𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠𝛥𝛥𝜶𝜶𝑏𝑏𝑏𝑏= −𝒓𝒓𝑠𝑠𝑠𝑠𝑠𝑠 𝑲 (26) where 𝑲𝑲 is the condensed stiffness matrix 𝑲𝑲𝑐𝑐𝑭𝑐𝑐𝑯𝑠𝑠𝑠𝑭𝑠𝑠𝑠 where 𝑲𝑲 is the condensed stiffness matrix 𝑲𝑲𝑐𝑐𝑭𝑐𝑐𝑯𝑠𝑠𝑠𝑭𝑠𝑠𝑠 where 𝑲𝑲 is the condensed stiffness matrix 𝑲𝑲𝑐𝑐𝑭𝑐𝑐𝑯𝑠𝑠𝑠𝑭𝑠𝑠𝑠 𝑲𝑲෡= 𝑲𝑲𝑐𝑐𝑐𝑐−𝑭𝑭𝑐𝑐𝑐𝑐(𝑯𝑯𝑠𝑠𝑠𝑠𝑠𝑠)−1𝑭𝑭𝑠𝑠𝑠𝑠𝑠𝑠 𝒅 (27) Equation (26a) is the macro-global equation that gives us the value of the concrete displacement for the fixed value of slip. With the value of 𝒅𝒅𝑐𝑐 in hand, we proceed to the macro-local equation (26b), where we update the value of the slip 𝜶𝜶𝑏𝑏𝑏𝑏. 6/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced oncrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. 3 NUMERICAL RESULTS The finite element mesh shown on Figure 4 consists of 800 CST elements, where the triangle sides of each element have the dimension 10 mm. There are 40 enriched elements along the reinforcement bar. 𝑥 The force-displacement diagram shown on Figure 5. plots the reaction in the 𝑥𝑥-direction at the left-hand side against the imposed displacement on the right-hand side. We can observe several particular phases of the composite behavior: the linear elastic phase is followed by a hardening phase when micro-cracks start to appear in concrete. After the ultimate load of about 500 N is reached, the crack develops in the weak zone in the middle of the specimen, so we enter the softening phase. After a while, the reaction starts ascending, due to the redistribution of stresses and the reinforcement activation. This resembles the typical diagrams that are found in the literature, which are obtained for a specimen with a single crack. 7/11 7/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Figure 5: Force-displacement diagram for the tension test. 𝛼 Figure 5: Force-displacement diagram for the tension test. 𝛼 On Figure 6. we can see the distribution of slip along the reinforcement bar, where the largest slips are taking place near the crack, and at the ends the slip is zero. On the left of the crack, 𝛼𝛼𝑏𝑏𝑏𝑏 has a positive value, and on the left side it is negative, because the reinforcement is moving (relatively to the concrete) from the ends to the centre (in other words, towards the crack). Figure 6: Distribution of slip 𝛼𝛼𝑏𝑏𝑏𝑏 along the reinforcement bar at the end of the analysis (nodal values). Figure 6: Distribution of slip 𝛼𝛼𝑏𝑏𝑏𝑏 along the reinforcement bar at the end of the analysis (nodal values). The bond stress has a very similar distribution (Figure 7a), according to the chosen bond-slip law for the interface. The evolution of bond stress in time gives rise to an interesting plot (Figure 7b), where the dashed lines represent the elements left of the crack, and the dotted lines represent the ones on the right. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 3 NUMERICAL RESULTS We can see that the elements on the left have a positive value of bond-stress, and for the elements on the right the bond stress is negative. The bold solid line represents the value of bond stress in the cracked element, that is very near to zero, since it is the inflection point of the curve shown on Figure 7a. The bold dashed and dotted line represent the elements nearest to the crack, where the bond stress is largest, and where the ultimate value of bond stress, 𝜏𝜏𝑦𝑦, has been reached. 8/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavina et al. Figure 7: Bond stress: (a) distribution of 𝜎𝜎𝑏𝑏𝑏𝑏 along the reinforcement bar (values at Gauss points); (b) evolution of 𝜎𝜎𝑏𝑏𝑏𝑏 in time for all enriched elements. Figure 7: Bond stress: (a) distribution of 𝜎𝜎𝑏𝑏𝑏𝑏 along the reinforcement bar (values at Gauss points); (b) evolution of 𝜎𝜎𝑏𝑏𝑏𝑏 in time for all enriched elements. Figure 8: Plastic slip: (a) distribution of 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝 along the reinforcement bar at the end of the analysis (values at Gauss points); (b) evolution in time for the enriched elements that have entered the plastic phase. Figure 8: Plastic slip: (a) distribution of 𝛼𝛼𝑏𝑏𝑏𝑏,𝑝𝑝 along the reinforcement bar at the end of the analysis (values at Gauss points); (b) evolution in time for the enriched elements that have entered the plastic phase. Related to that is the plastic slip development shown on Figure 8b, where it can be observed that plasticity occurs only at the end of the analysis, in five elements that are nearest to the crack (two on the left side, and three on the right). The same can be seen on Figure 8a, where the plastic slip is plotted in Gauss points along the reinforcement bar. The plastic slip is equal to zero in most of the enriched element, since they have not yet entered the plastic phase and the interface is behaving elastically. Related to that is the plastic slip development shown on Figure 8b, where it can be observed that plasticity occurs only at the end of the analysis, in five elements that are nearest to the crack (two on the left side, and three on the right). 3 NUMERICAL RESULTS The same can be seen on Figure 8a, where the plastic slip is plotted in Gauss points along the reinforcement bar. The plastic slip is equal to zero in most of the enriched element, since they have not yet entered the plastic phase and the interface is behaving elastically. Figure 9: Crack opening in concrete: (a) distribution of 𝛼𝛼𝑐𝑐 along the reinforcement bar at the end of the analysis (values at Gauss points); (b) evolution of 𝛼𝛼𝑐𝑐 in time for the cracked element in the middle of the specimen. Figure 9: Crack opening in concrete: (a) distribution of 𝛼𝛼𝑐𝑐 along the reinforcement bar at the end of the analysis (values at Gauss points); (b) evolution of 𝛼𝛼𝑐𝑐 in time for the cracked element in the middle of the specimen. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 9/11 Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Figure 9. gives the representation of the crack opening in concrete, that reaches the value of 𝛼𝛼𝑐𝑐=0.47 mm at the end of the analysis. As it has been already explained, the macro-crack starts to open when the material reaches the ultimate stress, which happens around time 0.25. There are also two small cracks in the elements right next to the main crack, but their size is negligible compared to the main crack. Figure 10: Comparison of the evolution in time of 𝛼𝛼𝑐𝑐 and 𝛼𝛼𝑏𝑏𝑏𝑏: the crack opening in concrete in the middle of the element is nearly equal to the sum of the absolute values of slip in the left and right node of the same element. Figure 10: Comparison of the evolution in time of 𝛼𝛼𝑐𝑐 and 𝛼𝛼𝑏𝑏𝑏𝑏: the crack opening in concrete in the middle of the element is nearly equal to the sum of the absolute values of slip in the left and right node of the same element. It is interesting to point out that the crack opening in concrete coincides with the slip activation, which can be compared on Figure 7b and Figure 9b. This is in accordance with our model assumption that there is no slip when the concrete and the steel have the same displacement. 3 NUMERICAL RESULTS So, the crack opening in concrete, 𝛼𝛼𝑐𝑐, is giving rise to the difference between the concrete and steel strain field, that is, in turn, activating the bond-slip 𝛼𝛼𝑏𝑏𝑏𝑏. Moreover, we can test this notion by plotting the crack opening evolution and the slip evolution on the same diagram, as has been done on Figure 10. We are plotting the values of 𝛼𝛼𝑐𝑐 in the Gauss point of the cracked element (represented by the solid line), and the values of 𝛼𝛼𝑏𝑏𝑏𝑏 in left and right node of the same element (dashed and dotted line, respectively). While we have already noted that the two nodes in question have the displacements of opposite sign, it is even more compelling that, quantitatively, the absolute values of the two slips add up to give nearly the same value as the crack opening at a given time. The sum of two slips is represented by a dotted line on Figure 10. Editor: Paulo de Mattos Pimenta. Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 4 CONCLUSIONS The methodology presented in this paper allows us to model the failure mechanisms in reinforced concrete happening on the different scales of the material, by taking into account the behavior of each of the constituents. The bond-slip is handled within a 1D elastoplastic framework, and we have shown that this simplification does not affect the model predictivity; hence it represents an improvement compared to previous works based on interface elements. We have given the multi-scale set-up for this kinds of problems, where multi-level operator-split solution procedures can be performed during the computation. In the numerical example we have shown the relationship between the crack opening and the bond-slip, which corresponds to the realistic processes happening during the failure of fiber-reinforced composites. The next step would be to extend the methodology to take into account the complete pull-out of the fiber, in order to capture the failure mechanisms taking place when the fiber’s ends are not anchored in the surrounding The next step would be to extend the methodology to take into account the complete pull-out of the fiber, in order to capture the failure mechanisms taking place when the fiber’s ends are not anchored in the surrounding material. Author’s Contributions: Conceptualization, A Ibrahimbegovic; Investigation, T Rukavina; Methodology, T Rukavina, A Ibrahimbegovic; Project administration, I Kozar, A Ibrahimbegovic; Software, T Rukavina; Supervision, A Ibrahimbegovic, I Kozar; Validation, T Rukavina, A Ibrahimbegovic, I Kozar; Visualisation, T Rukavina, Writing - original draft, T Rukavina; Writing - review & editing, A Ibrahimbegovic, I Kozar, T Rukavina. Editor: Paulo de Mattos Pimenta. 10/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Tea Rukavina et al. Multi-scale representation of plastic deformation in fiber-reinforced materials: application to reinforced concrete Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141 References Brancherie, D., Ibrahimbegovic, A. (2009). Novel anisotropic continuum-discrete damage model capable of representing localized failure of massive structures: Part I: theoretical formulation and numerical implementation, Engineering Computations 26(1/2): 100–127. Do, X.N., Ibrahimbegovic, A., Brancherie, D. (2017). Dynamics framework for 2D anisotropic continuum-discrete damage model for progressive localized failure of massive structures, Computers & Structures 183: 14–26. Dominguez, N., Brancherie, D., Davenne, L., Ibrahimbegovic, A. (2005). Prediction of crack pattern distribution in reinforced concrete by coupling a strong discontinuity model of concrete cracking and a bond-slip of reinforcement model. Engineering Computations 22(5/6): 558-582. Fries, T.-P., Belytschko, T. (2010). The extended/generalized finite element method: an overview of the method and its applications, International Journal for Numerical Methods in Engineering 84(3): 253-304. Ibrahimbegovic, A. (2009). Nonlinear solid mechanics: Theoretical formulations and finite element solution methods, Springer, Dodrecht. Ibrahimbegovic, A., Boulkertous, A., Davenne, L., Brancherie, D. (2010). Modelling of reinforced-concrete structures providing crack-spacing based on X-FEM, ED-FEM and novel operator split solution procedure. International Journal of Numerical Methods in Engineering 83(4): 452-481. Jukić, M., Brank, B., Ibrahimbegovic, A. (2014). Failure analysis of reinforced concrete frames by beam finite element that combines damage, plasticity and embedded discontinuity, Engineering Structures, 75: 507-527. Kohnehpooshi, O., Jaafar, M.S. (2017). Non-linear three dimensional finite elements for composite concrete structures, Latin American Journal of Solids and Structures, 14: 398-42. Melenk, J.M., Babuška, I. (1996). The partition of unity finite element method: Basic theory and applications, Computer Methods in Applied Mechanics and Engineering, 139(1-4): 289-314. Ožbolt, J., Lettow, S., Kožar, I. (2002). Discrete bond element for 3D finite element analysis of reinforced concrete structures, in book Beiträge aus der Befestigungstechnik und dem Stahlbetonbau, Lettow, S., Hoffman, J. (ed.), Ibidem Verlag, Stuttgart. Šćulac, P., Jelenić, G., Škec, L. (2014). Kinematics of layered reinforced-concrete planar beam finite elements with embedded transversal cracking, International Journal of Solids and Structures, 51: 74-92. Taylor, R. (2014). FEAP - Finite Element Analysis Program, University of California Berkeley, version 8.4. 11/11 Latin American Journal of Solids and Structures, 2019, 16(7 Thematic Section), e141
https://openalex.org/W2761536767
https://europepmc.org/articles/pmc5735200?pdf=render
English
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Spectrum of outcomes following traumatic brain injury—relationship between functional impairment and health-related quality of life
Acta neurochirurgica
2,017
cc-by
6,212
Abstract Background The outcome following traumatic brain injury (TBI) is heterogeneous and poorly defined and physical dis- ability scales like the extended Glasgow Outcome Score (GOSE) while providing valuation information in terms of broad categorisation of outcome are unlikely to capture the full spectrum of deficits. Quality of life questionnaires such as SF-36 are emerging as potential tools to help characterise factors important to patients’ recovery. This study assessed the association between physical disability and subjective health rating. The relationship is of value as it may help evaluate the impact of TBI on patients’ lives and facilitate the delivery of appropriate neuro-rehabilitation services. Results There is a statistically significant difference in the MCS and PCS scores based on patients’ GOSE scores. The mean scores of the eight SF-36 domains showed significant association with GOSE. GLMM demonstrated that GOSE was the strongest predictor of PCS and MCS. Age was an important variable in the PCS score while time following trau- ma was a significant predictor of MCS rating. Conclusions This study highlights that patients’ physical out- come following TBI is a strong predictor of the subjective mental and physical health. Nevertheless, there remains tre- mendous variability in individual SF-36 scores for each GOSE category, highlighting that additional factors play a role in determining quality of life. Methods A single-centre retrospective study was undertaken to assess the relationship between physical outcome as mea- sured by GOSE and quality of life captured by the SF-36 questionnaire. Cronbach’s alpha was calculated for each of the eight SF-36 domains to measure internal consistency of the test. Multivariate analysis of variance was conducted to look at the association between GOSE and the physical (PCS) and mental (MCS) component scores on the SF-36. Finally, we performed a generalised linear mixed model (GLMM) to Keywords Traumatic brain injury . Quality of life . SF-36 . Glasgow Outcome Scale Keywords Traumatic brain injury . Quality of life . SF-36 . Glasgow Outcome Scale Spectrum of outcomes following traumatic brain injury—relationship between functional impairment and health-related quality of life Anastasia Tsyben1,2 & Mathew Guilfoyle1,2 & Ivan Timofeev1,2 & Fahim Anwar3 & Judith Allanson3 & Joanne Outtrim4 & David Menon4 & Peter Hutchinson1,2 & Adel Helmy1,2 Anastasia Tsyben1,2 & Mathew Guilfoyle1,2 & Ivan Timofeev1,2 & Fahim Anwar3 & Judith Allanson3 & Joanne Outtrim4 & David Menon4 & Peter Hutchinson1,2 & Adel Helmy1,2 Received: 8 August 2017 /Accepted: 11 September 2017 /Published online: 7 October 2017 # The Author(s) 2017. This article is an open access publication assess the relative contribution of GOSE score, age at the time of trauma, sex and TBI duration towards MCS and PCS rating. Acta Neurochir (2018) 160:107–115 DOI 10.1007/s00701-017-3334-6 ORIGINAL ARTICLE - BRAIN INJURY * Anastasia Tsyben anastasia.tsyben@cantab.net 4 Division of Anaesthesia, Department of Medicine, Cambridge Biomedical Campus, Cambridge, UK 1 Department of Neurosurgery, Addenbrooke’s Hospital, Hills Road, Cambridge CB2 0QQ, UK 2 Division of Neurosurgery, Department of Clinical Neurosciences, Cambridge Biomedical Campus, Cambridge, UK 3 Department of Neurorehabilitation, Addenbrooke’s Hospital, Cambridge, UK 4 Division of Anaesthesia, Department of Medicine, Cambridge Biomedical Campus, Cambridge, UK Sample and measures A series of multivariate ANOVAs were conducted with GOSE scores as the independent variable and the two sum- mary scores, MCS and PCS, as the dependent variables. Interaction was also assessed between age of TBI and SF-36 domain scores at the first clinical appointment following discharge. This was a retrospective study of patients seen in Addenbrooke’s Hospital, Cambridge, Neurotrauma Outpatient Clinic between 2005–2013. The majority of indi- viduals underwent treatment for TBI at Addenbrooke’s, with a small proportion of patient’s being referred from other centres. Tests of physical function and subjective health status were administered at each clinic appointment. Statistical analysis Descriptive statistics were used to summarise the demograph- ic data. Normality of data was tested using the Shapiro-Wilk test while Levene’s and Bartlett’s tests were used to test ho- mogeneity of variance. Based on skewed distribution of the data, non-parametric testing of correlation among MCS, PCS and GOSE were examined using Spearman rank. The level of statistical significance was set at p < 0.05. All analyses were carried out in SPSS 24.0 (IBM SPSS, Chicago, IL, USA). The purpose of this study was to investigate the relation- ship between the functional score of patients with TBI and their subjective health status. The GOSE test was used as a measure of physical function, while the 36-Item Short Form Survery (SF-36) was used as an assessment of QoL. While both these assessment tools have been validated in the TBI population, there has been no definite characterisation of the relationship between physical disability and subjective per- ception of handicap [18, 24, 27]. Introduction Traumatic brain injury (TBI) is a leading cause of mortality and long-term disability among those under 45 and costs an estimated $75 billion each year in the USA alone [9, 12]. Despite improvements in the acute management of TBI, most patients are left with a degree of permanent disability affecting cognitive, psychological and physical function [39]. Commonly used outcome measures such as the functional independence measure (FIM), extended Glasgow Outcome Scale (GOSE) and Disability Rating Scale (DRS) while pro- viding broad categorisation of outcome are limited in their ability to capture the full spectrum of deficits following brain Acta Neurochir (2018) 160:107–115 108 injury [11]. In particular, such scales struggle to measure neu- robehavioral disability, which tends to affect a person’s per- sonality, cognition and character [43]. However, it is these aspects that often lead to disintegration of interpersonal rela- tionships, family burden and inability to return to vocation [3, 25]. 40]. In the scale, the higher scores indicate better subjective health. Two summary scores, physical component summary (PCS) and mental component summary (MCS), are derived by taking unweighted means of the corresponding domains. The SF-36 survey was only administered to patients with adequate communication skills who were able to respond to the questionnaire. Thus only patients with GOSE score of 3 or greater were included in the final analysis. Clinic appoint- ments with missing GOSE or SF-36 scores were excluded from analysis. In addition, subjects with incorrect or missing demographic details were also excluded, as these could not be incorporated into the final statistical analysis. Quality of life (QoL) questionnaires have gained increasing popularity, allowing for measurements of objective and sub- jective health indicators. When compared to objective clinical measures of physical function, QoL questionnaires are supe- rior at capturing the patients’ internal judgment of health and factors that may be important to the well being of the individ- ual. This emphasis on Patient Reported Outcome Measures (PROM) is drawing increased interest from both patient groups and funding bodies. Such information in turn may facilitate the delivery and evaluation of neuro-rehabilitation services as well as guide future clinical research. In the TBI population, QoL measures may have the added benefit of capturing the heterogeneity of outcomes and may delineate the natural history of this chronic condition. Measure of internal consistency of SF-36 domain scores and its association with physical disability Cronbach’s alpha was calculated for each of the eight domain scores for all patients during their first visit. The minimum threshold for the coefficient was set at 0.7 and preferable above 0.8 [13, 30]. A domain was considered distinct if its respective alpha coefficient exceeded inter-domain correlation of all other scales. Descriptive statistics A total of 513 adults above the age of 16 who were seen in neurosurgery clinic between 2005–2013 met the inclusion criteria and were included in the final analysis. Table 1 shows the demographic characteristics of the study cohort as well as the disability GOSE score at the first follow-up clinic. Mean age at the time of traumatic brain injury was 39.7 years (range: 16–91 years; SD = 16.9). The range of clinic follow-up dates was 0–611 months, with a total of 922 individual visits col- lected over this time period. The average number of follow-up clinics for a patient was two, although these were inconsistent Analysis using univariate ANOVAs found that GOSE scores had statistically significant effect on both PCS (F = 202.9; p < 0.005; partial η2 = 0.53) and MCS (F = 14.9; p < 0.005; partial η2=0.41) scores. Likewise, the mean scores of the eight domains of SF-36 also showed a significant associ- ation with GOSE scores (Pillai’s trace = 0.68; F = 17.8, p < 0.005; partial η2 = 0.14). Comparisons of GOSE and PCS and MCS scores are plotted in Fig. 1. Table 1 Demographic characteristics of the study population n 513 Sex Female 152 (30%) Male 361 (70%) Age at TBI (years) Median 38 Mean 39.7 SD 16.9 Range 16–91 GOSE (%) at first follow-up Upper good recovery 12.9 Lower good recovery 11.6 Upper moderate disability 30.8 Lower moderate disability 17.9 Upper severe disability 19.1 Lower severe disability 7.7 GOS, Extended Glasgow Scale. SD, standard deviation n 513 Sex Female 152 (30%) Male 361 (70%) Age at TBI (years) Median 38 Mean 39.7 SD 16.9 Range 16–91 GOSE (%) at first follow-up Upper good recovery 12.9 Lower good recovery 11.6 Upper moderate disability 30.8 Lower moderate disability 17.9 Upper severe disability 19.1 Lower severe disability 7.7 GOS, Extended Glasgow Scale. SD, standard deviation Contribution of independent variables to subjective QOL scores pp The physical disability was assessed using GOSE, an eight- scale global measure of function, which has been validated in TBI [13, 30]. The SF-36 questionnaire was used to assess the patient-reported QoL. The test consists of 36 multiple choice questions that are grouped into eight domains: PF, physical functioning; RP, role limitation due to physical problems; BP, bodily pain; RE, role limitations due to emotional problems; VT, vitality; GH, general health perception; MH, mental health; SF, social functioning. The domain scores were calcu- lated by transforming the raw data into a scale of 0–100 and using Likert’s method of unweighted summed ratings [26, A generalised mixed model (GLMM) was used to assess the relatively contribution of GOSE score, age at the time of trau- ma, sex and TBI duration on MCS and PCS score. Patient hospital numbers were identified as random effects, while sex, age at the time of trauma, TBI duration and GOSE were fixed effects. GLMM was used to account for the variable time points of questionnaire collections and the unequal sam- ple size observed for each follow-up visit. R2s were calculated to determine the goodness of fit of the model. Acta Neurochir (2018) 160:107–115 109 as they were based on the clinical need of the individual patients. To look at the general spread of responses on MCS and PCS for each GOSE category, a box and whisker plot was constructed. While there may be a positive relationship be- tween GOSE and SF-36 responses, there may be variability in responses highlighting individual differences in perceived QOL. Association between GOSE and SF-36 scores Based on the result of the Shapiro-Wilk test of normality, PCS and MCS results were found to be non-parametric (p < 10−14). The correlation among PCS, MCS and GOSE was significant- ly positive, indicating that as the GOSE score of disability improved, the patients’ subjective rating of health also rose. PCS showed the highest positive Spearman rank correlation of 0.67 (p < 0.001) with GOSE. MCS had a correlation of 0.61 (p < 0.001) with GOSE and 0.71 (p < 0.001) with PCS. Reliability of SF-36 domain score Summary scores for the eight domains of SF-36 at the first clinic visit as well as intra-class correlations are summarised in Table 2. The Cronbach’s alpha for the eight SF-36 domains was 0.90 suggesting high internal consistency of the question- naire. Individual alpha coefficients ranged from 0.87–0.89 and were substantially greater than the correlations between domains. Most domains had a strong inter-item correlation and were worthy of inclusion, resulting in a lower coefficient if deleted. Temporal variation in GOSE and SF-36 parameters To explore the relative concordance of GOSE and SF36 in identifying changes (improvement or deterioration) in out- come, we identified patients who attended the neurotrauma clinic on at least two occasions. Patients were divided into cohorts in which there was a change in GOSE category and the mean change in SF-36 calculated. Relationship among GOSE, SF-36 domain scores, age at trauma, gender and time since injury The generalised linear mixed model was used to examine the relationship among GOSE, PCS/MCS, gender, age at the time of trauma and time since injury. The PCS and MCS scores both increased as the GOSE rose. The regression coefficient shows that, all other things equal, for every 1-point rise in GOSE, the score on PCS would rise from 4.1–41.9 points (p < 0.0005). However, relative to GOSE 3, patients with a GOSE 4 score rated 2.6 lower on PCS, although this was not statistically significant (p = 0.266). This suggests a plateau effect at lower GOSE scores, such that physical disability no longer contributes to further declines in GOSE score below 4, while variables contributing to the MCS (which are likely to include cognitive, psychological and psychiatric symptoms) do. In addition, the age at the time of trauma was also statis- tically significant at p = 0.03, indicating that the older the patient was at the time of TBI, the worse they would score Acta Neurochir (2018) 160:107–115 110 Table 2 Domain characteristics including mean and standard deviation (SD) with reliability statistics: Cronbach’s alpha coefficient and inter-domain correlations Domain characteristics Reliability SF-36 domain Mean SD Alpha Inter-domain correlation mean (range) PF 67.0 30.9 0.89 0.47 (0.38–0.52) RP 32.5 41.3 0.89 0.53 (0.43–0.61) BP 59.7 31.2 0.89 0.54 (0.49–0.59) GH 58.3 25.0 0.89 0.57 (0.50–0.63) VI 44.7 25.6 0.88 0.59 (0.43–0.72) SF 52.7 31.5 0.87 0.62 (0.52–0.71) RE 44.3 45.5 0.89 0.56 (0.46–0.67) MH 58.8 24.6 0.88 0.57 (0.38–0.72) physical status likely influence patients’ mental and physical health perception. The mean score on MCS was 51.77 (SD 25.60) and PCS 52.46 (SD 19.46) (Table 3). on the PCS domain of SF-36 by approximately 0.07 points per year of age. The clinical significance of this small but statisti- cally significant difference is unclear. A comparison between scores on concurrent clinic visits demonstrated that both PCS and MCS correlated positively with GOSE score. If a patient’s GOSE score dropped during the subsequent visit, their SF-36 score also tended to drop and vice versa (Table 4 and Fig. 3), suggesting that both functional impairment and health-related quality of life metrics change together as patients recover or deteriorate. Similarly, the MCS score increased by 8.4–54.7 points for every 1 point rise in GOSE group (p < 0.005). Discussion Although GOSE had a high explanatory power on PCS and MCS, there was great inter-patient variability of scores as demonstrated in Fig. 2. Within each GOSE score, there is a large spectrum of physical and mental component scores underlining that, on an individual basis, factors other than The aim of this study was to characterise the relationship between physical outcome following TBI and subjective health scores, as measured by the SF-36 questionnaire. To our knowledge, this is one of the largest cohorts of patients cited in the TBI literature, with a total of 513 patients. The GOSE score had a significant impact on a patient’s PCS and MCS scores and the SF-36 proved to be a robust metric for assessing improvement in GOSE. As the GOSE score in- creased the subjective rating on MCS and PCS also rose by an average of 33.8 (p < 0.005) points on the MCS and 33.2 (p < 0.005) points on the PCS. These trends are consistent with the well-documented finding that poor physical function and reduced mobility lead to increased pain and decreased inde- pendence, as well as precluding return to vocation [22]. The finding of the positive relationship between physical function and QOL is important because it suggests that managing re- sidual physical deficits could lead to an increase in subjective health status. Interestingly, at lower GOSE categories (GOSE 3 and GOSE 4), there appears to be little difference in average PCS scores; however, the MCS continues to decline. Targeted rehabilitation has clearly shown that rehabilitation can de- crease disability and improve the quality of living [1, 2, 8, 19]. Even patients with moderate to severe TBI demonstrate some continued neuropsychological recovery several years 0.0 25.0 50.0 75.0 100.0 3 4 5 6 7 8 Mean Score (95% CI) eGOS MSC PSC Fig. 1 Comparison of GOSE to the two summary scores, PCS and MCS on SF-36. Mean MCS and PCS scores are plotted against GOSE categories (error bars: 95% confidence interval). All summary measures showed increasing scores with more favourable GOSE (multivariate ANOVA all p < 10−93) [3=Lower severe disability 4=Upper severe disability 5=Lower moderate disability 6=Upper moderate disability 7=Lower good recovery 8=Upper good recovery] 0.0 25.0 50.0 75.0 100.0 3 4 5 6 7 8 Mean Score (95% CI) eGOS MSC PSC Fig. 1 Comparison of GOSE to the two summary scores, PCS and MCS on SF-36. Relationship among GOSE, SF-36 domain scores, age at trauma, gender and time since injury Time since injury showed an increase in MCS score of 0.04 points per year and this was statistically significant at p = 0.018. With a significance value greater than 0.05, there is not enough evi- dence to conclude whether the gender or progression of time from TBI had an effect on the PCS outcome. Approximately 48% of the variability in the model was explained by the independent variables included in the analysis for PCS and 39% for MCS. Discussion Mean MCS and PCS scores are plotted against GOSE categories (error bars: 95% confidence interval). All summary measures showed increasing scores with more favourable GOSE (multivariate ANOVA all p < 10−93) [3=Lower severe disability 4=Upper severe disability 5=Lower moderate disability 6=Upper moderate disability 7=Lower good recovery 8=Upper good recovery] 111 Acta Neurochir (2018) 160:107–115 Fig. 2 Box and whisker plot showing the spread of MCS and PCS responses for each GOSE category. [3=Lower severe disability 4=Upper severe disability 5=Lower moderate disability 6=Upper moderate disability 7=Lower good recovery 8=Upper good recovery] more on factors such as neuropsychological function rather than physical disability [32]. after injury—particularly in the domain of cognitive speed, visuospatial skills and verbal memory [28]. When combined with age, sex and progression over time since TBI, the model predicted 48% of variability observed in PCS and 39% in MCS scores. Although the physical function played a critical role in QOL rating, it was not the sole deter- minate and other factors not included in the model influence subjective health. This is in line with Ruff and colleagues who found that social function and return to vocation depended The relationship between SF-36 and GOSE is consistent with findings reported by Wilson et al., who found a positive correlation between GOSE and eight subscales of SF-36, par- ticularly in the social functioning domain [42]. A meta- analysis of 49 studies found SF-36 to be the most widely used and robust tool for assessing outcome following TBI, with strong internal consistency and interpretability [31]. Table 3 Generalised linear mixed model of PCS and MCS scores. Discussion SE, standard error PCS MCS β SE CI (95%) p β SE CI (95%) p GOSE Upper good recovery 41.9 2.5 37.3–46.7 0.000 54.7 2.5 49.8–59.5 0.000 Lower good recovery 27.7 2.5 22.7–32.7 0.000 44.7 3.0 38.9–50.5 0.000 Upper moderate disability 13.2 2.3 8.6–17.8 0.000 25.0 2.6 19.9–30.0 0.000 Lower moderate disability 4.1 2.4 −0.6–8.7 0.049 13.0 2.9 7.3–18.7 0.000 Upper severe disability −2.6 2.4 −7.3–2.0 0.266 8.4 2.9 2.6–14.2 0.005 Age at trauma −0.07 0.03 −0.13– (-0.07) 0.03 0.06 0.05 −0.04–0.16 0.24 Gender Male 0.10 1.1 −2.1–2.3 0.93 −0.42 2.0 −4.4–3.6 0.84 Months since injury 0.02 0.01 −0.01–0.04 0.08 0.04 0.02 0.01–0.08 0.018 R2 0.48 0.39 Acta Neurochir (2018) 160:107–115 112 Table 4 GOSE and PCS/MCS score changes between the 1st and 2nd clinic appointment Table 4 GOSE and PCS/MCS score changes between the 1st and 2nd clinic appointment GOSE PCS MCS Score change Number of subjects Mean (SD) Range Median Mean (SD) Range Median −4 1 −16.6 −5.8 −3 1 −31.61 −13.36 −2 5 −6.68 (21.8) −31.3–27.6 −6.7 5.28 (19.2) −14.9–30.8 −0.08 −1 25 −5.25 (20.8) −42.7–41.7 −8.3 −6.19 (22.5) −42.4–48.9 −8.7 0 114 0.87 (15.5) −41.22–36.9 2.52 1.40 (17.9) −50.5–57.4 1.25 1 46 13.25 (15.6) −23.6–52.1 16.85 13.31 (20.4) −20.16–76.02 8.83 2 16 23.85 (16.1) −3.6–51.2 25.2 22.08 (17.7) −14.8–65.2 20.78 3 9 19.70 (29.8) −24.4–59.7 11.47 20.66 (20.8) −20.1–52.1 21 4 4 24.02 (36.9) −18.03–66 24.06 22.73 (22.3) −8.8–41.7 29 SD, standard deviation SD, standard deviation However, as mentioned by the authors, SF-36 may not be a sensitive tool for detecting emotional and cognitive distur- bances. In addition to the previous reported research, this study highlights that GOSE is the strongest predictor of PCS and MCS scores. There is also a statistically significant differ- ence in the MCS and PCS scores based on patients’ GOSE scores. 36, 41]. In contrast, other studies found reverse results with subjects having higher MCS than PCS [15, 17, 21, 34]. They argued that the discrepancy between the mental and physical scores could result from lack of awareness in patients with severe TBI. However, in the study by Steinbuechel et al. the number of patients with severe TBI was double of those with mild TBI. In terms of distribution of GOSE scores, we had a relatively equal number of subjects with GOSE below and above a score of 5. Fig. 3 Box and whisker plot showing the change in GOSE score and PCS/MCS score between the 1st and 2nd clinic appointment. Only time points with ten or more subjects are illustrated Conclusion The aftermath of TBI is heterogeneous, leaving patients with a spectrum of physical, cognitive and psychological sequelae. This study highlights that patients’ physical outcome follow- ing TBI is a strong predictor of their subjective mental and physical health. Nevertheless, this is not the only factor to predict QOL post trauma, as there is a tremendous variation in scores observed across the GOSE spectrum. Thus, despite poor physical function, some patients rate their quality of life as high and vice versa. Further analysis of data would include additional factors such education, employment status, family network and cognitive ability to better model the predictors and help understand the natural history of recovering follow- ing TBI. This in turn could facilitate delivery of appropriate and individualised rehabilitation to ensure the best possible outcome for patients with TBI. Despite the positive relationship between GOSE and SF-36 domain scores, there is tremendous variability in MCS and PCS scores within each of the GOSE categories. This is con- sistent with the findings of Polinder et al., whose meta- analysis found significant heterogeneity in SF-36 summary scores [31]. GOSE is a global score, which captures only a limited component of a subject’s overall health. Factors such as degree of cognitive ability, educational background and psychological health have all been found to affect QOL post TBI. On the other hand, the majority of studies have found no gender differences in physical and cognitive function follow- ing discharge [4, 29, 33, 35]. This is in keeping with our results that show gender to be a non-significant variable in influencing SF-36 rating. Interpersonal differences in social and economic status following trauma may also be an impor- tant contributing factor to outcome. For instance, economic advantage can allow better provision of home support, both physical and mental. It could assist patients in paying for private therapies to enhance psychological and physical health. In addition, the family network and meaningful inter- personal relationship play a crucial part in the support and rehabilitation of patients following TBI. Acknowledgements The authors would like to thank Helen Seeley and Liz Corteen for their assistance in data collection. Funding This study was supported by National Institute for Healthcare Research (NIHR, UK) through the Acute Brain Injury and Repair theme of the Cambridge NIHR Biomedical Research Center to PH and AH, an NIHR Senior Investigator Award to DM and an NIHR Research Professorship to PH. Discussion For instance, targeted re- habilitation programmes have been found to increase memory in patients with mild to moderate Alzheimer’s disease [10] and focused training of motor skills in Parkinson’s patients lead to increased activity within motor regions of the brain [37]. These studies provide compelling evidence that targeted ther- apy and rehabilitation in TBI patients, despite their age, may yield positive benefits on the cognitive function. These in turn may facilitate patients’ return to vocation and improving their emotional health. Nevertheless, there are additional challenges following trauma in the elderly associated with increased co- morbidities and not only with neuronal changes [6]. As such, this group of individuals may require additional in-hospital support and physiotherapy to promote their return to the community. were not included in the statistical analysis because of incom- plete information. These factors have been found to predict outcome following TBI and may explain some of the variabil- ity seen in the scores of MCS and PCS observed for our cohort. For example, research has shown that pre-injury em- ployment status and educational level are strong predictors of QOL and return to vocation [23]. Likewise, cognitive ability has been shown to account for 21% to 30% of the variability observed in the functional ability post trauma [7]. Our patients were not subdivided based on the severity of the trauma. As mentioned earlier, some studies have found that subjects with severe TBI lacked awareness and thus ranked higher on QOL questionnaires. Self-awareness is di- rectly associated with motivation and change of behavior, which may facilitate better functional outcomes following trauma. On the other hand, it is also more likely to result in depression and other psychological conditions further de- creasing perceived QOL [14]. Conclusion Authors were also supported by a European Union Framework Program 7 grant (CENTER-TBI; Grant Agreement No. 602150). AH is supported by MRC (G0802251). This study has several limitations. First, it is a retrospective single-centre study from a specialised tertiary neurotrauma clinic in which the population demographics, socioeconomic background and availability of rehabilitation services may be skewed and may not necessarily reflect the UK general TBI population. In addition, there were a limited number of fixed effects included in the study. Factors such as education, em- ployment status, relationship status and cultural background Discussion Our cohort of patients had higher average PCS scores (M = 52.46, SD ± 19.46) compared to MCS (M = 51.77, SD ± 25.60). This is in agreement with results of the study by Steinbuechel et al., Hawthorne et al. and Wilson et al. [20, Age at the time of trauma was a positive predictor of PCS score but the effect size was small. For every additional year, Fig. 3 Box and whisker plot showing the change in GOSE score and PCS/MCS score between the 1st and 2nd clinic appointment. Only time points with ten or more subjects are illustrated Acta Neurochir (2018) 160:107–115 113 the PCS score decreased by 0.07 points (p < 0.05). On the other hand the MCS score tended to rise with increasing age, although this finding was insignificant (B = 0.04, p = 0.36). This is in accordance with other studies, which found that the proportion of poor outcome following TBI increased with age [16, 38]. Despite the changes observed in the ageing brain, including modification in electrical coupling and cell connec- tions, it retains remarkable ability to respond to stimulation and improve working memory [5]. For instance, targeted re- habilitation programmes have been found to increase memory in patients with mild to moderate Alzheimer’s disease [10] and focused training of motor skills in Parkinson’s patients lead to increased activity within motor regions of the brain [37]. These studies provide compelling evidence that targeted ther- apy and rehabilitation in TBI patients, despite their age, may yield positive benefits on the cognitive function. These in turn may facilitate patients’ return to vocation and improving their emotional health. Nevertheless, there are additional challenges following trauma in the elderly associated with increased co- morbidities and not only with neuronal changes [6]. As such, this group of individuals may require additional in-hospital support and physiotherapy to promote their return to the community. the PCS score decreased by 0.07 points (p < 0.05). On the other hand the MCS score tended to rise with increasing age, although this finding was insignificant (B = 0.04, p = 0.36). This is in accordance with other studies, which found that the proportion of poor outcome following TBI increased with age [16, 38]. Despite the changes observed in the ageing brain, including modification in electrical coupling and cell connec- tions, it retains remarkable ability to respond to stimulation and improve working memory [5]. Compliance with ethical standards Conflict of Interest All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing 114 Acta Neurochir (2018) 160:107–115 arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. 11. Edwards SGM, Playford ED, Hobart JC, Thompson AJ (2002) Comparison of physician outcome measures and patients’ percep- tion of benefits of inpatient neurorehabilitation. BMJ 324:1493 12. Faul M, Coronado V (2015) Epidemiology of traumatic brain inju- ry. Handbook of Clin Neurol 127:3–13 Ethical approval All procedures performed in studies involving hu- man participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. For this retrospective study formal consent was not required. 13. Fayers P, Hays RD (2005) Assessing quality of life in clinical trials. Oxford University Press, Oxford 14. Fleming JM, Strong J, Ashton R (1998) Cluster analysis of self- awareness levels in adults with traumatic brain injury and relationshipto outcome. J Head Trauma Rehabil 13(5):39–51 15. Forslund MV, Roe C, Sigurdardottir S, Andelic N (2013) Predicting health-related quality of life 2?years after moderate-to-severe trau- matic brain injury. Acta Neurol Scand 128(4):220–227 Informed consent Data for the research were collected as part of a registered audit at Addenbrooke’s Hospital, Cambridge. Informed con- sent was obtained from all individual participants included in the study. 16. Gómez PA, Lobato RD, Boto GR, De la Lama A, González PJ, de la Cruz J (2000) Age and outcome after severe head injury. Acta Neurochir (Wien) 142(4):373–380 Abbreviations: DRS Disability Rating Scale, FIM Functional indepen- dence measure, GLMM Generalised linear mixed model, GOSE Extended Glasgow Outcome Score, MCS Mental Component Score (SF-36), PCS Physical Component Score (SF-36), PROM Patient Reported Outcome Measures, QoL Quality of life, SF-36 36-Item Short Form Surverym, TBI Traumatic brain injury 17. Grauwmeijer E, Heijenbrok-Kal MH, Ribbers GM (2014) Health- related quality of life 3 years after moderate to severe traumatic brain injury: a prospective cohort study. Arch Phys Med Rehabil 95(7):1268–1276 18. References 22. Johnstone B, Schopp LH, Harper J, Koscuilek J (1999) Neuropsychological impairments, vocational outcomes, and finan- cial costs for individuals with traumatic brain injury receiving state vocational rehabilitation services. J Head Trauma Rehabil 14(3): 220–232 1. Ashley MJ, Krych DK, Lehr RP (1990) Cost/benefit analysis for post-acute rehabilitation of the traumatically brain-injured patient. J Insur Med 22(2):156–161 2. Ben-Yishay Y, Silver SM, Piasetsky E, Rattok J (1987) Relationship between employability and vocational outcome after intensive holistic cognitive rehabilitation. J Head Trauma Rehabil 2(1):35–48 23. Keyser-Marcus LA, Bricout JC, Wehman P, Campbell LR, Cifu DX, Englander J, High W, Zafonte RD (2002) Acute predictors of return to employment after traumatic brain injury: a longitudinal follow-up. Arch Phys Med Rehabil 83(5):635–641 3. Brooks DN, McKinlay W (1983) Personality and behavioural change after severe blunt head injury—a relative’s view. J Neurol Neurosurg Psychiatry 46(4):336–344 24. Levin HS, Boake C, Song J, McCauley S, Contant C, Diaz-Marchan P, Brundage S, Goodman H, Kotrla KJ (2001) Validity and sensitivity to change of the extended glasgow outcome scale in mild to moderate traumatic brain injury. J Neurotrauma 18(6):575–584 4. Brown AW, Moessner AM, Mandrekar J, Diehl NN, Leibson CL, Malec JF (2011) A survey of very-long-term outcomes after trau- matic brain injury among members of a population-based incident cohort. J Neurotrauma 28(2):167–176 25. Lezak MD (1978) Living with the characterologically altered brain injured patient. J Clin Psychiatry 39(7):592–598 5. Burke SN, Barnes CA (2006) Neural plasticity in the ageing brain. Nat Rev Neurosci 7(1):30–40 26. Likert R (1932) A technique for the measurement of attitudes. Arch Psychol 22:1–55 6. Callaway DW, Wolfe R (2007) Geriatric trauma. Emerg Med Clin North Am 25(3):837–860 27. McCauley SR, Wilde EA, Moretti P et al (2013) Neurological out- come scale for traumatic brain injury: III. Criterion-related validity and sensitivity to change in the NABIS hypothermia-II clinical trial. J Neurotrauma 30(17):1506–1511 7. Chaytor N, Temkin N, Machamer J, Dikmen S (2007) The ecolog- ical validity of neuropsychological assessment and the role of de- pressive symptoms in moderate to severe traumatic brain injury. J Int Neuropsychol Soc 13(3):377–385 28. Millis SR, Rosenthal M, Novack TA, Sherer M, Nick TG, Kreutzer JS, High WM, Ricker JH (2001) Long-term neuropsychological outcome after traumatic brain injury. J Head Trauma Rehabil 16(4):343–355 8. Cope DN, Cole JR, Hali KM, Barkan H (1991) Brain injury: anal- ysis of outcome in a post-acute rehabilitation system. Compliance with ethical standards Guilfoyle MR, Seeley HM, Corteen E, Harkin C, Richards H, Menon DK, Hutchinson PJ (2010) Assessing quality of life after traumatic brain injury: examination of the short form 36 health survey. J Neurotrauma 27(12):2173–2181 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 19. Hajfey WJ, Abrams DL (1991) Employment outcomes for partici- pants in a brain injury work reentry program: preliminary findings. Head Trauma Rehabil 20. Hawthorne G, Gruen RL, Kaye AH (2009) Traumatic brain injury and long-term quality of life: findings from an Australian study. J Neurotrauma 26(10):1623–1633 21. Jacobsson LJ, Westerberg M, Lexell J (2010) Health-related quality-of-life and life satisfaction 6-15 years after traumatic brain injuries in northern Sweden. Brain Inj 24(9):1075–1086 References Part 1: general analysis. Brain Inj 5(2):111–125 29. Moore DW, Ashman TA, Cantor JB, Krinick RJ, Spielman LA (2010) Does gender influence cognitive outcome after traumatic brain injury? Neuropsychol Rehabil 20(3):340–354 9. Corso P, Finkelstein E, Miller T, Fiebelkorn I, Zaloshnja E (2006) Incidence and lifetime costs of injuries in the United States. Inj Prev 12(4):212–218 30. Nunnally JH, Bernstein IH (1994) Psychometric theory. McGraw- Hill, New York 10. Cotelli M, Manenti R, Brambilla M, Petesi M, Rosini S, Ferrari C, Zanetti O, Miniussi C (2014) Anodal tDCS during face-name asso- ciations memory training in Alzheimer’s patients. Front Aging Neurosci 6:38 31. Polinder S, Haagsma JA, van Klaveren D, Steyerberg EW, van Beeck EF (2015) Health-related quality of life after TBI: a Acta Neurochir (2018) 160:107–115 115 systematic review of study design, instruments, measurement prop- erties, and outcome. Popul Health Metr 13:4 37. Subramanian L, Hindle JV, Johnston S, Roberts MV, Husain M, Goebel R, Linden D (2011) Real-time functional magnetic reso- nance imaging neurofeedback for treatment of Parkinson’s disease. J Neurosci 31(45):16309–16317 32. Ruff RM, Marshall LF, Crouch J et al (1993) Predictors of outcome following severe head trauma: follow-up data from the traumatic coma data bank. Brain Inj 7(2):101–111 38. Thompson HJ, McCormick WC, Kagan SH (2006) Traumatic brain injury in older adults: epidemiology, outcomes, and future implica- tions. J Am Geriatr Soc 54(10):1590–1595 33. Schonberger M, Ponsford J, Reutens D, Beare R, Clarke D, O’Sullivan R (2011) The relationship between mood disorders and MRI findings following traumatic brain injury. Brain Inj 25(6):543–550 39. Thornhill S, Teasdale GM, Murray GD, McEwen J, Roy CW, Penny KI (2000) Disability in young people and adults one year after head injury: prospective cohort study. BMJ 320:1631 34. Scholten AC, Haagsma JA, Andriessen TMJC, Vos PE, Steyerberg EW, van Beeck EF, Polinder S (2015) Health-related quality of life after mild, moderate and severe traumatic brain injury: patterns and predictors of suboptimal functioning during the first year after in- jury. Injury 46(4):616–624 40. Ware JE, Snow KK, Kosinski M, Gandek B (1993) F- 36 health survey: manual and interpretation guide. Nimrod Press, Boston 41. Wilson L, Marsden-Loftus I, Koskinen S et al (2017) Interpreting quality of life after brain injury scores: cross-walk with the short form-36. J Neurotrauma 34(1):59–65 35. References Slewa-Younan S, van den Berg S, Baguley IJ, Nott M, Cameron ID (2008) Towards an understanding of sex differences in functional outcome following moderate to severe traumatic brain injury: a systematic review. J Neurol Neurosurg Psychiatry 79(11):1197– 1201 42. Wilson JT, Pettigrew LE, Teasdale GM (2000) Emotional and cog- nitive consequences of head injury in relation to the glasgow out- come scale. J Neurol Neurosurg Psychiatry 69(2):204–20s9 43. Wood RL (2013) Recognising and assessing neurobehavioural dis- ability after traumatic brain injury. NeuroRehabilitation 32(4):699– 706 36. von Steinbuechel N, Covic A, Polinder S et al (2016) Assessment of health-related quality of life after TBI: comparison of a disease- specific (QOLIBRI) with a generic (SF-36) instrument. Behav Neurol 2016:1–14
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Detecting Tuberculosis in Prisons: Switching Off the Disease at Its Source
Clinical infectious diseases/Clinical infectious diseases (Online. University of Chicago. Press)
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© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source Dr. Marc Woodman MD1, Dr. Louis Grandjean MD PhD1 Dr. Marc Woodman MD1, Dr. Louis Grandjean MD PhD1 1. Department of Infection, Immunity and Inflammation, Institute of Child Health, University College London, London, UK, WC1N 1EH Corresponding author: l.grandjean@ucl.ac.uk Phone: 020 7905 2215 Fax: 020 7905 2882 Corresponding author Institute of Child Health, 30 Guilford Street, University College London, WC1N 1EH Corresponding author: l.grandjean@ucl.ac.uk Phone: 020 7905 2215 Fax: 020 7905 2882 Corresponding author Institute of Child Health, 30 Guilford Street, University College London, WC1N 1EH Corresponding author: l.grandjean@ucl.ac.uk © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Mycobacterium tuberculosis is estimated to have killed 1 billion people over the last 200 years (1) and remains the worlds' most deadly human pathogen (2). In order to improve upon the current 2% annual decline in tuberculosis incidence and get anywhere near the 90% by 2035 reduction target of the World Health Organization (WHO) "End Tuberculosis Strategy" (3), tuberculosis must be stopped at source. There are few sources of tuberculosis worthier of intervention than tuberculosis disease arising in prisons. In resource poor settings, the incidence of tuberculosis is on average 23 times greater inside prisons than in the surrounding community (4). The incidence in the Brazilian prisons in this study was 3,900 per 100,000, one hundred times that of the general population. Unless tuberculosis disease is diagnosed and treated within the prison, on release, prisoners are more likely to return to high risk transmission networks and fuel the epidemic further (5). Prison settings often compound the problem by facilitating transmission through overcrowding, inadequate ventilation, incomplete treatment, late case detection and high prisoner turnover (6). Thus, the case for prison intervention is compelling. It is naïve to think that tuberculosis disease is contained within the walls of the prison. As a case in point, Sarita Colonia prison in the province of Callao, Peru according to publicly available figures is overcrowded by 483% (7). Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source In 2018, it was shown that living in close proximity to the prison significantly increased the probability of sharing identical tuberculosis pathogen genotypes with those inside the prison (8). Prison intervention will therefore diminish the incidence of tuberculosis cases in the surrounding community. Arguably even more concerning; due to prisoner exchange between countries, new cases of identical transmitted strains from Sarita Colonia prison in Peru have now emerged in both Florence and Madrid (9). Therefore, prison intervention may even act to contain international tuberculosis spread. Although much research time and many publications have focussed on the benefits of intervening in prisons to diagnose and treat tuberculosis, there remains a lack of evidence to determine which - of all the options available - is the best employ (10). Santos and co-authors in this edition of Clinical Infectious Diseases present the results of an ambitious yet well delivered study that helps to address the gaps in this important field of research. The authors intensively and prospectively screened consenting prisoners in three Brazilian prisons for tuberculosis using symptom screening, GeneXpert, sputum culture and chest radiography with Computer-Aided Detection for Tuberculosis (CAD4TB). They then retrospectively applied four alternative less comprehensive screening algorithms to the data to determine which was most cost effective per case detected. They found that 84% of tuberculosis cases were detectable by a single sputum sample for Xpert MTB/RIF, and that systematic screening with this method had a cost per case of US$234. By comparison, symptom screening had a similar cost per case detected at US$235, but missed twice as many cases, while algorithms involving CXR screening were more expensive and did not increase overall yield compared to testing with sputum Xpert/MTBRIF alone. This is a clear, well written paper drawn from research undertaken in extremely difficult field conditions that has generated clinically applicable results. It demonstrates that the most sensitive and effective algorithm - of those that were tested - to detect tuberculosis disease in prisons is to apply GeneXpert to any prisoner who is able to produce sputum. Considering the challenging environment that the authors were working in, the screening participation rate of 89.9% is impressive and adds to the generalizability of the study. Only 31% of patients were able to produce a sputum sample, which, as the authors conclude themselves likely underestimates the true burden of tuberculosis disease. Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source This raises the question of whether interferon gamma release assays and/or tuberculin skin testing together with chest radiography could have a role in detecting these cases that were potentially missed. While many studies have demonstrated the utility of GeneXpert as a point of care test and it is therefore expected that it would improve screening algorithms wherever it is applied, there are few studies that examine its use head to head with other algorithms in prisons. Overall, the merit of this paper rests on the quality of its intensive screening strategy and the fact that this was applied to all prisoners in an extremely high burden setting. It clearly demonstrates the utility of GeneXpert in prisons - one of the major sources of new tuberculosis disease - and argues correctly for the implementation of this test in similar settings worldwide. If the WHO End TB targets are to be achieved then well-informed, well-funded and widespread scaling-up tuberculosis control in prisons is a good place to start. Neither author has any potential conflicts to disclose. References 1. Paulson T. Epidemiology: A mortal foe. Nature. 2013 Oct 10;502(7470):S2-3. 2. Gordon SV, Parish T. Microbe Profile: Mycobacterium tuberculosis: Humanity’s deadly microbial foe. Microbiol Read Engl. 2018;164(4):437–9. 3. Uplekar M, Weil D, Lonnroth K, Jaramillo E, Lienhardt C, Dias HM, et al. WHO’s new end TB strategy. Lancet Lond Engl. 2015 May 2;385(9979):1799–801. 4. Baussano I, Williams BG, Nunn P, Beggiato M, Fedeli U, Scano F. Tuberculosis Incidence in Prisons: A Systematic Review. PLoS Med [Internet]. 2010 Dec 21 [cited 2020 Jan 31];7(12). Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3006353/ 5. Altice FL, Azbel L, Stone J, Brooks-Pollock E, Smyrnov P, Dvoriak S, et al. The perfect storm: incarceration and the high-risk environment perpetuating transmission of HIV, hepatitis C virus, and tuberculosis in Eastern Europe and Central Asia. Lancet Lond Engl. 2016 Sep 17;388(10050):1228–48. 6. Escombe AR, Oeser CC, Gilman RH, Navincopa M, Ticona E, Pan W, et al. Natural ventilation for the prevention of airborne contagion. PLoS Med. 2007 Feb;4(2):e68. 7. INPE. Informe Estadistico Penetenciario [Internet]. Ministerio de Justicia y Derechos Humanos; 2016 [cited 2020 Jan 31]. Available from: https://www.inpe.gob.pe/revistas/estadistica/2016/octubre2016/mobile/index.htm l 8. Warren JL, Grandjean L, Moore DAJ, Lithgow A, Coronel J, Sheen P, et al. Investigating spillover of multidrug-resistant tuberculosis from a prison: a spatial and molecular epidemiological analysis. BMC Med. 2018 Aug 3;16(1):122. 9. Perez Garcia L. Detecting Tuberculosis in Prisons: Switching Off the Disease at its Source Tuberculosis en un escenario global: Nuevas estrategias transnacionales para optimizar la vigilancia de su transmission. XV Congreso Argentino de Microbiologia CAM 2019; 2019 Sep 26; Buenos Aires. 10. Systematic review on the diagnosis, treatment, care and prevention of tuberculosis in prison settings [Internet]. European Centre for Disease Prevention and Control. 2017 [cited 2020 Jan 31]. Available from: https://www.ecdc.europa.eu/en/publications-data/systematic-review-diagnosis- treatment-care-and-prevention-tuberculosis-prison
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Developing selective media for quantification of multispecies biofilms following antibiotic treatment
PloS one
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Developing selective media for quantification of multispecies biofilms following antibiotic treatment Eva Vandeplassche, Tom Coenye, Aure´lie Crabbe´* Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium Eva Vandeplassche, Tom Coenye, Aure´lie Crabbe´* Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium * Aurelie.Crabbe@UGent.be * Aurelie.Crabbe@UGent.be OPEN ACCESS Citation: Vandeplassche E, Coenye T, Crabbe´ A (2017) Developing selective media for quantification of multispecies biofilms following antibiotic treatment. PLoS ONE 12(11): e0187540. https://doi.org/10.1371/journal.pone.0187540 Editor: Eduard Torrents, Institute for Bioengineering of Catalonia (IBEC), SPAIN Received: July 5, 2017 Accepted: October 20, 2017 Published: November 9, 2017 Copyright: © 2017 Vandeplassche et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Vandeplassche E, Coenye T, Crabbe´ A (2017) Developing selective media for quantification of multispecies biofilms following antibiotic treatment. PLoS ONE 12(11): e0187540. https://doi.org/10.1371/journal.pone.0187540 Editor: Eduard Torrents, Institute for Bioengineering of Catalonia (IBEC), SPAIN Copyright: © 2017 Vandeplassche et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Abstract a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 The lungs of cystic fibrosis (CF) patients are chronically colonized by a polymicrobial biofilm community, leading to difficult-to-treat infections. To combat these infections, CF patients are commonly treated with a variety of antibiotics. Understanding the dynamics of polymi- crobial community composition in response to antibiotic therapy is essential in the search for novel therapies. Culture-dependent quantification of individual bacteria from defined multi- species biofilms is frequently carried out by plating on selective media. However, the influ- ence of the selective agents in these media on quantitative recovery before or after antibiotic treatment is often unknown. In the present study we developed selective media for six bacterial species that are frequently co-isolated from the CF lung, i.e. Pseudomonas aer- uginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans. We show that certain supplementations to selective media strongly influence quantitative recovery of (un)treated biofilms. Hence, the developed media were optimized for selectivity and quantitative recovery before or after treatment with antibiotics of four major classes, i.e. ceftazidime, ciprofloxacin, colistin, or tobramycin. Finally, in a proof of concept experiment the novel selective media were applied to determine the community composition of multispecies biofilms before and after treatment with tobramycin. Introduction Chronic respiratory tract infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients [1]. In the CF lung environment, a polymicrobial community is the source of these continuing infections, which results in an inflammatory response and eventu- ally leads to lung deterioration [2–5]. Treatment of chronic lung infections in CF patients is particularly challenging as members of the bacterial community form biofilms [6] which are highly resistant to antimicrobial therapy [7,8]. The presence of biofilm-related infections has a significant impact on clinical health outcomes, more specifically on lung function [9]. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was funded by an Odysseus grant from the Research Foundation Flanders (AC, TC). Interspecies interactions in polymicrobial communities have been shown to influence vari- ous phenotypic characteristics of individual bacteria, such as antimicrobial susceptibility and Competing interests: The authors have declared that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 1 / 15 Selective media for quantification of multispecies biofilms production of virulence factors [10]. Hence, the importance of studying the role of complex bacterial communities in disease progression and antibiotic susceptibility is generally acknowl- edged [10,11]. To further elucidate the role of interspecies interactions in antibiotic suscepti- bility of defined polymicrobial communities, in vitro studies have provided valuable insights [12–14]. To this end, quantification of bacteria in multispecies consortia before and after anti- biotic treatment is performed using culture-dependent and -independent techniques. One of the most commonly used culture-dependent quantification methods for biofilms is determina- tion of CFU (colony forming unit) counts on selective media. Most bacteria in the CF lung are culturable, making this an effective approach for quantification of CF-related biofilms [15,16]. While selective media are available for a broad range of microorganisms, many are focused solely on isolation from clinical or environmental samples and not on quantitative recovery [17–20]. Few studies have investigated how the quantitative recovery on selective media com- pares to that on a general medium [21–23], although this is essential to obtain an accurate pic- ture of the amounts of particular taxa in bacterial communities. Furthermore, the influence of antibiotic treatment on the recovery on a selective medium, which is often supplemented with various antimicrobial substances, has not yet been investigated. Introduction This is important, as prior antibiotic exposure could increase susceptibility to the selective agent(s) in the medium, and therefore affect quantitative recovery. The present study aims at developing selective media for six bacterial species commonly found together in the CF lung in order to allow accurate quantification of individual species in complex biofilm communities, before and after antibiotic treatment. Furthermore, previously described selective media were evaluated in the process as well. Our approach for the develop- ment of a selective medium for each of the species of interest consists of three steps. First, the selectivity for each species was assessed using planktonic cultures. Second, the quantitative recovery of single-species biofilms was compared between different media. Third, the single- species biofilms were treated with antibiotics of four different classes after which the recovery was evaluated again. Based on results obtained in each step, media composition was optimized to improve quantitative recovery on the selective medium. Finally, the developed media were tested in various experiments, to confirm their applicability for quantification of bacteria from planktonic cultures and from biofilms, before and after antibiotic treatment. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 General media For each of the six bacterial species included, a general medium with a theoretical recovery of 100% was used: Luria Bertani (LB; Lab M) agar for P. aeruginosa and S. aureus, Brain Heart Infusion (BHI; Lab M) agar for S. anginosus, Nutrient agar (Lab M) for A. xylosoxidans and R. mucilaginosa, and Columbia agar (CA; Lab M) or Columbia Blood Agar (CBA; Columbia Agar base, Lab M + 5% defibrinated sheep blood, Biotrading) for G. haemolysans. Bacterial strains and culturing conditions Six bacterial species (clinical strains and one type strain; listed in Table 1) that are frequently co-isolated from CF patients were selected for the development of selective media: Pseudomo- nas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans [24–27]. For each species, at least one strain was included that was isolated from the respiratory tract or, when this was not readily available in public repositories, a type strain was used. For all strains, liquid cultures were grown in BHI (Brain Heart Infusion) broth (Lab M) at 37˚C with shaking at 250 rpm until stationary phase. For S. anginosus and G. haemolysans, cul- tures were incubated in microaerophilic conditions (±5% O2, ±15% CO2). When indicated, microaerophilic conditions (±5% O2, ±15% CO2) were obtained using the CampyGen Compact system (Oxoid, Thermo Fisher Scientific). To create an anaerobic envi- ronment (<1% O2), AnaeroGen Compact sachets (Oxoid, Thermo Fischer Scientific) or can- dle jars with the Anaerocult A system (Merck Millipore) were used. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 2 / 15 Selective media for quantification of multispecies biofilms Table 1. Overview of strains used in this study and isolation sites. Species Strain Isolation site Pseudomonas aeruginosa AA2 Lung; early CF infection [28] AA44 Lung; late CF infection [28] PAO1 Wound Staphylococcus aureus SP123 Sputum of extubated patient [29] Mu50 Wound Streptococcus anginosus LMG 14696 Human throat LMG 14502 Human respiratory tract Achromobacter xylosoxidans LMG 26680 Human blood LMG 14980 Sputum—Belgian CF patient Rothia mucilaginosa DSM 20746 Bronchial secretion ATCC 49042 Throat Gemella haemolysans LMG 18984 Unknown LMG 1068 Type strain https://doi.org/10.1371/journal.pone.0187540.t001 Table 1. Overview of strains used in this study and isolation sites. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 Formation of single- and multispecies biofilms All biofilms were grown in a PVC flat-bottomed 96-well microtiter plate (Thermo Fisher) as described previously, with modifications [35,36]. Stationary phase liquid cultures were diluted in BHI supplemented with 2.5% lysed horse blood (horse blood from Biotrading, protocol for lysed horse blood according to EUCAST). For single-species biofilms, 100 μL of a 2.5 x 107 CFU/mL suspension was transferred to a 96-well plate. For multispecies biofilms, 2.5 x 106 CFU of each strain were added per well to obtain an equal number of cells per species per well. The inoculated 96-well plates were incubated at 37˚C for 24 hours in a microaerophilic envi- ronment, thereby mimicking the low oxygen conditions in the CF lung [37,38]. Media and chemicals used for development of the selective media Previously described selective media. Previously described selective media were exam- ined for selectivity and adjusted when necessary. For all media, each selective component was examined to distinguish which components were essential for selective inhibition. For P. aeru- ginosa, Pseudomonas Isolation Agar (PIA; BD Diagnostics) is described which uses 25 μg/mL triclosan (Irgasan, Sigma Aldrich) as a selective agent since most bacterial species are suscepti- ble while P. aeruginosa is resistant [30,31]. For S. aureus, Mannitol Salt Agar is widely used for selection, based on a 7.5% NaCl concentration [32,33]. For S. anginosus, several media were considered: NAS (nalidixic acid sulfamethazine) agar [19], Mitis Salivarius Agar (Sigma- Aldrich) and Edwards medium (Oxoid, Thermo Fisher Scientific). For A. xylosoxidans, MCXVAA medium is described [18], based on MacConkey agar supplemented with 5 mg/mL xylose, 20 μg/mL vancomycin, 20 μg/mL aztreonam and 5 μg/mL amphotericin. For R. mucila- ginosa, Rothia Mucilaginosa Selective Medium (RMSM) was previously developed, containing 50 μg/mL sodium selenite and 10 μg/mL colistin [22]. Finally, for G. haemolysans, a supple- mented Edwards medium with 5 μg/mL colistin sulphate and 2.5 μg/mL oxolinic acid was described [20]. Antimicrobial susceptibility testing. To identify potential selective or discriminating antibiotic agents, an antibiotic susceptibility screening was performed. Susceptibility of the six species to 25 antibiotics was assessed using the disk diffusion assay (S1 Table). To this end, antibiotic disks were placed on inoculated BHI agar, according to the manufacturer’s instruc- tions. Subsequently, the MIC90 of selected antibiotics (S2 Table) was determined using the broth microdilution method according to EUCAST guidelines [34]. For all antibiotics, a range of 0.5 – 256 μg/mL was tested in BHI or nutrient broth, depending on the general medium for the intended strain. 3 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 Selective media for quantification of multispecies biofilms Selectivity testing The ability of the medium to select for each of the six species was checked by plating. A plank- tonic culture of each species, grown to stationary phase in BHI medium, was brought to an OD 590 nm corresponding to approximately 5 x 107 CFU/mL, serially diluted and then plated on the respective general medium as a positive control (theoretical recovery of 100%) and in parallel on each of the candidate selective media (detection limit = 102 CFU/mL). The medium was considered selective if distinct, countable colonies (clear growth) could be observed for the intended species only. All media were incubated at 37˚C for 16 hours or until colonies were of sufficient size for counting. https://doi.org/10.1371/journal.pone.0187540.t002 Concentrations in italic show additional concentrations tested when either no effect or complete removal of biofilm growth occurred with the initial concentrations. Antibiotic treatment of biofilms After 24 h, biofilms were treated with antibiotics as described previously [35]. Briefly, the bio- films were rinsed with physiological saline solution (0.9% NaCl in milliQ water). Afterwards, 100 μL of medium (BHI + 2.5% lysed horse blood) was added to the control biofilms or 100 μL of antibiotic solution in the same medium was added to the biofilms. Four antimicrobials from four major antibiotic classes were selected. An overview of the antibiotics and their concentra- tions used is shown in Table 2. As a starting point, one set of antibiotic concentrations that gave a decrease of one to two logs of P. aeruginosa biofilm was used on all strains. Other con- centrations of antibiotics were tested when no effect was observed for the other bacterial spe- cies or in case of complete biofilm killing (detection limit of 102 CFU/mL) with the initial test Table 2. Overview of antibiotic concentrations used for quantitative recovery experiments. Ceftazidime Ciprofloxacin Colistin Tobramycin Pseudomonas aeruginosa 2000 μg/mL 0.5 μg/mL 500 μg/mL 100 μg/mL Staphylococcus aureus 2000 μg/mL 0.5 μg/mL 500 μg/mL 100 μg/mL 200 μg/mL 400 μg/mL Streptococcus anginosus 2000 μg/mL 0.5 μg/mL 500 μg/mL 100 μg/mL 4 μg/mL Achromobacter xylosoxidans 2000 μg/mL 0.5 μg/mL 500 μg/mL 100 μg/mL 1000 μg/mL 4 μg/mL Rothia mucilaginosa 2000 μg/mL 0.5 μg/mL 500 μg/mL 100 μg/mL 1000 μg/mL 4 μg/mL Gemella haemolysans 2000 μg/mL 0.5 μg/mL 500 μg/mL 100 μg/mL 4 μg/mL 25 μg/mL Concentrations in italic show additional concentrations tested when either no effect or complete removal of biofilm growth occurred with the initial concentrations. https://doi.org/10.1371/journal.pone.0187540.t002 Overview of antibiotic concentrations used for quantitative recovery experiments. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 4 / 15 Selective media for quantification of multispecies biofilms concentration. All biofilms were incubated at 37˚C under microaerophilic conditions for an additional 24 hours. concentration. All biofilms were incubated at 37˚C under microaerophilic conditions for an additional 24 hours. Biofilm quantification and recovery testing Following antibiotic treatment of single or multispecies biofilms, biofilms were rinsed with physiological saline. To quantify the number of colony forming units, biofilms were homoge- nized by two rounds of vortexing (900 rpm, 5 min) and sonication (5 min; Branson Ultrasonic bath), as described previously [35]. The resulting suspensions were serially diluted and plated on the appropriate general and/or selective media. Plates were incubated at 37˚C overnight (16 hours) or until colonies were of sufficient size for counting. Recovery was considered quantita- tive when no significant difference was observed between the number of CFU on the general medium compared to the selective medium. Statistics All experiments were carried out at least in biological triplicate. Biofilm experiments contained three technical replicates. Statistical analyses were performed in SPSS 23.0. Normality of the data was checked through the Shapiro-Wilk test. Differences between the means of normally distributed data were assessed by ANOVA-testing, followed by a Dunnett’s post hoc analysis, or by an independent samples t-test. When normality was not confirmed, a Kruskal-Wallis non-parametric test or Mann-Whitney test was performed. Means and standard deviations are shown in each graph. Statistical significance is assumed when p-values are < 0.05. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 Selective media for quantification of multispecies biofilms Fig 1. Overview of selectivity experiments. The first column shows the intended species and an abbreviated name of the medium, the second column (O2) shows aerobic (A) or anaerobic (AN) incubation, and the third column (t) shows incubation times. Strains belonging to the same species showed the same results. Pa: P. aeruginosa AA2, PAO1, and AA44; Sa: S. aureus SP123 and Mu50; Sag: S. anginosus LMG 14696 and LMG 14502; Ax: A. xylosoxidans LMG 26680 and LMG 14980; Rm: R. mucilaginosa DSM 20746 and ATCC 49042; Gh: G. haemolysans LMG 18984 and LMG 1068. Results are shown for all tested media: Pseudomonas Isolation Agar (PIA), LB + triclosan (LB tricl), LB + 7.5% NaCl (LB NaCl), nalidixic acid sulphamethazine agar (NAS), Mitis-salivarius agar (MSA), McConkey + 5 mg/mL xylose + 20 μg/mL vancomycin (McXV), McConkey + 5 mg/mL xylose + 20 μg/mL vancomycin (McXV) + 20 μg/mL aztreonam (McXVA), McConkey + aztreonam (McA), Rothia mucilaginosa selective medium (RMSM), nutrient agar + 5 μg/mL mupirocin + 10 μg/ mL colistin sulphate (NMC), Edwards medium + 10 μg/mL colistin sulphate (Edwards + col), Columbia blood agar + 32/ 6.4 μg/mL co-trimoxazole (CBA + co32), Columbia agar + 32/6.4 μg/mL co-trimoxazole (CA + co32). https://doi org/10 1371/journal pone 0187540 g001 Fig 1. Overview of selectivity experiments. The first column shows the intended species and an abbreviated name of the medium, the second column (O2) shows aerobic (A) or anaerobic (AN) incubation, and the third column (t) shows incubation times. Strains belonging to the same species showed the same results. Pa: P. aeruginosa AA2, PAO1, and AA44; Sa: S. aureus SP123 and Mu50; Sag: S. anginosus LMG 14696 and LMG 14502; Ax: A. xylosoxidans LMG 26680 and LMG 14980; Rm: R. mucilaginosa DSM 20746 and ATCC 49042; Gh: G. haemolysans LMG 18984 and LMG 1068. Results are Fig 1. Overview of selectivity experiments. The first column shows the intended species and an abbreviated name of the medium, the second column (O2) shows aerobic (A) or anaerobic (AN) incubation, and the third column (t) shows incubation times. Strains belonging to the same species showed the same results. Pa: P. aeruginosa AA2, PAO1, and AA44; Sa: S. aureus SP123 and Mu50; Sag: S. anginosus LMG 14696 and LMG 14502; Ax: A. xylosoxidans LMG 26680 and LMG 14980; Rm: R. mucilaginosa DSM 20746 and ATCC 49042; Gh: G. Assessment of selectivity and quantitative recovery of selective media The results of the selectivity testing using planktonic cultures on previously described and modified selective media are summarized in Fig 1. Following a positive outcome for selectivity, the selective media were evaluated for quantitative recovery of cells from untreated biofilms (Fig 2). P. aeruginosa. We confirmed selectivity of Pseudomonas Isolation Agar (PIA, containing 25 μg/mL triclosan). In order to reduce the concentration of the selective agent, lower triclosan concentrations (5 – 0.31 μg/mL in LB agar) were tested. All tested concentrations of triclosan inhibited growth of all the other bacteria while allowing growth of P. aeruginosa after aerobic overnight (16h) incubation. At concentrations of 1.25 μg/mL triclosan or higher, S. anginosus showed growth of small colonies after 16h incubation which did not interfere with quantifica- tion of P. aeruginosa. It is important to notice that longer incubation times (24-48h) lead to interference of S. anginosus and A. xylosoxidans growth for quantification of P. aeruginosa, highlighting the importance of selecting appropriate incubation times. Subsequently, recovery of a single-species biofilm was tested on triclosan-supplemented LB agar. Addition of 5 μg/mL triclosan resulted in a significant decrease of P. aeruginosa recovery (p < 0.05), while addition of 2.5 and 1.25 μg/mL triclosan did not affect recovery. Hence, LB agar with 1.25 μg/mL triclo- san (LB tricl 1.25) was used for further experiments. S. aureus. Mannitol Salt Agar contains 7.5% NaCl, and we evaluated LB agar supple- mented with the same amount of NaCl (LB NaCl). Aerobic overnight incubation showed that only S. aureus was able to grow on this medium, confirming its selectivity. Furthermore, recov- ery of biofilm cells on LB NaCl showed no difference in CFU counts compared to LB agar. S. anginosus. NAS agar and Mitis Salivarius Agar combined with anaerobic incubation were examined for selectivity but both media also supported growth of S. aureus and R. 5 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 haemolysans LMG 18984 and LMG 1068. Results are shown for all tested media: Pseudomonas Isolation Agar (PIA), LB + triclosan (LB tricl), LB + 7.5% NaCl (LB NaCl), nalidixic acid sulphamethazine agar (NAS), Mitis-salivarius agar (MSA), McConkey + 5 mg/mL xylose + 20 μg/mL vancomycin (McXV), McConkey + 5 mg/mL xylose + 20 μg/mL vancomycin (McXV) + 20 μg/mL aztreonam (McXVA), McConkey + aztreonam (McA), Rothia mucilaginosa selective medium (RMSM), nutrient agar + 5 μg/mL mupirocin + 10 μg/ mL colistin sulphate (NMC), Edwards medium + 10 μg/mL colistin sulphate (Edwards + col), Columbia blood agar + 32/ 6.4 μg/mL co-trimoxazole (CBA + co32), Columbia agar + 32/6.4 μg/mL co-trimoxazole (CA + co32). https://doi.org/10.1371/journal.pone.0187540.g001 https://doi.org/10.1371/journal.pone.0187540.g001 mucilaginosa in these conditions. Since 1.25 μg/mL triclosan permitted growth of S. anginosus, LB supplemented with 1.25, 0.625 and 0.31 μg/mL triclosan was tested. Incubation took place under anaerobic conditions to inhibit P. aeruginosa growth, as P. aeruginosa cannot grow anaerobically without an alternative terminal electron acceptor [39,40]. This approach allowed growth of S. anginosus only. In addition, LB with triclosan (LB 1.25, 0.625, 0.31) also allowed 6 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 Selective media for quantification of multispecies biofilms Fig 2. Quantification of untreated biofilms of the six bacterial species on candidate selective media compared to a general medium. (A) P. aeruginosa AA2 (B) S. aureus SP 123 (C) S. anginosus LMG 14696 (D) A. xylosoxidans LMG 26680 (E) R. mucilaginosa DSM 20476 (F) G. haemolysans LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations. * p < 0.05, n  3. Fig 2. Quantification of untreated biofilms of the six bacterial species on candidate selective media compared to a general medium. (A) P. aeruginosa AA2 (B) S. aureus SP 123 (C) S. anginosus LMG 14696 (D) A. xylosoxidans LMG 26680 (E) R. mucilaginosa DSM 20476 (F) G. haemolysans LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations. * p < 0.05, n  3. https://doi.org/10.1371/journal.pone.0187540.g002 https://doi.org/10.1371/journal.pone.0187540.g002 quantitative recovery of cells from single-species S. anginosus biofilms. Based on these results, LB tricl 0.31 (with anaerobic incubation) was selected for further experiments. A. xylosoxidans. MCXVAA (based on MacConkey agar supplemented with 5 μg/mL xylose, 20 μg/mL vancomycin, 20 μg/mL aztreonam and 5 μg/mL amphotericin) [18], was con- firmed to be a selective medium for A. xylosoxidans. The selective value of each of these com- ponents (except for the anti-fungal amphotericin) was subsequently tested. MacConkey agar inhibited growth of the Gram-positive bacteria, while addition of 20 μg/mL aztreonam inhib- ited growth of P. aeruginosa. MacConkey agar with 1 μg/ml (or less) aztreonam also supported growth of P. aeruginosa, while at concentrations of 10 μg/mL or higher, partial growth inhibi- tion of A. xylosoxidans was observed. MacConkey supplemented with 5 μg/mL aztreonam (McA5) was tested further and this medium allowed quantitative recovery of cells from untreated A. xylosoxidans single-species biofilms. R. mucilaginosa. RMSM only inhibited growth of P. aeruginosa, while the other five spe- cies, including R. mucilaginosa, showed at least partial growth on this medium. Growth inhibi- tion of P. aeruginosa was due to 10 μg/mL colistin sulphate, while sodium selenite did not inhibit growth of any of the species investigated. Results from disk diffusion assays suggested that mupirocin could be a suitable selective agent (S1 Table). Based on MIC values (S2 Table), 5 μg/mL mupirocin was added to nutrient agar, together with 10 μg/mL colistin sulphate. This medium (NMC) consisting of nutrient agar, mupirocin and colistin sulphate, was selective for R. mucilaginosa and allowed quantitative recovery of R. mucilaginosa from untreated biofilms. G. haemolysans. We could not confirm selectivity for the previously described supple- mented Edwards medium [20] as it did not allow growth of G. haemolysans in our experi- ments. To develop a new selective medium for G. haemolysans, co-trimoxazole was used as this antibiotic did not inhibit G. haemolysans growth in the disk diffusion assay, while inhibi- tion zones were observed for all other species (S1 Table). Based on MIC values (S2 Table) for all strains, 32/6.4 μg/mL co-trimoxazole was added to CBA. However, besides G. haemolysans, S. aureus also showed growth on CBA agar with co-trimoxazole. Yet, in the absence of blood complete growth inhibition was observed for all bacteria except for G. haemolysans, which lead to the use of Columbia agar with 32/6.4 μg/mL co-trimoxazole (CA + co32) for further testing. Our data showed that the CA + co32 medium allowed quantitative recovery of G. haemolysans from untreated biofilms. Selective media for quantification of multispecies biofilms Selective media for quantification of multispecies biofilms PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 7 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 7 / 15 Quantitative recovery of biofilm cells after antibiotic treatment Treated biofilms were plated on the developed selective media and on general media to assess quantitative recovery following antibiotic treatment. Results are summarised in Fig 3. The optimized selective media are presented in Table 3. For P. aeruginosa, no significant difference was observed between the recovery of biofilm cells on LB, LB with 1.25 μg/mL triclosan, or PIA after treatment with ceftazidime, ciprofloxa- cin, colistin or tobramycin. Thus, LB tricl 1.25 with aerobic overnight incubation was an appropriate medium for quantitative recovery of P. aeruginosa biofilm cells after antibiotic treatment. 8 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 Selective media for quantification of multispecies biofilms Fig 3. Quantification of biofilms after antibiotic treatment on selective media compared to a general medium. (A) P. aeruginosa AA2 (B) S. aureus SP 123 (C) S. anginosus LMG 14696 (D) A. xylosoxidans LMG 26680 (E) R. mucilaginosa DSM 20476 (F) G. haemolysans LMG 18984. PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl; McA5 = McConkey agar supplemented with 5 μg/mL aztreonam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sulphate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations. * p < 0.05, n  3. https://doi.org/10.1371/journal.pone.0187540.g003 Similarly, in terms of recovery of S. aureus biofilms cells, there was no significant difference between LB and LB + 7.5% NaCl after treatment with ceftazidime, ciprofloxacin, colistin or tobramycin. After treatment with ceftazidime, ciprofloxacin, colistin, or tobramycin, complete recovery of S. anginosus biofilm cells was observed on LB supplemented with 1.25 μg/mL, 0.625 μg/mL or 0.31 μg/mL triclosan (incubated anaerobically) as no differences with recovery on BHI were observed. For further experiments LB with 0.31 μg/ml triclosan (LB tricl 0.31) was used together with anaerobic incubation. On McConkey agar with 5 μg/mL aztreonam, no significant difference in A. xylosoxidans recovery was observed after treatment with ciprofloxacin, colistin or tobramycin, compared to nutrient agar. However, after treatment with 2000 μg/mL ceftazidime, a significant decrease (p < 0.05) in recovery on McA5 was noted compared to nutrient agar. Hence, a lower concen- tration of aztreonam in MacConkey agar (1 μg/mL; McA1) was tested but this did not restore recovery of the A. xylosoxidans biofilm treated with ceftazidime. To test whether this effect was dependent on the concentration of the antibiotic, the concentration of ceftazidime was low- ered to 1000 μg/mL, which did improve biofilm recovery on both tested media (McA1 and McA5). However, as seen in the previous selectivity experiments, McA1 showed suboptimal inhibition of P. aeruginosa and furthermore did not lead to increased recovery compared to McA5. Taken together, McA5 was used as a selective medium for A. xylosoxidans. However, it remains important to keep in mind that recovery on McA5 is affected when biofilms are treated with ceftazidime, which varies dependent on the ceftazidime concentration. Selective media for quantification of multispecies biofilms Selective media for quantification of multispecies biofilms PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 9 / 15 Validation of the selective media For further validation of the six novel selective media, an additional strain (another clinical iso- late or type strain) of each species was tested (S1 and S2 Figs). The developed selective media allowed quantitative recovery of all the additional strains, with the exception of A. xylosoxidans LMG 14980 after ceftazidime treatment, and G. haemolysans LMG 1068 for both the untreated and treated (with ceftazidime and ciprofloxacin) single-species biofilm. Furthermore, a mixed planktonic culture with a defined composition of the six bacteria (± 5 x 107 CFU/mL of each) was plated on the six selective media and recovery was compared to the recovery of single-spe- cies cultures. The results show no significant decrease in recovery of the strains in the mixed culture compared to the single-species cultures showing that no growth inhibition occurred and that the mixed culture did not decrease recovery of the individual strains (S3 Fig). It is there- fore important to consider that highly concentrated supplementations to selective media may strongly influence quantitative recovery, in particular after preceding antibiotic treatment. For R. mucilaginosa, no significant difference between the recovery on NMC and nutrient agar was observed after treatment with either ceftazidime, ciprofloxacin, colistin or Table 3. Composition of optimized selective media. Selective medium Composition Pseudomonas aeruginosa LB tricl 1.25 LB agar 1.25 μg/mL triclosan Staphylococcus aureus LB NaCl LB agar 7.5% NaCl Streptococcus anginosus LB tricl 0.31 (anaerobic) LB agar 0.31 μg/mL triclosan Achromobacter xylosoxidans McA5 McConkey agar 5 μg/mL aztreonam Rothia mucilaginosa NMC Nutrient agar 5 μg/mL mupirocin 10 μg/mL colistin sulphate Gemella haemolysans CA + co32 Columbia agar 32/6.4 μg/mL co-trimoxazole https://doi.org/10.1371/journal.pone.0187540.t003 Table 3. Composition of optimized selective media. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 10 / 15 Selective media for quantification of multispecies biofilms Fig 4. Proof of concept: Quantification of multispecies biofilms. A multispecies biofilm of the six bacterial species was grown for 24 hours and subsequently incubated with fresh medium as an untreated control or treated with 100 μg/mL tobramycin in medium for an additional 24 hours in microaerophilic conditions. Graphs show mean recovery and standard deviations. The detection limit of 102 CFU/mL is represented by a dashed line. * p < 0.05, n  3. htt //d i /10 1371/j l 0187540 004 Fig 4. Proof of concept: Quantification of multispecies biofilms. A multispecies biofilm of the six bacterial species was grown for 24 hours and subsequently incubated with fresh medium as an untreated control or treated with 100 μg/mL tobramycin in medium for an additional 24 hours in microaerophilic conditions. Graphs show mean recovery and standard deviations. The detection limit of 102 CFU/mL is represented by a dashed line. * p < 0.05, n  3. https://doi.org/10.1371/journal.pone.0187540.g004 https://doi.org/10.1371/journal.pone.0187540.g004 tobramycin. The NMC medium was used for quantitative recovery of R. mucilaginosa follow- ing antibiotic treatment in further experiments. After treatment with ceftazidime, ciprofloxacin, colistin or tobramycin, no significant dif- ference in recovery of G. haemolysans on the developed selective medium (CA + co32) com- pared to Columbia agar was observed, confirming the developed selective medium for quantification after antibiotic treatment. Conclusion In this study, novel selective media were developed that allow quantitative recovery of (un) treated multispecies biofilms comprised of six bacteria frequently co-isolated from CF samples. We observed that incubation conditions, including incubation time and oxygen concentration, were essential to enable selective recovery. Furthermore, evaluation of the developed selective media for quantitative recovery of biofilms before and after antibiotic treatment revealed that certain selective agents profoundly affect recovery. These findings highlight the importance of keeping antibiotic supplementation in selective media to a minimum and to evaluate and potentially modify available selective media to ensure optimal recovery. In conclusion, six selective media were optimized for multispecies biofilm recovery, and we demonstrated that if a selective medium is to be used for quantitative recovery of untreated or antibiotic-treated cultures, prior recovery assessment is indispensable to avoid introducing biases for data interpretation. Supporting information S1 Table. Antibiotic disk diffusion assay. Mean inhibition zones and standard deviations (SD) are shown in mm per strain, ‘z’ shows that a clearer zone could be observed, yet no com- plete inhibition occurred. This assay was performed for P. aeruginosa PAO1, AA2, and AA44; S. aureus SP123; S. anginosus LMG 14696; A. xylosoxidans LMG 26680; R. mucilaginosa DSM 20746; G. haemolysans LMG 18984. (PDF) S2 Table. MIC data overview. Minimal Inhibitory Concentrations (range of 256–0.5 μg/mL) were determined for gentamicin and mupirocin in nutrient broth, and for co-trimoxazole (SMT-TMP) in BHI broth. -, not determined. S2 Table. MIC data overview. Minimal Inhibitory Concentrations (range of 256–0.5 μg/mL) were determined for gentamicin and mupirocin in nutrient broth, and for co-trimoxazole (SMT-TMP) in BHI broth. -, not determined. (PDF) (PDF) S1 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (A) P. aeruginosa PAO1 and AA44, (B) S. aureus Mu50, PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl. Graphs show mean recovery and standard devia- tions.  p < 0.05, n  3. (TIF) S2 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (C) S. anginosus LMG 14502, (D) A. xylosoxidans LMG 26680, (E) R. mucilaginosa ATCC 49042, and (F) G. haemolysans LMG 18984. LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); McA5 = McConkey agar supplemented with 5 μg/mL aztreo- nam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sul- phate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations.  p < 0.05, n  3. (TIF) S1 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (A) P. aeruginosa PAO1 and AA44, (B) S. aureus Mu50, PIA = Pseudomonas Isolation Agar; LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); LB NaCl = LB supplemented with 7.5% NaCl. Graphs show mean recovery and standard devia- tions.  p < 0.05, n  3. (TIF) S2 Fig. Quantification of untreated (left) and treated (right) biofilms of additional strains. (C) S. anginosus LMG 14502, (D) A. xylosoxidans LMG 26680, (E) R. mucilaginosa ATCC 49042, and (F) G. haemolysans LMG 18984. Proof of concept: Multispecies biofilms Finally, as a proof of concept experiment to show that the media can be used to isolate strains grown in a biofilm, the six bacterial species were quantified in multispecies biofilms, before and after treatment with 100 μg/mL tobramycin. Biofilms were grown for 24h and subse- quently treated for 24h in microaerophilic conditions (Fig 4). The results indicate that in the multispecies biofilm P. aeruginosa, S. anginosus and G. haemolysans become less abundant after treatment with tobramycin (p < 0.05), while S. aureus, A. xylosoxidans and R. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 11 / 15 Selective media for quantification of multispecies biofilms mucilaginosa are not affected by the treatment. Furthermore, biofilms were also grown for 48h and subsequently treated for 24h (S4 Fig). All species, except for G. haemolysans, could be iso- lated from this multispecies biofilm before and after treatment as well. These findings show that community composition can be altered by antibiotic treatment. Hence, deciphering the dynamics of multispecies consortia in response to antibiotic treatment using in vitro tools may help improve our understanding of how multispecies communities respond to antibiotics in vivo. Acknowledgments The authors would like to thank Charlotte Rigauts, Petra Rigole, Lisa Ostyn and Inne D’Hondt for their practical assistance. Supporting information LB tricl = LB agar supplemented with triclosan in various concentrations (μg/mL); McA5 = McConkey agar supplemented with 5 μg/mL aztreo- nam; NMC = nutrient agar supplemented with 5 μg/mL mupirocin and 10 μg/mL colistin sul- phate; CA + co32 = Columbia agar with 32/6.4 μg/mL co-trimoxazole. Graphs show mean recovery and standard deviations.  p < 0.05, n  3. (TIF) 12 / 15 PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 Selective media for quantification of multispecies biofilms S3 Fig. Validation of the novel selective media. Planktonic mono-species cultures and a mixed culture were plated on the newly developed selective media and showed equal recovery. Graphs show mean recovery and standard deviations. (TIF) S4 Fig. Proof of concept: Quantification of multispecies biofilms. A multispecies biofilm of the six bacterial species was grown for 48 hours and subsequently incubated with fresh medium as an untreated control or treated with 100 μg/mL tobramycin in medium for an additional 24 hours in microaerophilic conditions. Graphs show mean recovery and standard deviations. The detection limit of 102 CFU/mL is represented by a dashed line.  p < 0.05, n  3. (TIF) Conceptualization: Aure´lie Crabbe´. Data curation: Eva Vandeplassche, Aure´lie Crabbe´. Formal analysis: Eva Vandeplassche. Formal analysis: Eva Vandeplassche. Funding acquisition: Tom Coenye, Aure´lie Crabbe´. Project administration: Aure´lie Crabbe´. Supervision: Tom Coenye, Aure´lie Crabbe´. Supervision: Tom Coenye, Aure´lie Crabbe´. Writing – original draft: Eva Vandeplassche. Writing – original draft: Eva Vandeplassche. Writing – original draft: Eva Vandeplassche. Writing – review & editing: Tom Coenye, Aure´lie Crabbe´. Writing – review & editing: Tom Coenye, Aure´lie Crabbe´. Author Contributions Conceptualization: Aure´lie Crabbe´. PLOS ONE | https://doi.org/10.1371/journal.pone.0187540 November 9, 2017 References Insights into cystic fibrosis polymicrobial consortia: the role of species interactions in biofilm development, phenotype, and response to in-use antibiotics. Front Micro- biol. 2017; 7:1–11. 15. Sibley CD, Grinwis ME, Field TR, Eshaghurshan CS, Faria MM, Dowd SE, et al. Culture enriched molecular profiling of the cystic fibrosis airway microbiome. PLoS One. 2011; 6(7):25–7. 16. 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Diagnosis-specific sickness absence among injured working-aged pedestrians: a sequence analysis
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Diagnosis‑specific sickness absence among injured working‑aged pedestrians: a sequence analysis Linnea Kjeldgård1*, Helena Stigson1,2,3, Eva L. Bergsten1,4, Kristin Farrants1 and Emilie Friberg BMC Public Health BMC Public Health Kjeldgård et al. BMC Public Health (2023) 23:367 https://doi.org/10.1186/s12889-023-15259-w Open Access Abstract Keywords  Sick leave, Disability pension, Pedestrians, Fall accidents, Traffic injury, Population-base 3 Folksam Research, Folksam Insurance Group, Stockholm, Sweden 4 Faculty of Health and Occupational Studies, Department of Occupational Health Sciences and Psychology, University of Gävle, 801 76 Gävle, Sweden 3 Folksam Research, Folksam Insurance Group, Stockholm, Sweden 4 Faculty of Health and Occupational Studies, Department of Occupational Health Sciences and Psychology, University of Gävle, 801 76 Gävle, Sweden *Correspondence: Linnea Kjeldgård linnea.kjeldgard@ki.se 1 Division of Insurance Medicine, Department of Clinical Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden 2 Division of Vehicle Safety, Mechanics and Maritime Sciences, Chalmers University of Technology, Gothenburg, Sweden Linnea Kjeldgård linnea.kjeldgard@ki.se 1 Division of Insurance Medicine, Department of Clinical Neuroscience, Karolinska Institutet, 171 77 Stockholm, Sweden 2 Division of Vehicle Safety, Mechanics and Maritime Sciences, Chalmers University of Technology, Gothenburg, Sweden 3 Folksam Research, Folksam Insurance Group, Stockholm, Sweden 4 Faculty of Health and Occupational Studies, Department of Occupational Health Sciences and Psychology, University of Gävle, 801 76 Gävle, Sweden Abstract Background  The knowledge about the long-term consequences in terms of sickness absence (SA) among pedestri- ans injured in a traffic-related accident, including falls, is scarce. Therefore, the aim was to explore diagnosis-specific patterns of SA during a four-year period and their association with different sociodemographic and occupational factors among all individuals of working ages who were injured as a pedestrian. Methods  A nationwide register-based study, including all individuals aged 20–59 and living in Sweden, who in 2014–2016 had in- or specialized outpatient healthcare after a new traffic-related accident as a pedestrian. Diagnosis- specific SA (> 14 days) was assessed weekly from one year before the accident up until three years after the accident. Sequence analysis was used to identify patterns (sequences) of SA, and cluster analysis to form clusters of individuals with similar sequences. Odds ratios (ORs) with 95% confidence intervals (CIs) for association of the different factors and cluster memberships were estimated by multinomial logistic regression. Results  In total, 11,432 pedestrians received healthcare due to a traffic-related accident. Eight clusters of SA patterns were identified. The largest cluster was characterized by no SA, three clusters had different SA patterns due to injury diagnoses (immediate, episodic, and later). One cluster had SA both due to injury and other diagnoses. Two clusters had SA due to other diagnoses (short-term and long-term) and one cluster mainly consisted of individuals with disability pension (DP). Compared to the cluster “No SA”, all other clusters were associated with older age, no university education, having been hospitalized, and working in health and social care. The clusters “Immediate SA”, “Episodic SA” and “Both SA due to injury and other diagnoses” were also associated with higher odds of pedestrians who sustained a fracture. Conclusions  This nationwide study of the working-aged pedestrians observed diverging patterns of SA after their accident. The largest cluster of pedestrians had no SA, and the other seven clusters had different patterns of SA in terms of diagnosis (injury and other diagnoses) and timing of SA. Differences were found between all clusters regard- ing sociodemographic and occupational factors. This information can contribute to the understanding of long-term consequences of road traffic accidents. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Background W02.4, W03.4, W04.4, W05.4, W10.4, W15.4, W17.4, W18.4, W19.4, W51.4) during two years before the acci- dent, were included. The date of the accident, denoted as ­T0, refers to the first date of the in- or specialized outpa- tient healthcare visit/hospitalization, as the actual date of their accident/fall is not included in the registers. g Walking as physical activity and active transportation is good for public health and accordingly encouraged by different stakeholders [1–3]. However, walking in a road traffic environment (i.e. being a pedestrian) also involves some risk. Pedestrians can slip, trip, stumble, or collide with other road users. Globally, about a fifth of all fatali- ties within the road transport system are represented by pedestrians [4, 5]. The UN’s global sustainable develop- ment goals strive both for more sustainable and safer transportation [5, 6]. Likewise, the Swedish Vision Zero has the long-term vision that no one should die or suf- fer injuries leading to long-term consequences within the road transport system [7].fif The SA-status of the study population was assessed weekly during a period of four years; one year before and three years after the accident date, ­T0. Individuals who died (n = 213) or emigrated (n = 58) during the follow- up period were excluded. In addition, those with dis- ability pension (DP) (full- or part-time) during all of the 209  weeks of the four-year study period were excluded (n = 1167). This resulted in a study population of 11,432 injured pedestrians. Being injured in a road traffic accident may affect the individual’s work ability and lead to sickness absence (SA) or disability pension (DP). We have previously shown that many (20%) injured pedestrians had a new SA spell in connection to the accident [8]. However, the knowledge of long-term SA and the patterns of SA among injured pedestrians is limited. In addition, diverse work require- ments for different types of work (e.g. occupational sec- tors and type of occupation) could impact the injured pedestrian’s need of SA or DP. However, the impact of the occupational factors in relation to SA and DP among pedestrians involved in a traffic-related accident have, to the best of our knowledge, not yet been explored. Background Microdata from several nationwide registers were used and linked at the individual level, using the unique personal identity number assigned to all residents in Sweden [10].h - The in- and specialized outpatient registers, from the National Board of Health and Welfare, were used to iden- tify the study population as well as for medical informa- tion related to the injury.h - The Cause of Death Register, from the National Board of Health and Welfare, was used to identify those who had died within three years from the accident date.h - The Longitudinal Integration Database for Health Insurance and Labour Market Studies (LISA), from Sta- tistics Sweden, was used to identify the source popula- tion, all individuals living in Sweden 31 December the year before the accident, and information for the study population regarding sociodemographic factors (sex, age, educational level, country of birth, type of living area, and marital status) and occupational factors (occupa- tional sector, private/public, and type of occupation) also measured 31 December the year before the accident. To get a broader understanding of the long-term con- sequences in terms of SA among pedestrians injured in traffic-related accidents, including falls within the road transport system, the aim of this study was to explore patterns of diagnosis-specific (i.e. injury diagnoses or other diagnoses) SA during a four-years period and their association with different background factors including occupational factors among all individuals of working ages who were injured as a pedestrian. - Micro-data for Analyses of the Social Insurance (MiDAS), from the Swedish Social Insurance Agency, was used for information on the dates and diagnoses of SA and DP. Open Access T Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Page 2 of 16 Methods using a modified version of the Barell matrix [11], into categories of injured body region and type of injury. Most of the pedestrian had only one injury diagnosis recorded, but for those individuals who had several, the main diagnosis was prioritized before any of the second- ary diagnoses. Some pedestrians had up to three visits/ hospitalizations at ­T0. In these cases, the injuries requir- ing inpatient healthcare were prioritized over the injuries treated in outpatient healthcare. In addition, an injury with ICD10: S00-S99 was prioritized over an injury with ICD10: T00-T88.h All individuals living in Sweden, ≥ 16  years old, and with income from work, unemployment, or parental- leave benefits can apply for SA benefits from the Social Insurance Agency if having a disease or injury that leads to reduced work capacity [17]. The first day of a SA spell is an unreimbursed qualifying day (more days for self- employed). A physician’s certificate is required after day 7. For employees, day 2–14 are reimbursed by the employer [17]. For others, e.g., unemployed, the Social Insurance Agency administers the benefits from the sec- ond day of SA, with information on shorter SA spells available for these individuals. Therefore, in order not to introduce a bias, only information on SA spells > 14 days was used. All individuals aged 19–64 can be granted DP if disease or injury leads to long-term or permanent work incapacity. Both SA and DP can be granted for full- or part-time (100, 75, 50, 25%) of ordinary work hours. Accordingly, someone on part-time DP can at the same time have part-time SA. For young individuals (19– 30 years old) DP can be time limited. In general, it is very uncommon but possible to go from DP to non-DP.i The injured body region was categorized into the fol- lowing six groups: head, face, and neck (reference group); vertebral column and spinal cord; torso; upper extremi- ties; lower extremities; and “other and unspecified”. The type of injury was categorized into six groups: fracture; dislocation; sprains and strains; internal (brain, spinal cord, and other internal organs); external (open wounds, contusions, and superficial injuries) (reference group); and “other and unspecified”. Similar categorizations have been used in recent studies on injuries among different groups of road traffic users [8, 12–15]. fi Healthcare at ­T0 was also used to categorize inpatient healthcare: no (only specialized outpatient healthcare) (reference group); yes. Methods If someone had both specialized outpatient healthcare and inpatient healthcare they were categorized as inpatient healthcare.h Weekly states of SA were defined for the study popula- tion. To do this, the four years of follow-up were divided into 209 weeks, 52 weeks prior ­(W−52) through 156 weeks after ­(W+156) the week of the accident ­W0, defined as ­T0 ± 3 days. For each week, individuals were assigned a state (one out of four non overlapping states) based on their SA situation during that week: No SA or DP (no SA or DP during the week); SA due to injury diagnosis (any SA due to an injury diagnosis (ICD10: S00-T98) during the week, and no DP); SA due to other diagnoses (any SA due to other diagnoses than injuries, no DP, and no SA due to an injury diagnosis during the week); and DP (any DP, regardless of extent or diagnosis, during the week). The sociodemographic factors were categorized as: sex (women; men (reference group)), age group (20–24; 25–34 (reference group); 35–44; 45–54; 55–59  years), level of education (elementary school (≤ 9 years includ- ing missing); high school (9–12  years); university/col- lege (> 12  years) (reference group)), country of birth (Sweden (reference group); not Sweden), type of living area (cities (reference group); town and suburbs; rural areas), marital status (married (reference group); not married). These have all been demonstrated to be com- mon risk factors for SA in general [16] and after a traffic related injury [8, 12–15]. Several occupational factors were also included. Occupational sector was categorized according to the Swedish Standard of Industrial Classification SNI 2007 (Manufacturing, agriculture, forestry & fishing (G01, G02, G03); Construction (G04); Trade, transport, hotels & restaurants (G05, G06, G07); Finance, communica- tion & cultural service (G08, G09, G10, G11, G12, G15) (reference group); Education (G13); Health & social care (G14); Not in work/Unknown (G99)), Private/pub- lic employer (private (reference group); public; Not in work/Unknown), and also type of occupation, according to the Swedish Standard for Occupational Classification SSYK (white collar (reference group); blue collar; Not in work/Unknown). Methods A prospective cohort study was conducted, includ- ing all pedestrians, 20–59  years of age (which is to say within working ages throughout the follow-up) who had at least one hospitalization or visit to specialized outpa- tient healthcare due to an injury (International Statistical Classification of Diseases and Related Health Problems; ICD-10 [9]: S00-T88) sustained in a new traffic-related accident (including falls and bumping into other pedes- trians) (ICD-10: V01-V09, W00.4, W01.4, W02.4, W03.4, W04.4, W05.4, W10.4, W15.4, W17.4, W18.4, W19.4, W51.4) between 1 January 2014 and 31 December 2016 (n = 12,870) and living in Sweden. Individuals who were living in Sweden the 31 December the year before the accident and did not have any traffic related in- or out- patient healthcare (ICD-10: V01-V99, W00.4, W01.4, Reference groups for the below factors were chosen based on size of the groups and expected proportions with SA, with larger groups or groups expected to have lower proportions of SA being used as reference groups.h The type of accident was categorized into the follow- ing six groups: collision with pedestrian/bicyclist (V01, W03.4, W04.4, W51.4); collision with motor vehicle (V02- V06, V09.0, V09.2); unspecified (V09.1, V09.3, V09.9, W19.4); fall: snow and ice, street and highway (W00.4); fall: slipping, tripping, and stumbling, street and highway (W01.4) (reference group); and fall: other, street and high- way (W02.4, W05.4, W10.4, W15.4, W17.4, W18.4).h The main diagnosis and the secondary diagnoses for the healthcare visit/hospitalization were categorized Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Page 3 of 16 Lastly, season was based on ­T0 and categorized as win- ter (December, January, February), spring (Mars, April, May), summer (June, July, August) (reference group), autumn (September, October, November). using a modified version of the Barell matrix [11], into categories of injured body region and type of injury. Most of the pedestrian had only one injury diagnosis recorded, but for those individuals who had several, the main diagnosis was prioritized before any of the second- ary diagnoses. Some pedestrians had up to three visits/ hospitalizations at ­T0. In these cases, the injuries requir- ing inpatient healthcare were prioritized over the injuries treated in outpatient healthcare. In addition, an injury with ICD10: S00-S99 was prioritized over an injury with ICD10: T00-T88. Resultsh All clusters other than “No SA” were associated with older age, no university education, having been hospital- ized, and working in health and social care. There were 11,432 pedestrians with in- or specialized out- patient healthcare due to a new traffic accident including fall accidents 2014–2016 aged 20–59 years (Table 1). The median age was 45  years among women and 39  years among men. The most common type of accident was fall: slipping, tripping, and stumbling, followed by fall: snow and ice. The most common types of injuries were, frac- tures, external injuries, and sprains and strains injuries. A quarter of the injured pedestrians worked in Finance, communication & cultural service. Among women, 29% worked in Health & social care and 14% worked in Trade, transport, hotels & restaurants; the corresponding num- bers in men were 5%, and 24% respectively. The cluster “Immediate SA” was associated with the types of accidents collision with motor vehicle and Fall: slipping tripping and stumbling (OR (95% CI): 1.54 (1.26–1.87), and 1.25 (1.05–1.48) respectively). Whereas the clusters “Long-term or later SA” and “Both SA due to injury and other diagnoses” were associated with injuries sustained in a collision with pedestrian/bicyclists (1.60 (1.03—2.48), and 1.70 (1.18—2.45) respectively). Fractures, dislocations, sprains and strains, and inter- nal injuries had high OR for the clusters “Immediate SA”, “Episodic SA”, and “Both SA due to injury and other diagnoses”. Diverse patterns of SA during the study period were observed among the pedestrians (4358 unique sequences). By far the most common sequence observed, for both women and men, was to have no SA or DP during all four years ­(W−52 to ­W+156) (38.8% for women and 52.5% for men) (Fig. 1). Several of the most common sequences included no SA prior to the accident and then SA of various durations due to injury diagnoses starting from the week of the accident ­(W0). Another common sequence observed among the individuals was to have SA due to other diag- nosis (i.e. not injury) during the entire study period (0.6% among women and 0.2% among men) (Fig. 1). Upper extremities had high OR for all cluster but “Dis- ability pension”. Lower extremities had high OR for all cluster but “Disability pension” and “Long-term or later SA”. Injuries to the torso were associated with the clusters “Episodic SA”, “Both SA due to injury and other diagno- ses”, and “Other diagnoses short-term SA”. Resultsh Injuries to the vertebral column and spinal cord were associated with the clusters “Episodic SA”, “Long-term or later SA”, and “Other diagnoses short-term SA”. Regarding occupational factors, blue-collar work was associated with all clusters but “Disability pension” compared to the cluster “No SA” and working in Health & social care was associated with all clusters compared to “No SA”. Working in Construction had high ORs for being in the clusters “Immediate SA” (1.62 (1.24–2.13)) and “Episodic SA” (2.35 (1.51–3.65)), whereas working in Education had high ORs for being in the clusters “Both SA due to injury and other diagnoses” (1.48 (1.08–2.02)) and “Other diagnoses short-term SA” (1.45 (1.16–1.80)). Cluster analysis was then used to form groups of indi- viduals who had similar sequences. The cluster partition quality measures for the different numbers of clusters are presented in the appendix (Table A.1). Eight clus- ters were identified: “No SA” (including 47% of the study population), “Immediate SA” (18%), “Episodic SA” (4%), “Long-term or later SA” (3%), “Both SA due to injury and other diagnoses” (7%), “Other diagnoses short-term SA” (17%), “Other diagnoses long-term SA” (2%), and “Disa- bility pension” (3%). In the description of the clusters, SA is referred to SA due to an injury diagnosis unless other- wise stated. The eight clusters are illustrated using den- sity plots in Fig. 2 and using index plots in Fig. 3. Sensitivity analyses including the 1167 individuals who had DP during the entire follow-up did not alter the results substantially (data not shown). Statistical analyses Descriptive statistics of the study population were con- ducted including stratified by sex. i The patterns of SA states during a four-year period (from 1 year before and through 3 years after ­W0 ­(W−52 to ­W+156)) were analyzed using sequence analysis with TraMineR in R [18]. Thereafter, cluster analysis with optimal matching spell algorithm[19] was used to iden- tify different clusters of individuals who had similar sequences of SA-states. A cluster tree and several meas- ures of cluster partition quality [18] were used to choose the number of clusters (Table A.1). Density plots and index plots for each cluster are shown. Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 Page 4 of 16 Page 4 of 16 Multinomial logistic regression models were used to analyze the association between sociodemographic and occupational factors, type of accident, type of health- care, type of injury, injured body region, and SA-clusters. Crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) were calculated. Sensitivity analyses were conducted including the 1167 individuals who had DP during the entire follow-up. The statistical analyses were performed using SAS (version 9.4) and R (version 3.6.1). Almost all of the 5339 individuals in the cluster “No SA” were not on SA or DP during the entire follow up. In this cluster there were 53% men and 8% were identi- fied through the in-patient healthcare. The most com- mon type of injuries among individuals belonging to this cluster were external injuries (41%) and fractures (29%). Characteristics of the clusters and adjusted ORs for the other clusters compared to the cluster “No SA” are pre- sented in Table 3. Descriptive statistics of all factors of the clusters and unadjusted and adjusted ORs can be found in the appendix (Table A.2 and Table A.3). Discussion Characteristics of each of the eight identified clusters are summarised in Table 2 in terms of sequences, soci- odemographic characteristics, occupational characteris- tics and injury related characteristics. In this nationwide register study exploring diagnosis- specific patterns of SA among injured pedestrians there were in total 11,432 working-aged pedestrians Kjeldgård et al. Discussion BMC Public Health (2023) 23:367 Page 6 of 16 1 column percent 2 row percent Table 1  (continued) All Women Men n %1 n %1 n %1   Summer 2329 20.37 1135 18.27 1194 22.87   Autumn 2565 22.44 1402 22.57 1163 22.28 Year of accident   2014 3393 29.68 1788 28.78 1605 30.75   2015 3956 34.60 2172 34.96 1784 34.18   2016 4083 35.72 2252 36.25 1831 35.08 Occupational sector   Manufacturing, agriculture, forestry & fishing 1143 10.00 309 4.97 834 15.98   Construction 586 5.13 63 1.01 523 10.02   Trade, transport, hotels & restaurants 2160 18.89 886 14.26 1274 24.41   Finance, communication & cultural service 2897 25.34 1568 25.24 1329 25.46   Education 1035 9.05 836 13.46 199 3.81   Health & social care 2076 18.16 1792 28.85 284 5.44   Not in work/Unknown 1535 13.43 758 12.20 777 14.89 Private/Public   Private sector 6118 53.52 2651 42.68 3467 66.42   Public sector 2946 25.77 2390 38.47 556 10.65   Not in work/Unknown 2368 20.71 1171 18.85 1197 22.93 Type of occupation   White collar 4539 39.70 3219 51.82 1320 25.29   Blue collar 3295 28.82 1295 20.85 2000 38.31   Not in work/Unknown 3598 31.47 1698 27.33 1900 36.40 [8]. This could partly be explained by that the winter seasons during 2010 were colder and with more snow- fall than during the rather mild winters when the pre- sent study was conducted in 2014–2016 [22–24]. This difference in proportion of accidents related to snow and ice highlights the importance of road environment maintenance during the winter season to reduce the risk for these types of accidents, which is also called for by authors of other studies [25, 26]. that received in- or specialized outpatient healthcare due to a traffic-related accident. Of them, 71% were due to falls and a third of the falls related to snow and ice. Eight different clusters of SA were identified with different patterns of SA due to an injury diagnosis or whether due to other diagnoses. Compared to the clus- ter “No SA” all the other clusters were associated with older age, no university education, having been hos- pitalized, and working in health and social care. The clusters “Immediate SA”, “Episodic SA” and “Both SA due to injury and other diagnoses” were also associ- ated with e.g. higher odds of individuals who sustained a fracture. The present study showed a rather diverse popula- tion is involved in pedestrian road traffic accidents. Discussion BMC Public Health (2023) 23:367 Page 5 of 16 Table 1  Characteristics of all pedestrians aged 20–59 with a road traffic injury (including falls) in 2014–2016, by sex All Women Men n %1 n %1 n %1 All 11,432 6212 54.342 5220 45.662 Age group, years   20–24 1737 15.19 752 12.11 985 18.87   25–34 2306 20.17 1093 17.59 1213 23.24   35–44 2243 19.62 1156 18.61 1087 20.82   45–54 3144 27.50 1867 30.05 1277 24.46   55–59 2002 17.51 1344 21.64 658 12.61 Level of education   Elementary school 1778 15.55 770 12.40 1008 19.31   High school 5796 50.70 2925 47.09 2871 55.00   University/College 3858 33.75 2517 40.52 1341 25.69 Country of birth   Sweden 9200 80.48 4983 80.22 4217 80.79   Not Sweden 2232 19.52 1229 19.78 1003 19.21 Type of living area   Cities 4752 41.57 2585 41.61 2167 41.51   Towns and suburbs 4723 41.31 2597 41.81 2126 40.73   Rural areas 1957 17.12 1030 16.58 927 17.76 Married   No 7466 65.31 3755 60.45 3711 71.09   Yes 3966 34.69 2457 39.55 1509 28.91 Type of accident   Collision with pedestrian/bicyclist 591 5.17 286 4.60 305 5.84   Collision with motor vehicle 1595 13.95 734 11.82 861 16.49   Unspecified 1166 10.20 547 8.81 619 11.86   Fall: snow and ice 2529 22.12 1582 25.47 947 18.14   Fall: slipping, tripping, and stumbling 4136 36.18 2491 40.10 1645 31.51   Fall: other 1415 12.38 572 9.21 843 16.15 Inpatient healthcare   No 9923 86.80 5442 87.60 4481 85.84   Yes 1509 13.20 770 12.40 739 14.16 Type of injury   Fracture 4587 40.12 2648 42.63 1939 37.15   Dislocation 366 3.20 145 2.33 221 4.23   Sprains and strains 1724 15.08 932 15.00 792 15.17   Internal 794 6.95 404 6.50 390 7.47   External 3749 32.79 1961 31.57 1788 34.25   Other and unspecified 212 1.85 122 1.96 90 1.72 Injured body region   Head, face and neck 2345 20.51 1146 18.45 1199 22.97   Vertebral column and spinal cord 215 1.88 107 1.72 108 2.07   Torso 630 5.51 295 4.75 335 6.42   Upper extremities 4005 35.03 2308 37.15 1697 32.51   Lower extremities 4185 36.61 2330 37.51 1855 35.54   Other and unspecified 52 0.45 26 0.42 26 0.50 Season   Winter 4133 36.15 2444 39.34 1689 32.36   Spring 2405 21.04 1231 19.82 1174 22.49 Table 1  Characteristics of all pedestrians aged 20–59 with a road traffic injury (including falls) in 2014–2016, by sex Kjeldgård et al. Discussion Higher proportions of older women yet more younger men were injured pedestrians. Women were more often injured in falls due to slipping, tripping, and stumbling as well as falls due to snow and ice, while men were more often injured in falls due to other rea- sons and collisions with a motor vehicle. In addition, more women were working in Health & social care and more men were working in Trade, transport, hotels & restaurants. Moreover, a higher proportion of women had university/college education than men. These dif- ferences among the injured pedestrians are important to consider, as suggest different measures might be The most common injuries were fractures and exter- nal injuries, which has also been reported for pedes- trians in previous studies [1, 8, 20, 21]. Injuries to the lower and upper extremities and to the head, face and neck were the most common type of injured body region, also in line with previous studies [1, 8, 20, 21]. In contrast, the proportion of injuries from falls due to snow and ice was lower in the present study compared to a previous study conducted during 2010, 22% com- pared to 36% of all pedestrian accidents, respectively Kjeldgård et al. BMC Public Health (2023) 23:367 Page 7 of 16 Fig. 1  The 20 most common sequences of sickness absence states/week from one year before until three years after ­(W-52 to ­W+156) the week of the pedestrian accident ­(W0) (marked as week 0 in the figure) for women and men Fig. 1  The 20 most common sequences of sickness absence states/week from one year before until three years after ­(W-52 to ­W+156) the week of the pedestrian accident ­(W0) (marked as week 0 in the figure) for women and men accidents [27–30]. Sickness absence and DP have so far only been studied for pedestrians in our previous study, a cross sectional study from Sweden, examin- ing the risk of new SA in connection to the accident, but with no follow-up or information on the duration of SA [8]. That study found that 20% had a new SA in connection to the accident and that 18% had ongoing SA or full-time DP at the time of the accident [8]. Even though those that were on DP throughout follow-up were excluded in the present study, the eight clusters of SA and DP could be comparable to our cross-sectional study [8]. Discussion Furthermore, the present study provides a much clearer picture of the SA and DP development during the years after the accident. Separating SA diag- noses into injury diagnoses and other diagnoses also helps in understanding what SA is due to the accident and what could be due to other circumstances. The dis- tinction between the cluster “Episodic SA” and “Both SA due to injury diagnoses and other diagnoses” could not have been made without the possibility to identify SA with injury diagnoses. The pedestrians in the clus- ter “Episodic SA” had a SA spell due to injury diagno- sis in connection to the accident and then one more SA spell due to an injury diagnosis, while the pedestrians called for in order to reduce the number of individu- als involved in a pedestrian accident for women and men. For example, personal devices such as reflectors, to prevent collisions with other road traffic groups, and anti-slip shoes to prevent falls due to snow and ice, or related to the traffic environment such as gritting/salt- ing slippery roads or improving the separation between road user groups. Another established difference is also related to the possibility to return to work in different types of occupations. Rather few studies have reported on long-term conse- quences of being involved in an accident as a pedestrian. The long-term consequences can be investigated from several different perspectives, the present study inves- tigated the long-term consequences in terms of SA and DP. A study from Sweden reported the long-term conse- quences, in terms of risk of Permanent Medical Impair- ment (PMI), showing that 25% of the pedestrian falls and 20% of the pedestrian collisions led to a permanent reduction with a PMI of above 1% [21].h The long-term consequences of pedestrian injuries in terms of SA and DP to the best of our knowledge has not been previously studied. There are only a few stud- ies investigating SA and DP in relation to road traffic Kjeldgård et al. BMC Public Health (2023) 23:367 Page 8 of 16 Fig. 2  Density plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading Fig. Discussion 2  Density plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading Fig. 2  Density plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading Fig. 2  Density plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading Fig. 2  Density plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading It could be in relation to late effects of the accident (e.g. injuries, musculoskeletal disease, thrombosis, pneumonia or PTSD) but also a new accident (lead- ing to injury) or even another health issue not related to the accident. In relation to other road user groups, a previous study on bicyclists investigated SA and DP in the cluster “Both SA due to injury diagnoses and other diagnoses” had first SA due to an injury diagno- ses at the time of the accident and then had later SA due to other diagnoses. The SA spell that occurred later during follow-up does not necessarily need to be con- nected to the accident, especially for the latter cluster. Kjeldgård et al. BMC Public Health (2023) 23:367 Page 9 of 16 Fig. 3  Index plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading and every line represents one individual. The plots are ordered by the first state in the sequences Fig. Discussion 3  Index plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading and every line represents one individual. The plots are ordered by the first state in the sequences Fig. 3  Index plots of sickness absence (SA) states/week during the year before through three years after ­(W−52 to ­W+156) the week of the pedestrian accident (marked with 0 in the figure), for the eight identified clusters. The number of individuals in each cluster are stated in each cluster heading and every line represents one individual. The plots are ordered by the first state in the sequences traffic accidents in terms of SA and DP, future studies are needed, especially those with a comparison to the general population. after a bicycle crash but did not make distinctions in SA diagnoses [15]. In addition, other studies investi- gating SA after a road traffic accident did not separate the analyses for different road user groups or did not differentiate different SA diagnoses in the analysis [27– 30]. To elucidate the long-term consequences of road g The relationship between occupational factors and SA after a pedestrian accident has also not been inves- tigated in detail. However, an Australian study found Page 10 of 16 Kjeldgård et al. BMC Public Health (2023) 23:367 Table 2  Summary of sociodemographic, injury, and occupational characteristics in the eight identified clusters of SA sequences Cluster 1. No SA 2. Immediate SA 3. Episodic SA 4. Long-term or later SA 5. Both SA due to injury and other diagnoses 6. Other diagnoses short- term SA 7. Other diagnoses long- term SA 8. Discussion Disability pension N 5339 individuals 2046 individuals 445 individuals 369 individuals 796 individuals 1901 individuals 233 individuals 303 individuals Characterisation of SA sequences No SA or DP during the entire study period SA due to injury diagnoses in connection to the accident Two or more SA spells due to injury diagnoses, one at the time of the acci- dent and one prior to or later during the study period One or several SA spells due to injury diagnoses later dur- ing follow-up One SA spell due to an injury diagnosis starting in connec- tion to the accident and also one or sev- eral SA spells due to other diagnoses during the study period One or several short-term SA spells due to other diag- noses spread out during the study period Long-term SA due to other diagnoses DP Characterisation of sociodemographic differences More young men More individuals not born in Sweden, living in cities, and with higher levels of education More women. More individuals born in Sweden, married, and living in towns or rural areas More older women More often married, and living in towns or rural areas More older men More often married, born in Sweden, and with lower levels of education More older women More often married, born in Sweden, and living in towns or rural areas More women More often born in Sweden More older women More often born in Sweden, living in cities, and with lower levels of education More often either younger and older individuals More often unmar- ried, living in towns or rural areas, and with lower levels of education Characterisation of occupation differ- ences More often not in work/unknown work More often working in Construction, Trade, transport, hotels & restaurants, and Health & social care More often working in Construction, and Health & social care. More often blue- collar worker More often working in Health & social care. More often blue-collar worker More often working in Education, and Health & social care. More often white- collar worker More often working in Education, and Health & social care More often work- ing in Health & social care. More often not in work/ unknown work More often working in Health & social care. Discussion More often not in work/unknown work Characterisation of the injuries More often external injuries and injuries to head, face and neck More often fractures and injuries to upper and lower extremities More often fractures and injuries to upper and lower extremities More often external injuries and injuries to vertebral column & spinal cord More often fractures and injuries to upper and lower extremities More often injuries to torso and vertebral column & spinal cord More often internal injuries and injuries to head, face and neck Kjeldgård et al. BMC Public Health (2023) 23:367 Page 11 of 16 Table 3  Distributions and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for different factors in each of the eight clusters of sickness absence (SA) sequences spanning 1 year before to 3 years after the pedestrian accident ­(W−52 to ­W+156) among 11,432 individuals aged 20–59 years injured in a traffic-related accident in 2014–2016, using the cluster “No SA” as the reference 1. No SA 2. Immediate SA 3. Episodic SA 4. Long-term or later SA 5. Both SA due to injury and other diagnoses 6. Other diagnoses short- term SA 7. Other diagnoses long- term SA 8. Discussion BMC Public Health (2023) 23:367 Page 12 of 16 Table 3  (continued) 1. No SA 2. Immediate SA 3. Episodic SA 4. Long-term or later SA 5. Both SA due to injury and other diagnoses 6. Other diagnoses short- term SA 7. Other diagnoses long- term SA 8. Discussion Disability pension n (%) Adj ­OR1 (95% CI) n (%) Adj ­OR1 (95% CI) n (%) Adj ­OR1 (95% CI) n (%) Adj ­OR1 (95% CI) n (%) Adj ­OR1 (95% CI) n (%) Adj ­OR1 (95% CI) n (%) Adj ­OR1 (95% CI) All 5339 (46.7) 2046 (17.9) 445 (3.89) 369 (3.23) 796 (6.96) 1901 (16.63) 233 (2.04) 303 (2.65) Type of accident   Colli- sion with pedestrian/ bicyclist 307 (5.75) 89 (4.35) 1.28 (0.96–1.70) 21 (4.72) 1.36 (0.83–2.24) 28 (7.59) 1.60 (1.03–2.48) 44 (5.53) 1.70 (1.18–2.45) 79 (4.16) 0.78 (0.60–1.03) 10 (4.29) 0.86 (0.44–1.69) 13 (4.29) 0.87 (0.47–1.60)   Collision with motor vehicle 777 (14.55) 286 (13.98) 1.54 (1.26–1.87) 57 (12.81) 1.39 (0.98–1.98) 59 (15.99) 1.23 (0.87–1.73) 79 (9.92) 1.12 (0.84–1.50) 256 (13.47) 0.95 (0.79–1.14) 33 (14.16) 0.87 (0.56–1.35) 48 (15.84) 0.91 (0.62–1.33)   Unspeci- fied 587 (10.99) 163 (7.97) 1.06 (0.85–1.32) 42 (9.44) 1.23 (0.85–1.79) 35 (9.49) 0.93 (0.63–1.39) 61 (7.66) 1.03 (0.76–1.41) 220 (11.57) 1.08 (0.90–1.30) 25 (10.73) 0.93 (0.59–1.49) 33 (10.89) 0.93 (0.61–1.42)   Fall: snow and ice 1005 (18.82) 600 (29.33) 1.25 (1.05–1.48) 122 (27.42) 1.20 (0.89–1.61) 78 (21.14) 1.08 (0.77–1.50) 249 (31.28) 1.25 (1.00–1.57) 370 (19.46) 0.93 (0.79–1.11) 47 (20.17) 0.79 (0.52–1.19) 58 (19.14) 0.85 (0.58–1.24)   Fall: slip- ping, trip- ping, and stumbling 1938 (36.30) 670 (32.75) ref 150 (33.71) ref 130 (35.23) ref 279 (35.05) ref 753 (39.61) ref 102 (43.78) ref 114 (37.62) ref   Fall: other 725 (13.58) 238 (11.63) 1.06 (0.87–1.29) 53 (11.91) 1.12 (0.79–1.58) 39 (10.57) 0.86 (0.59–1.25) 84 (10.55) 1.10 (0.83–1.45) 223 (11.73) 0.97 (0.81–1.16) 16 (6.87) 0.57 (0.33–0.99) 37 (12.21) 0.93 (0.62–1.40) Type of injury   Fracture 1549 (29.01) 1477 (72.19) 6.81 (5.71–8.13) 290 (65.17) 4.91 (3.57–6.75) 109 (29.54) 0.91 (0.68–1.23) 503 (63.19) 4.65 (3.63–5.96) 464 (24.41) 0.73 (0.63–0.85) 86 (36.91) 0.94 (0.66–1.35) 109 (35.97) 1.11 (0.80–1.54)   Disloca- tion 177 (3.32) 73 (3.57) 2.98 (2.13–4.15) 16 (3.60) 2.37 (1.29–4.35) 13 (3.52) 0.94 (0.51–1.74) 29 (3.64) 2.60 (1.63–4.16) 48 (2.52) 0.75 (0.53–1.06) -2 -2 -2 -2   Sprains and strains 890 (16.67) 182 (8.90) 1.45 (1.15–1.83) 57 (12.81) 1.69 (1.13–2.52) 70 (18.97) 1.12 (0.79–1.58) 119 (14.95) 1.90 (1.41–2.57) 353 (18.57) 0.93 (0.78–1.10) 23 (9.87) 0.62 (0.37–1.03) 30 (9.90) 0.90 (0.56–1.43)   Internal 392 (7.34) 82 (4.01) 3.08 (2.17–4.37) 17 (3.82) 3.04 (1.52–6.08) 33 (8.94) 1.32 (0.82–2.11) 34 (4.27) 3.10 (1.86–5.17) 172 (9.05) 1.27 (1.01–1.61) 20 (8.58) 1.57 (0.86–2.87) 44 (14.52) 1.93 (1.23–3.03)   External 2213 (41.45) 220 (10.75) ref 58 (13.03) ref 136 (36.86) ref 103 (12.94) ref 820 (43.14) ref 88 (37.77) ref ref ref   Other and unspeci- fied 118 (2.21) 12 (0.59) 1.44 (0.76–2.75) -2 -2 -2 -2 -2 -2 44 (2.31) 1.15 (0.79–1.67) 9 (3.86) 2.38 (1.09–5.18) -2 -2 Kjeldgård et al. Discussion Disability pension Injured body region   Head, face and neck 1394 (26.11) 155 (7.58) ref 32 (7.19) ref 83 (22.49) ref 61 (7.66) ref 474 (24.93) ref 49 (21.03) ref 97 (32.01) ref   Vertebral column and spinal cord 89 (1.67) 35 (1.71) 1.53 (0.92–2.55) 10 (2.25) 2.74 (1.16–6.46) 15 (4.07) 3.13 (1.59–6.15) 13 (1.63) 1.79 (0.86–3.70) 41 (2.16) 1.77 (1.16–2.72) -2 -2 -2 -2   Torso 312 (5.84) 68 (3.32) 1.41 (0.97–2.04) 18 (4.04) 2.11 (1.08–4.12) 22 (5.96) 1.27 (0.75–2.13) 31 (3.89) 1.98 (1.18–3.33) 147 (7.73) 1.64 (1.28–2.09) 15 (6.44) 1.65 (0.87–3.15) 17 (5.61) 0.90 (0.50–1.62)   Upper extremities 1654 (30.98) 938 (45.85) 3.26 (2.48–4.28) 206 (46.29) 4.44 (2.60–7.58) 129 (34.96) 1.74 (1.20–2.52) 344 (43.22) 3.27 (2.18–4.92) 557 (29.30) 1.24 (1.03–1.49) 84 (36.05) 2.01 (1.25–3.26) 93 (30.69) 1.21 (0.81–1.81)   Lower extremities 1856 (34.76) 847 (41.40) 3.26 (2.49–4.28) 179 (40.22) 4.10 (2.41–6.99) 119 (32.25) 1.32 (0.90–1.92) 347 (43.59) 3.65 (2.44–5.48) 674 (35.46) 1.26 (1.05–1.52) 76 (32.62) 1.64 (1.01–2.65) 87 (28.71) 0.93 (0.62–1.41)   Other and unspeci- fied 34 (0.64) -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 Occupational sector   Manu- facturing, agriculture, forestry & fishing 556 (10.41) 230 (11.24) 0.97 (0.78–1.20) 48 (10.79) 1.11 (0.74–1.65) 49 (13.28) 1.10 (0.74–1.63) 77 (9.67) 0.96 (0.70–1.31) 165 (8.68) 0.85 (0.68–1.06) -2 -2 12 (3.96) 0.52 (0.27–1.01)   Construc- tion 240 (4.50) 147 (7.18) 1.62 (1.24–2.13) 40 (8.99) 2.35 (1.51–3.65) 29 (7.86) 1.46 (0.91–2.35) 38 (4.77) 1.38 (0.92–2.09) 79 (4.16) 1.04 (0.77–1.39) -2 -2 -2 -2   Trade, transport, hotels & restaurants 993 (18.60) 427 (20.87) 1.20 (1.00–1.44) 85 (19.10) 1.25 (0.89–1.75) 63 (17.07) 0.86 (0.60–1.24) 150 (18.84) 1.22 (0.94–1.59) 386 (20.31) 1.12 (0.94–1.32) 34 (14.59) 1.17 (0.72–1.91) 22 (7.26) 0.51 (0.30–0.87)   Finance, commu- nication & cultural service 1447 (27.10) 520 (25.42) ref 95 (21.35) ref 85 (23.04) ref 178 (22.36) ref 475 (24.99) ref 45 (19.31) ref 52 (17.16) ref   Education 409 (7.66) 210 (10.26) 1.19 (0.94–1.51) 44 (9.89) 1.20 (0.79–1.83) 33 (8.94) 1.41 (0.89–2.25) 96 (12.06) 1.48 (1.08–2.02) 215 (11.31) 1.45 (1.16–1.80) 21 (9.01) 1.45 (0.80–2.61) -2 -2   Health & social care 656 (12.29) 456 (22.29) 1.85 (1.53–2.25) 124 (27.87) 2.45 (1.76–3.41) 75 (20.33) 1.97 (1.36–2.86) 234 (29.40) 2.38 (1.85–3.07) 443 (23.30) 1.73 (1.44–2.07) 46 (19.74) 1.99 (1.24–3.22) 42 (13.86) 1.75 (1.10–2.79)   Not in work/ Unknown 1038 (19.44) 56 (2.74) 0.45 (0.31–0.65) 9 (2.02) 0.32 (0.14–0.72) 35 (9.49) 0.99 (0.57–1.73) 23 (2.89) 0.48 (0.27–0.84) 138 (7.26) 0.61 (0.47–0.81) 75 (32.19) 2.94 (1.57–5.52) 161 (53.14) 1.63 (1.06–2.52) Kjeldgård et al. Discussion To elucidate this, further studies are also needed to investigate different jobs, physical work- load, and other work demands in more detail. not only those involving a motor vehicle. Several recent studies stress that pedestrian injuries should be included in the traditional definition of traffic accidents [1, 21, 33]. The present study further strengthens this argument as 71% of the pedestrian accident were due to fall with no other road user involved. The large number of included pedestrians allowed for more detailed anal- yses investigating important factors for the subsequent patterns of SA. One limitation is that no data from primary health- care (to also capture the impact of minor injuries) was included, on the other hand, all injuries from traffic accidents severe enough to require in- or outpatient healthcare were included. which are those more likely to require SA. Another limitation is that only informa- tion from one injury diagnosis per pedestrian is taken into consideration, however, the majority had only one such injury (82%). Another limitation is that only indi- viduals that survived the entire follow-up period were included in the study and that those with DP during the entire study  period were excluded. These require- ments might potentially lead to an underestimation of the total number of injuries and the shorter-term con- sequences, there were however few that died or emi- grated during follow-up (during the three years after the accident). The focus here was the consequences in terms of SA and DP and as such the included individu- als should be at risk of receiving SA or DP. In the present study, the proportion of missing infor- mation on occupation was higher in the three clusters “No SA”, “Other diagnoses Long-term SA”, and “DP”. This could be due to that the individuals in the clus- ter “No SA” to a lower extent have a job and hence are not eligible to SA benefits (e.g. no income from work, unemployment and parental leave) the individuals in this cluster are also slightly younger than in the other clusters (e.g. still studying or not yet having begun their first job). The high proportion of missing in the other two clusters could be owing to the high proportion of these individuals were already on long-term SA and DP the year before the accident, when the information on occupation was assessed. Conclusions h This nationwide register study of the 11,432 working- aged pedestrians that received in- or specialized out- patient healthcare due to a traffic-related accident observed diverse patterns of SA due to injury diagno- ses and SA due to other diagnoses following the acci- dent. Almost half of the pedestrians had no SA during the entire follow up. Compared to this cluster of SA sequences, all other clusters were associated with older age, no university education, having been hospitalized, and working in health and social care. The three clus- ters of sequences characterised by SA due to an injury diagnosis in direct connection to the accident were associated with women, fractures, lower and upper extremities. A rather divergent population with more older women and more younger men was observed. The distribution of type of injuries as well as occupa- tional sectors were also distinctly different for women and men. These observed differences can be utilized to improve understanding of long-term consequences and prevention of road-traffic accidents among pedestrians. Discussion BMC Public Health (2023) 23:367 Page 13 of 16 1 Adjusted for: Sex, Age group, Level of education, Country of birth, Type of living area, Married, Type of accident, Inpatient healthcare, Type of injury, Injured body region, Season, Year of accident, Occupational sector, Private/Public, and Type of occupation 1. No SA 2. Immediate SA 3. Episodic SA 4. Long-term or later SA 5. Both SA due to injury and other diagnoses 6. Other diagnoses short- term SA 7. Other diagnoses long- term SA 8. Disability pension Type of occupation   White collar 1974 (36.97) 919 (44.92) ref 181 (40.67) ref 133 (36.04) ref 377 (47.36) ref 828 (43.56) ref 72 (30.90) ref 55 (18.15) ref   Blue collar 1314 (24.61) 733 (35.83) 1.93 (1.63–2.27) 175 (39.33) 2.51 (1.88–3.34) 141 (38.21) 1.86 (1.37–2.52) 277 (34.80) 2.03 (1.62–2.53) 555 (29.20) 1.35 (1.15–1.58) 51 (21.89) 1.99 (1.29–3.07) 49 (16.17) 1.48 (0.94–2.33)   Not in work/ Unknown 2051 (38.42) 394 (19.26) 1.05 (0.87–1.26) 89 (20.00) 1.42 (1.02–1.96) 95 (25.75) 1.17 (0.83–1.66) 142 (17.84) 1.05 (0.81–1.35) 518 (27.25) 1.23 (1.04–1.46) 110 (47.21) 2.48 (1.62–3.78) 199 (65.68) 2.13 (1.38–3.29) Kjeldgård et al. BMC Public Health (2023) 23:367 Page 14 of 16 Page 14 of 16 that individuals working as plant and machine opera- tors and drivers had a longer duration of work dis- ability after a road traffic accident [28]. In our study, working in Health & social care was associated with all clusters of SA sequences compared to the cluster “No SA”. Accordingly suggesting an association between this occupational sector and SA following an injury as a pedestrian. In addition, working in Construction was associated with the clusters “Immediate SA” and “Episodic SA” and working in Education was associ- ated with the clusters “Both SA due to injury and other diagnoses” and “Other diagnoses long-term”. This may be explained by the difference in workload and vari- ous demands the different occupational sectors have. Individuals in some occupations are more likely to have reduced work capacity in relation to an injury, e.g. due to work requirements of being physical active and/ or requiring mobility to complete work tasks. Accord- ingly, sustaining a fracture to the lower extremities in an occupation where you are required to walk and stand up more often lead to SA. It would be beneficial to pay attention to the physical workload of different jobs in interventions to reduce SA and DP following a pedestrian injury. Discussion The proportion of individuals in each occupational sector in this study corresponds well to those of the general population in Sweden[31], with the exception of women working in Health & social care where a higher proportion were observed among the injured pedestrians. Funding O Open access funding provided by Karolinska Institute. The study was finan- cially supported by AFA insurance, and we utilised data from the REWHARD consortium supported by the Swedish Research Council (VR; grant number 2017–00624). 13. Kjeldgård L, Ohlin M, Elrud R, Stigson H, Alexanderson K, Friberg E. Bicycle crashes and sickness absence - a population-based Swedish register study of all individuals of working ages. BMC Public Health. 2019;19(1):943. 14. Ohlin M, Kjeldgård L, Elrud R, Stigson H, Alexanderson K, Friberg E. Dura- tion of sickness absence following a bicycle crash, by injury type and injured body region: A nationwide register-based study. J Transp Health. 2018;9:275–81. Strengths and limitations One of the main strengths of this study is the use of high-quality nationwide register data, with total popu- lation coverage, several years of follow-up, and that the results were not hampered by recall bias [32]. Another strength is that all pedestrian injuries are included, Page 15 of 16 Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health Abbreviations DP Disability pension SA Sickness absence OR Odds ratio CI Confidence interval Abbreviations DP Disability pension SA Sickness absence OR Odds ratio CI Confidence interval Consent for publication Not applicable. 22. Winter 2010-2011 in numbers (In Swedish: Vintern 2010-2011 i siffror) [https://​www.​smhi.​se/​data/​vinte​rn-​2010-​2011-i-​siffr​or-1.​15194] 22. Winter 2010-2011 in numbers (In Swedish: Vintern 2010-2011 i siffror) [https://​www.​smhi.​se/​data/​vinte​rn-​2010-​2011-i-​siffr​or-1.​15194] Not applicable. 23. The snow situation in winter 2015–2016 (In Swedish: Snösituationen vintern 2015–2016) [https://​www.​smhi.​se/​klimat/​klima​tet-​da-​och-​nu/​ arets-​vatten/​snosi​tuati​onen-​vinte​rn-​2015-​2016-1.​114940] 23. The snow situation in winter 2015–2016 (In Swedish: Snösituationen vintern 2015–2016) [https://​www.​smhi.​se/​klimat/​klima​tet-​da-​och-​nu/​ arets-​vatten/​snosi​tuati​onen-​vinte​rn-​2015-​2016-1.​114940] Declarations 18. Gabadinho A, Ritschard G, Muller NS, Studer M. Analyzing and Visualizing State Sequences in R with TraMineR. J Stat Softw. 2011;40(4):1–37. Availability of data and materials The data cannot be made publicly available, according to privacy regula- tions. According to the General Data Protection Regulation, the Swedish law SFS 2018:218, the Swedish Data Protection Act, the Swedish Ethical Review Act, and the Public Access to Information and Secrecy Act, data can only be made available, after legal review, for researchers who meet the criteria for access to this type of sensitive and confidential data. Read- ers may contact professor Kristina Alexanderson (kristina.alexanderson@ ki.se) regarding the data. 15. Kjeldgård L, Stigson H, Alexanderson K, Friberg E. Sequence analysis of sickness absence and disability pension in the year before and the three years following a bicycle crash; a nationwide longitudinal cohort study of 6353 injured individuals. BMC Public Health. 2020;20(1):1710. 16. Allebeck P, Mastekaasa A. Swedish Council on Technology Assessment in Health Care (SBU). Chapter 5. Risk factors for sick leave - general studies. Scand J Public Health Suppl. 2004;63:49–108. 17. Social Insurance in Figures 2016. Swedish Social Insurance Agency; 2016. Acknowledgements Not applicable 10. Ludvigsson JF, Otterblad-Olausson P, Pettersson BU, Ekbom A. The Swed- ish personal identity number: possibilities and pitfalls in healthcare and medical research. Eur J Epidemiol. 2009;24(11):659–67. Authors’ contributions All authors (LK, HS, ELB, KF, and EF) contributed to the design of the study. LK performed the data management and the statistical analyses. LK wrote the first draft of the manuscript and revised it with input from all other authors (HS, ELB, KF, and EF). All authors (LK, HS, ELB, KF, and EF) read and approved the final version of the manuscript. 11. Barell V, Aharonson-Daniel L, Fingerhut LA, Mackenzie EJ, Ziv A, Boyko V, Abargel A, Avitzour M, Heruti R. An introduction to the Barell body region by nature of injury diagnosis matrix. Inj Prev. 2002;8(2):91–6. 12. Elrud R, Stigson H, Ohlin M, Alexanderson K, Kjeldgård L, Friberg E. 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Kjeldgård L, Stigson H, Klingegard M, Alexanderson K, Friberg E. Sickness absence and disability pension among injured working-aged pedes- trians - a population-based Swedish register study. BMC Public Health. 2021;21(1):2279. 9. International Statistical Classification of Diseases and Related Health Prob- lems, Tenth Revision (ICD-10). Geneva: World Health Organization; 1993. 9. International Statistical Classification of Diseases and Related Health Prob- lems, Tenth Revision (ICD-10). Geneva: World Health Organization; 1993. Competing interests The authors declare that they have no competing interests. Page 16 of 16 Kjeldgård et al. BMC Public Health (2023) 23:367 Kjeldgård et al. BMC Public Health (2023) 23:367 24. Andersson AK. Winter Road Conditions and Traffic Accidents in Sweden and UK - Present and Future Climate Scenarios. Göteborgs universitet; 2010. 25. Elvik R, Bjørnskau T. Risk of pedestrian falls in Oslo, Norway: Rela- tion to age, gender and walking surface condition. J Transp Health. 2019;12:359–70. 26. Schepers P, den Brinker B, Methorst R, Helbich M. Pedestrian falls: A review of the literature and future research directions. J Safety Res. 2017;62:227–34. 27. Gray SE, Collie A. Work absence following road traffic crash in Victoria, Australia: A population-based study. Injury. 2019;50(7):1293–9. 28. Berecki-Gisolf J, Collie A, McClure R. Work disability after road traffic injury in a mixed population with and without hospitalisation. Accident; analy- sis and prevention. 2013;51:129–34. 29. Rissanen R, Liang Y, Moeller J, Nevriana A, Berg H-Y, Hasselberg M. Trajec- tories of sickness absence after road traffic injury: a Swedish register- based cohort study. BMJ Open. 2019;9(7): e031132. 29. Rissanen R, Liang Y, Moeller J, Nevriana A, Berg H-Y, Hasselberg M. Trajec- tories of sickness absence after road traffic injury: a Swedish register- based cohort study. BMJ Open. 2019;9(7): e031132. 30. Hansson PG. Sick-leave after road traffic accidents. Scand J Soc Med. 1976;4(2):103–7. 30. Hansson PG. Sick-leave after road traffic accidents. Scand J Soc Med. 1976;4(2):103–7. 31. Employees aged 16–64 in the country by occupation (3-digit SSYK 2012), industry SNI2007 (rough level), age and gender. New time series. Year 2019 - 2020 (In Swedish: Anställda 16–64 år i riket efter yrke (3-siffrig SSYK 2012), näringsgren SNI2007 (grov nivå), ålder och kön. Ny tidsserie. År 2019 - 2020 [https://​www.​stati​stikd​ataba​sen.​scb.​se/​pxweb/​sv/​ssd/​ START__​AM__​AM020​8__​AM020​8B/​YREG6​1N/] 32. Ludvigsson JF, Andersson E, Ekbom A, Feychting M, Kim JL, Reuterwall C, Heurgren M, Olausson PO. External review and validation of the Swedish national inpatient register. BMC Public Health. 2011;11:450. 33. Methorst R, Schepers P, Christie N, Dijst M, Risser R, Sauter D, van Wee B. ’Pedestrian falls’ as necessary addition to the current definition of traffic crashes for improved public health policies. J Transp Health. 2017;6:10–2. Kjeldgård et al. 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https://cronfa.swan.ac.uk/Record/cronfa31775/Download/0031775-17022017135318.pdf
English
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Bisphenol A causes reproductive toxicity, decreases <i>dnmt1</i> transcription, and reduces global DNA methylation in breeding zebrafish <i>(Danio rerio)</i>
Epigenetics
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cc-by
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Cronfa - Swansea University Open Access Repository This is an author produced version of a paper published in : E i ti Cronfa URL for this paper: http://cronfa.swan.ac.uk/Record/cronfa31775 This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by the terms of the repository licence. Authors are personally responsible for adhering to publisher restrictions or conditions. When uploading content they are required to comply with their publisher agreement and the SHERPA RoMEO database to judge whether or not it is copyright safe to add this version of the paper to this repository. http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/ Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R., Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538. http://dx.doi.org/10.1080/15592294.2016.1182272 Cronfa - Swansea University Open Access Repository _____________________________________________________________ This is an author produced version of a paper published in : Epigenetics Cronfa URL for this paper: http://cronfa.swan.ac.uk/Record/cronfa31775 _____________________________________________________________ Paper: Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R., Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538. http://dx.doi.org/10.1080/15592294.2016.1182272 Cronfa - Swansea University Open Access Repository Epigenetics ISSN: 1559-2294 (Print) 1559-2308 (Online) Journal homepage: http://www.tandfonline.com/loi/kepi20 Paper: ape Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R., Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538. http://dx.doi.org/10.1080/15592294.2016.1182272 p Laing, L., Viana, J., Dempster, E., Trznadel, M., Trunkfield, L., Uren Webster, T., van Aerle, R., Paull, G., Wilson, R., Mill, J. & Santos, E. (2016). Bisphenol A causes reproductive toxicity, decreasesdnmt1transcription, and reduces global DNA methylation in breeding zebrafish(Danio rerio). Epigenetics, 11(7), 526-538. http://dx.doi.org/10.1080/15592294.2016.1182272 This article is brought to you by Swansea University. Any person downloading material is agreeing to abide by the terms of the repository licence. Authors are personally responsible for adhering to publisher restrictions or conditions. When uploading content they are required to comply with their publisher agreement and the SHERPA RoMEO database to judge whether or not it is copyright safe to add this version of the paper to this repository. http://www.swansea.ac.uk/iss/researchsupport/cronfa-support/ Epigenetics Date: 17 February 2017, At: 05:50 CONTACT L. V. Laing ll292@exeter.ac.uk; E. M. Santos e.santos@exeter.ac.uk Supplemental data for this article can be accessed on the publisher’s website. Published with license by Taylor & Francis Group, LLC © L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill, and E. M. Santos. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted. ABSTRACT ARTICLE HISTORY Received 22 February 2016 Revised 18 April 2016 Accepted 19 April 2016 KEYWORDS Aquatic; endocrine; meth- ylation; plasticizers; tele- ost; vertebrate; waste BS C Bisphenol A (BPA) is a commercially important high production chemical widely used in epoxy resins and polycarbonate plastics, and is ubiquitous in the environment. Previous studies demonstrated that BPA activates estrogenic signaling pathways associated with adverse effects on reproduction in vertebrates and that exposure can induce epigenetic changes. We aimed to investigate the reproductive effects of BPA in a fish model and to document its mechanisms of toxicity. We exposed breeding groups of zebrafish (Danio rerio) to 0.01, 0.1, and 1 mg/L BPA for 15 d. We observed a significant increase in egg production, together with a reduced rate of fertilization in fish exposed to 1 mg/L BPA, associated with significant alterations in the transcription of genes involved in reproductive function and epigenetic processes in both liver and gonad tissue at concentrations representing hotspots of environmental contamination (0.1 mg/L) and above. Of note, we observed reduced expression of DNA methyltransferase 1 (dnmt1) at environmentally relevant concentrations of BPA, along with a significant reduction in global DNA methylation, in testes and ovaries following exposure to 1 mg/L BPA. Our findings demonstrate that BPA disrupts reproductive processes in zebrafish, likely via estrogenic mechanisms, and that environmentally relevant concentrations of BPA are associated with altered transcription of key enzymes involved in DNA methylation maintenance. These findings provide evidence of the mechanisms of action of BPA in a model vertebrate and advocate for its reduction in the environment. Abbreviations: E2, 17b-estradiol; 5Fc, 5-formylcytosine; 5hmC, 5-hydroxymethylcytosine; ar, androgen receptor; amh, anti-M€ullerian hormone; cyp19a1a, aromatase; BPA, bisphenol A; dnmt3, DNA (cytosine-5)-methyltransferase 3; dnmt1, DNA methyltransferase 1; esr1, estrogen receptor 1; esr2a, estrogen receptor 2a; esr2b, estrogen receptor 2b; ER, estrogen receptor; GSI, gonadosomatic index; HSI, hepatosomatic index; hdac1, histone deacetylase 1; hdac3, histone deacetylase 3; mecp2, methyl CpG binding protein 2; mbd2, methyl-CpG-binding domain protein 2; mbd3a, methyl-CpG-binding domain protein 3a; 5mC, methylcytosine; PCA, principal component analysis; rpl8, ribosomal protein L8; vtg1, vitellogenin 1 Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio) L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill & E. M. Santos To cite this article: L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill & E. M. Santos (2016) Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio), Epigenetics, 11:7, 526-538, DOI: 10.1080/15592294.2016.1182272 To link to this article: http://dx.doi.org/10.1080/15592294.2016.1182272 © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC© L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill, and E. M. Santos. View supplementary material Accepted author version posted online: 27 Apr 2016. Published online: 27 Apr 2016. Submit your article to this journal Article views: 1504 View related articles View Crossmark data Citing articles: 4 View citing articles To link to this article: http://dx.doi.org/10.1080/15592294.2016.1182272 © 2016 The Author(s). Published with license by Taylor & Francis Group, LLC© L. V. Laing, J. Viana, E. L. Dempster, M. Trznadel, L. A. Trunkfield, T. M. Uren Webster, R. van Aerle, G. C. Paull, R. J. Wilson, J. Mill, and E. M. Santos. View supplementary material Accepted author version posted online: 27 Apr 2016. Published online: 27 Apr 2016. Submit your article to this journal Article views: 1504 View related articles View Crossmark data Citing articles: 4 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=kepi20 Date: 17 February 2017, At: 05:50 Download by: [Swansea University] Download by: [Swansea University] EPIGENETICS 2016, VOL. 11, NO. 7, 526–538 http://dx.doi.org/10.1080/15592294.2016.1182272 EPIGENETICS 2016, VOL. 11, NO. 7, 526–538 http://dx.doi.org/10.1080/15592294.2016.1182272 Bisphenol A causes reproductive toxicity, decreases dnmt1 transcription, and reduces global DNA methylation in breeding zebrafish (Danio rerio) L. V. Lainga, J. Vianab, E. L. Dempsterb, M. Trznadela, L. A. Trunkfielda, T. M. Uren Webstera, R. van Aerlec, G. C. Paulla, R. J. Wilsona, J. Millb,d, and E. M. Santosa aBiosciences, College of Life & Environmental Sciences, Geoffrey Pope Building, University of Exeter, Exeter, EX4 4QD, United Kingdom; bUniversity of Exeter Medical School, RILD building, University of Exeter, Exeter, EX2 5DW, United Kingdom; cCentre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, United Kingdom; dInstitute of Psychiatry, Psychology & Neuroscience (IoPPN), King’s College London, Denmark Hill, London, SE5 8AF, UK Introduction A study in Gobiocypris rarus found BPA exposure to be associated with altered DNA methylation in the 50 flanking region of cyp19a1a (aromatase), and the effects to be time-dependent.19 In addition, a significant decrease in the expression of DNA methyltransfer- ase 1 (dnmt1) in ovarian tissue has been reported, with a signifi- cant decrease in global DNA methylation.19 BPA has been shown to act as an estrogen receptor (ER) ago- nist,14,15 able to bind to ERs, resulting in feminizing effects.16,17 A study using the human cell line HepG2, found that BPA strongly activated estrogen receptor 1 (ESR1; previously known as ERa) mediated responses, but did not activate ESR2 (previ- ously known as ERb), while in the cell line HeLa, BPA was found to activate both ESR1 and ESR2.14 In fish, BPA induced esr1 expression in the livers of male fathead minnows (Pimephales promelas) exposed for 4 d to 10 mg BPA/L, consistent with an estrogenic mode-of-action.18 BPA has also been shown to alter the transcriptional profile of steroidogenic enzyme genes in a time-dependent manner, including aromatase (cyp19a1a), which is responsible for the irreversible conversion of androgens into estrogens and is a key regulator of estrogen synthesis in the gonads. This enzyme was significantly upregulated in both the ovary and testis of Gobiocypris rarus exposed to 15 mg/L BPA for 7d, followed by suppression after 35 d exposure.19 Given the extensive use and ubiquity of BPA, it is important to understand the mechanisms mediating its toxic effects and the impacts these can have on both wild populations and human health. The present study aims to investigate the effects of BPA on reproduction in the zebrafish model and identify epigenetic and transcriptional changes associated with BPA exposure. We exposed breeding groups of zebrafish to BPA for 15 d to determine if reproduction was affected by the exposure. The concentrations tested included environmentally relevant concentrations found world-wide (0.01 mg/L) and at point sources (0.1 mg/L).12,38 The highest concentration tested (1 mg/L) has only been reported in landfill leachate and is unlikely to occur in surface waters, but it was included to enable a mechanistic analysis of BPA toxicity. We quantified the tran- scription of genes involved in epigenetic signaling and repro- ductive function, together with global and locus-specific DNA methylation in exposed fish. Adverse impacts on reproduction have been observed in several fish models. Introduction ranging from 0.2 to 17.6 mg/g.6 BPA has been detected in the urine of »95% of adults in the USA and Asia.7,8 It has also been measured in the serum of adult men and women 9 and in breast milk, fetal plasma, and placental tissue, raising concerns about human exposures during critical periods of development.1,10 Bisphenol A (BPA) is a commercially important high produc- tion chemical widely used in the production of epoxy resins, utilized in food and beverage packaging, dental sealants, and as a monomer component of polycarbonate plastics.1,2 With over three million tons produced globally per annum, environmen- tal exposure is common,3 and in the USA BPA was measurable in 75% of food products tested.4 Human exposure occurs pre- dominantly via ingestion of contaminated food, caused by leaching of BPA from linings of canned goods and polycarbon- ate packaging. BPA has also been detected in drinking water at concentrations up to 15 ng/L.5 In addition, inhalation is thought to be a plausible secondary route of exposure,3 with BPA present in 86% of domestic dust samples at concentrations BPA is moderately water soluble, entering the environment via direct discharge from BPA production and processing industries, wastewater treatment plants and leachate from land- fill sites.11 Its presence is ubiquitous in the aquatic environment and surface water concentrations have been detected up to the low mg/L range, with peak concentrations reaching up to 21 mg/L.12 Concentrations in landfill leachate have been reported to reach up to 17,200 mg/L.1 Due to its ubiquitous EPIGENETICS 527 EPIGENETICS 527 527 nature, the potential for environmental exposure in wildlife populations, including fish, is very significant. Levels of BPA reported in fish vary; values of 1-11 ng BPA/g dry weight in muscle and 2-75 ng BPA/g dry weight in liver have been reported.13 To date, few studies have investigated the potential for BPA to induce epigenetic and transcriptional changes in fish. Water chemistry The mean measured concentrations of BPA in the tank water were between 100 and 139% of the nominal concentrations for all treatments, and are presented in Supporting Information Table S1. Introduction A multi-generational study in fathead min- now showed that BPA reduced gonadal growth in males and females, reduced hatching in F1 offspring of fish exposed to 640 mg/L and induced the estrogen regulated egg yolk protein, vitellogenin, a well established biomarker of xenoestrogen exposure, in the liver of male fish exposed to 640 and 1280 mg/ L BPA.20 Further multigenerational studies have demonstrated the potential adverse effects associated with exposure to BPA.21,22 Exposure to BPA in guppies has been associated with reduced sperm quality,23 and the presence of necrotic cells in the seminiferous tubules of Xiphophorus helleri was also reported.24 Together, these studies demonstrate the potential reproductive consequences following exposure to relatively high concentrations of BPA in fish. Effects of BPA on morphometric parameters The mean mass and length of male and female fish were 460.0 § 0.008 mg and 36.5 § 0.02 mm, and 480.6 § 0.01 mg and 35.7 § 0.03 mm, respectively. There were no significant differences in size or condition factor (mean 0.95 and 1.05 for males and females, respectively) between treatment groups. g Evidence also exists supporting the involvement of BPA in the etiology of a range of human disease phenotypes including cardiovascular disease,25 altered behavior in children,26 prostate cancer27 and recurrent miscarriage.28 In addition to the well- established estrogenic mode-of-action, additional mechanisms have been proposed, including potential anti-androgenic activ- ity.29 Low dose effects and non-monotonic dose response curves have been reported.30,31 More recently, increasing evi- dence suggests that BPA may alter the epigenetic regulation of gene expression; for example, altered DNA methylation pat- terns have been observed both globally (i.e., changes to the total genomic content of DNA methylation) and at the regulatory regions of specific genes (i.e., locus-specific) in mammals.32-36 In humans, exposure to BPA in the workplace has been associ- ated with hypomethylation of LINE-1 in spermatozoa, a marker of global DNA methylation levels in the genome.37 Understanding the effects of BPA exposure on epigenetic pro- cesses, and how these alterations perturb expression of genes that are related to development and reproduction, are impor- tant to the evaluation of adverse effects associated with BPA exposure, both in humans and wildlife, particularly for expo- sures at environmentally relevant concentrations. No alterations in general feeding and swimming behavior were observed in any spawning group, with the exception of the mortality of one female in the 0.1 mg/L BPA treatment. The egg output calculations for that group were adjusted accordingly. Hepatosomatic index (HSI; the ratio of liver weight to body weight) in males was significantly increased in fish exposed to 1 mg/L BPA, but no effects of BPA were observed in females (Supporting Information Fig. S1). There were no significant differences in the gonadosomatic index (GSI; the ratio of gonad weight to body weight) of males or females as a result of the BPA exposure. Effects of bisphenol A on gene transcription Analysis of genes involved in reproductive processes in the liver revealed that vtg1 and esr2b were significantly upregulated in males following exposure to 1 mg/L BPA when compared to the solvent control group (fold-change D 172.90, PD0.009 and fold- change D 5.40, PD0.014, respectively). In females, esr2b was sig- nificantly upregulated following exposure to 0.01 mg/L BPA (P D 0.044). For genes involved in epigenetic regulation, the most pro- nounced changes observed were for dnmt1, which was signifi- cantly downregulated in the livers of females exposed to 0.01 mg/ L BPA (P D 0.040) and in both males and females exposed to 0.1 (males: P D 0.020; females: P D 0.005) and 1 mg/L BPA (males: P D 0.020; females: P D 0.005). In addition, changes were also observed for histone deacetylase 3 (hdac3), methyl-CpG-binding domain protein 2 (mbd2) and methyl CpG binding protein 2 (mecp2) in males, and for mbd2 in females (Fig. 2A and B; Sup- porting Information Figs. S2 and 3). In the gonads, BPA exposure was also associated with signif- icant changes in transcription for genes involved in reproduc- tive function and on epigenetic pathways (Figs. 2, 3). Principal component analysis (PCA) for the testis indicated clear separa- tions between the transcription profiles of fish exposed to the solvent control and fish exposed 1 mg/L BPA, based on the data for all genes quantified (Fig. 3). For ovaries, changes were more pronounced and PCA revealed a separation between fish exposed to 0.1 and 1 mg/L BPA and the solvent control (Fig. 3). In the testis, the transcript encoding esr2a and cyp19a1a were significantly downregulated in response to 1 mg/L BPA (P D 0.002 and 0.018, respectively; Fig. 2; Supporting Information Fig. S4). There was also a significant association between the con- centration of BPA and the level of transcription for cyp19a1a (P D 0.025; Supporting Information Table S4), which decreased with increasing concentrations of BPA. In addition, for anti- M€ullerian hormone (amh), BPA affected gene transcription (P  0.05) and a decreasing trend across all concentrations was observed, but this was not statistically significant (P D 0.094; Sup- porting Information Fig. S4). Similarly to the testis, in the ovaries of exposed females, the transcript encoding esr2a was significantly downregulated following exposure to 1 mg/L BPA (P  0.001). Effects of bisphenol A on gene transcription In addition, there were similar (but non-significant) trends for other genes involved in reproductive function including esr1 and ar, which appeared to decrease with increasing exposure concentra- tions (Fig. 2; Supporting Information Fig. S5). Figure 1. A) Cumulative number of eggs per female per day in breeding groups exposed to 0.01, 0.1, and 1 mg/L BPA. Data is presented for a 10 d pre-exposure followed by a 15 d chemical exposure periods (n D 3 replicate groups per treat- ment). Statistical comparisons were conducted in R (version 3.0.2), and the lme4 package was used to fit mixed effects linear models, followed by repeated meas- ures ANOVA and Chi-squared Wald test to determine the effects of the exposure to BPA compared to the solvent control. B) Mean fertilization success (%) during the 15 d chemical exposure period (n D 3 replicate groups per treatment). Statisti- cal analyses were conducted using R (version 3.0.2); the Regression coefficient (R2) was calculated using linear modeling. Asterisks indicate significant differences between treatment groups (P < 0.01; P < 0.001). BPA spawned a significantly greater number of eggs per female when compared to all other treatment groups (P  0.01); this increased egg production intensified throughout the exposure period (Fig. 1A). During the pre-exposure, fertilization success remained consistently high with no significant differences between groups and an overall mean fertilization rate of 85.6%. During the 15 d exposure, fertilization success in colonies exposed to 1 mg/L BPA significantly declined (P D 0.001; Fig. 1B). Additionally, for this treatment group, there was a sig- nificant negative correlation between the length of the exposure (number of days) and the average percentage of fertilization (R2 D 0.80; P  0.001), indicating that the effects of BPA on fertilization became progressively more pronounced over the exposure period. BPA spawned a significantly greater number of eggs per female when compared to all other treatment groups (P  0.01); this increased egg production intensified throughout the exposure period (Fig. 1A). During the pre-exposure, fertilization success remained consistently high with no significant differences between groups and an overall mean fertilization rate of 85.6%. During the 15 d exposure, fertilization success in colonies exposed to 1 mg/L BPA significantly declined (P D 0.001; Fig. 1B). Effects of bisphenol A on reproduction During the 10 d pre-exposure period there were no differences in cumulative egg production between treatment groups (P D 0.098). During the exposure, groups treated with 1 mg/L 528 L. V. LAING ET AL. 528 Figure 1. A) Cumulative number of eggs per female per day in breeding groups exposed to 0.01, 0.1, and 1 mg/L BPA. Data is presented for a 10 d pre-exposure followed by a 15 d chemical exposure periods (n D 3 replicate groups per treat- ment). Statistical comparisons were conducted in R (version 3.0.2), and the lme4 package was used to fit mixed effects linear models, followed by repeated meas- ures ANOVA and Chi-squared Wald test to determine the effects of the exposure to BPA compared to the solvent control. B) Mean fertilization success (%) during the 15 d chemical exposure period (n D 3 replicate groups per treatment). Statisti- 2 Effects of bisphenol A on gene transcription Additionally, for this treatment group, there was a sig- nificant negative correlation between the length of the exposure (number of days) and the average percentage of fertilization (R2 D 0.80; P  0.001), indicating that the effects of BPA on fertilization became progressively more pronounced over the exposure period. As in the liver, dnmt1 was significantly downregulated in ova- ries following exposure to all three BPA concentrations tested (P D 0.032, 0.032, 0.032, respectively). Although no significant group-wise changes in dnmt1 transcription were observed in the testis (Fig. 2; Supporting Information Fig. S4), the expression of dnmt1 in the testis was associated with BPA exposure concentra- tion (R2 D 0.110; P D 0.046; Supporting Information Table S4). In addition, changes in mbd2 transcription were observed in the testis, with a significant increase in transcription measured in males exposed to 0.01 mg/L BPA (P D 0.020), but reduced expres- sion in males exposed to 1 mg/L BPA (P D 0.030; Fig. 2; Support- ing Information Fig. S4). EPIGENETICS 529 Figure 2. Transcript profiles for target genes in the livers of females (A) and males (B), and in the ovary (C) and testis (D) following exposure to 0.01, 0.1, and 1 mg/L BPA for 15 d. Data were collected for 6-8 fish per treatment, and data points classified as outliers (using the Chauvenet’s criterion) and for which the expression was below the detection limit of the assay were excluded from analysis. Where amplification was detected in more than 70% of individuals, data are represented as fold-change rela- tive to the expression in the solvent control group. Where amplification was detected in less than 70% of individuals, data are presented as the proportion of individuals for which the target genes were amplified. Asterisks represent significant differences between treatment groups compared to the solvent control group (P < 0.05, P < 0.01, P < 0.001). Figure 2. Transcript profiles for target genes in the livers of females (A) and males (B), and in the ovary (C) and testis (D) following exposure to 0.01, 0.1, and 1 mg/L BPA for 15 d. Data were collected for 6-8 fish per treatment, and data points classified as outliers (using the Chauvenet’s criterion) and for which the expression was below the detection limit of the assay were excluded from analysis. Effects of bisphenol A on gene transcription Where amplification was detected in more than 70% of individuals, data are represented as fold-change rela- tive to the expression in the solvent control group. Where amplification was detected in less than 70% of individuals, data are presented as the proportion of individuals for which the target genes were amplified. Asterisks represent significant differences between treatment groups compared to the solvent control group (P < 0.05, P < 0.01, P < 0.001). Effects of bisphenol A on global DNA methylation Analysis of global DNA methylation in the gonads revealed sig- nificant decreases in the proportion of global methylation fol- lowing exposure to 1 mg/L BPA in both males (by 3.2%; P D 0.029; Fig. 4A) and females (by 4.9%; P D 0.041; Fig. 4B). Targeted DNA methylation profiling in the promoter region of amh revealed that exposure to 1 mg/L BPA caused a small but significant increase in methylation compared to the solvent control for the first of the three CpG sites assessed in the testes Figure 3. Principal components (PC) score plots showing the relative similarity of gonadal transcription profiles for zebrafish exposed to solvent, 0.01, 0.1, and 1 mg/L BPA for 15 d. A) Ovary. B) Testis. Points represent PC scores for individual fish along PCs 1 and 2. Circles represent a general characterization of the PC space occupied by each treatment group and were calculated using the prcomp package in R (version 3.0.2). Figure 3. Principal components (PC) score plots showing the relative similarity of gonadal transcription profiles for zebrafish exposed to solvent, 0.01, 0.1, and 1 mg/L BPA for 15 d. A) Ovary. B) Testis. Points represent PC scores for individual fish along PCs 1 and 2. Circles represent a general characterization of the PC space occupied by each treatment group and were calculated using the prcomp package in R (version 3.0.2). 530 L. V. LAING ET AL. 30 L. V. LAING ET AL. Figure 4. Global DNA methylation profiles in the gonads of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Graphs present the percentage of global DNA methylation in ovaries (A) and testis (B). Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 4. Global DNA methylation profiles in the gonads of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Graphs present the percentage of global DNA methylation in ovaries (A) and testis (B). Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). testes (males). Although group-wise comparisons of this region revealed no significant differences in the female ovaries (Figs. Effects of bisphenol A on global DNA methylation 6 and 7), dnmt1 promoter methylation was significantly corre- lated with BPA exposure at various sites (positions 4, 5, 6 and 8; Supporting Information Table S4). (P D 0.032; Fig. 5, see Supporting Information Fig. S6 for the position of this CpG site), with DNA methylation at this site being significantly correlated with BPA exposure concentration (R2 D 0.1625; P D 0.013). No differences in DNA methylation were seen for this region in ovaries from exposed female fish (Fig. 5). BPA was also not associated with altered DNA methyl- ation at two CpG sites in the 50 flanking region of the esr1 gene in either the liver or gonads (Supporting Information Fig. S7). The analysis of 11 CpG sites across the promoter of dnmt1 identified significant increases in DNA methylation for a num- ber of sites in the liver (in both males and females) and the Discussion Exposure to BPA resulted in a consistent downregulation of dnmt1 transcription in the ovary and in the liver of both males and females following exposure to BPA, including at Figure 5. Gene specific DNA methylation profiles for three CpG sites in the promoter region of anti-M€ullerian hormone (amh) in the ovaries (A) and testes (B) of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of three CpG sites in the 50 flanking regions of the amh gene. Data are presented as box- plots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 5. Gene specific DNA methylation profiles for three CpG sites in the promoter region of anti-M€ullerian hormone (amh) in the ovaries (A) and testes (B) of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of three CpG sites in the 50 flanking regions of the amh gene. Data are presented as box- plots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). EPIGENETICS 531 EPIGENETICS 531 fic DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the ovaries (A) and testis (B) owing exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of 11 CpG sites in the 50 flanking regions of the dnmt1 gene. Data are presented or each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 6. Gene-specific DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the ovaries (A) and testis (B) of adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. C) Example pyrogram of 11 CpG sites in the 50 flanking regions of the dnmt1 gene. Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). pathways, as previously reported for a range of organisms.39–41 We have investigated the effects of BPA on the expression of transcripts involved in reproductive function and known to be directly or indirectly regulated by estrogens. environmentally relevant concentrations in females. Discussion In associa- tion with this, we found a reduction in global DNA methylation, probably due to the decrease in dnmt1 expression. At the highest concentration tested, BPA caused reduced fertilization, poten- tially via estrogenic mechanisms. Together, our data provide evi- dence of the molecular mechanisms of action of BPA and the potential for it to cause adverse health impacts in vertebrates. We found no evidence for significant alterations in the tran- scription of esr1 or DNA methylation across the esr1 promoter in the gonads and livers of both sexes, but a significant associa- tion between BPA concentration and decreased transcription was found for the livers of females, and a trend for reduced expression was also observed in the ovaries and testis, similar to that described previously.31 Disruption of ESR1 has been associated with alterations of spermatogenesis and subse- quently infertility in mice,42,43 therefore suggesting that the apparent decrease in esr1 transcript in the testis may contribute toward the observed decline in fertilization success at this concentration. Reproductive effects of BPA on adult zebrafish In parallel, BPA induced a significant increase in the transcription of the egg yolk protein, vtg1, and an increase in HSI in males, likely as a result of increased vitello- genin production in hepatocytes, indicating an association between the induction of esr2b in males and the induction of vtg1, as previously reported for fathead minnows.44 Together, these findings suggest that the effects of BPA on reproduction involve disruption of estrogen receptor signaling principally via esr1 and esr2b in the liver, and esr2a in the gonads. transcription was reported in ovaries of Gobiocypris rarus exposed to 0.05 mg/L BPA for 35, d and was associated with disruption of oogenesis and the occurrence of atretic follicles.31 These findings concur with previous studies reporting that esr2a is more sensitive compared to esr1, to the natural estro- gen, 17b-estradiol (E2).41 In contrast, BPA caused increased transcription of esr2b in the livers of males and females but not in the gonads, and, importantly, for females this effect was observed at the environmentally relevant concentration of 0.01 mg/L BPA. In parallel, BPA induced a significant increase in the transcription of the egg yolk protein, vtg1, and an increase in HSI in males, likely as a result of increased vitello- genin production in hepatocytes, indicating an association between the induction of esr2b in males and the induction of vtg1, as previously reported for fathead minnows.44 Together, these findings suggest that the effects of BPA on reproduction involve disruption of estrogen receptor signaling principally via esr1 and esr2b in the liver, and esr2a in the gonads. In the testis, a decrease in amh transcription was associated with increased BPA exposure concentrations. Similarly, in mammals, downregulation of AMH has been reported follow- ing exposure to BPA.46,47 Exposure to 1 mg/L BPA also caused significant DNA hypermethylation in the amh promoter in the testis (CpG 1), demonstrating that exposure to BPA caused epi- genetic alterations at this specific gene locus. There was also a significant correlation between the level of methylation in CpG 1 and amh transcription, and with BPA exposure concentra- tion. This suggests that epigenetic mechanisms may be playing a role in the observed decline in amh transcript in testis tissue, which in turn could have consequences for the functioning of the testis, resulting in de-masculinization. Fertilization success decreased over time with the mean fer- tilization rate dropping from 89% on day 1 to 69% by day 15. Reproductive effects of BPA on adult zebrafish We provide evidence that BPA exposure results in an impairment of reproductive function in breeding zebrafish. These effects included an increase in the number of eggs spawned and a decrease in fertilization success in groups exposed to 1 mg/L BPA. A number of mechanisms may con- tribute to the observed effect of BPA on reproduction, includ- ing stimulation of estrogen responsive processes via the interaction of BPA or its metabolites with estrogen signaling BPA was found to downregulate esr2a in both ovaries and testes, but not in the liver. Similarly, a decrease in esr2a 532 L. V. LAING ET AL. 532 L. V. LAING ET AL. Figure 7. Gene specific DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the livers of female (A) and male (B) adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). Figure 7. Gene specific DNA methylation profiles for 11 CpG sites in the promoter region of DNA (cytosine-5)-methyltransferase 1 (dnmt1) in the livers of female (A) and male (B) adult zebrafish following exposure to 0.01, 0.1, and 1 mg/L BPA. Data are presented as boxplots (n D 6-8 for each group). Asterisks indicate significant differences compared to the solvent control (P < 0.05, P < 0.01, P < 0.001). following a long term exposure to BPA,19 and studies using the aromatase knockout (ArKO) mouse found ArKO males to have reduced fertility,45 demonstrating the critical role of aro- matase in gametogenesis in males. transcription was reported in ovaries of Gobiocypris rarus exposed to 0.05 mg/L BPA for 35, d and was associated with disruption of oogenesis and the occurrence of atretic follicles.31 These findings concur with previous studies reporting that esr2a is more sensitive compared to esr1, to the natural estro- gen, 17b-estradiol (E2).41 In contrast, BPA caused increased transcription of esr2b in the livers of males and females but not in the gonads, and, importantly, for females this effect was observed at the environmentally relevant concentration of 0.01 mg/L BPA. Reproductive effects of BPA on adult zebrafish These findings are consistent with those of Haubruge et al., who reported declines in sperm count of 40-75% in guppies exposed to 0.274 or 0.549 mg/L BPA.23 BPA exposure has been linked to male sexual dysfunction in humans, and urinary con- centrations of BPA have been associated with declines in sperm concentration, motility, and morphology in men.48 The mecha- nism by which disruption of normal spermatogenesis takes place is hypothesized to be via disruption of the Sertoli cells, which are directly sensitive to xenobiotic chemicals, and whose functions are essential during spermatogenesis.23 Our data are in agreement with these findings and further document the In addition to the disruption in estrogen receptor signaling, changes in sex steroid biosynthesis may have contributed to the observed disruption of reproduction in colonies exposed to 1 mg/L BPA. We found a significant decrease in cyp19a1a tran- script in the testis of males exposed to 1 mg/L BPA, and a sig- nificant association between transcription and BPA exposure concentration. In ovaries, a decreasing trend was also observed. These findings suggest potential feedback mechanisms were activated to counteract the estrogen/androgen ratio imbalance caused by BPA, through reducing the irreversible conversion of testosterone into estrogens. Similar findings have recently been reported for the Chinese rare minnow (Gobiocypris rarus) EPIGENETICS 5 EPIGENETICS 533 importance of Sertoli cells as targets for BPA toxicity, by dem- onstrating its effects on amh and cyp19a1a, both expressed in these cells in the testis. measured a significant decrease in global DNA methylation in the testis of fish exposed to 1 mg/L BPA, suggesting that the BPA- induced reduction in global methylation is likely to be functionally linked to the decrease in dnmt1 transcription. These data align with the reported hypomethylation of sperm associated with the presence of BPA in urine, in a study of male factory workers in China.37 There is evidence to suggest that DNA demethylation and methylation establishment events during early development are guided by the paternal DNA methylation program instructed by the sperm chromosomes.55,56 Therefore, it is plausible that changes to the global DNA methylation pattern in testes such as those reported for fish exposed to 1 mg/L BPA may have the potential to impact on the epigenetic reprogramming of embryos, with poten- tial consequences for their subsequent development. Effects of BPA on epigenetic regulation There is now strong evidence demonstrating that BPA has the potential to induce changes in DNA methylation at both gene- specific and genome-wide levels in exposed organisms32,33; however, this has rarely been studied in fish. It is important to note that global DNA methylation in this study, measured using the LUMA assay, provides only an esti- mate of the total DNA methylation across all areas of the genome and all cell types in a given tissue. Decreased dnmt1 transcription may be causing demethylation of specific areas of the genome or within specific cell types, but this may not be detectable by a global measurement of DNA methylation, including all cell types simultaneously. This may explain why dnmt1 transcription appears to be more sensitive to BPA expo- sure compared to global methylation measurements. In our study, we found a significant decrease in the expression of the DNA methylation maintenance enzyme, dnmt1, for all three BPA concentrations tested in ovaries of females—including at environmentally relevant concentrations—and the DNA methyla- tion pattern in the promoter region of the dnmt1 gene was found to be significantly associated with BPA exposure concentrations for four CpG sites. The expression of dnmt1 is known to be associated with changes in global DNA methylation, and inactivation of dnmt1 has been shown to cause global demethylation of the genome.52 In this regard, it was interesting that global DNA meth- ylation levels were significantly decreased in ovarian tissue of fish exposed to 1 mg/L BPA, potentially as a consequence of the sup- pression in dnmt1 transcription. In contrast, previous studies in Gobiocypris rarus, have reported global DNA hypermethylation in ovaries exposed to 0.015 mg/L BPA for 35, d19 suggesting these epi- genetic effects may be concentration- and time-dependent, and potentially vary across vertebrate species. Importantly, dnmt1 is reported to be an important maternal transcript involved in the regulation of DNA methylation during the first stages of embryo development, particularly prior to the zygote genome activa- tion.53,54 Therefore, the significant decrease in the expression of dnmt1 observed in ovaries of females exposed to all three concen- trations of BPA could have potential consequences for the appro- priate development of offspring, in addition to influencing the level of DNA methylation in the ovary of exposed females. The transcript profile for mbd2 was significantly altered fol- lowing exposure to BPA in both male testis and female livers. Reproductive effects of BPA on adult zebrafish Changes in fertilization success may have occurred not only due to effects of BPA on spermatogenesis but also due to BPA- induced alterations in egg quality. Females exposed to 1 mg/L BPA produced an increased number of eggs, but these eggs may have lacked the quality required for fertilization success and embryo survival. Many factors contribute to egg quality, of which the hormonal environment during oogenesis is a critical one.49 The observed changes in the expression of estrogen recep- tors and the trends observed for cyp19a1a in females indicate a disruption of the estrogen/androgen balance within ovaries and consequent alterations in sex steroid signaling pathways, puta- tively leading to alterations in oogenesis and oocyte quality. This hypothesis is supported by previous studies in which BPA was shown to affect oogenesis.50 In addition, a study in pregnant mice exposed to BPA found gross abnormalities in the meiotic prophase of oogenesis, including synaptic defects, which were suggested to occur via Esr2 (ERb) signaling.51 Interestingly, in the present study, changes were also observed in the expression of an ERb subtype (esr2a) in the gonads of both sexes, suggesting similar mechanisms could be occurring. In the liver, we observed a significant decrease in dnmt1 transcription in males and females, including at environmen- tally relevant concentrations, demonstrating the very significant impact of BPA on the expression of this key DNA methylation maintenance enzyme. In addition, we report significant hyper- methylation of the promoter region of the dnmt1 gene in both male and female livers. Based on the positive association between the expression of this gene and global DNA methyla- tion, it is plausible that the suppression of dnmt1 may impact on global methylation as seen in other tissues. However, this could not be measured in the liver due to technical limitations related to the amount of DNA obtained from this tissue. The fact that changes in the transcript and methylation profile for dnmt1 occur at environmentally relevant concentrations high- lights the potential for BPA to cause epigenetic effects in exposed organisms within current exposure scenarios. Exposures of breeding zebrafish to bisphenol A The selected 15 groups that showed consistent breeding and behavioral patterns during the initial acclimation period were subjected to a 10 d pre-exposure period, followed by a 15 d expo- sure period. Reproductive data for the 10 d pre-exposure period were collected to ensure that all breeding groups were reproduc- ing consistently and there were no differences between reproduc- tive measurements for any of the breeding groups prior to the chemical exposure period. Three independent replicate breeding groups were assigned at random to each treatment. A flow- through system was used to dose the tanks for 15 d with three concentrations of BPA (0.01, 0.1, and 1 mg/L) using ethanol (0.0005%) as a solvent. An absolute control receiving water alone and a solvent control receiving the same concentration of ethanol as the chemical exposures were also included. Effects of BPA on epigenetic regulation mbd2 belongs to a family of nuclear proteins capable of binding specifically to methylated DNA, and may also function to repress transcription from methylated gene promoters.57 We found also a significant decrease in mecp2 transcription in male livers, a gene involved in transcriptional repression by associat- ing with methylated CpG dinucleotides where it silences tran- scription by recruiting histone deacetylases, resulting in chromatin remodeling.58 In addition, in male livers a significant decrease in hdac3 transcription was also observed. These find- ings suggest that BPA is not only interacting with the processes linked to DNA methylation, but also has the potential to dis- rupt processes linked to chromatin structure and potentially impact on gene function via these mechanisms. For males, dnmt1 transcription was also negatively associated with BPA exposure concentrations and a significant hypermethyla- tion of two CpG sites in the promoter region of the dnmt1 gene in fish exposed to 0.1 mg/L BPA was observed. In addition, we Despite the advances in our understanding of the epigenetic and transcriptional consequences of BPA in a model vertebrate, there are some limitations to the methodologies used: the locus- specific DNA methylation measurements conducted were 534 L. V. LAING ET AL. 534 based on the sodium bisulphite treatment of genomic DNA and, therefore, cannot distinguish between DNA modifications such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and methylcytosine (5mC), which have unknown func- tional significance.59 In addition, we explored the methylation status of specific CpG positions, within the regulatory regions of select target genes, hypothesized to be targets of BPA toxic- ity. This hypothesis-driven approach was successful in identify- ing some important mechanisms of BPA toxicity but may have missed other interesting effects outside these targeted regions, as suggested by the effects of BPA on global methylation levels. In addition, the global and locus-specific methylation measure- ments reported in this study are single measurements of DNA methylation across multiple cellular populations and cell types within each tissue. Both the gonad and liver are comprised of a mixture of cell types, whose genomic methylation and tran- scriptional activity is unique to the function of each cell type. In the testis for example, a large percentage of the cellular com- position is made up of sperm cells containing very little cyto- plasm and limited transcriptional activity, and the genomic DNA of sperm cells is also known to be hypermethylated. Fish husbandry Wild type WIK strain adult zebrafish (originating from a stock population at the University of Exeter) were maintained according to the conditions reported in Paull et al.60 Prior to the start of the experiment, fish were randomly allocated into 18 breeding groups of 4 males and 4 females, kept in individual 15-L flow-through tanks and were allowed to breed naturally during an acclimation period of 7 d. After this period, colonies that failed to spawn consistently were removed prior to the start of the experiment. Mains tap water was filtered by reverse osmosis [Environmental Water Systems (UK) Ltd.] and recon- stituted with Analar-grade mineral salts to standardized syn- thetic freshwater (final concentrations to give a conductivity of 300 mS: 122 mg/L CaCl22H2O, 9.4 mg/L NaHCO3, 50 mg/L MgSO47H2O, 2.5 mg/L KCl, 50 mg/L Tropic Marin Sea Salt), aerated, and heated to 28C in a reservoir, before it was sup- plied to each aquarium using a flow-through system. Tanks were aerated and supplied with a flow rate of 48 L/day of water.60 Tank water was maintained at 28 § 0.5C and pH 7- 7.5 and fish were maintained under a 12 h light:dark cycle, including dawn and dusk transition periods of 30 min. Fish were fed live Artemia nauplii once daily (ZM Premium Grade Artemia; ZM Ltd.) and TetraMin tropical flake food (Tetra; Melle, Germany) twice daily, to satiation. Effects of BPA on epigenetic regulation In contrast, the ovary contains oocytes characterized by very large cytoplasm where transcripts are stored to support the initial stages of embryogenesis before embryonic genome activation. Therefore, the datasets collected for these tissues are strongly dependent on the cellular composition of the tissue. In future studies, a genome-wide approach to measure methylation and also histone modifications, as well as analysis of single cells or pure populations of cells, may help to further characterize the effects of BPA on epigenetic signaling pathways. Fish husbandry Conclusions Overall, we have found evidence that BPA caused significant disruption to reproduction in breeding zebrafish exposed to 1 mg/L BPA, likely via estrogenic mechanisms. The potential for BPA to cause disruption of reproduction shown here raises concerns for its toxicity when organisms are exposed to BPA in environments affected by other stressors, including other envi- ronmental endocrine disruptors with similar mechanistic path- ways that may act additively to cause reproductive disruption. Importantly, BPA also caused significant alterations in the tran- scription of a number of genes involved in epigenetic regulation in both liver and gonad tissue, most notably on dnmt1, which occurred in conjunction with decreases in global DNA methyl- ation. Of note, some changes were observed after exposure to environmentally relevant concentrations of BPA (0.01 mg/L), corresponding to current exposure scenarios for both humans and wildlife. These findings provide evidence of the adverse effects of BPA in a model vertebrate and advocate for BPA’s replacement within consumer products and its reduction in the environment. On day one of the exposure period, tanks were spiked with the appropriate amount of BPA to achieve the required expo- sure concentrations. Flow rates were monitored daily to ensure the chemical concentrations remained consistent and dosing stocks were replaced every day. Water samples from each tank were collected on days 5, 10, and 15 of the exposure, and were stored at ¡20C until chemical analysis. The effects of BPA on reproduction were determined by measuring the egg production and fertilization success of indi- vidual groups. Eggs were collected each morning approximately one hour post-fertilization (hpf), washed and transferred to petri dishes for analysis. The numbers of fertilized and unfertil- ized eggs were determined by visual inspection for each treat- ment using a dissection microscope (Motic DM143, Hong Kong). Water chemistry For analysis of the concentrations of BPA in the exposure water, methanol, acetonitrile and water, both HPLC and LC- MS grade, HiPerSolv CHROMANORM®, were purchased from VWR Int. One mL of each water sample was added to a glass vial and mixed with 1 mL of HPLC-grade acetonitrile. Before LC-MS/MS analysis, aliquots were vortexed and diluted in a mixture of acetonitrile and water (1:3 v/v). Analyses were per- formed using a Surveyor MS Pump Plus HPLC pump with an HTC PAL autosampler coupled to a TSQ Vantage triple quad- rupole mass spectrometer equipped with heated electrospray Transcript profiling Transcript profiling of genes encoding epigenetic regulatory proteins and genes involved in reproductive function was con- ducted using real-time quantitative PCR (RT-QPCR) as previ- ously described.61 Beacon Designer 3.0 software (Premier Biosoft International, Paulo Alto, CA) was used for designing primers for each target gene using zebrafish NCBI RefSeq sequences, and primers were purchased from MWG-Biotech (Ebersburg, Germany). Assays for each transcript were opti- mized and standard curves were generated as previously described.61 Primer specificity was confirmed by observation of a single amplification product of the expected melting tempera- ture throughout the range of detection. The linear correlation (R2) between the mean Ct and the logarithm of the cDNA dilu- tion was > 0.99 in each case, and efficiencies were between 1.86-2.24. The primer sequences, annealing temperatures, PCR product sizes and PCR efficiencies for each primer pair are shown in Supporting Information Table S2. Template preparation and pyrosequencing was carried out as described by Tost and Gut (2007) 66 on bisulfite-treated DNA from 8 individual fish (gonads and livers) per treatment group. Briefly, genomic DNA (500ng) was treated with sodium bisulfite using the EZ-96 DNA Methylation-Gold Kit (Zymo Research, CA, USA) according to the manufacturers’ standard protocol. Water negative controls were run in duplicate to ver- ify the absence of DNA contamination. Bisulfite-PCR amplifi- cation was performed in duplicate using the primers and assay conditions provided in Supporting Information Table S3. Unmodified DNA samples were included during primer opti- mization to confirm primer specificity for bisulfite-modified DNA. Chemicals All chemicals were obtained from Sigma-Aldrich, UK, unless stated otherwise. g On day 15 of the exposure period, all fish were sacrificed humanely using a lethal dose of benzocaine followed by EPIGENETICS EPIGENETICS 535 et al. 2015) 63 using the Biomart portal.64 Zebrafish esr1 (ENS- DARG00000004111) has 3 known transcripts [esr1-001 (3449 bp), esr1-201 (3502 bp) and esr1-202 (212 bp)] and 2 transcription start sites (TSSs). The dnmt1 gene (ENS- DARG00000030756) also has 2 TSSs and 3 transcripts [dnmt1- 001 (4896 bp), dnmt1-201 (4893 bp) and dnmt1-202 (5031 bp)]. amh (ENSDARG00000014357) has one transcript (amh-001, 3243 bp) and one TSS (Supporting Information Fig. S6). Target sites within the promoter sequences were cho- sen based on their proximity to the TSSs and estrogen-respon- sive elements (EREs), identified using JASPAR,65 and the matrix models ESR1 (MA0112) and ESR2 (MA0258). PCR and bisulfite pyrosequencing assays were designed using the Pyro- Mark Assay design software (Qiagen, Hilden, Germany). Pyro- sequencing primers and their corresponding target sequences are shown in Supporting Information Table S3. destruction of the brain, in accordance with UK Home Office regulations. The wet weight and fork length were recorded, and the condition factor for each fish was calculated (k) D [weight (g) £ 100]/[fork length (cm)]3. The gonads and livers were dis- sected and weighed, and the gonadosomatic index (GSI) D gonad weight (mg)/[total weight (mg)- gonad weight (mg)] £ 100 and hepatosomatic index (HSI) D liver weight (mg)/[total weight (mg)- liver weight (mg)] £ 100 were calculated. Gonads and livers were collected, snap frozen in liquid nitrogen and stored at ¡80C until analysis for transcript profiling and DNA methylation. Luminometric-based assay (LUMA) for global DNA methylation pp g RNA and DNA were extracted together from the livers and gonads of 8 male and 8 female fish from each treatment group using the AllPrep DNA/RNA Micro Qiagen Kit (Qiagen, Hil- den, Germany) according to the manufacturer’s instructions, which allows for extraction of both RNA and DNA from the same tissue sample. NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, USA) was used to assess RNA and DNA purity and concentration. RNA was treated with DNase I (Qiagen) to remove any potential DNA contamination. cDNA was synthesized from 2 mg of total RNA using random hexamers (MWG-Biotech, Ebersberg, Germany) and M-MLV reverse transcriptase (Promega, Madison, USA), according to manufacturer’s instructions. cDNA was then diluted 1:2 and RT-QPCR was performed in duplicate using an iCycler iQ Real-time Detection System (Bio-Rad Laboratories, Hercules, CA) and SYBR Green chemistry, as previously described.61 On each plate, a template-minus negative control was run in duplicate to verify the absence of cDNA contamina- tion. Efficiency-corrected relative expression levels were deter- mined after normalization to a control gene, ribosomal protein l8 (rpl8), which has been shown to have stable expression in the livers and gonads following exposures to estrogens in another cyprinid fish species.44,62 The LUMA assay was performed as described by Karimi et al. (2006) using DNA extracted from gonad samples from 8 indi- vidual fish per treatment.67 Sufficient quantities of DNA were not available to perform the LUMA assay in liver samples; therefore, analysis of global DNA methylation were conducted only for gonad samples. Each DNA sample (250 ng) was digested in duplicate with HpaII and MspI, and data were nor- malized to the EcoRI peak to account for any technical differ- ences between samples.68 Global DNA methylation values were calculated according to the formula [HpaII(G)/EcoRI(T)]/ [MspI(G)/EcoRI(T)], where G and T refer to the peak heights for HpaII or MspI (methylation) and EcoRI (input DNA), respectively. Bisulfite pyrosequencing DNA sequence data for the promoter regions of esr1, amh, and dnmt1 were obtained from Ensembl (release 83; Cunningham 536 L. L. V. LAING ET AL. 536 (HESI II) source (ThermoFisher Scientific, Hemel Hempstead, UK). Chromatographic separation was achieved using a reversed-phase, 3 mm particle size, C18 Hypersil GOLD col- umn 50 mm £ 2.1 mm i.d. (Thermo Scientific, San Jose CA, USA). Analytes were separated using a linear gradient of water and methanol. The initial conditions for the gradient consisted of 10% methanol, which was increased to 100% in 4.5 min and maintained for 1 min before returning to the initial 10% metha- nol. The flow rate was 500 mL/min. The temperature of the autosampler was set at 8C, and the column was kept at a room temperature. The HESI probe was operating in the negative mode and an ion-spray voltage of ¡4.0 kV was applied. The heated capillary temperature was set at 275C and the vaporizer temperature was 60C. Nitrogen was employed as sheath and auxiliary gas at a pressure of 30 and 5 arbitrary units, respec- tively. The argon CID gas was used at a pressure of 1.5 mTorr and the optimum collision energy (CE) for each transition was selected. Quantification of BPA was performed using two char- acteristic multiple reaction monitoring (MRM) transitions of precursor ion 227.1! 212.1 (CE: 20 V) and 227.1 ! 133.1 (CE: 28 V). For transcript profiles, data points classified as outliers (using Chauvenet’s criterion) and data points for which the expression was below the assay detection limit were excluded from analysis. Where amplification was detected in more than 70% of individuals, data were represented as fold-change rela- tive to the expression in the water control group and groups were then compared using one-way ANOVA and Kruskal- Wallis tests with post-hoc tests as described previously. Where amplification was detected in less than 70% of individuals, data were represented as the proportion of individuals for which the target genes were detected, and analysis was conducted using a binomial generalized linear model. In the gonadal data sets, PCA was also performed using the prcomp function to identify the main trends in gene expression. In order to determine if there were associations between the methylation levels for specific loci in the promoter regions of genes of interest and their transcription, correlation analysis was conducted. Bisulfite pyrosequencing Where data was normally distributed Pearson corre- lation was used, and where data did not meet the assumptions of parametric testing, Spearman correlation analysis was per- formed. Correlation analyses were also conducted to determine the relationship between global methylation and dnmt1 tran- scription, as above. The relationship between BPA concentration and transcript expression or methylation was also determined using regression analysis, calculated using linear modeling. Funding We thank the Aquatic Resources Center technical team for support with zebrafish husbandry. This work was funded by a PhD studentship from the Fisheries Society of the British Isles (http://www.fsbi.org.uk/) and the University of Exeter (http://www.exeter.ac.uk/) to LVL and EMS. TMUW was funded by a Natural Environment Research Council CASE PhD stu- dentship (grant no. NE/I528326/1) and the Salmon & Trout Association (http://www.salmon-trout.org/). No potential conflicts of interest were disclosed. No potential conflicts of interest were disclosed. Statistical analysis Statistical analyses were carried out using R (version 3.0.2).69 Prior to analysis, data were tested for equal variance and for normality using the Shapiro–Wilk test. Proportional data and variables with non-Gaussian distributions or non-homoge- neous variances were subjected to variance-stabilizing arcsine transformations or log transformations. Non-parametric statis- tics were used when transformations did not result in distribu- tions meeting the assumptions for parametric tests. All graphs were plotted using untransformed data for ease of interpreta- tion. For the mean fertilization rates, comparisons between treatments were performed using Kruskal-Wallis tests followed by the Wilcoxon signed rank test. The Regression coefficient (R2) was calculated using linear modeling for fertilization rates. Linear mixed effects models were generated using the lme4 package 70 in order to explore the effect of BPA concentration and length of exposure on egg numbers. Non-significant terms were removed from models; models were compared based on likelihood ratio testing to give the appropriate minimum ade- quate model. Model results were inspected to ensure residuals were normally distributed. All graphs were plotted using untransformed data for ease of interpretation, and were created using the R packages ggplot2,71 gplots,72 beeswarm,73 and ggbiplot.74 References Reduced sperm counts in guppies ( Poecilia reticulata) following exposure to low levels of tributyltin and bisphenol A. Proc R Soc Lind B 2000; 267:2333-7; PMID:11413652; http://dx.doi.org/10.1098/rspb.2000.1288 7. Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J, Needham LL. Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. Environ Health Persp 2004; PMID:15811827; 113:391-5; http://dx.doi.org/10.1289/ehp.7534 p g p 24. Kwak H, Bae M, Lee M, Lee Y, Lee B, Kang K, Chae CH, Sung HJ, Shin JS, Kim JH, et al. Effects of nonylphenol, bisphenol A, and their mixture on the viviparous swordtail fish ( Xiphophorus helleri). Envi- ron Toxicol Chem 2001; 20:787-95; PMID:11345455; http://dx.doi. org/10.1002/etc.5620200414 p g p 8. 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Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems
Građevinar
2,023
cc-by
8,869
DOI: https://doi.org/10.14256/JCE.3767.2023 Primljen / Received: 7.4.2023. Ispravljen / Corrected: 15.6.2023. Prihvaćen / Accepted: 4.8.2023. Dostupno online / Available online: 10.11.2023. Građevinar 10/2023 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems Authors: Research Paper İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems İbrahim Hüner, PhD. CE Pamukkale University, Denizli, Turkey Department of Civil Engineering fsm-1453@hotmail.com Corresponding author This study determines the effects of different types of base isolator systems on the seismic performance of liquefied natural gas (LNG) storage tanks. Nonlinear time-history analyses of the non-isolated and three different isolated models were performed for the average acceleration of seven ground motions scaled to achieve a specified safe shutdown earthquake. The ANSYS Workbench program was used in the modelling studies of the LNG liquid, inner steel tank, outer shell, ring beam, roof and concrete foundation and side wall insulation. The LS-DYNA program was used for the nonlinear analyses of the LNG liquid, inner steel tank and concrete foundation. The results of the total base shear force, sloshing height, steel tank stresses and lateral deflection were compared. The results indicated that there was no difference between the convective and impulsive modes for the LNG tanks with isolators. It was concluded that the wave motion of the liquid was different from the oscillation of the structure and the earthquake isolation times did not affect the sloshing motion. In the non-isolated system, the stress reached 400 MPa, whereas it was 350 MPa on average in the LNG tanks with isolators. Prof. Bülent Akbaş, PhD. CE Key words: Gebze Institute of Technology, Gebze, Kocaeli, liquefied natural gas tanks, HDRB, LRB, FPS, LS-DYNA, ANSYS workbench, nonlinear analyses Turkey Department of Civil Engineering akbasb@gyte.edu.tr Prethodno priopćenje İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Nelinearno ponašanje spremnika za ukapljeni prirodni plin s različitim sustavima potresne izolacije Prof. Abdullah Cem Koç, PhD. CE Pamukkale University, Denizli, Turkey Department of Civil Engineering a_c_koc@pau.edu.tr Ovaj rad pobliže određuje učinke različitih tipova izolacijskih sustava na ponašanje spremnika za ukapljeni prirodni plin (engl. liquefied natural gas - LNG) tijekom potresa. Provedene su nelinearne analize primjenom vremenskog zapisa neizoliranih i triju različitih izoliranih modela za prosječno ubrzanje sedam vrsta gibanja tala stupnjevanih kako bi se postigla određena moguća sigurna obustava rada postrojenja tijekom potresa. Program ANSYS Workbench primijenjen je za modeliranje tekućine ukapljenog plina, unutarnjeg čeličnog spremnika, vanjske stijenke, serklaže, krova, betonskih temelja i izolacije stražnje stijenke. Program LS-DYNA primijenjen je za nelinearne analize tekućine LNG-a, unutarnjeg čeličnog spremnika i betonskih temelja. Uspoređeni su rezultati ukupne poprečne potresne sile u podnožju, visine zapljuskivanja, naprezanja čeličnog spremnika i bočnog pomaka. Rezultati su pokazali da nema razlike između konvektivnih i impulsnih modova za izolirane spremnike LNG-a. Zaključeno je da se valno gibanje tekućine razlikuje od osciliranja konstrukcije, a razdoblja protupotresnih izolacija nisu utjecala na zapljuskivanje. U neizoliranom su sustavu vrijednosti naprezanja dosegle 400 MPa, dok su te vrijednosti u izoliranim spremnicima za LNG prosječno iznosile 350 MPa. Ključne riječi: spremnik za ukapljeni prirodni plin, gumeni ležajevi s velikim prigušenjem (HDRB), gumeni ležajevi s olovnom jezgrom (LRB), klizni ležajevi s njihalom (FPS), nelinearne analize GRAĐEVINAR 75 (2023) 10, 979-995 979 Građevinar 10/2023 1. Introduction Natural gas is a hydrocarbon-based gas that consists largely of methane and has a very low density (0.66 kg/m3). The transportation of natural gas, which plays an important role in meeting the energy demands of the world, from source to end user, is a critical process. Road transport has become an important alternative to natural gas supply owing to problems such as natural disasters and security issues in natural gas transmission lines located at sea and on land. Liquefied natural gas (LNG) (430–480 kg/m3) is used in land and sea transportation and it is obtained by the condensation of natural gas at −168 °C temperature and normal atmospheric pressure conditions. It should be noted that there is a difference of approximately 600 times between the densities of liquid natural gas and gaseous gas. Different types of storage and transport tanks are used to provide reasonably fast and predictable transportation of LNG. These tanks are generally divided into different classes, such as heavytonnage pickup truck, ship, terminal and storage tanks [1]. The tank type covered in this study is the fixed LNG storage tank with very large storage volume. These tanks, which are called fully contained storage tanks, consist of an inner tank made of cryogenic steel (9 % nickel) and an outer cylindrical shell wall made of typical post-tensioned reinforced concrete along the vertical and radial directions. The base slab and spherical roof dome are made of reinforced concrete. Adequate thermal insulation is provided between the tanks [2]. In the more common cases, where the sole plate is in direct contact with the ground, freezing is prevented using electric heating plates [1]. In API 620 [3], API625 [4] and NFPA 59A [5], the design requirements for LNG tanks are stringent because they store a high-energy chemical substance. LNG storage tanks are designed according to three levels of seismic action. The operational basis earthquake (OBE) ground motion is represented by an acceleration response spectrum with a 10 % probability of exceedance over a 50 years period (mean return interval of 475 years). The tank system should be designed to continue operating during and after the OBE. After a safe shutdown earthquake (SSE), no deformation should occur in the tank support systems, insulation layers, or isolators. The accepted maximum considered earthquake (MCER), determined based on site-specific research, has a 2 % probability of exceedance within a 50 years period (mean recurrence interval of 2475 years). The aftershock level earthquake (ALE) ground motion is defined as half the SSE. An LNG tank system subjected to an ALE should maintain the primary container volume at its maximum operating level with no loss from the secondary container [3-5]. The behaviour of structures exposed to fluid pressure caused by earthquakes was first investigated by Westergaard in 1933 [6]. In the studies carried out by Jacobsen [7, 8], the rigid tank containing the liquid and the support legs carrying 980 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç the horizontally accelerated liquid tank were analysed. In studies conducted by Housner [9, 10], the hydrodynamic pressure created by the liquid in the tank was separated into its components and the effects of the impulsive pressure caused by the liquid part accelerating with the tank and the convective pressure caused by the agitated liquid part were investigated. The convective component was then modelled using a single degree-of-freedom (DOF) oscillator [11, 12]. Haroun and Housner [13] developed a 3-DOF model for ground-supported deformable tanks, including flexible behaviour. Veletsos and Tang [14] also conducted similar studies. Malhatro et al. [15] considered the pulsed and convective modes and modified the properties of the mechanical analogue to include higher modes in the resulting base shear and base overturning moments. The seismic risks of LNG storage tanks are higher than those of traditional buildings because they can lead to secondary disasters such as explosions and environmental pollution, which would result in significant property damage or loss of life. For example, the destruction of an LNG tank during the 1964 Niigata earthquake in Japan caused fires and explosions, resulting in serious societal losses and pollution [16]. Since the 1990s, several studies have been conducted on the dynamic analysis of LNG storage tanks [17, 18]. Basic isolation techniques, such as lead-core rubber bearings (LRBs), highdamping rubber bearings (HDRBs), steel hysteretic shock absorbers and friction pendulum bearings are used in the seismic design of LNG tanks and in many other applications in modern structural design [19-22]. Three different LNG tank structural configurations, that is, with a fixed bottom, seismically isolated with rubber bearings only and seismically isolated with rubber bearings and steel hysteretic dampers, were tested in [19]. The test results confirmed that the use of steel hysteretic dampers drastically decreased large displacements. Christovasilis and Whittaker [23] investigated the seismic response of a conventional and isolated 150,000 m3 capacity vertical cylindrical LNG tank by applying finite element analysis to mechanical models. The base shear and overturning moment in the seismically isolated LNG tank were 10 %–15 % of the values computed for the conventional tank and the wave heights were unaffected by the use of a seismic isolation system. Gregoriou et al. [20, 21] analysed the seismic response of three typical LNG tanks isolated using high-damping rubber bearings and lead-core rubber bearings. As a result of these processes, problems related to the base shear force and deflection of the inner steel were reduced by approximately 70 %–60 %. However, an increase in the agitation height was observed compared to the nonisolated tank, especially in the case of high-damping rubber support insulation. Marti et al. [1] studied a typical modern LNG tank with a capacity of 160,000 m3 and they reported that seismically isolated tanks can be used when the design peak ground accelerations are in the range of approximately 0.30–0.90 g. Ruifu et al. [22] analysed the seismic response of GRAĐEVINAR 75 (2023) 10, 979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems a vertical, cylindrical, extra-large and insulated LNG tank with a multiple friction pendulum system (MFPS). They presented statistically classified data, including pile shear, wave height, impulsive acceleration, convective acceleration and outer tank acceleration and showed that the isolation system could adapt perfectly to different liquid levels and was very effective in controlling the seismic response of extra-large LNG tanks. Datoli et al. [24] examined an LNG tank with a capacity of approximately 172,000 m3 during an earthquake of 7.1 Richter magnitude. They constructed a finite element model (FEM) model consisting of a flat anchored base and a cylindrical metal wall in contact with the LNG and reported that the fluid motion and fluid–structure interactions were responsible for a failure type known as the elephant’s foot. Zhao et al. [25] investigated the effects of liquid filling rate and earthquake motion periods on the efficiency of a lead– rubber bearing system using the ABAQUS program. Zhao et al. [26] investigated the liquid filling ratio and earthquake motion periods to determine the efficiency of a lead–rubber bearing system using the general finite element code ABAQUS. They analysed a 160,000 m3 LNG prestressed storage tank for 12 earthquake waves under four site classes using stress distributions on the outer and inner tanks, as well as tip displacement and base shear. The maximum stress of the inner tank was greater than 500 MPa at the 100 % liquid level in all four site classes, creating significant safety hazards. Design optimisation and establishment of an early warning system are imperative for controlling high liquid levels. Barone and Sartori [27] investigated two elevated LNG tanks with a full working capacity of 10,130 m3 and 91 pieces of friction pendulum systems (FPSs) isolated at the Corsini port of Ravenna, Italy. In their experiments, they observed that a high lateral flexibility and a high friction coefficient effectively separated the motion of the ground from the structure and dissipated some of the seismic energy. Kilic et al. [28] studied the performance of one broad tank, one medium tank and one slender tank with two- and threedimensional elastomeric bearings, which were isolated along only the horizontal direction and in both directions (horizontal and vertical), respectively, by performing nonlinear dynamic time history analyses. It was observed that 3D isolators provided more efficient results than 2D isolators. Chen et al. [29] performed shake table tests and numerical models of an LRB isolated LNG tank. Although the base shear force, overturning moment and acceleration spectra of the tank Građevinar 10/2023 were significantly reduced, the displacement of the tank posed a danger to the piping system connected to the tank. Sharari et al. [30] examined a 160,000 m3 full containment LNG tank considering the soil structure and fluid–structure interactions while assessing the impacts of the depth of soil liquefaction on the performance of different components of the system. According to the nonlinear time history results, the seismic forces on the inner steel and outer reinforced concrete tank walls decreased as the liquefaction depth increased. However, increases in the lateral displacements, shear forces and bending moments of the pile head were observed. The positive effects of the isolators on the LNG tank in earthquakes of 0.4 g and above were compared. To calculate the sloshing height in the wide and high LNG tanks with a long period of 9.80 s, earthquakes with a minimum of 27 s and above were selected. The effects of the isolators on the sloshing height of the LNG liquid were investigated. The analysis results of medium and strong earthquakes in anchored and isolated LNG tanks were evaluated. This paper presents a case study analysing the behaviour of an LNG tank without seismic isolation and with three different types of base seismic isolation. 2. Description and modelling of the liquefied natural gas (LNG) tank For this study, an LNG tank with a volume of 232.000 m3 constructed for high-seismic hazard areas was selected. The effects of the insulation type on the spherical bottom slip, LNG sloshing wave height, lateral displacements on the steel tank wall, stresses in the tank and earthquake isolation force–displacement diagrams were investigated. In this LNG tank, the height-to-radius ratio is 1. The LNG tank consists of an inner layer, insulation layer and outer layer. The height of the inner and outer tanks is 45 m and their diameters are 84 and 88 m, respectively. The maximum height of the liquid is 42 m. The outer shell has a constant thickness (0.8 m along its height), whereas the thickness of the inner shell increases from top to bottom, from 12 to 32 mm. The space (1.20 m) between the two shells was filled with perlite to provide thermal insulation. While the outer shell was placed directly on a circular foundation slab (1.80 m), a layer of foam glass (0.70 m) was placed between the inner shell base and foundation slab for thermal insulation. Analyses were performed considering the geometric properties listed Table 1. Material properties of the LNG tank Description Young modulus E (MPa) [MPa] Poisson ratio ν Mass density γ [kg/m3] Inner steel 210000 0.30 7850 Concrete 37000 0.20 2500 LNG 2000 - 480 Perlite isolation 7500 - 240 Foam glass isolation 1200 - 150 GRAĐEVINAR 75 (2023) 10, 979-995 981 Građevinar 10/2023 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Table 2. Geometric dimensions of the LNG tank Description Dimension Height of inner steel tank 45.00 m Diameter of inner steel tank 84.00 m Thickness of inner steel tank wall 32 mm-12 mm Inner steel tank bottom thickness 5 mm Height of LNG 42.00 m Height of outer concrete tank 61.00 m Diameter of outer concrete tank 88.00 m Wall thickness of outer concrete tank 0.80 m Sphere thickness of outer concrete tank 0.40 m Outer concrete wall with between inner steel tank perlite insulation thickness 1.20 m Thickness of bottom base insulation (foam glass) 0.70 m Thickness of foundation 1.80 m in Table 1 and the material properties listed in Table 2. The steel tank forming the inner layer is composed of a nickelcontaining steel alloy (9 % Ni). Because of this steel alloy, the inner layer tank exhibits excellent low-temperature resistance, good weldability and low susceptibility to cold cracks [3, 26]. As shown in Fig. 1, the inner tank consists of six different layers with a height of 8 m. DOF rectangular shell elements. The fibre glass and pearlite isolation layers were modelled using eight-nodded 12-DOF solid elements. The fluid content was modelled using eightnodded 12-DOF fluids. The fluid–structure interaction was approximated by determining the appropriate coupling equations at the nodes of the fluid–structure interface. The outer concrete wall, inner steel tank, concrete dome and ring beam, foundation slab, bottom foam glass and side perlite insulation were modelled using quadrilateral shell elements. In the analyses, the fluid side was considered an acoustic element and was combined with the structural side with the combining option to obtain results consistent with the theoretical calculations. Table 3. Thickness of each layer in the inner tank Layer Level [m] Height [m] Thickness [mm] 1 +2.50 to +10.50 8.00 32 2 +10.50 to +18.50 8.00 28 3 +18.50 to +25.50 8.00 24 Figure 1. Schematic view of the basic design section of the liquefied 4 +25.50 to +33.50 8.00 20 natural gas (LNG) storage tank with a volume of 232,000 m3 5 +33.50 to +41.50 8.00 16 For example, the R1/32 Layer refers to the first layer with a thickness of 32 mm at the bottom. The thicknesses of these layers gradually increase from top to bottom, as presented in Table 3. When the inner tank is filled with 100 % LNG, the maximum stress in it exceeds 500 MPa in the four site classes. Considering that the yield strength of the alloy steel used is between 500 and 600 MPa, this stress value poses a risk to structural safety [26, 31]. The selected LNG tanks were modelled using the ANSYS Workbench finite element program [32]. The outer and inner shells as well as the dome and foundation plate were modelled with four-point, 24- 6 +41.50 to +47.50 5.00 12 982 The seismic analysis FEM developed in ANSYS Workbench was converted to LS-DYNA and seismic transient nonlinear analyses were carried out in LS-DYNA. The shell and solid elements had similar DOFs and types. The MAT_24 piecewise multilinear material model was used for the metal parts and the MAT_01 elastic model was adopted for the concrete and isolation parts. For the fluid side, MAT_NULL with Lagrangian options was used with automatic node-to-surface coupling of the sloshing effects have been presented. GRAĐEVINAR 75 (2023) 10, 979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems Građevinar 10/2023 Figure 2. Finite element network structure for the LNG tank system 3. Modelling of the isolation systems The base isolation systems used in this study included a series of similar rubber bearings homogeneously distributed under the foundation slab. The total number of required supports was calculated considering that a single element supports a maximum of 10 to 12 m2 of foundation slab area [20, 21]. As this study aimed to examine the behavioural differences of isolator types under the same system, combined isolator placement was not performed. Therefore, the estimated number of bearings for a tank with a volume of 232,000 m3 was 597. The frequency for extra-large LNG tanks is generally between 2 and 10 Hz, which is the specific resonance range for earthquake-induced ground movements [18]. used to model the seismic isolation system [32]. Using the SpaceClaim program, the bearings were converted to beam/ link elements to maintain proper locations and the connection points were linked to the concrete base to create bonding. In the ANSYS Workbench program, it was assumed that the link elements were rigidly connected to the concrete and a fixed boundary condition was applied on the other side. For the seismic simulations, these link elements were converted to bushing elements using Beam ELFORM 6. The beam elements in LS-DYNA were MAT_197_SEISMIC_ISOLATOR, whose characteristics are described below.. Figure 3. Details A and B An FEM of the previously described LNG tank was developed to introduce seismic isolators at the base of the structure during the modal analyses. The bearings are assumed to have infinite vertical stiffness. Based on this assumption, the displacement constraint in the direction of the earthquake motion at the base of the tank walls was relaxed and nonlinear horizontal springs were placed to connect the base of the walls to the ground. In the ANSYS Workbench program, link elements (LINK8) that act as truss elements and combine nonlinear materials were GRAĐEVINAR 75 (2023) 10, 979-995 Figure 4. Earthquake isolation arrangement plan for high-damping rubber bearing (HDRB), lead-core rubber bearing (LRB) and friction pendulum system (FPS) isolators 983 Građevinar 10/2023 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Table 4. Engineering characteristics and bilinear spring parameters of HDRB SI-N 900/204 and LRB SI-N 900/225-185 Description HDRB SI-N 900/204 LRB SI-N 900/225-185 Vertical stiffness Kv [kN/mm] 2317 2198 Effective horizontal stiffness Ke [kN/mm] 2.50 2.47 Elastic horizontal stiffness K1 [kN/mm] 19.67 17.33 Yield displacement d1 [mm] 12 18 Plastic horizontal stiffness K2 [kN/mm] 1.97 1.77 Maximum seismic displacement d2 [mm] 400 400 Maximum vertical load at load combinations including the seismic action [kN] 7980 6630 Elastomer stiffness [mm] 900 900 Elastomer thickness te [mm] of total design 204 225 Dynamic shear modulus Gdin na γ =1 [MPa] 0.8 0.6 Table 5. Engineering characteristics and bilinear spring parameters of FPS FIP-D M 1600/800 (3700) Description Vertical stiffness Kv [kN/mm] 36715 Restoring stiffness Kr [kN/mm] 0.6376 Effective horizontal stiffness Ke [kN/mm] 1.145 Friction force developed by the isolator F0 [kN] 203.5 Maximum horizontal force Fmax [kN] 458 Minimum friction coefficient µ [%] 4.872 Effective viscous damping ratio ξe 0.2826 Equivalent radius of curvature R [mm] 3700 Maximum seismic displacement d [mm] 400 Isolator diameter excluding anchoring elements D [mm] 940 Isolator height excluding dowels H [mm] 199 3.1. High damping rubber bearings (HDRB) HDRBs are one of three main subtypes of steel-reinforced elastomeric bearings [33]. They consist of thin rubber layers reinforced with steel plates. According to a preliminary design [34], the number of high damping rubber bearings (SI-N 900/204) that should be used for an LNG tank with a volume of 232,000 m3 is 597. The properties used in this study are listed in Table 4. 3.2. Lead core rubber bearings (LRB) Lead-core rubber bearings are composed of steel plate layers, rubber layers and a lead core. Similar to the steel shims in natural rubber bearings, the steel layers provide vertical stiffness, the rubber layers provide high lateral flexibility and the lead core provides these devices with extra stiffness and damping properties. According to a preliminary 984 FIP-D M 1600/800 (3700) design [34], the number of lead core rubber bearings (SI-N 900/225-185) that should be used for an LNG tank with a volume of 232,000 m3 is 597. The properties used in this study are listed in Table 4. 3.3. Friction pendulum system (FPS) Friction pendulum bearings are curved surface sliding bearings consisting of an articulated slider and a cover plate. The slider used in the system is covered with a selflubricating composite liner. During an earthquake, the articulated slider on the bearing side along the concave surface enables the structure to move with gentle pendulum movements. According to a preliminary design [34], the number of the friction pendulum bearings [FIP-D M 1600/800 (3700)] that should be used for an LNG tank with a volume of 232,000 m3 is 597. The properties used in this study are listed in Table 5. GRAĐEVINAR 75 (2023) 10, 979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems Građevinar 10/2023 Table 6. Earthquake specifications used for the time history analyses No Earthquake ground motions (PEER register number) Station Abbrev. Year Mw Time [s] SSE scale factor PGA (SSE) 1 Imperial Valley / SAD (RSN6) El Centro Array IV2 1940 6.95 53.72 4.85 1.022 2 Kern County / SAD (RSN15) Taft Lincoln School KC 1952 7.36 54.35 5.75 1.037 3 Borrego Mtn / SAD (RSN36) El Centro Array BM 1968 6.80 79.99 7.20 0.414 4 Imperial Valley-I / SAD (RSN162) Calexico Fire Station IV06-1 1979 6.53 37.86 6.30 1.282 5 Imperial Valley-II / SAD (RSN169) Delta IV06-2 1979 6.53 70.00 3.95 0.931 6 Victoria / Meksiko (RSN266) Chihuahua VM 1980 6.33 27.00 4.50 0.679 7 Irpinia / Italija (RSN286) Bisaccia IITA 1980 6.90 38.26 5.05 0.482 4. Analysis results 4.1. Earthquake ground motions Analyses of the LNG tank system and the filling systems added to this system were conducted for three types of seismic ground motions: OBEs, SSEs and ALEs. According to the definitions in API 625 [4] and NFPA 59A [5] the ground motion of the OBE is represented by an acceleration response spectrum with a 10 % probability of deflection over a 50-year period (an average return interval of 475 years). An SSE is defined as the ground motion of an accepted maximum earthquake (MCER) over a 50-year period (mean recurrence interval of 2475 years) based on site-specific research. The ALE ground motion is defined as half of the SSE. The magnitudes used for the analyses of past earthquakes are given in Table 6, including the year, time, scale factor of the SSE, peak ground acceleration of the SSE and ground motion. A linear scaling approach was used to match the acceleration time series during the period of interest. The specified period range was selected as 0.1 and 12 s. This range represents a very broad spectrum band that matches the target acceleration spectrum, owing to the long agitation period (Tc1 = ~9.7 s) of the stored LNG. Seven pairs of earthquake ground motions, called initial or core, were used to match the SSE spectrum. The calculations indicated that the impulsive mode of the median spectral coordinates matched well with the design spectrum for the specified periods, whereas the convective mode was significantly lower than the design spectrum for these periods. The adopted soil class was D and the ground motion and design level acceleration parameter values were determined as Ss = 1.926, S1 = 0.660, SDS = 1.926 (SSE) and SD1 = 1.123 (SSE). Seven pairs of earthquake ground motions were used as seeds to match the SSE spectra. 4.2. Modal analysis results 4.2.1. Inner steel tank and LNG fluid The results of the inner steel tank and LNG fluid modal analyses for the non-isolated (anchored bottom) tank and systems with HDRB, LRB and FPS isolators are listed in Table 7. Figure 6 shows the first, second, third and fourth convective modes of the FEM. The convective mode shapes represent the agitation of the liquid in the tank without the tank wall. The impulsive mode shapes represent the combined motion of the tank and liquid. Figure 7 shows the first, second, third and fourth impulsive modes of the FEM. The results of static and dynamic analyses of the LNG tank are presented in this section. The first horizontal convective and impulsive mode periods were 10 and 0.2302 s, respectively. The high-period (low-frequency) convective mode caused the agitation of the liquid and damaged the tank roof. The difference between the impulsive and convective mode frequencies indicated that the interaction effects between Figure 5. Acceleration site-specific response spectra for the design earthquake increased with the safe shutdown earthquake (SSE) scale factor these modes were not significant. GRAĐEVINAR 75 (2023) 10, 979-995 985 Građevinar 10/2023 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Figure 6. Convective mode types of the finite element model (FEM) for the non-isolated LNG tank: a) First convective mode T1 = 9.8232 s; b) Second convective mode T2 = 7.4460 s; c) Third convective mode T3 = 6.6269 s; d) Fourth convective mode T4 = 6.3291 s Figure 7. Impulsive mode types of the FEM for the non-isolated LNG tank: a) First impulsive mode T1 = 0.2302 s; b) Second impulsive mode T2 = 0.2301 s; c) Third impulsive mode T3 = 0.2178 s; d) Fourth impulsive mode T4 = 0.2158 s 986 GRAĐEVINAR 75 (2023) 10, 979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems Građevinar 10/2023 Table 7. Natural periods of the inner model Steel tank and LNG fluid LNG Fluid Part Mode number Non-isolated HDRB isolator LRB isolator FPS isolator Frequency [Hz] Period [s] Frequency [Hz] Period [s] Frequency [Hz] Period [s] Frequency [Hz] Period [s] Mod 1 0.1018 9.8232 0.1011 9.8912 0.1011 9.8912 0.1006 9.9404 Mod 2 0.1343 7.4460 0.1343 7.4460 0.1343 7.4460 0.1343 7.4460 Mod 3 0.1509 6.6269 0.1509 6.6269 0.1509 6.6269 0.1509 6.6269 Mod 4 0.1580 6.3291 0.1580 6.3291 0.1580 6.3291 0.1580 6.3291 Mod 5 0.1780 5.6180 0.1780 5.6180 0.1780 5.6180 0.1780 5.6180 Mod 6 0.1784 5.6054 0.1782 5.6117 0.1782 5.6117 0.1781 5.6148 Mod 1 4.3436 0.2302 3.9718 0.2518 3.9706 0.2519 3.4718 0.2880 Mod 2 4.3445 0.2301 4.0986 0.2440 4.0975 0.2441 3.8823 0.2576 Mod 3 4.5913 0.2178 4.1754 0.2395 4.1741 0.2396 3.9885 0.2507 Mod 4 4.633 0.2158 4.7762 0.2094 4.7417 0.2109 4.099 0.2440 Mod 5 5.016 0.1994 5.0602 0.1976 5.0593 0.1977 4.8246 0.2073 Mod 6 5.301 0.1886 5.7193 0.1748 5.7174 0.1749 5.010 0.1996 4.2.2. Outer concrete Modal analyses were also performed for the external prestressed concrete walls, ring beams, domes and mat foundations. The results of the outer concrete modal analyses for the non-isolated tank and systems with HDRB, LRB and FPS isolators are listed in Table 8. Figure 8 shows the first, second and third mode shapes of the FEM. In the first mode, Figure 8. Outer concrete mode shapes of the FEM for the non-isolated tank: a) First mode T1 = 0.2302 s; b) Second mode T2 = 0.2178 s; c) Third mode T3 = 0.1994 s GRAĐEVINAR 75 (2023) 10, 979-995 987 Građevinar 10/2023 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Table 8. Natural periods of the outer concrete model Non-isolated HDRB isolator LRB isolator FPS isolator Part Mode No Outer concrete tank Mod 1 4.3445 0.2302 3.9718 0.2518 3.9716 0.2518 3.4718 0.2880 Mod 2 4.5913 0.2178 4.7762 0.2094 4.7174 0.2120 3.8823 0.2576 Mod 3 5.0163 0.1994 5.7193 0.1748 5.7174 0.1749 4.8246 0.2073 Frequency [Hz] Period [s] Frequency [Hz] the walls were deformed in the radial direction; however, the roof diaphragm did not move laterally. In the second mode, the walls were displaced laterally, whereas the ring beam, dome and foundation were rigid. In the third mode, the prestressed concrete walls moved along their axes in the positive and negative directions. 4.3. Times history analysis results The ground motion acceleration was defined using the LOAD_BODY_X option in the LS-DYNA program. The ground was fixed to provide the structure with inertia. Additionally, LOAD_BODY_Z was defined to conserve gravity. Because a large structure with a fluid domain was analysed, an inclined interface time was used to neglect the initial gravitational effect and avoid unnecessary strain on the structure. The LS-DYNA software was used to simulate the fluid–structure interaction in a tank filled with fluid. This software offers significant advantages, particularly Period [s] Frequency [Hz] Period [s] Frequency [Hz] Period [s] for solving dynamic contact problems. In addition, the LSDYNA program provides materials and standardised contact formulations that can be used to model fluids and represent the interaction between the tank shell and fluid during seismic excitation. 4.3.1. Inner steel tank stresses The inner tank maximum von Mises stress values (MPa) and percentages (%) of reduction with respect to the nonisolated values occurring during a seven-scale earthquake in the non-isolated and HDRB-, LRB- and FPS-isolated LNG tanks fully filled with LNG are given in Table 9. The maximum stress distributions of the non-isolated and HDRB-, LRBand FPS-isolated LNG tanks for the 1940 Imperial Valley Earthquake El Centro Array Base Station data are shown in Fig. 9. Compared with conventional tanks, HDRB, LRB and FPS seismic isolators provide an average reduction of 15 % to 5 % in repulsive modes. Considering the fixed base Figure 9. Inner steel tank wall tension stresses for non-isolated and HDRB-, LRB- and FPS-isolated tanks: a) Non-isolated; b) HDRB isolator; c) LRB isolator; d) FPS isolator 988 GRAĐEVINAR 75 (2023) 10, 979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems Građevinar 10/2023 Table 9. Inner tank maximum von Mises stress values (MPa) and reduction rates (%) with respect to the non-isolated values Earthquake ground motions Base type Non-isolated HDRB isolator LRB isolator FPS isolator IV2-1940 406 369 9% 358 12 % 347 15 % KC-1952 378 354 6% 354 6% 349 8% BM-1968 380 348 8% 348 8% 346 9% IV06-I-1979 392 357 9% 346 12 % 350 11 % IV06-II-1979 405 348 14 % 355 12 % 345 15 % VM-1980 382 352 8% 352 8% 345 10 % IITA-1980 375 351 6% 356 5% 328 12 % conditions, the reduction in the maximum stresses in the inner shell of approximately 15 % compared with the nonisolated situation reveals the importance of isolator use. Dynamic loads generate much greater stress than static loads, which is critical for LNG tanks. Considering that the yield strength of 9 % Ni steel is between 515 MPa and 585 MPa, the maximum stress in the inner tank of the anchored LNG tank exceeding 400 MPa poses a risk to the safety of the structure [26, 31]. 4.3.2. LNG fluid maximum sloshing wave height The LNG fluid maximum sloshing wave height values (mm) and percentages (%) of reduction with respect to the nonisolated values occurring during a seven-scale earthquake in the non-isolated and HDRB-, LRB- and FPS-isolated LNG tanks fully filled with LNG are given in Table 10. The maximum sloshing wave heights of the non-isolated and HDRB-, LRBand FPS-isolated LNG tanks for the 1979 Imperial Valley earthquake Calexico Fire Station data are shown in Fig. 10. The FPS and LRB earthquake isolators exhibited the best performance at the height of the agitated liquid in the LNG tank, which was examined for seven different earthquakes and four different systems. This result can be explained by a friction force of approximately 200 kN when the FPS-type earthquake isolator was used. This frictional force absorbs the horizontal forces that occur during an earthquake. In an LRB-type isolator, the lead core in the centre absorbs horizontal forces. Higher sloshing heights than those in the LRB, FPS and fixed-bottom LNG tanks were observed when the HDRB-type isolator was used. The LNG tank oscillated because of the horizontal force originating Figure 10. LNG fluid maximum sloshing wave height for non-isolated and HDRB-isolated, LRB-isolated and FPS-isolated tanks: a) Non-isolated; b) HDRB isolator; c) LRB isolator; d) FPS isolator GRAĐEVINAR 75 (2023) 10, 979-995 989 Građevinar 10/2023 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Table 10. LNG fluid maximum sloshing wave height values (mm) Structural bearing type IV2-1940 KC-1952 BM-1968 IV06-I-1979 IV06-II-1979 VM-1980 IITA-1980 Fixed 2463 2119 3317 3421 2006 2567 3355 HDRB 2577 1920 3638 3433 1959 2836 3488 LRB 2379 1934 3560 3428 1880 2470 3438 FPS 2090 1932 3550 3348 1899 2760 3276 from the rubber system. As a result, the LNG liquid height in the convective mode increased during an earthquake. The sloshing height in conventional tanks was higher than that in LNG tanks with isolators in the Kern County and Imperial Valley-II earthquakes and the earthquake oscillations in the convective mode continued with high accelerations. 4.3.3. The maximum lateral displacement of inner steel tank wall The maximum lateral displacement values (mm) and reduction rates (%) of the inner steel tank with respect to the non-isolated values occurring during a seven-scale earthquake in the non- Figure 11. Maximum lateral displacement values (mm) at the steel wall for non-isolated and HDRB-, LRB- and FPS-isolated tanks: a) Nonisolated; b) HDRB isolator; c) LRB isolator; d) FPS isolator Table 11. Maximum lateral displacement values (mm) at the steel tank wall and reduction rates (%) with respect to the non-isolated values Base type Earthquake ground motions Non-isolated IV2-1940 898 496 45 % 424 53 % 604 33 % KC-1952 578 361 38 % 369 36 % 329 43 % 990 HDRB isolator LRB isolator FPS isolator BM-1968 369 322 13 % 278 25 % 345 7% IV06-I-1979 706 553 22 % 430 39 % 299 58 % IV06-II-1979 1120 287 74 % 287 74 % 401 64 % VM-1980 449 444 1% 312 30 % 440 2% IITA-1980 171 263 -53 % 253 -48 % 297 -74 % GRAĐEVINAR 75 (2023) 10,979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems Građevinar 10/2023 isolated and HDRB-, LRB- and FPSisolated LNG tanks fully filled with LNG are given in Table 11. The maximum lateral displacements of the inner steel tank wall values of the non-isolated and HDRB-, LRB- and FPS-isolated LNG tanks for the 1979 Imperial Valley Earthquake Delta Station data are shown in Fig. 11. It was observed that the horizontal displacement movements of the inner tanks in the isolated LNG tank were less than those of the fixed system during high-acceleration and Figure 12. LNG tank base shear force non-isolated and HDRB-, LRB- and FPS-isolated tanks long oscillating earthquakes. However, the elephant foot deformation type, which is observed in lowintensity earthquakes, is less common in LNG tanks fixed to the principle, the base shear forces that occur in these systems foundation with anchor bolts than in structures with isolators. during an earthquake are less than those of the LRB and HDRB When using an LRB-type isolator in severe earthquakes, less isolator systems. Consequently, LNG tanks with frictionlateral deformation occurs in the inner tank compared with that based pendulum-type isolators performed much better than generated with other isolator systems. This is because of the other rubber-based isolator systems when compared to the ability of the lead core in the centre of the LRB-type isolators to base shear forces occurring during earthquakes. inhibit lateral displacement. The comparative analysis results of the maximum inner steel tank von Mises stresses, LNG fluid sloshing wave heights, inner steel tank lateral displacements and base shear forces are 4.3.4. Maximum base shear force of inner steel tank shown in Fig. 13. The maximum base shear forces of the inner steel tank (kN) and reduction rates (%) with respect to the non-isolated 4.3.5. Force–displacement diagrams for LNG isolation types values occurring during a seven-scale earthquake for nonisolated and HDRB-, LRB- and FPS-isolated LNG tanks fully filled with LNG are given in Table 12. The maximum base shear The force (kN) and displacement (mm) values during a force values of the non-isolated and HDRB-, LRB- and FPSseven-scale earthquake for the HDRB-, LRB- and FPSisolated LNG tanks for the 1979 Imperial Valley earthquake isolated LNG tanks fully filled with LNG are listed in Table Delta Station data are shown in Fig. 12. The maximum base 13. The force–displacement graphics of the LNG tank base shear forces of the inner steel tank for the HDRB and LRB isolators of the HDRB-, LRB- and FPS-isolated LNG tanks isolator systems were between 50 % and 10 % lower than for the 1940 Imperial Valley earthquake El Centro Array Base those of the non-isolated LNG tanks. It has been observed Station data are shown in Fig. 14. While the LRB earthquake that the base shear forces formed in LNG tanks with an FPS isolator shows less displacement than the other isolators are 67 %–49 % lower than those in systems without isolators. under the effect of a large shear force coming from the LNG Because systems with FPS operate according to the friction tank in high-acceleration and long-oscillating earthquakes, Table 12. Maximum base shear forces (kN) and reduction rates (%) with respect to the non-isolated tanks for HDRB-, LRB- and FPS-isolated tanks Base type Earthquake ground motions Non-isolated IV2-1940 6.78 × 105 5.26 × 105 22 % 4.48 × 105 34 % 3.10 × 105 54 % KC-1952 5 5.92 × 10 3.98 × 10 33 % 3.94 × 10 5 34 % 2.08 × 10 5 65 % BM-1968 4.87 × 10 5 3.70 × 10 24 % 3.38 × 10 5 31 % 2.18 × 10 5 55 % IV06-I-1979 6.08 × 10 5 5.47 × 10 10 % 4.33 × 10 5 29 % 2.01 × 10 5 67 % IV06-II-1979 6.44 × 10 5 3.19 × 10 50 % 3.27 × 10 5 49 % 2.36 × 10 5 63 % VM-1980 6.29 × 10 5 4.72 × 10 25 % 3.56 × 10 5 43 % 2.53 × 10 5 60 % IITA-1980 3.89 × 10 3.21 × 10 17 % 3.09 × 10 5 21 % 1.97 × 10 5 49 % 5 GRAĐEVINAR 75 (2023) 10, 979-995 HDRB isolator 5 5 5 5 5 5 LRB isolator FPS isolator 991 Građevinar 10/2023 İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Figure 13. Nonlinear analysis results: a) Von Mises stress (MPa); b) sloshing wave height (mm); c) lateral displacement (mm); d) base shear force (kN) Figure 14. LNG tank base isolators force–displacement graphics: ) HDRB isolator; b) LRB isolator; c) FPS isolator Table 13. LNG tank base isolators maximum force–displacement values Earthquake ground motions 992 Isolator type HDRB LRB FPS Force [kN] Displacement [mm] Force [kN] Displacement [mm] Force [kN] Displacement [mm] IV2-1940 880 340 746 286 506 499 KC-1952 656 225 658 232 345 229 BM-1968 615 205 564 173 363 256 IV06-I-1979 904 360 710 274 336 210 IV06-II-1979 534 164 544 164 395 302 VM-1980 791 296 595 194 422 346 IITA-1980 536 165 518 145 328 195 GRAĐEVINAR 75 (2023) 10, 979-995 Nonlinear behaviour of liquefied natural gas tanks with different seismic isolation systems the FPS isolators perform better in moderate-intensity and low-oscillation earthquakes. This is because the horizontal displacement force–displacement angle ratio of the LNG tank in earthquakes when FPS-type isolators are used and the friction-damped force is exceeded is higher than that with LRB- and HDRB-type isolator systems. Compared to the HDRB isolator system, the lead in the LRB core limited the horizontal displacement movement of the LNG tank. The horizontal displacement of LNG tanks during an earthquake is critical because of the mechanical and carrier pipe systems; in this respect, the LRB isolator type is more advantageous than the FPS and HDRB. 5. Conclusions In this study, an LNG tank with a volume of 232,000 m3 placed on four different base types was analysed under seven different earthquake effects. The height-to-radius ratio was selected as 1 for maximum LNG fluid. The base shear force, sloshing height, inner steel tank lateral deflection, von Mises stresses in the inner steel tank and force–displacement of the isolators were investigated. -- When comparing the LNG tanks with the HDRB, LRB and FPS isolators with that fixed to the foundation with anchor bolts, no difference was observed between the convective and impulsive modes. It was concluded that the wave motion of the liquid was different from the oscillation of the structure and the earthquake isolation times did not affect the sloshing motion. -- An average stress of 270 MPa occurred in the inner steel tank owing to the static loading. The stresses varied depending on the intensity of the earthquake and whether the system was isolated or fixed. While a stress value of 400 MPa occurred in the fixed system, these values were 350 MPa on average for the LNG tanks with the HDRB, LRB and FPS-type earthquake isolator systems. The yield strength of the steel tank is between 515 and 585 MPa. According to the API 620 standard, 80 % of this value (412– 468 MPa) should not be exceeded for the SSE. Otherwise, there is a significant risk to LNG tanks. Therefore, base isolator systems should be used in structures under risk. -- The sloshing time varies between 9.82–9.94 s for tanks without isolator and with HDRB-, LRB- and FPS-type isolators. For the isolated and anchored systems, the wave height varies depending on the agitation time of the LNG liquid. The sloshing wave height of the liquid is proportional to the harmonic movements of the earthquake acceleration of the structure rather than the numerical magnitude of the earthquake accelerations. For example, the sloshing wave height for acceleration values varying between +0.3 g and −0.3 g of the BM-1968 earthquake within a period of 10 s is greater than that for the IV06-II-1979 earthquake. -- Except for earthquakes such as IV06-I-1979 with very high GRAĐEVINAR 75 (2023) 10, 979-995 Građevinar 10/2023 acceleration values, the sloshing wave height observed as a result of the regular harmonic motion acceleration values formed in anchored LNG tanks was less than that in structures with isolators. This result can be attributed to the damping function oscillating between high and low acceleration values. However, the acceleration values decrease owing to the damping provided by the lead core in the LRB centre and the frictional force generated in the FPS, resulting in a lower sloshing height in tanks using these types of isolators than that in tanks with the HDRB isolator. -- In moderate and weak earthquakes, such as the Irpina earthquake, the pressure of the impulsive mass in the inner steel tank is blocked by the anchors and the lateral displacement movement of the structure is not allowed. This is why there is buckling with less movement than that in structures with isolators. However, in earthquakes of +0.4 g and above, the lateral displacement of the inner steel tank wall in the anchored LNG tank is greater than that in tanks with LRB, HDRB and FPS isolators. Therefore, elastoplastic buckling of the inner steel tank, which we call the elephant foot strain, was observed in the anchored tanks. -- With an increase in average ground acceleration, the total bottom shear force acting on the LNG tank increased. Moreover, with an increase in the maximum shear force on the base, the percentage of shear force reduction in systems with isolators also increased. The bottom shear forces were found to be similar in the LNG tanks with HDRB and LRB isolators. However, better performance was obtained in LNG tanks with an FPS system compared with that of the other two isolator types. This can be attributed to the fact that each FPS isolator generated a frictional force that absorbed a base shear force of 200 kN. -- LNG transported by pipes from a ship docking at a port is discharged from the roof of the LNG tank to the inner steel tank. During this time, it is desirable that the horizontal displacement of the LNG tank be limited during an earthquake to avoid damage to the mechanical and piping systems. When the force–displacement graphs of the HDRB, LRB and FPS isolator-supported LNG tanks are examined, it can be observed that, compared with the other earthquake isolators, the LRB isolator has the least horizontal displacement. When the acceleration–time graphs of seven earthquakes with 0.4 g and above ground movements are examined, the FPS showed more lateral displacement than the HDRB and LRB LNG tanks because of the amount of horizontal force acting on each isolator. This can be attributed to the earthquake forces exceeding the friction-damped force and reaching a higher horizontal displacement motion. This situation is undesirable for LNG tanks. Meanwhile, the LRB system provides a 10 %–15 % advantage over the HDRB isolator owing to the horizontal damping of the middle lead core. 993 Građevinar 10/2023 -- It was observed that earthquake isolators used in LNG storage tanks perform well even during very strong earthquakes, which may occur once every 2475 years. The use of isolator systems is recommended for the earthquake safety of LNG storage tanks, as they may cause major environmental disasters and destruction. Systems with LRB isolators outperformed those with FPS and HDRB isolators and fixed support systems in all criteria, except for the base shear force. İbrahim Hüner, Bülent Akbaş, Abdullah Cem Koç Acknowledgments This study was funded by the Pamukkale University Scientific Research Project Coordination Unit (grant number 2019FEBE055). The authors would like to thank to Associate Prof. Temel Varol from the Metallurgical and Materials Engineering Department, Karadeniz Technical University, Ph.D. student Murat Çelik from the Civil Engineering Department, Istanbul Technical University and M.Sc. Mechanical Engineer Hakan Balaban for their valuable contributions. REFERENCES [1] Marti, J., Crespo, M., Martinez, F.: Seismic isolation of liquefied natural gas tanks: A comparative assessment, The Journal of the Anti-Seismic Systems International Society, 1 (2010) 1, pp. 125140, doi:10.2140/siaps.2010.1.125. [13] Haroun, M.A., Housner, G.W.: Dynamic interaction of liquid storage tanks and foundation soil, in Dynamic Response of Structures: Experimentation: Observation, Prediction and Control, American Society of Civil Engineers, ASCE, New York, USA, 1981. [2] Summers, P.B., Castellano, M.G., Bergamo, G., Gatti, F., Marti, J., Poggianti, A.: Seismic risk reduction at petrochemical and LNG acilities: Main results from in-depth project, 14th World Conference on Earthquake Engineering, Beijing, China, October, 2006. [14] Veletsos, A.S., Tang, Y.: Soil-structure interaction effects for laterally excited liquid-storage tanks., Earthquake Engineering and Structural. Dynamics, 19 (1990) 4, pp. 473-496, doi:10.1002/ eqe.4290190402. [3] API Standard 620, Design and construction of large, welded, lowpressure storage tanks, American Petroleum Institute Publishing Services, 12th Edition, Addendum 1, API, Washington D.C., USA, 2014. [4] API Standard 625, Tank systems for refrigerated liquefied gas storage, American Gas Association Publishing Services, 1st Edition, Addendum 2, AGA, Washington D.C., USA, 2014. [5] NFPA 59A, Standard for the production, storage and handling of liquefied natural gas LNG, National Fire Protection Association Publishing Services, NFPA , Washington D.C., USA, 2016. [17] Bomhard, H., Stempniewski, L.: LNG tanks for seismically highly affected sites, Intl. Post SMiRT Conference Seminar on Isolation, Energy Dissipation and Control of Vibrations of Structures, IAEA, Capri, Italy, 1993. [6] Westergaard, H.M.: Water pressures on dams during earthquakes, Transactions of the American Society of Civil Engineers, 98 (1933) 2, Washington D.C., USA. [18] Tajirian, F.F.: Base isolation design for civil components and civil structures, in Proceedings of the Structural Engineers World Conference, San Francisco, California, USA, July, 1998. [7] Jacobsen, L.S.: Impulsive hydrodynamics of fluid inside a cylindrical tank and of fluid surrounding a cylindrical pier, Bulletin of the Seismological Society of America, 39 (1949) 3, pp. 189-204, doi:10.1785/BSSA0390030189. 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https://openalex.org/W2160681063
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English
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Computing all hybridization networks for multiple binary phylogenetic input trees
BMC bioinformatics
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14,072
METHODOLOGY ARTICLE Open Access © 2015 Albrecht. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Correspondence: benjamin.albrecht@bio.ifi.lmu.de Institut für Informatik, Ludwig-Maximilians-Universität, Amalienstr. 17, 80333 München, Germany Abstract Background: The computation of phylogenetic trees on the same set of species that are based on different orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific hybridization events recombining genes of different species. An important approach to analyze such events is the computation of hybridization networks. Results: This work presents the first algorithm computing the hybridization number as well as a set of representative hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of hybridization networks including their computation and visualization. Hybroscale is freely available1 and runs on all three major operating systems. Conclusion: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0. Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding certain features to its graphical representation. Keywords: Hybridization networks, Maximum acyclic agreement forests, Phylogenetics Albrecht BMC Bioinformatics (2015) 16:236 DOI 10.1186/s12859-015-0660-7 METHODOLOGY ARTICLE Open Access Computing all hybridization networks for multiple binary phylogenetic input trees Benjamin Albrecht Abstract Background: The computation of phylogenetic trees on the same set of species that are based on different orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific hybridization events recombining genes of different species. An important approach to analyze such events is the computation of hybridization networks. Results: This work presents the first algorithm computing the hybridization number as well as a set of representative hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of hybridization networks including their computation and visualization. Hybroscale is freely available1 and runs on all three major operating systems. Conclusion: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0. Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding certain features to its graphical representation. Keywords: Hybridization networks, Maximum acyclic agreement forests, Phylogenetics Albrecht BMC Bioinformatics (2015) 16:236 DOI 10.1186/s12859-015-0660-7 METHODOLOGY ARTICLE Open Access Computing all hybridization networks for multiple binary phylogenetic input trees Benjamin Albrecht Abstract Background: The computation of phylogenetic trees on the same set of species that are based on different orthologous genes can lead to incongruent trees. One possible explanation for this behavior are interspecific hybridization events recombining genes of different species. An important approach to analyze such events is the computation of hybridization networks. Results: This work presents the first algorithm computing the hybridization number as well as a set of representative hybridization networks for multiple binary phylogenetic input trees on the same set of taxa. To improve its practical runtime, we show how this algorithm can be parallelized. Moreover, we demonstrate the efficiency of the software Hybroscale, containing an implementation of our algorithm, by comparing it to PIRNv2.0, which is so far the best available software computing the exact hybridization number for multiple binary phylogenetic trees on the same set of taxa. The algorithm is part of the software Hybroscale, which was developed specifically for the investigation of hybridization networks including their computation and visualization. Hybroscale is freely available1 and runs on all three major operating systems. Conclusion: Our simulation study indicates that our approach is on average 100 times faster than PIRNv2.0. Moreover, we show how Hybroscale improves the interpretation of the reported hybridization networks by adding certain features to its graphical representation. Keywords: Hybridization networks, Maximum acyclic agreement forests, Phylogenetics Albrecht BMC Bioinformatics (2015) 16:236 DOI 10.1186/s12859-015-0660-7 Computing all hybridization networks for multiple binary phylogenetic input trees Benjamin Albrecht Background its parents, which ensures a reduction of the gene flow between its parental genotypes. Due to these circum- stances, homoploid hybrid speciation is considered as a rare phenomenon. Note that, after such a new hybrid species has successfully established itself, there can still occur gene flow back from the hybrid species to their parent species, which is denoted as introgression [1]. Recombinational or homoploid hybrid speciation [1] is a mechanism influencing the evolution of species by merg- ing a sizable percentage of the genomes of two different species. It has been discovered especially in plants [2, 3], but also in certain animals [4]. If two individuals each belonging to different species hybridize, a new species, containing genes from both parental individuals, can arise under the following certain circumstances. First of all, the resulting hybrid has to produce viable gametes, which is often a problem due to the two genetically different parental sets of chromosomes preventing a correct mei- otic pairing. Second, if these two sets are similar enough and, thus, the hybrid is able to produce any progeny, its early recombinants have to find and successfully colonize its own unexploited niche that is different from either of Now, regarding a well-established homoploid hybrid species resulting from such a evolutionary process as described above, we can reconstruct its evolutionary his- tory by taking two different scenarios each corresponding to one of its parental species into account. This is typ- ically done by, first, computing two rooted phylogenetic trees each based on those genes corresponding to one of both parental gene sets and, second, by reconciling these two topologically different trees into one rooted phylogenetic network, whose reticulate nodes (nodes of in-degree ≥2) represent certain putative hybridization events. Because of those major hurdles a hybrid species has to face, hybridization events rarely happen and, thus, Albrecht BMC Bioinformatics (2015) 16:236 Page 2 of 15 from a biological point of view, only those networks con- taining a minimum number of reticulate nodes are of high interest. regarding the interpretation of hybridization networks, we decided to discuss those rather complex theoretical issues in a forthcoming paper [13]. Due to hybridization, the genome of hybrid species, however, can obviously contain more than just two genes having different evolutionary histories. Background Thus, given a set T of rooted binary gene trees sharing the same set of taxa, the general problem is to compute a rooted phylo- genetic network displaying T by a minimum hybridiza- tion number as defined later by Eq. 2. Unfortunately, this is a well-known NP-hard problem, which is how- ever fixed-parameter tractable, even for the simplest case when only just two binary input trees are given [5]. In the general case, however, if the input consists of more than two rooted binary trees, the problem still remains fixed-parameter tractable as recently shown by van Iersel and Linz [6]. More precisely, this means that the problem is exponential in some parameter related to the problem itself, namely the hybridization num- ber, and only polynomial in the size of the input trees. Note that this is an important feature, which facili- tates the development of exact algorithms as it is used by our algorithm for some subproblems to maximize efficiency. Given a hybridization network displaying several input trees, it is often visually challenging for a user to figure out the embedding of those trees. Thus, we have devel- oped the software Hybroscale providing a function for highlighting each input tree by coloring its corresponding edges within a resulting network, which makes it easier for a biologist to analyze hybridization events. Moreover, Hybroscale sorts the set of computed networks by support values indicating how often a certain hybridization event occurs in the set of representative networks. To demonstrate the efficiency of our implementation, we computed the hybridization number for a specific syn- thetic dataset and compared the respective runtime with the best currently available software PIRNv2.0 [7, 8]. Note that there are two main differences between our approach and the one corresponding to PIRN. On the one hand, our software provides the better practical runtime for com- puting hybridization numbers because of parallelization, certain reduction steps, and other algorithmic issues as discussed in the upcoming part of this paper. On the other hand, our approach additionally enables the computation of all representative networks allowing the assignment of meaningful support values to each internal node repre- senting a putative hybridization event which helps biolo- gists to figure out hybridization events that might played an important role. Background Note that the networks reported by PIRN2.0 are either also calculated by our approach or are not considered as being relevant because there exist other networks representing these networks as described in the upcoming part of this paper. y In this work, we tackle this NP-hard problem by pre- senting the first algorithm that is able to compute the exact hybridization number as well as a certain set con- taining all representative networks for, not just only two, but an arbitrary number of rooted binary phylogenetic X -trees all sharing the same set of taxa. Note that, until now, the software PIRNv2.0 [7, 8] is the most efficient software that guarantees the computation of the exact hybridization number for multiple input trees. In most cases, however, PIRN runs only reasonable efficient if the number of hybridization events is relatively small and, moreover, PIRN does usually output only a small sub- set of those networks that are computed by our method which plays an important role for the interpretation of the networks as shown later. The algorithm, presented in this work, is based on previous work of Albrecht et al. [9] describing an algorithm for just two input trees, which itself is based on several works including Baroni et al. [10], Bordewich and Semple [11], and Whidden et al. [12]. Moreover, this previous approach could only com- pute a subset of all representative networks and, thus, the motivation for this work was to extend this former algo- rithm such that now all of those networks for an arbitrary number of input trees can be computed. Methods In this section, we first introduce the notation and termi- nology that is used throughout the paper and then present the algorithm ALLHNETWORKS. Albrecht BMC Bioinformatics (2015) 16:236 Additionally, by T|X ′ we define the subgraph that is obtained from T(X ′) by suppressing all nodes of both in- and out-degree 1. Moreover, given a tree T, through- out this paper we use T to denote the tree that is obtained from T by suppressing each node of both in- and out- degree 1. Now, given a rooted phylogenetic X -tree T and a taxa set X ′ ⊆ X , we define T(X ′) as the minimal con- nected subgraph of T whose leaf set contains each taxon in X ′. Additionally, by T|X ′ we define the subgraph that is obtained from T(X ′) by suppressing all nodes of both in- and out-degree 1. Moreover, given a tree T, through- out this paper we use T to denote the tree that is obtained from T by suppressing each node of both in- and out- degree 1. Hybridization networks. A hybridization network N is a rooted phylogenetic network, which is a rooted acyclic digraph not containing nodes of both in- and out-degree 1 and whose leaves are all labeled one-to-one by a taxon of the taxa set X (cf. Fig.1(a)). Each node v of in-degree greater than 1 is called a hybridization node and each edge directed into v is called a reticulation edge or, in the context of hybridization, a hybridization edge. We say a hybridization network N on X displays a rooted phyloge- netic X ′-tree T′, with X ′ ⊆X , if we can delete a set of hybridization edges E′ followed by suppressing each node of both in- and out-degree 1 such that the resulting rooted phylogenetic X -tree T contains T′ as restricted subtree on X ′. In such a case, we say that E′ refers to T′ (cf. Fig.1(b)). Since a network can display a tree in potentially several ways, E′ is not necessarily unique. To quantify the num- ber of reticulation events of a network N, the reticulation number r(N) is defined by Agreement forests. Let T1 and T2 be two rooted binary phylogenetic X -trees. For technical purpose, we regard the root of both trees T1 and T2 as being a node that has been attached to the original roots and to a taxon ρ ̸∈X . Now, an agreement forest for T1 and T2 is a set of compo- nents F = {Fρ, F1, . . . Albrecht BMC Bioinformatics (2015) 16:236 , Fk} on X ∪{ρ} with the following properties. (1) Each component Fi with taxa set Xi refers to the restricted subtree T1|Xi and T2|Xi, respectively. i i (2) There is exactly one component, denoted as Fρ, containing ρ. (3) Let Xρ, X1, . . . , Xk be the taxa sets corresponding to Fρ, F1, . . . , Fk. Then, all trees in {T1(Xi)|i ∈{ρ, 1, . . . , k}} and {T2(Xi)|i ∈{ρ, 1, . . . , k −1}} are node disjoint subtrees of T1 and T2, respectively. r(N) =  v∈V:δ−(v)>0 (δ−(v) −1), (1) (1) (1) A maximum agreement forest is an agreement forest of minimum size, which implies there does not exist a smaller set of components fulfilling each property listed above. Moreover, we call an agreement forest F for two rooted binary phylogenetic X -trees T1 and T2 acyclic, if its underlying ancestor-descendant graph AG(T1, T2, F) does not contain any directed cycles (cf. Fig. 2). More specifically, this graph AG(T1, T2, F) contains one node corresponding to exactly one component of F. Moreover, where δ−(v) denotes the in-degree of node v. Moreover, for a set T of rooted phylogenetic X -trees, we define the hybridization number h(T ) as where δ−(v) denotes the in-degree of node v. Moreover, for a set T of rooted phylogenetic X -trees, we define the hybridization number h(T ) as h(T ) = min{r(N) : N displays each T ∈T }. (2) h(T ) = min{r(N) : N displays each T ∈T }. (2) h(T ) = min{r(N) : N displays each T ∈T }. (2) (2) Fig. 1 a A hybridization network N with taxa set X = {a, b, c, d, e} whose reticulation edges are consecutively numbered. b A phylogenetic X-tree T that is displayed by N. Based on N, both edge sets {3, 6, 1} and {3, 6, 2} refer to T. c The restricted network N|E′,X ′ with E′ = {3, 6, 1} and X ′ = {b, c, d, e} still containing nodes of both in- and out-degree 1 Fig. 1 a A hybridization network N with taxa set X = {a, b, c, d, e} whose reticulation edges are consecutively numbered. b A phylogenetic X-tree T that is displayed by N. Based on N, both edge sets {3, 6, 1} and {3, 6, 2} refer to T. Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 3 of 15 Page 3 of 15 Page 3 of 15 Now, given a hybridization network N on X and an edge set E′ referring to an embedded rooted phylogenetic X ′- tree T′ in N, the reduced network N|E′,X ′, with X ′ ⊆X , is computed as follows. First, E′ is deleted and, second, each node of out-degree 0 that is unlabeled or not labeled by a taxon in X ′ is removed repeatedly. The resulting directed graph corresponds to T′|X ′ but still contains nodes of both in- and out-degree 1, and, thus, each node in N|E′,X ′ can be mapped back to exactly one specific node of the unre- stricted network N (cf. Fig.1(c)). Moreover, the network N(v) denotes a network rooted at v that is computed by, first, removing each node that cannot be reached from v and, second, by suppressing each node of both in- and out-degree 1. is contained in the subtree rooted at v. Given a set F of trees, the label set L(F) is simply the union of each label set L(Fi) of a tree Fi ∈F. is contained in the subtree rooted at v. Given a set F of trees, the label set L(F) is simply the union of each label set L(Fi) of a tree Fi ∈F. Now, given a rooted phylogenetic X -tree T and a taxa set X ′ ⊆ X , we define T(X ′) as the minimal con- nected subgraph of T whose leaf set contains each taxon in X ′. Additionally, by T|X ′ we define the subgraph that is obtained from T(X ′) by suppressing all nodes of both in- and out-degree 1. Moreover, given a tree T, through- out this paper we use T to denote the tree that is obtained from T by suppressing each node of both in- and out- degree 1. is contained in the subtree rooted at v. Given a set F of trees, the label set L(F) is simply the union of each label set L(Fi) of a tree Fi ∈F. Now, given a rooted phylogenetic X -tree T and a taxa set X ′ ⊆ X , we define T(X ′) as the minimal con- nected subgraph of T whose leaf set contains each taxon in X ′. Preliminaries h The upcoming definitions used for describing and dis- cussing our algorithm follow the work of Huson et al. [14]. We assume that the user is familiar with general graph-theoretic concepts. Phylogenetic trees. A rooted phylogenetic X -tree T is a directed tree, whose edges are directed from the root to the leaves and whose nodes, except the root, have a degree not equal to 2. If T is a binary tree its root has in-degree 0 and out-degree 2, each inner node an in-degree of 1 and an out-degree of 2, and each leaf an in-degree of 1 and an out-degree of 0. Moreover, each leaf is labeled one-to- one by a taxon of the taxa set X , which usually consists of certain species or genes and is also denoted by L(T). For a node v of T, the label set L(v) contains each taxon that As we state that our algorithm guarantees the compu- tation of the exact hybridization number, we are aware of the fact that this algorithm raises some questions regard- ing its correctness. However, since in this paper we want to focus on the efficiency of the presented algorithm as well as on the advantages of our software Hybroscale Albrecht BMC Bioinformatics (2015) 16:236 c The restricted network N|E′,X ′ with E′ = {3, 6, 1} and X ′ = {b, c, d, e} still containing nodes of both in- and out-degree 1 Albrecht BMC Bioinformatics (2015) 16:236 Page 4 of 15 Fig. 2 An agreement forest F of two phylogenetic X-trees T1 and T2 together with the corresponding ancestor-descendant graph AG(T1, T2, F). Note that, as AG(T1, T2, F) does not contain any directed cycles, F is acyclic Fig. 2 An agreement forest F of two phylogenetic X-trees T1 and T2 together with the corresponding ancestor-descendant graph AG(T1, T2, F). Note that, as AG(T1, T2, F) does not contain any directed cycles, F is acyclic two nodes Fi and Fj with i ̸= j are connected via a directed edge (Fi, Fj) if either two nodes Fi and Fj with i ̸= j are connected via a directed edge (Fi, Fj) if either as already described in Baroni et al. [10], as follows. First, select the node vρ of in-degree 0, which corresponds to Fρ, and remove vρ by deleting this node together with all its incident edges. Next, again choose a node v1 with in- degree 0 and remove this node. By continuing this way until finally all nodes have been removed, one receives the ordering  = (vρ, v1, . . . , vk). In the following, we call the ordering of components corresponding to , denoted by (Fρ, F1, . . . , Fk), an acyclic ordering of F. As during each of those steps there can occur multiple nodes of in-degree 0, especially if F contains components only consisting of isolated nodes, such an acyclic ordering is in general not unique. (i) the root of T1(Xi) is an ancestor of the root of T1(Xj) or (ii) the root of T2(Xi) is an ancestor of the root of T2(Xj), (ii) the root of T2(Xi) is an ancestor of the root of T2(Xj), where Xi, Xj ⊆X denotes the taxa set of Fi and Fj, respectively. Again, we call an acyclic agreement forest of minimum size a maximum acyclic agreement forest. Note that for a maximum acyclic agreement forest for two rooted binary phylogenetic X -trees T1 and T2 containing k components there exists a hybridization network whose reticulation number is k −1 [15]. This means, in particu- lar, if a maximum acyclic agreement forest for T1 and T2 contains only one component, both trees are equal. where Xi, Xj ⊆X denotes the taxa set of Fi and Fj, respectively. Again, we call an acyclic agreement forest of minimum size a maximum acyclic agreement forest. Note that for a maximum acyclic agreement forest for two rooted binary phylogenetic X -trees T1 and T2 containing k components there exists a hybridization network whose reticulation number is k −1 [15]. This means, in particu- lar, if a maximum acyclic agreement forest for T1 and T2 contains only one component, both trees are equal. Representative networks. As mentioned above, our algorithm ensures the computation of all representative networks, which are those hybridization networks with minimum hybridization number (cf. Eq. two nodes Fi and Fj with i ̸= j are connected via a directed edge (Fi, Fj) if either (2)) fulfilling an additional property that is based on the following If F is acyclic and, thus, AG(T1, T2, F) does not contain any directed cycles, one can compute an acyclic ordering, If F is acyclic and, thus, AG(T1, T2, F) does not contain any directed cycles, one can compute an acyclic ordering, Albrecht BMC Bioinformatics (2015) 16:236 Page 5 of 15 the hybridization number or all representative networks showing the embedding of those input trees. Similar to the approach described in the work of Albrecht et al. [9], ALLHNETWORKS can be separated into three phases. The reduction phase (consisting of a subtree reduction follow- ing the work of Bordewich and Semple [16] and a cluster reduction following the work of Baroni et al. [10] and Linz [17]), the exhaustive search phase, and the output phase (combining the result of all clusters and undoing each sub- tree reduction). Whereas the reduction and the output phase can be conducted in polynomial time, the second phase solves an NP-hard problem and, thus, its runtime is exponential [5]. However, as recently shown by van Iersel and Linz [6], certain parts of the problem still remain fixed-parameter tractable, which, as already noted in the introduction, is an important feature that is exploited by our algorithm to maximize its efficiency. observation. Given a hybridization network containing a node v with in-degree of at least 3, one can generate fur- ther networks by simply dragging some of its hybridiza- tion edges upwards resulting in a stack of hybridization nodes. More precisely, such a stack is a path (v1, . . . , vn) in which each hybridization node vi is connected through a hybridization edge to vi+1 (cf. Fig. 3). From a biologi- cal point of view, such a stack implies that a hybridiza- tion event belonging to a hybridization node vi happened before those corresponding to the in-edges of a hybridiza- tion node vj with i < j. However, as for each of those networks there exists a network where each stack is fully compressed, we only consider those compressed networks as being relevant. Consequently, the set of representative networks con- sists only of those networks with minimum hybridization number not containing any stacks of hybridization nodes leaving the interpretation of the ordering of the hybridiza- tion events open. two nodes Fi and Fj with i ̸= j are connected via a directed edge (Fi, Fj) if either Moreover, just for simplicity, we claim that each of those networks has to be binary not contain- ing any nodes of out-degree greater than 2. By introducing multifurcating nodes, which are nodes having an out- degree of at least 3, the set of representative networks typically shrinks because due to those nodes a network can display several binary networks. At this point, we have to give a remark regarding the cor- rectness of the cluster reduction. The well-known work of Baroni et al. [10] contains a proof showing that the exact hybridization number of two binary phylogenetic X -trees can also be computed by adding up the exact hybridiza- tion numbers of its minimum common clusters. A more general proof, showing that this concept also holds for multiple binary phylogenetic X -trees, can be found in our forthcoming paper [13]. Lastly, given two representative networks N1 and N2, we say that N1 differs from N2 if either their graph topologies (disregarding edge labels) are not isomorphic or their edge sets indicating the embedding of each input tree differ. In the upcoming part, we will briefly discuss the exhaus- tive search phase and its parallelization. A description of the other two phases is omitted but can be looked up in the work of Albrecht et al. [9]. The exhaustive search phase runs for an increasing parameter k bounding the reticulation number of each computed network. If a hybridization network with reticulation number less than or equal to k does not exist, the search is continued with k + 1 until a hybridization network displaying all input trees can be computed. The algorithm ALLHNETWORKS For each edge set Ei referring to the embedded tree Ti|L(F′) in N, the set VA s of source nodes of Type A contains all nodes v with N|Ei,L(F′)(v) iso- morphic to Ti|L(F′)(vsib), where vsib denotes the sibling of the node v′ with L(v′) = L(Fi) in Ti|L(F′)∪L(Fi). Note that, due to the restriction of the network to L(F′), this set usually consists of more than one node. Source Nodes of Type B. The set VB s of source nodes of Type B is computed such that it contains each node v of a subtree, whose root is a sibling of a node in VA s and which does not contain any taxa of L(F′). Moreover, its leaf set L(v) has to consist only of several subsets representing the total taxa set L(F) of a component F in F, which means that v must not be part of a subtree corresponding to a component that is added afterward. The input tree Ti is added to a so far computed net- work N by adding hybridization edges connecting certain parts of N. Given an edge set E′ referring to a phyloge- netic X -tree T′ that is displayed by N, such parts can be derived from the components of a maximum acyclic agreement forest for T′ and Ti. Again, in order to guar- antee the computation of all representative networks, the insertion of Ti has to be performed for all maximum acyclic agreement forests referring to Ti and each phylo- genetic X -tree T′ that is embedded in N, and, additionally, for all edge sets E′ referring to T′. Note that, given two rooted binary phylogenetic X -trees, the computation of all maximum acyclic agreement forests follows the algo- rithm ALLMAAFS [18]. For a better understanding the definitions of source and target nodes are illustrated in Fig. 4. Now, given a valid pair (s, t) of source and target nodes, a new hybridization edge is inserted as follows. The in- edge of the source node s is split in a way that there is a new node s′ that is connected to s and to the parent of s. If the parent of t is of in-degree one the in-edge of t is split in the same way. Otherwise, its parent node acts as t′ which allows the computation of networks contain- ing nodes of in-degree greater than 2. The algorithm ALLHNETWORKS In this section, we give a high level description of our algo- rithm ALLHNETWORKS. More information, involving a more detailed description of the upcoming steps as well as some theoretical issues, will be discussed in a forthcoming paper [13]. The input of the algorithm is a set T of rooted binary phylogenetic X -trees and its output is either just Exhaustive search phase. Given a set T consisting of n rooted binary phylogenetic X -trees and a parameter Fig. 3 An illustration of a stack of hybridization nodes. The hybridization node with in-degree 4 of the left tree T1 can be resolved, amongst others, into two different stacks of hybridization nodes (v1, v2, v3) as demonstrated by T2 and T3, respectively. Note that by resolving a hybridization node into a stack of hybridization nodes the set of trees that are displayed in the original network remains unchanged Fig. 3 An illustration of a stack of hybridization nodes. The hybridization node with in-degree 4 of the left tree T1 can be resolved, amongst others, into two different stacks of hybridization nodes (v1, v2, v3) as demonstrated by T2 and T3, respectively. Note that by resolving a hybridization node into a stack of hybridization nodes the set of trees that are displayed in the original network remains unchanged Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 6 of 15 Page 6 of 15 k ∈N, in a first step we choose an ordering of T , which is for convenience (T1, T2, . . . , Tn) in the following. Second, each tree of this ordering is added sequentially to a set N of networks in all possible ways. At the beginning, N only consists of the first tree of the ordering, which is T1 in this case. By adding an upcoming input tree Ti (i > 1), the size of N grows rapidly, because in general there exist multi- ple ways of how this can be achieved (cf. Fig. 6). Since we do not delete any edges from a so far computed network N, we can disregard those networks whose reticulation number exceeds k. Note that, in order to guarantee the computation of all representative networks, this step must be performed for each possible ordering of T . Source Nodes of Type A. The algorithm ALLHNETWORKS Note that this is an optional step that is necessary to ensure that each com- puted hybridization network does not contain any stacks of hybridization nodes such that it applies to the definition of a representative network. Finally, the two nodes s′ and t′ are connected through a path P consisting of two edges. This is done because, on the one hand, we only allow nodes of in-degree one as source nodes, but, on the other hand, in order to compute all representative networks, we have to enable that a target node can additionally be attached to hybridization edges. Due to this fact, however, before reporting a network embedding all input trees, one still has to suppress all nodes of both in- and out-degree 1. By referring to the terminology used above, in Fig. 5 we A maximum acyclic agreement forest F of Ti and T′ is added to N by, first, computing an acyclic ordering (Fρ, F1, . . . , Fk) of F which can be done with the help of the directed graph AG(T′, Ti, F) as previously described. Next, each component Fi, beginning with F1, is added to N by inserting a new hybridization edge connecting a cer- tain source and target node such that, after all components of F have been inserted, N displays the considered input tree Ti. In order to guarantee the computation of the exact hybridization number, all acyclic orderings and all valid combinations of source and target nodes, as described below, have to be taken into account. More precisely, in order to avoid directed cycles, we consider a pair (s, t) of source and target nodes as being valid if the source node s cannot be reached from t. Note that the way of how we add a tree to a network is similar to the algorithm HYBRIDPHYLOGENY [10]. Fig. 4 An illustration of the definitions of target (left) and source nodes (right) for a component Fi (p, q > i) in which red nodes correspond to target nodes, blue nodes to source nodes of Type A, and green nodes to source nodes of Type B. The algorithm ALLHNETWORKS Moreover, dashed edges and dotted edges are those edges that are disregarded when considering the restricted network in terms of the chosen embedded tree and the taxa set of the so far added components, respectively The set of target and source nodes corresponding to a component Fi in F is defined as follows. Let F′ = {Fρ, F1, . . . , Fi−1} ⊂F = {Fρ, F1, . . . , Fk} be the set of components that has been added so far. Note that, since N is initialized with Fρ, at the beginning L(F′) equals L(Fρ) and the first component that is added is F1. Fig. 4 An illustration of the definitions of target (left) and source nodes (right) for a component Fi (p, q > i) in which red nodes correspond to target nodes, blue nodes to source nodes of Type A, and green nodes to source nodes of Type B. Moreover, dashed edges and dotted edges are those edges that are disregarded when considering the restricted network in terms of the chosen embedded tree and the taxa set of the so far added components, respectively Target Nodes. The set Vt of target nodes contains all nodes v with N|E′,L(F′)∪L(Fi)(v) isomorphic to Ti|L(Fi). Due to the restriction of the network to L(F′), this set usually contains more than one node. Target Nodes. The set Vt of target nodes contains all nodes v with N|E′,L(F′)∪L(Fi)(v) isomorphic to Ti|L(Fi). Due to the restriction of the network to L(F′), this set usually contains more than one node. Albrecht BMC Bioinformatics (2015) 16:236 Page 7 of 15 give a short example of how a certain input tree is added to a network. representative networks, one still has to compute for each network all possible combinations of edge sets each refer- ring to the embedding of one input tree. For clarity, the algorithm ALLHNETWORKS has been described so far with respect to one single embedding of each input tree. Given n input trees, to generate all Lastly, we give some high level ideas why the algo- rithm ALLHNETWORKS is correct, i.e., calculates all p p g Fig. 5 An illustration of how an input tree Ti is inserted into a network Ni−1 with the help of an embedded tree T′. a The network Ni−1 together with an embedded tree T′. The algorithm ALLHNETWORKS b The input tree Ti, which will be embedded into Ni−1 by inserting the maximum acyclic agreement F forest of Ti and T′ consisting of three components Fρ, F1, and F2. c, d All important elements that have to be considered during the insertion of both components F1 and F2, respectively. Blue dots correspond to source nodes and red nodes to target nodes. Note that, regarding N(1) i , there is only one valid pair of source and target nodes. Dashed edges are those edges that are disregarded when considering the restricted network in terms of T′ and the taxa set of the so far added components of F. e The resulting network Ni, which is obtained from N(3) i by suppressing each node of both in- and out-degree 1 Fig. 5 An illustration of how an input tree Ti is inserted into a network Ni−1 with the help of an embedded tree T′. a The network Ni−1 together with an embedded tree T′. b The input tree Ti, which will be embedded into Ni−1 by inserting the maximum acyclic agreement F forest of Ti and T′ consisting of three components Fρ, F1, and F2. c, d All important elements that have to be considered during the insertion of both components F1 and F2, respectively. Blue dots correspond to source nodes and red nodes to target nodes. Note that, regarding N(1) i , there is only one valid pair of source and target nodes. Dashed edges are those edges that are disregarded when considering the restricted network in terms of T′ and the taxa set of the so far added components of F. e The resulting network Ni, which is obtained from N(3) i by suppressing each node of both in- and out-degree 1 Albrecht BMC Bioinformatics (2015) 16:236 Page 8 of 15 hybridization number k in parallel. As mentioned above, the insertion of an input tree Ti to a so far computed network results in several new networks, which are then processed by inserting the next input tree Ti+1 of the chosen ordering (cf. Fig. 6). Since the processing of net- works runs independently from each other, these steps can be parallelized in a simple manner. Based on the num- ber of hybridization edges of a so far computed network, each of those steps is more or less likely to result in a representative network. The algorithm ALLHNETWORKS Thus, we set up a priority queue to process the most promising networks first, which, on the one hand, depends on the number of so far inserted input trees and, on the other hand, on its reticulation number. representative networks for a set of rooted binary phy- logenetic X -trees. We refer readers who are interested in a detailed proof to our forthcoming paper [13]. Let N′ be a representative network displaying a subset T ′ of all input trees T and let N be a hybridization net- work (not containing any stacks of hybridization nodes) that is based on N′ and displays a further input tree Ti ̸∈T . Then, one can obtain N from N′ by insert- ing a set E′ of reticulation edges whose source and target nodes can be derived from an acyclic agreement for- est F for Ti and a certain tree displayed by N′. This is due to the fact that N′ must contain such an agree- ment forest F so that each of its components, except Fρ, is rooted at a target node of an edge in E′ whose inci- dent nodes are contained in Vt and VA s ∪VB s , respectively. Moreover, one can show that by constructing networks for all possible orderings of the input trees, it suffices to take only maximum acyclic agreement forests into account. Such a priority queue, however, does only speed up the computation of the hybridization number, since in this case all computational paths can be aborted immediately as far as the first minimum hybridization network could be computed successfully. For the computation of all rep- resentative networks, however, each computational path has to be processed anyway until either it can be early aborted (which is the case if the reticulation number of the corresponding network exceeds k) or it leads to a representative network. Parallelization In order to improve the practical runtime of the algorithm ALLHNETWORKS, our implementation is able to run the exhaustive search looking for hybridization networks with Fig. 6 An illustration of how the insertion of the input trees is conducted by the algorithm ALLHNETWORKS in respect of the parameter k bounding the maximal reticulation number of resulting networks. Beginning with the first input tree T1, repeatedly, first, an embedded tree T′ of a so far computed network N is extracted, and, second, the current input tree Ti is inserted into N by sequentially adding the components of a maximum acyclic agreement forest for T′ and Ti. As soon as the reticulation number of a so far computed network exceeds k one can be sure that this network cannot lead to a network with reticulation number smaller or equal to k and, thus, the corresponding computational path can be aborted Fig. 6 An illustration of how the insertion of the input trees is conducted by the algorithm ALLHNETWORKS in respect of the parameter k bounding the maximal reticulation number of resulting networks. Beginning with the first input tree T1, repeatedly, first, an embedded tree T′ of a so far computed network N is extracted, and, second, the current input tree Ti is inserted into N by sequentially adding the components of a maximum acyclic agreement forest for T′ and Ti. As soon as the reticulation number of a so far computed network exceeds k one can be sure that this network cannot lead to a network with reticulation number smaller or equal to k and, thus, the corresponding computational path can be aborted Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 9 of 15 Moreover, as the algorithm computes networks for all different orderings of input trees and all different acyclic orderings of maximum acyclic agreement forests, a rep- resentative network can be computed multiple times. As a consequence, to ensure that the output only consists of unique networks, one has to filter the set of networks obtained from the exhaustive search step. For this pur- pose, we first group this set after the sum of support values computed for each network (as defined later) and then check each of those subgroups for isomorphic networks in parallel. Due to the typically large number of computed networks (cf. Additional features Given just the extended newick format [19] of a hybridiza- tion network, its topology is in general hard to interpret. Although there exist software packages, which are able to display rooted phylogenetic networks, e.g., the software Dendroscope [14], most of them are not able to visualize the embedding of all input trees, which is a preferable fea- ture for studying hybridization events. In order to close this gap, we have developed the software Hybroscale, which is specifically designed for studying hybridization networks. Besides the computation of a graphical lay- out of rooted trees and rooted networks, which is opti- mized by minimizing the number of crossings between all hybridization edges, Hybroscale can additionally highlight each hybridization edge that is necessary for displaying all embedded input trees by assigning a specific color to each tree (cf. Fig 7). Thus, Hybroscale is a software that, on the one hand, enables an easy handling of our algorithm and, on the other hand, ensures the readability of the computed networks. Our synthetic dataset is freely available2 and consists of several tree sets each containing multiple rooted phy- logenetic X -trees. Each X -tree is generated by ranging over all different combinations of four parameters, namely the number of input trees n, the number of leaves ℓ, an upper bound for the hybridization number k, and the clus- ter degree c as defined below. Each of the n input trees is obtained from a bicombining network N, which means that N only contains hybridization nodes of in-degree 2. This network N is computed in respect to these four dif- ferent parameters as follows. In a first step, a random binary tree T with ℓleaves is computed which is done in the following way. First, at the beginning, two nodes u and v of a specific set V, which is initialized by ℓnodes of both in- and out-degree 0, are randomly selected. Those two selected nodes u and v are then connected to a new node w and, finally, V is updated by replacing u and v by its par- ent node w. This process is repeated until V consists only of one node corresponding to the root of T. In a second step, k hybridization edges are created in T with respect to parameter c such that the resulting network N contains exactly k hybridization nodes of in-degree 2. Simulation study T h h To show the efficiency of our implementation, we have integrated our algorithm into the Java software Hybroscale and conducted a simulation study compar- ing its runtime to PIRNv2.0 [7, 8], which is so far the best available software for computing exact hybridization numbers for multiple rooted binary phylogenetic X -trees. 2 Results and discussion In this section, we first report a simulation study indicat- ing that our approach is much faster than other existing methods and then illustrate how Hybroscale can be used for studying hybridization networks by applying the soft- ware to a well known grass (Poaceae) dataset. Parallelization Tables 1, 2), the restriction of the filtering step to small subgroups usually provokes a large speedup. Note that, as already mentioned above, we consider two networks as being different if either their graph topologies (disregarding edge labels) are not isomorphic or their sets of edges that are necessary for displaying each input tree differ. is {austrodant, karoochloa}. Second, we determine the fraction of networks containing (v). This step ensures that the user can instantly look at those networks con- taining the most promising hybridization events, which is an important feature, because usually a large number of networks is reported (cf. Tables 1, 2). Additional features Furthermore, Hybroscale assigns each hybridization node a support value indicating the fraction of networks containing this node and, additionally, sorts the reported networks by the sum of those values in decreasing order. More specifically, the computation of support values is done as follows. Given a network N, each edge set Ei refer- ring to one of the input trees Ti, and a certain hybridiza- tion node v, we, first, compute the following ordering of taxa sets (v) = (L(N(v)|E1,X ), . . . , L(N(v)|En,X )). More precisely, each element L(N(v)|Ei,X ) consists of those taxa adhering to each leaf that can be reached from v by directed paths only crossing those hybridization edges in Ei indicating the embedding of Ti. For example, regarding Fig. 7, the set referring to the hybridization edges indicat- ing the embedding of Tree 2 and the node labeled by 22 % In this context, the cluster degree is an ad hoc con- cept influencing the computational complexity of a tree set similar to the concept of the tangling degree intro- duced in the work of Scornavacca et al. [18]. When adding a hybridization edge e with target node v2 and source node v1, we say that e respects cluster degree c, if v1 can- not be reached from v2 and there is a path of length less than or equal to c leading from v2 to a certain node p such that v1 can be reached from p. Consequently, net- works providing a small cluster degree in general contain more minimum common clusters than networks of large Albrecht BMC Bioinformatics (2015) 16:236 Page 10 of 15 Fig. 7 Our software Hybroscale showing a hybridization network displaying the embedding of four input trees by the colors blue, red, green, and orange Fig. 7 Our software Hybroscale showing a hybridization network displaying the embedding of four input trees by the colors blue, red, green, and orange Fig. 7 Our software Hybroscale showing a hybridization network displaying the embedding of four input trees by the colors blue, red, green, and orange cluster degrees and, thus, typically can be processed quite fast when applying a cluster reduction beforehand. For a better understanding, in Fig. 8 an example of this concept is depicted. Additional features 4 6 8 10 12 14 16 0 200 400 600 800 1000 1200 Upper Bound of the Hybridization Number Mean REAL Runtime (s) 89 % 26 % 17 % 100 % 100 % 97 % PIRN Hybroscale n=3 Fig. 9 The figure shows the mean average runtime corresponding to Hybroscale and PIRNv2.0 grouped by parameter k denoting the hybridization number of the network that was used to obtain the tree set T from. Thus, this parameter k acts as an upper bound of the hybridization number of T . Each percentage indicates the proportion of tree sets that could be computed within the time limit of 20 minutes To compare the efficiency, both programs have been run on a grid computer providing 16 cores and 40 GB RAM for our synthetic dataset containing tree sets with param- eters n ∈{3, 4, 5}, ℓ∈{10, 25, 50}, k ∈{5, 10, 15}, and c ∈ {1, 3, 5}. More precisely, we have generated for all 81 com- binations of the four parameters 30 tree sets as described above resulting in 2430 tree sets in total. The results for three input trees (n = 3) are presented in Figs. 9, 10 4 6 8 10 12 14 16 0 200 400 600 800 1000 1200 Mean REAL Runtime (s) 89 % 26 % 17 % 100 % 100 % 97 % PIRN Hybroscale Fig. 8 An illustration of the cluster degree parameter c = 1. When inserting an in-going edge e to node v2 that is respecting c, each node that is marked green or is part of a green marked subnetwork forms a potential source node Fig. 8 An illustration of the cluster degree parameter c = 1. When inserting an in-going edge e to node v2 that is respecting c, each node that is marked green or is part of a green marked subnetwork forms a potential source node Upper Bound of the Hybridization Number Fig. 9 The figure shows the mean average runtime corresponding to Hybroscale and PIRNv2.0 grouped by parameter k denoting the hybridization number of the network that was used to obtain the tree set T from. Thus, this parameter k acts as an upper bound of the hybridization number of T . Each percentage indicates the proportion of tree sets that could be computed within the time limit of 20 minutes Fig. Additional features The numbers inside the plot indicate how many tree sets could be computed for the corresponding hybridization number within the time limit of 20 minutes. Note that for the hybridization numbers 0 to 3 all corresponding tree sets could be computed by Hybroscale and PIRNv2.0 within comparable runtimes Fig. 10 The figure shows the mean average runtime of all tree sets grouped by the computed hybridization numbers. The numbers inside the plot indicate how many tree sets could be computed for the corresponding hybridization number within the time limit of 20 minutes. Note that for the hybridization numbers 0 to 3 all corresponding tree sets could be computed by Hybroscale and PIRNv2.0 within comparable runtimes Fig. 11 A scatterplot of the runtimes generated by PIRNv2.0 (x-axis) against the runtimes generated by Hybroscale (y-axis) of all 810 data sets consisting of three input trees. Note that PIRNv2.0 is not able to compute the result for 449 tree sets corresponding to each dot in the figure whose x-value is 1200. From those tree sets just 6 according to the dots whose y-value is also 1200 could not be computed by Hybroscale Fig. 11 A scatterplot of the runtimes generated by PIRNv2.0 (x-axis) against the runtimes generated by Hybroscale (y-axis) of all 810 data sets consisting of three input trees. Note that PIRNv2.0 is not able to compute the result for 449 tree sets corresponding to each dot in the figure whose x-value is 1200. From those tree sets just 6 according to the dots whose y-value is also 1200 could not be computed by Hybroscale and 11, whereas the results for four and five input trees (n = 4, 5) can be found in the Additional file 1. Due to time limitations, if the hybridization number of a certain tree set could not be computed within 20 minutes, the compu- tation of this tree set was aborted. In Figs. 9, 11, 12, and 13 those unfinished tree sets were taken into account with a runtime of 20 minutes whereas in Fig. 10 these tree sets were omitted. #Tree Sets 0 200 400 600 800 1000 <1200 <1100 <1000 <900 <800 <700 <600 <500 <400 <300 <200 <100 <50 <10 n=3 Runtime (s) PIRN Hybroscale REAL Runtime Hybroscale USER Runtime Fig. Additional features 8 An illustration of the cluster degree parameter c = 1. When inserting an in-going edge e to node v2 that is respecting c, each node that is marked green or is part of a green marked subnetwork forms a potential source node Fig. 8 An illustration of the cluster degree parameter c = 1. When inserting an in-going edge e to node v2 that is respecting c, each node that is marked green or is part of a green marked subnetwork forms a potential source node Albrecht BMC Bioinformatics (2015) 16:236 Page 11 of 15 Fig. 11 A scatterplot of the runtimes generated by PIRNv2.0 (x-axis) against the runtimes generated by Hybroscale (y-axis) of all 810 data sets consisting of three input trees. Note that PIRNv2.0 is not able to compute the result for 449 tree sets corresponding to each dot in the figure whose x-value is 1200. From those tree sets just 6 according to the dots whose y-value is also 1200 could not be computed by Hybroscale #Tree Sets 0 200 400 600 800 1000 <1200 <1100 <1000 <900 <800 <700 <600 <500 <400 <300 <200 <100 <50 <10 n=3 Runtime (s) PIRN Hybroscale REAL Runtime Hybroscale USER Runtime Fig. 12 The figure shows the number of tree sets that could be computed within the runtime given on the x-axis by considering the real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization with 16 cores can significantly improve the runtime of Hybroscale in this case. Note that this is, on the one hand, due to the low time limit of just 20 minutes and, on the other hand, due to the low computational complexity of the considered tree sets Fig. 10 The figure shows the mean average runtime of all tree sets grouped by the computed hybridization numbers. The numbers inside the plot indicate how many tree sets could be computed for the corresponding hybridization number within the time limit of 20 minutes. Note that for the hybridization numbers 0 to 3 all corresponding tree sets could be computed by Hybroscale and PIRNv2.0 within comparable runtimes Fig. 10 The figure shows the mean average runtime of all tree sets grouped by the computed hybridization numbers. Additional features 12 The figure shows the number of tree sets that could be computed within the runtime given on the x-axis by considering the real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization with 16 cores can significantly improve the runtime of Hybroscale in this case. Note that this is, on the one hand, due to the low time limit of just 20 minutes and, on the other hand, due to the low computational complexity of the considered tree sets #Tree Sets 0 200 400 600 800 1000 <1200 <1100 <1000 <900 <800 <700 <600 <500 <400 <300 <200 <100 <50 <10 n=3 Runtime (s) PIRN Hybroscale REAL Runtime Hybroscale USER Runtime Each of the simulation results given in Figs. 9, 10, 11, 12, and 13, which are now discussed in more detail, clearly demonstrates that our implementation is much faster than PIRN. Figure 9 shows that, by increasing the upper bound of the hybridization number k, the mean average run- time of the datasets computed by PIRN increases up to 1000 seconds whereas the mean average runtime corre- sponding to Hybroscale is always below 100 seconds. Note that, as the runtime of each unfinished dataset was set to 1200 seconds, if we would set the time limit to a higher value, the maximal mean average runtime produced by PIRN is expected to be even higher — otherwise, to pro- duce a reasonable comparison between both programs, we would have to leave out each dataset, which could not be computed by one of both programs, which means that we would end up with only those non representative datasets that are quite easy to compute. Figure 10 shows that Hybroscale, in comparison to PIRN, can compute more datasets within the time limit and datasets having a significant larger hybridization number. Whereas PIRN Runtime (s) Fig. 12 The figure shows the number of tree sets that could be computed within the runtime given on the x-axis by considering the real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization with 16 cores can significantly improve the runtime of Hybroscale in this case. Additional features Note that this is, on the one hand, due to the low time limit of just 20 minutes and, on the other hand, due to the low computational complexity of the considered tree sets Fig. 12 The figure shows the number of tree sets that could be computed within the runtime given on the x-axis by considering the real-runtime of PIRN and both real- and user-runtime of Hybroscale. Only the rightmost bar group reveals that the massive parallelization with 16 cores can significantly improve the runtime of Hybroscale in this case. Note that this is, on the one hand, due to the low time limit of just 20 minutes and, on the other hand, due to the low computational complexity of the considered tree sets Albrecht BMC Bioinformatics (2015) 16:236 Page 12 of 15 Fig. 13 Distribution of the speedups of Hybroscale versus PIRN computed for each tree set of our synthetic dataset. For three input trees Hybroscale is on mean average about 110 times faster than PIRN, for four input trees on mean average about 170 times, and for five input trees on mean average about 190 times higher computational complexity and, on the other hand, the time limit would be set to a higher value. A possi- ble explanation for the speedup without taking advan- tage of parallelization is, on the one hand, the proven method allMAAFs [18] that is used for solving the NP- hard problem of computing all maximum acyclic agree- ment forests. The efficiency of this method has been indicated recently in the work of Albrecht et al. [9]. On the other hand, in contrast to our approach, we assume that PIRN does only apply a subtree reduction and not additionally a cluster reduction to the set of initial input trees. Finally, we have computed the speedup of Hybroscale versus PIRN by comparing its runtimes produced for each tree set within our synthetic dataset. More pre- cisely, for each tree set d we have computed the speedup s(d) = RP(d)/RH(d), where RP and RH denotes the real- runtime produced by PIRN and Hybroscale, respectively. Application to a grass dataset As mentioned above our algorithm computes all repre- sentative networks for a set of input trees. In particular, given only two input trees, this means that Hybroscale in general outputs multiple networks for each maximum acyclic agreement forest instead of only one as it is the case for the method described in the work of Albrecht Thus, it is obvious that our implementation outper- forms PIRN which becomes even clearer by looking at Fig. 11 showing a scatterplot of the runtimes produced by both programs. The figure shows that for each runtime of a specific data set produced by PIRN the correspond- ing runtime of Hybroscale is smaller or equal. Moreover, looking at the bottom right of the figure, there exist a lot of data sets that could be computed by Hybroscale quite fast in less than 200 seconds, whereas PIRN is not able to come up with a result in less than 1200 seconds. Table 1 Output produced by Hybroscale applied to two phylogenetic trees belonging to a well known grass (Poaceae) dataset Genes Taxa HNumber #MAAFs #HNetworks ndhf phyB 40 8 459 2079 ndhf rbcl 36 8 72 1488 ndhf rpoc 34 9 144 264 ndhf waxy 19 6 46 599 phyB its 30 8 21 195 phyB rbcl 21 4 4 6 phyB rpoc 21 4 5 9 phyB waxy 14 3 6 10 rbcl rpoc 26 7 18 111 rbcl waxy 12 4 10 84 rpoc its 31 12 12 3480 rpoc waxy 10 2 1 1 waxy its 15 5 6 15 Table 1 Output produced by Hybroscale applied to two phylogenetic trees belonging to a well known grass (Poaceae) dataset p By comparing real- with user-runtimes, Fig. 12 demon- strates that the better performance of Hybroscale is not only due to the applied massive parallelization. As the user-runtime indicates the total CPU time, which means that the time spent on all available cores is simply added up, this time indication corresponds to the runtime pro- duced by a program that is executed on a system only providing a single core with no parallel execution tak- ing place. Figure 12 shows the number of tree sets that could be computed within the runtime given at the x- axis. Additional features Figure 13, showing the distribution of the speedups corre- sponding to each of those tree sets, reveals that for three input trees Hybroscale is on mean average about 110 times faster than PIRN, for four input trees on mean average about 170 times, and for five input trees on mean average about 190 times. Fig. 13 Distribution of the speedups of Hybroscale versus PIRN computed for each tree set of our synthetic dataset. For three input trees Hybroscale is on mean average about 110 times faster than PIRN, for four input trees on mean average about 170 times, and for five input trees on mean average about 190 times Fig. 13 Distribution of the speedups of Hybroscale versus PIRN computed for each tree set of our synthetic dataset. For three input trees Hybroscale is on mean average about 110 times faster than PIRN, for four input trees on mean average about 170 times, and for five input trees on mean average about 190 times is just able to compute hybridization numbers up to 5, Hybroscale is able to compute hybridization numbers up to 13. Application to a grass dataset set of representative networks) within 20 minutes (Continued) Computing HNumbers Computing HNetworks Genes #Taxa HNumber Runtime #HNetworks Runtime ndhf phyB rbcl waxy 7 2 2.581 1 2.594 ndhf phyB rpoc its 19 9 984.937 - - ndhf phyB rpoc waxy 5 0 0.056 1 0.071 ndhf phyB waxy its 10 5 4.159 8016 26.434 ndhf rbcl rpoc its 24 - - - - ndhf rbcl rpoc waxy 9 4 3.165 396 14.864 ndhf rbcl waxy its 11 6 54.399 2 159.99 ndhf rpoc waxy its 10 5 4.213 324 16.663 phyB rbcl rpoc its 14 - - - - phyB rbcl rpoc waxy 4 0 0.057 1 0.06 phyB rbcl waxy its 6 2 2.574 3 2.589 phyB rpoc waxy its 5 0 0.064 1 0.065 rbcl rpoc waxy its 9 5 7.205 333 38.471 ndhf phyB rbcl rpoc its 14 - - - - ndhf phyB rbcl rpoc waxy 4 0 0.066 1 0.084 ndhf phyB rbcl waxy its 6 3 4.232 135 22.506 ndhf phyB rpoc waxy its 5 0 0.059 1 0.083 ndhf rbcl rpoc waxy its 9 5 35.899 235 587.54 phyB rbcl rpoc waxy its 4 0 0.066 1 0.076 ndhf phyB rbcl rpoc waxy its 4 0 0.062 1 0.083 et al. [9]. As a consequence, the output usually consists of a huge number of different hybridization networks, which is demonstrated by Tables 1 and 2 presenting the results of our software Hybroscale applied to a well known grass (Poaceae) dataset3 consisting of three nuclear loci and three chloroplast genes. This dataset, which is also used in the work of van Iersel et al. [20], was originally pub- lished by the Grass Phylogeny Working Group (2001) and Table 2 Output produced by Hybroscale applied to phylogenetic trees belonging to a grass (Poaceae) dataset. Each runtime given in this table is stated in seconds. A missing result for a certain tree set means that our software Hybroscale could not compute the exact hybridization number (resp. Application to a grass dataset set of representative networks) within 20 minutes (Continued) Computing HNumbers Computing HNetworks Genes #Taxa HNumber Runtime #HNetworks Runtime ndhf phyB rbcl waxy 7 2 2.581 1 2.594 ndhf phyB rpoc its 19 9 984.937 - - ndhf phyB rpoc waxy 5 0 0.056 1 0.071 ndhf phyB waxy its 10 5 4.159 8016 26.434 ndhf rbcl rpoc its 24 - - - - ndhf rbcl rpoc waxy 9 4 3.165 396 14.864 ndhf rbcl waxy its 11 6 54.399 2 159.99 ndhf rpoc waxy its 10 5 4.213 324 16.663 phyB rbcl rpoc its 14 - - - - phyB rbcl rpoc waxy 4 0 0.057 1 0.06 phyB rbcl waxy its 6 2 2.574 3 2.589 phyB rpoc waxy its 5 0 0.064 1 0.065 rbcl rpoc waxy its 9 5 7.205 333 38.471 ndhf phyB rbcl rpoc its 14 - - - - ndhf phyB rbcl rpoc waxy 4 0 0.066 1 0.084 ndhf phyB rbcl waxy its 6 3 4.232 135 22.506 ndhf phyB rpoc waxy its 5 0 0.059 1 0.083 ndhf rbcl rpoc waxy its 9 5 35.899 235 587.54 phyB rbcl rpoc waxy its 4 0 0.066 1 0.076 ndhf phyB rbcl rpoc waxy its 4 0 0.062 1 0.083 Table 2 Output produced by Hybroscale applied to phylogenetic trees belonging to a grass (Poaceae) dataset. Each runtime given in this table is stated in seconds. A missing result for a certain tree set means that our software Hybroscale could not compute the exact hybridization number (resp. set of representative networks) within 20 minutes (Continued) Table 2 Output produced by Hybroscale applied to phylogenetic trees belonging to a grass (Poaceae) dataset. Each runtime given in this table is stated in seconds. A missing result for a certain tree set means that our software Hybroscale could not compute the exact hybridization number (resp. set of representative networks) within 20 minutes not compute the exact hybridization number (resp. Application to a grass dataset For example, the leftmost bar-group shows that PIRN could only finish 360 of 810 tree sets consisting of three trees within 1200 seconds whereas Hybroscale could fin- ish 804 by taking parallelization into account and 793 by not taking parallelization into account. Note that the difference between both bars corresponding to the real- and user-runtime of Hybroscale would be even larger if, on the one hand, the dataset would contain tree sets of Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 13 of 15 Page 13 of 15 Table 2 Output produced by Hybroscale applied to phylogenetic trees belonging to a grass (Poaceae) dataset. Each runtime given in this table is stated in seconds. A missing result for a certain tree set means that our software Hybroscale could not compute the exact hybridization number (resp. Application to a grass dataset set of representative networks) within 20 minutes Computing HNumbers Computing HNetworks Genes #Taxa HNumber Runtime #HNetworks Runtime ndhf its 46 17 3.262 - - ndhf phyB 40 8 0.199 2079 33.035 ndhf rbcl 36 8 0.175 1488 32.1 ndhf rpoc 34 9 0.197 264 5.353 ndhf waxy 19 6 0.179 599 5.693 phyB its 30 8 0.238 195 8.304 phyB rbcl 21 4 0.083 6 1.65 phyB rpoc 21 4 0.091 9 1.678 phyB waxy 14 3 0.071 10 1.615 rbcl its 29 12 4.41 - - rbcl rpoc 26 7 0.147 111 3.836 rbcl waxy 12 4 0.126 84 4.338 rpoc its 31 12 4.5 3480 217.575 rpoc waxy 10 2 0.07 1 1.582 waxy its 15 5 0.118 15 2.712 ndhf phyB its 30 13 243.411 - - ndhf phyB rbcl 21 9 7.226 - - ndhf phyB rpoc 21 8 6.189 36948 206.114 ndhf phyB waxy 14 4 1.599 54 2.87 ndhf rbcl its 28 - - - - ndhf rbcl rpoc 26 11 7.223 46946 511.296 ndhf rbcl waxy 12 5 4.198 114 5.577 ndhf rpoc its 31 - - - - ndhf rpoc waxy 10 3 2.583 14 2.632 ndhf waxy its 15 8 4.213 6490 26.697 phyB rbcl its 17 8 9.437 8661 233.768 phyB rbcl rpoc 15 6 4.652 40 4.867 phyB rbcl waxy 7 2 2.568 11 2.592 phyB rpoc its 19 7 3.774 57 4.633 phyB rpoc waxy 5 0 0.045 1 0.075 phyB waxy its 10 4 3.122 204 3.844 rbcl rpoc its 24 - - - - rbcl rpoc waxy 9 3 1.585 5 1.62 rbcl waxy its 11 6 6.224 63 7.49 rpoc waxy its 10 4 2.626 4 2.635 ndhf phyB rbcl its 17 - - - - ndhf phyB rbcl rpoc 15 9 224.934 1517 403.728 Table 2 Output produced by Hybroscale applied to phylogenetic trees belonging to a grass (Poaceae) dataset. Each runtime given in this table is stated in seconds. A missing result for a certain tree set means that our software Hybroscale could not compute the exact hybridization number (resp. et al. [9]. As a consequence, the output usually consists of a huge number of different hybridization networks, which is demonstrated by Tables 1 and 2 presenting the results of our software Hybroscale applied to a well known grass (Poaceae) dataset3 consisting of three nuclear loci and three chloroplast genes. This dataset, which is also used in the work of van Iersel et al. [20], was originally pub- lished by the Grass Phylogeny Working Group (2001) and reanalyzed in Schmidt (2003). Application to a grass dataset Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Page 14 of 15 Page 14 of 15 Again, we ran Hybroscale on a grid computer providing 16 cores and 40 GB RAM for each tree set within the grass dataset and summarized the respective results in Table 2. This table shows that Hybroscale is able to calculate the hybridization number for 50 out of 57 tree sets. This means, in particular, that for seven tree sets Hybroscale cannot produce a result within a time limit of 20 minutes. Moreover, even though for 5 tree sets the hybridiza- tion number could be calculated, the respective entire set of representative networks could not be calculated as in this case a time limit of 20 minute is not sufficient to explore the whole solution space. Consequently, this biological example demonstrates that, although our algo- rithm seems to be faster than all so far existing methods, calculating minimum hybridization networks remains a computationally hard problem, which is still not solved sufficiently. the computation of meaningful support values indicat- ing which of the computed hybridization events might have played an important role during evolution. Addi- tionally, in combination with our software Hybroscale, we improve the interpretation of the reported hybridization networks by assigning support values to each hybridiza- tion node and by highlighting the embedding of all input trees. p Moreover, even though for 5 tree sets the hybridiza- tion number could be calculated, the respective entire set of representative networks could not be calculated as in this case a time limit of 20 minute is not sufficient to explore the whole solution space. Consequently, this biological example demonstrates that, although our algo- rithm seems to be faster than all so far existing methods, calculating minimum hybridization networks remains a computationally hard problem, which is still not solved sufficiently. Additionally, our reported simulation study indicates that our algorithm is much faster than the only so far existing software PIRNv2.0 [7, 8] for computing the exact hybridization number for more than two binary phyloge- netic trees on the same set of taxa. As shown in Fig. 12, the better performance is not only due to paralleliza- tion but apparently also due to algorithmic issues and, presumably, due to the application of certain reduction rules. Application to a grass dataset Availability and requirements Availability and requirements • Project name: Hybroscale • Project home page: www.bio.ifi.lmu.de/ softwareservices/hybroscale • Operating system(s): Platform independent • Programming language: Java • Other requirements: Java 7 or higher A b d Conclusion As already discussed in the work of Albrecht et al. [9], it makes sense to consider hybridization if there is a signif- icant difference between certain gene trees and if other effects, as for example incomplete lineage sorting, could be excluded. The number of genes affected by hybridiza- tion, however, is of course not limited to a fixed value, e.g., two, and, thus, a method computing hybridization net- works for an arbitrary number of input trees is of high interest. Application to a grass dataset set of representative networks) within 20 minutes Computing HNumbers Computing HNetworks Genes #Taxa HNumber Runtime #HNetworks Runtime ndhf its 46 17 3.262 - - ndhf phyB 40 8 0.199 2079 33.035 ndhf rbcl 36 8 0.175 1488 32.1 ndhf rpoc 34 9 0.197 264 5.353 ndhf waxy 19 6 0.179 599 5.693 phyB its 30 8 0.238 195 8.304 phyB rbcl 21 4 0.083 6 1.65 phyB rpoc 21 4 0.091 9 1.678 phyB waxy 14 3 0.071 10 1.615 rbcl its 29 12 4.41 - - rbcl rpoc 26 7 0.147 111 3.836 rbcl waxy 12 4 0.126 84 4.338 rpoc its 31 12 4.5 3480 217.575 rpoc waxy 10 2 0.07 1 1.582 waxy its 15 5 0.118 15 2.712 ndhf phyB its 30 13 243.411 - - ndhf phyB rbcl 21 9 7.226 - - ndhf phyB rpoc 21 8 6.189 36948 206.114 ndhf phyB waxy 14 4 1.599 54 2.87 ndhf rbcl its 28 - - - - ndhf rbcl rpoc 26 11 7.223 46946 511.296 ndhf rbcl waxy 12 5 4.198 114 5.577 ndhf rpoc its 31 - - - - ndhf rpoc waxy 10 3 2.583 14 2.632 ndhf waxy its 15 8 4.213 6490 26.697 phyB rbcl its 17 8 9.437 8661 233.768 phyB rbcl rpoc 15 6 4.652 40 4.867 phyB rbcl waxy 7 2 2.568 11 2.592 phyB rpoc its 19 7 3.774 57 4.633 phyB rpoc waxy 5 0 0.045 1 0.075 phyB waxy its 10 4 3.122 204 3.844 rbcl rpoc its 24 - - - - rbcl rpoc waxy 9 3 1.585 5 1.62 rbcl waxy its 11 6 6.224 63 7.49 rpoc waxy its 10 4 2.626 4 2.635 ndhf phyB rbcl its 17 - - - - ndhf phyB rbcl rpoc 15 9 224.934 1517 403.728 Computing HNumbers Computing HNetworks Genes #Taxa HNumber Runtime #HNetworks Runtime et al. [9]. As a consequence, the output usually consists of a huge number of different hybridization networks, which is demonstrated by Tables 1 and 2 presenting the results of our software Hybroscale applied to a well known grass (Poaceae) dataset3 consisting of three nuclear loci and three chloroplast genes. This dataset, which is also used in the work of van Iersel et al. [20], was originally pub- lished by the Grass Phylogeny Working Group (2001) and reanalyzed in Schmidt (2003). Additional file Additional file 1: Supplementary material. Supplementary Material contains Supplementary Figures. Endnotes 1www.bio.ifi.lmu.de/softwareservices/hybroscale 2www.bio.ifi.lmu.de/softwareservices/hybroscale 3www.sites.google.com/site/cassalgorithm/data-sets 1www.bio.ifi.lmu.de/softwareservices/hybroscale 2www.bio.ifi.lmu.de/softwareservices/hybroscale While some approaches only focus on reconciling two binary phylogenetic X -trees [9, 21], in this article, we present the algorithm ALLHNETWORKS that is able to cope with multiple input trees. Moreover, instead of reporting just the hybridization number or only a small number of hybridization networks, our approach is based on the first algorithm that is able to output all represen- tative networks, which is an important feature enabling Application to a grass dataset Finally, we would like to mention that in the mean- time we have extended the algorithm ALLHNETWORKS such that it can be applied to multiple rooted multifur- cating phylogenetic trees sharing an overlapping set of taxa. Moreover, to make Hybroscale applicable to larger input sizes, we have added an option allowing to gener- ate certain constraints for either limiting the search space of all representative networks before running our algo- rithm or to filter the set of reported networks after its computation. This mechanism was motivated by the pre- vious work of Kelk et. al [22] suggesting to come up with a general method for generating these constraints, which is absolutely meaningful in our point of view. In Fig. 7, one out of 324 possible hybridization networks reconciling four different binary phylogenetic trees corre- sponding to the sequences ndhf, rpoC, waxy, and ITS is given. The embedding of the trees is demonstrated by the four colors blue, red, green, and orange. This means, for example, that we can simply determine the embedding of the tree corresponding to rpoC, which is denoted as Tree 1 in this case, by taking the red colored edges into account. h l d h h b d In Fig. 7, one out of 324 possible hybridization networks reconciling four different binary phylogenetic trees corre- sponding to the sequences ndhf, rpoC, waxy, and ITS is given. The embedding of the trees is demonstrated by the four colors blue, red, green, and orange. This means, for example, that we can simply determine the embedding of the tree corresponding to rpoC, which is denoted as Tree 1 in this case, by taking the red colored edges into account. Moreover, the support values assigned to each hybridiza- tion node reveal that a hybridization event involving the two species oryza and lygeum occurs in 97 % of all 324 net- works, which could be a strong signal that this event is also part of the true underlying evolutionary history. How- ever, the reader should be aware of the fact that there still exist other mechanisms explaining such inconsistencies, as for example incomplete lineage sorting. Hence, such networks just help to build hypothesis that still have to be tested by applying further experiments. References 1. Mallet J. Hybrid speciation. Nature. 2007;446:279–83. 2. Rieseberg LH. Hybridization, introgression, and linkage evolution. Plant Mol Biol. 2000;42:205–24. 3. Soltis P, Soltis D. The role of hybridization in plant speciation. J Comput Biol. 2009;60:561–88. 4. Schwenk K, Brede N, Streitl B. Introduction. extent, processes and evolutionary impact of interspecific hybridization in animals. Phil Trans R Soc B Biol Sci. 2008;363(1505):2805–811. 5. Bordewich M, Semple C. Computing the minimum number of hybridization events for a consistent evolutionary history. Discrete Appl Math. 2007;155:914–28. 6. van Iersel L, Linz S. A quadratic kernel for computing the hybridization number of multiple trees. Inform Process Lett. 2013;113(9):318–23. 7. Wu Y. Close lower and upper bounds for the minimum reticulate network of multiple phylogenetic trees. Bioinformatics. 2009;26(12):140–8. p p y g ; ( ) 8. Wu Y. An algorithm for constructing parsimonious hybridization networks with multiple phylogenetic trees. J Comput Biol. 2013;20(10):792–804. 9. Albrecht B, Scornavacca C, Cenci C, Huson DH. Fast computation of minimum hybridization networks. Bioinformatics. 2011;28(2):191–7. l l b d l l p p y g 8. Wu Y. An algorithm for constructing parsimonious hybridization networks with multiple phylogenetic trees. J Comput Biol. 2013;20(10):792–804. p p y g p 9. Albrecht B, Scornavacca C, Cenci C, Huson DH. Fast computation of minimum hybridization networks. Bioinformatics. 2011;28(2):191–7. y 10. Baroni M, Semple C, Steel M. Hybrids in real-runtime. Syst Biol. 2006;55(1):46–56. y 10. Baroni M, Semple C, Steel M. Hybrids in real-runtime. Syst Biol. 2006;55(1):46–56. 11. Bordewich M, Semple C. Computing the hybridization number of two phylogenetic tress is fixed-parameter traceable. IEEE/ACM Trans Comput Biol Bioinformatics. 2007;4:458–66. 12. Whidden C, Beiko R, Zeh N. Fast FPT algorithms for computing rooted agreement forests: Theory and experiments In: Festa P, editor. In: Proceedings of the 9th International Symposium on Experimental Algorithms, SEA 2010. Heidelberg, Germany: Springer; 2010. p. 141–53. 13. Albrecht B. Computing hybridization networks for multiple rooted binary phylogenetic trees by maximum acyclic agreement forests. arXiv:1408.3044. 2014. 13. Albrecht B. Computing hybridization networks for multiple rooted binary phylogenetic trees by maximum acyclic agreement forests. arXiv:1408.3044. 2014. 14. Huson DH, Rupp R, Scornavacca C. Phylogenetic Networks: Concepts, Algorithm and Applications. Shaftesbury Road, Cambridge: Cambridge University Press; 2011. doi:ISBN:9780521755962. 14. Huson DH, Rupp R, Scornavacca C. Phylogenetic Networks: Concepts, Algorithm and Applications. Shaftesbury Road, Cambridge: Cambridge University Press; 2011. doi:ISBN:9780521755962. 15. Baroni M, Gruenewald S, Moulton V, Semple C. Competing interests Th h d l h Competing interests The authors declare that they have no competing interests. Page 15 of 15 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Albrecht BMC Bioinformatics (2015) 16:236 Authors’ contributions h h l k d The whole work was done by BA involving the development of the algorithm, the implementation of the software Hybroscale, the conduction of the simulation study, and finally drafting the manuscript. Acknowledgements We would like to thank Cuong Van Than and Daniel H. Huson for helpful discussion about certain theoretical issues regarding the presented algorithm. We also gratefully acknowledge the critical review by an anonymous reviewer on an earlier version of the manuscript. Received: 15 April 2015 Accepted: 3 July 2015 Received: 15 April 2015 Accepted: 3 July 2015 Received: 15 April 2015 Accepted: 3 July 2015 References Bounding the number of hybridisation events for a consisten evolutionary history. Math Biol. 2005;51:171–82. 15. Baroni M, Gruenewald S, Moulton V, Semple C. Bounding the number of hybridisation events for a consisten evolutionary history. Math Biol. 2005;51:171–82. 16. Bordewich M, Semple C. On the computational complexity of the rooted subtree prune and regraft distance. Ann Combinator. 2005;8:409–23. 16. Bordewich M, Semple C. On the computational complexity of the rooted subtree prune and regraft distance. Ann Combinator. 2005;8:409–23. 17. Linz S. Reticulation in evolution. Germany: PhD thesis, Mathematisch-Naturwissenschaftliche Fakultät der Heinrich-Heine-Universität Düsseldorf; 2008. 17. Linz S. Reticulation in evolution. Germany: PhD thesis, Mathematisch-Naturwissenschaftliche Fakultät der Heinrich-Heine-Universität Düsseldorf; 2008. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: 18. Scornavacca C, Linz S, Albrecht B. A first step towards computing all hybridization networks for two rooted binary phylogenetic trees. J Comput Biol. 2010;19(11):1227–42. 19. Cardona G, Rossello F, Valiente G. Extended newick: It is time for a standard representation of phylogenetic networks. BMC Bioinformatics. 2008;9:532. 19. Cardona G, Rossello F, Valiente G. Extended newick: It is time for a standard representation of phylogenetic networks. BMC Bioinformatics. 2008;9:532. 20. van Iersel L, Kelk S, Rupp R, Huson DH. Phylogenetic networks do not need to be complex: using fewer reticulations to represent conflicting clusters. Bioinformatics. 2010;26(12):124–31. 21. Chen ZZ, Wang L. Hybridnet: a tool for constructing hybridization networks. Bioinformatics. 2010;26:2912–1913. 21. Chen ZZ, Wang L. Hybridnet: a tool for constructing hybridization networks. Bioinformatics. 2010;26:2912–1913. 22. Kelk S, Linz S, Morrison DA. Fighting network space: it is time for an sql-type language to filter phylogenetic networks. arXiv:1310.6844. 2013. 22. Kelk S, Linz S, Morrison DA. Fighting network space: it is time for an sql-type language to filter phylogenetic networks. arXiv:1310.6844. 2013.
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Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms
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To cite this version: Zakaria Amarouchi, Qassim Esmaeel, Lisa Sanchez, Cédric Jacquard, Majida Hafidi, et al.. Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms. Microor- ganisms, 2021, 9 (7), ￿10.3390/microorganisms9071386￿. ￿hal-03435746￿ All content following this page was uploaded by Essaïd Ait Barka on 25 June 2021. The user has requested enhancement of the downloaded file. HAL Id: hal-03435746 https://hal.science/hal-03435746v1 Submitted on 18 Nov 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/352748855 Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms Zakaria Amarouchi 1,2, Qassim Esmaeel 1, Lisa Sanchez 1, Cédric Jacquard 1, Majida Hafidi 2, Nathalie Vaillant-Gaveau 1 and Essaid Ait Barka 1,* Zakaria Amarouchi 1,2, Qassim Esmaeel 1, Lisa Sanchez 1, Cédric Jacquard 1, Majida Hafidi Nathalie Vaillant-Gaveau 1 and Essaid Ait Barka 1,* 1 Université de Reims Champagne-Ardenne, RIBP EA4707 USC INRAE 1488, SFR Condorcet FR CNRS 3417, 51100 Reims, France; zakariaamarouchi@gmail.com (Z.A.); qassim.esmaeel@univ-reims.fr (Q.E.); lisa.sanchez@univ-reims.fr (L.S.); cedric.jacquard@univ-reims.fr (C.J.); nathalie.gaveau@univ-reims.fr (N.V.-G.) 1 Université de Reims Champagne-Ardenne, RIBP EA4707 USC INRAE 1488, SFR Condorcet FR CNRS 3417, 51100 Reims, France; zakariaamarouchi@gmail.com (Z.A.); qassim.esmaeel@univ-reims.fr (Q.E.); lisa.sanchez@univ-reims.fr (L.S.); cedric.jacquard@univ-reims.fr (C.J.); nathalie.gaveau@univ-reims.fr (N.V.-G.) 1 Université de Reims Champagne-Ardenne, RIBP EA4707 USC INRAE 1488, SFR Condorcet FR CNRS 3417, 51100 Reims, France; zakariaamarouchi@gmail.com (Z.A.); qassim.esmaeel@univ-reims.fr (Q.E.); lisa.sanchez@univ-reims.fr (L.S.); cedric.jacquard@univ-reims.fr (C.J.); nathalie.gaveau@univ-reims.fr (N.V.-G.) nathalie.gaveau@univ-reims.fr (N.V.-G.) 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Ressources, Faculté des Sciences, Université Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr * Correspondence: ea.barka@univ-reims.fr; Tel: +33-326913221 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Ressources, Faculté des Sciences, Université Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr * Correspondence: ea barka@univ-reims fr; Tel: +33-326913221 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Ressources, Faculté des Sciences, Université Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr * Correspondence: ea.barka@univ-reims.fr; Tel: +33-326913221 2 Laboratoire de Biotechnologie Végétale et Valorisation des Bio-Res Moulay Ismail, Meknès B.P 11201, Morocco; hafidimaj@yahoo.fr y j y * Correspondence: ea.barka@univ-reims.fr; Tel: +33-326913221 Abstract: In many vineyards around the world, Botrytis cinerea (B. cinerea) causes one of the most serious diseases of aerial grapevine (Vitis vinifera L.) organs. The control of the disease relies mainly on the use of chemical products whose use is increasingly challenged. To develop new sustainable methods to better resist B. cinerea, beneficial bacteria were isolated from vineyard soil. Once screened based on their antimicrobial effect through an in vivo test, two bacterial strains, S3 and S6, were able to restrict the development of the pathogen and significantly reduced the Botrytis-related necrosis. The photosynthesis analysis showed that the antagonistic strains also prevent grapevines from considerable irreversible PSII photo-inhibition four days after infection with B. cinerea. The 16S rRNA gene sequences of S3 exhibited 100% similarity to Bacillus velezensis, whereas S6 had 98.5% similarity to Enterobacter cloacae. On the other hand, the in silico analysis of the whole genome of isolated strains has revealed the presence of “biocontrol-related” genes supporting their plant growth and biocontrol activities. Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms The study concludes that those bacteria could be potentially useful as a suitable biocontrol agent in harvested grapevine. Citation: Amarouchi, Z.; Esmaeel, Q.; Sanchez, L.; Jacquard, C.; Hafidi, M.; Vaillant-Gaveau, N.; Barka, E.A. Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms. Microorganisms 2021, 9, 1386. https://doi.org/10.3390/ microorganisms9071386 Academic Editor: Fred O. Asiegbu Received: 3 June 2021 Accepted: 23 June 2021 Published: 25 June 2021 Citation: Amarouchi, Z.; Esmaeel, Q.; Sanchez, L.; Jacquard, C.; Hafidi, M.; Vaillant-Gaveau, N.; Barka, E.A. Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms. Microorganisms 2021, 9, 1386. https://doi.org/10.3390/ microorganisms9071386 Keywords: gray mold; biocontrol; grapevine Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms Article  in  Microorganisms · June 2021 DOI: 10.3390/microorganisms9071386 DOI: 10.3390/microorganisms9071386 CITATIONS 0 READS 112 7 authors, including: Some of the authors of this publication are also working on these related projects: BioScreen Project View project BioScreen Project: New biosourced and multifunctional molecules for the control of phytopathogenic agents in crops in the transboundary region View project Zakaria Amarouchi Université de Reims Champagne-Ardenne 1 PUBLICATION   0 CITATIONS    SEE PROFILE Qassim Esmaeel Université de Reims Champagne-Ardenne 36 PUBLICATIONS   219 CITATIONS    SEE PROFILE Lisa Sanchez Université de Reims Champagne-Ardenne 49 PUBLICATIONS   1,315 CITATIONS    SEE PROFILE Cédric Jacquard Université de Reims Champagne-Ardenne 65 PUBLICATIONS   1,756 CITATIONS    SEE PROFILE 7 authors, including: Some of the authors of this publication are also working on these related projects: BioScreen Project: New biosourced and multifunctional molecules for the control of phytopathogenic agents in crops in the transboundary region View project All content following this page was uploaded by Essaïd Ait Barka on 25 June 2021. The user has requested enhancement of the downloaded file. Citation: Amarouchi, Z.; Esmaeel, Q.; Sanchez, L.; Jacquard, C.; Hafidi, M.; Vaillant-Gaveau, N.; Barka, E.A. Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms. Microorganisms 2021, 9, 1386. https://doi.org/10.3390/ microorganisms9071386 Citation: Amarouchi, Z.; Esmaeel, Q.; Sanchez, L.; Jacquard, C.; Hafidi, M.; Vaillant-Gaveau, N.; Barka, E.A. Beneficial Microorganisms to Control the Gray Mold of Grapevine: From Screening to Mechanisms. Microorganisms 2021, 9, 1386. https://doi.org/10.3390/ microorganisms9071386 Academic Editor: Fred O. Asiegbu Received: 3 June 2021 Accepted: 23 June 2021 Published: 25 June 2021 1. Introduction The second stage of perception uses the recognition of microbial effectors, the virulence factors that suppress MTI to initiate effector-triggered immunity (ETI), triggering a cascade of complex signaling events, leading to suppression of pathogen assaults. Botrytis cinerea is one of the highest broadly studied necrotrophic fungal pathogens. B. cinerea has no apparent host specificity infecting therefore, more than 1000 plant species [9]. The gray mold (GM) caused by B. cinerea has a devastating impact on various economically important crops, including grape, strawberry, and tomato [10] with annual economic losses exceeding USD 10 to 100 billion worldwide [9,11,12]. Several disease controlling approaches have been implemented in the past and present to control B. cinerea. Currently, pesticides remain the main method used to fight the pathogen, and in some instances, the only option, involving significant financial costs. Until lately, the use of chemical fungicides to protect plant was thought to be fairly safe. Nevertheless, more than ever before, chemical fungicides use faces multiple challenges namely the development of resistance to fungicides resulting in the decline or even failure of control effect [13–16], increased consumers desire of food free of pesticide residues [17], enhanced concern regarding environmental pollution, and stricter regulatory policies are being imposed on the use of synthetic chemical fungicides [17,18] To overcome the difficulties previously stated, it is urgent to search for alternative, effective, and eco-friendly strategy of disease control [19–21]. Thus, in recent years, the use of microbes as a biocontrol agent is gaining interest in agriculture [22]. Plant growth promoting rhizobacteria (PGPR) are bacteria that inhabit the rhizosphere and can improve the extent or quality of plant growth directly and or indirectly. The direct promotion by PGPR involves either delivering plant with a plant growth promoting substances or helping plants to mobilize and acquire nutrients from the rhizosphere. The indirect effect occurs when PGPR prevent the harmful effect of pathogens. In the last few decades, bacteria including species of Arthobacter, Azospirillum, Azotobacter, Bacillus, Burkholderia, Enterobacter, Klebsiella, Pseudomonas, and Serratia have reported to enhance plant fitness. Various mechanisms for antagonism have been implicated, such as competition for nutrients and space, secretion of cell wall degrading enzymes, siderophores [23,24], parasitism of the pathogen, biofilm formation, induction of host defenses via production of various pathogenesis related proteins (PR) [25], and the involvement of reactive oxygen species (ROS) in the defense response are responsible for their antagonistic activity [18]. 1. Introduction Pathogens cause a devastating impact on crops varying from economic hardship to poisoning of food supplies (such as ergotism) and horrendous famines such as the Irish potato famine that lasted from 1845 to 1852. After their assaults, pathogens might trigger substantial changes to the host physiology, which can occur directly by secreting toxins and lytic enzymes or indirectly through inducing host responses stimulated by the pathogen. Among the significant physiological processes, the photosynthesis is the principal process affected by foliar diseases [1]. The photosynthesis decline might be proportional to decrease in green leafy tissue. Furthermore, the decline in photosynthesis, infections can trigger other physiological changes such as limited water use efficiency, which in turn, excessive water restriction may further induce a lower rate of photosynthesis (as reviewd in [2]). The pathogen might also impact the net carbon assimilation rate by enhancing the leaf respiration, which is requested to supply the demand initiated by the accelerated cells host metabolic activity [3]. The photosynthesis decreases through the infection process as a result of repression of photosynthetic gene expression [4,5]. Despite various struggles to decipher mechanisms by which pathogens can disturb photosynthetic capacity, current knowledges of the subject remain far from inclusive. Received: 3 June 2021 Accepted: 23 June 2021 Published: 25 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses /by/4.0/). www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1386. https://doi.org/10.3390/microorganisms9071386 2 of 19 2 of 19 Microorganisms 2021, 9, 1386 Plants do not have a circulatory system and adaptive immune system like animals. To block pathogen progress, plants have evolved a two-layered innate immune system. The first line of plants defense is accomplished via a set of defined receptors, namely pattern recognition receptors (PRRs), which able to identify conserved microbe-associated molecular patterns (MAMPs) [6]. Following MAMPs recognition, MAMP-triggered immunity (MTI) primary defense responses are triggered including mitogen-activated protein kinase (MAPK) phosphorylation cascades, cell wall alterations, callose deposition, defense genes expression, and defense-related proteins accumulation [7,8]. When perceived by intracellular immune receptors, pathogen effectors trigger the effector-triggered immunity (ETI; [8]). 2.2. Isolation, Purification, and Enrichment of Antagonistic Bacteria The isolation of bacteria was carried out according to the protocol previously described by Nally et al. [29], with some modifications. Portions of 15 g of Rhizospheric soils were suspended in 250 mL of sterile Luria-Bertani (LB) liquid medium (tryptone 10 g/L; yeast extract 5 g/L; NaCl 10 g/L; pH 7.2). The enrichment culture was incubated on a rotary shaker (180 rpm) at 28 °C for 24 h. This operation was repeated for the soil recovered under the vines showing the GM symptoms. After shaking, the cultures were kept undisturbed for 30 min. Then, isolation of viable bacterial cultures from soil suspensions was done by serial dilution plate count in phosphate-buffer saline (PBS 10 mM, pH 6.5). Aliquots of 100 μL from the five different dilutions of the sequential enrichment were spread in triplicate on LB medium and incubated at 28 °C for 24–72 h until colony development. Colonies with distinct morphologies were picked and purified using the streaking method. The purified isolates were used to screen antagonistic bacteria against B. cinerea. Afterwards, pure bacterial cultures were maintained in cryovials containing LB broth with 25% glycerol and preserved at −80°C. 1. Introduction PR proteins regulated resistance in response to B. cinerea in grapes. Therefore, nitrogen and carbon metabolisms play critical roles in the resistance of grapes against B. cinerea [26]. However, the main inhibitory action of antagonistic bacteria is to produce antifungal metabolites and antibiotics [27,28]. Given the wide-ranging and the importance of B. cinerea in agriculture, the control of GM is of great concern. In this context, our strategy was to develop new biotechnologies allowing grapevine to better resist parasitic pressures of B. cinerea through the isolation and screening of beneficial bacteria. Once screened, the mechanisms contributing to the biocontrol effect of selected bacteria was deciphered by determining their antimicrobial effect by in vivo test, and finally chlorophyll fluorescence imaging after Botrytis challenge. On the other hand, the in silico analysis of the whole genome of isolated strains has Microorganisms 2021, 9, 1386 3 of 19 revealed the presence of “biocontrol-related” genes supporting their plant growth and biocontrol activities. revealed the presence of “biocontrol-related” genes supporting their plant growth and biocontrol activities. 2.1. Soil Sampling Soil samples were collected from different distinct locations of vineyard (Chardonnay cv.) in Meknes-Morocco. Sampling was carried out at the end of the growing season, after the occurrence of the asexual multiplication of B. cinerea and the appearance of symptoms of gray rot detectable on the grapevine. Although isolation of antagonistic bacterial strains was taken from soil samples of healthy grapevine, the bacteria coming from soil collected under the infected plants were selected as control, to compare the communities of both populations. At each sampling points, soil was collected aseptically from healthy and severely infected grapevines with B. cinerea. Samples were kept in paper bags placed in ice and processed within 24 h. 2.5. Biochemical Characterization of Biocontrol Isolates Biochemical characteristics of isolated strains such as carbohydrate assimilation and fermentation were performed using BIOLOG GENIII microtiter plate (Hayward CA, USA) as recommended by manufacturers. 2.4. Identification of Antagonistic Bacteria All potential antagonistic bacteria were identified by 16S rRNA gene analysis. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) as previously described by [30]. Briefly, genomic DNA was extracted from a pure colony using the Wizard Genomic Purification DNA Kit (Promega Corp., Madison, WI, USA), according to the manufacturer’s instructions. Next, the bacterial 16S rRNA gene was amplified by PCR using FD2 (5- AGAGTTTGATCATGGCTCAG -3) and RP1(5- ACGGTTACCTTGTTACGACTT -3) primers. The PCR was carried out with a 50-µL final volume, containing 25-µL of Master Mix (Thermo Scientific Fermentas, Villebon sur Yvette, France), 2.5 µL of each primer, 15 µL sterile water, and 5 µL template DNA in a PTC-200 Thermocycler (C1000 touch thermal cycler, Bio-Rad, Hercules, CA, USA). The PCR conditions used were as follows: an initial denaturation step at 94 ◦C for 5 min, followed by 30 cycles of denaturation at 94°C for 45 s, primer annealing at 55 °C for 45 s, and elongation at 72 °C for 1.5 min, and a final elongation step at 72 ◦C for 10 min. g g p The PCR product was subjected to electrophoresis on agarose gel with 0.5X TAE buffer (Tris Acetate-EDTA) at a ratio of 1% (weight/volume). Gels were stained with ethidium bromide, visualized under UV light (300 nm), and were then excised and purified using the Gene JET Gel Extraction Kit (Thermo Scientific Fermentas, Waltham, USA), as recommended by the manufacturer. A 1000-bp DNA ladder marker served as the standard size. The PCR product was commercially sequenced by Genewiz Co., Ltd. (Leipzig, Germany). The data output was analyzed, and the sequences were compared with sequences in the National Center for Biotechnology Information (NCBI) database using the BLAST search program (http://www.ncbi.nlm.nih.gov/). Alignment of 16S rRNA gene sequences from GenBank database was performed using ClustalX 1.8.3 with default settings [31]. Phylogenesis was analyzed by MEGA version 7. Distances were calculated using the Kimura two parameter distance model. The tree was built by the neighbor- joining method. The dataset was boot- strapped 1000 times [32]. 2.3. In Vitro Screening of Potential Antagonistic Bacteria Isolated strains were tested for antifungal activity against B. cinerea. Assays were performed by patching, in the middle of PDA medium (Sigma-Aldrich, MO, USA) plates, 5 mm of agar plug carrying freshly grown culture of the fungal pathogen. After that, a volume of 5 µL of suspension of each isolate was drooped at four sites approximately 1 cm from the rim of the plate and incubated at 22 °C for five days at which point we start measurements. Plates inoculated with B. cinerea were also used as control. The plates were visually inspected for the presence of inhibition zones between the fungus and the colonies considered to be potential antagonistic bacteria. The antifungal effect was estimated by calculating the percentage of inhibition (%) of mycelial growth measured as follows: I(%) = (1 − Cn / Co) × 100 (1) (1) where “Cn” is the average diameter of the mycelial in the presence of the antagonists and “Co” the average diameter of the control. Experiments were conducted in duplicate and the results reported are averages of three independent experiments. The diameter of the clear zones depends on the performance of the bacteria. Thus, the screened strains with high zones of clearing were selected for molecular identification and in vivo assays. Microorganisms 2021, 9, 1386 4 of 19 2.4. Identification of Antagonistic Bacteria 2.6. Cellular Fatty Acid Analysis Cellular fatty acid analysis was carried out at BCCM/LMG (the Belgian Co-ordinated Collections of Microorganisms, Ghent University, Ghent, Belgium). Bacterial isolates were grown for 24 h at 30 °C under aerobic conditions on LB medium. Inoculation and harvesting of the cells, and the extraction and analysis were performed conform to the recommendations of the commercial identification system MIDI (Microbial Identification System, Inc., Newark, DE, USA). The whole-cell fatty acid composition was determined gas chromatographically on an Agilent Technologies 6890N gas chromatograph (Santa Clara, CA, USA). The peak naming table MIDI TSBA 5.0 was used. 2.10. Inoculation of In Vitro Plantlets with Antagonistic Bacteria and Infection by B. cinerea Isolates with higher percentage of inhibition (%) during in vitro screening were tested for antagonistic activity against B. cinerea on sterile grapevine plantlets. Briefly, roots of 6- weeks-old grapevine plantlets were gently removed from the MS agar medium (Sigma- Aldrich, France) and transferred into magenta boxes containing 120 g of soil. Plantlets were then inoculated with bacterial inoculum at a concentration of 108 CFU per g of soil. Control was treated with sterile PBS. Bacterized and non-bacterized plantlets were then grown for an additional 10 days. Thereafter, the upper side of each leaf was inoculated by a drop of 5 μL of B. cinerea germinated spore suspension. This protocol was used for measures of necrosis surfaces. The antifungal effect was estimated by calculating the percentage of inhibition (%) of mycelial growth measured as describes in in vitro tests. For IMAGING-PAM analysis, plantlets were sprayed with the germinated spore suspension of B. cinerea to have a homogenous application. Plantlets were then placed in growth chamber at 22 °C.. All the experiments were performed in triplicate. 2.8. Grapevine In Vitro Plantlets Plantlets of Vitis vinifera cv. Chardonnay (clone 7535) were micro-propagated by nodal explants grown on 15 mL of Murashige-Skoog (MS) agar medium in 25 mm-culture tubes as described by Ait Barka et al. [36]. Cultures were performed in a growth chamber under white fluorescent light (200 µmol/m2/s), with 16 h/8 h day/night photoperiod at a constant temperature of 26 °C. 2.9. Bacterial Isolates and Inoculum Preparation Bacterial suspensions were prepared by inoculating 100 mL of Luria-Bertani (LB) liquid culture medium and incubated on a rotary shaker at 180 rpm at 28 °C for 18 h at which point they reached the late exponential growth phase. After incubation, cells were harvested by centrifugation at 4500 g at 4 °C for 15 min. Each culture was washed three times and resuspended in 20 mL of phosphate-buffered saline (PBS). The density of bacterial cultures was determined by spectrophotometry and adjusted at approximately 108 colony-forming units (CFU) mL-1 with an optical density 0.8 at 600 nm (OD600). For the inoculum preparation, the fungal pathogen, B. cinerea strain 630 was grown on potato dextrose agar (PDA) (Sigma-Aldrich, MO, USA) at 22 °C. The conidia were collected from 20-day-old culture plates by scratching the Petri dishes surface with sterile potato dextrose broth (PDB 24 g/L) medium and filtered to remove hyphae. Conidial concentrations were measured by a hemocytometer and the final density was adjusted to 105 conidia/mL. After incubation during 3 h at 22 °C and 150 rpm, the resulting germinated spore suspension was used for plant inoculation. 2.7. Whole Genome Sequencing, Assembly, and Annotation 2.7. Whole Genome Sequencing, Assembly, and Annotation The genomic DNA of strains was isolated using the Wizard Genomic Purification DNA Kit (Promega Corp., Madison, WI, USA) according to the manufacturer’s instructions. The integrity of extracted DNA was assessed by running the sample on 1% agarose gel. The genome sequence of the strain was sequenced at MicrobesNG (http://www.microbesng.uk) using the method summarized in Table S1. The draft genome sequences were used for annotation using the Rapid Annotation Subsystem Technology (RAST) server (http://rast.nmpdr.org) [33]. The annotated genes were analyzed using SEED database [34]. To predict the presence of secondary metabolites (SMs) gene clusters associated with biocontrol activity, the draft genome sequence was analyzed by Microorganisms 2021, 9, 1386 5 of 19 5 of 19 antiSMASH software online (https://antismash.secondarymetabolites.org/#!/start) [35]. The Whole Genome Shotgun projects of the strains have been deposited in GenBank under the accession numbers JAFETM000000000 and JAFETL000000000. The version described in this paper version JAFETM010000000 and JAFETL010000000, respectively. antiSMASH software online (https://antismash.secondarymetabolites.org/#!/start) [35]. The Whole Genome Shotgun projects of the strains have been deposited in GenBank under the accession numbers JAFETM000000000 and JAFETL000000000. The version described in this paper version JAFETM010000000 and JAFETL010000000, respectively. 2.12. Statistical Analysis The experimental design used was performed in triplicates. Statistical analyses were carried out using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA) (www.graphpad.com). For lesion diameter Student’s t-tests (α > 0.05) was used to compare lesion area between inoculated and non-inoculated plants. 2.11. IMAGING-PAM Analysis Chlorophyll fluorescence parameters were measured with an IMAGING-PAM measuring system (Heinz Walz, Germany) using the saturation pulse method [37]. Control and bacterized plantlets were dark adapted for 30 min to establish the minimal fluorescence level (F0) and the maximal fluorescence (Fm) after a saturating flash (1 s; 13,000 µmol/m2/s). Each leaf was detached from the plantlet then exposed immediately to an actinic illumination of 79 µmol/m2/s. After fluorescence stabilization, a second saturating flash was imposed to determine the maximal fluorescence (Fm’) of light- adapted inflorescences. The effective PSII quantum yield, ΦPSII, is calculated according to the equation of Genty et al. [38]. The quantum yield of regulated energy dissipation in Microorganisms 2021, 9, 1386 6 of 19 6 of 19 PSII, ΦNPQ, and the quantum yield of nonregulated energy dissipation in PSII, ΦNO, was calculated according to Kramer et al. (2004) [39]. Please note that ΦPSII + ΦNPQ + ΦNO = 1. The data were collected taking in consideration the entire leaf area including necrosis area. Measurements were taken 24 h before inoculation of antagonistic bacteria, 24 h before infection with B. cinerea, and 4 consecutive days after infection. The means ± standard deviations originated from three independent experiments realized in duplicates, each replicate consisted of four plantlets. 3.1. Isolation and In Vitro Screening of Antagonistic Bacteria As a result of multiple inoculations and purification, 42 pure cultures of potential antagonistic bacteria were successfully enriched and isolated from vineyard soil. These freshly isolated strains were purified on LB plates, selected based on their morphology, and used as objects of investigation. All isolates were screened for their ability to inhibit the mycelial growth of B. cinerea by direct confrontation tests in PDA medium plates. Out of 42 tested strains, only two isolates were estimated as antagonistic potential bacteria against this fungus. They were nominated S3 and S6 which were the most active against fungal culture, by showing the strong percentage of inhibition I (%) (Figure 1A). These isolates were tested for their ability to protect grapevines against GM in planta. 3.2. Disease Symptoms were Significantly Reduced in Root-Bacterized Plantlets To test the ability of S3 and S6 to protect grapevine in our system, we performed infection on whole potted-plant with B. cinerea strain 630 in control versus root-bacterized plantlets. Assays performed showed that the two antagonistic bacteria had high inhibiting ability against B. cinerea in grapevines three days after inoculation of the pathogen. They significantly reduced Botrytis-related necrosis by approximately 50% at 72 hpi (Figure 1B). In addition, disease symptoms were significantly less developed in bacterized plants, confirming the protective impact of S3 and S6 against B. cinerea (Figure 1C). Non-bacterized plantlets showed severe symptoms typical of GM, manifesting as necrosis around the infection spot (Figure 1C). In contrast, plants treated with the antagonistic bacteria exhibited a reduction in disease symptoms, displayed by a smaller size of necrosis diameter compared to the control. These isolates were selected for molecular investigation. 7 of 19 Microorganisms 2021, 9, 1386 Percentage of inhibition I(%) 0% 10% 20% 30% 40% Bacillus velezensis Enterobacter cloacae S3 S6 A C B Figure 1. Impact of isolates on B. cinerea growth inhibition. Direct confrontation tests of isolates against B. cinerea (A). Results indicated are the mean of percent inhibition of mycelial growth of B. cinerea after 72 h of incubation in PDA medium (A). The Ability of isolate to protect grapevines (cv. Chardonnay), in planta, against B. cinerea (B, C). In vitro grapevine plantlets inoculated or not with S3 and S6, 72 hours post infection (hpi) with B. cinerea (B). (Control, a; B. cinerea, b; S3 + Bc, c; S6 + Bc, d). Diameter of necrosis measured on leaves infected with B. cinerea (C). Percentage of inhibition I(%) a b c d 0% 10% 20% 30% 40% Bacillus velezensis Enterobacter cloacae S3 S6 Bc S3+Bc S6+Bc Diameter of necrosis in cm2 0.1 0.5 0.0 1.5 A C B a A b B b c c us ve S3 C sbacte S6 S3 S6 Bc S3+Bc S6+Bc Diameter of necrosis in cm2 0.1 0.5 0.0 1.5 C Figure 1. Impact of isolates on B. cinerea growth inhibition. Direct confrontation tests of isolates against B. cinerea (A). Results indicated are the mean of percent inhibition of mycelial growth of B. cinerea after 72 h of incubation in PDA medium (A). The Ability of isolate to protect grapevines (cv. Chardonnay), in planta, against B. cinerea (B, C). 3.2. Disease Symptoms were Significantly Reduced in Root-Bacterized Plantlets In vitro grapevine plantlets inoculated or not with S3 and S6, 72 hours post infection (hpi) with B. cinerea (B). (Control, a; B. cinerea, b; S3 + Bc, c; S6 + Bc, d). Diameter of necrosis measured on leaves infected with B. cinerea (C). 3.3. Identification of Antagonistic Bacteria The molecular identification using 16S rRNA gene sequences of the antagonistic rhizobacteria show that S3 and S6 are closely related to Bacillus velezensis (100%), and Enterobacter cloacae (98.5%), respectively (Figure 2). Strains S3 and S6 also shared sequence identity with other species including B. subtilis (99%), and Pantoea agglomerans (98%), respectively (Figure 2). Both bacterial strains (S3 and S6) isolated under healthy canopy, were used for further characterization. In addition, to calculate the pair-wise average nucleotide identity (ANI) values of both strains with their closest known relatives, the draft genome sequences of strains S3 and S6 were compared against all type strain genomes available in the microbial genomes atlas (MiGA) webserver [40]. Results showed that strains S3 belongs to Bacillus velezensis (99% ANI). For strain S6, the closest relatives found was Enterobacter cloacae (98 % ANI). Moreover, digital DDH values of both strains were compared against all type strain genomes available in the TYGS database [41]. The analysis generated DDH values more than 70% % for both strains and the closet relatives were Bacillus velezensis and Enterobacter cloacae for strains S3 and S6, respectively. 8 of 19 Microorganisms 2021, 9, 1386 Figure 2. Neighbor-joining phylogenetic tree based on the 16SrRNA sequences of antagonistic isolates S3, and S6. showing the relationship with the genus Bacillus (A) and Enterobacter (B). The sequence of B. licheniformis NBRC 12200 and Aeromonas hydrophila ATCC 7966 were chosen as an out-group. Antagonistic strains are shown in bold. 3.4. Characterization of Biocontrol Isolates A B A A B B Figure 2. Neighbor-joining phylogenetic tree based on the 16SrRNA sequences of antagonistic isolates S3, and S6. showing the relationship with the genus Bacillus (A) and Enterobacter (B). The sequence of B. licheniformis NBRC 12200 and Aeromonas hydrophila ATCC 7966 were chosen as an out-group. Antagonistic strains are shown in bold. 3.4. Characterization of Biocontrol Isolates The data presented in Table 1 showed some biochemical characteristics of isolated strains. The selected antagonistic isolates (S3 and S6) were characterized by biochemical methods. The optimum growth conditions of isolates are at 28°C, pH 6.0, and in the presence of 1% NaCl. Although cells of strain S3 were sensitive to fusidic acid, minocycline, naldixic acid, rifamycin SV, lncomycin, and vancomycin, strain S6 was resistant to them. Isolates were able to grow in the presence of sodium butyrate, guanidine HCl, lithium chloride, potassium tellurite, and tetrazolium violet, but did not grow in the presence of sodium bromate, D-serine, and niaproof 4 except E. cloacae, which was resistant (Table 1). The two isolates failed to hydrolyze gelatin. Furthermore, the strains showed different abilities to use different carbon sources (Table 1). They were able to assimilate, D-cellobiose, mannose, mannitol, and N-acetyl-glucosamine, L-glutamic acid, sucrose, L-aspartic acid, D-maltose, D-fucose (except B. velezensis S3), and L-histidine, as Microorganisms 2021, 9, 1386 9 of 19 9 of 19 sole carbon sources whereas, 3-methylglucose, α-ketobutyric acid, and D-aspartic acid were not used by both strains. The isolates exhibited different patterns of cellular fatty acids profile features characterized by different level of C15: 0 iso-anteiso, C17: 0 iso- anteiso, summed feature 2 (comprising C12: 0 aldehyde, C14: 0 3-OH/ C16: 1 iso I and/or unknown ECL 10.928), C18:1 ω7c, C16: 0, C17:0 cyclo, C14:0, summed feature 3 (comprising C16:1 ω7c/15 iso 2OH), C12: 0, and C19: 0 cyclo ω8c. Isolate S3 was characterized by a fatty acid profile dominated to an unusual extent (> 98%) by saturated fatty acids (Figure S1). Hence, cells had less iso odd-numbered fatty acid and more anteiso odd-numbered fatty acid, with the major fatty acid being anteiso-C15:0(37.18%) (Table 1). Cells of strain S6 exhibited only 37,88% of saturated fatty acids but revealed other structure as branched chain (29,51%), cyclopropane (16,90%), and hydroxy unsaturated (14.68%) fatty acids that was deficient in strain S3 (Figure S2). The major fatty acid for S6 were C16:0 (18.07%) (Table 2). Table 1. Biochemical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate (Hayward CA, USA). 3.4. Characterization of Biocontrol Isolates Oxidation of Oxidation of Oxidation of S6 S3 S6 S3 S6 S3 3-methylglucose - - D-melibiose + + L-lacticacid + + Aceticacid + - D-raffinose + - L-malicacid + + acetoaceticacid - - D-saccharicacid + ± L-pyroglutamicacid ± ± bromo-succinicacid + - D-salicin ± - L-rhamnose + - Citricacid + ± D-serine - - L-serine + - D-arabitol ± - D-sorbitol + + Methylpyruvate + ± D-asparticacid - - D-trehalose + + mucicacid + ± D-cellobiose + + D-turanose ± ± myo-inositol + ± Dextrin ± + Formicacid ± - N-acetylneuraminicacid - ± D-fructose + + Gelatin - - N-acetyl-D-galactosamine + ± D-fructose-6-PO4 + ± Gentiobiose + - N-acetyl-D-glucosamine + + D-fucose ± - Glucuronamide + + N-acetyl-β-D-mannosamine + + D-galactose + - Glycerol + ± Pectin ± ± D-galacturonicacid + + Glycyl-L-proline + - p-hydroxyphenylaceticacid ± - D-gluconicacid + + Inosine + ± propionicacid - - D-glucose-6-PO4 + ± L-alanine + + Quinicacid - ± D-glucuronicacid + + L-arginine ± + Stachyose + - D-lacticacidmethylester ± ± L-asparticacid + + Sucrose + + D-malicacid - - L-fucose ± - Tween 40 - ± D-maltose + + L-galactonicacid lactone + + Α-D-glucose + + D-mannitol + + L-glutamicacid + + D-mannose + + L-histidine + + Growth in the presence of S6 S3 Growth in the presence of S6 S3 Growth in the presence of S6 S3 1% Nacl + + Lincomycin + - Rifamycin SV + - 4% Nacl + + Lithiumchloride + + Sodium bromate - - 8% Nacl ± ± Minocycline ± - Sodium butyrate + + 1% sodium lactate + + Nalidixicacid ± - Tetrazoliumblue + - Aztreonam ± - Niaproof 4 + - Tetrazolium violet + ± D-serine - - pH 5 ± - Troleandomycin + - Fusidicacid ± - pH 6 + + Vancomycin + - Guanidinehcl + + Potassium tellurite + + Table 1. Biochemical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate (Hayward CA, USA). emical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate USA). Table 1. Biochemical characteristics for the antagonistic strains based on BIOLOG GENIII microtiter plate (Hayward CA, USA). 10 of 19 Microorganisms 2021, 9, 1386 Table 2. Major cellular fatty acid content of S3 and S6 strains. 3.4. Characterization of Biocontrol Isolates Structure Fatty acid Systematic name Saturated % In isolated strains S3 S6 Saturated C12: 0 Dodecanoic 4.23 7.52 C13: 0 Tridecanoic - 1.68 C13: 0 ANTEISO 0.40 - C14: 0 Tetradecanoic 1.01 8.23 C14: 0 ISO 1,11 - C15: 0 ISO 11.95 - C15: 0 ANTEISO 37.18 - C16: 0 Hexadecanoic 17.16 18.07 C16: 0 ISO 2.41 - C17: 0 Heptadecanoic 0.57 2.38 C17: 0 ISO 9.44 - C17: 0 ANTEISO 11.64 - C18: 0 Octadecenoic 1.21 - Hydroxy Unsaturated C15: 0 3-OH 3- Hydroxy- pentadecenoic - 0.58 C16: 1 ω 5c cis-11-Hexadecenoic - - C16: 1 ω 11c 1.69 - C17:1 ω 8c - 0.60 C18: 1 ω 7c cis-11- Octadecenoic - 13.49 Cyclopropane C17: 0 cyclo Cyclo-heptadecanoic - 15.04 C19: 0 cyclo ω 8c 9-(2-eptylcyclopropyl) Nonanoic - 1.86 Branched chain Summed feature 1 C 15:1 ISO H/ C 13:0 3OH C 15:1 ISO I/ C 13:0 3OH - 3.28 Summed feature 2 C12: 0 aldehyde, C 16:1 ISO I/ C 14:0 3OH and/or unknown ECL 10.928 - 17.85 Summed feature 3 C 16:1 ω7c/ 15 iso 2OH - 8.38 Table 2. Major cellular fatty acid content of S3 and S6 strains. 3.5. Genomic Feature and In Silico Analysis 3.5. Genomic Feature and In Silico Analysis As summarized in Table 3, the draft genome sequence of the strain S3, assembled into 21 contigs, consists of 4,157, 680p with a 46.35% G+C. Strain S6, grouped into 30 contigs exhibited genome size of 4, 604, 658bp, and the GC content was 55.90%. The total number of predicted protein-coding sequences and RNAs was 4316 and 98 of RNAs for S3 versus 4228 and 116 of RNAs in S6, respectively. The genome characteristics of each isolate were detailed in Table 3. The predicted coding sequences (CDS) of S6 were classified into 560 subsystems, most of which were involved in amino acids and derivatives synthesis, carbohydrate and protein metabolism, cofactors, vitamins, prosthetic groups and pigment formations, and stress response (Figure S2). Based on phylogenic analysis, chemical characteristics, and genotypic data described in this report, the isolate S3 belongs to B. velezensis while S6 is attributed to E. cloacae species. The draft genome sequence of each isolate was subject to multiple genomic analyses with the aim to identify all the genes potentially responsible for its antimicrobial activity especially those produced by non- ribosomal peptides synthetases (NRPSs). The in silico analysis using antiSMASH server revealed the presence of different secondary metabolites gene clusters (SMGCs) involved in biocontrol. The draft genome of S3 predicted the presence of many SMGCs, including bacillibactin, fengycin, surfactin and bacillaene. In our analysis of S6 genome, we detected the presence of non-ribosomal peptide (NRPs) and bacteriocin. Microorganisms 2021, 9, 1386 11 of 19 Table 3. Genomic future and in silico analysis of draft genome sequences of bacterial isolates. Attribute S3 S6 Size (bp) 4.15 Mb 4.60 Mb G+C content (%) 46.35 55.90 RNA genes 98 101 Protein-coding genes 3983 4258 N50 959,830 319,447 L50 2 4 Number of Subsystems 328 560 Most frequently species B. velezensis E. cloacae Number of contigs 21 30 3.6. Antagonistic Strains Prevent Plants from Considerable Photo-Inhibition of PSII after Pathogen Challenge Table 3. Genomic future and in silico analysis of draft genome sequences of bacterial isolates. 3.6. Antagonistic Strains Prevent Plants from Considerable Photo-Inhibition of PSII after Pathogen Challenge To evaluate the effect of root inoculation with B. velezensis S3 and E. cloacae S6 on photosynthesis before and 24, 48, 72, and 96 h post-infection with B. cinerea, changes in excitation flux at PSII were monitored. 3.5. Genomic Feature and In Silico Analysis Photosynthetic parameters including effective PSII quantum yield Y(II), quantum yield of nonregulated energy dissipation Y(NO), quantum yield of regulated energy dissipation Y(NPQ), and maximum PSII quantum yield (Fv/Fm) were evaluated. The false-color scales shown at the bottom of the fluorescence images indicate the amplitude of the particular parameter (Figure 3a). Before infection with the pathogen, no significant difference between bacterized and non-bacterized plantlets was observed regarding the monitored photosynthetic parameters. The value Fv/Fm were around 0.8 before the infection (Figure 3b). Furthermore, no significant fluctuation was occurred in Fv/Fm value in plantlets during 3 days after infection. However, considerable PSII photo-inhibition was observed when plants were not treated with bacteria and were exposed to a prolonged infection with B. cinerea (Figure 3b), while at the same time, in response to B. cinerea, bacterized plantlets exhibit indistinct symptoms (Figure 3a), and Fv/Fm value remain constant during kinetics (Figure 3b). Best results were displayed by the two strains. In the case of bacterized plantlets, the higher effective quantum yield of photochemical energy conversion in PSII Y(II) was maintained before as well at 96 hpi with B. cinerea in comparison to the non-bacterized plant, which decreased. The quantum yield of regulated energy n PSII Y(NPQ) was down-regulated before infection, compared to control. In contrast, four days after infection, the bacterized plantlets dissipated a higher Y(NPQ) than control (Figure 3c). Although no difference in the quantum yield of nonregulated energy loss in PSII Y(NO) was observed between plantlets before infection, this response resulted in a lower Y(NO) in bacterized plantlets after a prolonged infection with B. cinerea. 12 of 19 Microorganisms 2021, 9, 1386 Figure 3. B. velezensis S3 and E. cloacae S6 prevent grapevines from PSII photo-inhibition four days after infection with B. cinerea. Images of the effective PSII quantum yield Y(II), the quantum yield of regulated energy dissipation Y(NPQ) and of nonregulated energy dissipation Y(NO), from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea. The pixel value display is based on a false-color scale ranging from black (0.000) via red, yellow, green, blue, to purple (ending at 1.00). The figure shows representative images of one from two independent experiments (A). Evolution of the maximum PSII quantum yield (Fv/Fm) from grapevine leaves inoculated or not with B. velezensis S3 and E. Y(II) Y(NPQ) Y(NO) Figure 3. B. velezensis S3 and E. cloacae S6 prevent grapevines from PSII photo-inhibition four days after infection with B. cinerea. Images of the effective PSII quantum yield Y(II), the quantum yield of regulated energy dissipation Y(NPQ) and of nonregulated energy dissipation Y(NO), from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea. The pixel value display is based on a false-color scale ranging from black (0.000) via red, yellow, green, blue, to purple (ending at 1.00). The figure shows representative images of one from two independent experiments (A). Evolution of the maximum PSII quantum yield (Fv/Fm) from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea (B). Changes in chlorophyll fluorescence parameters (Y(II), Y(NO), and Y(NPQ)) from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea. Excitation flux at PSII in infected leaves (C). Values shown are means ± SD of two independent repetitions (each repetition was realized in triplicates). 3.5. Genomic Feature and In Silico Analysis cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea (B). Changes in chlorophyll fluorescence parameters (Y(II), Y(NO), and Y(NPQ)) from grapevine leaves inoculated or not with B. velezensis S3 and E. cloacae S6 0, 24, 48, 72, and 96 hpi with B. cinerea. Excitation flux at PSII in infected leaves (C). Values shown are means ± SD of two independent repetitions (each repetition was realized in triplicates). Control Control + Bc 630 B. velesenzis + Bc 630 E. cloacea + Bc 630 YII YNPQ YNO YII YNPQ YNO YII YNPQ YNO YII YNPQ YNO 24hPI 72hPI 96hPI 0% 20% 40% 60% 80% 100% Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc 0 hpi 24 hpi 48 hpi 72 hpi 96 hpi Y(II) Y(NPQ) Y(NO) 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0 24 48 72 96 Fv/Fm Hours after infection Control + Bc B. velezensis + Bc E. cloacae + Bc 0 1 A B C Control Control + Bc 630 B. velesenzis + Bc 630 E. cloacea + Bc 630 YII YNPQ YNO YII YNPQ YNO YII YNPQ YNO YII YNPQ YNO 24hPI 72hPI 96hPI A A 0% 20% 40% 60% 80% 100% Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc Control + Bc B. velezensis+ Bc E. cloacae+ Bc 0 hpi 24 hpi 48 hpi 72 hpi 96 hpi 1 C 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0 24 48 72 96 Fv/Fm H ft i f ti 0 B 1 C B 4. Discussion Rhizosphere is an extremely competitive environment, where diverse genera of microorganisms are constantly competing for resources and with each other to survive [42]. This work was undertaken to screen efficient competitive strains from vineyard to control grapevine GM. Among 42, two strains (S3 and S6) have been showed the best performance against the B. cinerea. When plantlets were previously bacterized with S3 or S6 then infected with the B. cinerea, the symptoms of gray mold were reduced compared to control, confirming therefore the beneficial effect of these strains as reported in in vitro confrontation test. Similar results were reported by Miotto-Vilanova et al., [37] when grapevine plantlets were previously bacterized with Burkholderia phytofirmans PsJN before infection with B. cinerea. Microorganisms 2021, 9, 1386 13 of 19 13 of 19 The 16S rRNA gene sequences of S3 exhibited 100% similarity to B. velezensis, while the strain S6 showed 98.5% similarity to E. cloacae. B. velezensis and E. cloacae have been frequently reported as plant growth promoting bacteria and/or biocontrol agents [43–50]. The fact that many Bacilus species have very close phenotypic and physiological properties as well as 16S rRNAs gene sequences makes their classification very difficult [51]. The Bacillus genus encompasses a large genetic biodiversity [52], and in addition to the “original members” B. subtilis, B. licheniformis, and B. pumilus, earlier described by Gordon et al. [53], many novel species belonging to the B. subtilis species complex have been described in recent decades, among them B amyloliquefaciens [54], B. velezensis [55], and B. methylotrophicus [56]. Recently, these Bacillus species have been reclassified by genome comparisons and phylogenomic analyses [57]. In particular, B. methylotrophicus and B. amyloliquefaciens subsp. plantarum were later confirmed as heterotypic synonyms of B. velezensis [58]. Hence, many studies have suggested that B. amyloliquefaciens subsp. Plantarum, B. methylotrophicus, and B. velezensis formed a monophyletic group [59,60]. y p p y g p The plant growth-promoting ability has been related to distinctive physiological activities and molecular changes that might have an intense impact on the fitness (growth and/or health) of plants. Both B. velezensis S3 and E. cloacae S6 have triggered the resistance of grapevine toward GM disease. B. velezensis is a heterotypic synonym of B. methylotrophicus, B. amyloliquefaciens subsp. plantarum, and B. oryzicola, and is used to control plant fungal diseases [61] such as B. cinerea [62–64]. 4. Discussion Bacillus species are promising agent for the biological control of postharvest diseases [52], particularly B. velezensis, which is widely used in agriculture [65]. Additionally, Morales-Cedeño et al. [66] showed that B. velezensis BLE7 showed similar activities to thiabendazole, a commonly used fungicide for B. cinerea [67]. In cells of B. velezensis, the composition of iso and anteiso fatty acids was higher. The a-C15:0 became even more prominent component of the fatty acids. Furthermore, the major changes observed were a sharp decrease in a-C17:0 content in parallel with a significant increase in a-C15:0. The shift to a fatty acid profile dominated by a-C15:0 draws attention to the critical role of this fatty acid in low temperatures (4 °C) growth [68], presumably due to its physical properties and their effects in maintaining a fluid, liquid-crystalline state of membrane lipids [69], making this bacterium a potential effective biocontrol agent in extreme environments. Enterobacter cloacae is perhaps best known as an opportunistic human pathogen that is commonly found in hospitals causing a wide range of infections, although some lineages have also been described as plant endophytes [49]. Indeed, several Enterobacter species can colonize internal plant tissues, improve plant growth and prevent from pathogens attacks [70–74]. Thus, Entrobacter cloacae was found to halt fungal phytopathogens growth such as Phytium debaryanum by 35.13% and Rhizoctonia solani with pathogen growth inhibition up to 60% [75]. Additionally, E. cloacae inhibited the development of Pythium myriotyum, Gaeumannomyces graminis and Heterobasidion annosum [52]. y g The observed inhibition is due to production of several antifungal metabolites such as H2S, ammonia and volatile compounds such as phenylethyl alcohol, 4,5-dimethy l-1- hexene, and butyl acetate that halt the growth of fungal phytopathogens [50,76]. Recently, it has been reported that E. cloacae is able to produce inorganic volatile substances such as ammonia, IAA and hydroxamate siderophore, hydrogen cyanide and salicylic acid [74,77], in addition to chitinase, cellulase, and beta-glucosidase enzyme all of which may participate to the biocontrol activity [73,78–80]. Furthermore, Chaouachi et al. [81] reported for the first time volatile organic compounds (VOCs) with antifungal activity produced by E. cloacae against B. cinerea decay on tomato fruit. Similarly, Bacillus species such as B. velezensis [63,82] are known to produce antifungal VOCs against several phytopathogens including B. cinerea. In addition, Patel et al. [83] and Agbodjato et al. 4. Discussion [84] reported that AIA with ammonia production prevents the development of various plant pathogenic fungi and enhance the plant growth. Additionally, solubilization of phosphate and potassium occurs due to the production of protons and oxalic, tartaric acid and polysaccharidic capsules by bacteria Microorganisms 2021, 9, 1386 14 of 19 14 of 19 [76,85]. Romero et al. [86] have reported that antimicrobial compounds produced by Bacillus spp. are mainly classified into two categories: ribosome-synthesized peptides such as bacteriocin, and small microbial peptides enzymatically synthesized by non-ribosomal pathways, mainly cyclic lipopeptides (CLPs). Since B. velezensis is not pathogenic to humans, different strains of B. velezensis, which is a typical PGPR, have received significant attention in the last decade. [76,85]. Romero et al. [86] have reported that antimicrobial compounds produced by Bacillus spp. are mainly classified into two categories: ribosome-synthesized peptides such as bacteriocin, and small microbial peptides enzymatically synthesized by non-ribosomal pathways, mainly cyclic lipopeptides (CLPs). Since B. velezensis is not pathogenic to humans, different strains of B. velezensis, which is a typical PGPR, have received significant attention in the last decade. So far, B. velezensis was described to halt the growth of many pathogenic fungi, such as Aspergillus flavus [87], Cylindrocladium quinqueseptatum, Cryphonectria parasitica and Helicobasidium purpureum [88], Fusarium oxysporum and Ralstonia solanacearum [89], and Rhizoctonia solani AG1-IB [90], by the biosynthesis of β-1,3-1,4-glucanase, lipopeptide antibiotics (surfactin, iturin, and fengycin, for example), polyketides (actinomycin D, bacitracin, and cyclosporin A, for example), siderophores, and NH3 [44,91–93]. These data are in line with our in silico analysis of both bacteria B. velezensis S3 and E. cloacae S6. Thus, analysis of E. cloacae S6 genome pointed out the presence of non-ribosomal peptide synthetase (NRPS) and bacteriocin, while the genome of strain B. velezensis S3 revealed different SMGCs, such as bacillibactin, fengycin, surfactin and bacillaene. Hence, it was hypothesized that these strains might produce a variety of antifungal compounds that participate in the control of the grapevine GM disease. p p g p The photosynthesis provides near 90–95 % of plants dry matter and the metabolic energy needed for plant’s development. Pathogen attack not only impact plant defenses reactions but can also lead to changes in the rate of photosynthesis and therefore the carbohydrates metabolism [37]. 4. Discussion Hence, four days after infection, it appears that under Botrytis exposure, thermal dissipation in control was down-regulated not due to an increased PSII quantum efficiency, but due to an increased nonregulated energy loss in PSII, suggesting that both photochemical energy conversion and protective regulatory mechanism were insufficient [94]. Consequently, the larger portion of absorbed light energy is allocated to nonregulated energy loss in PSII [95]. The latter parameter indicates an irreversible damage of photosynthetic apparatus as confirmed by the decreased Fv/Fm ratio, since the Y(NO) leads to the formation of singlet oxygen via the triplet state of chlorophyll (3chl*) [96,97]. In line with our results, several reports on photosynthesis have indicated that photosynthesis rates are altered after infection with several plant pathogens [4,95]. We also have observed that the regulation of mechanisms involved in nonphotochemical dissipation of energy was blocked, making grapevine plantlets unable to protect themselves against damage from excessed illumination [37]. A significant decline of effective quantum yield of PSII(Y(II)) complemented by a quantum yield of regulated energy dissipation nonphotochemical chlorophyll fluorescence quenching [Y(NPQ)] increase was observed after the B. cinerea infection. The [Y(NPQ)] is a molecular adaptation that represents the fastest response of the photosynthetic membrane to excess light. It is a protective process in which excess absorbed light energy is dissipated into heat [98,99] and prevent the photosynthetic apparatus from oxidative damage [100]. However, the irreversible damages described above were prevented/attenuated when grapevine plantlets were bacterized either with B. velezensis S3 and E. cloacae S6, probably by restricting mycelial development, and therefore protecting photosynthesis apparatus. Therefore, finding safe and eco-friendly alternatives to synthetic fungicides is urgently needed to control postharvest diseases of fruit [101]. Hence, this study provided new biotechnologies by reporting for the first time that following root inoculation, B. velezensis S3 and E. cloacae S6 allowing grapevine to better resist aerial parasitic pressures of B. cinerea while at the same time prevent grapevine from considerable photo-inhibition. 5. Conclusions and Z.A.; writing—review and editing, E.A.B., Q.E., N.V.- G., Z.A., and M.H.; supervision, E.A.B., N.V.-G., C.J., and M.H.; project administration, E.A.B., N.V.- G. and M.H.; funding acquisition, E.A.B., N.V.-G. and M.H. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, E.A.B., N.V.-G. and M.H.; methodology, E.A.B., N.V.-G., M.H., L.S. and Q.E.; software, Q.E.; validation, E.A.B., N.V.-G., and M.H.; formal analysis, E.A.B., N.V.-G., L.S., Z.A., and Q.E.; investigation, E.A.B., Q. E., N.V.-G., and M.H.; resources, E.A.B., Q.E., N.V.-G. and M.H.; data curation, E.A.B., N.V.-G, Z.A., and M.H.; writing—original draft preparation, E.A.B., Q.E., N.V.-G., M.H. and Z.A.; writing—review and editing, E.A.B., Q.E., N.V.- G., Z.A., and M.H.; supervision, E.A.B., N.V.-G., C.J., and M.H.; project administration, E.A.B., N.V.- G. and M.H.; funding acquisition, E.A.B., N.V.-G. and M.H. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the University of Reims Champagne-Ardenne, and the University Moulay Ismail-Faculty of Sciences via the attribution of the doctoral grant. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In conclusion, both bacteria screened as efficient anti-Botrytis were identified as B. velezensis S3 and E. cloacae S6. In silico analysis of draft genome sequences of B. velezensis S3 indicates the presence of gene clusters involved in amino acids and derivatives synthesis, carbohydrates and proteins metabolism, cofactors, vitamins, prosthetic groups Microorganisms 2021, 9, 1386 15 of 19 15 of 19 and pigment formations, and stress response; while that of the E. cloacae S6 revealed the presence of different secondary metabolites gene clusters, including bacillibactin, fengycin, surfactin and bacillaene and the presence of non-ribosomal peptide synthetase and bacteriocin. Further work is obviously required to characterize how these bacteria trigger the biotic stress resistance in plants to establish a set of biotic stress biomarkers that might help to predict efficacy of induced resistance for different crops. In addition, it would also be very interesting to analyze the plant’s response to colonization, i.e., lipid content, fatty acid composition as well as secondary metabolites profiles. p y p Nevertheless, genus Enterobacter is a member of the ESKAPE group, which contains the major opportunistic and multi-resistant bacterial pathogens for humans during recent decades in hospital wards. Therefore, a deeper progress in genome sequencing of E. cloacae S6 is critical before a potential use of this strain for plant protection. Additionally, deciphering the mechanisms of horizontal gene transfer that may occur in specific microhabitats may be a key step in the development of a regulatory framework for the environmental release of these bacteria. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/article/10.3390/microorganisms9071386/s1. Table S1. Project information of the draft genome sequences of the strains. Figure S1. Proportion of different structure in a fatty acid profile of isolated strains. Figure S2. Subsystem information of the strain B. velezensis S3 and E. cloacae S6 and predicted by SEED viewer, most of which were involved in amino acids and derivatives synthesis, carbohydrate metabolism, cofactors, vitamins, prosthetic groups and pigment formations, and stress response. Author Contributions: Conceptualization, E.A.B., N.V.-G. and M.H.; methodology, E.A.B., N.V.-G., M.H., L.S. and Q.E.; software, Q.E.; validation, E.A.B., N.V.-G., and M.H.; formal analysis, E.A.B., N.V.-G., L.S., Z.A., and Q.E.; investigation, E.A.B., Q. 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Genome-wide identification of microsatellite markers from cultivated peanut (Arachis hypogaea L.)
BMC genomics
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Lu et al. BMC Genomics (2019) 20:799 https://doi.org/10.1186/s12864-019-6148-5 Lu et al. BMC Genomics (2019) 20:799 https://doi.org/10.1186/s12864-019-6148-5 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Genome-wide identification of microsatellite markers from cultivated peanut (Arachis hypogaea L.) Qing Lu1†, Yanbin Hong1†, Shaoxiong Li1, Hao Liu1, Haifen Li1, Jianan Zhang2, Haofa Lan2, Haiyan Liu1, Xingyu Li1, Shijie Wen1, Guiyuan Zhou1, Rajeev K. Varshney3, Huifang Jiang4, Xiaoping Chen1* and Xuanqiang Liang1* Abstract Background: Microsatellites, or simple sequence repeats (SSRs), represent important DNA variations that are widely distributed across the entire plant genome and can be used to develop SSR markers, which can then be used to conduct genetic analyses and molecular breeding. Cultivated peanut (A. hypogaea L.), an important oil crop worldwide, is an allotetraploid (AABB, 2n = 4× = 40) plant species. Because of its complex genome, genomic marker development has been very challenging. However, sequencing of cultivated peanut genome allowed us to develop genomic markers and construct a high-density physical map. Results: A total of 8,329,496 SSRs were identified, including 3,772,653, 4,414,961, and 141,882 SSRs that were distributed in subgenome A, B, and nine scaffolds, respectively. Based on the flanking sequences of the identified SSRs, a total of 973,984 newly developed SSR markers were developed in subgenome A (462,267), B (489,394), and nine scaffolds (22,323), with an average density of 392.45 markers per Mb. In silico PCR evaluation showed that an average of 88.32% of the SSR markers generated only one in silico-specific product in two tetraploid A. hypogaea varieties, Tifrunner and Shitouqi. A total of 39,599 common SSR markers were identified among the two A. hypogaea varieties and two progenitors, A. duranensis and A. ipaensis. Additionally, an amplification effectiveness of 44.15% was observed by real PCR validation. Moreover, a total of 1276 public SSR loci were integrated with the newly developed SSR markers. Finally, a previously known leaf spot quantitative trait locus (QTL), qLLS_T13_A05_7, was determined to be in a 1.448-Mb region on chromosome A05. In this region, a total of 819 newly developed SSR markers were located and 108 candidate genes were detected. Conclusions: The availability of these newly developed and public SSR markers both provide a large number of molecular markers that could potentially be used to enhance the process of trait genetic analyses and improve molecular breeding strategies for cultivated peanut. Keywords: Genome sequence, Simple sequence repeats, Molecular breeding, Peanut (Arachis hypogaea L.) Background fighting malnutrition and ensuring food security. In China, peanut accounts for almost half of the total output of all oil crops and is increasingly important as an oil and pro- tein crop. Therefore, it is critical to improve peanut pro- duction and quality to ensure an edible oil supply. Cultivated peanut or groundnut (Arachis hypogaea L.) is a globally important legume that is widely planted in Asia, Africa, America, and other areas because it is rich in seed oil and protein. Thus, peanut has great significance for However, peanut production is often constrained by factors such as drought, salinity, and disease [1]. During the past 10 years, with the development of peanut gen- omics, there have been several successful achievements in peanut trait mapping [2–5] and molecular breeding [6, 7]. Owing to the limited available genetic markers * Correspondence: chenxiaoping@gdaas.cn; liangxuanqiang@gdaas.cn †Qing Lu and Yanbin Hong contributed equally to this work. 1Crops Research Institute, Guangdong Academy of Agricultural Sciences, South China Peanut Sub-Center of National Center of Oilseed Crops Improvement, Guangdong Provincial Key Laboratory for Crop Genetic Improvement, Guangzhou 510640, China Full list of author information is available at the end of the article Whole genome identification of SSRs In this study, the whole genome sequence of A. hypo- gaea cv. Fuhuasheng was used to identify SSRs with dif- ferent repeat motifs, from mono- to hexa-nucleotide. A total of 8,329,496 SSRs were obtained, with a density of ~ 3264.31 SSRs per Mb (Table 1; Additional file 1: Table S1). Penta-nucleotide was the most common type, accounting for more than half of all identified SSRs (57.75%), followed by hexa-nucleotide (26.61%) (Table 1). In addition, more SSRs were identified in subgenome B (4,414,961) than in subge- nome A (3,772,653), and 141,882 SSRs were identified on the nine scaffolds of the peanut reference genome assembly (Table 1). Analysis of SSR distribution on each chromosome re- vealed that the largest number of SSRs was present on chromosome A07 (538,928), followed by chromosome B09 (532,326) (Additional file 1: Table S1; Additional file 2: Fig- ure S1). Pearson correlation analysis revealed that chromo- some length was significantly positively associated with the number of SSRs of each chromosome (r = 0.996, p < 0.01) (Additional file 1: Table S1). Furthermore, the average dens- ity of SSRs was 3283.89 SSRs per Mb, ranging from 3164.75 SSRs per Mb on chromosome A10 to 3843.36 SSRs per Mb on chromosome A08 (Additional file 1: Table S1; Additional file 2: Figure S1). Moreover, the densities of SSRs on chro- mosomes A08 and B03 were the highest in subgenomes A and B, respectively (Additional file 2: Figure S1). g Cultivated peanut is an allotetraploid (AABB, 2n = 4× = 40) that probably derived from hybridization be- tween two diploids, A. duranensis and A. ipaensis [18, 19]. Recently, breakthroughs have been made in peanut genome sequencing. From 2016 to 2018, the genomes of the two diploid progenitors and one allotetraploid wild species, A.monticola, were successfully sequenced [20– 23]. Based on the genomic sequences of the two diploid progenitors, genome-wide g-SSRs were identified and developed, and a high-density SSR physical map of wild peanut species was constructed [24]. Importantly, in 2019, cultivated peanut genomics research experi- enced a substantial milestone when genome sequen- cing was completed for three cultivated peanut : Fuhuasheng [25], Shitouqi [26], and Tifrunner [27]. The high-quality genome assemblies provide the opportunity for developing genome-wide g-SSR markers in cultivated peanut. p y ( g ) A total of 501 types of SSR motifs were identified in the peanut genome (Additional file 1: Table S2). Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 Page 2 of 9 novel, newly developed g-SSR markers could be helpful for advancing agronomic trait mapping, gene cloning, and molecular breeding of cultivated peanut in the future. and low-density genetic maps, those studies could not provide optimal resolution of trait dissection and identify candidate genes. Therefore, the development of a high- density genetic map is particularly urgent for peanut trait mapping and breeding. Whole genome identification of SSRs The re- peat number of all of these motifs ranged from 2 to 335, most of which were concentrated in the top of 50s (Additional file 2: Figure S2a). The proportions of penta- and hexa-nucleotides with two repeats were much greater than those of other types in both subgenome A and B, and ranged from ~ 20 to 50% (Additional file 2: Figure S2b). Of the SSR motif types, the penta-nucleotide type, AAAAT/ATTTT, had the highest occurrence, and accounted for 9.55% of all types, followed by AAATT/ AATTT and AAAAG/CTTTT, which accounted for 4.97 and 4.85%, respectively (Additional file 2: Figure S3). For each type of SSR motif, from mono- to hexa-nucleotide, the richest motifs were T (1.48%), TA (1.25%), AAT (0.37%), AAAT (0.34%), AAAAT (1.44%), and ACGCGT (0.64%). Here, we identified genome-wide g-SSRs and devel- oped g-SSR markers from the genome assembly of A. hypogaea cv. Fuhuasheng, a landrace from North China that was sequenced in our previous work [25]. The aims of this study were to: (1) identify genome-wide SSRs and show the distribution of motif length, type, and repeat number between the two subgenomes (A and B); (2) de- velop g-SSR markers and construct a high-density SSR physical map of cultivated peanut; and (3) evaluate the application of these SSR markers and validate the poly- morphisms in different cultivated peanut species. These Results and discussion Simple sequence repeats (SSRs) are genomic fragments that consist of tandemly repeated units that are present in both coding and non-coding regions of the genome [8, 9]. SSR markers, designed by flanking sequences, are useful for and widely applied in plant genetic analyses and marker-assisted selection breeding. SSRs derived from expressed sequence tags (ESTs), transcriptome se- quences, and genomic DNA sequences are referred to as EST-SSRs, transcriptome-SSRs, and g-SSRs, respectively. In the past decade, several hundred EST-SSR markers were developed by investigating ESTs [10, 11], and thou- sands of transcriptome-SSR markers were identified based on different transcriptome libraries of cultivated peanut [12–14]. Meanwhile, two integrated consensus genetic maps with thousands of different types of markers, such as EST-SSRs, transcriptome-SSRs, and g- SSRs, were constructed [15, 16]. Limited by the poly- morphisms, these available SSRs were insufficient for constructing high-density genetic maps and enhancing molecular breeding [17]. Genome-wide SSR marker development The flanking sequences of all identified SSRs were used to design suitable forward and reverse primer pairs. A total of 973,984 SSR markers were successfully devel- oped on 20 chromosomes and nine scaffolds in peanut Lu et al. BMC Genomics (2019) 20:799 Page 3 of 9 Table 1 Different types of SSRs identified in A. hypogaea L SSR types SSR Number Proportion (%) Subgenome A Proportion (%) Subgenome B Proportion (%) Scaffolds Mono-nucleotide 255,774 3.07 112,466 2.98 138,624 3.14 Di-nucleotide 376,173 4.52 167,574 4.44 202,132 4.58 Tri-nucleotide 337,026 4.05 155,918 4.13 175,197 3.97 Tetra-nucleotide 334,132 4.01 143,250 3.8 185,051 4.19 Penta-nucleotide 4,810,032 57.75 2,189,218 58.03 2,538,767 57.5 Hexa-nucleotide 2,216,359 26.61 1,004,227 26.62 1,175,190 26.62 Total 8,329,496 3,772,653 4,414,961 141,882 Table 1 Different types of SSRs identified in A. hypogaea L density in the middle of each chromosome except for chromosome B07 (Figs. 1 and 2). In addition, for all SSR markers in subgenome A, subgenome B, and the scaf- folds, penta-nucleotide SSRs were the most abundant (491,927) (Additional file 2: Figure S4a) and accounted for more than half of all SSRs (~ 50.51%) (Additional file 2: Figure S4b). Hexa-nucleotide SSRs were the second most abundant (199,408),and represented 20.47% of the SSRs in the two subgenomes and scaffolds, followed by compound and di-nucleotide SSRs. Of the repeat motif types, TA/AT was most abundant, accounting for more than 4.0% of all repeat motifs (Additional file 2: Figure S5). The second highest number of repeat motifs was AAAAT/TTTTA, which represented 3.4% of all repeat motifs. These analyses showed that the AT repeat patterns were the dominant re- peat motifs of SSR markers, whereas GC repeat patterns were rare. (Additional file 1: Table S1), which accounted for 11.69% of all identified SSRs. Of these newly developed SSR markers, a total of 462,267 and 489,394 SSR markers were located in subgenomes A (Additional file 3: Table S3 and Additional file 4: Table S4) and B (Additional file 5: Table S5 and Additional file 6: Table S6), which accounted for 47.46 and 50.25% of all SSR markers, respectively (Additional file 1: Table S1). In addition, 22,323 SSR markers were mined on the nine scaffolds, and only accounted for 2.29% of all SSR markers (Additional file 7: Table S7). Based on the start positions of SSR markers, we suc- cessfully anchored these markers to the reference gen- ome physical map (Fig. 1). Genome-wide SSR marker development Currently, g-SSR markers are common and popular for such analyses, and they have wide applications in mo- lecular genetics and breeding, because they have multiple advantages, such as simplicity, abundance, ubiquity, vari- ation, co-dominance, and multi-allelism [28]. Recently, several studies were devoted to developing different types of SSR markers in peanut, such as EST-SSRs [11, 29], transcriptome-SSRs [12–14], and g-SSRs [30], even though the peanut genome had not yet been resolved. With the recent completion of genome sequencing of pea- nut and two diploid progenitor species, A. duranensis and A. ipaensis, a large number of genome-wide g-SSRs were identified [20–22]. Furthermore, tens of thousands of g- SSR markers (51,354 for A. duranensis and 60,893 for A. ipaensis) were also developed from the two progenitor species in 2017 [24]. However, there was limited reports on the development of a large number of genome-wide g- SSR markers from allotetraploid cultivated peanut because of the challenging in its genome sequencing. Fortunately, the genomes of allotetraploid A. hypogaea cv. Fuhuasheng, Shitouqi, and Tifrunner were successfully sequenced [25–27]. Here, large-scale genome-wide g-SSR markers were developed from the A. hypogaea cv. Fuhuasheng genome to help enhance genetic and genomic analyses and molecular breeding of peanut. crops. Currently, g-SSR markers are common and popular for such analyses, and they have wide applications in mo- lecular genetics and breeding, because they have multiple advantages, such as simplicity, abundance, ubiquity, vari- ation, co-dominance, and multi-allelism [28]. Recently, several studies were devoted to developing different types of SSR markers in peanut, such as EST-SSRs [11, 29], transcriptome-SSRs [12–14], and g-SSRs [30], even though the peanut genome had not yet been resolved. With the recent completion of genome sequencing of pea- nut and two diploid progenitor species, A. duranensis and A. ipaensis, a large number of genome-wide g-SSRs were identified [20–22]. Furthermore, tens of thousands of g- SSR markers (51,354 for A. duranensis and 60,893 for A. ipaensis) were also developed from the two progenitor species in 2017 [24]. However, there was limited reports on the development of a large number of genome-wide g- SSR markers from allotetraploid cultivated peanut because of the challenging in its genome sequencing. Fortunately, the genomes of allotetraploid A. hypogaea cv. Fuhuasheng, Shitouqi, and Tifrunner were successfully sequenced [25–27]. Here, large-scale genome-wide g-SSR markers were developed from the A. hypogaea cv. Fuhuasheng genome to help enhance genetic and genomic analyses and molecular breeding of peanut. Genome-wide SSR marker development For the physical map, the marker density significantly differed among chromo- somes, and ranged from 338.38 per Mb on chromosome B01 to 699.92 per Mb on chromosome A08, with an average density of 392.45 per Mb (Additional file 1: Table S1; Fig. 2). Moreover, the distribution of each chromosome showed that there was lower marker Development of new markers can help improve genetic analysis, gene/QTL mapping, and molecular breeding of Fig. 1 Overview of the high-density SSR physical map in peanut (A. hypogaea L.).The bar represents the number of SSR markers within a 1-Mb window Fig. 1 Overview of the high-density SSR physical map in peanut (A. hypogaea L.).The bar represents the number of SSR markers within a 1-Mb window Lu et al. BMC Genomics (2019) 20:799 Page 4 of 9 Fig. 2 Genome-wide distribution of SSR markers on different chromosomes of the A. hypogaea genome. From the outer edge inward, circles represent the (a) gene position; (b) distribution of all genes; (c) gene density per Mb; (d) distribution of the 1276 integrated public SSR markers; (e) distribution of the 188 SSRs tested by PCR amplification; (f) distribution of all newly developed SSR markers; and (g) SSR marker density per Mb. Blue and red lines represent homologous loci in different and the same chromosomes, respectively Fig. 2 Genome-wide distribution of SSR markers on different chromosomes of the A. hypogaea genome. From the outer edge inward, circles represent the (a) gene position; (b) distribution of all genes; (c) gene density per Mb; (d) distribution of the 1276 integrated public SSR markers; (e) distribution of the 188 SSRs tested by PCR amplification; (f) distribution of all newly developed SSR markers; and (g) SSR marker density per Mb. Blue and red lines represent homologous loci in different and the same chromosomes, respectively In silico evaluation of the newly developed SSR markers To evaluate the amplification specificity of the newly de- veloped SSR markers, the forward and reverse primers of each SSR marker were used for in silico analysis based on the cultivated peanut genome sequences of A. hypo- gaea cv. Tifrunner and Shitouqi, and genome sequences of its two progenitors, A. duranensis and A. ipaensis (Table 2). crops. Integration of publicly available SSR markers g p y Previously, an integrated consensus genetic map, which contained 5874 markers, was successfully constructed with 20 linkage groups [16]. Based on forward and re- verse primer sequences, a total of 5125 markers were mapped to the reference genome, A. hypogaea cv. Fuhuasheng. Finally, 3304 markers with 6838 loci were mined from 20 chromosomes (6699 loci) and nine scaf- folds (139 loci) (Additional file 2: Figure S7). In addition, most of the markers (2927), which accounted for 88.6% of all markers, only matched one or two loci. For ex- ample, approximately 1832 markers only matched a sin- gle locus (Additional file 7: Table S10), and a total of 1095 markers were double loci identification on the refer- ence genome (Additional file 2: Figure S7). The remaining markers had more than three loci, and one marker even matched 258 loci (Additional file 2: Figure S7). Further- more, most of the multiple loci of these markers were lo- cated in the corresponding subgenomic chromosomes, such as A01 vs. B01 and A02 vs. B02 (Fig. 2). This may have been caused by co-evolution of the two peanut subgenomes. After clumped, a total of 39,599 SSR markers were shared among the four Arachis species, which indicates that these common markers were very conservative in different Arachis (Fig. 3; Additional file 7: Table S8). Moreover, 9334 and 210,058 markers overlapped be- tween the two progenitors and two cultivated varieties, respectively. In particular, numerous specific SSR markers were also obtained for each genome, which in- dicates genomic polymorphism among the four different Arachis species. Genome-wide SSR marker development The number of in silico products indicated that 87.35% (855,422) and 89.29% (874,384) of the SSR markers generated only one in silico-specific product in the twotetraploids A. hypogaea cv. Tifrunner and Shi- touqi, respectively. Approximately 4% of markers were mismatched in the two cultivated varieties, and less than 8% of markers generated two products. In addition, less than 0.8% of SSR markers generated more than two products; in particular, only 0.08 and 0.11% of the markers generated ≥6 in silico products. For the two progenitors, a total of 356,778 and 410,764 SSR markers, which accounted for 36.43 and 41.59% of all SSR markers, respectively, generated only one in silico- specific product. However, more than half of total markers were mismatched in the two progenitors. Ap- proximately 3% of all markers generated two in silico products, and less than 0.5% of the markers generated Lu et al. BMC Genomics (2019) 20:799 Page 5 of 9 Table 2 In silico PCR products in A. hypogaea and its two progenitors specise 0 1 2 3 4 5 ≥6 A. hypogaea cv. Tifrunner 40,116 (4.10%) 855,422 (87.35%) 72,710 (7.42%) 7789 (0.80%) 1850 (0.19%) 600 (0.06%) 804 (0.08%) A. hypogaea cv. Shitouqi 40,210 (4.11%) 874,384 (89.29%) 53,994 (5.51%) 7162 (0.73%) 1777 (0.18%) 667 (0.07%) 1097 (0.11%) A. duranensis 581,706 (59.4%) 356,778 (36.43%) 34,490 (3.52%) 4668 (0.48%) 933 (0.10%) 293 (0.03%) 423 (0.04%) A. ipaensis 532,523 (54.38%) 410,764 (41.59%) 30,814 (3.15%) 4120 (0.42%) 607 (0.06%) 199 (0.02%) 264 (0.03%) more than two in silico products (Table 2). In total, 1, 729,806 and 767,542 markers could generate only one in silico product in cultivated peanut and its two progeni- tors, respectively. These SSR markers that only gener- ated one in silico product would be potentially useful for molecular breeding in the future. varieties. These results indicated that the newly developed SSR markers were available and could be useful for molecu- lar breeding strategies, such as true hybrid F1 offspring selec- tion, in the future. PCR validation of the newly developed SSR markers in different species To validate amplification of the newly developed SSR markers, a total of 188 SSR markers with motifs ≥5 repeats were arbitrarily and evenly selected for PCR amplification in two cultivars, Fuhuasheng and Yueyouhei4hao (Additional file 7: Table S9). In total, 83 of the SSR markers (44.15%) could amplify clear target products in at least one of the two varieties (Additional file 2: Figure S6). Moreover, 25 of the 83 SSR markers displayed polymorphism between the two Fig. 3 Venn diagram of SSR markers in different Arachis species Fig. 3 Venn diagram of SSR markers in different Arachis species Page 6 of 9 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 Page 6 of 9 Page 6 of 9 Of these 1832 unique loci that matched markers, a total of 1276 SSR markers were located in the corre- sponding genetic linkage groups (Additional file 7: Table S10). Based on the physical positions of the 1276 pub- licly available SSR markers, they were successfully merged with the physical map and anchored to the draft genome sequence of tetraploid cultivated peanut (Fig. 2). This map, which has the highest marker density and the most uniform genetic background, will be of great bene- fit to molecular breeding, and gene and QTL mining of peanut in the future. near this QTL, GNB464 and TC40D04 were successfully anchored in a 1.448-Mb region on chromosome A05 in the A. hypogaea cv. Fuhuasheng reference genome (Additional file 2: Figure S8d). Searching this region in the high-density SSR physical map revealed a total of 819 newly developed markers (Additional file 2: Figure S8d). Gene identification of this region indicated that there were 108 genes, and functional annotation showed that multiple genes were re- lated to disease resistance, such as the TMV resistance pro- tein and NAC domain (Additional file 7: Table S11). Genetic linkage mapping is an effective strategy for QTL identification in crops. However, a high-density gen- etic map is a prerequisite for QTL fine mapping of mul- tiple phenotypic traits. In peanut, most QTLs were only preliminarily mapped because of the lack of sufficiently available genetic markers in the last decade [33–37]. Therefore, the development of molecular markers has be- come increasingly important. Then, high-density inte- grated consensus maps with thousands of markers were constructed in 2013 and 2018 [15, 16]. PCR validation of the newly developed SSR markers in different species However, in culti- vated peanut, development of high-density physical markers was rare. Here, we developed millions of SSR markers in cultivated peanut, and this large number of newly developed SSR markers, such as multiple QTLs on chromosome A05 or even on the region of qLLS_T13_ A05_7, could provide more possibilities for further fine mapping or even gene cloning. A high-density physical map with uniform genomic positions and coverage is necessary for conducting high- resolution gene/QTL mapping in crops. During the past years, several available QTLs for yield, disease resistance, and other traits were detected in peanut (https://www. peanutbase.org/). However, most of the QTLs were de- tected across different genetic backgrounds and environ- ments, and only a few QTLs have been used for molecular breeding; therefore, the use of the QTLs in molecular breeding has been limited [31]. Thus, consen- sus genetic map construction was needed to improve the use of QTLs in molecular breeding. Therefore, two inte- grated consensus genetic maps with thousands of markers were constructed in 2013 and 2018 [15, 16], and multiple independent consensus QTLs were identi- fied [16]. Here, to obtain a comprehensive consensus map, we integrated the published public SSR markers with the newly developed SSR physical map (Fig. 1). The availability of a high-density physical map would provide an opportunity to generate high-throughput genotyping data for different types of populations, and accelerate mapping and breeding applications of different traits. Application of SSR markers in peanut High-density SSR physical maps can be used to screen more markers for QTL fine mapping. For example, a previous study reported that multiple novel QTLs for re- sistance to leaf spots and tomato spotted wilt virus were identified in peanut based on an improved genetic link- age map with a total of 418 markers, and multiple resist- ance QTL clusters were detected on linkage A05, which only contained 24 markers [32]. Based on the physical position of the marginal linkage markers (PM179– TC40D04), a total of 6804 new SSR markers were ob- tained in this region (Additional file 2: Figure S8a). On the A05 linkage, a total of 11 leaf spot QTLs were de- tected, especially for the QTL qLLS_T13_A05_7, which had the largest LOD value (5.26) and phenotypic vari- ation explanation (15.55%) (Additional file 2: Figure S8b). Moreover, this QTL overlapped with our previous meta-QTL MQTL_LLS_A05.1, sharing the common marker GNB464 (Additional file 2: Figure S8b and c) [16]. Based on the sequences of the flanking markers Conclusions Development of new genome-wide markers and construc- tion of a physical map with uniformly distributed genome- wide physical markers would aid in the elucidation of complex traits and improvement of molecular breeding. In this study, we identified a total of 8,329,496 genomic SSRs and developed 973,984 newly genomic SSR markers from the cultivated peanut reference genome “Fuhua- sheng” using MISA software with default parameters. Moreover, we mined a number of 39,599 common SSR markers from two A. hypogaea cv. Tifrunner and Shitouqi and two progenitors, A. duranensis and A. ipaensis. In addition, we integrated 1276 public SSR loci with the newly developed physical map, and obtained the first high-density genomic physical map for peanut. Finally, we fine mapped a leaf spot quantitative trait locus to a 1.448- Mb region by marker encryption and identified 108 candi- date genes. In summary, these newly developed and inte- grated public SSR markers are an important genomic resource for both accelerating genetic analyses of complex traits and molecular breeding applications in peanut. Supplementary information S l i f i Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-019-6148-5. pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-019-6148-5. Additional file 1: Table S1. Number of SSRs and SSR markers identified on different chromosomes. Table S2. Summary of SSR motifs and repeats. Additional file 1: Table S1. Number of SSRs and SSR markers identified on different chromosomes. Table S2. Summary of SSR motifs and repeats. y Additional file 2: Figure S1. Chromosome-wide distribution of SSRs in A. hypogaea cv. Fuhuasheng genome. Figure S2. Number of SSR repeat motifs. Figure S3. Abundance of the top 30 different types of SSR motifs. Figure S4. Number (A) and percentage (B) of different types of SSR markers. Figure S5. Summary of SSR types of the developed SSR markers. Figure S6. Product size of 188 SSR markers tested by PCR amplification. Figure S7. Number of loci in the public SSR markers as determined by e-PCR remapping. Figure S8. Comparison of known QTLs in genetic and physical maps. An R package (http://www.R-project.org), Cmplot, was used to draw the high-density physical map of the newly developed SSR markers, and another R package, Rcircos, was used to visualize various features of each chromo- some, such as gene position and distribution, the density of all SSR markers, and the distribution of the tested SSR markers. Additional file 3: Table S3. Summary of SSR markers in subgenome A01–A05. Additional file 3: Table S3. Summary of SSR markers in subgenome A01–A05. Additional file 4: Table S4. Summary of SSR markers in subgenome A06–A10. Additional file 4: Table S4. Summary of SSR markers in subgenome A06–A10. In silico evaluation and PCR validation of SSR markers Four whole genome sequences, including those of two cultivated peanut landraces, A. hypogaea cv. Shitouqi [26] and Tifrunner [27], and their two diploid progeni- tors, A. duranensis and A. ipaensis [20], were used as templates for in silico evaluation of the designed SSR markers using electronic PCR (e-PCR) with the follow- ing parameters: 4-bp mismatch, 1-bp gap, and 0–2000- bp product size [40]. The paired primers should meet Additional file 5: Table S5. Summary of SSR markers in subgenome B01–B05. Additional file 5: Table S5. Summary of SSR markers in subgenome B01–B05. Additional file 6: Table S6. Summary of SSR markers in subgenome B06–B10. Additional file 7: Table S7. Summary of SSR markers on nine scaffolds. Table S8. Plant materials and DNA isolation Plant materials and DNA isolation A cultivated peanut that was de novo sequenced in our previous study [25], A. hypogaea cv. Fuhuasheng, was Page 7 of 9 Page 7 of 9 Page 7 of 9 Lu et al. BMC Genomics (2019) 20:799 the following criteria: (1) only one in silico PCR product generated from any one template; (2) the SSR basic motif was as expected; and (3) the PCR product length was also as expected. used as the reference genome for SSR identification. Two peanut varieties, Fuhuasheng and Yueyouhei4hao, were used for amplification and validation of the newly developed SSR markers by polymerase chain reaction (PCR). All of the materials were planted in a field in the summer season (July to November) at the experimental station of Guangdong Academy of Agricultural Sciences, Guangzhou, China. High-quality genomic DNA was ex- tracted from young leaves using a Plant Genomic DNA Extraction Kit (BioTeke Corporation, Beijing, China) ac- cording to the manufacturer’s handbook (http://www. bioteke.com/en/?c=show&m=view&id=99). p A total of 188 newly developed SSR primer pairs were arbitrarily selected and then synthesized for validation by PCR amplification. The PCR mixture was prepared in a 10-μl volume that contained 1 μl template DNA (~ 100 ng), 0.4 μl of each primer, 3.2 μl ddH2O, and 5 μl 2× Power Taq PCR Master Mix (BioTeke Corporation, Beijing, China). The PCR amplification program was as follows: (1) 95 °C for 5 min; (2) total of 35 cycles, each cycle was as below: 30 s at 95 °C, 30s at 55 °C for anneal- ing, 30 s at 72 °C for extension; (3)10 min at 72 °C, and then storage at 4 °C. PCR products were separated and tested in Fragment Analyzer™Automated CE System (Advanced Analytical Technologies, Inc., Beijing, China), and PROSize 2.0 was used to analyze and visualize the data (https://www.aati-us.com/support/software/). Identification of SSRs and design for SSR markers Identification of SSRs and design for SSR markers The reference genome sequence of A. hypogaea cv. Fuhuasheng was downloaded from GenBank under ac- cession number SDMP00000000 [25]. Perl scripts from MISA were used to perform SSR identification with the default parameters (http://pgrc.ipk-gatersleben.de/misa/ ). The identification criteria were as follows: mono- nucleotide repeat motifs with at least 12 repeats, di- nucleotide repeat motifs with five repeats, tri-nucleotide repeat motifs with four repeats, tetra-nucleotide repeat motifs with three repeats, and penta- and hexa- nucleotide repeat motifs with two repeats. Compound SSRs were defined as those with a < 100-nt interval be- tween two repeat motifs [24, 38]. Integrating new and previously published SSR markers In a previous study, we constructed an integrated con- sensus genetic map with published markers, including EST-SSR, transcriptome-SSR, and g-SSR markers [16]. Based on the forward and reverse primer sequences of each marker, these public SSR markers were mapped to the A. hypogaea cv. Fuhuasheng reference genome for integration with the newly developed SSR markers using e-PCR software with default parameters [40]. The forward and reverse primers of each SSR were de- signed based on the flanking sequences of SSR repeat motifs using Primer 3 [39]. Two Perl scripts, p3_in.pl and p3_out.pl, were used for the programmer-to- programmer data interchange between MISA and Pri- mer 3.0 (http://pgrc.ipk-gatersleben.de/misa/primer3. html). The primer design criteria were as follows: primer length was between 18 and 27 bp, melting temperature was 57 to 63 °C, GC content ranged from 30 to 70%, and product size was from 100 to 300 bp. Funding 10. Guo B, Chen X, Hong Y, Liang X, Dang P, Brenneman T, et al. Analysis of gene expression profiles in leaf tissues of cultivated peanuts and development of EST-SSR markers and gene discovery. Int J Plant Genomics. 2009;715605. https://doi.org/10.1186/1471-2229-9-35. This work was supported by the National Natural Science Foundation of China (31771841, 31801401), the National key research and development program (2018YFD0201009), the Science and Technology Program of Guangzhou, China (201904010190), the Open Fund of Guangdong Key Laboratory of Crop Genetic Improvement (2017B030314090), the Natural Science Foundation of Guangdong Province (2017A030311007), the Modern Agro-industry Technology Research System (CARS-13), the teamwork projects funded by Guangdong Natural Science Foundation (No.2017A030312004), “Jinying Star” (2018) and “Youth Associate Research Fellow” (2019) Talent Pro- ject of Guangdong Academy of Agricultural Sciences. These funding bodies had no role in the design of the study, extraction, analysis, and interpretation of data, or in writing the manuscript. 11. Liang X, Chen X, Hong Y, Liu H, Zhou G, Li S, et al. Utility of EST derived SSR in cultivated peanut (Arachis hypogaea L.) and Arachis wild species. BMC Plant Biol. 2009;9:35. 12. Zhang J, Liang S, Duan J, Wang J, Chen S, Cheng Z, et al. De novo assembly and characterization of the transcriptome during seed development, and generation of genic-SSR markers in Peanut (Arachis hypogaea L.). BMC Genomics. 2012;13:90. 13. Huang L, Wu B, Zhao J, Li H, Chen W, Zheng Y, et al. Characterization and transferable utility of microsatellite markers in the wild and cultivated Arachis species. PLoS One. 2016;11:e0156633. Supplementary information S l i f i Summary of common SSR markers in four Arachis species. Table S9. Summary of 188 SSR markers tested by PCR amplification. Table S10. Summary of the public SSR markers with only a single locus match to the reference genome. Table S11. Candidate gene identification and annotation for the target QTL, qLLS_T13_A05_7. Page 8 of 9 Page 8 of 9 Page 8 of 9 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics Acknowledgments 7. Pandey MK, Roorkiwal M, Singh VK, Ramalingam A, Kudapa H, Thudi M, et al. Emerging genomic tools for legume breeding: current status and future prospects. Front Plant Sci. 2016;7:455. Consent for publication Consent for publication Not applicable. 16. Lu Q, Liu H, Hong YB, Li H, Liu HY, Li XY, et al. Consensus map integration and QTL meta analysis narrowed a locus for yield traits to 0.7 cM and refined a region for late leaf spot resistance traits to 0.38 cM on linkage group A05 in peanut (Arachis hypogaea L.). BMC Genomics. 2018;19:887. Not applicable. group A05 in peanut (Arachis hypogaea L.). BMC Genomics. 2018;19 Received: 27 July 2019 Accepted: 29 September 2019 Received: 27 July 2019 Accepted: 29 September 2019 22. Lu Q, Li H, Hong Y, Zhang G, Wen S, Li X, et al. Genome sequencing and analysis of the peanut B-genome progenitor (Arachis ipaensis). Front Plant Sci. 2018;9:1099. Abbreviations 5. Mondal S, Badigannavar AM. Mapping of a dominant rust resistance gene revealed two R genes around the major rust_QTL in cultivated peanut (Arachis hypogaea L.). Theor Appl Genet. 2018;131:1671–81. ESTs: Expressed sequence tags; PCR: Polymerase chain reaction; QTL: Quantitative trait locus; SSR: Simple sequence repeats 6. Varshney RK, Mohan SM, Gaur PM, Gangarao NV, Pandey MK, Bohra A, et al Achievements and prospects of genomics-assisted breeding in three legume crops of the semi-arid tropics. Biotechnol Adv. 2013;31:1120–34. Authors’ contributions XPC and XQL designed the experiments. QL, YBH, JNZ and HFL1 analyzed the data. HL and HFL2 developed SSR markers. HYL, XYL and SW performed PCR experiments. QL and YBH and HFJ drafted the manuscript. SXL, GYZ and Varshney RK revised the manuscript, respectively. All authors have read and approved the final manuscript. 1 Haofa Lan; 2 Haifen Li. 8. Gupta PK, Balyan HS, Sharma PC, Ramesh B. Microsatellites in plants: a new class of molecular markers. Curr Sci. 1996;70:45–54. 9. Haq SU, Jain R, Sharma M, Kachhwaha S, Kothari SL. Identification and characterization of microsatellites in expressed sequence tags and their cross transferability in different plants. Int J Genomics. 2014; 2014:863948. See: https://www.ncbi.nlm.nih.gov/pubmed/25389527. Author details 1 18. Kochert G, Halward T, Branch WD, Simpson CE. RFLP variability in peanut (Arachis hypogaea L.) cultivars and wild species. Theor Appl Genet. 1991;81:565–70. 1Crops Research Institute, Guangdong Academy of Agricultural Sciences, South China Peanut Sub-Center of National Center of Oilseed Crops Improvement, Guangdong Provincial Key Laboratory for Crop Genetic Improvement, Guangzhou 510640, China. 2MolBreeding Biotechnology Co., Ltd., Shijiazhuang, China. 3Center of Excellence in Genomics & Systems Biology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India. 4Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan 430062, China. 19. Moretzsohn MC, Gouvea EG, Inglis PW, Leal-Bertioli SC, Valls JF, Bertioli DJ. A study of the relationships of cultivated peanut (Arachis hypogaea) and its most closely related wild species using intron sequences and microsatellite markers. Ann Bot. 2013;111:113–26. 20. Bertioli DJ, Cannon SB, Froenicke L, Huang G, Farmer AD, Cannon EK, et al. The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut. Nat Genet. 2016;48:438–46. 21. Chen X, Li H, Pandey MK, Yang Q, Wang X, Garg V, et al. Draft genome of the peanut A-genome progenitor (Arachis duranensis) provides insights into geocarpy, oil biosynthesis, and allergens. Proc Natl Acad Sci U S A. 2016;113: 6785–90. Competing interests p g The authors declare that they have no competing interests. 17. Cho YG, Ishii T, Temnykh S, Chen X, Lipovich L, McCouch SR, et al. Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.). Theor Appl Genet. 2000;100:713–22. 25. Chen X, Lu Q, Liu H, Zhang J, Hong Y, Lan H, et al. Sequencing of cultivated peanut, Arachis hypogaea, yields insights into genome evolution and oil improvement. Mol Plant. 2019;12:920–34. 3. Chen Y, Ren X, Zheng Y, Zhou X, Huang L, Yan L, et al. Genetic mapping of yield traits using RIL population derived from Fuchuan Dahuasheng and ICG6375 of peanut (Arachis hypogaea L.). Mol Breed. 2017;37:17. 4. Shasidhar Y, Vishwakarma MK, Pandey MK, Janila P, Variath MT, Manohar SS, et al. Molecular mapping of oil content and fatty acids using dense genetic maps in groundnut (Arachis hypogaea L.). front. Plant Sci. 2017;8:794. Availability of data and materials 14. Zhong R, Zhou M, Zhao C, Hou L, Li C, Wang X, et al. SSR marker development from peanut gynophore transcriptome sequencing. Plant Breed. 2016;135:111–7. The datasets supporting the conclusions of this article are included within the article and its additional files. 15. Shirasawa K, Bertioli DJ, Varshney RK, Moretzsohn MC, Leal-Bertioli SC, Thudi M, et al. Integrated consensus map of cultivated peanut and wild relatives reveals structures of the a and B genomes of Arachis and divergence of the legume genomes. DNA Res. 2013;20:173–84. Ethics approval and consent to participate Not applicable. Not applicable. 3. Chen Y, Ren X, Zheng Y, Zhou X, Huang L, Yan L, et al. Genetic mapping of yield traits using RIL population derived from Fuchuan Dahuasheng and ICG6375 of peanut (Arachis hypogaea L.). Mol Breed. 2017;37:17. 4. Shasidhar Y, Vishwakarma MK, Pandey MK, Janila P, Variath MT, Manohar SS, et al. Molecular mapping of oil content and fatty acids using dense genetic maps in groundnut (Arachis hypogaea L.). front. Plant Sci. 2017;8:794. References Genome-wide characterization of microsatellites in Triticeae species: abundance, distribution and evolution. Sci Rep. 2016;6:32224. 38. Deng P, Wang M, Feng K, Cui L, Tong W, Song W, et al. Genome-wide characterization of microsatellites in Triticeae species: abundance, distribution and evolution. Sci Rep. 2016;6:32224. 39. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3—new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115. 40. Schuler GD. Electronic PCR: bridging the gap between genome mapping and genome sequencing. Trends Biotechnol. 1998;16:456–9. References 1. Krishna G, Singh BK, Kim EK, Morya VK, Ramteke PW. Progress in genetic engineering of peanut (Arachis hypogaea L.)—a review. Plant Biotechnol J. 2015;13:147–62. 1. Krishna G, Singh BK, Kim EK, Morya VK, Ramteke PW. Progress in genetic engineering of peanut (Arachis hypogaea L.)—a review. Plant Biotechnol J. 2015;13:147–62. 23. Yin D, Ji C, Ma X, Li H, Zhang W, Li S, et al. Genome of an allotetraploid wild peanut Arachis monticola: a de novo assembly. Gigascience. 2018;7:1–9. 24. Zhao C, Qiu J, Agarwal G, Wang J, Ren X, Xia H, et al. Genome-wide discovery of microsatellite markers from diploid progenitor species, Arachis duranensis and A. ipaensis, and their application in cultivated peanut (A. hypogaea). Front Plant Sci. 2017;8:1209. 2. Pandey MK, Wang ML, Qiao L, Feng S, Khera P, Wang H, et al. Identification of QTLs associated with oil content and mapping FAD2 genes and their relative contribution to oil quality in peanut (Arachis hypogaea L.). BMC Genet. 2014;15:133. 25. Chen X, Lu Q, Liu H, Zhang J, Hong Y, Lan H, et al. Sequencing of cultivated peanut, Arachis hypogaea, yields insights into genome evolution and oil improvement. Mol Plant. 2019;12:920–34. 3. Chen Y, Ren X, Zheng Y, Zhou X, Huang L, Yan L, et al. Genetic mapping of yield traits using RIL population derived from Fuchuan Dahuasheng and ICG6375 of peanut (Arachis hypogaea L.). Mol Breed. 2017;37:17. 26. Zhuang W, Chen H, Yang M, Wang J, Pandey MK, Zhang C, et al. The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution and crop domestication. Nat Genet. 2019;51:865–76. 4. Shasidhar Y, Vishwakarma MK, Pandey MK, Janila P, Variath MT, Manohar SS, et al. Molecular mapping of oil content and fatty acids using dense genetic maps in groundnut (Arachis hypogaea L.). front. Plant Sci. 2017;8:794. Page 9 of 9 Lu et al. BMC Genomics (2019) 20:799 Lu et al. BMC Genomics (2019) 20:799 27. Bertioli DJ, Jenkins J, Clevenger J, Dudchenko O, Gao D, Seijo G, et al. The genome sequence of segmental allotetraploid peanut Arachis hypogaea. Nat Genet. 2019;51:877–84. 28. Powell W, Machray GC, Provan J. Polymorphism revealed by simple sequence repeats. Trends Plant Sci. 1996;1:215–22. 29. Song GQ, Li MJ, Xiao H, Wang XJ, Tang RH, Xia H, et al. EST sequencing and SSR marker development from cultivated peanut (Arachis hypogaea L.). ElectrJ Biotechnol. 2010;13:7–8. 30. References Wang H, Penmetsa RV, Yuan M, Gong L, Zhao Y, Guo B, et al. Development and characterization of BAC-end sequence derived SSRs, and their incorporation into a new higher density genetic map for cultivated peanut (Arachis hypogaea L.). BMC Plant Biol. 2012;12:10. 31. Zhao T, Wang J, Xu G, Weng B. Advance on peanut breeding of high- yielding and good-quality and the application of biological technology. Chin J Trop Crop. 2011;32:2187–95 (in Chinese with English abstract). 32. Pandey MK, Wang H, Khera P, Vishwakarma MK, Kale SM, Culbreath AK, et al. Genetic dissection of novel QTLs for resistance to leaf spots and tomato spotted wilt virus in peanut (Arachis hypogaea L.). Front Plant Sci. 2017;8:25. 33. Hong YB, Liang XQ, Chen XP, Liu HY, Zhou GY, Li SX, et al. Construction of Genetic Linkage Map Based on SSR Markers in Peanut (Arachis hypogaea L.). Agr Sci China. 2008;7:915–21. 34. Varshney RK, Bertioli DJ, Moretzsohn MC, Vadez V, Krishnamurthy L, Aruna R, et al. The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.). Theor Appl Genet. 2009;118:729–39. 35. Khedikar YP, Gowda MV, Sarvamangala C, Patgar KV, Upadhyaya HD, Varshney RK. A QTL study on late leaf spot and rust revealed one major QTL for molecular breeding for rust resistance in groundnut (Arachis hypogaea L. ). Theor Appl Genet. 2010;121:971–84. 35. Khedikar YP, Gowda MV, Sarvamangala C, Patgar KV, Upadhyaya HD, Varshney RK. A QTL study on late leaf spot and rust revealed one major QTL for molecular breeding for rust resistance in groundnut (Arachis hypogaea L. ). Theor Appl Genet. 2010;121:971–84. 36. Sarvamangala C, Gowda MVC, Varshney RK. Identification of quantitative trait loci for protein content, oil content and oil quality for groundnut (Arachis hypogaea L.). Field Crop Res. 2011;122:49–59. 37. Sujay V, Gowda MVC, Pandey MK, Bhat RS, Khedikar YP, Nadaf HL, et al. Quantitative trait locus analysis and construction of consensus genetic map for foliar disease resistance based on two recombinant inbred line populations in cultivated groundnut (Arachis hypogaea L.). Mol Breeding. 2012;30:773–88. 37. Sujay V, Gowda MVC, Pandey MK, Bhat RS, Khedikar YP, Nadaf HL, et al. Quantitative trait locus analysis and construction of consensus genetic map for foliar disease resistance based on two recombinant inbred line populations in cultivated groundnut (Arachis hypogaea L.). Mol Breeding. 2012;30:773–88. 38. Deng P, Wang M, Feng K, Cui L, Tong W, Song W, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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How does climate change adaptation affect public budgets? Development of an assessment framework and a demonstration for Austria
Mitigation and adaptation strategies for global change
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How does climate change adaptation affect public budgets? Development of an assessment framework and a demonstration for Austria Gabriel Bachner1 & Birgit Bednar-Friedl1,2 & Nina Knittel1 Received: 24 September 2018 /Accepted: 15 January 2019 # The Author(s) 2019 /Published online: 4 2019 February ORIGINAL ARTICLE How does climate change adaptation affect public budgets? Development of an assessment framework and a demonstration for Austria ORIGINAL ARTICLE * Gabriel Bachner gabriel.bachner@uni-graz.at https://doi.org/10.1007/s11027-019-9842-3 Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 https://doi.org/10.1007/s11027-019-9842-3 Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11027-019- 9842-3) contains supplementary material, which is available to authorized users. 1 Introduction In the last decade, national strategies for adaptation to climate change have been developed and implemented in numerous countries (Lesnikowski et al. 2015). It is widely held that, unlike mitigation, adaptation is a private good and therefore should be carried out by private actors (Tol 2005), whereas the role of the public sector is limited to providing the right incentives (Jones et al. 2013) and to correct for market failure (Osberghaus et al. 2010). However, the state owns public infrastructure, acts as service provider (e.g., by means of early warning systems), and plays a significant role as information broker (e.g., to coordinate extreme weather event preparedness and response) (Eakin and Patt 2011). This view is also supported when looking at national adaptation strategies, since a considerable share of adaptation is initiated by legislation, as well as financed and implemented by the public sector (Urwin and Jordan 2008; Biesbroek et al. 2010; McDonald 2011; Mees et al. 2012). Motivated by that, this paper takes a closer look at the direct and indirect consequences of public adaptation for federal budgets and develops a general modeling framework for quantification. The public household is affected by climate change impacts and public adaptation through various channels (Bräuer et al. 2009; Bachner and Bednar-Friedl 2018). The impacts of climate change lead to direct costs for the public household, such as higher expenditures for disaster relief payments to private households or reconstruction of damaged public infrastructure. The benefit of adaptation is that it reduces some of these direct impact costs. Additionally, indirect effects on public budgets arise from both impacts and adaptation measures. This is because of changes in the tax base (e.g., due to changed economic activity and structure) and thus also tax revenues (cf. Lis and Nickel 2010; Schinko et al. 2016). Public adaptation is usually integrated into the government’s general practices to Bclimate proof^ investment decisions and to mainstream adaptation into other policy fields (Bierbaum et al. 2013). However, the economic literature on adaptation until now has mostly ignored this complexity and modeled adaptation in a very stylized way, e.g., in the form of generic adaptation capital formation to identify an optimal adaptation level (de Bruin et al. 2009; Agrawala et al. 2011a, b; Bosello et al. 2013). Abstract Public adaptation to climate change affects government budgets directly on the expenditure side, but also indirectlyviachangesin the tax base and governmentconsumption patterns. While such indirect effects have been analyzed intensively for mitigation policies, similarly detailed model-based frame- works and studies for adaptation policy are still missing. The objectives of the present paper are (i) to fill this gap by proposing a general modeling framework that allows for a comprehensive analysis of effects of adaptation on federal budgets, both on the expenditure and the revenue side, as well as of macroeconomiceffectsand(ii)todemonstrateitsusefulnessbyapplyingthisframeworktothecaseof Austria.Wefindthatpublicadaptationcanleadtosubstantialpositivemacroeconomiceffectsongross domesticproduct(GDP),welfare,andemployment.Theresultsarerobustwithrespecttoassumptions abouttheeffectivenessofadaptation.Also,wedemonstratethatitisessentialforanalysistocoverboth the expenditure and revenue side, as overall government revenues can increase due to adaptation, offsetting additional direct public expenses for adaptation, thus increasing the budget balance. This is becauseoflesssevereclimatechangeimpactsandthecorrespondinglowerpaymentsforpost-disaster relief and unemployment benefits as well as higher tax revenues. We thus strongly recommend makinguseofeconomy-widemodelingframeworkswhenplanningfor adaptation,astheyshedlight on the true costs and benefits of adaptation. Keywords Climate change . Public adaptation . Indirect effects . Public budgets . Computable general equilibrium Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11027-019- 9842-3) contains supplementary material, which is available to authorized users. * Gabriel Bachner gabriel.bachner@uni-graz.at * Gabriel Bachner gabriel.bachner@uni-graz.at Birgit Bednar-Friedl birgit.friedl@uni-graz.at 1 Wegener Center for Climate and Global Change, University of Graz, Brandhofgasse 5, 8010 Graz, Austria 2 Institute of Economics, University of Graz, Universitätsstraße 15, 8010 Graz, Austria Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1326 1 Introduction For practical decision-making on public adaptation, however, these generalized insights from integrated assessment models are insuf- ficient in that they do not allow for assessing the effects and usefulness of specific adaptation measures. This paper intends to fill this gap by developing a modeling framework that allows for a more detailed analysis of the economy-wide and budgetary effects of public adaptation. As shown in the literature, climate change leads to significant losses in a broad range of economic sectors and of overall economic performance (Ciscar et al. 2011, 2012; Bosello et al. 2012; Sussman et al. 2014; Bosello and De Cian 2014; OECD 2015; Steininger et al. 2015; Dellink et al. 2017). Economy-wide effects can be much larger than direct impacts within sectors, due to indirect effects (Hallegatte et al. 2007). As argued above, adaptation also influences public budgets indirectly via sectoral spillovers and macroeconomic feedback effects, which affect the tax base and thereby alter the budget balance and the fiscal position of a country. Yet while the fiscal implications of mitigation have been addressed (see Siegmeier et al. 2018 for a review), there is little known about the implications of public adaptation on government budgets. We argue that there is an essential difference between mitigation and adaptation, which requires a separate and detailed analysis to better understand the effects of adaptation on government budgets. While mitigation policies have the potential to generate revenues (e.g., via carbon taxes), the direct effects of adaptation arise mostly on the expendi- ture side. Mitigation therefore allows for cutting distortionary taxes or increasing expenditures Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1327 (such as on carbon-free technologies) but financing adaptation requires either cutting other public expenditures or raising taxes in order to ensure sustainable government finances. (such as on carbon-free technologies) but financing adaptation requires either cutting other public expenditures or raising taxes in order to ensure sustainable government finances. In general, the literature on budgetary implications of climate change impacts and adapta- tion is scarce. Regarding the direct consequences of climate change impacts for public budgets, some papers describe effects qualitatively and provide empirical estimates for changes in expenditures (Bräuer et al. 2009; Margulis and Narain 2010; Osberghaus and Reif 2010; Jones et al. 2013; Gilmore and St. Clair 2018). 1 Introduction Complementary to these are econometric studies that estimate the effects of climate variability and extreme weather events on public finances (Lis and Nickel 2010; Ouattara and Strobl 2013; Melecky and Raddatz 2015; Leppänen et al. 2015); however, they are limited to direct effects on public expenditures, disregarding indirect effects. Indirect effects to public budgets have been analyzed for climate change impacts—but not for public adaptation—in a computable general equilibrium framework by Bachner and Bednar-Friedl (2018). Since the quantification of adaptation-induced effects on public budgets is still underde- veloped, the objectives of this paper are as follows. First, we present a general modeling framework that allows for a comprehensive analysis of effects of adaptation on federal budgets, both on the expenditure and the revenue side, as well as of macroeconomic effects. The proposed framework can be applied to any country or region, even though adaptation is very location specific. Second, we demonstrate its usefulness by applying this framework to the case of Austria. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1328 Baseline Impact Adaptation Tax revenue loss Other transfers Unemployment benefits Additional adaptation costs Reconstruction and relief payments Non-climate related public services Government consumption Increased government consumption with adaptation Decrease in other transfers to balance revenue and expenditure Total government expenditure Fig. 1 Conceptual model of effects of climate change impacts and adaptation on government expenditures. Magnitudes of expenditure categories in the Baseline are set to reflect the EU-wide public expenditure structures (EUROSTAT 2018). The effects in the Impact and Adaptation scenarios are depicted exaggeratedly for illustrative purposes Baseline Impact Adaptation Government consumption Increased government consumption with adaptation Decrease in other transfers to balance revenue and expenditure Total government expenditure Fig. 1 Conceptual model of effects of climate change impacts and adaptation on government expenditures. Magnitudes of expenditure categories in the Baseline are set to reflect the EU-wide public expenditure structures (EUROSTAT 2018). The effects in the Impact and Adaptation scenarios are depicted exaggeratedly for illustrative purposes Fig. 1 Conceptual model of effects of climate change impacts and adaptation on government expenditures. Magnitudes of expenditure categories in the Baseline are set to reflect the EU-wide public expenditure structures (EUROSTAT 2018). The effects in the Impact and Adaptation scenarios are depicted exaggeratedly for illustrative purposes for expenditure but also increasing the need for unemployment benefits (lower economic activity implies higher unemployment). To ensure no increase of the public deficit, govern- ment expenditures need to be re-distributed within the now smaller total available budget. To achieve this aim, two adjustments are possible: cutting either transfers to households or other non-climate-related government consumption, or both. It is plausible to assume for many countries that the level of government consumption is fixed by multi-annual budgeting rules. As a consequence, the total level of government consumption would remain constant but is restructured towards more disaster relief payments and less other government consumption, leaving other transfers to balance revenue and expenditure (as indicated in Fig. 1). for expenditure but also increasing the need for unemployment benefits (lower economic activity implies higher unemployment). To ensure no increase of the public deficit, govern- ment expenditures need to be re-distributed within the now smaller total available budget. To achieve this aim, two adjustments are possible: cutting either transfers to households or other non-climate-related government consumption, or both. 2.1 Scenarios and conceptual model In the modeling framework, we distinguish between three types of scenarios for 2050: (i) the Baseline scenario, describing general socioeconomic development; (ii) the Impact scenario, which adds climate change impacts to the Baseline; and (iii) the Adaptation scenario, which adds adaptation measures to the Impact scenario, reducing impacts but also inducing new costs for adaptation. By comparing these scenarios to each other, we are able isolate effects of climate change impacts (Impact versus Baseline scenario) and of adaptation (Adaptation versus Impact scenario). In the Impact and Adaptation scenarios, the federal government’s budget is affected both on the expenditure and on the revenue side, however, in different ways. Figure 1 conceptually illustrates the effects of climate change impacts and adaptation on government expenditures. In the Baseline scenario, expenditures consist of four major items: (i) non-climate-related government consumption of goods and services (including wages of public workers), (ii) expenditures on reconstruction (investment) and relief payments for damages caused by extreme weather events, (iii) unemployment benefits to be paid as social security, and (iv) other transfers to households. The first two items represent general govern- ment consumption. With climate change, i.e., in the Impact scenario, the composition, and the level of government expenditures change. As a direct effect of climate change, reconstruction and relief payments increase due to higher damages, but several indirect effects also emerge. Depending on the sectoral and macroeconomic impact of climate change, tax revenues change. As most macroeconomic climate change impact studies find a negative effect on general economic activity (GDP), it is plausible to assume a loss in tax revenues, reducing the means 2.2 Quantitative model and analysis To analyze and quantify the economy-wide and budgetary effects of climate change impacts and adaptation, including indirect effects, we suggest a stepwise approach. Step 1 aims to isolate adaptation (relevant) expenditures from total public expenditures. To do so, budget and government expenditure reports need to be collected and screened for adaptation-relevant keywords that indicate adaptation actions. The selection of adaptation-relevant keywords can be supported by reviewing national adaptation strategies, implementation plans, or submis- sions to national climate assessments (Bierbaum et al. 2013). After the identification of adaptation-relevant budgetary items, it is necessary to narrow down adaptation-relevant expenditure to adaptation expenditure. This can be done by applying shares, which is not an easy endeavor, especially when adaptation is not the primary objective but a co-benefit (Gilmore and St. Clair 2018). Experts in ministerial departments often have valuable knowl- edge concerning the exact purposes of budgetary items and can thus aid in estimating the shares of adaptation. Alternatively, the OECD Development Assistance Committee (2016) and the European Commission (2016) developed a set of attribution rules that indicate which share of total costs should be assigned to adaptation expenditures for specific budgetary items. In step 2, starting from these current expenditures, a realistic adaptation pathway is co- developed for the future (e.g., 2050) together with experts and stakeholders, ideally from key ministries engaged in adaptation action as well as finance ministries. Often, however, the capacity and the willingness to engage in such a process is limited. An alternative procedure is therefore to develop this pathway based on general recommendations from the literature, such as the concept of adaptation phasing (Watkiss et al. 2015). To deal with uncertainties and limits to specific adaptation actions, such a pathway should not be regarded as set in stone, but rather be regularly re-assessed in an iterative manner, such as is proposed by Haasnoot et al. (2013) who present the approach of Bdynamic adaptive policy pathways.^ In step 3, the direct and indirect consequences of the adaptation pathway for the public budget are analyzed. In principle, several approaches could be used to assess these impacts: projections based on econometric models (Lis and Nickel 2010; Ouattara and Strobl 2013; Leppänen et al. 2015), government financial risk simulation models like CATSIM (Hochrainer- Stigler et al. 2014b), or multi-sectoral computable general equilibrium (CGE) models. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 It is plausible to assume for many countries that the level of government consumption is fixed by multi-annual budgeting rules. As a consequence, the total level of government consumption would remain constant but is restructured towards more disaster relief payments and less other government consumption, leaving other transfers to balance revenue and expenditure (as indicated in Fig. 1). In the Adaptation scenario, direct and indirect effects again impact government expendi- tures. The direct effect is caused by additional public adaptation costs. We assume that multi- annual budgeting accounts for public (planned) adaptation and thus adaptation increases total government consumption. The indirect effects arise again via macroeconomic feedback effects. Impacts are reduced by adaptation and economic activity increases; therefore, total tax revenue losses are less severe compared to the Impact scenario. Since reconstruction and relief payments are smaller with adaptation, other government consumption is expanded as compared to the Impact scenario (however, not reaching the Baseline level due to residual damages also in the Adaptation scenario). Due to higher employment, which can be caused by both higher economic activity and typically high labor intensity of government consumption, expenditure for unemployment benefits is lower compared to the Impact scenario. Depending on the size of the of tax revenue loss and the effect on employment, other transfers can then either be increased (as shown in Fig. 1) or cut to keep the balance between revenue and expenditure. In this context, it is important to consider government budgeting rules. Many countries have committed themselves to avoiding excessive deficits and pursuing fiscal sustainability. For instance, the European Stability and Growth Pact (European Union 2008, Art. 121 and 126) requires that all member states keep the government deficit below 3% of GDP, with govern- ment debt not exceeding 60% of GDP. In our analysis and as illustrated in Fig. 1, we therefore assume that government consumption in the Impact scenario is fixed at the Baseline level, and that this fixation is established via adjustments in transfers to households. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1329 2.2 Quantitative model and analysis Econo- metric models are based on time-series or panel data, which allows for an assessment of budgetary effects over time, but they usually neglect macroeconomic feedback effects that alter the tax base. Financial risk simulation models are particularly adept at studying loss distribu- tions and effects on budgetary gaps, but the representation of the overall economy is highly aggregated. CGE models have the advantage of capturing these macroeconomic and cross- sectoral feedback effects as they distinguish between different economic sectors and agents (Fisher-Vanden et al. 2013) and also capture the revenue side. However, CGE models are more suitable for capturing long-term equilibrium effects, not short-term effects. 3 Model implementation and data In the following analysis, we apply the described modeling framework to the case of Austria. We focus on the impact fields with the highest federal budgetary importance and with potentially strong impacts for the Austrian economy (Bednar-Friedl et al. 2017): Agriculture, Forestry, and Catastrophe Management (including protection from natural hazards). Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1330 Agriculture is heavily subsidized and thus public resources indirectly fund adaptation in this sector. Forestry is of high relevance since its expected contribution to macroeconomic damages is relatively high (−0.8% GDP loss in 2050; Bachner et al. 2015). In addition, the government owns a large share of the protection forests in Austria. Catastrophe Management is closely connected to the public domain, since the Austrian disaster fund is fully financed out of tax revenue (Schinko et al. 2016). Our scenarios are in line with the RCP-SSP framework, which is standard in climate change research. Representative Concentration Pathways (RCPs) de- scribe different global emission trajectories, while Shared Socioeconomic Pathways (SSPs) describe different narratives for socioeconomic development. By combining RCPs and SSPs, different states of the world emerge in which climate change impacts materialize (Moss et al. 2010; O’Neill et al. 2014). For the Baseline scenario, we choose a Bmiddle-of-the-road^ shared socioeconomic pathway (SSP2; O’Neill et al. 2014). For details on the Baseline calibration, see Appendix A.1. 1 This co-development took place within the research project PACINAS (http://anpassung.ccca.at/pacinas/en/), funded by the Austrian Climate and Energy Fund. 3.1 Current and future public adaptation at the federal level in Austria To identify current federal expenditures on public adaptation (step 1), we screened the federal budget in the base year (2016) for adaptation-relevant expenditure items and categorized them into gray, green, or soft measures (see Appendix A.2 for details). Soft measures comprise information measures such as early warning systems; gray measures are comprised of struc- tural protection, for example, flood protection dams; and green measures are ecosystem-based measures such as natural flood retention areas or forest management. Additionally, we include a separate category for research and development (R&D). The result of the screening is shown in the base year (2016) in Fig. 2, with the largest expenditure items being gray measures in the Catastrophe Management impact field (CATM, € 471 million p.a.) and green measures in the Agriculture impact field (AGRI, € 257 million p.a.). Expenditures on soft measures and R&D play only a minor role. In total, current annual adaptation-relevant expenditures sum up to € 908 million (15% of the screened budgetary subdivisions). Starting from 2016, we co-developed1 an indicative adaptation expenditure pathway until 2050 (step 2). This pathway combines expert judgment on the additional resources needed for single adaptation measures, on limits to further increase gray adaptation measures and international recommendations on the phasing of gray, green, and soft measures (Watkiss et al. 2015). For the Baseline expenditure path, the growth rate of the mid-term budget forecast for the Federal State of Austria is used (1.65% annually; assuming the same shares as in 2016) (BMF 2015). Figure 2 illustrates the pathway by type of adaptation measure and impact field in which adaptation is carried out and funded. On average, expenditures of this indicative Adaptation scenario rise by 3% p.a. over the period 2016–2050, which is above the 1.65% assumed in the Baseline. For details on this co-developed pathway, see Appendix A.2. 3.2 Macroeconomic model and scenario implementation For the macroeconomic analysis (step 3), we use a single-country, comparative static CGE model of Austria (Bachner et al. 2015; Steininger et al. 2016; Bachner and Bednar-Friedl Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1331 - 250 500 750 1,000 1,250 1,500 1,750 2,000 2,250 2,500 2,750 Mio. € R&D: R&D gray: CATM gray: FORE green: CATM green: FORE green: AGRI soft: CATM soft: FORE soft: AGRI Baseline 1.65% p.a. Fig. 2 Indicative adaptation pathway for the Austrian federal budget until 2050 (R&D= Research and Devel- opment; CATM= Catastrophe Management; FORE= Forestry; AGRI= Agriculture) Fig. 2 Indicative adaptation pathway for the Austrian federal budget until 2050 (R&D= Research and Devel- opment; CATM= Catastrophe Management; FORE= Forestry; AGRI= Agriculture) 2018). The model covers 40 economic sectors and one representative private household, which is endowed with labor and capital. The respective factor income is spent for consumption or is saved. In addition, there is a public household providing public services, financed by the following taxes: sales taxes on output, tax on capital gains, labor tax, value added tax, and export tax. All tax rates are fixed, thus determining flexible government income, which in turn gives the total amount of available public budget to be spent. The model includes classical unemployment and international trade is depicted via the Armington (1969) assumption, where domestic goods are imperfect substitutes for imports. The foreign balance is fixed at the share of the model’s benchmark year (2008). Regarding the development of the budget deficit, we assume a constant deficit-to-GDP ratio, which is an empirically well-supported assumption for the structural deficit and also in accordance with the criteria of the EU Stability and Growth Pact. For more details and the algebraic formulation, see Bachner (2017). 3.2.1 Implementation of the impact scenario Climate change is implemented as average changes for the future 30-year climatic period 2036–2065 (i.e., 2050), relative to the average of the reference climatic period 1981–2010. Specifically, we use the SRES (Special Report on Emissions Scenarios) A1B emissions scenario (Nakićenović and Swart 2000), which corresponds to the RCP6.0 scenario with + 2.5 °C global mean temperature by the end of the century (Knutti and Sedláček 2013). For details, see Appendix A.1. Climate change impacts are implemented in ten impact fields. For each of these fields, different types of impacts are quantified using a range of (bio)physical models. These impacts are implemented into the macroeconomic CGE model by (i) changes in production cost structures (e.g., a different production process in Agriculture), (ii) changes in productivity (e.g., yield changes in Agriculture and Forestry), (iii) changes in investments (e.g., reconstruc- tion of infrastructure after flood events), and/or (iv) changes in public expenditures (e.g., more post-disaster relief payments in Catastrophe Management). Table 1 summarizes the impacts for the three impact fields under consideration (for all other, see Appendix A.1). As explained in Section 2, to keep expenditures on public service provision (i.e., government consumption) at the same level as in the Baseline scenario, we assume in the Impact scenario that transfers to private households are adjusted accordingly. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1332 Table 1 Summary of impacts and (bio-)physical impact models Impact field Impacts (Bio)physical impact model used f quantifying impact Agriculture Crop productivity of main crops (grain maize, winter wheat, winter rape, soybean, temporary grassland) and grassland due to changes in temperature and precipitation Regression analysis (Mitter et al. 2 based on simulations with the b process model EPIC (Izaurralde 2006) and the farm optimizatio PASMA (Schmid 2004) Forestry Biomass productivity in commercial production forests due to changed precipitation and temperature, bark beetle disturbances on productivity of commercial forests and protection functionality of protection forests Estimation of productivity change forestry revenue model PICUS (Schörghuber et al. 2010) and o from spruce bark beetles with F scenario model (Seidl et al. 200 impact of bark beetle disturbanc tection functionality based on e (Lexer et al. 2015) Catastrophe Management Building damages due to riverine floods Simulation of riverine flooding da hybrid convolution approach (Hochrainer-Stigler et al. 2014a builds on results of the LISFLO the Climate Cost project (Feyen Watkiss 2011; Rojas et al. 3.2.1 Implementation of the impact scenario 2013) Adam Cost project (Kundzewic 2010; Lugeri et al. 2010) Table 1 Summary of impacts and (bio-)physical impact models Impact field Impacts (Bi qua Agriculture Crop productivity of main crops (grain maize, winter wheat, winter rape, soybean, temporary grassland) and grassland due to changes in temperature and precipitation Re Forestry Biomass productivity in commercial production forests due to changed precipitation and temperature, bark beetle disturbances on productivity of commercial forests and protection functionality of protection forests Es Catastrophe Management Building damages due to riverine floods Sim (Bio)physical impact model used for quantifying impact Regression analysis (Mitter et al. 2015b) based on simulations with the biophysical process model EPIC (Izaurralde et al. 2006) and the farm optimization model PASMA (Schmid 2004) Regression analysis (Mitter et al. 2015b) based on simulations with the biophysical process model EPIC (Izaurralde et al. 2006) and the farm optimization model PASMA (Schmid 2004) Estimation of productivity changes with forestry revenue model PICUS 3G (Schörghuber et al. 2010) and of damages from spruce bark beetles with FISCEN scenario model (Seidl et al. 2009, 2011); impact of bark beetle disturbances on pro- tection functionality based on expert guess (Lexer et al. 2015) Simulation of riverine flooding damages in a hybrid convolution approach (Hochrainer-Stigler et al. 2014a) (which builds on results of the LISFLOOD model, the Climate Cost project (Feyen and Watkiss 2011; Rojas et al. 2013) and on the Adam Cost project (Kundzewicz et al. 2010; Lugeri et al. 2010) Simulation of riverine flooding damages in a hybrid convolution approach (Hochrainer-Stigler et al. 2014a) (which builds on results of the LISFLOOD model, the Climate Cost project (Feyen and Watkiss 2011; Rojas et al. 2013) and on the Adam Cost project (Kundzewicz et al. 2010; Lugeri et al 2010) Watkiss 2011; Rojas et al. 2013) and on the Adam Cost project (Kundzewicz et al. 2010; Lugeri et al. 2010) 3.2.2 Implementation of the adaptation scenario The Adaptation scenario builds on the Impact scenario but additionally incorporates direct costs and benefits (i.e., avoided impacts) of public adaptation measures in the three impact fields under consideration. Adaptation costs are divided into operating costs (e.g., labor costs, contracting to spatial planning bureaus, maintenance costs for public infrastructure) and investment costs. Changes in sectoral operating costs are modeled as shifts within the production cost structures while holding unit costs constant (but having a different composition of costs). Changes in government consumption patterns and levels (which are also a part of operating costs) are implemented as additional consumption, financed via cuts in transfers to the private household (and hence reduced private consumption). Accumulation effects of annual investment changes are accounted for, resulting in a changed capital stock in 2050 with associated changed annual capital costs (depreciation). Note that the deployed CGE model is not dynamic but comparative static. The development of the capital stock is therefore no explicit part of the model but accounted for when developing the adaptation pathway. Changes in investment are financed via changed savings and thus corresponding changes in private consumption. Table 3 shows how the indicative adaptation pathway (Fig. 2) translates into annual changes of sectoral costs as well as changes in government consumption (annualized for 2050, relative to the Impact scenario). For details on the calculations, see Appendix A.2 (Table 2). The benefit of adaptation is avoided damage. The respective assumptions on the effective- ness of different measures (i.e., by how much damages can be reduced) are summarized in Table 3. Following the literature and expert estimates, we assume that agricultural crop yields can be increased by 10% and damages in Forestry can be reduced by 30–40%. For the Catastrophe Management impact field (flood protection), we use benefit-cost ratios to quantify effectiveness. For the Adaptation scenario, we use mean values for a central simulation run, Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1333 but also provide results for a bandwidth (lower and upper bound) to address uncertainty. For details, see Appendix A.3. i ll h l i h Ad i i h di f bli i Table 2 Translation of the adaptation path into annual cost vectors for modeled sectors (agriculture, forestry, water) and agents in 2050 (in million €) Agriculture Forestry Water Government consumption Aggregate investment Labor + 25 + 69 + 48 Capital (incl. land) + 126 + 32 Research and development + 60 Civil engineering and planning + 165 Machinery + 213 + 102 Construction (Investment) + 39 −45 −6 Total + 239 + 336 + 93 + 226 −6 le 2 Translation of the adaptation path into annual cost vectors for modeled sectors (agriculture, forestry, er) and agents in 2050 (in million €) but also provide results for a bandwidth (lower and upper bound) to address uncertainty. For details, see Appendix A.3. Finally, note that also in the Adaptation scenario, we assume that expenditures of public service provision are maintained at the Baseline level by adjusting transfers to households. In addition, however, we allow for increased consumption for adaptation measures (as indicated in Fig. 1). 4.1 Economy-wide effects of public adaptation In 2050, climate change-induced annual GDP losses in the Impact scenario are −0.15% relative to the Baseline (see Appendix A.4 for the detailed results). In the mean Adaptation In 2050, climate change induced annual GDP losses in the Impact scenario are 0.15% relative to the Baseline (see Appendix A.4 for the detailed results). In the mean Adaptation Table 3 Description, assumption, and sources of effectiveness of adaptation measures in the impact fields CATM (Catastrophe Management), AGRI (Agriculture), and FORE (Forestry) Impact field Adaptation measure/purpose [lower] mean [upper] value for analysis Description of unit of measurement Source CATM Soft flood protection measures [1.5] 9.2 [10] Benefit/cost-ratio (Kuik et al. 2016) Green flood protection measures [1.2] 1.8 [4.3] Lower bound: 10th percentile Gray flood protection measures [1] 3.9 [7.7] Upper bound: 90th percentile AGRI Change in agricultural crop management practices [5%] 10% [15%] Yield increase potential in response to adaptation measures (Mitter et al. 2015a; Schönhart et al. 2016) FORE Educational measures against bark beetles; more intensive tending of forests; better infrastructure in forests [20%] 32% [45%] Damage reduction of damages to protective forest (Kolström et al. 2011; Lexer 2017) Educational measures against bark beetles; more intensive tending of forests; better infrastructure in forests [35%] 40% [45%] Damage reduction of damages to commercial timberland Genetics: Better usage of autochthonous species to increase resilience of forests [0%] 0.03% [0.03%] Reduction in timber growth loss Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1334 scenario, these losses can be reduced by public adaptation to only −0.06%. To put it differently, adaptation leads to a net benefit of + 0.09%-points of annual GDP compared to not adapting; however, there is still a residual loss when compared to the Baseline scenario with no climate change. This benefit from adaptation is illustrated in Fig. 3 (black diamonds). The reasons for this GDP effect are twofold: First, there are positive effects from adaptation- specific productivity gains (Agriculture) as well as positive employment effects. Second, there are reductions of direct and indirect climate change impacts from the Forestry and Catastrophe Management impact fields. The welfare loss, measured as Hicksian equivalent variation, in the Impact scenario is − 0.48% (again, see Appendix A.4 for the detailed results), which in the Adaptation scenario can be reduced to −0.27%. Public adaptation therefore creates a welfare benefit of + 0.2%-points (Fig. 3). 4.1 Economy-wide effects of public adaptation Note that the size of the welfare effect is larger than the GDP effect in the Impact and the Adaptation scenarios. This is because some consumption expenditure is Bforced^ to shift from generic (welfare enhancing) consumption to the renewal of destroyed assets. This shift however only restores the original state (e.g., prior to an extreme event) and is thus reducing other consumption possibilities (i.e., welfare). In contrast, the GDP effect is neutral to this re- distribution within the consumption structure. Figure 3 furthermore shows how the total benefit of public adaptation on GDP, welfare, and unemployment is distributed across impact fields. We find that the positive effect of adaptation on GDP arises from Agriculture (due to productivity gains) and Forestry (especially due to the -0.1% 0.0% +0.1% +0.2% +0.3% +0.4% GDP Welfare Unemployment Agriculture Forestry Catastrophe Management Total effect of adaptation Fig. 3 Effects of adaptation on GDP, welfare, and unemployment for 2050 (Adaptation scenario relative to Impact scenario, given in percentage point differences), distinguished by impact field and in total. Error bars indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts -0.1% 0.0% +0.1% +0.2% +0.3% +0.4% GDP Welfare Unemployment Agriculture Forestry Catastrophe Management Total effect of adaptation Total effect of adaptation Unemployment Fig. 3 Effects of adaptation on GDP, welfare, and unemployment for 2050 (Adaptation scenario relative to Impact scenario, given in percentage point differences), distinguished by impact field and in total. Error bars indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1335 reduction of damages to protection forests). For Catastrophe Management, the shift from a capital-intensive to a more labor-intensive structure results in only a marginal GDP effect from adaptation, as the effects offset each other. Substantial positive effects on welfare emerge from reduced damage costs in Catastrophe Management and the associated reduction in forced consumption. A positive contribution of adaptation to welfare also arises from Agriculture, due to productivity gains and the moderating effect on food prices. In Forestry, we find positive welfare effects primarily caused by the reduced damage to protection forests and thus more public finances available to increase transfers to households. We also see that unemployment is reduced by adaptation in all impact fields, which is driven by the focus on green and soft adaptation as well as by higher expenditures for labor-intensive government consumption. 2 Note that the effect of adaptation in Catastrophe Management to transfers would be negative when performed in isolation (since there is no large positive effect from these measures on employment and GDP), as expensive structural measures need to be financed by cuts in transfers. However, the overall positive contribution of adaptation in Forestry and Agriculture more than compensates for these transfer cuts. 4.2 Effects of public adaptation on government budgets Beginning with a description of results on the revenue side of the budget, the effect of adaptation on the main tax revenue categories is shown in Fig. 4a, which compares the Adaptation and Impact scenario in absolute terms (and with %-points differences on the secondary axis). With adaptation, tax revenues increase for labor and production taxes (due to higher economic activity and employment), but decline in value added tax, since there is less forced consumption for restoring damaged assets and because the govern- ment needs to finance its adaptation expenditure (which is taxed at lower rates than private consumption). Figure 4b shows the expenditure side (again, Adaptation compared to the Impact scenario). In the Adaptation scenario, relief payments to households can be reduced substantially; thus, non-climate-related government consumption can be increased. How- ever, direct public adaptation expenditures amount to additional € 219 million (0.1% of total expenditure). Moreover, due to higher tax revenues and less payments for unem- ployment benefits, transfers to households can be increased in the Adaptation scenario. Only when the effectiveness of adaptation is very low, transfers to households need to be reduced to ensure stability of government consumption (service provision). Eventually, the analysis allows for matching numerical assessment to the conceptual model from Fig. 1, which is presented in Figure A.1. Finally, Fig. 4 also shows the decomposition of the effects of adaptation across the three impact fields. On the revenue side, we find that the relative contribution to higher labor taxes is particularly strong for public adaptation in Forestry, as forest management is comparatively labor-intensive. Revenues from value-added tax decline, however, as a result of adaptation in Catastrophe Management, due to less forced consumption, leading to a slightly negative contribution of adaptation in that regard. On the expenditure side, we find a significant reduction in relief payments to private households from Catastrophe Management in the form of structural flood protection. Adaptation expenditures increase particularly due to adaptation in Catastrophe Management and Forestry, because both sectors are recipients of subsidies for, e.g., ensuring the function of protection forests.2 2 Note that the effect of adaptation in Catastrophe Management to transfers would be negative when performed in isolation (since there is no large positive effect from these measures on employment and GDP), as expensive structural measures need to be financed by cuts in transfers. 4.1 Economy-wide effects of public adaptation Regarding the uncertainty of effectiveness of adaptation (see the error bars in Fig. 3), we find that the results are robust in terms of the direction of the effects. 4.2 Effects of public adaptation on government budgets However, the overall positive contribution of adaptation in Forestry and Agriculture more than compensates for these transfer cuts. Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1336 Discussion and conclusions he first part of the paper we presented a modeling framework that allows for a more -0.20% -0.10% 0.00% +0.10% +0.20% +0.30% +0.40% +0.50% -100 -50 0 50 100 150 200 250 Production tax Labor tax Capital tax Value added tax Other taxes Mio. €2008 -2.50% -2.00% -1.50% -1.00% -0.50% 0.00% +0.50% +1.00% +1.50% +2.00% +2.50% -500 -400 -300 -200 -100 0 100 200 300 400 500 Non-climate related government consumption Additional adaptation expenditures Relief payments to households Transfers to households Unemployment benefits Mio. €2008 Agriculture Forestry Catastrophe Management Total in Mio. € %-points change (secondary axis) (a) Revenue side (b) Expenditure side 4 Decomposition of effects of public adaptation on tax revenue (a) and public expenditure (b) for the three tation fields for 2050 (Adaptation scenario relative to Impact scenario). Diamonds show absolute total effect riangles show percentage point differences between Adaptation and Impact scenarios (secondary axis). Error indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts. that %-point changes are not shown for relief payments (−81%) and for additional adaptation expenditures ch would be infinite when calculated since its value in the Baseline and Impact scenario would be zero) g p g g ( ) -0.20% -0.10% 0.00% +0.10% +0.20% +0.30% +0.40% +0.50% -100 -50 0 50 100 150 200 250 Production tax Labor tax Capital tax Value added tax Other taxes Mio. €2008 (a) Revenue side -2.50% -2.00% -1.50% -1.00% -0.50% 0.00% +0.50% +1.00% +1.50% +2.00% +2.50% -500 -400 -300 -200 -100 0 100 200 300 400 500 Non-climate related government consumption Additional adaptation expenditures Relief payments to households Transfers to households Unemployment benefits Mio. €2008 Agriculture Forestry Catastrophe Management Total in Mio. € %-points change (secondary axis) (b) Expenditure side (b) Expenditure side Fig. 4 Decomposition of effects of public adaptation on tax revenue (a) and public expenditure (b) for the three adaptation fields for 2050 (Adaptation scenario relative to Impact scenario). Diamonds show absolute total effect and triangles show percentage point differences between Adaptation and Impact scenarios (secondary axis). Error bars indicate different assumptions on effectiveness of adaptation measures to reduce climate change impacts. 4.2 Effects of public adaptation on government budgets Note that %-point changes are not shown for relief payments (−81%) and for additional adaptation expenditures (which would be infinite when calculated since its value in the Baseline and Impact scenario would be zero) 5 Discussion and conclusions In the first part of the paper, we presented a modeling framework that allows for a more detailed analysis of the budgetary effects of public adaptation to climate change, taking account of economy-wide feedback effects. Until now, such analyses have not been available, but are urgently needed for informed policy making, including the phasing and prioritization of Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1337 adaptation measures. The proposed framework can be applied to any country or region; however, it involves considerable effort with respect to data preparation and stakeholder consultation, as adaptation is often integrated in general public sector activities. adaptation measures. The proposed framework can be applied to any country or region; however, it involves considerable effort with respect to data preparation and stakeholder consultation, as adaptation is often integrated in general public sector activities. In the second part of the paper, we demonstrated the usefulness of the proposed framework for the case of Austria. We show that public adaptation already involves considerable costs for the federal budget (15% of the pre-selected budget is adaptation-relevant). These costs are likely to rise in the future, due to ongoing climate change but also due to re-investment cycles in public infrastructure. For requirements of robust government finances, these higher expen- ditures on adaptation require that other public expenditures are reduced accordingly; however, the extent depends on how tax revenues and social benefit payments are affected by adapta- tion. To also capture these indirect effects of public adaptation, we investigated how public adaptation in Agriculture, Forestry, and Catastrophe Management affects GDP, welfare, and unemployment. The first key lesson learned is that due to positive effects on GDP, welfare and employment, public adaptation can lead to a net benefit from a macroeconomic perspective. While the effectiveness of adaptation measures to reduce climate change impacts is still subject to uncertainty, we find that the direction of our results is highly robust with regard to different assumptions on the effectiveness of adaptation measures. When looking more closely at the government budget, we find that public adaptation has the potential to increase government revenues (in total and for all categories except valued added tax) because of higher economic activity as compared to a scenario with only the impacts of climate change. 5 Discussion and conclusions On the expenditure side, public adaptation leads to a partial re- distribution of government expenditures towards adaptation but also to a reduced need for disaster relief payments. Moreover, reduced unemployment translates into less unemployment payments. The effect on transfers tends to be positive but can be slightly negative when adaptation measures’ effectiveness is very low. Overall, the second key lesson learned is that it is possible that the positive indirect effects of public adaptation outweigh the direct public costs of adaptation, with a resulting increase in the overall government budget, another insight offered by the proposed framework. Regarding global adaptation strategy recommendations, our findings imply that adaptation assessments should take a comprehensive perspective based on quantitative economic model- ing, and include both the revenue and expenditure side as well as indirect effects. Computable general equilibrium models are very useful in that respect. As we have shown, the macroeco- nomic benefits of adaptation can outweigh the costs, because of indirect effects, e.g., on economic activity in other sectors and employment. Such effects may turn cost-benefit assessments in favor for adaptation measures with high direct costs, but strong positive indirect effects. This is the case, e.g., for labor-intensive green adaptation measures. We also demonstrate the advantage of a close collaboration between academia and admin- istration (especially from finance and treasury), in order to develop realistic and policy- compatible adaptation pathways. Such a collaboration also raises awareness within the policy domain and enhances the mainstreaming of climate change adaptation across the board. Regarding the generalizability of our results for other countries, we conclude the following. For countries with (i) similar budgetary structures as well as (ii) similar patterns of impacts (as in Austria), the revealed mechanisms can lead to the same qualitative effects. Regarding the first similarity—the budgetary structure—we know that across EU member states, these structures are very homogenous (EUROSTAT 2018). Among OECD countries, the variations are larger, yet the dominant revenue and expenditure categories are still the same (OECD 2018a, b). Similar impact patterns relate to high damages from flooding and forestry (loss of Mitigation and Adaptation Strategies for Global Change (2019) 24:1325–1341 1338 ecosystem services) and moderate positive effects in Agriculture. As this is the case for many developed regions and industrialized countries, the qualitative insights and mechanisms as obtained from our case might also carry over to many other OECD and EU countries. References Agrawala S, Bosello F, Carraro C et al (2011a) Adapting to climate change: costs, benefits, and modelling approaches. Int Rev Environ Resour Econ 5:245–284. https://doi.org/10.1561/101.00000043 Agrawala S, Bosello F, Carraro C et al (2011a) Adapting to climate change: costs, benefits, and modelling approaches. Int Rev Environ Resour Econ 5:245–284. https://doi.org/10.1561/101.00000043 Agrawala S, Bosello F, Carraro C et al (2011b) Plan or react? Analysis of adaptation costs and benefits using integrated assessment models. Clim Chang Econ 02:175–208. https://doi.org/10.1142/S2010007811000267 Agrawala S, Bosello F, Carraro C et al (2011b) Plan or react? Analysis of adaptation costs and benefits using integrated assessment models. Clim Chang Econ 02:175–208. https://doi.org/10.1142/S2010007811000267 Armington PS (1969) A theory of demand for products distinguished by place of production (Une theorie de la demande de produits differencies d’apres leur origine) (Una teoria de la demanda de productos distinguiendolos segun el lugar de produccion). Staff Papers - International Monetary Fund 16:159. https://doi.org/10.2307/3866403 Armington PS (1969) A theory of demand for products distinguished by place of production (Une theorie de la demande de produits differencies d’apres leur origine) (Una teoria de la demanda de productos distinguiendolos segun el lugar de produccion). Staff Papers - International Monetary Fund 16:159. https://doi.org/10.2307/3866403 Bachner G (2017) Assessing the economy-wide effects of climate change adaptation options of land transport systems in Austria. Reg Environ Chang 17:929–940. https://doi.org/10.1007/s10113-016-1089-x Bachner G (2017) Assessing the economy-wide effects of climate change adaptation options of land transport systems in Austria. Reg Environ Chang 17:929–940. https://doi.org/10.1007/s10113-016-1089-x Bachner G, Bednar-Friedl B (2018) The effects of climate change impacts on public budgets and implication fiscal counterbalancing instruments. Environ Model Assess. https://doi.org/10.1007/s10666-018-9617-3 Bachner G, Bednar-Friedl B (2018) The effects of climate change impacts on public budgets and implications of fiscal counterbalancing instruments. Environ Model Assess. https://doi.org/10.1007/s10666-018-9617-3 h d i dl b i i i l i li h Bachner G, Bednar-Friedl B, Nabernegg S, Steininger KW (2015) Macroeconomic evaluation of climate change in Austria: a comparison across impact fields and total effects. In: Steininger KW, König M, Bednar-Friedl B et al (eds) Economic evaluation of climate change impacts: development of a cross-sectoral framework and results for Austria. Springer, Berlin, pp 415–440 Bachner G, Bednar-Friedl B, Nabernegg S, Steininger KW (2015) Macroeconomic evaluation of climate change in Austria: a comparison across impact fields and total effects. 5 Discussion and conclusions As this paper is the first to analyze not only the direct but also the indirect effects of public adaptation on government budgets, limitations and several directions for future research should naturally be discussed. First, a better understanding of how the effectiveness of adaptation can change over time is definitely needed. Second, while we develop an indicative and plausible adaptation pathway together with professionals from relevant government departments, several alternative adaptation pathways could be developed and compared with regard to their macroeconomic and budgetary consequences. Third, it would be interesting to investigate in more detail how different budgetary rules, such as flexible tax rates or additional foreign debt, interact with public adaptation spending, an exercise which would require a more dynamic modeling setting. Acknowledgements We thank Keith Williges for careful proof-reading. Funding information Open access funding provided by University of Graz. Funding for this research was granted by the Austrian Climate and Energy Fund under its 6th Call of the Austrian Climate Research Program (grant number B36862; project PACINAS). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and repro- duction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References https://doi.org/10.1007/s10584-011-0340-1 Bosello F, Carraro C, De Cian E (2013) Adaptation can help mitigation: an integrated approach to post-2012 climate policy. Environ Dev Econ 18:270–290. https://doi.org/10.1017/S1355770X13000132 Bosello F, Carraro C, De Cian E (2013) Adaptation can help mitigation: an integrated approach to post-2012 climate policy. 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https://zenodo.org/records/1631369/files/article.pdf
German
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Krankenpflege
Deutsche medizinische Wochenschrift/Deutsche Medizinische Wochenschrift
1,900
public-domain
429
Krankenpflege. Die oft anerkannte Thatsache, dass Berlin sich über die Grenzen Deutschlands hinaus durch eine ungewöhnlich grosse Zahl vorzüglicher Privatkliniken auszeichnet, hat durch die am 11. d. M. eröffnete Privat-Frauenklinik von Dr. K. Ab e 1 (IPotsdamerstrasse 92) eine neue Bestätigung erfahren. Die nicht nur zweckmässig eingerichtete, sondern auch architektonisch schöne Anstalt kann vollen Anspruch auf den Bei- fall der Fachgenossen wie der sie aufsuchenden Patienten erheben. Die ganz aus Stein und Eisen erbaute Klinik verfügt über eine erfreu- liche Fülle von Licht und Luft; beide, besonders für Kranke so überaus wohlthätige Factoren werden namentlich von den an der Hinterfront der Anstalt gelegenen Gärten der benachbarten Häuser in einer ungewöhn- lichen Weise begünstigt. Die Vertheilung der Räumlichkeiten ist die in neuerer Zeit übliche: Im obersten (IV.) Stock die Operationsräume, im Souterrain bezw. Parterre die Wirthschaftsräume, im Hochparterre Sprech- und Wartezimmer, Bureau, Zimmer der Oberin, in den übrigen Stockwerken die Krankenzimmer, Laboratorium etc. Zu den Räumen der mit der Anstalt verbundenen Poliklinik führt zweckmässiger Weise ein Nebeneingang, so dass die ambulanten Patienten die eigentliche Klinik nicht zu betreten brauchen, ebenso sind der Poliklinik besondere Wartezimmer, Closets u. s. w. zugewiesen. Die Verbindung zwischen den einzelnen Stockwerken wird durch einen elektrischen Aufzug, construirt von Stiegler, hergestellt. Der durchweg vorhandene Cementfussboden ist überall mit Linoleum bedeckt; hierdurch, sowie durch die an den meisten Zimmern vorhandenen Doppelthüren wird möglichst jedes störende Geräusch von den Patienten ferngehalten. Die Heizung findet durch centrale Warmwasserleitung, die Beleuchtung theils durch Gasglühlicht, theils elektrisch durch Nernstlampen statt. Im Winter kann den Zimmern vorgewärmte Luft zugeführt werden, wodurch gleichzeitig ventilirt wird. An Luftraum und innerer Ausstattung bieten die Krankenräume - insbesondere diejenigen für die TTT. Klasse - das äusserste dar, was von einer Privatklinik mit Recht verlangt werden kann. Der Operationsraum entspricht modernen Anforderungen der Antiseptik. Bemerkenswerth ist, dass in der Anstalt nicht nur Nan- heimer, sondern auch Moor-Bäder angewandt werden können. Vor- trefflich ausgestattet ist das zu mikroskopischen, chemischen und bacteriologischen Untersuchungen eingerichtete, mit Arbeitsplätzen für Curse versehene Laboratorium. Die Anstalt umfasst zur Zeit 28 Betten, kann aber auf 45 Betten gebracht worden. Dio Pensions- preise stellen sich täglich für Kassenkranke auf 2,50 Mark, sonst für die III. Klasse auf 3,50 Mark, für die II. auf 6-7,50 Mark, für die I. auf 10 Mark und mehr. Die von der ärztlichen Leitung völlig ge- trennte Verwaltung ruht in den Händen der von der Martin'schen Frauenklinik her vortheilhaft bekannten Oberin Frau Horn. DEUTSCBIF MIEDiCIIflSCIIIE WOC}TEINSCHPJFP. DEUTSCBIF MIEDiCIIflSCIIIE WOC}TEINSCHPJFP. No. 46 No. 46 748 Druck von G. Bern8tein in J3erlin. Krankenpflege. J. S. J. S. Druck von G. Bern8tein in J3erlin.
https://openalex.org/W1972077906
https://bmcimmunol.biomedcentral.com/counter/pdf/10.1186/1471-2172-10-6
English
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Chemokines are secreted by monocytes following OK-432 (lyophilized Streptococcus pyogenes) stimulation
BMC immunology
2,009
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BioMed Central BioMed Central This article is available from: http://www.biomedcentral.com/1471-2172/10/6 This article is available from: http://www.biomedcentral.com/1471-2172/10/6 © 2009 Olsnes et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: OK-432, penicillin-killed Streptococcus pyogenes, is used in treating lymphangiomas and carcinomas. We have studied in vitro the role of mononuclear phagocytes (MNPs), including purified monocytes (MOs), in the immune response to OK-432. MIP-1α/β and MCP-1 secretions were assessed in whole blood (WB), peripheral blood mononuclear cells (PBMCs) and purified MOs, after in vitro stimulation with OK-432 with or without adherence for 24 hours. Results: OK-432 stimulated MNPs to secrete MCP-1 and MIP-1α/β in healthy individuals and in head and neck squamous cell carcinoma (HNSCC) patients, except for OK-432 stimulation of WB giving a minimal MIP-1α/β response. Upon culture on low-attachment wells, a spontaneous chemokine secretion was observed, with an unchanged secretion following OK-432 stimulation. Inhibition of Syk kinase and/or PI-3 kinase did not significantly change the chemokine response to OK-432, except for MIP-1α production being increased upon Syk inhibitor addition and an increased MCP-1 response upon addition of both inhibitors. Adhesion may possibly involve β1 and/ or β3 integrins, not β2, whereas β1–3 integrins may act as co-stimulatory receptors for OK-432. Based on direct blockage of CD36 or CD18 by antibodies, MCP-1 production may be mediated by CD18 while MIP-1β and MCP-1 production may occur upon binding to CD36. Conclusion: Adherent human MOs produce MCP-1 and MIP-1α/β upon stimulation with OK-432. CD36 modulates MIP-1β and MCP-1 response. Thus, to some extent OK-432 acts as a substance whereby only MOs adhered to surfaces secrete MCP-1 and MIP-1α/β, in part explaining why OK- 432 is suited as a biological response modifying drug. Open Research article Chemokines are secreted by monocytes following OK-432 (lyophilized Streptococcus pyogenes) stimulation Carla Olsnes*1, Helen Stavang1,2, Karl Brokstad3, Jan Olofsson1,2 and Hans J Aarstad1,2 Open Access Address: 1Department of Surgical Sciences, Faculty of Medicine, University of Bergen, Jonas Lies vei 66, 5021, Bergen, Norway, 2Department of Head and Neck Surgery, Haukeland University Hospital, 5021, Bergen, Norway and 3Broegelmann Research Laboratory, Gades Institute, University of Bergen, 5021, Bergen, Norway Email: Carla Olsnes* - carla.olsnes@ore.uib.no; Helen Stavang - helen.stavang@ore.uib.no; Karl Brokstad - karl.brokstad@gades.uib.no; Jan Olofsson - jan.olofsson@ore.uib.no; Hans J Aarstad - hans.jorgen.aarstad@helse-bergen.no * Corresponding author Email: Carla Olsnes* - carla.olsnes@ore.uib.no; Helen Stavang - helen.stavang@ore.uib.no; Karl Brokstad - karl.b Jan Olofsson - jan.olofsson@ore.uib.no; Hans J Aarstad - hans.jorgen.aarstad@helse-bergen.no arla.olsnes@ore.uib.no; Helen Stavang - helen.stavang@ore.uib.no; Karl Brokstad - karl.brokstad@gades.uib.no; on@ore.uib.no; Hans J Aarstad - hans.jorgen.aarstad@helse-bergen.no * Corresponding author Received: 16 December 2008 Accepted: 28 January 2009 Received: 16 December 2008 Accepted: 28 January 2009 Published: 28 January 2009 Published: 28 January 2009 BMC Immunology 2009, 10:6 doi:10.1186/1471-2172-10-6 BMC Immunology 2009, 10:6 doi:10.1186/1471-2172-10-6 Page 1 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 cancer [1,2] and lymphangiomas [3]. Killed bacterial tox- ins [4] along with bacillus Calmette-Guerin (BCG) [5], β- glucan [6], interferons [7] and monoclonal antibodies [8] are examples of BRMs used in cancer treatment. from tonsillitis, impetigo, necrotizing fasciitis, and scarlet fever to lethal toxic shock syndrome [13]. Thus, studying the interactions of OK-432 with the immune system may shed light into the biology of these diseases as well. One major MNP function is secretion of chemokines, such as monocyte chemoattractant protein (MCP) -1 and macrophage inflammatory protein (MIP) -1α/β [14]. Chemokines have been shown to be secreted in response to OK-432 stimulation [15] and have the main effect of attracting new leucocytes to the site from where they are secreted [16]. It should be of interest, to determine the modality of chemokine secretion from MOs and Mϕs upon OK-432 stimulation. We have therefore found it pertinent to address this issue in the present investigation. Japan has a long standing tradition in using penicillin- killed lyophilized Streptococcus pyogenes, denominated OK-432 or picibanil, as a biological response modifier (BRM) for treatment of cancer [1,2]. Sakamoto et al. [1] published in 2001 a meta-analysis showing a 20% 5-year survival improvement with immunochemotherapy, com- pared to chemotherapy alone, following OK-432 treat- ment in patients diagnosed with non-small-cell lung cancer. This meta-analysis was based on 1,520 patients enrolled in 11 randomized clinical phase III trials. Fur- thermore, Oba et al [2] published in 2007 a meta-analysis including 8009 gastric carcinoma patients from 8 rand- omized clinical phase III trials and concluded that com- pared to control conditions, addition of OK-432 treatment increased survival. There are also reports sug- gesting that patients with other cancers, such as head and neck squamous cell carcinoma (HNSCC) [9], may benefit from OK-432 treatment. OK-432 has also been used as a maturation factor for DCs cells as part of vaccination ther- apy of cancer patients [10]. Priming provided by adherence, presumably associated with MNP differentiation from MO to Mϕ, contributes to interleukin secretion following OK-432 MNP stimulation [17]. Therefore, it is of interest to study to what extent the MNP chemokine response to OK-432 depends on adher- ence. Molecularly, we have found it relevant to investigate the role of β-integrin receptors, as these receptors partici- pate during differentiation of MNP from MO to Mϕ. http://www.biomedcentral.com/1471-2172/10/6 Fur- thermore, these receptors may be functionally blocked by addition of piceatannol (Syk kinase inhibitor) and/or LY294002 (phosphoinositide (PI)-3 kinase inhibitor) [18-22]. OK-432 may stimulate TLR receptors. In addition to OK-432, we have studied chemokine secretion upon stimulation with the known TLR2 agonist LTA and TLR4 agonist LPS. Moreover, we explored the role of the TLR2 co-receptor CD36 [23] in OK-432 stimulation. Lymphangiomas are benign neoplasias of lymphatic ori- gin, often congenital, that may extend around vital struc- tures [11]. Surgical removal of lymphangiomas has been the standard treatment, but may be technically difficult. Injection of OK-432 into cystic lymphangioma lesions may lead to shrinkage and subsequent cure as first reported by Ogita [3]. Today, injection with OK-432 is established as a major treatment option for lymphangi- omas [11], and is an important example of the use of BRMs in medicine. We have determined that in vitro purified adherent MNPs may secrete chemokines MCP-1, MIP-1α and MIP-1β fol- lowing OK-432 stimulation. MOs deprived of adherence did not respond to OK-432 stimulation as assessed by chemokine secretion. Inhibition of Syk and PI-3 kinase did not block the stimulatory effect of OK-432. MIP-1α/β production in WB and MOs was higher upon stimulation with LTA or LPS while OK-432 stimulation gave a higher MCP-1 secretion in WB and MOs in comparison with LTA or LPS. CD36, and to some extent CD18, participate in the OK-432-modulated MNP chemokine response. OK-432 presumably exerts its effect by activating the immune system to secrete toxic substances, which in turn eliminate tumor cells [12]. It is, however, relatively little known about the mechanisms of action of OK-432. The principal cells responding to the drug, the engaged recep- tors or signal transduction pathways, are to a surprisingly large extent unknown. This should be an important area of study. The efficiency of OK-432 treatment needs to be improved in order to make OK-432 a better cancer treat- ment drug. Additional drugs, which could improve OK- 432's response, as well as studying to what extent other (cancer) drugs potentially interfere with the receptors and signal transduction pathways driving the OK-432 response, should therefore be determined as to optimize and improve treatment. Background as monocytes (MOs) in blood and become, e.g., tissue macrophages (Mϕs) or dendritic cells (DCs) upon extravasation into tissues. The innate immune system is presumably involved when biological response modifiers (BRMs) are utilized in the treatment of diseases such as g The innate immune system detects and eliminates invad- ing foreign material through non-specific defense mecha- nisms elicited by, e.g., mononuclear phagocytes (MNP). MNPs originate as monoblasts in the bone marrow, reside Page 1 of 11 (page number not for citation purposes) Page 1 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 In vitro chemokine production in unstimulated WB, PBMCs or MOs Figure 1 In vitro chemokine production in unstimulated WB, PBMCs or MOs. Human WB, PBMCs or MOs were plated in parallel on regular (R) or low attachment (L) plates and left unstimulated for 24 h (A). Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP-1). WB (n = 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6). In itro chemok or MOs Figure 1 chemokine response profile as healthy controls (Fig. 2). With respect to MCP-1, close correlation between protein levels and mRNA levels was observed in three healthy donors (Fig. 3A) as well as two HNSCC patients, tested on two separate days (Fig. 3C) suggesting a pre-transcrip- tional regulation upon in vitro OK-432 stimulation of MOs. Chemokine production following OK-432 versus LTA or LPS stimulation In separate experiments, MOs, stimulated with piceatannol (120 μM) (B) or wortman- nin (100 nM) (D) for 30 min followed by stimulation with OK-432 for an additional 24 h, were analyzed by ELISA or real-time PCR with respect to MCP-1 production and gene expression, respectively. A, B, and D: n = 3; C: n = 2. OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and correlates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin Figure 3 OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and corre- lates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin. MCP-1 gene expression was assessed in MOs stimulated with OK-432 for 24 h from three randomly selected healthy donors (A) or two HNSCC patients on two separate days (C). In separate experiments, MOs, stimulated with piceatannol (120 μM) (B) or wortman- nin (100 nM) (D) for 30 min followed by stimulation with OK-432 for an additional 24 h, were analyzed by ELISA or real-time PCR with respect to MCP-1 production and gene expression, respectively. A, B, and D: n = 3; C: n = 2. MIP-1α/β upon stimulating PBMCs versus MOs (Fig. 2). Stimulated WB gave a lower MIP-1α secretion compared to PBMCs or MOs (Fig. 2). The effect of OK-432 was also tested with MOs from HNSCC patients giving the same OK-432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients Figure 2 OK-432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients. Human WB, PBMCs or MOs were stimulated with media or OK-432 (0.01 KE/ml) for 24 h (A). MOs isolated from four HNSCC patients were stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12); MO (n = 27); B: HNSCC (n = 4). l f MO f OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and correlates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin Figure 3 OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and corre- lates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin. Chemokine production following OK-432 versus LTA or LPS stimulation Tested were WB (Fig. 4) and MOs (Fig. 5) stimulated in parallel with OK-432 or LTA (TLR2 ligand) or LPS (TLR4 ligand). In WB stimulated with OK-432 or LTA, the response was primarily seen with MCP-1, and only to some extent with MIP-1α/β, whereas the LPS-stimulated response was determined by MIP-1α/β secretion. In puri- fied MOs, OK-432 and LTA stimulated secretion of MCP- 1. LPS inhibited MCP-1 production. Thus, it appears that OK-432 stimulation is more akin to LTA than to LPS stim- ulation. In vitro che or MOs Figure 1 p , g In vitro chemokine production in unstimulated WB, PBMCs or MOs. Human WB, PBMCs or MOs were plated in parallel on regular (R) or low attachment (L) plates and left unstimulated for 24 h (A). Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP-1). WB (n = 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6). MIP-1α/β upon stimulating PBMCs versus MOs (Fig. 2). Stimulated WB gave a lower MIP-1α secretion compared to PBMCs or MOs (Fig. 2). The effect of OK-432 was also tested with MOs from HNSCC patients giving the same = 8); PBMC (MIP-1α/β: n = 7; MCP-1: n = 4); MO (n = 6). OK-432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients Figure 2 OK-432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients. Human WB, PBMCs or MOs were stimulated with media or OK-432 (0.01 KE/ml) for 24 h (A). MOs isolated from four HNSCC patients were stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12); MO (n = 27); B: HNSCC (n = 4). OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and correlates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin Figure 3 OK-432 augments MCP-1 gene expression in MOs from healthy donors and HNSCC patients and corre- lates with MCP-1 protein levels upon inhibition with piceatannol and wortmannin. MCP-1 gene expression was assessed in MOs stimulated with OK-432 for 24 h from three randomly selected healthy donors (A) or two HNSCC patients on two separate days (C). Results In vitro chemokine production in unstimulated cultures Upon adherence, only MCP-1 was to some extent secreted (Fig. 1). Upon low adherence conditions, MCP-1 and MIP-1α/β were secreted at all studied conditions (Fig. 1). Chemokine production following OK-432 stimulation At all tested regular growth conditions, OK-432 stimu- lated the release of MIP-1α/β, and MCP-1 (all p < 0.05) (Fig. 2). We determined a similar secretion for MCP-1 and Furthermore,S. pyogenes is an important pathogen causing human disease. The diseases caused by S. pyogenes range Page 2 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 Chemokine stimulation Figure 4 Chemokine production follows OK-432, LTA or LPS in vitro stimulation of WB. Human WB was stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP- 1). Control, OK-432, LPS: n = 8; LTA: n = 4. Chemokine production following OK-432 versus LTA or LPS stimulation MCP-1 gene expression was assessed in MOs stimulated with OK-432 for 24 h from three randomly selected healthy donors (A) or two HNSCC patients on two separate days (C). In separate experiments, MOs, stimulated with piceatannol (120 μM) (B) or wortman- nin (100 nM) (D) for 30 min followed by stimulation with OK-432 for an additional 24 h, were analyzed by ELISA or real-time PCR with respect to MCP-1 production and gene expression, respectively. A, B, and D: n = 3; C: n = 2. OK-432 st and isolate patients Figure 2 OK 432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients Figure 2 OK-432 stimulates release of chemokines in WB, PBMCs, and isolated human MOs from healthy donors and HNSCC patients. Human WB, PBMCs or MOs were stimulated with media or OK-432 (0.01 KE/ml) for 24 h (A). MOs isolated from four HNSCC patients were stimulated for 24 h with media or OK-432 (0.01 KE/ml) (B). Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). A: WB (n = 8); PBMC (n = 12); MO (n = 27); B: HNSCC (n = 4). Page 3 of 11 (page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 Chemokine production follows OK-432, LTA or LPS in vitro stimulation of WB Figure 4 Chemokine production follows OK-432, LTA or LPS in vitro stimulation of WB. Human WB was stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multiplex (MIP) or ELISA (MCP- 1). Control, OK-432, LPS: n = 8; LTA: n = 4. more, MIP-1β response was reduced by 74% (19 ± 3 vs 5 ± 2 ng/ml; p < 0.02), and the MCP-1 response by 94% (54 ± 9 vs 3.1 ± 1.7 ng/ml; p < 0.02). In MOs, MCP-1 (19.8 ± 6.4 vs -0.2 ± 0.2 ng/ml; p < 0.02) response was completely blocked, while MIP-1α was reduced by 92% 17.9 ± 10.7 vs 1.5 ± 1.1 ng/ml; p < 0.05) and MIP-1β by 98% 12.1 ± 3.8 vs 0.2 ± 0.6 ng/ml; p < 0.03). Thus, it appears that chemokine responses following OK-432-stimulation are almost absent if the cells are minimally allowed to adhere to a surface. Stimulation of MOs with OK-432 is independent of Syk and PI-3 kinase In order to determine whether Syk kinase activation was necessary for the observed chemokine production in OK- 432-stimulated MOs, the Syk kinase inhibitor piceatannol (120 μM, (Fig. 3B), 30 μM (not shown), 1 μM (Fig. 8) or 0.5 μM (not shown), or diluent control, was added for 30 minutes to adherent MOs prior to addition of OK-432 for 24 hours. Western blot (Wb) analysis (Fig. 9) shows that Chemokine production following OK-432 stimulation of PBMC, MO-depleted PBMCs or MOs Chemokine secretion was determined from PBMCs, MOs separated by adherence and MO-depleted PBMCs (Fig. 7). MO-depleted PBMCs (PB) and MOs secreted 18% and 36% of the PBMC-derived MCP-1, respectively (Figure 7). Thus, MOs are the major source for MCP-1 production. In contrast, PBMCs stimulated with LPS gave a negative MCP-1 production since stimulation with LPS gives lower MCP-1 secretions compared to media controls. Chemokine production following OK-432 stimulation of WB, PBMCs and MOs cultured on regular or low- attachment culture wells Chemokine secretion was compared in WB and MNPs cul- tured on regular (R) or low-attachment (L) wells (Fig. 6). In WB, the MCP-1 response was reduced by 90% at the L compared to the R condition (48 ± 4 vs 7 ± 27 ng/ml; p < 0.02). In PBMCs, MIP-1α response was abolished at the L condition 24 ± 9.7 vs -1 ± 5.5 ng/ml; p < 0.04). Further- Page 4 of 11 0.02). In PBMCs, MIP-1α response was abolished at the L condition 24 ± 9.7 vs -1 ± 5.5 ng/ml; p < 0.04). Further- OK-432, LTA or LPS in vitro stimulation of MOs Figure 5 OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432, LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n = 10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n = 9; LPS: n = 16). OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs Figure 6 OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs. WB, PBMCs or MOs were stimulated with OK-432 (0.01 KE/ml) or left unstimulated on regular (R) or low-attachment (L) plates for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8; PBMC: n = 8; MO: n = 6. OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs Figure 6 OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs. WB, PBMCs or MOs were stimulated with OK-432 (0.01 KE/ml) or left unstimulated on regular (R) or low-attachment (L) plates for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8; PBMC: n = 8; MO: n = 6. OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs Figure 6 OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs. WB, PBMCs or MOs were stimulated with OK-432 (0.01 KE/ml) or left unstimulated on regular (R) or low-attachment (L) plates for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8; PBMC: n = 8; MO: n = 6. Syk and PI- lation of M Figure 8 With respect to mRNA, MCP-1 levels are also reduced when exposed for 30 min to 120 μM piceatannol followed by 24-hour incubation with OK-432 (Fig. 3B, bot- tom). Thus, piceatannol, at high, likely unspecific, concen- trations (120 μM) lowers MCP-1 secretion while at low, specific, concentrations (0.5, 1 or 30 μM), has no effect on the tested chemokines, despite exhibiting efficacy in lower- ing Syk kinase phosphorylation. Syk kinase becomes phosphorylated following adherence of MOs and is further phosphorylated following OK-432 stimulation but this phosphorylation is substantially reduced in MOs exposed to piceatannol (30 μM) for 30 min before stimulation with OK-432 for an additional 15 min. Addition of piceatannol had no effect on chemokine production, as measured by MCP-1, in human MOs unless it was used at high, unspecific concentrations (120 μM) (Fig. 3B, top). With respect to mRNA, MCP-1 levels are also reduced when exposed for 30 min to 120 μM piceatannol followed by 24-hour incubation with OK-432 (Fig. 3B, bot- tom). Thus, piceatannol, at high, likely unspecific, concen- trations (120 μM) lowers MCP-1 secretion while at low, specific, concentrations (0.5, 1 or 30 μM), has no effect on the tested chemokines, despite exhibiting efficacy in lower- ing Syk kinase phosphorylation. did not significantly reduce MIP-1α, MIP-1β, or MCP-1 production in OK-432-stimulated MOs. Furthermore, a combination of piceatannol (Pic) (30 μM), and LY294002 (LY) (50 μM), or diluent control (ethanol, EtOH), was used throughout the entire MO isolation pro- cedure as well as during incubation with media (Fig. 8C) or OK-432 for an additional 24 hours (Fig. 8D). Incuba- tion with media blocked spontaneous in vitro secretion of all tested chemokines, but no significant effect was observed in the ability of MOs to produce MIP-1α, and MIP-1β upon OK-432 stimulation. On the contrary, MCP- 1 production appears to be augmented following OK-432 stimulation, pointing to the existence of an additional MCP-1 pathway, dependent on Syk and PI-3 kinase, throughout the isolation and adherence protocol. The role of PI-3 kinase in OK-432's ability to stimulate MOs was tested where LY294002 (Fig. 8) or Wortmannin (Fig. 3D) was added for 30 min to freshly isolated MOs followed by incubation with OK-432 for 24 hours. West- ern blot analysis (Fig. 9) shows that PI-3 kinase is not phosphorylated following adherence of MOs but becomes phosphorylated following OK-432 stimulation. Chemokine production following OK-432 stimulation of WB, PBMCs and MOs cultured on regular or low- attachment culture wells OK-432, LTA or LPS in vitro stimulation of MOs Figure 5 OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432, LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n = 10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n = 9; LPS: n = 16). OK-432, LTA or LPS in vitro stimulation of MOs Figure 5 OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432, LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n = 10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n = 9; LPS: n = 16). , g OK-432, LTA or LPS in vitro stimulation of MOs. MOs were stimulated in parallel with OK-432 (0.01 KE/ml), LTA (0.5 μg/ml), LPS (1 μg/ml), or left unstimulated for 24 h. Supernatants were then collected and analyzed using Multi- plex (MIP) or ELISA (MCP-1). MIP-1α (Control, OK-432, LPS: n = 9; LTA: n = 7).MIP-1β (Control, OK-432, LPS: n = 10; LTA: n = 8); MCP-1 (Control, OK-432: n = 19; LTA: n = 9; LPS: n = 16). OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs Figure 6 OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs. WB, PBMCs or MOs were stimulated with OK-432 (0.01 KE/ml) or left unstimulated on regular (R) or low-attachment (L) plates for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8; PBMC: n = 8; MO: n = 6. OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs Figure 6 OK-432 stimulation of adherent or suspended WB, PBMCs, and MOs. WB, PBMCs or MOs were stimulated with OK-432 (0.01 KE/ml) or left unstimulated on regular (R) or low-attachment (L) plates for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). WB: n = 8; PBMC: n = 8; MO: n = 6. Syk and PI- lation of M Figure 8 This OK-432-dependent PI-3 kinase phosphorylation is decreased when MOs are exposed to LY294002 for 30 min before stimulation with OK-432 for 15 min. Addition of LY294002 (Fig. 8B) or Wortmannin (results not shown) Syk and PI- lation of M Figure 8 Syk and PI- lation of M Figure 8 Syk and PI 3 kinase independent signaling for OK 432 stimu lation of MOs Figure 8 Syk and PI-3 kinase-independent signaling for OK- 432 stimulation of MOs. Piceatannol (1 μM) or diluent control was added to MO cultures for 30 min prior to addi- tion of OK-432 for 24 h (A). LY294002 (50 μM) was added to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50 μM) and piceatannol (30 μM) was added to whole blood and throughout the monocyte isolation procedure (C) and fol- lowed by stimulation with OK-432 or left unstimulated for 24 h (D). Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4. Syk and PI 3 kinase independent signaling for OK 432 stimu lation of MOs Figure 8 Syk and PI-3 kinase-independent signaling for OK- 432 stimulation of MOs. Piceatannol (1 μM) or diluent control was added to MO cultures for 30 min prior to addi- tion of OK-432 for 24 h (A). LY294002 (50 μM) was added to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50 μM) and piceatannol (30 μM) was added to whole blood and throughout the monocyte isolation procedure (C) and fol- lowed by stimulation with OK-432 or left unstimulated for 24 h (D). Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4. p pg MO-depleted PBMCs secrete less chemokines than PBMCs upon OK-432 stimulation. PBMCs, MOs, or PBs (MO-depleted PBMCs) were left unstimulated or stimulated in parallel with OK-432 (0.01 KE/ml) or LPS (1 μg/ml) for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). n = 2. Syk kinase becomes phosphorylated following adherence of MOs and is further phosphorylated following OK-432 stimulation but this phosphorylation is substantially reduced in MOs exposed to piceatannol (30 μM) for 30 min before stimulation with OK-432 for an additional 15 min. Addition of piceatannol had no effect on chemokine production, as measured by MCP-1, in human MOs unless it was used at high, unspecific concentrations (120 μM) (Fig. 3B, top). Chemokine production following OK-432 stimulation of WB, PBMCs and MOs cultured on regular or low- attachment culture wells Page 4 of 11 (page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 Syk and PI-3 kinase-independent signaling for OK-432 stimu- lation of MOs Figure 8 Syk and PI-3 kinase-independent signaling for OK- 432 stimulation of MOs. Piceatannol (1 μM) or diluent control was added to MO cultures for 30 min prior to addi- tion of OK-432 for 24 h (A). LY294002 (50 μM) was added to MOs for 30 min prior to addition of OK-432 for 24 h (B). In separate experiments, a combination of LY294002 (50 μM) and piceatannol (30 μM) was added to whole blood and throughout the monocyte isolation procedure (C) and fol- lowed by stimulation with OK-432 or left unstimulated for 24 h (D). Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). A: n = 4; B: n = 5; C: n = 4; D: n = 4. MO-depleted PBMCs secrete less chemokines than PBMCs upon OK-432 stimulation Figure 7 MO-depleted PBMCs secrete less chemokines than PBMCs upon OK-432 stimulation. PBMCs, MOs, or PBs (MO-depleted PBMCs) were left unstimulated or stimulated in parallel with OK-432 (0.01 KE/ml) or LPS (1 μg/ml) for 24 h. Supernatants were analyzed using Multiplex (MIP) or ELISA (MCP-1). n = 2. Role of CD36 on stimulation of MOs with OK-432 f f Anti-CD36 antibody or isotype control was added to wells followed by addition of PBMCs, incubated for 40 min, and OK-432 was added for an additional 24 hours (Fig. 10B). CD36 addition decreased MIP-1β and MCP-1 (both p = 0.028) but not MIP-1α secretion. Thus, in MOs, OK- 432 elicits both MIP-1β and MCP-1 production in part via CD36 acting as a co-receptor. All studied chemokines were secreted without particular stimulation factors at low adherence conditions, whereas MIP-1α/β secretion was completely inhibited by adherence. At low adherence conditions, the secretion response of MIP-1α/β and MCP-1 from PBMCs and MOs was abolished upon OK-432 stimulation. This might indicate that MNPs undergoing adherence fundamen- tally alter their chemokine secretion responses and sup- ports that MO to Mϕ differentiation interferes with the regulation of chemokine secretion. This is also sup- ported by our observations of MIP-1α-derived OK-432 stimulated secretion being lower in WB compared to PBMCs and MOs. Diapedesis depends on, e.g., integrin receptor engagement co-triggering differentiation of MOs to Mϕs [18]. Such stimulation may be provided when MOs are isolated by in vitro adherence. Syk phos- phorylation is a necessary part of β1–3 integrin-generated activation [19-21], whereas PI-3 kinase phosphorylation is required in β2 integrin-generated activation [20]. We have presently shown that adherence of MOs stimulated phosphorylation of Syk but not PI-3 kinase, as deter- mined by Western blots. Thus, it appears that possibly both β1 and αvβ3 integrins, but not β2 integrin, play a role in mediating MO adhesion. Based on Western blots, binding of OK-432 to MOs elicited a higher Syk phos- phorylation compared to unstimulated MOs, possibly suggesting an adherence-mediated priming effect. PI-3 Page 6 of 11 (page number not for citation purposes) Role of β2-integrin (CD18) during stimulation of MOs with OK-432 Treating MOs with a neutralizing F(ab) – anti-CD18 anti- body (Fig. 10A) prior to OK-432 stimulation, the secre- tion of MCP-1 (statistically nonsignificant), but not the other tested chemokines, decreased. Thus, the β2 integrin (CD18) may possibly be involved in MCP-1 secretion fol- lowing OK-432 activation of human isolated MOs. Page 5 of 11 (page number not for citation purposes) Page 5 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 OK-432 promotes Syk or PI-3 kinase phosphorylation in MOs Figure 9 OK-432 promotes Syk or PI-3 kinase phosphorylation in MOs. MOs, isolated by the monocyte negative isolation kit, were incubated either with piceatannol, Pic. (30 μM) or diluent control (A) or LY294002, LY (50 μM) (B) and either left unstimulated or further stimulated with OK-432 for 15 min. Blots shown are representative of two separate experi- ments performed with pooled MOs obtained from two dif- ferent blood donors. Role of CD18 and CD36 in OK-432 activation of MOs Figure 10 Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti- CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incu- bated either with isotype control or anti-CD36 (2.5 μg/ml) (B) prior to incubation with OK-432 for 24 h. Supernatants were analyzed for shown chemokines by Multiplex (MIP) or ELISA (MCP-1). A: n = 3; B: n = 6. CD18: MCP-1 (NS). chemokine response in purified MOs from HNSCC OK-432 promotes Syk or PI-3 kinase phosphorylation in MOs Figure 9 OK-432 promotes Syk or PI-3 kinase phosphorylation in MOs. MOs, isolated by the monocyte negative isolation kit, were incubated either with piceatannol, Pic. (30 μM) or diluent control (A) or LY294002, LY (50 μM) (B) and either left unstimulated or further stimulated with OK-432 for 15 min. Blots shown are representative of two separate experi- ments performed with pooled MOs obtained from two dif- ferent blood donors. Role of CD18 and CD36 in OK-432 activation of MOs Figure 10 Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti- CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incu- bated either with isotype control or anti-CD36 (2.5 μg/ml) (B) prior to incubation with OK-432 for 24 h. Supernatants were analyzed for shown chemokines by Multiplex (MIP) or ELISA (MCP-1). A: n = 3; B: n = 6. Role of β2-integrin (CD18) during stimulation of MOs with OK-432 CD18: MCP-1 (NS). Role of CD18 and CD36 in OK-432 activation of MOs Figure 10 Role of CD18 and CD36 in OK-432 activation of MOs. MOs were incubated either with isotype control or anti- CD18/F(ab) (12.5 μg/ml) for 24 h (A). PBMCs were incu- bated either with isotype control or anti-CD36 (2.5 μg/ml) (B) prior to incubation with OK-432 for 24 h. Supernatants were analyzed for shown chemokines by Multiplex (MIP) or ELISA (MCP-1). A: n = 3; B: n = 6. CD18: MCP-1 (NS). OK-432 pr MOs Figure 9 OK-432 promotes Syk or PI-3 kinase phosphorylation in MOs. MOs, isolated by the monocyte negative isolation kit, were incubated either with piceatannol, Pic. (30 μM) or diluent control (A) or LY294002, LY (50 μM) (B) and either left unstimulated or further stimulated with OK-432 for 15 min. Blots shown are representative of two separate experi- ments performed with pooled MOs obtained from two dif- ferent blood donors. chemokine response in purified MOs from HNSCC patients. Discussion We have studied in vitro OK-432-stimulated mononuclear MNPs chemokine secretion. Purified MOs, PBMCs or WB secreted MCP-1, MIP-1α, and MIP-1β following OK-432 stimulation at a similar rate, except for a substantially lower MIP-1α secretion from WB. This is in line with other investigators, e.g., showing that S. pyogenes directly acti- vates Mϕs to secrete chemokines [24]. Cytokine mRNA quantification of MCP-1 transcripts showed that OK-432 stimulation was followed by cytokine mRNA synthesis within hours. There was a correlation between measured cytokine mRNA levels and cytokine-secreted levels sug- gesting that OK-432 mainly exerts its effects at the pre- transcriptional level. We furthermore separated by adher- ence most of the MOs from a sample of PBMCs. Upon OK-432 stimulation both these cell fractions secreted MCP-1 at about twice the level of the MO-depleted frac- tion. These findings support that MNPs are the main source for MCP-1 within PBMCs. OK-432 also elicited a Page 6 of 11 (page number not for citation purposes) Page 6 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 kinase phosphorylation occurred only upon stimulation with OK-432. We have shown that addition of either one of these inhibitors to OK-432-stimulated MOs did not change chemokine secretion. On the contrary, with addi- tion of both of these inhibitors, MCP-1 secretion was increased, pointing to the existence of other possible sig- naling pathways. In addition, presence of both inhibi- tors throughout the isolation and separation of monocytes blocked the unstimulated secretion of chem- okines supporting that β integrins are important for chemokine secretion during the adhesion process. Our observed Syk phosphorylation upon OK-432 binding pointed towards an integrin dependence and resonated with Cuzzola et al [22], who have shown that the MO interleukin response to Group A Streptococci (GAS) was dependent on the β2 integrin. We explored whether this applied to the OK-432-mediated activation of MOs by adding a blocking anti-CD18 antibody. MCP-1 produc- tion appears to be partially inhibited by the anti-CD18 antibody. Thus, the β2 integrin may play a role in medi- ating the effects of OK-432 on MOs. Nevertheless, other receptors may phosphorylate either Syk or PI-3 kinase or both and their nature has yet to be established. upon OK-432 stimulation after initiation of MO differen- tiation [17]. The present report thus adds more support to the notion that OK-432 is a target-seeking substance. Discussion Only when MNPs are stimulated by, e.g., tumor cells or intercel- lular substances [29], one observes a chemokine response. The present results argue in favor of chemokine secretion being an important effector function of MNPs during OK- 432 stimulation. This is important as to designing future BRM treatments with OK-432. Platinum-based drugs, e.g., negatively interfere more with monocyte than lymphocyte function [31] and should thus be avoided in combination with OK-432 treatment. MIP-1α/β and MCP-1 secretion is induced by stimulation of MOs with OK-432. These chemokines are ligands of the receptors CXCR1, CCR1, CCR2 and CCR5, present on pol- ymorphonuclear granulocytes, basophiles, granulocytes, MOs, Mϕs, DCs, NK cells, T, and B cells [32]. Thus, the shown chemokine secretion after OK-432 stimulation of MOs may co-explain the systemic effects of OK-432 on the immune system. Similar findings were made by Veckman et al [24] upon stimulating Mϕs with pathogenic live bac- teria (serotype T1M1 (IH32030)) isolated from a child with bacteremia. In contrast, we have stimulated mono- cytes with a heat- and penicillin G-killed, low-virulence SU strain of S. pyogenes and found very low levels of RANTES (CCL5). Therefore, it is plausible that virulence status/degree, strain, and preparation of bacteria influence chemokine production and MOs may have a different chemokine profile compared to Mϕs. This becomes espe- cially interesting when the emphasis is on OK-432 being a lyophilized, killed S. pyogenes. S. pyogenes is an impor- tant pathogen causing human disease. The diseases caused by S. pyogenes range from tonsillitis, impetigo, and scarlet fever to lethal toxic shock syndrome [13]. The shown mechanisms of MNP activation by OK-432 thus shed light on these diseases as well. OK-432 probably stimulates MOs via TLR2 [15] and/or TLR4 [25]. In order to study the role of these receptors, MOs were stimulated in parallel with OK-432, and the known TLR2 or TLR4 agonists, LTA and LPS, respectively. Results showed that MOs respond to OK-432 in a similar fashion to LTA stimulation, thus, arguing for primary TLR2-dependent stimulation of MOs by OK-432. A potential candidate for co-stimulation of MOs is CD36. This receptor has been found to function as a co- receptor for TLR2 receptor [26]. We determined a reduced MIP-1β and MCP-1, but an unchanged MIP-1α response upon employing blocking anti-CD36 antibod- ies. Thus, CD36 may function as an important receptor in the OK-432 stimulation of MOs with respect to chem- okine secretion. Conclusion h f We have found that MCP-1 and MIP-1α/β production by OK-432-stimulated human purified adherent MOs occurs in most healthy individuals. MIP-1α secretion is mostly found following differentiation stimuli of MOs to become Mϕs. Furthermore, MOs stimulated with OK-432 respond with chemokine secretion dependent on adherent growth conditions. OK-432 may rely on β2 integrin stimulation. CD36 modulates the MIP-1β and MCP-1 response to OK- 432. To some extent OK-432 may act as a target-seeking substance whereby only MOs adhered, e.g., to a target, secrete substantial amounts of chemokines in part explaining why OK-432 is suited as a BRM drug. Other co-stimulating receptors than the presently shown ones may be important for chemokine production following OK-432 stimulation of MNPs. Examples include CD62L (L- selectin receptor), a mediator of MOs rolling on and interact- ing with endothelial cells [27] and CD162 (P-selectin glyco- protein ligand-1), a promoter in the interaction between MOs and platelets [28]. Future studies are needed to estab- lish their roles in OK-432 stimulation in MOs. The shown MNP stage-dependence for MIP-1α secretion and adherence-dependent chemokine response to OK- 432 supports the hypothesis that chemokine secretion is less favoured when MOs are stimulated by OK-432 in pb compared to extravasal stimulation. This is in line with our previous observation that TNF-α is primarily secreted Page 7 of 11 (page number not for citation purposes) Experimental conditions In different experimental series, MOs (1 × 105 cells/well) were cultured either with piceatannol (0.5, 1, 30 or 120 μM – final concentration), LY294002 (50 μM – final con- centration), wortmannin (100 nM – final concentration) or anti-CD36 (2.5 μg/ml – final concentration) or anti- mouse IgG1 isotype control (2.5 μg/ml – final concentra- tion) or pepsin-degraded (Fab) anti-CD18 (12.5 μg/ml – final concentration) or anti-rat IgG2B isotype control (12.5 μg/ml – final concentration) for 30 min before OK-432 (0.01 KE/ml – final concentration) was added and after 24 hours the supernatants were collected and stored at -80°C for further analysis. Whole blood, PBMC, and monocyte preparation Whole blood was diluted 1:1 with Ultraculture (Lonza, Basel, Switzerland), allocated to a 24-well plate (Nunc A/ S, Roskilde, Denmark) and upon experiment comple- tion, the supernatant was collected after the whole blood/Ultraculture mixture was centrifuged. PBMCs were separated by density gradient centrifugation with Lymphoprep® (Nycomed, Oslo, Norway) as density gra- dient medium in LeukoSep tubes (Greiner-Bio One GmbH, Frickenhausen, Germany). MOs were, unless Reagents and antibodies OK-432 (Picibanil) (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan) was dissolved in sterile water as a stock Page 7 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-2172/10/6 BMC Immunology 2009, 10:6 otherwise stated, isolated from blood by gradient centrif- ugation followed by adherence to plastic. The PBMC yield of 7 ml blood (6 × 105 cells/well) was allocated to a 24-well plate (Nunc) with RPMI 1640 (Lonza) supple- mented with amphotericin B (2.5 μg/ml) and glucose (2% v/v) (both Sigma), HEPES (1% v/v), L-glutamine (1% v/v), penicillin (100 IU/ml), streptomycin (100 μg/ ml), sodium bicarbonate (2.7% v/v), sodium pyruvate (1% v/v)(all from Lonza) and 20% AB serum to a total volume of 0.5 ml/well. After 40 minutes pre-incubation, the adherent MOs were purified by washing, and then cultured in complete RPMI 1640 (Lonza)/20% AB serum with 0.5 ml/well. The method yields in general cultures which contain at least 75% MOs, as determined by non- specific esterase stain (NSES), and more than 90% viable cells, as tested by tryphan blue stain. In some experi- ments, MOs were also isolated using the negative isola- tion kit (Dynal Biotech, Oslo, Norway) according to the manufacturer's protocol. Briefly, PBMCs, isolated by gra- dient centrifugation using Lymphoprep-containing Leu- koSep tubes, were resuspended in PBS/0.1% bovine serum albumin (BSA) (Sigma) and exposed to an equal mixture of blocking reagent and antibody mix for 10 min at 4°C. After 10 min, PBMCs were washed, added fresh PBS/0.1% BSA, added to washed beads, and incubated for 15 min with gentle tilting and rotation at 4°C. After incubation, resuspended rosettes were pipetted and placed in the Dynal MPC magnet for 5 min. The super- natant was collected and placed in the Dynal MPC mag- net for an additional 2 min. The collected MO-pure supernatant (95% purity – NSES) was washed, cells counted, and added to a 24-well plate. solution with a concentration of 1 KE (Klinische Einheit)/ ml = 0.1 mg/ml. LTA from S. pyogenes and LPS from E.coli 026:B6 (Sigma, St.Louis, MO, USA) were dissolved in PBS as a stock solution of 5 mg/ml (LTA) and 25 μg/ml (LPS) and used at a final concentration of 0.5 μg/ml and 1 μg/ ml, respectively. Reagents and antibodies F(ab) (degraded by pepsin (Sigma)), azide-free, blocking rat anti-human CD18 (Chemicon International, Temecula, CA, USA) was dissolved in sterile PBS as a 100 μg/ml stock solution. Anti-rat IgG2B isotype control (R&D Systems Europe Ltd.) was dissolved in sterile PBS as a 500 μg/ml stock solution. Patients and controls Patients admitted to surgery for head and neck squamous cell carcinoma (HNSCC) gave written consent before par- ticipating in the study. All patients were males. Patient 1 was 60 years and had tonsillar carcinoma (T3N0M0G1). Patient 2 had carcinoma of the tongue (T2N2M0G2). Patient 3 had carcinoma of the basal tongue (T2N2M0G2). Patient 4 had laryngeal carcinoma (T4N1M0). All patients donated blood approximately 2 weeks post-operatively as is appropriate in the event of starting treatment with OK- 432. Whole blood from controls was obtained from ran- domly selected, healthy donors at Haukeland University Hospital. As selection was done randomly, age and sex of donors were not taken into consideration, i.e. not recorded. The study was approved by the Regional Com- mittee for Medical Ethics. Reagents and antibodies The Syk inhibitor piceatannol (Calbio- chem, La Jolla, CA, USA) was dissolved in ethanol as a stock solution with a concentration of 2 mg/ml (8000 μM) and used at a final concentration of 0.5, 1, 30 or 120 μM [18,19,33]. The PI-3 kinase inhibitor, LY294002 (Sigma) was dissolved in sterile water at a concentration of 5 mg/ml (14.5 mM) and used at a final concentration of 50 μM. The PI-3 kinase inhibitor, wortmannin (Sigma) was dissolved in DMSO at a concentration of 1 mg/ml (2.3 mM) and used at a final concentration of 100 nM. Azide-free, blocking mouse anti-human CD36 (HyCult, Uden, The Netherlands) came as a 100 μg/ml stock solu- tion and anti-mouse rat IgG1 isotype control (R&D Sys- tems Europe Ltd., Abingdon, Great Britain) was dissolved in sterile PBS as a 250 μg/ml stock solution. F(ab) (degraded by pepsin (Sigma)), azide-free, blocking rat anti-human CD18 (Chemicon International, Temecula, CA, USA) was dissolved in sterile PBS as a 100 μg/ml stock solution. Anti-rat IgG2B isotype control (R&D Systems Europe Ltd.) was dissolved in sterile PBS as a 500 μg/ml stock solution. solution with a concentration of 1 KE (Klinische Einheit)/ ml = 0.1 mg/ml. LTA from S. pyogenes and LPS from E.coli 026:B6 (Sigma, St.Louis, MO, USA) were dissolved in PBS as a stock solution of 5 mg/ml (LTA) and 25 μg/ml (LPS) and used at a final concentration of 0.5 μg/ml and 1 μg/ ml, respectively. The Syk inhibitor piceatannol (Calbio- chem, La Jolla, CA, USA) was dissolved in ethanol as a stock solution with a concentration of 2 mg/ml (8000 μM) and used at a final concentration of 0.5, 1, 30 or 120 μM [18,19,33]. The PI-3 kinase inhibitor, LY294002 (Sigma) was dissolved in sterile water at a concentration of 5 mg/ml (14.5 mM) and used at a final concentration of 50 μM. The PI-3 kinase inhibitor, wortmannin (Sigma) was dissolved in DMSO at a concentration of 1 mg/ml (2.3 mM) and used at a final concentration of 100 nM. Azide-free, blocking mouse anti-human CD36 (HyCult, Uden, The Netherlands) came as a 100 μg/ml stock solu- tion and anti-mouse rat IgG1 isotype control (R&D Sys- tems Europe Ltd., Abingdon, Great Britain) was dissolved in sterile PBS as a 250 μg/ml stock solution. Reverse transcription TaqMan RT reagents (Applied Biosystems, Foster City, CA, USA) were used to reverse transcribe total RNA from OK- 432-stimulated MOs obtained from normal blood donors. Final concentration of the RT reaction was 1 × TaqMan RT buffer, 5.5 mM MgCl 2, 2 mM dNTP mixture (500 μM of each dNTP), 2.5 μM random hexamers, 0.4 units/μl RNase inhibitor, 2.5 units/μl Multiscribe RT, 5– 25 ng RNA, and DEPC water to a total volume of 50 μl. The RT thermal cycling was performed on a Mastercycler gradient thermal cycler (Eppendorf, Hamburg, Germany) under the following conditions: primer incubation for 10 min at 25°C, reverse transcription for 30 minutes at 48°C, and inactivation of reverse transcriptase for 5 minutes at 95°C. Real-time quantitative RT-PCR analysis Real-time quantitative RT-PCR analysis The ABI PRISM 7700 Sequence Detection System (Applied Biosystems) with 96-well plates was used to per- form real-time quantitative RT-PCR. Reactions were car- ried out in a total volume of 25 μl containing 12.5 μl of 2 × TaqMan Universal PCR Master Mix (Applied Biosys- tems), 300 nM (0.25 μl) forward primer, 300 nM (0.25 μl) reverse primer, 200 nM (1 μl) TaqMan probe, 8 μl water, and 3 μl cDNA template. Thermocycler conditions were as follows: incubation for 2 minutes at 50°C, fol- lowed by incubation for 10 minutes at 95°C and 40 cycles of two-step (denaturing for 15 seconds at 95°C followed by annealing/extension for 1 minute at 60°C) PCR. As a reference gene, CD68 was used to normalize the gene Western blot analysis y MOs from 7 ml blood were isolated by gradient centrifu- gation in Lymphoprep-containing LeukoSep tubes, fol- lowed by the monocyte negative isolation kit (Dynal Biotech), allowed to adhere for 40 minutes, after which piceatannol or LY294002 or diluent control was added for 30 min, followed by incubation with OK-432 for 15 min. Upon experiment completion, cells were washed once with cold PBS, added lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and 1× complete proteinase inhibitor (Roche, Nutley, NJ, USA)) and scraped gently using a cell scraper. The cell lysate was clarified by centrif- ugation at 16,000 g for one minute at 4°C. Protein levels were measured using the Bio-Rad BCA Protein Assay (Bio- Rad, Hercules, CA, USA). Samples were added 1× loading buffer and the lysate was boiled for 5 minutes. Samples (6 μg/lane) and a Precision Plus Protein Kaleidoscope stand- ard (Bio-Rad) were electrophoresed on a 10% SDS poly- acrylamide gel for 60 minutes at 200 V and transferred to nitrocellulose membranes (Bio-Rad) in electroblotting buffer (25 mM Tris base, 0.2 M glycine, and 20% metha- nol) for 60 minutes at 100 V. Membrane was blocked with 5% BSA in TBS-Tween for one hour at RT with gentle tilt- ing. The blots were exposed either to a polyclonal anti- body against phosphorylated Syk or PI3 kinase p85/p55 or total Syk or total PI3 kinase p85 (all: Cell Signaling, Beverly, MA, USA) (all: 1:1000 in 5% BSA in TBS-Tween) overnight at 4°C with gentle shaking. After washing, an HRP-linked goat anti-rabbit IgG (H + L) antibody (Bio- Rad) (1:2000) was added for one hour at room tempera- ture with gentle tilting. Proteins were detected using lumi- nol/enhancer/hydrogen peroxide – based ECL and visualized on the Molecular Imager ChemiDoc XRS Sys- tem (Bio-Rad). http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 expression of MCP-1. To generate a standard curve for rel- ative gene expression determination, cDNA synthesized from pooled total RNA of MOs obtained from healthy blood donors was used. The standard curves generated for the cDNA analyzed had slopes with a mean of -3.3. (buffer RLT containing beta-mercaptoethanol (Sigma)), homogenized by vortexing for 30 s, and the homogenate was applied to an Rneasy spin column after addition of ethanol in order to bind total RNA to the membrane. Contaminants were removed by washing with buffers RW1 and RPE, and total RNA was eluted using water. In addition, total RNA was isolated from MOs of normal blood donors and subsequently pooled for use in generat- ing the standard curve. Primer and probe design Primer and probes for the TaqMan assays were designed using the Primer Express software (Applied Biosystems). Intron-spanning primers were used to prevent amplifica- tion of genomic DNA. The candidate genes were MCP-1 [GenBank: NM_002982] (70 bp-amplicon); primers: (F) 5'-CTCTCGCCTCCAGCATGAA-3', (R) 5'-GGAATGAAG- GTGGCTGCTATGA-3', probe: 5'-CCGCCCTTCTGT- GCCTGCTGC-3'and the housekeeping gene used was CD68 [GeneBank: BC015557] (67 bp-amplicon); prim- ers: (F) 5'-CCCCACGCAGCAAAGTG-3', (R) 5'-CCAG- GGGTGCTTGGAGATCT-3, probe: 5'-TCTCGGCTCAGAA TGCATCCCTTCG-3'). Designed primers and probes were purchased from MedProbe (Oslo, Norway). Page 9 of 11 (page number not for citation purposes) Total RNA extraction In some experiments, total RNA was isolated immediately after harvesting MOs (24 hours post-stimulation), using the RNeasy Minikit's (Qiagen, Hilden, Germany) protocol for isolation of total RNA from animal cells grown in monolayer, as recommended by the manufacturer. Briefly, adherent MOs were added 350 μl lysis buffer Page 8 of 11 (page number not for citation purposes) Page 8 of 11 (page number not for citation purposes) BMC Immunology 2009, 10:6 Luminex fluorescent bead-based technology (Luminex Corporation Austin, TX, USA). Luminex fluorescent bead-based technology (Luminex Corporation Austin, TX, USA). Luminex fluorescent bead-based technology (Luminex Corporation Austin, TX, USA). Statistical analyses The statistical program package Statistical Package for the Social Sciences (Ver. 16 SPSS, Chicago, Ill., USA) was used. Chemokine levels and expression were compared by the Wilcoxon signed-rank test (two-tailed). Differences were considered significant at p < 0.05. 8. Knecht R, Peters S, Solbach C, Baghi M, Gstottner W, Hambek M: EGFR antibody-supplemented TPE-chemotherapy. Preclini- cal investigations to a novel approach for head and neck can- cer induction treatment. Anticancer Res 2003, 23:4789-95. 9. 9. Kumazawa H, Yamashita T, Tachikawa T, Minamino M, Nakata Y: Local injection of OK-432/fibrinogen gel into head and neck carcinomas. Eur J Cancer 1994, 30A:1741-4. 10. Naito K, Ueda Y, Itoh T, Fuji N, Shimizu K, Yano Y, Yamamoto Y, Imura K, Kohara J, Iwamoto A, et al.: Mature dendritic cells gen- erated from patient-derived peripheral blood monocytes in one-step culture using streptococcal preparation OK-432 exert an enhanced antigen-presenting capacity. Int J Oncol 2006, 28:1481-9. References 1. Sakamoto J, Teramukai S, Watanabe Y, Hayata Y, Okayasu T, Naka- zato H, Ohashi Y: Meta-analysis of Adjuvant Immunochemo- therapy Using OK-432 in Patients With Resected Non- Small-Cell Lung Cancer. J Immunother 2001, 24:250-256. 1. Sakamoto J, Teramukai S, Watanabe Y, Hayata Y, Okayasu T, Naka- zato H, Ohashi Y: Meta-analysis of Adjuvant Immunochemo- therapy Using OK-432 in Patients With Resected Non- Small-Cell Lung Cancer. J Immunother 2001, 24:250-256. g J 2. Oba K, Teramukai S, Kobayashi M, Matsui T, Kodera Y, Sakamoto J: Efficacy of adjuvant immunochemotherapy with polysaccha- ride K for patients with curative resections of gastric cancer. Cancer Immunol Immunother 2006, 56(6):905-911. ( ) 3. Ogita S, Tsuto T, Deguchi E, Tokiwa K, Nagashima M, Iwai N: OK- 432 therapy for unresectable lymphangiomas in children. J Pediatr Surg 1991, 26:263-8. g 4. Okamoto M, Ohe G, Oshikawa T, Nishikawa H, Furuichi S, Yoshida H, Matsuno T, Saito M, Sato M: Induction of Th1-type cytokines by lipoteichoic acid-related preparation isolated from OK- 432, a penicillin-killed streptococcal agent. Immunopharmacol- ogy 2000, 49:363-76. gy , 5. Baran J, Baj-Krzyworzeka M, Weglarczyk K, Ruggiero I, Zembala M: Modulation of monocyte-tumour cell interactions by Myco- bacterium vaccae. Cancer Immunol Immunother 2004, 53:1127-34. 6. Ooi VE, Liu F: Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr Med Chem 2000, 7:715-29. gy 5. Baran J, Baj-Krzyworzeka M, Weglarczyk K, Ruggiero I, Zembala M: Modulation of monocyte-tumour cell interactions by Myco- bacterium vaccae. Cancer Immunol Immunother 2004, 53:1127-34. 6. Ooi VE, Liu F: Immunomodulation and anti-cancer activity of polysaccharide-protein complexes. Curr Med Chem 2000, 7:715-29. 7. Moschos SJ, Edington HD, Lan SR, Rao UN, Jukic D, Shipe-Spotloe J, Kirkwood JM: Neoadjuvant treatment of regional stage IIIB melanoma with high-dose interferon alfa-2b induces objec- tive tumor regression in association with modulation of tumor infiltrating host cellular immune responses. J Clin Oncol 2006, 24:3164-71. Acknowledgements This work has been supported by the Norwegian Cancer Society and the Strategic Research Program at Helse Bergen, Norway. We thank Dagny Ann Sandnes for expert ELISA analysis, Petra Vogelsang for assistance with the Western blot/Molecular Imager ChemiDoc XRS System, Broegelmann Research Laboratory for providing lab and instruments for Western blot analysis and The Research Core Facility (FFS) at Haukeland University Hos- pital for general lab and instrument access. Competing interests h h d l h h The authors declare that they have no competing interests. The authors declare that they have no competing interests. py 13. Cleary PP: Streptococcus moves inward. Nat Med 2006, 12:384-6. 14. Van Ginderachter JA, Movahedi K, Hassanzadeh Ghassabeh G, Meer- schaut S, Beschin A, Raes G, De Baetselier P: Classical and alterna- tive activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion. Immunobiology 2006, 211:487-501. Abbreviations BRM: biological response modifier; HNSCC: head and neck squamous cell carcinoma; MIP: macrophage inflam- matory protein; MCP: monocyte chemoattractant protein; pb: peripheral blood; PI: phosphoinositide; pSyk: phos- phoSyk; TLR: Toll-like receptor. 11. Knipping S, Goetze G, Neumann K, Bloching M: Sclerotherapy of cervical cysts with Picibanil (OK-432). Eur Arch Otorhinolaryngol 2006, 264:423-7. 12. Ryoma Y, Moriya Y, Okamoto M, Kanaya I, Saito M, Sato M: Biolog- ical effect of OK-432 (picibanil) and possible application to dendritic cell therapy. Anticancer Res 2004, 24:3295-301. MCP-1 protein analysis The contents of MCP-1 in supernatants were determined by enzyme-linked immunosorbant assay kit (ELISA) man- ufactured by R&D Systems (R&D Systems). All procedures were performed according to the specifications of the manufacturer. In short, 96-well microtiter plates (Costar Corning, NY, USA) were coated overnight at room tem- perature with monoclonal mouse anti-human MCP-1 capture antibodies. Diluted samples and recombinant human MCP-1 standard were added and incubated for 2 h at room temperature followed by addition of bioti- nylated polyclonal goat anti-human MCP-1. The plates were incubated for 20 minutes at room temperature with streptavidin-conjugated horseradish peroxidase. Tetra- methyl-benzidine (TMB) (Sigma) and H2O2 were used as substrate. Absorbency values were measured at 450 nm using Softmax Pro version 4.0 on an Emax Precision microtiter plate reader (Molecular Devices, Sunnyvale, CA, USA). The lower detection level was 15.62 pg/ml. Chemokine levels obtained with media controls (after 24 h) were substracted from those obtained with OK-432-, LTA-, LPS-stimulation. All media controls tested directly after monocyte purification had nondetectable chemok- ine levels. Multiplex cytokine analysis Chemokines in supernatants were detected using the Luminex immunobead technology. A 25-plex or 5-plex chemokine kit (all from Invitrogen/Biosource, Carlsbad, CA, USA), were used to analyze cytokine levels in super- natants from successfully completed experiments. In short, antibody-coupled beads were incubated with target analyte after which they were incubated with biotinylated detection antibody before finally being incubated with streptavidin-phycoerythrin. Standard sensitivity was 10 pg/ml (MIP-1α, MIP-1β). Samples were then read by the Bio-Plex array reader (Invitrogen/Biosource), using Page 9 of 11 (page number not for citation purposes) Page 9 of 11 (page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 Authors' contributions CO performed the Western blot analysis, Multiplex analy- sis, all the experiments and data analysis. HS did the RT- PCR experiments and analysis. KB operated the Luminex machine. JO provided OK-432. CO and HJA were respon- sible for the concept and design of the experiments and primarily writing the manuscript. All authors have read and approved the manuscript. 15. Graveline R, Segura M, Radzioch D, Gottschalk M: TLR2-depend- ent recognition of Streptococcus suis is modulated by the presence of capsular polysaccharide which modifies macro- phage responsiveness. Int Immunol 2007, 19:375-89. p g p 16. Moser B, Wolf M, Walz A, Loetscher P: Chemokines: multiple levels of leukocyte migration control. Trends Immunol 2004, 25:75-8. Page 10 of 11 (page number not for citation purposes) Page 10 of 11 (page number not for citation purposes) BMC Immunology 2009, 10:6 http://www.biomedcentral.com/1471-2172/10/6 http://www.biomedcentral.com/1471-2172/10/6 17. Olsnes C, Stavang H, Olofsson J, Aarstad HJ: TNF-alpha is secreted by monocytes in transit to become macrophages, but not by peripheral blood monocytes, following OK-432 (lyophilized S. pyogenes) stimulation. Scand J Immunol 2007, 66:684-93. 18. Schenkel AR, Mamdouh Z, Muller WA: Locomotion of monocytes on endothelium is a critical step during extravasation. Nat Immunol 2004, 5:393-400. 19. Vines CM, Potter JW, Xu Y, Geahlen RL, Costello PS, Tybulewicz VL, Lowell CA, Chang PW, Gresham HD, Willman CL: Inhibition of beta 2 integrin receptor and Syk kinase signaling in mono- cytes by the Src family kinase Fgr. Immunity 2001, 15:507-19. 20 G G C 2 g y 20. Reyes-Reyes M, Mora N, Gonzalez G, Rosales C: beta1 and beta2 integrins activate different signalling pathways in mono- cytes. Biochem J 2002, 363:273-80. y J 21. Butler B, Williams MP, Blystone SD: Ligand-dependent activation of integrin alpha vbeta 3. J Biol Chem 2003, 278:5264-70. 22. Cuzzola M, Mancuso G, Beninati C, Biondo C, Genovese F, Tomasello F, Flo TH, Espevik T, Teti G: Beta 2 integrins are involved in cytokine responses to whole Gram-positive bacteria. J Immu- nol 2000, 164:5871-6. 23. Collot-Teixeira S, Martin J, McDermott-Roe C, Poston R, McGregor JL: CD36 and macrophages in atherosclerosis. Cardiovasc Res 2007, 75:468-77. 24. Veckman V, Miettinen M, Matikainen S, Lande R, Giacomini E, Coccia EM, Julkunen I: Lactobacilli and streptococci induce inflamma- tory chemokine production in human macrophages that stimulates Th1 cell chemotaxis. J Leukoc Biol 2003, 74:395-402. 25. Authors' contributions Okamoto M, Oshikawa T, Tano T, Ahmed SU, Kan S, Sasai A, Akashi S, Miyake K, Moriya Y, Ryoma Y, et al.: Mechanism of anticancer host response induced by OK-432, a streptococcal prepara- tion, mediated by phagocytosis and Toll-like receptor 4 sign- aling. J Immunother 2006, 29:78-86. g J 26. Triantafilou M, Gamper FG, Haston RM, Mouratis MA, Morath S, Har- tung T, Triantafilou K: Membrane sorting of toll-like receptor (TLR)-2/6 and TLR2/1 heterodimers at the cell surface determines heterotypic associations with CD36 and intracel- lular targeting. J Biol Chem 2006, 281:31002-11. g g J 27. Tacke F, Randolph GJ: Migratory fate and differentiation of blood monocyte subsets. Immunobiology 2006, 211:609-18. 28. Ma YQ, Plow EF, Geng JG: P-selectin binding to P-selectin glyc- oprotein ligand-1 induces an intermediate state of alphaMbeta2 activation and acts cooperatively with extra- cellular stimuli to support maximal adhesion of human neu- trophils. Blood 2004, 104:2549-56. p 29. Coussens LM, Werb Z: Inflammation and cancer. Nature 2002, 420:860-7. 30. Otto F, Schmid P, Mackensen A, Wehr U, Seiz A, Braun M, Galanos C, Mertelsmann R, Engelhardt R: Phase II trial of intravenous endotoxin in patients with colorectal and non-small cell lung cancer. Eur J Cancer 1996, 32A:1712-8. J 31. Briegert M, Kaina B: Human monocytes, but not dendritic cells derived from them, are defective in base excision repair and hypersensitive to methylating agents. Cancer Res 2007, 67:26-31. 32. Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M: The chemokine system in diverse forms of macrophage activa- tion and polarization. Trends Immunol 2004, 25:677-8. p 33. Huang ZY, Barreda DR, Worth RG, Indik ZK, Kim MK, Chien P, Sch- reiber AD: Differential kinase requirements in human and mouse Fc-gamma receptor phagocytosis and endocytosis. J Leukoc Biol 2006, 80:1553-62. Authors' contributions Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 11 of 11 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Page 11 of 11 (page number not for citation purposes)
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LXVI.—<i>On the special protection of appendages in process of regeneration after artificial mutilation among insects</i>
Annals & magazine of natural history
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LXVI.—On the special protection of appendages in process of regeneration after artificial mutilation among insects Edmond Bordage Published online: 22 Sep 2009. LXVI.—On the special protection of appendages in process of regeneration after artificial mutilation among insects Edmond Bordage Published online: 22 Sep 2009. To cite this article: Edmond Bordage (1900) LXVI.—On the special protection of appendages in process of regeneration after artificial mutilation among insects , Annals and Magazine of Natural History: Series 7, 5:30, 501-503, DOI: 10.1080/00222930008678323 This article was downloaded by: [York University Libraries] On: 31 December 2014, At: 13:47 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions ity Libraries] at 13:47 31 December 2014 ork University Libraries] at 13:47 31 December 2014 Special Protection of Appeltdages among [nsects. 50I LXVI.--On the Special Protection o( Appendages 52 Process of Refeneration a])er Artificial Mutilation amon.q Insects. By EDMOND BORDAGE *. WHE:N a limb is removed owing to self-mutilation from the body of' an Arthropod, such as a crab or a Phasmid for in- stanc% regeneration starts from tho very surface of the section so produced. The same thing, however, rarely happens when, on the other hand, regeneration follows the artificial severance of a limb. * Translated from ' Comptes Rendus,' exxix. (1899) pp: 501-504, by Wilfred Mark Webb, F.L,~. From a separate impressmn communi- cated by the Author. t I1. H. Brindley, ' On certain Character.~ of Reproduced Appendages in Arthropoda,' 1898. * Translated from ' Comptes Rendus,' exxix. (1899) pp: 501-504, by Wilfred Mark Webb, F.L,~. From a separate impressmn communi- cated by the Author. 1 i dl ' O i Ch f d d A d y t I1. H. Brindley, ' On certain Character.~ of Reproduced Appendages in Arthropoda,' 1898. PLEASE SCROLL DOWN FOR ARTICLE Among Mantidm, B]attidmt, and the Orthoptera saltatoria, for exampl% artificial cuts are followed by contraction and by more or less marked displace- ment of the divided muscles, which are retracted within the chitinous covering of the limb, so that, if regeneration follow~ the part in process of growth remains entirely hidden until the next moult. The chitinous ease therefore in this instance plays the part of a protector. York University Libraries] at 13:47 31 December 2014 p y p p It may even happen among the Mantid~e~ which possess the power ot" self mutilation developed in a high degree, or among the Blattidee, where it is much less marked, that contraction of the muscles may be produced in the interior of the tro- chanter, or even inside the joint, if developed, that forms the haunch (coxa)~ after self-mutilation along the trochantero- femoral suture. In this ease, if there be regeneration the part in process of growth will remain hidden until the next moult. Most often, however, among the Manti&e, and more rarely in the Blatti&e~ this contraction does not take place after self mutilation. In this case one can very soon see whether there will be regenerat~ion without its being necessary to wait for the next moult. It is true that one cannot perceive the various parts which make up the rudiment in process of growth, for it is coiled upon itself~ and, what is more~ covered over by the non-ehitinized cuticle, which protects it after the manner of a pocket. This cuticle lacks transparency on account of its brownish coloration ; but the very slightly marked projection which this protective pouch forms at the end of the trochani~er shows nevertheless that the work of regeneration is going on. g g g In the Phasmidm the rudiment in process of growth destined 502 Special Protection of Appemlages among Insects. to replace a limb detached by self-mutilation sometimes shows i~se]f~ but in an indistinct manner, coiled up under the pro- tective cuticle, which has some degree of transparency in certain species. p So far as the Mantidse and Blattid~e are concerned, I noticed that the amount of withdrawal of the muscles divided by self-mutilation within the chitinous covering was propof tional to the violence of the efforts made by the insects in getting rid of the limb. When self-mutilation took place easily the contraction was practically nil. York University Libraries] at 13:47 31 December 2014 y p y It now remains for us to examine the particular case pre- sented by the Phasmida~. y When artificial separation takes place in the region com- prising the femur and the upper two thirds of the tibia, cou- traction of the severed muscles is most marked. When~ again, similar cuts are made in the upper part of the region formed by the lower third of the tibia, contraction is still produced, and as it is exactly there that the power of regeneration begins to show itself, the part in process of growth remains hidden until the following moult. Then in proportion as the cuts approach the tarsus~ contraction becomes less and less evident, until in the neighbourhood of the tibio-tarsal articulation the divided muscles practically remain m position ; so that the part in process of regeneration may be visible before a moult takes place. The result is the same when the cuts are made on any of the first three joints of the tarsus. y j On examining the internal structure of the limb we find that it is precisely in the spot under discussion--upon the lower portion of the tibia and the first joints of the tarsus-- that the muscles are inserted which move the tarsus as a whole and its various parts. Cuts made in this region pass through the surfaces ot the chitinous covering to which these muscles are attached and t¥om which they run towards the joints below which they have to move. Under these circum- stances one can understand why the contraction will be slight or not produced at all. It is only possible to produce it when the cut is more or less remo~e from the upper attachment- surface of the divided muscles: which is not the case. In other insects there is complete withdrawal of the cut muscles when t},e section passes through the tarsal region. It is evident that these particular features point to differences in the number and position of the attachment-surfaces of the muscles in question, differences which are revealed upon careful dissection. It follows, then, that among the Phasmidm par~s in process of regeneration in the region we are considering are more or _New Species of Prionocalus,]'~'om Ecuador and Peru. 503 less apparent from the outset of their formation. * In the most perfectly regenerated limb~ there is a tetramerous tarsus. I have nevertheless obtained after cuts made upon the third joint of the t 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. Tetramery after regeneration has been determined anaong the Phasmi&e not only in the four genera I quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa- phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria me.vicana, the total nmnber of species of Orthoptera cursoria in which tetramery has been determined being thus twenty-five tarsus :-- 1 A pentamerous tarsus with incompletely separated joints 1. A pentamerous tarsus with incompletely separated 2 A pentamerous tarsus as perfect as the normal one York University Libraries] at 13:47 31 December 2014 It is interesting to compare the slowness of the growth of parts in process of regeneration after artificial removal~ as well as that--much less marked nevertheless--of limbs in- tended to replace those removed by self-mutilation, among Phasmida~ with the marvellous rapidity of such growth which has been iound among the Mantidze and Blattid~e. Whilst among the latter limbs regenerated after self- or artificial mutilation may begin to be of use to the insect immediately after the next moult, in the Phasmi&e limbs in course of regeneration cannot become useful until after the second or third moult. I have noted that the same holds good for the Orthoptera saltatoria. Downloaded by [York University Libraries] at 13:47 31 Downloaded by [York University Libr LXVII.~New Species of the Coleopterous Geuus Prionocalus from Ecuador and ff eru. By CIIAS. O. WATI~I~HOUSE V P E S THE British Museum has recently received a few specimens of Longicorns of the genus Prionocalus. One I refer with a slight doubt to P. cacicus~ Whit% but in the type the tubercle 1. A pentamerous tarsus with incompletely separated 2 A pentamerous tarsus as perfect as the normal one 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. Tetramery after regeneration has been determined anaong the Phasmi&e not only in the four genera I quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa- phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria me.vicana, the total nmnber of species of Orthoptera cursoria in which tetramery has been determined being thus twenty five 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. Tetramery after regeneration has been determined anaong the Phasmi&e not only in the four genera I quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa- phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria me.vicana, the total nmnber of species of Orthoptera cursoria in which tetramery has been determined being thus twenty five 2. A pentamerous tarsus as perfect as the normal one. 3. A bent and monstrous tarsus of six incompletely separated joints. But these are rare exceptions to the rule. York University Libraries] at 13:47 31 December 2014 As I have already said, bowever~ growth proceeds with the greatest slowness; it fbllows therefore that during the time which elapses before the next moult the part in process of growth barely forms a minute projection from 1 to 2 millim, in length. It is covered by a thin protective cuticle of a brown eolour~ moulding itself exactly upon the rudiment of the limb~ which up to the present shows no separation into joints. It is only after the next moult that the limb, beginning to be of appre- ciable lengtl b will show any distinct traces of division into tarsal joints. The growth is so slow that it is only after two or even three moults that the mutilated limb is completed arid becomes serviceable to the insect *. York University Libraries] at 13:47 31 December 2014 Tetramery after regeneration has been determined anaong the Phasmi&e not only in the four genera I quoted in my previous papers~ but also in Anehiale, Acanthoderus, Lopa- phus, .Diapheromera~ and probably in Cyphocrania, Diura~ and .Bacteria me.vicana, the total nmnber of species of Orthoptera cursoria in which tetramery has been determined being thus twenty-five
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Correction: Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility
PLOS computational biology/PLoS computational biology
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Correction Correction: Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility Correction Correction: Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility Correction A number of figure legends are associated with the wrong figures in the published article. g p The legend of Figure 6 should be associated with Figure 4. The legend of Figure 6 should be associated with Figure 4. The legend of Figure 4 should be associated with Figure 5. The legend of Figure 5 should be associated with Figure 6. The legend of Figure 5 should be associated with Figure 6. Reference 1. Loving KA, Lin A, Cheng AC (2014) Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility. PLoS Comput Biol 10(7): e1003741. doi:10.1371/journal.pcbi.1003741 Citation: The PLOS Computational Biology Staff (2014) Correction: Structure- Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility. PLoS Comput Biol 10(9): e1003875. doi:10. 1371/journal.pcbi.1003875 1. Loving KA, Lin A, Cheng AC (2014) Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility. PLoS Comput Biol 10(7): e1003741. doi:10.1371/journal.pcbi.1003741 PLOS Computational Biology | www.ploscompbiol.org Correction: Structure-Based Druggability Assessment of the Mammalian Structural Proteome with Inclusion of Light Protein Flexibility The PLOS Computational Biology Staff The PLOS Computational Biology Staff The PLOS Computational Biology Staff A number of figure legends are associated with the wrong figures in the published article. PLOS Computational Biology | www.ploscompbiol.org Published September 9, 2014 Published September 9, 2014 Copyright:  2014 The PLOS Computational Biology Staff. This is an open- access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PLOS Computational Biology | www.ploscompbiol.org 1 September 2014 | Volume 10 | Issue 9 | e1003875 September 2014 | Volume 10 | Issue 9 | e1003875
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Review on Stochastic Approach to Inflation
Universe
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27,324
universe universe universe Review Review Diego Cruces 1,2 Diego Cruces 1,2 1 Institut de Ciencies del Cosmos (ICCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain; dcruces@ub.edu; Tel.: +34-93-402-0817 1 Institut de Ciencies del Cosmos (ICCUB), Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain; dcruces@ub.edu; Tel.: +34-93-402-0817 2 Departament de Física Quàntica i Astrofísica, Faculty of Physics, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain 2 Departament de Física Quàntica i Astrofísica, Faculty of Physics, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain 2 Departament de Física Quàntica i Astrofísica, Faculty of Physics, Universitat de Barcelona, Martí i Franquès 1, 08028 Barcelona, Spain Abstract: We present a review on the state-of-the-art of the mathematical framework known as stochastic inflation, paying special attention to its derivation, and giving references for the readers interested in results coming from the application of the stochastic framework to different inflationary scenarios, especially to those of interest for primordial black hole formation. During the derivation of the stochastic formalism, we will emphasise two aspects in particular: the difference between the separate universe approach and the true long wavelength limit of scalar inhomogeneities and the generically non-Markovian nature of the noises that appear in the stochastic equations. Keywords: stochastic inflation; primordial black holes; early universe; inflation 1. Introduction Although cosmological inflation was first introduced as a possible solution to the hot Big Bang model problems [1,2], the study of vacuum fluctuations during this accelerated expansion of the universe also gives an explanation to the anisotropies observed in the Cosmic Microwave Background (CMB). The idea is that the different scales of fluctuations leave the observable universe during inflation, long after it, they reenter the observable universe at different times, being the scales reentering the cosmological horizon (horizon from now on) during the time of recombination the responsible for the CMB anisotropies. In fact, the almost gaussianity and scale invariance of the vacuum quantum fluctuations predicted by Slow Roll (SR) inflation has been remarkably well confirmed by the recent Plank satellite mission [3,4]. Citation: Cruces, D. Review on Stochastic Approach to Inflation. Universe 2022, 8, 334. https:// doi.org/10.3390/universe8060334 Academic Editor: Yi-Fu Cai Received: 28 March 2022 Accepted: 15 June 2022 Published: 17 June 2022 Citation: Cruces, D. Review on Stochastic Approach to Inflation. Universe 2022, 8, 334. https:// doi.org/10.3390/universe8060334 Academic Editor: Yi-Fu Cai Received: 28 March 2022 Accepted: 15 June 2022 Published: 17 June 2022 universe universe Citation: Cruces, D. Review on Stochastic Approach to Inflation. Universe 2022, 8, 334. https:// doi.org/10.3390/universe8060334 It is however true that, although inflation provides a causal mechanism to generate CMB anisotropies, the CMB is only accessible to a restricted range of scales, constraining the inflationary phase only during a limited time interval. In order to be accessible to smaller scales of inflation, that reenter the horizon before the recombination epoch, we must then seek for any other hint that help us to extend the time interval of inflation that we can constraint. Primordial Black Holes (PBHs) is one of those hints [5]. In addition to the fact that PBHs can probe the missing scales of inflation, they represent natural candidates not only for dark matter (DM) [6], but also as the seed of supermassive BHs at the center of massive galaxies [7] and even as the progenitors of some events that radiate the gravitational waves detected by the LIGO/VIRGO collaboration [8]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. g y PBHs are expected to form if the amplitude of density perturbations from inflation is big enough such that when they reenter the horizon they will collapse into a Black Hole (BH). This is not the case for CMB scales, for which the SR approximation holds and where the amplitude of inhomogeneities is too small to form PBHs; however, for smaller scales, this amplitude is less constrained and it could grow until PBHs are possible to form, in this case, SR is not a good approximation and other inflationary regimes arise such as Ultra Slow Roll (USR) and Constant Roll (CR). Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Since the over-densities responsible for the formation of PBHs must be large enough, they will be found at the tail of the probability distribution of density perturbations, which https://www.mdpi.com/journal/universe Universe 2022, 8, 334. https://doi.org/10.3390/universe8060334 Universe 2022, 8, 334 2 of 39 makes them exponentially rare [9]. At the same time, a large over-density at small scales can modify the large-scale dynamics of the universe in a non-perturbative way. Thus, in order to predict the abundance of PBHs, a precise statistical knowledge of inflationary perturbations is highly desirable. The hope of the stochastic approach to inflation is that it incorporates quantum cor- rections to the inflationary dynamics in a non-perturbative way [10]. In this approach, wavelengths that are well outside the horizon are approximated in powers of spatial gradi- ents rather than on amplitudes (as in linear theory). At the same time though, those modes are influenced by the quantum sector by receiving quantum-kicks from stochastic forces generated by the perturbative sub-horizon modes. The success of the stochastic formalism resides in the fact that it allows to reduce a quantum problem into a statistical one and it has been widely used in the literature [10–68]. Since this manuscript is mainly devoted to stochastic inflation, we will briefly review the long history behind it, which will also help us to understand why the paper is organized as it is. Soon after Starobisnky first proposed the idea of treating the short-wavelength part of the field as a white noise which is included as a source term in the equation of motion for the classical (long-wavelength) field in [10], stochastic inflation immediately gained huge popularity [11–24], manly because of the possibility of solving the Fokker-Planck equation that govern the evolution of the probability distribution of the amplitude of the perturbations of the scalar field during inflation in an exact way for some specific form of the potential, or even for generic potentials with the help of the first time passage analysis [69] as firstly done in [24]. In the references above, the heuristic derivation of Starobinsky, which was only valid in single-field slow-roll inflationary models, was ex- tended beyond SR and multifield inflation. At the same time, Morikawa et al. put the stochastic formalism on a more firm ground [25,26], where the heuristic derivation of the equations of motion of stochastic inflation done by previous authors was refined. More concretely, they were able to derive the same stochastic equations integrating out the short-wavelength part of the scalar field in the path integral. Although this derivation lead to the same equations of motion as the heuristic argument followed by previous authors, it is very useful in order to understand what are the approximations that lead to the stochastic equations first presented by Starobinsky and its consequences. y y q The most important of these approximations is probably to compute the variance of the noises in a pure de-Sitter background. This approach has been vastly used in mort applica- tions of the stochastic formalism until today. Examples of this can be found in [27,29–33,35,37–39,50,54,56], but more importantly, with the aid of this approach, the well-known δN formalism [70–73] was extended to an stochastic δN formalism [36,41,46,47,53,61,62,66] which allow us to compute the probability distribu- tion of the curvature perturbation and hence to connect the results of stochastic inflation with PBH formation as done for example in [52,58,59,63,64,67]. p However, computing the noises in a pure de-Sitter background has important con- sequences, in fact, it was checked in [35,37,40,45,47–49] that, under this approximation, stochastic formalism recovers the standard result of Quantum Field Theory (QFT) for test scalar fields on a fixed inflationary background, which give us a hint that thos ap- proach does not correctly take into account the backreaction of the scalar field in the metric. As pointed out in [74], this method misses the coupling between the long- and short-wavelength sectors; more concretely, the background in which the noises should be computed is not exactly de-Sitter, but it is both slow-roll and stochastically corrected. This means that the results obtained with a stochastic formalism that computes the noises in a pure de-Sitter background should not be taken seriously if we are looking for deviations from de-Sitter shuch as in the spectral index. This important limitation of the stochastic formalism as presented by Starobinsky (which will be further studied in Section 6.1.1), led to some authors to present alternative ways of solving the equations of motion of stochastic Universe 2022, 8, 334 3 of 39 3 of 39 inflation beyond the usual Fokker-Plank equation with de-Sitter noises, some examples are the recursive stategy presented in [42–44] or numerical codes as in [67]. inflation beyond the usual Fokker-Plank equation with de-Sitter noises, some examples are the recursive stategy presented in [42–44] or numerical codes as in [67]. Another important simplification that is usually performed in the stochastic framework is to split long- and short-wavelengths modes via a sharp window function (a step function). This choice leads to white noises which are very easy to handle both analytically and numerically. However, it was noted in [28] that this choice of window function leads to some problems in the asymptotic of the noise correlator at large spatial distances. This is why some works with smooth window functions that lead to coloured noises have also been proposed [34,68]. p p If we now go back to the historical controversies of stochastic inflation, we can find the issue of the time variable in which this formalism must be formulated. Already in one of the first works by Starobinsky [24], it was pointed out that the number of e-folds, (defined as N = R Hdt, where t is the cosmic time and H is the Hubble parameter) seem to be a very appropriate time variable. This choice was later justified by connecting stochastic inflation with results form QFT in curved space times [35,37,47]; however, as also pointed out by these references, the choice of N as time variable is only a consequence of writing the stochastic equations in terms only of the scalar field. In fact, one can check that the only way to write all the scalar degrees of freedom in terms solely of the scalar field is to use the uniform-N gauge in the separate universe approach [60], which justifies the use of N as time variable since, by definition, in this gauge it is unperturbed. It was not until recently [65] when the stochastic formalism was formulated in another gauge, making it clear that the choice of N as time variable is only a consequence of the gauge choice. Finally, it was lately noticed that the stochastic formalism of inflation was leading to some inconsistencies at next-to-leading order in SR parameters even at linear level. For example, it was checked in [55] that the linearization of the stochastic equations do not exactly reproduce the well-known equation of motion of the scalar linear perturbations in the long-wavelength limit (the Mukhanov-Sasaki equation, see Section 4.1). Furthermore, in [57] it was demonstrated that, during an ultra-slow-roll (USR) inflationary regime, some noises that appear in the stochastic equations of motion were incompatible with the rest of the system. The origin of these inconsistencies can be traced back to the use of the separate universe approach in the construction of the stochastic formalism and they can be solved by including the momentum constraint in the separate universe approach [65]. This review will give a detailed derivation of the stochastic formalism to inflation rather than giving a list of results obtained within this formalism. During the derivation we will emphasise all controversial aspects that have been indicated in the above summary of the long history of the stochastic formalism, from the difference between the separate universe approach and the true long-wavelength limit of scalar inhomogeneities to the construction of the noises using an exact de-Sitter background. g g The review is organized as follows: after presenting the basic inflationary concepts with the aid of the homogeneous picture of inflation in Section 2, we will start study- ing inflationary inhomogeneities, focusing on its long-wavelength behaviour. In order to do so we will present different approximations to the exact Arnowitt-Deser-Misner (ADM) equations of Section 3. These approximations are linear perturbation theory (in Section 4), gradient expansion (in Section 5) and the stochastic formalism (in Section 6), which combines the two approximation schemes presented before. Finally, in Section 7 we will give some conclusions. 2. Homogeneous Inflationary Scenarios and Slow-Roll Parameters As we have already mentioned, PBHs represent natural candidates for dark matter (DM) (latest constraints on this idea can be found in [75]). However, one possibility to statistically generate enough PBHs for this to hold one needs, at least, a power spectrum of primordial curvature perturbations several order of magnitudes larger than the one observed in the cosmic microwave background (CMB). Note that this is not the only possibility; one could statistically generate enough PBHs with a large non-gaussianity in Universe 2022, 8, 334 4 of 39 the probability distribution of fluctuations even without enhancing the power spectrum. This scenario is discussed in [76]. It is known that a period of Slow-Roll (SR), of which the predictions of the CMB are based upon, cannot lead to the appropriate power spectrum necessary to generate enough PBHs to match the DM abundance [77] ( For the non-linear relation between the inflationary power spectrum and PBHS abundance, under the assumption of gaussianity, the interested reader can see [78]). Thus, one necessarily needs an inflationary epoch evolving beyond SR. A possibility is the introduction of an inflection point in the inflationary potential [79]. This leads the inflaton to undergo a so-called Ultra-Slow-Roll (USR) phase of inflation [80,81]. Taking into account that the statistics of PBHs from non-gaussian fluctuations has yet to be fully developed. The single field USR option with standard kinetic term seems then to be the best [76]. Such a system is described by the Einstein-Hilbert action with a minimally coupled scalar field i.e.,: S = 1 2 Z p−g h M2 PLR −(∇φ)2 −2V(φ) i , (1) (1) whose homogeneous solution is an universe described by a Friedman-Lemaitre-Robertson- Walker (FLRW) metric: ds2 = −dt2 + a(t)2d⃗x · d⃗x , ds2 = −dt2 + a(t)2d⃗x · d⃗x , (2) (2) where a(t) represents the scale factor. where a(t) represents the scale factor. The equation of motion of the scalar field in the universe described by (2) has the following equation of motion: ¨φb + 3Hb ˙φb + Vφb  φb = 0 , (3) (3) where Vφb ≡∂V(φb) ∂φb , Hb ≡˙a a is the Hubble parameter, and a dot denotes a derivative with respect to the cosmic time t. Finally, the super-script “b” stands for “background”, the meaning of which will be clarified later on. 2. Homogeneous Inflationary Scenarios and Slow-Roll Parameters (9) (9) As we will show later on, an exponential decrease of ϵ1 makes the power spectrum of curvature perturbation increase. • Both SR and USR are, at least approximately, sub-cases of Constant-Roll (CR). Here ¨φb Hb ˙φb = κ where κ is a constant. SR is realized when κ = 0 while USR when κ = −3. We will not analyse further this generic case. • Both SR and USR are, at least approximately, sub-cases of Constant-Roll (CR). Here ¨φb Hb ˙φb = κ where κ is a constant. SR is realized when κ = 0 while USR when κ = −3. We will not analyse further this generic case. It is important to remark that, given a potential related to PBH formation, the SR and USR phases alternate. Thus, the equations of motion (6) and (8) will always be an approximation of the system. 2. Homogeneous Inflationary Scenarios and Slow-Roll Parameters The Friedmann equation is The Friedmann equation is  Hb2 = 1 3M2 PL     ˙φb2 2 + V  φb   . (4) (4) The Slow-Roll (SR) parameters ϵi define the rate of change of the Hubble parameter: ϵ1 ≡− ˙Hb Hb2 =  ˙φb2 2H2M2 PL ≪1 ; ϵi+1 ≡ ˙ϵi Hϵi with i ≥1, (5) (5) where, to write the final expressions, we have used the Friedmann equation and the equation of motion of the field. q We can now define different inflationary regimes depending on the values of the ϵis: • Slow-Roll (SR): The field is slowly rolling down a potential with an almost constant velocity, which makes the acceleration negligible. In this case the equation of motion (3) is approximately   • Slow-Roll (SR): The field is slowly rolling down a potential with an almost constant velocity, which makes the acceleration negligible. In this case the equation of motion (3) is approximately b b  b 3Hb ˙φb + Vφ  φb ≃0 . (6) (6) The SR parameters are much smaller than one (ϵi ≪1) and can be written in terms of the potential as ϵSR 1 ≃ 1 2M2 PL Vφb V !2 ; ϵSR 2 ≃ 2 M2 PL Vφbφb V ! −4ϵSR 1 . (7) (7) Universe 2022, 8, 334 5 of 39 • Ultra-Slow-Roll (USR): The field is moving along an exactly flat potential Vφ = 0  , which makes the acceleration relevant. In this case the equation of motion (3) is • Ultra-Slow-Roll (USR): The field is moving along an exactly flat potential Vφ = 0  , which makes the acceleration relevant. In this case the equation of motion (3) is ¨φb + 3Hb ˙φb = 0 . (8) (8) From (8) one can infer that the velocity of the field (and hence ϵ1) exponentially decreases, which makes some ϵi ∼O(1). More precisely: From (8) one can infer that the velocity of the field (and hence ϵ1) exponentially decreases, which makes some ϵi ∼O(1). More precisely: ϵUSR i = −6 + 2ϵUSR 1 when i even. ϵUSR i = 2ϵUSR 1 when i>1 and odd. (9) ϵUSR i = −6 + 2ϵUSR 1 when i even. ϵUSR i = 2ϵUSR 1 when i>1 and odd. (9) ϵUSR i = 2ϵUSR 1 when i>1 and odd. 3. Inhomogeneities during Inflation: The ADM Formalism Although it is very useful in order to understand the inflationary dynamics, the homogeneous picture of inflation presented in Section 2 is not very realistic and some inhomogeneities must be introduced in order to explain the universe we currently observe. Thus, we must define a general metric which is not restricted to homogeneity and isotropy. An option which is very useful in the context of inflation is to work in the so-called Arnowitt- Deser-Misner (ADM) formalism [82]. This framework supposes that the four-dimensional space-time is foliated into a family of three-dimensional hypersurfaces Σt, labeled by its time coordinate t. In order to work in the ADM formalism, it is convenient to write the metric as: ds2 = gµνdxµdxν = −α2dt2 + γij(dxi + βidt)(dxj + βjdt) , (10) (10) and the action (1) presented in the previous section becomes: and the action (1) presented in the previous section becomes: γ h M2 PL  αR(3) + α  KijKij −K2 −2αV(φ) + α−1 ˙φ −βi∂iφ −αγij∂iφ∂jφ i , (11) (11) where we have introduced many new terms: where we have introduced many new terms: • α is the lapse function, which measures the rate of flow of proper time with respect to coordinate time t as one moves normally to Σt. • α is the lapse function, which measures the rate of flow of proper time with respect to coordinate time t as one moves normally to Σt. • βi is the shift vector, which measures how much the local spatial coordinate system shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt. • γij represents the induced metric on the hypersurface Σt, that we we will decompose • βi is the shift vector, which measures how much the local spatial coordinate system shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt • βi is the shift vector, which measures how much the local spatial coordinate system shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt. • βi is the shift vector, which measures how much the local spatial coordinate system shifts tangential to Σt, when moving from Σt to Σt+δt along the normal direction to Σt. 3. Inhomogeneities during Inflation: The ADM Formalism • γij represents the induced metric on the hypersurface Σt, that we we will decompose as γij = a2e2ζ ˜γij with det ˜γij = 1 so that the scale factor a is explicitely present. (3) (3) g g t+δt g • γij represents the induced metric on the hypersurface Σt, that we we will decompose as γij = a2e2ζ ˜γij with det ˜γij = 1 so that the scale factor a is explicitely present. p y p R(3) ij is the Ricci tensor of the spatial metric, and hence R(3) ≡γijR(3) ij . • R(3) ij is the Ricci tensor of the spatial metric, and hence R(3) ≡γijR(3) ij . j j • Finally, Kij is the extrinsic curvature (K ≡γijKij), which is defined as: j j • Finally, Kij is the extrinsic curvature (K ≡γijKij), which is defined as: Kij ≡−∇inj = −1 2α ∂tγij −Diβj −Djβi  , (12) (12) where ni ≡(−α, 0, 0, 0) is the unit vector normal to the spatial hypersurfaces and ∇µ and Di are the covariant derivatives with respect to gµν and γij, respectively. Universe 2022, 8, 334 6 of 39 It is also convenient to decompose the extrinsic curvature into its trace and traceless part as: Kij = γij 3 K + a2e2ζ ˜Aij , (13) (13) where ˜γij ˜Aij = 0. Note that,in the homogeneous limit, i.e., when α = 1, βi = 0 and γij = a2δij and hence the metric (10) reduces to (2), we can identify the extrinsic curvature with the background Hubble parameter, namely K = −3 ˙a a = −3Hb. We will then define a more general inhomogeneous Hubble parameter as H ≡−K 3 . This makes sense because K represents the expansion rate of the constant time hypersurfaces. In the ADM formalism, the lapse function and the shift vector act as Lagrange multi- pliers for the action (11), and hence they generate two constraints: the Hamiltonian and the momentum constraints, which are, respectively: R(3) −˜Aij ˜Aij + 2 3K2 = 2 M2 PL E , (14) Dj ˜Aij −2 3 DiK = 1 M2 PL Ji , (15) (14) (15) where E ≡Tµνnµnν and Ji ≡Tµjnµγj i and Tµν is, in our case, the stress-energy tensor of the scalar field, i.e., Tµν = ∇µφ∇νφ −1 2 gµν(∇αφ∇αφ + 2V(φ)) . 3. Inhomogeneities during Inflation: The ADM Formalism (16) (16) The two remaining variables, γij and Kij are the dynamical ones and their evolution equations are given by: • For γij: • For γij: (∂t −βk∂k)ζ + ˙a a = −1 3(αK −∂kβk), (17) (∂t −βk∂k) ˜γij = −2α ˜Aij + ˜γik∂jβk + ˜γjk∂iβk −2 3 ˜γij∂kβk. (18) (17) (18) • for Kij: (∂t −βk∂k)K = α  ˜Aij ˜Aij + 1 3K2  −DkDkα + 4πGα(E + Sk k), (19) (19) (∂t −βk∂k) ˜Aij = e−2ζ a2  α  R(3) ij −γij 3 R(3)  −  DiDjα −γij 3 DkDkα  + α(K ˜Aij −2 ˜Aik ˜Ak j ) + ˜Aik∂jβk + ˜Ajk∂iβk −2 3 ˜Aij∂kβk −8πGαe−2ζ a2  Sij −γij 3 Sk k  , (2 (20) where Sij = Tij and Sk k = γklSlk. where Sij = Tij and Sk k = γklSlk. where Sij = Tij and Sk k = γklSlk. where Sij = Tij and Sk k = γklSlk. Finally, and although it can be recovered using the ADM equations just presented, it is also worthy to present here the Klein-Gordon equation for the evolution of the scalar field: Finally, and although it can be recovered using the ADM equations just presented, it is also worthy to present here the Klein-Gordon equation for the evolution of the scalar field: 1 √−g∂µ p−ggµν∂νφ  −Vφ = 0 . (21) (21) We will present more detail in the following section, but it is worth remarking here that the homogeneous equations of Section 2 are straightforwardly recovered setting α = 1, βi = 0 and γij = a2δij g βi = 0 and γij = a2δij. g βi = 0 and γij = a2δij. β γ j j Since the ADM equations are equivalent to the Einstein equations but much easier to implement numerically, they are very useful to study inhomogeneous space-times in an Universe 2022, 8, 334 7 of 39 7 of 39 exact way; however, the numerical methods capable of solving exactly these equations are computationally expensive [83]. Fortunately there exist some very useful approximations that can be done when studying the inflationary universe that, not only allow us to con- siderably simplify the numerical way of solving the ADM equations, but they even admit some analytical solutions as we will see in the following. 4. Linear Perturbation Theory Since, as explained in the introduction, the deviations from an exactly homogeneous and isotropic FLRW universe that we observe are very small, it makes sense to solve the system of ADM Equations (14)–(21) by expanding them around a FLRW background. If this expansion is done up to first order, we call it linear perturbation theory. There are many reviews on this topic [84–88]; however, since we are taking a slightly different point of view from most of them, we will go through linear perturbation theory in some detail. With this in mind, the ADM metric (10) will be written as: gµν ≃gb µν + δgµν , (22) (22) where gb µν is the homogeneous and isotropic metric of Section 2 and δgµν represent the perturbation. This implies that the lapse function, the shift vector and the spatial metric are approximated as: α ≃1 + A , βj ≃aBj , e2ζ ≃1 + 2D , ˜γij ≃δij −2Eij (23) , ˜γij ≃δij −2Eij (23) (23) where Eij is traceless. The reason why Eij is traceless is because is the perturbation of ˜γij, which has unit determinant. Precisely, any matrix with unit determinant can be written as: ˜γij = e−2Mij , where Mij is traceless. Note that the last two linearizations in (23) leads to here Mij is traceless. Note that the last two linearizations in (23) leads to γij ≃a2(1 + 2D)δij −2Eij  . (24) (24) Finally, the scalar field responsible for inflation must also be linearized, i.e., φ ≃φb + δφ . (25) (25) It can be easily shown that, of the linear variables introduced above, A, D and δφ transform as scalars under rotations in the background space-time coordinates, Bi as a 3-vector and Eij as a 3D-tensor. This does not mean that the only scalar components are A, D and δφ. In fact, we know from Euclidean 3D vector calculus that a vector can be decomposed as: Bi = BS i + BV i with ∂iBS j −∂jBS i = 0 and ∂iBV i = 0 , (26) (26) and hence and hence BS i = ∂iB , (27) BS i = ∂iB , (27) where B is some scalar field. where B is some scalar field. 4. Linear Perturbation Theory Similarly, for a tensor field we have Similarly, for a tensor field we have Eij = ES ij + EV ij + ET ij , (28) Eij = ES ij + EV ij + ET ij , Eij = ES ij + EV ij + ET ij , (28) Universe 2022, 8, 334 8 of 39 where where where ES ij =  ∂i∂j −1 3δij∇2  E , EV ij = 1 2 ∂jEi + ∂iEj  with ∂iEi = 0 , ∂iET ij = 0 , (29) δijET ij = 0 , (30) (29) (30) where E is again a scalar field. where E is again a scalar field. The procedure explained above allow us to decompose the perturbations into a scalar, vector and tensor sector. These sectors have the characteristic of evolve independently one from each other at linear order in perturbation theory, which make them easier to handle. During this review we will be mostly focused on scalar perturbations of the metric since they are the ones that couple to the scalar field perturbation δφ. This allows us to write the perturbed metric of (23) as: ds2 = −(1 + 2A)dt2 + 2a∂iBdxidt + a2  (1 + 2D)δij −2  ∂i∂j −1 3δij∇2  E  dxidxj . (31) (31) Similarly to what we have just done with the metric, we can split each one of the ADM equations presented in Section 3 into an homogeneous and a perturbed part as follows: Similarly to what we have just done with the metric, we can split each one of the ADM equations presented in Section 3 into an homogeneous and a perturbed part as follows: • Hamiltonian constraint (14). • Hamiltonian constraint (14). 0 =R(3) −˜Aij ˜Aij + 2 3K2 − 2 M2 PL E ≃  6  Hb2 − 2 M2 PL     ˙φb2 2 + V(φb)      + " −12Hb  HbA −˙D + 1 3 ∇2 a B  −4∇2 a2  D + 1 3∇2E  − 2 M2 PL  ˙φb ˙δφ −  ˙φb2 A + Vφbδφ # . (32) (32) Note that in (32) the term inside the first square brackets corresponds to the back- ground Hamiltonian constraint (4) of Section 2, being the term inside the second square brackets the linearization of the Hamiltonian constraint. 4. Linear Perturbation Theory We will follow this same notation for the rest of the ADM equations. • Momentum constraint (15). 0 = Dj ˜Aij −2 3 DiK − 1 M2 PL Ji ≃[0] + " ∂i −2HbA + 2 ˙D + 2 3∇2 ˙E + 1 M2 PL ˙φbδφ !# . (33) (33) As it can be seen in (33), the momentum constraint do not have a contribution at the background level, which is logical due to the presence of spatial derivatives, which cannot appear in an exactly homogeneous and isotropic global background. However, it does have an homogeneous contribution in the perturbative sector due to the presence of a total spatial derivative, this is of crucial importance for the rest of the review. Universe 2022, 8, 334 9 of 39 • Evolution equation for ζ (17). 0 = (∂t −βk∂k)ζ + ˙a a = −1 3(αK −∂kβk) ≃  −Hb + ˙a a  +  ˙D −HbA −δH −1 3 ∇2 a B  , (34) (34) where we have used the identification K ≡−3H explained below Equation (13). From (34) we can easily identify the perturbation of the Hubble parameter, i.e., H = Hb + δH = Hb +  −HbA + ˙D −1 3 ∇2 a B  . (35) (35) • Evolution equation for ˜Aij (18). • Evolution equation for ˜Aij (18). • Evolution equation for ˜Aij (18). 0 = (∂t −βk∂k) ˜γij = −2α ˜Aij + ˜γik∂jβk + ˜γjk∂iβk −2 3 ˜γij∂kβk ≃[0] +  2 ˜Aij −2  ∂i∂j −1 3δij∇2  B a + ˙E  . (36) +  2 ˜Aij −2  ∂i∂j −1 3δij∇2  B a + ˙E  . (36) (36) • Evolution equation for K (19). • Evolution equation for K (19). • Evolution equation for K (19). 0 = (∂t −βk∂k)K = α  ˜Aij ˜Aij + 1 3K2  −DkDkα + 4πGα(E + Sk k) ≃ " −3 ˙Hb −3  Hb2 − 1 M2 PL  ˙φb2 −V(φb) # +  6 ˙HbA + 3Hb ˙A −3 ¨D + ∇2 a ˙B + ∇2 a2 A + 6  Hb2 A −6Hb ˙D + Hb ∇2 a B − 1 M2 PL  2 ˙φb ˙δφ −2  ˙φb2 A −Vφbδφ  . (37) (37) • Evolution equation for ˜Aij (20). • Evolution equation for ˜Aij (20). • Evolution equation for ˜Aij (20). 0 = (∂t −βk∂k) ˜Aij −e−2ζ a2  α  R(3) ij −γij 3 R(3)  −  DiDjα −γij 3 DkDkα  −α(K ˜Aij −2 ˜Aik ˜Ak j ) −˜Aik∂jβk −˜Ajk∂iβk + 2 3 ˜Aij∂kβk + 8πGαe−2ζ a2  Sij −γij 3 Sk k  [ ] 0 = (∂t −βk∂k) ˜Aij −e−2ζ a2  α  R(3) ij −γij 3 R(3)  −  DiDjα −γij 3 DkDkα  −α(K ˜Aij −2 ˜Aik ˜Ak j ) −˜Aik∂jβk −˜Ajk∂iβk + 2 3 ˜Aij∂kβk + 8πGαe−2ζ a2  Sij −γij 3 Sk k  ≃[0] + " ∂i∂j −1 3δij∇2  ˙B a + 2 HbB a + ¨E + 3Hb ˙E + A + D + 1 3∇2E !# . + " ∂i∂j −1 3δij∇2  ˙B a + 2 HbB a + ¨E + 3Hb ˙E + A + D + 1 3∇2E !# . (38) + " ∂i∂j −1 3δij∇2  ˙B a + 2 HbB a + ¨E + 3Hb ˙E + A + D + 1 3∇2E !# . (38) (38) • Evolution equation for the scalar field (21). • Evolution equation for the scalar field (21). 0 = 1 √−g∂µ p−ggµν∂νφ  −Vφ ≃ h ¨φb + 3Hb ˙φb + Vφb(φb) i +  ¨δφ + 3Hb ˙δφ + Vφbφbδφ −∇2 a2 δφ + 2Vφb A −˙φb  ˙A −3 ˙D + ∇2 a B  . (39) (39) Universe 2022, 8, 334 10 of 39 10 of 39 Once we have seen how linear perturbation theory works and what are the linear equations that describe small inhomogeneities during an inflationary epoch, it is important to have a physical intuition about what a perturbation of the metric really means. By definition, a perturbation is the difference between the value of a quantity in the real and inhomogeneous space-time and its value on the idealized FLRW background. This seems trivial; however, in order to make such a comparison, it is necessary to compute these two values at the same space-time point. Since the quantities to compare live in different space-times, we require a pointwise correspondence between them, which is given by a coordinate system xµ such that the point Pb in the background space-time and the point P in the perturbed space-time, which have the same coordinate values, correspond to each other. • Evolution equation for ˜Aij (20). The freedom in the choice among these coordinate systems is called the gauge choice. The way the different gauges are related in linear perturbation theory (for gauge trans- formations beyond linear perturbation theory see for example [89]) is via an infinitesimal gauge transformation of the coordinates: ˜xµ = xµ + δxµ (40) (40) We can split the vector δxµ into its time an space components δxµ = λ0, λi , and following the same idea as when we decomposed the perturbations in the metric, λi can be written as λi = λi ⊥+ ∂iη, where λi ⊥is a 3-dimensional divergenless vector and η is a scalar function. In terms of these functions, we can impose the gauge invariance of the perturbed metric (31) to deduce the gauge transformation of each one of the scalar variables in the metric: D →˜D = D + aHbλ0 + 1 3∇2η , A →˜A = A + aHbλ0 + a ˙λ0 , E →˜E = E −η , B →˜B = B + a ˙η −λ0 . (41) (41) B →˜B = B + a ˙η −λ0 . (41) Finally, the scalar field perturbation will transform as: Finally, the scalar field perturbation will transform as: Finally, the scalar field perturbation will transform as: δφ →˜δφ + a ˙φλ0 . (42) (42) From (41) and (42) we can clearly see that the freedom on the choice of the gauge allow us to set two out of the five scalar perturbations to zero by choosing η and λ0 accordingly. This reduces the scalar degrees of freedom to three (which further reduces to two when using the ADM equations for a single scalar field), which can be written in terms of gauge invariant, and hence physical, variables: the two Bardeen potentials [90] Ψ ≡−D −1 3∇2E −aHbB + a ˙E  , Φ ≡A + aHb(B + a ˙E) + a d dt(B + a ˙E) , (43) (43) and the Mukhanov-Sasaki (MS) variable and the Mukhanov-Sasaki (MS) variable Q ≡δφ − ˙φb Hb  D + 1 3∇2E  . (44) (44) During this review we will pay special attention to the MS variable. • Evolution equation for ˜Aij (20). In fact, it can be shown that, by rearranging the linearized ADM equations of (32)–(39), the MS variable Universe 2022, 8, 334 11 of 39 follows a simple equation of motion which, written in terms of the SR parameters defined in Section 2, is: follows a simple equation of motion which, written in terms of the SR parameters defined in Section 2, is: ¨Q + 3Hb ˙Q +  −∇2 a2 +  Hb2 −3 2ϵ2 + 1 2ϵ1ϵ2 −1 4ϵ2 2 −1 2ϵ2ϵ3  Q = 0 . (45) (45) In order to solve (45), we must take into account that subhorizon scales during inflation are microscopic, and hence we must study the behavior of the inflationary scalar field using quantum field theory (QFT). The way to proceed is similar to what we do when quantizing the harmonic oscillator: ˆQ(x, t), which is now a quantum operator, can be expressed in Fourier space as: ˆQ(x, t) = Z dk (2π)3/2 ˆQk(t) , (46) (46) where where ˆQk(t) = e−ik·xQk(t)ak + e−ik·xQ∗ k(t)a† k , (47) (47) and ak and a† k are the usual creation and annihilation operators, that satisfy the usual commutation relation: and ak and a† k are the usual creation and annihilation operators, that satisfy the usual commutation relation: [ak, a† k’] = δ(3)(k −k’) (48) (48) With this construction, the variable Qk of (47) is the solution of the MS Equation (45) in Fourier space, i.e.,: ¨Qk + 3Hb ˙Qk +  k2 a2 +  Hb2 −3 2ϵ2 + 1 2ϵ1ϵ2 −1 4ϵ2 2 −1 2ϵ2ϵ3  Qk = 0 (49) (49) If we impose the Bunch-Davies vacuum [91] to be recovered at early time, we can only write analitically a solution for (49) if a specific condition is satisfied. In order to see what is this condition we will define the variable z as z = a ˙φb Hb and the conformal time τ as adτ = dt, with this, the analytical solution of (49) in terms of the conformal time only exists if ν2 ≡1 4 + τ2 1 z d2z dτ2 (50) (50) is a constant. It is easy to check that this is the case generically; in fact we can integrate by parts τ = R dt a to get: 1 is a constant. • Evolution equation for ˜Aij (20). It is easy to check that this is the case generically; in fact we can integrate by parts τ = R dt a to get: 1 τ ≃−1 aHb (1 + O(ϵ1)) . See for example Appendix E of [65] for the derivation. The equation above, together with the fact that 1 z d2z dτ2 = a2 Hb2 2 −ϵ1 + 3 2ϵ2 −1 2ϵ1ϵ2 + 1 4ϵ2 2 + 1 2ϵ2ϵ3  , makes ν defined in (50) to be a constant generically up to O(ϵ1). Note that, since in SR, ϵi is also a constant up to O(ϵ1), in this case we have that νSR is constant up to O ϵSR i 2 . The analytical solution in this case is: Qk = e i 2 π(ν+ 1 2) √π 2a √ −τH(1) ν (−kτ) , (51) (51) where H(1) ν is the Hankel function of first class. where H(1) ν is the Hankel function of first class. In order to finish this reminder of linear perturbation theory, let us introduce a very useful quantity that characterizes the properties of the perturbations: the power spectrum. We will give a physical interpretation of the power spectrum later on and for the moment Universe 2022, 8, 334 12 of 39 12 of 39 we will focus only on its mathematical definition. The power spectrum of a generic quantity X(x, t) is defined as the Fourier transform of the two-point correlation function, i.e.: ⟨0|X(x1, t)X(x2, t)|0⟩= Z dk (2π)3 |Xk(t)|2 ≡ Z dk k PX(k, t)sin(kr) kr , (52) (52) where r = |x1 −x2| and k = |k|. From (52) it is clear that the power spectrum is defined as: PX(k, t) ≡ k3 2π2 |Xk|2 . (53) (53) Another interesting quantity derived from the power spectrum is the spectral index X 1 hi h i d fi d Another interesting quantity derived from the power spectrum is the spectral index nX s −1, which is defined as: Another interesting quantity derived from the power spectrum is the spectral index nX s −1, which is defined as: g q nX s −1, which is defined as: nX s −1, which is defined as: nX s −1 ≡d log PX d log k . (54) (54) 4.1. Long Wavelength Limit of Linear Perturbation Theory As explained in the introduction, many quantities of interest such as the power spec- trum of the scalar tilt are usually computed in the long wavelength limit, or superhorizon scales. This makes sense because, as long as inflation is taking place, the exponential expansion of the universe stretch the perturbations of the quantum fields from microscopic to cosmological scales. In this way, the inhomogeneities that re-enter the horizon once inflation has finished and hence the ones of interest today, were in its long-wavelength limit when inflation ended. In this section we will take a close look to this limit and its physical consequences. We will study the behaviour of perturbations with characteristic wavelength L much larger that a local Hubble radius H(−1) l in such a way that if we consider L to be infinitely large compared with H(−1) l , we can interpret the region inside H(−1) l as a local uni- verse without perturbations, i.e., homogeneous and isotropic. In other words, if L H(−1) l →∞, or equivalently, if k alHl ≃kτl →0, then the region inside H(−1) l represents a local FLRW universe. With this in mind, let us study the long wavelength limit of perturbations during inflation. In order to do so we will take two different point of view: the first one is very intuitive and we will call it the k →0 limit, the second one is slightly less intuitive but it is very useful when dealing with non-linear perturbations, this is the so-called linear separate universe approach of k = 0 case. Although they are very similar, the two point of view are not exactly equivalent as we will see in the following. or equivalently, if k alHl ≃kτl →0, then the region inside H(−1) l represents a local FLRW universe. With this in mind, let us study the long wavelength limit of perturbations during inflation. In order to do so we will take two different point of view: the first one is very intuitive and we will call it the k →0 limit, the second one is slightly less intuitive but it is very useful when dealing with non-linear perturbations, this is the so-called linear separate universe approach of k = 0 case. Although they are very similar, the two point of view are not exactly equivalent as we will see in the following. 4.1.1. K →0 Limit 4.1. Long Wavelength Limit of Linear Perturbation Theory . Long Wavelength Limit of Linear Perturbation Theory 4.1.1. K →0 Limit Since each spatial derivative introduces a factor k in Fourier space, it would be logical to think that in the k →0 limit of the ADM equations we must neglect any term that contains a spatial derivative (This will be done in the next subsection). However, by doing that, we would be neglecting terms that actually contribute in the k →0 limit. g g y In the following we will enumerate the two cases in which neglecting a term with a spatial derivative would be too naive: 1. Non-local terms. 1. Non-local terms. As an example, let us imagine a re-scaling of the spatial coordinates xi →˜xi = (1 + δ)xi . (55) (55) It is easy to realize that if we rewrite δ in terms of the transformations parameters of (41) we get: 2 It is easy to realize that if we rewrite δ in terms of the transformations parameters of (41) we get: 2 ∇2η = δ . (56) (56) The fact that a transformation as the one described in (55) is perfectly allowed in a FLRW universe together with (56) and the transformations rules of the perturbed Universe 2022, 8, 334 13 of 39 13 of 39 metric variables of (41) immediately tell us that variables like ∇2E cannot generically be neglected in the long wavelength limit even if they contain a Laplacian. In order to avoid this problem, we will only neglect terms that contain extra spatial derivatives, by extra we mean that we will neglect a term like ∂iX if and only if it is compared with X, but we will not neglect it if it appear alone. 2. Equations with overall spatial derivatives. Equations with overall spatial derivatives. A clear example is the momentum constraint of (33). Because it contains a total spatial derivative, it gives non-trivial information even in the k →0 limit, namely: A clear example is the momentum constraint of (33). Because it contains a total spatial derivative, it gives non-trivial information even in the k →0 limit, namely: HbA −˙D −1 3∇2 ˙E − 1 2M2 PL ˙φbδφ = 0 . (57) (57) The same happens with the evolution equation for ˜Aij (38). 4.1.2. K = 0 or Linear Separate Universe Approach 4.1.2. K = 0 or Linear Separate Universe Approach 4.1.2. K = 0 or Linear Separate Universe Approach The second point of view to solve linear perturbation theory in the long wavelength limit is the linear separate universe approach [93–95], in this case we will forget for a moment about the perturbed ADM equations and we will take advantage of the fact that, in the long wavelength limit, each local patch represents an homogeneous and isotropic universe such that its line element is: ds2 l = −dt2 l + al(tl)2δijdxi ldxj l , (63) (63) where, as before, the subscript l stands for local. As a consequence, the constraints and equations of motion will be the same as the ones presented in Section 2, i.e., where, as before, the subscript l stands for local. As a consequence, the constraints and equations of motion will be the same as the ones presented in Section 2, i.e., 3M2 PLH2 l = 1 2  d dtl φl 2 + V(φl) , d dtl Hl + H2 l = − 1 3M2 PL " d dtl φl 2 −V(φl) # , d2φl dt2 l + 3Hl d dtl φl + Vφl(φl) = 0 . (64) (64) The first thing to realize here is that the momentum constraint is missing. This is because in the separate universe approach we are not only assuming that each patch is homogeneous and isotropic (we were also making this assumption in the k →0 limit where the momentum constraint plays a role), but we are also assuming that each patch evolve independently from each other. Since the momentum constraint gives information about the interaction between the different FLRW patches due to the presence of a spatial derivative, it makes sense that it does not appear in the linear separate universe approach. Before proceeding, let us clarify here that when we talk about the information about the interaction between different patches encoded in the momentum constraint, which makes these patches evolve in a not completely independent way, we do not claim that what happens in one patch will affect the others, but rather that all the patches as an ensemble must satisfy some conditions (given by the momentum constraint) that are absent if we look to a single patch but that are important when comparing them. The same happens with the evolution equation for ˜Aij (38). Universe 2022, 8, 334 14 of 39 14 of 39 The same happens with the evolution equation for ˜Aij (38). Taking these two important point into account one can re-derive the equation of motion for the MS variable Q in the k → 0 limit. The result is, as expected, the Equation (45) but without the Laplacian, i.e., ¨Q + 3Hb ˙Q +  Hb2 −3 2ϵ2 + 1 2ϵ1ϵ2 −1 4ϵ2 2 −1 2ϵ2ϵ3  Q = 0 . (58) (58) If we want to know the solution for Equation (58) we can simply take the kτ →0 limit of the solution (51) obtained before (we will do that later on); however, this would be restricted to ν (defined in (50)) being a constant. In this section we will solve independently the long wavelength equation of motion for Q without any assumption, i.e., valid at all orders in slow-roll parameters. The price to pay is that, since the initial conditions are given at sub-horizon scales and (58) is only valid at super-horizon scales, we will not specify any initial condition here. It can be checked that (58) can be written as a total derivative as follows: 2M2 PLHb a3 ˙φb d dt " a3 ˙φb 2M2 PL  ˙Q Hb −ϵ2 2 Q # = 0 . (59) (59) For convenience, we will define the comoving curvature perturbation as: Rc ≡−Hb ˙φb Q . (60) (60) It terms of Rc, (59) takes a very simple form: It terms of Rc, (59) takes a very simple form: a3ϵ1 ˙Rc = C1 , (61) (61) where C1 is a constant. Therefore, the solution of the MS equation in the long wavelength limit (58) is Q = ˙φb Hb Rc = C2 ˙φb Hb + C1 ˙φb Hb Z dt a3ϵ1 , (62) (62) where C2 is also a constant. Solution (62) is the well-known exact solution in the k →0 limit for the single component scalar field case [85,92]. The term proportional to C1 is usually known as decaying mode, name inherited from its SR behaviour where ˙φb is roughly constant, since a ∼e−Hbt during inflation, we can clearly see that the term proportional to C1 decays as e−3Hbt during SR. However, this is not the case beyond SR, for example in USR we have ˙φb ∼e−3Hbt (see (8)) and hence ϵ1 ∼e−6Hbt, which makes the term proportional to C1 be approximately constant whereas the one proportional to C2 decays, contrary to what happens in SR. 4.1.2. K = 0 or Linear Separate Universe Approach Within this approximation, we can only see the effect of perturbations if we compare different patches between them or with the global background. Knowing that the differ- ences must be perturbative, we can follow our results from linear perturbation theory and write: Hl ≃Hb + δH = Hb −HbA + ˙D −1 3 ∇2 a B , dtl ≃(1 + A)dt , φl ≃φb + δφ . (65) (65) The term 1 3 ∇2 a B can be set to zero without loss of generality, this is because since each patch is independent from each other, we can always choose an orthogonal threading for all of them, in which βi (and hence B) is zero. Another way of reasoning is that, as checked in the k →0 limit of perturbation theory, a term with a Laplacian can only be important in the long wavelength limit if it contains non-local information, but at the same time, non-local terms would give some information about the interaction between local patches, which is in contradiction with the absence of the momentum constraint (and hence with the separate universe assumption) as explained above. If we also take into account that the scalar perturbation related with the traceless part of the spatial metric (i.e., E) does not appear in (65) so neither in (64), we can set ∇2Bsep = ∇2Esep = 0 . (66) (66) Universe 2022, 8, 334 15 of 39 15 of 39 in the separate universe approach (as indicated with the superscript “sep”). Note that, although (66) coincides with the Newtonian (or longitudinal) gauge, this is not a gauge choice, but a consequence of the separate universe approach. q p pp With this in mind, we can write the equations in (64) in terms of the global background and perturbations over it as follows: −6  Hb2 Asep + 6Hb ˙Dsep = 1 M2 PL  ˙φbδφsep −  ˙φb2 Asep + Vφbδφsep  , 6 ˙HbAsep + 3Hb ˙Asep −3 ¨Dsep + 6  Hb2 Asep −6Hb ˙Dsep = 1 M2 PL  2 ˙φb ˙δφsep −  ˙φb2 Asep −Vφbδφsep  , ¨δφsep + 3Hb ˙δφsep + Vφbφbδφsep = −2Vφb Asep + ˙φb ˙Asep −3 ˙Dsep . 4.1.2. K = 0 or Linear Separate Universe Approach (67) (67) As one can see from (67), the linear separate universe approach does not use the whole system of ADM equations presented in Section 4, it only uses the long-wavelength version of: (a) the Hamiltonian constraint (32), (b) the evolution equation for the trace of the extrinsic curvature (37) and (c) the equation of motion of the scalar field (39). All of them setting both ∇2Bsep and ∇2Esep to zero accordingly with the separate universe assumption. In order to see what are the consequences of this reduction in the number of equations and variables when comparing with the k →0 limit of Section 4.1.1, we can follow a similar procedure to what we did when defining the MS variable and write down a “separate- universe” gauge invariant variable [96,97], meaning that it is gauge invariant under time reparametrizations. Qsep ≡δφsep − ˙φb Hb Dsep . (68) (68) Similarly to what we did with the MS Equation (45), we can rearrange the equations of the linear separate universe approach (67) and write a single equation of motion for Qsep, the result, in terms of the SR parameters, is: ϵ1ϵ2 3(3 −ϵ1)  ˙Qsep +  Hb2 −3 2ϵ2 + 1 2ϵ1ϵ2 −1 4ϵ2 2 −1 2ϵ2ϵ3 − ϵ1ϵ2 2 2(3 −ϵ1) ! Qsep = 0 . (69) (69) Comparing (69) with (58) we can now clearly identify two extra terms that appear when assuming that each local patch evolves independently from each other, i.e., when using the separate universe approach. These terms are O(ϵ1ϵ2) [55], being strongly depen- dent on the inflationary regime, for example, they are O(ϵUSR 1 ) in USR (where ϵ2 ≃−6) and O ϵSR 1 2 in SR.   In order to better quantify this difference we will solve (69) in a similar way as done with (58) and compare the results. We can again write (69) as a total derivative as follows 2V 3a3Hb ˙φb d dt   3a3 Hb2 ˙φb 2V  ˙Qsep Hb −ϵ2 2 Qsep   = 0 . 4.1.2. K = 0 or Linear Separate Universe Approach (70) (70) Defining a “separate universe” comoving curvature perturbation as Defining a “separate universe” comoving curvature perturbation as Rsep c ≡−Hb ˙φb Qsep , (71) we can write (70) as 3a3 ˙φb2 2V = C′ 1 , (72) Rsep c ≡−Hb ˙φb Qsep , (71) 3a3 ˙φb2 2V = C′ 1 , (72) Rsep c ≡−Hb ˙φb Qsep , (71) (71) we can write (70) as 3a3 ˙φb2 2V = C′ 1 , (72) (72) Universe 2022, 8, 334 16 of 39 16 of 39 where C′ 1 is a constant. Hence, the solution for Qsep is: where C′ 1 is a constant. Hence, the solution for Qsep is: where C′ 1 is a constant. Hence, the solution for Qsep is: Qsep = C′ 2 ˙φb Hb + C′ 1 ˙φb Hb Z  1 a3ϵ1 −1 3a3  . (73) (73) We can see that the solutions for Qsep of (73) and for Q of (62) differ by an extra term in the decaying mode, which is obviously due to the difference of O(ϵ1ϵ2) in the equation of motion. The importance of this extra term depends on the inflationary regime, being more important beyond SR, where the term proportional to C′ 1 does not decay (see discussion below (62)). Since the main difference between the linear separate universe approach and the k →0 limit is the inclusion of the non-trivial information of the momentum constraint at large scales, which is not taken into account in the former, one could think that the two solutions will coincide if we impose the solution (73) to satisfy the momentum constraint; however, this is not the case. In fact, in the following we are going to check that the mode proportional to C′ 1, which contains the new extra term, does not represent the k →0 limit of some solution to the perturbations equations with k ̸= 0. This should not be a surprise since the correct solution for the MS variable in the long wavelength limit is given by (62). In order to check that, let us rearrange equations (65) to write HbAsep −˙Dsep − ˙φb 2M2 PL δφsep = −Hb ˙φb 2V  ˙Qsep Hb −ϵ2 2 Qsep  . 4.1.2. K = 0 or Linear Separate Universe Approach (74) (74) If we want solution (73) to be the k →0 solution to the perturbation equations with k ̸= 0 we must be sure that our solution satisfies the momentum constraint (although it is lost in the separate universe approach.) because, as we know, this constraint gives non-trivial information even at large scales. What would be the momentum constraint in the linear separate universe approach is HbAsep −˙Dsep − ˙φb 2M2 PL δφsep = 0 , (75) (75) which, comparing with (74), immediately implies: ˙Qsep Hb −ϵ2 2 Qsep = 0 . (76) (76) From condition (76) and the equation of motion (70) we can see that, if the momentum constraint of (75) is satisfied at initial time, it will be always satisfied so one could think that the problem of ignoring the momentum constraint in the linear separate universe approach is simply solved by an appropriate choice of initial conditions; however, the only mode that satisfies (76) is the mode proportional C′ 2 in the solution (73), which means that the mode proportional to C′ 1 therein does not represent a k →0 solution of the perturbation equations with k ̸= 0 and it must be set to zero. However, setting C′ 1 = 0 means that we are losing the term proportional to C1 in the correct solution of (62), which can be important beyond SR. Thus, imposing that the momentum constraint must be satisfied in the separate universe approach does not give the correct solution for the long-wavelength limit of the MS variable and hence we have to use the whole set of ADM equations in the long wavelength limit to describe the correct dynamics (at all orders in SR parameters) of the MS variable at super-horizon scales. Let us indicate here that although there exist some ways to recover the correct k →0 limit of the MS variable using only the linear separate universe approach (see [96,97]), we will not study them here because they are restricted to linear perturbation theory and, as we will see later on, we are planning to use the long wavelength limit of inhomogeneities beyond perturbation theory. 4.1.2. K = 0 or Linear Separate Universe Approach For clarity purposes, let us summarize the findings of this section until now: Universe 2022, 8, 334 17 of 39 17 of 39 • Although both the k →0 and the linear separate universe approach consider local homogeneous and isotropic universes, the k →0 limit allow some interaction between them whereas the linear separate universe approach assume that they evolve inde- pendently. Mathematically speaking, the linear separate universe approach ignore possible non-local terms and equations with overall spatial derivatives, both present in the k →0 limit. • As a consequence, the equation of motion for Qsep differ by terms of O(ϵ1ϵ2) from the equation of motion for Q in the long wavelength limit. This difference induces an extra decaying term, namely: • As a consequence, the equation of motion for Qsep differ by terms of O(ϵ1ϵ2) from the equation of motion for Q in the long wavelength limit. This difference induces an extra decaying term, namely: Qsep = Q(k →0) + C′ 1 ˙φb Hb Z 1 3a3 dt (77) (77) • The difference (77) always decays so one could think that it can be safely ignored; however, its importance strongly depends on the inflationary regime. For example, in USR we have Qsep −Q(k →0) ∼O(ϵ1) so if we want to be precise enough when studying the long-wavelength limit of perturbation theory, we should use the k →0 limit rather than the linear separate universe approach. • The difference (77) always decays so one could think that it can be safely ignored; however, its importance strongly depends on the inflationary regime. For example, in USR we have Qsep −Q(k →0) ∼O(ϵ1) so if we want to be precise enough when studying the long-wavelength limit of perturbation theory, we should use the k →0 limit rather than the linear separate universe approach. • The difference (77) always decays so one could think that it can be safely ignored; however, its importance strongly depends on the inflationary regime. For example, in USR we have Qsep −Q(k →0) ∼O(ϵ1) so if we want to be precise enough when studying the long-wavelength limit of perturbation theory, we should use the k →0 limit rather than the linear separate universe approach. p pp • Finally, imposing the momentum constraint to be satisfied in the separate universe approach not only does not solve the difference between Qsep and Q(k →0), but it makes it worse. 4.2. Linear Perturbation Theory and Pbhs In order to finish this section we will study the behaviour of the power spectrum of the inflationary regimes that can lead to the formation of PBHs in the long wavelength limit (The reader interested in PBHs as probes for the physics of the very early universe in a more detailed way can read the following reviews: [98,99]). We will do that in a very qualitative way and at leading order in SR parameters, this is why we will simply use the kτ →0 limit of solution (51), where the initial conditions are properly specified. If we expand the Hankel function of first order we have: lim kτ→0 Qk = lim kτ→0 e i 2(ν+ 1 2) √π 2a √ −τH(1) ν (−kτ) ≃−ie i 2(ν+ 1 2) 2ν−1 a√π √ −τ(−kτ)−νΓ[ν] , (78) (78) where Γ[ν] is the Euler gamma. where Γ[ν] is the Euler gamma. g For the formation of PBH we are interested in the power spectrum of the comoving curvature perturbation, i.e., For the formation of PBH we are interested in the power spectrum of the comoving curvature perturbation, i.e., PRc = k3 2π2 Hb ˙φb !2 |Qk|2 ≃  Hb4 π3 ˙φb2  k 2 3−2ν 1 aHb 3−2ν Γ[ν]2 , (79) (79) where we have used τ ≃− 1 aHb . aH For USR and SR we have ν ≃3 2 and hence the power spectrum is roughly scale invariant: 4 2 aH For USR and SR we have ν ≃3 2 and hence the power spectrum is roughly scale invariant: 4 2 PSR Rc ≃PUSR Rc ≃  Hb4 4π2 ˙φb2 =  Hb2 8π2M2 PLϵ1 . (80) (80) However, and although the k dependence of the comoving curvature power spectrum for USR and SR is roughly the same, and hence nRc s −1 ∼O(ϵ1) for both, this is not true for the time dependence. In fact we have PSR Rc ∼constant , PUSR Rc ∼e6Hbt . (81) (81) Universe 2022, 8, 334 18 of 39 18 of 39 An exponential growth of the power spectrum at super-horizon scales as it happens in USR has important consequences for the formation of PBH. In fact, by definition of the power spectrum, PRc give us the variance of the probability distribution that follow the amplitude of the perturbations Rk1  c with characteristic wavenumber k1, therefore, a growth in the power spectrum can be interpreted as a growth in the variance or, equivalently, a spreading of the probability distribution. This means that high values for the amplitude of the perturbations placed at the tail of the probability distribution are much more probables if the power spectrum grows. Finally, and since the high values for the amplitude of the curvature perturbations are the ones that collapse to form a PBH when they re-enter the horizon, we can conclude that inflationary regimes beyond SR favor the creation of PBH. The argument given above is very hand-waving but it is enough to remark the impor- tance of inflationary regimes beyond SR when talking about PBH. where Γ[ν] is the Euler gamma. However, if we want to make actual predictions about the mass and abundances of PBH we need to study inflation- ary dynamics in a much more precise way, in fact, the tail of the probability distribution for the amplitude of perturbations, which is where the perturbations responsible for the formation of PBH are located, is very sensitive to any small change in the power spectrum (or in the higher order correlators as the bispectrum). This is the reason why a description of the perturbations at all orders in SR is highly desirable, making the linear separate universe approach not the best approximation to study the tail of the probability distribution of the amplitude of perturbations generated during a inflationary regime beyond SR, i.e., to study the formation of PBH. The reason is that, generically, Qsep −Q(k →0) ∼O(ϵ1) as explained above. Finally, another problem arises when dealing with inhomogeneities large enough to form a PBH, namely, non-linear or even non-perturbative effects can play an impor- tant role, reason why we need to go beyond linear perturbation theory. The rest of the review is devoted to the study of inhomogeneities generated during inflation beyond linear perturbation theory. 5. Gradient Expansion Having a quantity whose leading order is gradient expansion is O(σ−1) could seem problematic; however this is only telling us that βi is generically a non-local quantity, in fact, its linearization give us the non-local variable B (see (23)) studied in Section 4.1.1. Furthermore, we will see that βi always appear t th ith ti l d i ti i th ti f ti where we are using the subscript (0) to remind the reader that we are at leading order in gradient expansion It is important to remark here that the leading order in gradient expansion of each quantity can be different, for example, the leading order for α and ζ is O(σ0) whereas the leading order for βi is O(σ−1). Having a quantity whose leading order is gradient expansion is O(σ−1) could seem problematic; however this is only telling us that βi is generically a non-local quantity, in fact, its linearization give us the non-local variable B (see (23)) studied in Section 4.1.1. Furthermore, we will see that βi always appear together with a spatial derivative in the equations of motion. expansion of each quantity can be different, for example, the leading order for α and ζ is O(σ0) whereas the leading order for βi is O(σ−1). Having a quantity whose leading order is gradient expansion is O(σ−1) could seem problematic; however this is only telling us that βi is generically a non-local quantity, in fact, its linearization give us the non-local variable B (see (23)) studied in Section 4.1.1. Furthermore, we will see that βi always appear together with a spatial derivative in the equations of motion. One could be worried about the fact that a homogeneous and isotropic metric con- tains terms outside the diagonal; however, following [110] we know that a space-time is homogeneous and isotropic if: 1. All constant time hypersurfaces Σt are constant curvature spaces. In our case the hypersurfaces Σt are simply Euclidean and this condition is trivially satisfied. 1. All constant time hypersurfaces Σt are constant curvature spaces. In our case the hypersurfaces Σt are simply Euclidean and this condition is trivially satisfied. 2. The extrinsic curvature of the hypersurfaces is homogeneous and isotropic. 5. Gradient Expansion The gradient expansion is a non-perturbative, in terms of the amplitude of the inhomo- geneities, expansion of the ADM equations valid when the characteristic wavelength of the inhomogeneities L is much larger that the Hubble horizon H−1 l [100–109]. Since this is the same approximation that we do when studying the long wavelength behaviour of the linear perturbations, we will follow the same idea and we will take advantage of the fact that, when L ≫H−1 l , the universe is locally homogeneous and isotropic to define an expansion parameter σ ≡ H−1 l L such that at leading order in σ, each local patch of the universe of size (σH−1 l ) (we will call this scale the coarse-grained scale) is approximately described by a FLRW universe. Higher order terms in the σ will instead capture local inhomogeneities. Contrary to the linear theory approach to cosmological perturbations, the gradient expansion is valid for any amplitude of local over-densities as long as the patch is taken small enough for the gradients to be negligible. Note that this assumption on which the gradient expansion is based on implies that a patch can be found such that any spatial gradient would only introduce an order σ. In other words, for any generic function X, ∂iX ∼X × O(σ). This is because a function which is approximately homogeneous in local coordinates can be written as X(t, σxi) with σ ≪1. Thus, we have ∂iX(t, σxi) = σ ∂ ∂(σxi) X(t, σxi) = σ ∂ ∂(σxi) X(t, σxi) σxi=0 + O(σ2) , and since ∂ ∂(σxi) X(t, σxi) σxi=0 c 0 can be of the same order as X(t, σxi) we can generically write: nce ∂ ∂(σxi) X(t, σxi) σxi=0 can be of the same order as X(t, σxi) we can generically write ∂iX ∼X × O(σ) . ∂iX ∼X × O(σ) . Universe 2022, 8, 334 19 of 39 19 of 39 When we were studying linear perturbation theory, the way of relating different patches was to define a global background over which each patch represented a perturba- tion. In this case we do not have a physical background metric because we are no longer perturbing anything; however we will still define a fictitious global background metric with coordinates t and xi, i.e., ds2 b = −dt2 + a(t)2δijdxidxj . 5. Gradient Expansion (82) (82) We are then interested in writing each local FLRW patch, which at leading order in σ and in terms of local coordinates is simply (63), in terms of the coordinates t and xi. At leading order in gradient expansion and considering only scalar perturbations (note that, if we want to study inflation in a fully non-perturbative way, we should also take into account vector and tensor perturbations. This is because, although they are independent at linear order in perturbation theory, this is no longer true at higher orders. The reason why we do not include vector and tensor perturbations here is because, although at this level it would be straightforward, it is not possible when applying gradient expansion to stochastic inflation as we will see) we have: ds2 l = −(0)α2dt2 + (0)γij  dxi + (0)βidt  dxj + (0)βj , (83) (83) with the conditions: with the conditions: 1. (0)α = (0)α(t) , 2. (0)βi = b(t)xi , 3. (0)γij = γ(t)δij = a(t)2e2 (0)ζ(t)δij , 1. (0)α = (0)α(t) , 2. (0)βi = b(t)xi , 3. (0)γij = γ(t)δij = a(t)2e2 (0)ζ(t)δij , where we are using the subscript (0) to remind the reader that we are at leading order in gradient expansion It is important to remark here that the leading order in gradient expansion of each quantity can be different, for example, the leading order for α and ζ is where we are using the subscript (0) to remind the reader that we are at leading order in gradient expansion It is important to remark here that the leading order in gradient expansion of each quantity can be different, for example, the leading order for α and ζ is O(σ0) h th l di d f βi i O(σ−1) H i tit h l di d where we are using the subscript (0) to remind the reader that we are at leading order in gradient expansion It is important to remark here that the leading order in gradient expansion of each quantity can be different, for example, the leading order for α and ζ is O(σ0) whereas the leading order for βi is O(σ−1). 5. Gradient Expansion Focusing only in the scalar part of Mij we can then write ˜γij −δij ≃−2  ∂i∂j −1 3δij∇2  C + O(σ2) , (87) (87) where C is a scalar function. This immediataly implies that  ∂i∂j −1 3δij∇2 C ∼O(σ). Note that, as we will see later on, this condition is not in contradiction with ∇2C ∼O(σ0). 5. Gradient Expansion Using the definition of extrinsic curvature of (12) together with the conditions for (0)α, (0)βi and (0)γij specified below (83), we can see that the extrinsic curvature only depends on time and hence this condition is also satisfied. Different functions for (0)α, (0)βi and (0)ζ will give different FLRW patches as long as they satisfy the conditions given below (83). We can then relate the different locally homogeneous and isotropic patches by knowing the different non-perturbative functions for (0)α, (0)βi and (0)ζ that lead to each one of them. Of course, in the same way as in perturbation theory, the value of (0)α, (0)βi and (0)ζ will depend on the gauge choice and on the solution for the ADM equations. We can generalize the idea of writing a FLRW metric in terms of xi and t to writ- ing quasi-FLRW metrics using the same coordinates. Following the ADM formalism we can write a metric valid at all orders in gradient expansion. With the identification γij = a(t)2e2ζ ˜γij we have: (84) ds2 = gµνdxµdxν = −α2dt2 + a(t)2e2ζ ˜γij(dxi + βidt)(dxj + βjdt) , Universe 2022, 8, 334 20 of 39 where the leading order in gradient expansion for each variable is: where the leading order in gradient expansion for each variable is: (0)α ∼O(σ0) , (0)ζ ∼O(σ0) , (0)βi ∼O(σ−1) , ˜γij −δij ∼O(σ) , (0)φ ∼O(σ0) . (85) The last term has been added to take into account the expansion of the scalar field, which is generically non-zero at the background level. It is important to realize here that the condition ˜γij −δij ∼O(σ) implies a further condition on the scalar part of ˜γij, in fact, using the expansion of the exponential of a matrix we can write The last term has been added to take into account the expansion of the scalar field, which is generically non-zero at the background level. It is important to realize here that the condition ˜γij −δij ∼O(σ) implies a further condition on the scalar part of ˜γij, in fact, using the expansion of the exponential of a matrix we can write ˜γij = e−2Mij ≃δij −2Mij + O(σ2) . (86) (86) Now, Mij must be traceless by definition (see see the paragraph under (23)). Focusing only in the scalar part of Mij we can then write Now, Mij must be traceless by definition (see see the paragraph under (23)). 5.1. O(σ0) in Gradient Expansion Having specified the leading order gradient expansion for each one of the non- perturbative variables, we can now expand the ADM equations in terms of σ. As an example we will expand the Hamiltonian constraint at O σ0 using the spatially flat gauge, i.e., (γf)ij = a2δij , (88) (88) where the subscript f stands for “flat”. Since in this gauge we also have R(3) f = 0 we can write the Hamiltonian constraint from (14) as: −(Af)ij(Af)ij + 2 3K2 f − 2 M2 PL (Tf)µν(nf)µ(nf)ν = 0 , (89) (89) where (nf)µ = gµν(nf)ν =  1 αf , −(βf)i αf  . i where (nf)µ = gµν(nf)ν =  1 αf , −(βf)i αf  .   Equation (89) can be written in terms of the metric variables αf and (βf)i of (84) and the scalar field φf. Using the results for (Af)ij and Kf given by (18) and (17) respectively we have: −1 4α2 f  δik∂j(βf)k + δjk∂i(βf)k −2 3δij∂k(βf)k  δik∂j(βf)k + δjk∂i(βf)k −2 3δij∂k(βf)k  +2 3 −3 Hb αf + 1 αf ∂k(βf)k !2 − 2 M2 PL " ˙φ2 f 2α2 f − ˙φf(βf)i∂iφf α2 f + (βf)i(βf)j∂iφf∂jφf 2α2 f + ∂iφf∂iφf 2a2 + V(φf) # = 0 , (90) (90) which is valid at all orders in gradient expansion. Keeping only the O(σ0) terms we get: 21 of 39 21 of 39 Universe 2022, 8, 334 2 3 −3 Hb (0)αf + 1 αf ∂k  (0)βf k !2 − 2 M2 PL    (0) ˙φf 2 2  (0)αf 2 − (0) ˙φf  (0)βf i (0)(∂iφf)  (0)αf 2 +  (0)βf i (0)βf j (0)(∂iφf) (0) ∂jφf  2  (0)αf 2 + V(φf)  = 0 , (91) (91) where by (0)(∂iφf) we mean σ ∂ ∂(σxi) φ(t, σxi) σxi=0 as explained before. where by (0)(∂iφf) we mean σ ∂ ∂(σxi) φ(t, σxi) σxi=0 as explained before. Note that the first line of (90) is O(σ) because of the condition of (0)βi below Equation (83). 5.1. O(σ0) in Gradient Expansion Following this procedure, it is straightforward to write all the ADM equations at order O(σ0); however, we know from linear perturbation theory that there are equations with global spatial derivatives that play a role in the k →0 limit, which translated to the gradient expansion way of thinking means that there are equations that contain an overall factor σ and hence in order to extract its contribution at O(σ0) they must be written up to O(σ). We know that the momentum constraint is one of these equations so let us analyze it. In spatially flat gauge, the momentum constraint (15) is: ∂j ˜Af  ij −2 3∂iKf = 1 M2 PL Ji (92) (92) If we expand (92) up to O(σ) we get: (0)∂i −3 Hb αf ! = 1 M2 PL   − 1 (0)αf (0) ˙φf (0)(∂iφf) +  (0)βf k (0)αf (0)(∂kφf) (0)(∂lφf)   . (93) If we expand (92) up to O(σ) we get: −2 3 (0)∂i −3 Hb αf ! = 1 M2 PL   − 1 (0)αf (0) ˙φf (0)(∂iφf) +  (0)βf k (0)αf (0)(∂kφf) (0)(∂lφf)   . (93) (93) An important aspect regarding the gradient expansion is worthy to remark at this point, note that, in the derivation of (93) we have used ∂j ˜Af  ij ∼O(σ), which seems in contradiction with ˜Af  ij ∼O(σ) (as we saw when deriving the Hamiltonian constraint at O(σ0)) and the fact that each spatial gradient introduces an order in σ. However, in this case we have an exception due to the traceless nature of ˜Af  ij. Let us see why: from the condition 2 below (83) we have (βf)i ≃b(t, σxl)xi and from (18) in spatially flat gauge: ∂j ˜Af  ij = δik∂j∂j(βf)k + ∂i∂k(βf)k −2 3∂i∂k(βf)k = 4 3∂i∂k(βf)k = 4∂ib(t, σxl) + O(σ2) , which is clearly O(σ). y ( ) The linear version of (93) obviously corresponds to the linear momentum constraint in spatially flat gauge, i.e., ∂i  HbAf  = ∂i ˙φb 2M2 PL δφf ! . (94) (94) As already stressed during Section 4.1.1, although (94) is an equation that appears at O(σ), it contains information at O(σ0), namely HbAf = ˙φb 2M2 PL δφf . 5.2. Non-Perturbative Separate Universe Approach 5.2. Non-Perturbative Separate Universe Approach The O(σ0) expression for the Hamiltonian constraint (91) seems very difficult to solve, and we have just argued the difficulties that arise when trying to extract the O(σ0) information from the O(σ) momentum constraint. This is why a further approximation in addition to the O(σ0) gradient expansion is usually performed in the literature [20,111,112]. This new approximation is the separate universe approach, which assumes that each FLRW patch of the universe evolve independently one from each other. We have already studied the linear version of the separate universe approach in Section 4.1.2, where we have seen that this assumption implies that neither non-local terms nor the momentum constraint will be present, which is equivalent to state that βi ∼O(σ3) as done in [109]. Within this approximation, the Hamiltonian constraint in spatially flat gauge of (91) takes a much simpler form: Hb (0)αsep f !2 = 1 3M2 PL    (0) ˙φsep f 2 2  (0)αsep f 2 + V  (0)φsep f   , (96) (96) which reminds us of the background Hamiltonian constraint (4). In fact, this coincidence extends also to the equation of motion of the scalar field as one could expect since we are assuming every FLRW to evolve independently from the others. A famous application of the separate universe approach is the δN formalism in order to compute the uniform- density curvature perturbation in a non perturbative way [70–73]. As a reminder, the linear uniform-density curvature perturbation is defined as Rud ≡−Hb ˙ρb δρf , Rud ≡−Hb ˙ρb δρf , 2 where ρ is the energy density of the scalar field, which in inflation is ρ = 3M2 PL  Hb2 and δρf is the perturbation of the energy density in spatially flat gauge. and In this section we have presented the non-perturbative generalization of the long wavelength linear perturbation theory presented in Section 4, being the O(σ0) in gradient expansion of Section 5.1 the generalization of the k →0 limit for scalar perturbations of Section 4.1.1 and being the separate universe approach the non-linear generalization of the k = 0 (or linear separate universe approach) case of Section 4.1.2. 5.1. O(σ0) in Gradient Expansion (95) (95) Unfortunately, things are not that easy when dealing with the fully non-perturbative momentum constraint at O(σ), in fact, if we look at (93), we will easily realize that the total spatial derivative is no longer present. How the O(σ0) information is encoded in (93) in a fully non-perturbative way is beyond the scope of this review, neverthe- Universe 2022, 8, 334 22 of 39 22 of 39 less, it is very important to be aware that we must take this information into account if we want to correctly describe the non-perturbative and long wavelength dynamics of the inhomogeneities, otherwise we would be making a mistake equivalent as when us- ing the linear separate universe approach of Section 4.1.2 instead of the k →0 limit of Section 4.1.1. Nevertheless, due to its usefulness, in the following section we will present the non-perturbative version of the linear separate universe approach. 6. Stochastic Approach to Inflation The stochastic approach to inflation combines the two approximations schemes pre- sented until now to study the evolution of inhomogeneities in a non-perturbative way. The idea is to split the variables of interest (let us say X) into two parts: an infrared (IR) part that contains all the inhomogeneities with characteristic wavelength larger that some coarse-grained scale (σH)−1 (σ is the same parameter as the one used in gradient expan- sion) and a ultraviolet (UV) part, which encompasses inhomogeneities with characteristic scale smaller than (σH)−1 (or characteristic wavenumber k bigger than σaH). Since the UV part starts evolving well inside the Hubble horizon, we will assume that it is perturbatively small. Thus, one can use linear perturbation theory to describe it, where initial conditions are well defined. The IR part instead can be large; however, since the IR part only contains long wavelengths, the gradient expansion can be used there. As we will see, whenever an UV mode exits the coarse-grained scale, it will act as a kick for the IR part, solving the initial condition problem of gradient expansion. Note that, although we will only study stochastic inflation in the context of Einstein’s gravity, the only requirements for the construction of a stochastic formalism are the possibility of a separation between IR and UV modes and a well-behaved perturbative expansion. There is then no reason a priori to think that the stochastic framework can not be applied to modifications of Einstein’s gravity such as massive gravity [113,114]. The stochastic formalism is then a mathematical framework that, in principle, allows us to study the inhomogeneities generated during inflation in a non-perturbative way, reason why it is widely used when studying PBHs formation (see for example [115,116]). To see how stochastic inflation works and why it is called “stochastic” we will derive the formalism step by step. From Section 5 it should be clear now that we will have different stochastic equations depending on if we are using the separate universe approach or the O(σ0) gradient expansion. The stochastic formalism that uses the separate universe approach is the most widely used in the literature (see for example [10,14–19,23,27,35,37,38, 51–53,63,66,115,116]) so we will start with its derivation. After that, we will take advantage of the stochastic equations just derived to present a stochastic formalism based on the O(σ0) gradient expansion [65]. 5.2. Non-Perturbative Separate Universe Approach For this reason, it is obvious that the O(σ0) gradient expansion and the separate universe approach will give different “decaying” terms, being the ones of the O(σ0) gradient expansion the correct ones, as it happened in linear theory. Using the results from linear theory again, we can conclude that the error made when using the separate universe approach instead of the O(σ0) gradient expansion will be generically of O(ϵ1), strongly depending on the inflationary regime. This conclusion is deduced of course assuming that higher orders in perturbation theory will not spoil the results at leading order, assumption that can be problematic if the inhomogeneity under study is non-perturbative. We will finish this section by reminding the reader that setting initial conditions for long wavelength perturbations is problematic because we cannot use the Bunch-Davies vacuum, which is only well defined when k aH →∞. This is why, although the gradient expansion is a very useful way to study inhomogeneities in a non-perturbative way, we need some other tool to set the initial conditions. Universe 2022, 8, 334 23 of 39 23 of 39 6. Stochastic Approach to Inflation Before starting with the derivation and, since it is not as trivial as the spatially flat gauge, let us present the gauge we will be working with: the uniform-N gauge. We define the number of e-folds N as the integrated expansion rate of Σt hypersurfaces, i.e., N ≡−1 3 Z Kdtl , (97) (97) where K ≡−3Hl (being Hl the local Hubble parameter) is the extrinsic curvature defined in (12), which coincides with the expansion rate of Σt hypersurfaces as shown in Section 3 and tl is the local time of (63). In terms of the variables and coordinates of the ADM metric (84) N can be written as:   N ≡ Z  Hb + ˙ζ −1 3 Diβi  dt . (98) (98) The uniform-N gauge is defined such that N coincides with the number of e-folds defined in the global background of (82), i.e., N ≡R Hbdt. From (98), this immediately implies ζδN = 0 and (βδN)i = 0 (where the subscript δN specifies the gauge), or DδN = 0 and BδN = 0 in its linear limit (see Equation (23)). The reason behind this gauge choice will be clarified along the following two subsections. 6.1. Stochastic Formalism Based on the Separate Universe Approach As we have already indicated, this is the stochastic formalism most widely used in the literature due to its simplicity; however, as we will see, it has some problems. First of all, it is very important to realize that in the separate universe approach, both the uniform-N gauge and the spatially flat gauge are equivalent, which leads to many authors to use the Universe 2022, 8, 334 24 of 39 24 of 39 uniform-N gauge for the IR part and the spatially flat gauge for the UV part [60]. Let us see why: uniform-N gauge for the IR part and the spatially flat gauge for the UV part [60]. Let us see why: y As we know, in the separate universe approach, due to the absence of non-local terms, both ( ˜γsep)ij = δij and ∂i(βsep)i = 0 are automatically satisfied (see (66)). This means that, under this assumption, we have to add the condition ∂i  βsep f i = 0 to the spatially flat gauge where ( ˜γf) = δij and ζf = 0. In the same way, we have to add the condition  ˜γsep δN  ij = δij to the uniform-N gauge, where ∂i(βδN)i = 0 and ζδN = 0. The main consequence of this is that, under the separate universe condition, we can express all the scalar fluctuations only in terms of the field inhomogeneities not only in the spatially flat gauge, but also in the uniform-N gauge. This can be clearly seen when looking at the linear “separate-universe” gauge invariant MS variable of (68). For non-linear generalizations of this variables see [117,118]. Before continuing, let us remind the reader that the equivalence between these two gauges in only valid under the separate universe approach, which, as seen in Section 4.1.2, it generically fails at O(ϵ1). During this section, and although it is not compulsory, we will work using the number of e-folds N ≡R Hbdt as time variable (Note that we can use any time variable we want because we will use the coordinates of a fictitious global background, i.e., the coordinates of (84). If we would instead use local coordinates (as in (63)), we would be interested in using an unperturbed time variable, being N the natural choice in the uniform-N gauge). 6.1. Stochastic Formalism Based on the Separate Universe Approach −3 Hb αIR ∂ ∂N Hb αIR ! −3 Hb αIR !2 + DkDkαIR − 1 M2 PL   Hb αIR !2∂φIR ∂N 2 −V  φIR   = −3  Hb2 (αIR)3 ∂αUV ∂N +    9  Hb2 (αIR)4 ∂αIR ∂N − 6Hb (αIR)3 ∂Hb ∂N !  αUV − 6  Hb2 (αIR)3 αUV −∇2 a2 αUV + 1 M2 PL  2 Hb αIR !2 ∂φIR ∂N ∂φUV ∂N −2  Hb2 (αIR)3 ∂φIR ∂N 2 αUV −Vφ  φIR φUV  . (101) (101) Note that this is the same as we did in linear perturbation theory of Section 4 but using the metric (63) (or equivalently (84)) as background metric. Now, since the IR variables are well outside the Hubble horizon, we will use the separate universe approach for them. Since αIR ∼O(σ0) and hence DkDkαIR ∼O(σ2) we have: −3 Hb (0)αIR ∂ ∂N Hb (0)αIR ! −3 Hb (0)αIR !2 − 1 M2 PL   Hb (0)αIR !2 ∂(0)φIR ∂N !2 −V  (0)φIR   = −3  Hb2  (0)αIR 3 ∂αUV ∂N +    9  Hb2  (0)αIR 4 ∂(0)αIR ∂N − 6Hb  (0)αIR 3 ∂Hb ∂N !  αUV − 6  Hb2  (0)αIR 3 αUV −∇2 a2 αUV + 1 M2 PL  2 Hb (0)αIR !2 ∂(0)φIR ∂N ∂φUV ∂N −2  Hb2  (0)αIR 3 ∂(0)φIR ∂N !2 αUV −Vφ  (0)φIR φUV  , (102) (102) where we have inserted an extra subindex (0) to indicate that we are at leading order in gradient expansion. Using Fourier analysis we can now define more rigorously the IR and UV modes. If we choose the Heaviside theta as a window function (The choice of the Heaviside theta as window function in the stochastic formalism is the most common one. However it can lead to some problems as indicated in [28]) we have the following decomposition for a generic function X. 6.1. Stochastic Formalism Based on the Separate Universe Approach Another important aspect is that, in order not to overload notation, we will suppress the subscript δN indicating the gauge we are using, such that in the following, unless otherwise stated, a variable without a subscript that indicates the gauge is a variable in the uniform-N gauge. As indicated before, the stochastic formalism presented in this subsection is based on the separate universe approach for the IR part and on linear perturbation theory for the UV part. To illustrate this we will consider in detail the equation of motion for the trace the extrinsic curvature (19) in uniform-N gauge, which can be written using the variables of the ADM metric (84) as: − Hb 2α !2 ∂˜γij ∂N ∂˜γij ∂N −3 Hb α !2 + DkDkα − 1 M2 PL   Hb α !2 ∂φ ∂N 2 −V(φ)  = 0 . (99) (99) The first thing to do is to split the variables of interest into their IR and UV part. In this case we only have two variables to split: The first thing to do is to split the variables of interest into their IR and UV part. In this case we only have two variables to split: α = αIR + αUV , φ = φIR + φUV . (100) (100) In (100) we are not considering ∂˜γij ∂N as a variable of interest not only because ˜γij = δij in the separate universe approach, but also because ∂˜γij ∂N ∂˜γij ∂N ∼O(σ2) in gradient expansion and quadratic in perturbation theory so it does not play any role even if we were using O(σ0) gradient expansion. Due to the perturbative nature of the UV variables, we will expand (99) keeping only linear terms in UV and isolate them in the right hand side of the equation getting 25 of 39 Universe 2022, 8, 334 −3 Hb αIR ∂ ∂N Hb αIR ! −3 Hb αIR !2 + DkDkαIR − 1 M2 PL   Hb αIR !2∂φIR ∂N 2 −V  φIR   = −3  Hb2 (αIR)3 ∂αUV ∂N +    9  Hb2 (αIR)4 ∂αIR ∂N − 6Hb (αIR)3 ∂Hb ∂N !  αUV − 6  Hb2 (αIR)3 αUV −∇2 a2 αUV + 1 M2 PL  2 Hb αIR !2 ∂φIR ∂N ∂φUV ∂N −2  Hb2 (αIR)3 ∂φIR ∂N 2 αUV −Vφ  φIR φUV  . 6.1. Stochastic Formalism Based on the Separate Universe Approach XIR(t, x) ≡ Z dk (2π)3/2 Θ(σal(N)Hl(N) −k)X IR k (t, x) , XUV(t, x) ≡ Z dk (2π)3/2 Θ(k −σal(N)Hl(N))X UV k (t, x) , (103) (103) where, similarly as in linear perturbation theory (see (47)),X UV k (t, x) is define as the follow- ing hermitian operator: X UV k (t, x) = e−ik·xXk(N)ak + eik·xX∗ ka† k , (104) (104) where Xk(N) is the solution of the evolution equation for the perturbation X over the local background defined by (83) and ak and a† k are the usual creation and annihilation operators which follow the commutation relation given in (48). Note that, in the spirit of gradient expansion, the splitting is done in the local cosmolog- ical coarse-grained scale (σHl)−1, which generically differs form the one of the background, Universe 2022, 8, 334 26 of 39 for example in uniform-N gauge we have Hl = Hb (0)αIR . Inserting the definition of XUV of (103) into (102) we get: for example in uniform-N gauge we have Hl = Hb (0)αIR . Inserting the definition of XUV of (103) into (102) we get: −3 Hb (0)αIR ∂ ∂N Hb (0)αIR ! −3 Hb (0)αIR !2 − 1 M2 PL   Hb (0)αIR !2 ∂(0)φIR ∂N !2 −V  (0)φIR   3  Hb2  (0)αIR 3 ∂ ∂N σa Hb (0)αIR ! Z dk (2π)3/2 δ k −σa Hb (0)αIR ! αUV k − 2 M2 PL Hb (0)αIR !2 ∂(0)φIR ∂N ∂ ∂N σa Hb (0)αIR ! Z dk (2π)3/2 δ k −σa Hb (0)αIR ! ϕUV k + Z dk (2π)3/2 Θ k −σa Hb (0)αIR !( −3  Hb2  (0)αIR 3 ∂αUV k ∂N +    9  Hb2  (0)αIR 4 ∂(0)αIR ∂N − 6Hb  (0)αIR 3 ∂Hb ∂N !  αUV k − 6  Hb2  (0)αIR 3 αUV k + k2 a2 αUV k 1 M2 PL  2 Hb (0)αIR !2 ∂(0)φIR ∂N ∂ϕUV k ∂N − 2  Hb2  (0)αIR 3 ∂(0)φIR ∂N !2 αUV k −Vφ  (0)φIR ϕUV k   ) , (105) (105) where αUV k and ϕUV k are operators defined as in (104). where αUV k and ϕUV k are operators defined as in (104). 6.1. Stochastic Formalism Based on the Separate Universe Approach If we now follow the procedure for the evolution equation of the trace of the extrinsic curvature just explained with the Hamiltonian constraint we get: 6 Hb (0)αIR !2 − 2 M2 PL   Hb (0)αIR !2 ∂(0)φIR ∂N !2 + V  (0)φIR  = 2 M2 PL Hb (0)αIR !2 ∂(0)φIR ∂N ξ1 , (108) 2 6 Hb (0)αIR !2 − 2 M2 PL   Hb (0)αIR !2 ∂(0)φIR ∂N !2 + V  (0)φIR  = 2 M2 PL Hb (0)αIR !2 ∂(0)φIR ∂N ξ1 , (108) which can be solved for  Hb (0)αIR 2 , i.e., Hb (0)αIR !2 = V  (0)φIR 3M2 PL −1 2  ∂(0)φIR ∂N 2 − ∂(0)φIR ∂N ξ1 . (109) (108) Hb (0)αIR !2 = V  (0)φIR 3M2 PL −1 2  ∂(0)φIR ∂N 2 − ∂(0)φIR ∂N ξ1 . (109) (109) Finally, the stochastic equation of motion for the field is obtained in the same way: ∂2 (0)φIR ∂N2 +    3 + ∂ ∂N  Hb (0)αIR  Hb (0)αIR     ∂(0)φIR ∂N + Vφ  (0)φIR  Hb (0)αIR 2 ∂2 (0)φIR ∂N2 +    3 + ∂ ∂N  Hb (0)αIR  Hb (0)αIR     ∂(0)φIR ∂N + Vφ  (0)φIR  Hb (0)αIR 2 = −∂ξ1 ∂N −ξ2 −    3 + ∂ ∂N  Hb (0)αIR  Hb (0)αIR    ξ1 + ∂(0)φIR ∂N ξ3 (0)αIR , (110) = −∂ξ1 ∂N −ξ2 −    3 + ∂ ∂N  Hb (0)αIR  Hb (0)αIR    ξ1 + ∂(0)φIR ∂N ξ3 (0)αIR , (110) (110) where ξ2 is defined similarly to ξ1 and ξ3: where ξ2 is defined similarly to ξ1 and ξ3: ξ2 ≡−∂ ∂N σa Hb (0)αIR ! Z dk (2π)3/2 δ k −σa Hb (0)αIR ! ∂ϕUV k ∂N . (111) (111) As anticipated before, the usage and uniform-N gauge and the separate universe approach ensures that all the scalar inhomogeneities are encoded in the scalar field. This becomes clearer once we realize that we can write the system (106)–(110) in terms only of the scalar field. 6.1. Stochastic Formalism Based on the Separate Universe Approach k k The right-hand side of (105) has two different terms: k k The right-hand side of (105) has two different terms: 1. The second integral (terms multiplying the Heaviside theta) is the evolution equation for the extrinsic curvature linearized over a local FLRW patch defined by (0)αIR and (0)φIR. Once the Bunch-Davies vacuum is chosen for that patch, this term will be automatically satisfied so it can be consistently set to zero. Note that the solution of this part equalized to zero is precisely what give us the functions Xk in (104). p q p y g 2. The first two integrals, proportional to a Dirac delta, can be seen as boundary con- ditions and hence they will act as the initial conditions missing when using only gradient expansion. 2. The first two integrals, proportional to a Dirac delta, can be seen as boundary con- ditions and hence they will act as the initial conditions missing when using only gradient expansion. We then get: We then get: −3 Hb (0)αIR ∂ ∂N Hb (0)αIR ! −3 Hb (0)αIR !2 − 1 M2 PL   Hb (0)αIR !2 ∂(0)φIR ∂N !2 −V  (0)φIR   = −3  Hb2  (0)αIR 3 ξ3 + 2 M2 PL Hb (0)αIR !2 ∂(0)φIR ∂N ξ1 , (106) 3 + 2 M2 PL Hb (0)αIR ! ∂(0)φIR ∂N ξ1 , (106) (106) where we have defined ξ1 and ξ3 as: where we have defined ξ1 and ξ3 as: where we have defined ξ1 and ξ3 as: ξ1 ≡−∂ ∂N σa Hb (0)αIR ! Z dk (2π)3/2 δ k −σa Hb (0)αIR ! ϕUV k , ξ3 ≡−∂ ∂N σa Hb (0)αIR ! Z dk (2π)3/2 δ k −σa Hb (0)αIR ! αUV k . (107) (107) Universe 2022, 8, 334 27 of 39 Universe 2022, 8, 334 27 of 39 For the moment we will not characterize the quantities ξ1 and ξ3. 6.1.1. Characterization of the Noises 6.1.1. Characterization of the Noises 6.1.1. Characterization of the Noises 6.1.1. Characterization of the Noises The interpretation of ξ1 and ξ2 (note that ξ3 no longer appears in the final equation of motion) as classical noises is not trivial because they are, strictly speaking, quantum operators. In order to see how they are effectively classical, we can compute the two-point correlation function of ξ1 for example at equal space point, the result is: ⟨0|ξ1(N1)ξ1(N2)|0⟩= ∂ ∂N σa Hb (0)αIR ! σa Hb (0)αIR !2 δφ(N1) k= σa Hb (0)αIR ! 2 δ(N1 −N2) , (114) (114) where we have used (104) together with the commutation relation (48). From (114) we see that δφk, which is the solution for the field perturbation over the local patch of size  σa Hb (0)αIR −1 , is evaluated at the coarse-grained scale, i.e., well outside the Hubble horizon. It can then be shown that at those scales, any perturbation that started from a coherent vacuum state has evolved into a highly squeezed state [119,120], which means that we where we have used (104) together with the commutation relation (48). From (114) we see that δφk, which is the solution for the field perturbation over the local patch of size  σa Hb (0)αIR −1 , is evaluated at the coarse-grained scale, i.e., well outside the Hubble horizon. It can then be shown that at those scales, any perturbation that started from a coherent vacuum state has evolved into a highly squeezed state [119,120], which means that we can consider δφ(N) k= σa Hb (0)αIR ! 2 as the power spectrum of a classical random variable, can consider δφ(N) k= σa Hb (0)αIR ! as the power spectrum of a classical random variable, can consider δφ(N) k= σa Hb (0)αIR ! ! whose time evolution is consistent with probabilities conserved along classical trajectories. whose time evolution is consistent with probabilities conserved along classical trajectories. Once this is clarified, we are now in position to describe ξ1 as a classical white noise (Its “white” nature is due to the presence of δ(N1 −N2) in the two-point correlator. Note that this is a consequence of the the choice of the Heaviside theta function as Window function, any other choice would lead to coloured noises, which are much more difficult to deal with, both analytically and numerically) with variance given in (114). 6.1. Stochastic Formalism Based on the Separate Universe Approach Inserting (106) and (109) into (110) and neglecting ξ2 i terms because they are quadratic in perturbation theory we get: ∂2 (0)φIR ∂N2 +  3 − 1 2M2 PL ∂(0)φIR ∂N !2 ∂(0)φIR ∂N +  3M2 PL −1 2 ∂(0)φIR ∂N !2  Vφ  (0)φIR V  (0)φIR  −∂ξ1 ∂N −ξ2 −  3 − 1 2M2 PL ∂(0)φIR ∂N !2 − 1 M2 PL ∂(0)φIR ∂N !2 − Vφ  (0)φIR V  (0)φIR  ∂(0)φIR ∂N ! ξ1 , (112) (112) which can be conveniently written if we use an auxiliary variable (0)πIR: (0)πIR = ∂(0)φIR ∂N + ξ1 , ∂(0)πIR ∂N +   3 −  (0)πIR2 2M2 PL   (0)πIR +   3M2 PL −  (0)πIR2 2    Vφ  (0)φIR V  (0)φIR  = −ξ2 . (113) (113) Universe 2022, 8, 334 28 of 39 6.1.1. Characterization of the Noises Furthermore, since the field fluctuations are Gaussian to a good level of approximation, the variance computed in (114) is enough to fully characterize ξ1. Finally, in order to characterize the system (113) we also need: ⟨0|ξ1(N1)ξ2(N2)|0⟩= ⟨0|ξ2(N1)ξ1(N2)|0⟩∗= ∂ ∂N σa Hb (0)αIR ! σa Hb (0)αIR !2     δφ(N1) k= σa Hb (0)αIR ! ∂δφ∗(N1) k= σa Hb (0)αIR ! ∂N     δ(N1 −N2) , ⟨0|ξ1(N1)ξ1(N2)|0⟩= ∂ ∂N σa Hb (0)αIR ! σa Hb (0)αIR !2 ∂δφ(N1) k= σa Hb (0)αIR ! ∂N 2 δ(N1 −N2) . (115) (115) The characterization of ξ1 and ξ2 as white noises give us now a intuitive picture of the physics behind the stochastic formalism. As explained before, different functions for (0)αIR and (0)φIR (in uniform-N gauge) describe the evolution of different FLRW patches, the way of getting these different functions is now clear if we see ξ1 and ξ2 as random variables. For example, the evolution of a specific patch, let us call it 1FLRW, will be given by 1 (0)αIR and 1 (0)φIR, whose specific form will be determined by the random values that the noises 1ξ1 and 1ξ2 will pick at each time step. Now, if we want to describe a second patch 2FLRW, we just have to solve again the stochastic equation with different random values for the noises 2ξ1 and 2ξ2, always satisfying the statistics described by (114) and (115). Like this, we are then able to describe the evolution of an ensemble of FLRW patches by solving many times the same stochastic equation with different random values for the noises. The correlators between these patches are then simply described by statistical moments of the IR variables. Universe 2022, 8, 334 29 of 39 29 of 39 The stochastic system is already fully characterized by (113)–(115); however, this system is very difficult to solve because, in order to compute the variance of the noises ξ1 and ξ2, we need to solve the perturbation equations and compute δφk over a stochastic background every time step, which makes the systen non-Markovian. Although there are numerical algorithms capable of doing so [67], it would be very convenient if we were able to write an analytical solution for δφk in terms of the IR variables, that would make the system Markovian and easily solvable. 6.1.1. Characterization of the Noises Let us try to solve the equation for δφk over the stochastic local background. We know that, as a consequence of the separate universe approach used here, the linearized equation for the field perturbation in the uniform-N gauge will be the same as the equation for the “separate-universe” gauge invariant variable Qsep defined in (68), in other words, Qsep = δφf = δφδN under the separate universe assumption. Now, since the linearized equation for Qsep only gives the correct solution for the true gauge-invariant MS variable Q defined in (44) if we set ϵ1 = 0 as checked in Section 4.1.2, the equation we will try to solve here is the linearized equation for the gauge invariant quantity Q over a local background in which all the SR parameters have been set to zero (note that, in order for this approximation to work we need −3 2ϵ2 −1 4ϵ2 2 ∼O(ϵ1) , which is true for SR and USR. This is because we are setting the combination of SR parameters multiplying Q in (45) to zero), in other words, we will try to solve the equation for Q as if the local background were an exact de-Sitter, which is: ∂2δφk ∂N2 + 3∂δφk ∂N +  (0)αIR2 k2 a2Hb2 δφk = 0 , (116) (116) or, using the Hamiltonian constraint of (109): ∂2δφk ∂N2 + 3∂δφk ∂N + k2 a2   3M2 PL −( (0)πIR) 2 2 V  (0)φIR   δφk = 0 . (117) (117) Now, both the functions (0)πIR and V  (0)φIR are stochastic so, in order to know them we must already know the value for the variable ξ1. This makes Equation (117) not analitically solvable, more concretely, the stochastic system of (113) is non-Markovian, meaning that the value of the noises at each time N will depend on the whole evolution of the stochastic patch up to N. The only option remaining to solve the stochastic system of (113) seems then to be numerically; however, a further and very important approximation is usually done, which consists on assuming that the IR (and stochastic) quantities do not differ much from their global background (and deterministic) counterpart, namely YIRXUV ≃YbXUV + O XUV2 . Here XUV and YIR are any UV and IR functions, we then define Yb as the equivalent background function of YIR. 6.1.1. Characterization of the Noises Under this approximation, the last term of (116) is: k2 (0)αIR aHb !2 δφk ≃  k aHb 2 δφk + O  δφ2 k  , (118) (118) where we have substituted (0)αIR by its background value, i.e., 1. Under this approximation we can write a analytical solution for (116), that, once evaluated at coarse-grained scale, will correspond to the long-wavelength limit of solution (51) with ν = 3 2, i.e., δφ(N)k=(σaHb) 2 =  Hb2 2 σaHb3 . (119) (119) Universe 2022, 8, 334 30 of 39 and therefore the correlators (114) and (115) can be written as follows: and therefore the correlators (114) and (115) can be written as follows: ⟨ξ1(N1)ξ1(N2)⟩= Hb 2π !2 δ(N1 −N2) , (120) ⟨ξ1(N1)ξ2(N2)⟩= ⟨ξ2(N1)ξ2(N2)⟩= 0 , (121) (120) (121) Under this approximation, the stochastic system of (113) simplifies considerably and becomes a Markovian process with additive noises, meaning that ξ1 depends only on time, which is true as long as we are using the global background to characterize it. The system is: (0)πIR = ∂(0)φIR ∂N + Hb 2π ξ(N) , ∂(0)πIR ∂N + 3 (0)πIR + M2 PL Vφ  (0)φIR V  (0)φIR  = 0 , (122) (122) where ⟨ξ(N1)ξ(N2)⟩= δ(N1 −N2) and we have dropped O(ϵ1) terms in order to be consistent with the computation of the noises and the reasoning above (116). Note that in SR the acceleration of the field is also of higher order in SR parameters so in this case the stochastic equation would simplify even more: ∂(0)φIR ∂N + M2 PL Vφ  (0)φIR V  (0)φIR  = −Hb 2π ξ(N) . (123) (123) Although the approximation used in order to arrive to (122) seems very useful, it has important consequences, in fact, it is equivalent to state that any YIR −Yb ∼O XUV . Thus, we immediately see that if this approximation holds, the system in (122) can only reproduce the results of linear theory at leading order in SR parameters, and hence it does not give any non-perturbative (or even non-linear) information. In fact, (122) is slightly inconsistent. 6.1.1. Characterization of the Noises The point is that, by the same approximation adopted on the right hand side, the left hand side should also be linearized (in fact the linearization of the left-hand side of (122) is sometimes performed when recursive methods are used in order to solve the stochastic system as it can be seen in [42–44]). This inconsistency can however give some information when comparing the results from the stochastic formalism with the ones of linear perturbation theory, in fact, since we know that the correlations functions calculated with the stochastic system of (122) will coincide, up to second order in perturbation theory, to the ones calculated in linear perturbation theory with QFT methods, any inconsistency between the two approaches will signal the break-down of perturbation theory. pp g p y Since the stochastic Equation (116) and its deterministic counterpart with (0)αIR = 1 have the same structure, one could be tempted to write the solution of (116) in a similar way as the solution (119) but substituting Hb by Hb (0αIR , i.e: δφ(N) k= σa Hb (0αIR ! 2 = Hb (0αIR !2 1 2  σa Hb (0αIR 3 , (124) (124) Universe 2022, 8, 334 31 of 39 and hence the stochastic system of (113) would approximately be: and hence the stochastic system of (113) would approximately be: (0)πIR = ∂(0)φIR ∂N + Hb 2 (0)αIRπ ξ(N) , ∂(0)πIR ∂N + 3 (0)πIR + 3M2 PL Vφ  (0)φIR V  (0)φIR  = 0 . (125) (125) The system of (125) represents a Markovian process with non-additive noises (the term proportional to ξ does no longer depends only on time), which is, provided that (124) holds, able to describe the inhomogeneities generated during inflation in a non-perturbative way. g g g p y Unfortunately, we cannot trivially generalize the solution (119) for a deterministic equation to a solution (124) for a stochastic equation, this is due to the differences between stochastic and deterministic integrals (see for example [121]). This is why, although it is the most common approach found in the literature, we will not trust (125) to describe non-linear inflationary effects in this review. y Having discarded this option, we are left with three different ways of solving the stochastic system of (113): 1. 6.1.1. Characterization of the Noises We can use the system (113) where the noises are computed numerically over the stochastic local background, for example, using the algorithm described in [67]. • Pros: It describe non-linear inflationary dynamics. • Cons: It is very difficult to solve due to the non-Markovianity of the process. Furthermore, it is only valid up to leading order in SR parameters due to the use of the separate universe approach. 2. We can use the system of (122) • Pros: It is a Markovian process with additive noises for which even analytical solutions can be obtained (see Section VI of [65]). • Cons: It only describes linear perturbations whenever they are approximately described by solution (119), which is not the case during some interesting regimes for PBH formation, such as a SR-USR transition where ν ̸= 3 2. It is then even less precise than the linear separate universe approach. 3. Finally, we can solve the MS equation over a global background at all orders in SR parameters, i.e., we can solve (45) and characterize the noises as in (114) with this solution. This can be done because, as we have already indicated, under the separate universe assumption Q = δφδN. Once the noises are characterized in this way, we can use the stochastic system (113) and solve the dynamics. • Pros: It is a Markovian process with additive noises able to describe the linear dynamics of the inflationary perturbations even when they are not approximately described by (119). y ( ) • Cons: It is not capable of describing any non-linear effects and it is inconsistent generically at leading order in ϵ1 due to the use of the separate universe approach. This inconsistency can be clearly seen in the term proportional to ξ1 in (112), which in the background can be written as  3 −ϵ1 + ϵ1ϵ2 3−ϵ1  and not (3 −ϵ1) as it should be if it came from the MS equation. In fact  3 −ϵ1 + ϵ1ϵ2 3−ϵ1  is precisely the term that appears in the equation for Qsep (69) derived in Section 4.1.2, making it clear that this inconsistency is a consequence of the separate universe approach. 6.1.1. Characterization of the Noises Due to the problems that the stochastic formalism presented in this section has (namely its validity only up to leading order in ϵ1 due to the separate universe assumption and its difficulty to describe non-linear dynamics), it does not seem very promising if we want to describe the non-perturbative dynamics of the inohomogeneities generated during inflationary regimes of interest for PBH formation with enough precision. However, in the Universe 2022, 8, 334 32 of 39 32 of 39 next subsection we will present a stochastic formalism (firstly presented in [65]) which is at least able to reproduce linear perturbation theory at all orders in SR parameters, and with the potential to correctly describe the non-perturbative dynamics of the inhomogeneities during any inflationary regime. However, before doing so, and in order to finish this subsection, it is important to know that the stochastic system of (113) can be straightforwardly derived by splitting into a IR and an UV only the scalar field in the separate universe equations of (64). In this case we would have used the metric (63) for the local patch instead of the metric (83) as we have done here. We have not chosen this option in this review because of three main reasons: 1. It does not make it clear that we have used gradient expansion, and hence the problem of not using the momentum constraint that we solve in the next subsection is difficult to remark. 2. Since it only works if the time variable is unperturbed, it could lead us to think that the number of e-folds N is the only allowed time variable for a stochastic formalism that describes all the scalar inhomogeneities in terms of the inflaton field. On the contrary, the derivation used in this review is valid for any time variable and makes it clear that the description of inhomogeneities in terms solely of the inflaton field is only a gauge choice. y g g 3. It does not explicitly obtains the linear equations used for the characterization of δφk, more concretely, for example this derivation would miss the k2 a2 αUV k term that multiplies the Heaviside theta in (105). 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion Splitting (127) into IR and UV and using the decomposition of ˜γij explained around (87) to keep only O(σ) terms in the IR part (remember that the O(σ0) information from the momentum constraint Universe 2022, 8, 334 33 of 39 can only be extracted if we write the momentum constraint up to O(σ)), we can write the stochastic equation for the momentum constraint: can only be extracted if we write the momentum constraint up to O(σ)), we can write the stochastic equation for the momentum constraint: (0)∂i  ∂ ∂N 1 3∇2CIR  −(0)∂iαIR (0)αIR + ∂(0)φIR ∂N (0)∂iφ 2M2 PL = −∂iξ4 , (128) (128) where ξ4 is defined similarly to ξ1, ξ2 and ξ3, i.e., ξ2 ≡−∂ ∂N σa Hb (0)αIR ! Z dk (2π)3/2 δ k −σa Hb (0)αIR ! −k2 3 CUV k  . (129) (129) With the addition of the stochastic Equation (128) to the system of (113) obtained before, we have a stochastic formalism able to describe the non-linear evolution of scalar inhomogeneities at all orders in SR parameters. With the addition of the stochastic Equation (128) to the system of (113) obtained before, we have a stochastic formalism able to describe the non-linear evolution of scalar inhomogeneities at all orders in SR parameters. However, it is not all good news: firstly, since the construction of the gradient expan- sion in Section 5, we have been neglecting possible interactions scalar-tensor or scalar-vector, reason why the stochastic formalism constructed here will not take these interactions into account either. Secondly, we do not exactly know how to extract the O(σ0) information form (128) in a fully non linear way. Finally, we do not know which is the combination of (0)φIR and ∇2CIR that give us the correct and non-perturbative and gauge invariant quan- tity that describe scalar inhomogeneities, i.e., we do not have a non-linear generalization of the MS variable. The first issue is beyond the scope of this review. With respect to the third one, it is true that a non-linear gauge invariant variable at leading order in gradient expansion has been defined in [117,118] as: ∂iQNL ≡∂iφ + 1 α ∂φ ∂N ∂iζ However, the variable above does not include the term proportional to ∇2E in its linearization. Reason why it can be only interpreted as a non-linear generalization of Qsep. 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion As many times claimed during the review, the separate universe approach generically fails to give the correct long-wavelength evolution of the inhomogeneities at O(ϵ1). The O(σ0) gradient expanision solves this problem by including both non-local terms and the momentum constraint, this is why in this section we will construct a stochastic formalism based on O(σ0) gradient expansion. First of all, it is important to remark that some of the affirmations we did about the uniform-N gauge at the beginning of Section 6.1 are no longer correct, more concretely, we cannot longer study the scalar inhomogeneities in terms solely of the inflaton field. This is clear form linear perturbation theory where the MS variable can be written as Q = δφf = δφδN −∂φb ∂N 1 3∇2EδN , (126) (126) so if we insist on using the uniform-N gauge we must also take into account the contribution from E when studying scalar perturbations. We could also use spatially flat gauge in this case and forget about E; however, in this case we should take into account all the terms proportional to  (0)βf i that appear for example in (90), this is why we will keep using the uniform-N gauge, where βi = 0. One can easily check that the stochastic equations for the evolution of the extrinsic curvature (106), for the Hamiltonian constraint (109) and for the evolution of the field (110) do not change when including E and hence the stochastic system is still the one given by (113). The only difference will then be given by the inclusion of the momentum constraint (15), which, in uniform-N gauge can be written as: Dj −Hb 2α ∂˜γij ∂N ! −2 3 DiK = − 1 M2 PLα ∂φ ∂N ∂iφ , (127) (127) where we have used the evolution Equation for ˜γij (18) to eliminate ˜Aij. 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion This is the reason we will not use the non-linear variable defined above and we will solve the second and third issues, at least approximately, by imposing that both the momentum constraint and the non-linear generalization of the MS variable match their linear counterpart when the global background is subtracted. In this way, the O(σ0) information of (128) can be straightforwardly extracted and the whole system of stochastic equations based on the O(σ0) gradient expansion and hence valid at all orders in SR parameters is: (0)πIR = ∂(0)φIR ∂N + ξ1 , ∂(0)πIR ∂N +   3 −  (0)πIR2 2M2 PL   (0)πIR +   3M2 PL −  (0)πIR2 2    Vφ  (0)φIR V  (0)φIR  = −ξ2 , ∂ ∂N 1 3 (0)∇2CIR  −   Hb v u u u t3M2 PL −( (0)πIR) 2 2 V  (0)φIR  −1   + 1 22 PL ∂(0)φIR ∂N  (0)φIR −φb = −ξ4 , (130) (0)πIR = ∂(0)φIR ∂N + ξ1 , ∂(0)πIR ∂N +   3 −  (0)πIR2 2M2 PL   (0)πIR +   3M2 PL −  (0)πIR2 2    Vφ  (0)φIR V  (0)φIR  = −ξ2 , ∂ ∂N 1 3 (0)∇2CIR  −   Hb v u u u t3M2 PL −( (0)πIR) 2 2 V  (0)φIR  −1   + 1 22 PL ∂(0)φIR ∂N  (0)φIR −φb = −ξ4 , (130) (130) where we have used the Hamiltonian constraint to eliminate (0)αIR in the last equation. Note that ξ1,ξ2 and ξ4 are constructed in the uniform-N gauge, which is no longer equiva- lent to the spatially flat gauge. To see the gauge transformation between spatially flat and uniform-N gauges in linear theory one can see [60], where it is claimed that the differences between those two gauges is always of higher order in gradient expansion; however, this conclusion is reached by Universe 2022, 8, 334 34 of 39 considering the value of ϵ1 at horizon crossing (ϵ∗ 1 there) to be constant with k, which is generically not a good approximation beyond SR. 6.2. Stochastic Formalism Based on O(σ0) Gradient Expansion In fact in [67] it is shown numerically that the difference between δφf and δφδN can be O(ϵ1) in regimes of interest for PBH formation, in agreement with the differences between the separate universe approach and O(σ0) gradient expansion remarked in this paper. Finally, as suggested in [122–124], we can define the non-linear counterpart of the MS variable of (44) at leading order in gradient expansion as: QIR = (0)φIR −φb − ∂(0)φIR ∂N 1 3 (0)∇2CIR , (131) (131) where we remind the reader that (0)φIR and (0)∇2CIR are both in the uniform N gauge. 0 where we remind the reader that (0)φIR and (0)∇2CIR are both i The stochastic formalism based on the O(σ0) gradient expansion presented in this section was introduced for the first time, although in a different gauge, in [65], where the authors show numerically that, when computing the noises over a global background, i.e., when using the approximation described around (118), the two point correlator com- puted using the stochastic formalism perfectly matches with the one computed in linear perturbation theory at all orders in SR parameters, as expected. This already represents an important improvement with respect to any stochastic formalism based on the separate universe approach as the one explained in Section 6.1. 6.3. Stochastic Formalism Versus Linear Perturbation Theory Once the stochastic formalism has been properly introduced, the simplest way to know if there exists any important non-perturbative effect during inflation is to compare the results obtained within the stochastic framework with the results coming from linear perturbation theory explained in Section 4. In order to do so, it is crucial to realize that the results coming from the stochastic formalism are in real space whereas the result for the linear MS variable in terms of Hankel function of (51) is in Fourier space. Due to the difficulty when trying to express stochastic correlators in Fourier space, we will compare the stochastic two-point correlator with the linear two-point correlator, both in real space. As explained below (115), the correlators between different FLRW patches are de- scribed by statistical moments of IR variables, so the two point correlator of the scalar inhomogeneities is: ⟨(0)φIR (0)φIR⟩= Var  (0)φIR , (132) (132) if we are using the separate universe approach or if we are using the separate universe approach or ⟨QIRQIR⟩= Var  QIR , (133) (133) if we are using O(σ0) in gradient expansion. if we are using O(σ0) in gradient expansion. if we are using O(σ0) in gradient expansion. O th th h d th t i t l t i li t b ti th i d fi d On the other hand, the two-point correlator in linear perturbation theory is defined as (see (52)): On the other hand, the two-point correlator in linear perturbation theory is defined as (see (52)): ⟨Q(N)Q(N)⟩= Z σa(N)Hb(N) σa(N=0)Hb(N=0) dk k PQ(k, N) , (134) (134) where we are now using the power spectrum evaluated at the same spatial point PQ(k, N) ≡ k3 2π2 |Qk(N)|2 , (135) (135) where Qk in uniform-N gauge is given by (126). The limits of (134) correspond to the selection of modes inside the coarse-grained scale defined by k = σa(N)Hb(N) and they are necessary to correctly compare the two point correlators obtained in both formalisms, in fact, in the stochastic formalism the IR part of the field recieves stochastic kicks from N = 0 onwards. Thus the first k-mode from which the IR field recieves a kick is the one with k = σa(N = 0)Hb(N = 0). 6.3. Stochastic Formalism Versus Linear Perturbation Theory Universe 2022, 8, 334 35 of 39 Whenever PQ(k, N) does not depend on N, one can do a very useful approximation, which consist on evaluating the power spectrum at coarse-grained scale crossing, i.e., at k = σaHb, and assume that this value does not change with time. This would allow us to write (134) as: N ⟨Q(N)Q(N)⟩= Z N 0 P  k = σa(N′)Hb(N′)  dN′ , (136) (136) In this case one could write the power spectrum as the derivative with respect to the number of e-folds N of the correlator in real space. PQ(k) = d dN ⟨Q(N)Q(N)⟩. (137) (137) However, and although it has been sometimes wrongly used for a time-dependent power spectrum [33], this technique cannot be used if the power spectrum evolves with time, which makes it only valid at zeroth order in O(ϵ1). Finally, and due to the difficulty in the definition of non-linear gauge invariant vari- ables (remember for example that the “trivial” non-linear generalization of the MS vari- able of (131) is not guaranteed to be the correct one), some authors have used the so- called stochastic-δN formalism to compute the non-linear curvature perturbation, which is very useful when dealing with PBHs, by studying the statistics in the number of e-folds [36,41,46,50,51]. However, and similarly to what happens with the usual δN for- malism, the stochastic-δN formalism uses the separate universe approach and hence it is generically only valid at leading order in SR parameters. 7. Conclusions The stochastic approach to inflation seems the most promising way to study, in a non-perturbative way, the probability distribution of the scalar inhomogeneities generated during inflation and responsible for the creation of PBHs. However, as remarked during the review, there is still a lot of work to do in that direction. Indeed, although very useful, the most common and widely used stochastic formalism has some difficult problems to solve, which can be summed up in three points: 1. As we have seen during the review, the stochastic formalism uses a gradient expansion for the IR part and a perturbative expansion for the UV part in such a way that the IR part, due to the large wavelength of the characteristic inhomogeneities that form this sector, can be described as a local FLRW universe. This description, that we called O(σ0) gradient expansion, relates the different local FLRW patches via non-local terms and the momentum constraint, and describes at all orders in SR parameters the correct dynamics of long wavelength scalar inhomogeneities. However, it presents some problems such as the extraction of the O(σ0) information from the momentum constraint. This is the reason why it is usually assumed that the different local FLRW universes evolve independently from each other, which is an assumption known as the separate universe approach. Under this approximation, the problem with the momentum constraint disappears (the momentum constraint itself disappears); nevertheless, we checked in Section 4.1.2 that this assumption fails to describe the correct long- wavelength dynamics of scalar perturbations generically at O(ϵ1) already in its lin- ear limit. For this reason, the stochastic formalism commonly used presented in Section 6.1, which is based on the separate universe approach will fail to describe the non-perturbative dynamics of the scalar inhomogeneities at O(ϵ1). p y g ( ) A stochastic formalism that does not uses the separate universe approach can also be constructed, as we did in Section 6.2. However, this option is not without its difficulties, more concretely, it has problems when extracting the long wavelength information from the momentum constraint and when describing the scalar inhomo- geneities with a gauge invariant generalization of the MS variable. Universe 2022, 8, 334 36 of 39 2. On the other hand, the UV part of the inhomogeneities is assumed to behave pertur- batively, having as a background the local FLRW background defined by the IR part. 7. Conclusions Since the UV part acts as a stochastic noise for the IR part and the IR part is necessary to characterize the UV noises, the stochastic approach to inflation is generically a non-Markovian process, meaning that the value of the noises depend on the whole history of the local patch over which they are computed. Thi d t t h bl h l i th t h ti ti 2. On the other hand, the UV part of the inhomogeneities is assumed to behave pertur- batively, having as a background the local FLRW background defined by the IR part. Since the UV part acts as a stochastic noise for the IR part and the IR part is necessary to characterize the UV noises, the stochastic approach to inflation is generically a non-Markovian process, meaning that the value of the noises depend on the whole history of the local patch over which they are computed. This does not represent a huge problem when solving the stochastic equations nu- merically; however, in order to have analytical results we need to do some other approximation that makes the process Markovian. This approximation consists of computing the stochastic noises over the global background instead of over the local one Unfortunately doing so is equivalent to assume that all the IR inhomogeneities history of the local patch over which they are computed. This does not represent a huge problem when solving the stochastic equations nu- merically; however, in order to have analytical results we need to do some other approximation that makes the process Markovian. This approximation consists of computing the stochastic noises over the global background instead of over the local one. Unfortunately, doing so is equivalent to assume that all the IR inhomogeneities are linear in perturbation theory. This does not represent a huge problem when solving the stochastic equations nu- merically; however, in order to have analytical results we need to do some other approximation that makes the process Markovian. This approximation consists of computing the stochastic noises over the global background instead of over the local one. Unfortunately, doing so is equivalent to assume that all the IR inhomogeneities are linear in perturbation theory. p y 3. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 7. Conclusions Finally, and although we have not paid too much attention to this problem, any stochastic approach that aims to describe the non-perturbative behaviour of scalar in- homogeneities at the long wavelength limit should also take into account scalar-vector and scalar-tensor interactions, which no longer decouple beyond linear perturba- tion theory. 3. References 1. Starobinsky, A.A. 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English
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Action prediction modulates both neurophysiological and psychophysical indices of sensory attenuation
Frontiers in human neuroscience
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ORIGINAL RESEARCH ARTICLE published: 28 February 2014 doi: 10.3389/fnhum.2014.00115 published: 28 February 2014 doi: 10.3389/fnhum.2014.00115 Frontiers in Human Neuroscience INTRODUCTION are perceived as less intense than externally generated stimuli, a phenomenon known as sensory attenuation. Sensory attenuation has been demonstrated in the somatosensory (Blakemore et al., 1998), the auditory (Sato, 2008) and the visual domain (Cardoso- Leite et al., 2010; Roussel et al., 2013). Cardoso-Leite et al. (2010), for example, studied the influence of the congruency between anticipated and actual action effect (tilted Gabor stimuli) on the detection of the latter. Detection performance in the congruent condition (i.e., when the effect anticipated by the action and the true effect matched) was worse than in the neutral and incongru- ent conditions, demonstrating sensory attenuation. Studies inves- tigating sensory attenuation as a perceptual phenomenon have been complemented by studies investigating neurophysiological correlates of anticipated action effects (e.g., Schafer and Marcus, 1973; Blakemore et al., 1998; Bäss et al., 2008; Aliu et al., 2009; Gentsch and Schütz-Bosbach, 2011; Hughes and Waszak, 2011; Hughes et al., 2013b). To give an example, Bäss et al. (2008) found a reduced auditory N1 component for action-triggered tones compared to externally triggered tones, suggesting that cortical activity was attenuated for the former. The ability to produce intended effects in the environment is an important aspect of action control. The ideomotor the- ory of action claims that bidirectional associations are formed between an action’s motor code and the ensuing sensory effects. These associations can be used to select an action by anticipat- ing or internally activating their perceptual consequences (e.g., Greenwald, 1970; Prinz, 1997; Elsner and Hommel, 2001; Herwig et al., 2007; Waszak et al., 2012). A similar approach has also been employed in the development of forward models of action, which claim that an efference copy, generated during action selection, is used to predict future behavioral state of the system as well as the sensory consequences of that behavior (Wolpert et al., 1995; Wolpert and Miall, 1996). Many computational models also include inverse models that provide the motor command which, given the particular current state, would result in a desired end state, for example, a particular sensory effect (Wolpert et al., 1995). These principles of action prediction have been investigated using a number of different methodologies. One line of evidence comes from paradigms studying processing of anticipated action effects. Cedric Roussel 1,2*, Gethin Hughes 3 and Florian Waszak1,2 1 Laboratoire de Psychologie de la Perception, Université Paris Descartes, Sorbonne Paris Cité, Paris, France 2 Laboratoire Psychologie de la Perception, Centre National de la Recherche Scientifique-Université Paris Descarte 3 Department of Psychology, University of Essex, Colchester, UK Sensory attenuation refers to the observation that stimuli that are predicted based on one’s action are attenuated. This phenomenon has primarily been observed as a neurophysiological phenomenon, with reduced Event-Related Potential (ERP) (e.g., Bäss et al., 2008) and BOLD (e.g., Blakemore et al., 1998). However, psychophysical investigations (e.g., Sato, 2008; Cardoso-Leite et al., 2010; Roussel et al., 2013) have confirmed that action prediction also influences the perception of sensory action effects. The present study recorded both neurophysiological and psychophysical measures in a Sensory attenuation refers to the observation that stimuli that are predicted based on one’s action are attenuated. This phenomenon has primarily been observed as a neurophysiological phenomenon, with reduced Event-Related Potential (ERP) (e.g., Bäss et al., 2008) and BOLD (e.g., Blakemore et al., 1998). However, psychophysical investigations (e.g., Sato, 2008; Cardoso-Leite et al., 2010; Roussel et al., 2013) have confirmed that action prediction also influences the perception of sensory action effects. The present study recorded both neurophysiological and psychophysical measures in a single experiment, to confirm whether the two phenomena are related. In addition, by measuring the ERP modulations of both stimulus contrast and prediction congruency, we sought to directly relate the neurophysiological phenomenon to the magnitude of sensory processing in the brain. Participants performed left- and right-hand voluntary actions that were previously associated with the letters A and H. In the test phase, participants were presented with these same two letters, at one of two possible contrasts. Participants were required to report which of the two possible contrasts had been presented. We observed both reduced contrast discrimination (in line with Roussel et al., 2013) and a reduced ERP response for congruent action-effects. Furthermore, our congruency modulation was observed on the same component that differed as a function of stimulus contrast. Taken together these results strongly suggest that neurophysiological indices of sensory attenuation reflect reduced sensory processing of voluntary action effects. *Correspondence: *Correspondence: Cedric Roussel, Laboratoire Psychologie de la Perception, Centre National de la Recherche Scientifique-Université Paris Descartes, UMR 8158, Centre Biomédical des Saints-Pères, 45 rue des Saints-Pères, 75006 Paris, France e-mail: cedric_roussel@live.fr Keywords: action prediction, effect prediction, psychophysycs, neurophysiology, contrast discrimination Reviewed by: Reviewed by: Amanda Therrien, Johns Hopkins University School of Medicine, USA Mikkel C. Vinding, Aarhus University, Denmark Edited by: Harriet Brown, University College London, UK HUMAN NEUROSCIENCE HUMAN NEUROSCIENCE Keywords: action prediction, effect prediction, psychophysycs, neurophysiology, contrast discrimination HUMAN NEUROSCIENCE HUMAN NEUROSCIENCE ORIGINAL RESEARCH ARTICLE bli h d F b INTRODUCTION Notably, it has been shown that self-generated stimuli Both attenuated phenomenological and neurophysiological responses are usually interpreted along the same lines in terms February 2014 | Volume 8 | Article 115 | 1 www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. of forward models (e.g., Miall and Wolpert, 1996), as if they reflect the same mechanism. At the same time, this mechanism has usually been considered to be relatively low-level (“sen- sory”). However, a systematic investigation of the relationship between sensory attenuation as a perceptual phenomenon, on the one side, and as a neurophysiological phenomenon, on the other side, is missing. As a consequence, it is impossible to tell how the attenuation of perceptual awareness is related to the attenuation of cortical responses that have been observed a number of times in separate experiments. Moreover, con- cerning the locus of the effect, differences in cortical responses between conditions with and without effect anticipation cannot always be unequivocally attributed to sensory processing. Often they may also be caused by other differences in attentional and cognitive processing (cf., Waszak et al., 2012; Hughes et al., 2013a). As a consequence, different studies do not always con- verge to the same conclusions. For example, Bäss et al. (2008) observed attenuated fronto-central negativity when comparing action-triggered vs. externally triggered auditory stimuli. They concluded that early sensory processing in the auditory cortex is reduced. By contrast, Hughes and Waszak (2011) compared Event-Related Potential (ERPs) to action-triggered vs. exter- nally triggered visual stimuli. They observed an increased, not a decreased, visual P1 component. In this study, attenuated cortical responses were observed in a frontoparietal network, starting 150 ms after stimulus. This result would be in line with the findings of Del Cul et al. (2007) showing that sub- jective thresholds of visual stimuli is reflected in later process- ing in a fronto-parietal network, rather than in early visual areas. components and reduced awareness of the action effects interrelate. MATERIALS AND METHODS STIMULI The stimuli were two white letters (A and H) presented within a virtual square of 3.3◦of visual angle and displayed on a 24 inch LED monitor at a 60 Hz refresh. These two stimuli were presented at two different contrast values (C0 and C1, determined for each subject; see Section Contrast Determination Phase) at the center of the screen. In the test phase we used a uniform noise texture to increase perceptual variance. This noise texture was re-sampled on each screen refresh with always the same number of white and black pixels. The mean luminance of the noise was equal to the gray background. CONTRAST DETERMINATION PHASE In order to determine individual contrast values C0 and C1 yield- ing a discrimination d′ of about 1.5, every participant completed a psychophysical staircase converging on 90% correct responses in a letter identification task (A vs. H). This correct response rate was used to ensure that the stimuli were supraliminal and that we could then independently manipulate discrimination. We used the resulting contrast value as the referential contrast C0 in a Two Alternative Forced Choice (2AFC) paradigm (with constant stimuli ranging from C0 to C0 + 12%) in order to calculate the contrast value of C1 yielding 80% correct responses in a luminance discrimination task (C0 vs. C1). For an ideal observer, this contrast yields a discrimination d′ of around 1.5 (Macmillan and Creelman, 1991). This phase lasted on average 5 min. The aim of the current experiment was to shed new light on two interrelated questions. First, we investigated whether neurophysiological indices of sensory attenuation reflect early, low-level or later, higher-level mechanisms. Second, we explored how neurophysiological and perceptual indices of sensory atten- uation relate. To do so, using Electro Encephalography (EEG), we adapted a luminance discrimination protocol that has been used before successfully to assess perceptual sensitivity and response bias of anticipated and unanticipated visual action effects (Roussel et al., 2013). Roussel et al. made participants learn an association between left and right key presses and the presentation of the letters A and H, respectively. They then made participants perform left and right key presses that ran- domly triggered presentation of either an H or and A at one of two possible contrasts. Participants were required to make discrimination judgment between the two contrasts. They showed contrast discrimination to be worse when the prediction (H or A, as learned during the association phase of the experiment) matches the true stimulus. Importantly, this paradigm does not only manipulate whether an action effect is predicted or not, but also the action effects’ physical energy (contrast, as we used visual stimuli). It, thus, allows us to test whether or not prediction influ- ences the same early components in the EEG as physical stimulus energy. If this is the case, then the effect of prediction (sensory attenuation) is likely an early, low-level phenomenon. Moreover, assessing both psychophysical and neurophysiological measures of sensory attenuation enables us to tell how neurophysiological Frontiers in Human Neuroscience TEST PHASE Participants fixated at a square at the center of the screen, just as in the association phase. They were asked to produce, at random, right and left key presses. Again, the key presses triggered presen- tation of letter stimuli 200 ms after the key press. In this phase, however, Hs and As were presented randomly after each key press, such that 47.5% of the generated stimuli were congruent with the previous association (i.e., the letter corresponded to the one associated to that key press in the association phase), and 47.5% were incongruent. On the remaining 5% of trials, no stimulus was presented. The stimuli appeared randomly (but in equal proportions) with the luminance C0 or the luminance C1. Participants were told that there were two categories of luminance ranging from the value 0–49 for the C0 category and from 51 to 100 for the C1 category. In order to maintain this uncertainty about the contrast on 5% of trials stimuli appeared with a random contrast between C0 – 15% contrast and C1 + 15% contrast. After the stimulus had disappeared participants were required to judge the luminance value of the stimulus on a luminance response bar. On this bar participants could place the cursor on the perceived contrast value with values of 49 and under corresponding to C0 and 51 and over corresponding to C1. Participants completed 3 tests blocks of 44 trials (on average 25 min) before running a re- association phase composed of one of each type of association block (10 min on average). Thereafter, they ran another tests blocks. In total participants responded to 264 test trials. The experiment lasted on average 1.30 h. Participants could take short breaks prior to each of the association phases. An ANOVA with the factors congruency (congruent, incon- gruent) and stimulus contrast (C0, C1) was run on ERPs aver- aged for each participant. Since our task involved visual stimuli, we focused our analysis on a region of interest at the occip- ital electrodes (O1 Oz O2). Since our stimuli were degraded and presented in a continuous stream of background visual noise, we postulated that this might influence the latency of the visual response. Therefore we inspected the ERPs over our region of interest to determine the time window corresponding to an apparent peak for the visual stimulus. TEST PHASE The time window for analysis was selected around the peak of this visual com- ponent. Importantly, this time-window was not selected based on the difference between our conditions but rather on the presence of the component itself. Analysis of the modulation of this component by contrast, would then provide further jus- tification for the time-window, since stimulus contrast should influence the magnitude of the visual response. It is impor- tant to note that the main comparison of interest—the congru- ency effect—was orthogonal to the contrast effect, and therefore the selection of the time-window would not unduly bias this comparison. EEG RECORDING AND DATA PREPROCESSING have to generate an action given a desired outcome, the action- effect relationship will be encoded particularly strongly. EEG was recorded with 64 electrodes (actiCAP, Brain Prod- uctsGmbH, Germany). The EEG was digitized at 500 Hz. EEG analysis was conducted using EEGLAB (Delorme and Makeig, 2004) and custom-built Matlab scripts. The data were resampled offline to a 250 Hz sample rate, low band-pass filtered at 45 Hz to remove line noise. Epochs were generated from −500 to 980 ms relative to stimulus onset, with a 200 ms prestimulus baseline correction. Initial artifact rejection was conducted in a semiautomatic manner (in EEGLAB) by rejecting epochs with activity above 100 µV or below −100 µV, as well as rejecting trials where activity at any time point for any electrode was more than 5 standard deviations from the mean activity for that epoch. Any channels that contributed to the rejection of many epochs were considered for removal and later interpola- tion. Frontal channels that showed large amplitude blink activity were also excluded from further analysis during this this first semiautomatic artifact rejection. Ocular artifact correction was conducted in EEGLAB in Matlab using independent component analysis (Delorme and Makeig, 2004). Following removal of eye blinks and eye movements, noisy channels were replaced by an interpolated weighted average from surrounding electrodes. Data were then re-referenced to the common average. A final round of semiautomatic artifact rejection with a threshold of +/− 80 µV was used to remove any remaining artifacts. All ERPs are presented with a low-pass filter of 20 Hz for visual presentation purposes. The association phase consisted of three FreeAsso blocks and two MemoryAsso blocks. Each FreeAsso block contained 50 trials. Each MemoryAsso block contained 30 sequences of, on average, 5 items. Each Participant ran three FreeAsso and two MemoryAsso blocks. This phase took on average 25 min. ANALYSIS OF DISCRIMINATION PERFORMANCE The luminance discrimination task was considered to be a yes/no protocol, with C1 being the target. That is, a C1 response to a C1 stimulus is a hit, a C1 response to a C0 stimulus is a false alarm, etc. According to Signal Detection Theory (Green and Swets, 1966), d′ (perceptual sensitivity) and c (response bias) are calculated using d′ = z(hit rate) −z(false alarm rate) and c = −0.5 × [z(hit rate) + z(false alarm rate)])1. Since participants provided their judgments of contrast using a continuous scale from 1 to 100 this allowed us to analyze not only their overall contrast judgment (C0 or C1) but also their rating of the per- ceived intensity (contrast) of the stimulus. We split the ratings into 10 bins to compute ROC (receiver operating characteristic) curves for each participant. The area under the curve, A′ was calculated such that A′ = 1/2 ∗6(Fi+1 −Fi)(Hi+1 + Hi), with F and H being False alarms and Hits respectively (Macmillan and Creelman, 1991). D′, c and A were calculated separately for congruent and incongruent trials. ASSOCIATION PHASE Participants fixated on a 3.34◦visual angle square located at the center of the screen. They were asked to press with their right/left index finger one of two keys (right and left on a response pad), each key press triggering presentation of a visual effect (A or H). The key-letter mapping was counterbalanced across participants. The letters appeared 200 ms after the key press at full contrast in the square at the center of the screen. There were two types of association phase. First, in the free association blocks (“FreeAsso”) a sequence of 50 actions (left/right) was freely generated by the participants at a pace of about 1 key press per second. The experimenter demonstrated the pace to the participants before the experiment. We also measured the time participants needed for each block to control their pace. In 5% of the trials the visual effect was a W. In these catch trials, the participant had to press both buttons within 1 s of the appear- ance of the stimulus. Catch trials were meant to ensure that par- ticipants paid attention to the effect stimuli. Second, in memory association blocks (“MemoryAsso”), random lists of As and Hs were presented to the participants (the average list size was 5 going from 3 to 8 items adapted to the participants performance with a simple 1up 1down rule). The lists were presented via headphones as spoken letters. After the lists were presented, participants had to reproduce the sequence by pressing the corresponding button sequence. This phase was meant to foster the learning of the action-effect associations. We reasoned that when participants Frontiers in Human Neuroscience February 2014 | Volume 8 | Article 115 | 2 www.frontiersin.org www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. Frontiers in Human Neuroscience Neurophysiological and psychophysical indices of sensory attenuation Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. FIGURE 1 | ROC curves. Mean of participants ROC curves for congruent and incongruent trials. For calculation detail of the ROC see Analysis of Discrimination Performance section. FIGURE 2 | Contrast effect: ERP and Topography. This figure presents the mean ERPs on O1 Oz O2 for C0 (in blue) and C1 (in red) from −500 to 980 ms relative to stimulus apparition. The blued surface represents the analysis time window (from 180 to 320 ms, centered on the pic around 250 ms). In the top left corner the topography of the difference (C1 – C0) is presented for the analysis time window. * p < 0.05. FIGURE 1 | ROC curves. Mean of participants ROC curves for congruent and incongruent trials. For calculation detail of the ROC see Analysis of Discrimination Performance section. FIGURE 2 | Contrast effect: ERP and Topography. This figure presents the mean ERPs on O1 Oz O2 for C0 (in blue) and C1 (in red) from −500 to 980 ms relative to stimulus apparition. The blued surface represents the analysis time window (from 180 to 320 ms, centered on the pic around 250 ms). In the top left corner the topography of the difference (C1 – C0) is presented for the analysis time window. * p < 0.05. FIGURE 2 | Contrast effect: ERP and Topography. This figure presents the mean ERPs on O1 Oz O2 for C0 (in blue) and C1 (in red) from −500 to 980 ms relative to stimulus apparition. The blued surface represents the analysis time window (from 180 to 320 ms, centered on the pic around 250 ms). In the top left corner the topography of the difference (C1 – C0) is presented for the analysis time window. * p < 0.05. FIGURE 1 | ROC curves. Mean of participants ROC curves for congruent and incongruent trials. For calculation detail of the ROC see Analysis of Discrimination Performance section. participants had action-effect mapping 1 (left →A, right →H), and 8 had mapping 2 (8 women, 7 men; mean age = 24 years, SEM = 3.69 years). stimulus onset. This peak appears to be greater for C1 than C0, such that it likely reflects processing of the visual stimulus (a delayed visual N1 component) (Ciesielski and French, 1989; Mangun and Hillyard, 1991; Johannes et al., 1995). RESULTS In order to ensure that the data were equivalent between the EEG and the behavioral analysis only trials free from EEG artifacts were analyzed. The amount of rejected data was less than 10% of the total number of trials. PSYCHOPHYSICAL RESULTS We analyzed our data dependent on participants’ contrast dis- crimination. Discrimination performance (d′) was lower in the congruent condition (d′ congruent: 1.22, SD = 0.46) than in the incongruent (d′ incongruent: 1.42, SD = 0.45) condition. A one factor repeated measures analysis of variance including the factor of congruency showed this effect of congruency on d′ to be significant (F(1,14) = 5.36, p = 0.03). At the same time, the criterion (c) was not different in the two congruency conditions (1 factor repeated measures ANOVA; F(1,14) = 0.165, p = 0.69). A bias free measure of sensitivity (A′) also confirms our finding. Sensitivity appears to be better for incongruent trials (A′: 0.81, SD = 0.075) than for congruent trials (A′: 0.79, SD = 0.067) (F(1,14) = 4.670, p = 0.04) (see Figure 1). In contrast the criterion (bias) appeared not to differ between conditions (Congruent: 0.39, SD = 0.46; Incongruent: 0.41, SD = 0.54; F(1,14) = 0.165, p = 0.691). In line with the aim of the current experiment, it appears that this visual ERP peaks as a function of congruent motor prediction. Since we also observed attenuated sensitivity for congruent trials in the behavioral analysis presented above, this provides evidence that neural and behavioral measures of sensory attenuation are likely related. Neurophysiological and psychophysical indices of sensory attenuation To quantify these effects we took the average amplitude of each condition in a 140 ms time window centered on this peak (180–320 ms). A repeated measure analysis of variance including the factor of contrast and congruency revealed a significant main effect of Contrast (F(1,14) = 6.54, p = 0.023), confirming significantly greater amplitude for C1 (mean = −1.67; std = 1.55, CI: +/− 1.21) compared to C0 (mean = −0.81; std = 1.8, CI: +/−1.63). The topography of this difference is consistent with modulation of an occipital ERP component. We also observed a significant main effect of Congruency (F(1,14) = 11.36, p = 0.005), such that our visual component was of significantly smaller amplitude in the congruent condition (mean = −0.99; std = 1.57, CI: +/− 1.24), compared to the incongruent condition (mean = −1.50; std = 1.64, CI: +/−1.36). The topography of this difference is also consistent with a modulation of visual processing as a function of action prediction. However the interaction between both factors was not significant (F(1,14) = 0.02, p = 0.885). EEG RESULTS In this section we focus on the effect of motor prediction on the neurophysiological indices of visual processing to determine the degree to which behavioral and neural sensory attenuation are related. The ERPs and the topographies for the different condi- tions are presented in Figures 2, 3. A large negative deflection is apparent in all the waveforms, peaking at around 250 ms after PARTICIPANTS Nineteen participants took part in the experiment. They were naive to the purpose of the experiment. Four of these nineteen participants were excluded from the analysis as their luminance discrimination d′s were below 0.5 (for 2 of them) or because the ratio of right left key presses during the test phases exceeded a 75% 25% ratio (for one of them). One was rejected because of the poor quality of the EEG recordings. Seven of the remaining Frontiers in Human Neuroscience February 2014 | Volume 8 | Article 115 | 3 www.frontiersin.org www.frontiersin.org www.frontiersin.org Frontiers in Human Neuroscience DISCUSSION FIGURE 3 | Congruency effect: ERP and Topography. This figure presents the mean ERPs on O1 Oz O2 for congruent (in blue) and incongruent (in red) from −500 to 980 ms relative to stimulus apparition. The blued surface represents the analysis time window (from 180 to 320 ms, centered on the pic around 250 ms). In the top left corner the topography of the difference (Incongruent—Congruent) is presented for the analysis time window. * p < 0.05. be observed in the visual domain. More importantly, one of the aims of the experiment presented above was to investigate the locus of sensory attenuation. This was done by way of comparing the influence of prediction and stimulus contrast, respectively, on the ERPs triggered by visual action effects. We observed that an N1 component was clearly modulated by stimu- lus contrast, with larger contrasts resulting in a larger deflection. Importantly, congruency affected the very same component, with congruent trials resulting in smaller amplitudes than incongruent trials. As concerns the effect of congruency on ERPs, we observed that the contrast-sensitive visual component was significantly smaller in the congruent condition compared to the incongruent condition. We, thus, observed, to our knowledge for the first time, sensory attenuation in psychophysical and neurophysiolog- ical indices at the same time, suggesting that the two measures of sensory attenuation are likely related. However, note that the psychophysical effect corresponds to an interaction between contrast and congruency: the discrimination between the two contrast levels is more difficult in congruent than in incon- gruent trials. If sensory attenuation assessed with psychophys- ical methods were a direct reflection of the ERPs assessed at the same time, we would have expected to see an interaction between these two factors in our ERP data as well. However, this was not the case. Of course, it might be that the ERP data simply lack sufficient statistical power. However, it is also possible that psychophysical and ERP indices of sensory atten- uation (at least those assessed in the current experiment) are not in a simple one-to-one relationship. Perception might be dependent not only on early cortical responses, but also on later processing and/or recurrent processing, tweaking the relationship between perceptual measures and observable neurophysiological measures. Of course, our data cannot show which processing stage precisely the two factors manipulated in the current experiment influence. DISCUSSION First of all, our experiment shows that neurophysiological sen- sory attenuation is a phenomenon that is not restricted to the auditory and somatosensory modality, but that it can also February 2014 | Volume 8 | Article 115 | 4 www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. FIGURE 3 | Congruency effect: ERP and Topography. This figure presents the mean ERPs on O1 Oz O2 for congruent (in blue) and incongruent (in red) from −500 to 980 ms relative to stimulus apparition. The blued surface represents the analysis time window (from 180 to 320 ms, centered on the pic around 250 ms). In the top left corner the topography of the difference (Incongruent—Congruent) is presented for the analysis time window. * p < 0.05. the fact that the stimuli used in the current experiment were not presented with a sharp onset, but embedded in dynamic pixel noise, such that the detection of a pattern is more time- consuming. This evidence of reafferent attenuation in the visual modality is also important regarding to the literature of saccadic sup- pression (Bridgeman et al., 1975; Deubel et al., 1996) thought to rely on a “corollary discharge” from the motor command affecting the perceptual network (Sperry, 1950; Paus et al., 1995). Nonetheless the link between sensory attenuation and saccadic suppression must be more thoroughly investigated since some essential differences separate both phenomena, notably the timing of the effect. For example sensory attenuation has been shown to occur on the final consequence of the action (Blakemore et al., 1998) while saccadic suppression has been shown to occur during the saccade (Bridgeman et al., 1975; Deubel et al., 1996). The second aim of the present study was to explore how neuro- physiological and perceptual indices of sensory attenuation relate. We used a luminance discrimination protocol to assess perceptual sensitivity and response bias of anticipated and unanticipated visual action effects, assessing EEG activity at the same time. The psychophysical results show that discrimination performance (d′, A′) was better in the incongruent condition than in the congruent condition. At the same time, the criterion was not different in the two congruency conditions. The results, thus, are in line with the findings of Roussel et al. (2013) and Cardoso-Leite et al. (2010). 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Detection Theory: A User’s Guide. Mahwah, New Jersey: Lawrence Erlbaum Associates, Inc. Mangun, G. R., and Hillyard, S. A. (1991). Modulations of sensory-evoked brain potentials indicate changes in perceptual processing during visual-spatial prim- ing. J. Exp. Psychol. Hum. Percept. Perform. 17, 1057–1074. doi: 10.1037/0096- 1523.17.4.1057 DISCUSSION The current paradigm differs in important aspects from other studies investigating visual evoked potentials. Notably, we presented stimuli in visual noise resampled at each screen refresh. The actions’ effects were, therefore, not presented with a sharp onset. Moreover, in our experiment, stimuli were trig- gered by an action. It is therefore difficult to compare our results to ERPs found in previous studies. However, previous research seems to suggest that contrast-dependent processes take place rather early in the visual processing stream (e.g., Schadow et al., 2007), while later components are rather modulated by motion and form perception (Bach and Ullrich, 1997; Göpfert et al., 1998). Importantly, our experiment allowed us to directly compare the effect of contrast and prediction. As it demon- strates that motor prediction influences the same processing stage as visual contrast, we assume that motor prediction as manipulated in our experiment influences an early processing stage that is otherwise still modulated by basic stimulus-features. This interpretation is corroborated by the fact that the N1 component in question has an occipital topography. That this component has a relatively late latency is probably due to In conclusion, in our experiment ERP effects of visual sensory attenuation were found to correspond to contrast-dependent processing stages. We conclude that motor prediction, thus, influ- ences quite early processes. Moreover, we demonstrated that both February 2014 | Volume 8 | Article 115 | 5 www.frontiersin.org www.frontiersin.org www.frontiersin.org Neurophysiological and psychophysical indices of sensory attenuation Roussel et al. psychophysical and ERP indices of sensory attenuation can be observed in the visual modality. However, the exact relationship between the two types of measure needs to be further clarified, as there are not only commonalities, but also differences. Hughes, G., Desantis, A., and Waszak, F. (2013b). Attenuation of auditory N1 results from identity-specific action-effect prediction. Eur. J. Neurosci. 37, 1152– 1158. doi: 10.1111/ejn.12120 Hughes, G., and Waszak, F. (2011). ERP correlates of action effect prediction and visual sensory attenuation in voluntary action. 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Citation: Roussel C, Hughes G and Waszak F (2014) Action prediction modulates both neurophysiological and psychophysical indices of sensory attenuation. Front. Hum. Greenwald, A. G. (1970). Sensory feedback mechanisms in performance control: with special reference to the ideo-motor mechanism. Psychol. Rev. 77, 73–99. doi: 10.1037/h0028689 Neurosci. 8:115. Received: 05 November 2013; accepted: 17 February 2014; published online: 28 February 2014. REFERENCES doi: 10.3389/fnhum.2014.00115 f This article was submitted to the journal Frontiers in Human Neuroscience. This article was submitted to the journal Frontiers in Human Neuroscience. This article was submitted to the journal Frontiers Herwig, A., Prinz, W., and Waszak, F. (2007). Two modes of sensorimotor integra- tion in intention-based and stimulus-based actions. Q. J. Exp. Psychol. (Hove) 60, 1540–1554. doi: 10.1080/17470210601119134 Hughes, G., Desantis, A., and Waszak, F. (2013a). Mechanisms of intentional binding and sensory attenuation: the role of temporal prediction, temporal control, identity prediction and motor prediction. Psychol. Bull. 139, 133–151. doi: 10.1037/a0028566 February 2014 | Volume 8 | Article 115 | 6 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org
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Abnormal Development of Dendrites in Adult-Born Rat Hippocampal Granule Cells Induced by Cyclophosphamide
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Abnormal Development of Dendrites in Adult-Born Rat Hippocampal Granule Cells Induced by Cyclophosphamide Lin Wu 1, Dandan Guo 1, Qi Liu 1, Fei Gao 1, Xiaochen Wang 1, Xueying Song 1, Fuwu Wang 2 and Ren-Zhi Zhan 1* 1Department of Physiology, Shandong University School of Medicine, Jinan, China, 2Department of Histology and Embryology, Shandong University School of Medicine, Jinan, China Although development of cognitive decline in cancer patients who receive chemotherapy is common, the underlying mechanism(s) remains to be identified. As abnormalities in adult hippocampal neurogenesis may serve as substrate for cognitive dysfunction, the present study examines the effect of cyclophosphamide (CPP), a widely prescribed chemotherapeutic agent, on dendritic development of adult-born hippocampal granule cells in the rat. CPP was intraperitoneally injected into male Sprague-Dawley rats once a week for four consecutive weeks. Four weeks and 1 week after the last dose of CPP, Morris water maze test and doublecortin (DCX) immunohistochemistry were carried out to determine the effects of CPP on cognitive function and the rate of hippocampal neurogenesis, respectively. Adult newborn hippocampal granule cells were labeled at the same day as the first dose of CPP and were examined 10 weeks after labeling. Results showed that cognitive decline induced by CPP was associated with both suppressed adult hippocampal neurogenesis and abnormal development of dendrites of newborn granule cells. The abnormalities of dendrites in newborn granule cells after CPP exposure included less dendritic branching, shorter total dendritic length, thinner and torturous dendritic shafts with intermittent appearances of varicosities, and lower spine densities of stubby and thin types along dendritic shafts, but an increased density of mushroom-like spines. Adult-born granule cells in the presence of CPP, a widely used anti-cancer medication, display abnormal dendritic morphologies and fewer dendritic spines which may underlie cognitive dysfunction. Citation: Wu L, Guo D, Liu Q, Gao F, Wang X, Song X, Wang F and Zhan R-Z (2017) Abnormal Development of Dendrites in Adult-Born Rat Hippocampal Granule Cells Induced by Cyclophosphamide. Front. Cell. Neurosci. 11:171. doi: 10.3389/fncel.2017.00171 Edited by: Antonio Gambardella, Magna Græcia University, Italy Edited by: Antonio Gambardella, Magna Græcia University, Italy Reviewed by: Jennifer Larimore, Agnes Scott College, United States Carmelo Sgobio, German Center for Neurodegenerative Diseases (HZ), Germany *Correspondence: Ren-Zhi Zhan zhan0001@gmail.com *Correspondence: Ren-Zhi Zhan zhan0001@gmail.com Received: 09 March 2017 Accepted: 06 June 2017 Published: 21 June 2017 Keywords: adult neurogenesis, chemotherapy, cognition, dendrite, granule cell, hippocampus, spine Keywords: adult neurogenesis, chemotherapy, cognition, dendrite, granule cell, hippocampus, spine Edited by: Edited by: Antonio Gambardella, Magna Græcia University, Italy ORIGINAL RESEARCH published: 21 June 2017 doi: 10.3389/fncel.2017.00171 Doses and Delivery of CPP y CPP monohydrate (Sigma, St. Louis, MO, USA) was dissolved in sterile saline to make a 50 mg/ml of stock solution. The stock solution was stored at −80◦C until use. CPP was intraperitoneally injected into animals once a week for four consecutive weeks at either 25 mg/kg or 50 mg/kg. Animals received equivalent amount of saline in a schedule same to CPP-treated ones were used as controls. As a subcortical structure, the hippocampus functions in learning, memory formation and mood regulation. Granule cells in the hippocampal dentate gyrus are continuously to be generated throughout life in this brain region from neural stem/progenitor cells that reside in the subgranular zone. Ultimately, nearly a quarter of newborn granule cells survive to become mature neurons under normal conditions (Christian et al., 2014). Studies have shown that newborn granule cells integrate into the pre-existing dentate network and participate in almost all of the known functions of the hippocampus (Clelland et al., 2009; Sahay et al., 2011; Aimone et al., 2014; Christian et al., 2014; Opendak and Gould, 2015). The rate of adult hippocampal neurogenesis is under intense regulatory control of internal and external environments (Aimone et al., 2014). Studying in animals has shown that cyclophosphamide (CPP), a commonly used chemotherapeutic agent, suppresses adult hippocampal neurogenesis (Yang et al., 2010; Briones and Woods, 2011; Lyons et al., 2011; Christie et al., 2012; Dietrich et al., 2015). Given the fact that it takes 6∼8 weeks for newborn granule cells to mature (Aimone et al., 2014; Christian et al., 2014; Bergami et al., 2015), a better understanding of detrimental effects of chemotherapeutic agents on cognitive function should focus not only on the rate of adult neurogenesis but also on the integration of newborn granule cells. The normal integration process needs newborn granule cells to be positioned appropriately, to have fully developed dendrites, and to innervate onto right targets. Maturation of newborn granule cells may be delayed by reduced physical or exploring activities (Ambrogini et al., 2010) which are present during and even after chemotherapy (Irwin et al., 2004). In the present study, we explored possible abnormalities of dendritic morphologies of newborn granule cells of the rat hippocampus after exposure to CPP. INTRODUCTION Systemic cancer treatment is associated with cognitive decline that manifests as impairments in learning, memory formation, attention, information processing speed and executive functioning (Anderson-Hanley et al., 2003; Minisini et al., 2004; Vardy and Tannock, 2007; Janelsins et al., 2011; Dietrich et al., 2015; Iyer et al., 2015; Ono et al., 2015; Wefel et al., 2015). Multiple mechanisms June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 1 Cyclophosphamide and Dendritic Development Wu et al. including biological and neuropsychiatric responses to cancers (Andreotti et al., 2015; Wefel et al., 2015), toxic effects of chemotherapeutic agents (Ahles and Saykin, 2007; Hutchinson et al., 2012; Dietrich et al., 2015) and side effects of adjuvant hormones (Rugo and Ahles, 2003; Castellon et al., 2004) have been proposed for the development of cognitive dysfunction. Although both direct and indirect detrimental effects of chemotherapeutic agents on brain cells may contribute to chemotherapy-induced cognitive dysfunction, specific pathways have yet to be identified (Ahles and Saykin, 2007; Dietrich et al., 2015). and during the experiments. In total, 34 animals were used in the present study; two animals died after CPP monohydrate (CPP) injection (all were in the 3rd week after initiation of injection). Three sets of experiments were conducted in 32 animals: 14 for Morris water maze testing, six for assessment of adult neurogenesis with doublecortin (DCX) immunohistochemistry and 12 for dendritic measurements following intradentate retroviral vector injection. Transcardial Perfusion and Tissue Preparation Each animal was deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg). After complete paralysis, the animal was perfused through the aorta with 50 ml heparinized saline, followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 6.8) over a period of 40 min. The brain was removed from the skull upon completion of perfusion, and then post-fixed in the same fixative at 4◦C overnight. Thereafter, the brain was sequentially immersed in 10% sucrose in 0.1 M PBS for 4 h, 15% sucrose in 0.1 M PBS for 8 h, and finally 20% sucrose in 0.1 M PBS at 4◦C overnight. Tissue block including the hippocampus was made with the aid of an appropriate brain matrix (WPI, Sarosata, FI, USA). After the tissue block was firmly embedded with a medium that consisted of 30% (w/v) chicken egg albumin (Sigma, St. Louis, MO, USA), 0.5% (w/v) gelatin, and 0.9% (v/v) glutaraldehyde in 0.1 M PBS as described previously (Zhan and Nadler, 2009), coronal sections were cut into 40 µm or 110 µm in thickness (depending on experimental aims) with a vibratome (VT1000 S, Leica Biosystems, Wetzlar, Germany). Doublecortin (DCX) Immunohistochemistry The effect of CPP on the rate of neurogenesis was compared in saline- and CPP-injected rats (three animals each group) by staining and counting DCX-positive newborn granule cells. Brain sections were prepared 1 week after the final dose of CPP. Sections (40 µm in thickness) localized between 3.0 mm and 3.6 mm posterior to the bregma were serially cut. Sections with the order of number 3, 9 and 15 were subjected to DCX immunohistochemical staining with the free-floating procedure. After the collected sections were washed with 0.1 M PBS, they were incubated in a solution that consisted of 30% methanol and 3% H2O2 in water for 30 min to inactivate endogenous peroxidase. After rinsing, sections were incubated in a blocking solution that consisted of 2.5% BSA (Sigma, St. Louis, MO, USA), 0.2% Triton X-100 and 5% normal donkey serum in 0.1 M PBS at 4◦C for 2.5 h to minimize non-specific reactions. Without rinsing, the sections were then incubated with a goat Behavioral Assessments For animals to be used for behavioral studies, open field test assessing general locomotor activity was conducted to qualify animals before CPP exposure. To this aim, a 90 cm × 90 cm white board, divided into 36 equal squares by red lines was used as a field apparatus. Each side of the board was fenced to prevent animals to jump out. Animals were individually placed in the central square and allowed to explore the area freely. The numbers of crossing (squares crossed), rearing (standing on hind limbs), grooming (washing face) and urination were recorded for 5 min. After each trial, the apparatus was cleaned with ethanol. Qualified rats were randomly divided into two groups, receiving intraperitoneal injection of saline (control group) or CPP (CPP group); both were carried out once a week for a consecutive 4 weeks. Four weeks after the last injection, the Morris water maze was conducted to assess animals’ spatial learning and memory abilities. The water maze task was carried out for seven consecutive days in which the first 6 days were used to train the animals to find the hidden platform, whereas the last day was used to probe how well the animals remembered the position of the platform. A cylindrical pool (120 cm in diameter), filled with water (22–25◦C) was placed in a quiet testing room. The cylindrical pool was equally divided into four quadrants with a removable platform (10 cm in diameter) to be located in the center of the first quadrant. The platform was set 2 cm below the surface of water to make the platform invisible to animals. A digital camera was mounted above the pool and linked to an automatic tracking system (SMART polyvalent video-tracking system, Panlab, Spain) to track and record the performance of individual animals. Several bright-colored cues posted on the wall of the maze were used to help animals to find the platform. In the training sessions, each rat was put into the pool from Construction of CAG–GFP Retroviral Vector Vector Concentrated CAG–GFP retroviral vector was made from an established stable virus-producing platinum-E cell line (Gao et al., 2015) through two-step ultraspeed centrifugation detailed below. After the frozen cells were thawed quickly in a 37◦C water bath, 1 ml of cell suspension was immediately transferred into a 10 ml tube that contained 4 ml of Dulbecco’s modified eagle’s medium (DMEM). The cell suspension mixed with DMEM was centrifuged at 1000 rpm for 5 min. After discarding the supernatant, the cell pellet was re-suspended with 2 ml of M10 (10% serum-contained DMEM). The resulted cell suspension was transferred into a 10 ml culture dish in which 8 ml culture medium was pre-added. After mixing, cells were cultured in a 5% CO2 incubator at 37◦C. Upon ∼90% confluence was reached, culture medium was removed and the cells were rinsed twice with autoclaved PBS. After cells were digested in 1 ml of 0.25% trypsin-contained solution for 3–4 min, 4 ml of M10 was added into the dish. After thorough mixing, 2 ml of cell suspension was added into an 8 ml M10-contained 10 ml culture dish. At this stage, 12 dishes were prepared each time. Dishes were cultured in a 5% CO2 incubator at 37◦C and the supernatant was collected 24 and 48 h after culture. Supernatants collected from all dishes were pooled together and centrifuged (18,000 rpm) at 4◦C for 2 h. The pellet was suspended with 1 ml Dulbecco’s phosphate- buffered saline and then centrifuged (12,000 rpm) at 4◦C for 1.5 h. After removal of supernatant, the pellet was suspended in 30 µl of Dulbecco’s phosphate-buffered saline. After being kept at 4◦C overnight, the viral suspension was gently mixed, divided into 5–10 µl aliquots, and kept at −80◦C until use. Animals After several washes with 0.1 M PBS, immunoreactions were visualized by an incubation with 3,3-diaminobenzidine, enhanced by the addition of 0.08% ammonium nickel sulfate (Vector Labs., Burlingame, CA, USA). The reaction was terminated by incubating the section with water. Sections were then flatly mounted on slide glasses and left to be air-dried for at least an hour. After dehydration with a series of graded ethanol solutions, the sections were then cleared in xylene and coverslipped with neutral balsam. DCX-positive cells in the dentate gyrus were counted and the lengths of the superior and inferior pyramidal blade of the dentate gyrus were measured in each section by using Image-Pro- Plus (Media Cybernetics, Rockville, MD, USA). The density of DCX-positive cells was calculated by dividing the number of cells with the total length of granule cell layer and expressed as the number of cells/mm. Values in three sections each animal were averaged. the four different quadrants with its head facing the wall to prevent the animal seeing the platform directly. If a rat could find the platform within 60 s, it would be allowed to stay onto the platform for 20 s before removal from the pool. Instead, if a rat failed to find the platform within 60 s, it would be guided onto the platform. The time taken to find the hidden platform (latency to escape) was recorded. On the 7th day, the platform was removed and each rat was put into the pool from a start point (the opposite quadrant of the first quadrant) to probe its memory. The rat was allowed to swim freely in the pool for 60 s and the path of navigation was traced. Swimming speed and the time spent in the target quadrant and non-target quadrants were collected. Animals All animal experiments were approved by the Animal Ethics Committee of Shandong University School of Medicine and performed according to the guides for the care and use of laboratory animals set by National Research Council (US). Male Sprague-Dawley rats (160–180 g, 6–8 weeks old) purchased from Beijing HFK Bioscience Company (Beijing, China) were housed in a university animal facility with a 12 h light/dark cycle and free access to food and water, and were weighed weekly before June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 2 Cyclophosphamide and Dendritic Development Wu et al. Wu et al. anti-DCX (Santa Cruz, San Jose, CA, USA) that was diluted with the blocking solution (1:400) at 4◦C overnight. After three washes with 0.1 M PBS, the sections were incubated with biotinylated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA) in a dilution of 1:400 at 4◦C for 1.5 h. After rinsing with 0.1 M PBS, the sections were incubated with the avidin-peroxidase complex (Vector Labs., Burlingame, CA, USA) at 4◦C for 60 min. After several washes with 0.1 M PBS, immunoreactions were visualized by an incubation with 3,3-diaminobenzidine, enhanced by the addition of 0.08% ammonium nickel sulfate (Vector Labs., Burlingame, CA, USA). The reaction was terminated by incubating the section with water. Sections were then flatly mounted on slide glasses and left to be air-dried for at least an hour. After dehydration with a series of graded ethanol solutions, the sections were then cleared in xylene and coverslipped with neutral balsam. DCX-positive cells in the dentate gyrus were counted and the lengths of the superior and inferior pyramidal blade of the dentate gyrus were measured in each section by using Image-Pro- Plus (Media Cybernetics, Rockville, MD, USA). The density of DCX-positive cells was calculated by dividing the number of cells with the total length of granule cell layer and expressed as the number of cells/mm. Values in three sections each animal were averaged. anti-DCX (Santa Cruz, San Jose, CA, USA) that was diluted with the blocking solution (1:400) at 4◦C overnight. After three washes with 0.1 M PBS, the sections were incubated with biotinylated donkey anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA) in a dilution of 1:400 at 4◦C for 1.5 h. After rinsing with 0.1 M PBS, the sections were incubated with the avidin-peroxidase complex (Vector Labs., Burlingame, CA, USA) at 4◦C for 60 min. Frontiers in Cellular Neuroscience | www.frontiersin.org Enhancement of GFP Fluorescence with Anti-GFP Antibody for Dendritic Analyses Anti GFP Antibody for Dendritic Analyses For dendritic measurements, transcardial perfusion was performed 10 weeks after retroviral vector injection. Coronally cut hippocampal sections (110 µm in thickness) in which GFP-labeled granule cell(s) presented were subjected to GFP immunostaining by the free-floating procedure. Sections were first washed with 1XPBS, and then incubated in a blocking solution that consisted of 2.5% BSA, 0.2% Triton X-100 and 5% goat serum in 1XPBS to minimize non-specific reactions. Thereafter, sections were incubated with a mouse anti-GFP (Invitrogen, Carlsbad, CA, USA) in a dilution of 1:1000 at 4◦C overnight. After three washes with 1XPBS, the sections were incubated with an Alexa fluor 488-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) in a dilution of 1:600. After another three washes with 1XPBS, sections were mounted on glass slides with 75% glycerol in PBS and coverslipped. Sections in which GFP was immunofluorescently amplified were scanned with the Zeiss 780 laser scanning microscope (Carl Zeiss, Jena, Germany). Images yielded were used for quantitative measurements of dendrite length, the complexity of dendritic arborization and dendrite spine. Statistical Analysis Data are expressed as mean ± standard error of the mean (SEM). Two-group data are compared with unpaired Student’s t-test. One-way analysis of variance (ANOVA) was used to compare the means of three or more groups. Follow one-way ANOVA, Dunnett’s test was used for individual comparisons. Latency to escape was analyzed with repeated measures ANOVA, whereas data for Sholl analysis were compared with two-way ANOVA. A p ≤0.05 is considered to be significantly different. Measurements of Dendrite Length and Complexity Complexity To measure dendrite length and the complexity of dendritic arborization, individual GFP-labeled cells residing in the suprapyramidal blade were z-stacked under a 20× objective with a z-series interval of 1 µm and zoom 1. A 3-dimensional image created from the z-series file each cell was compressed into a 2-demnesional image. Only cells with relatively intact dendritic arborizations were quantitatively analyzed further. After GFP-positive soma and dendritic arborization were manually traced under ImageJ1 with the NeuronJ plugin2, the length of each dendritic segment was measured. Total dendrite length was calculated by summing the lengths of all segments. After tracing, the complexity of dendritic arborization for individual cells was carried out by using Sholl analysis with the ‘‘Sholl plugin’’. For Sholl analysis, the interval between concentric circles was 25 µm with the center point at the center of soma. For each rat, 3 cells were randomly picked for either of analysis and the values were averaged to represent the animal. 1http://imagej.nih.gov/ij/ 2http://www.imagescience.org/meijering/software/neuronj// Intradentate Injection of CAG–GFP Retroviral Vector After an animal was anesthetized with an intraperitoneal injection of sodium pentobarbital (25 mg/kg) and ketamine (60 mg/kg), the animal was placed on a stereotaxic frame suitable for rats (Stoelting, Wood Dale, IL, USA). After the skin was sterilized with a sequential application of 7.5% povidone iodine and 75% alcohol, the scalp was incised and a skull hole on the right side was drilled. One microliter of CAG–GFP retroviral vector was injected into the right dentate gyrus at a rate of 0.1 µl/min using a 1 µl Hamilton June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 3 Wu et al. Cyclophosphamide and Dendritic Development Quantification of Dendritic Spines syringe (Bonaduz, GR, Switzerland) that was connected to an automatic injection pump (Stoelting, Wood Dale, IL, USA). The injection site was located 3.2 mm posterior from the bregma and 2.5 mm right to the midline, in a depth of 2.4 mm (measured from the surface of the cortex). The needle was left in the place for additional 15 min after the completion of injection to prevent possible backflow before removal. All surgical procedures were performed under sterile conditions. Animals were returned to cages after recovery from anesthesia. Quantification of Dendritic Spines z-stacks for dendritic segments located in the middle molecular layer were carried by using a 63× oil objective. The other parameters set for z-stacks were as follows: 0.12 µm of z-series thickness, zoom 4, and a spatial resolution of 0.03 µm × 0.03 µm × 0.12 µm. A 3-dimensional image was reconstructed from individual z-stack file. Spines were classified into stubby, thin or mushroom type based on spine length, head diameter to neck width ratio, and the size of head specific to dentate granule cells (Tyler and Pozzo-Miller, 2003; Boda et al., 2004; Zhao et al., 2014). A stubby spine had a length that was similar to the diameter of the neck as well as the diameter of the head. A mushroom spine displayed a great head diameter to neck width ratio in addition to a head surface area that was ≥0.4 µm2. The head surface area was estimated by using the function 0.25 × π × dx (µm) × dy (in µm); dx and dy were the lengths of shortest and longest axes of the spine head which were measured under ImageJ manually (Zhao et al., 2014). A spine that could not be classified into either stubby type or mushroom form with or without a bulbous head was referred to as the thin type. The numbers of total and spines in different forms on each segment were counted and the densities of total and individual types were calculated by dividing the numbers of spines with the length of corresponding dendritic segment. Six to nine dendritic segments (30–50 µm in length each) from at least three cells each animal located in the middle molecular layer were randomly scanned and analyzed. Total, stubby, thin and mushroom spine densities from all counted segments each animal were averaged and used to represent the animal. Long-Lasting Cognitive Decline after CPP Exposure Is Associated with a Suppression of Hippocampal Neurogenesis Morris water maze test revealed that 4-week CPP treatment significantly reduced learning and memory abilities, even though the test was held 4 weeks after the last dose of CPP. Times spent for finding the platform were analyzed with repeated measures ANOVA (Figure 1A). Since Chi-analysis revealed a violation of sphericity, data were analyzed with Huynh-Feldt correction. Statistical significances were found between the two groups (F(1) = 22.9, p = 0.0004) and at different time points (F(5) = 16.1, p < 0.0001) but no significant interaction between ‘‘group’’ and ‘‘time’’ (F(5) = 0.84, p = 0.5247). At the first day of training, the time needed to find the platform (latency to escape) between the control (Control) and CPP groups was similar; however, as training days increased, animals in the control group spent less time finding the platform. A day after completion of training, the animals were tested on how firm they intended to find the platform for escape. As shown in Figure 1B, the swimming speeds were not statistically different between the control and CPP groups. However, animals in the control group stayed significantly longer in the target quadrant searching for the platform, whereas the same trend did not exist in the CPP-treated group (Figures 1C,D). in Figure 2A, at both doses, CPP significantly reduced the density of DCX-positive cells in the hippocampal dentate gyrus. The density of DCX-positive cells was reduced significantly from 46.0 ± 4.1 cells/mm in the control group (n = 3) to 31.2 ± 3.3 cells/mm in 25 mg/kg group (n = 3) and 23.8 ± 0.9 cells/mm in 50 mg/kg group (n = 3), respectively (Figure 2B). CPP Exposure Results in Less Dendritic Arborization and Complexity in Newborn Granule Cells At 10 weeks after retroviral vector injection, GFP-labeled cells from control animals were found to reside in the border between the granule cell layer and the hilus with apical dendrites extending throughout the molecular layer and tips ending at the hippocampal fissure (Figure 3A). The morphology of dendritic tree of newborn granule cells obtained from CPP-treated animals appeared different from control rats in at least two aspects: they appeared to be beading (Figure 3B) and branched to a lesser extent (Figure 3C). Total dendrite length in the CPP group is 1279.0 ± 62.5 µm (n = 6), which is significantly shorter than 1921.0 ± 110.5 µm (n = 6) observed in the control group (p = 0.005, by unpaired t-test; Figure 3D). Sholl analysis revealed that dendritic arborization of newborn granule cells after CPP treatment is significantly less complex than that in the control group (F(14,140) = 39.7, p < 0.0001), as shown in Figure 3E. Noticeably, the number of intersection indicating dendritic complexity was significantly less in CPP-treated group in dendritic segments (between 135 µm and 270 µm away from the soma). To determine if cognitive dysfunction induced by CPP is associated with a slower rate of adult hippocampal neurogenesis, we performed DCX immunohistochemistry. In addition to 50 mg/kg of CPP, a smaller dose was tested also. As shown FIGURE 1 | Reduced learning and memory abilities after cyclophosphamide (CPP) treatment revealed by Morris water maze test. Animals were tested 4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of the mean (SEM). (A) Latency to escape recorded during the training days. ∗p < 0.01 (t = 2.82 at 4th and 2.99 at 6th, df = 72), in comparison with “control” at the same time revealed by by Dunnett post hoc test following repeated measures analysis of variance (ANOVA). (B) Swimming speed recorded in the testing day. (C) Time spent in the target quadrant in the testing day. ∗p < 0.05–0.01 (df = 24), in comparison with that in the target quadrant. Comparisons were done with Dunnett’s test following one-way ANOVA. TQ, NT1, NT2 and NT3 indicate the target quadrant, non-target quadrant 1, non-target quadrant 2 and non-target quadrant 3, respectively. (D) Time spent in the target quadrant between the control (Control) and CPP groups in the testing day (t = 2.81, df = 12). CPP Reduces Total Dendritic Spines but Increases Mushroom-Like Spines ∗p < 0.01 (t = 2.82 at 4th and 2.99 at 6th, df = 72), in comparison with “control” at the same time revealed by by Dunnett post hoc test following repeated measures analysis of variance (ANOVA). (B) Swimming speed recorded in the testing day. (C) Time spent in the target quadrant in the testing day. ∗p < 0.05–0.01 (df = 24), in comparison with that in the target quadrant. Comparisons were done with Dunnett’s test following one-way ANOVA. TQ, NT1, NT2 and NT3 indicate the target quadrant, non-target quadrant 1, non-target quadrant 2 and non-target quadrant 3, respectively. (D) Time spent in the target quadrant between the control (Control) and CPP groups in the testing day (t = 2.81, df = 12). Long-Lasting Cognitive Decline after CPP Exposure Is Associated with a Suppression of Hippocampal Neurogenesis pp p g To observe the effect of CPP on cognitive function, a dose of 50 mg/kg reported by a previous study (Christie et al., 2012) was applied. Before Morris water maze testing, body weight was measured and open field test was conducted in individual animals. Body weight was significantly lower in CPP group (379.0 ± 9.7 g, n = 7) in comparison with the control group (430.9 ± 4.8 g, n = 7; p < 0.005 via unpaired t-test). Open field test revealed that rats in CPP group (n = 7) had a trend to be less active than control group (n = 7). Events summed up from the number of crossing, rearing, glooming and urination in a 1http://imagej.nih.gov/ij/ 2http://www.imagescience.org/meijering/software/neuronj// June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 4 Cyclophosphamide and Dendritic Development Wu et al. Wu et al. 5 min-period was 51.3 ± 19.7 in CPP group and 107.0 ± 20.7 in the control group (p = 0.075, unpaired t-test). Morris water maze test revealed that 4-week CPP treatment significantly reduced learning and memory abilities, even though the test was held 4 weeks after the last dose of CPP. Times spent for finding the platform were analyzed with repeated measures ANOVA (Figure 1A). Since Chi-analysis revealed a violation of sphericity, data were analyzed with Huynh-Feldt correction. Statistical significances were found between the two groups (F(1) = 22.9, p = 0.0004) and at different time points (F(5) = 16.1, p < 0.0001) but no significant interaction between ‘‘group’’ and ‘‘time’’ (F(5) = 0.84, p = 0.5247). At the first day of training, the time needed to find the platform (latency to escape) between the control (Control) and CPP groups was similar; however, as training days increased, animals in the control group spent less time finding the platform. A day after completion of training, the animals were tested on how firm they intended to find the platform for escape. As shown in Figure 1B, the swimming speeds were not statistically different between the control and CPP groups. However, animals in the control group stayed significantly longer in the target quadrant searching for the platform, whereas the same trend did not exist in the CPP-treated group (Figures 1C,D). 5 min-period was 51.3 ± 19.7 in CPP group and 107.0 ± 20.7 in the control group (p = 0.075, unpaired t-test). CPP Reduces Total Dendritic Spines but Increases Mushroom-Like Spines After revealing abnormalities in the dendritic tree of newborn granule cells after CPP treatment, we then quantified spines in dendritic segments of the middle molecular layer. In the control group, dense protrusions in variable shapes appeared along the relatively regular dendritic shaft (Figure 4A, left panel). CPP treatment resulted in unusual appearances of dendrites—shafts appeared to be thinner with intermittent varicosities, and in addition, fewer protrusions were present (Figure 4A, right panel). Total spine density in the control group (Control) is ∼1.2 spines per µm length (n = 5), which was significantly denser than 0.7 spines per µm length (n = 5) in CPP-treated group (Figure 4B). In contrast to the density of total, stubby and thin spines, the density of mushroom-like spines, defined as having a ≥0.4 µm2 in head surface area with shorter neck, increased significantly in CPP-treated animals compared to the control animals (Figure 4B). FIGURE 1 | Reduced learning and memory abilities after cyclophosphamide (CPP) treatment revealed by Morris water maze test. Animals were tested 4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of the mean (SEM). (A) Latency to escape recorded during the training days. ∗p < 0.01 (t = 2.82 at 4th and 2.99 at 6th, df = 72), in comparison with “control” at the same time revealed by by Dunnett post hoc test following repeated measures analysis of variance (ANOVA). (B) Swimming speed recorded in the testing day. (C) Time spent in the target quadrant in the testing day. ∗p < 0.05–0.01 (df = 24), in comparison with that in the target quadrant. Comparisons were done with Dunnett’s test following one-way ANOVA. TQ, NT1, NT2 and NT3 indicate the target quadrant, non-target quadrant 1, non-target quadrant 2 and non-target quadrant 3, respectively. (D) Time spent in the target quadrant between the control (Control) and CPP groups in the testing day (t = 2.81, df = 12). FIGURE 1 | Reduced learning and memory abilities after cyclophosphamide (CPP) treatment revealed by Morris water maze test. Animals were tested 4 weeks after the last dose of CPP (50 mg/kg). Error bars are standard error of the mean (SEM). (A) Latency to escape recorded during the training days. DISCUSSION In addition to confirming that persistent cognitive decline induced by CPP is associated with a suppression of adult hippocampal neurogenesis, we found that CPP treatment leads June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 5 Wu et al. Wu et al. Cyclophosphamide and Dendritic Development FIGURE 2 | CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. (A) Representative images showing DCX-positive cells in sections obtained from the control (left panel) and CPP-treated rats (middle and right panels). Upper row: imaged under a lower objective; Bottom row: areas indicated by the black boxes in the upper row were visualized under a higher magnification lens. (B) The densities of DCX-positive cells in the absence (Control) and the presence of two doses of CPP. The number of animals is three in each group. ∗p < 0.05 and ∗∗p < 0.01 (df = 6), in comparison with “Control” by Dunnett’s test following one-way ANOVA. FIGURE 2 | CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. (A) Representative images showing DCX-positive cells in sections obtained from the control (left panel) and CPP-treated rats (middle and right panels). Upper row: imaged under a lower objective; Bottom row: areas indicated by the black boxes in the upper row were visualized under a higher magnification lens. (B) The densities of DCX-positive cells in the absence (Control) and the presence of two doses of CPP. The number of animals is three in each group. ∗p < 0.05 and ∗∗p < 0.01 (df = 6), in comparison with “Control” by Dunnett’s test following one-way ANOVA. FIGURE 2 | CPP reduces doublecortin (DCX)-positive cells in the hippocampal dentate gyrus. (A) Representative images showing DCX-positive cells in sections obtained from the control (left panel) and CPP-treated rats (middle and right panels). Upper row: imaged under a lower objective; Bottom row: areas indicated by the black boxes in the upper row were visualized under a higher magnification lens. (B) The densities of DCX-positive cells in the absence (Control) and the presence of two doses of CPP. The number of animals is three in each group. ∗p < 0.05 and ∗∗p < 0.01 (df = 6), in comparison with “Control” by Dunnett’s test following one-way ANOVA. to remarkable abnormal development of dendrites in newborn granule cells. DISCUSSION (B) Newborn granule cells obtained from a rat treated with CPP (scale bar = 50 µm). (C) A comparison of the number of dendritic branches between the control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (D) A comparison of total dendrite length between the control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (E) Sholl analysis compares the complexity of dendritic arborization between the control (Control) and CPP groups. The numbers of animals studied are shown inside the figure. For each animal, the value was averaged from three cells. Statistical comparisons were done with two-way ANOVA. Sidak’s multiple comparisons were used for individual comparisons. ∗Indicates p < 0.05∼0.001 as compared to the control (df = 150). FIGURE 3 | Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence (Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the retroviral vector. (A) A newborn granule cell obtained from a control rat (scale bar = 50 µm). (B) Newborn granule cells obtained from a rat treated with CPP (scale bar = 50 µm). (C) A comparison of the number of dendritic branches between the control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (D) A comparison of total dendrite length between the control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (E) Sholl analysis compares the complexity of dendritic arborization between the control (Control) and CPP groups. The numbers of animals studied are shown inside the figure. For each animal, the value was averaged from three cells. Statistical comparisons were done with two-way ANOVA. Sidak’s multiple comparisons were used for individual comparisons. DISCUSSION Multiple clinical surveys have provided solid evidence to show that chemotherapy can lead to cognitive decline, although the to remarkable abnormal development of dendrites in newborn granule cells. Multiple clinical surveys have provided solid evidence to show that chemotherapy can lead to cognitive decline, although the FIGURE 3 | Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence (Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the retroviral vector. (A) A newborn granule cell obtained from a control rat (scale bar = 50 µm). (B) Newborn granule cells obtained from a rat treated with CPP (scale bar = 50 µm). (C) A comparison of the number of dendritic branches between the control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (D) A comparison of total dendrite length between the control (Control) and CPP groups. Each data point represents one animal in which the value was averaged from three newborn granule cells. Data are compared by unpaired student’s t-test. (E) Sholl analysis compares the complexity of dendritic arborization between the control (Control) and CPP groups. The numbers of animals studied are shown inside the figure. For each animal, the value was averaged from three cells. Statistical comparisons were done with two-way ANOVA. Sidak’s multiple comparisons were used for individual comparisons. ∗Indicates p < 0.05∼0.001 as compared to the control (df = 150). FIGURE 3 | Representative images and statistical comparisons show the number of branch, total dendrite length and the complexity of arborization in the absence (Control) and presence of CPP. The first dose of CPP (50 mg/kg) was given at the same day as the CAG-GFP retroviral vector labeling and was continued for consecutive 4 weeks. Animals in the control group received equivalent amounts of saline. Animals in both groups were sacrificed 10 weeks after the injection of the retroviral vector. (A) A newborn granule cell obtained from a control rat (scale bar = 50 µm). DISCUSSION ∗Indicates p < 0.05∼0.001 as compared to the control (df = 150). June 2017 | Volume 11 | Article 171 6 Frontiers in Cellular Neuroscience | www.frontiersin.org Cyclophosphamide and Dendritic Development Wu et al. in the present study on the basis of the same dose which has been used in a previous study (Christie et al., 2012) and this dose resulted in nearly 50% reduction in DCX-labeled newborn granule cells (Figure 2). Since CPP was applied for a consecutive 4 weeks and dendritic morphological changes were examined more than 2 months after birth, dendritic changes observed in the present study are considered to be chronic response. To avoid the influence of intradentate retroviral vector injection on behavioral tests and behavioral training on dendritic developments, cognitive function, alteration in hippocampal neurogenesis and dendritic morphologies were not studied in the same set of animals rather than three separated sets of animals. FIGURE 4 | CPP reduces total but increases mushroom-like dendritic spines in adult-born granule cells. (A) Images of dendritic segments in 10 week-old newborn granule cells in a control animal (left panel) and a CPP (50 mg/kg)-treated animal (CPP, right panel). Mushroom-like spines are indicated by arrowheads. Scale bar = 5 µm. Note that dendritic segment obtained from a CPP-treated animal appears to be thinner with irregular varicosities (right panel). (B) Bar graphs show that CPP treatment reduces total, thin and stubby dendritic spines but increases mushroom-like spines. Error bars are SEMs. Statistical comparisons are done with unpaired t-test. “n” indicates the number of animals. “a”, “b”, “c” and “d” indicates p values at 0.045 (t = 2.37, df = 8), 0.038 (t = 2.48, df = 8), 0.043 (t = 2.41, df = 8) and 0.009 (t = 3.49, df = 8), respectively. The Morris Water Maze was an established method for testing hippocampus-dependent learning, acquisition of spatial memory and long-term spatial memory. The substrate for those hippocampus-dependent cognitive deficits during and after CPP treatment has been firmly established. A recent study revealed that CPP treatment was associated with abnormal dendritic structures in hippocampal principal cells (Acharya et al., 2015). By combining retroviral labeling and confocal imaging, CPP was found to cause an abnormal development of dendrites in newborn granule cells. DISCUSSION The characteristics of dendritic abnormality include fewer branches and shorter total length of dendrites, irregularity of dendritic shafts especially intermediate dendritic varicosities along the thinner and tortuous shafts, and sparser dendritic spines but increased mushroom-like spines. Because the morphology of dendrites and spines attached to dendritic shafts are critical in receiving excitatory projections and in cellular signal integration (Šišková et al., 2014), the abnormalities observed after CPP treatment are expected to result in lower excitability and insufficient integration of newborn granule cells, which could contribute to the development of cognitive decline. In comparison with dendrites of newborn granule cells in the controlled animals, dendrites of newborn granule cells in the presence of CPP appeared to be intermediately thinner and torturous. Since these features were not obvious in pre-existing granule cells in a similar treatment regimen (Acharya et al., 2015), dendritic changes in newborn granule cells are unlikely to be caused by direct toxic effect but developmental defect. While total spine density (due to decreased thin and stubby spines) was found to be lower in CPP-treated animals, the mushroom-like spine density was rather increased. As larger spines are generally considered to be composed by more excitatory amino acid receptors (Matsuzaki et al., 2001; Noguchi et al., 2005) and transformed from small spines in response to sensory stimuli (Grutzendler et al., 2002; Yasumatsu et al., 2008), the increase in mushroom-like spines after CPP treatment is puzzling. Several mechanisms may underlie the increased density in mushroom spines in the presence of CPP. First, it is possible that the increased density of mushroom-like spines after CPP exposure might have been a compensatory response, since persistent lack of excitatory synapses is known to result in increased head diameter and strength of remaining spines after naturation (Turrigiano, 2007). In addition, changes in dendritic geometry may alter integration of excitatory inputs, resulting in only FIGURE 4 | CPP reduces total but increases mushroom-like dendritic spines in adult-born granule cells. (A) Images of dendritic segments in 10 week-old newborn granule cells in a control animal (left panel) and a CPP (50 mg/kg)-treated animal (CPP, right panel). Mushroom-like spines are indicated by arrowheads. Scale bar = 5 µm. Note that dendritic segment obtained from a CPP-treated animal appears to be thinner with irregular varicosities (right panel). (B) Bar graphs show that CPP treatment reduces total, thin and stubby dendritic spines but increases mushroom-like spines. Error bars are SEMs. DISCUSSION Statistical comparisons are done with unpaired t-test. “n” indicates the number of animals. “a”, “b”, “c” and “d” indicates p values at 0.045 (t = 2.37, df = 8), 0.038 (t = 2.48, df = 8), 0.043 (t = 2.41, df = 8) and 0.009 (t = 3.49, df = 8), respectively. underlying mechanism remains to be fully elucidated (Anderson- Hanley et al., 2003; Minisini et al., 2004; Vardy and Tannock, 2007; Janelsins et al., 2011; Jim et al., 2012; Lindner et al., 2014; Dietrich et al., 2015; Wefel et al., 2015). Animal studies have found that chemotherapeutic agents, including CPP, profoundly slow the rate of adult hippocampal neurogenesis (Mustafa et al., 2008; Seigers et al., 2008, 2009, 2010; Yang et al., 2010; Briones and Woods, 2011; Lyons et al., 2011; Christie et al., 2012; ElBeltagy et al., 2012; Nokia et al., 2012). Because adult hippocampal neurogenesis is implicated in functions that overlap with those general functions of hippocampus (Clelland et al., 2009; Sahay et al., 2011; Aimone et al., 2014; Christian et al., 2014; Opendak and Gould, 2015), in addition to studying the rate of neurogenesis, studying the intrinsic electrophysiological properties and integration of newborn granule cells under the influence of chemotherapeutic agents is needed. For newborn granule cells to be integrated into the existing neural network properly, normal developments of dendrites and dendritic spines are essential. The integration process takes 6∼8 weeks in mice (Aimone et al., 2014; Christian et al., 2014; Bergami et al., 2015), and it may be even longer in other species (Kohler et al., 2011), making this process potentially susceptible to environmental changes. underlying mechanism remains to be fully elucidated (Anderson- Hanley et al., 2003; Minisini et al., 2004; Vardy and Tannock, 2007; Janelsins et al., 2011; Jim et al., 2012; Lindner et al., 2014; Dietrich et al., 2015; Wefel et al., 2015). Animal studies have found that chemotherapeutic agents, including CPP, profoundly slow the rate of adult hippocampal neurogenesis (Mustafa et al., 2008; Seigers et al., 2008, 2009, 2010; Yang et al., 2010; Briones and Woods, 2011; Lyons et al., 2011; Christie et al., 2012; ElBeltagy et al., 2012; Nokia et al., 2012). Frontiers in Cellular Neuroscience | www.frontiersin.org DISCUSSION Because adult hippocampal neurogenesis is implicated in functions that overlap with those general functions of hippocampus (Clelland et al., 2009; Sahay et al., 2011; Aimone et al., 2014; Christian et al., 2014; Opendak and Gould, 2015), in addition to studying the rate of neurogenesis, studying the intrinsic electrophysiological properties and integration of newborn granule cells under the influence of chemotherapeutic agents is needed. For newborn granule cells to be integrated into the existing neural network properly, normal developments of dendrites and dendritic spines are essential. The integration process takes 6∼8 weeks in mice (Aimone et al., 2014; Christian et al., 2014; Bergami et al., 2015), and it may be even longer in other species (Kohler et al., 2011), making this process potentially susceptible to environmental changes. Clinical dosages of CPP vary according to the regimens of treatment. For intravenous injection, 40–50 mg/kg of CPP has been recommended for a consecutive 4 days (Cyclophosphamide FDA Packet Insert, 2013) in which several cycles of treatment may be given. Fifty milligram per kilogram of CPP was chosen for the examinations of cognitive functions and dendritic changes June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 7 Cyclophosphamide and Dendritic Development Wu et al. Wu et al. few spines to be more active and consequently larger. Indeed, increases in mushroom-like dendritic spines have been observed in later stages of Alzheimer’s disease-like pathology (Dickstein et al., 2010) and in Fyn defect mice (Babus et al., 2011) as well. It remains unknown if direct damage to CA1 pyramidal cells by CPP might have affected the development of newborn granule cells. (Bergami et al., 2008; Chan et al., 2008; Wang et al., 2015). CPP-induced decrease in total spine density with an increase in mushroom spine density is also opposite to what has been observed after BDNF application (Tyler and Pozzo-Miller, 2003). It is reasonable to consider that the abnormal development of dendrites in newborn granule cells in the presence of CPP might have been at least partially mediated by the down-regulation of BDNF expression. However, future studies are needed to examine if endogenously generated or externally applied BDNF could rescue detrimental developmental changes caused by CPP. The development of dendrites in newborn granule cells is under strong regulatory control of the internal and external environments (Aimone et al., 2014). REFERENCES Bergami, M., Masserdotti, G., Temprana, S. G., Motori, E., Eriksson, T. M., Göbel, J., et al. (2015). A critical period for experience-dependent remodeling of adult-born neuron connectivity. Neuron 85, 710–717. doi: 10.1016/j.neuron. 2015.01.001 Acharya, M. M., Martirosian, V., Chmielewski, N. N., Hanna, N., Tran, K. K., Liao, A. C., et al. (2015). Stem cell transplantation reverses chemotherapy- induced cognitive dysfunction. Cancer Res. 74, 676–686. doi: 10.1158/0008- 5472.CAN-14-2237 Bergami, M., Rimondini, R., Santi, S., Blum, R., Götz, M., and Canossa, M. (2008). Deletion of TrkB in adult progenitors alters newborn neuron integration into hippocampal circuits and increases anxiety-like behavior. Proc. Natl. Acad. Sci. U S A 105, 15570–15575. doi: 10.1073/pnas.0803702105 Ahles, T. A., and Saykin, A. J. (2007). Candidate mechanisms for chemotherapy- induced cognitive changes. Nat. Rev. Cancer 7, 192–201. doi: 10.1038/nrc2073 Boda, B., Alberi, S., Nikonenko, I., Node-Langlois, R., Jourdain, P., Moosmayer, M., et al. (2004). The mental retardation protein PAK3 contributes to synapse formation and plasticity in hippocampus. J. Neurosci. 24, 10816–10825. doi: 10.1523/JNEUROSCI.2931-04.2004 Aimone, J. B., Li, Y., Lee, S. W., Clemenson, G. D., Deng, W., and Gage, F. H. (2014). Regulation and function of adult neurogenesis: from genes to cognition. Physiol. Rev. 94, 991–1026. doi: 10.1152/physrev.00004.2014 Ambrogini, P., Cuppini, R., Lattanzi, D., Ciuffoli, S., Frontini, A., and Fanelli, M. (2010). Synaptogenesis in adult-generated hippocampal granule cells is affected by behavioral experiences. Hippocampus 20, 799–810. doi: 10.1002/hipo. 20679 Briones, T. L., and Woods, J. (2011). Chemotherapy-induced cognitive impairment is associated with decreases in cell proliferation and histone modifications. BMC Neurosci. 12:e124. doi: 10.1186/1471-2202 -12-124 Anderson-Hanley, C., Sherman, M. L., Riggs, R., Agocha, V. B., and Compas, B. E. (2003). Neuropsychological effects of treatments for adults with cancer: a meta-analysis and review of the literature. J. Int. Neuropsychol. Soc. 9, 967–982. doi: 10.1017/s1355617703970019 Castellon, S. A., Ganz, P. A., Bower, J. E., Petersen, L., Abraham, L., and Greendale, G. A. (2004). Neurocognitive performance in breast cancer survivors exposed to adjuvant chemotherapy and tamoxifen. J. Clin. Exp. Neuropsychol. 26, 955–969. doi: 10.1080/138033904905 10905 Andreotti, C., Root, J. C., Ahles, T. A., McEwen, B. S., and Compas, B. E. (2015). Cancer, coping, and cognition: a model for the role of stress reactivity in cancer-related cognitive decline. Psychooncology 24, 617–623. doi: 10.1002/ pon.3683 Chan, J. P., Cordeira, J., Calderon, G. A., Iyer, L. K., and Rios, M. (2008). DISCUSSION It remains unclear what mechanism(s) may underlie abnormal development of new born granule cells after CPP exposure. BDNF, along with nerve growth factor (NGF), neurotrophin-3 (NT3) and neurotrophin-4/5 (NT4/5), belongs to a family of closely related, small, secreted proteins called neurotrophins. Mainly through acting on tyrosine receptor kinases, BDNF affects neuronal proliferation, differentiation, survival and morphological maintenance, as well as dendrite outgrowth (Waterhouse and Xu, 2009). Additionally, BDNF is known to be involved in numerous processes of functional and structural synaptic plasticity (Zagrebelsky and Korte, 2014). Evidence obtained from the hippocampal dentate gyrus shows that global over-expression of BDNF promotes growth of dendrites in dentate granule cells (Tolwani et al., 2002), while deficits in BDNF signaling lead to abnormal dendritic development of newborn granule cells (Bergami et al., 2008; Chan et al., 2008; Wang et al., 2015). In addition to dendritic morphology, it appears that following BDNF deficits, the dendritic spines display more dramatic and specific changes than dendrite morphology (Zagrebelsky and Korte, 2014). The features of dendritic abnormality after CPP treatment are very similar to those caused by BDNF deficits, which include decreased dendrite branching, length and complexity In conclusion, adult-born granule cells in the presence of CPP, a widely used anti-cancer medication, display abnormal dendritic morphologies and fewer dendritic spines, which may relate to chemotherapeutic agents-induced cognitive dysfunction. ACKNOWLEDGMENTS This study was supported by a grant from the National Natural Science Foundation of China (No: 31371183) to R-ZZ. The authors would like to thank Prof. Yanxun Liu and Mrs. Qian Zhu in the Shandong University School of Public Health for their help in statistical analyses. AUTHOR CONTRIBUTIONS LW did most of animal experiments, behavioral studies and imaging data processing. DG and QL did immunohistochemistry and data analysis. FG, XS and XW did confocal imaging. FW helped in experimental design. R-ZZ designed the study and wrote the article. 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Enhanced tonic GABA current in normotopic and hilar ectopic dentate granule cells after pilocarpine-induced status epilepticus. J. Neurophysiol. 102, 670–681. doi: 10.1152/jn.00147.2009 Frontiers in Cellular Neuroscience | www.frontiersin.org REFERENCES Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092. doi: 10.1038/nn736 Yang, M., Kim, J. S., Song, M. S., Kim, S. H., Kang, S. S., Bae, C. S., et al. (2010). Cyclophosphamide impairs hippocampus-dependent learning and memory in adult mice: possible involvement of hippocampal neurogenesis in chemotherapy-induced memory deficits. Neurobiol. Learn. Mem. 93, 487–494. doi: 10.1016/j.nlm.2010.01.006 Minisini, A., Atalay, G., Bottomley, A., Puglisi, F., Piccart, M., and Biganzoli, L. (2004). What is the effect of systemic anticancer treatment on cognitive function? 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Neuropharmacology 76, 628–638. doi: 10.1016/j.neuropharm.2013. 05.029 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Zhan, R.-Z., and Nadler, J. V. (2009). Enhanced tonic GABA current in normotopic and hilar ectopic dentate granule cells after pilocarpine-induced status epilepticus. J. Neurophysiol. 102, 670–681. doi: 10.1152/jn.00147.2009 Copyright © 2017 Wu, Guo, Liu, Gao, Wang, Song, Wang and Zhan. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. p y j Zhao, C., Jou, J., Wolff, L. J., Sun, H., and Gage, F. H. (2014). Spine morphogenesis in newborn granule cells is differentially regulated in the outer and middle molecular layers. J. Comp. Neurol. 522, 2756–2766. doi: 10.1002/cne. 23581 June 2017 | Volume 11 | Article 171 Frontiers in Cellular Neuroscience | www.frontiersin.org 10
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DOI https://doi.org/10.36059/978-966-397-287-9-116 DOI https://doi.org/10.36059/978-966-397-287-9-116 КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ Попович Ольга Станіславівна кандидат юридичних наук, науковий співробітник відділу дослідження проблем кримінального права Науково-дослідного інституту вивчення проблем злочинності імені академіка В. В. Сташиса Національної академії правових наук України м. Харків, Україна Сучасний міжнародний тероризм є одним з нових факторів впливу на світову політику, стан і розвиток міжнародної системи постбіполярного часу. Терористичні акти дедалі частіше спрямовані на масові людські жертви, спричиняють руйнування матеріальних і підрив духовних цінностей, провокують війни, недовіру та ненависть між соціальними, національними і релігійними групами, що сприяє виникненню і поглибленню системних криз. Зарубіжний досвід боротьби з терористичними актами був предметом наукового інтересу багатьох вчених. Іспанський досвід боротьби з терористичною діяльністю є предметом наукових досліджень багатьох українських і зарубіжних правознавців. Їх надбання становлять собою цінне підґрунтя для набуття наукових знань у цьому напрямку. Кримінальну відповідальність за низку терористичних злочинів містить книга ІІ «Злочини та покарання» в розділу XXII «Злочини проти публічного порядку» (глава VII «Про терористичні організації і групи та терористичні злочини») КК Королівства Іспанія [1]. Ця глава складається з 2-х частин «Про терористичні організації і групи» (ст. 571) і «Про терористичні злочини» (статті 572‒580). Особливо корисним для пізнання вважаємо ч. 1 ст. 573 КК Іспанії, де терористичним злочином визнано: (1) будь-який тяжкий злочин проти Корони, життя, свободи, фізичної недоторканості, моральної цілісності особи, статевої свободи й недоторканості, її власності; (2) проти природних ресурсів, навколишнього природного середовища, суспільного здоров’я; (3) злочинне діяння, що може спричинити ризики катастрофи, пожежі; (4) злочини, що становить собою збройний напад, володіння, зберігання і торгівлю зброї, боєприпасів і вибухових речовин, передбачених цим Кодексом; (5) захоплення кораблів, літаків чи інших транспортних засобів, якщо воно вчинено з такими цілями, як-от: (а) повалення конституційного ладу, припинення або тяжка дестабілізація 432 діяльності економічних, політичних інститутів або соціальних структур держави; (б) примушування публічної влади вчинити певну дію чи не вчиняти її; (в) серйозне порушення громадського спокою; (г) серйозна дестабілізація діяльності міжнародної організації; (д) провокація стану страху серед населення або його частини. діяльності економічних, політичних інститутів або соціальних структур держави; (б) примушування публічної влади вчинити певну дію чи не вчиняти її; (в) серйозне порушення громадського спокою; (г) серйозна дестабілізація діяльності міжнародної організації; (д) провокація стану страху серед населення або його частини. р у р Згідно з ч. 2 ст. 573 розглядуваного Кодексу терористичними злочинами також вважаються інформаційні злочини, передбачені у статтях 197bis та 197 [2, с. 80, 81]. Згідно з ч. 2 ст. 573 розглядуваного Кодексу терористичними злочинами також вважаються інформаційні злочини, передбачені у статтях 197bis та 197 [2, с. 80, 81]. КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ 3 цієї ж статті за розроблення біологічної або хімічної зброї, перевезення, володіння чи будь-яке сприяння іншому поводженню з ядерними речовинами чи матеріалами, які призначені для вироблення радіоактивного випромінювання, або відповідними пристроями. України. Частиной 2 ст. 574 Кодексу підвищено кримінальну відповідальність у виді позбавлення волі на строк від десяти до двадцяти років за ті ж дії, скоєні з метою тероризму, стосовно ядерної, хімічної, радіоактивної або біологічної зброї, боєприпасів, речовин до неї або пристроїв. Таке ж покарання передбачено у ч. 3 цієї ж статті за розроблення біологічної або хімічної зброї, перевезення, володіння чи будь-яке сприяння іншому поводженню з ядерними речовинами чи матеріалами, які призначені для вироблення радіоактивного випромінювання, або відповідними пристроями. України. Частиной 2 ст. 574 Кодексу підвищено кримінальну відповідальність у виді позбавлення волі на строк від десяти до двадцяти років за ті ж дії, скоєні з метою тероризму, стосовно ядерної, хімічної, радіоактивної або біологічної зброї, боєприпасів, речовин до неї або пристроїв. Таке ж покарання передбачено у ч. 3 цієї ж статті за розроблення біологічної або хімічної зброї, перевезення, володіння чи будь-яке сприяння іншому поводженню з ядерними речовинами чи матеріалами, які призначені для вироблення радіоактивного випромінювання, або відповідними пристроями. р р За ч. 1 ст. 575 КК Іспанії карається позбавленням волі на строк від двох до п’яти років особа, яка пройшла бойове або військове навчання (підготовку), а також навчання (підготовку) для розроблення біологічної або хімічної зброї, виготовлення вогненебезпечних, вибухових, задушливих, запалювальних чи інших небезпечних речовин, скоєне з метою вчинення терористичних злочинів. Згідно з частинами 2 і 3 цієї ж статті таке ж покарання встановлене за різного роду підготовку до вчинення злочинів, передбачених ч. 1 ст. 575 цього Кодексу, зокрема, за надання послуг зв’язку (в мережі Інтернет тощо), за підготовку документів, якщо ці діяння вчинені умисно і навіть на території інших держав. Стаття 576 передбачено покарання у виді позбавлення волі на строк від п’яти до десяти років і штрафу від трьох до п’яти його мінімальних розмірів за будь-яке пряме, а також непряме, використання будь-яких товарів чи цінностей для сприяння терористичній діяльності, тобто фінансування тероризму. Посилене покарання може бути призначено, якщо товари чи цінності передано особі, яка є відповідальною за вчинення терористичного злочину. За фінансування терористичної діяльності у ст. 576 КК Іспанії передбачено кримінальну відповідальність і юридичної особи у виді штрафу. Відповідно до ч. 1 ст. 577 КК Іспанії будь-яке співробітництво з терористичною групою або організацією з метою вчинення терористичного злочину карається позбавленням волі на строк від п’яти до десяти років. У ч. КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ Статтею 573bis КК Іспанії встановлено покарання, що призначаються за вчинення терористичних злочинів, передбачених у попередній нормі. Відповідно до § 1 – 4 ч. 2 ст. 573bis цього Кодексу скоєння терористичного злочину карається позбавленням волі: Статтею 573bis КК Іспанії встановлено покарання, що призначаються за вчинення терористичних злочинів, передбачених у попередній нормі. Відповідно до § 1 – 4 ч. 2 ст. 573bis цього Кодексу скоєння терористичного злочину карається позбавленням волі: − на максимальний строк у випадку спричинення смерті хоча б одній особі; – на строк від двадцяти до двадцяти п’яти років, якщо злочинні дії супроводжувалися викраденням або незаконним позбавленням волі людини, внаслідок яких особу не знайдено; – строком від п’ятнадцяти до двадцяти років, якщо злочинні дії призвели до аборту або наслідків, передбачених ст. 149 (тілесні ушкодження тяжкі), ст. 150 (тілесні ушкодження середньої тяжкості), ст. 157 або 158 (пошкодження плода людини або його захворювання), ст. 346 (вчинені загальнонебезпечним способом різного роду руйнування) чи ст. 351 цього Кодексу (небезпечної для життя або здоров’я особи пожежі); – строком від десяти до п’ятнадцяти років, якщо злочинні дії заподіяли інше тілесне ушкодження або супроводжувалися насильством до особи чи погрозами його заподіяти. У ст. 571 розглядуваного Кодексу наведено дефініцію терористичних груп або організацій. Ними визнано угруповання, які характеризуються визначеннями, наведеними у § 2 ч. 1 ст. 570bis та § 2 ч. 1 ст. 570ter і які мають намір або мету вчинення терористичних злочинів. Згідно з ч. 1 ст. 572 цього Кодексу позбавленням волі від восьми до чотирнадцяти років, забороною займати публічну посаду на строк від шести до чотирнадцяти років караються діяння особи, яка засновує, організовує чи керує терористичною групою чи організацією чи сприяє цьому. Частина 1 ст. 574 КК Іспанії містить кримінальну відповідальність у виді позбавлення волі на строк від восьми до п’ятнадцяти років за зберігання зброї, боєприпасів, володіння або зберігання вибухових, запалювальних, легкозаймистих або задушливих речовин, пристроїв чи їх компонентів, виготовлення, транспортування, продаж, або поставку зазначених предметів у будь-якій формі, їх використання чи розміщення з метою вчинення терористичних злочинів, перелічених у ч. 1 ст. 573 КК 433 України. Частиной 2 ст. 574 Кодексу підвищено кримінальну відповідальність у виді позбавлення волі на строк від десяти до двадцяти років за ті ж дії, скоєні з метою тероризму, стосовно ядерної, хімічної, радіоактивної або біологічної зброї, боєприпасів, речовин до неї або пристроїв. Таке ж покарання передбачено у ч. КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ Київ, 30 верес. 2016 р.). Київ : Нац. акад. прокуратури України, 2016. 432 с. DOI https://doi.org/10.36059/978-966-397-287-9-117 КРИМІНАЛЬНА ВІДПОВІДАЛЬНІСТЬ ЗА ТЕРОРИСТИЧНУ ДІЯЛЬНІСТЬ В ІСПАНІЇ 3 цієї ж норми визначено покарання у виді штрафу за вчинення цього злочину з необережності. Згідно з ч. 1 ст. 578 позбавленням волі на строк від одного до трьох років карається: (а) публічне виправдання або звеличення терористичних злочинів, передбачених статтями 572 – 577 цього Кодексу Іспанії, осіб, які їх учинили; (б) зневага, дискредитація або приниження жертв зазначених злочинів, а також членів їх сімей. Відповідно до ч. 1 ст. 579 поширення повідомлень і гасел з метою підбурювання інших осіб до вчинення терористичних злочинів карається штрафом. Крім того, у ст. 579bis Кодексу Іспанії передбачено можливість призначення особам, засудженим за терористичні злочини, додаткових покарань у виді позбавлення права займатися певними видами діяльності [2, с. 80‒82]. Статтею 580 досліджуваного Кодексу спеціально закріплено 434 обов’язок визнання винесених раніше вироків іноземних судів за скоєння терористичних злочинів з обтяжуючою обставиною у виді рецидиву [1, с. 245]. Як відмічають фахівці-дослідники, проаналізовані вище норми КК Іспанії забезпечують сувору кримінальну відповідальність за вчинення злочинів, пов’язаних з тероризмом [49, с. 82]. Література: 1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин- ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну діяльність в Іспанії. Протидія терористичній діяльності: міжнародний досвід і його актуальність для України : матеріали між нар. наук.-практ. конф. (м. Київ, 30 верес. 2016 р.). Київ : Нац. акад. прокуратури України, 2016. 432 с. Література: 1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин- ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну діяльність в Іспанії. Протидія терористичній діяльності: міжнародний досвід і його актуальність для України : матеріали між нар наук практ Література: 1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин- ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. Література: 1. Кримінальний кодекс Королівства Іспанія / під ред. В. Л. Менчин- ського. Перекл. на укр. мову О. В. Лішевської. Київ : ОВК, 2017. 284 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну діяльність в Іспанії. Протидія терористичній діяльності: міжнародний досвід і його актуальність для України : матеріали між нар. наук.-практ. конф. (м. Київ, 30 верес. 2016 р.). Київ : Нац. акад. прокуратури України, 2016. 432 с. 2. Гуркевич В. Кримінальна відповідальність за терористичну діяльність в Іспанії. Протидія терористичній діяльності: міжнародний досвід і його актуальність для України : матеріали між нар. наук.-практ. конф. (м. Пустова Наталія Олександрівна у др аспірантка кафедри кримінального права і кримінології факультету № 1 Інституту з підготовки фахівців для підрозділів Національної поліції Львівського державного університету внутрішніх справ м. Львів, Україна Кримінальне право розвивається циклічно під впливом двох тенденції, які споконвіку конкурують між собою. Перша – посилює кримінальну відповідальність, яка виражається у розширенні її підстав шляхом криміналізації, надмірної диференціації, а також у підвищенні суворості покарання, скороченні можливостей для звільнення від кримінальної відповідальності та покарання тощо. Друга – пом’якшує кримінальну відповідальність, що полягає у декриміналізації та депеналізації, пом’якшенні інших кримінально-правових наслідків вчинення злочину. Динамізм кримінального законодавства залежить від низки різноманітних чинників: політичних, соціальних, економічних, міжнародно- правових. Неабиякий вплив також має наука кримінального права і практика застосування приписів Кримінального кодексу України (далі – КК 435
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The Role of the Arabidopsis Exosome in siRNA–Independent Silencing of Heterochromatic Loci
PLOS genetics
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Abstract The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: chekanovaj@umkc.edu * E-mail: chekanovaj@umkc.edu . These authors contributed equally to this work. . These authors contributed equally to this work. The exosome complex plays a central role in RNA metabolism in eukaryotes. Evolutionarily conserved from archaea to humans, the exosome is a stable complex of RNase-like and RNA binding proteins that catalyzes 39 to 59 processing and decay of various RNA substrates [15]. The current view of eukaryotic exosome structure is based mostly on studies done in yeast and human. The eukaryotic exosome has nuclear and cytoplasmic forms that share ten components. The key structural feature is a nine-subunit donut-shaped structure called the exosome ring. Six of the subunits, RNase PH domain-containing proteins Rrp41, Rrp42, Rrp43, Rrp45, Rrp46 and Mtr3, are organized into a hexameric ring, capped on one side by a trimer of subunits that contain S1 and KH RNA binding domains (Rrp40, Rrp4 and Csl4) [16,17]. The 9-subunit ring structure has no catalytic activity in yeast and human, due to amino acid replacements that disable binding of RNA, phosphate ion, or catalysis [16,17]. The exosome active sites are contributed by the tenth protein, Rrp44 (Dis3), which has endonucleolytic and exonucleolytic activities and considered to be the tenth subunit of the exosome core [18,19]. In addition to Rrp44, the nuclear form of the eukaryotic exosome is associated with a second active 39 to 59 exonuclease, Rrp6 [20,21]. Most functions of the exosome are dependent on cofactors. One of the notable complexes associated with the nuclear exosome is the Trf- Air-Mtr4 polyadenylation (TRAMP) complex endowed with a poly(A) polymerase activity that stimulates degradation [22–24]. Jun-Hye Shin., Hsiao-Lin V. Wang., Jinwon Lee, Brandon L. Dinwiddie, Dmitry A. Belostotsky, Julia A. Chekanova* School of Biological Sciences, University of Missouri–Kansas City, Kansas City, Missouri, United States of America Abstract The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In Arabidopsis, exosome regulates expression of a ‘‘hidden’’ transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation. Citation: Shin J-H, Wang H-LV, Lee J, Dinwiddie BL, Belostotsky DA, et al. (2013) The Role of the Arabidopsis Exosome in siRNA–Independent Silencing of Heterochromatic Loci. PLoS Genet 9(3): e1003411. doi:10.1371/journal.pgen.1003411 Received December 19, 2012; Accepted February 11, 2013; Published March 28, 2013 pyright:  2013 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, whic e, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright:  2013 Shin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, use, distribution, and reproduction in any medium, provided the original author and source are credited. et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted roduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by grants from USDA to DAB and JAC (grant #35301-18207), from MLSRB to JAC (grant #13230), NSF to DAB and JAC (grant #0724168), and NIH to DAB and JAC (#GM073872). The Role of the Arabidopsis Exosome in siRNA– Independent Silencing of Heterochromatic Loci Jun-Hye Shin., Hsiao-Lin V. Wang., Jinwon Lee, Brandon L. Dinwiddie, Dmitry A. Belostotsky Julia A. Chekanova* Author Summary To maintain genomic stability and prevent expansion of invasive genomic sequences such as transposable ele- ments (TEs), eukaryotes have evolved defensive mecha- nisms to control them. Here, we examine the role of the Arabidopsis exosome complex in such mechanisms. Evo- lutionarily conserved from archaea to humans, the exosome is a stable complex of RNase-like and RNA binding proteins that plays a central role in RNA metabolism in eukaryotes. Depletion of the exosome allows some repetitive sequences to escape from silencing. Most of these transcripts emanate from centromeric and pericentromeric chromosomal regions and other hetero- chromatic loci, and many derive from repetitive and transposable elements. In plants, TEs are targeted for de novo DNA methylation by smRNA–mediated pathways. However, we found that exosome depletion has only minor effects on smRNA populations that are acting in the main silencing mechanism in Arabidopsis, siRNAs–depen- dent DNA methylation RdDM. Instead, exosome depletion affects histone H3K9 dimethylation, an epigenetic mark that affects chromatin structure and thus alters transcrip- tion. Our data suggest that the exosome collaborates in gene silencing, likely acting in a parallel pathway to other mechanisms. We also propose that the Arabidopsis exosome may coordinate the transcriptional interplay of different RNA polymerases to modulate repression of some repetitive sequences. The model system in which siRNA-mediated silencing is the best understood mechanistically is fission yeast. In S. pombe RNA Pol II carries out the functions attributed to Pol IV and Pol V in plants, therefore, it generates both siRNA precursors and scaffold transcripts to which siRNAs bind at loci that are subject to siRNA- mediated silencing. Exosome defects in S. pombe were reported to result in the loss of transcriptional silencing from centromeric, silent mating type, and telomeric loci [38–40]. In S. pombe, in the absence of exosome-mediated degradation, abundant aberrant RNA species enter the RNAi pathway and interfere with heterochromatic silencing through competition for RNAi biogen- esis machinery, resulting in the dramatic decrease in centromeric siRNAs [38–40]. Recently, it was also shown that exosome plays an important role in remodeling of facultative heterochromatin [41]. Earlier work in plants also suggested that aberrant RNAs could enter RNAi pathways unless they are degraded by the 59 to 39 pathway [42]. However, the role of the exosome complex in smRNA metabolism in Arabidopsis has not been examined. It is also not known whether the Arabidopsis exosome complex interacts with the RdDM silencing pathway. Introduction High-throughput analyses have revealed that eukaryotic genomes are pervasively transcribed [1–4], and the majority of the transcriptional activity takes place outside of protein-coding genes, producing non-coding RNAs (ncRNAs) derived from genome regions once thought to be transcriptionally silent, including intergenic and heterochromatic regions [1–3,5]. Per- vasive transcription constitutes a risk for the cell, as it can be associated with expansion of TEs, loss of genomic stability and defects in gene expression. However, recent studies have also shown that ncRNAs themselves can have important regulatory functions, including the establishment and maintenance of the epigenetic architecture of eukaryotic genomes. In some cases, long ncRNAs serve directly as molecular scaffolds for recruiting chromatin modifiers [6,7], whereas in other cases ncRNAs are processed by the RNAi machinery into short interfering siRNAs that guide DNA methylation and chromatin modifications to homologous regions of the genome [8,9]. Thus, RNA-mediated heterochromatin formation requires an affected region to be transcribed for transcriptional silencing to occur. Many of the ncRNA transcripts earned the term ‘‘hidden’’ because they remain invisible unless RNA degradation is prevented, for example, by inactivation of the degradation machinery [1,3,4,10–14], raising the important question of how these ncRNAs are regulated. March 2013 | Volume 9 | Issue 3 | e1003411 1 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing biogenesis, RNA Pol IV transcripts are made double-stranded by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), processed into 24 nt siRNA by DICER-LIKE 3 (DCL3), and then incorporated into ARGONAUTE (AGO4 and possibly AGO6) to form an AGO-siRNA complex [32–35]. The AGO- siRNA complex and other RdDM effectors [31,35–37], assemble on scaffold RNA to form a guiding complex that recruits DNA methyltransferases and histone methyltransferases to direct the silencing of specific genomic loci through a mechanism that is not fully understood. Pol IV is thought to initiate RdDM pathway, whereas Pol V and AGO4-associated siRNAs function down- stream from Pol IV to promote DNA methylation by recruiting the silencing complex to targeted loci. RNA Pol IV, Pol V and Pol II activities in RdDM are functionally diversified and coordinated; however, it is not yet clear how they are functionally integrated in heterochromatin silencing. Author Summary The plant exosome might differ from yeast and human exosomes, as its ring subunit Atrrp41p appears to retain an active site and was also shown to have catalytic activity in vitro [1,25]. Our previous genome-wide study using tiling microarrays to examine exosome targets in Arabidopsis revealed that a large number of exosome substrates correspond to ncRNAs originated from promoters, 59UTRs, intergenic regions, repetitive elements and TEs [1]. Many of these ncRNAs derive from centromeric and pericentromeric regions and other heterochromatic loci known to give rise to smRNAs that participate in silencing of these loci [26]. In Arabidopsis, the main and most-studied pathway for transcrip- tional gene silencing of repetitive elements and transposons is the siRNA-based silencing mechanism known as RNA-dependent DNA methylation (RdDM) [9,27–29]. The effects of exosome depletion on these ncRNAs and, potentially, on smRNAs are unlikely to be attributable to indirect effects of exosome depletion on the expression of RdDM pathway components, since no genes acting in siRNA biogenesis, siRNA-mediated transcriptional gene silencing (TGS), DNA methylation or demethylation, or histone H3K9 modifications were found to be affected in these lines [1]. To answer these questions we employed next-generation sequencing to profile populations of smRNAs in exosome-depleted plants, and in mutants of RdDM pathway genes. Unexpectedly, we found that loss of the exosome subunits had little effect on the global populations of smRNAs and had no affect on the level of DNA methylation in examined RdDM loci; rather, it resulted in a reduction of histone H3K9 dimethylation. We propose that the Arabidopsis exosome may coordinate the transcriptional interplay of RNA polymerases Pol II, Pol V and Pol IV, to achieve the appropriate level of transcriptional repression of heterochromatic loci. Results RdDM induces de novo methylation of cytosines in all sequence contexts at the region of siRNA–DNA or siRNA-RNA sequence homology. This silencing pathway requires two plant-specific RNA polymerases, Pol IV and Pol V, specializing in transcrip- tional gene silencing (TGS) [28], although transcriptional activity of Arabidopsis Pol II was also reported to be involved in siRNA- directed gene silencing [30]. The mechanistic details of RNA- dependent silencing are not fully understood and also appear to vary from one genomic location to another, but the RdDM pathway likely consists of three main steps: (i) siRNA production from transcripts that are likely transcribed by RNA Pol IV [9], (ii) synthesis of non-coding RNAs that could serve as scaffolds by RNA Pol V and/or Pol II at some of the loci [30,31], and (iii) assembly of AGO-siRNA effector complexes to recruit methyla- tion machinery to complementary sequences [9]. In siRNA March 2013 | Volume 9 | Issue 3 | e1003411 Exosome depletion does not affect smRNA profiles Previously, we found that the majority of transcripts upregulated in RRP4 and RRP41 exosome depletion mutants originate from the promoters, repeats, intergenic, and siRNA generating regions [1]. Most of these regions harbor repeats and TEs that are known to be silenced by RdDM through siRNAs. Since microarray experiments allow estimation of only the length of affected regions, but not the exact length of affected transcripts, we set out to examine whether the exosome is involved in down regulation of these regions through regulating either quantity or quality of smRNAs. To characterize any changes in smRNA populations that occur in response to exosome depletion, we employed next-generation sequencing to deep sequence the smRNA populations in depletion mutants of exosome subunits March 2013 | Volume 9 | Issue 3 | e1003411 2 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing and smRNAs between 15- and 32 nt in length were selected and mapped to the Arabidopsis genome (TAIR version 9). RRP4 and RRP41. Null T-DNA insertion mutations in RRP4 and RRP41 are lethal; therefore, we used inducible RNA-interference (iRNAi) constructs to reduce RRP4 and RRP41. The seedlings of RRP4 (rrp4-i) or RRP41 (rrp41-i) transgenic plants grown on estradiol-containing medium to induce the RNAi constructs subsequently exhibit a growth arrest ([1], Figure 1A). We selected the earliest time-point of estradiol treatment corresponding to the accumulation of underprocessed 5.8S rRNA species (the hallmark of the exosome defect), but before growth retardation, to minimize changes in gene expression that did not result directly from exosome depletion [1]. Small RNA libraries for Illumina sequencing were generated from the seedlings of rrp4-i and rrp41-i iRNAi lines grown with and without estradiol (Table S1) We first examined the smRNAs from the iRNAi transgenes used for inactivation of RRP4 or RRP41 [1]. As expected, these silencing cassettes generate silencer sequences corresponding to RRP4 or RRP41 (mapping to AT1G03360 and AT3G61620 loci). Profiling silencer sequences by size and by first nucleotide revealed that the majority of the silencer sequences are 21, 22 and 24 nt and start with 59U or 59A (Figure S1), suggesting that they are preferentially loaded into Ago1, Ago2 and Ago4 complexes [43] to silence their target. Silencer sequences produced from iRNAi transgenes were filtered out and libraries without silencer reads were termed FLR, for filtered reads (Table S1). Each library was normalized either to Figure 1. Exosome depletion does not affect populations of smRNAs corresponding to repeats and transposable elements To further investigate whether the exosome participates in gene silencing and interacts with the RdDM pathway, we examined the transcription patterns of several specific loci regulated through RdDM. solo LTR and AtSN1 are the heterochromatic loci for which the role of RdDM players in their silencing and interactions between them are best-understood [30,31,50–52]. Transcriptional silencing of solo LTR and AtSN1 heterochromatic loci are dependent on Pol IV and Pol V [30,31,50–52]. Based on previous studies, both solo LTR and AtSN1 loci can be subdivided into region A and an adjacent region B [30,31]. Region A represents the siRNA-generating region likely transcribed by Pol IV, and region B gives rise to the ncRNAs that are proposed to serve as a scaffold for recruiting siRNA-mediated complexes that form heterochromatin (Figure 4A). Pol V was proposed to produce transcripts which serve as the scaffolds [31], although in case of solo LTR, Pol II was also shown to be involved [30]. In Arabidopsis, repeats and TEs are silenced by siRNAs through RdDM; therefore, we examined the effect of exosome loss on 20–25 nt smRNAs corresponding specifically to TEs and repeats. Surprisingly, we saw no changes in the groups of smRNAs mapped to tandem repeats (TR), inverted repeats (IR), dispersed repeats (DR) or the group of TEs in both exosome mutants (Figure 2A and 2B). The diverse heterochromatic siRNAs participating in TE silencing are mostly 24-mers and are Pol IV- and/or Pol V-dependent [9]. Most siRNA production relies on Pol IV, but there are also Pol V-dependent and Pol IV-independent siRNA-generating loci [44,45]. Therefore, to examine whether the exosome complex functionally overlaps with the components of the RdDM pathway, we constructed lines containing rrp4-i or rrp41-i iRNAi and mutations affecting Pol IV, Pol V, RDR2 and DCL3, which are nrpd1, nrpe1, dcl3 and rdr2 respectively (allele numbers provided in Methods). This approach also allowed us to confirm that smRNAs observed in exosome depletion lines are siRNAs produced by components of the RdDM pathway and not short RNA degradation products accumulated in the absence of functional exoribonucleolytic complex. We then used real-time RT–PCR to examine the levels of transcript produced from region A, as a measure of the silencing status of each locus. We found that exosome defects resulted in accumulation of polyadenylated ncRNA produced from both regions A and B of solo LTR (Figure 4B). Exosome depletion does not affect smRNA profiles Profiling repeat- and transposable element-generated smRNAs by their size confirmed that the exosome defect did not affect the group of 20–25 nt smRNAs even in Pol IV, Pol V, RDR2 and DCL3 deficient genetic backgrounds. Typically, siRNAs participating in RdDM are 24 nt long; therefore we profiled smRNAs mapping to transposable elements by length, but observed no change in abundance of 24 nt smRNAs (Figure 2E). Further analysis of the 24 nt smRNAs mapped specifically to the different transposable element superfamilies led to the same conclusion (Figure 2F and 2G). We therefore concluded that there are no significant changes in the populations of siRNAs corresponding to repeats and TE superfamilies in exosome depletion mutants. We also did not observe any significant differences in amounts of mature 21-mer miRNAs. The results of our sequencing analysis were confirmed by Northern blot analysis (Table S3, Figure 3, Figure S2). Together, these data suggest that the Arabidopsis exosome complex is not involved in siRNA metabolism on a global scale. Nevertheless, we can not exclude the possibility that exosome might control a small number of smRNA precursor transcripts at a few specific loci that would have been missed in our experiments and with the data processing approach we took while dissecting differences on genomic level. the total number of mapped non-redundant reads or to the total number of non-redundant filtered reads (FLR), multiplied by 106 (RPM, reads per million). Both methods of normalization produced similar results; therefore, only data normalized using filtered reads (FLR) are presented graphically in this study. We then classified smRNAs based on their size, the nature of their first nucleotide, and their genomic features. The majority of functional smRNAs in A. thaliana range from 21 to 24 nt. Our libraries were constructed using 15–32 nt smRNAs; therefore, we were able to detect any effect exosome depletion might have on smRNA metabolism. We found that exosome defect does not lead to changes in smRNAs in the 15–19 nt and 26–32 nt categories (data not shown). Importantly, the group of 20–25 nt smRNAs, which contains the majority of functional smRNAs, was present in similar proportions, although with minor variations, relative to the number of total reads in the libraries of both of exosome depletion mutants and in their corresponding non-induced lines, and constituted about half of total smRNAs mapped to the genome (Table S1, Figure 1B). Exosome depletion does not affect smRNA profiles Characterization of up-regulated loci and smRNA populations upon depletion of exosome subunits RRP4 and RRP41. (A) Phenotypes of rrp41 iRNAi/nrpd1, rrp41 iRNAi/nrpe1, rrp41 iRNAi/dcl3, rrp41 iRNAi/rdr2 double mutants. RRP4 and RRP41 correspond to the iRNAi lines grown without estradiol and rrp4-i and rrp41-i correspond to lines grown on estradiol-containing medium, to induce the RNAi-mediated knockdown of RRP4 and RRP41, respectively. (B) 20–25 nt smRNAs sequences profiled by size in exosome depletion mutants rrp4-i and rrp41-i. (C) The relative frequency of each 59 terminal nucleotide among populations 20–25 nt smRNAs in rrp4-i and rrp41-i mutants. (D) Genomic features of loci generating 20–25 nt small RNAs upon depletion of exosome subunits, according to TAIR9 annotation units. doi:10.1371/journal.pgen.1003411.g001 Figure 1. Characterization of up-regulated loci and smRNA populations upon depletion of exosome subunits RRP4 and RRP41. (A) Phenotypes of rrp41 iRNAi/nrpd1, rrp41 iRNAi/nrpe1, rrp41 iRNAi/dcl3, rrp41 iRNAi/rdr2 double mutants. RRP4 and RRP41 correspond to the iRNAi lines grown without estradiol and rrp4-i and rrp41-i correspond to lines grown on estradiol-containing medium, to induce the RNAi-mediated knockdown of RRP4 and RRP41, respectively. (B) 20–25 nt smRNAs sequences profiled by size in exosome depletion mutants rrp4-i and rrp41-i. (C) The relative frequency of each 59 terminal nucleotide among populations 20–25 nt smRNAs in rrp4-i and rrp41-i mutants. (D) Genomic features of loci generating 20–25 nt small RNAs upon depletion of exosome subunits, according to TAIR9 annotation units. doi:10.1371/journal.pgen.1003411.g001 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 3 SmRNA–Independent Exosome Silencing (Figure 2C) and the rrp41/rdr2 and rrp41/dcl3 double mutants (Figure 2D). Similar to previous reports, we observed a significant reduction in the amount of smRNAs corresponding to TEs, TRs and IRs in nrpd1, nrpe1, rdr2, and dcl3 mutants [27,44,47,48]. Depletion of the exosome in nrpd1, nrpe1 and rdr2 mutants had no effect on the amount of TE and repeat-associated smRNAs produced in these mutants (Table S2, Figure 2C and 2D). Depletion of rrp41 in dcl3 led to a minor restoration of this defect in all groups of repeats and TEs. In the absence of dcl3, other Arabidopsis Dicer proteins are known to process dcl3 substrates [49]; therefore this minor restoration most likely resulted from compensatory effects of other DICER proteins (Table S2, Figure 2D). Exosome depletion does not affect smRNA profiles Therefore, for simplicity we graphed only data corresponding either to 20–25 nt smRNAs, or to smRNAs of one specific length. In addition, the depletion of either RRP4 or RRP41, which are both essential for exosome function, with slight variations, had no effect on the smRNA size distribution (Figure 1B) or the frequencies of their first nucleotide (Figure 1C). All together, these results suggest that defects in exosome function do not lead to accumulation of un-degraded smRNA fragments or to any changes in the cleavage bias of Dicer proteins. Also, exosome depletion did not change proportions of smRNAs mapped to different classes of RNAs, such as mRNAs, tRNAs, rRNAs, and snoRNAs (Figure 1D). Therefore, unlike the situation in S. pombe, where exosome acts as a negative regulator of siRNA biogenesis, Arabidopsis exosome does not act to prevent spurious RNAs from entering RNAi pathway. Exosome depletion does not affect populations of smRNAs corresponding to repeats and transposable elements We then compared the amplitudes of the region A derepression in the rrp41, with rrp41 iRNAi/nrpd1 and rrp41 iRNAi/nrpe1 double mutants relative to the respective single mutants. As previously reported by others [30,31], we observed solo LTR to be significantly derepressed in Pol IV and Pol V single mutants (Figure 4C and 4F). Interestingly, only the combination of exosome defect with mutation of Pol V, Pol IV, Pol V, RDR2 and DCL3 are not essential for viability [27,29,46]. Combining mutations in nrpd1, nrpe1, dcl3 and rdr2 with rrp41-i iRNAi knock-down line did not exacerbate the phenotypes of single exosome depletion mutants (Figure 1A). We next analyzed the smRNAs corresponding to repeats and TEs produced in the rrp41/nrpd1 and rrp41/nrpe1 double mutants March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 4 SmRNA–Independent Exosome Silencing Figure 2. Characterization of 20–25 nt smRNAs corresponding to transposons and repeats in exosome and RdDM mutants (TE = transposable element; TR = Tandem repeat; IR = Inverted repeat; DR = Dispersed repeat) (A) Results of depletion of exosome rrp4 subunit. (B Results of depletion of rrp41 exosome subunit. (C) Results of depletion of rrp41 in nrpd1 and nrpe1 genetic backgrounds. (D) Results of depletion o rrp41 in dcl3 and rdr2 mutants. (E) Characterization of smRNAs mapped to repeats and transposable elements in rrp4-i and rrp41-i libraries profiled based on the reads length (F, G). Classification of 24 nt smRNAs corresponding to the different superfamilies of TEs in rrp4-i (F) and rrp41-i mutants (G [76]. doi:10.1371/journal.pgen.1003411.g002 Figure 2. Characterization of 20–25 nt smRNAs corresponding to transposons and repeats in exosome and RdDM mutants (TE = transposable element; TR = Tandem repeat; IR = Inverted repeat; DR = Dispersed repeat) (A) Results of depletion of exosome rrp4 subunit. (B Figure 2. Characterization of 20–25 nt smRNAs corresponding to transposons and repeats in exosome and RdDM mutants. (TE = transposable element; TR = Tandem repeat; IR = Inverted repeat; DR = Dispersed repeat) (A) Results of depletion of exosome rrp4 subunit. (B) Results of depletion of rrp41 exosome subunit. (C) Results of depletion of rrp41 in nrpd1 and nrpe1 genetic backgrounds. (D) Results of depletion of rrp41 in dcl3 and rdr2 mutants. (E) Characterization of smRNAs mapped to repeats and transposable elements in rrp4-i and rrp41-i libraries profiled based on the reads length (F, G). Exosome depletion does not affect populations of smRNAs corresponding to repeats and transposable elements Classification of 24 nt smRNAs corresponding to the different superfamilies of TEs in rrp4-i (F) and rrp41-i mutants (G) [76]. doi:10.1371/journal.pgen.1003411.g002 doi:10.1371/journal.pgen.1003411.g002 increased in abundance, we carried out strand-specific RT-PCR for the A and B regions. but not with mutation of Pol IV, resulted in the synergistic increase of region A transcript (Figure 4C). Reverse transcription with oligo dT primers does not discriminate between transcripts originating from either DNA strand; thus an elevated level of polyadenylated transcript could result from transcription from either one of the DNA strands. Therefore, to find out which of the transcripts Following standard nomenclature, the top transcript (also called top strand RNA) corresponds to the transcript identical to the sequence of the DNA top strand (and therefore produced from the bottom DNA strand), and the bottom transcript is identical to the March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 5 SmRNA–Independent Exosome Silencing Figure 3. Expression of miRNAs in exosome mutants. miRNA families, miR-159a, miR-167a, miR-173 and variations in sequence length in each family. smRNAs mapped to matching mature miR-159, miR-167miR-173, and miR-167 sequences [94](miRBase release 18) were plotted versus the sum of their normalized reads per million (rpm) from smRNA libraries constructed from RRP4, rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1, RRP4 iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants. Detection of miRNAs by Northern Blot analysis demonstrates that mature miRNA levels are not affected by exosome depletion, and confirms the results of bioinformatic analysis. Total RNA stained with ethidium bromide was used as a loading control. doi:10.1371/journal.pgen.1003411.g003 Figure 3. Expression of miRNAs in exosome mutants. miRNA families, miR-159a, miR-167a, miR-173 and variations in sequence length in each family. smRNAs mapped to matching mature miR-159, miR-167miR-173, and miR-167 sequences [94](miRBase release 18) were plotted versus the sum of their normalized reads per million (rpm) from smRNA libraries constructed from RRP4, rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1, RRP4 iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants. Detection of miRNAs by Northern Blot analysis demonstrates that mature miRNA levels are not affected by exosome depletion, and confirms the results of bioinformatic analysis. Total RNA stained with ethidium bromide was used as a loading control. d i 10 1371/j l 1003411 003 doi:10.1371/journal.pgen.1003411.g003 sequence of DNA bottom strand. The scaffold RNAs were reported to correspond to region B top strand [30,31]. Exosome depletion does not affect populations of smRNAs corresponding to repeats and transposable elements Pol V mutants had no additive or synergistic effect on the level of region A top transcript. Surprisingly, the level of expression of region A top transcript was even somewhat decreased in rrp41 iRNAi/nrpd1 and rrp41 iRNAi/nrpe1, compared to nrpd1 and nrpe1 single mutants, opposite to the pattern we observed for the bottom strand (Figure 4E). Production of scaffold transcripts is central in silencing of the locus and it was reported that even in the presence of functional Pol IV and siRNAs, silencing of solo LTR fails when scaffold RNAs are not produced [30,31]. Similar to previous results [30,31], we observed region A top and bottom transcripts to be significantly derepressed in Pol IV and Pol V single mutants (Figure 4D and 4E), and depletion of RRP41 lead to increased accumulation of the region A top and bottom transcripts (inserts in Figure 4D and 4E). Interestingly, we found that the bottom transcript was synergistically derepressed in rrp41 iRNAi/nrpe1 double mutants relative to nrpe1 and rrp41 iRNAi single mutants, while no change was observed in rrp41 iRNAi/nrpd1 double mutants (Figure 4D). Despite the fact that the exosome defect equally affected the levels of both top and bottom region A transcripts, combining the exosome defect with either Pol IV or We therefore examined the scaffold-producing region B and found that the exosome also affects the amount of region B top transcript, but there is no synergistic increase of this transcript in rrp41 iRNAi/nrpe1 double mutants (Figure 4F and 4G). When we March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 6 SmRNA–Independent Exosome Silencing Figure 4. Effect of exosome subunits depletion on expression of ncRNA transcripts from RdDM-regulated loci. (A) Diagrams of solo LTR and AtSN1 loci, based on analysis of transcription units by Wierzbicki et al. (2008). Region A corresponds to the siRNA-producing region of solo LTR, region B corresponds to the adjacent to solo LTR region that produces scaffold RNA, and red lines mark regions amplified by RT-PCR and qPCR. The dotted line corresponds to the region of scaffold RNAs hypothesized to be complementary to the siRNAs produced from region A. (B) Depletion PLOS Genetics | www.plosgenetics.org 7 March 2013 | Volume 9 | Issue 3 | e1003411 Figure 4. Effect of exosome subunits depletion on expression of ncRNA transcripts from RdDM-regulated loci. RRP41 depletion does not affect de novo DNA methylation in solo LTR and AtSN1 loci The solo LTR, AtSN1 and IGN5 loci are silenced primarily by RdDM, through siRNA mediated de novo methylation of DNA [9,30,31]. We reasoned that if the exosome is involved in controlling the amount of RNA expressed from these loci in a siRNA-dependent manner, then the exosome defect might affect the amount of siRNAs generated from these regions. To address this question, we first compared solo LTR and AtSN1-specific smRNAs. We found that production of smRNAs from the siRNA- generating A regions was not altered in rrp4-i or rrp41-i mutants relative to WT (Figure 5A and 5B), similar to the results of the global smRNA analysis we described above. The increased amount of smRNAs observed in dcl3 mutants is because in the absence of DCL3, the other Dicer proteins process DCL3 substrates [49]. In order to make sure that the smRNAs produced from one strand of region A are not masking the smRNAs produced from the opposite strand in exosome depletion mutants, we also analyzed these smRNA populations in a strand-specific manner. However, the patterns of strand-specific siRNAs were very similar to the patterns we observed previously and siRNAs were not affected by exosome depletion (Figure 5C and 5D). We examined an additional region controlled by RdDM, the IGN5 locus [31], and found that IGN5-specific smRNAs are also not affected in exosome mutants, similar to solo LTR and AtSN1 loci (Figure S3C). This implies that the increase in accumulation of transcripts in exosome-depleted plants was not a result of siRNA defect. To verify this directly, we examined the patterns of DNA methylation in these regions by using methylation sensitive restriction enzymes (Figure 5E). The DNA of the solo LTR region was examined by two different assays (Figure 5E and 5F). We found that, consistent with the results of the region-specific siRNA analysis, de novo DNA methylation was not changed in rrp41-i plants (Figure 5A–5D). Taken together, these results indicate that an increase in transcript accumulation is not caused by the loss of de novo methylation and the region is still silenced by RdDM. It also suggests that in the examined loci, the exosome complex functions independently of RdDM. Interestingly, the increased amount of transcripts accumulated in these regions does not contribute to increased smRNA amounts in the exosome- depleted plants. This was observed regardless of whether these transcripts originated from siRNA-generating regions, or adjacent regions. Exosome depletion does not affect populations of smRNAs corresponding to repeats and transposable elements (A) Diagrams of solo LTR and AtSN1 loci, based on analysis of transcription units by Wierzbicki et al. (2008). Region A corresponds to the siRNA-producing region of solo LTR, region B corresponds to the adjacent to solo LTR region that produces scaffold RNA, and red lines mark regions amplified by RT-PCR and qPCR. The dotted line corresponds to the region of scaffold RNAs hypothesized to be complementary to the siRNAs produced from region A. (B) Depletion March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org 7 SmRNA–Independent Exosome Silencing SmRNA–Independent Exosome Silencing of exosome subunits RRP4 and RRP41 leads to an increase in noncoding transcripts generated from siRNA-producing region A and scaffold RNA- producing region B of solo LTR. RT was primed with oligo(dT). (C, D, E) Expression of region A of solo LTR in exosome depleted plants and various mutants. (C) Combining depletion of RRP41 with mutation in Pol V leads to a synergistic increase in accumulation of transcripts from region A. Strand-specific RT-PCR analysis revealed that both top and bottom transcripts of region A are affected by depletion of RRP41 subunit, but only bottom transcript is synergistically affected in rrp41-i/nrpe1 double mutants (D), while the amount of top transcript is decreased in both rrp41-i/nrpd1 and rrp41-i/nrpe1 double mutants (E). (F, G) Expression of region B of solo LTR in exosome depleted plants and various mutants. (F) Depletion of rrp41 leads to increased amounts of transcript produced from both strands of region B. (G) RT-PCR analysis of solo LTR top transcript. (H, I) Depletion of exosome subunit RRP41 leads to increase in ncRNA transcripts generated from the AtSN1 region. (H) Combining depletion of RRP41 with mutation in Pol V leads to synergistic increase in accumulation of polyadenylated transcript from region A. (I) Amount of the region A bottom strand of AtSN1 is synergistically increased in rrp41-i/nrpe1 double mutants. doi:10.1371/journal.pgen.1003411.g004 A transcript in iRNAi/nrpe1 mutants (Figure 4B, 4C, 4G and 4H) does not produce any increase in the amount of siRNAs (Figure 5A–5D). A transcript in iRNAi/nrpe1 mutants (Figure 4B, 4C, 4G and 4H) does not produce any increase in the amount of siRNAs (Figure 5A–5D). examined AtSN1, we observed a very similar synergistic increase in the level of the siRNA-producing region A of bottom strand transcript of AtSN1 in rrp41 iRNAi/nrpe1 mutants (Figure 4H and 4I). H3K9me2 levels are affected in exosome-depleted plants H3K9me2 levels are affected in exosome depleted plants DNA methylation and histone modification are two major epigenetic marks regulating gene expression and chromatin state in plants. Monomethylated histone H3 lysine 27 (H3K27me1) and dimethylated histone H3 lysine 9 (H3K9me2) are hallmarks of heterochromatin, and silencing of solo LTR, AtSN1 and IGN5 loci also involves histone modifications [30,31]. Although de novo methylation does not directly affect the level of H3K9me2, it does affect the level of H3K27me1 [31], suggesting that in addition to histone modification pathways, which are dependent on RdDM, other, RdDM-independent, pathways also contribute to transcrip- tional silencing of these regions. We therefore used chromatin immunoprecipitation (ChIP) to examine whether the exosome is involved in regulation of histone modifications in these loci. Exosome depletion does not affect populations of smRNAs corresponding to repeats and transposable elements Together, these results suggest that the exosome participates in controlling the amount of top transcripts emanating from the scaffold-producing region B of solo LTR, and thus may contribute to the repression of region A through regulating the level of region B transcripts. RRP41 depletion does not affect de novo DNA methylation in solo LTR and AtSN1 loci Indeed, even several thousand-fold upregulation of region Similar to the results reported by others [30,31], we observed a significant decrease in the level of H3K9me2 in the solo LTR locus in nrpd1 and nrpe1 mutants, which affect Pol IV and Pol V, respectively. We found that RRP41 depletion also led to a decrease in H3K9me2 but less than observed in nrpd1 and nrpe1 mutants (Figure 6A). The decrease in level of this repressive histone modification also correlated with a mild increase in RNA Pol II occupancy in the solo LTR region, as would be expected with a release of transcriptional block (Figure 6B). The rrp41 iRNAi/nrpe1 double mutant did not exhibit any additive or synergistic effect on the loss of H3K9me2 relative to respective single mutants. When we examined AtSN1, we found that the level of H3K9me2 was mildly decreased in all mutants tested (Figure 6A). For AtSN1, it was previously suggested that RNA Pol III is the main RNA polymerase transcribing the region when the region is in a derepressed state [31], although RNA Pol II was also reported to be associated with this region [30]. We found that RNA Pol II occupancy in AtSN1 was very low but it increased significantly in rrp41 iRNAi/nrpe1 double mutants (Figure 6B), in accordance with the synergistic increase of the transcript level we observed (Figure 4H and 4I). Depletion of another exosome subunit, RRP4, caused a similar loss of H3K9me2 at solo LTR and AtSN1 loci (Figure 6C). We then chose several additional regions, termed REG3 and REG4 (Figure S3A), that are mildly upregulated in exosome mutants according to our previous microarray analysis [1], and examined them using ChIP. REG3 harbors a MuDR transposon, and REG4 is situated in a tandem repeat area. Neither of these loci produces smRNAs or is controlled by DNA methylation (Figure 6E and data not shown). We found that the H3K9me2 in these loci was similarly affected by exosome depletion (Figure 6C), while the level of H3K27 methylation in these regions didn’t show any difference (Figure 6D). Exosome associates with transcripts produced from a scaffold-generating area adjacent to solo LTR g g j We then examined exosome association with ncRNA loci. Detection of some protein–nascent mRNA interactions by ChIP were reported previously for proteins working on RNA, but the results of our attempts to localize tagged exosome subunits at solo LTR locus have proven inconclusive. Transcripts from region A are normally below the level of detection in wild-type plants, but transcription from the region B adjacent to solo LTR has been previously documented in wild-type plants [1,30,31]. In order to confirm that the exosome directly associates with these transcripts, we conducted RNA immunoprecipitation (RIP) using plants carrying a transgene expressing RRP41-TAP, and examined the ncRNAs associated with the exosome by RT-PCR. No region A transcripts were detected in immunoprecipitates, but we found that region B transcripts were co-precipitated with exosome (Figure 7A). These data suggest that in wild-type plants, exosome physically associates with polyadenylated transcripts produced from region B of solo LTR. We found that, similar to depletion of the core subunits RRP4 and RRP41, rrp6l1-2 and rrp6l2-2 mutants exhibited increased accumulation of transcripts produced from region A. As would be expected based on cytoplasmic localization of RRP6L3 protein, no effect was observed in rrp6l3-1 mutants. To our surprise, we observed a dramatic derepression of region A in rrp6l1-2/rrp6l2-2 double mutants, suggesting that both RRP6L1 and RRP6L2 proteins are involved in the silencing of this region and might have a redundant function in this process. We also examined the status of solo LTR DNA methylation in rrp6l1-2, rrp6l2-2, and rrp6l1-2/rrp6l2-2 double mutants. We found that methylation was not affected in these mutants regardless of the extent of derepression of the region (Figure 7D), consistent with the results obtained using rrp4-i and rrp41-i depletion mutants. Taken together, these results indicate that the observed increase in transcript accumulation is not caused by the loss of de novo methylation and the region is still methylated by RdDM. This further confirms that the exosome complex functions indepen- dently of the RdDM pathway. In contrast to solo LTR, we did not detect a physical association of exosome with AtSN1 region B transcript (Figure 7A). This implies that exosome depletion may not directly affect the silencing of AtSN1. However, we observed that exosome depletion resulted in accumulation of transcript in the AtSN1 locus and we detected a synergistic derepression of the locus in rrp41/nrpe1 mutants, similar to solo LTR locus (Figure 4H and 4I). RRP41 depletion does not affect de novo DNA methylation in solo LTR and AtSN1 loci These results suggest that the exosome may participate in maintaining chromatin structure in these regions March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 8 SmRNA–Independent Exosome Silencing March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing Figure 5. smRNA accumulation and DNA methylation in solo LTR and AtSN1 loci is unaltered upon exosome depletion. (A, B) 20– 25 nt smRNAs produced from region A of solo LTR (A) and region A of AtSN1 (B) in rrp4-i, rrp41-i exosome depletion lines and RdDM mutants. All locus-specific datasets of 20–25 nt smRNAs are plotted versus the sum of their normalized reads per million (rpm). (C, D) Strand-specific analysis of smRNAs generated at regions A and B of AtSN1 (C) and solo LTR (D) loci in different mutants. (E, F) DNA methylation analysis of AtSN1 and solo LTR loci by digestion of purified DNA with the methylation-sensitive endonucleases HaeIII for AtSN1 (E), AluI for solo LTR (E), and McrBC for solo LTR (D), followed by PCR. doi:10 1371/journal pgen 1003411 g005 y doi:10.1371/journal.pgen.1003411.g005 as well, and does so by specifically affecting the level of H3K9me2 in addition to controlling the level of transcripts. our preparations. In addition, RRP6L2 was later shown to have at least some commonalities with core exosome substrates [54]. We therefore examined whether the Arabidopsis RRP6-like proteins control the amount of ncRNA at the solo LTR locus. To determine this, we used T-DNA insertion alleles in RRP6L1, RRP6L2 and RRP6L3. We isolated the rrp6l1-2 allele from the University of Wisconsin BASTA population (Ws ecotype), and the alleles of the rrp6l2-2 and rrp6l3-1 are SALK alleles (Col-0 ecotype). To control for effects of ecotype, we compared the amount of region A transcript in rrp6l3-1, rrp6l2-2, rrp6l1-2/rrp6l2- 2 mutants to Col-0 wild type plants, and rrp6l1-2, rrp6l1-2/rrp6l2-2 mutants to Ws ecotype plants (Figure 7B and 7C). The exosome and smRNA metabolism The exosome functions in virtually all aspects of RNA metabolism and it appears to also have a prominent role in transcriptional gene silencing in different species [1,10,38–41,55– 59]. This study examined the role of the exosome complex in metabolism of smRNAs and explored the possible relationship between the exosome and the RdDM pathway in gene silencing in Arabidopsis. Our results showed that exosome-mediated silencing did not produce global changes in smRNA profiles, nor in DNA methylation at specific loci. However, we did find effects on histone methylation, indicating that the exosome may regulate chromatin structure, thereby playing an important role in maintenance of gene silencing on a much broader scale than the RdDM pathway. It is clear from our results using suppression of key exosome components that plants have an exosome-dependent pathway that relies on ncRNAs to target heterochromatin. Exosome associates with transcripts produced from a scaffold-generating area adjacent to solo LTR Most likely the regulation of AtSN1 is more complex because an additional RNA polymerase, RNA Pol III, is involved. AtSN1 is transcribed mostly by RNA Pol III [31,53], suggesting that the double deficiency in exosome and Pol V may increase both Pol II and Pol III access to the locus. We also observed the increased Pol II association with AtSN1 in rrp41/nrpe1 mutants by ChIP assay using anti-Pol II (Figure 6B), which is consistent with the results of qRT-PCR. Therefore, it is also possible that the loss of exosome function may lead to the alteration of chromatin structure in regions adjacent to AtSN1 and thus affect the stability of silencing in AtSN1 indirectly. Nevertheless, these results are similar to the interplay between exosome and Pol V observed for solo LTR. March 2013 | Volume 9 | Issue 3 | e1003411 RRP6 is involved in controlling levels of ncRNA from the solo LTR locus The 9-subunit exosome complex is catalytically inactive in yeast and human. Instead, active sites are contributed by Rrp44 (Dis3) and by the subunit Rrp6, which is substoichiometric, nuclear- specific, and not essential for viability. Degradation of S. cerevisiae nuclear ncRNAs depends on polyadenylation by the TRAMP complex and involves Rrp6, the subunit that is also responsible for elimination of heterochromatic RNAs in S. pombe [20,22–24,39– 41]. In Arabidopsis there are three RRP6-like proteins – nuclear localized RRP6L1 and RRP6L2, and cytoplasmic RRP6L3; these were suggested to be functional homologues of RRP6 [54]. None of the RRP6-like proteins co-purified with the exosome complex in our proteomic studies [1], but may have been underrepresented in Our finding that the increase in ncRNA transcribed from heterochromatic loci in exosome-depleted plants did not lead to an increase in levels of smRNA indicates that exosome function in Arabidopsis differs from that in fission yeast. In fission yeast, exosome defects have a dramatic effect on siRNAs leading to redistribution of the spectrum of Ago1-associated siRNAs, from March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 10 SmRNA–Independent Exosome Silencing Figure 6. The effect of the exosome subunits depletion on the levels of H3K9me2 in different loci. (A, B) The levels of H3K9me2, and Pol II occupancy at solo LTR and AtSN1 examined by ChIP in RRP41, rrp41-i, RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, and nrpd1/nrpe mutants using ib di i H K (A) d RNA P l II (B) i l (C) Eff f RRP d l i l l f H K i d b ChIP l LTR Figure 6. The effect of the exosome subunits depletion on the levels of H3K9me2 in different loci. (A, B) The levels of H3K9me2, and Pol II occupancy at solo LTR and AtSN1 examined by ChIP in RRP41, rrp41-i, RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, and nrpd1/nrpe mutants using antibodies against H3K9me2 (A), and RNA Pol II (B), respectively. (C) Effect of RRP4 depletion on levels of H3K9me2 examined by ChIP at solo LTR, IGN5, REG 3, and REG 4 (C), and on levels of H3K27me1 at solo LTR, AtSN1, and IGN5 loci (D). No Ab, ChIP with no antibody, is used as a negative control. RRP6 is involved in controlling levels of ncRNA from the solo LTR locus (D) Analysis of DNA methylation in solo LTR by McrBC treatment in Col-0, Ws, rrp6l1-2, rrp6l2-2, double mutant rrp6l1-2/rrp6l2-2, rrp6l3-1mutant plants. RRP41/nrpd1 mutant DNA is used as a control. doi:10.1371/journal.pgen.1003411.g007 reported [63–71]. One of the DNA methylation-independent gene silencing pathways is mediated by MOM1 (Morpheus’ molecule 1) protein [63,65], which predominantly silences transposons and loci harboring sequences related to gypsy-like transposons. Activa- tion of transcription in mom1 mutants occurs with no change in DNA methylation, histone modifications or chromatin condensa- tion, and the investigation of the relationship between RdDM and MOM1 revealed a very complex interplay between these two pathways [63,69,72–74]. However, a reduction in H3K9 dimethy- lation was reported in some loci in mom1 mutants and it was suggested that MOM1 may transduce RdDM signals to repressive histone modifications by an unknown mechanism [75]. mostly repeat-associated to those derived predominantly from exosome substrates such as rRNA and tRNA [39], indicative of exosome acting as a negative regulator of siRNA biogenesis. Our data indicate that the Arabidopsis exosome most likely lost this function during evolution, meaning that exosome substrates do not compete with siRNA precursors for siRNA biogenesis machinery and spurious transcripts do not enter RNAi pathways in plants. Additionally, it suggests that perhaps only very few of the ncRNA transcripts controlled by the exosome could be bona fide siRNA precursors. One of the reasons for this could be the fact that plants evolved two plant-specific RNA polymerases, Pol IV and Pol V, which specialize in siRNA-mediated TGS. Pol IV is required for biogenesis of the majority of 24-nt siRNAs and is supported by Pol V, which is responsible for production of a subset of siRNAs [31,44,45,60]. It is also plausible that there might be other unknown plant-specific ribonucleases that specialize in controlling stability of siRNAs or the amount of siRNA precursors generated by Pol IV and/or Pol V in plants. We also cannot rule out the possibility that some of the transcripts controlled by the exosome in a small subset of loci are legitimate siRNA precursors; this definitely warrants further in-depth investigation. Also, a recent study of MORC family ATPases revealed that mutation of AtMORC1 or AtMORC6 caused derepression of DNA methylated genes and TEs without any loss of DNA methylation, change in histone methylation or alteration of siRNA levels [71]. These proteins are involved in alteration of chromosome superstructure and are likely to act downstream of DNA methylation. RRP6 is involved in controlling levels of ncRNA from the solo LTR locus (E) Analysis of DNA methylation in REG 3 and REG 4 regions by McrBC treatment in RRP41, rrp41-i, RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, and nrpd1/nrpe1 mutant plants. REG 3 and REG 4 are not methylated in wild type plants and no changes were observed in mutants. The error bars in ChIP experiments represent the standard error of the mean (SE) and correspond to the difference between 2 biological replicates. doi:10.1371/journal.pgen.1003411.g006 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 11 SmRNA–Independent Exosome Silencing Figure 7. Exosome associates with transcripts produced from the region adjacent to the solo LTR scaffold-generating area. (A). RT- PCR of RNA-immunoprecipitation using plants carrying a functional RRP41-TAP transgene and empty-TAP transgene to examine the association of exosome with noncoding transcripts produced at siRNA and scaffold RNA producing loci. Region B transcripts were co-precipitated with exosome, while no region A transcripts were detected in immunoprecipitates. Transgenic plants are in Ws ecotype. (B, C) Two homologous rrp6 catalytic subunits of exosome are involved in controlling the amount of ncRNA emanating from the solo LTR locus. (C) Expression pattern of region A of solo LTR locus in rrp6l3-1, rrp6l2-2, 6l1-2, and the double mutant rrp6l1-2/rrp6l2-2 compared relative to the RNA expression in Col-0 ecotype wild-type (B), compared relative to the RNA expression in Ws ecotype wild-type (C). (D) Analysis of DNA methylation in solo LTR by McrBC treatment in Col-0, Ws, rrp6l1-2, rrp6l2-2, double mutant rrp6l1-2/rrp6l2-2, rrp6l3-1mutant plants. RRP41/nrpd1 mutant DNA is used as a control. doi:10.1371/journal.pgen.1003411.g007 Figure 7. Exosome associates with transcripts produced from the region adjacent to the solo LTR scaffold-generating area. (A). RT- PCR of RNA-immunoprecipitation using plants carrying a functional RRP41-TAP transgene and empty-TAP transgene to examine the association of exosome with noncoding transcripts produced at siRNA and scaffold RNA producing loci. Region B transcripts were co-precipitated with exosome, while no region A transcripts were detected in immunoprecipitates. Transgenic plants are in Ws ecotype. (B, C) Two homologous rrp6 catalytic subunits of exosome are involved in controlling the amount of ncRNA emanating from the solo LTR locus. (C) Expression pattern of region A of solo LTR locus in rrp6l3-1, rrp6l2-2, 6l1-2, and the double mutant rrp6l1-2/rrp6l2-2 compared relative to the RNA expression in Col-0 ecotype wild-type (B), compared relative to the RNA expression in Ws ecotype wild-type (C). RRP6 is involved in controlling levels of ncRNA from the solo LTR locus These results indicate that there are multiple parallel pathways for DNA methylation-independent gene silencing in Arabidopsis. The exosome-mediated silencing we observed here bears some similarities to the silencing observed for MOM1 and MORC; for example, they show effects on repetitive sequences and an absence of effects on siRNAs, although there are notable differences as well. Here we show that, similar to MOM1 and MORC mechanisms, exosome-dependent gene silencing also affects repetitive sequences and acts independent of RdDM, although our results are limited in scope. Characterization of the Exosome and DNA methylation-independent gene silencing How can exosome function in gene silencing in Arabidopsis? There are multiple mechanisms by which the exosome can be envisioned to participate in gene silencing in Arabidopsis. Heterochromatin assembly is used by all eukaryotes in gene silencing. In addition to repressive histone modifications employed by all organisms, humans and plants widely use DNA methylation as well, and ncRNAs play a central role in the control of chromatin structure in all organisms. While ncRNA-mediated silencing proceeds through multiple mechanisms some of which are organism-specific, the end result appears to be the same repressive histone modifications. For example, budding yeast, which lacks RNAi machinery, employs strategies that include, but not limited to, the use of antisense, cryptic or read-through transcripts, as well as transcripts originating from divergent promoters to guide histone modifications. Fission yeast is more similar to higher eukaryotes and uses all of the above strategies in addition to utilizing RNAi as well. However, DNA methylation is not used by budding and fission yeast. Plants, on the other hand, evolved very sophisticated epigenetic mechanisms that include the use of both RNAi-dependent and RNAi-independent pathways to guide DNA methylation and histone modifications for gene silencing [9,31–33,44,45,47,61,68,70,75,77,78]. Exosome com- plex proved to be amazingly versatile in impacting gene silencing in budding and fission yeasts. In fission yeast, the organism which takes full advantage of RNAi machinery to regulate its gene expression, the exosome is involved in silencing of both facultative and constitutive heterochromatin by acting in several different pathways through smRNAs, produced in either an RNAi- dependent or RNAi-independent manner [38,39,79,80]. It was also found to act through surveillance of RNA quantity and quality as well as by collaborating with termination machinery [40,41,57,80,81], similarly to the manner exosome participates in gene silencing in bakers yeast, which lacks RNAi machinery [55,58,59]. Our results showed that the exosome depletion produced no effect on siRNAs and DNA methylation of solo LTR, AtSN1 and IGN5 loci, arguing that the exosome complex functions indepen- dently of RdDM. However, our findings also indicated that the exosome is involved in the silencing of these loci and does interact with the RdDM pathway, possibly through its functional interaction with RNA Pol V. The converging transcripts we observed in the rrp41-i and rrp4-i mutants in solo LTR and AtSN1 suggest that the exosome is involved in regulation of either processing or level of RNA from these loci (Figure 4A–4I, and model Figure 8). Exosome and DNA methylation-independent gene silencing siRNA-dependent RdDM is thought to be the main pathway for transcriptional gene silencing of repetitive elements and transpo- sons in plants [27,28,31,61,62], although existence of other DNA methylation-independent gene silencing pathways have also been March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 PLOS Genetics | www.plosgenetics.org 12 SmRNA–Independent Exosome Silencing relationship between these pathways remains an interesting topic for future study. subunit AtRrp44a [J. Lee and J. Chekanova unpublished data] is involved in this process, and whether components of the TRAMP complex also participate. The different silencing pathways likely have different functions, depending on the genomic region, the nature of the regulated sequences, and the precision and dynamics of silencing required. For example, methylated sequences can affect the expression of nearby genes. The expression of nearby genes is negatively correlated with the density of methylated, but not unmethylated TEs. Methylated TEs are preferentially removed from gene-dense regions over time and TE families that have a higher proportion of methylated insertions are distributed farther from genes [76], arguing that positional effects and the surrounding landscape most likely contributes to the choice of silencing mechanisms and the interplay between them. p p p We also observed that the exosome physically associates with the polyadenylated ncRNA transcripts from scaffold producing regions. We could not reliably crosslink the exosome to the DNA of the target locus by ChIP (data not shown), although this could simply reflect the difficulty of reliably crosslinking proteins to DNA through RNA, or it could mean that the exosome binds to the transcripts after they are released from the locus and that exosome-mediated regulation of the transcripts may be important for maintenance of chromatin structure around the locus. H3K9 dimethylation was reported to be disturbed and lost when isolated Arabidopsis nuclei were treated with RNase A [82], meaning that histone modification may be affected by RNA level and/or RNA in close proximity to the target loci. In fission yeast, the mutation of Cid14, one of the subunits of the TRAMP complex, results in accumulation of aberrant heterochromatic RNA close to the target loci and leads to a mild decrease in H3K9 methylation. Exosome and DNA methylation-independent gene silencing It was recently shown that decrease of H3K9 methylation in yeast is the result of HP1 protein (Heterochromatin Protein1), which binds to H3K9me2 heterochromatin and propagates H3K9me2 spreading, being titrated by an excess of heterochromatic RNA [83]. In our study, we also observed a combination of the transcripts accumulation in exosome mutants relative to WT with a weak decrease in H3K9me2 levels in solo LTR (Figure 6A). Taken together, these data could suggest that a similar mechanism to regulate the stability of chromatin structure might operate in plants. However, LHP1 (Like-HP1), the closest Arabidopsis homolog of yeast HP1, has specificity for H3K27me3 [84], not H3K9me2, and the rrp41 iRNAi/nrpe1 double mutant did not exhibit any additive or synergistic effect on the loss of H3K9me2 relative to respective single mutants as well, suggesting that the loss of H3K9me2 observed in the exosome mutants is unlikely to result from an unknown functional homolog of Arabidopsis HP1 simply titrating an excess of ncRNA off chromatin, as reported in fission yeast. How can exosome function in gene silencing in Arabidopsis? We found that production of smRNAs from the siRNA-generating A regions was totally abolished in rrp41/nrpd1 double mutant (Figure 5A–5D), ruling out a possibility for these transcripts to serve as a double stranded precursors for RNA Pol IV-independent siRNAs. We also found that the exosome physically associates with the polyadenylated transcripts produced from the scaffold region (region B) and exhibits synergistic derepression of the locus (region A) when combined with a Pol V mutant, while there was no change in the derepression in rrp41/ nrpd1 double mutants (Figure 4B, 4C, 4H and 4I). Based on these results, we speculate that RNA polymerase V may function in gene silencing of these loci in two ways, the first acting in the DNA- methylation-dependent RdDM pathway, and the second acting independently of a DNA-methylation. Indeed, RdDM- indepen- dent roles of Pol V in silencing of 5S rDNA [31,85] and several other loci [82] were previously reported. A recent genome-wide study of Pol V-associated loci also hints at the possibility of Pol V In Arabidopsis, silencing of repetitive elements involves siRNA- dependent DNA methylation guided by homologous siRNAs [9]. Repressive histone modifications always appear to accompany DNA methylation, however, the mechanistic link between them is not yet fully understood. In budding and fission yeasts, degrada- tion of nuclear ncRNAs depends on polyadenylation by the TRAMP complex and involves Rrp6. We also found that mutations in two RRP6-like proteins AtRRP6 L1and AtRRP6 L2 led to significant dereperession of solo LTR (Figure 7B, 7C and 7D) and occurred in a DNA methylation-independent manner as in rrp4 and rrp41 (Figure 7F). These results suggest that Atrrp6s may be true nuclear catalytic subunits of Arabidopsis exosome, or may also work independently of core exosome. It will be interesting to examine whether another putative exosome catalytic March 2013 | Volume 9 | Issue 3 | e1003411 13 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing Figure 8. Model for the role of the exosome complex in gene silencing at solo LTR in Arabidopsis. The process of silencing of the solo LTR locus is substantially more complex than portrayed here, but for simplicity only the factors examined in this study are represented. No modifications to the prevailing views on the roles of Pol IV, Pol V, Pol II, RDR2, DCL3, and AGO4 are proposed [9,28,30,31,60]. A. How can exosome function in gene silencing in Arabidopsis? Either Pol II transcripts or the process of transcription from region B recruits Pol V (blue), complexed with AGO4 and siRNA, to the scaffold-producing region B. Due to the sequence complementarity between siRNAs, which are derived from region A only, and the portion of the scaffold transcripts that partially overlaps with region A, AGO4/siRNA RISC localizes to region A and recruits other components of the silencing machinery. Both Pol II and Pol V were implicated in producing region B scaffold transcripts [30,31]. Exosome is not involved in siRNA metabolism and does not contribute to DNA methylation. Exosome participates in controlling the amount of top transcripts emanating from the scaffold-producing region B of solo LTR, and thus may contribute to the repression of region A through regulating the level of region B transcripts. The exosome associates with transcripts emanating from the scaffold-producing region and plays a role in locus silencing through maintaining or establishing chromatin structure. B. More than one silencing pathway controls the solo LTR locus. The exosome associates with transcripts emanating from the adjacent scaffold-producing region, and plays a role in locus silencing through maintaining or establishing chromatin structure by affecting histone methylation (H3K9), in parallel to the RdDM pathway, which affects siRNAs and DNA methylation (‘‘M’’ in red hexagons). doi:10 1371/journal pgen 1003411 g008 Figure 8. Model for the role of the exosome complex in gene silencing at solo LTR in Arabidopsis. The pr y g doi:10.1371/journal.pgen.1003411.g008 transcripts, one of which is controlled by the exosome, and the exosome functions by regulating the Pol II transcripts that are distinct from the transcripts that are used in RdDM pathway. This possibility would be very interesting to examine, particularly in light of the yeast exosome involvement in gene silencing through regulation of cryptic transcripts, transcripts originating from divergent promoters and read-through transcripts [4,55,58,59]. How the Arabidopsis exosome complex and the exosome controlled ncRNAs facilitate recruitment of chromatin modifiers in order to enforce silencing through repressive histone modifica- tions remains an interesting topic of future studies. We suggest that the exosome may coordinate the transcriptional interplay of RNA polymerases Pol II and Pol V to achieve the right level of transcriptional repression of heterochromatic loci (Figure 8). having unknown functions in addition to the function it plays in the RdDM pathway [45]. How can exosome function in gene silencing in Arabidopsis? The exosome complex is not involved in the regulation of quality or quantity of siRNAs produced from region A. RNA Pol II (green) generates transcripts from region B of solo LTR. It is also possible that Pol II transcribes both A and B regions in opposite directions. Either Pol II transcripts or the process of transcription from region B recruits Pol V (blue), complexed with AGO4 and siRNA, to the scaffold-producing region B. Due to the sequence complementarity between siRNAs, which are derived from region A only, and the portion of the scaffold transcripts that partially overlaps with region A, AGO4/siRNA RISC localizes to region A and recruits other components of the silencing machinery. Both Pol II and Pol V were implicated in producing region B scaffold transcripts [30,31]. Exosome is not involved in siRNA metabolism and does not contribute to DNA methylation. Exosome participates in controlling the amount of top transcripts emanating from the scaffold-producing region B of solo LTR, and thus may contribute to the repression of region A through regulating the level of region B transcripts. The exosome associates with transcripts emanating from the scaffold-producing region and plays a role in locus silencing through maintaining or establishing chromatin structure. B. More than one silencing pathway controls the solo LTR locus. The exosome associates with transcripts emanating from the adjacent scaffold-producing region, and plays a role in locus silencing through maintaining or establishing chromatin structure by affecting histone methylation (H3K9), in parallel to the RdDM pathway, which affects siRNAs and DNA methylation (‘‘M’’ in red hexagons). doi:10 1371/journal pgen 1003411 g008 Figure 8. Model for the role of the exosome complex in gene silencing at solo LTR in Arabidopsis. The process of silencing of the solo LTR locus is substantially more complex than portrayed here, but for simplicity only the factors examined in this study are represented. No modifications to the prevailing views on the roles of Pol IV, Pol V, Pol II, RDR2, DCL3, and AGO4 are proposed [9,28,30,31,60]. A. The exosome complex is not involved in the regulation of quality or quantity of siRNAs produced from region A. RNA Pol II (green) generates transcripts from region B of solo LTR. It is also possible that Pol II transcribes both A and B regions in opposite directions. March 2013 | Volume 9 | Issue 3 | e1003411 Plant materials and growth conditions g iRNAi lines of exosome subunits RRP4 and RRP41, RNA Pol IV (SALK_128428.20.10, nrpd1a-3, nrpd1-3), RNA Pol V (SALK_029919, nrpd1b-11, nrpe1-11), RDR2 ( SAIL_1277808, rdr2-1), and DCL3 ( SALK_005512.38.70.x0, dcl3-1) mutants were described previously [1,27,33,86]. rrp41 iRNAi/nrpd1-3, rrp41 iRNAi/nrpe1-11, rrp4 iRNAi/nrpd1-3,and rrp4 iRNAi/nrpe1-11 dou- ble mutants were obtained by crossing of rrp41 iRNAi and rrp4 iRNAi with nrpd1/nrpe1-11 line. rrp41 iRNAi/dcl3-1, rrp41 iRNAi/ rdr2-1 double mutants were obtained by crossing. The small RNA reads with 20 to 25 nt length were calculated and plotted versus the sum of their normalized reads per million (rpm). The relative frequencies of each 59 terminal nucleotide of the small RNAs were calculated (Tables S1, S2 ) and represented graphically. Repetitive genomic features were classified using TAIR9 Tandem Repeat Finder (version 4.04) [95] and Inverted Repeat Finder (version 3.05) [96]. Annotation of dispersed repeats was done with Repeat Masker (version 3-3-0) [97]. The alleles of the rrp6l2-2 and rrp6l3-1 correspond to SALK_011429 and SALK_122492 lines, respectively. The rrp6l1-2 allele was isolated from the University of Wisconsin BASTA population. The ecotype background is Col-0 for all Salk alleles and Ws for University of Wisconsin alleles. To induce iRNAi, seedlings were germinated and grown for 7 days on K6 MS plates with 8 mM 17b-estradiol, as described before [1]. For analysis of locus-specific expression of smRNAs (solo LTR, AtSN1, IGN5, REG3, and REG4), the expressed normalized reads per million (rpm) were calculated for respective genomic locus and locus-specific datasets were plotted for comparisons. Analysis of DNA methylation Genomic DNA was isolated from 7-day-old seedlings using a DNeasy kit (QIAGEN). The methylation analysis using DNA sensitive methylation enzymes was followed as described [27,31,77]. SmRNA–Independent Exosome Silencing AT1G03360 and AT3G61620 loci), respectively. Therefore, silencer sequences produced from iRNAi transgenes were filtered out from each library and libraries were analyzed separately to ensure accurate interpretations. The remaining smRNA reads, termed FLR for filtered reads, were used for further analysis. ases to modulate repression of heterochromatic sequences. The mechanisms that link this RNA metabolic complex, the epigenetic modification of histone methylation, and heterochromatic silenc- ing in plants remain to be elucidated. Our results indicate that there is no one-size-fits-all pathway or mechanism that exclusively governs silencing of all loci; rather, different loci and different players in RdDM interact with different pathways and are silenced by different, likely overlapping mechanisms. The positional effects and the surrounding landscape most likely also play important roles in the choice of silencing mechanisms and the interplay between them. This may reflect the crucial importance of silencing in developmental gene regulation and in maintenance of genomic stability by suppression of invasive sequences. Each library was normalized either to the total number of mapped non-redundant reads or to the total number of non- redundant filtered reads (FLR), multiplied by 106 (rpm, reads per million). Both methods of normalizations were compared and found to produce results which lead to identical interpretations, therefore, only data analyzed using filtered reads are presented in this study. Classification of small RNAs was performed by BEDTools (v2.10.0) [93] and in-house UNIX shell programming using the following databases: TAIR9 annotations for protein coding and non-coding features (tRNA, rRNA, ncNRA, miRNA, snRNA, snoRNA, and transposable elements [76]), miRBase (release 18) [94] or mature miRNA annotations. Some smRNAs match more than one annotation category; therefore the sum of the numbers is bigger than the total input number. Bioinformatic analysis of small RNAs Data processing was done using available tools and custom in- house UNIX shell programming [43,75,87–90]. The raw sequences in Illumina GAIIx and demultiplexed HiSeq 2000 sequencing reads were trimmed removing adapter using ‘‘fas- tx_clipper’’ in the FASTX-Toolkit (version 0.0.13) [91] and smRNAs with lengths between 15- and 32-nt were selected and mapped to the Arabidopsis genomic sequences (TAIR9 version) using BOWTIE (version 0.12.7) [92]. Reads that failed to perfectly map to the nuclear genome with no mismatches, and reads present in fewer than two counts were discarded. All Arabidopsis lines used in this study carried iRNAi cassette transgenes used for inactivation of either RRP4 or RRP41 exosome subunit genes [1]. These silencing cassettes generate a number of 21-, 22- and 24-nt silencer sequences corresponding to RRP4 or RRP41 genes (mapping to RNA analysis Total RNA was isolated from 7-day-old seedlings using the mirVana miRNA isolation kit (Ambion) according to the manufacturer’s protocol. The total RNA sample was used for sequencing library construction using the Small RNA sample Prep v1.5 kit and TruSeq Small RNA Sample Prep kit (Illumina, San Diego, CA) according to the manufacturer’s instructions. The smRNA libraries were sequenced using the Illumina Genetic Analyzer II (by DNA Core Facility, University of Missouri) and Illumina HiSeq 2000 (by Biotechnology Center, University of Wisconsin) according to the manufacturer’s instructions. HiSeq 2000 sequencing reads were demultiplexed using Casava v 1.8 (by Bioinformatic Resource Center, University of Wisconsin) before further bioinformatic analysis Total RNA was isolated from 7-day-old seedlings using the Trizol method. For RT-qPCR, 1–4 mg of total RNA digested with DNase I (Fermentas) was reverse transcribed 1 hour either at 50uC (for oligo-dT primer) or 55uC (for specific primers) using 60–100 units SuperScript III Reverse Transcriptase (Invitrogen). Tran- scripts were quantified by RT-qPCR using the comparative threshold cycle method (DDCt, primers listed in Table S4), using Actin2 (At3g18780) as endogenous reference. Polyacrylamide Northern Blot analyses were performed as described [25]. How can exosome function in gene silencing in Arabidopsis? Alternative possibility is that Pol II produces two distinct pools of In summary, our data suggest that the exosome likely acts in a parallel pathway to RdDM pathways in gene silencing, possibly affecting the transcriptional interplay of different RNA polymer- March 2013 | Volume 9 | Issue 3 | e1003411 March 2013 | Volume 9 | Issue 3 | e1003411 14 PLOS Genetics | www.plosgenetics.org SmRNA–Independent Exosome Silencing How can exosome function in gene silencing in Arabidopsis? The DNA-methylation-independent function of Pol V may then be in addition to its function in RdDM, and may operate in parallel to the exosome pathway. If this is the case, the depletion of both rrp41 and nrpd1 may not lead to synergistic derepression because it would be compensated by the RdDM-independent function of Pol V. However, deficiencies in exosome and Pol V would result in synergistic desilencing due to the loss of three different pathways. Both Pol II and Pol V were reported to be responsible for the transcription of scaffold RNA and be required for silencing [30,31], although it is not known how their activities are functionally integrated. It is also not known how Pol V initiation sites are chosen, but they appear to be promoter independent [31]. Perhaps transcription by Pol II helps maintain open chromatin architecture at this site, and together with the resulting noncoding RNAs facilitates Pol V transcription initiation. Alternative possibility is that Pol II produces two distinct pools of having unknown functions in addition to the function it plays in the RdDM pathway [45]. The DNA-methylation-independent function of Pol V may then be in addition to its function in RdDM, and may operate in parallel to the exosome pathway. If this is the case, the depletion of both rrp41 and nrpd1 may not lead to synergistic derepression because it would be compensated by the RdDM-independent function of Pol V. However, deficiencies in exosome and Pol V would result in synergistic desilencing due to the loss of three different pathways. Both Pol II and Pol V were reported to be responsible for the transcription of scaffold RNA and be required for silencing [30,31], although it is not known how their activities are functionally integrated. It is also not known how Pol V initiation sites are chosen, but they appear to be promoter independent [31]. Perhaps transcription by Pol II helps maintain open chromatin architecture at this site, and together with the resulting noncoding RNAs facilitates Pol V transcription initiation. Author Contributions Conceived the experiments: JAC DAB. Designed the experiments: JAC J- HS. Performed the experiments: J-HS. Analyzed the data: JAC J-HS H- LVW. Contributed reagents/materials: JL. Wrote the paper: JAC. Bioinformatic pipeline development: H-LVW JAC. Bioinformatic analysis: H-LVW. Contributed to bioinformatics tool development/writing: BLD JHS H-LVW. Conceived the experiments: JAC DAB. Designed the experiments: JAC J- HS. Performed the experiments: J-HS. Analyzed the data: JAC J-HS H- LVW. Contributed reagents/materials: JL. Wrote the paper: JAC. Bioinformatic pipeline development: H-LVW JAC. Bioinformatic analysis: H-LVW. Contributed to bioinformatics tool development/writing: BLD JHS H-LVW. Figure S3 Effects of exosome deletion, RdDM, and other mutants. (A) Diagrams of IGN5, REG 3 and REG 4 genomic loci, based on analysis of transcription units by Wierzbicki et al. (2008) [1,31]. Region A corresponds to siRNA producing region, region Supporting Information Table S2 Summary of smRNA sequence reads in the libraries of RRP41/nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, rrp41-i/nrpe1, RRP41/rdr2, rrp41-i/rdr2, RRP41/dcl3 and rrp41-i/dcl3 plants. (XLS) Figure S1 iRNAi silencer sequences produced by rrp4-i and rrp41-i cassettes in response to estradiol treatment. (A, B) 20–25 nt smRNAs corresponding RRP4 in rrp4-i (A) and corresponding and to RRP4 in rrp41-i (B) depletion mutants profiled based on the length of the reads. (C, D) 20–25 nt smRNAs produced from in rrp4-i (C) and rrp4-i (D) depletion mutants profiled based on both their length and the terminal 59 nucleotide. The major silencer sequences are 59U and 59A smRNA species. (TIF) Table S3 Expression profiling of known mature miRNAs in the libraries of RRP4, rrp4-i, RRP41, and rrp41-i mutant plants. (XLS) Table S4 Oligonucleotides used in this study. (XLS) Figure S2 miRNA families, miR-158a, miR-158b, miR-860, miR-823, miR-841, miR-5561 and variations in sequence length. miRNA families miR-158a, miR-158b, miR-860, miR-823, miR- 841, and miR-5561 and variations in sequence length in each family. smRNAs mapped to matching mature miR-158, miR-860, miR-823, miR-841, and miR-5561 sequences [94](miRBase release 18) were plotted versus the sum of their normalized reads per million (rpm) from smRNA libraries constructed from RRP4, rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1, RRP4 iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants. (TIF) Acknowledgments We thank Aaron Stevens for IT assistance, 3D artist Thal Syhabout for preparing the digital artwork for our model, and Xuemei Chen and Steven Jacobsen for RdDM mutants. This paper is dedicated to the memory of Dima Belostotsky, an eminent scientist and a valued colleague whose untimely death in March 2009 is a big loss for the field of RNA biology. 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Zhang et al. Journal of Neuroinflammation (2021) 18:215 https://doi.org/10.1186/s12974-021-02230-y Zhang et al. Journal of Neuroinflammation (2021) 18:215 https://doi.org/10.1186/s12974-021-02230-y (2021) 18:215 Zhang et al. Journal of Neuroinflammation https://doi.org/10.1186/s12974-021-02230-y The role of interferon regulatory factor 8 for retinal tissue homeostasis and development of choroidal neovascularisation Peipei Zhang1, Anja Schlecht1,2, Julian Wolf1, Stefaniya Boneva1, Yannik Laich1, Jana Koch1, Franziska Ludwig1, Myriam Boeck1, Adrian Thien1, Carmen Härdtner3,4, Katrin Kierdorf5,6,7, Hansjürgen Agostini1, Günther Schlunck1, Marco Prinz5,7,8, Ingo Hilgendorf3,4, Peter Wieghofer5,9*† and Clemens Lange1*† Abstract Background: Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. Methods: In this study, we examined interferon-regulatory factor 8 (Irf8)–deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). Results: Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. Conclusions: Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation. Keywords: Irf8, Interferon regulatory factor 8, Retinal microglia, Choroidal neovascularisation, RNA sequencing * Correspondence: peter.wieghofer@medizin.uni-leipzig.de; clemens.lange@uniklinik-freiburg.de †Peter Wieghofer and Clemens Lange both contributed equally to the work. 5Medical Faculty, Institute of Neuropathology, University Hospital, University of Freiburg, Freiburg im Breisgau, Germany 1Medical Faculty, Eye Center, University Hospital, University of Freiburg, Freiburg im Breisgau, Germany Full list of author information is available at the end of the article * Correspondence: peter.wieghofer@medizin.uni-leipzig.de; clemens.lange@uniklinik-freiburg.de †Peter Wieghofer and Clemens Lange both contributed equally to the work. 5Medical Faculty, Institute of Neuropathology, University Hospital, University of Freiburg, Freiburg im Breisgau, Germany 1Medical Faculty, Eye Center, University Hospital, University of Freiburg, Freiburg im Breisgau, Germany Full list of author information is available at the end of the article Background flow cytometry, immunohistochemistry and RNA se- quencing (RNA-seq). We found that Irf8-deficient MG exhibited functionally relevant alterations in gene ex- pression patterns that were associated with a significant disruption of microglial development, normal postnatal retinal vascular and functional development, and in- creased CNV lesion size in the adult situation. flow cytometry, immunohistochemistry and RNA se- quencing (RNA-seq). We found that Irf8-deficient MG exhibited functionally relevant alterations in gene ex- pression patterns that were associated with a significant disruption of microglial development, normal postnatal retinal vascular and functional development, and in- creased CNV lesion size in the adult situation. g Retinal microglia cells (rMG) constitute the resident myeloid cell population in the neuroretina and are crit- ical for retinal development, tissue homeostasis and re- sponse to cell damage. During development, rMG are in intimate contact to endothelial tip cells and contribute to postnatal vascular development [16] as well as neur- onal survival by modulating programmed cell death and trophic influences [56]. During adulthood, rMG interact closely with synapses to maintain synaptic structure and electroretinal function and continuously scan the local environment for danger signals associated with injury or pathogens [56]. In response to tissue damage or infec- tion, rMG rapidly attain an activated phenotype, migrate towards the site of injury and contribute to phagocytosis, inflammation and pathological events [4, 52, 59]. As such, activated microglia cells have been found in the subretinal space of patients with age-related macular de- generation (AMD) and in particular at sites of choroidal neovascularisation (CNV) in neovascular AMD [11, 20] which is a common cause of irreversible blindness in the elderly [10, 60]. Studies on the role of rMG in the devel- opment of CNV, however, revealed conflicting results, and both detrimental and protective roles of MG for the progression of CNV have been discussed in the past [2, 14, 36, 49, 51]. Methods Mice All animal experiments were authorized by the local ani- mal care and use committee under the respective EU, national, federal and institutional regulations for animal experiments (ethical protocol numbers G14/89, G20/13). Mice were bred on a C57BL/6J background and devoid of the Crb1 mutation. Cx3cr1GFP/GFP mice were crossed with C57BL/6J mice to generate Cx3cr1GFP/+ (Irf8 WT) mice. Irf8-/- mice were crossed with Irf8-/-Cx3cr1GFP/GFP mice to obtain Cx3cr1GFP/+:Irf8-/- (Irf8 KO) mice [24, 26]. Phage artificial chromosome-transgenic Irf8-VENUS reporter mice were used to trace the expression of IRF8 [53]. CAG::mRFP1 mice were purchased from the Jack- son Laboratory (Bar Harbor, ME). Laser-induced choroidal neovascularisation (CNV) Laser-induced choroidal neovascularisation (CNV) The laser-induced CNV model was used as previously described [15, 31, 50]. In brief, mice were anaesthetized by intraperitoneal administration of ketamine hydro- chloride (100 mg/kg, Pharmacia & Upjohn, Erlangen, Germany) and xylazine (6 mg/kg, Bayer Vital GmbH, Le- verkusen, Germany). Pupillary dilatation was achieved by applying 0.5% tropicamide (Bausch + Lomb, Berlin, Germany) and 5% phenylephrine hydrochloride (URSA- PHARM Arzneimittel GmbH, Saarbrücken, Germany). After covering the cornea with a coverslip coated with dexpanthenol eye gel (50 mg/g, Bausch + Lomb, Berlin, Germany), three to six laser spots (488 nm, 150 mW, 100 μm and 100 ms) were applied to each eye using the VISULAS 532s Laser System (Carl Zeiss, Jena, Germany) in combination with ZEISS Laser Slit Lamp 532s (Carl Zeiss, Jena, Germany). Only laser spots with visible for- mation of vaporisation bubbles were included in this study. The aim of this study was to determine the function of IRF8 in retinal microglia in the healthy as well as per- turbed retina. Specifically, we aimed to investigate whether IRF8 is involved in microglial cell homeostasis, neuroretinal function and pathological CNV formation. The latter is of particular interest, as PU.1 and CSF1R signalling acting upstream and downstream of IRF8, re- spectively, are critical for postnatal angiogenesis and for- mation of pathological neovascularisation in the eye [16, 54]. For this purpose, we analysed Irf8 reporter and knockout mice by in vivo imaging, functional studies, © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 2 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 2 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Genotyping Transgenic mice were genotyped according to the primers and programs shown in supplemental table 1. The interferon regulatory factor (IRF) family of tran- scription factors consists of nine members that are in- volved in hematopoietic differentiation, oncogenesis, Toll-like and purinergic receptor signalling and expres- sion of interferons and interferon-inducible genes [39, 57]. In particular, Irf8 plays a pivotal role in the regula- tion of lineage commitment and MG cell maturation during brain development [27, 47]. Besides its essential role during development, Irf8 is crucial for the function of resident myeloid cells in the adult steady state. As such, the deletion of Irf8 in mice leads to a disturbed homeostasis of resident tissue macrophages in the liver, the kidney and brain including microglia and other CNS-associated macrophages [19, 25, 48, 58]. The role of Irf8 in regulating rMG gene expression and its influ- ence on retinal development and neuroretinal function, however, are currently unknown. Bone marrow transplantation Stainings were imaged using the Nano Zoomer S60 digital slide scanner (Hamamatsu, Herrsching am Ammersee, Germany) and analysed with NDP viewer software (Ha- mamatsu, Herrsching am Ammersee, Germany) or with a confocal laser scanning microscope (Zeiss LSM 510 or Leica TCS SP8 or Olympus FV1000), Zen software (Carl Zeiss, Jena, Germany), LAS X software (Leica, Nussloch, Germany) or Fluoview FV1000 (Olympus, Tokyo, Japan). For a detailed list of antibodies used, see supplementary table 2. Meanwhile, bone marrow cells (BMCs) were collected from the tibias and femurs of CAG-mRFP1 mice and re- suspended in phosphate-buffered saline (PBS). The re- cipient mice were intravenously injected with 3 × 106 BMCs via the tail vein. Nine weeks after bone marrow transplantation, the efficiency of reconstitution was assessed by flow cytometry which will be explained below. Three-dimensional reconstruction of retinal microglia Three-dimensional reconstruction of retinal microglia Imaging for 3D reconstruction was performed using a Zeiss LSM 510 confocal laser scanning microscope with a 20× objective, 3× zoom and 1024 × 1024 pixel reso- lution. The interval thickness of the z-stacks was set to 1.0 μm. The morphology of retinal microglia in the inner plexiform layer (IPL) and outer plexiform layer (OPL) was determined by a three-dimensional reconstruction using the filament mode of IMARIS software (Bitplane, Zurich, Switzerland). Three cells per layer and mouse were reconstructed and analysed. Bone marrow transplantation Bone marrow transplantation experiments were carried out as previously described [22]. In brief, a total of 12 re- cipient control and 14 Irf8 knockout mice were head- shielded and lethally irradiated (RS2000 irradiator, Rad Source, Kanas, USA) in two independent experiments. Page 3 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 to embedding in Tissue-Tek O.C.T. compound (Sakura, Aplphen aan den Rijn, The Netherlands). Seven micrometre-thick cryosections were cut using a cryostat (Leica CM1950, Leica, Nussloch, Germany). Following blocking in Ultra V block for 10 min at room temperature, the sections were incubated with primary antibodies against ßIII tubulin (1:500, ab18207, Abcam, Cambridge, UK), collagen type IV (1:1000, ab6586, Abcam, Cambridge, UK), Ceh-10 homeo domain con- taining homolog (CHX10, 1:200, ab16141, Abcam, Cam- bridge, UK), GFP (1:500, 600-101-215, ROCKLAND, Limerick, PA, USA), glial fibrillary acidic protein (GFAP, 1:500, 087A1005RE, Fremont, CA, USA) or Iba1 (1:500) for 60 min with corresponding Alexa Fluor® 568- conjugated secondary antibodies (1:500, Life technolo- gies, Eugene, OR, USA). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Stainings were imaged using the Nano Zoomer S60 digital slide scanner (Hamamatsu, Herrsching am Ammersee, Germany) and analysed with NDP viewer software (Ha- mamatsu, Herrsching am Ammersee, Germany) or with a confocal laser scanning microscope (Zeiss LSM 510 or Leica TCS SP8 or Olympus FV1000), Zen software (Carl Zeiss, Jena, Germany), LAS X software (Leica, Nussloch, Germany) or Fluoview FV1000 (Olympus, Tokyo, Japan). For a detailed list of antibodies used, see supplementary table 2. to embedding in Tissue-Tek O.C.T. compound (Sakura, Aplphen aan den Rijn, The Netherlands). Seven micrometre-thick cryosections were cut using a cryostat (Leica CM1950, Leica, Nussloch, Germany). Following blocking in Ultra V block for 10 min at room temperature, the sections were incubated with primary antibodies against ßIII tubulin (1:500, ab18207, Abcam, Cambridge, UK), collagen type IV (1:1000, ab6586, Abcam, Cambridge, UK), Ceh-10 homeo domain con- taining homolog (CHX10, 1:200, ab16141, Abcam, Cam- bridge, UK), GFP (1:500, 600-101-215, ROCKLAND, Limerick, PA, USA), glial fibrillary acidic protein (GFAP, 1:500, 087A1005RE, Fremont, CA, USA) or Iba1 (1:500) for 60 min with corresponding Alexa Fluor® 568- conjugated secondary antibodies (1:500, Life technolo- gies, Eugene, OR, USA). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Immunohistochemistry and imaging Immunohistochemistry and imaging After intracardiac perfusion with PBS and 4% parafor- maldehyde (PFA), eyes were fixated in 4% PFA for 45 min at room temperature and processed to RPE- choroidal-scleral and retinal flat mounts. After incuba- tion in PBST/BSA blocking buffer overnight, the flat mounts were incubated with primary antibodies against collagen type IV (1:500, AB769, Merck Millipore, Darm- stadt, Germany), Iba1 (1:500, #019-19741, Wako, Neuss, Germany) or alpha smooth muscle actin (SMA, 1:500, ab5694, Abcam, Cambridge, UK) for two nights at 4 °C, followed by incubation with Alexa Fluor® 568 or 647- conjugated secondary antibodies overnight at 4 °C (1: 500, Life technologies, Eugene, OR, USA). Eyes of Irf8- VENUS mice were fixated in 4% PFA for 1 h and incu- bated in 10%, 20% and 30% sucrose for 24 h each prior In vivo characterisation and analysis Fundus morphology, retinal structure and physiological function were investigated using fundus photography, fundus fluorescein angiography (FFA), optical coherence tomography (OCT) and electroretinography (ERG) as previously described [32]. Fundus photography, FFA and OCT were performed using a Micron III retinal micro- scope (Phoenix Technology Group, Pleasanton, CA, USA) and the StreamPix software (Norpix Inc., Mon- treal, Canada). For FFA, 10% sodium fluorescein (Alcon, Freiburg, Germany) was diluted to a concentration of 50 μL/mL in 0.9% sodium chloride for injection (VWR, Leuven, Belgium) and administered intraperitoneally (2 μL/g). Ninety seconds after dye injection, the angiograms were recorded. For quantification of CNV size, hyper- fluorescent areas in early-phase angiograms were mea- sured in pixels using ImageJ. Image-guided OCT was performed using the OCT2 scan head. In OCT images, the thickness of the inner nuclear layer (INL) (200 pixels from the optic nerve head) was measured using ImageJ (https://imagej.nih.gov/ij/). For ERG, mice were dark- adapted overnight and anaesthetized by intraperitoneal injection of ketamine hydrochloride (66.8 mg/kg) and xylazine (12.76 mg/kg). ERG signals were amplified, re- corded and analysed automatically using Ganzfeld Q450 (Roland-Consult, Brandenburg, Germany) with the inte- grated software developed by Prof. Dr. rer. nat. Michael Bach (Eye Center, University of Freiburg, Germany). RNA sequencing First-strand cDNA was generated using SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech La- boratories, Inc., Mountain View, CA, USA). Double- standed cDNA was amplified with LD PCR and purified with AMPure XP beads. Library preparation was con- structed conforming to the Illumina Nextera XT Sample Preparation Guide (Illumina, San Diego, CA, USA). In brief, 150 pg of input cDNA was tagmented via Nextera XT transposome. The products were purified and ampli- fied with a limited-cycle PCR program to construct se- quencing libraries. The libraries were quantified with the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems, Wobum, MA, USA). Equimolar amounts of each library were pooled for cluster Statistical analysis Statistical analysis was performed using GraphPad Prism v6 (La Jolla, USA) as follows: an unpaired t test was ap- plied if the normality was given by the Kolmogorov- Smirnov test. Otherwise, the Mann–Whitney U test was used. Difference with significance was defined as p < 0.05. Protein analysis P i Protein was extracted from the choroid and the retinae using RIPA buffer (#R2078, Sigma Aldrich) containing protease in- hibitor (cOmplete, Mini; edta-FREE Protease inhibitor Cock- tail, Roche Diagnostics, Manheim, Germany) and phosphatase inhibitors (PhosSTOP, Roche Diagnostics, Man- heim, Germany), respectively, for preservation. Total protein concentration for each sample was measured with the Pier- ceTM Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scienticis, Inc., Rockland, IL, USA). RNA extraction RNA extraction, RNA library preparation and RNA se- quencing were performed in collaboration with the Gen- omics Core Facility "KFB-Center of Excellence for Fluorescent Bioanalytics" (University of Regensburg, Germany). RNA extraction was performed according to manufacturer’s instructions using the RNeasy Plus Mini Kit (QIAGEN, Hilden, Germany). After pelleting the sample by centrifugation, the RNA stabilisation reagent was removed and replaced by RLT Plus buffer for lysing retinal microglia. Genomic DNA was removed select- ively and efficiently by using gDNA Eliminator spin col- umns for RNA purification. After adding Ethanol to the flow-through, the sample was applied to an RNeasy MinElute spin column to collect RNA. Finally, after washing the column, total purified RNA was eluted in RNase-free water. The quality and integrity of total RNA was assessed with a Agilent 2100 Bioanalyser in combin- ation with the RNA 6000 Pico LabChip Kit (Agilent, Palo Alto, CA, USA). Fluorescence-activated cell sorting Fluorescence-activated cell sorting Following transcardial perfusion with 1× PBS and enu- cleation, eyes were dissected in ice-cold 1× PBS to iso- late the retinae of Irf8+/+Cx3cr1GFP/+ or Irf8-/-Cx3cr1GFP/+. For the lasered mice, the central parts (70%) of the retinae were used for FACS while the per- ipheral parts were omitted. After tissue homogenisation and filtration through a 50-μm cell strainer (Sysmex, Goerlitz, Germany), dead cell exclusion was performed by incubation with fixable viability dye 780 (1:1000, 65- 0865-14, eBioscience, Waltham, MA, USA). Anti-CD16/ CD32 (Fc) receptor (1:200, 553142, BD Biosciences, Hei- delberg, Germany) was used to avoid unspecific binding. Following staining with anti-CD45 (1:200, 103133, Page 4 of 18 Page 4 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation BioLegend, San Diego, CA, USA), anti-CD11b (1:200, 17-0112-83, eBioscience, Waltham, MA, USA), anti- Ly6C (1:200, 560593, BD Bioscience, Heidelberg, Germany) and anti-Ly6G (1:200, 560601, BD Biosci- ences, Heidelberg, Germany) for 20 min at 4 °C, retinal microglia characterised as CD45lowCD11b+Cx3cr1GF- P/+Ly6C-Ly6G- were analysed and sorted into RNA sta- bilisation reagent (QIAGEN, Hilden, Germany) using a MoFlo Astrios EQ High Speed Cell Sorter (Beckman Coulter, Munich, Germany). For RNA sequencing, an average number of 10,000 retinal microglia per sample was obtained from pooling 3 to 4 mice. Flow cytometric purification of wild-type retinal and brain microglia to- gether with bone marrow–derived monocytes was de- scribed before [59]. Data are available under the GSE accession number GSE160845. For a detailed list of anti- bodies used, see supplementary table 2. generation on the cBot using the Illumina TruSeq SR Cluster Kit v3. The sequencing run was performed on a HiSeq1000 instrument with TruSeq SBS Kit v3 accord- ing to the Illumina HiSeq 1000 System User Guide. Illu- mina image analysis and base calling were recorded in library base call format (.bcl) and further converted to Fastq files via the CASAVA1.8.2 software. RNA sequencing data analysis q g y Quality control and transcriptome profiling including reads mapping, annotation, quantification and normal- isation were performed by GenXPro (GenXPro, Frank- furt, Germany). Briefly, FastQC was performed to assess sequencing quality. After removing reads containing adapter sequences and duplicate reads via cutadapt soft- ware (GitHub, San Francisco, CA, USA) and FastUniq, the filtered reads were mapped to the mouse genome from ENSEMBL (https://www.ensembl.org/Mus_ musculus/Info/Index) using bowtie2. The transcripts were functionally annotated with gene transfer format file version 90, quantified using HTSeq and normalised as transcripts per kilobase million (TPM) via DESeq2. Differential gene expression analysis with threshold (log2 fold change greater than 1.5 or less than −1.5, p < 0.05, TPM ≥100 in at least one of the two compared groups) was performed using DESeq2. Data was visua- lised using RStudio (v1.2.1335) and R (v3.5.3). Volcano plots were created using the ggplot2 package, and Gene Ontology (GO) analysis was performed using R with the clusterProfiler 3.10.1 package [62]. IRF8 is the most abundantly expressed member of the IRF family in retinal microglia The interferon regulatory factor (IRF) family is critical for the development, maturation and function of Page 5 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation CD45loCD11b+ rMG in the steady state (Fig. 1C). In line with this finding, immunofluorescence analysis of retinal flat mounts and cryosections confirmed that IRF8 is mostly expressed in IBA1-positive microglial cells in the steady state (Fig. 1D and Supplementary Figure 1C). Since expression of Irf family members may change in response to stress and inflammation, we next assessed the expression levels of all IRF family members (1–9) and of common MG signature genes, such as Tmem119 and P2ry12, in retinal MG in 2–3 months old Cx3cr1GFP/+ mice in the steady state and upon MG acti- vation in the laser-induced CNV model. While the ex- pression of the signature genes P2ry12 and Tmem119 decreased in retinal MG after laser injury, activation markers such as Cd74 were increased, as reported before [59]. However, most members of the IRF family were expressed at the same level in the rMG in the context of myeloid cells [40]. Using flow cytometry and RNA se- quencing, we first determined the expression levels of different IRF family members in adult retinal microglia (rMG), brain microglia (bMG) and bone marrow (BM) monocytes (Fig. 1A, B). In general, genes belonging to the IRF family were expressed at different levels in rMG, bMG and BM monocytes (Fig. 1B). Compared with other IRF family members, Irf5 and Irf8 exhibited the highest expression levels in rMG, bMG and BM mono- cytes. In the retina, Irf8 emerged as the most prominent IRF member in rMG. Interestingly, the expression of Irf8 in rMG exceeded the expression in bMG and BM mono- cytes indicating a distinct function of Irf8 for rMG. To validate Irf8 expression in adult rMG, we next analysed Irf8-VENUS reporter mice by flow cytometry and immu- nohistochemistry. Flow cytometry analysis revealed a strong VENUS expression (98.7% ± 0.5%) in Fig. 1 Irf8 is predominantly expressed in retinal microglia (MG) and blood monocytes. A Flow chart of retinal MG RNA sequencing. B Differential expression of IRF family members in retinal MG (rMG), brain MG (bMG) and bone marrow (BM) monocytes. The gene expression profile was generated from four CD45loCD11b+Ly6C-Ly6G- rMG samples, four CD45loCD11b+Ly6C-Ly6G- bMG samples and four CD45+CD11b+SscloCD115+Ly6C+ bone marrow (BM) monocytes. morphology, distribution and transcriptional activity of retinal MG in the steady state To assess the role of IRF8 in distribution and cell morph- ology of rMG in the adult situation, we next examined ret- inal flat mounts from 8-week-old Irf8 knockout (KO) mice by immunofluorescence microscopy (Fig. 2A,B). Irf8 KO mice revealed a strikingly altered rMG distribution and morphology compared with wild-type (WT) animals, characterised by slightly reduced MG numbers in the inner plexiform layer (IPL, Irf8 WT: 104 ± 7 cells/mm2, Irf8 KO: 88 ± 5 cells/mm2, p = 0.09) and highly significant decrease of cell numbers in the outer plexiform layer (OPL, Irf8 WT: 116 ± 6 cells/mm2; Irf8 KO: 43 ± 1 cells/ mm2, p < 0.0001, Fig. 2B,C). Quantitative morphometric analysis using IMARIS revealed a severely altered morph- ology of retinal microglia in Irf8 KO mice, including sig- nificantly shorter length of dendrites (IPL: Irf8 KO: 356.5 ± 14.3 μm, Irf8 WT 1043.0 ± 59.4 μm, p = 0.009; OPL: Irf8 KO: 297.7 ± 35.6 μm, Irf8 WT: 788.8 ± 26.1 μm, p = 0.01), and reduced number of dendrite segments (IPL: Irf8 KO: 62 ± 4, Irf8 WT: 167 ± 14, p = 0.009; OPL: Irf8 KO: 44 ± 6, Irf8 WT: 139 ± 6, p = 0.01), branch points (IPL: Irf8 KO: 30 ± 2, Irf8 WT: 82 ± 7, , p = 0.009; OPL: Irf8 KO: 21 ± 3, Irf8 WT: 68 ± 3, p = 0.01) and terminal points (IPL: Irf8 KO: 33 ± 2, Irf8 WT: 86 ± 7, p = 0.009; OPL: Irf8 KO: 23 ± 3, Irf8 WT: 71 ± 3, p = 0.01, Fig. 2D,E) com- pared with controls. Having established a profoundly al- tered rMG distribution and phenotype in the adult situation, we next explored rMG cell numbers at earlier stages of postnatal development. Interestingly, reduced microglia cell numbers were already present in the neuro- blast layer at postnatal day 1 (P1) and later in the OPL at P7 but only transiently in the IPL at P7 that could be com- pensated until adulthood (Suppl. Figure 2A, B). These findings suggest an impaired MG distribution specifically in the deeper layers of the retina which is already present shortly after birth and persists into adulthood. morphology, distribution and transcriptional activity of retinal MG in the steady state Taken together these results strongly suggest that IRF8 plays a critical role in maintaining the distribution, morphology and homeostasis of retinal microglia and has a substantial impact on the transcriptional activity of rMG in the steady state. IRF8 is the most abundantly expressed member of the IRF family in retinal microglia In ac- cordance with the RNA-seq data, we found a strong im- munoreactivity for TMEM119 and P2RY12 in Irf8- competent rMG cells, which was almost absent in Irf8- deficient rMG. Conversely, we found an increased im- munoreactivity for the mannose receptor (CD206, Mrc1) in Irf8-deficient rMG, consistent with and further sup- porting the RNA-seq results (Fig. 3D). In addition, flow cytometry analysis of homeostatic CD45+CD11b+ rMG confirmed our RNA-seq results showing a reduced but still detectable protein expression of CX3CR1 and CD64 in Irf8-deficient rMG compared with controls with a trend towards lower MERTK and higher F4/80 expres- sion as reported before in bMG [44] (Fig. 3E). laser-induced inflammation compared with controls. Only Irf2 and Irf5 were slightly downregulated in retinal MG, whereas Irf7 was modestly upregulated. Notably, the expression of Irf8 in retinal MG remained stable in the laser-induced CNV model compared with controls (data not shown). IRF8 is the most abundantly expressed member of the IRF family in retinal microglia C Flow cytometry analysis of Irf8-VENUS expression in CD45loCD11b+ retinal MG (98.7% ± 0.5, green solid line) in comparison with a negative control (grey dotted line). D Immunohistochemistry of the retinal flat mounts (upper panel) and cryosections (lower panel) from Irf8-VENUS mice reveal that all IBA1+ (red) rMG express Irf8-VENUS (green) Fig. 1 Irf8 is predominantly expressed in retinal microglia (MG) and blood monocytes. A Flow chart of retinal MG RNA sequencing. B Differential expression of IRF family members in retinal MG (rMG), brain MG (bMG) and bone marrow (BM) monocytes. The gene expression profile was generated from four CD45loCD11b+Ly6C-Ly6G- rMG samples, four CD45loCD11b+Ly6C-Ly6G- bMG samples and four CD45+CD11b+SscloCD115+Ly6C+ bone marrow (BM) monocytes C Flow cytometry analysis of Irf8-VENUS expression in CD45loCD11b+ retinal MG Fig. 1 Irf8 is predominantly expressed in retinal microglia (MG) and blood monocytes. A Flow chart of retinal MG RNA sequencing. B Differential expression of IRF family members in retinal MG (rMG), brain MG (bMG) and bone marrow (BM) monocytes. The gene expression profile was generated from four CD45loCD11b+Ly6C-Ly6G- rMG samples, four CD45loCD11b+Ly6C-Ly6G- bMG samples and four CD45+CD11b+SscloCD115+Ly6C+ bone marrow (BM) monocytes. C Flow cytometry analysis of Irf8-VENUS expression in CD45loCD11b+ retinal MG (98.7% ± 0.5, green solid line) in comparison with a negative control (grey dotted line). D Immunohistochemistry of the retinal flat mounts (upper panel) and cryosections (lower panel) from Irf8-VENUS mice reveal that all IBA1+ (red) rMG express Irf8-VENUS (green) Page 6 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 processes such as “cell migration” (GO:0016477, p.adj. < 1.3 × 10-5), “cell motility” (GO:0048870, p.adj. < 4.8 × 10-5), “localization of cells” (GO:0051674, p.adj. < 4.8 × 10-5), “regulation of cell proliferation” (GO:0032944, p.adj. < 3.5 × 10-3) and “cell adhesion” (GO:0007155, p.adj. < 7.7 × 10-4) suggesting a reduced migratory po- tential of retinal microglia in the Irf8 KO mice compared with controls (Fig. 3B). Among the downregulated DEGs, we found numerous microglia signature genes es- sential for microglia homeostasis, including the spalt like transcription factor 1 (Sall1, log2FC = −10.69, −log10p = 24.57), allograft inflammatory factor 1 (Aif1 or Iba1, log2FC = −2.73, −log10p = 35.04), purinergic receptor P2Y, G-Protein couple 12 (P2ry12, log2FC = −2.50, − log10p = 45.06) and transmembrane 119 (Tmem119, log2FC = −2.13, −log10p = 24.57) (Fig. 3A,C). IRF8 is not required for the development of the retinal structure, vasculature and function Having established the quantitative and qualitative changes in Irf8-deficient rMG, we next investigated whether Irf8-deficiency influences retinal structure and electroretinal function during steady state. To this end, we examined the retina of adult Irf8-deficient and con- trol mice using color fundus photography (CF), fluores- cein angiography (FA), optical coherence tomography (OCT), electroretinography (ERG) and immunohisto- chemical staining of retinal flat mounts for retinal ves- sels (Suppl. Figure 3). To explore the associated transcriptional changes in Irf8-deficient rMG, we next performed RNA sequencing (RNA-seq) of FACS-sorted rMG from adult Irf8 KO mice and controls. We determined 277 differentially expressed genes (DEGs) of which 142 were significantly upregulated, and 135 genes were downregulated in Irf8 KO microglia compared with microglia of control mice (Fig. 3A). Gene ontology (GO) cluster analysis revealed that most of these downregulated genes contribute to In general, Irf8 KO mice showed regular retinal struc- ture, vasculature and function compared with controls. Qualitative assessment of retinal structure and vessels on CF and FA images of Irf8-deficient and control mice revealed no gross abnormalities, particularly no vascular dye leakage as an indicator of disturbed vascular archi- tecture or spontaneous neovascularisation (Suppl. Figure Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 7 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 7 of 18 Fig. 2 Irf8 deficiency alters retinal MG distribution, morphology and transcriptional profile. A Breeding scheme. B Irf8 KO mice demonstrate reduced density of Cx3cr1-GFP+ (green) retinal MG. Quantitative analysis are shown in (C). Pictures are representative for n = 5 mice in each group. C Quantification in microglial numbers in the inner plexiform (IPL) and outer plexiform layer (OPL). n = 5 mice were analysed per group. Data are shown as mean ± SEM. D 3D reconstruction of retinal MG by IMARIS reveals that Irf8 deficiency alters morphology of retinal MG, resulting in shorter total length of dendrites, less dendrite segments and attenuated branching. E Quantitative analysis of retinal morphology. Four to six cells were reconstructed per mouse per IPL and OPL separately. Statistics were performed with the mean values per mouse (n = 6–7 in the Irf8 WT and n = 3–4 in the Irf8 KO group). Data are shown as mean ± SEM Fig. 2 Irf8 deficiency alters retinal MG distribution, morphology and transcriptional profile. A Breeding scheme. B Irf8 KO mice demonstrate reduced density of Cx3cr1-GFP+ (green) retinal MG. IRF8 is not required for the development of the retinal structure, vasculature and function The mannose receptor CD206 (encoded by Mrc1) is absent under homeostatic conditions but detectable under Irf8-deficient conditions. E Myeloid expression levels shown as transcripts per million (TPM) and analogue surface marker expression, as determined by flow cytometry, of Irf8 WT (blue) and Irf8 KO (red) mice, expressed as mean fluorescence intensity (MFI) (left). Representative histograms are shown (right) including fluorescence minus one controls (grey line). Six mice per group were analysed for CX3CR1, CD64 and MERTK, three mice per group for F4/80. Data are shown as mean ± SEM controls (Suppl. Figure 3D). Specifically, no significant difference was detected between Irf8 KO mice and con- trols with respect to scotopic a-waves emanating from rods, scotopic b-waves corresponding to depolarisation of bipolar cells, and photopic b-waves arising from cones. Immunohistochemical staining of retinal flat mounts revealed a regular retinal vasculature in Irf8 KO mice, including equal numbers of arteries labelled by smooth muscle actin (SMA, Irf8 WT 6.2 ± 0.4, Irf8 KO: 5.9 ± 0.3 per animal) and major vessels stained with Isolectin-B4 (IB4, Irf8 WT: 11.3 ± 0.5, Irf8 KO: 11.0 ± 0,5 per retinal per animal, Suppl. Figure 3E). Addition- ally, branch points in the central superficial vascular plexus (Irf8 WT: 17.0 ± 2.7, Irf8 KO 22.1 ± 2.4 per ani- mal), central deep plexus (Irf8 WT: 8.7 ± 4.7, Irf8 KO: 74.0 ± 3.4 per animal), peripheral superficial plexus (Irf8 WT: 29.0 ± 3.5, Irf8 KO: 28.1 ± 2.0 per animal) and per- ipheral deep plexus (Irf8 WT: 59.0 ± 5.1, Irf8 KO: 63.9 ± 5.7 per animal) were similar between both groups (Suppl. Figure 3F). revealed more than 2-fold larger CNV lesions in Irf8 KO mice (8603 ± 1309 pixels per animal) compared with con- trols (3697 ± 425 pixels per animal, p < 0.005). Measure- ment of collagen type IV-labelled CNV area on RPE/ choroidal flat mounts confirmed significantly enlarged CNV lesions in Irf8 KO mice (52143 ± 7670 μm2) com- pared with Irf8 WT (25203 ± 4156 μm2, p < 0.005, Fig. 4B). As expected, microscopic evaluation of RPE/choroidal flat mounts revealed that activated amoeboid Cx3cr1- GFP+ cells accumulate at CNV lesions in Irf8 KO mice as well as in controls. IRF8 is not required for the development of the retinal structure, vasculature and function Quantitative analysis are shown in (C). Pictures are representative for n = 5 mice in each group. C Quantification in microglial numbers in the inner plexiform (IPL) and outer plexiform layer (OPL). n = 5 mice were analysed per group. Data are shown as mean ± SEM. D 3D reconstruction of retinal MG by IMARIS reveals that Irf8 deficiency alters morphology of retinal MG, resulting in shorter total length of dendrites, less dendrite segments and attenuated branching. E Quantitative analysis of retinal morphology. Four to six cells were reconstructed per mouse per IPL and OPL separately. Statistics were performed with the mean values per mouse (n = 6–7 in the Irf8 WT and n = 3–4 in the Irf8 KO group). Data are shown as mean ± SEM 3A,B). In OCT images, the thickness of the inner nuclear layer (INL) and the outer nuclear layer (ONL) contain- ing the photoreceptors (PR) was similar in Irf8-deficient mice (INL: 15.2 ± 0.5 μm, ONL: 69.8 ± 1.0 μm) compared with control animals (INL: 15.8 ± 0.4 μm, ONL: 72.3 ± 1.0 μm, Suppl. Figure 3C). ERG measure- ments demonstrated similar dark-adapted scotopic and light-adapted photopic responses in Irf8 KO mice and Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 8 of 18 Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Fig. 3 (See legend on next page.) Page 9 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 (See figure on previous page.) Fig. 3 Irf8 deficiency leads to expression loss of homeostatic signature genes. A Volcano plot of differentially expressed genes in Irf8 KO retinal MG (n = 3) compared with control (n = 5). Significantly up- and downregulated genes are shown in red and blue, respectively. The top significantly up- and downregulated genes are labelled. B The top 5 downregulated GO clusters in Irf8 KO retinal MG. Significance is represented as p.adjust, the size of each data circle indicates the number of genes involved in each enriched GO term. C Representative signature genes found to be highly expressed in competent retinal MG are significantly downregulated in the Irf8 KO mice. D Immunohistochemistry of retinal flat mounts demonstrate a strong immunoreactivity for P2RY12 and TMEM119 shown as colour-coded signal intensity in Irf8-competent retinal MG that is reduced or absent in Irf8 KO mice. IRF8 is not required for the development of the retinal structure, vasculature and function The number of Cx3cr1-GFP+ cells around CNV lesions, however, was significantly decreased in Irf8 KO mice (34 ± 7 cells per lesion per animal) com- pared with control animals (84.6 ± 8.8 cells per lesion per animal, p < 0.001, Fig. 4C). In addition, the number of rMG significantly increased in the IPL above CNV areas in controls, whereas no such increase was observed in Irf8 KO mice, further pointing to a defect in rMG migration (Suppl. Figure 4). Taken together, Irf8-deficient mice revealed reduced MG cell numbers suggesting an impaired MG migratory behaviour under physiological and pathological condi- tions which was associated with increased CNV lesion size in the laser-CNV model. Overall, these data show that Irf8 is not essential for the development and maintenance of homeostatic retinal structure, vascular network and function. This is particu- larly surprising given the significant changes in rMG cell numbers in the Irf8 KO mice during development and in the adult. Thus, the impaired retinal MG cell morph- ology and expression profile in otherwise unremarkable retinal homeostasis in Irf8-deficient mice provide a unique opportunity to investigate the role of retinal MG in the development of CNV. Retinal microglia rather than infiltrating monocytes account for the larger CNV lesions in the Irf8 KO mice Since Cx3cr1 is expressed in retinal microglia and infil- trating monocyte-derived macrophages from the blood [26], the observed Cx3cr1GFP/+ positive cells around CNV lesions could belong to both cell populations. However, Irf8-deficient mice are characterized by a low number of peripheral monocytes, which suggests that very few monocytes from the blood infiltrated the CNV lesion (Terry et al, 2015 PMID: 25277331). To investi- gate the influence of peripheral monocytes in our model, we next performed bone marrow transplantation experi- ments with bone marrow from CAG-RFP reporter animals to restore the peripheral monocyte pool in Irf8- deficient mice with Irf8 potent monocytes (Fig. 5A). Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ Irf8-po- tent peripheral monocytes in Irf8-deficient animals Irf8 deficiency aggravates CNV formation Retinal MG change their phenotype and transcriptional profile after tissue injury and modulate the development of pathological CNV, which represents a hallmark of neovascular AMD [59]. To investigate the role of IRF8 in microglial cell activation after tissue injury and formation of CNV, we next studied Irf8-deficient and control mice in the laser-induced CNV model. Both Irf8 KO and Irf8 WT mice developed typical laser-induced CNV 7 days after laser photocoagulation visible, as hyperfluorescent lesions with clear demarcation in FA images (Fig. 4A). Quantifica- tion of hyperfluorescent CNV areas in angiograms Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 10 of 18 Page 10 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Fig. 4 Irf8-deficiency aggravates CNV formation. A Fundus fluorescein angiography at day 7 following laser treatment demonstrates enlarged CNV lesions in Irf8 KO mice (n = 11) compared with Irf8 WT (n = 13). Data are presented as mean ± SEM. B 3D reconstruction of collagen type IV- labelled CNV lesions using IMARIS. IHC of RPE-choroid-scleral flat mounts confirms that Irf8 deficiency (n = 11) increases CNV severity in comparison with control mice (n = 12). Data are presented as mean ± SEM. C Less Cx3cr1GFP/+ (green) cells around CNV lesions (red) at day 7 after laser treatment were observed in the Irf8 KO mice (n = 6) compared with control mice (n = 7). Data are presented as mean ± SEM Fig. 4 Irf8-deficiency aggravates CNV formation. A Fundus fluorescein angiography at day 7 following laser treatment demonstrates enlarged CNV lesions in Irf8 KO mice (n = 11) compared with Irf8 WT (n = 13). Data are presented as mean ± SEM. B 3D reconstruction of collagen type IV- labelled CNV lesions using IMARIS IHC of RPE choroid scleral flat mounts confirms that Irf8 deficiency (n = 11) increases CNV severity in Fig. 4 Irf8-deficiency aggravates CNV formation. A Fundus fluorescein angiography at day 7 following laser treatment demonstrates enlarged CNV lesions in Irf8 KO mice (n = 11) compared with Irf8 WT (n = 13). Data are presented as mean ± SEM. B 3D reconstruction of collagen type IV- labelled CNV lesions using IMARIS. IHC of RPE-choroid-scleral flat mounts confirms that Irf8 deficiency (n = 11) increases CNV severity in comparison with control mice (n = 12). Data are presented as mean ± SEM. Irf8 deficiency aggravates CNV formation C Less Cx3cr1GFP/+ (green) cells around CNV lesions (red) at day 7 after laser treatment were observed in the Irf8 KO mice (n = 6) compared with control mice (n = 7). Data are presented as mean ± SEM mice (Mildner et al, 2007 PMID: 18026096). Interest- ingly, the reconstituted Irf8-deficient animals still dem- onstrated increased CNV lesion size that was approximately twice as large compared with reconsti- tuted Irf8 WT animals (Irf8 KO 82,009 ± 16,242 μm2; Irf8 WT 53,386 ± 4793 μm2, p = 0.06) which was associ- ated with slightly reduced numbers of RFP-GFP+ micro- glia at sites of CNV (Irf8 KO 74 ± 16.5; Irf8 WT 197.3 ± 12.1, p = 0.14) (Fig. 5C,D). The numbers of reconstituted RFP+GFP- monocyte-derived macrophages, in contrast, were around twofold increased in Irf8-deficient mice compared with controls by using flow cytometry (Irf8 KO: 95.78 ± 0.59% (RFP+Ly6Chi), 97.42 ± 0.14% (RFP+Ly6Clo), Irf8 WT: 31.19 ± 4.85% (RFP+Ly6Chi), 40.24 ± 5.5% (RFP+Ly6Clo), Fig. 5B). The observed higher recombination efficiency of Ly6Chi and Ly6Clo monocytes in Irf8 KO is likely due to the initially low abundance of these cells and their respective progenitors in Irf8 KO mice. Of note, due to the head shielding, the recipients’ bone marrow in the skull is still active and not substituted by the donor cells that could explain the comparably lower recombination efficiency in Irf8 WT Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 11 of 18 (2021) 18:215 Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8 WT (n = 8) mice were used per group. Data are presented as mean ± SEM. Irf8 deficiency aggravates CNV formation C,D Following bone marrow transplantation, Irf8-deficient mice exhibit a 2-fold increase of laser-induced CNV compared with control mice, while the number of Cx3cr1GFP/+ GFP-positive and RFP-negative microglia at sites of CNV was similar in both groups. The number of reconstituted RFP-positive and GFP-negative blood-derived monocytes were increased in Irf8-deficient mice compared with controls. Irf8 KO (N = 12) and Irf8 WT (n = 14) mice were used per group. Data are presented as mean ± SEM Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8 WT (n = 8) mice were used per group. Data are presented as mean ± SEM. C,D Following bone marrow transplantation, Irf8-deficient mice exhibit GFP/+ Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8 WT (n = 8) mice were used per group. Data are presented as mean ± SEM. Irf8 deficiency aggravates CNV formation C,D Following bone marrow transplantation, Irf8-deficient mice exhibit a 2-fold increase of laser-induced CNV compared with control mice, while the number of Cx3cr1GFP/+ GFP-positive and RFP-negative microglia at sites of CNV was similar in both groups. The number of reconstituted RFP-positive and GFP-negative blood-derived monocytes were increased in Irf8-deficient mice compared with controls. Irf8 KO (N = 12) and Irf8 WT (n = 14) mice were used per group. Data are presented as mean ± SEM Fig. 5 Bone marrow chimera experiments: reconstituted Irf8-deficient mice contain similar monocyte numbers as controls and reveal increased CNV lesion size. A Experimental setup. After head-shielded irradiation, bone marrow of CAG-RFP mice was transplanted intravenously to irradiated control (Irf8+/+:Cx3cr1GFP/+) and Irf8-deficient mice (Irf8-/-:Cx3cr1GFP/+) at the age of 8 weeks, respectively. Nine weeks after transplantation, flow cytometry was performed to check the reconstitution of blood cells. Ten weeks after transplantation, all mice underwent laser treatment to induce CNV. Analysis was performed at day 7 after laser induction. B After bone marrow transplantation, reconstitution of the blood cells was analyzed. Following head-shielded bone marrow transplantation, we observed a successful reconstitution of RFP+ peripheral CD45+CD11b+CD115+SScloLy6Chi/lo monocytes in Irf8-deficient animals compared with controls by using flow cytometry. Irf8 KO (n = 6) and Irf8 WT (n = 8) mice were used per group. Data are presented as mean ± SEM. C,D Following bone marrow transplantation, Irf8-deficient mice exhibit a 2-fold increase of laser-induced CNV compared with control mice, while the number of Cx3cr1GFP/+ GFP-positive and RFP-negative microglia at sites of CNV was similar in both groups. The number of reconstituted RFP-positive and GFP-negative blood-derived monocytes were increased in Irf8-deficient mice compared with controls. Irf8 KO (N = 12) and Irf8 WT (n = 14) mice were used per group. Data are presented as mean ± SEM Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 12 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 12 of 18 Fig. 6 Transcriptional profiling of Irf8 KO mice during CNV formation. A Volcano plot of differentially expressed genes in Irf8 KO retinal MG (n = 4) compared with control (n = 5) after laser photocoagulation. Significantly up- and downregulated genes are shown in red and blue, respectively. The top significantly up- and downregulated genes are labelled. B The top 5 upregulated GO clusters in retinal MG of lasered Irf8 KO mice. Irf8 deficiency aggravates CNV formation Significance is represented as p.adjust, the size of each data circle indicates the number of genes involved in each enriched GO term. C Differential expression of purinergic receptors in rMG from Irf8 KO compared with Irf8 WT mice. D Cnet-plot links Fibronectin (Fn1) to the GO terms inflammatory response, angiogenesis and cell adhesion. Genes written in bold belong to the top differentially upregulated genes (see A). E Expression of Fn1 is significantly increased on transcriptional level in Irf8-deficient microglia, shown as transcripts per million (Irf8 KO N = 4, Irf8 WT n = 5). Data are presented as mean ± SEM. F Expression of FN1 is significantly increased on protein level in Irf8-deficient microglia, measured by ELISA. N = 5 mice per group. Data are presented as mean ± SEM. G Expression of Fn1 can be traced back to myeloid Cx3cr1-GFP+ cells at the boarder of the CNV lesion at day 7 following laser. H Fibronectin and Collagen IV are strongly associated in the CNV lesions in both Irf8 WT and Irf8 KO mice Fig. 6 Transcriptional profiling of Irf8 KO mice during CNV formation. A Volcano plot of differentially expressed genes in Irf8 KO retinal MG (n = 4) compared with control (n = 5) after laser photocoagulation. Significantly up- and downregulated genes are shown in red and blue, respectively. The top significantly up- and downregulated genes are labelled. B The top 5 upregulated GO clusters in retinal MG of lasered Irf8 KO mice. Significance is represented as p.adjust, the size of each data circle indicates the number of genes involved in each enriched GO term. C Differential expression of purinergic receptors in rMG from Irf8 KO compared with Irf8 WT mice. D Cnet-plot links Fibronectin (Fn1) to the GO terms inflammatory response, angiogenesis and cell adhesion. Genes written in bold belong to the top differentially upregulated genes (see A). E Expression of Fn1 is significantly increased on transcriptional level in Irf8-deficient microglia, shown as transcripts per million (Irf8 KO N = 4, Irf8 WT n = 5). Data are presented as mean ± SEM. F Expression of FN1 is significantly increased on protein level in Irf8-deficient microglia, measured by ELISA. N = 5 mice per group. Data are presented as mean ± SEM. G Expression of Fn1 can be traced back to myeloid Cx3cr1-GFP+ cells at the boarder of the CNV lesion at day 7 following laser. Discussion The interferon regulatory factor 8 (IRF8) is an essential transcription factor for the development, maturation and homeostasis of microglia (MG) in the brain and other tissue macrophages [19, 21, 27]. However, the role of IRF8 for retinal MG (rMG) during homeostasis and neo- vascular eye disease has not been elucidated so far. In this study, we show that Irf8 is essential for a mature rMG gene expression profile and influences MG morph- ology, migration and the response to pathological neovascularisation. Our results show that Irf8 is strongly expressed in rMG in the steady state compared with other IRF family members. The expression of Irf8 in rMG was even sub- stantially higher than in brain MG (bMG), suggesting a distinct and tissue-specific function of Irf8 in microglia of the retina. Morphological analysis revealed that Irf8- deficient mice exhibited an overall reduced branching of rMG as well as a decreased MG cell number specifically in the outer plexiform layer, which was already observed during postnatal development. These findings recapitu- late findings in the brain [21, 27, 39, 44] and point to a migratory defect that is already present during postnatal retinal layering and maintained into adulthood. To gain further insight into the molecular changes in Irf8-defi- cient rMG, we isolated rMG by flow cytometry and ana- lysed their transcriptional profile by RNA sequencing. Our analysis revealed significant transcriptional differ- ences between Irf8-potent and Irf8-deficient microglia, which is consistent with several in vitro studies [25, 40]. We found that Sall1 was the most downregulated MG signature gene in the retina of Irf8-deficient mice. This is of particular functional importance since SALL1 is a critical transcription factor for maintaining the p yp Next, we analysed the DEG that were upregulated in Irf8-deficient MG by GO cluster analysis and found an activation of biological processes such as “defense re- sponse” (GO:0006952, p.adj. < 8.4 × 10-6), “biological ad- hesion” (GO:0022610, p.adj. < 2.9 × 10-4), “cell adhesion” (GO:0007155, p.adj. < 6.1 × 10-4), “inflammatory re- sponse” (GO:0006954, p.adj. < 9.5 × 10-6) and “positive regulation of cell substrate adhesion” (GO:0010811, p.adj. < 1.9 × 10-3) in Irf8-deficient MG (Fig. 6C). Irf8 deficiency aggravates CNV formation H Fibronectin and Collagen IV are strongly associated in the CNV lesions in both Irf8 WT and Irf8 KO mice Page 13 of 18 Page 13 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 compared with controls (Irf8 KO 96.6 ± 20.1; Irf8 WT 49.25 ± 5.3, p = 0.07) (Fig. 5D). retinal MG in Irf8-deficient mice compared with control mice (data not shown). Consistent with the increased number of Fn1 transcripts in Irf8-deficient MG, we found significantly increased FN1 protein levels in the RPE/choroid of Irf8-deficient mice (3314 ± 587.1 pg/μg protein) compared with controls (1550 ± 268.5 pg/μg protein, p < 0.05) using ELISA on tissue lysates (Fig. 6E, F). Immunohistochemical studies showed that FN1 ex- pression was restricted to the area of CNV lesions and expressed by microglia at sites of CNV (Fig. 6G). In addition to the enlarged collagen IV-positive CNV lesion described above, Irf8-deficient mice showed a markedly enlarged FN1-positive CNV lesion compared with WT mice, indicating increased fibrosis (Fig. 6H). Transcriptional profile of Irf8 KO retinal microglia during CNV formation In order to decipher the molecular mediators of en- hanced CNV formation in Irf8 KO mice, we next iso- lated CNV-associated rMG from Irf8 KO and control mice by flow cytometry and analysed the cells using RNA-seq. In total, we found 84 genes that were differen- tially upregulated and 78 genes that were downregulated in Irf8-deficient microglia 7 days following laser injury. In line with the RNA-seq analysis under homeostatic conditions, we identified similar DEG that were down- or upregulated in Irf8-deficient microglia after tissue in- jury, such as Sall1, P2yr12 and Mrc1 (Fig. 6A). The downregulation of P2ry12 in laser-treated Irf8 KO mice prompted us to explore the expression of other puriner- gic receptors which are critical for MG cell activation and migration. Here, we found several other genes en- coding purinergic receptors to be strongly downregu- lated in Irf8-deficient MG, such as Adora1, P2ry12 and P2ry13, underlining the proposed migration defect upon laser injury (Fig. 6B). Furthermore, we analysed the ex- pression of key M1 (Cd86, H2-Ab1, Tlr2) and M2 signa- ture genes (CD163, Mrc1) in isolated CNV-associated MG in Irf8-deficient mice and control animals (Suppl. Figure 5). We found that common M1 markers such as Cd86, H2-Ab1 and Tlr2 were significantly downregu- lated in Irf8-deficient MG compared with Irf8-potent MG in the laser CNV model. On the other hand, some of the common M2 markers, such as Cd163 and Mrc1, were significantly upregulated in Irf8-deficient retinal MG compared with Irf8-potent MG at sites of CNV sug- gesting a M1 to M2 polarization in Irf8-deficient MG compared with wild-type MG in the laser CNV model. Taken together, these studies show that Irf8-deficient microglia exhibit a significantly altered expression profile in the laser CNV model with downregulated migratory genes, such as purinergic receptors, and upregulated pro-fibrotic factors, such as Fn1. Discussion Furthermore, proliferation of myeloid cells relies on IRF8 signalling [61] which could explain the insufficient compensatory expansion of MG in the OPL and contribute to the niche-dependent phenotype with reduced microglial density especially in the OPL in adult Irf8-deficient mice. Despite the aforementioned significant changes in MG cell density and transcriptional profile, Irf8-deficient mice exhibited a normal retinal structure, vascular sup- ply, and physiological function. This is particularly sur- prising as retinal MG are in close contact with retinal vessels, especially during development, and are known to shape the mature retinal vasculature [9, 13, 16, 18]. This suggests that Irf8-deficient MG are functionally suffi- cient to accompany physiological retinal development and that minor developmental disturbances, which we cannot completely rule out, can be compensated over time. The fact that Irf8-deficient microglia exhibit tran- scriptional changes that reduce their responsiveness while maintaining a normal retinal phenotype, provides an ideal setting to study the role of microglia in the de- velopment of choroidal neovascularisation, which is known to be associated with significant microglia activa- tion and migration [35, 52, 59]. In the laser-induced CNV model, which mimics as- pects of neovascular AMD, we found significantly in- creased CNV lesion size under Irf8-deficient conditions. At the same time, the overall numbers of Irf8-deficient Cx3cr1-GFP+ cells were significantly reduced around the lesions compared with wild type. In line with this find- ing, MG density in the IPL above CNV lesions was sig- nificantly decreased in Irf8 KO mice compared with wild type. These observations may be mediated either by a migration defect caused by impaired purinergic signal- ling [45] or by reduced microglial proliferation of Irf8- deficient MG, or both, as discussed above. Interestingly, the present study shows that impaired migration and a lower number of MG in the vicinity of CNV are associ- ated with a more severe CNV phenotype in Irf8-deficient mice. This finding is in contrast to previous studies showing that depletion of retinal or circulating myeloid cells by clodronate is associated with reduced CNV size suggesting an anti-angiogenic effect of myeloid cells on CNV [14, 34, 51]. On the other hand, however, our re- sults are consistent with reports showing that increased accumulation of myeloid cells in neovascular lesions is associated with decreased CNV and that myeloid cells use FasL (CD95L) to inhibit CNV formation [2]. Discussion A closer look at the DEG of three key GO clusters essential for CNV development, namely "angiogenesis", "inflam- matory response" and "cell adhesion", revealed Fibronec- tin, a profibrotic mediator encoded by Fn1, as a prominent upregulated gene in Irf8-deficient MG linking all three biological processes (Fig. 6D). In addition, we found a marked increase in the expression of other pro- fibrotic factors such as Fgf2 and Spp1 and a subtle in- crease in the expression of Tgfb1 in CNV-associated Page 14 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 homeostatic gene expression pattern of MG and thus regulates, for example, the expression of MG signature genes such as Aif1, P2ry12 and Tmem119 [6, 33, 55]. Ac- cordingly, we found Tmem119 and P2ry12 as signifi- cantly reduced in Irf8-deficient rMG recapitulating the findings from human brain microglia which suggest a dependence of P2RY12 expression on IRF8 signalling [5]. Notably, because SALL1 is not exclusively expressed by microglia but also by astrocytes and oligodendrocytes [8, 38, 48], non-cell autonomous effects of this gene can- not be excluded. Further, single-cell RNA-seq (scRNA- Seq) of conditional Irf8 KO mice (Fcgr1-Cre:Irf8fl/fl) re- vealed a strong downregulation of Sall1, Aif1, Tmem119 and P2ry12 in bMG while other genes, typically expressed in macrophages, like Mrc1, encoding the man- nose receptor CD206, were upregulated [58]. In line with this study, we found a strong upregulation of Mrc1 on transcriptional level and the encoded mannose receptor CD206 on protein level in Irf8-deficient rMG which is consistent with data from Irf8-deficient bMG [44]. In addition, other myeloid genes like Fcgr1, also known as CD64, were downregulated on RNA and protein level. These findings strongly support common transcriptional changes between retinal MG and brain MG under Irf8- deficient conditions, as shown before in a direct com- parison of wild-type rMG and bMG by scRNA-seq [59]. Furthermore, it underscores that retinal microglial dif- ferentiation and maturation are highly dependent on a defined transcriptional program instructed by PU.1 [16], SALL1 [29] and IRF8. pathways of P2RY12 [40]. This hypothesis is supported by in vitro studies showing a diminished entry of rMG into retinal explant cultures following an interruption of purinergic signalling [37]. Discussion These seemingly contradictory contributions of myeloid cells to CNV development could be reconciled by the poten- tially different roles of resident MG and infiltrating To identify functionally related gene signatures, we performed a gene ontology (GO) enrichment analysis that revealed a downregulation of the clusters “cell mi- gration“, “cell motility”, “cell adhesion” and “regulation of cell proliferation” in adult Irf8-deficient rMG in the steady state. The downregulation of these GO terms was consistent with the reduced number of rMG in the OPL in adult Irf8 KO mice, supporting previous in vitro data describing IRF8 as an essential transcription factor for microglial motility and migration [28, 39]. The signifi- cant downregulation of genes relevant for cell migration could also explain the observed developmental pheno- type and point to an impaired Irf8-dependent perception of guiding cues. During development, neurons con- stantly release guiding cues, such as purines and other extracellular nucleotides which guide microglia to colon- ise the developing outer retina [1, 37]. Irf8-dependent downregulation of sensors that detect these guiding cues, such as Adora1 or P2yr12, may be responsible for the disruption of purinergic signalling in Irf8-deficient rMG, which attenuates their migratory capacity. Indeed, in vitro cultivated brain MG lacking Irf8 had a strongly reduced phosphorylation of AKT after ATP treatment, thereby diminishing the ATP-mediated signalling Page 15 of 18 Page 15 of 18 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation deficient retinal microglia accumulating at CNV showed less M1 and more M2 signature gene expression com- pared with wild-type MG at CNV, suggesting increased M1 to M2 polarization in Irf8-deficient MG. This is of particular clinical interest as some studies have sug- gested that more M2-like macrophages, which are as- sumed to be pro-angiogenic, accumulate at the site of wet compared with dry AMD and that the pathological shift of macrophage polarization may contribute to the pathogenesis of CNV in neovascular AMD [7]. The ex- pression signatures or myeloid cells at the lesion site are likely to be more complex than the aforementioned polarization state; however, it confirms an altered signa- ture of a set of well-known and characterized markers. Discussion Furthermore, GO cluster analysis showed that Irf8-defi- cient rMG exhibit increased activity of biological pro- cesses that are critical for inflammation and cell adhesion, which points to an interaction of microglia with extracellular matrix components driving CNV for- mation. We hereby identified Fibronectin, encoded by the Fn1 gene, as being significantly higher expressed in Irf8 KO rMG and linking the GO terms "cell adhesion", "inflammatory response" and "angiogenesis". The immu- nohistochemical and ELISA analyses confirmed that the expression of Fibronectin protein was also significantly increased at sites of CNV, suggesting that Irf8-deficient MG or other cells affected by the loss of Irf8 contribute to increased Fibronectin abundance at sites of CNV. Of note, collagen IV fibers were reported to depend on an established Fibronectin matrix directly co-localizing with Fibronectin fibers in vitro that could be functionally re- lated to our observation of increased CNV lesion size promoted by higher Fibronectin expression in rMG [42, 43]. Furthermore, we found a marked increase in the expression of the pro-fibrotic factors Fgf2 and Spp1 and a subtle increase in the expression of Tgfb1 in CNV-associated retinal MG in Irf8-deficient mice compared with control mice. Since FGF2 and SPP1 are important mediators of scarring and have been identified in human choroidal neovascular membranes [17, 52], overexpression of these factors in retinal MG may have further contributed to increased CNV lesion size in Irf8-deficient mice. This is of particular inter- est as pharmaceutical inhibition of SPP1 has been shown to modulate CNV formation and inhibition of FGF2 was associated with reduced CNV and subret- inal fibrosis in a laser-induced mouse CNV model [3, 41, 52]. monocyte-derived macrophages. To investigate the dis- tinct roles of these cellular populations, this study ex- ploits Irf8-deficient mice which are characterized by a near absence of circulating monocytes and, at the same time, altered but present resident retinal microglia. The enlarged CNV lesions in Irf8-deficient mice associated with reduced numbers of Cx3cr1-GFP+ resident micro- glia cells may thus be interpreted as a consequence of dysfunctional pro-fibrotic Irf8-deficient MG or as a re- sult of insufficient numbers of CNV-suppressing micro- glia in Irf8-deficient animals. The latter may in turn indicate a general protective role of wild-type microglia which would be in line with previous studies [2, 46]. Discussion The hypothesis that deficient microglia substantially influ- ence CNV size in Irf8-deficient mice is further supported by the head-shielded bone marrow transplantation ex- periments performed in this study, which showed en- larged CNV in Irf8-deficient animals compared with controls despite successful reconstitution of Irf8-potent peripheral monocytes. However, we cannot exclude the possibility that other Irf8-expressing cell types in the ret- ina or from the blood contribute to the increased CNV lesion size in Irf8-deficient mice. Since Irf8 expression in the retina was mainly restricted to retinal microglial cells, which represent the most numerous myeloid cell population in CNV, in contrast to a low number of infil- trating peripheral monocytes [59], we consider this pos- sibility rather unlikely. Furthermore, heterozygous Cx3cr1GFP/+ expression could have an impact on mye- loid cells and be associated with an inflammatory pheno- type. However, since both Irf8 wild-type and knockout mice in our study were consistently heterozygous for Cx3cr1, we consider this effect negligible and used the Cx3cr1GFP/+ line to visualise Irf8 wild-type and knockout mice in a comparable manner, since other standard markers such as IBA1 are dysregulated and cannot be used [44]. To explore potential mechanisms in Irf8-deficient ret- inal MG contributing to CNV formation, we next per- formed RNA-sequencing on sorted retinal MG at sites of CNV. Among others, we found that purinergic receptors, encoded by genes such as P2ry12, P2ry13 and Adora1, were significantly downregulated in Irf8 KO rMG after laser injury. Since these receptors are critical for the rec- ognition of ATP released during tissue injury and thus control cell migration, their downregulation may be at least partly responsible for the reduced MG cell number at sites of CNV in Irf8-deficient mice [12, 23, 30]. This hypothesis is in line with in vitro work by Masuda et al. showing a downregulation of genes encoding purinergic receptors, including P2ry12 and P2rx4, in Irf8-deficient brain microglia which was associated with a migration defect in vitro [39]. It is interesting to note that Irf8- Availability of data and materials The online version contains supplementary material available at https://doi. org/10.1186/s12974-021-02230-y. RNA sequencing data are available under the GSE accession number GSE160845 and GSE182504. The online version contains supplementary material available at https://doi. org/10.1186/s12974-021-02230-y. The online version contains supple org/10.1186/s12974-021-02230-y. Author details 1 1Medical Faculty, Eye Center, University Hospital, University of Freiburg, Freiburg im Breisgau, Germany. 2Institute of Anatomy, Wuerzburg University, Wuerzburg, Germany. 3Cardiology and Angiology, University Heart Center, University of Freiburg, Freiburg im Breisgau, Germany. 4Medical Center and Faculty of Medicine, Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg Bad Krozingen, University of Freiburg, Freiburg, Germany. 5Medical Faculty, Institute of Neuropathology, University Hospital, University of Freiburg, Freiburg im Breisgau, Germany. 6CIBSS-Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany. 7Medical Faculty, Center for Basics in NeuroModulation (NeuroModulBasics), University of Freiburg, Freiburg im Breisgau, Germany. 8Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg im Breisgau, Germany. 9Institute of Anatomy, Leipzig University, Leipzig, Germany. Additional file 3. Supplemental figure 3 Irf8 deficiency does not affect the retinal structure, function and vasculature. A-C Representative color fundus images (A), fluorescein angiography (B) and optical coherence tomography (OCT) images of Irf8 WT and Irf8 KO mice. C) Both Irf8 WT (blue, n=12) and Irf8 KO (red, n=12) mice displayed a regular retinal struc- ture, a similar thickness of the inner nuclear layer and the outer retina at 100 and 200 μm from the optic nerve head in the optical coherence tomographs. Data are shown as mean ± SEM. ONH = Optic nerve head. D Electroretinography (ERG). No significant difference was found between the Irf8 WT (blue, n=9) and Irf8 KO (red, n=9) mice concerning the dark- adapted scotopic and light-adapted photopic ERG measurements at dif- ferent flash intensities. Data are shown as mean ± SEM. E Staining against smooth muscle actin (SMA, red) reveals a comparable number of arteries (Irf8 WT (n=5); Irf8 KO (n=7)) and major vessels (Irf8 WT (n=11); Irf8 KO (n= 13)) between both groups. Data are shown as mean ± SEM. F No signifi- cant differences in vessel branch formation in the superficial (upper panel) or deep plexus (lower panel) in the central or peripheral area of the retina could be observed, compared between Irf8 WT (n=6) and Irf8 KO (n=7). Data are presented as mean ± SEM. Additional file 3. Supplemental figure 3 Irf8 deficiency does not affect the retinal structure, function and vasculature. A-C Representative color fundus images (A), fluorescein angiography (B) and optical coherence tomography (OCT) images of Irf8 WT and Irf8 KO mice. Declarations Additional file 1. Supplemental figure 1 Irf8 is expressed predominantly in retinal MG and in some bipolar or Müller cells. No Irf8-VENUS expression could be detected co-localised with GFAP (A), βIII-Tubulin (B) and Collagen IV (E), indicating that IRF8 is not expressed in retinal astro- cytes, ganglion cells or vessels. All IBA1+ cells exhibited a strong Irf8- VENUS signal (C) suggesting that all retinal MG express IRF8. Some CHX10+ cells could be co-localised with Irf8-VENUS expression (D) dem- onstrating that some bipolar cells or Müller cells express VENUS. Funding KK was supported by a project grant of the Fritz-Thyssen Foundation. CL, IH, KK and MP were supported by the SFB/TRR167. Open Access funding en- abled and organized by Projekt DEAL. Abbeviations Ad 1 Ad Adora1: Adenosine A1 receptor; Aif1: Allograft inflammatory factor 1; AMD: Age-related macular degeneration; ATP: Adenosine triphosphate; CNS: Central nervous system; CNV: Choroidal neovascularization; CSF1R: Colony stimulating factor 1 receptor; ELISA: Enzyme-linked immunosorbent assay; ERG: Electroretinography; FFA: Fundus fluorescein angiography; FN: Fibronectin; GO: Gene ontology; INL: Inner nuclear layer; IRF: Interferon regulatory factor; MG: Microglia; OCT: Optical coherence tomography; ONL: Outer nuclear layer; P2ry12: Purinergic receptor P2Y, G- Protein couple 12; PR: Photoreceptors; Sall1: Spalt-like transcription factor 1; Tmem119: Transmembrane 119; WT: Wild type Authors’ contributions PZ, AS, SB, JK, FL, YL, JK, FL, MB, AT, CH and PW performed experiments and analysed data. JW analysed RNA-seq data. HA, GS, IH, KK and MP provided scientific input. CL and PW supervised the project and wrote the manuscript. The authors read and approved the final manuscript. Conclusions In conclusion, this study identifies IRF8 as a critical me- diator for the morphology, distribution and expression profile of retinal microglia, and for transformation to a reactive phenotype. The niche-dependent phenotype Zhang et al. Journal of Neuroinflammation (2021) 18:215 Zhang et al. Journal of Neuroinflammation (2021) 18:215 Page 16 of 18 Zhang et al. Journal of Neuroinflammation already present during postnatal development, the al- tered morphology and the disturbed rMG distribution did not lead to any impairment of retinal morphology and function in the steady state, but to enlarged lesions in the laser CNV model. This highlights the importance of IRF8 and retinal MG for the development of patho- logical neovascularisation in the eye and highlights the potential of immunomodulatory therapeutic interven- tions of rMG recruitment in retinal disease. Additional file 5. Supplemental figure 5: Polarisation markers expressed by CNV-associated microglia. The M1 and M2 polarisation markers Cd86, H2-Ab1, Tlr2, Cd163 and Mrc1 (CD206) are shown as transcripts per million in comparison between Irf8 WT and KO under CNV conditions. Additional file 6. Supplemental table 1: List of primer sequences. Additional file 7. Supplemental table 2: List of antibodies used for immunohistochemistry and flow cytometry. Additional file 5. Supplemental figure 5: Polarisation markers expressed by CNV-associated microglia. The M1 and M2 polarisation markers Cd86, H2-Ab1, Tlr2, Cd163 and Mrc1 (CD206) are shown as transcripts per million in comparison between Irf8 WT and KO under CNV conditions. Additional file 7. Supplemental table 2: List of antibodies used for immunohistochemistry and flow cytometry. Acknowledgements ld l k h We would like to thank Gabriele Prinz, Michael Bach and Daniel Böhringer for excellent technical and bioinformatical assistance. We are grateful to Frank Rosenbauer for providing Irf8-VENUS reporter mice. Competing interests h h d l The authors declare no conflict of interest in this study. The authors declare no conflict of interest in this study. Author details 1 C) Both Irf8 WT (blue, n=12) and Irf8 KO (red, n=12) mice displayed a regular retinal struc- ture, a similar thickness of the inner nuclear layer and the outer retina at 100 and 200 μm from the optic nerve head in the optical coherence tomographs. Data are shown as mean ± SEM. ONH = Optic nerve head. D Electroretinography (ERG). No significant difference was found between the Irf8 WT (blue, n=9) and Irf8 KO (red, n=9) mice concerning the dark- adapted scotopic and light-adapted photopic ERG measurements at dif- ferent flash intensities. Data are shown as mean ± SEM. E Staining against smooth muscle actin (SMA, red) reveals a comparable number of arteries (Irf8 WT (n=5); Irf8 KO (n=7)) and major vessels (Irf8 WT (n=11); Irf8 KO (n= 13)) between both groups. Data are shown as mean ± SEM. F No signifi- cant differences in vessel branch formation in the superficial (upper panel) or deep plexus (lower panel) in the central or peripheral area of the retina could be observed, compared between Irf8 WT (n=6) and Irf8 KO (n=7). Data are presented as mean ± SEM. Received: 31 March 2021 Accepted: 4 August 2021 Received: 31 March 2021 Accepted: 4 August 2021 Ethics approval and consent to participate All animal experiments were authorized by the local animal care and use committee under the respective EU, national, federal and institutional regulations for animal experiments. Consent for publication All authors consented to publish this article. Additional file 2. Supplemental figure 2 Temporal and spatial distribution of retinal microglia during development. A Representative pictures showing the numbers of microglia per field of view in Irf8 WT and Irf8 KO mice in comparison between the ganglion cell and inner plexiform layer (GCL/IPL) and the developing neuroblast layer (NBL) or outer plexiform layer (OPL), respectively, at postnatal day (P) 1, P7 and in adult mice. B Quantification thereof. Data are presented as mean ± SEM. 1. Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of three key innate immune pathways for the most common retinal degenerative diseases. EMBO Mol Med. 2018;10(10). https://doi.org/10.152 52/emmm.201708259. References 1. Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of three key innate immune pathways for the most common retinal degenerative diseases. EMBO Mol Med. 2018;10(10). https://doi.org/10.152 52/emmm.201708259. 1. Akhtar-Schäfer I, Wang L, Krohne TU, Xu H, Langmann T. Modulation of three key innate immune pathways for the most common retinal degenerative diseases. 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https://openalex.org/W2476895969
https://bmcmedresmethodol.biomedcentral.com/track/pdf/10.1186/s12874-016-0194-3
English
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Minimax and admissible adaptive two-stage designs in phase II clinical trials
BMC Medical research methodology
2,016
cc-by
10,551
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shan et al. BMC Medical Research Methodology (2016) 16:90 DOI 10.1186/s12874-016-0194-3 Shan et al. BMC Medical Research Methodology (2016) 16:90 DOI 10.1186/s12874-016-0194-3 Open Access Abstract Background: Simon’s two-stage design is the most widely implemented among multi-stage designs in phase II clinical trials to assess the activity of a new treatment in a single-arm study. In this two-stage design, the sample size from the second stage is fixed regardless of the number of responses observed in the first stage. Methods: We develop a new minimax adaptive design for phase II clinical trials, by using the branch-and-bound intelligent algorithm based on conditional error functions. Results: We compare the performance of the proposed design and competitors, including Simon’s minimax design, and a modified Simon’s design that allows early stopping for futility or efficacy. The maximum sample size of the proposed minimax adaptive design is guaranteed to be less than or equal to those from other existing designs. When the proposed design has the same maximum sample size as others, it always has the smallest expected sample size. In addition to the minimax adaptive design, we also introduce admissible adaptive designs determined from a Bayesian perspective. Conclusions: The proposed adaptive minimax design can save sample sizes for a clinical trial. The minimum required sample size is critical to reduce the cost of a project. Keywords: Adaptive design, Admissible design, Efficacy, Futility, Minimax design, Simon’s design : Adaptive design, Admissible design, Efficacy, Futility, Minimax design, Simon’s design when the treatment is indeed ineffective. For this reason, a multi-stage design is often implemented, and among these designs the most popular is Simon’s two-stage design [2]. Simon [2] proposed two optimal designs: the optimal design with the expected sample size under the null (ESS0) minimized, and the minimax design having the smallest ESS0 among the designs with the maximum sample size (MSS) minimized. Simon’s design allows early stopping in the first stage for futility only. Recently, Mander and Thompson [3] extended Simon’s design to allow stopping for efficacy or futility by introducing an additional design parameter that represents the stopping criteria for effi- cacy in the first stage. It is guaranteed that the MSS of the modified design is less than or equal to that of Simon’s. Minimax and admissible adaptive two-stage designs in phase II clinical trials Guogen Shan1, Hua Zhang2 and Tao Jiang3* Methods Simon [2] proposed the widely used two-stage designs for early phase II clinical trials with binary endpoints by test- ing the response rate to make a conclusion of go or no-go to the next trial phase of the study. In this study design, the unacceptable response rate πu can be estimated from his- torical data, and the acceptable response rate πa is often the targeted response rate of a new treatment or ther- apy, where πu < πa. For example, in the clinical trial for urothelial cancer with neoadjuvant therapy [10], the unac- ceptable and acceptable response rates are πu = 35 % and πa = 50 %, respectively. The hypotheses to be tested are In phase II clinical trials, it is desirable to achieve the primary goal of the study with the number of patients minimized, as the cost of the study highly depends on the number of patients. In addition, Institutional Review Boards approve proposed studies based on the maxi- mum possible number of patients that are needed to address the scientific questions. Therefore, the minimax design is preferable by researchers with the smallest MSS as compared to the optimal design when the MSS dif- ference between the two designs is not small. To our best knowledge, no adaptive design based on the mini- max criteria has been proposed for use in practice. Due to the importance of such designs, we develop a new minimax adaptive design with the monotonic property respected in this article by using the branch-and-bound algorithm [7] based on conditional type I and II error rates. H0 : π ≤πu, against against against Ha : π ≥πa. Ha : π ≥πa. The null hypothesis is rejected for a large response rate. Let n1, n2, and n be the number of subjects enrolled in the first stage, the second stage, and both stages combined, respectively, and x1, x2, and x are the associated number of responses observed from the study. In the clinical trial of the neoadjuvant therapy for urothelial cancer [10], Simon’s minimax design was used for sample size determination to achieve 80 % power (β = 0.2) at the significance level of α = 0.1 when the response rates were πu = 35 % and πa = 50 %. The design was calculated as: (r1/n1, r/n) = (10/31, 21/49) with the ESS0=40.8. Background In phase II clinical trials, a new treatment or a new therapy is often assessed by measuring activity with dichotomized endpoints, responding ’yes’ or ’no’ to the intervention. For Oncology clinical trials, the response criteria may be determined by the Response Evaluation Criteria In Solid Tumours (RECIST) [1]. The traditional experiment in phase II Oncology trials is often conducted in a single arm study, which is also popular in other studies, such as AIDS. All patients enrolled in the study are treated with the same treatment, and their measurements are obtained at the end of the study and compared to the priori estimate from historical studies with the similar condition of experiment and patients. From ethical and economical considerations, a trial should be allowed to stop earlier after an interim analysis to better protect patients, especially in situations In Simon’s design and the modified Simon’s design due to Mander and Thompson [3], the second stage sample size is always fixed and is not allowed to be modified as the result observed from the first stage. To make a design flex- ible and efficient, adaptive designs have been developed to allow the second stage sample size to depend on first *Correspondence: jtao@263.net 3Department of Statistics, Zhejiang Gongshang University, Hangzhou, 310018 Zhejiang, China Full list of author information is available at the end of the article Shan et al. BMC Medical Research Methodology (2016) 16:90 Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 2 of 14 stage responders. It is easy to show that Simon’s opti- mal design is a special case of adaptive designs, therefore, the expected sample size of an optimal adaptive design is always less than or equal to that of Simon’s design. Several optimal adaptive designs have been developed for phase II clinical trials, and the majority of them are based on the optimal criteria with the smallest ESS0. Banerjee and Tsi- atis [4] developed an optimal adaptive two-stage design by using a Bayesian decision-theoretic construct to mini- mize the expected loss through backward induction, with type I and II error rates respected. The sample size savings are small to modest when compared to Simon’s optimal design. Later, Englert and Kieser [5] developed an opti- mal adaptive two-stage design based on conditional error functions [6] and an efficient search strategy [7]. Background Although these adaptive designs guarantee the type I and II error rates, these designs suffer from a counter-intuitive fea- ture that the second stage sample size may increase as the number of responses observed from the first stage increases. Very recently, Shan et al. [8] developed an optimal adaptive two-stage design with the monotonic- ity property respected; the second stage sample size is a non-increasing function of the first stage responders. This improvement makes it practical to use the optimal adaptive design. The remainder of this article is organized as follows. In “Methods” Section, we introduce the detailed search method for the optimal adaptive design when the first stage and the MSS of the second stage sample size are fixed, then present the approach to find the minimax adaptive design. In “Results” Section we compare the MSS and the ESS0 of the proposed minimax adaptive design with competitors. A real clinical trial from a cancer study is used to illustrate the proposed design at the end of “Results” Section. Finally, we provide some remarks in “Discussion and conclusions” Section. Methods A deci- sion can not be made in the first stage when 11 < x1 ≤16, and additionally n2 = n −n1 = 49 −32 = 17 patients will be enrolled in the second stage. At the end of the study, the null hypothesis will be rejected if x > 21. Otherwise, it is concluded that the new treatment is not promising enough to warrant further investigation. The ESS0 of the minimax-EF design is 39.2 as compared to the ESS0 = 40.8 from Simon’s minimax design. In the two aforementioned designs, the second stage sample size is not allowed to change with the number of responses observed from the first stage. To improve the efficiency and flexibility of a study, we propose a new adaptive two-stage design based on the minimax crite- ria from Simon’s, the design with the smallest ESS0 and Table 1 The proposed adaptive minimax design for the urothelial cancer trial with the neoadjuvant therapy with (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5) S n2(S) n(S) r(S) Minimax-EF design ≤11 0 32 0 12 17 49 21 13 17 49 21 14 17 49 21 15 17 49 21 16 17 49 21 ≥17 0 32 0 Minimax adaptive design ≤9 0 28 0 10 21 49 21 11 21 49 21 12 21 49 21 13 21 49 21 14 19 47 20 15 18 46 20 ≥16 0 28 0 MSS. In the proposed adaptive design, the second stage sample size, n2(S), depends on the number of first stage responses, S, and n2(S) is a non-increasing function of S, specifically, n2(S1) ≥n2(S2) when S1 < S2. For a given first stage sample size n1, the value of S ranges from 0 to n1: S = 0, 1, . . . , n1. Moreover, the associated critical value for each S, r(S), also needs to be determined for the adaptive design. Then, the proposed design is presented as design is less than that of the minimax design (35.2 agaisnt 40.8), but the MSS is much larger for the optimal design as compared to the minimax design (58 against 49). p g ( g ) A modified Simon’s design that allows early stopping for futility or efficacy, was proposed by Mander and Thompson [3] who introduced another design param- eter, r2, as ((r1, r2)/n1, r/n). This design is referred to as the Minimax-EF design. Methods For the aforementioned can- cer study, the design can be calculated by using the Stata package, simon2stage [3], as ((r1, r2)/n1, r/n) = ((11, 16)/32, 21/49), see Table 1. With n1 = 32 patients enrolled in the first stage of the study, the study will be stopped for futility if x1 ≤11 or efficacy if x1 > 16. A deci- sion can not be made in the first stage when 11 < x1 ≤16, and additionally n2 = n −n1 = 49 −32 = 17 patients will be enrolled in the second stage. At the end of the study, the null hypothesis will be rejected if x > 21. Otherwise, it is concluded that the new treatment is not promising enough to warrant further investigation. The ESS0 of the minimax-EF design is 39.2 as compared to the ESS0 = 40.8 from Simon’s minimax design. n1 and (n2(S), r(S)) , S = 0, 1, 2, · · · , n1, with a total of 2n1 + 3 unknown parameters. As pointed out by many researchers [5, 8], it becomes quickly impos- sible to estimate these parameters by enumerating all attainable values of n2(S) and r(S), even after controlling for the upper bound of the second stage sample size. Conditional error functions are frequently used in adap- tive designs to fully use the information from the previous stage, specifically, P(s|r(s), n2(s), π) = 1 −B (r(s) −s : n2(s), π) , where s is the observed first stage response under the design parameters n2(s) and r(s), and B(x : y, z) is the cumulative probability function of a binomial distribution for observed value x with size y and probability z. Note that P(s|π) = 0 or 1 when the study is terminated after the first stage for futility or efficacy, respectively. As a spe- cial case, it is always reasonable to stop the trial when no response is observed from the first stage, P(0|π) = 0. It should be noted that the proposed design allows early stopping in the first stage for either futility or efficacy just as existing adaptive designs. In the two aforementioned designs, the second stage sample size is not allowed to change with the number of responses observed from the first stage. Methods The trial was allowed to stop for futil- ity at the first stage if the number of first stage responses x1 ≤10 was observed from a total of n1 = 31 patients. Otherwise, an additional n2 = n −n1 = 49 −31 = 18 patients were enrolled in the second stage, and at least 22 responses should be observed from total 49 patients, x ≥22, in order to claim that the neoadjuvant therapy had sufficient activity. The MSS of the minimax design was 49. An alterntive to the minimax design is Simon’s opti- mal design whose ESS0 was the smallest among all designs that met the design criteria. The design parameters for the optimal design are: (r1/n1, r/n) = (7/20, 24/58) with the ESS0 = 35.2. As expected, the ESS0 of the optimal Both minimax and optimal designs have been widely used in clinical trials. It is often the case that the expected sample size of the minimax design is much larger than that of the optimal design, although the minimax design has a smaller maximum sample size. To compromise between the maximum sample size and the expected sample size under the null, an admissible adaptive design was pro- posed by Jung et al. [9], which was implemented in Java language by them. By using the Bayes risk function as in Jung et al. [9], we propose a new admissible adaptive two- stage design that is between the minimax adaptive design and the optimal adaptive design. Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 3 of 14 Page 3 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 design is less than that of the minimax design (35.2 agaisnt 40.8), but the MSS is much larger for the optimal design as compared to the minimax design (58 against 49). A modified Simon’s design that allows early stopping for futility or efficacy, was proposed by Mander and Thompson [3] who introduced another design param- eter, r2, as ((r1, r2)/n1, r/n). This design is referred to as the Minimax-EF design. For the aforementioned can- cer study, the design can be calculated by using the Stata package, simon2stage [3], as ((r1, r2)/n1, r/n) = ((11, 16)/32, 21/49), see Table 1. With n1 = 32 patients enrolled in the first stage of the study, the study will be stopped for futility if x1 ≤11 or efficacy if x1 > 16. Methods (1) is used in two folders. The function is first used to find all satisfied designs with the smallest MSS, minmax(n1+n2(s),s=0,1,...,n1). The second is to identify the minimax adaptive design as the one from these in the previous step with the smallest ESS0. function. Two procedures are recursively utilized in the algorithm to identify the optimal design. The first pro- cedure is the branching process that splits the problem into several complement problems. The conditional type I error functions are used in this step to split problems. Although it is not a requirement to sort the elements in  in the design search, it helps to reduce the computational intensity to sort them by n2(S) in an ascending order, and P(S|πu) in an increasing order. The ordering of n2(S) is used to meet the monotonicity feature of the proposed design. We start the design search with a fixed first stage sam- ple size n1 and the MSS n. Then, the MSS of the second stage is n2,max = n −n1. It is easy to show that n2(S) ≤n2,max and r(S) ≤n2(S). The optimal design needs to be searched over a triangle space for each S, φ(S) = {(n2(S), r(S)) : r(S) ≤n2(S) ≤n2,max}, where S = 0, 1, 2, · · · , n1. The complete search space is a product of these triangle spaces, The second procedure, the bounding procedure, com- putes boundary values of constraint functions. Let O(S, WS) be the WS-th conditional error function in the  when the number of responses is S in the first stage. Sup- pose the current branching outcome from the branching procedure is at S = k (φ(0) × φ(1) × · · · × φ(n1)) . As the first stage sample size n1 increases, the size of this complete search space increases exponentially. Therefore, it is not feasible to conduct this naive search to identify the optimal design. O(0, 1),O(1, W1),O(2,W2), . . . ,O(k,Wk), O(k+1,1), . . . ,O(n1,1). (2) (2) When no response is observed from the first stage, the trial is assumed to be stopped for futility, that is repre- sented by O(0, 1). Note that we use n2(S, WS) and r(S, WS) to replace n2(S) and r(S) in the design search. Methods The objec- tive function at the current branching step is calculated as It is much more complicated to search for an opti- mal solution over a two-dimensional space than a one- dimensional space. For this reason, Englert and Kieser [5] suggested using the union of all type I conditional error functions and (0, 1), referred to as , as the parameter space. For each element in , it contains the informa- tion of n2(S) and r(S) as in the two-dimensional space. That said, it is equivalent to determine the conditional type I error value for S and (r(S), n2(S)). It is still not fea- sible to conduct a grid search over the parameter space (a(n1 + 1) −dimensional space) due to the fact that the size of the parameter space increases very quickly as n1 and n2,max go up. f = k  s=0 (n1 + n2(s, ws))×b(s : n1, πu)+ n1  s=k+1 n1b(s : n1, πu). The overall goal is to find the values of W for each S in O(S, W) that minimizes the objective function as min O(S,WS),S=0,1,...,n1 f . min O(S,WS),S=0,1,...,n1 f . In order to overcome the computational burden, the branch-and-bound algorithm [7], an intelligent algorithm, is considered when searching for the optimal design over a one-dimensional space on each S. This algorithm can be used to search for the optimal design with or with- out constraints [5, 8]. The monotonicity restriction in the optimal adaptive design search by Shan et al. [8] is an important feature that makes a design usable in practice. The second stage sample size is a non-increasing func- tion of the number of responses observed from the first stage: n2(S1) ≥n2(S2) when S1 < S2. This monotonicity restriction is respected in the proposed minimax adaptive design. Two constraints need to be satisfied in the design search Two constraints need to be satisfied in the design search αmin = k  s=0 P (s|r(s, ws), n2(s, ws), πu) × b(s : n1, πu), and βmin = 1 − k  s=0 P (s|r(s, ws), n2(s, ws), πa) × b(s : n1, πa) − n1  s=k+1 b(s : n1, πa). These two constraints help to determine the set of feasi- ble solutions, and discard the candidates that do not lead to the optimal design. Methods To improve the efficiency and flexibility of a study, we propose a new adaptive two-stage design based on the minimax crite- ria from Simon’s, the design with the smallest ESS0 and Table 1 The proposed adaptive minimax design for the urothelial cancer trial with the neoadjuvant therapy with (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5) S n2(S) n(S) r(S) Minimax-EF design ≤11 0 32 0 12 17 49 21 13 17 49 21 14 17 49 21 15 17 49 21 16 17 49 21 ≥17 0 32 0 Minimax adaptive design ≤9 0 28 0 10 21 49 21 11 21 49 21 12 21 49 21 13 21 49 21 14 19 47 20 15 18 46 20 ≥16 0 28 0 Table 1 The proposed adaptive minimax design for the urothelial cancer trial with the neoadjuvant therapy with For each design, the type I and II error rates are then calculated from conditional error functions as α = n1  s=0 P(s|r(s), n2(s), πu) × b(s : n1, πu), and β = 1 − n1  s=0 P(s|r(s), n2(s), πa) × b(s : n1, πa), β = 1 − n1  s=0 P(s|r(s), n2(s), πa) × b(s : n1, πa), where b(.) is the density function of a binomial distribu- tion. All designs with guaranteed type I and II error rates, are candidates for the optimal design. Often, multiple designs meet the design criteria, and an additional criteria should be applied in order to find the optimal design. The criteria used in the proposed adaptive minimax design is the smallest ESS0 and MSS, where b(.) is the density function of a binomial distribu- tion. All designs with guaranteed type I and II error rates, are candidates for the optimal design. Often, multiple designs meet the design criteria, and an additional criteria should be applied in order to find the optimal design. The criteria used in the proposed adaptive minimax design is the smallest ESS0 and MSS, min max(n1+n2(s),s=0,1,...,n1) ESS0, (1) (1) where ESS0 = n1 s=0[ n1 + n2(s)] ×b(s : n1, πu) is the expected sample size under the null for the design with n1 and max(n1 + n2(s), s = 0, 1, . . . , n1) as the first stage Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 4 of 14 sample size and the MSS. The min in Eq. Methods As pointed out, it is time consuming to compute the actual type I and II error rates for each element in the parameter space, and the branch-and-bound algorithm is able to finish the design search in a timely manner by dis- carding elements that do not lead to the optimal design, which is the key idea of this intelligent algorithm. When the sample sizes (n1, n) are given, the ESS0 is the objective The minimax-EF design is a special case of the mini- max adaptive design, therefore, the MSS of the minimax- EF design is the upper bound of the proposed adaptive design. For this reason, we start the search with the MSS, Page 5 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 in parameter space , is useful to meet the monotonic relationship between the n2(S) and S in searching for the design. Among these obtained optimal adaptive designs, the one with the smallest ESS0 is the adaptive mini- max design when the MSS is nt. From the relationship between the proposed design and the minimax-EF design, it is guaranteed that an optimal adaptive design will be obtained when MSS is nt. The MSS is then decreased by 1, and the optimal adaptive design is searched again with the MSS=nt −1. This procedure will be continued until no optimal design is obtained from three consecutive MMS values, say n∗−1, n∗−2, and n∗−3. Then, n∗is the nt, which is the MSS of the minimax-EF design. For this given MSS, say nt, the possible number of subjects from the first stage, n1, ranges between 1 and nt −1. The search for n1 = 1 and nt −1 as the first stage sample sizes are excluded for practical reasons: it is not realistic to enroll only one patient to make a decision. For each sample size configuration (n1, nt), the algo- rithm is applied for the design search. If the study is stopped for futility when S ≤s −1, then we assign n2(s, Ws) = n −n1 to guarantee that the MSS is exactly nt. It should be noted that the MSS could occur at mul- tiple S values. Methods The ascending order of n2(S) for elements Table 2 Comparison between three optimal designs for expected sample size ESS0 at α = 0.05 given πa −πu = 0.2 and 0.15 Minimax Optimal Simon Minimax-EF Adaptive Adaptive πu πa Power n ESS0 n ESS0 n ESS0 n ESS0 πa −πu = 20 % 0.1 0.3 0.8 25 19.51 24 20.30 23 20.94 29 14.85 0.9 33 26.18 33 23.96 33 23.93 35 22.38 0.2 0.4 0.8 33 22.25 32 24.93 32 23.22 37 20.48 0.9 45 31.23 44 35.68 44 33.39 53 29.74 0.3 0.5 0.8 39 25.69 36 30.68 36 29.31 46 23.45 0.9 53 36.62 50 42.47 50 41.03 60 34.08 0.4 0.6 0.8 39 34.44 39 34.33 39 26.86 46 24.39 0.9 54 38.06 54 38.03 53 42.65 66 35.64 0.5 0.7 0.8 37 27.74 37 26.90 37 26.87 43 23.33 0.9 53 36.11 51 41.14 51 37.74 59 33.45 0.6 0.8 0.8 35 20.77 33 23.97 33 22.13 38 20.28 0.9 45 35.90 45 33.30 45 31.36 52 28.74 0.7 0.9 0.8 26 23.16 26 23.11 25 18.00 27 14.82 0.9 32 22.66 32 22.66 32 22.64 36 20.80 πa −πu = 15 % 0.1 0.25 0.8 40 28.84 38 33.94 38 28.87 43 24.49 0.9 55 40.03 53 47.87 53 41.29 62 36.45 0.2 0.35 0.8 53 40.44 53 40.41 53 40.33 63 34.87 0.9 77 58.42 76 66.51 74 59.58 87 50.80 0.3 0.45 0.8 65 49.63 64 51.32 64 48.08 77 41.33 0.9 88 78.51 88 78.45 88 68.29 104 59.96 0.4 0.55 0.8 70 60.07 69 54.17 69 49.84 82 44.05 0.9 94 78.88 94 76.30 94 74.20 106 63.84 0.5 0.65 0.8 68 66.11 68 66.05 67 58.41 81 43.01 0.9 93 75.00 93 72.20 93 69.84 109 61.87 0.6 0.75 0.8 62 43.79 62 42.89 61 45.26 69 38.53 0.9 84 73.20 84 73.13 84 64.00 97 54.99 0.7 0.85 0.8 49 34.44 49 34.36 49 33.00 59 29.78 0.9 68 48.52 65 50.46 65 48.78 78 42.60 Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 6 of 14 Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Methods Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference Power = 80 % Power = 90 % S n2(S) n(S) r(S) S n2(S) n(S) r(S) πu = 0.2 Simon:(4/18, 10/33) Simon:(5/24, 13/45) Minimax-EF:((2, 6)/15, 10/32) Minimax-EF:((4, 9)/25, 13/44) New:n1 = 19 New:n1 = 23 ≤4 0 19 0 ≤4 0 23 0 5 13 32 10 5 21 44 12 6 13 32 10 6 21 44 13 7 13 32 9 7 21 44 13 8 13 32 10 8 21 44 13 9 11 30 10 9 21 44 13 ≥10 0 19 0 10 15 38 11 ≥11 0 23 0 πu = 0.3 Simon:(6/19, 16/39) Simon:(7/24, 21/53) Minimax-EF:((8, 13)/27, 15/36) Minimax-EF:((11, 17)/37, 20/50) New:n1 = 20 New:n1 = 32 ≤5 0 20 0 ≤9 0 32 0 6 16 36 14 10 18 50 19 7 16 36 15 11 18 50 20 8 16 36 15 12 18 50 20 9 16 36 15 13 18 50 20 10 16 36 15 14 18 50 20 11 16 36 15 15 18 50 20 12 14 34 15 16 18 50 20 ≥13 0 20 0 17 11 43 18 ≥18 0 32 0 πu = 0.4 Simon:(17/34, 20/39) Simon:(12/29, 27/54) Minimax-EF:((17, 19)/34, 20/39) Minimax-EF:((12, 19)/29, 27/54) New:n1 = 16 New:n1 = 35 ≤6 0 16 0 ≤14 0 35 0 7 23 39 20 15 18 53 26 8 23 39 20 16 18 53 27 9 23 39 20 17 18 53 27 10 23 39 20 18 18 53 27 11 23 39 21 19 18 53 26 12 22 38 20 20 17 52 26 13 16 32 18 21 17 52 26 14 9 25 16 22 17 52 26 15 5 21 15 23 17 52 27 16 3 19 16 ≥24 0 35 0 Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference Page 7 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Methods Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) p p p g a u + g ( 1/ 1, / ), stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) πu = 0.5 Simon:(12/23, 23/37) Simon:(14/27, 32/53) Minimax-EF:((10, 15)/20, 23/37) Minimax-EF:((17, 23)/34, 31/51) New:n1 = 20 New:n1 = 28 ≤10 0 20 0 ≤14 0 28 0 11 17 37 23 15 23 51 30 12 17 37 23 16 23 51 31 13 17 37 23 17 23 51 31 14 17 37 23 18 23 51 31 15 15 35 22 19 23 51 31 ≥16 0 20 0 20 23 51 31 21 21 49 29 22 6 34 22 ≥23 0 28 0 πu = 0.6 Simon:(8/13, 25/35) Simon:(15/26, 32/45) Minimax-EF:((10, 14)/17, 24/33) Minimax-EF:((15, 20)/25, 32/45) New:n1 = 15 New:n1 = 23 ≤9 0 15 0 ≤14 0 23 0 10 18 33 24 15 22 45 32 11 18 33 24 16 22 45 32 12 17 32 23 17 21 44 31 13 16 31 22 18 21 44 31 14 14 29 21 19 21 44 31 15 14 29 21 20 10 33 24 21 10 33 25 22 8 31 24 ≥23 0 23 0 πu = 0.7 Simon:(19/23, 21/26) Simon:(13/18, 26/32) Minimax-EF:((19, 20)/23, 21/26) Minimax-EF:((13, 18)/18, 26/32) New:n1 = 13 New:n1 = 18 ≤9 0 13 0 ≤13 0 18 0 10 12 25 21 14 14 32 26 11 12 25 21 15 14 32 26 12 12 25 20 16 14 32 26 13 7 20 16 17 14 32 26 ≥17 3 21 18 minimum MSS, and the optimal design associated with n∗is the final minimax adaptive two-stage design. It is For the candidates of an admissible design, the f step is to identify the MSS values of the minimax ad minimum MSS, and the optimal design associated with n∗is the final minimax adaptive two-stage design. It is obvious that n∗≤nt. For the candidates of an admissible design, the first step is to identify the MSS values of the minimax adap- tive design and the optimal adaptive design by Shan et al. Shan et al. BMC Medical Research Methodology (2016) 16:90 Page 8 of 14 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference Power=80 % Power=90 % S n2(S) n(S) r(S) S n2(S) n(S) r(S) πu = 0.1 Simon:(2/22, 7/40) Simon:(3/31, 9/55) Minimax-EF:((4, 6)/33, 7/38) Minimax-EF:((6, 8)/47, 9/53) New:n1 = 18 New:n1 = 33 ≤1 0 18 0 ≤3 0 33 0 2 20 38 7 4 20 53 8 3 20 38 7 5 20 53 9 4 19 37 6 6 20 53 9 5 19 37 6 7 18 51 8 6 18 36 6 8 17 50 8 ≥7 0 18 0 ≥9 0 33 0 πu = 0.2 Simon:(6/31, 15/53) Simon:(8/42, 21/77) Minimax-EF:((6, 13)/31, 15/53) Minimax-EF:((13, 18)/62, 21/76) New:n1 = 31 New:n1 = 47 ≤6 0 31 0 ≤9 0 47 0 7 22 53 15 10 27 74 20 8 22 53 15 11 27 74 20 9 22 53 15 12 27 74 20 10 22 53 15 13 27 74 20 11 22 53 15 14 26 73 20 12 21 52 15 15 26 73 20 ≥13 0 31 0 16 26 73 20 17 25 72 20 18 14 61 18 ≥19 0 47 0 πu = 0.3 Simon:(16/46, 25/65) Simon:(27/77, 33/88) Minimax-EF:((13, 19)/43, 25/64) Minimax-EF:((27, 33)/77, 33/88) New:n1 = 32 New:n1 = 51 ≤9 0 32 0 ≤15 0 51 0 10 32 64 24 16 37 88 33 11 32 64 25 17 37 88 33 12 32 64 25 18 37 88 33 13 32 64 25 19 37 88 33 14 32 64 25 20 37 88 33 15 31 63 24 21 37 88 33 16 30 62 24 22 37 88 33 17 29 61 24 23 37 88 33 18 24 56 22 24 37 88 34 ≥19 0 32 0 25 36 87 33 26 34 85 33 27 34 85 33 ≥28 0 51 0 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference Page 9 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Table 3 Proposed optimal adaptive designs for πa = πu + 0.2 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) πu = 0.4 Simon:(28/59, 34/70) Simon:(24/62, 45/94) Minimax-EF:((16, 23)/41, 34/69) Minimax-EF:((21, 31)/55, 45/94) New:n1 = 37 New:n1 = 52 ≤15 0 37 0 ≤20 0 52 0 16 32 69 33 21 42 94 44 17 32 69 34 22 42 94 45 18 32 69 34 23 42 94 45 19 32 69 34 24 42 94 45 20 32 69 34 25 42 94 45 21 31 68 33 26 42 94 45 22 31 68 33 27 42 94 45 23 31 68 33 28 42 94 45 24 21 58 29 29 42 94 45 ≥25 0 37 0 30 42 94 45 31 39 91 43 ≥32 0 52 0 πu = 0.5 Simon:(39/66, 40/68) Simon:(28/57, 54/93) Minimax-EF:((39, 40)/66, 40/68) Minimax-EF:((30, 38)/59, 54/93) New:n1 = 54 New:n1 = 55 ≤28 0 54 0 ≤28 0 55 0 29 13 67 39 29 38 93 54 30 13 67 40 30 38 93 54 31 13 67 40 31 38 93 54 32 13 67 40 32 38 93 54 33 13 67 40 33 38 93 54 34 13 67 39 34 38 93 54 35 13 67 39 35 38 93 53 36 13 67 39 36 38 93 54 37 9 63 38 37 38 93 53 ≥38 0 54 0 ≥38 0 55 0 πu = 0.6 Simon:(18/30, 43/62) Simon:(48/72, 57/84) Minimax-EF:((16, 22)/27, 43/62) Minimax-EF:((48, 53)/72, 57/84) New:n1 = 32 New:n1 = 58 ≤19 0 32 0 ≤37 0 58 0 20 29 61 42 38 26 84 57 21 29 61 42 39 26 84 57 22 29 61 42 40 26 84 57 23 28 60 42 41 26 84 57 24 28 60 42 42 25 83 57 25 28 60 42 43 25 83 57 26 27 59 41 44 23 81 56 27 27 59 41 ≥45 0 58 0 28 15 47 34 ≥29 0 32 0 Page 10 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Table 4 Proposed optimal adaptive designs for πa = πu + 0.15 at α = 0.05. Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) Simon’s minimax design (r1/n1, r/n), and the minimax that stops for futility and efficacy ((r1, r2)/n1, r/n), are provided as reference (Continued) πu = 0.7 Simon:(16/23, 39/49) Simon:(33/44, 53/68) Minimax-EF:((16, 21)/23, 39/49) Minimax-EF:((29, 35)/41, 51/65) New:n1 = 25 New:n1 = 37 ≤18 0 25 0 ≤26 0 37 0 19 24 49 39 27 28 65 51 20 24 49 39 28 28 65 51 21 24 49 39 29 28 65 51 22 23 48 38 30 28 65 51 23 8 33 26 31 27 64 50 ≥24 0 25 0 32 27 64 50 33 24 61 48 ≥34 0 37 0 q with n −ESS0 as the slope and ESS0 as the intercept. As ESS0 is always less than n, T is an increasing function of q. [8], nmin and nopt that are in the range of the MSS of an admissible design. Secondly, for each given MSS, n, between nmin and nopt, the optimal design with the small- est ESS0 is calculated by using the algorithm aforemen- tioned. The sample size information, n and ESS0, are used in calculating the Bayes risk function [8], nmin and nopt that are in the range of the MSS of an admissible design. Secondly, for each given MSS, n, between nmin and nopt, the optimal design with the small- est ESS0 is calculated by using the algorithm aforemen- tioned. The sample size information, n and ESS0, are used in calculating the Bayes risk function Results BMC Medical Research Methodology (2016) 16:90 Page 11 of 14 Table 5 Probability of early termination at the first stage for the designs with πa = πu + 0.2 and 80 % power πu Simon Minimax-EF New adaptive design 0.2 0.716 0.402 0.674 0.3 0.666 0.582 0.417 0.4 0.913 0.921 0.527 0.5 0.661 0.589 0.589 0.6 0.647 0.554 0.597 0.7 0.946 0.949 0.579 Table 5 Probability of early termination at the first stage for the designs with πa = πu + 0.2 and 80 % power the minimax-EF design. When the proposed design has the same MSS as either of the two minimax designs, the ESS0 of the proposed design is always smaller. For exam- ple, the saving of the ESS0 from the proposed design as compared to the minimax-EF design when they have the same MSS in Table 2, ranges from 0.03 to 10.16, with an average of 3.07 patients. The optimal adaptive design is included as a reference, and the MSS of this design is generally larger than that of the proposed minimax adaptive design, with a range from 2 to 16, and an aver- age of 9.3 patients from all the configurations studied in Table 2. We present the proposed minimax adaptive design with specific design parameters in Tables 3 and 4 for πa = πu + 0.2 and πa = πu + 0.15, respectively. The pre- specified type I error rate is set as α = 0.05, and two type II error rates are studied, β = 0.1 and 0.2. We present the minimum adaptive design for various values of πu, from 0.2 to 0.7. For example, for the design to achieve 90 % power with πu = 0.6, πa = 0.8 as in Table 3, Simon’s min- imax design, the minimax-EF design and the proposed adaptive minimax design are displayed in Fig. 1: the MSS of the study (n(S)) VS the number of responses from the first stage (S). Simon’s minimax design is calculated as (n1, n, r1, r) = (26, 45, 15, 32) with the MSS=45 and the ESS0 = 35.90. The minimax-EF design is found to be ((r1, r2)/n1, r/n) = ((15, 20)/25, 32/45) with 25 patients enrolled in the first stage, and a possible total sample size of 25. Results We compare performance of the proposed minimax adap- tive design, Simon’s minimax design, the minimax-EF design, and the optimal adaptive design due to Shan et al. [8]. The first three designs are minimax designs, while the last one is under the optimal criteria. The first T = q × n + (1 −q) × ESS0 = (n −ESS0)q + ESS0, where q is a pre-specified weight value, q ∈[ 0, 1]. It can be seen that the Bayes risk function T is a linear function of The number of responses in the first stage: S Maximum sample size: n(S) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 20 23 26 29 32 35 38 41 44 47 50 53 Proposed adaptive minimax design Simon’s minimax design Minimax−EF design Fig. 1 The comparison among Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design for the design with parameters (α, β, πu, πa) = (0.05, 0.1, 0.6, 0.8). The maximum sample size, n(S), is plotted as a function of the number of responses from the first stage, S 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 The number of responses in the first stage: S n among Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design for the design with πa) = (0.05, 0.1, 0.6, 0.8). The maximum sample size, n(S), is plotted as a function of the number of responses from the first Fig. 1 The comparison among Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design for the design with parameters (α, β, πu, πa) = (0.05, 0.1, 0.6, 0.8). The maximum sample size, n(S), is plotted as a function of the number of responses from the first stage, S Shan et al. Results The trial will be stopped for futitlity when S ≤ 15 or efficacy when S > 20 out of n1 = 25 patients in the first stage. The ESS0 is 33.3 for this design. For the proposed minimax adaptive design, the second stage design and the last design are adaptive designs. To the best of our knowledge, we do not find a direct com- petitor in the category of adaptive two-stage designs under the minimax criteria. Simon’s minimax design is the most commonly used design under the minimax criteria, thus it is included in the comparison. The minimax- EF design stops for either futility or efficacy in the first stage. This stopping rule is also applied in the proposed design, thus, this design is also included in the compar- ison. Simon’s design only allows stopping of the trial at the first stage for futility, and the other three designs allows the stoppage for either futility or efficacy in the first stage. The MSS and the ESS0 are compared in Table 2 for the proposed minimax adaptive design, and the other three competitors when πa −πu = 0.2, and 0.15 at the significance level of α = 0.05. As expected, the pro- posed minimax adaptive design has a smaller or the same MSS as compared to the Simon’s minimax design and 0.0 0.2 0.4 0.6 0.8 1.0 30 40 50 60 q The Bayes risk function: T Design with n=50 Design with n=51 Design with n=52 Design with n=53 Design with n=54 Design with n=55 Design with n=56 Design with n=57 Design with n=58 Design with n=59 Design with n=60 Fig. 2 The Bayes risk function as a function of the weight value q in searching for an admissible adaptive design for (α, β, πu, πa) = (0.05, 0.1, 0.3, 0.5) Fig. 2 The Bayes risk function as a function of the weight value q in searching for an admissible adaptive design for (α, β, πu, πa) = (0.05, 0.1, 0.3, 0.5) Shan et al. Application pp We revisit the urothelial cancer trial with the neoad- juvant therapy [10]. Simon’s minimax design was used for study design to attain 80 % power at the signifi- cance level of α = 0.1. The research team expected a 15 % increase in response rate as compared to the priori estimated response rate πu = 35 %. The design param- eters using Simon’s minimax design are: (n1, n, r1, r) = (31, 49, 10, 21) with the ESS0=40.8. The minimax-EF design is: ((r1, r2)/n1, r/n) = ((11, 16)/32, 21/49). The design parameters, (n1, n(S), r(S)), for the proposed adap- tive minimax design are presented in Table 1, and also plotted in Fig. 3. They all have the same maximum sample size 49, but the expected sample size under the null for the proposed design is smaller, 38.9 VS 40.8(Simon’s design), and 39.2 (the minimax-EF design). The adaptive design is also flexible to allow the second stage sample size and its associted critical value to depend on the result from the first stage. As suggested by one of the reviewers, we compare the probability of early termination (PET) at the first stage for these designs. The PET is defined as the probability of a study that is stopped at the first stage due to either futil- ity or efficacy. We present the PET of the three designs with πa = πu + 0.2 and 80 % power in Table 5. The PET of the new adaptive design is always less than that by Simon’s minimax design in these cases. There is no clear relationship between the minimax-EF design and the new design with regard to the PET. It can be seen that the PET of the new design is more consistent as compared to the competitors. Results BMC Medical Research Methodology (2016) 16:90 Page 12 of 14 Table 6 Admissible adaptive designs for (α, β, πu, πa) = (0.05, 0.1, 0.3, 0.5) Table 6 Admissible adaptive designs for (α, β, πu, πa) = (0.05, 0.1, 0.3, 0.5) Interval of q n ESS0 Comment [ 0.000, 0.040] 60 34.08 Optimal design [ 0.040, 0.105] 59 34.12 [ 0.105, 0.132] 57 34.36 [ 0.132, 0.468] 54 34.81 [ 0.468, 0.580] 53 35.69 [ 0.580, 0.721] 51 38.45 [ 0.721, 1.000] 50 41.03 Minimax design sample size is allowed to change as a function of the first stage responders, and the relationship is monotonic. The first stage sample size is n1 = 23 and the maximum sam- ple size is 45 and this maxmum sample size only occurs for the cases with S = 15 and 16 responders observed from the first stage. The trial is terminated at the end of the first stage for futility or efficacy for S ≤14 or S ≥23, respectively. In such cases, the MSS is the first stage sam- ple size, which is 23. When the first stage response is between 15 and 22, the corresponding second stage sam- ple size n2(S) is presented in Table 3. As compared to the other designs, the adaptive minimax design has the smallest expected sample size and the smallest first stage sample size in this particular example. In the proposed design, the second stage sample size is a non-increasing function of the number of responses from the first stage, not a constant as in Simon’s design and the Minimax-EF design. It can be seen that although the adaptive optimal design has the smallest expected sample size as compared to others, the MSS of the adaptive optimal design is often much larger than that of the proposed adaptive minimax. Abbreviations ESS h ESS0, the expected sample size under the null; MMS, maximum sample size Discussion and conclusions To reduce the computational time, one may use a backward search method as in this article, starting with the maximum sam- ple size from the minimax-EF design. In addition, when the proposed design and other designs have the same MSS, the expected sample size under the null of the proposed design is always smaller than others. g Randomized clinical trials are used in clinical tri- als by comparing the new treatment or therapy to the best available treatment for disease. Randomized clin- ical trials are preferable in the majority of studies to reduce the selection bias and confounding effects, thus capturing the true effectiveness of the new treatment. The widely used two-stage design for a two-arm study with binary outcomes is the one due to Thall et al. [16], that does not allow the second stage sample size to change from the results of the first stage. We will extend the adaptive approach from the one-arm study to this two-arm study to develop a new adaptive two-stage design for a randomized clinical trial with dichotomous endpoints. The proposed adaptive design assumes a monotonic relationship between the second stage sample size and the first stage result. In practice, an investigator may want to accrue more patients in the second stage when the number of response from the first stage is large, to obtain as much information as possible from the clini- cal study. In this case, an additional constraint can be added during the design search to meet the investiga- tor’s requirement: the second stage sample sizes are the same when S is above Sc, where Sc can be determined by the new constraint from the investigator. The new con- straint added in the design search should be clinically meaningful. Discussion and conclusions Figure 2 displays the lines of the Bayes risk function T as a function of q for each design to attain 90 % power at the significance level of 0.05, with πu = 0.3 and πa = 0.5. The minimax adaptive design and the optimal adaptive design are presented in Table 3, with nmin = 50 and nopt = 60. Therefore, a total of 11 lines are displayed in the figure to represent the optimal designs when n is between 50 and 60. In order to identify an admissible design for a given range of q, one has to first compute the intersections among these 11 lines, and the maximum number of inter- sections between 0 and 1 is 11 2  = 55. Within these 55 intersections, 2 of them are out of the range of [ 0, 1], this leads to a total of 53 intersections between 0 and 1. After sorting the x-values of these intersections, the design among these 11 designs is the admissible design for a given range of q when this design has the smallest T over this range, see Table 6. It can be seen that the optimal adaptive design is the admissible design when q is close to 0, and the minimax design is the admissible design when q is close to 1. We develop a new minimax adaptive two-stage design for use in phase II clinical trials to assess the new treat- ment’s activity. The software program to implement the adaptive designs in this article is written in the statisti- cal language, R [11–14], and it is available per request from the first author (guogen.shan@unlv.edu) or the cor- responding author (jtao@263.net). We are also working together to develop a new R package to implement the adaptive minimax and admissible designs from this arti- cle and the adaptive optimal design by Shan et al. [8]. The proposed design allows the second stage sample size and its associated critical value to depend on the result from the first stage. The proposed design satisfies the mono- tonicity property of the relationship between the second stage sample size and the first stage responders, which is an important feature for a practical application. The MSS of the proposed adaptive minimax design is always less than or equal to that of the minimax-EF design. We consider this as an important advantage of Shan et al. Acknowledgment g The authors are very grateful to the Editor, and two referees for their insightful comments that help improve the manuscript. The naive point estimate for the probability of response rate is calculated as the number of responses divided by the total number of patients, and it is well known that this estimate is biased. In the traditional Simon’s design, Jung and Kim [15] derived the uniformly minimum variance unbiased estimate for the probability of response based on the Rao-Blackwell theorem. To the best of our knowledge, no unbiased estimate for the probability of response Discussion and conclusions BMC Medical Research Methodology (2016) 16:90 Page 13 of 14 The number of responses in the first stage: S Maximum sample size: n(S) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 22 25 28 31 34 37 40 43 46 49 52 55 58 Proposed adaptive minimax design Simon’s minimax design Minimax−EF design Fig. 3 For the urothelial cancer trial with parameters (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5), the design parameters for Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design. The maximum sample size, n(S), is plotted as a function of the number of responses from the first stage, S Fig. 3 For the urothelial cancer trial with parameters (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5), the design parameters for Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design. The maximum sample size, n(S), is plotted as a function of the number of responses from the first stage, S Fig. 3 For the urothelial cancer trial with parameters (α, β, πu, πa) = (0.1, 0.2, 0.35, 0.5), the design parameters for Simon’s minimax design, the minimax-EF design and the proposed adaptive minimax design. The maximum sample size, n(S), is plotted as a function of the number of responses from the first stage, S has been proposed in an adaptive two-stage design set- ting. This may be due to the complexity of an adaptive design as compared to the traditional sample size fixed design. the minimax adaptive design to reduce the computational intensity as compared to adaptive designs based on the optimal criteria [8], where the upper bound of the sample size has to be set in the design search process. To reduce the computational time, one may use a backward search method as in this article, starting with the maximum sam- ple size from the minimax-EF design. In addition, when the proposed design and other designs have the same MSS, the expected sample size under the null of the proposed design is always smaller than others. the minimax adaptive design to reduce the computational intensity as compared to adaptive designs based on the optimal criteria [8], where the upper bound of the sample size has to be set in the design search process. Funding g Shan’s research is partially supported by grants from the National Institute of General Medical Sciences from the National Institutes of Health: P20GM109025, P20GM103440, and 5U54GM104944. Zhang’s work was supported by the Zhejiang Provincial Natural Science Foundation of China (grant no. LY15F020001) and the National Natural Science Foundation of China (grant no. 61170099). Page 14 of 14 Page 14 of 14 Shan et al. BMC Medical Research Methodology (2016) 16:90 Shan et al. BMC Medical Research Methodology (2016) 16:90 13. Shan G, Ma C. Unconditional tests for comparing two ordered multinomials. Stat Methods Med Res. 2016;25(1):241–54. doi:10.1177/0962280212450957. http://dx.doi.org/10.1177/ 0962280212450957. 14. Shan G, Moonie S, Shen J. Sample size calculation based on efficient unconditional tests for clinical trials with historical controls. J Biopharm Stat. 2016;26(2):240–9. http://view.ncbi.nlm.nih.gov/pubmed/25551261. 15. Jung SHH, Kim KMM. On the estimation of the binomial probability in multistage clinical trials. Stat Med. 2004;23(6):881–96. doi:10.1002/sim.1653. http://dx.doi.org/10.1002/sim.1653. 16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing Designs for Comparative Clinical Trials. Biometrika. 1988;75(2):303–10. http://www.jstor.org/stable/2336178. 13. Shan G, Ma C. Unconditional tests for comparing two ordered multinomials. Stat Methods Med Res. 2016;25(1):241–54. doi:10.1177/0962280212450957. http://dx.doi.org/10.1177/ 0962280212450957. Availability of data and materials This is a manuscript to develop novel study designs, therefore, no real data is involved. Authors’ contributions The idea for the paper was originally developed by GS and TJ. GS and HZ computed and analysed the adaptive minimax and admissible two-stage designs in this paper. GS, HZ and TJ drafted the manuscript, revised the paper critically and approved the final version. 15. Jung SHH, Kim KMM. On the estimation of the binomial probability in multistage clinical trials. Stat Med. 2004;23(6):881–96. doi:10.1002/sim.1653. http://dx.doi.org/10.1002/sim.1653. Received: 18 March 2016 Accepted: 22 July 2016 Received: 18 March 2016 Accepted: 22 July 2016 Competing interests 16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing Designs for Comparative Clinical Trials. Biometrika. 1988;75(2):303–10. http://www.jstor.org/stable/2336178. 16. Thall PF, Simon R, Ellenberg SS. Two-Stage Selection and Testing Designs for Comparative Clinical Trials. 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Stat Med. 2004;23(4):561–9. http://view.ncbi. nlm.nih.gov/pubmed/14755389. 9. Jung SHH, Lee T, Kim K, George SL. Admissible two-stage designs for phase II cancer clinical trials. Stat Med. 2004;23(4):561–9. http://view.ncbi. nlm.nih.gov/pubmed/14755389. • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p 10. Siefker-Radtke AO, Dinney CP, Shen Y, Williams DL, Kamat AM, Grossman HB, Millikan RE. A phase 2 clinical trial of sequential neoadjuvant chemotherapy with ifosfamide, doxorubicin, and gemcitabine followed by cisplatin, gemcitabine, and ifosfamide in locally advanced urothelial cancer. Cancer. 2013;119(3):540–7. doi:10.1002/cncr.27751. http://dx.doi.org/10.1002/cncr.27751. 10. Siefker-Radtke AO, Dinney CP, Shen Y, Williams DL, Kamat AM, Grossman HB, Millikan RE. A phase 2 clinical trial of sequential neoadjuvant chemotherapy with ifosfamide, doxorubicin, and gemcitabine followed by cisplatin, gemcitabine, and ifosfamide in locally advanced urothelial cancer. Cancer. 2013;119(3):540–7. doi:10.1002/cncr.27751. http://dx.doi.org/10.1002/cncr.27751. 11. Shan G, Wang W. ExactCIdiff: An R Package for Computing Exact Confidence Intervals for the Difference of Two Proportions. The R Journal. 2013;5(2):62–71. 12. Shan G. 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Non-AIDS Associated Kaposi's Sarcoma: Clinical Features and Treatment Outcome
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Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: claus.garbe@med.uni-tuebingen.de To date it remains unclear whether KS itself is a true malignancy or rather just a reactive proliferation [5]. Introduction Kaposi’s sarcoma (KS) was first described in 1872 by the Hungarian dermatologist Moritz Kaposi [1]. It is a rare neoplasm of lymphatic endothelial cells frequently evident as multiple vascular cutaneous and mucosal nodules. Lymph node and visceral manifestation is seen in cases of strong immunosuppres- sion or aggressive disease. This study focuses on non-AIDS related KS of the skin i.e. cutaneous manifestations of KS in HIV-negative patients present- ed to the Department of Dermatology, University of Tuebingen Medical Center between 1987 and 2009. Lena Jakob, Gisela Metzler, Ko-Ming Chen, Claus Garbe* Department of Dermatology, Eberhard-Karls-University, Tuebingen, Germany Department of Dermatology, Eberhard-Karls-University, Tuebingen, Germany PLoS ONE | www.plosone.org Abstract Background: Kaposi’s sarcoma (KS) in HIV negative patients is rare and has to be distinguished from AIDS associated KS. Two groups are at risk to develop non-AIDS related KS: elderly men mainly of Mediterranean origin and persons with iatrogenic immunosuppression. Patients and Methods: In order to define risk-groups and major clinical features we retrospectively evaluated clinical data of all patients with non-AIDS associated KS presenting to the Department of Dermatology, University Hospital Tuebingen between 1987 and 2009. Data were extracted from the tumor registry of the Comprehensive Cancer Center Tuebingen and from patient records. Results: 20 patients with non-AIDS KS have been identified. The average age at KS onset was 66.6 years; the male-to-female- ratio was 3:1. Most of the patients were immigrants from Mediterranean or Eastern European countries (60%). 15 cases of classic KS versus 5 cases of iatrogenic KS were observed. In 95% of the cases, KS was limited to the skin, without mucosal, lymph node or visceral manifestation. KS lesions were in all cases multiple and mostly bilateral, the most common localization was the skin of the lower extremities. Tumor control was achieved in nearly all cases by the use of local or systemic therapy. No patient died from KS. Conclusions: Unlike KS in AIDS patients, non-AIDS associated KS is a rather localized process which rarely involves lymph nodes or organs. It is mostly seen in elderly males from Mediterranean or Eastern European countries and in most cases responsive on local or systemic therapeutic strategies. Citation: Jakob L, Metzler G, Chen K-M, Garbe C (2011) Non-AIDS Associated Kaposi’s Sarcoma: Clinical Features and Treatment Outcome. PLoS ONE 6(4): e18397. doi:10.1371/journal.pone.0018397 r G, Chen K-M, Garbe C (2011) Non-AIDS Associated Kaposi’s Sarcoma: Clinical Features and Treatment Outcome. PLoS ONE 6(4): e18397 0018397 Editor: H. Peter Soyer, The University of Queensland, Australia Editor: H. Peter Soyer, The University of Queensland, Australia Received January 1, 2011; Accepted February 28, 2011; Published April 12, 2011 Copyright:  2011 Jakob et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. Materials and Methods Four groups are at risk to develop KS: elderly males of Mediterranean and Eastern European lineage; children and adults from central Africa; persons who are iatrogenically immunocom- romised; and homosexual men infected with human immunode- ficiency virus (HIV) [2]. Major differences in clinical presentation and in prognosis among those groups have lead to the following classifications: classical KS, endemic or so called African KS, iatrogenic KS and AIDS-KS. This is a retrospective descriptive study of a series of 20 patients admitted consecutively for KS, from 1987 to 2009 in the Department of Dermatology, University of Tuebingen Medical Center. Informed consent was not obtained for all patients as the entire data was analyzed anonymously. This applies according to the German Medical Association’s professional code of conduct and has been approved by the Ethics Committee, University of Tuebingen (Figure S1). Chang et al. discovered human herpes virus 8, (HHV-8), also known as Kaposi’s sarcoma associated herpes virus, which is strongly implicated in the pathogenesis of all types of KS [3]. The herpes virus is considered necessary but not sufficient for the development of KS, which is a multistep process including not only HHV-8 infection, but also genetic and angiogenic factors, as well as the production of several inflammatory cytokines [4]. In order to define risk-groups and major clinical features we retrospectively identified cases registered by the tumor registry of the Comprehensive Cancer Center Tuebingen and evaluated clinical data documented in the hospital records. All cases presented histopathologically approved KS lesions of the skin and negative HIV-1/2 screening by macro enzyme 1 April 2011 | Volume 6 | Issue 4 | e18397 Non-AIDS Associated Kaposi’s Sarcoma Non-AIDS Associated Kaposi’s Sarcoma Figure 1. Brownish macules and plaques on the foot of a patient with classic KS. doi:10.1371/journal.pone.0018397.g001 immunoassay. Histological diagnosis was in most cases completed by immunohistochemical tests such as HHV-8 staining, or immunostaining with antibodies against endothelial markers D2- 40, CD31 and CD34. Most patients underwent tumor staging by lymph node and abdominal ultrasound as well as chest x-ray. Demographic features such as origin, age at onset, gender of the patient, as well as clinical features such as clinical subtype of KS and localization of lesions were evaluated. Furthermore treatment modalities, results and tumor recurrence in the time of observation were recorded. Treatment outcome was classified according to the Response Evaluation Criteria in Solid Tumors (RECIST Guide- lines) [6]. Results vascular channels in the dermis below an integral epidermis. The so-called promontory sign is sometimes found in patch stage lesions and denotes vascular spaces surrounding pre-existing blood vessels (Figure 3). Perivascular lymphoplasmocytic cells as well as extravasated erythrocytes and hemosiderin deposits are charac- teristic for patch stage. 20 cases of non-AIDS KS were identified in this study. Mean age at diagnosis of the group was 66.6 year-old (SD = 15.36). The youngest patient was 36-year-old and iatrogenically immunosup- pressed; the oldest developed his classic KS by the age of 90. Mean age at onset of patients with classic KS was 69.6 years (SD = 12.09). Mean age at onset of patients with classic KS was 69.6 years (SD = 12.09). As KS progresses to the plaque stage bizarre vessels with an atypical endothelial lining dissect the collagen tissue (Figure 4). In the nodular stage spindled endothelial cells predominate forming slit-like vascular spaces containing erythrocytes and hyaline globules. Figure 5 and 6 demonstrate a well-circumscript spindle-cell tumor with poorly defined slit-like vascular spaces. A male predominance in KS – 75% male patients (n = 15) versus 25% female patients (n = 5) was shown with a male/female ratio of 3:1. 60% (n = 12) of the patients observed in Tuebingen were immigrants from Mediterranean and Eastern European countries (all first generation) versus eight German patients. Figure 7 shows D2-40 immune staining of lymphatic endothelial cells revealing arborizing vascular structures and lymphocytic cell infiltration. Figure 8 displays HHV-8 stained atypical endothelial cells. Regarding the clinical Subtype 15 cases of classic KS and five cases of iatrogenic KS were identified. Iatrogenic immunosuppression was in one case used for a liver transplant-patient and four patients were under immunosuppres- sive therapy due to autoimmune diseases i.e. Behc¸et’s disease, myasthenia gravis, chronic membranous glomerulonephritis and systemic sarcoidosis. Immunosuppressive medication included systemic corticosteroids in all five cases in addition to azathioprine in one case and ciclosporin A in two cases. Treatment modalities included local therapy such as surgery and radiotherapy as well as systemic immune therapy with interferon-a-2a. Interferon-a-2a was applied by subcutaneous injection using 3 million international units three times weekly. One patient developed neutralizing antibodies and therefore his therapy was switched to Interferon alphacon (consensus interferon 9 mg daily, then every second day). The duration of administration One patient was categorized as classic KS even though presenting a primary nodal peripheral T-cell lymphoma causing immunosuppression. Materials and Methods One patient had a stable disease course and therefore did not need treatment and one patient refused therapy. Median time of follow-up for all patients were four years (SEM = 7.86 months). In 7 cases time of observation was determined by intercurrent death. Causes of death were other than KS in all cases. Statistical package of social sciences 16.0 software (SPSS Inc., Chicago, IL, U.S.A.) was used to calculate means and standard deviations. Statistical package of social sciences 16.0 software (SPSS Inc., Chicago, IL, U.S.A.) was used to calculate means and standard deviations. Figure 1. Brownish macules and plaques on the foot of a patient with classic KS. doi:10.1371/journal.pone.0018397.g001 PLoS ONE | www.plosone.org Discussion of interferon ranged from five weeks to six years. In one case with post-transplant KS immunosuppressive medication was switched from ciclosporin A to sirolimus resulting in a partial response. One patient had a stable disease course and therefore did not need treatment and one patient refused therapy. Non-AIDS KS is considered a rare disease, but incidence varies according to individual factors such as origin, sex, age and immune status of the patient. Between 1987 and 2009, only 20 cases of non-AIDS related KS were observed at the Department of Dermatology in Tuebingen, Germany. This is in line with the low incidence of cases appearing in North-Western Europe. Treatment outcome was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST-Guidelines). The term ‘‘complete response’’ means clearance of KS lesions on later visits when compared with the first lesions on admission. ‘‘Partial response’’ equals at least 30 percent decrease in the sum of the longest diameter of target lesions whereas ‘‘stable disease’’ involves less than 30 percent decrease. The terms complete and partial response as well as stable disease implicate the absence of new lesions or of progressive lesions. This was achieved in all cases. No patient presented a progressive disease course initially nor died of KS. Four patients developed multiple local recurrences. Two of them had primarily received a local therapy and one interferon-a-2a. No data regarding the incidence of KS in Germany currently exists. Iscovich et al. analyzed reports of classic KS from different cancer registries throughout the world between 1988 and 1992. Low incidence rates were found in Denmark (0.18 per million men aged over 65 per year), intermediate rates in France, and especially high rates in Sardinia and Sicily (13.2 per million men aged over 65 per year) [7]. It is worth mentioning that the majority of the non-AIDS KS in Tuebingen (12 out of 20) have been found in first generation immigrants from Mediterranean countries with presumably higher incidence rates e.g. in Turkey and Italy (1 per 100,000 men per Clinical features and treatment outcome of non-AIDS KS are presented in Table 1. Figure 4. Plaque stage KS with bizarre vessels dissecting the upper dermis. There is erythrocyte extravasation and hemosiderin pigmentation. doi:10.1371/journal.pone.0018397.g004 Figure 6. Tumor stage KS: Close up view. doi:10.1371/journal.pone.0018397.g006 Figure 4. Plaque stage KS with bizarre vessels dissecting the upper dermis. There is erythrocyte extravasation and hemosiderin pigmentation. doi:10.1371/journal.pone.0018397.g004 Figure 6. Results However, immunosuppression was in this case not iatrogenically induced. Figure 2. Patch stage classic KS: Red to brownish irregularly- shaped macules and plaques. doi:10.1371/journal.pone.0018397.g002 In 95% (n = 19) of the cases, KS was limited to the skin, without mucosal, lymph node or visceral manifestation. One patient presented mucosal lesions of the oral and genital region as well as inguinal lymph node invasion. KS lesions were multiple in all patients (n = 20), no patient presented just one singular lesion. 70% of the patients (n = 14) were affected on both hemispheres of the body and 30% (n = 6) presented unilateral lesions. The most frequent manifestation was the skin of the lower leg (n = 8), six patients were affected on their feet and five patients on all four extremities. All in all 95% of the patients were affected on the skin of the lower extremities. Figure 1 and 2 present a patch stage classic KS characterized by several brownish irregularly-shaped maculae as well as the existence of a few partly indurated plaques. Figure 2. Patch stage classic KS: Red to brownish irregularly- shaped macules and plaques. doi:10.1371/journal.pone.0018397.g002 All stages of KS lesions were observed within the group: The initial patch stage exhibits an irregular proliferation of jagged April 2011 | Volume 6 | Issue 4 | e18397 2 Figure 3. Patch stage KS with promontory sign. Dilated irregular vascular channels surround a pre-existing vessel. doi:10.1371/journal.pone.0018397.g003 Non-AIDS Associated Kaposi’s Sarcoma Figure 5. Tumor stage KS: Well-circumscript spindle-cell tumor. Erythrocytes lie within poorly defined slit-like vascular spaces. doi:10.1371/journal.pone.0018397.g005 Figure 5. Tumor stage KS: Well-circumscript spindle-cell tumor. Erythrocytes lie within poorly defined slit-like vascular spaces. doi:10.1371/journal.pone.0018397.g005 Figure 3. Patch stage KS with promontory sign. Dilated irregular vascular channels surround a pre-existing vessel. doi:10.1371/journal.pone.0018397.g003 Discussion Patients with widespread disease may need systemic chemotherapeutic or immunologic medication. Positive results have been found for pegylated liposomal doxorubicin, danaurubicin, paclitaxel and interferon a [24,25]. In patients with iatrogenic KS, immunosuppressive medication may be reduced or modified with the considerate possibility of grafts being rejected with insufficient immunosup- pression [26]. Gender presents another factor which strongly influences KS manifestation. Former male-to-female ratios from 10:1 to 15:1 had been reported in classic KS [9]. More recent studies showed gender ratios ranging from 1:1 in England up to 4:1 in Italy [10,11]. Iscovich and colleagues ascertained a lower gender difference in populations with lower incidence rates [7]. The Tuebingen case group showed a significant male predominance with a male-to-female ratio of 3:1. Just as other investigations classic KS was the most frequent subtype we found. Iatrogenic KS was with 25% a considerately highly represented subtype. This may be an effect of mismatch in classification. Other studies call this subtype post transplant-KS and patients with KS under immunosuppressive therapy for Between 1987 and 2009 single KS lesions have been surgically removed and irradiated. The immune stimulating, antiviral and anti-proliferative properties of interferon-a-2a have been used for both systemic and intralesional therapy. In one patient with post- transplant KS immunosuppressive medication was switched from ciclosporin to the m-TOR inhibitor sirolimus. Sirolimus has immunosuppressive, anti-angiogenic and anti-neoplastic potential [27]. Current studies confirm it’s positive effects on iatrogenic KS [28]. Figure 8. HHV-8 staining of atypical endothelial cells. doi:10.1371/journal.pone.0018397.g008 We achieved complete responses in half of the patients. The rest presented partial remissions or stable disease courses. No patient died of KS. Due to the small number of cases we were not able to demonstrate differences between treatments, neither were we able to provide outcome diagnosis between different subtypes. Four patients developed multiple local recurrences. Three of them had primarily received a surgical therapy and one had primarily been treated with interferon-a-2a. In summary this study analyzed the clinical manifestation, treatment and outcome of all patients with non-AIDS associated KS patients presenting to the Department of Dermatology, University Hospital Tuebingen, between 1987 and 2009. In addition to HHV-8 infection, individual factors like origin, age, sex and immune status of the patient seem to have an impact on the development of KS. Discussion Tumor stage KS: Close up view. doi:10.1371/journal.pone.0018397.g006 April 2011 | Volume 6 | Issue 4 | e18397 April 2011 | Volume 6 | Issue 4 | e18397 PLoS ONE | www.plosone.org 3 Non-AIDS Associated Kaposi’s Sarcoma Figure 7. D2-40 staining of arborizing vascular structures; irregular lymphatic vessels. doi:10.1371/journal.pone.0018397.g007 autoimmune disorders may not be listed in this group. However the increased risk of KS in iatrogenically immunosuppressed patients is well documented [12–14]. One of our patients presented with a nodal peripheral T-cell lymphoma, a hematological malignancy that possibly implicates immunosuppression. Over fourfold significant increase of Kaposi’s sarcoma in patients with lymphohematopoietic malignancies have been reported in the literature [15]. Presumably immunodeficien- cy of any kind; iatrogenic, malignant or HIV-induced is a considerable factor in the development of KS. Clinically, non-AIDS KS mostly presents itself as multiple bilateral cutaneous lesions of the lower limb [7]. We found the lesions to be multiple in 100% of the cases, they were mostly bilateral (70%) and the lower extremity was clearly the most affected localization (95%). Only one patient with extracutaneous lesions was identified. This is in line with the results of Hong and Lee who compared characteristics of KS in HIV positive and negative subjects [16]. Figure 7. D2-40 staining of arborizing vascular structures; irregular lymphatic vessels. doi:10.1371/journal.pone.0018397.g007 KS can be seen as a systemic disease with mutilocular occurrence of vascular tumors. Thus the therapeutic administra- tion of KS differs essentially from the management of most other neoplastic diseases. In comparison with other tumors KS therapy comprises growth control rather than elimination without presenting a palliative situation. A standard therapeutic guideline does not exist as the therapeutic options have to be chosen depending on subtype and stage of the disease as well as on the immune status of the patient [17]. year [8]). Immigrants from those two countries also represent the two largest ethnic minorities in Germany. Mean age at onset of our 20 patients was 66.6 years. Focusing exclusively on classic KS an average age of 69.6 years was found, which is comparable to more comprehensive investigations like the Italian 870-cases study conducted by Dal Maso et al. showing a mean age of 72 years [8]. Treatment modalities comprise local therapy for example surgery, radiotherapy and local chemotherapy such as injections of vinca alkaloids or local immune therapy by interferon,9 cis retinoid acid or imiquimod [18–23]. April 2011 | Volume 6 | Issue 4 | e18397 Discussion In contrast to AIDS-associated KS, KS in HIV negative patients appears less aggressive, mostly limited to the skin and well-responsive on local or systemic therapeutic strategies. Figure 8. HHV-8 staining of atypical endothelial cells. doi:10.1371/journal.pone.0018397.g008 April 2011 | Volume 6 | Issue 4 | e18397 PLoS ONE | www.plosone.org April 2011 | Volume 6 | Issue 4 | e18397 4 Non-AIDS Associated Kaposi’s Sarcoma Table 1. Clinical features and treatment outcome. Patient Age Origin Subtypea Localizationb Treatmentc Resultd Recurrence P1 44 Italy immune lower legs sirolimus PR - P2 87 Germany classic lower legs IFNa SD - P3 76 Romania classic lower legs Rx, IFNa SD - P4 70 Germany immune lower legs Rx, IFNa CR - P5 59 Turkey classic extremities excs. CR multiple P6 69 Italy classic lower leg No need - - P7 48 Egypt immune lower legs excs. CR multiple P8 78 Germany classic extremities, mucosa, LN Rx, excs. SD multiple P9 51 Turkey classic foot excs. CR - P10 45 Italy classic foot IFNa PR - P11 89 Germany classic feet Rx, excs. SD - P12 36 Turkey immune extremities IFNa CR - P13 69 Italy classic extremities drop-out - - P14 71 Germany classic extremities IFNa SD - P15 70 Italy immune feet Rx PR - P16 68 Germany classic feet IFNa CR multiple P17 62 Turkey classic feet IFNa CR - P18 70 Italy classic lower leg IFNa, excs. CR - P19 79 Germany classic nose excs. CR - P20 90 Germany classic lower legs Rx CR - p p p doi:10.1371/journal.pone.0018397.t001 Author Contributions Conceived and designed the experiments: CG LJ. Performed the experiments: LJ. Analyzed the data: LJ KMC. Contributed reagents/ materials/analysis tools: GM CG. Wrote the paper: LJ CG KMC GM. Figure S1 Ethical vote for the study. References 1. Kaposi M (1872) Idiopathisches multiples Pigmentsarkom der Haut. Arch Dermatol Syph 4: 265–272. 1. Kaposi M (1872) Idiopathisches multiples Pigmentsarkom der Haut. Arch Dermatol Syph 4: 265–272. 14. Penn I (1979) Kaposi’s sarcoma in organ transplant recipients: report of 20 cases. Transplantation 27: 8–11. 2. Antman K, Chang Y (2000) Kaposi’s sarcoma. N Engl J Med 342: 1027–1038. 15. Royle JS, Baade P, Joske D, Fritschi L (2010) Risk of second cancer after lymphohematopoietic neoplasm. Int J Cancer. 3. Chang Y, Moore PS (1996) Kaposi’s Sarcoma (KS)-associated herpesvirus and its role in KS. Infect Agents Dis 5: 215–222. 16. Hong A, Lee CS (2002) Kaposi’s sarcoma: clinico-pathological analysis of human immunodeficiency virus (HIV) and non-HIV associated cases. Pathol Oncol Res 8: 31–35. 4. Ensoli B, Sgadari C, Barillari G, Sirianni MC, Sturzl M, et al. (2001) Biology of Kaposi’s sarcoma. Eur J Cancer 37: 1251–1269. 5. Goh SG, Calonje E (2008) Cutaneous vascular tumours: an update. Histopathology 52: 661–673. 17. Vogt T, Brockmeyer N, Kutzner H, Schofer H (2008) Short German guidelines: angiosarcoma and Kaposi sarcoma. J Dtsch Dermatol Ges 6 Suppl 1: S19–S24. 18. Trakatelli M, Katsanos G, Ulrich C, Kalabalikis D, Sotiriadis D, et al. (2010) Efforts to counteract locally the effects of systemic immunosupression: a review on the use of imiquimod, a topical immunostimulator in organ transplant recipients. Int J Immunopathol Pharmacol 23: 387–396. 6. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, et al. (2000) New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 92: 205–216. 19. Celestin Schartz NE, Chevret S, Paz C, Kerob D, Verola O, et al. (2008) Imiquimod 5% cream for treatment of HIV-negative Kaposi’s sarcoma skin lesions: A phase I to II, open-label trial in 17 patients. J Am Acad Dermatol 58: 585–591. 7. Iscovich J, Boffetta P, Franceschi S, Azizi E, Sarid R (2000) Classic kaposi sarcoma: epidemiology and risk factors. Cancer 88: 500–517. 8. Dal ML, Polesel J, Ascoli V, Zambon P, Budroni M, et al. (2005) Classic Kaposi’s sarcoma in Italy, 1985–1998. Br J Cancer 92: 188–193. . Dal ML, Polesel J, Ascoli V, Zambon P, Budroni M, et al. (20 20. FDA (1998) KS drug goes to FDA. Food and Drug Administration. GMHC Treat Issues 12: 7. 28. Stallone G, Schena A, Infante B, Di PS, Loverre A, et al. (2005) Sirolimus for Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 352: 1317–1323. Non-AIDS Associated Kaposi’s Sarcoma 27. Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 35: 7S–14S. 25. Brambilla L, Romanelli A, Bellinvia M, Ferrucci S, Vinci M, et al. (2008) Weekly paclitaxel for advanced aggressive classic Kaposi sarcoma: experience in 17 cases. Br J Dermatol 158: 1339–1344. 26. Montagnino G, Bencini PL, Tarantino A, Caputo R, Ponticelli C (1994) Clinical features and course of Kaposi’s sarcoma in kidney transplant patients: report of 13 cases. Am J Nephrol 14: 121–126. 25. Brambilla L, Romanelli A, Bellinvia M, Ferrucci S, Vinci M, et al. (2008) Weekly paclitaxel for advanced aggressive classic Kaposi sarcoma: experience in 17 cases. Br J Dermatol 158: 1339–1344. 26. Montagnino G, Bencini PL, Tarantino A, Caputo R, Ponticelli C (1994) Clinical features and course of Kaposi’s sarcoma in kidney transplant patients: report of 13 cases. Am J Nephrol 14: 121–126. 27. Sehgal SN (2003) Sirolimus: its discovery, biological properties, and mechanism of action. Transplant Proc 35: 7S–14S. 28. Stallone G, Schena A, Infante B, Di PS, Loverre A, et al. (2005) Sirolimus for Kaposi’s sarcoma in renal-transplant recipients. N Engl J Med 352: 1317–1323. PLoS ONE | www.plosone.org References Kaposi’s sarcoma in Italy, 1985–1998. Br J Cancer 92: 188–193. 9. Ronchese F, Kern AB (1953) Kaposi’s sarcoma (angioreticulomatosis). Postgrad Med 14: 101–111. 21. Schwartz RA, Micali G, Nasca MR, Scuderi L (2008) Kaposi sarcoma: a continuing conundrum. J Am Acad Dermatol 59: 179–206. 10. Grulich AE, Beral V, Swerdlow AJ (1992) Kaposi’s sarcoma in England and Wales before the AIDS epidemic. Br J Cancer 66: 1135–1137. g J 22. Szajerka T, Jablecki J (2007) Kaposi’s sarcoma revisited. AIDS Rev 9: 230–236. 11. Geddes M, Franceschi S, Barchielli A, Falcini F, Carli S, et al. (1994) Kaposi’s sarcoma in Italy before and after the AIDS epidemic. Br J Cancer 69: 333–336. 23. Brambilla L, Bellinvia M, Tourlaki A, Scoppio B, Gaiani F, et al. (2010) Intralesional vincristine as first-line therapy for nodular lesions in classic Kaposi sarcoma: a prospective study in 151 patients. Br J Dermatol 162: 854–859. 12. Hoshaw RA, Schwartz RA (1980) Kaposi’s sarcoma after immunosuppressive therapy with prednisone. Arch Dermatol 116: 1280–1282. sarcoma: a prospective study in 151 patients. Br J Dermatol 162: 8 24. Di LG, Kreuter A, Di TR, Guarini A, Romano C, et al. (2008) Activity and safety of pegylated liposomal doxorubicin as first-line therapy in the treatment of non-visceral classic Kaposi’s sarcoma: a multicenter study. J Invest Dermatol 128: 1578–1580. 13. Klepp O, Dahl O, Stenwig JT (1978) Association of Kaposi’s sarcoma and prior immunosuppressive therapy: a 5-year material of Kaposi’s sarcoma in Norway. Cancer 42: 2626–2630. PLoS ONE | www.plosone.org April 2011 | Volume 6 | Issue 4 | e18397 April 2011 | Volume 6 | Issue 4 | e18397 5 PLoS ONE | www.plosone.org April 2011 | Volume 6 | Issue 4 | e18397 6
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Within-Canopy Experimental Leaf Warming Induces Photosynthetic Decline Instead of Acclimation in Two Northern Hardwood Species
Frontiers in forests and global change
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Within-canopy experimental leaf warming induces photosynthetic Within-canopy experimental leaf warming induces photosynthetic decline instead of acclimation in two northern hardwood species decline instead of acclimation in two northern hardwood species Kelsey R. Carter Kelsey R. Carter Michigan Technological University Molly A. Cavaleri Michigan Technological University Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp Part of the Forest Sciences Commons Recommended Citation Recommended Citation Carter, K. R., & Cavaleri, M. A. (2018). Within-canopy experimental leaf warming induces photosynthetic decline instead of acclimation in two northern hardwood species. Frontiers in Forests and Global Chang 1(11). http://dx.doi.org/10.3389/ffgc.2018.00011 Retrieved from: https://digitalcommons.mtu.edu/forestry-fp/64 Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp Part of the Forest Sciences Commons Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp Part of the Forest Sciences Commons Part of the Forest Sciences Commons Edited by: Kouki Hikosaka, Tohoku University, Japan Reviewed by: Mary A. Heskel, Macalester College, United States Joseph Ronald Stinziano, University of New Mexico, United States *Correspondence: Kelsey R. Carter kcarter@mtu.edu Specialty section: This article was submitted to Forest Ecophysiology, a section of the journal Frontiers in Forests and Global Change Received: 03 September 2018 Accepted: 03 December 2018 Published: 19 December 2018 Specialty section: This article was submitted to Forest Ecophysiology, a section of the journal Frontiers in Forests and Global Change Received: 03 September 2018 Accepted: 03 December 2018 Published: 19 December 2018 Within-Canopy Experimental Leaf Warming Induces Photosynthetic Decline Instead of Acclimation in Two Northern Hardwood Species Northern hardwood forests are experiencing higher temperatures and more extreme heat waves, potentially altering plant physiological processes. We implemented in-situ leaf-level warming along a vertical gradient within a mature forest canopy to investigate photosynthetic acclimation potential of two northern hardwood species, Acer saccharum and Tilia americana. After 7 days of +3◦C warming, photosynthetic acclimation was assessed by measuring differences between heated and control photosynthetic rates (Aopt) at leaf optimum temperatures (Topt). We also measured the effects of warming and height on maximum rates of Rubisco carboxylation, stomatal conductance, transpiration, and leaf traits: leaf area, leaf mass per area, leaf nitrogen, and leaf water content. We found no evidence of photosynthetic acclimation for either species, but rather Aopt declined with warming overall. We found slight shifts in LMA and Narea, leaf traits associated with photosynthetic capacity, after 1 week of experimental warming. T. americana LMA and Narea was lower in the upper canopy heated leaves than in the control leaves, contributing a shift in Narea height distribution in the heated leaves. T. americana showed evidence of greater resiliency to warming, with greater thermoregulation, physiological plasticity, and evapotranspiration. As expected, Aopt of A. saccharum increased with height, but Aopt of T. americana was highest in the sub canopy, possibly due to constraints on leaf water balance and photosynthetic capacity in the upper canopy. Thus, models relying on canopy height or light environment may incorrectly estimate vertical variation of photosynthetic capacity. If these species are not able to acclimate to warmer temperatures, we could see alteration of plant carbon balance of these two key northern hardwood species. Keywords: Acer saccharum, canopy, experimental warming, leaf traits, photosynthesis, Tilia americana, thermal acclimation Recommended Citation Recommended Citation Carter, K. R., & Cavaleri, M. A. (2018). Within-canopy experimental leaf warming induces photosynthetic decline instead of acclimation in two northern hardwood species. Frontiers in Forests and Global Change, 1(11). http://dx.doi.org/10.3389/ffgc.2018.00011 Retrieved from: https://digitalcommons.mtu.edu/forestry-fp/64 Follow this and additional works at: https://digitalcommons.mtu.edu/forestry-fp Part of the Forest Sciences Commons Part of the Forest Sciences Commons ORIGINAL RESEARCH published: 19 December 2018 doi: 10.3389/ffgc.2018.00011 Citation: Carter KR and Cavaleri MA (2018) Within-Canopy Experimental Leaf Warming Induces Photosynthetic Decline Instead of Acclimation in Two Northern Hardwood Species. Front. For. Glob. Change 1:11. doi: 10.3389/ffgc.2018.00011 Heat waves are expected to become more frequent in the northern hemisphere compared to the southern hemisphere with continued climate change (Meehl and Tebaldi, 2004; Gershunov et al., 2009). Northern hardwood forests are already experiencing more extreme temperature events, potentially altering both plant photosynthetic and respiratory capacities and reducing ecosystem level primary productivity (Ciais et al., 2005; Bastos et al., 2014; Filewod and Thomas, 2014). December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org Northern Hardwood Experimental Warming Carter and Cavaleri In addition, there is evidence that upper canopy leaves in mid-latitude ecosystems are currently operating near their thermal thresholds (O’Sullivan et al., 2017; Mau et al., 2018), and photosynthetic decline could be exacerbated in temperate ecosystems due to their characteristically hot, dry summers. Both the overall elevated temperatures and increased number of heat waves could also contribute to shifts or reductions of species ranges (Thomas et al., 2004; Jump et al., 2006). Specifically, climate change is expected to impact the abundance and distribution of northern hardwood key species, such as Acer saccharum, Fagus grandifolia, and Tilia americana, with some evidence predicting a decline or shift to higher latitudes (Iverson et al., 2008; Tang and Beckage, 2010; Treyger and Nowak, 2011), while other models predict the expansion of species, such as A. saccharum (Walker et al., 2002). Understanding how warmer temperatures will affect northern hardwood species’ physiology will give us a better understanding of how these forests will respond to climate change. plants with higher rates of stomatal conductance tend to have a higher capacity for temperature regulation due to higher rates of evapotranspiration (Lu et al., 1998), which could, in turn, allow for maintained rate of photosynthesis with higher air temperatures (Michaletz et al., 2016) or prevent longer term damage that can occur when temperatures exceed high temperature thresholds (Urban et al., 2017; Drake et al., 2018). A plant’s ability to cope with stressors, either through photosynthetic acclimation or thermal regulation, can vary between species and across canopy vertical gradients. Species with low leaf area, complex leaves, and high stomatal conductance, and therefore high thermoregulation capacity, can maximize carbon gain by preventing excessively high leaf temperatures (Michaletz et al., 2016; Fauset et al., 2018). Citation: Species that have high trait plasticity may hold a thermal regulation advantage if they are able to adjust traits in their upper canopy leaves. In addition to differences in thermal regulation across a vertical gradient, leaves in the upper canopy may have a greater ability to acclimate than leaves lower in the canopy. Leaves that are exposed to one stressor (e.g., light, drought) have been shown to have a higher plasticity to respond to another stressor (e.g., temperature) (Havaux, 1992; Niinemets et al., 1999). This has important implications for canopy gradients, as upper canopy leaves are often exposed to multiple stressors at a given time. p g Photosynthetic thermal acclimation could help alleviate some of the negative impacts of supraoptimal temperatures. Photosynthesis rates generally increase with increasing measurement temperatures up to an optimum rate (Aopt), after which rates will decline (Berry and Bjorkman, 1980). Declines in net photosynthesis above this optimum temperature (Topt) occur as a result of several processes, including increased thylakoid membrane permeability (Bukhov et al., 1999; Zhang et al., 2009), Rubisco activase dysfunction (Wang and Portis, 1992; Salvucci et al., 2001; Zhang et al., 2002), higher rates of photorespiration (Ku and Edwards, 1978), stomatal closure (Farquhar and Sharkey, 1982), and higher rates of daytime respiration [reviewed in (Sage and Kubien, 2007)]. Photosynthetic acclimation occurs either through a shift in Topt to a higher temperature, or through a greater capacity to photosynthesize at optimum temperatures (i.e., higher rates of Aopt) (Way and Yamori, 2014). Mechanisms involved in photosynthetic thermal acclimation include increased membrane stability through physical changes in the thylakoid membrane structure (Huner, 1988; Havaux et al., 1996) and production of more stable isoforms of Rubisco activase (Salvucci et al., 2001; Portis, 2003). In addition to physiological acclimation through positive shifts in Topt and/or Aopt, some plants can regulate their leaf temperatures through convective cooling. Leaf traits, such as leaf area and shape can influence leaf thermal regulation (Vogel, 1970). Leaves with smaller areas and high complexity, for example, can have lower boundary layer thickness, which can lead to increased evaporative cooling (Gurevitch and Shuepp, 1990; Nicotra et al., 2008; Leigh et al., 2017). Abbreviations: Amax, light saturated net photosynthesis (µmol m−2 s−1); Aopt, photosynthetic rate at the temperature optima (µmol m−2 s−1); Eleaf , leaf evapotranspiration; gs, stomatal conductance (µmol m−2 s−1); LMA, leaf mass per area (g cm−2); Narea, nitrogen per leaf area (g m−2); Nmass, nitrogen per leaf mass (mg g−1); Tair, air temperature (◦C); Tleaf , leaf temperature (◦C); TLeafMax, maximum leaf temperature (◦C); Topt, optimum temperature (◦C); WUE, instantaneous water use efficiency (Amax/Eleaf ); WUEint, intrinsic water use efficiency (Amax/gs); ˆV cmax, maximum rate of Rubisco carboxylation; VPD, vapor pressure deficit (kPa); 1T, temperature difference between heated and control leaf (◦C). Frontiers in Forests and Global Change | www.frontiersin.org Citation: In addition, Although northern hemisphere ecosystems are expected to experience drastic changes in their temperature regimes, there is convincing evidence that temperate forests will be able to photosynthetically acclimate to moderate climate warming (Gunderson et al., 2000, 2010; Cunningham and Read, 2002; Turnbull et al., 2004; Way and Oren, 2010; Sendall et al., 2015b). In experiments lasting from 2 weeks to three full growing seasons, seedlings and saplings in temperate hardwoods have been shown to acclimate to moderate temperature increases (Gunderson et al., 2000, 2010; Cunningham and Read, 2002; Sendall et al., 2015b); however, photosynthetic acclimation in a mature temperate hardwood forest canopy is yet to be experimentally examined. Globally, acclimation most commonly occurs through positive shifts in Topt and through upregulated photosynthesis at the new growth temperature (Way and Yamori, 2014). The modes of photosynthetic acclimation vary in temperate species, with most studies finding a positive shift in Topt (Cunningham and Read, 2002; Gunderson et al., 2010; Sendall et al., 2015b). More specifically, studies on A. saccharum seedlings have found acclimation either through positive shifts in Topt (Sendall et al., 2015b) or Aopt (Gunderson et al., 2000). Understanding within- canopy physiological acclimation is particularly important within the global carbon cycle, as upper canopy leaves have high photosynthetic capacity (Carswell et al., 2000; Meir et al., 2002), and can cycle a disproportional amount of carbon in closed- canopy ecosystems (Ellsworth and Reich, 1993). Barriers to canopy warming studies include cost and energy required for mature tree-scale warming; therefore, most in-situ temperate ecosystem warming studies have focused on warming the forest understory (de Frenne et al., 2010; Melillo et al., 2011; Fu et al., 2013; Jarvi and Burton, 2013; also reviewed in Chung et al., 2013; Marchin et al., 2016; Noh et al., 2016) or early successional growth (Rollinson and Kaye, 2012; Rich et al., 2015). December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 2 Northern Hardwood Experimental Warming Carter and Cavaleri Environmental Information, 2018). Mean annual rainfall was 86.6 cm and mean annual snowfall is 564 cm. The soil is classified as Michigamme coarse loam (NRCS Soil Survey Staff, 2018). Site description can be found in Potvin and Lilleskov (2017). Fewer studies have looked at the effects of warming on temperate forest canopies beyond the seedling developmental stage. Leaf Warming g Individual leaves were heated using 100 watt, 120VAC silicon heating pads (24100, Kat’s, Five Star Manufacturing Group Inc., Springfield, TN). Leaf temperature was monitored using 30 AWG copper-constantan thermocouple wire [TT-T-30 SLE(ROHS)], OMEGA Engineering, Inc., Norwalk, CT, USA) wired to a solid- state thermocouple multiplexer (AM25T, Campbell Scientific Inc., Logan, UT, USA) connected to a data logger (CR1000, Campbell Scientific Inc.). Leaf thermocouples were adhered on the abaxial side of the heated and control leaves using breathable medical tape (Slot et al., 2016). Thermocouples were extended using 24 AWG copper- constantan thermocouple wire or 20 AWG for thermocouples that extended more than 15 meters, to ensure that current resistance did not exceed 100  (TT-T-20 and TT-T-24 OMEGA Engineering Inc.) using thermocouple connectors (SMPW-CC-T-MF, OMEGA Engineering Inc.). Heating pad temperature was controlled using a 24-380VAC SSR-25 DA solid state relay module (SSR-25 DA, Fotek Controls Co., Taiwan) wired into a digital output module (SDM-CD 16D, Campbell Scientific Inc.). The heating pad turned offwhen the heated leaf temperature reached more than 3◦C above the control leaf temperature. A datalogger heating program monitored leaf temperature every 15 s, and instantaneous leaf temperatures were recorded every 2 min. The heating pads were attached to a metal frame 7–12 cm below the leaf and secured to the metal frame using metal mesh, which also prevented direct exposure of thermocouple to the heating pad (Figure 1). Control and heated leaves were selected to ensure that the heated leaf was Design g Experimental warming was conducted on two species, T. americana and A. saccharum, at three canopy positions: understory, sub canopy, and upper canopy (0–2, 6–8, and 12– 14 m). Canopy scaffolding (Contur Modular Scaffold, BilJax, Archbold, OH, USA) enabled access to one A. saccharum tree [12.5 m height, 15.6 cm diameter at breast height (DBH)] and two T. americana trees (7.5 and 14 m height; 7.0 and 16.9 cm DBH, respectively). T. americana sub and upper canopy warming was conducted on separate individuals, while A. saccharum sub and upper canopy warming was on the same individual tree. Understory measurements were conducted on three individual saplings per species, ranging from 0.3 to 2.1 m height, located adjacent to the canopy scaffolding. The upper canopy leaves sampled were partially shaded late in the day by an adjacent 16 m tall emergent tree. g y g We had two primary study objectives: (1) to develop and test a novel leaf warming device, and (2) to assess whether two northern hardwood species, T. americana and A. saccharum, could acclimate to 7 days of leaf-level +3◦C warming. While short-term warming treatments may result in more conservative acclimation responses than warming for longer time periods, studies have found that temperate tree Topt can adjust to seasonal temperature variations within 1–5 days (Gunderson et al., 2010; Sendall et al., 2015b). In addition, Smith and Dukes (2017) recently found photosynthetic biochemical acclimation to experimental warming after 7 days. We hypothesized that (i) both species would be able to photosynthetically acclimate to warmer temperatures through shifts in both Topt and Aopt, (ii) T. americana would have a higher resiliency, through higher thermoregulation and higher trait plasticity, to warming in the upper canopy leaves due to characteristic higher stomatal conductance and lower leaf area (Thomas, 2010), and (iii) leaves in the upper canopy for both species would have a higher capacity to acclimate than leaves at lower heights. Three fully-developed leaves per species per height (18 total) were heated day and night +3.0◦C above a nearby control leaf for 7 days. Understory and sub canopy warming was conducted from July 14–21, 2016, and upper canopy warming was conducted August 23–30, 2016. As a result of heater malfunction, one T. americana upper canopy leaf experienced a total of 19 fewer hours of warming than the other heated leaves. Citation: Studies have implemented warming on immature trees using warming chambers (Gunderson et al., 2010), by pumping heated air through tubes and into an immature tree canopy (Bauerle et al., 2009), or through passive heating (Yamaguchi et al., 2016). Smaller scale within-canopy warming, either through branch or leaf warming, is a practical method to investigate the plant physiological effects of warming in forest canopies (Cavaleri et al., 2015). Heated cables have been used to warm mature temperate tree branches (Nakamura et al., 2010), and large, infrared heaters have been implemented within canopies to warm branches and leaves (Nakamura et al., 2016). As far as we are aware, there have only been two examples of leaf-level warming in mature forests canopies, both in tropical ecosystems (Doughty, 2011; Slot et al., 2014). These studies used resistance wires covered in aluminum foil (Doughty, 2011) or heat rope and infrared reflective frames (Slot et al., 2014) to heat upper canopy leaves. Leaf-level warming studies can give us important information on the physiological responses of forest ecosystem upper canopies that are currently unattainable through in-situ ecosystem-level warming studies. Study Site y This study was conducted in a mature secondary growth northern hardwood stand dominated by white ash (Fraxinus americana L), sugar maple (Acer saccharum Marshall), basswood (Tilia americana L), and northern red oak (Quercus rubra L), located at the USDA Forest Service Northern Research Station, Forestry Sciences Laboratory in Houghton, MI, USA (N47◦6′ 52.884′′, W 88◦32′ 52.332′′). In 2013, the basal area was 27 m2 ha−1 and the stand density was 2,960 trees ha−1. Mean stand height was 14 m, with a stand age from 50 to 60 years. A. saccharum comprised 10% of the stand basal area, while T. americana comprised 20% stand basal area. Site elevation is 243 m. The previous 30 years average monthly air temperature ranged from −8.65 to 15.57◦C (NOAA National Centers for Frontiers in Forests and Global Change | www.frontiersin.org December 2018 | Volume 1 | Article 11 3 Northern Hardwood Experimental Warming Carter and Cavaleri temperatures above 33◦C, VPD often reached above 2 kPa. At temperatures below 20◦C, VPD was often slightly below 1 kPa. FIGURE 1 | Example of a leaf heater positioned underneath an understory Tilia americana leaf. To gain insight into mechanistic drivers of temperature response and acclimation, we measured a variety of leaf traits on all experimental leaves, including leaf mass per area (LMA), leaf water content, leaf nitrogen on both area and mass bases (Narea and Nmass, respectively), % leaf carbon (%C), maximum rate of Rubisco carboxylation (Vcmax), stomatal conductance (gs), and leaf evapotranspiration (Eleaf ). Stomatal conductance and evapotranspiration were measured concurrently with photosynthesis using the LI6400 (Li-COR Inc.). After completing gas exchange measurements, sampled leaves were placed in a sealed plastic bag in an ice cooler for no longer than 2 h, weighed for fresh mass, and immediately placed in a −20◦C freezer. At the conclusion of the experiment, frozen leaves were thawed and measured for leaf area using a desktop scanner (HP Deskjet 4480) and ImageJ v1.50 image analysis software (Schneider et al., 2012). Leaves were placed in a 60◦C drying oven for at least 72 h for dry mass (g). LMA was obtained by dividing the dry mass (g) by total leaf area (cm2). Leaf water content was calculated by subtracting dry mass (g) from fresh mass (g), dividing by fresh mass (g), and multiplying by 100 to calculate percent water content (%). Study Site Dried leaves were ground to a fine powder in a ball bearing grinder (8000 M Mixer/Mill, Spex Sample Prep, Metuchen, NJ, USA) and analyzed for % carbon (C) and % nitrogen (N) using a combustion analyzer (ESC 4010, Costech Analytical Technologies Inc., Valencia, CA, USA). FIGURE 1 | Example of a leaf heater positioned underneath an understory Tilia americana leaf. exposed to a similar ambient environment to the control leaf, including height and shade. Leaf Scorching Due to an artifact of this leaf warming method, the temperature difference between paired heated and control leaves (1T) was often >3◦C before the digital output module turned the heater off. Leaf scorching, defined as visible leaf necrosis, was assessed on all heated leaves to account for possible damage to photosynthetic apparatus caused by spikes in leaf temperature. Percent leaf area scorched was calculated on scanned leaf images using ImageJ software. Environmental Monitoring Air temperature and relative humidity were monitored using HOBO sensors (U23 Pro V2, Onset Corp, Bourne, MA, USA) placed on the canopy access scaffolding at heights of 0.5, 6.25, and 12.5 m. In July, the air temperature sensors were placed on the southeast side of the scaffolding and were moved to the northwest side of the tower in August. Air temperature and relative humidity were also measured in an adjacent open field (Vaisala temperature and relative humidity probe HMP50-L, Campbell Scientific Inc.). Gas Exchange and Leaf Traits g After 1 week of experimental warming, gas exchange (net photosynthesis, stomatal conductance, and leaf evapotranspiration) response to temperature was measured on each individual heated and control leaf. Gas exchange measurements were conducted using an open-system LI6400 infrared gas analyzer fitted with a 6400-88 expanded temperature kit (Li-COR Inc., Lincoln, NE, USA). Photosynthetic response to temperature was measured at nine temperatures (17, 20, 23, 25, 30, 33, 35, 37◦C); although we were unable to reach 37◦C for some measurements. Due to difficulties reaching a low enough temperature to extract the parameter Topt, we included an additional 15◦C temperature measurement to A. saccharum understory and sub canopy temperature curves. Based on photosynthetic light response curves measured prior to leaf warming (data not shown), photosynthetic photon flux density was controlled at 800 µmol m−2 s−1 for the understory and sub canopy, and 1,200 µmol m−2 s−1 for the upper canopy leaves. CO2 concentration was controlled at 400 ppm. Flow was controlled between 200 and 500 µmol m−2 s−1 to keep the vapor pressure deficit (VPD) between 1 and 2 kPa; although, at December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org Data Analysis Differences between Aopt, Topt = −b 2a (2) (2) Aopt is extracted by setting Tleaf = Topt in Equation (1) and solving for Amax. The polynomial regression model was not able to capture Topt of 9 of the 18 A. saccharum curves. For these curves, we took the temperature at the maximum Amax value from each individual curve and treated this value as Topt. The inability to fit positive polynomial curves to these data is likely due to the very low response to temperature in A. saccharum understory and sub canopy leaves (Figure 2A); which, can likely, in part, be due to the very low stomatal conductance recorded in these leaves (Figure S1A). To examine if the response of water use efficiency (WUE) to temperature varied between treatments, species, and canopy position, we calculated instantaneous water use efficiency (WUE), calculated as Amax/Eleaf , and intrinsic water use efficiency (WUEint), calculated as Amax/gs. WUE and WUEint response to temperature was modeled using a mixed effects model where Tleaf , species, treatment, and canopy position were the fixed effects and individual tree was the random effect. WUE slopes and intercepts were extracted and compared with post- hoc contrasts using the FSA package (Ogle, 2018) in R Statistical software (R Core Team, 2015). In order to explain any possible differences in photosynthetic rates between heated and control leaves, stomatal conductance at the photosynthetic optimum temperature (gs at Topt), maximum rate of Rubisco carboxylation (Vcmax) at the photosynthetic optimum temperature, and equation parameters of evapotranspiration (Eleaf ) response to temperature was compared between treatment, species, and canopy position. gs was modeled using Equation (1) and substituting gs for Amax (Figures S1A,B). In the cases where Topt was extracted at the maximum Amax value, gs at Topt was also taken as this leaf temperature. To identify environmental differences between species and across canopy vertical gradients, we investigated maximum and mean leaf temperatures between species and maximum and mean leaf and air temperatures along the canopy vertical gradient. Average and maximum leaf temperatures of control leaves were compared for each canopy position and species using two-way ANOVAs and post-hoc contrasts. Average and maximum air temperatures were compared at each canopy position using a one-way ANOVA and Tukey’s post-hoc mean separation. The canopy air temperature and relative humidity sensors were moved between the July and August sampling. Data Analysis Warming device efficacy was determined by examining the average 1T across species, canopy position, sample month, and time of day (daytime or nighttime). To assess the effect of species and height on temperature spiking, heated leaf maximum temperature (TLeafMax) and the frequency of time points where heated leaves were 10◦C higher than control leaves (1T > 10◦C) for each leaf pair were compared between species and canopy positions using two-way ANOVAs and post-hoc contrasts. p g y p Photosynthetic acclimation is denoted by a positive shift in Topt or an increase in the photosynthetic rate at Topt (Aopt). In order for a Topt shift to result in enhanced photosynthetic performance, an upregulation of photosynthesis at the new growth temperature must also occur. For example, a positive shift in Topt could occur at the same time as decreased Aopt, which is considered a detractive adjustment to photosynthesis, as opposed to constructive adjustments that would occur with positive shifts in Topt and Aopt (Way and Yamori, 2014). Topt and Aopt were determined by fitting individual temperature response curves to December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 4 Northern Hardwood Experimental Warming Carter and Cavaleri regression equation where the leaf temperature was exponentially transformed to each response curve (Figures 2C,D): regression equation where the leaf temperature was exponentially transformed to each response curve (Figures 2C,D): the second order polynomial regression model (Cavieres et al., 2000) (Figures 2A,B): Amax = aTleaf 2 + bTleaf + c (1) (1) Eleaf = β0 + β1eTleaf (4) Eleaf = β0 + β1eTleaf (4) (4) where Amax is light saturated CO2 assimilation (µmol m−2 s−1) at leaf measurement temperature (Tleaf ) (◦C). Topt is calculated from the first derivative of the polynomial equation: where β0 is the intercept and β1 describes the exponential rise in Eleaf with increasing temperature. Differences between Aopt, Topt, ˆV cmax at Topt, gs at Topt, Eleaf (intercept, β0), and Eleaf (exponential rise, β1) values between treatment, species, and canopy position were compared using mixed effects models that accounted for individual tree as the random effect and species, treatment, and canopy position as the fixed effects. Mean separation was compared using post-hoc contrasts. where β0 is the intercept and β1 describes the exponential rise in Eleaf with increasing temperature. Data Analysis To account for differences in canopy temperatures between sample months, we used a Welch’s t-test to measure the difference between in July and August using air temperature measurements collected in an adjacent open field. Vcmax was calculated for each Amax value using the one point method (De Kauwe et al., 2016a,b). The one-point method works under the assumption that light saturated photosynthesis (Amax) is limited by Rubisco carboxylation instead of RuBP regeneration, or photosynthetic electron transport. The constants for the apparent Vcmax ( ˆV cmax) were estimated based on Bernacchi et al. (2001) estimation of Michalis constants for CO2 and O2 temperature dependencies. The CO2 compensation point was estimated from Crous et al. (2013). Because the one-point method uses internal CO2 concentration to calculate ˆVcmax, we removed all data points that had CO2 concentration <50 and >500 ppm, which resulted in the removal of 12 out of 192 data points (4.1% of the data). ˆV cmax was fitted to Tleaf using an Arrhenius equation (Medlyn et al., 2002) (Figures S1 C,D): Leaf trait differences between treatments, species, and canopy positions were analyzed to investigate possible drivers of photosynthetic rates across canopy positions and photosynthetic acclimation. Leaf traits (Nmass, Narea, leaf area, LMA, %C, and leaf water content) were compared for differences across treatment, species, and canopy position using a mixed effects model where treatment, species, and canopy position were the fixed effects and individual tree was the random effect and post-hoc contrasts. In order to elucidate drivers of photosynthetic rates and leaf water content, Aopt responses to leaf traits (LMA, Narea, and Nmass) and leaf water content correlation with Eleaf (Intercept) were assessed for differences in species and treatment using mixed effects models with tree as the random effect. All statistical analyses were performed using R Statistical Software (R Core Team, 2015). Mixed effects models were analyzed using the “nlme” package in R (Pinheiro et al., 2018). f (Tk) = k25exp Ea(Tk −298) (298RTk)  (3) (3) Where Tk is the temperature in Kelvin, k25 is the rate of ˆV cmax at 25 ◦C, and Ea is the activation energy, or exponential rise, of the ˆV cmaxto increasing temperature. ˆV cmax at Topt was calculated by substituting k25 and Ea, estimated from individual temperature response curves, and substituting Topt (K) into Equation (3). Frontiers in Forests and Global Change | www.frontiersin.org RESULTS Mean differences between heated and control leaves (1T) across species, canopy position, sample month (July and August), and daytime vs. nighttime ranged from 2.91 to 3.14◦C (Table S2). Temperatures of the warmed leaves were more variable than controls (Figure 3A); however, average 1T values were close to 3◦C at all canopy positions (Table S2; Figure 3B). Data Analysis p Evapotranspiration (Eleaf ) parameters were estimated for each individual Eleaf -Tleaf response curve by fitting a December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 5 Northern Hardwood Experimental Warming Carter and Cavaleri FIGURE 2 | Photosynthetic (Amax) and leaf evapotranspiration (Eleaf) response to leaf temperature (Tleaf). Individual temperature response curves were fit to polynomial equations for (A) A. saccharum and (B) T. americana photosynthetic response. Exponential transformations of leaf temperature were fit to the evapotranspiration response for (C) A. saccharum and (D) T. americana. Dotted lines depict individual curves in the upper canopy, dashed lines depict curves in the sub canopy, and solid lines represent understory temperature response curves. Control leaves are represented by blue lines while red lines represent heated leaves. FIGURE 2 | Photosynthetic (Amax) and leaf evapotranspiration (Eleaf) response to leaf temperature (Tleaf). Individual temperature response curves were fit to polynomial equations for (A) A. saccharum and (B) T. americana photosynthetic response. Exponential transformations of leaf temperature were fit to the evapotranspiration response for (C) A. saccharum and (D) T. americana. Dotted lines depict individual curves in the upper canopy, dashed lines depict curves in the sub canopy, and solid lines represent understory temperature response curves. Control leaves are represented by blue lines while red lines represent heated leaves. Frontiers in Forests and Global Change | www.frontiersin.org Air Temperature, Leaf Temperature, and Warming Device Performance Maximum and mean daily leaf and air temperatures were consistent across all canopy heights for both of our study species. Neither daily maximum nor daily mean air temperatures differed across canopy positions (Figures S2A,C; Table S1), and we found no difference in mean daily Tair between canopy months when measured in an adjacent open field [20.15 ± 1.31◦C for July, 19.56 ± 0.80◦C for August (mean ± SEM)] (Table S1). Mean daily Tleaf of unheated foliage did not differ across canopy position or between species (Figure S2B; Table S1). Control leaf maximum daily temperatures (TLeafMax) showed an almost significant species × canopy position interaction; however, post-hoc mean separation found no significant differences (Figure S2D; Table S1). Moderate temperature spiking did occur in all heated leaves, and we found some evidence of leaf scorch, primarily in A. saccharum. The occurrences of temperature spiking differed across canopy positions and between species. While A. saccharum heated leaf TLeafMax was higher in the upper canopy than the sub canopy and understory, T. americana TLeafMax did not vary with canopy height (Figure 4A). There was no difference in A. saccharum and T. americana heated TLeafMax in the understory or sub canopy, but A. saccharum heated TleafMax was higher than T. americana in the upper canopy by ∼9◦C. The mean % frequency 1T > 10◦C was <1.2% for all canopy positions for both species (Figure 4B), and there were no effects of species, canopy position, or their interaction (Table 1). Leaf scorching was found on 5 of the 18 total heated leaves: four A. saccharum and one T. The leaf-level warming device effectively increased temperatures of treated leaves +3◦C compared to paired control leaves over 24 h for both species at all canopy positions. December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 6 Northern Hardwood Experimental Warming Carter and Cavaleri FIGURE 3 | Leaf heater performance. (A) Average of heated and control leaf temperatures (Tleaf) at three canopy positions (Acer saccharum and Tilia americana combined) and (B) example of the variation in Tleaf for one heated and one control T. americana leaf over 24 h on August 25, 2016. Control Tleaf is depicted by the black lines and heated Tleaf is depicted by gray lines. Upper canopy is represented by dotted lines, sub canopy is depicted by solid lines, and understory is depicted by dashed lines. Air Temperature, Leaf Temperature, and Warming Device Performance FIGURE 3 | Leaf heater performance. (A) Average of heated and control leaf temperatures (Tleaf) at three canopy positions (Acer saccharum and Tilia americana combined) and (B) example of the variation in Tleaf for one heated and one control T. americana leaf over 24 h on August 25, 2016. Control Tleaf is depicted by the black lines and heated Tleaf is depicted by gray lines. Upper canopy is represented by dotted lines, sub canopy is depicted by solid lines, and understory is depicted by dashed lines. americana. A. saccharum showed some degree of scorching on one leaf in the upper canopy (9% of leaf area scorched), two leaves in the sub canopy (10% and 2%), and one leaf in the understory (17%). Only one T. americana leaf, located in the understory, exhibited scorching on 1% of its leaf area. canopy positions and species (Figures 6, 7). Neither the mixed effects model nor post-hoc contrast detected any differences between species or between canopy positions in the values of Topt (Figure 6A; Table 2). A. saccharum upper canopy Aopt was twice that of understory and sub canopy levels, while T. americana sub and upper canopy Aopt values were more than double understory Aopt (Figure 6B). Rates of Aopt of T. americana were greater than rates of A. saccharum in the upper and sub canopy (Figure 6B). ˆV cmax at Topt was higher in T. americana than A. saccharum at all canopy positions. T. americana sub canopy ˆV cmax was double the rate of the understory and upper canopy and A. saccharum ˆV cmax at Topt was consistent throughout all canopy positions (Figure 6C). Stomatal conductance at optimum temperatures (gs at Topt) showed similar patterns with species and canopy position as did Aopt; however, there were no differences between species in the understory and upper canopy (Figure 7A). A. saccharum Eleaf intercept followed a similar pattern to A. saccharum gs at Topt, where the upper canopy had higher rates of Eleaf compared to the understory and sub canopy. T. americana Eleaf intercept was highest in the sub canopy and the upper canopy Eleaf intercept was higher than the understory (Figure 7B). Frontiers in Forests and Global Change | www.frontiersin.org Leaf Level Acclimation and Within-Canopy Differences in Gas Exchange Parameters g There was no evidence of photosynthetic acclimation for either A. saccharum or T. americana after 1 week of experimental warming, but there was evidence of overall reduced rates of photosynthetic capacity in the warmed leaves, indicating detractive adjustment with experimental warming. We found no warming treatment effects on optimum temperature for photosynthesis (Topt), stomatal conductance (gs) at Topt, maximum rate of Rubisco carboxylation ( ˆV cmax) at Topt, or leaf evapotranspiration (Eleaf ) parameters for either study species (Table 2; Figure 5). We did, however, find a significant overall treatment effect for rates of photosynthesis at optimum temperatures, where warmed leaves showed slightly lower Aopt than control leaves (p = 0.020; Table 2; Figure 5B). The mixed effects model comparing the effects of Tleaf , treatment, species, and canopy position on both instantaneous water use efficiency (WUE, Amax/Eleaf ) and intrinsic water use efficiency (WUEint, Amax/gs) showed interaction effects across most variables (Table S3); however, post-hoc analyses primarily distinguished differences between A. saccharum sub canopy heated and control leaves (Figure S3). Overall, WUE and WUEint Optimum temperature was consistent throughout all canopy positions, while patterns with height of both optimum rates of photosynthesis, stomatal conductance, evapotranspiration, and the rate of Rubisco carboxylation differed by species. There were no significant treatment interactions for Topt, Aopt, ˆV cmax at Topt, gs at Topt, or the intercept term of Eleaf (Table 2); therefore, treatments were pooled and analyzed for differences between December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 7 Northern Hardwood Experimental Warming Carter and Cavaleri TABLE 1 | Two-way ANOVA p-value results of heated leaf temperature spiking. Max daily heated Tleaf % Frequency 1T > 10◦C Species 0.002** 0.907 Canopy position 0.271 0.296 Species × Canopy position <0.001*** 0.447 Heated leaf daily maximum leaf temperature and percentage of occurrences where the difference heated and control Tleaf was >10◦C (1T > 10◦C). p-value results of treatment, canopy position, and the interaction effect of species and canopy position. **p < 0.01, ***p < 0.001. Understory and sub canopy daily maximum Tleaf n = 9, where n is the number of days used to calculate the mean. A. saccharum upper canopy n = 8, T. americana upper canopy n = 10. FIGURE 4 | Summary of heated leaf temperature spiking. Leaf Traits ***Denotes results of contrast between species at each canopy position, where p < 0.001. Overall, both LMA and Narea increased with canopy height and had opposing treatment responses between A. saccharum and T. americana, whereas, Nmass and %C had no treatment effect and the height response differed between species. Canopy position had a strong positive effect on both A. saccharum and T. americana LMA and Narea (Figures 8A–D). LMA showed evidence of a ∼22% decrease with warming, but only in the upper canopy and only for T. americana (near significant 3-way interaction; Table 3; Figure S4B), while A. saccharum showed no treatment effect on LMA at any canopy position (Table 3; Figure S4A). Both species showed Narea treatment effects in the upper canopy only, but in opposite directions (Figures S4C,D), a pattern which mirrored LMA (Figures S4A,B). A. saccharum upper canopy Narea was greater than sub canopy or understory for both heated and control leaves (Figures 8C,D). T. americana control leaf Narea steadily increased from the understory to the upper canopy (Figure 8C), while heated leaf Narea increased from the understory to sub canopy but did not increase from the sub to upper canopy (Figure 7D). This resulted in a 39% higher Narea for T. americana upper canopy control leaf than the sub canopy leaf (p = <0.001, Figure 8B); however, heated sub and upper canopy leaves did not differ in Narea (Figure 8C). Heated upper canopy Narea was slightly greater than control leaf Narea (14%, p = 0.019) in A. saccharum leaves (Figure S4C). A. saccharum Nmass did not change with height, while Nmass of T. americana was greatest in the sub canopy (Figures 8E,F). T. americana Nmass was significantly greater than A. saccharum at all canopy positions (Figures 8E,F). There were weakly correlated with Tleaf , and significant correlations only occurred for some species, canopy position, and treatment combinations (Table S4). The only significant A. saccharum WUE vs. Tleaf correlation occurred in heated leaves located in sub (WUE p = 0.016 and WUEint p < 0.001) and upper (WUE p = 0.011) canopy, which all had decreasing slopes (Table S4; Figures S3A,C). T. americana water use efficiency tended to have a negative response to temperature, where only the sub canopy heated leaf (both WUE and WUEint) and the upper canopy heated leaf (WUEint only) did not decrease with increasing temperature (Table S4; Figures S3B–D). Leaf Level Acclimation and Within-Canopy Differences in Gas Exchange Parameters (A) Maximum daily heated leaf temperature (Tleaf) and (B) percent frequency of time points where the difference in control and associated heated leaf was >10◦C (1T > 10◦C) for Acer saccharum (open circles) and Tilia americana (filled circles) at each canopy position. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. ***Denotes results of contrast between species at each canopy position, where p < 0.001. TABLE 1 | Two-way ANOVA p-value results of heated leaf temperature spiking. leaf intercept was higher than the control leaf (Figure S3A). There were very few differences detected between species (Table S3). Leaf Traits Experimental warming had no effect on leaf area or leaf water content; however, both leaf traits were higher for T. americana than A. saccharum. A. saccharum leaf area did not vary with height (Table 3; Figure 7C); however, leaf area in the T. americana sub canopy was ∼60% higher than in the understory or upper canopy and was greater than that of A. saccharum in both sub and upper canopy (Figure 7C). For both species, leaf water content declined linearly with increasing canopy height, and was greater for T. americana than A. saccharum at all canopy positions (Table 3; Figure 7D). FIGURE 4 | Summary of heated leaf temperature spiking. (A) Maximum daily heated leaf temperature (Tleaf) and (B) percent frequency of time points where the difference in control and associated heated leaf was >10◦C (1T > 10◦C) for Acer saccharum (open circles) and Tilia americana (filled circles) at each canopy position. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. ***Denotes results of contrast between species at each canopy position, where p < 0.001. FIGURE 4 | Summary of heated leaf temperature spiking. (A) Maximum daily heated leaf temperature (Tleaf) and (B) percent frequency of time points where the difference in control and associated heated leaf was >10◦C (1T > 10◦C) for Acer saccharum (open circles) and Tilia americana (filled circles) at each canopy position. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. ***Denotes results of contrast between species at each canopy position, where p < 0.001. FIGURE 4 | Summary of heated leaf temperature spiking. (A) Maximum daily heated leaf temperature (Tleaf) and (B) percent frequency of time points where the difference in control and associated heated leaf was >10◦C (1T > 10◦C) for Acer saccharum (open circles) and Tilia americana (filled circles) at each canopy position. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. Leaf Traits Treatment 0.489 0.020* 0.319 0.546 0.173 0.364 Species 0.244 <0.001*** 0.012* <0.001*** 0.001** 0.364 Canopy position 0.573 <0.001*** <0.001*** 0.001** <0.001*** 0.386 Treatment × Species 0.112 0.741 0.272 0.419 0.862 0.330 Treatment × Canopy Position 0.971 0.817 0.886 0.836 0.187 0.385 Species × Canopy position 0.858 0.001** 0.032* 0.006** 0.015 0.430 Treatment × Species × Canopy position 0.923 0.925 0.879 0.851 0.572 0.387 *Denotes significance at p < 0.05. ** Denotes significance at p < 0.01. *** Denotes significance at p < 0.001. The full names for the symbols are provided in the abbreviations list. n = 3 (n = 2 for A. saccharum heated understory and sub canopy ˆV cmax at Topt). FIGURE 5 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), the maximum rate of Rubisco carboxylase ( ˆV cmax) at Topt, and stomatal conductance (gs) at Topt for the heated and control leaves. (A) A. saccharum and T. americana Topt for control (filled) and heated (no fill) leaves, (B) Aopt for both species, (C) ˆV cmax) at Topt for both species, and (D) gs at Topt for both species. Heated and control leaves are pooled across all canopy positions for each species individually. Error bars denote SEM. *denotes results mixed effects model comparing treatment, species, and canopy position. *p < 0.05, ***p < 0.001. n = 18. FIGURE 5 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), the maximum rate of Rubisco carboxylase ( ˆV cmax) at Topt, and stomatal conductance (gs) at Topt for the heated and control leaves. (A) A. saccharum and T. americana Topt for control (filled) and heated (no fill) leaves, (B) Aopt for both species, (C) ˆV cmax) at Topt for both species, and (D) gs at Topt for both species. Heated and control leaves are pooled across all canopy positions for each species individually. Error bars denote SEM. *denotes results mixed effects model comparing treatment, species, and canopy position. *p < 0.05, ***p < 0.001. n = 18. species differences in the slope response of any gas exchange parameters to any leaf traits; however, there were differences in intercepts (Table S6). T. americana had a higher Aopt per LMA intercept (4.03 µmol m−2 s−1) than A. saccharum (−0.938 µmol m−2 s−1, p = 0.003). A. saccharum had higher Aopt per Nmass intercept (−1.94 µmol m−2 s−1) than T. Leaf Traits The mixed effects model showed some treatment effects; however, post- hoc analysis only distinguished treatment differences between A. saccharum sub canopy (WUE slope p = 0.030, WUE intercept p < 0.001, WUEint slope p = 0.004, and WUEint intercept p < 0.001) and T. americana upper canopy WUE intercept (p = 0.050) (upper case letters, Table S4). A. saccharum sub canopy heated leaf had a more negative slope than the control leaf, while T. americana upper canopy heated December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 8 Northern Hardwood Experimental Warming Carter and Cavaleri TABLE 2 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as the random effect, for Topt, Aopt, gs at Topt, and ˆV cmax at Topt. TABLE 2 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as the random effect, for Topt, Aopt, gs at Topt, and ˆV cmax at Topt. TABLE 2 | P value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as the random effect, for Topt, Aopt, gs at Topt, and ˆV cmax at Topt. Topt Aopt gs at Topt Vcmax at Topt Eleaf intercept (β0) Eleaf exponential rise (β1) Treatment 0.489 0.020* 0.319 0.546 0.173 0.364 Species 0.244 <0.001*** 0.012* <0.001*** 0.001** 0.364 Canopy position 0.573 <0.001*** <0.001*** 0.001** <0.001*** 0.386 Treatment × Species 0.112 0.741 0.272 0.419 0.862 0.330 Treatment × Canopy Position 0.971 0.817 0.886 0.836 0.187 0.385 Species × Canopy position 0.858 0.001** 0.032* 0.006** 0.015 0.430 Treatment × Species × Canopy position 0.923 0.925 0.879 0.851 0.572 0.387 *Denotes significance at p < 0.05. ** Denotes significance at p < 0.01. *** Denotes significance at p < 0.001. The full names for the symbols are provided in the abbreviations list. n = 3 (n = 2 for A. saccharum heated understory and sub canopy ˆV cmax at Topt). Frontiers in Forests and Global Change | www.frontiersin.org Leaf Traits FIGURE 6 | Optimum temperature for photosynthesis (Topt), the FIGURE 6 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), maximum rate of Rubisco carboxylation ( ˆV cmax) at Topt at each canopy position for each species. (A) Topt, (B) Aopt, (C) ˆV cmax at Topt for Acer saccharum (open circles) and Tilia americana (filled circles) leaves in thee understory, sub canopy, and upper canopy. Variables at each canopy position includes both heated and control leaves because there were no significant interactions present in the mixed effects model. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. ** and *** denote results of contrasts between species at each canopy position. **p < 0.01, ***p < 0.001. n = 6 for Topt, Aopt, ˆV cmax at Topt (T. americana and A. saccharum upper canopy), gs at Topt, and Eleaf intercept. n = 5 for Vcmax at Topt understory and sub canopy. FIGURE 6 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), maximum rate of Rubisco carboxylation ( ˆV cmax) at Topt at each canopy position for each species. (A) Topt, (B) Aopt, (C) ˆV cmax at Topt for Acer saccharum (open circles) and Tilia americana (filled circles) leaves in thee understory, sub canopy, and upper canopy. Variables at each canopy position includes both heated and control leaves because there were no significant interactions present in the mixed effects model. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. ** and *** denote results of contrasts between species at each canopy position. **p < 0.01, ***p < 0.001. n = 6 for Topt, Aopt, ˆV cmax at Topt (T. americana and A. saccharum upper canopy), gs at Topt, and Eleaf intercept. n = 5 for Vcmax at Topt understory and sub canopy. FIGURE 7 | Leaf water traits and fluxes for each species at different canopy positions. (A) gs at Topt, (B) Eleaf intercept, (C) Leaf area, and (D) leaf water content for Acer saccharum (open circles) and Tilia americana (filled circles) by canopy position. Leaf Traits Leaf area and leaf water content for each canopy position includes both heated and control leaves because there were no significant interactions present in the mixed effects model. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. *denotes results of contrast between species at each canopy position. *p < 0.05, **p < 0.01, ***p < 0.001. n = 6. heated leaf had double the slope of the control leaf (p = 0.043, Table S6). Leaf Traits americana (−4.49 µmol m−2 s−1, p = 0.032); however, there were no differences in Aopt per Narea between the two species (p = 0.485). T. americana also had a slightly higher leaf water content per Eleaf (intercept) than A. saccharum (p = 0.026; Figure S5). The only significant treatment interaction occurred for Aopt response to Narea where post-hoc comparison showed that T. americana were no significant treatment effects or treatment interaction effects found for Nmass (Table 3; Figures S4E,F). A. saccharum had a higher %C than T. americana in the understory and mid canopy control leaves, but not in the heated leaves. %C tended to increase with canopy height for A. saccharum and T. americana control leaves but was consistent throughout the canopy in the heated leaves (Figures 8G,H). There were also no significant treatment or treatment interaction effects for %C (Table 3; Figures S4G,H). All photosynthetic leaf gas exchange parameters and leaf traits (Aopt vs. LMA, Narea, and Nmass) were correlated, while leaf water content and Eleaf intercept was not (Table S5). There were no December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 9 Northern Hardwood Experimental Warming Carter and Cavaleri FIGURE 6 | Optimum temperature for photosynthesis (Topt), the photosynthetic rate at Topt (Aopt), maximum rate of Rubisco carboxylation ( ˆV cmax) at Topt at each canopy position for each species. (A) Topt, (B) Aopt, (C) ˆV cmax at Topt for Acer saccharum (open circles) and Tilia americana (filled circles) leaves in thee understory, sub canopy, and upper canopy. Variables at each canopy position includes both heated and control leaves because there were no significant interactions present in the mixed effects model. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. ** and *** denote results of contrasts between species at each canopy position. **p < 0.01, ***p < 0.001. n = 6 for Topt, Aopt, ˆV cmax at Topt (T. americana and A. saccharum upper canopy), gs at Topt, and Eleaf intercept. n = 5 for Vcmax at Topt understory and sub canopy. Potential Resilience to Future Warming: Comparing Species While there are disadvantages to heating individual leaves instead of whole plants, leaf-level warming studies can give us important information on the physiological responses of forest ecosystem upper canopies that are currently unattainable through in-situ ecosystem-level warming studies. Physiological acclimation has also been found to occur more readily in plant tissues that are not fully developed (Turnbull et al., 1993); however, warming individual plant tissues, instead of whole plants, introduces carbon sink-source interactions that are difficult to parse from one another. Fully- developed leaves lose their capacity to import carbohydrates upon maturation (Turgeon, 2006). As such, mature leaves can only use the carbohydrates they produce themselves for both maintenance and growth. In contrast, developing leaves are able to import carbohydrates from other parts of the plant. Therefore, our leaves were treated as independent units with respect to whole-tree source-sink dynamics, and by warming mature leaves our results were not affected by changes in sink activity of our experimental leaves. In addition to canopy gradient plasticity, our results could have been confounded with ontogeny. All of our understory measurements were conducted on saplings, while the canopy warming was implemented on reproductively mature trees. Ontogeny could affect our results because trees that are fully shaded might allocate their resources differently than canopy trees with both sun and shade leaves. Both of our study species have been found to have high photosynthetic capacities and leaf area at intermediate size classes (Thomas, 2010). A. saccharum leaf area and photosynthesis have been found to increase with increasing plant size (Sendall et al., 2015a), a result which is similar to what we found in our A. saccharum leaves. LMA also tends to increase with increasing plant age, focusing leaf construction toward longer-live leaves with high photosynthetic capacity (Valladares and Niinemets, 2008). Particularly with shade tolerant species, resource allocation differs as trees mature and become less light limited (Sendall et al., 2015a). This suggests that plant age, in combination with canopy position, could have played a role in our results between the understory and canopy trees. While leaf area could have been affected by ontogeny Instead of Aopt thermally acclimating to warmer temperatures, we found evidence of photosynthetic decline in the heated leaves (Figure 5B). Other warming studies conducted on A. Potential Resilience to Future Warming: Comparing Species T. americana showed a greater resiliency to warming compared to A. saccharum, possibly through more efficient thermoregulation. Maximum temperatures of A. saccharum heated leaves were higher in the upper canopy, while T. americana maximum temperatures were consistent between canopy positions (Figure 4A). A. saccharum also had more leaf scorching than T. americana. Lower leaf temperatures and less evidence of leaf scorch suggests that T. americana may have a greater thermoregulation ability than A. saccharum. This is further supported by species differences in leaf area, stomatal conductance at Topt, and evapotranspiration. Smaller leaves can promote convective cooling by allowing higher transpiration rates induced through a thinner boundary layer (Michaletz et al., 2016; Fauset et al., 2018). T. americana leaf area is lower in the upper canopy compared with the sub canopy, while A. saccharum leaf area is consistent across all canopy positions (Figure 7C). Lower T. americana leaf area combined with high Eleaf could allow higher thermoregulation (Figure 7B). These results are similar to other studies, which have found that Tilia species have high leaf morphological plasticity (Lichtenthaler et al., 2007; Legner et al., 2014), leaf area that decreases with canopy height (Koike et al., 2001), and high rates of stomatal conductance (Thomas, 2010). In addition, while not significant, T. americana had a trend of higher Topt and greater Topt plasticity, where Topt declined with increasing canopy position. While canopy gradient differences in Topt might be influenced by seasonality, we did not find any differences in Tair between our sample months (Table S1). This suggests that ambient temperature variation, or seasonality, likely did not have a strong impact on within-canopy temperature differences. It is also possible that thermal acclimation is more likely to occur when experimental warming is applied to entire plants as opposed to individual leaves or that acclimation is more likely to occur when the heated treatment is applied to leaves that are not fully developed. When warming individual leaves as opposed to entire plants, there is a potential to miss important aspects of plant physiological acclimation. For example, stomatal closure is induced through the signaling of the hormone abscisic acid, a reaction often induced by leaf importation of abscisic acid produced in roots (Davies and Zhang, 1991; but not always Sampaio Filho et al., 2018). This suggests a possibility that warming of leaves might not induce the same amount of stomatal closure as whole-plant warming. Photosynthetic Acclimation and Responses to Warming 2017). In addition, our low sample size might have limited our ability to detect statistical significance. Most warming studies measure acclimation response after a set time that leaves are exposed to warmer temperatures, instead of throughout the warming experiment, making it difficult to pinpoint an acclimation timeframe. Gunderson et al. (2000) showed that A. saccharum seedlings have the capacity to acclimate to +4◦C Contrary to our hypotheses, neither study species showed evidence of photosynthetic acclimation (either an increase in Topt or Aopt) at any canopy position. It is possible that 1 week of warming was not enough time to allow for acclimation; although, photosynthetic acclimation has been found to occur after 1 week of experimental warming (e.g., Smith and Dukes, December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 10 Northern Hardwood Experimental Warming Carter and Cavaleri plant-scale carbon gain. If CO2 release through respiration does not equally acclimate to the declines in photosynthesis, plant carbon balance could be negatively affected (Drake et al., 2016). warmer temperatures through a shift in Aopt. While we did not see any positive shifts in Aopt or Topt with our experiment (Figure 5), a study conducted on taller trees during a different growing season that was located about 72 kilometers away from our study site (that experienced maximum daily temperatures similar to our study) found A. saccharum Topt to be much higher than the leaves in our study site (∼27◦C at 12.5 m, compared to our average of 20.2◦C) (Mau et al., 2018), suggesting that higher Topt can be found within this species. December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org Drivers of Within-Canopy Photosynthetic Rates Photosynthetic optimization with height differed between the two species, and this optimization was likely driven by different leaf traits. A. saccharum LMA and control leaf Narea were highest in the upper canopy leaves (Figures 8A–D). High LMA in the upper canopy is a common trend in canopy gradients and is due to higher leaf thickness and/or density which can maximize photosynthetic capacity in high light environment (Niinemets, 1999; Zhang et al., 2011; Coble et al., 2014). Our results are supported by other studies which have found that A. saccharum LMA does increase with height (Ellsworth and Reich, 1993; Coble et al., 2014; Filewod and Thomas, 2014). This is consistent with other studies where Narea of leaves exposed to high irradiance is predictive of photosynthetic capacity (Meir et al., 2002), and agrees with our results which found positive correlations between Narea and Aopt for both of our study species (Figure S5B). A. saccharum Nmass is distributed evenly throughout the understory and canopy (Figures 7E,F), a result often found in A. saccharum canopies (Ellsworth and Reich, 1993; Niinemets and Tenhunen, 1997; Coble and Cavaleri, 2015). T. americana Aopt was higher than A. saccharum in the sub and upper canopy (Figure 6B), which can likely be attributed to high Narea, leading to a capacity for high rates of Rubisco carboxylation ( ˆV cmax), in T. americana leaves (Figures 6C, 8C,D). This is further supported by the higher Aopt per Narea found in T. americana sub and upper canopy leaves (Figure S5B). In contrast to A. saccharum, T. americana photosynthetic rates and Nmass were maximized in the sub canopy (Figures 6B, 8E,F). In shade tolerant species, leaf nitrogen is prioritized to more shaded leaves to maximize the light harvesting capacity (Niinemets, 1997; Schoettle and Smith, 1999; Koike et al., 2001). Nitrogen is a major component In addition to differences in nitrogen optimization, the two species also had different patterns of water-associated leaf traits at different canopy positions. T. americana photosynthesis could be limited in the upper canopy due to hydraulic constraints, as well as stomatal and mesophyll conductance restrictions that occur with increasing canopy height (Niinemets and Tenhunen, 1997; Bond et al., 1999; Ryan et al., 2006; Duursma and Medlyn, 2012; Buckley et al., 2013). Lowered mesophyll conductance can limit Amax through decreased CO2 diffusion through cells and through alterations in intercellular membrane structure due to tissue shrinkage (Lawlor and Tezara, 2009). Potential Resilience to Future Warming: Comparing Species saccharum have found evidence of photosynthetic decline in both seedlings (Filewod and Thomas, 2014) and saplings (Gunderson et al., 2000), even in some cases where the trees show positive Topt acclimation (Gunderson et al., 2000). In addition, the only other leaf-level canopy warming experiment to study photosynthesis also found evidence of photosynthetic decline (Doughty, 2011). In our study, the decline in Aopt is likely due to a decline in the functioning of photosynthetic machinery because we did not detect a decline in either ˆV cmax or gs with experimental warming (Table 2; Figures 5C,D). Leaf scorching discovered in our heated leaves shows further evidence of potential photosynthetic apparatus damage. While our study only investigated photosynthetic acclimation and responses at the leaf level, declines in Aopt have important implications for larger, December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 11 Northern Hardwood Experimental Warming Carter and Cavaleri el comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as eaf area, and leaf water content. TABLE 3 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as the random effect, for LMA, Narea, Nmass, %C, leaf area, and leaf water content. TABLE 3 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as the random effect, for LMA, Narea, Nmass, %C, leaf area, and leaf water content. TABLE 3 | P-value results for mixed effect model comparing treatment, species, canopy position, and the interactions between all three variables, with individual tree as the random effect, for LMA, Narea, Nmass, %C, leaf area, and leaf water content. LMA Narea Nmass %C Leaf area Leaf water content Treatment 0.516 0.007** 0.336 0.806 0.676 0.420 Species 0.348 <0.001*** <0.001*** 0.011* 0.024* 0.001** Canopy position <0.001*** <0.001*** <0.001*** 0.004** 0.048* 0.001** Treatment × Species 0.366 0.001** 0.210 0.684 0.301 0.301 Treatment × Canopy position 0.695 0.015* 0.784 0.730 0.386 0.622 Species × Canopy Position 0.722 <0.001*** 0.002** 0.548 0.136 0.899 Treatment × Species × Canopy position 0.062 <0.001*** 0.444 0.330 0.972 0.113 *Denotes significance at p < 0.05. **Denotes significance at p < 0.01. ***Denotes significance at p < 0.001. The full names for the symbols are provided in the abbreviations list. n = 3. Potential Resilience to Future Warming: Comparing Species of chlorophyll and photosynthetic enzymes, and the high Nmass found in the sub canopy likely contributed to high photosynthetic rates in the sub canopy (Evans, 1989) (Figure S5C). and light availability, this is an interaction that occurs in most forest ecosystems, as trees existing in the overstory would be unlikely to develop branches within the understory. In addition, while there is evidence that suggests differing thermoregulation between T. americana canopy positions, it should be noted that T. americana upper canopy leaf area is higher than A. saccharum, and TLeafMax does not differ between canopy positions for A. saccharum control leaves (Figure S2D). A. saccharum upper canopy heated leaf Narea was higher than the control leaves, while T. americana had lower Narea in the upper canopy heated leaves. There were no treatment differences for T. americana Nmass (Table 3); therefore, this pattern was largely driven by a reduction in LMA in the upper canopy heated leaves (Figure 8B). Lowered Narea and LMA with warming suggests the possibility of lowered substrate availability in T. americana upper canopy leaves. While we did not measure respiration, declined LMA in T. americana upper canopy leaves may be attributed to higher rates of respiration in the heated leaves. If respiration was higher in the heated leaves, non- structural carbohydrates could have been used more quickly, leading to lower leaf mass; although, we had high variability in T. americana upper canopy heated leaf %C and did not find differences between treatments in the upper canopy (Figure 8H). A. saccharum had the opposite trend of Narea in the upper canopy leaves, where Narea was higher in the control leaves compared to the heated leaves (Figures S4C). Shifts in Narea suggest possible acclimation of other physiological processes, such as respiration, which is closely associated with leaf nitrogen (Turnbull et al., 2003) and has been found occur in A. saccharum (Gunderson et al., 2000; Reich et al., 2016). Other studies have found little evidence of Narea acclimation to experimental warming in temperate trees (Sendall et al., 2015b; Scafaro et al., 2017; Sharwood et al., 2017); however, a tropical leaf warming study found that LMA and Narea can increase within 1 week of experimental warming (Slot et al., 2014). Frontiers in Forests and Global Change | www.frontiersin.org Drivers of Within-Canopy Photosynthetic Rates Hydraulic restrictions on gs can limit Amax by decreasing intercellular CO2 concentrations, Frontiers in Forests and Global Change | www.frontiersin.org December 2018 | Volume 1 | Article 11 12 Northern Hardwood Experimental Warming Carter and Cavaleri Heated and control leaf functional traits at different canopy positions. (A) Control leaf mass per area (LMA) of Acer saccharum (open circles) and Tilia osed circles) leaves by canopy position, (B) heated LMA, (C) control nitrogen per area (Narea), (D) heated Narea, (E) control nitrogen per mass (Nmass), mass, (G) control % carbon, (H) heated % carbon. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. e results of post-hoc contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by tters. *denotes results of contrast between species at each canopy position. *p < 0.05, ***p < 0.001. n = 3. FIGURE 8 | Heated and control leaf functional traits at different canopy positions. (A) Control leaf mass per area (LMA) of Acer saccharum (open circles) and Tilia americana (closed circles) leaves by canopy position, (B) heated LMA, (C) control nitrogen per area (Narea), (D) heated Narea, (E) control nitrogen per mass (Nmass), (F) heated Nmass, (G) control % carbon, (H) heated % carbon. Error bars denote SEM. Letters above and below error bars denote results of post-hoc contrasts. Letters denote results of post-hoc contrasts between canopy position of A. saccharum is represented by capital letters, T. americana contrasts are represented by lower case letters. *denotes results of contrast between species at each canopy position. *p < 0.05, ***p < 0.001. n = 3. thereby limiting CO2 fixation in the Calvin cycle (Farquhar and Sharkey, 1982). T. americana gs at Topt and leaf water content were high in the sub canopy (Figures 7A,D); however, leaf water content was low in the upper canopy, which could have limited upper canopy photosynthesis. T. americana also had a more negative WUE relationship with temperature compared to A. saccharum, which suggests that this species might maintain higher rates of Eleaf even at a detriment to short-term carbon December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 13 Northern Hardwood Experimental Warming Carter and Cavaleri FUNDING Funding for this project was provided by the National Institute of Food and Agriculture U.S. Department of Agriculture McIntire- Stennis Cooperative Forestry Research Program Grant #1001534 and Department of Energy award DE-SC-0011806. Funding was also provided by the DeVlieg Foundation Fellowship and Ecosystem Science Center at Michigan Technological University. Leaf Heating Device Performance Plant acclimation to one type of stress can improve protection from other stressors (Havaux, 1992). Niinemets et al. (1999) found that electron transport in temperate tree leaves acclimated to high light environments is more stable under high temperature conditions. Upper canopy leaves are acclimated to high light conditions, possibly inducing stress acclimation in the upper canopy leaves in our study. The single instance of leaf scorching in the upper canopy occurred in A. saccharum, where the heated TleafMax was 43.5◦C. This maximum leaf temperature was at least 5◦C higher than maximum temperatures found at all canopy positions for both study species (Figure 4). High temperatures experienced by A. saccharum upper canopy leaves could have contributed to leaf scorching. • Our study supports our hypothesis that T. americana will likely have greater resiliency to climate warming due to a higher thermoregulation ability and higher trait plasticity between canopy positions of traits associated thermoregulation. • Canopy position photosynthetic optimization differed between our study species and these differences can, in part, be explained by the species’ leaf traits. Higher rates of photosynthesis in A. saccharum upper canopy leaves can be attributed to higher Narea and LMA. Higher T. americana photosynthetic rates in the sub canopy can be linked to high Nmass and gs in the sub canopy leaves, as well as hydraulic limitations on leaf mesophyll experienced by the upper canopy leaves. Our results suggest that models that predict canopy photosynthesis based on canopy height or leaf traits, such as LMA, may incorrectly estimate photosynthesis for species that do not optimize photosynthesis in their upper canopy. AUTHOR CONTRIBUTIONS KC and MC designed the experiment and wrote the manuscript. KC collected data, performed the data analysis, and drafted the manuscript. g In addition, A. saccharum leaves were slightly more affected by scorching than T. americana. The higher scorch damage in A. saccharum could have occurred for several reasons. As mentioned previously, higher Eleaf might have allowed greater thermoregulation in T. americana leaves (Figure 7B). T. americana also had higher leaf water content (Figure 7D), which might lead to higher tolerance to heat damage. Additionally, while both species are shade tolerant, T. americana is considered less so than A. saccharum (Crow, 1990; Baltzer and Thomas, 2007; Thomas, 2010). Species with higher shade tolerance have an overall lower plasticity to be able to adapt to high stress environments encountered in upper canopies (Reich et al., 2003). This suggests that A. saccharum could be less tolerant of the temperature fluctuations associated with the heating device. Another possibility for higher leaf scorching in A. saccharum is an artifact of the leaf heating device. T. americana petioles position their leaves so that they are relatively parallel to the ground compared to A. saccharum leaves. This makes T. americana easier to consistently heat over the entire surface of the leaf, likely preventing leaf scorch along leaf margins. Leaf Heating Device Performance Leaf Heating Device Performance Overall, the novel heating device worked well for both study species. The leaves were heated successfully +3 ± 0.14◦C above ambient leaf temperature across canopy positions, times of day, and sample months (Table S2). Higher heating device efficacy in the understory may be because the understory leaves were less exposed to temperature fluctuations due to direct radiation and sun flecks, allowing fewer spikes in temperature and, therefore, more consistent heating. While the heaters performed well, there was evidence of scorch damage to some of the heated leaves, and slightly more damage in the understory and sub canopy than the upper canopy. This suggests that shaded leaves may be more susceptible to damage at supraoptimal temperatures than upper canopy leaves. Leaves located higher in the canopy are exposed to more severe environments; i.e., high irradiance, temperatures, and wind. Plant acclimation to one type of stress can improve protection from other stressors (Havaux, 1992). Niinemets et al. (1999) found that electron transport in temperate tree leaves acclimated to high light environments is more stable under high temperature conditions. Upper canopy leaves are acclimated to high light conditions, possibly inducing stress acclimation in the upper canopy leaves in our study. The single instance of leaf scorching in the upper canopy occurred in A. saccharum, where the heated TleafMax was 43.5◦C. This maximum leaf temperature was at least 5◦C higher than maximum temperatures found at all canopy positions for both study species (Figure 4). High temperatures experienced by A. saccharum upper canopy leaves could have contributed to leaf scorching. Leaf Heating Device Performance Overall, the novel heating device worked well for both study species. The leaves were heated successfully +3 ± 0.14◦C above ambient leaf temperature across canopy positions, times of day, and sample months (Table S2). Higher heating device efficacy in the understory may be because the understory leaves were less exposed to temperature fluctuations due to direct radiation and sun flecks, allowing fewer spikes in temperature and, therefore, more consistent heating. While the heaters performed well, there was evidence of scorch damage to some of the heated leaves, and slightly more damage in the understory and sub canopy than the upper canopy. This suggests that shaded leaves may be more susceptible to damage at supraoptimal temperatures than upper canopy leaves. Leaves located higher in the canopy are exposed to more severe environments; i.e., high irradiance, temperatures, and wind. Conclusions gain (Table S4). Stomatal conductance was high in A. saccharum upper canopy leaves (Figure 7A), likely contributing to high rates of photosynthesis in the upper canopy. While leaf water content was lower in the upper canopy than in the understory, A. saccharum photosynthetic rates was highest in the upper canopy (Figure 6B). This suggests that leaf water is not a limiting factor for A. saccharum upper canop leaves. • We demonstrated that our novel leaf warming device successfully heated individual leaves 3.02 ± 0.01◦C above control leaf temperatures; however, there was evidence of leaf scorching, suggesting that there is room for improvement with this method. A simple improvement is to select individual leaves that are positioned parallel to the ground, which helps keep the heater in the correct position below the leaf. • We demonstrated that our novel leaf warming device successfully heated individual leaves 3.02 ± 0.01◦C above control leaf temperatures; however, there was evidence of leaf scorching, suggesting that there is room for improvement with this method. A simple improvement is to select individual leaves that are positioned parallel to the ground, which helps keep the heater in the correct position below the leaf. • Our results showed that our two study species were not able to photosynthetically acclimate to 1 week of leaf warming; instead, we found declines in photosynthesis. If neither of these species are able to acclimate to longer-term elevated temperatures, we could see a decline in CO2 sequestration in northern hardwood ecosystems. Frontiers in Forests and Global Change | www.frontiersin.org REFERENCES J., Reich, P. B., Barton, C. V., Medlyn, B. E., et al. (2016). Does physiological acclimation to climate warming stabilize the ratio of canopy respiration to photosynthesis? New Phytol. 211, 850–863. doi: 10.1111/nph.13978 y g g Buckley, T. N., Cescatti, A., and Farquhar, G. D. (2013). What does optimization theory actually predict about crown profiles of photosynthetic capacity when models incorporate greater realism? 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E., Bakkenes, M., Beaumont, L. J., Colllingham, Y. C., et al. (2004). Extinction risk from climate change. Nature 427, 145–148. doi: 10.1038/nature02121 Thomas, S. C. (2010). Photosynthetic capacity peaks at intermediate size in temperate deciduous trees. Tree Physiol. 30, 555–573. doi: 10.1093/treephys/tpq005 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Treyger, A. L., and Nowak, C. A. (2011). Changes in tree sapling composition within powerline corridors appear to be consistent with climatic changes in New York State. Glob. Chang. Biol. 17, 3439–3452. doi: 10.1111/j.1365-2486.2011.02455.x Copyright © 2018 Carter and Cavaleri. REFERENCES This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. j Turgeon, R. (2006). Phloem loading: how leaves gain their independence. Bioscience 56, 15. doi: 10.1641/0006-3568(2006)056[0015:PLHLGT]2.0.CO;2 Turnbull, M. H., Doley, D., and Yates, D. J. (1993). The dynamics of photosynthetic acclimation to changes in light quantity and quality in three December 2018 | Volume 1 | Article 11 Frontiers in Forests and Global Change | www.frontiersin.org 17
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https://journals.openedition.org/factsreports/pdf/4419
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Hubgrade Smart Monitoring Centers: Measuring Resource Consumption and Moving towards a Circular Economy
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Hubgrade Smart Monitoring Centers: Measuring Resource Consumption and Moving towards a Circular Economy Electronic version URL: http://journals.openedition.org/factsreports/4419 ISSN: 1867-8521 Publisher Institut Veolia Printed version Date of publication: 31 December 2017 Number of pages: 32-37 ISSN: 1867-139X Printed version Date of publication: 31 December 2017 Number of pages: 32-37 ISSN: 1867-139X Field Actions Science Reports The journal of field actions Special Issue 17 | 2017 Artificial Intelligence and Robotics in the City Field Actions Science Reports The journal of field actions Special Issue 17 | 2017 Artificial Intelligence and Robotics in the City Electronic reference Electronic reference Antonio Neves Da Silva and Patrice Novo, « Hubgrade Smart Monitoring Centers: Measuring Resource Consumption and Moving towards a Circular Economy », Field Actions Science Reports [Online], Special Issue 17 | 2017, Online since 31 December 2017, connection on 21 April 2019. URL : http:// journals.openedition.org/factsreports/4419 Creative Commons Attribution 3.0 License www.factsreports.org How can cities, businesses and industries boost growth in the face of resource scarcity? Firstly, they can start by measuring their consumption in order to manage it more effectively. Then, all of us can to move away from a linear model of consumption. We can accelerate this transition today, thanks to IoT1, the digital revolution. At Veolia, we are the fi rst to monitor and to optimize water, energy and material fl ows in real-time. We are developing smart monitoring centers called Hubgrade relying on connected products and artifi cial intelligence. With these centers, we are creating new jobs and business opportunities to save resources. At Veolia, we are the fi rst to monitor and to optimize water, energy and material fl ows in real-time. We are developing smart monitoring centers called Hubgrade relying on connected products and artifi cial intelligence. With these centers, we are creating new jobs and business opportunities to save resources. Hubgrade boosts energy effi ciency and water conservation measures. It optimizes material recovery and maximizes the use of renewable energy. However, this is only possible, with the focus on the human factor. 1 IoT – Internet of Things Hubgrade in Madrid, Spain 1.1 BOOSTING ECONOMIC GROWTH WHILE FACING RESOURCE SCARCITY Global urbanization is increasing and cities are putting the planet under enormous pressure. Since the 1970s, humanity has been consuming more natural resources than the planet can provide and renew in a year. Today, humanity actually needs one and a half planets to be sustainable. By 2050, at current rates, we would need almost three planets. Our demand for water, energy, food and goods is rapidly increasing. The Take-Make-Dispose model of the past has also led to extreme pollution, price volatility and biodiversity collapse. Besides the impact on climate change an on the environment, this causes evident effects on the economy and society. The challenge now is to decouple economic growth from resource consumption. We need to move towards a Circular Economy where nothing is wasted. In practice, this means not only preserving energy and water, while minimizing waste. We also need to improve energy efficiency, increase recycling and boost renewable energies. Moreover, we need business opportunities to accelerate this change. End-user End-user ‘Doing more with less’ is conceptually simple but quantifying resource effi ciency is more complex in practice. INTRODUCTION Hubgrade is the name of Veolia’s smart monitoring centers for water, energy and waste management. In these centers, Veolia’s analysts leverage real-time data to optimize resource consumption of municipal, commercial and industrial clients. This innovation relies on a dedicated organization, disruptive digital tools and new business models. Hubgrade is a tremendous opportunity to introduce a cultural change in our organization. We can revolutionize the way we operate and become more effi cient. At the same time, we are off ering new services and an enhanced customer experience to our clients. We can equip each of these centers to manage data from a multitude of facilities: from municipal water networks to waste collection systems, to buildings, to industrial sites, to district energy systems and more. Today, we have 15 Hubgrades already accelerating the transition towards a Circular Economy. They guarantee us that no precious resources are wasted. 32 AI in the city, the age of prediction and anticipation www.factsreports.org AI in the city, the age of prediction and anticipation www.factsreports.org 1.2 SMART URBAN METABOLISM: HIGHLIGHTING OPPORTUNITIES TO SAVE RESOURCES IN REAL-TIME models, using real-time data. Such virtual models represent the interconnectivity between different subsystems, which can be urban infrastructures, offices, schools, hospitals, industries and even households. The new generation of data solutions allows us a deeper study of urban metabolism. Urban metabolism is a model representing the transformation of natural resources in products and services. This model quantifi es economic value of what we use and what we waste. So, what are the benefits of smart urban metabolism models? These models make it is easier to replicate sustainable solutions to other subsystems. This happens when these subsystems have similar challenges. For example, an urban infrastructure needs to become more resilient, livable or even inclusive. The same applies to buildings and to industries that must become greener and more resource effi cient. Now it is possible to have a higher temporal resolution of consumption. This enables us to build smart urban metabolism Ecological footprint - http://www.footprintnetwork.org Ecological footprint - http://www.footprintnetwork.org The resources we are wasting will end up being either emissions or discharges to land or to water sources. In order to avoid such waste, a holistic approach on resource saving is required. Smart urban metabolism offers exactly that. It highlights the value of a subsystem’s waste to other economic sectors. This facilitates a business transaction so that waste can be reused, recovered or recycled by other subsystems. Emissions and discharges also cost money and become evident opportunities to save. These predictive models to analyze resource consumption are something very concrete to us at Veolia. We convert these models into performance contracts with our clients, with guaranteed savings. It may seem that we have always operated contracts like these. So what has changed? 33 www.factsreports.org Hubgrade, smart monitoring center in Paris, launched 2016 Now, we can commit to save significantly more. We understand the value chain of resources outside our traditional operations. Sensors, smart products and other digital technologies extend our capabilities. Besides implementing and operating effi cient systems, we engage end-users to play a major role in the solution. They take responsibility to change their behaviors because they foresee the benefits for them. Through smart solutions, citizens and other end-users receive the insight they need to save more. Veolia cannot monitor every resource fl ow in a city, and that is not the point. However, we do commit to improve resource effi ciency for the perimeter we operate. 1.2 SMART URBAN METABOLISM: HIGHLIGHTING OPPORTUNITIES TO SAVE RESOURCES IN REAL-TIME This is why the municipality of Pudong in Shanghai has chosen Veolia to manage its water networks. Hubgrade, smart monitoring center in Paris, launched 2016 2.1 WHAT IS HUBGRADE? Energy and water savings, waste minimization and recycling rates, carbon emission reduction – Veolia has always tracked these key performance indicators. Now, we can track them in real-time and from anywhere we want. However, this requires a major organizational change. As a result, clients and end users become more aware of how they can make savings. 2.2 HOW DOES HUBGRADE WORK? More recently in Shanghai, Veolia launched a Hubgrade for Water, Energy and Waste management. We can now offer higher level of commitments to existing customers. We can also offer these innovative services to new clients all over China. This way, they too, can save even more. Clients want control over costs and consumption, so their systems are fitted with sensors. These sensors transmit data in real time to Hubgrade. Then, our analysts manage this data to identify savings. This can result into immediate action or a roadmap for improvements. We transmit recommendations to clients and our teams, depending on the type of contract. This helps them identify and prioritize resource saving measures. From an online dashboard, clients can monitor their own indicators and compare them to benchmarks. This way, they can clearly measure their progress and see the reduction in their bills. 1 EMS – Energy Management Systems 2 BMS – Building Management Systems 3 CMMS – Computerized Maintenance Management Systems 4 SCADA – Supervisory control and data acquisition 2.3 DEDICATED TEAM OF EXPERTS Hubgrade relies on a team of experts that share a common goal: improving resource effi ciency together with operational units on- site and customers. In order to get the full potential out of Hubgrade, Veolia developed new engineering profi les: For this reason, Veolia deploys a dedicated organization, digital tools and new business models. This is how we created Hubgrade, our smart monitoring center. Through Hubgrade, Veolia is bringing operational synergies to all our water, energy and waste activities. p g g p • Data analyst with expertise in energy, water and waste management • Auditor-coach who ensures operations on-site follow the analysts’ recommendations • Systems expert setting up the right data from sensors into the software applications In Hubgrade, we combine data management with our technical expertise on the field. This results in signifi cant risk mitigation. At the same time, we are much closer and more responsive to customers needs. The capability of the Hubgrade team relies on the ubiquity of digital technologies. Hubgrade enables Veolia to respond quickly and in a targeted manner. Whenever there is an alert reported on the systems, there can be a work order generated. Hubgrade is a real asset for change management to boost operation performance and to offer new services. The Hubgrade team can act remotely or dispatch a team on site. If this is necessary, they schedule an intervention and dispatch it to operational units. The customer can follow up all the process in parallel through a reporting application. Hubgrades: Dublin, IRELAND; Paris, Marseille, FRANCE; Brussels, BELGIUM; Bilbao, Madrid, Barcelona, SPAIN; Birmingham, UK; Milan, ITALY; Amsterdam, NETHERLANDS; Dubaï, UAE; Stockholm, SWEDEN; Budapest, HUNGARY; Shanghai, CHINA; Sydney, AUSTRALIA These new roles ensure the implementation of change management in our traditional activities. With this team, Veolia closes the loop on the resource value chain. Thus, Hubgrade guarantees we deliver more savings. 34 AI in the city, the age of prediction and anticipation www.factsreports.org AI in the city, the age of prediction and anticipation www.factsreports.org Hubgrade, a new organization to guarantee more savings Hubgrade, a new organization to guarantee more savings 2.4 DIGITAL SOLUTIONS H u b g r a d e re li e s o n s t a tis ti c a l m o d e ls , optimization algorithms, geographic information and forecasting tools. Data mining through meta-heuristic algorithms allows us to predict customer needs. We analyze correlations between consumption patterns and production profi les to identify improvements. Ensuring different systems communicate with each other is a major challenge in the digital transformation. At the core of Hubgrade, various systems are integrated such as EMS1, Waste Management Platforms, Water Quality Monitoring and Control Systems, BMS2, CMMS3, Asset Management software, SCADA4 systems, and even Carbon Footprint, Indoor Air Quality monitoring applications and more. These systems also cover functionalities such as financial analysis and benchmarking. In addition, machine learning is making Hubgrade more powerful and autonomous. It brings new capabilities to help Veolia’s clients switch to an “industry 4.0 mindset” by bringing them valuable information for the resources they need to operate throughout their entire production chain. In practice, we are not just looking at utilities anymore. We also developed reporting dashboards to share with our analysts the most important information from digital systems. This helps them in decision-making and in communicating performance indicators to operational teams on the fi eld. Additionally, we provide the client with an online access to reports, as well as awareness- raising information to end-users. The way in which these systems work can be broken down into four stages: • 1. Collection of information from sensors “HUBGRADE IS ALREADY A MAJOR “ONE VEOLIA” ACHIEVEMENT .” “CREATING NEW JOBS FOR THE ROLES OF ANALYSTS, AUDITORS AND SYSTEMS EXPERTS THAT OPERATE IN HUBGRADE AND INVESTING IN THEIR CAREER DEVELOPMENT IS A MAJOR HUMAN RESOURCES ACCOMPLISHMENT OF VEOLIA.” “HUBGRADE IS ALREADY A MAJOR “ONE VEOLIA” ACHIEVEMENT .” “CREATING NEW JOBS FOR THE ROLES OF ANALYSTS, AUDITORS AND SYSTEMS EXPERTS THAT OPERATE IN HUBGRADE AND INVESTING IN THEIR CAREER DEVELOPMENT IS A MAJOR HUMAN RESOURCES ACCOMPLISHMENT OF VEOLIA.” AI in the city, the age of prediction and anticipation www.factsreports.org 5 Deloitte, Smart Cities How rapid advances in technology are reshaping our economy and society Version 1.0, November 2015 “HUBGRADE IS ALREADY A MAJOR “ONE VEOLIA” ACHIEVEMENT .” • 2. Supply of information to databases • 3. Data visualization through dashboards and reports • 4. Reporting to operational teams, client and end-users 35 www.factsreports.org digital businesses. Moving from a TCO6 model to a SaaS7 model has infl uenced Veolia. We offer Performance as a service on top of our core activities. Hubgrade in Dubai We are analyzing the consumption at the heart of our customers’ industrial process. For example, through machine-learning solutions, we can monitor the consumption of individual equipment. Moreover, we do not need meters for all of them. We can apply this machine learning technologies to commercial buildings, too. One single high- frequency meter powered by machine-learning algorithms enables us to breakdown electric consumption per type of equipment: lighting, air conditioning, computers, appliances and others. In the near future, with a single meter and some sensors we can even precisely measure the electricity use by each tenant. Our client will be able to send his tenants invoices for their individual consumptions without additional meters. These solutions will be cost effective enough for the complete switch from readings. Hubgrade in Dubai Hubgrade in Dubai digital businesses. Moving from a TCO6 model to a SaaS7 model has infl uenced Veolia. We offer Performance as a service on top of our core activities. digital businesses. Moving from a TCO6 model to a SaaS7 model has infl uenced Veolia. We offer Performance as a service on top of our core activities. Crossing data, from customer activities and from ours, highlights the direct value we create for them. We monitor and report indoor air quality and comfort conditions in real-time. This way, customers know in transparency that quality is guaranteed, while consumption is kept to a minimum. Although sustainability demands a long-term view, product lifecycles are getting shorter. Anyway, it does not alter our mission. We just need to be continuously innovating as demand varies and clients’ expectations change. The fi rst main real-time solutions with innovative business models offered by Veolia are applicable to all our energy, water and waste management activities: Through an online application, the circular economy seems more tangible. Clients know how much money they are saving. They can also check the emissions they reduced and the waste diverted from landfi ll. • Monitoring, analysis and optimization • Interactive reporting • End-user apps • Information modeling 6 TCO – Total Cost of Ownership 7 SaaS – Software as a Service 2.5 INNOVATIVE BUSINESS MODELS • Predictive maintenance and condition monitoring “Everything that can be digital, will be digital”5. This influences business models as well. We are adding a major layer of digital services on top of our core activities. Consequently, our business models have to change. 6 TCO – Total Cost of Ownership 7 SaaS – Software as a Service Hubgrade in Milan Hubgrade in Milan The unique value of the new digital component of our services has to be unleashed. For that, we need to integrate new business models in our offer. These are similar to the ones used in “CHINA IS THE FIRST COUNTRY TO RECEIVE ALL THIS POTENTIAL WITH A HUBGRADE THAT DELIVERS ENERGY, WATER AND WASTE MANAGEMENT OPTIMIZATION TO MUNICIPAL, COMMERCIAL AND INDUSTRIAL CUSTOMERS.” 36 AI in the city, the age of prediction and anticipation www.factsreports.org AI in the city, the age of prediction and anticipation www.factsreports.org We demonstrate all of these services to clients when visiting a Hubgrade. Everywhere there are Hubgrades, we offer an enhanced customer experience. Hubgrade provides all the transparency clients expect to trust expertise in data and our commitment on results. Citizens of China Let us now discover how it all started. Let us now discover how it all started. Well, we started by optimizing buildings. Human beings spend 90% of their time in buildings10 - that is why these are the first places where we need to save resources opportunity11. In our globe, buildings consume around 40% of energy, 25% of water and 40% of materials12. Simultaneously, they account for the biggest share of greenhouse gas emissions on the planet. This represents approximately 1/3 of the whole globe. To address carbon emissions and tackle resource consumption, Europe established an energy effi ciency directive in 2012. beings spend 90% of their time in buildings10 - that is why these are the first places where we need to save resources opportunity11. In our globe, buildings consume around 40% of energy, 25% of water and 40% of materials12. Simultaneously, they account for the biggest share of greenhouse gas emissions on the planet. This represents approximately 1/3 of the whole globe. To address carbon emissions and tackle resource consumption, Europe established an energy effi ciency directive in 2012. Back then, to address the challenges of our customers, Veolia started to develop new digital solutions for energy management. Shared Value creation8 Although one can argue that artificial intelligence will eradicate jobs thus harming society, this argument remains vague. We believe innovation is the best way to create societal value. It has been through artificial intelligence that we are improving safety conditions to innumerous workers in the fi eld. This is the case with the implementation of our waste sorting solution I-Sorter. Workers received training and new career progression opportunities. This example is a real value lever for sustainability. In 2014, as part of the group’s reorganization, Veolia took the opportunity to extent this acquired expertise in these smart monitoring centers, to its water and waste management activities, and created Hubgrade. The fi rst Hubgrades also optimize, in addition to buildings, the efficiency of Waste-to-Energy facilities and the electricity consumption of wastewater treatment plants. Creating new jobs for the roles of analysts, auditors and systems experts that operate in Hubgrade and investing in their career development is a major human resources accomplishment of Veolia. The main idea is to combine human and digital capabilities to boost a social and economic dynamic while preserving the planet. Today, Veolia’s digital solutions apply to all our activities. We use them for route optimization in waste collection. With them, we reduce leaks in water networks. We also use them to improve the effi ciency of buildings, industries and more. Small and medium IT services companies are developing the digital tools we use in Hubgrade. With these SMEs9 we establish partnerships with a long-term view. We commit to create this value and deliver it to our clients so they can keep sharing it with local communities. Hubgrade is already a major “One Veolia” achievement. It enables us to address the needs of our customers very closely. China is the first country to receive all this potential with a Hubgrade that delivers energy, water and waste management optimization to municipal, commercial and industrial customers. Thanks to Hubgrade, customers and citizens interact with us in real-time. We can now provide them with the services and information they need so that together we can do more for a sustainable future. 8 M. Porter & M. Kramer – Harvard Business Review – January February 2011 [Note: Creating Shared Value is not included in Corporate Social Responsibility, which is separate from profi t maximization. CSV is rather a transition and expansion form the concept of CSR.] 9 SME – Small and Medium Enterprises 2.5 INNOVATIVE BUSINESS MODELS With these solutions, we generated an average 15% savings in energy consumption. This is the case of Indra Systems, one of our 160 Energy Performance Contracts, optimized via a real-time monitoring center. Indra, which is the IT and Defense systems leader in Spain reduced by 15% the energy consumption of its 65 buildings, together with Veolia. Citizens of China We demonstrate all of these services to clients when visiting a Hubgrade. Everywhere there are Hubgrades, we offer an enhanced customer experience. Hubgrade provides all the transparency clients expect to trust expertise in data and our commitment on results. 10 Source : https://www.buildinggreen.com/blog/we-spend-90-our- time-indoors-says-who 11 McKinsey – Resource Revolution 2011 12 Source : https://www.euenergycentre.org/images/unep%20 info%20sheet%20-%20ee%20buildings.pdf 8 M. Porter & M. Kramer – Harvard Business Review – January February 2011 [Note: Creating Shared Value is not included in Corporate Social Responsibility, which is separate from profi t maximization. CSV is rather a transition and expansion form the concept of CSR.] 9 SME – Small and Medium Enterprises 9 SME – Small and Medium Enterprises 3. OVER THREE MILLION DATA POINTS BEING MONITORED BY VEOLIA Veolia monitors over three million sensors, from which one million are smart meters. The manner to leverage data from them is far from optimal. The good news is that Veolia is deploying Hubgrades worldwide in an industrialized way. We already monitor over 300 thousand data points in our Hubgrades. 37
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https://hal.inria.fr/hal-00690868/file/978-3-642-21040-2_20_Chapter.pdf
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FIRE: Fault Injection for Reverse Engineering
Lecture notes in computer science
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FIRE: fault injection for reverse engineering To cite this version: Manuel San Pedro, Mate Soos, Sylvain Guilley. FIRE: fault injection for reverse engineering. 5th Workshop on Information Security Theory and Practices (WISTP), Jun 2011, Heraklion, Crete, Greece. pp.280-293, ￿10.1007/978-3-642-21040-2_20￿. ￿hal-00690868￿ To cite this version: Manuel San Pedro, Mate Soos, Sylvain Guilley. FIRE: fault injection for reverse engineering. 5th Workshop on Information Security Theory and Practices (WISTP), Jun 2011, Heraklion, Crete, Greece. pp.280-293, ￿10.1007/978-3-642-21040-2_20￿. ￿hal-00690868￿ Distributed under a Creative Commons Attribution 4.0 International License FIRE: Fault Injection for Reverse Engineering Manuel San Pedro1, Mate Soos2, and Sylvain Guilley1 1 Institut TELECOM 2 INRIA, Security Research Labs Abstract. In this paper, we propose a new technique that uses fault injection to reverse-engineer a private block cipher implemented with an unknown S-box. The private algorithm we wish to retrieve differs from a known algorithm in the choice of the S-Box, which we find using a novel, fault-injecting technique. The main idea is to consider the components of the S-Box as the solutions of a linear boolean system, whose equations stem from the faults injected, using existing fault models. We focus on two well-known block ciphers, DES and AES, and prove it to be feasible to retrieve the the S-Box for both cases. We present the fault models used, the equations extracted from the faults injected, and analyse the final results. Given the detailed analysis, the technique can be applied with ease to most ciphers employing an S-box. HAL Id: hal-00690868 https://inria.hal.science/hal-00690868v1 Submitted on 24 Apr 2012 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction According to Kerckhoffs’s principle, a cryptosystem should be secure even if everything about the system except the secret key is public knowledge [9]. Even though this became a fundamental principle of modern cryptology, it is moderately common for companies and sometimes even standards bodies to keep the inner workings of a system secret [1, 6]. We then talk about security through obscurity, or black-box cryptography. Under Kerckhoffs’s principle, cryptanalysis consists in retrieving the cipher key. But when dealing with security through obscurity, the goal is now modified to also retrieve information on the private algorithm. This is called reverse- engineering. Nowadays, with the omnipresence of embedded cryptography, it has become crucial to be able to perform attacks on electronic devices embedding unknown cryptosystems. Previous attempts at reverse-engineering unknown cryptosystems were either through (electro-)optical means, such as the discovery of the MIFARE algorithm [6], or through the use of side-channel analysis [11]. Side-channel analysis was originally devised to find the secret key through the measurement of physical characteristics of the chip such as power intake. Guilley et al. [7] employed this technique to retrieve the internals of black-box ciphers. This is called the side-channel analysis for reverse-engineering (SCARE) attack. In this paper, we present a new type of attack employing the principle of fault injection [2] to retrieve the unknown S-box of a black-box cipher. Fault injection was originally devised to retrieve the secret key through injection of faults into the chip executing the algorithm and observing the modified output. Our attack injects faults into the chip, collects the output from the chip, performs analysis of this data and finally converts the data into a set of equations in binary variables, which are finally solved using Gaussian elimination to retrieve the S-box. This new type of attack we call fault injection for reverse engineering (FIRE). The rest of the paper is organised as follows. In Sect. 2, we describe the state of the art, such as physical attacks of cryptosystems and linear systems solving. In Sect. 3, we present a DES-based cryptosystem, and a FIRE attack on it. Then, in Sect. 4, we describe an AES-based cryptosystem, and its corresponding FIRE attack. Finally, in Sect. 5 we conclude this paper. 2.2 Solving linear boolean systems 2.2 Solving linear boolean systems If we consider an S-box as a boolean function fn→m (i.e. a boolean function from {0, 1}n to {0, 1}m), we can split it into m and fn→1, called the components. Each one of the components will be considered as a vector s ∈{0, 1}2n, being the solution of a linear system in 2n variables. Each one of the faults injected brings a certain l number of equations (depending on the fault model), that the component s must satisfy. This means that each component s is one of the solutions of the system in {0, 1} of l equations: A · X = B. (1) (1) where A is a l ×2n boolean matrix, and both X and B are vectors of 2n elements. The equations are of the form L2n−1 i=0 ai ·xi = bi. Let L be the set of solutions of the system L = {s ∈{0, 1}2n : A · s = B}. Let us note that where A is a l ×2n boolean matrix, and both X and B are vectors of 2n elements. The equations are of the form L2n−1 i=0 ai ·xi = bi. Let L be the set of solutions of the system L = {s ∈{0, 1}2n : A · s = B}. Let us note that s ∈L ⇔¯s ∈L (2) (2) It stems from the fact that if α and β are boolean variables, then α ⊕β = ¯α ⊕¯β. This property will be important for the rest of the study, since the minimum of candidates returned will be 2. To solve this linear system of equations, we have used the Sage software [14] to perform the Gaussian elimination, but any mathematical software is adequate for the job, as the matrices are typically quite small. 2.1 Physical attacks on cryptographic systems Most of the cryptographic algorithms used in serious applications are supposed to be secure against algorithmic attacks. However, they are implemented on physical components, and hence become vulnerable against physicals attacks. Once such algorithms are implemented, either on dedicated hardware or as software on a micro-controller, the different physical properties of the algorithm can be observed. Over the years, sophisticated attacks have been developed to attack cryptographic devices through such observations. Side-channel attack The physical implementation of a cipher may reveal useful information about the secret key in an indirect way. Kocher in [10] and in [11] published two novel attack techniques exploiting side channel leakage of cryptographic devices. Computation requires time, consumes power and causes electromagnetic radiations: all these are possible sources of information related to the secret key. These techniques are powerful, as they allow to reduce the complexity of a brute-force attack by several orders of magnitude. However, they require physical access to the device to collect the necessary measurements. Fault-injection attack Fault attacks is the active way of attacking the physical implementation of an algorithm. During the proper functioning of the device, the attacker perturbs it by injecting hardware faults which produce an erroneous (or faulted) output. The attacker then exploits this to retrieve secret information. As explained in [8], the most common ways to carry out such an attack are manipulating the supply voltage or the the external clock, or applying laser or X-ray beams. The SCARE attack More recently it has been shown ([3, 5, 7]) that side-channel attacks could be used to retrieve secret parts of private algorithms. This is called side-channel attack for reverse-engineering, or simply SCARE. when a side-channel is used to retrieve an S-box on a private block cipher such as DES or AES, the attacker studies the transition y = SB(x⊕k). In a classical side-channel attack, SB is known and we wish to retrieve k. In SCARE, we assume to know k and wish to retrieve SB. 3 The case of DES We first give a description of a FIRE attack on a DES-like cryptosystem. Even if the attack has already been shown by Biham & Shamir in [2], it gives us a good foundation to proceed during the more complex case of a SPN such as AES in Sect. 4. The Data Encryption Standard (DES) was developed in the 1970s by the National Bureau of Standards with the help of the National Security Agency. Its purpose was to provide a standard method for protecting sensitive commercial and unclassified data. IBM created the first draft of the algorithm, calling it LUCIFER. DES officially became a federal standard in November of 1976 [12]. DES is a symmetric cryptosystem, specifically a 16-round Feistel cipher. It has a 64-bit block size and uses a 56-bit key. From this key, 16 sub-keys are created and are used at each round. The input is split in two halves. The progression of the cipher is described in Fig. 1. The round function, applied to a 32 bits register R and a 48 bits round Key K, F(R, K), consists in the succession of 4 sub-functions: first, E is an expansion function applied to R which returns a 48 bits output. The key K is then XOR-ed to E(R). S is the substitution function. It consists in 8 S-Boxes SB0, . . . , SB7 each of which map a 6-bit input to a 4-bit output. A 32-bit permutation P is finally applied to the output of S. IP m L0 R0 F L1 = R0 R1 = L0 ⊕F(R0, K1) F R1 = L0 ⊕F(R0, K1) R2 = L1 ⊕F(R1, K2) R14 = L13 ⊕F(R13, K14) R16 = L15 ⊕F(R15, K16) L2 = R1 L15 = R14 L16 = R15 F IP −1 c Fig. 1. The DES cipher, a 16-round Feistel cipher. IP is a 64 bit permutation. The round function applies F to the right half of the register, XORs the result to the left half, and exchanges the roles of the halves. Fig. 1. The DES cipher, a 16-round Feistel cipher. IP is a 64 bit permutation. The round function applies F to the right half of the register, XORs the result to the left half, and exchanges the roles of the halves. 3 The case of DES We consider the fault model introduced by Biham and Shamir in [2]: it assumes that the attacker is able to inject faults at the last round, round no. 15, on the right register R15. We consider that the substitution function S has been modified and kept secret. We then wish to retrieve SB0, . . . , SB7, the 8 S-Boxes which compose it. Let c = (L16, R16) be the correct and c⋆= (L⋆ 16, R⋆ 16) be the faulty ciphertext, resulting from the same plaintext m and secret key K. If we consider that the secret key is not known but fixed to a certain value, we will not retrieve the exact S-boxes, instead we will retrieve the function x 7→SBi(x ⊕ki), where ki is key input of the ith S-Box. Without loss of generality, let us consider that the key is known for the attack, hence we can ignore it for our present discussion. We thus have: R16 = L15 ⊕F(R15) = L15 ⊕F(L16) and R⋆ 16 = L15 ⊕F(R⋆ 15) = L15 ⊕F(L⋆ 16). hence we get: R16 ⊕R⋆ 16 = F(L16) ⊕F(L⋆ 16) R16 ⊕R⋆ 16 = P[S(E(L16))] ⊕P[S(E(L⋆ 16))] P −1[R16 ⊕R⋆ 16] = S(E(L16)) ⊕S(E(L⋆ 16)). Since c and c⋆are known, the only unknown register, L15, disappears once R16 is XOR-ed with R⋆ 16. The intrinsic design of Feistel block-ciphers allows us to have the knowledge of the fault injected, and its effect during the cipher, giving us the difference at the input and output of the S-Boxes. We note ∆in and ∆out, those differences: ∆in = E(L16) ⊕E(L⋆ 16) ∆out = P −1[R16 ⊕R⋆ 16]. where ∆in and ∆out are 48 and 32 bits long. However, if we focus on the ith S-box Sbi for instance, we can consider ∆i in and ∆i out as 6 and 4 bits long. We know x = E(L16)[6 ∗i : 6 ∗(i + 1)], the 6 bits input of SBi during the unaltered cipher, x⋆= E(L⋆ 16)[6 ∗i : 6 ∗(i + 1)], the 6 bits input of SBi during the faulty cipher. We have the relation: Sbi(x) ⊕Sbi(x⋆) = ∆i out. Sbi(x) ⊕Sbi(x⋆) = ∆i out. Our goal is to retrieve Sbi, which is a boolean function from {0, 1}6 to {0, 1}4. Let’s consider it component-wise, i.e. as 4 functions from {0, 1}6 to {0, 1}: (s0, s1, s2, s3). 3 The case of DES From each injected fault, we must have: For j = 0, . . . , 3, sj,x ⊕sj,x⋆= ∆i out(j), (3) (3) where ∆i out(j) is the jth bit of ∆i out. where ∆i out(j) is the jth bit of ∆i out. out( ) out For each injected fault and for each component j, sj must satisfy the previous equation. It is then added to the final system. We now have a distinguisher, we can define Li,j,N as the set of candidates for the jth component of the ith S-box. Considering N fault injections, giving us (xk, x⋆ k, ∆out,k) ( k from 1 to N), we have: Li,j,N = {s ∈{0, 1}64 such that ∀k, k ≤N : sxk ⊕sx⋆ k = ∆i out,k(j)}. Simulating an error perturbing randomly one single input bit of an S-box of DES, we reach the final set of two candidates mentioned at eq. (4) after Fig. 2. Attacking component 0 of the first S-Box of DES: On the x axis, the number of faults injected, on the y-axis the mean of #L1,0,x after 1000 tries. In the end, we only have 2 candidates. Fig. 2. Attacking component 0 of the first S-Box of DES: On the x axis, the number of faults injected, on the y-axis the mean of #L1,0,x after 1000 tries. In the end, we only have 2 candidates. approximately 130 fault injections. Fig. 2 illustrates the mean progression of #L1,0,N with 1000 experiments. This attack converges to the expected solution, meaning that, since we have the property (2), ∃n0 such that ∀n > n0, Li,j,n = {sj, ¯sj}. (4) (4) Note that in order to fully retrieve the 8 S-boxes, one has to test both candidates for all the 32 components. This leads to an exhaustive search in 232, which is trivially feasible. 4 The case of AES AES is a widely used symmetric-key encryption by Daemen and Rijman [4], adopted as a standard by the National Institute of Standards and Technology of the US. It is based on a design principle known as a Substitution Permutation Network (SPN). AES has a fixed block size of 128 bits and a key size of 128, 192, or 256 bits. It operates on a 4 × 4 array of bytes, termed the state (where 1 byte = 8 bits). Most calculations carried out by the cipher are done in the finite field of GF(28). The AES cipher is specified as a number of repetitions of transformation rounds, each round made up with 4 round transformations: SubBytes, Mix- Columns, ShiftRows and AddRoundKey. Note that the last round is exempt from MixColumns. Without loss of generality, since we consider that the cipher key is known, we set it to 0, and we also discard the final ShiftRows operation since it can trivially be inverted. Hence we only consider operations MixColumns and SubBytes, as explained below in detail. MixColumns applies a linear transformation to a column of the state: MixColumns       x y z t      =   02 03 01 01 01 02 03 01 01 01 02 03 03 01 01 02  ·   x y z t  , where the operations are performed in GF(28). SubBytes is a non linear transformation which is applied to each byte of the state. It is traditionally implemented as a S-box, which can be seen as a boolean function SB from 8 bits to 8 bits. Note that SubBytes is a bijection. SubBytes       x y z t      =   SB(x) SB(y) SB(z) SB(t)  . In our attack, this function is unknown, and the goal is to retrieve it. In our attack, this function is unknown, and the goal is to retrieve it. 4.1 Fault injection Let us assume that we are able to inject a fault on one byte of the block, just before the last MixColumns, during the 9th round. The attack is column-wise, meaning that we only care about the column on which the fault is injected. For example, let us look at the first column of a regular cipher, from the last MixColumns until the end of the cipher. We have:   α β γ δ   MC −−→ MC       α β γ δ       SB −→   x y z t  = c. (5) (5) Now, the same data is processed, but with a fault ϵ injected before the last MixColumns. Fig. 3 illustrates the propagation of the error. Now, the same data is processed, but with a fault ϵ injected before the last MixColumns. Fig. 3 illustrates the propagation of the error. Round 9 Round 10 MixCol SubByte Output Fig. 3. Propagation of the fault on our simplified AES: we perturb a byte just before the last MixColumns. The error propagates to the whole column. Fig. 3. Propagation of the fault on our simplified AES: we perturb a byte just before the last MixColumns. The error propagates to the whole column. We thus have: α α ⊕ϵ α ⊕ϵ x⋆ We thus have:   α β γ δ   FI −→   α ⊕ϵ β γ δ   MC −−→ MC       α ⊕ϵ β γ δ       SB −→   x⋆ y⋆ z⋆ t⋆  = c⋆. (6) (6) Now that we have a triplet (c, c⋆, ϵ). Let us examine how we could exploit Fault Injection to extract information on SB. We start the attack from the ciphertexts, we retrieving SB−1, which is exactly the same since SB is bijective in SPNs. We have, from eq. (5) and eq. (6): We have, from eq. (5) and eq. (6): ve, from eq. (5) and eq. 4.1 Fault injection (6): SB−1(c) ⊕SB−1(c⋆) = MC       α β γ δ      ⊕MC       α ⊕ϵ β γ δ       = MC       ϵ 0 0 0      =   02 · ϵ ϵ ϵ 03 · ϵ  , because MixColumns is linear. It translates into the system because MixColumns is linear. It translates into the system SB−1(x) ⊕SB−1(x⋆) = 02 · ϵ SB−1(y) ⊕SB−1(y⋆) = ϵ SB−1(z) ⊕SB−1(z⋆) = ϵ SB−1(t) ⊕SB−1(t⋆) = 03 · ϵ . (7) (7) 4.2 Translation of the FI into equations 4.2 Translation of the FI into equations Let us remind ourselves that SB−1 is a boolean function from {0, 1}8 to {0, 1}8. Considering it component-wise, i.e. as 8 independent functions from {0, 1}8 to {0, 1}: SB−1 = {SB−1 0 , SB−1 1 , . . . , SB−1 7 } with SB−1 i : {0, 1}8 7→{0, 1}}. Now, SB−1 i can be seen as a set of 256 boolean variables: SB−1 i = {si,0, si,1, . . . , si,255}. If we consider bit-wise the equations given in (7) then for a fault injected, we know that, necessarily, for i = 0 . . . 7, SB−1 i has to satisfy si,x ⊕si,x⋆= (02 · ϵ)i si,y ⊕si,y⋆= ϵi si,z ⊕si,z⋆= ϵi si,t ⊕si,t⋆= (03 · ϵ)i . (8) (8) (8) These four equations are to be manipulated according to the fault model, and used to build the final system that is solved with Gaussian elimination to finally give the solutions. 4.3 Random and unknown faults First, we discuss the fault model that is close to the one presented by Piret and Quisquater in [13]. The error is injected on the first byte of the state, just before the last MixColumns. It is random and unknown. By adding lines of the system (7), without any knowledge of the value of ϵ, we have: SB−1(x) ⊕SB−1(x⋆) ⊕SB−1(y) ⊕SB−1(y⋆) ⊕SB−1(t) ⊕SB−1(t⋆) = 0 SB−1(x) ⊕SB−1(x⋆) ⊕SB−1(z) ⊕SB−1(z⋆) ⊕SB−1(t) ⊕SB−1(t⋆) = 0 , since 03 · ϵ ⊕02 · ϵ ⊕ϵ = 0. The operations are made on GF(28). 1 since 03 · ϵ ⊕02 · ϵ ⊕ϵ = 0. The operations are made on GF(28). Each one of the 8 components of SB−1 has to satisfy these equations. Now that we have removed ϵ, we can inject them into the system. Once solved, this system returns all the satisfying candidates, including the eight solutions. Considering N fault injections, giving us (ck, c⋆ k) (k from 1 to N), we can define the distinguisher LN for the attack of SB−1: LN =  s ∈{0, 1}256 such that ∀k < N, sxk ⊕sx⋆ k ⊕syk ⊕sy⋆ k ⊕stk ⊕st⋆ k = 0 sxk ⊕sx⋆ k ⊕szk ⊕sz⋆ k ⊕stk ⊕st⋆ k = 0,  LN =  s ∈{0, 1}256 such that ∀k < N, sxk ⊕sx⋆ k ⊕syk ⊕sy⋆ k ⊕stk ⊕st⋆ k = 0 sxk ⊕sx⋆ k ⊕szk ⊕sz⋆ k ⊕stk ⊕st⋆ k = 0,  It so happens that after n0 ≈400 faults injected, we have a constant set of solutions S: ∀n > n0, Ln = S. More precisely, the attack converges to a set S with 512 candidates. First we describe in detail this set S, and then we discuss the possible conclusion of the attack through exhaustive search. To account for the 512 solutions, we consider S as an orbit of the 8 compo- nents of SB−1: we have always SB−1 0 , SB−1 1 , . . . , SB−1 7 ∈S. But we also have (0, 0, . . . , 0) and (1, 1, . . . , 1) in S (they indeed satisfy all the equations brought by the distinguisher), we then state that: Proposition 1. u, v ∈S ⇒u ⊕v ∈S Proposition 1. u, v ∈S ⇒u ⊕v ∈S Proposition 1. u, v ∈S ⇒u ⊕v ∈S Proof. Without loss of generality, we shorten the definition of S to a single boolean equation, which does not change with the real context. For instance: S = {s ∈{0, 1}256 such that sy ⊕s⋆ y ⊕sz ⊕s⋆ z = 0}. S = {s ∈{0, 1}256 such that sy ⊕s⋆ y ⊕sz ⊕s⋆ z = 0}. S = {s ∈{0, 1}256 such that sy ⊕s⋆ y ⊕sz ⊕s⋆ z = 0}. Now let u, v ∈S. uy ⊕uy⋆⊕uz ⊕uz⋆= 0 , and vy ⊕vy⋆⊕vz ⊕vz⋆= 0. Then uy ⊕uy⋆⊕uz ⊕uz⋆⊕vy ⊕vy⋆⊕vz ⊕vz⋆= 0. Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. Then uy ⊕uy⋆⊕uz ⊕uz⋆⊕vy ⊕vy⋆⊕vz ⊕vz⋆= 0. Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. y y y y Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. Then (u ⊕v)y ⊕(u ⊕v)y⋆⊕(u ⊕v)z ⊕(u ⊕v)z⋆= 0. Finally u ⊕v ∈S. We now can define S such that: S = {a0 · SB−1 0 ⊕. . . a7 · SB−1 7 ⊕a8 · (1, . . . , 1), ai ∈{0, 1}}. We can remove from S the trivial solution (1, . . . , 1) and (0, . . . , 0): in fact, it is mandatory for a SPN S-box to be bijective, and it would not be the case if (1, . . . , 1) or (0, . . . , 0) was one of the components. From this set, how can the full S-box be efficiently retrieved? We have 510 candidates that must be replaced into the correct position out of 8 possible choices. A naive exhaustive search would lead to C510 8 × 8! ≈271 possibilities. However, as we have already noticed, ∀s ∈S, ¯s ∈S. We can form 255 groups of elements of S, each of them including a candidate and its complement. For an optimal exhaustive search, one has to select 8 of those groups, and then test the 256 possibilities. This would lead to 28 × C255 8 ≈257 possibilities to finish the attack. This computational complexity is moderately high, but can be achieved with a large set of modern GPUs and/or FPGAs, and is not out of reach of any major organisation such as multinational companies or governments. However, we also propose another solution by finishing the attack using the SCARE method. 4.4 SCARE conclusion of a FIRE attack In this section, we propose a finishing of a FIRE attack when we are in the context described in Sect. 4.3. We have a set S, of 510 candidates containing the 8 component of SB−1. In order to use side-channel information to finish the attack, we use the curves of the DPA-Contest [15] to find SB−1. The context is the following. We have – N power traces corresponding of the functioning of the components with known inputs/outputs/cipher keys. 1 – N power traces corresponding of the functioning of the components with known inputs/outputs/cipher keys. 1 – The set S of a reduced amount of candidates for the components of SB−1. Here, 510. It is well-known that the power consumption of components strongly depend on the data processed, and more exactly the number of bit-flips completed. This number is given by the hamming distance between a register at a time t and t + 1. We then talk about Hamming distance model. We study here the transition during the last SubBytes of the AES chiper. For every candidates s ∈S, for every component j of SB−1, we compute what would be the hamming distance between c (which is known) and the state at the input of the last SubBytes, if we would have s = SB−1 j . We then use a distinguisher (Pearson’s correlation) in order to measure the dependence between those hamming distances and the power traces. On Fig. 4, the correlation traces resulting from the attack of the 7th component by using SCARE. This means that we are looking for SB−1 7 amongst the 510 members of S. On the figure, by using 10000 traces, we clearly can identify SB−1 7 in red and bold, ¯ SB−1 7 , the symmetric below, and the 508 bad candidates, giving a correlation close to zero. An adversary able to perform fault injections on a Fig. 4. SCARE on the 7th component of SB−1, with N = 10000 power traces. We have the 510 correlation traces: on the x-axis the time samples of the power traces, on the y-axis, the value of the correlation. We clearly identify here the solution (on the top) and its complementary (on the bottom) Fig. 4. SCARE on the 7th component of SB−1, with N = 10000 power traces. 4.4 SCARE conclusion of a FIRE attack We have the 510 correlation traces: on the x-axis the time samples of the power traces, on the y-axis, the value of the correlation. We clearly identify here the solution (on the top) and its complementary (on the bottom) component is very likely to be able to get a campaign of acquisition of power traces in order to conclude the attack this way. Hence it alleviates the burden on the attacker of making the exhaustive search in 257 as munitioned at the end of Sec. 4.3. Note that the SCARE attack is feasible here since we have a very restricted number of candidate for the solutions. When dealing with SCA, the number of candidates to test is very important: 256 hypothesis to test when we want to retrieve a key byte, but 228 hypothesis to test when we are looking for a single component of SB−1. Here the FIRE attack carried out most of the job by reducing the 228 to 510. On Fig. 4, we have the results with N = 10000 power curves. However, from N = 5000 curves (taken randomly from the ones available for the DPA Contest), the attack is feasible, meaning that we are able to extract the solutions. 4.5 Results with various fault models and contexts In this section, we present several other realistic fault models, or context allowing us to perform a FIRE attacks. Random and known faults Let us consider the strongest fault model: we are able to inject a random and known fault during the cipher execution. 100 110 120 130 140 150 160 170 180 190 200 0 10 20 30 40 50 60 70 80 90 100 Number of faults injected Number of Solutions returned Case of AES Fig. 5. Attacking component 0 of AES S-Box inverse. On the x-axis, the number of faults injected, on the y-axis the mean of #L0,x after 100 tries. In the end, we only have 2 candidates. 100 110 120 130 140 150 160 170 180 190 200 0 10 20 30 40 50 60 70 80 90 100 Number of faults injected Number of Solutions returned Case of AES Fig. 5. Attacking component 0 of AES S-Box inverse. On the x-axis, the number of faults injected, on the y-axis the mean of #L0,x after 100 tries. In the end, we only have 2 candidates. The advantage with this model, is that, since we know ϵ, we are able to target which one of the components of SB−1 we are attacking. k Considering N fault injections, giving us (ck, c⋆ k, ϵk), k = 1, . . . N, we can define the distinguisher Li,N for the attack of the ith component of SB−1: Li,N =        s ∈{0, 1}256 such that ∀k < N, sxk ⊕sx⋆ k = (02 · ϵk)i syk ⊕sy⋆ k = ϵk i szk ⊕sz⋆ k = ϵk i stk ⊕st⋆ k = (03 · ϵk)i        . This model, combined with the technique described in Sect. 4.2, allows us to retrieve the full SB−1 in less than 180 faults injected. Fig. 5 illustrates the progression of L0,N, simulating an error occurring randomly on the first byte of the state just before the last MixColumns. Stuck-at model It has been shown that it is possible for an attacker to force a byte to a certain value, that it can choose. 5 Conclusion In this paper, we have introduced a new tool to reverse-engineer a private algorithm. This new FIRE attack allows us to retrieve the S-Box of private block-ciphers in a reasonable number of faults injected and under plausible and existing fault models. For the sake of practical demonstration, we have carried out the attack on two major ciphers, AES and DES, but the attack can be made to work on almost any cipher containing and unknown S-Box. In the case of the DES S-boxes, around 1000 Fault Injections are needed and a final exhaustive search in 232 is necessary to fully retrieve all the 8 S-Boxes. For AES, under the most plausible model, around 400 fault injections suffice and lead to a finite set of 510 candidates. We can then either conclude the attack using exhaustive search in 257, or perform a data acquisition campaign and finish the attack using SCARE. ∃ϵ ∈GF(28) such that τ = α ⊕ϵ. ∃ϵ ∈GF(28) such that τ = α ⊕ϵ. It hence leads to the fault model presented at Sec. 4.3, just as if we would have injected an unknown and random ϵ. 3. Clavier, C. An improved scare cryptanalysis against a secret a3/a8 gsm algorithm. In ICISS (2007), P. D. McDaniel and S. K. Gupta, Eds., vol. 4812 of Lecture Notes in Computer Science, Springer, pp. 143–155. 2. Biham, E., and Shamir, A. Differential fault analysis of secret key cryptosystems. In CRYPTO (1997), B. S. K. Jr., Ed., vol. 1294 of Lecture Notes in Computer Science, Springer, pp. 513–525. 1. Anderson, R. A5 (was: Hacking digital phones). Newsgroup Communication, 1994. 4.5 Results with various fault models and contexts If we suppose that, just before the last SubBytes, one can force the first byte to a given value τ:   α β γ δ   FI −→   τ β γ δ   SB −→   x⋆ y⋆ z⋆ t⋆  = c⋆. Hence he has access to x⋆which is equal to SB(τ). It leads to a trivial attack, since with 256 accurate stuck-at injections, one can retrieve the full S-Box. Note that even one single stuck-at injection, we get a lot of information to bring into the system, if we decide do use different models during the attack. In the case where it is not possible to inject a stuck-at fault at the input of the last SubBytes, but that it can be done just before the last MixColumns:   α β γ δ   FI −→   τ β γ δ   MC −−→ MC       τ β γ δ       SB −→   x⋆ y⋆ z⋆ t⋆  = c⋆. In that case, even if we know the value of τ, α is assumed to be random. But we have: 4. Daemen, J., and Rijmen, V. The block cipher rijndael. In CARDIS (1998), J.-J. Quisquater and B. Schneier, Eds., vol. 1820 of Lecture Notes in Computer Science, Springer, pp. 277–284. References 2. Biham, E., and Shamir, A. Differential fault analysis of secret key cryptosystems. In CRYPTO (1997), B. S. K. Jr., Ed., vol. 1294 of Lecture Notes in Computer Science, Springer, pp. 513–525. 3. Clavier, C. An improved scare cryptanalysis against a secret a3/a8 gsm algorithm. In ICISS (2007), P. D. McDaniel and S. K. Gupta, Eds., vol. 4812 of Lecture Notes in Computer Science, Springer, pp. 143–155. 4. Daemen, J., and Rijmen, V. The block cipher rijndael. In CARDIS (1998), J.-J. Quisquater and B. Schneier, Eds., vol. 1820 of Lecture Notes in Computer Science, Springer, pp. 277–284. 13 5. Daudigny, R., Ledig, H., Muller, F., and Valette, F. Scare of the des. In ACNS (2005), pp. 393–406. 6. Garcia, F. D., and et al. Dismantling MIFARE Classic. In ESORICS (2008), S. Jajodia and J. L´opez, Eds., vol. 5283 of LNCS, Springer, pp. 97–114. 7. Guilley, S., Sauvage, L., Micolod, J., R´eal, D., and Valette, F. Defeating any secret cryptography with scare attacks. In LATINCRYPT (2010), M. Abdalla and P. S. L. M. Barreto, Eds., vol. 6212 of Lecture Notes in Computer Science, Springer, pp. 273–293. 8. Hamid, H. B.-E., Choukri, H., Tunstall, D. N. M., and Whelan, C. The sorcerer’s apprentice guide to fault attacks. 9. Kerckhoffs, A. La cryptographie militaire. Journal des sciences militaires IX (January 1883), 5–83. 10. Kocher, P. C. Timing attacks on implementations of diffie-hellman, rsa, dss, and other systems. 104–113. 11. Kocher, P. C., Jaffe, J., and Jun, B. Differential power analysis. In CRYPTO (1999), M. J. Wiener, Ed., vol. 1666 of Lecture Notes in Computer Science, Springer, pp. 388–397. 12. National Bureau of Standards. Data Encryption Standard, 1977. 13. Piret, G., and Quisquater, J.-J. A differential fault attack technique against spn structures, with application to the aes and khazad. In Cryptographic Hardware and Embedded Systems - CHES 2003, vol. 2779 of Lecture Notes in Computer Science. Springer Berlin / Heidelberg, 2003, pp. 77–88. 14. Stein, W., et al. Sage Mathematics Software. http://www.sagemath.org. 15. VLSI Research Group TELECOM ParisTech. The DPA contest, 2008/2009. http://www.dpacontest.org/. 14
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Flight State Identification of a Self-Sensing Wing via an Improved Feature Selection Method and Machine Learning Approaches
Sensors
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Received: 31 March 2018; Accepted: 24 April 2018; Published: 29 April 2018 Abstract: In this work, a data-driven approach for identifying the flight state of a self-sensing wing structure with an embedded multi-functional sensing network is proposed. The flight state is characterized by the structural vibration signals recorded from a series of wind tunnel experiments under varying angles of attack and airspeeds. A large feature pool is created by extracting potential features from the signals covering the time domain, the frequency domain as well as the information domain. Special emphasis is given to feature selection in which a novel filter method is developed based on the combination of a modified distance evaluation algorithm and a variance inflation factor. Machine learning algorithms are then employed to establish the mapping relationship from the feature space to the practical state space. Results from two case studies demonstrate the high identification accuracy and the effectiveness of the model complexity reduction via the proposed method, thus providing new perspectives of self-awareness towards the next generation of intelligent air vehicles. Keywords: self-sensing wing; feature extraction; feature selection; flight state identification; machine learning Xi Chen 1,*, Fotis Kopsaftopoulos 2 ID , Qi Wu 3, He Ren 1 and Fu-Kuo Chang 4 1 Shanghai Engineering Research Center of Civil Aircraft Health Monitoring, Shanghai Aircraft Customer Service Co., Ltd., Shanghai 200241, China; renhe@comac.cc 2 Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; kopsaf@rpi.edu 3 School of Electronic, Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China; wuqi7812@sjtu.edu.cn 3 School of Electronic, Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China; wuqi7812@sjtu.edu.cn 4 Department of Aeronautics and Astronautics, Stanford University, Stanford, CA 94305, USA; fkchang@stanford.edu g * Correspondence: chenxi1@comac.cc or aero.x.chen@gmail.com; Tel.: +86-21-20875861 sensors sensors sensors rticle Flight State Identification of a Self-Sensing Wing via an Improved Feature Selection Method and Machine Learning Approaches Xi Chen 1,*, Fotis Kopsaftopoulos 2 ID , Qi Wu 3, He Ren 1 and Fu-Kuo Chang 4 1. Introduction In the active mode, they can be used for damage detection and structural health monitoring while in passive mode, the wing structural vibration during flying can be captured to reflect the air dynamic characteristics [11]. The wing configuration is shown in Figure 1. the current structural design and data processing methods with a departure from the existing technologies. Recent years have seen the development of different sensing network architectures and simulations [3–6], among which, an expandable network made of polymer-based substrates was designed by the Structure and Composites Lab (SACL) at Stanford University. This network contains many micro-nodes which have the potential to integrate micro-sensors, actuators and electronics for different applications [7]. Based on the development of integration and fabrication techniques [8–10], a smart structure with the sensor network monolithically embedded in the layup of a composite UAV wing was successfully fabricated [11]. This smart wing consists of four sensor networks and each network is integrated with strain gauges, resistive temperature detectors (RTD) and piezoelectric lead zirconate titanate (PZT) transducers. Specifically, the strain gauge is used to measure the wing strain distribution and identify any potentially dangerous areas. RTD detects the temperature distribution in order to provide the temperature compensation [12]. PZT transducers can be used for both active and passive measurements. In the active mode, they can be used for damage detection and structural health monitoring while in passive mode, the wing structural vibration during flying can be captured to reflect the air dynamic characteristics [11]. The wing configuration is shown in Figure 1. Figure 1. The self-sensing composite wing design [2]. Figure 1. The self-sensing composite wing design [2]. Figure 1. The self-sensing composite wing design [2]. Figure 1. The self-sensing composite wing design [2]. After realizing sensing ability through multi functional structures development, the next step is to equip the smart wing with thinking and judging capability, i.e., the structure is expected to be aware of surroundings and identify its current flying state. There have been studies devoted to addressing the related identification problem based on either strain or vibration signals obtained from experiments. Huang et al. studied the active flutter control and closed-loop flutter identification and a fast-recursive subspace method was applied in high-dimensional aero-servo-elastic system. The wind tunnel test showed that the natural frequency and modal damping ratios of the flutter modes can be precisely tracked [13]. 1. Introduction Pang and Cesnik employed non-linear least squares fit and Kalman filtering to obtain wing shape information and rigid body attitude. Results revealed that the Kalman filter has good performance in the presence of sensor noise [14]. For elastic deformation, Sodja et al. conducted a dynamic aeroelastic wind tunnel experiment under harmonic pitching excitations, experimental data including the bending and torsion deformation were consistent with the elastic analysis model developed by the Delft University of Technology [15]. For more general flight states, Kopsaftopoulos and Chang established a stochastic global identification method using PZT signals from both time and frequency domain based on developed Vector-dependent Functionally Pooled (VFP) model [2,16,17]. A large range of airspeeds and angles of attack were considered in the VFP-based identification framework and the structural dynamics of the composite wing could be captured and predicted. Overall, the above data processing approaches mainly belong to state space methods and improved time series analysis Based on the previous study yet from another perspective if we can After realizing sensing ability through multi-functional structures development, the next step is to equip the smart wing with thinking and judging capability, i.e., the structure is expected to be aware of surroundings and identify its current flying state. There have been studies devoted to addressing the related identification problem based on either strain or vibration signals obtained from experiments. Huang et al. studied the active flutter control and closed-loop flutter identification and a fast-recursive subspace method was applied in high-dimensional aero-servo-elastic system. The wind tunnel test showed that the natural frequency and modal damping ratios of the flutter modes can be precisely tracked [13]. Pang and Cesnik employed non-linear least squares fit and Kalman filtering to obtain wing shape information and rigid body attitude. Results revealed that the Kalman filter has good performance in the presence of sensor noise [14]. For elastic deformation, Sodja et al. conducted a dynamic aeroelastic wind tunnel experiment under harmonic pitching excitations, experimental data including the bending and torsion deformation were consistent with the elastic analysis model developed by the Delft University of Technology [15]. For more general flight states, Kopsaftopoulos and Chang established a stochastic global identification method using PZT signals from both time and frequency domain based on developed Vector-dependent Functionally Pooled (VFP) model [2,16,17]. 1. Introduction The current state sensing and awareness of flight vehicles relies on traditional sensors and detection devices mounted on different locations of the vehicle, e.g., Pitot tubes installed in front of the nose for airspeed measurement, transducers located on each side of the fuselage for angle of attack detection. Inspired by the unsurpassed flight capabilities of birds, a novel “fly-by-feel” (FBF) concept has been recently proposed for the development of the next generation of intelligent air vehicles that can “feel”, “think”, and “react” [1,2]. Such bio-inspired systems will not only be able to sense the environment (temperature, pressure, aerodynamic forces, etc.), but also be able to think in real-time and be aware of their current flight state and structural health condition. Further, such systems will react intelligently under various situations and achieve superior performance and agility. Compared with the traditional approaches, this FBF concept has the following advantages: (1) structural complexity reduction by integrated structures with self-sensing ability, (2) structural health on-line monitoring through embedded multi-functional materials, (3) autonomous flight control and decision-making Sensors 2018, 18, 1379; doi:10.3390/s18051379 www.mdpi.com/journal/sensors 2 of 21 Sensors 2018, 18, 1379 based on self-awareness [2]. Towards this end, great challenges have been posed to the current structural design and data processing methods with a departure from the existing technologies. Sensors 2018, 18, x 2 of 21 decision-making based on self-awareness [2]. Towards this end, great challenges have been posed to Recent years have seen the development of different sensing network architectures and simulations [3–6], among which, an expandable network made of polymer-based substrates was designed by the Structure and Composites Lab (SACL) at Stanford University. This network contains many micro-nodes which have the potential to integrate micro-sensors, actuators and electronics for different applications [7]. Based on the development of integration and fabrication techniques [8–10], a smart structure with the sensor network monolithically embedded in the layup of a composite UAV wing was successfully fabricated [11]. This smart wing consists of four sensor networks and each network is integrated with strain gauges, resistive temperature detectors (RTD) and piezoelectric lead zirconate titanate (PZT) transducers. Specifically, the strain gauge is used to measure the wing strain distribution and identify any potentially dangerous areas. RTD detects the temperature distribution in order to provide the temperature compensation [12]. PZT transducers can be used for both active and passive measurements. 1. Introduction A large range of airspeeds and angles of attack were considered in the VFP-based identification framework and the structural dynamics of the composite wing could be captured and predicted. improved time series analysis. Based on the previous study yet from another perspective, if we can Overall, the above data processing approaches mainly belong to state space methods and improved time series analysis. Based on the previous study yet from another perspective, if we can Sensors 2018, 18, 1379 3 of 21 extract distinguished features from the continuous coupled structural aerodynamic behavior, it is possible to identify the flight state directly using the limited features instead of detailed characterization of the structural responses. Machine learning techniques can be employed to establish the mapping relationship from the feature space to the practical state space. Facing a series of signals generated from the embedded sensor network, one of the main challenges is what kind of features should be extracted and whether these features are useful for classification. A set of features without careful selection and evaluation may lead to poor results whatever superior machine learning models are applied. Feature engineering is such a research field including feature extraction and selection. For a period of time series signals with noise, various statistical features can be calculated such as the mean value, standard deviation, peak value, kurtosis, etc. from both time domain and frequency domain [18], a feature pool is then created with different number of features depending on the characteristics of the signals [19–21]. More features are encouraged to avoid missing important candidates with superior classification performance. The next step is feature selection in which a limited subset is obtained by eliminating less effective features. It reduces model dimension and computational time [22]. Generally, feature selection can be divided into three categories as filter, wrapper and embedded. Filter methods rank the variables completely separate to the model used for classification. The assignment of feature importance is based on information generated by some statistical algorithms. Filter methods are computationally simple and fast without the interaction with the classifier and feature dependencies [23]. Embedded solutions select salient features as part of the learning process of the model, which can be linear regression, support vector machine, decision tree, random forest, etc. These methods integrate the subset selection into the model construction but are difficult to adjust for the optimal search [24]. 1. Introduction The third category is wrapper, in which features are selected based on the performance of a given model by searching the possible subsets space and assessing the performance of the given model on each subset, models can be various learning machines [25]. Although wrapper methods often achieve sound classification performance by considering the feature dependencies, the frequent interactions between feature subset search and the classifier cause high computational costs [26]. We have demonstrated the effectiveness of establishing the mapping relationship from the feature space to the flight state space through neural networks modelling [27]. This paper significantly improves the previous work by creating a much larger feature pool and considering the co-linearity among various features. To sum up, the objective of this paper is the introduction and evaluation of a novel feature selection method for accurate flight state identification of a self-sensing wing structure based on experimental vibration data recorded by piezoelectric sensors under multiple flight states. The developed method belongs to the filter family and is capable of obtaining a group of most important features for classification with low mutual dependency. The framework of the data acquisition, methodology development, evaluation and application is shown in Figure 2. The rest of the paper is organized as follows: Section 2 presents the problem statement. Section 3 focuses on the feature extraction and feature selection in which the novel filter algorithm is introduced. Two case studies including the general flight state identification and the stall detection and alerting are conducted in Section 4 followed by their results and discussions in Section 5. Concluding marks are made in the last section. 4 of 21 loyed to Sensors 2018, 18, 1379 haracterization of Data Acquisition Self-sensing wing PZT signal preparation Methodology Development Feature extraction Feature selection Application: Stall Alerting 2 1 VIF 1 j j R   Modified distance evaluation ( ) , 1 1 K w j k j k d d K    ( ) ( ) b j j j w j d d    Variance inflation factor ( ) , , , 1 1 ( 1) K b j e j k j k e d u u K K     Feature pool Time domain Frequency domain Information domain Evaluation: Flight State Identification 3D visulizaiton Correlation Analysis Accuracy comparison Stall phenomenon Accuracy comparison Figure 2. Framework of the proposed methodology. 1. Introduction Facing a series of signals generated from the embedded sensor network, one of t enges is what kind of features should be extracted and whether these features are u ification. A set of features without careful selection and evaluation may lead to poo tever superior machine learning models are applied. Feature engineering is such a resea di f i d l i F i d f i i i l i h i Figure 2. Framework of the proposed methodology. Problem Statement The problem statement of this work is as follows: based on signals collected from th nsors embedded in the self-sensing wing through a series of experiments under varying ates develop a feature selection method that is capable of obtaining limited useful featur Data Acquisition Self-sensing wing PZT signal preparation Methodology Development Feature extraction Feature selection 2 1 VIF 1 j j R   Modified distance evaluation ( ) , 1 1 K w j k j k d d K    ( ) ( ) b j j j w j d d    Variance inflation factor ( ) , , , 1 1 ( 1) K b j e j k j k e d u u K K     Feature pool Time domain Frequency domain Information domain Data Acquisition Methodology Development Methodology Development Evaluation: Flight State Identification 3D visulizaiton Correlation Analysis Accuracy comparison Application: Stall Alerting Evaluation: Flight State Identification 3D visulizaiton Correlation Analysis Accuracy comparison Stall phenomenon Accuracy comparison Figure 2. Framework of the proposed methodology. Figure 2. Framework of the proposed methodology. Evaluation: Flight State Identification Application: Stall Alerting Figure 2. Framework of the proposed methodology Figure 2. Framework of the proposed methodology. Facing a series of hallenges is what kind 2. Problem Statement hallenges is what kind of features should be extracted and whether these features are useful fo lassification. A set of features without careful selection and evaluation may lead to poor result whatever superior machine learning models are applied. Feature engineering is such a research field ncluding feature extraction and selection. For a period of time series signals with noise, variou The problem statement of this work is as follows: based on signals collected from the PZT sensors embedded in the self-sensing wing through a series of experiments under varying flight states, develop a feature selection method that is capable of obtaining limited useful features for flight state identification with high accuracy and low model complexity. Specifically, the coupled 5 of 21 Sensors 2018, 18, 1379 aerodynamic-mechanical responses represent different flight states, with each state characterized by a specific angle of attack (AoA) and airspeed and kept constant during the data collection. The first problem is that whether a few salient features can be extracted from a period of vibrational time series (e.g., thousands of data points) as a representation of the corresponding flight state. In this way, we can skip the investigation into the detailed aeroelastic behavior and use the limited features to identify the specific flight state directly instead of using the entire lengthy signal. This would significantly reduce the complexity of the flight state characterization. The second problem is how to guarantee the effectiveness of selected features. If the selected strong features are highly correlated with each other, they will exhibit similar identification ability which are still away from the optimal subset. The above two problems constitute the motivation of this study and are addressed in the following approaches: firstly, a large number of features is extracted to cover a wide range of descriptions of the flight state. Then, a modified distance evaluation algorithm is conducted to obtain a subset of individually powerful features followed by the combination of a variance inflation factor algorithm to reduce high dependency among features in the subset. Machine learning models are employed to evaluate the above method for multiple flight states identification as well as a specific case of stall detection and alerting. The main novel aspects of this study include: The main novel aspects of this study include: (1) A large feature pool is created covering up to 47 different features from the time, frequency and information domains. Facing a series of hallenges is what kind 2. Problem Statement (1) A large feature pool is created covering up to 47 different features from the time, frequency and information domains. (2) A novel filter feature selection method is developed by combining a modified distance evaluation algorithm and a variance inflation factor. (2) A novel filter feature selection method is developed by combining a modified distance evaluation algorithm and a variance inflation factor. (3) The flight state identification is treated as a classification problem by establishing the mapping relationship from the feature space to the physical space characterized by varying angle of attack and airspeed of the self-sensing wing structure in wind tunnel experiments. (3) The flight state identification is treated as a classification problem by establishing the mapping relationship from the feature space to the physical space characterized by varying angle of attack and airspeed of the self-sensing wing structure in wind tunnel experiments. (4) The application on stall detection and alerting with high identification accuracy provides new perspectives for autonomous flight control with real-time flight state monitoring. (4) The application on stall detection and alerting with high identification accuracy provides new perspectives for autonomous flight control with real-time flight state monitoring. 3. Methodology Development In this section, a novel filter feature selection method is proposed via the combination of a modified distance evaluation algorithm and a variance inflation factor. In order to obtain sufficient feature candidates, a large feature pool is firstly created by extracting features covering a wide range. The output of this method is a feature subset consisting of most salient features with low correlation, which is able to represent a lengthy time-series signal of the wing structural response under certain flight state. 3.1. Feature Extraction t4 = ∑N n=1 (x(n)−t1)4 N t10 = min(x(n)) t16 = t9 t7 t5 = ∑N n=1(x(n)−t1)2 N t11 = t9 −t10 t17 = t3 t63 t6 = q ∑N n=1 (x(n))2 N t12 = ∑N n=1|x(n)−t1| N t18 = t4 t64 t25 = ∑N n=1 (x(n)−t1)9 N·t29 Note: x(n) is a signal series for n = 1, 2, . . . , N, N is the number of data points. Table 1. Features in time domain. Time Domain Feature Parameters Un-Dimensional t1 = ∑N n=1 x(n) N t7 =  ∑N n=1 √ |x(n)| N 2 t13 = t9 t6 t19 = ∑N n=1 (x(n)−t1)3 N·t23 t2 = q ∑N n=1(x(n)−t1)2 N t8 = ∑N n=1|x(n)| N t14 = t6 t8 t20 = ∑N n=1 (x(n)−t1)4 N·t24 t3 = ∑N n=1 (x(n)−t1)3 N t9 = max(x(n)) t15 = t9 t8 . . . t4 = ∑N n=1 (x(n)−t1)4 N t10 = min(x(n)) t16 = t9 t7 t5 = ∑N n=1(x(n)−t1)2 N t11 = t9 −t10 t17 = t3 t63 t6 = q ∑N n=1 (x(n))2 N t12 = ∑N n=1|x(n)−t1| N t18 = t4 t64 t25 = ∑N n=1 (x(n)−t1)9 N·t29 Note: x(n) is a signal series for n = 1, 2, . . . , N, N is the number of data points. Table 2. Features in the frequency domain. Table 2. Features in the frequency domain. Frequency Domain Feature Parameters f1 = ∑K k=1 y(k) N f6 = q ∑K k=1 ( f rk−f5)2y(k) K f10 = f6 f5 f2 = ∑K k=1 (y(k)−f1)2 K f7 = r ∑K k=1 f rk2y(k) ∑K k=1 y(k) f11 = ∑K k=1 ( f rk−f5)3y(k) K· f63 f3 = ∑K k=1 (y(k)−f1)3 K(√ f2) 3 f8 = r ∑K k=1 f rk4y(k) ∑K k=1 f rk2y(k) f12 = ∑K k=1 ( f rk−f5)4y(k) K· f64 f4 = ∑K k=1 (y(k)−f1)4 K· f22 f9 = ∑K k=1 f rk2y(k) √ ∑K k=1 y(k)∑K k=1 f rk4y(k) f13 = ∑K k=1 √ | f rk−f5|y(k) K√ f6 f5 = ∑K k=1 ( frk·y(k)) ∑K k=1 y(k) Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of the kth spectrum line. 3.1. Feature Extraction Feature extraction relies heavily on experts’ knowledge, it is encouraged to extract different kinds of features, as many as possible in case of missing useful ones. In this study, we intend to create a large feature pool from three main sources, namely the time, frequency and information domains. In time domain, 25 statistical features are calculated including 12 commonly used features such as mean, standard deviation, variance, peak, mean absolute deviation, etc. and 13 un-dimensional features such as crest factor, shape factor and a series of normalized central moments. The expressions of all time domain features are listed in Table 1. In terms of their physical insights, t1–t12 may reflect the vibration amplitude and energy while t13–t25 may represent the series distribution of the signal in time domain. Previous studies employed Fast Fourier Transform (FFT) to convert the time series into frequency spectrum [19,20]. However, the signal instances from the wind tunnel experiments are samples of a stochastic process with considerable noise. Welch’s method improves FFT by shortening the signals and averaging, and thus the peaks are smoothed for noise reduction [28]. Herein, a sample-long 6 of 21 Sensors 2018, 18, 1379 Hamming data window with 90% overlap is used for the Welch-based spectral estimation. A series of power spectrum y(k) without log transformation is then used for frequency domain feature extraction. Thirteen statistical features such as mean spectrum, spectrum center, root mean square spectrum, etc. and their mathematical expressions are shown in Table 2. f 1 may indicate the vibration energy in the frequency domain. f 2–4, f 6, f 10–13 may describe the convergence of the spectrum power. f 5, f 7–9 may show the position change of the main frequency. Hamming data window with 90% overlap is used for the Welch-based spectral estimation. A series of power spectrum y(k) without log transformation is then used for frequency domain feature extraction. Thirteen statistical features such as mean spectrum, spectrum center, root mean square spectrum, etc. and their mathematical expressions are shown in Table 2. f 1 may indicate the vibration energy in the frequency domain. f 2–4, f 6, f 10–13 may describe the convergence of the spectrum power. f 5, f 7–9 may show the position change of the main frequency. Table 1. Features in time domain. 3.1. Feature Extraction Time Domain Feature Parameters Un-Dimensional t1 = ∑N n=1 x(n) N t7 =  ∑N n=1 √ |x(n)| N 2 t13 = t9 t6 t19 = ∑N n=1 (x(n)−t1)3 N·t23 t2 = q ∑N n=1(x(n)−t1)2 N t8 = ∑N n=1|x(n)| N t14 = t6 t8 t20 = ∑N n=1 (x(n)−t1)4 N·t24 t3 = ∑N n=1 (x(n)−t1)3 N t9 = max(x(n)) t15 = t9 t8 . . . t4 = ∑N n=1 (x(n)−t1)4 N t10 = min(x(n)) t16 = t9 t7 t5 = ∑N n=1(x(n)−t1)2 N t11 = t9 −t10 t17 = t3 t63 t6 = q ∑N n=1 (x(n))2 N t12 = ∑N n=1|x(n)−t1| N t18 = t4 t64 t25 = ∑N n=1 (x(n)−t1)9 N·t29 Note: x(n) is a signal series for n = 1, 2, . . . , N, N is the number of data points. Table 2. Features in the frequency domain. Frequency Domain Feature Parameters f1 = ∑K k=1 y(k) N f6 = q ∑K k=1 ( f rk−f5)2y(k) K f10 = f6 f5 f2 = ∑K k=1 (y(k)−f1)2 K f7 = r ∑K k=1 f rk2y(k) ∑K k=1 y(k) f11 = ∑K k=1 ( f rk−f5)3y(k) K· f63 f3 = ∑K k=1 (y(k)−f1)3 K(√ f2) 3 f8 = r ∑K k=1 f rk4y(k) ∑K k=1 f rk2y(k) f12 = ∑K k=1 ( f rk−f5)4y(k) K· f64 f4 = ∑K k=1 (y(k)−f1)4 K· f22 f9 = ∑K k=1 f rk2y(k) √ ∑K k=1 y(k)∑K k=1 f rk4y(k) f13 = ∑K k=1 √ | f rk−f5|y(k) K√ f6 f5 = ∑K k=1 ( frk·y(k)) ∑K k=1 y(k) Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of he kth spectrum line. Table 1. Features in time domain. Table 1. Features in time domain. Time Domain Feature Parameters Un-Dimensional t1 = ∑N n=1 x(n) N t7 =  ∑N n=1 √ |x(n)| N 2 t13 = t9 t6 t19 = ∑N n=1 (x(n)−t1)3 N·t23 t2 = q ∑N n=1(x(n)−t1)2 N t8 = ∑N n=1|x(n)| N t14 = t6 t8 t20 = ∑N n=1 (x(n)−t1)4 N·t24 t3 = ∑N n=1 (x(n)−t1)3 N t9 = max(x(n)) t15 = t9 t8 . . . 3.1. Feature Extraction Its mathematical expression using normalized singular spectrum is: FI = ∑ M−1 i=1 (σi+1 −σi)2 σi (5) (5) where σi is the normalized value through σi = σi/∑M j=1 σj, and M is the number of singular value j j Approximate Entropy (ApEn) quantifies the amount of regularity and the unpredictability of fluctuations of a signal [36], which is computed in the following procedures: j j Approximate Entropy (ApEn) quantifies the amount of regularity and the unpredictability of fluctuations of a signal [36], which is computed in the following procedures: (1) Set the input as [x1, x2, . . . , xN]. (1) Set the input as [x1, x2, . . . , xN]. (2) Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N −m, where m is the subsequence length. (3) C t t t f b {x(j m)} {x(j m)|j ∈[1 N m]} h x(j m) i d fi d ( ) p [ 1 N] (2) Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N −m, where m is the subsequence length. (2) Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N −m, where m is the subsequence length. (2) Construct the subsequence x(i, m) = [xi, xi+1, . . . , xi+m−1] for 1 ≤i ≤N m, where m is the subsequence length. (3) Construct a set of subsequences {x(j, m)} = {x(j, m)|j ∈[1, . . . N −m]}, where x(j, m) is defined in Step (2). q g (3) Construct a set of subsequences {x(j, m)} = {x(j, m)|j ∈[1, . . . N −m]}, where x(j, m) is defined in Step (2). (3) Construct a set of subsequences {x(j, m)} = {x(j, m)|j ∈[1, . . . N −m]}, where x(j, m) is define in Step (2). (4) For each x(i, m) ∈{x(j, m)}, C(i, m) = ∑N−m j=1 kj N−m , where kj = ( 1 if |x(i, m) −x(j, m)|< r 0 otherwise (4) For each x(i, m) ∈{x(j, m)}, C(i, m) = ∑N−m j=1 kj N−m , where kj = ( 1 if |x(i, m) −x(j, m)|< r 0 otherwise . 3.1. Feature Extraction . . , MSE(12)]. where Ske = 3(MSEa −MSEb)/MSEc, MSEa, MSEb, MSEc represent mean, median and standard deviation of MSE(τ) = [MSE(1), MSE(2), . . . , MSE(12)]. Fractal dimension characterizes the space filling capacity of a pattern that changes with the scale at which it is measured [33]. Herein, two approaches are used as Petrosian Fractal Dimension (PFD) and Higuchi Fractal Dimension (HFD). PFD is calculated as: PFD = log10 N log10 N + log10(N/(N + 0.4Nδ)) (3) (3) where N is the length of the signal and Nδ is the number of sign changes in the signal derivative [30]. In terms of HFD, firstly k new series are constructed from the original signal [x1, x2, . . . , xN] by [xm, xm+k, xm+2k, . . . , xm+⌊(N−m)/k⌋k], where m = 1, 2, . . . , k. Secondly the length L(m, k) for each new series is calculated as: where N is the length of the signal and Nδ is the number of sign changes in the signal derivative [30]. In terms of HFD, firstly k new series are constructed from the original signal [x1, x2, . . . , xN] by [xm, xm+k, xm+2k, . . . , xm+⌊(N−m)/k⌋k], where m = 1, 2, . . . , k. Secondly the length L(m, k) for each new series is calculated as: L(m, k) = ∑⌊(N−m)/k⌋ i=2 xm+ik −xm+(i−1)k (N −1) ⌊(N −m)/k⌋k (4) (4) and the average length L(k) = j ∑k i=1 L(i, k) k /k. After kmax repetitions, a least-squares method is used to obtain the best slope that fits the curve of ln(L(k)) versus ln(1/k), which is defined as the Higuchi Fractal Dimension. For details, please refer to [34]. and the average length L(k) = j ∑k i=1 L(i, k) k /k. After kmax repetitions, a least-squares method is used to obtain the best slope that fits the curve of ln(L(k)) versus ln(1/k), which is defined as the Higuchi Fractal Dimension. For details, please refer to [34]. Fisher Information (FI) measures the expected value of the observed information [35]. 3.1. Feature Extraction Frequency Domain Feature Parameters f1 = ∑K k=1 y(k) N f6 = q ∑K k=1 ( f rk−f5)2y(k) K f10 = f6 f5 f2 = ∑K k=1 (y(k)−f1)2 K f7 = r ∑K k=1 f rk2y(k) ∑K k=1 y(k) f11 = ∑K k=1 ( f rk−f5)3y(k) K· f63 f3 = ∑K k=1 (y(k)−f1)3 K(√ f2) 3 f8 = r ∑K k=1 f rk4y(k) ∑K k=1 f rk2y(k) f12 = ∑K k=1 ( f rk−f5)4y(k) K· f64 f4 = ∑K k=1 (y(k)−f1)4 K· f22 f9 = ∑K k=1 f rk2y(k) √ ∑K k=1 y(k)∑K k=1 f rk4y(k) f13 = ∑K k=1 √ | f rk−f5|y(k) K√ f6 f5 = ∑K k=1 ( frk·y(k)) ∑K k=1 y(k) Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of the kth spectrum line. Frequency Domain Feature Parameters Note: y(k) is a spectrum for k = 1, 2, . . . , K, K is the number of spectrum components;f rk is the frequency value of the kth spectrum line. In electroencephalograph (EEG) analysis for neural diseases diagnosis and vibration analysis for mechanical defects, fractal dimensions from computational geometry and entropies from information theory have demonstrated effectiveness in early diseases/fault diagnosis [29,30]. Inspired by that, a group of complex features are employed and their terminologies are Multi-Scale Entropy, Partial Mean of Multi-Scale Entropy, Petrosian Fractal Dimension, Higuchi Fractal Dimension, Fisher Information, Approximate Entropy, and Hurst Exponent, respectively. pp py p p y Multi-Scale Entropy (MSE) introduces the scale factor based on the sample entropy to measure the complexity of signal under different scale factors [31]. It is calculated as: MSE = {τ SampEn(τ, m, r) = −ln[Cr,m+1(r)/Cr,m(r)]} (1) (1) where τ is the scale factor, m is the embedding dimension and r is the threshold. Here m = 2, r = 0.2 * standard deviation, τ = 12. Sensors 2018, 18, 1379 7 of 21 The first three values are selected due to the relatively high distinction among different classes. Also, an integrated non-linear index called Partial Mean of Multi-Scale Entropy (PMMSE) is used to simultaneously reflect the mean value and variation trend of MSE [32], which is expressed as: PMMSE = (1+|Ske|/3) · MSEa (2) (2) where Ske = 3(MSEa −MSEb)/MSEc, MSEa, MSEb, MSEc represent mean, median and standard deviation of MSE(τ) = [MSE(1), MSE(2), . 3.1. Feature Extraction (5) ApEn is calculated as: (5) ApEn is calculated as: ApEn(m, r, N) = 1 N −M " N−m ∑ i=1 ln C(i, m) C(i, m + 1) # (6) (6) Hurst Exponent (HST) measures the long-term memory of a signal. It is used to quantify the relative tendency of the signal either to regress to the mean or to cluster in a direction [37]. For time series 8 of 21 Sensors 2018, 18, 1379 X = [x1, x2, . . . , xN], its accumulated deviation within range T is calculated as X(t, T) = ∑t i=1 (xi −x), where x = 1 T ∑T i=1 xi, t ∈[1, 2, . . . , N]. Then: R(T) S(T) = max(X(t, T)) −min(X(t, T)) q (1/T)∑T t=1 [x(t) −x]2 (7) (7) The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Ex The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Exponent. The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Exponent. In summary, abbreviations of the complex features extracted from information domain are listed in Table 3. The slope of ln(R(n)/S(n)) versus ln(n) for n ∈[2, 3, . . . , N] is defined as the Hurst Exponent. In summary, abbreviations of the complex features extracted from information domain are listed in Table 3. In summary, abbreviations of the complex features extracted from information domain are listed in Table 3. In summary, abbreviations of the complex features extracted from information domain are listed able 3. Table 3. Features in information domain. Table 3. Features in information domain. Table 3. Features in information domain. Table 3. Features in information domain. Information Domain Feature Parameters I1 = MSE [1] I4 = PMMSE I7 = FI I2 = MSE [2] I5 = PFD I8 = ApEn I3 = MSE [3] I6 = HFD I9 = HST 3 2 Feature Selection Information Domain Feature Parameters I1 = MSE [1] I4 = PMMSE I7 = FI I2 = MSE [2] I5 = PFD I8 = ApEn I3 = MSE [3] I6 = HFD I9 = HST Information Domain Feature Parameters 3.2. Feature Selection 3.2. Feature Selection , K, k ̸= e (11) (11) 9 of 21 Sensors 2018, 18, 1379 (3) Calculate the variance factor of d(b) j as: v(b) j = sum( ue,j −uk,j ) min( ue,j −uk,j ) (12) v(b) j = sum( ue,j −uk,j ) min( ue,j −uk,j ) (12) (12) (4) Calculate the compensation factor as: (4) Calculate the compensation factor as: (4) Calculate the compensation factor as: δj = sum(vb j ) v(b) j (13) δj = sum(vb j ) v(b) j δj = sum(vb j ) v(b) j (13) (13) (5) Calculate the ratio d(b) j and d(w) j considering the compensation factor: αj = δj d(b) j d(w) j (14) (14) then normalize αj and obtaining the feature importance criteria: then normalize αj and obtaining the feature importance criteria: then normalize αj and obtaining the feature importance criteria: then normalize αj and obtaining the feature importance criteria: αj = αj sum(αj) (15) (15) A higher αj indicates that the corresponding feature j has greater importance. Features can be ranked in terms of the αj values in Equation (15) in descending order. This algorithm is referred to as Modified Distance Evaluation algorithm (MDE). Although the top ranked features have superior discriminative capability, they may suffer from high multi-collinearity, which refers to the non-independence among features [39]. Herein, the variance inflation factor (VIF) is used to avoid high collinearity. Assuming a training sample set X with J features X1, X2, . . . , XJ and class Y, the VIF of feature j is calculated as: VIFj = 1 1 −R2 j (16) (16) where R2 j is the R-squared value of the regression equation Xj = β0 + βX′, in which X′ contains all features except Xj. An improved algorithm combining MDE and VIF is presented in Algorithm 1 and is abbreviated as MDV (Modified Distance evaluation and variance inflation Factor). Set the selected future subset Fsub = ∅, j = 1; (1) Set the selected future subset Fsub = ∅, j = 1; (1) Set the selected future subset Fsub = ∅, j = 1; (2) Rank the J features in terms of the αj defined in Equation (15) in descending order. Set Fr to represent t index list of the ranked features. (1) Set the selected future subset Fsub = ∅, j = 1; Algorithm 1: MDV Algorithm. 3.2. Feature Selection 3.2. Feature Selection Feature extraction guarantees a wide coverage of the object descriptions from various aspects while feature selection ensures that a set of most salient descriptions can be utilized. For large-scale models, feature selection is of utter importance in computation reduction and efficiency improvement. The distance evaluation technique ranks the feature importance independent of the model used for classification, which belongs to the filter category as mentioned in the Introduction. Salient features result in minimum inner-class distances of the same class while have maximum margins for different classes. It has been widely used in fault diagnosis of rotating machinery [20,21,38]. Suppose a feature set has K conditions,  qi,k,j, i = 1, 2, . . . , Ik; k = 1, 2, . . . , K; j = 1, 2, . . . , J , where qi,k,j is the jth eigenvalue of the ith sample under the kth condition, Ik is the sample number of the kth condition, and J is the feature number of each sample. Totally Ik × K × J features are obtained in the feature set  qi,k,j . Herein, a modified distance evaluation algorithm is presented as follows: e average distance of the same condition samples: (1) Calculate the average distance of the same condition samples: dk,j = 1 Ik × (Ik −1)∑ Ik l,i=1 qi,k,j −ql,k,j , l, i = 1, 2, . . . , Ik, l ̸= i (8) (8) then obtain the average distance of K conditions: then obtain the average distance of K conditions: d(w) j = 1 K∑ K k=1 dk,j (9) d(w) j = 1 K∑ K k=1 dk,j (9) (2) Calculate the average eigenvalue of all samples under the same condition: (2) Calculate the average eigenvalue of all samples under the same condition: (2) Calculate the average eigenvalue of all samples under the same condition: (2) uk,j = 1 Ik∑ Ik i=1 qi,k,j (10) (10) then obtain the average distance between condition samples: then obtain the average distance between condition samples: then obtain the average distance between condition samples: d(b) j = 1 K(K −1)∑ K k,e=1 ue,j −uk,j , k, e = 1, 2, . . . 3.2. Feature Selection Add the first feature in Fr to Fsub, j = j + 1; (3) while j < J : calculate the VIFj of the jth feature in Fr with the features in Fsub; if VIFj < 10: add the jth feature in Fr to Fsub; The MDV algorithm describes the feature-subset selection for multi-class classification based on the filter method with the MDE and VIF. The threshold of 10 in MDV is an empirical value. A larger 10 of 21 Sensors 2018, 18, 1379 threshold will result in a higher correlation of the selected feature in Fr with the existing features in Fsub [23]. threshold will result in a higher correlation of the selected feature in Fr with the existing features in Fsub [23]. 4.1. Data Prepraration A series of wind tunnel experiments of the self-sensing composite wing were conducted under various angles of attack (AoAs) and freestream velocities at Stanford University. The open-loop wind tunnel with a square test section of 0.76 m by 76 m was used and a basis was designed to supported the composite wing allowing adjustments in the angle of attack (AoA). The composite wing dimension is outlined in Table 4. Table 4. Wing Dimension. Wing Geometry Chord 0.235 m Span 0.86 m Area 0.2 m2 Aspect ratio 3.66 Table 4. Wing Dimension. Compared with the size of the wind tunnel test section, the additional 0.1 m extension of the wing span was attached to the wing fixture. The AoAs range from 0 degree up to 18 degrees with an incremental step of 1 degree. At each degree, data were collected for all velocities ranging from 9 to 22 m/s (incremental step of 1 m/s). For experimental details, please refer to [2]. PZT signals reflect the coupled airflow-structural dynamics through the wing structural vibration and each time series contains coupled behavior with repeated patterns of a certain flight state. This study focuses on the usage of PZT sensor signals for flight state identification. In each experiment, the structural vibration responses (60,000 data points) were recorded from the PZT located near the wing root at 1000 Hz sampling frequency. For each flight state, data are prepared in two steps: (1) the entire signal of 60,000 data points is divided into 60 segments (1000 data points for each segment) to ensure enough samples for training while each segment has sufficient data points for feature extraction; (2) the first order difference and zero-mean are conducted for each sample sequence in order to eliminate the influence of zero drift. To evaluate the effectiveness of the proposed method and apply it for dangerous state pre-warning, two sets of data are collected for general flight state identification and stall detection and alerting. 4.2. General Flight State Identification To evaluate the effectiveness of the proposed method and apply it for dangerous state pre-warning, two sets of data are collected for general flight state identification and stall detection and alerting. (4) Stability selection is a kind of wrapper method, in which features are selected based on the established models using different subsets, model could be of various types and structures such as logistic regression, SVM, etc. By calculating the frequency of a feature ended up being selected as important from a feature subset being tested, powerful features are expected to have high scores close to 100%, weaker features will have lower score and the least useful ones will close to zero [44]. Herein, a randomized logistic regression is used as the selection model. state identification and stall detection and alerting. 4.2. General Flight State Identification The first data set includes PZT signals with a coarse resolution covering the range of 16 flight states corresponding to combinations of four AoAs (1, 5, 9, 13 degrees) and four airspeeds (10, 13, 16, 19 m/s). Four signal segments are shown in Figure 3 under a series of AoAs and a fixed airspeed of 10 m/s as an example Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. Figure 3. Indicative signals under a set of AoAs and a constant velocity of 10 m/s. 4.2. General Flight State Identification For experimental details, please refer to [2]. PZT signals reflect the coupled airflow-structural dynamics through the wing structural It measures the mutual dependence between variable X and Y. Features with low rankings are removed. incremental step of 1 degree. At each degree, data were collected for all velocities ranging from 9 to 22 m/s (incremental step of 1 m/s). For experimental details, please refer to [2]. PZT signals reflect the coupled airflow structural dynamics through the wing structural (2) SVM_L1 is one of the embedded methods, which selects salient features as part of the learning system [18]. Support Vector Machine (SVM) is a popular machine learning method based on structural risk minimization principle. It constructs a hyperplane that has the largest distance to the nearest training data points, which are so called support vectors. An appropriate separation can reduce the generalization error of the classifier [41]. L1 is a regularization item added to the loss function as |W|, where W standards for the parameter matrix of the learning model [42]. This is a penalty item to make the model sparse with fewer useful input dimensions. PZT signals reflect the coupled airflow-structural dynamics through the wing structural vibration and each time series contains coupled behavior with repeated patterns of a certain flight state. This study focuses on the usage of PZT sensor signals for flight state identification. In each experiment, the structural vibration responses (60,000 data points) were recorded from the PZT located near the wing root at 1000 Hz sampling frequency. For each flight state, data are prepared in two steps: (1) the entire signal of 60,000 data points is divided into 60 segments (1000 data points for each segment) to ensure enough samples for training while each segment has sufficient data points for feature extraction; (2) the first order difference and zero-mean are conducted for each sample (3) GBDT is a tree-based model belonging to the embedded category. It combines weak decision trees in an iterative manner based on gradient descent through additive training. Trees are added at each iteration with modified parameters learned in the direction of residual loss reduction [43]. for feature extraction; (2) the first order difference and zero mean are conducted for each sample sequence in order to eliminate the influence of zero drift. 4.2. General Flight State Identification The first data set includes PZT signals with a coarse resolution covering the range of 16 flight states corresponding to combinations of four AoAs (1, 5, 9, 13 degrees) and four airspeeds (10, 13, 16, 19 m/s). Four signal segments are shown in Figure 3 under a series of AoAs and a fixed airspeed of 10 m/s as an example. It is noticed that the flight state with AoA of 13 degrees and velocity of 10 m/s can be obviously identified since the amplitude of the voltage distinguishes it from other signals (it is because this flight state is close to the stall condition which will be discussed later). The second largest amplitude comes with 9 degrees which can be separated to a certain extent but already has overlaps with the rest two. In the study, the identification of the different flight states relies on the features selected by the developed method in Section 3. To compare the feature selection effectiveness, four other feature selection methods are employed including Univariate Feature Selection based on mutual information (UFS_m), Support Vector Machine with L1 regularization (SVM_L1), Gradient Boosted Decision Tree (GBDT) and Stability selection (STAB). These methods cover three main feature selection categories. A brief introduction is presented as follows: rs 2018, 18, 1379 11 of 2 11 of 21 Sensors 2018, 18, 1379 (1) UFS_m is a commonly used filter method. It performs test on each feature by evaluating the relationship between the feature and the response variable based on mutual information [40], which is defined as p(x y) Sensors 2018, 18, x 10 of 21 (1) UFS_m is a commonly used filter method. It performs test on each feature by evaluating the relationship between the feature and the response variable based on mutual information [40], which is defined as p(x y) Sensors 2018, 18, x 10 of 21 I(X, Y) = ∑y∈Y ∑x∈X p(x, y) log( p(x, y) p(x)p(y)) (17) e of the wind tunnel test section, the additional 0.1 m extension of the he wing fixture. The AoAs range from 0 degree up to 18 degrees with an (17) f the th an It measures the mutual dependence between variable X and Y. Features with low rankings are removed. incremental step of 1 degree. At each degree, data were collected for all velocities ranging from 9 to 22 m/s (incremental step of 1 m/s). It is noticed that the flight state with AoA of identified since the amplitude of the voltage dis 4.3. Application to Stall Detection and Identification From the wind tunnel experiments, the mean values of the signal energy for a series of AoAs (from 0 to 17 degrees) under four airspeeds (10, 13, 16, 19 m/s) are obtained and shown in Figure 4. Figure 4. Signal energy under various flight states. Figure 4. Signal energy under various flight states. Figure 4. Signal energy under various flight states. Figure 4. Signal energy under various flight states. It is noticed that the flight state with AoA of identified since the amplitude of the voltage dis 4.3. Application to Stall Detection and Identification Moreover, it is observed that at 12 degrees, the signal energy for some flight states has certain increase compared with the rest small angles. This degree is defined as the alert region as the transition between the safe region marked in light green and the critical stall region. When the self-sensing wing comes to this region, warnings should be provided to the flight control for angle reduction. The second data set covers a higher resolution of flight states (AoAs: 11, 12, 13 degrees, airspeeds: 10, 13, 16, 19 m/s) for critical states alerting. In aerodynamics, stall phenomenon is one of the dangerous conditions wherein a sudden reduction of the lift coefficient occurs as the angle of attack increases beyond a critical point. According to previous analysis [2], the signal energy can be used as an indicator of the lift loss of the self-sensing wing. From the wind tunnel experiments, the mean values of the signal energy for a series of AoAs (from 0 to 17 degrees) under four airspeeds (10, 13, 16, 19 m/s) are obtained and shown in Figure 4. 13 degrees with the high speed of 19 m/s, which is reflected in the red line with zero energy starting from 14 degrees. Therefore, we define the orange shaded area starting from 13 degrees as the stall region which should be avoided. Moreover, it is observed that at 12 degrees, the signal energy for some flight states has certain increase compared with the rest small angles. This degree is defined as the alert region as the transition between the safe region marked in light green and the critical stall region. When the self-sensing wing comes to this region, warnings should be provided to the flight control for angle reduction. The second data set covers a higher resolution of flight states (AoAs: 11, 12, 13 degrees, airspeeds: 10, 13, 16, 19 m/s) for critical states alerting. In aerodynamics, stall phenomenon is one of the dangerous conditions wherein a sudden reduction of the lift coefficient occurs as the angle of attack increases beyond a critical point. According to previous analysis [2], the signal energy can be used as an indicator of the lift loss of the self-sensing wing. It is noticed that the flight state with AoA of identified since the amplitude of the voltage dis 4.3. Application to Stall Detection and Identification p g g g ( flight state is close to the stall condition which will be discussed later). The second largest amplitude comes with 9 degrees which can be separated to a certain extent but already has overlaps with the rest two. In the study, the identification of the different flight states relies on the features selected by the developed method in Section 3. To compare the feature selection effectiveness, four other feature selection methods are employed including Univariate Feature Selection based on mutual information (UFS_m), Support Vector Machine with L1 regularization (SVM_L1), Gradient Boosted Decision Tree (GBDT) and Stability selection (STAB). These methods cover three main feature selection categories. The second data set covers a higher resolution of flight states (AoAs: 11, 12, 13 degrees, airspeeds: 10, 13, 16, 19 m/s) for critical states alerting. In aerodynamics, stall phenomenon is one of the dangerous conditions wherein a sudden reduction of the lift coefficient occurs as the angle of attack increases beyond a critical point. According to previous analysis [2], the signal energy can be used as an indicator of the lift loss of the self-sensing wing. From the wind tunnel experiments, the mean values of the signal energy for a series of AoAs (from 0 to 17 degrees) under four airspeeds (10, 13, 16, 19 m/s) are obtained and shown in Figure 4. A brief introduction is presented as follows: (1) UFS_m is a commonly used filter method. It performs test on each feature by evaluating the relationship between the feature and the response variable based on mutual information [40], which is defined as The signal energy variation with respect to the angle of attack is similar under four different airspeeds. It is noticed that for relatively low velocities (10 m/s, 13 m/s & 16 m/s), the significant increase occurs approximately after 14 degrees while for the relatively high speed (19 m/s), stall happens much early at 13 degrees. It should be noted that the data were stopped recording after 12 of 21 Sensors 2018, 18, 1379 [ ] 13 degrees with the high speed of 19 m/s, which is reflected in the red line with zero energy starting from 14 degrees. Therefore, we define the orange shaded area starting from 13 degrees as the stall region which should be avoided. ai pee I i o i e a o e a increase occurs approximately afte 5.1. General Flight State Identification happens much early at 13 degrees. It should be noted that the data were stopped recording after 13 degrees with the high speed of 19 m/s, which is reflected in the red line with zero energy starting The first data set with a relatively low resolution of 16 flight states is used to evaluate the performance of six feature selection methods, which include Univariate Feature Selection based on mutual information (UFS_m), Support Vector Machine with L1 regularization (SVM_L1), Gradient Boosted Decision Tree (GBDT) and Stability selection (STAB), Modified Distance Evaluation (MDE), and our proposed filter method Modified Distance Evaluation with Variance Inflation Factor (MDV). Feature rankings are obtained and the top 10 features for different methods are listed in Table 5 and their detailed expressions are listed in Appendix A. Table 5. Top 10 ranking matrix. Ranking UFS_m SVM_L1 GBDT STAB MDE MDV 1 F25 F41 F47 F47 F35 F35 2 F34 F43 F40 F12 F26 F30 3 F6 F39 F46 F21 F2 F5 4 F2 F25 F14 F20 F6 F28 5 F5 F46 F39 F19 F31 F42 6 F4 F19 F44 F18 F30 F45 7 F40 F33 F41 F17 F12 F41 8 F23 F13 F1 F16 F8 F46 9 F42 F44 F21 F15 F36 F14 10 F17 F10 F45 F14 F10 F23 Table 5. Top 10 ranking matrix. Sensors 2018, 18, 1379 13 of 21 13 of 21 It is observed from the table that the ranking results vary with the different methods. An intuitive evaluation is to simply visualize the features distribution under various flight states. For example, four features are plotted in Figure 5 including: F1 (mean value in time domain), F29 (spectrum kurtosis in frequency domain), F35 (spectrum power convergence in frequency domain), and F47 (Hurst Exponent in information domain). The x axis denotes the 16 flight states while the y axis is the feature value before normalization. The shaded area along each vertical line segment represents the feature distribution in a single flight state and each subplot of Figure 5 describes a feature distribution on 16 flight states. As mentioned in Section 3, F1 (mean value) has no effects in classification. Correspondingly, F1 has the highest overlap among flight states. Similarly, F47 has large overlaps which exhibits pool classification capability. Theoretically, the ranking of F1 and F47 should be low but they are ranked high in GBDT and STAB. ai pee I i o i e a o e a increase occurs approximately afte 5.1. General Flight State Identification Correlation between features by MDV (a) and MDE (b). can be seen that the feature subset through MDV sel mpared to the entire feature pool p y g y mparison, the overall collinearity of the features in MDV is much lower except for the small regio the top three. To visualize the feature selection performance by MDV, t-Distributed Stochastic Neighb mbedding (t-SNE) is employed which is a relatively new method of dimension reductio rticularly suitable for non-linear and high-dimensional datasets. It is a kind of manifold learnin hnique by mapping to probability distributions through affine transformation. For detaile gorithm, please refer to [45]. The 3D visualization by t-SNE is shown in Figure 7. The left figure e visualization using the entire feature pool while the right figure uses only top six featur (a) (b) gure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selecte atures Figure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selected features. p mparison, the overall collinearity of the fea he top three. To visualize the feature selection perf bedding (t-SNE) is employed which is ticularly suitable for non-linear and high- hnique by mapping to probability distr orithm, please refer to [45]. The 3D visual visualization using the entire feature p (a) much lower except for the sm DV is much lower except for the small regi by MDV, t-Distributed Stochastic Neighb vely new method of dimension reducti nal datasets. It is a kind of manifold learni hrough affine transformation. For detail t-SNE is shown in Figure 7. The left figure the right figure uses only top six featur (b) (b) (a) ure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selec Figure 7. 3D visualization by t-SNE: (a) t-SNE using original features; (b) t-SNE using selected features. Further, machine learning techniques are used to quantify the flight state identification proce each feature selection method, the most salient 6 features are obtained as model inputs and light states are set as model outputs. Five supervised learning models are employed includ istic Regression (LR), Support Vector Machine (SVM), Naïve Bayes (NB), Random Forest (R Neural Network (NN). ai pee I i o i e a o e a increase occurs approximately afte 5.1. General Flight State Identification In comparison, F30 and F35 show smaller overlap and thus have better classification performance. This may provide some physical insights of the effectiveness of different feature selection methods. sors 2018, 18, x 13 Figure 5. Pool and superior features against 16 flight states. Figure 5. Pool and superior features against 16 flight states. igure 5. Pool and superior features against 16 flight stat Figure 5. Pool and superior features against 16 flight states. The last column MDV in Table 4 is an improvement of MDE for preventing high collinearity amine the effects of the proposed algorithm, Correlation analysis is conducted for MDV and M shown in Figure 6. The last column MDV in Table 4 is an improvement of MDE for preventing high collinearity. To examine the effects of the proposed algorithm, Correlation analysis is conducted for MDV and MDE as shown in Figure 6. It is obvious that the top 10 features selected by MDE are highly correlated with each other. In comparison, the overall collinearity of the features in MDV is much lower except for the small region of the top three. To visualize the feature selection performance by MDV, t-Distributed Stochastic Neighbor Embedding (t-SNE) is employed which is a relatively new method of dimension reduction particularly suitable for non-linear and high-dimensional datasets. It is a kind of manifold learning technique by mapping to probability distributions through affine transformation. For detailed algorithm, please refer to [45]. The 3D visualization by t-SNE is shown in Figure 7. The left figure is the visualization using the entire feature pool while the right figure uses only top six features obtained by MDV. It can Sensors 2018, 18, 1379 14 of 21 14 of 21 be seen that the feature subset through MDV selection exhibits better classification effects compared to the entire feature pool. p p g g y examine the effects of the proposed algorithm, Correlation analysis is conducted for MDV and MDE as shown in Figure 6. (a) (b) Figure 6. Correlation between features by MDV (a) and MDE (b). Figure 6. Correlation between features by MDV (a) and MDE (b). 2018, 18, x 1 ned by MDV. It can be seen that the feature subset through MDV selection exhibits fication effects compared to the entire feature pool. (a) 018, 18, x (b) (b) (a) Figure 6. Correlation between features by MDV (a) and MDE (b). Figure 6. ai pee I i o i e a o e a increase occurs approximately afte 5.1. General Flight State Identification Cross-validation is used in each model and the average accuracy va ve tests is computed to reduce the unbalance influence between training and testing samples uld be noted that since the objective of the case study is to compare the effects of different feat ction methods instead of obtaining the optimized parameter setting for each machine learn del to achieve the highest accuracy level, default parameter settings in Python scikit-learn pack LR, SVM, NB and RF are used and remain the same for all feature selection methods while the parameter setting is as follows: {hidden layer size = 20 solver = ‘lbfgs’ activation functio Further, machine learning techniques are used to quantify the flight state identification process. For each feature selection method, the most salient 6 features are obtained as model inputs and the 16 flight states are set as model outputs. Five supervised learning models are employed including Logistic Regression (LR), Support Vector Machine (SVM), Naïve Bayes (NB), Random Forest (RF), and Neural Network (NN). Cross-validation is used in each model and the average accuracy value of five tests is computed to reduce the unbalance influence between training and testing samples. It should be noted that since the objective of the case study is to compare the effects of different feature selection methods instead of obtaining the optimized parameter setting for each machine learning model to achieve the highest accuracy level, default parameter settings in Python scikit-learn package for LR, SVM, NB and RF are used and remain the same for all feature selection methods while for NN, the parameter setting is as follows: {hidden layer size = 20, solver = ‘lbfgs’, activation function = ’relu’, learning rate = 0.001, maximum iteration = 100}. The identification results are shown in Figure 8. 15 of 21 Sensors 2018, 18, 1379 ’relu’, learning Fi 8 Figure 8. Identification accuracy against different feature selection methods. Figure 8. Identification accuracy against different feature selection methods. Figure 8. Identification accuracy against different feature selection methods. Figure 8. Identification accuracy against different feature selection methods. It can be observed that our proposed method MDV achieves the highest identification accuracy in all five machine learning models and particularly, there is a significant improvement in Logistic It can be observed that our proposed method MDV achieves the highest identification accuracy in all five machine learning models and particularly, there is a significant improvement in Logistic Regression. ai pee I i o i e a o e a increase occurs approximately afte 5.1. General Flight State Identification This demonstrates the superior effectiveness of MDV. The comparison between MDV and MDE shows that a group of individually powerful features with low collinearity can lead to better results. 5.2. Stall Detection and Alerting So far, the developed MDV algorithm has achieved the best performance in feature selection and the final flight state identification accuracy is up to 100%. Herein, the second dataset with higher resolution is used for the application of stall detection and alerting. Similarly, totally 47 features as discussed in Section 3 are extracted and the most salient 6 features are selected by MDV as model inputs. A neural network is employed with the same parameter settings as the first case. The split rule is 80% samples for training and 20% samples for testing. The classification report is shown in Table 6 including three criteria: Precision, Recall and F1-score. Precision is the ratio of correctly predicted positive observations to the total predicted positive observations while Recall is the ratio of correctly predicted positive observations to the all observations in the actual class. F1-Score is the weighted average of Precision and Recall: F1-Score = 2 * (Recall * Precision)/(Recall + Precision) [46]. Safe, Alert, and Stall regions are divided with corresponding flight states. The overall identification accuracy is 98%. Table 6. Classification report. States ID AoA (deg) Speed (m/s) Precision Recall F1-Score Safe 1 11 10 0.92 1.00 0.96 2 11 13 0.92 1.00 0.96 3 11 16 1.00 0.92 0.96 4 11 19 1.00 0.92 0.96 Alert 5 12 10 1.00 1.00 1.00 6 12 13 1.00 0.92 0.96 7 12 16 0.92 1.00 0.96 8 12 19 1.00 1.00 1.00 Stall 9 13 10 1.00 1.00 1.00 10 13 13 1.00 1.00 1.00 11 13 16 1.00 1.00 1.00 12 13 19 1.00 1.00 1.00 Table 6. Classification report. Table 6. Classification report. Sensors 2018, 18, 1379 16 of 21 16 of 21 To facilitate detailed analysis, a normalized confusion matrix is presented in Figure 9. Each row of the matrix represents the test samples in a true class label while each column indicates the samples in a predicted class label [47]. As can be observed from Table 6, for stall states (ID: 9, 10, 11, 12), Recall values all equal to 100%, meaning that all the critical states can be successfully identified and there is no safety risk. In terms of alert states (ID: 5, 6, 7, 8), Recall value of State 6 is 0.92, which means 92% samples in State 6 are correctly predicted. 5.2. Stall Detection and Alerting By examining the 6th row in the confusion matrix, the rest 8% samples are misclassified as State 1, which is in the safe region. This situation may lead to dangerous results since the wing is already in the alert states yet there is no warning. From the other perspective, the precision value of State 7 is 0.92, which indicates that among all samples predicted as State 7, there are 8% samples actually belonging to State 4 as shown in the 7th column of the confusion matrix. This value can be interpreted as the false-alarm ratio that the wing flying in the safe region yet receives a false alert. For safe states (ID: 1, 2, 3, 4), the misclassified samples are for State 3 and State 4, in which 8% samples of State 3 are predicted as State 2 while 8% samples of State 4 are identified as State 7, which is the false alarm. Sensors 2018, 18, x 16 of 21 For safe states (ID: 1, 2, 3, 4), the misclassified samples are for State 3 and State 4, in which 8% samples of State 3 are predicted as State 2 while 8% samples of State 4 are identified as State 7, which is the false alarm. Sensors 2018, 18, x 16 of 21 Figure 9. Confusion matrix of flight state identification. Figure 9. Confusion matrix of flight state identification. Figure 9. Confusion matrix of flight state identification. Figure 9. Confusion matrix of flight state identification. Further, we select the different number of features from the modified distance evaluation (MDE) method and use the same neural network structure for training and testing. The comparison on the overall identification accuracy between MDV and various MDE is shown in Figure 10. The x axis denotes number of top ranked features selected. Further, we select the different number of features from the modified distance evaluation (MDE) method and use the same neural network structure for training and testing. The comparison on the overall identification accuracy between MDV and various MDE is shown in Figure 10. The x axis denotes number of top ranked features selected. It can be seen that if we use the same number of input as MDV, features selected by MDE lead to a pool result of 0.33. The identification accuracy reaches the same level as MDV until the number of top ranked features selected from MDE increases to 20. 5.2. Stall Detection and Alerting This shows that our proposed method MDV is able to address the collinearity problem and uses fewer features to achieve superior performance with a considerable model complexity reduction. 17 of 21 the axis Sensors 2018, 18, 1379 method and us overall identifi Figure 10. Identification accuracy between MDV and various MDE. Figure 10. Identification accuracy between MDV and various MDE. Figure 10. Identification accuracy between MDV and various MDE. Figure 10. Identification accuracy between MDV and various MDE. to a pool res f k 6. Conclusions of top ranked features selected from MDE increases to 20. This shows that our proposed method MDV is able to address the collinearity problem and uses fewer features to achieve superior performance with a considerable model complexity reduction. This paper focuses on the feature engineering in structural vibration signals obtained from a self-sensing composite wing through wind tunnel experiments. In addition to common statistical features from the time domain and frequency domain, complex features from the information domain inspired by electroencephalograph analysis and mechanical fault diagnosis are also extracted, some of which exhibit good classification ability. A novel filter feature selection method (MDV) is proposed by combining the modified distance evaluation (MDE) algorithm and the variance inflation factor (VIF). MDE is able to select individually powerful features but cannot address high collinearity. VIF is then applied for each top ranked feature to remove highly correlated elements. Results from both general flight state identification and stall detection & alerting demonstrate that this method can reduce the model complexity with fewer features while maintain a high identification accuracy. Knowledge can be gained by calculating the limited important features obtained by MDV efficiently for flight state identification using light-weight machine learning models. This would save considerable efforts in feature extraction and feature selection by manpower and has the potential to provide autonomous control with real-time flight state monitoring. For multi-sensor utilizations, this method can be applied to each sensor and ensemble methods can be developed to fuse multi-source results for more robust identification. Author Contributions: X.C. analyzed the data and developed the feature selection method; F.K. and F.-K.C. designed the self-sensing wing and performed the wind tunnel experiments; Q.W. provides feature extraction algorithms; H.R. and F.-K.C. coordinated the research and revised the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 51705242), Shanghai Sailing Program (Grant No. 16YF1404900), and the U.S. Air Force Office of Scientific Research (AFOSR) program “Avian-Inspired Multi-functional Morphing Vehicles” under grant FA9550-16-1-0087 with Program Manager Byung-Lip (“Les”) Lee. Acknowledgments: The authors would like to thank Pengchuan Wang, Ravi Gondaliya, Jun Wu and Shaobo Liu for their help during the wind tunnel experiments. Also, the authors would like to acknowledge the support of Lester Su and John Eaton in the wind tunnel facility at Stanford University. Conflicts of Interest: The authors declare no conflict of interest. References 1. NASA Fly-By-Feel Systems Represent The Next Revolution In Aircraft Controls. Available online: https://www.nasa.gov/centers/dryden/news/X-Press/aerovations/2011/fly-by-feel.html (accessed on 16 May 2017). 2. Kopsaftopoulos, F.; Nardari, R.; Li, Y.H.; Chang, F.K. 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In Proceedings of the 10th International Workshop on Structural Health Monitoring, Stanford, CA, USA, 1–3 September 2015. 12. Roy, S.; Lonkar, K.; Janapati, V.; Chang, F.-K. A novel physics-based temperature compensation model for structural health monitoring using ultrasonic guided waves. Struct. Heal. Monit. Int. J. 2014, 13, 321–342. [CrossRef] 13. Huang, R.; Zhao, Y.; Hu, H. Wind-Tunnel Tests for Active Flutter Control and Closed-Loop Flutter Identification. AIAA J. 2016, 54, 1–11. [CrossRef] 14. Pang, Z.Y.; Cesnik, C.E.S. Strain state estimation of very flexible unmanned aerial vehicle. In Proceedings of the 57th AIAA/ASCE/AHS/ASC Structures, Structural Dynamics, and Materials Conference, San Diego, CA, USA, 4–8 January 2016. 15. Appendix A The expressions of selected features by different feature selection methods are shown in Table A1. 18 of 21 Sensors 2018, 18, 1379 Table A1. Top 10 ranking feature expressions. R UFS_m SVM_L1 GBDT STAB MDE MDV 1 t25 = ∑N n=1 (x(n)−t1)9 N·t29 I3 = MSE[3] I9 = HST I9 = HST f10 = f6 f5 f10 = f6 f5 2 f9 = ∑K k=1 frk2y(k) √ ∑K k=1 y(k)∑K k=1 frk4y(k) I5 = PFD I2 = MSE[2] t12 = ∑N n=1|x(n)−t1| N f1 = ∑K k=1 y(k) N f5 = ∑K k=1 ( frk·y(k)) ∑K k=1 y(k) 3 t6 = q ∑N n=1 (x(n))2 N I1 = MSE[1] I8 = ApEn t21 = ∑N n=1 (x(n)−t1)5 N·t25 t2 = q ∑N n=1(x(n)−t1)2 N t5 = ∑N n=1(x(n)−t1)2 N 4 t2 = q ∑N n=1(x(n)−t1)2 N t25 = ∑N n=1 (x(n)−t1)9 N·t29 t14 = t6 t8 t20 = ∑N n=1 (x(n)−t1)4 N·t24 t6 = q ∑N n=1 (x(n))2 N f3 = ∑K k=1 (y(k)−f1)3 K(√ f2) 3 5 t5 = ∑N n=1(x(n)−t1)2 N I8 = ApEn I1 = MSE[1] t19 = ∑N n=1 (x(n)−t1)3 N·t23 f6 = q ∑K k=1 ( f rk−f5)2y(k) K I4 = PMMSE 6 t4 = ∑N n=1 (x(n)−t1)4 N t19 = ∑N n=1 (x(n)−t1)3 N·t23 I6 = HFD t18 = t4 t64 f3 = ∑K k=1 (y(k)−f1)3 K(√ f2) 3 I7 = FI 7 I2 = MSE[2] f8 = r ∑K k=1 f rk4y(k) ∑K k=1 f rk2y(k) I3 = MSE[3] t17 = t3 t63 t12 = ∑N n=1|x(n)−t1| N I3 = MSE[3] 8 t23 = ∑N n=1 (x(n)−t1)7 N·t27 t13 = t9 t6 t1 = ∑N n=1 x(n) N t16 = t9 t7 t8 = ∑N n=1|x(n)| N I8 = ApEn 9 I4 = PMMSE I6 = HFD t21 = ∑N n=1 (x(n)−t1)5 N·t25 t15 = t9 t8 f11 = ∑K k=1 ( f rk−f5)3y(k) K· f63 t14 = t6 t8 10 t17 = t3 t63 t10 = min(x(n)) I7 = FI t14 = t6 t8 t10 = min(x(n)) t23 = ∑N n=1 (x(n)−t1)7 N·t27 19 of 21 19 of 21 Sensors 2018, 18, 1379 References Kohavi, R.; John, G.H. 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